Sustained Energy for Enhanced Human Functions and Activity [1 ed.] 0128054131, 9780128054130

Sustained Energy for Enhanced Human Functions and Activity addresses the basic mechanistic aspects of energy metabolisms

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Sustained Energy for Enhanced Human Functions and Activity [1 ed.]
 0128054131, 9780128054130

Table of contents :
Cover
Sustained Energy for Enhanced Human Functions and Activity
Copyright
Dedication
List of Contributors
Preface
Section 1: Introduction
1. Information Theory and the Thermodynamic Efficiency of Biological Sorting Systems: Case Studies of the Kidney and of Mitoch ...
Introduction
A Summary of the Working of the Kidney
The Paradox of the Thermodynamic Efficiency of the Kidney
Maxwell's Demon, Its Exorcism, and the Entropic and Energetic Cost of Sorting
The Kidney as a Maxwell's Demon
Mitochondrial Adenosine 5′-Triphosphate (ATP)-Synthase as a Maxwell's Demon
Closing Remarks
Acknowledgments
References
2. Roles of AMP, ADP, ATP, and AMPK in Healthy Energy Boosting and Prolonged Life Span
Introduction
Adenosine Monophosphate/5′-Adenylic Acid
Adenosine Diphosphate/Adenosine Pyrophosphate
Adenosine Triphosphate
5′-Adenosine Monophosphate-Activated Protein Kinase
Cell Signaling
Mechanistic Target of Rapamycin/Mammalian Target of Rapamycin/FK506-Binding Protein 12-Rapamycin-Associated Protein 1
Role of Mechanistic Target of Rapamycin/Mammalian Target of Rapamycin in Widely Studied Diseases
Autophagy
Endoplasmic Reticulum Stress (Unfolded Protein Response)
Mitochondrial Stress
Existing Energy Boosters
Energy Boosting in Clinical Medication and Their Mechanisms
Possibilities of Other Medicinal Herbals in Ayurveda
Advanced Research Strategies for Developing Healthy Energy Boosters
Acknowledgments
References
3. An Overview of Nitrite and Nitrate: New Paradigm of Nitric Oxide
Nitric Oxide Biochemistry and Physiology
Endothelial Dysfunction and Loss of NO Production
l-Arginine Supplementation
Nitrate–Nitrite–Nitric Oxide Pathway
Summary
References
4. An Overview on Nitric Oxide and Energy Metabolism
Introduction
Biochemistry of Nitric Oxide
Regulation of Mitochondrial Respiration
Direct Effects of Nitric Oxide on Mitochondrial Respiration
Indirect Effect of Nitric Oxide on Mitochondrial Respiration
Nitric Oxide Regulates Energy Metabolism and Body Composition
Nitric Oxide and Oxygen Consumption in Association With Physical Activity and Fitness
Nitric Oxide Governs Metabolism of the Proximate Principles of Food
Conclusion
References
5. Antioxidants and Mitochondrial Bioenergetics
Introduction
Experimental Studies
Therapeutic Potential of Antioxidants
Mosquito-Borne Diseases and Cancer
Clinical Significance of Charnoly Body Theranostics
Clinical Significance of Charnoly Body Formation in Zika Virus and Other Diseases
Personalized Nanotheranostics
Fetal Alcohol Syndrome and Zika Virus Disease
Autophagy Versus Charnolophagy
Phagolysosome Versus Charnolophagosome
Clinical Significance of Charnoly Body and Charnolophagy
Metallothioneins Provide Mitochondrial Neuroprotection
Therapeutic Potential of Antioxidants
Significance of Charnolopharmacotherapy
Conclusion
Acknowledgments
References
Further Reading
6. Protein, Carbohydrates, and Fats: Energy Metabolism
Introduction
Carbohydrates
Sugar in Foods
Sugar in Drinks
Specific Dynamic Action of Carbohydrates
Glycemic Index
Obesity and Carbohydrates
Protein
Protein–Energy Malnutrition
Lipids
Fat and Energy in the Diet and Obesity
Dietary Fatty Acid and Lipid Metabolism
Diet-Induced Thermogenesis/Specific Dynamic Action
Energy Metabolism and Obesity
Conclusion
Abbreviations
Acknowledgments
References
Further Reading
Section 2: Botanicals and Herbal Indgredients and Marine Nutraceuticals
7. Role of Selected Medicinal Plants in Sports Nutrition and Energy Homeostasis
Introduction
Diverse Medicinal Plants
Citrus aurantium
Rhodiola rosea
Schisandra chinensis
Tribulus terrestris
Vitis vinifera
Withania somnifera
Conclusions
References
Further Reading
8. Withania somnifera: Ethnobotany, Pharmacology, and Therapeutic Functions
Introduction
Chemical Composition
Toxicologic Studies of Withania somnifera
Pharmacokinetic Profile of Withania somnifera
Neuroprotective Effects of Withania somnifera
Anti-Parkinson Effects of Withania somnifera
Anti-Alzheimer Effects of Withania somnifera
Antiischemic and Antihypoxic Effects of Withania somnifera
Cardioprotective Effects of Withania somnifera
Anticancer Effects of Withania somnifera
Antiinflammatory Effects of Withania somnifera
Antimicrobial Effects of Withania somnifera
Antiarthritic Effects of Withania somnifera
Antistress Effects of Withania somnifera
Antidiabetic Effects of Withania somnifera
Aphrodisiac Effects of Withania somnifera
Conclusion
Acknowledgments
References
9. An Overview on Tribulus terrestris in Sports Nutrition and Energy Regulation
Introduction
Aphrodisiac Activity
Supplementation in Sport
Contamination and/or Adulteration of Dietary Supplements
Recommendations for Sport Nutrition With Supplements
Risk of Violating Antidoping Rules
References
10. The Use of Maca (Lepidium meyenii) for Health Care: An Overview of Systematic Reviews
Introduction
Methods
Data Sources
Study Selection
Type of Study
Type of Participants
Type of Intervention
Type of Control Intervention
Type of Outcome Measures
Data Extraction
Assessing the Methodological Quality of Systematic Reviews
Results
Discussion
References
11. An Overview on Rhodiola rosea in Cardiovascular Health, Mood Alleviation, and Energy Metabolism
Introduction/Overview
Active Constituents
Rhodiola rosea and Exercise Performance
Rhodiola rosea Combined With Other Substances
Mechanisms of Action
How to Take
Side Effects
Conclusions and Gap Analysis
References
Further Reading
12. Energy and Health Benefits of Shilajit
Introduction
Chemistry
Safety Studies
Research Studies
Human Studies
Animal Studies
In Vitro Studies
Discussion and Summary
References
13. An Overview on Ginseng and Energy Metabolism
Review
Introduction of Ginseng
Active Chemical Constituents
Ginsenosides/Saponins
Polysaccharides
Polyacetylenes
Alkaloids
Glucosides
Phenolic Acid
Others
Pharmacological Effects
Anticancer Activity
Antidiabetic Activity
Lipid-Regulating and Antithrombotic Activities
Immunoregulatory Activity
Wound and Ulcer Healing Activity
Neuroregulation Activity
Health Effects
Antiaging Activity
Antifatigue
Others
Conclusions
References
14. Glycyrrhiza glabra (Licorice): Ethnobotany and Health Benefits
Effect on Respiratory System
Hepatoprotective Effect
Effect on Cardiovascular System
Immunity Regulation and Antiinflammation Effects
Antitumor Activities
Breast Cancer
Hepatocellular Carcinoma
Prostate Cancer
Effect on Gastrointestinal Tract
Effect on Endocrine System
Side Effects and Cautions
Conclusion and Perspective
Abbreviations
References
15. An Overview of Yohimbine in Sports Medicine
Introduction
Mechanisms of Action
What Are the Needs for Sports Enhancement?
Theoretical Reflections
Animal Model Research
Human Correlates
Metabolic Effects in Humans
Obesity
Normal Males
Athletes
Proven and Potential Toxicities
Would Yohimbine Be Unique in Its Activity for Sports?
Has Yohimbine Been Adequately Studied for Sports?
References
16. Black Ginger Extract Enhances Physical Fitness Performance and Muscular Endurance
Introduction
Mechanisms of Kaempferia parviflora Extract Underlying Increases in Physical Fitness by Kaempferia parviflora Extract
Effects of Kaempferia parviflora Extract on Physical Fitness and Muscular Endurance in Mice
Clinical Trials
Conclusion
References
17. Role of Marine Nutraceuticals in Cardiovascular Health
Introduction to Angiotensin-I–Converting Enzyme Inhibition
Marine-Derived Angiotensin-I–Converting Enzyme Inhibitors
Bioactive Peptides
Chitooligosaccharide Derivatives
Phlorotannins
Fucoxanthin
Concluding Remarks
References
18. Royal Jelly in Medicinal to Functional Energy Drinks
Introduction
Royal Jelly
Chemical Composition
Standardization
Safety
Energy-Enhancing Actions
Antifatigue Effect
Sustainability of Physical Performance
Formulations
Medicinal Energy Drinks
Soft Energy Drinks
Marketing Challenges
Conclusion
References
19. Role of Caffeine in Sports Nutrition
Introduction
An Overview on Caffeine Metabolism
Absorption
Metabolism
Factors Affecting Metabolism and Pharmacokinetics
Summary of Caffeine Metabolism
Effects of Caffeine on Endurance Performance
Effects of Caffeine on Strength Performance
Maximal Strength
Strength Endurance
Effects of Caffeine on Power Performance
Caffeine Ingestion: Source and Dosage
Mechanisms of Action of Caffeine
Side Effects, Health Risks, and Cautions
Summary
References
Further Reading
20. Beneficial Roles of Caffeine in Sports Nutrition and Beverage Formulations
Caffeine: History
Sources and Consumption
Mechanisms of Action
Effects on Performance
Caffeine Responders
Caffeine Habituation
Caffeine Abstinence Period
Timing of Caffeine Administration
Caffeine Dosage
Caffeine Effects and Training Status/Specificity of Training
Summary
References
Section 3: Amino Acids
21. Amino Acids and Energy Metabolism: An Overview
Introduction
Amino Acid Metabolism
Glucogenic Amino Acids and Ketogenic Amino Acids
Catabolism of Amino Acid Carbon Skeletons
Amino Acids That Produce Pyruvic Acid
Amino Acids That Produce α-Ketoglutaric Acid via Glutamic Acid
Amino Acids That Produce Succinyl Coenzyme A
Amino Acids That Produce Acetyl Coenzyme A or Acetoacetic Acid
Amino Acids That Produce Fumaric Acid
Amino Acids That Produce Oxaloacetate
Control of Body Temperature and Energy Metabolism
Inhibition of Core Temperature Hypothermia and Synthesis of Skeletal Muscle Proteins by Administration of Amino Acids Under ...
Amino Acids That Contribute to the Reduction of Hypothermia
Conclusion
References
22. Branched Chain Amino Acids and Sports Nutrition and Energy Homeostasis
Biological Bases of Branched Chain Amino Acids
Branched Chain Amino Acids and Athletic Performance
Central Fatigue
Muscle Damage and Subjective Perception of Exertion
Anabolic Response in Muscle Recovery
Scientific Opinions and Guidelines of Governing Bodies and Institutions
Effects of Administration of Branched Chain Amino Acids in Different Sports
Practical Applications and Concluding Remarks
References
23. HMB Supplementation: Clinical and Performance-Related Effects and Mechanisms of Action
Introduction
Metabolism
Pharmacokinetics
Clinically Related Effects
Ergogenic-Related Effects
Mechanisms of Action
Increased Sarcolemmal Integrity
Increased Metabolic Efficiency
Upregulation of Insulin-like Growth Factor-I
Increased Activation of Satellite Cells and Myogenic Factors
Stimulation of Protein Synthesis Through Mammalian Target of Rapamycin Signaling Pathway Activation
Suppression of Proteolysis by Inhibition of Proteolytic Systems
Conclusions
References
Further Reading
Section 4: Antioxidants and B-Vitamins
24. Antioxidants and Vitamins: Roles in Cellular Function and Metabolism
Antioxidants
Endogenous Antioxidants
Ascorbic Acid
Vitamin E
Thiol Antioxidant
Nucleotide-Reduced Equivalent Molecules
Other Endogenous Antioxidants
Exogenous Antioxidants
Dietary Polyphenols
Phenolic Acids
Flavonoids
Stilbenes
Lignans
Antioxidant Enzymes
Vitamins
Effects of Antioxidant and Vitamin Supplementation
Oxidative Stress
Reactive Oxygen Species
Mitochondria
Peroxisomes
Cytochrome P450
Consequences of Antioxidant and Vitamin Deficiency: Oxidative Stress in Cellular System
Oxidative Damage to Lipids
Oxidative Damage to Proteins
Oxidative Damage to Nucleic Acid
Antioxidant and Oxidative Stress Hypothesis of Aging
Conclusion
Acknowledgments
References
Section 5: Design a Novel Beverage Formulation
25. Salient Features for Designing a Functional Beverage Formulation to Boost Energy
Introduction
Designing a Functional Energy Beverage
Energy Beverage Formulations
Additives, Thickeners, Soluble Dietary Fiber, and Carbohydrates
Energy Formulations and Future Trends
Conclusion
References
Section 6: Safety and Toxicity
26. Caffeine-Containing Energy Drinks/Shots: Safety, Efficacy, and Controversy
Introduction
Caffeine's Prevalence in Society and the Emergence of CEDS
Pharmacology of Caffeine
Safety of CEDS
Efficacy of CEDS
Controversy
Conclusion
References
Further Reading
27. An Overview on the Constituents and Safety of Energy Beverages
Introduction
Epidemiology
Constituents
Extracardiac Effects
Neurologic
Gastrointestinal
Renal
Endocrine
Psychiatric
Cardiac Effects
Vascular Wall Effects
Endothelial Dysfunction
Aortic Dissection
Hemodynamics
Elevated Blood Pressure
Increased Heart Rate
Postural Orthostatic Tachycardia Syndrome
Electrical Effects
Outline placeholder
Increased Corrected QT Interval
Supraventricular Arrhythmia
Ventricular Arrhythmia
Coronary Disease
Outline placeholder
Coronary Artery Spasm
Sudden Cardiac Death
Myocardial Ischemia
Heart Muscle Disease
Outline placeholder
Takotsubo (Stress) Cardiomyopathy
Effects in Specific Populations
Long-Term Effects
Conclusions
References
28. Interactions of Commonly Used Prescription Drugs With Food and Beverages
Introduction
Taking Medications on a Full or Empty Stomach
Food–Drug Interactions
Effects of Calcium on Antibiotics and Other Medications
Soy-Containing Products and Anticoagulants
Garlic and Anticoagulants and Protease Inhibitors
Effects of Foods Containing Vitamin K on Warfarin Anticoagulation
Effects of Ginger on Warfarin Anticoagulation
Effects of Caffeine on Drug Metabolism
Effects of Tyramine on Monoamine Oxidase Inhibitor Therapy
Drug–Beverage Interactions
Effects of Acidic Beverages on Drug Absorption
Effects of Citrus Juice on Drug Metabolism
Effects of Alcohol on Drug Metabolism and Efficacy
Effects of Cranberry on Drug Metabolism
Green Tea Reduces Anticoagulant Effects
Conclusion
References
Section 7: Commentary and Future Directions
Novel Energy Beverage Formulations: Efficacious, Healthy, and Safe
References
Energy: A High School Student's Perspective: Fossil Fuel to Renewables
Fossil Fuel to Renewables
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Z
Back Cover

Citation preview

Sustained Energy for Enhanced Human Functions and Activity Edited by

Debasis Bagchi

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2017 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-805413-0 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre G. Wolff Acquisition Editor: Megan R. Ball Editorial Project Manager: Billie Jean Fernandez Production Project Manager: Julie-Ann Stansfield Designer: Matthew Limbert Typeset by TNQ Books and Journals

Dedicated to my respected and beloved Raj Bhaiya, Mr. Raj Kumar Kaushal, Chandigarh, India, who is always full of energy and inspiration.

List of Contributors Muzamil Ahmad Indian Institute of Integrative Medicine (CSIR), Srinagar, India; Academy of Scientific and Innovative Research, Indian Institute of Integrative Medicine (CSIR), Jammu, India

Asif Ali Aligarh Muslim University, Aligarh, India Carlos E. Neves Amorim Federal University of Maranhão (UFMA), São Luís-MA, Brazil

Dawn E. Anderson Taylor University, Upland, IN, United States Debasis Bagchi University of Houston College of Pharmacy, Houston, TX, United States; Cepham Research Center, Piscataway, NJ, United States

Manashi Bagchi Dr. Herbs LLC, Concord, CA, United States Karan Bhatti The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, United States

Nathan S. Bryan Baylor College of Medicine, Houston, TX, United States Christian E.T. Cabido Federal University of Maranhão (UFMA), São Luís-MA, Brazil Jason M. Cholewa Coastal Carolina University, Conway, SC, United States Nevio Cimolai University of British Columbia, Vancouver, BC, Canada Neil D. Clarke Coventry University, Coventry, United Kingdom Nawab J. Dar Indian Institute of Integrative Medicine (CSIR), Srinagar, India; Academy of Scientific and Innovative Research, Indian Institute of Integrative Medicine (CSIR), Jammu, India

Amitava Das The Ohio State University Wexner Medical Center, Columbus, OH, United States

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LIST OF CONTRIBUTORS

Michael Duncan Coventry University, Coventry, United Kingdom Elia Salinas García Nutriresponse, Madrid, Spain Lucas Guimarães-Ferreira Federal University of Espirito Santo, Vitoria, Brazil Bill J. Gurley University of Arkansas for Medical Sciences, Little Rock, AR, United States

Safia Habib Aligarh Muslim University, Aligarh, India Zhang Han Tianjin University of Traditional Chinese Medicine, Tianjin, China Kohsuke Hayamizu Yokohama University of Pharmacy, Yokohama, Japan John P. Higgins The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, United States; Memorial Hermann Ironman Sports Medicine Institute, Houston, TX, United States; Lyndon B. Johnson General Hospital, Houston, TX, United States; HEARTS (Houston Early Age Risk Testing & Screening Study), Houston, TX, United States

Daniel A. Jaffe United States Military Academy, West Point, NY, United States Raj K. Keservani Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, India Rajesh K. Kesharwani NIMS University, Janupur, India Se-Kwon Kim Pukyong National University, Busan, Republic of Korea Tae-Hun Kim Kyung Hee University, Seoul, Republic of Korea Rick Kingston University of Minnesota, Bloomington, MN, United States Marijana Zovko Koncic University of Zagreb, Zagreb, Croatia  ski National Centre for Sports Medicine, Warsaw, Poland Jarosław Krzywan Temitope O. Lawal University of Illinois at Chicago, Chicago, IL, United States; University of Ibadan, Ibadan, Nigeria

Hye Won Lee Korea Institute of Oriental Medicine, Daejeon, Republic of Korea Myeong Soo Lee Korea Institute of Oriental Medicine, Daejeon, Republic of Korea

LIST OF CONTRIBUTORS

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Fernanda Lima-Soares Federal University of Maranhão (UFMA), São Luís-MA, Brazil

Gail B. Mahady University of Illinois at Chicago, Chicago, IL, United States Lou Massa City University of New York, New York, NY, United States Chérif F. Matta Mount Saint Vincent University, Halifax, NS, Canada; Dalhousie University, Halifax, NS, Canada; Saint Mary’s University, Halifax, NS, Canada; Université Laval, Quebec, QC, Canada

Moinuddin Aligarh Muslim University, Aligarh, India Barbara Morawin University of Zielona Gora, Zielona Gora, Poland Hiroyoshi Moriyama The Japanese Institute for Health Food Standards, Tokyo, Japan

Arundathi Nair Laramie High School, Laramie, WY, United States Aurora Norte Nursing Department, University of Alicante, Alicante, Spain Kassiana Araújo Pessôa Federal University of Maranhão (UFMA), São Luís-MA, Brazil

Andrzej Pokrywka University of Zielona Gora, Zielona Gora, Poland; National Centre for Sports Medicine, Warsaw, Poland

Yufeng Qin National Engineering Technology Research Center of Glue of Traditional Medicine, Dong’e, China; Shandong Dong-E-E-Jiao Co., Ltd., Dong’e, China

Nishikant A. Raut University of Illinois at Chicago, Chicago, IL, United States; Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur, India

Sashwati Roy The Ohio State University Wexner Medical Center, Columbus, OH, United States

Wenwen Ru National Engineering Technology Research Center of Glue of Traditional Medicine, Dong’e, China; Shandong Dong-E-E-Jiao Co., Ltd., Dong’e, China

José Miguel Martínez Sanz Nursing Department, University of Alicante, Alicante, Spain

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LIST OF CONTRIBUTORS

Chandan K. Sen The Ohio State University Wexner Medical Center, Columbus, OH, United States

Sushil Sharma Saint James School of Medicine, St. Vincent, West Indies Hiroshi Shimoda Oryza Oil & Fat Chemical Co. Ltd., Ichinomiya, Japan Kanhaiya Singh The Ohio State University Wexner Medical Center, Columbus, OH, United States

Prabhakar Singh Veer Bahadur Singh Purvanchal University, Jaunpur, India Isabel Sospedra Nursing Department, University of Alicante, Alicante, Spain Sidney J. Stohs Creighton University School of Pharmacy and Health Professions, Omaha, NE, United States

Anand Swaroop Cepham Research Center, Piscataway, NJ, United States Sheila L. Thomas University of Arkansas for Medical Sciences Library, Little Rock, AR, United States

Kazuya Toda Oryza Oil & Fat Chemical Co. Ltd., Ichinomiya, Japan Eric T. Trexler University of North Carolina, Chapel Hill, NC, United States Yamini B. Tripathi Banaras Hindu University, Varanasi, India Dongliang Wang National Engineering Technology Research Center of Glue of Traditional Medicine, Dong’e, China; Shandong Dong-E-E-Jiao Co., Ltd., Dong’e, China

Sheila M. Wicks Rush University, Chicago, IL, United States Isuru Wijesekara University of Sri Jayewardenepura, Nugegoda, Sri Lanka Wang Xiaoying Tianjin University of Traditional Chinese Medicine, Tianjin, China Durgavati Yadav Banaras Hindu University, Varanasi, India Wang Yu Tianjin University of Traditional Chinese Medicine, Tianjin, China Nelo E. Zanchi Federal University of Maranhão (UFMA), São Luís-MA, Brazil

LIST OF CONTRIBUTORS

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 -Lacny University of Zielona Gora, Zielona Gora, Poland Agnieszka Zembron Haojun Zhang Qilu University of Technology, Jinan, China Xiangshan Zhou National Engineering Technology Research Center of Glue of Traditional Medicine, Dong’e, China; Shandong Dong-E-E-Jiao Co., Ltd., Dong’e, China

Preface The term “energy” represents the strength, vigor, enthusiasm, vitality, tenacity, power, or zest required for sustained physical or mental activity. Sustainable or sustained energy fulfills the needs of the present as well as future needs. Sustained energy has two major components: (1) renewable energy and (2) energy efficiency. Thus, the provision of continuous energy resources should be distributed in a way such that they meet the needs of the present requirements of the body without compromising the ability to meet the future needs of the body. Human energy comes from the foods that we consume and the liquids/beverages that we drink. The three main nutrients used for energy are carbohydrates, proteins, and fats. The human body can also use protein and fats for energy once carbohydrates have been depleted. Once food is consumed, the human body breaks down nutrients into smaller components and absorbs them to use as energy or fuel. This process is known as metabolism. This book will discuss the physiological aspects of the human body and highlight how nutraceuticals/ functional foods, botanicals and herbal supplements, structurally diverse antioxidants, B-vitamins, and diverse amino acids, including branched chain amino acids (BCAA), marine nutraceuticals, caffeine, and vital nutrients, can boost human energy safely and effectively for enhanced functions. Regulating energy level is a fundamental process in every living organism. Adenosine triphosphate (ATP), a key, vital component of sustained energy, should be maintained at optimal levels in the cells in order to regulate the metabolic process. Maintaining a balance between energy supply and energy expenditure is very important for the whole body to function effectively. On the other hand, failing to regulate energy metabolism results in a huge increase in the prevalence of metabolic disorders. The human body needs to maintain an optimal level of ATP for routine energy production and physical performance. This supply of ATP is absolutely vital for the heart, lungs, and skeletal muscles to have all of the energy required for maximum strength and endurance output. The ATP level is also decreased during myocardial ischemiaereperfusion injury. Recent studies have demonstrated that proper nutrition, including antioxidants, vitamins, micronutrients, and selected amino acids, can enhance the energy level in the body and protect the heart as well as lungs from ischemiaereperfusion injury. Selected nutraceuticals and functional foods, including standardized extracts of Withania somnifera, Tribulus terrestris, Lepidium meyenii (maca), Trigonella foenumgraecum, Glycyrrhiza glabra (liquorice), Kaempferia parviflora (black ginger), Camellia sinensis (black and green tea), Rhodiola rosea, Schisandra chinensis, shilajit, ginseng, green coffee bean, cocoa leaves, white willow, yohimbine, guarana, and cola nuts; caffeine in requisite amounts; amino acids, especially BCAA and including leucine, isoleucine, and valine; as well as phenylalanine, taurine and glutamine, chromium(III), magnesium, zinc, and copper supplements, are well demonstrated to promote sustained energy, reduce stress and exhaustion, improve endurance and immune function, decrease anxiety, and exhibit antiinflammatory, nerve-relaxant, and adaptogenic properties.

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PREFACE

This book will address the basic mechanistic aspects of energy metabolism, the chemistry, biochemistry, and pharmacology of a variety of botanical or herbal supplements, micronutrients, antioxidants, structurally diverse amino acids, and other nutraceuticals, which have demonstrated a boost in and sustained functional energy. There are a number of chapters on caffeine so that readers may thoroughly understand the pros and cons. Also, a chapter highlighting the detrimental interactions of nutraceuticals and functional foods with commonly used prescriptions medications is included in this book. The Commentary and Future Directions section summarizes the earlier chapters, highlights paradigm shifts in the field, and envisions future directions. Finally, a talented high school student, Arundathi Nair, who received numerous state and national awards and recognitions, provided a section of her innovative thoughts on energy. Today, it is very important to maintain good health and an optimal quality of life with effective energy. This new book will discuss the basic biochemistry and physiology of the human body and will demonstrate how exercise, proper nutrition, and selected ingredients can help humans to remain healthy, happy, and survive with sustained energy. A growing body of evidence demonstrates that nutraceutical and functional foods may naturally normalize this delicate balance. This book demonstrates how proper nutrient balance is useful to boost sustained energy. Finally, I extend my sincere regards and gratitude to the esteemed authors for their valuable contributions to this book. I also wish to sincerely acknowledge Nancy Maragioglio, Megan R. Ball, Billie Jean-Fernandez, Namrata Bagchi, and Anirban Misra for their tremendous encouragement, helpful suggestions, criticisms, and technical support. Debasis Bagchi, PhD, MACN, CNS, MAIChE

1

Information Theory and the Thermodynamic Efficiency of Biological Sorting Systems: Case Studies of the Kidney and of Mitochondrial ATP-Synthase

Chérif F. Matta1, 2, 3, 4, Lou Massa5 1 MOUNT SAINT V INCENT UN IVERSITY, HALIFAX, NS, CANADA; 2 DALHOUSIE UNIVERSITY, H A L I F A X, NS , C ANADA; 3 S AI N T M AR Y’S UNIVERSITY, HALIFAX, NS, CANADA; 4 UNIV ER SIT É L AV A L , Q U E BE C , QC , C A N AD A ; 5 CITY UNIVERSITY OF NEW Y ORK, NEW Y ORK, NY, UNITED STATES

Physiologically, the work represented by the composition of the final urine is an almost negligible fraction of the work it is known the kidney must do. . [It] represents only about one per cent of the probable metabolism of the kidney as calculated from the oxygen consumption. This is not to say that the efficiency of the kidney is only one percent. . [T]he kidney’s efficiency may be very high indeeddit is the thermodynamic approach that is only one per cent efficient.1 Smith (1951)

Finally comes the universality of the gravitational law, and the fact that it extends over such enormous distances that Newton, in his mind, worrying about the solar system, was able to predict what would happen in an experiment of Cavendish, where Cavendish’s little model of the solar system, two balls attracting, has to be expanded ten million million times to become the solar system, Then ten million million times larger again we find galaxies attracting each other by exactly the same law. Nature uses only the longest threads to weave her patterns, so each small piece of her fabric reveals the organization of the entire tapestry.1 Feynman (1965)

1

Emphasis/bold type by the current authors.

Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00001-6 Copyright © 2017 Elsevier Inc. All rights reserved.

3

4 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Introduction It happens that the kidney, which is an organ, and the mitochondrion, which is an organelle, are both physical realizations of Maxwell’s demon. To understand how this occurs, an analysis of the operation of the demon construct is given as it applies to the kidney and the mitochondrion as information-gathering regulatory devices. Remarkably, information is not free, but costs a minimum of kT ln 2 per bit irrespective of the mechanism at play. H.A. Johnson and K.D. Knudsen pioneered an accounting of the cost of information and resolved the long-held paradox that the kidney’s thermodynamic efficiency appears to be significantly lower than that of comparable organs. Applying this approach to mitochondrial adenosine 5’-triphosphate (ATP)-synthase, the efficiency of oxidative phosphorylation is shown to approach 90% rather than the oft-quoted value of w60%. The parallel of the kidney and the mitochondrion as two realizations of Maxwell’s demon of substantially different sizes is a biological illustration of an almost poetic observation of Feynman to the effect that nature’s patterns in the small are found to be repeated in the large, and are reminiscent of repetitions characteristic of fractals.

A Summary of the Working of the Kidney The kidney is the principal organ for osmoregulation in higher organisms. It regulates osmolarity by first filtering water and small solutes and then selectively reabsorbing what is needed, leaving out the rest as waste in the urine. The “functional unit” of the kidney is the nephron, which constitutes its repeating building block (Fig. 1.1); there are on the order of 106 nephrons in a kidney. Arterial blood flowing into the nephron’s glomerulus (via the afferent arteriole) gets partly filtrated in Bowman’s capsule pushed through the pores by arterial blood pressure. The filtrate containing ions and small molecules is then channeled to the proximal convoluted tubule. The remainder of the blood is channeled away through the efferent arteriole (Fig. 1.1). Many of the ions (e.g., Naþ) and small molecules (e.g., glucose, amino acids) in the lumen of the proximal convoluted tubule are important for the organism and need to be reabsorbed. Reabsorption to the nearby blood capillaries occurs through the tubule cells (forming the wall of the tubule) against a concentration gradient (that is, going from low to high concentration), and hence is done actively with consumption of energy in the form of adenosine 50 -triphosphate (ATP). The manner by which this is achieved is described next. A cell lining the lumen of the tubule, a tubule cell, has a side facing the tubule lumen called the luminal surface and a side facing the interstitial medium away from the tubule called the basal surface. Naþ ions penetrate a given tubule cell through its luminal surface in the direction of the concentration gradient via cotransporters that drag along with it other crucial solutes such as glucose and amino acids into the cell. Once inside a tubule cell, the Naþ ions need to cross the opposite (far) side (that is, the basal surface) to reach the interstitial fluid. The movement of Naþ ions crossing the basal

Chapter 1  Information Theory and Thermodynamic Efficiency

5

Proximal Distal convoluted convoluted Bowman’s tubule tubule capsule Glomerulus

Cortex

Medulla

Loop of Henle Collecting tubule

to ureter

FIGURE 1.1 Simplified diagram of a nephron showing its location within the tissue of a human kidney. www. bioweb.uncc.edu; http://player.slideplayer.com/38/10766277/#.

cell toward the interstitial fluid is in a direction opposite to the concentration gradient, and hence necessitates the expenditure of energy (active transport). That active transport is catalyzed by the basal surface’s membrane-bound Naþ-Kþ pump (Naþ-Kþ-ATPase) that uses one ATP to pump three Naþ ions out of the cell, each Naþ being cotransported with one Kþ and two Cl ions (Naþ/2Cl/Kþ cotransport system) (Greger and Schlatter, 1983). This pumping maintains low intracellular Naþ concentration encouraging more to be reabsorbed from the filtrate residing in the lumen of the tubule. Glucose, amino acids, and other small crucial molecules have their concentration build up inside the cell (they are cotransported with Naþ from the lumen across the luminal surface) and diffuse passively on the other (basal) side down their concentration gradients. The filtrate then enters the loop of Henle, first into the descending limb and then into the ascending limb of this loop. The ascending limb is primarily permeable to small ions such as Naþ, Kþ, and Cl, which are actively transported, again using ATP, into the renal medulla to maintain its hypertonicity. Although permeable to salts, the ascending limb of the loop of Henle is impermeable to water in contrast to the descending limb which is permeable to water but not to salts.

6 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Because the medullary interstitium is hypertonic, water leaks from the filtrate in the descending limb of the loop of Henle into the medulla by passive transport under osmotic pressure created by the ascending limb. This is the mechanism nature has fine-tuned to regain a large portion of the water (99%) that has been filtered in Bowman’s capsule, because ATP-coupled transporters are optimized to operate on ions rather than water. The fluid contained in the upper segments of the loop of Henle (whether the descending or ascending limbs) near the outer border of the medulla have a concentration on the order of 0.10.3  103 osmol/L. The fluid in the bottom segment, the farthest from the outer surface of the medulla, has a significantly higher osmolarity to the tune of 1.2  103 osmol/L. The fluid then passes to the distal convoluted tubule, where further and final reabsorption of ions, small molecules, and water occurs before fluid from different nephrons is eventually pooled into collecting ducts that pass through the medulla again. The collecting ducts have an adjustable permeability to water that is modulated by the action of antidiuretic hormone (ADH), also called vasopressin, and secreted by the posterior pituitary gland. ADH is secreted in the bloodstream when osmoreceptors located in the hypothalamus sense a rise in the tonicity of the blood’s plasma, e.g., after a salty meal, in which case the organism must retain water to regain the osmotic balance for its optimal vital functions. When the levels of ADH are high, permeability of the collecting ducts to water increases, allowing its passive diffusion into the medulla in view of its hypertonicity. This reabsorption in the medullary interstitium has the effect of concentrating the fluid (urine) before it is collected into the urinary bladder for eventual excretion.

The Paradox of the Thermodynamic Efficiency of the Kidney The thermodynamic efficiency of an organ or of an organelle can be defined as the ratio of the work output w to the free energy input (DG), that is: h¼

w . DG

(1.1)

The free energy input is usually taken as the product of the free energy of hydrolysis of ATP (DGATP ) by the amount of ATP used to produce w. The accepted value of DGATP under standard biochemical conditions is about 60 kJ/mol but under cellular conditions DGATP is about 50 kJ/mol (Garrett and Grisham, 2013; Gue´rin, 2004). For a reversible heat engine (only for a heat engine and not just any type of engine), efficiency is governed by the second law which, according to Kelvin’s equation, cannot exceed: h¼1

Tcold ; Thot

(1.2)

where the second term is the ratio of the absolute temperatures (in K) of the cold and hot reservoirs. Because most if not all known biochemical processes occur under

Chapter 1  Information Theory and Thermodynamic Efficiency

7

approximately isothermal conditions, Eq. (1.2) would imply a null efficiency (Pereira and Cuesta, 2016) which is nonsensical. This is so because biological systems are not thermal engines but rather electrochemical and/or regulatory ones with theoretical efficiencies that can approach 100% if efficiency is measured with respect to the conversion of DG (Pereira and Cuesta, 2016). If, however, efficiency refers to DH, that is: w ; DH

(1.3)

ε ; ðDG=DHÞ

(1.4)

ε¼

then: h¼

which implies that efficiency in this case can be higher than 100% for entropy-generating processes (DS > 0) (Pereira and Cuesta, 2016; Nelson et al., 2011). The partial osmotic work w exerted by the kidney owing to one given solute (in the mixture of solutes constituting urine) can be estimated by an equation derived in 1905 by von Rhorer, (1905) [or by its improved form for ions (Eggleton et al., 1940)]: 

w ¼ nRT

  cplasma  curine cplasma ;  ln curine curine

(1.5)

where n is the total number of moles of this solute extracted from blood to urine when the blood plasma concentration is cplasma and the concentration of this solute in urine is curine. Using this equation and after several simplifications and assumptions [described in detail in Eggleton et al. (1940), examples of which are the use of electrical conductivity to estimate the total electrolyte concentration or the use of vapor pressure measurements to estimate the total molar concentration], the estimated work is slightly underestimated, but by no more than w5%e15% (Eggleton et al., 1940). In a nutshell, this is how the energy output is evaluated experimentally. To evaluate efficiency, we now need the energy input. The chemical energy use in isolated dog kidney preparations was estimated by Eggleton et al. (1940) by measuring oxygen consumption. This is accomplished by measuring the oxygen tension in the arterial blood in both the afferent and efferent arterioles, with the drop in O2 tension being the measure of its use by the kidney (Eggleton et al., 1940). The energetic conversion factor is taken to be an energy of 5 kcal for every liter of consumed O2 [5 kcal/ L(O2)]. With the osmotic work output estimated as described earlier and with an energy input obtained from measuring the change in O2 tension, the kidney’s thermodynamic efficiency was found to be on the order of 1% (Eggleton et al., 1940), a value that corroborates earlier estimates such as that of Borsook and Winegarden (1931). These efficiency evaluations of isolated dog kidney are also in close agreement with estimates performed on anaesthetized human subjects (Clark and Barker, 1951). An energy transduction efficiency of 1% is extremely low compared with, say, muscles with a transduction efficiency of the order of 45%e66% (taking into account both the

8 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

heat emitted and the mechanical work produced versus ATP consumed by the muscle) (Wilkie, 1975). Commenting on their calculated efficiency of human kidneys, Clark and Baker (1951) list among their principal conclusions that: These data suggest that the bulk of oxygen consumed by the kidney is used for purposes not measured by external work. In making this statement, the authors were possibly referring to their previous observation that a large expenditure of energy is spent to maintain steady-state conditions in the kidney (Clark and Barker, 1951). Their remark however, does not preclude other contributions to this disproportionate energy expenditure compared with the osmotic work output of the kidney. This is exactly what Johnson and Knudsen (JK) argued in their 1965 article in Nature (Johnson and Knudsen, 1965), and (Johnson) in two following publications (Johnson, 1970, 1987). It all hinges on how thermodynamic efficiency is defined. JK cite Wiener’s observation that an electronic valve is energetically inefficient because it produces no mechanical or chemical work; its function is mainly to allow an oscillating current to pass in only one direction (Wiener, 1961). Most of the energy consumed by the valve is “wasted” in heating the filament to emit electrons that are channeled in only one direction. The electronic valve has not been designed to perform mechanical or chemical work but rather it constitutes an open control loop device: that is, a cybernetic rather than a mechanical or electrochemical machine. Control devices can have substantial energetic expenditures in sorting actions such as unidirectional allowance or forbiddance of the passage of a given ion through a channel, the selection of a particular solute from a solution with multiple components, etc. To perform this type of sorting, the device first has to “acquire information” about the target that allows the control device to distinguish the “right” target from the “wrong” one. In this aspect, such a control device can be regarded as a realization of Maxwell’s demon, and hence must experience an increase in entropy that needs to be dissipated to recover the demon’s initial state for a subsequent measuring act.

Maxwell’s Demon, Its Exorcism, and the Entropic and Energetic Cost of Sorting Maxwell discovered that the individual molecular speeds (v) at any given temperature T differ greatly from one molecule to another. The probability distribution of molecular speeds at a certain temperature is known as the MaxwelleBoltzmann distribution, which, in modern standard notation, is written:    m 32 mv2 PðvÞ ¼ 4p v2 exp ; 2kT 2pkT

where k is the Boltzmann constant and m is the molecular mass of the ideal gas.

(1.6)

Chapter 1  Information Theory and Thermodynamic Efficiency

0.0025

9

T = 200 K

Probability (v)

0.0020

0.0015 T = 700 K

0.0010

0.0005

0.0000 0

500 1000 Molecular speed (v, ms-1)

1500

FIGURE 1.2 MaxwelleBoltzmann distribution of molecular speeds of O2 molecules (molecular weight ¼ 32 g/mol) at two different temperatures.

Fig. 1.2 displays this distribution for O2 molecules at two different temperatures. Enshrined within this distribution is the second law of thermodynamics itself because if we bring a hot and cold body into contact, molecular collisions at their interface (solid) or in the bulk (fluid mixture) will cause those of the hotter body to decelerate and those of the colder body to move faster on average. That is, heat flows spontaneously from the hotter to the colder body as required by the second law. The distribution can be split at the median speed so that 50% of the molecules will be slower (colder) and 50% faster (hotter) than this speed. If we could somehow have a pair of ultrafine tweezers to pick and separate these molecules into a “colder group” and a “hotter group,” we would have introduced order, reduced entropy of the system, and created a system capable of performing useful work for nothing. This is the gist of what became known as Maxwell’s demon’s apparent violation of the second law of thermodynamics. In a section entitled “Limitation of the Second Law of Thermodynamics,” Maxwell (1872) writes in his classic 1872 monograph Theory of Heat: One of the best established facts in thermodynamics is that it is impossible in a system enclosed in an envelope which permits neither change of volume nor passage of heat, and in which both the temperature and the pressure are everywhere the same, to produce any inequality of temperature or of pressure without the expenditure of work. This is the second law of thermodynamics, and it is undoubtedly true as long as we can deal with bodies only in mass, and have no power of perceiving or handling the separate molecules of which they are made up. But if we conceive a being whose faculties are so sharpened that he can follow every molecule in its course, such a being, whose attributes are still as essentially finite as our own, would be able to do what is at present impossible to us. For we have seen that the molecules in a vessel full of air at uniform

10 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

temperature are moving with velocities by no means uniform, though the mean velocity of any great number of them, arbitrarily selected, is almost exactly uniform. Now let us suppose that such a vessel is divided into two portions, A and B, by a division in which there is a small hole, and that a being, who can see the individual molecules, opens and closes this hole, so as to allow only the swifter molecules to pass from A to B, and only the slower ones to pass from B to A. He will thus, without expenditure of work, raise the temperature of B and lower that of A, in contradiction to the second law of thermodynamics. The entire thought experiment described by Maxwell hinges on the act of “seeing,” i.e., observing the molecules’ paths as they approach the supernatural being. The being, later called by William Thomson (Lord Kelvin) “Maxwell’s demon,” needs to determine the speed (momentum) of the incoming molecules to determine if they are “cold” or “hot” and also continuously measure their positions to open the shutter to allow them to the “correct” portion, A or B, respectively. With today’s core knowledge that includes quantum mechanics, one has to consider Heisenberg’s indeterminacy principle in such acts of observation of gas molecules because according to quantum mechanics position x and velocity (momentum p ¼ mv) are noncommuting observables and hence cannot be simultaneously observed with infinite precision. These two incompatible observables can be determined only within a combined indeterminacy that does not exceed the order of the reduced Planck’s constant Z ¼ ðh=2pÞ: Z Dx Dp  . 2

(1.7)

Although this principle is a fundamental limitation on the precision of any observation irrespective of any experimental setup, it is insignificant in most cases of relevance to Maxwell’s thought experiment. Heisenberg’s indeterminacy principle can be more important in the case of light atoms and high pressures but not for heavier atoms, the low pressures typically found in biology, or molecules with masses that are several thousands of times larger than the mass of an electron. [See the discussion of this point on page 163 of Brillouin (2004) and the references cited therein.] Thus, ignoring any role of quantum indeterminacy, we depict Maxwell’s thought experiment in the diagram of Fig. 1.3, which is self-explanatory. Maxwell’s apparent contradiction of the second law was addressed by several workers for over 60 years (Brillouin, 2004; Szilard, 1929; Leff and Rex, 1990, 2003; Bennett, 1987) until it was resolved by L. Szilard in his important publication in 1929. Szilard (1929) realized that the demon’s act of “seeing” or “observing” these molecules, a first indispensable step to enable it to sort them, will result in the rapid increase in the entropy of the demon, and that as a result the entropy of the entire closed system (container plus demon) will increase as required by the second law. To use Brillouin’s words, the demon has been “exorcised” (Brillouin, 2004). Let us now briefly retrace Brillouin’s argumentation, exorcism, and derivation of the principal result (Brillouin, 2004).

Chapter 1  Information Theory and Thermodynamic Efficiency

11

FIGURE 1.3 A representation of Maxwell’s demon. The demon observes the molecules in the two compartments and allows fast (hot) molecules (red circles) to go only from the right partition to the left partition and the slow (cold) molecules (blue squares) only from left to right. After some time, all “hot” molecules will be found at the left and all “cold” molecules at the right. By observing the particles and sorting them based on speed using a frictionless door the demon appears to have violated the second law creating order out of disorder.

Consider an isolated thermodynamic system (a system that does not exchange matter or energy with its surroundings) that consists of a gas at constant temperature T0 in a partitioned container and a demon operating a shutter at the hole in the partition, as displayed in Fig. 1.3. Included in the system is also an electric bulb (with a battery as its power supply) that the demon will use to illuminate the incoming molecules to “see” them. To produce light, the filament is heated by the current flowing from the battery and is brought to a temperature T1 > T0. The light produced in this manner will have a frequency such that it is visible against the blackbody radiation thermal background (kT0), the condition being: hn1 > kT0 /b h

hn1 > 1: kT0

(1.8)

As it produces current, the battery itself produces energy E but no change in the entropy of the system, although the filament loses entropy to the gas by radiating E: DSfilament ¼ 

E . T1

(1.9)

12 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

The energy radiated by the filament is absorbed by the gas at the lower temperature T0, and hence the entropy of the gas increases by: DSgas ¼ þ

E . T0

(1.10)

In the absence of any action by the demon, the overall change in the entropy of this isolated system must be positive, i.e., DS ¼ DSgas þ DSfilament ¼

E E  > 0: T0 T1

(1.11)

The demon must detect (absorb, by whatever mechanism) at least one scattered photon from the molecule. Hence, given the condition expressed by inequality Eq. (1.8), the demon’s entropy will increase by: DSdemon ¼

  hn1 hn1 ¼k ¼ kb. T0 kT0 |fflfflfflffl{zfflfflffl ffl}

(1.12)

b>1

Before any of these operations, the initial entropy of system S0 can be expressed in terms of the total number of indistinguishable microstates P0 according to Boltzmann’s formula: S0 ¼ k ln P0 .

(1.13)

Once the demon has gained information about the system, that information can be used to decrease the entropy of the gas by more completely specifying its microscopic configuration or “microstate” (which is termed, in older parlance, “complexion”). Stated differently, the information gained by the demon decreases the number of possible microstates, say by p. Hence, the reduced number of microstates is now: P1 ¼ P0  p;

(1.14)

and the resulting change in the entropy of the system owing to the gain of information is: DSinformation ¼ S  S0 ;

(1.15a)

  p . DSinformation ¼ k ln P1  k ln P0 ¼ k lnðP0  pÞ  k ln P0 ¼ k ln 1  P0

(1.15b)

Remembering the Taylor series expansion: lnð1  xÞ ¼ 

N X xn n¼1

n

;

1  x < 1;

(1.16)

and because p 0; DStotal ¼ DSdemon þ DSinformation ¼ k b  P0

(1.18)

Chapter 1  Information Theory and Thermodynamic Efficiency

13

because b > 1 (Eq. 1.8) and p/P0 < 1, as required to satisfy the second law. This remarkable result is a generalization of Carnot’s second law of thermodynamics, which can now be stated in a more suggestive notation as: DS0  I  0;

(1.19)

because information entropy has the reverse sign of thermodynamic entropy. Acquiring information by the demon while reducing the entropy of the gas by sorting hot and cold molecules produces more entropy within the demon so that the overall change complies with the second law. Because entropy and energy are closely related, the production of entropy in the demon is tantamount to absorption of heat. This is made clear by writing the definition of the change in entropy: Final Z

state

DS ¼

dQ ; T

(1.20)

Initial state

where dQ indicates an infinitesimal quantity of heat that is an inexact differential, as indicated by the d symbol. Every binary decision the demon undertakes (allowing a cold molecule to the right or a hot one to the left) halves the total number of possible microstates, and Eq. (1.13) is modified to (Johnson, 1987): S ¼ k ln

  P0 ; 2

(1.21)

so that the entropy change in the gas per binary decision is: S  S0 ¼ k ln

  P0  k ln P0 ¼ k ln 2: 2

(1.22)

Brillouin (2004) [and before him, (Szilard, 1929)] proceed to show that there exists a minimum principle that is a law of nature (analogous to the Heisenberg indeterminacy principle) irrespective of the particular mechanism by which the sorting or information gathering is accomplished. In Brillouin’s words: every physical measurement requires a corresponding entropy increase, and there is a lower limit, below which the measurement becomes impossible. This limit corresponds to a change in entropy of the order of k, Boltzmann’s constant. A more accurate discussion will prove later that the exact limit is k ln 2, or approximately 0.7 k for 1 bit of information obtained. As Gabor states it: [D. Gabor, M. I. T. Lectures, 1951] “We cannot get anything for nothing, not even an observation.”2 To be able to continue functioning, the demon must dissipate his gained entropy outside the system, perhaps in the form of heat to the surroundings, which is possible 2

The bold type by the current authors emphasizes Gabor’s statement.

14 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

only if the system is no longer isolated. Overall, if the system is to continue operating, it must dissipate the energy from the demon. The amount of information necessary to achieve one binary sorting decision is 1 bit (e.g., the amount of information upon receiving a message that has a binary message such as yes/no, 1/0, heads/tails, etc.). Gaining 1 bit of information is accompanied by a minimal increase in the entropy given by: DSmin ¼ k ln 2;

(1.23)

in J/K (SI units), or equivalently, a gain of εmin ¼ T DSmin ¼ kT ln 2

(1.24)

of heat [in joules per bit (SI units)], which cannot be converted to any useful work and must be dissipated by the system to regain its initial state (Brillouin, 2004; Szilard, 1929). We term the energy spent in recognition the “sorting work output” (SWO) because SWO must be taken into account in any efficiency calculation of a regulatory apparatus, organ, or organelle. Thus a Maxwell’s demon is essentially a device converting negative entropy [or “negentropy,” a word coined by Schro¨dinger in What Is Life? (Schro¨dinger, 1944)], into information. The negative entropy the demon converts is by radiating light to shine on the molecules. The demon then converts this negentropy into information, reducing the entropy of the system by reducing its configurational Boltzmann entropy [Eqs. (1.13) and (1.14)]. The demon uses the information to reduce the gas’ entropy but inevitably increasing its own entropy, completing Brillouin’s cycle (Brillouin, 2004): negentropy/information/negentropy

(1.25)

In an important article that appeared in 1961, to erase 1 bit of information from a computer’s memory requires the expenditure of at least the same minimum amount of energy as in Eq. (1.24) (Landauer, 1961; Smith, 2008). This limit is again completely independent of the mechanism of information erasure and is also a fundamental principal of nature. In Table 1.1 we provide the energetic cost of 1 bit of information (kT ln2) at various temperatures (some extreme temperatures as well as temperatures of typical relevance to biochemistry). The Landauer principle places a limit on the speed of any future supercomputer owing to excessive heat production even though today’s computers are still far from reaching the Landauer limit. There may be another limit on computer speed, reflecting quantum uncertainty. Because 1 bit of information is associated with a minimum energy below which it cannot be read or erased (listed in Table 1.1 for a few selected temperatures), that energy minimum dictates the lifetime of the interaction between the observed physical system and the observing demon.

Chapter 1  Information Theory and Thermodynamic Efficiency

15

Table 1.1 Temperature Dependence of the Energy Equivalent of the Bit (εmin) and of the Uncertainty in the Characteristic Time to Discern 1 Bit of Information (Dt) t o

εmin ðT Þ ¼ kT In 2

T

( C)

(K)

(J)

(eV)

270

3

2:9  10

0

273

2:6  1021

25

298

37

310

100

373

3000 30,000

23

Dt z ℏ/2kT ln 2 (kcal/mol)

4

(s)

1:8  10 0.016

4:1  10 0.38

1:8  1012

21

0.018

0.41

1:9  1014

21

0.019

0.43

1:8  1014

21

0.022

0.51

1:5  1014

w3300

20

3:2  10

0.20

4.5

1:7  1015

w30,300

2:9  1019

1.80

42

1:8  1016

2:9  10 3:0  10 3:6  10

3

2:0  1014

The timescale for reading or erasing 1 bit of information can be found by applying Heisenberg’s timeeenergy indeterminacy relation: Z DE Dt  ; 2

(1.26)

where the product of the uncertainties in energy and in the characteristic time must exceed 1/2 of the reduced Planck’s constant. Using εmin as a measure of the order of magnitude of the uncertainty in energy, we get: Dt 

Z ; 2kT ln 2

(1.27)

with representative values illustrated in Table 1.1. The values listed in this table show that at biological temperature (2537oC) the temporal uncertainty in the acquisition of 1 bit is of the order of 1014 s (w10 fs), which is consistent with a fast chemical reaction, as expected in the combination of two reactants in the process of molecular recognition. From Table 1.1 it is clear that the cost of 1 bit of information is small (less than 0.5 kcal/mol at biologically relevant temperatures such as the human body temperature of 37 C). Finally, we also provide what we term the mass equivalent of the bit. Given the special relativity masseenergy equivalence, one can calculate the smallest mass equivalent to the minimum energetic cost of information. At a few different temperatures, 1 terabyte of information (8  1012 bits): m¼

kT ln 2  8  1012 bits=terabyte ; c2

(1.28)

is equivalent to a mass of 2:3  104 , 2:3  102 , 0.23, and 2.3 mg at temperatures of 3, 3  102 , 3  103 , and 3  104 K, respectively. The mass equivalent of the bit at room temperature is 2:8  1021 kg/bit (at T ¼ 293K).

16 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

The Kidney as a Maxwell’s Demon Johnson and Knudsen (1965), and Johnson (1970, 1987), demonstrated that the low thermodynamic efficiency of the kidney, alluded to earlier, hinges on how its efficiency is defined. If one defines the efficiency of the kidney strictly on the basis of osmotic work, it would appear to be inefficient with an efficiency to the tune of only 1% which is out of step with most remaining organs in the body. In its broader sense, efficiency is defined as: hð%Þ ¼

Wout  100%; Win

(1.29)

where the work output includes all useful work that the device performs averaged over a long time. However, the kidney is not just an osmotic pump; it is also, and primarily, a physical realization of Maxwell’s demon, i.e., an ion sorting machine. As a control organ, the sorting activity of the kidney is of prime importance, and by including the energetic penalty of sorting into its Wout, JK demonstrated that the kidney has an efficiency commensurate with other body organs such as muscles only when the cost of sorting is accounted for. Essentially, the calculation of efficiency concerns energy bookkeeping. In that bookkeeping the energetic cost of sorting by a control device cannot be ignored. This is especially true because the cost of sorting, which accompanies dissipation of free energy to restore the demon to its initial state, is the most useful form of work a control device can perform. The pioneering work of JK that resolved the paradox of the kidney’s thermodynamic inefficiency is now recapped briefly from the three main publications (Johnson and Knudsen, 1965; Johnson, 1970, 1987). To concentrate all attention on JK’s main arguments of thermodynamics, citations referring to experimental quantities are omitted in the remainder of this section as they can be found in JK’s articles (Johnson and Knudsen, 1965; Johnson, 1970, 1987). The bookkeeping distilled from JK’s articles can be summarized, ignoring osmotic work (introducing an error of only w1%), as:  From Table 1.1, at the normal human body temperature, εmin ¼ 0.019 eV.  The human renal tubules selectively reabsorb w18 mmol/min z1022 ions/min: Emin ¼ εmin ð37 CÞ  1022 ¼ 0:019  1022 eV=min ¼ 7:2 cal=min

(1.30)

 The known input power of the kidney is w100 cal/min.  Assuming a noiseless environment in which the sorting is performed by the demon, the efficiency is: hð%Þ z

7  100 z 7%. 100

(1.31)

Chapter 1  Information Theory and Thermodynamic Efficiency

17

 However the environment in the kidney is noisy, given the background thermal bath in which the operation of sorting takes place. To make a binary decision in a noisy environment with 95% reliability, the signal-to-noise ratio must be at least 3 (Brillouin, 2004). In the kidney, the background environmental noise is wkT and hence the minimum cost of measuring in such a noisy environment is 3kT z0:08 eV.  The realistic minimum energy to sort ions per minute in the kidney is thus: 0 Emin ¼ ε0min ð37 CÞ  1022 ¼ 0:08  1022 eV=min z 31 cal=min

(1.32)

 Thus in a noisy environment, the efficiency of the sorting work is revised to: hð%Þ z 30%.

(1.33)

The kidney is the second highest consumer of power per unit tissue mass after the heart muscle. Its apparent inefficiency is an aberration that puzzled physiologists for decades. The puzzle is resolved if we enlarge the definition of efficiency to include the energetic penalty of measuring and sorting. The 30% efficiency obtained when this is done aligns the kidney’s efficiency as a control organ with that of the heart muscle as a mechanical pump and that of a contemporary gasoline engine as a heat engine.

Mitochondrial Adenosine 50 -Triphosphate (ATP)-Synthase as a Maxwell’s Demon The mitochondrion is a cell organelle of utmost importance in sustaining life. There could be anywhere from a few hundreds to a few thousands mitochondria in any given eukaryotic cell. The mitochondrion is the location of central metabolism of all higher forms of life: that is, the Krebs [tricarboxylic acid (TCA)] cycle and oxidative phosphorylation. These two interconnected networks of reactions use respiratory O2 to “burn” the common end products of the metabolism of nutrients (carbohydrates, proteins, and lipids) into CO2 and metabolic water. The DG released from this gradual oxidation is eventually captured in adenosine 5’-triphosphate (ATP), the “universal energy currency” of living matter, the exergonic hydrolysis of which to adenosine 50 diphosphate (ADP) and inorganic phosphate pulls thousands of nonspontaneous biochemical reactions to completion. Incidentally, the kidney tissues are extremely rich in mitochondria given their enormous energy (ATP) demands. FoF1 ATP-synthase (referred to here as ATP-synthase for simplicity) catalyzes both directions of the following reaction, which we write in the thermodynamically unfavorable phosphorylation direction of (driven) ATP synthesis: Fo F1 ATP-Synthase ðmitochondrial inner membraneÞ

! ADP þ Pi ATP þ H2 O



DG 00 z þ 31 kJ=mol ; DGcell z þ 50 kJ=mol

(1.34)

18 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

where the first figure for DG refers to standard biochemical conditions, whereas the second refers to cellular conditions (with pH and concentrations of ions typically found in cells, particularly of the Mg2þ ion known to significantly alter the energy of this reaction) (Garrett and Grisham, 2013). The second value of DG is the one that will be used in our estimate of mitochondrial efficiency described subsequently. When the cell is energy depleted, ATP-synthase catalyzes the formation of ATP, but in case there is plenty of energy, the enzyme catalyzes the reverse reaction to create and maintain a proton gradient, in which case it is usually referred to as ATPase. Whereas in eukaryotic cells the mitochondrion is the main site of phosphorylation of ADP to ATP, the electron transport chain (ETC) reactions in bacteria occur in their plasma membranes. In this chapter we are primarily concerned with human physiology and biochemistry, and hence the focus is on eukaryotic cells. Just like the kidney, the mitochondrion performs sorting work output (SWO). This extraordinary microscopic power plant has a three-dimensional compartmentalized structure that strictly dictates its functions including its demon-like sorting function. To see how and why, it is advantageous to recap briefly some basic elements of Peter Mitchell’s chemiosmotic theory for which he received the 1978 Nobel Prize in Chemistry (Mitchell, 1961, 2011; Mitchell and Moyle, 1965; Reynafarje and Lehninger, 1978). The mitochondrion is a small cell organelle the size of a bacterium in the form of a “double-bag” a small sac in a larger sac of sorts. Thus, the mitochondrion has two bilayer phospholipid membranes, an outer membrane enclosing an inner one. The outer membrane is smooth and nonselective, whereas the inner membrane is strictly selective. The inner membrane, containing the mitochondrial matrix, is highly convoluted and corrugated to increase its effective surface. These corrugations of the inner membrane are omitted from the idealized diagram in Fig. 1.4 for clarity. The TCA cycle takes place in the mitochondrial matrix. The TCA cycle produces two types of electron carriers, namely, the reduced forms of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). NADH and FADH2 deliver their electrons to protein complexes (I and II, respectively) embedded in the inner mitochondrial membrane (Fig. 1.4). Electrons are then passed in a cascade of coupled 0 redox reactions of increasing standard reduction potential Eo , known as the ETC. ETC reactions occur roughly along the horizontal arrows in Fig. 1.4 within the inner membrane itself. 0 The passage of electrons from a reduced form of a redox couple of lower Eo to one o0 with a higher E is exergonic given that: 0

0

DG o ¼ nFDE o ;

(1.35)

n is the stoichiometric number of transferred electrons, F is the Faraday constant 0 (96,485 JV/mol) if G is in molar units, and where “o ” indicates biochemical standard state conditions (Weinman and Me´hul, 2004). The DG released from the ETC is coupled with the active pumping of protons against the concentration and electrical gradients out of the matrix and into the intermembrane gap. This pumping of Hþ out of the matrix and into the intermembrane space causes and

Chapter 1  Information Theory and Thermodynamic Efficiency

19

FIGURE 1.4 Idealized diagram of a mitochondrion. Nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) are oxidized by complexes I and II, respectively, and the electrons are channeled along a concatenation of coupled redox reactions the agents of which reside within the inner mitochondrial membrane. The last step is the reduction of O2 at complex IV. As the electrons cascade through the series of redox reactions of increasing standard reduction potentials, the free energy released is coupled with the active pumping of protons from the matrix into the gap between the two membranes (the intermembrane space), creating a proton excess in this gap. As a result, the outer surface of the inner membrane is maintained at higher (positive) electric potential with respect to the inner (matrix) face which is maintained at a low (negative) potential. The inner mitochondrial membrane is thus a nanoelectric capacitor storing electric energy as a potential difference. Discharging this capacitor releases this energy, whether this occurs through adenosine 50 -triphosphate (ATP)synthase (which captures this energy to synthesize ATP) or through a leak in the membrane leading to wasteful dissipation of heat. Pereira and Cuesta (2016) proposed the following analogy with a battery: Complex I and II represent the anode, complex IV represents the cathode, the mitochondrial matrix represents the electrolyte of the battery, the concatenation of redox reactions in the inner membrane is the wire connecting the anode and the cathode, and finally, the proton pumps are the load in the electrical circuit. Cyt c, cytochrome c; Pi, inorganic phosphate. Adapted from Wikipedia.

maintains stored free energy owing to the chemical and electrical (predominant) gradients that result. This is expressed as: 

out

in  DG ¼ DGchem: þ DGelec: ¼ 2:3 nRT log Hþ P  log Hþ N þ nFDj;

(1.36)

where P ¼ the positive and N ¼ the negative side of the membrane, and Dj ¼ voltage across the membrane. The definition of pH can be used to simplify this equation further, and we write: DG ¼ 2:3 nRT DpH þ nFDj . |fflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflffl} |fflfflffl{zfflfflffl} DGchem:

DGelec:

(1.37)

20 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

When protons reenter the matrix through a channel in ATP-synthase downhill with respect to both gradients, the free energy they release is used to drive the synthesis of ATP according to Eq. (1.34) (Nicholls, 1982). Several pharmacologically active agents, including some poisons, short circuit this proton circuit by allowing leakage of the protons directly into the mitochondrial matrix bypassing ATP-synthase. Such agents include, for example, HCN (poison), rotenone (fish poison and insecticide), amytal (a central nervous system depressant of the barbiturate family), antimycin A (an antibiotic), etc. Some thermoregulatory proteins found in hibernating animals such as polar bears allow a small amount of protons to bypass ATP-synthase and leak to the matrix passively (through a pore in the protein) along the electrochemical concentration gradient. Such controlled leakage allows some free energy to be released as heat to keep the animal warm, especially in the winter. The transfer of electrons from one reaction to the next in the concatenation of coupled reactions of the ETC in the inner mitochondrial membrane releases DG because these reactions have increasing standard reduction potentials. The released DG from the ETC is coupled with the active pumping of protons from the mitochondrial matrix to the intermembrane space. This generates a considerable electric and chemical gradient perpendicular to the inner membrane and constitutes stored energy (DG) often called the “proton-motive force” (PMF). The energy of the PMF is harnessed when (and only when) the protons are channeled back to the matrix through a rotary mechanism in ATPsynthase driving Eq. (1.34) to completion. The voltage across the inner mitochondrial membrane in most cells in normal conditions is around 150e200 mV (Kadenbach et al., 2010) (Nicholls, 1982; Navarro and Boveris, 2007; Romanovsky and Tikhonov, 2010). The membrane thickness measured in ˚ (Sjo¨strand and Cassell, rat heart muscle mitochondria by freeze fracturing is >165 A 1978). The electric field strength across the membrane is thus on the order of 107 V/m, which is sufficiently strong as to have observable chemical and spectroscopic effects (Sowlati-Hashjin and Matta, 2013). ATP-synthase must pick Hþ from a mixture of all sorts of solutes including neutral molecules and ions (e.g., Naþ, Kþ, Liþ, Mg2þ, Cl, OH, HCOe 3 ) and hence this enzyme is performing acts of information-based selection and in this sense is as much a control device as a Maxwell demon (Matta and Massa, 2015). There are at least two sorting and control options for ATP-synthase: (Hþ and allowed in)/(not Hþ and forbidden entry). Each such dichotomous decision is equivalent to the reading 0 of 1 bit of information (Matta and Massa, 2015) and hence must cost at least εmin 37 C ¼ kT ln 2z0:4 kcal mol (Table 1.1). Glycolysis occurs in the cytosol, whereas the Krebs cycle occurs inside the mitochondrial matrix. The connection between the two compartments happens by the passage of pyruvate from the cytosol to the mitochondrial matrix. Fig. 1.5 summarizes the chain of reactions in glycolysis and the following Krebs cycle with emphasis on the sites of production of NADH and FADH2 responsible for channeling the electrons obtained from the breakdown of foodstuff to the ETC.

Chapter 1  Information Theory and Thermodynamic Efficiency

21

FIGURE 1.5 The catabolic pathway from glycolysis to the Krebs cycle with emphasis on the number and sites of production of electron carriers NADH and FADH2. Enzymes and several intermediate steps are omitted for clarity. In eukaryotic cells, the yellow box (to the left) includes reactions occurring in the cytosol whereas the rest of the reactions occur inside the mitochondrial matrix. Because every glucose molecule (a 6 carbon unit) generates two molecules of glyceraldehyde-3-phosphate (G-3-P), a 3 carbon unit, all reactions following the generation G-3-P must be multiplied by 2 to keep the stoichiometry relative to one molecule of glucose (the dotted blue box indicates reactions and species that must be multiplied by 2). Thus, per molecule of glucose, we have a total of 2 NADH produced in the cytosol in glycolysis and 8 NADH + 2 FADH2 produced in the matrix of the mitochondrion. These reduced coenzymes eventually deliver 24 electrons to the electron transport chain. Redrawn with modification following Fig. 18.1 (p. 582) of Voet, D., Voet, J.G., Pratt, C.W., 2013. Fundamentals of Biochemistry: Life at the Molecular Level, fourth ed. John Wiley and Sons, Inc., Hoboken, NJ, (USA) with permission.

Because NADH delivers its two electrons to the ETC earlier (to complex I) than FADH2 (to complex II), NADH starts at a lower rung in the electrochemical ladder of the ETC series than FADH2. As a consequence, NADH releases more DG than FADH2. This has the net effect that, on average, NADH is equivalent to 2.5 ATP, whereas FADH2 is equivalent to only 1.5 ATP, as will shortly be explained. The two NADH molecules formed in glycolysis cannot cross the inner mitochondrial membrane to be channeled along with the Krebs cycleegenerated NADH molecules to the inner surface of the inner membrane where electrons start their journey across the ETC. For the electrons of these two NADH molecules to reach the inner side of the inner mitochondrial membrane, nature has devised two shuttle mechanisms: the malateaspartate shuttle (MAS) and the glycerol-phosphate shuttle (GPS). In the MAS, NADH electrons are used to reduce cytosolic oxaloacetate into malate (a reversible reaction). Malate crosses the inner mitochondrial membrane to the mitochondrial matrix where it joins the Krebs cycle. In the Krebs cycle, malate undergoes the

22 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

opposite reaction of that which happens in the cytosol: that is, malate is oxidized back to oxaloacetate while reducing mitochondrial NADþ into NADH via coupling of the two redox reactions. The net action of this shuttle is hence effectively to transport one NADH from the cytosol inside the mitochondrial matrix. The two electrons of one NADH molecule that delivers its electrons to the ETC releases DG used to pump sufficient Hþ to produce two to three ATP (often taken as w2.5 ATP molecules/molecule of NADH). Therefore, an NADH molecule imported into the mitochondrial matrix through the MAS is equivalent to 2.5 ATP molecules on average. The cytosolic NADH delivering its electrons via the GPS, on the other hand, is less efficient. The reason is that NADH delivers its two electrons masquerading as FADH2 inside the matrix because the GPS functions as follows: First, dihydroxyacetone phosphate is reduced by cytosolic NADH to glycerol-3-phosphate (G3P) (and NADþ); G3P then crosses the inner membrane to the matrix and then undergoes the reverse reaction, but this time instead of coupling with the NADþ/NADH redox reaction it couples with that of the FADþ/FADH2 redox couple. In this case, the net effect is that an NADH molecule outside the mitochondrion is “effectively” converted into an FADH2 molecule inside the mitochondrial matrix. But because FADH2 joins the ETC later than NADH, it eventually produces less ATP. Indeed, the DG released by FADH2 pumps sufficient protons for the formation of one to two molecules of ATP only, with a commonly accepted figure of w1.5 ATP/FADH2. Hence an NADH using the GPS will release only the equivalent of w1.5 ATP (and not 2.5 ATP as a NADH molecule shuttled via the MAS). In the ETC, complex I pumps about four Hþ, complex II does not contribute to creating the proton gradient directly, complex III pumps about four Hþ, and complex IV pumps around two Hþ. So one mitochondrial matrix NADH contributes on average w10 Hþ, whereas an FADH2 pumps only approximately six Hþ. It takes on average probably three to four Hþ to be channeled back through ATP-synthase to phosphorylate one ATP from ADP (Eq. 1.34), which yields about one NADH: 2.5 ATP and w1 FADH2: 1.5 ATP. Per molecule of glucose, we obtain 2 NADH in the cytosol and 8 NADH þ 2 FADH2 in the matrix of the mitochondrion (Fig. 1.5). If we take the average estimates (2.5 ATP/ NADH, 1.5 ATP/FADH2), we obtain: 8 > 2:5  ð2 þ 8Þ > MAS: > > < |fflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflffl} NADH

þ

8 1:5  2 > > > |fflfflfflffl{zfflfflfflffl} >
> > > : |fflfflfflffl{zfflfflfflffl} FADH2

> > 2:5  ð8Þ > GPS: > : |fflfflfflfflfflffl{zfflfflfflfflfflffl} FADH2 NADH |fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl} Oxydative phosphorylation ðvia ETCÞ

2 |{z} ATPðGTPÞ produced directly in the TCAcycle

þ

2 |{z}

¼

32 ATP 30 ATP

.

(1.38)

ATP produced directly in glycolysis

|fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl} Substrate level phosphorylation

The best current estimates based on more realistic conversion ratios place the range a little higher, between 32 and 34 molecules of ATP per glucose: the former figure if the GPS is used and the latter if the MAS is used (Garrett and Grisham, 2013).

Chapter 1  Information Theory and Thermodynamic Efficiency

23

We now follow steps similar to JK as we apply them to the mitochondrion to calculate the efficiency of the ETC and oxidative phosphorylation with the cost of sorting taken into account (Matta and Massa, 2015). We summarize our approach in the following points (Matta and Massa, 2015):  Efficiency is defined as the percent ratio of the Gibbs energy necessary to phosphorylate 32e34 ATP molecules from an equal number of ADP and Pi (Eq. 1.34) to the theoretical DG of oxidation of a mole of glucose: C6 H12 O6 þ 6O2 /6CO2 þ 6H2 O;

DG ¼ þ2937 kJ=mol

(1.39)

under given thermodynamic conditions.  The Gibbs energy of hydrolysis of ATP is about 50 kJ/mol under cellular conditions (Garrett and Grisham, 2013).  The formation of 32e34 ATP molecules requires 32  5034  50 ¼ 1600e1700 kJ/ mol, which is what 1 mole of glucose eventually delivers biochemically (Gue´rin, 2004).  Ignoring any SWO yields an efficiency of w55%e58% (Garrett and Grisham, 2013), the efficiency generally quoted in biochemistry textbooks.  As can be seen from Eq. (1.38), four ATP molecules are phosphorylated directly at the substrate level, leaving 28e30 formed by oxidative phosphorylation.  To be conservative, the lower limit of this range is chosen: that is, 28 ATP molecules are taken to be formed by oxidative phosphorylation.  Again leaning to the lower bound of accepted values, we assume a ratio of three Hþ/ATP molecule, the same step involving the recognition and sorting of either “Hþ and allowed” or “non-Hþ and forbidden.”  The recognition step can be accomplished with 98% fidelity with a signal-to-noise ratio of 4 (Brillouin, 2004).  Thermal noise at body temperature ¼ kT, multiplied by 4 yields 10.3 kJ/mol, which is taken as the minimal discernable signal.  Because three Hþ are necessary to form one ATP, every molecule of ATP formed requires three acts of observation of Hþ. The cost of recognizing protons by ATPsynthase per molecule of ATP formed is thus 3  10.3 z 31 kJ/mol.  The cost of proton recognition to form 28 ATP molecules is then DGinfo. z 866 kJ / mol.  The energy of phosphorylation of all 32 ATP molecules, 4 (at the substrate level) þ 28 (oxidatively), DGATP z 1600 kJ/mol.  The total useful work produced is equal to the energy spent directly to phosphorylate the 32 molecules in addition to the cost of recognizing the necessary protons for the formation of 28 of those 32 ATP molecules (the SWO). Thus, the Gibbs energy necessary to produce 32 ATP molecules, including the informational cost associated with 28 of them, is given by: DG ¼ DGATP þ DGinfo. ¼ 1600 þ 866 ¼ 2466 kJ=mol.

(1.40)

24 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Combining these results, we obtain an overall revised efficiency for the ETC: h¼

2466  100 z 84%; 2937

(1.41)

which is w30% higher than the typical textbook quoted value obtained ignoring the cost of sorting by ATP-synthase. Whether the enzyme operates as ATP-synthase or ATPase, it acts as a Maxwell’s demon because it must recognize Hþ as distinct from the background. The efficiency we have calculated is much closer to the known mechanical efficiency of the ATP-synthase rotorestator mechanism that is close to 100% with almost no losses for friction during rotation (Romanovsky and Tikhonov, 2010). The significantly higher efficiency of the mitochondrion when its SWO is accounted for is better aligned with the efficiency of the mechanical rotation that is necessary for the energy transduction by ATP-synthase.

Closing Remarks The cost of sorting is useful work; we propose to call it sorting work output (SWO). Whenever an organ or organelle performs regulatory function, SWO becomes crucial. To explicitly account for SWO one may add an extra term to the central equation of chemiosmotic theory (Eq. 1.37) and write the effective free energy available for the oxidative phosphorylation as: DGeffective ¼ 2:3 nRT DpH þ nFDj þ nkT ln 2 ; |fflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflffl} |fflfflffl{zfflfflffl} |fflfflfflfflfflffl{zfflfflfflfflfflffl} DGchem:

DGelec:

(1.42)

DGinfo:

where the third positive term signals the dissipative nature of energy that ATP-synthase spends to perform SWO, and n is the number of protons to be recognized. Examples of regulator organs performing SWO are abundant in biology. Obvious examples include membrane-bound gate proteins in neurons that select Kþ and Naþ ions to generate the action potential, specific membrane transporters, and the overall brain activity. What constitutes an act of sorting is a gray area because any act of molecular recognition could possibly constitute one. A topic for future investigation regards when and under what conditions one can clearly define an act of sorting. This ambiguity is nonexistent in the topics we discuss here because sorting in the context of the operation of a kidney or a mitochondrion is conceptually clear. We have given estimates, for the first time to our knowledge, of the characteristic time for measuring a bit of information in the biochemical context. One bit is taken in the mitochondrial context to be a sorting between Hþ and allowed/not Hþ and forbidden. Inserting the energetic cost of 1 bit into Heisenberg indeterminacy relation [Eqs. (1.26) and (1.27)] led us to predict the characteristic time to measure 1 bit at physiological temperature, found to be on the order of 10 fs, irrespective of any mechanistic realization of the recognition step.

Chapter 1  Information Theory and Thermodynamic Efficiency

25

The first step of proton entry through ATP-synthase, the actual step of recognition, involves the protonation of an aspartic acid residue that protrudes from ATP-synthase into the intermembrane gap (Romanovsky and Tikhonov, 2010) and acts as an antenna or a fishing line to catch wandering protons. The timescale of proton transfer reaction has been determined experimentally for the double-proton transfer reaction of a model of DNA base pair (the 7-azaindole dimer) and found to occur in two steps: One proton is transferred from monomer A to monomer B followed by the second proton transferred from monomer B to monomer A (Folmer et al., 1998). The timescale for the first step is reported to be 660 fs, and that of the second on the picosecond timescale (Folmer et al., 1998), both well above our predicted minimum. Quantum chemical calculations (including quantum mechanical tunneling corrections) of the double-proton transfer reaction in an isolated formic acid dimer, in the absence of any external field, is estimated to occur at a timescale of a w100 ps (Arabi and Matta, 2011), again well above our proposed lower limit. We mention in passing that one of the intellectual leaders in this field, S. Nath (2016), has taken notice of our work. He has given prominent attention to our results (Matta and Massa, 2015). We find his analysis of our work to be highly interesting, and therefore consider that it may be valuable to the reader if we take time to comment on the definition of efficiency as viewed in our work (Matta and Massa, 2015) and that of Nath (2016). The basic idea for the reader to take into account, to avoid any possibility of confusion, is to understand that the one word “efficiency” is being used in two different ways, i.e., in our work and that of Nath. Such recognition will avoid a conflict of understanding that could otherwise occur if only the magnitude of the two efficiencies are compared without realizing that the magnitudes are numerical measures of different experimental quantities, both perhaps unfortunately called by the same name. In general the word “efficiency” means the ratio of energy input to energy output. In our work, we emphasize the energy cost of information, as for example in the information cost of ATP-synthase in accepting protons but rejecting nonprotons. The energy for the cost of this information, a form of energy output we term SWO, is supplied by the oxidation of glucose. The remaining energy output is that stored in the ATP bonds that are the end product of the mitochondrial process. In emphasizing the role of the energy cost of information we are following the pioneering analysis of JK in accounting for the actual efficiency of the kidney. In this way, we arrive at an efficiency for the mitochondrial oxidative phosphorylation that approaches 90%, considerably higher than the corresponding textbook value of nearly 60%, but more in line with the mechanical efficiency of ATP-synthase’s rotatory mechanism. This increase in the estimate of efficiency of the mitochondria precisely parallels the increase in the estimate of the efficiency of the kidney achieved by JK, and for the same reason: that is, recognizing explicitly that there is an energy cost of information that must be paid for by whatever is the ultimate source of energy that is driving the process. Remarkably, information is not free, but must be accounted for. We are indebted to S. Nath (2016) for pointing this out explicitly:

26 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

. one has to be extremely careful in the exact definition of the overall efficiency of the energy transduction process. If over and above the stored energy captured in the pyrophosphate bonds of ATP, we consider the free energy spent in molecular recognition of protons, and include that informational energy and other energy terms in the calculation of useful work, then in principle we can approach an efficiency of 100% because of the inviolability of the principle of energy conservation. Now, we hasten to add that Nath (2016) obtains an efficiency value differing from ours, some 45%, precisely because the energy cost of information is eschewed in that calculation. But he gives reasons for omitting to consider the information cost: . by defining the efficiency of the OX PHOS process as the maximum output work that a molecule of ATP can deliver in a user molecule (e.g. muscle actomyosin) under isothermal conditions divided by the input (redox) energy donated by a pair of electrons moving down the respiratory chain in mitochondria. As stated earlier, two different definitions of efficiency can differ in magnitude without conflict, of course. Following the lead of JK, we are drawing attention to the energy cost of information, whereas in Nath (2016), that energy cost is purposely omitted for the reasons quoted previously. In retrospect the work of JK is of such obvious fundamental importance that one might wonder why it has gone so unnoticed all these years. We can only speculate that because information theory is much more recent than classical thermodynamics, its relative unfamiliarity may be a contributing factor. And then information is not such an obvious experimental quantity as are the measurable free energies stored in chemical bonds and exist almost as an unnoticed background to the formation of the bonds that occupy the front stage. Furthermore, JK argue that because there is quasisteady-state condition in the kidney there is no net accumulation or depletion of ions owing to the act of observing them, so it is easy for that energetic cost to be missed. The same applies in the case of the mitochondrion. We hope the contribution of this chapter might in a small way bring the pioneering contribution of JK to the forefront, as would be appropriate. Although the mitochondrion is roughly a million billionth the size of a kidney,3 both have much in common in terms of their thermodynamic operation and efficiency. Both the kidney tubule’s cells and the mitochondrion’s ATP-synthase are Maxwell’s demons, although the relative contribution of the SWO in the mitochondrion is smaller (but nonnegligible) compared with the kidney. In the mitochondrion, SWO is as much as (30% / 60%)  100 ¼ 50% of its generally accepted thermodynamic efficiency, whereas in the kidney it represents (40% / 1%)  100 ¼ 4000%, which prompted Homer Smith to 3

The size of a human kidney (an organ) is, say, of the order of 10 cm, whereas that of a mitochondrion (a cell organelle) is of the order of a micrometer (1 cm ¼ 10,000 mm), so the linear dimensions of the two have a ratio of 1:100,000, whereas their volumes have a ratio of 1:1015.

Chapter 1  Information Theory and Thermodynamic Efficiency

27

remark (in 1951, before Johnson’s report and the discovery of the role of SWO) that (Smith, 1951): [T]he kidney’s efficiency may be very high indeeddit is the thermodynamic approach that is only one per cent efficient. The parallel of the kidney and the mitochondrion as two realizations of Maxwell’s demon of substantially different size, in which the cell of one (the kidney) even contains thousands of the other (mitochondria) to meet its energetic demands, is a biological illustration of Feynman’s (1965) opening quotation to this chapter: Nature uses its longest threads to weave her patterns, so each small piece of her fabric reveals the organization of the entire tapestry.

Acknowledgments The authors thank Professor Debasis Bagchi for his kind invitation and for allowing us to contribute in a small way to this important collective work. The authors are indebted to Dr. Kelly Resmer and Dr. Eric Fisher for their critical reading of the manuscript. L.M. was funded by the United States Naval Research Lab (Project 47203-00 01) and by the Professional Staff Congress, CUNY (63842-00 41). C.F.M acknowledges Professor Amiram Goldblum, the Lady Davis Trust, and the Hebrew University of Jerusalem for a Lady Davis Visiting Professorship during which this chapter was written, and the Natural Sciences and Engineering Research Council of Canada, Canada Foundation for Innovation, and Mount Saint Vincent University for funding.

References Arabi, A.A., Matta, C.F., 2011. Effects of external electric fields on double proton transfer kinetics in the formic acid dimer. Phys. Chem. Chem. Phys. (PCCP) 13, 13738e13748. Bennett, C.H., 1987. Demons, engines and the second law. Sci. Am. 257, 108e116. Borsook, H., Winegarden, H.M., 1931. The energy cost of the excretion of urine. Proc. Natl. Acad. Sci. U.S. A. 17, 13e28. Brillouin, L., 2004. Science and Information Theory, second ed. Dover Publications, Inc., Mineola, New York. Clark, J.K., Barker, H.G., 1951. Studies of renal oxygen consumption in man. I. The effect of tubular loading (PAH), water diuresis and osmotic (mannitol) diuresis. J. Clin. Invest. 30, 745e750. Eggleton, M.G., Pappenheimer, J.R., Winton, F.R., 1940. The influence of diuretics on the osmotic work done and on the efficiency of the isolated kidney of the dog. J. Physiol. 97, 363e382. Feynman, R., 1965. The Character of Physical Law. The M.I.T. Press, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. Folmer, D.E., Poth, L., Wisniewski, E.S., Castleman Jr., A.W., 1998. Arresting intermediate states in a chemical reaction on a femtosecond time scale: proton transfer in model base pairs. Chem. Phys. Lett. 287, 1e7. Garrett, R.H., Grisham, C.M., 2013. Biochemistry, fifth ed. Brooks/Cole, Cengage Learning, Belmont.

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Greger, R., Schlatter, E., 1983. Properties of the basolateral membrane of the cortical thick ascending ¨ gers Arch. limb of Henle’s loop of rabbit kidney. A model for secondary active chloride transport. Pflu Eur. J. Physiol. 396, 325e334. Gue´rin, B., 2004. Bioe´nerge´tique. EDP Sci. Les Ulis (France). Johnson, H.A., 1970. Information theory in biology after 18 years. Science 168, 1545e1550. Johnson, H.A., 1987. Thermal noise and biological information. Quarter. Rev. Biol. 62, 141e152. Johnson, H.A., Knudsen, K.D., 1965. Renal efficiency and information theory. Nature 206, 930e931. Kadenbach, B., Ramzan, R., Vogt, S., 2010. New extension of the Mitchell Theory for oxidative phosphorylation in mitochondria of living organisms. Biochim. Biophys. Acta 1800, 205e212. Landauer, R., 1961. Irreversibility and heat generation in the computing process. IBM J. Res. Dev. 5, 183e191. Leff, H.S., Rex, A.F. (Eds.), 1990. Maxwell’s Demon: Entropy, Information, Computing. Princeton University Press, Princeton. Leff, H.S., Rex, A.F. (Eds.), 2003. Maxwell’s Demon 2: Entropy, Classical and Quantum Information, Computing. Institute of Physics Publishing, Bristol. Matta, C.F., Massa, L., 2015. Energy equivalence of information in the mitochondrion and the thermodynamic efficiency of ATP synthase. Biochemistry 54, 5376e5378. Maxwell, J.C., 1872. Theory of Heat. Longmans, Green, and Co., London. Mitchell, P., 1961. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191, 144e148. Mitchell, P., 2011. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biochim. Biophys. Acta 1807, 1507e1538 (This is a re-publication of a Research Report (No. 66/1) originally published by Glynn Research Ltd., Bodmin, Cornwall, May, 1966). Mitchell, P., Moyle, J., 1965. Stoichiometry of proton translocation through the respiratory chain and adenosine triphosphate systems of rat liver mitochondria. Nature 208, 147e151. Nath, S., 2016. The thermodynamic efficiency of ATP synthesis in oxidative phosphorylation. Biophys. Chem. 219, 69e74. Navarro, A., Boveris, A., 2007. The mitochondrial energy transduction system and the aging process. Am. J. Physiol. Cell Physiol. 292, C670eC686. Nelson, F.E., Ortega, J.D., Jubrias, S.A.C.K.E., Kushmerick, M.J., 2011. High efficiency in human muscle: an anomaly and an opportunity? J. Exptl. Biol. 214, 2649e2653. Nicholls, D.G., 1982. Bioenergetics: An Introduction to the Chemiosmotic Theory. Academic Press, Inc., New York. Pereira, E.C., Cuesta, A., 2016. A personal perspective on the role of electrochemical science and technology in solving the challenges faced by modern societies. J. Electroanal. Chem. 780, 355e359. Reynafarje, B., Lehninger, A.L., 1978. The Kþ/site and Hþ/site stoichiometry of mitochondrial electron transport. J. Biol. Chem. 253, 6331e6334. Romanovsky, Y.M., Tikhonov, A.N., 2010. Molecular energy transducers of the living cell. Proton ATP synthase: a rotating molecular motor. Phys.-Uspekhi 53, 893e914. Schro¨dinger, E., 1944. What Is Life? Cambridge University Press, Cambridge. Sjo¨strand, F.S., Cassell, R.Z., 1978. Structure of inner membranes in rat heart muscle mitochondria as revealed by means of freeze-fracturing. J. Ultrastruct. Res. 63, 111e137. Smith, H.W., 1951. The Kidney: Structure and Function in Health and Disease. Oxford University Press, New York.

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Smith, E., 2008. Thermodynamics of natural selection III: Landauer’s principle in computation and chemistry. J. Theor. Biol. 252, 213e220. Sowlati-Hashjin, S., Matta, C.F., 2013. The chemical bond is external electric fields: energies, geometries, and vibrational Stark shifts of diatomic molecules. J. Chem. Phys. 139, 144101 (Erratum: J. Chem. Phys. 141, 039902, 2014). ¨ ber die Entropieverminderung in einem thermodynamischen System bei Eingriffen Szilard, L., 1929. U intelligenter Wesen (English translation: on the decrease of entropy in a thermodynamic system by the intervention of intelligent beings). Z. Phys. 53, 840e856 (English Translation: Behav. Sci. 9, 301e310, 1964). Voet, D., Voet, J.G., Pratt, C.W., 2013. Fundamentals of Biochemistry: Life at the Molecular Level, fourth ed. John Wiley and Sons, Inc., Hoboken, NJ (USA). ¨ ber die osmotische arbeit der nieren (On the osmotic work of the kidneys). von Rhorer, L., 1905. U ¨ gers Arch. Eur. J. Physiol. 109, 355e374. Pflu Weinman, S., Me´hul, P., 2004. Toute la Biochimie. Dunod, Paris. Wiener, N., 1961. Cybernetics. The M.I.T. Press and John Wiley & Sons, Inc., New York. Wilkie, D.R., 1975. Muscle as a thermodynamic machine. Ciba Found. Symp. 31, 327e339.

2

Roles of AMP, ADP, ATP, and AMPK in Healthy Energy Boosting and Prolonged Life Span Durgavati Yadav1, Yamini B. Tripathi1, Prabhakar Singh2, Rajesh K. Kesharwani3, Raj K. Keservani4 1

BANARAS HINDU UNIVERSITY, VARANASI, INDIA; 2 VEER BAHADUR SINGH P URVANCHAL UN IV ER SIT Y, JAUNP UR , INDIA; 3 NIMS UNIVERSITY, JANUPUR, INDIA; 4 RAJIV GANDHI PROUDYOGIKI V ISHWAVIDYALAYA, B HOPAL, INDIA

Introduction Different genetic and dietary manipulations known to prolong the life span have been shown to both decrease and increase the production of adenosine triphosphate (ATP) in cells (Bratic and Trifunovic, 2010). The molecular mechanism behind this dualism is not known; undoubtedly more experiments are needed to clarify the role of mitochondrial biogenesis, the mitochondrial respiration rate, and reactive oxygen species (ROS) production in different aspects of aging. Increased mitochondrial respiration would induce low levels of ROS production that in turn would act to stimulate antioxidant defense systems of the cell (Schulz et al., 2007).

Adenosine Monophosphate/50 -Adenylic Acid Adenosine monophosphate/50 -adenylic acid (AMP) is a nucleotide used as a monomer in RNA. It is an ester of phosphoric acid and adenosine. It consists of a phosphate group, ribose sugar, and adenine (Nelson and Cox, 2008). AMP is produced in normal cells during various metabolic processes. It can be produced during ATP synthesis by the enzyme adenylate kinase by combining two adenosine diphosphate (ADP) molecules or by the hydrolysis of ADP and ATP. It is also formed in the living system when RNA is broken down. It is metabolically active in cellular signaling when converted into other forms. It can be converted into inosine monophosphate by the enzyme myoadenylate deaminase and is widely used as a flavor enhancer. Inosinate takes part in regulating purine nucleotide biosynthesis. It is first nucleotide formed during purine metabolism. In the catabolic pathway it is converted into uric acid and is excreted from the body. In Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00002-8 Copyright © 2017 Elsevier Inc. All rights reserved.

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32 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

intracellular signaling, enzyme adenylate cyclase converts ATP to cyclic AMP, which is involved in reactions regulated by hormone-like adrenaline, glucagon, etc.

Adenosine Diphosphate/Adenosine Pyrophosphate ADP is essential to the flow of energy in living organisms. It consists of a sugar backbone attached to a molecule of adenine and phosphate to the fifth carbon of the ribose sugar. Biosynthesis of ATP occurs from ADP and AMP. The two phosphates in ADP can be correlated with ATP and AMP. ADP and phosphate are the precursors for synthesizing ATP in the payoff reactions of glycolysis, the tricarboxylicacid cycle, and oxidative phosphorylation mechanisms (Liapounova et al., 2006). It has a central role in activating blood platelets stored in the dense bodies of these cells. ADP reacts with ADP receptor families found on the platelets (P2Y1, P2Y12, and P2X1) (Murugappa and Kunapuli, 2006). ADPeATP cycling supplies energy required by the biological system, a thermodynamic process of transferring energy from one source to another (Berg et al., 2013). A proton gradient generates a chemiosmotic potential in the mitochondria of the cell (also known as the proton motive force) driving ADP phosphorylation via ATP synthase (Fo-F1 ATPase-complex V). The Fo domain of ATPase couples a proton translocation across the inner mitochondrial membrane with the phosphorylation of ADP to ATP (Reid et al., 1966).

Adenosine Triphosphate ATP is the molecular currency of the cell and is present in high concentrations (w6 mM). Biosynthesis of ATP is achieved through oxidative phosphorylation, photophosphorylation, and substrate-level phosphorylation. Hydrolysis of ATP to ADP or AMP yields energy and is sufficient to drive many unfavorable processes in the direction required by the cell. It is a small packet of energy used by the cell. An active person (12-MJ diet) turns over about 75 k of ATP every day, so a typical ATP molecule is broken down into ADP and resynthesized 1000 times each day. In rapidly metabolizing tissues, the lifetime of each ATP molecule is only a few seconds. The concentration of free ADP is normally lower than that of ATP in the cytosol of eukaryotic cells (about 200 times lower). This situation is clouded by large amounts of bound ADP permanently attached to the actin cytoskeleton. A low concentration of free cytosolic ADP is essential for metabolism to work properly. The diffusion rate is proportional to the concentration, which makes it difficult to recycle ADP quickly in rapidly metabolizing tissues such as cardiac muscle. Shuttle systems have evolved that accelerate ADP transport within cells. Coenzymes [such as ATP, nicotinamide adenine dinucleotide and hydrogen, nicotinamide adenine dinucleotide phosphate (NADPH), and coenzyme A (CoASH)] do not move easily between cell compartments and allow cells to keep their cytosol more oxidizing than their mitochondria, which suppresses lactate production under aerobic conditions. There are few coenzyme transporters; instead, elaborate metabolite shuttle networks are used to move material from one compartment to another.

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Energy stored in the pH and potential gradients drives the manufacture of ATP. This molecule consists of a purine base (adenine) attached by the 90 nitrogen atom to the 10 carbon atom of a pentose sugar (ribose). Three phosphate groups are attached at the 50 carbon atom of the pentose sugar. The addition and removal of these phosphate groups interconverts ATP, ADP, and AMP. When ATP is used in DNA synthesis, the ribose sugar is first converted into deoxyribose by ribonucleotide reductase. ATP transports chemical energy within cells for metabolism. It is one of the end products of photophosphorylation, aerobic respiration, and fermentation, and is used by enzymes and structural proteins in many cellular processes including biosynthetic reactions, motility, and cell division. ATP concentration inside the cell is typically 1e10 mM (Imamura et al., 2009). ATP can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as an energy source. For complex fuels to be synthesized into ATP, they first need to be broken down into smaller and simpler molecules. Carbohydrates are hydrolyzed into simple sugars, and fats (triglycerides) are metabolized to give fatty acids and glycerol.

50 -Adenosine Monophosphate-Activated Protein Kinase 50 -Adenosine monophosphate-activated protein kinase (AMPK) is involved in the cell metabolism, synthesis, and active transport of cellular constituents. The role of AMPK in cell metabolism is shown in Fig. 2.1.

Cell Signaling AMPK is an enzyme that has an important role in energy homeostasis in eukaryotic cells. It consists of three protein subunits which together make a functional enzyme. It is conserved from yeasts to humans. Its heterotrimeric enzyme is composed of one catalytic (a1 or a2) subunit and two regulatory (b1 or b2 and g1, g2, or g3) subunits, encoded by separate genes, forming a total of 12 complexes (Hardie, 2008). It is expressed in various tissues such as brain, skeletal muscle, and liver. AMPK activation is involved in a variety of functions such as hepatic fatty acid oxidation and ketogenesis, cholesterol synthesis inhibition, lipid synthesis, and adipocyte lipolysis synthesis and inhibition. It causes stimulation of skeletal muscle fatty acid oxidation. It also modulates insulin secretion by pancreatic b cells and hence has a role in muscle glucose uptake. AMPK is activated in response to various acts of cellular metabolism including stress and hormones, acting as a metabolic sensor and master regulator of the signaling pathway. Maintaining sufficient levels of ATP (the immediate source of cellular energy) is essential for the proper functioning of all living cells. As a consequence, cells require mechanisms to balance energy demand with supply. AMPK is activated by a decrease in ATP that leads to the activation of catabolic pathways and the inhibition of anabolic pathways. Findings have proved that ADP and AMP cause activation of mammalian AMPK. Phosphorylated AMPK provides a mechanism for the

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Role of AMPK in Cell Metabolism Cellular Stress (Heat Shock, Hypoxia, Low glucose)

Leptin/Adiponectin (From Adepocytes)

Fasting (Reduce energy available to Cells)

[ATP/ATP] [ADP/ATP]

Ion channels

Excercise (More energy required by Cells)

β -oxidation of fatty acids Glycolysis

Cholestrol synthesis

Glucose transport

AMPK Activation

eNOS Fatty acid synthesis

Protein synthesis & Glycogen synthesis PGC-1 α (PPAR-r coactivator 1 transcription factor)

Cell survival

Glucose regulated gene expression

[NAD/NADH]

SIRT -1

FOXO (Fork Head TranscriptionFactor)

Cellular / Gene regulation restoration of AMP to normal level Increased expression of energy

FIGURE 2.1 50 -Adenosine monophosphate-activated protein kinase (AMPK) activation is behind the entire important energy-related signaling pathway in the cells. Both the anabolic and catabolic pathways are involved in cellular signaling. On the left side boxes show reactions blocked by AMPK activation; on the right side boxes show reactions activated by AMPK (arrows). ADP, adenosine diphosphate; ATP, adenosine triphosphate; PGC-1a, peroxisome proliferator activated receptor gamma coactivator 1 alpha; PPAR-r, peroxisome proliferator activated receptor gamma; eNOS, endothelial nitric oxide synthase; NAD/NADH, nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide with hydrogen.

regulation of AMPK in which AMP and ADP protect it against dephosphorylation and regulation of AMPK (Zhang et al., 2009). Fig. 2.2 illustrates different regulatory pathways involving AMPK. AMPK activation by pharmacological agents is a unique challenge and holds considerable potential to reverse metabolic abnormalities. They are activated by two different pathway signals: a calcium ion (Ca2þ)-dependent pathway mediated by calmodulin-dependent protein kinase kinase b and an AMP-dependent pathway mediated by liver kinase B1 (Sanders et al., 2007). With other upstream kinases, including transforming growth factor beactivated kinase-1 (Tak1), these phosphorylate phospho-

Chapter 2  Roles of AMP, ADP, ATP, and AMPK

35

Signalling Involving AMPK Abbott A769662

ase) am Kin (Upstre LKB1 CaMKK β

Cholestrol Synthesis AMP / ATP

PKA

TAK Ca2+

HMG-CoA reductase

EF2K

Protein Synthesis

DNA Damage PP2C

AMPK

DNA Repair

AS 160

RAB

GLU-4

P Thr 172

p53

COX-2 Cell Cycle Arrest

ACC1 / FAS Fatty Acid Synthesis

TSC2 Cancer Progression

P13T

AKT

Apoptosis p53

GSK3

mTOR

p27

Protein Synthesis / Cell Survival / Cell Growth Autophagy

Cellular Proliferation FIGURE 2.2 Various cellular molecules become activated by phosphorylating threonine residue causing 50 adenosine monophosphate-activated protein kinase (AMPK) activation. AMPK activation in turn causes changes in different cellular molecules, which again activate cellular processes important for cell survival. ACC-1, acetyl-CoA carboxylase; AKT, protein kinase B; AMP, adenosine monophosphate/50 -adenylic acid; AS160, Akt substrate of 160 kDa; ATP, adenosine triphosphate; CaMKKb, calmodulin-dependent protein kinase kinase b; COX-2, cyclooxygenase-2; EF2K, elongation factor 2 kinase; FAS, fatty acid synthase; GLU-4, glucose transporter type 4; GSK3, glycogen synthase kinase 3; HMG-CoA, hydroxymethylglutaryl coenzyme A; LKB1,liver kinase B1; mTOR, mechanistic target of rapamycin/mammalian target of rapamycin; P13T, activated phosphatidylinositol; PKA, protein kinase A; PP2C, protein phosphatase 2C; RAB, member of Ras superfamily of monomeric G proteins; TAK, thermostable adenylate kinase; TSC2, tuberous sclerosis complex 2.

AMPKa on a subunit. AMP binding to the g subunit leads to the allosteric activation of AMPK as well as protection of Thr-172 from dephosphorylation, maintaining the enzyme in the activated state. Activation of AMPK by Tak1 leads to cytoprotective autophagy in untransformed cells against tumor necrosis factorerelated apoptosis-inducing ligandeinduced apoptosis (Herrero-Martı´n et al., 2009). Muscle AMPK is activated in response to exercise (Long and Zierath, 2006; Reznick and Shulman, 2006); this has led to intense interest in developing potential therapies for obesity and type 2 diabetes (Hardie, 2008). Diseases caused by impaired AMPK are in Fig. 2.3.

36 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Diseases Caused By Impaired AMPK

Over Nutrition

Alzheimer’s Disease

Inactivity

Ghrelin/Cannabinoids Genetic Factors

Metabolic Dysregulation

Insulin Resistance Obesity

Atherosclerosis Cardiovascular diseases

AMPK Deficit

Mitochondrial Dysfunction PCOS

Diabetes Risks

Insulin Resistance

Chronic Inflammation

NAFLD Dyslipidemia

Cancers

FIGURE 2.3 Diseases caused by deficient or dysregulated 50 -adenosine monophosphateeactivated protein kinase (AMPK). Metabolic dysregulation causes metabolic syndrome, giving rise to a wide variety of functional disorders. Lifestyle diseases are also involved, affecting most of the urban population. NAFLD, nonalcoholic fatty liver disease; PCOS, polycystic ovary syndrome.

Mechanistic Target of Rapamycin/Mammalian Target of Rapamycin/FK506-Binding Protein 12-RapamycinAssociated Protein 1 Mechanistic target of rapamycin/mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinase protein family (Brown et al., 1994). It is a serine/ threonine protein kinase regulating cell growth, cell proliferation, cell motility, cell survival, protein synthesis, autophagy, and transcription (Choi et al., 2002). mTOR integrates input from upstream pathways, including insulin, growth factors such as insulin-like growth factor (IGF)-1 and IGF-2, and amino acids (Hay and Sonenberg, 2004) mTOR also senses cellular nutrient, oxygen, and energy levels (Tokunaga et al., 2004). The mTOR pathway is a central regulator of mammalian metabolism and physiology, with important roles in the function of tissues including liver, muscles, white and brown adipose tissue, and the brain, and is dysregulated in human diseases such as diabetes, obesity, depression, and certain forms of cancer (Beevers et al., 2006; Kennedy and Lamming, 2016). Rapamycin inhibits mTOR by associating with its

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37

intracellular receptor FKBP12. The FKBP12erapamycin complex binds directly to the FKBP12erapamycin binding domain of mTOR, inhibiting its activity (Huang and Houghton, 2001; Huang et al., 2003). mTOR is the catalytic subunit of two structurally distinct complexes: mTOR complex 1 (mTORC1) and mTORC2. Both complexes localize to different subcellular compartments, which affects their activation and function (Laplante and Sabatini, 2012). mTORC1 is composed of mTOR, the regulatory-associated protein (Raptor), mammalian lethal SEC13 protein 8 (MLST8), and noncore components PRAS40 and DEPTOR. This complex functions as a nutrient/energy/redox sensor and controls protein synthesis (Kim et al., 2002). The activity of mTORC1 is stimulated by insulin, growth factors, serum, phosphatidic acid, amino acids (particularly leucine), and oxidative stress. mTORC2 is composed of mTOR, rapamycin-insensitive companion of mTOR, MLST8, and mammalian stress-activated protein kinase interacting protein 1. mTORC2 functions as an important regulator of the cytoskeleton by stimulating F-actin stress fibers, paxillin, RhoA, Rac1, Cdc42, and protein kinase C-a (PKC-a) (Frias et al., 2006). mTORC2 also phosphorylates serine/threonine protein kinase Akt/protein kinase B at serine residue S473, thus affecting metabolism and survival (Berg et al., 2013). Phosphorylation of serine stimulates Akt phosphorylation at a threonine T308 residue by PDK1and leads to full Akt activation (Sarbassov et al., 2005; Stephens et al., 1998). Rapamycin inhibits mTORC1 and provides the beneficial effects of the drug (including life-span extension in animal studies). Rapamycin inhibits mTORC2 only in certain cell types under prolonged exposure in a complex way. Disruption of mTORC2 produces the diabetic-like symptoms of decreased glucose tolerance and insensitivity to insulin.

Role of Mechanistic Target of Rapamycin/Mammalian Target of Rapamycin in Widely Studied Diseases mTOR is implicated in the failure of a “pruning” mechanism of the excitatory synapses in autism spectrum disorders. Decreased mTOR activity has been found to increase the life span in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster (Jia et al., 2004; Kaeberlein et al., 2005; Kapahi et al., 2004; Powers et al., 2006). The rapamycin has been confirmed to increase life span in mice (Fok et al., 2014; Harrison et al., 2009; Popovich et al., 2014). It is hypothesized that some dietary regimes such as caloric restriction and methionine restriction cause life span extension by decreasing mTOR activity. Some studies suggested that mTOR signaling may increase during aging, at least in specific tissues such as adipose, and rapamycin may act in part by blocking this increase. Methionine restriction may act in part by limiting the levels of essential amino acids including leucine and methionine, which are potent activators of mTOR (Caron et al., 2015). Administration of leucine into the rat brain has been shown to decrease food intake and body weight via activation of the mTOR pathway. mTOR signaling intersects with Alzheimer’s disease (AD) pathology in several aspects, which suggests its potential role as a contributor to disease progression. General findings

38 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

demonstrate mTOR signaling hyperactivity in AD brains. Studies of human AD brain reveal dysregulation in phosphatase and tensin homolog (PTEN), Akt, S6K, and mTOR (Chano et al., 2007; Li et al., 2005; Rosner et al., 2008). mTOR signaling appears to be closely related to the presence of soluble amyloid beta (ab) and tau proteins aggregation and form two hallmarks of the disease, ab plaques and neurofibrillary tangles, respectively. mTOR is a negative regulator of autophagy (Diaz-Troya et al., 2008); therefore hyperactivity in mTOR signaling should reduce ab clearance in the AD brain. Autophagy disruptions may be a potential source of pathogenesis in protein misfolding diseases, including AD (Li et al., 2008; McCray and Taylor, 2007; Nedelsky et al., 2008). Studies using mouse models of Huntington’s disease demonstrate that treatment with rapamycin facilitates the clearance of huntingtin aggregates (Ravikumar et al., 2004). Perhaps the same treatment may be useful in clearing ab deposits as well. Overactivation of mTOR signaling contributes significantly to the initiation and development of tumors. mTOR activity was found to be dysregulated in many types of cancer including breast, prostate, lung, melanoma, bladder, brain, and renal carcinomas. PTEN (commonly mutated in most of the cancers) phosphatase negatively affects mTOR signaling by interfering with the effect of PI3K, an upstream effector of mTOR (Guertin and Sabatini, 2005). Similarly overexpression of downstream mTOR effectors 4E-BP1, S6K, and eIF4E leads to a poor cancer prognosis (Po´pulo et al., 2012). Also, mutations in tuberous sclerosis complex 2 protein inhibits the activity of mTOR; it may lead to a condition named tuberous sclerosis complex, resulting in benign lesions and increasing the risk of renal cell carcinoma. Increased mTOR activity was shown to drive cell cycle progression and increase cell proliferation. Moreover active mTOR supports tumor growth indirectly by inhibiting autophagy. Continuous and persistent inactivation of mTORC1 (Brook et al., 2016) signaling in skeletal muscle facilitates the loss of muscle mass and strength during muscle wasting in old age, cancer cachexia, and muscle atrophy from physical inactivity. mTORC2 activation appears to mediate neurite outgrowth in differentiated mouse neuron cells (Salto et al., 2015). Intermittent mTOR activation in prefrontal neurons by b-hydroxyb-methylbutyrate inhibits age-related cognitive decline associated with dendritic pruning in animals. Scleroderma is a chronic systemic autoimmune disease characterized by hardening of the skin that affects internal organs in severe forms. mTOR has a role in fibrotic diseases; blockade of the mTORC pathway is under investigation as a treatment for scleroderma. Various natural compounds, including epigallocatechin gallate, caffeine, curcumin, and resveratrol, have been reported to inhibit mTOR when applied to isolated cells in culture (Zhou et al., 2010). However there is not yet evidence that these substances inhibit mTOR when taken as dietary supplements. Some mTOR inhibitors (e.g., temsirolimus, everolimus) are beginning to be used to treat cancer (Faivre et al., 2006). mTOR inhibitors may also be useful to treat several age-associated diseases (Hasty, 2010) including neurodegenerative diseases such as AD and Parkinson’s disease (Bove´ et al., 2011). Ridaforolimus is another mTOR inhibitor; currently it is in clinical development.

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Autophagy Autophagy is a process in which there is orderly degradation of cellular components and their recycling. During this process, targeted cytoplasmic constituents are isolated from the cell in an autophagosome, a double-membrane vesicle. Then the autophagosome fuses with a lysosome and the content is degraded and finally recycled (Patel et al., 2012). Three different forms of autophagy are commonly described: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA; Peracchio et al., 2012). Macroautophagy is used primarily to eradicate damaged cell organelles or unused proteins (Levine et al., 2011). This involves the formation of a double membrane, known as an autophagosome, about the organelle marked for destruction (Mizushima et al., 2002). The autophagosome then travels through the cytoplasm of the cell to a lysosome,  and the two organelles fuse (Hafner Cesen et al., 2012). Within the lysosome, the contents of the autophagosome are degraded through acidic lysosomal hydrolases. Microautophagy, on the other hand, involves the direct engulfment of cytoplasmic material into the lysosome (Kroemer and Ja¨a¨ttela¨, 2005). This occurs by invagination or cellular protrusion. CMA is a complex and specific pathway that involves recognition by the heat shock 70 kDa (Hsc70)-containing complex (Bandyopadhyay et al., 2008). This means that a protein must contain the recognition site for this Hsc70 complex that will allow it to bind to this chaperone, forming the CMA-substrateechaperone complex (Kaushik and Cuervo, 2012). This complex then moves to the lysosomal membraneebound protein that will recognize and bind with the CMA receptor, allowing it to enter the cell (Arias et al., 2015). Upon recognition, the substrate protein becomes unfolded and is translocated across the lysosome membrane with the assistance of the lysosomal Hsc70 chaperone (Arias et al., 2015; Huber and Teis, 2016). CMA is significantly different from other types of autophagy because it translocates protein material one by one is extremely selective about what material crosses the lysosomal barrier. In the context of disease, autophagy has been seen as an adaptive response to stress that promotes survival, whereas in other cases it appears to promote cell death and morbidity (Patel et al., 2012). In the extreme case of starvation, the breakdown of cellular components promotes cellular survival by maintaining cellular energy levels. Autophagy is required for the life spaneprolonging effects of caloric restriction. A 2010 French study of nematodes, mice, and flies showed that inhibition of autophagy exposed cells to metabolic stress. Resveratrol and dietary restriction prolonged the life span of normal, autophagy-proficient nematodes but not of nematodes in which autophagy had been inhibited by knocking out Beclin 1 (a known autophagic modulator).

Endoplasmic Reticulum Stress (Unfolded Protein Response) Endoplasmic reticulum (ER) stress [unfolded protein response (UPR)] is a stress response found to be conserved among all mammalian species and yeast. ER stress is stimulated in response to the misfolded protein in the lumen of ER. Here, UPR has three objectives to perform to maintain the normal cell metabolism: first, to restore the normal

40 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

function of the cell by halting protein translation; second, to degrade misfolded proteins; and third, to activate the signaling pathways leading to the increased production of molecular chaperones involved in protein folding. If these objectives are not fulfilled within a time span, the UPR progresses to apoptosis. Prolonged excessive stimulation of the UPR has been implicated in prion disease as well as several other neurodegenerative diseases. Inhibition of the overactivated UPR could be a treatment for those diseases (Senft and Ronai, 2015). Diseases susceptible to UPR inhibition include CreutzfeldteJakob disease, AD, Parkinson’s disease, and Huntington’s disease (Xu et al., 2005). Protein folding includes all of the processes involved in the production of a protein after polypeptides are newly synthesized by the ribosomes. The proteins, which are secreted or sorted to other cell organelles, have an N-terminal signal sequence that will interact with a signal recognition particle (SRP). This SRP will lead the whole complex (ribosomes, RNA, and polypeptide) to the ER membrane. Once the sequence has been recognized, protein continues its translation and the resultant strand translocates directly into the ER lumen. Protein folding commences as soon as the polypeptide enters the luminal environment, even as translation of the remaining polypeptide continues. When one of the ER elements is impaired, as often occurs under pathological conditions, overall cellular homeostasis becomes upset. Furthermore, UPR activation could trigger mitochondrial function changes or autophagy, modulating the UPR and exemplifying cross-talk processes (Hetz, 2012). Chemical inducers cause ER stress; for example, brefeldin A (thapsigargin) leads to ER Ca2þ depletion as a result of inhibition of sarco/ER Ca2þ-ATPase. A23187 upregulates expression of ER stress proteins; 2-deoxyglucose and dithiothreitol reduce the disulfide bridges of proteins, thereby accumulating denatured proteins inside the ER. Fenretinide and bortezomib (Velcade) induce ER stress leading to apoptosis in melanoma cells via different cellular mechanisms. Tunicamycin inhibits N-linked glycosylation in the ER lumen.

Mitochondrial Stress Mitochondria have a central role in energy metabolism, ATP production relies on the electron transport (ET) chain. It is composed of respiratory chain complexes IeIV, in which electrons are transferred in a stepwise fashion until they finally reduce oxygen to form water. There is tight coupling between the process of ET and ATP synthesis, and therefore inhibition of ATP synthase will also inhibit ET and cellular respiration. Outside the mitochondria, ROS can be produced by plasma membrane NADPH oxidases and lipid peroxidation and by some cytosolic enzymes (Andreyev et al., 2005). ROS produced within mitochondria presents almost 90% of the total ROS produced in the cell. Mitochondrial ET is the major ROS production site that leads to the suggestion that mitochondria are a prime target for oxidative damage, and hence the mitochondrial theory of aging, a correlate to the free radical theory (Harman, 1972). Mitochondria are also the only organelle in animal cells that possess their own DNA, mitochondrial DNA (mtDNA), which is localized adjacent to the mitochondrial respiratory chain. A mouse

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model provided the first experimental evidence for a causative link between mtDNA mutations and aging phenotypes in mammals (Trifunovic et al., 2004). The increase in somatic mtDNA mutations is associated with premature onset of aging and related phenotypes such as weight loss, reduced subcutaneous fat, alopecia, kyphosis, osteoporosis, anemia, reduced fertility, heart enlargement, and reduced life span (Trifunovic et al., 2004). The mitochondrial theory of aging predicts that mtDNA mutations levels should increase exponentially as a consequence of accelerated oxidative stress. The link between the mitochondrial energy balance and aging, as well as a possible connection between mitochondrial metabolism and molecular pathways, is important for extension of the life span. The relationship between energy metabolism and longevity has been suggested by two seemingly opposing theories. In 1928 Pearl proposed the “rate of living hypothesis,” a direct link of the metabolic output of an organism to its longevity (Turturro et al., 1999). In the 20th century, scientists proposed a new twist on this old theory: Longevity is limited by consumption. In other words, an organism’s metabolic rate determines its life span (Turturro et al., 1999). Another theory, “uncoupling to survive,” proposes that energy metabolism is in a positive relation with longevity (Brand, 2000), citing a direct connection of increased mitochondrial proton conductance with lowered ROS production (Brand, 2000). It has been proposed that UCPs protect from oxidative damage by lowering the proton motive force, thus causing a “mild” uncoupling and thereby attenuating superoxide production from the E26 transformation-specific (ETS). During “mild” uncoupling, UCPs activate with superoxide and other ROS products from oxidation of membrane phospholipids, respiration rate is increased, and in parallel, ROS production is decreased (Xu et al., 2014). It has been proposed that a superoxide induces the uncoupling of mitochondria by interacting with the UCPs; this process requires fatty acids and can be inhibited with purine nucleotides (Echtay, 2007). One explanation for the extended life span of long-lived mitochondrial mutants proposed that these animals have an upregulated fermentative malate dismutation where fumarate is terminally reduced at complex II to succinate (Tissenbaum, 2012). This is an alternative anaerobic metabolic pathway found only in nematodes; it is upregulated and leads to the production of lower ROS. Several other explanations have been proposed, such as an antioxidative role of ubiquinone and reduced complex I activity (in the case of clk-1 mutant) and an endogenous protective system induction against ROS (superoxide dismutase, catalase, and glutathione peroxidase by stimulating responses), but a clear mechanism is still unknown (Butler et al., 2010). Most of these explanations need experimental confirmation. Results in other model organisms point to a general mechanism that cannot be by processes specific for C. elegans, such as in malate fermentation. Insulin/IGF-like signaling is an important evolutionary conserved pathway involved in the longevity determination. This pathway has pleotropic effects on growth, development, metabolic homeostasis, fecundity, and the regulation of life span (Broughton and Partridge, 2009). It always intersects with c-Jun N-terminal kinase and TOR pathways, which are, respectively, involved in regulating protein synthesis and growth in a response to different stress signals (Partridge et al., 2011). A study on aged

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rats showed increased intramitochondrial ROS production and oxidative damage, an increase in proton leak resulting in depletion of membrane potential, and a reduction in activities of ATPase and complex IV. Treatment of aged rats with the IGF1 corrected these parameters and indicated a protective effect of IGF1 that is closely related to mitochondrial protection (Richardson et al., 2004). Studies that linked mitochondrial respiration/ATP production and longevity have given conflicting results, and it is not an easy task to reconcile them in a unifying theory. Different genetic and dietary manipulations are known to prolong life span, and have shown both decreases and increases in the ATP production of cells. The molecular mechanism behind this dualism is not known; more experiments are needed to clarify the role of mitochondrial biogenesis, mitochondrial respiration rate, and ROS production in different features of aging. The increasing age of mammals is linked to increased levels of mtDNA mutations and a deteriorating ETS. Various other types of evidence have linked somatic mtDNA mutations in a variety of diseases and symptoms such as osteoporosis, decreased fertility, weight loss, hair loss, and the graying of hair. Mitochondria are an important regulator of longevity. It has been known for a long time that ETS chainedeficient cells easily undergo apoptosis and that there is an increased loss of cells in age-related mitochondria dysfunction. Mosaic respiratory chain deficiency is found in aged humans in the tissues of heart, skeletal muscles, colonic crypts or neurons, etc. Currently in the scientific arena mitochondria hold promise for future research on aging.

Existing Energy Boosters A few natural and organic foods are energy boosters used in our traditional foods. These include oat straw extract, a natural energy booster with no sugar or caffeine. Oat straw expands arteries in brain so that more blood pumps through it, revving brain function. Rhodiola, one of nature’s best energy builders, helps multiply energy molecules. It is a powerful source of protein, fiber, and antioxidants. Kiwis contain twice the potassium of bananas and twice the vitamin C of oranges; they keep a person fully energetic. Amino acids found in eggs, cottage cheese, and smoked salmon help some people feel more alert and help with physical and mental stamina. Water-rich foods such as cucumbers, celery, radishes, tomatoes, and peppers can help to obtain needed hydration. Coconuts are packed with healthy fats and oils that are good for the brain. Whole-grain foods such as oatmeal give sustained energy. Spinach and other leafy green vegetables supply the body with vitamins, fiber, and magnesium. They are rich in chlorophyll, which also acts as a liver-detoxifying agent (Kashiyama et al., 2012).

Energy Boosting in Clinical Medication and Their Mechanisms AMPK is found in blueberries, grapefruit, green tea, and cayenne peppers. It is an important factor to consider for developing supplements. AMPK helps body use sugar, which results in boosting the metabolism. It is generally activated during exercise to help muscles use stored sugar and fat for energy. However it can also be activated by food.

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The secret to activating the power of AMPK is to include foods that contain polyphenols, nootkatone, and capsaicin. AMPK and mTOR control whole-body energy status.

Possibilities of Other Medicinal Herbals in Ayurveda Resveratrol is important for longevity. It maintains general good health, provides antioxidant protection, restricts calories, and promotes upregulation of the SIRT1 gene. Resveratrol has extended the life span of a variety of organisms, as published in reputable journals (Carter et al., 2014; Kulkarni and Canto`, 2015; Patel et al., 2010; Szkudelska and Szkudelski, 2010). Study ranged from yeasts (fungi) to roundworms (nematodes), fruit flies (insects), fish (vertebrates), and mice (mammals) (Hector et al., 2012; Morselli et al., 2010; Timmers et al., 2012; Valenzano and Cellerino, 2006; Yu and Li, 2012). It blocks the accumulation of abnormal protein aggregates associated with Parkinson’s and Huntington’s diseases and increases organisms’ survival rates. It also blocks the accumulation of ab in AD patients (Dasgupta and Milbrandt, 2007). The SIRT1 protein protects organisms from the effects of stress and aging. SIRT1 is useful to scientists who study aging and how resveratrol is affected by other mechanisms, because it gives them new avenues. Curcumin is also an important biomolecule from herbs that is widely studied for its beneficial effects on longevity and inflammation. It works by AMPK activation to orchestrate gene signaling that enhances fat burning (Goel et al., 2008; Hsu and Cheng, 2007; Liao et al., 2011; Soh et al., 2013).

Advanced Research Strategies for Developing Healthy Energy Boosters The principle “Let food be thy medicine and medicine be thy food,” advocated by Hippocrates, the father of modern medicine, emphasizes the strong association between human health and nutrition. In the market, various products and supplements are available for human consumption for general and specific cases. The roles of dietary active compounds are an important area of investigation in the modern nutraceutical industry’s research and development sector.

Acknowledgments Durgavati Yadav acknowledges Vivek Kumar Yadav (M. Tech. Lovely Professional University, Punjab) for his helpful support and for providing literature to complete this chapter.

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48 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Kroemer, G., Mathew, R., Kongara, S., Beaudoin, B., Karp, C., Bray, K., Degenhardt, K., Chen, G., Jin, S., White, E., Qu, X., Yu, J., Bhagat, G., Furuya, N., Hibshoosh, H., Troxel, A., Rosen, J., Eskelinen, E., Mizushima, N., Ohsumi, Y., Cattoretti, G., Levine, B., Yue, Z., Jin, S., Yang, C., Levine, A., Heintz, N., Wu, T., Li, Y., Gong, L., Lu, J., Du, X., Zhang, W., He, X., Wang, J., Mathew, R., Karp, C., Beaudoin, B., Vuong, N., Chen, G., Chen, H., Bray, K., Reddy, A., Bhanot, G., Gelinas, C., Dipaola, R., KarantzaWadsworth, V., White, E., Bae, H., Guan, J., Lu, Z., Luo, R., Lu, Y., Zhang, X., Yu, Q., Khare, S., Kondo, S., Kondo, Y., Yu, Y., Mills, G., Liao, W., Bast, R., Feng, W., Marquez, R., Lu, Z., Liu, J., Lu, K., Issa, J., Fishman, D., Yu, Y., Bast, R., Fu, L., Wen, X., Bao, J., Liu, B., Zhu, H., Wu, H., Liu, X., Li, B., Chen, Y., Ren, X., Liu, C., Yang, J., Korkmaz, G., Sage, C., le Tekirdag, K., Agami, R., Gozuacik, D., Frankel, L., Wen, J., Lees, M., Høyer-Hansen, M., 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Chapter 2  Roles of AMP, ADP, ATP, and AMPK

49

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An Overview of Nitrite and Nitrate: New Paradigm of Nitric Oxide Nathan S. Bryan BAYLOR CO LLEGE OF ME DICINE, HOUSTON, T X, UNITED STATES

Nitric Oxide Biochemistry and Physiology Nitric oxide (NO) is a cell-signaling molecule produced by a family of enzymes, nitric oxide synthase (NOS). NOS1, or neuronal NOS, is constitutively expressed in neurons, generates low concentrations of NO upon stimulation, and is responsible for transmission of signals in the central and peripheral nervous system (Garthwaite et al., 1988). NOS2, or inducible NOS (iNOS), is expressed and upregulated during inflammation or infection from cytokine stimulation (Geller et al., 1993). iNOS generates much higher concentrations of NO that lead to inhibition of respiration in infectious agents. Prolonged expression and activity of iNOS can cause tissue damage due to oxidation reactions of NO. NOS3, or endothelial NOS (eNOS), is constitutively expressed in endothelial cells and is responsible for maintaining normal blood pressure and integrity of the cardiovascular system (Furchgott and Zawadzki, 1980). These heme- and flavin-containing enzymes utilize electrons from nicotinamide adenine dinucleotide phosphate and produce NO by the mixed-function oxidation of the guanidino nitrogen atoms of the amino acid L-arginine (Li and Poulos, 2005; Daff, 2010), yielding L-citrulline as a by-product. NO, being a gas under normal physiological pressure and temperature, can diffuse into three dimensions and activate its cellular targets. NO, being uncharged, can freely permeate membranes and activate intracellular targets inside the cell where it was produced or in nearby cells, autocrine and paracrine signaling, respectively. For years, the primary target of NO was thought to be soluble guanylyl cyclase (sGC), which, upon binding and activation by NO, converts guanosine triphosphate into cyclic guanosine monophosphate (cGMP) (Arnold et al., 1977). cGMP then acts as a secondary messenger that, through a calcium-dependent pathway, causes smooth muscle relaxation and inhibits platelet aggregation along with many other physiological functions (Radomski et al., 1987). Accumulation of cGMP due to activation by NO is a major therapeutic target, as this is the mechanism of action of phosphodiesterase inhibitors (PD5) for the treatment of erectile dysfunction (Harris et al., 1989). However, without sufficient NO production by NOS enzymes, there is no activation of sGC and no Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00003-X Copyright © 2017 Elsevier Inc. All rights reserved.

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accumulation of cGMP, and therefore PD5 inhibitors become ineffective. This reveals that erectile dysfunction is a condition of insufficient NO production. Over the past 30 years, NO-based nitrosylation of cysteine residues on protein has revealed a major cGMP-independent mechanism of NO-based signaling affecting protein structure and function akin to phosphorylation (Stamler et al., 1992; Lane et al., 2001). Protein Snitrosation constitutes a large part of the ubiquitous influence of NO on cellular signal transduction, and accumulating evidence indicates important roles for S-nitrosation, both in normal physiology and in a broad spectrum of human diseases. S-nitrosylation is a mechanism for dynamic, posttranslational regulation of most or all major classes of protein (Foster et al., 2003; Lima et al., 2010). Denitrosation of cellular proteins is just as important as the S-nitrosation events themselves and confirms the complexity and dynamics of NO signaling (Sanghani et al., 2009). It is known that denitrosylation of S-nitrosothiol (SNO) proteins in cells can be accomplished by simple chemistry, wherein intracellular glutathione or other thiols act as acceptors and effectively remove nitroso groups via transnitrosation reactions. In this system, the rate of SNO protein decomposition would be modulated by changes in intracellular thiol levels, and conditions that promote glutathione oxidation in cells would enhance steady-state levels of protein Snitrosation. This mechanism would put S-nitrosation under redox control within the cell. These pathways also have important therapeutic applications, as many drugs are being developed to affect this signaling pathway (Raffay et al., 2016; Luzina et al., 2015). Both cGMP and protein SNO formation are decreased and dysfunctional in the face of insufficient NO production. Therefore, therapeutically, we must focus first on restoring NO production/homeostasis to maintain both pathways.

Endothelial Dysfunction and Loss of NO Production The enzymatic production of NO normally proceeds very efficiently in young, healthy individuals. However, in disease characterized by oxidative stress where essential NOS cofactors become oxidized, NOS uncoupling, or conditions of hypoxia where oxygen is limiting, this process can no longer maintain NO production (Forstermann and Munzel, 2006). Aging and hypertension are well known cardiovascular risk factors that lead to a loss of NO production (Lakatta and Yin, 1982; Kannel et al., 1971). Most of the functional and structural vascular alterations that lead to cardiovascular complications are similar in aging and hypertension, and both lead to decreased NO production (Ross, 1999). Moreover, these vascular changes associated with hypertension are generally considered to be an accelerated form of the changes seen with aging (Soltis, 1987). When we are young and healthy, the endothelial production of NO through L-arginine is efficient and sufficient; however, as we age, we lose our ability to synthesize endothelial-derived NO. Most of the works on the activity of NO in cells and tissues agree that the bioavailability or the generation of NOS-derived NO decreases with aging. It has been proposed that superoxide can scavenge NO to form peroxynitrite and thereby reduce its effective concentrations in cells (van der Loo et al., 2000). Berkowitz et al. (2003)

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observed the upregulation of arginase (an enzyme that degrades the natural substrate for NOS, L-arginine) in aged blood vessels and the corresponding modulation of NOS activity. Taddei et al. (2001) have shown that there is a gradual decline in endothelial function due to aging, with greater than 50% loss in endothelial function in the oldest age group tested as measured by forearm blood flow assays. Egashira et al. (1993) reported more dramatic findings in the coronary circulation of aging adults, whereby there was a loss of 75% of endothelium-derived NO in 70e80-year-old patients compared to young, healthy 20-year olds. Vita et al. (1990) demonstrated that increasing age was one predictor of abnormal endothelium-dependent vasodilation in atherosclerotic human epicardial coronary arteries. Gerhard et al. (1996) concluded from their 1996 study that age was the most significant predictor of endothelium-dependent vasodilator responses by multiple stepwise regression analyses. Collectively, these important findings illustrate that endothelium-dependent vasodilation in resistance vessels declines progressively with increasing age. This abnormality is present in healthy adults who have no other cardiovascular risk factors, such as diabetes, hypertension, or hypercholesterolemia. Most of these studies found that impairment of endothelium-dependent vasodilation was clearly evident by the fourth decade. In contrast, endothelium-independent vasodilation does not change significantly with aging, demonstrating that the responsiveness to NO does not change, only the ability to generate it. These observations enable us to conclude that reduced availability of endothelium-derived NO from L-arginine conversion occurs as we age and to speculate that this abnormality may create an environment that is conducive to atherogenesis and other vascular disorders, including Alzheimer’s disease. It appears that normal aging interrupts NO signaling at every conceivable level, from production to inactivation, and this can be further exacerbated by poor diet and lack of physical exercise. Given that NO is a necessary molecule for maintenance of health and prevention of disease, restoration of NO homeostasis may provide a new treatment modality for age and age-related disease. The “Holy Grail” in cardiovascular medicine is to determine how best to do this.

L-Arginine

Supplementation

For years, scientists and physicians have investigated L-arginine supplementation as a means to enhance NO production. This strategy has been shown to work effectively in young, healthy individuals with functional endotheliums or in older patients with high levels of asymmetric dimethyl L-arginine (Porst et al., 2003) where the supplemental L-arginine can outcompete this natural inhibitor of NO production. Patients with endothelial dysfunction, however, by definition as described previously, are unable to convert L-arginine to NO, and therefore, this strategy has failed in clinical trials. In fact, for L-arginine therapy in acute myocardial infarction (MI): the Vascular Interaction With Age in Myocardial Infarction randomized clinical trial published in the JAMA in 2006 concluded that L-arginine, when added to standard postinfarction therapies, did not improve vascular stiffness measurements or ejection fraction and was associated

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with higher postinfarction mortality (Schulman et al., 2006). L-arginine should not be recommended following acute MI. Also in patients with peripheral artery disease, L-arginine failed to provide improvement and, in fact, made many patients worse (Wilson et al., 2007). However, there are also a number of studies showing benefit to patients taking L-arginine and just as many showing no benefit and no harm.

NitrateeNitriteeNitric Oxide Pathway Since the biochemical basis of endothelial dysfunction is the inability to convert L-Arginine to NO, how do we safely and effective restore NO homeostasis in humans since L-arginine is not the answer? This is a critically important consideration given the ubiquitous nature of NO in health. Although the L-arginineeNO pathway was the first to be discovered, it does not necessarily mean it is the primary pathway for the endogenous production of NO. In fact, nitrogen cycling in bacteria and production of NO as an intermediate in denitrification may be one of the most primitive pathways known, dating back to the Archean era (Moir, 2011). The now-recognized human nitrateenitriteeNO pathway that still relies on bacteria may be a redundant system for overcoming the body’s inability to make NO from L-arginine (Bryan and Loscalzo, 2011). This alternate route involves the provision of nitrate and nitrite reductively recycled to NO. The two-electron reduction of nitrate to nitrite occurs through symbiosis with facultative anaerobic bacteria that reside in the crypts of our tongue (Lundberg et al., 2004; Hyde et al., 2014). The circulation of nitrate back to the oral cavity for reduction to nitrite/NO has been termed the enterosalivary nitrateenitriteeNO pathway (Lundberg et al., 2008). This pathway in humans appears to serve as an alternative pathway that can provide an endothelium-independent source of bioactive NO compensating for insufficient host NO production. This pathway is dependent upon commensal oral bacteria of the tongue to perform the first step (twoelectron reduction), since mammals lack a functional nitrate reductase. Further, the presence of nitrate-reducing bacteria in the oral cavity, concentrated on the tongue, has been well documented (Duncan et al., 1995; Hyde et al., 2014; Doel et al., 2005). The bioactivation of nitrate from dietary (mainly green, leafy vegetables, and beets) or endogenous sources requires its initial reduction to nitrite, and because mammals lack specific and effective nitrate reductase enzymes, this conversion is mainly carried out by commensal bacteria (Duncan et al., 1995; Doel et al., 2005; Hyde et al., 2014). Dietary nitrate is rapidly absorbed in the upper gastrointestinal tract. In the blood, it mixes with the nitrate formed from the oxidation of endogenous NO produced from the NOS enzymes. After a meal rich in nitrate, the nitrate levels in plasma increase greatly and remain high for a prolonged period of time (the plasma half-life of nitrate is 5e6 h). The nitrite levels in plasma also increase after nitrate ingestion after approximately 90 min (Lundberg and Govoni, 2004). Although much of the nitrate is eventually excreted in the urine, up to 25% is actively taken up by the salivary glands and is concentrated up to 20-fold in saliva (Spiegelhalder et al., 1976; Lundberg and Govoni, 2004). In the mouth, commensal facultative anaerobic bacteria reduce salivary nitrate to nitrite during

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anaerobic respiration by the action of nitrate reductases (Duncan et al., 1995; Lundberg et al., 2004). After a dietary nitrate load, the salivary nitrate and nitrite levels can approach 10 mM and 1e2 mM, respectively (Lundberg and Govoni, 2004). When saliva enters the acidic stomach (1e1.5 L per day), much of the nitrite is rapidly protonated to form nitrous acid (HNO2; pKa w3.3), which decomposes further to form NO and other nitrogen oxides (Lundberg et al., 1994; Benjamin et al., 1994). A simplified human nitrogen cycle is illustrated in Fig. 3.1. Nitrite does not have to be protonated to be absorbed and is about 98% bioavailable when swallowed in an aqueous solution (Hunault et al., 2009). There is significant evidence in the literature that these bacterial communities provide the host a source of NO that may be able to overcome insufficient NO production from the endothelium (Bryan and Ivy, 2015). Increasing the dietary intake of nitrate-rich vegetables has been demonstrated in a metaanalysis to be effective in blood pressure reduction (Siervo et al., 2013). To obtain sufficient nitrate levels for blood pressure management, a minimum of 450e550 mg nitrate needs to be provided at least 90 min prior in order to allow sufficient time for entero-salivary circulation and reduction to nitrite by oral bacteria. Nitrate has been shown to lower blood pressure beginning at 90 min and lasting out for several hours (Coles and Clifton, 2012; Kapil et al., 2010; Larsen et al., 2006) Administration of sodium nitrate for 4 weeks to older patients with increased cardiovascular risk profiles can also reverse vascular dysfunction (Rammos et al., 2014). All biological effects of nitrate are abolished by antiseptic mouthwash that kills oral bacteria that impact conversion of nitrate to nitrite and, in some cases, result in

DIET NO3BACTERIA 2 e- reduction Oxyheme proteins

NO2Oxygen, ceruloplasmin

BACTERIA 1 e- reduction Acid Deoxy-hemoproteins

NO oxidation

NOS L-arginine

reduction

FIGURE 3.1 The human nitrogen cycle whereby nitrate is serially reduced to nitrite and nitric oxide (NO), providing the host with a source of bioactive NO.

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a transient increase in blood pressure (Kapil et al., 2012; Petersson et al., 2009; Woessner et al., 2016; McDonagh et al., 2015). Nitrite reduction to NO can occur in a much simpler mechanism than nitrate. The one-electron reduction of nitrite can occur by ferrous heme proteins (or any redox active metal) through the following reaction: (II) NO þ Hþ 4 NO þ Fe þ OH 2 þ Fe

This is the same biologically active NO as that produced by NOS, with nitrite rather than L-arginine as the precursor, and it is a relatively inefficient process (Feelisch et al., 2008). Much of the recent focus on nitrite physiology is due to its ability to be reduced to NO during ischemic or hypoxic events (Bryan et al., 2004; Zweier et al., 1995; Bryan, 2006). Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters et al., 1967; Kozlov et al., 1999), protonation (Zweier et al., 1995), deoxyhemoglobin (Cosby et al., 2003), and xanthine oxidase (Li et al., 2004; Webb et al., 2004). Therefore, for this reaction to occur, the tissues or biological compartment must have a sufficient pool of nitrite stored. Since plasma nitrite is a direct measure of NOS activity (Kleinbongard et al., 2003), a compromised NOS system can also affect downstream nitrite production and metabolism, which can perhaps exacerbate any condition associated with decreased NO bioavailability. Therefore, we must titrate up nitrite concentrations from our diet and reduction of nitrate by bacteria. Considerable published data support the notion that exogenous nitrite contributes to whole body NO production: NO produced from nitrite in the upper intestine is up to 10,000 times the concentrations that occur in tissues from enzymatic synthesis (McKnight et al., 1997), nitrite can act as a circulating NO donor (Dejam et al., 2004), and nitrite can itself perform many actions previously attributable to NO (Gladwin et al., 2005) without the intermediacy of NO (Bryan et al., 2005). Dietary nitrite and nitrate have been shown to protect from tissue injury and restore NO homeostasis in eNOS/ mice that cannot make NO enzymatically (Bryan et al., 2007, 2008). Orally administered nitrite also attenuates cardiac allograft rejection in rats (Zhan et al., 2009). Experiments in primates revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm (Pluta et al., 2005). Moreover, inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in vivo in sheep (Hunter et al., 2004). Topical application of nitrite improves skin infections and ulcerations (Hardwick et al., 2001). Furthermore, enriching dietary intake of nitrite and nitrate translates into significantly less injury from heart attack (Bryan et al., 2007). Previous studies demonstrated that nitrite therapy given intravenously prior to reperfusion protects against hepatic and myocardial ischemia/reperfusion injury (Duranski et al., 2005). Additionally, oral nitrite has also been shown to reverse L-NAMEeinduced hypertension and serves as an alternate source of NO in vivo (Tsuchiya et al., 2005). These results have since been corroborated in humans (DeVan et al., 2016; Sindler et al., 2011). Replenishing nitrate and nitrite through dietary means may then act as a protective measure to compensate for insufficient NOS activity under conditions of hypoxia or in a

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number of conditions characterized by NO insufficiency. Since a substantial portion of steady-state nitrite concentrations in blood and tissue are derived from dietary sources (Bryan et al., 2005), modulation of nitrite and/or nitrate intake may provide a first line of defense for conditions associated with NO insufficiency (Bryan, 2006). The recognition of this mammalian nitrogen cycle has led researchers to explore the role of dietary nitrate and nitrite in physiological processes that are known to be regulated by NO (Lundberg et al., 2008). Nitrite can transiently form nitrosothiols under both normoxic and hypoxic conditions (Bryan et al., 2004, 2005), and a recent study by Bryan et al. demonstrates that steady-state concentrations of tissue nitrite and nitroso are affected by changes in dietary NOx (nitrite and nitrate) intake (Bryan et al., 2005). Nitrite can also activate sGC and lead to an increase in cGMP (Bryan et al., 2005). Therefore, nitrite can restore both pathways of NO-based signaling. Nitrite and nitrate therapy may then offer an all-natural, over-the-counter, and cost-effective regimen for conditions associated with NO insufficiency. Studies using a patented formulation, Neo40 (US patents 8,303,995; 8,298,589; 8,435,5708; 8,962,038; 9,119,823; and 9,241,999), using 15e20 mg of sodium nitrite in the form of an orally disintegrating tablet, found that it could modify cardiovascular risk factors in patients over the age of 40, significantly reduce triglycerides, and reduce blood pressure (Zand et al., 2011). This same lozenge was used in a pediatric patient with argininosuccinic aciduria and significantly reduced his blood pressure when prescription medications were ineffective (Nagamani et al., 2012). A more recent clinical trial using the nitrite lozenge reveals that a single lozenge can significantly reduce blood pressure, dilate blood vessels, and improve endothelial function and arterial compliance in hypertensive patients (Houston and Hays, 2014). Furthermore, in a study of prehypertensive patients (BP > 120/80 < 139/89), administration of one lozenge twice daily leads to a significant reduction in blood pressure (12 mmHg systolic and 6 mmHg diastolic) after 30 days (Biswas et al., 2015). The same lozenge was used in an exercise study and was found to lead to a significant improvement in exercise performance (Lee et al., 2015). In patients with stable carotid plaque, 6 months of using Neo40 twice daily led to a significant 11% reduction in carotid intima media thickness (Lee, 2016). These studies provide evidence that dietary nitrite and nitrate is well tolerated, increases plasma nitrite concentrations, improves endothelial function, and lessens carotid artery stiffening in middle-aged and older adults, perhaps by altering multiple metabolic pathways, thereby warranting larger long-term clinical trials. This has the potential to provide the basis for new preventive or therapeutic strategies and new dietary guidelines for optimal health. From a public health perspective, we may be able to make better recommendations on diet and dramatically affect the incidence and severity of cardiovascular disease and the subsequent clinical events. All of these studies together, along with the observation that nitrite can act as a marker of NOS activity (Kleinbongard et al., 2003), opened a new avenue for the diagnostic and therapeutic application of nitrite, especially in cardiovascular diseases, using nitrite as a marker as well as an active agent.

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Summary As science advances and new discoveries and understanding are made apparent, it is important to be able to incorporate these new findings into meaningful guidelines that can enhance health, lengthen life, and reduce illness and disability. It is known that it takes approximately 17 years for new basic science discoveries to become standards of care or be fully implemented into public health. As Paracelsus exclaimed, “dose makes the poison.” There are clear and delineated doses of both nitrite and nitrate that provide indisputable evidence of promoting health and even treating serious medical conditions. Fortunately, these doses fall well below toxic and fatal doses. This provides a sufficient range that can safely be achieved from diet alone. At a time when the globe is faced with epidemics of heart disease, obesity, and metabolic syndrome, we can no longer ignore fundamental nutritional, biochemical, and physiological benefits of nutrients found in the most healthy and nutritious foods, nitrite and nitrate, especially since the etiology of the aforementioned diseases are based on poor diet and nutrition. As with any nutrient, it is time to consider the riskebenefit analysis of nitrite and nitrate. It is known that lowering blood pressure by just 5 mmHg reduces the risk of stroke by 35% and risk of ischemic heart disease by 21%, the top two killers of people worldwide. There is now clear, indisputable blood pressureelowering effects of dietary nitrite and nitrate by at least 5 mmHg. By recognizing these relatively new findings and implementing nutritional and dietary interventions with sufficient nitrite and nitrate, perhaps 35% or roughly 6 million deaths could be prevented each year. I have referred to nitrite as a vitamin previously and even proclaimed it “Vitamin N,” but perhaps it may fit the characteristics of a dietary mineral. A mineral is, by definition, a solid, inorganic substance of natural occurrence. After all, sulfates and phosphates are recognized minerals, and nitrite and nitrate are similar in structure and composition except replacing the sulfur and phosphorus with nitrogen, respectively, with different oxidation states. How we classify nitrite and nitrate may not be important at this stage other than to finally recognize them for nutrients that they are. The underlying chemistry of these two anions must be controlled to maximize the benefits while preventing any unwanted nitrosation chemistry causing N-nitrosamine formation. Nutritionists, physiologists, physicians, toxicologists, meat scientists, and dieticians from both academia and industry need to converge to recognize and establish nutrient guidelines for nitrite and nitrate, similar to other well-recognized nutrients. The data and facts are now available for such an initiative. It is time to change the dialogue from one of avoiding nitrite and nitrate in the diet to one of including sufficient amounts to achieve the well-documented health benefits from them.

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van der Loo, B., Labugger, R., Skepper, J.N., Bachschmid, M., Kilo, J., Powell, J.M., Palacios-Callender, M., Erusalimsky, J.D., Quaschning, T., Malinski, T., Gygi, D., Ullrich, V., Luscher, T.F., 2000. Enhanced peroxynitrite formation is associated with vascular aging. J. Exp. Med. 192 (12), 1731e1744. Vita, J.A., Treasure, C.B., Nabel, E.G., McLenachan, J.M., Fish, R.D., Yeung, A.C., Vekshtein, V.I., Selwyn, A.P., Ganz, P., 1990. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease. Circulation 81 (2), 491e497. Walters, C.L., Casselden, R.J., Taylor, A.M., 1967. Nitrite metabolism by skeletal muscle mitochondria in relation to haem pigments. Biochim. Biophys. Acta 143 (2), 310e318. Webb, A., Bond, R., McLean, P., Uppal, R., Benjamin, N., Ahluwalia, A., 2004. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc. Natl. Acad. Sci. U.S.A. 101 (37), 13683e13688. Wilson, A.M., Harada, R., Nair, N., Balasubramanian, N., Cooke, J.P., 2007. L-arginine supplementation in peripheral arterial disease: no benefit and possible harm. Circulation 116 (2), 188e195. Woessner, M., Smoliga, J.M., Tarzia, B., Stabler, T., Van Bruggen, M., Allen, J.D., 2016. A stepwise reduction in plasma and salivary nitrite with increasing strengths of mouthwash following a dietary nitrate load. Nitric Oxide 54, 1e7. Zand, J., Lanza, F., Garg, H.K., Bryan, N.S., 2011. All-natural nitrite and nitrate containing dietary supplement promotes nitric oxide production and reduces triglycerides in humans. Nutr. Res. 31 (4), 262e269. Zhan, J., Nakao, A., Sugimoto, R., Dhupar, R., Wang, Y., Wang, Z., Billiar, T.R., McCurry, K.R., 2009. Orally administered nitrite attenuates cardiac allograft rejection in rats. Surgery 146 (2), 155e165. Zweier, J.L., Wang, P., Samouilov, A., Kuppusamy, P., 1995. Enzyme-independent formation of nitric oxide in biological tissues. Nat. Med. 1 (8), 804e809.

An Overview on Nitric Oxide and Energy Metabolism

4

Safia Habib, Moinuddin, Asif Ali ALIGARH MUSLIM UNIVERS ITY, ALIGARH, INDIA

Introduction Nitric oxide was first identified by Ignarro in 1987. This oxide is an uncharged diatomic molecule capable of interacting with different biological macromolecules, both in cytoprotective and cytotoxic fashion. Exact in vivo concentration of nitric oxide is not clear, but by using porphyrinic-based nitric oxide selective electrodes, it was found to be 10 nMe5 mM. Nitric oxide is capable of exerting its effects directly as well as indirectly through its metabolites, which are produced by reacting with other free radicals like  superoxide O2 ,  , hydrogen peroxide (H2O2), and hydroxyl radical (OH). Nitric oxide is synthesized in human system in presence of various enzymes known as nitric oxide synthases (NOSs), and it mediates its action through an intracellular messenger, the cyclic guanosine monophosphate (cGMP) through which it exerts many normal physiological functions, which mainly include the following: 1. 2. 3. 4.

smooth muscle relaxation and maintenance of blood pressure signaling and regulating gastrointestinal activity inhibiting platelet aggregation killing invading bacteria and parasites

All these functions are carried out by physiological concentrations of nitric oxide (Table 4.1). The enzymes responsible for nitric oxide synthesis have three isoforms: neuronal NOS (type I), inducible NOS (type II), and endothelial NOS (type III). All of the three use substrates L-arginine, reduced nicotinamide adenine dinucleotide phosphate (NADPH), and oxygen, and produce citrulline, nicotinamide adenine dinucleotide phosphate (NADPþ), and nitric oxide. Besides this pathway, our system also produces nitric oxide through some minor pathways, like through mitochondrial NOS. Along with this, in human skeletal muscle, a splice variant of neuronal NOS is also reported to be present and is known to modulate muscle contraction and blood flow as well as metabolism. Nonenzymatic production of nitric oxide, though not so significant, takes place at low pH in the presence of reductants from nitrite. Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00004-1 Copyright © 2017 Elsevier Inc. All rights reserved.

67

68 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Table 4.1

Role of Nitric Oxide in Cellular Responses

Cytotoxic Role

Cytoprotective Role

1. Nitric oxide decreases protein synthesis. 2. It inhibits mitochondrial respiration.

1. Nitric oxide scavenges free radicals. 2. It decreases tumor cell growth and improves normal DNA synthesis. 3. It decreases pathogen proliferation. 4. It blocks the release of proinflammatory PGE2 (Prostaglandin E2). 5. It increases cerebral flow and oxygen to brain. 6. It improves T-cellemediated immune response.

3. It increases lipid peroxidations. 4. It combines with reactive oxygen species to produce highly reactive species. 5. It is associated with genotoxic effects. 6. Low concentration promotes tumor growth.

Along with normal physiological and biochemical characteristics and functions, nitric oxide has a role in relation to energy metabolism as well. We can divide the functions of nitric oxide into four main categories, all mediated through cGMP: 1. 2. 3. 4.

It regulates mitochondrial respiration. It regulates energy metabolism and body composition. It makes respiration sensitive to oxygen supply. It is known to govern the metabolism of the proximate principles of food and to modulate insulin sensitivity.

Each of the previously mentioned functions depends on the concentration of nitric oxide present and whether the action is a direct action of the molecule or an indirect one.

Biochemistry of Nitric Oxide Nitric oxide (NO) at physiological concentration is not so reactive, but since it is capable of rapid diffusion from one cell to another, it can mediate its physiological functions by binding to Feþ2 in the heme of soluble guanylyl cyclase, producing cGMP. However, nitric oxide may be converted to highly reactive nitrogen species (RNS), which exert multiple effects on cell physiology and also can cause cell death. Nitric oxide can also act on mitochondria and bring about inhibition of mitochondrial respiration. Also, it can protect against mitochondria-mediated cell death. Cells exposed to NO show immediate but reversible inhibition of respiration at cytochrome oxidase; however, after several hours of exposure to nitric oxide, an irreversible inhibition develops. The kinetically fast reactions occurring physiologically are considered relevant. NO does not react rapidly with amines or thiols, but its reaction with metal complexes can be considered as relevant. It reacts with metal complexes to form metal nitrosyls, e.g., FeeNO complex, which is quite stable. The radical is also capable of reacting with metallo-oxo as well as metal oxo complexes. NO can also directly interact with a

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hypervalent complex formed by agents such as H2O2 and can reduce it to lower valency state (Katherine et al., 2009; Christine et al., 2013; Sharron et al., 2010). Feþ

(2,3)

þ H2O2 / Feþ

(4,5)

¼ O þ H 2O

Feþ4 ¼ O þ NO/Feþ3 þ NO2 

The presence of NO results in scavenging superoxide, which, besides preventing enzyme inactivation, also converts any ferrous oxy adducts to active ferric state. It is also reported that at low concentration of NO, direct effects will predominate, while at  higher concentration, indirect effects are mediated by NO O2 ,  (Fig. 4.1). NO protects tissue from peroxide-mediated damage by scavenging metal oxo species. It has been shown to inhibit lipid oxygenase activity by reacting with nonheme iron at the active site. A heme protein, cyclooxygenase, involved in the conversion of arachidonic acid to prostaglandin, and other related enzymes are also influenced by NO radical reactions and metal NO interaction A possible mechanism accounted for cyclooxygenase inhibition by superoxide involves the reduction of ferric form to the inactive ferrous state. It has also been reported that NO generation results in nitrosative reactions at nucleophilic centers, resulting in the formation of S-nitrosothiols (Sangwon, 2011). Excess production of NO has been shown to mediate glutamate-induced neuronal toxicity in cortical and striatal neurons cultures. NO-mediated apoptosis has

1. 2. 3. 4. 5.

Cysteine-S-nitrosylation. Tyrosine nitration. Metal nitrosylation. Mutagenic. Stimulate prostaglandin synthesis. ACTION

O2 .

ACTION 1. 2. 3. 4. 5.

+

NO.

ONOOACTION

Cytokine release. Lipid peroxidation. DNA damage. Catecholamine inhibition. Redox sensitive transcription factors activated. SYNTHESIS

Nitrite reduction.

1. Nitrosative deamination of DNA.(GC AT) 2. Modification of proteins. 3. PARP activation. 4. Mitochondrial respiration inhibited irreversibly.

Polyamine pathway. L-Arginine oxidation. FIGURE 4.1 Direct and indirect effects of nitric oxide.

70 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

also been reported in murine peritoneal macrophages. NO also reacts with oxyhemoglobin and results in the formation of met-Hb and NO3  . Hb-Feþ2 (O2) þ NO / Hb-Feþ3 þ NO3 Rate constant ¼ 3  107 M/S

NO also interacts with deoxy-Hb and met-Hb by binding to heme iron center. The binding is hindered by a water molecule coordinated to heme Fe3þ atom in case of metHb, and it has been reported that the association rate constant is 100-fold less for ferrous deoxy-Hb. It has also been reported that binding of NO to heme moiety of soluble guanylyl cyclase results in a pentacoordinate complex, and the bond to the proximal histidine is lost.

Regulation of Mitochondrial Respiration We know that nitric oxide is available throughout the system and performs many important physiological functions. The direct effect of nitric oxide includes its protective as well as deleterious actions, such as that it is known to act as an agent where it induces metabolic derangements, energy losses, and even death of the target cell (Kindo Gerelli et al., 2016; Sun et al., 2016). Since mitochondria contains enzymes and proteins which are central to the regulation of energy expenditure and are considered to be the powerhouses of the cell where energy is released from the oxidation of food stuff, which is trapped as chemical energy in the form of adenosine triphosphate (ATP). The mitochondrial inner membrane or cristae contains the enzymes of the electron transport chain, whereas the fluid matrix contains the enzymes of citric acid cycle, urea cycle, heme synthesis, etc. Nitric oxide and its derivatives are known to influence mitochondrial respiration in a concentration-dependent manner, resulting in reversible or irreversible inhibition of ATP production. Nitric oxide interacts with mitochondria and regulates mammalian energy metabolism (Busija et al., 2016; Kautza et al., 2015). Physiological levels of nitric oxide reversibly reduce cytochrome oxidase, whereas high concentrations irreversibly inhibit complexes I, II, III, IV, and V in the mitochondrial respiratory chain. Here, physiological concentrations can be taken to be somewhat in nanomolar range and high concentrations to be in micromolar range. As already mentioned, all these effects of nitric oxide depend on its direct actions and those mediated indirectly. First, we consider the direct effects.

Direct Effects of Nitric Oxide on Mitochondrial Respiration Nitric oxide is known to inhibit the mitochondrial respiratory chain in many ways, targeting and compromising the production of ATP, causing an irreversible inhibition of ATP production by modulating cytochrome oxidase so that the binding of oxygen is compromised and the inhibition is due to its reversible binding to heme a2þ 3 that could be reversed by light and oxygen, thereby increasing the apparent Km for the respiratory requirement of oxygen, as is the case with competitive inhibitors (Borutaite et al., 2005;

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Simonin and Galina, 2013). It has been reported that cells that participate in immune responses, like macrophages, astrocytes, etc., under stress lead to the activation of inducible NOS due to the production of proinflammatory cytokines, thereby increasing the concentration of nitric oxide, which could diffuse to the nearby cells and could result in the inhibition of their own respiration as well as of the nearby cells (Ulrich and William, 2012). It has been also reported that 60-nM nitric oxide raises the apparent Km of respiration for oxygen from less than 1 mm to 30 mM. Also reported is that addition of 1e4 mM nitric oxide to isolated mitochondria caused reversible inhibition of oxygen consumption at low oxygen concentrations, leading to reversible depolarization of mitochondrial potential and efflux of mitochondrial calcium (Christelle et al., 2012), leading to diminished ATP synthesis. This effect is variable according to the type of the tissue mitochondria; for example, inhibition of mitochondrial respiration in the heart needs a higher level of nitric oxide as compared to isolated liver and brain mitochondria (Claude and Piantadosi, 2012). As we know, the cytochrome c oxidase is located in the inner mitochondrial membrane and catalyzes the reduction of oxygen to watergenerating membrane proton gradient, i.e., the reaction is coupled to the pumping of protons out of mitochondria and simultaneous oxidation of cytochrome c2þ to cytochrome c3þ, since this is the terminal complex of mitochondrial respiratory chain and is responsible for nearly 90% of oxygen consumption, and a compromise at this level leads to diminished energy levels, since almost all energy production essentially needs this complex (cytochrome aa3, complex IV). Inhibition of cytochrome c by nitric oxide could be explained at these levels (Fig. 4.2): 1. Cytochrome c is a protein of inner mitochondrial respiratory chain that undergoes nitration due to the presence of four tyrosine residues, thereby compromising the energy levels. 2. Nitric oxide gets bound to Cuþ1 or Cuþ2 of cytochrome oxidase, specifically forming Cuþ1Noþ, which ultimately forms nitrite that inhibits cytochrome oxidase. 3. Nitric oxide is also capable of binding to (Feþ2Cuþ2) and (Feþ3Cuþ1), which are partially reduced forms of cytochrome oxidase center. Overall, it could be said that nitric oxide competitively inhibits cytochrome oxidase by increasing apparent Km for oxygen and is also capable of regulating energy homeostasis and oxygen sensitivity in different tissues.

Indirect Effect of Nitric Oxide on Mitochondrial Respiration The response of nitric oxide also takes place in presence of other free radicals like   radical ðOH  Þ, and hydrogen peroxide (H2O2). superoxide radical O2 ,  , hydroxyl  Nitric oxide reacts with O2 ,  to form peroxynitrite (ONOO) at a constant rate of 6.7  109 L/S/mole. NO þ O2 ,  /ONOO þ Hþ ONOO þ Hþ 4ONOOH/OH þ NO2



72 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

RNSO

NO

.

Creatine Kinase. Aconitase.

ONOO-

RNS INHIBITS

Cytochrome Oxidase.

Cytochrome oxidase.

Complex III.

ComplexI,II,III.

Respiratory Inhibition.

ATP Synthase.

Cytochrome Release.

Proton Leak.

MTP ATP Depletion

OxidaƟve Stress

Energy DepleƟon

APOPTOSIS

NECROSIS

FIGURE 4.2 Role of nitric oxide in mitochondrial respiration.

 Upon protonation, ONOO can lead to the formation of NO2 ,  and OH. Peroxynitrite is a powerful oxidant that has a capability of reacting with many biological macromolecules, leading to alteration of their normal physiological properties and functions. The molecules may include proteins, nucleic acids, lipids, etc. Talking in terms of energy production and considering mitochondria to be a central energyproducing hub, nitric oxide indirectly acts mainly at the level of RNS, where nitric oxide is converted to strong oxidant NO2 and N2O3. Reaction of nitric oxide with superoxide leads to the formation of peroxynitrite, which is a far more potent oxidant, along with the formation of another species, i.e., S-nitrosothiol (Finocchietto et al., 2011; Benamar et al., 2008). RNS are known to cause irreversible changes at the level of electron transport chain through interaction with mitochondrial respiratory chain complexes to bring about S-nitrosation of reduced nicotinamide adenine dinucleotide (NADH)eubiquinone oxidoreductase system. Besides this, S-nitrosothiol inactivation of complex I also

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results along with the damage to iron sulfur centers; all these changes, in a cumulative way, ultimately lead to the inhibition of ATP production and making the cell compromised at energy levels, ultimately leading to cell death, as we know that electron flow occurs through successive dehydrogenase enzymes together, known as the electron transport chain. The electrons flow through electronegative potential to electropositive potential, and hence, with the help of the electron transport chain, the total energy change is released in small increments so that energy can be trapped as chemical bond energy in the form of ATP. Inhibition of the respiratory chain leads to a vicious cycle of oxidant production, leading to local accumulation of H2O2 and ONOO, which can damage the cell’s antioxidant defenses like glutathione (GSH), glutathione peroxidase, catalase, etc. (Antunes et al., 2007). In fact, it is suggested that peroxynitrite or nitrosothioleinduced complex I inhibition occurs only after cellular GSH depletion specifically in the case of astrocytes, whereas decline in the overall activity of complexes II, III, and IV has been reported in neurons and astrocytes. Peroxynitrite is also known to be neurotoxic, and being a highly active free radical, its toxicity at the level of energy compromise could also be explained in a way that it leads to prolonged activation of poly(adenosine diphosphate ribose) polymerase, along with the disruption of Caþ2 homeostasis (Ricardo et al., 2013; Celia et al., 2012). Peroxynitrite and S-nitrosothiols are also known to interrupt the mitochondrial permeability transition pore, which leads to its own deleterious effects due to the fact that it results in leakage of many enzymes, proteins, and cytochrome c, causing apoptosis, whereas depletion of ATP synthesis due to membrane depolarization leads to cellular necrosis (da Silva et al., 2014). Since cytochrome c is the mediator of apoptosis and is a peripheral membrane protein, it is loosely bound to mitochondria, so it is released when mitochondrial permeabilization occurs. The pore has a tendency to close itself if the injury is transient, but if it remains open, it results in dissipation of mitochondrial proton gradient release of cytochrome c and ATP depletion. Along with these mechanisms, both nitric oxide and peroxynitrite are known to cause protein modifications that can lead to persistent inhibition of the complexes of the respiratory chain. Also, peroxynitrite (ONOO) inhibits and damages mitochondrial DNA, superoxide dismutase, ATP synthase, and mitochondrial membrane; this brings about irreversible inhibition of mitochondrial complexes I and II. Therefore, it could be said that peroxynitrite (ONOO) inhibits mitochondrial function at many sites and may affect respiration in heart, skeletal muscles, neurons, etc., causing decline in overall activity (da Silva et al., 2014; James et al., 1999; Katrin et al., 2013). Mostly it is said that the cytotoxicity attributed to nitric oxide is due to peroxynitrite produced through a diffusion-controlled reaction between nitric oxide and superoxide. All these cytotoxic events trigger cellular responses, which lead to oxidative injury, committing cells to either necrosis or apoptosis, and playing an important role in the etiopathology of cancer, neurodegenerative diseases, circulatory shock, myocardial infarction, diabetes, etc.

74 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Nitric Oxide Regulates Energy Metabolism and Body Composition Nitric oxide and other oxides of nitrogen act to monitor and regulate the cellular functions by bringing about different changes, along with regulation of vascular tone, which ultimately lead to changes in body composition. Being a gaseous signaling molecule, NO.governs many functions like blood delivery, glucose uptake, food intake, and metabolism of lipid and glucose (Table 4.2). Nitric oxide controls blood flow during exercise through neuronal NOS and endothelial NOS. Human studies have reported that nitric oxide plays a role in short-term adaptations to exercise, whereas long-term exercise is not so dependent on basal nitric oxide production. Altogether, combining the actions of nitric oxide on different tissues and its regulation on blood flow contributes to better substrate delivery and better substrate utilization by different tissues, including muscles and myocardium, which is definitely going to increase and contribute to enhanced exercise performance. As far as metabolism is concerned, nitric oxide is reported to preserve intracellular energy stores by promoting glucose uptake. Inhibition of NOS by L-NMMA (L-NG-monomethyl arginine), a relatively nonselective inhibitor of all NOS isoforms, resulted in almost 48% decline in glucose uptake by skeletal muscles (Brian and Bradford, 2014; Thomas et al., 2011; Cassilda et al., 2013). Nitric oxide regulates carbohydrate, amino acid, and lipid metabolism by acting on enzymes involved in different metabolic pathways, such as that nitric oxide is known to inhibit glyceraldehyde 3-phosphatedehydrogenase and thus in turn inhibits glycolysis at physiological levels. Nitric oxide has the property to act as a signaling molecule, and

Table 4.2

Functions of Nitric Oxide Synthases (NOSs)

S No.

Enzymes

Activators

Functions

1.

Endothelial NOS (eNOS)

Ca2þ/phosphorylation

2.

Neuronal NOS (nNOS)

Ca2þ/Phosphorylation

3.

Inducible NOS (iNOS)

Cytokines/endotoxin

4.

Mitochondrial NOS (mtNOS)

Ca2þ

Increases lipid oxidation and decreases lipid synthesis Suppresses gluconeogenesis Improves insulin release and glucose uptake Regulates insulin level Affects cytochrome oxidase activity Increases rynodine receptor activity Increases cardiac contractility Promotes insulin resistance in liver and muscle Promotes hyperphagia Increases glucose output Promotes inflammation Presence doubtful Isolated function not clear Taken to be nNOS bound to mitochondria

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through cGMP, it stimulates glucose uptake, along with oxidation of glucose and fatty acid in different tissues, such as skeletal muscles, liver, heart, and adipose tissue. It inhibits the synthesis of glucose, glycogen, and fat in the target tissues, whereas above its physiological levels, NO acts as an oxidant and inhibits enzyme-catalyzed reactions through protein modifications. Nitric oxide decreases the level of malonyl-CoA via inhibition of acetyl-CoA carboxylase and activation of malonyl-CoA carboxylase; therefore, nitric oxide reduces fat mass and corrects hyperlipidemia. Nitric is known to induce hyperphagia, antagonize anorectic signals, and stimulate food intake, whereas inhibitors of nitric oxide, e.g., N(G)-nitro-L-arginine methyl ester (L-NAME), promote weight loss and reduce food intake. Systemic regulation of glucose metabolism is attributed to nitric oxide production by endothelial nitric oxide synthase, which is known to suppress hepatic gluconeogenesis. This might give an impact that by promoting food intake, nitric oxide may increase adiposity and insulin resistance, but human studies have shown that L-arginine supplementation had favorable effects on body composition, since it leads to improved body composition and even reduces serum concentration of glucose, free fatty acids, and triacylglycerides, and thus increases muscle mass, despite the fact that it induces hyperphagia (Francis et al., 2008; Changjie et al., 2013; Yueh-Ying et al., 2014).

Nitric Oxide and Oxygen Consumption in Association With Physical Activity and Fitness Physical activity during any sports event or, for that matter, intense exercise, needs appropriate supply of nutrients and oxygen to different tissues and skeletal muscles involved. Nitric oxide has to play an important role in this context, since it is reported to be involved in blood delivery, oxygen supply, and distribution of oxygen to different tissues, specifically to the ones that are far from blood vessels, and it is also governed by gradients of nitric oxide and oxygen. It has been reported that oxygen is the substrate of all the three isoforms of NOSs with different apparent Km values, e.g., Km for endothelial NOS is 4 mM, Km of inducible nitric oxide synthase is 130 mM, and that of neuronal NOS is 350 mM. At low concentrations, NO competes with oxygen, whereas at high concentrations, it interacts with respiratory chain enzymes through nitrosylation and oxidation. Under normoxia, nitric oxide is scavenged by oxyhemoglobin and oxymyoglobin to produce nitrate, methemoglobin and metmyoglobin so that nitric oxide interaction with cytochrome is minimized, but under the conditions of hypoxia, nitric oxide reversibly binds to deoxyhemoglobin and deoxymyoglobin due to less competition from oxygen, and it reaches cytochrome oxidase, as now nitric oxide will be produced by nitrate through reaction of deoxyhemoglobin and deoxymyoglobin. Basically, hemoglobin and myoglobin produce nitric oxide at low oxygen and consume nitric oxide at high oxygen concentration. Under the influence of local large concentration of nitric oxide produced in response to inducible NOS, cellular respiration and globin activity is inhibited, leading to loss in the ability of oxygen supply and transport (Cynthia et al., 2012; Andrea et al., 2015).

76 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

If we consider skeletal muscle activity during exercise, any sports activity, or any exhaustive physical activity in this context, there are two criteria that classify the enhanced physical performance. These are metabolic dilations and flow-mediated dilation of feed arteries induced by nitric oxide produced constitutively by neuronal and endothelial NOS. It is also postulated that there is a nitric oxideeindependent pathway that leads to vasodilations of the arteriole network during exercise and intense physical activity, where acetylcholine released from neuromuscular junction triggers hyperpolarization, which is conducted along the endothelial cell layer via gap junction between cells regulating blood flow and vascular tone. Besides this mechanism, metabolic dilation is under the influence of prostacyclin, magnesium, potassium ions, pO2, pCO2, and temperature, also including K-ATP channels (Tohru et al., 2000; Tolga et al., 2013).

Nitric Oxide Governs Metabolism of the Proximate Principles of Food Nitric oxide is known to modulate liver metabolism through its action on the portal vein and hepatic artery, overall influencing the synthetic and detoxification function of liver. Nitric oxide is synthesized at various sites and performs various functions. At the basal physiological levels, nitric oxide, as mentioned, retains its essential antitumor, antibacterial, antiviral, and other activities, but when the burst of nitric oxide is produced under cytokine action, it changes itself to a cytotoxic agent, acting directly and indirectly through its metabolites (Farkas et al., 2016; Sosa and Grubesic, 2016). In terms of metabolism, nitric oxide and its derivatives are known to induce metabolic dysfunction, such as that it interferes with Krebs cycle and inhibits glycolytic enzymes and mitochondrial enzymes with FeeS groups. Nitric oxide and its reactive species are also known to disturb metabolic pathways by interacting with the structure of proteins and nonprotein compounds through the sulfhydryl group of cysteine residues and nitration of tyrosine and tryptophan residues. Although the role of nitric oxide in human metabolism is not completely understood and variable findings are being reported, data analysis supports that nitric oxide plays an important role in governing carbohydrate metabolism. It has been reported that when animal subjects were treated with nitric oxide donors, it resulted in the decrease in the mRNA levels of glucose transporter GLUT 2 and phosphoenolpyruvate carboxykinase (Molina et al., 2016), but when animals were treated with lipopolysaccharide, a hyperglycemic peak was followed by hypoglycemia. In terms of exercise and physical activities, nitric oxide does not mediate glucose uptake during rest or moderate exercise, whereas intense exercise affects glucose metabolism and glucose uptake, as shown in Fig. 4.3. Nitric oxide is known to suppress fatty acid metabolism in resting human skeletal muscle. Many animal and human models have also reported that increased rate of free fatty acid metabolism lowers the efficiency of the muscle, but when fatty acids are exclusively used as an energy source, it overall results in energy compromise. In

Chapter 4  An Overview on Nitric Oxide and Energy Metabolism

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Epinephrine/Norepinephrine

Serotonin Ganalin

Leptin / Neuropeptide Y

Ghrelin

n NOS

Regulators

Hunger/ Satiety Lack of exercise

Loss of regulation

Oxidised LDL and Phospholipids. Insulin resistance. Metabolic complications.

Obesity

TNF

n NOS

e NOS

i NOS

NO.

Caveolin

Increases Ca2+ release. Increases cardiac output. Fat mass

Insulin release. Glucose transport. Increases Ca Gluconeogenesis.

Hyperglycemia. Hyperinsulinemia. Gluconeogenesis.

Pancreatic function

Stimulate islet cell

NO. Regulate metabolism. Regulatess body composition. Improves insulin sensitivity. FIGURE 4.3 Role of nitric oxide in metabolic control.

conclusion, at rest, nitric oxide does not change glucose uptake by human skeletal muscles, but it is known to increase the fatty acid uptake, both at rest and under moderate exercise. In heart, it is clearly reported that endogenous nitric oxide appears to stimulate fatty acid oxidation and inhibit carbohydrate oxidation while inhibiting oxygen consumption (Monti et al., 2015; Rabelo et al., 2016).

Conclusion Nitric oxide is synthesized in our body by NOSs and is known to exert its effects at different sites through cGMP-mediated pathways. In terms of its functions, nitric oxide plays an important role in metabolic control, mitochondrial respiration, and production of energy. Energy expenditure at the resting state, which involves basic cellular functions, e.g., cell turnover, DNA/RNA synthesis, protein synthesis, and thermogenesis, in all these conditions mitochondria are central to energy expenditure. Nitric oxide acts as regulator of cellular function, where it can act as a mediator of immune function and synaptic transmission, regulator of blood flow, etc. Nitric oxide is also known to regulate the binding and release of oxygen, thus affecting the metabolic capacity of different

78 SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

tissues. Nitric oxide from brain antagonizes anorectic signals and stimulates food intake, so nitric oxide dysfunction limits exercise capacity, whereas exercise at moderate levels enhances the activity of nitric oxide system. On the other hand, nitric oxide can react with cellular constituents through its RNS, like peroxynitrite, nitrosothiols, etc., to bring about irreversible inhibition of respiration, and it also can irreversibly react with cellular constituents to bring about their modifications. Therefore, depending upon the concentration and time of exposure, nitric oxide can be both cytoprotective and cytotoxic, i.e., it functions as a double-edged sword. A high concentration of nitric oxide inhibits mitochondrial respiration, resulting in the inhibition of heat production, which prevents heat-induced damage to different tissues. Hyperthermia leads to an increase in the activity of NOSs and ultimately vasodilatation and increased blood flow that contribute to the supply of substrate and oxygen during intense physical activity, thereby enhancing exercise capacity and performance. It also preserves intracellular energy stores by inhibiting glycolysis and Krebs cycle and regulating lipid metabolism.

References Andrea, P., Bianca, C., Andrielly, H.R.A., Barbara, M., 2015. Spotlights on immunological effects of reactive nitrogen species: when inflammation says nitric oxide. World J. Exp. Med. 5, 64e76. Antunes, F., Boveris, A., Cadenas, E., 2007. On the biologic role of the reaction of NO with oxidized cytochrome c oxidase. Antioxid. Redox Signal 10, 569e579. Benamar, A., Rolletschek, H., Borisjuk, L., Avelange-Machere, M.H., Curien, G., Mostefai, H.A., Andriantsitohaina, R., Macherel, D., 2008. Nitrite-nitric oxide control of mitochondrial respiration at the frontier of anoxia. Biochim. Biophys. Acta 1777, 1268e1275. Borutaite, V., Moncada, S., Brown, G.C., 2005. Nitric oxide from inducible nitric oxide synthase sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319e323. Brian, E.S., Bradford, G.H., 2014. Anti-obesogenic role of endothelial nitric oxide synthase. Vitamins Horm. 96, 323e346. Busija, D.W., Rutkai, I., Dutta, S., Katakam, P.V., 2016. Role of mitochondria in cerebral vascular function: energy production, cellular protection, and regulation of vascular tone. Compr. Physiol. 6, 1529e1548. Cassilda, P., Nuno, R.F., Ba´rbara, S.R., Rui, M.B., Joa˜o, L., 2013. The redox interplay between nitrite and nitric oxide: from the gut to the brain. Redox Biol. 1, 276e284. Celia, H.T., Gabriela, S.R., Rosely, O.G., 2012. Nitric oxide in skeletal muscle: role on mitochondrial biogenesis and function. Int. J. Mol. Sci. 13, 17160e17184. Changjie, H., Qingguo, Z., Baisong, L., 2013. The role of nitric oxide signaling in food intake; insights from the inner mitochondrial membrane peptidase 2 mutant mice. Redox Biol. 1, 498e507. Christelle, K., Suhas, K., Sruti, S., 2012. Myoglobin and mitochondria: a relationship bound by oxygen and nitric oxide. Nitric Oxide 26, 251e258. Christine, H., Daniel, B., Kim-Shapiro, 2013. Hemoglobin-mediated nitric oxide signaling. Free Radic. Biol. Med. 0, 464e472. Claude, A., Piantadosi, 2012. Regulation of mitochondrial processes by protein S-Nitrosylation. Biochim. Biophys. Acta 1820, 712e721.

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Cynthia, M.B., Daniel, L., Serpi, C.E., 2012. Nitric oxide in adaptation to altitude. Free Radic. Biol. Med. 52, 1123e1134. da Silva, M.P., Cedraz-Mercez, P.L., Varanda, W.A., 2014. Effects of nitric oxide on magnocellular neurons of the supraoptic nucleus involve multiple mechanisms. Braz. J. Med. Biol. Res. 47, 90e100. Farkas, I., Vastagh, C., Farkas, E., Ba´lint, F., Skrapits, K., Hrabovszky, E., Fekete, C., Liposits, Z., 2016. Glucagon-like Peptide-1 excites firing and increases GABAergic miniature postsynaptic currents (mPSCs) in Gonadotropin-releasing hormone (GnRH) neurons of the male mice via activation of Nitric oxide (NO) and suppression of endocannabinoid signaling pathways. Front. Cell. Neurosci. 10, 214. Finocchietto, P.V., Holod, S., Barreyro, F., Peralta, J.G., Alippe, Y., Giovambattista, A., Carreras, M.C., Poderoso, J.J., 2011. Defective leptin-AMP-dependent kinase pathway induces nitric oxide release and contributes to mitochondrial dysfunction and obesity in ob/ob mice. Antioxid. Redox Signal 15, 2395e2406. Francis, K., Matilda, P., Ezekiel, M., Norma, O.R., Gregory, J.M., Brent, E.W., Elizabeth, A.K., Alan, C., Michael, W.S., 2008. Vascular inflammation, insulin resistance and reduced nitric oxide production precede the onset of peripheral insulin resistance. Arterioscler. Thromb. Vasc. Biol. 28, 1982e1988. James, R.B., Paul, T.S., He Zhang, 1999. Nitric oxide acutely inhibits neuronal energy production. J. Neurosci. 19, 147e158. Katherine, J.H., Kari, T.C., Gordon, P.M., John, A.C., 2009. Nitric oxide mediates a shift from early necrosis to late apoptosis in cytokine-treated b-cells that is associated with irreversible DNA damage. Am. J. Physiol. Endocrinol. Metab. 297, E1187eE1196. Katrin, F.N., Volker, L., Rolf, H., Georges, von, D., 2013. Thrombin has Biphasic effects on the nitric oxide-cGMP pathway in endothelial cells and contributes to experimental pulmonary hypertension. PLoS One 8, e63504. Kautza, B., Gomez, H., Escobar, D., Corey, C., Ataya, B., Luciano, J., Botero, A.M., Gordon, L., Brumfield, J. , Martinez, S., Holder, A., Ogundele, O., Pinsky, M., Shiva, S., Zuckerbraun, B.S., 2015. Inhaled, nebulized sodium nitrite protects in murine and porcine experimental models of hemorrhagic shock and resuscitation by limiting mitochondrial injury. Nitric Oxide 51, 7e18. Kindo, M., Gerelli, S., Bouitbir, J., Hoang Minh, T., Charles, A.L., Mazzucotelli, J.P., Zoll, J., Piquard, F., Geny, B., 2016. Left ventricular transmural gradient in mitochondrial respiration is associated with increased sub-endocardium nitric oxide and reactive oxygen species productions. Front. Physiol. 7, 331. Molina, M.N., Ferder, L., Manucha, W., 2016. Emerging role of nitric oxide and heat shock proteins in insulin resistance. Curr. Hypertens. Rep. 18, 1. Monti, L.D., Galluccio, E., Fontana, B., Spadoni, S., Comola, M., Marrocco Trischitta, M.M., Chiesa, R., Comi, G., Bosi, E., Piatti, P., 2015. Pharmacogenetic influence of eNOS gene variant on endothelial and glucose metabolism responses to L-arginine supplementation: post hoc analysis of the Larginine trial. Metabolism 64, 1582e1591. Rabelo, L.A., Todiras, M., Nunes-Souza, V., Qadri, F., Szija´rto´, I.A., Gollasch, M., Penninger, J.M., Bader, M., Santos, R.A., Alenina, N., 2016. Genetic deletion of ACE2 induces vascular dysfunction in C57BL/6 mice: role of nitric oxide imbalance and oxidative stress. PLoS One 11, e0150255. Ricardo, C., Crabtree, M.J., Vidhya, S., Barbara, C., David, A.K., 2013. Nitric oxide synthases in heart failure. Antioxid. Redox Signal 18, 1078e1099. Sangwon, F.K., 2011. The role of nitric oxide in prostaglandin biology; update. Nitric Oxide 25, 255e264. Sharron, H.F., Jennifer, L.B., Jackie, D.C., 2010. cGMP-dependent protein kinases and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol. Rev. 62, 525e563. Simonin, V., Galina, A., 2013. Nitric oxide inhibits succinate dehydrogenase-driven oxygen consumption in potato tuber mitochondria in an oxygen tension-independent manner. Biochem. J. 449, 263e273.

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Sosa, I., Grubesic, A., 2016. Putative hormone with anti-obesogenic and insulin-sensitizing effect. Int. J. Immunopathol. Pharmacol. 1, 147e148. Sun, M., Liu, H., Xu, H., Wang, H., Wang, X., 2016. CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity. Mol. Cell Biochem. 420, 195e206. Thomas, P.J., Solomon, J.M., Li, H.Y., John, P.K., 2011. Progressive hyperglycemia across the glucose tolerance continuum in older obese adults is related to skeletal muscle capillarization and nitric oxide bioavailability. J. Clin. Endocrinol. Metab. 96, 1377e1384. Tohru, F., Martin, R.S., Masuko, U.-F., Yian, C., Georg, K., David, G.H., 2000. Regulation of the vascular extracellular superoxide dismutase by nitric oxide and exercise training. J. Clin. Invest. 105, 1631e1639. Tolga, A., Faruk, T., Emine, K., Mehmet, Z.O., Faik, V., 2013. The relationships between simulated tennis performance and biomarkers for nitric oxide synthesis. J. Sports Sci. Med. 12, 267e274. Ulrich, F., William, C.S., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829e837. Yueh-Ying, H., Erick, F., Juan, C.C., 2014. Adiposity, Fractional exhaled nitric oxide, and Asthma in U.S. Children. Am. J. Respir. Crit. Care Med. 190, 32e39.

5

Antioxidants and Mitochondrial Bioenergetics

Sushil Sharma SAINT JAMES SCHOOL OF ME DICINE, ST. VINCENT, WEST INDIES

Introduction As a universal preapoptotic biomarker of compromised mitochondrial bioenergetics, Charnoly body (CB) was initially discovered in developing undernourished rat Purkinje neurons. CB appears as a pleomorphic, electron-dense, multilamellar, quasicrystalline stack of degenerated mitochondrial membranes and downregulation of mitochondrial genome owing to free radical overproduction (Sharma, 1985; Sharma et al., 1986, 1987; Sharma, 1988). CB originates by the aggregation of degenerated mitochondrial membranes as a result of free radicaleinduced, compromised mitochondrial bioenergetics. Free radicals cause lipid peroxidation by the structural and functional breakdown of polyunsaturated fatty acids caused by nutritional stress, toxin exposure, and microbial infection [including Zika virus (ZIKV)]. In general, free radicals induce proteases to cause proteolysis, lipases to cause lipolysis, nucleases to cause DNA fragmentation, and apoptosis in highly vulnerable cells, such as neural progenitor cells (NPCs) derived from induced pluripotent cells. We divided the CB life cycle into four major phases: origin, development, maturation, and degradation (Sharma et al., 1993a,b), and defined CB autophagy as charnolophagy and lysosome-containing phagocytosed CB as charnolophagosome. These neurodegenerative changes at the subcellular level are triggered as a consequence of compromised mitochondrial bioenergetics and can be ameliorated by antioxidants including glutathione, metallothioneins (MTs), CoQ10, melatonin, selegiline, resveratrol, sirtuin, rutin, lycopene, and catechin by preventing CB pathogenesis. Thus, antioxidants serve as free radical scavengers and augment mitochondrial synthesis as well as their bioenergetics in different body organs, particularly in the central nervous system (CNS) and cardiovascular tissue. We have shown that CoQ10, melatonin, and MTs prevent thiol oxidation of brain mitochondrial complex-1, a-synuclein, and parkin to provide neuroprotection in Parkinson’s disease (PD) (Ebadi et al., 2001). By employing potent complex-1 inhibitors, 1-methyl,4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPPþ), rotenone, and salsolinol as Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00005-3 Copyright © 2017 Elsevier Inc. All rights reserved.

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experimental models of PD, it was established that CoQ10 provides neuroprotection by inhibiting mitochondrial oxidative and nitrative stress (Ebadi et al., 2001). In addition, a specific monoamine oxidase-B (MAO-B) inhibitor, selegiline, provides neuroprotection by MT induction in cultured dopaminergic (DA-ergic) (SK-N-SH) neurons (Ebadi et al., 2002). Furthermore, we established a functional relationship between a-synuclein and MTs in PD (Ebadi and Sharma, 2002) and discovered that MTs provide ubiquinone (CoQ10)-mediated neuroprotection by preventing free radical overproduction, whereas selegiline attenuates MPPþ-induced mitochondrial degeneration and CB formation to provide neuroprotection and apoptotic signal transduction by suppressing c-fos and c-jun proto-oncogene expression in cultured DA-ergic (SK-N-SH) neurons (Sharma et al., 2003). We determined that MT isoforms attenuate peroxynitrite (ONOO)-induced oxidative and nitrative stress in PD (Sharma and Ebadi, 2003; Ebadi and Sharma, 2003; Ebadi et al., 2004a,b; Sharma et al., 2004), and homozygous weaver (wv/wv) mice exhibit symptoms typical of progressive neurodegeneration and Parkinsonism owing to downregulation of mitochondrial complex-1 and a reduction in CoQ10 (Ebadi et al., 2004a,b). We also established that CoQ10 stabilizes MTs (Ebadi et al., 2004a,b), whereas MTs attenuate ONOO induced oxidative stress in PD (Ebadi et al., 2005a,b; Ebadi et al., 2006). This was further confirmed in various a-synuclein and MTs gene-manipulated mouse models of PD and aging (Ebadi et al., 2005a,b; Sharma and Ebadi, 2014a,b,c). We discovered that CoQ10 inhibits mitochondrial complex-1 downregulation and nuclear factor-kB activation in cultured SK-N-SH neurons as well as in wv/wv mice (Ebadi et al., 2004a,b). A potent mitochondrial complex-1 inhibitor, MPPþ, inhibited TRPC-1, a calcium channel, to cause apoptosis in cultured SH-S-Y5Y neurons (Bollimuntha et al., 2005), whereas cocaine and methamphetamine-induced neurotoxicity in SK-N-SH neurons as well as in C57BL/6J mice was attenuated by CoQ10, further confirming the therapeutic potential of CoQ10 in mitochondrial neuroprotection (Klongpanichapak et al., 2006). It is known that iron participates in the Fenton reaction to synthesize ONOO ions in the presence of OH and NO radicals, generated as a by-product of mitochondrial oxidative phosphorylation during adenosine triphosphate (ATP) synthesis in the electron transport chain. ONOO ions induce not only oxidative but also nitrative stress in physicochemically injured mitochondria to trigger CB formation. In addition, iron causes mitochondrial translocation of a-synuclein to induce oxidative and nitrative stress as a result of free radical overproduction (Sangchot et al., 2002). Iron-induced oxidative stress, mitochondrial aggregation, and a-synuclein translocation in SK-N-SH cells cause progressive neurodegeneration (Sangchot et al., 2002), whereas a potent iron chelator, deforoxamine, attenuated iron-induced oxidative stress by inhibiting a-synuclein translocation in the cultured SK-N-SH cells (Sangchot et al., 2002). Pretreatment with CoQ10 prevented iron-induced apoptosis in cultured SK-N-SH neurons (Kooncumchoo et al., 2006). We established that mitochondrial complex-1 activity and 18F-dihydroxyphenylalanine (18F-DOPA) uptake are significantly reduced

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in a genetically engineered mouse model of PD, whereas CoQ10 provides neuroprotection (Sharma et al., 2006). Furthermore, we determined that CoQ10 augments brain regional 18 F-DOPA and 18-fluorodeoxyglucose (18-FdG) uptake in MT-overexpressing wv/wv mice (Sharma and Ebadi, 2008a) and MTs provide therapeutic potential in PD (Sharma and Ebadi, 2008b). Hence MTs can be used as early and sensitive biomarkers of redox signaling in various neurodegenerative disorders (Sharma and Ebadi, 2011a) including ZIKV charnolopathies involved in diversified embryopathies (Sharma, 2016aee; Sharma et al., 2016a,b). We discovered for the first time interleukin (IL)-10 receptors on mouse cortical and hippocampal cultured neurons (Sharma et al., 2011) and established that IL-10 directly protects cortical neurons by activating phosphoinositide(PI)-3 kinase and signal transducer and activator of transcription (STAT)-3emediated signal transduction cascade. Similar to MTs, IL-10 provides antiapoptotic and antiinflammatory neuroprotection through zinc-mediated transcriptional activation of genes involved in growth, proliferation, differentiation, and development. Potential mitochondrial bioenergetics-based drugs described in this chapter include CB antagonists, charnolophagy agonists, charnolophagosome stabilizers, and CB sequestration inhibitors to prevent and/or cure diversified charnolopathies involved in various neurodegenerative disorders, cardiovascular disorders, and cancer. In general, mitochondrial-targeted drugs can be classified as follows: (1) CB agonists/antagonists; (2) charnolophagy agonists/antagonists, charnolophagosome stabilizers/destabilizers; and (3) CB sequestrants/desquestrants, in addition to charnolostatics, charnolocidals, and charnolomimetics, as described in this chapter.

Experimental Studies To establish the novel concept of charnolopharmacotherapeutics for the prevention and cure of progressive neurodegenerative diseases, cardiovascular diseases, multidrugresistant malignancies, and infections (including ZIKV disease), we performed several in vivo, ex vivo, and in vitro experiments on gene-manipulated animals and cultured cells. These experiments were accomplished by performing in vitro as well as in vivo experiments on developing normal and protein or pyridoxine (B6)-deficient developing rats, MTs, and a-synuclein gene-manipulated mice, aging mitochondrial genome knockout (RhOmgko) cultured human DA-ergic (SK-N-SH and SH-S-Y5Y) cells, and neuronal stem cells. Transmission electron microscopy, scanning electron microscopy, confocal microscopy, and digital fluorescence imaging were employed to discover different phases of CB life cycle, charnolophagy, charnolophagomes, and CB sequestration in the developing undernourished rat Purkinje neuron. First, the mitochondrial membrane potential (DJ) in response to toxic exposure of MPPþ, salsolinol, and rotenone was evaluated by conducting digital fluorescence microscopic and confocal microscopic analyses using JC-1 as a sensitive fluorochrome in cultured SK-N-SH and SH-S-Y5Y neurons. Subsequently, several nuclear and mitochondrial genes involved in

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growth, proliferation, differentiation, development, and apoptosis were analyzed at the translational and transcriptional levels by performing immunoblotting, reverse transcriptaseepolymerase chain reaction, and microarray analyses. Nuclear magnetic resonance spectroscopy and magnetic resonance imaging were performed to correlate and confirm molecular imaging data derived from micropositron emission tomography (microPET) neuroimaging. The PET radiopharmaceuticals 18FdG and 18F-DOPA were synthesized in the cyclotron and microPET imaging labs as molecular imaging biomarkers of brain regional mitochondrial bioenergetics and DA-ergic neurotransmission, respectively. A Siemens Molecular Solutions microPET Imaging System equipped with microPET Manager for data acquisition in List mode, and AsiPro for image reconstruction in multidimensional mode were used to acquire in vivo molecular images of brain regional mitochondrial bioenergetics and DA-ergic neurotransmission in normal control and toxin-exposed gene-manipulated mice. The detailed methodology is described in several of our publications.

Therapeutic Potential of Antioxidants We established that as potent free radical scavengers, MTs prevent CB formation and serve as antiinflammatory and apoptotic agents in polysubstance abuse (Sharma and Ebadi, 2011b; Sharma, 2015a,b,c). We established the clinical significance of MTs in CB prevention in nanomedicine and proposed three types of nanoparticles (NPs), based on their response to mitochondrial bioenergetics. Neutral NPs have no effect on CB formation; toxic NPs enhance CB formation, whereas neuroprotective NPs inhibit CB formation. Hence reactive oxygen species (ROS)-scavenging antioxidant-loaded NPs can be developed for therapeutic intervention in neurotoxin-induced charnolopathies involved in diversified embryopathies, including ZIKV microcephaly (Sharma et al., 2013; Sharma, 2015a,b; Sharma, 2016a-e). We established the significance of CB formation in kainic acide and domoic acide induced apoptosis of cultured neurons and hippocampal CA-3 and dentate gyrus neurons in the developing brain (Sharma et al., 2014a,b). In this chapter, the clinical significance of antioxidants as promising charnolopharmacotherapeutic agents for the safe and effective treatment of multidrug-resistant malignancies and other chronic diseases is highlighted (Sharma, 2014a-i, 2015a,b,c, 2016b). We reported that intrauterine exposure of ethanol causes fetal alcohol syndrome (FAS) represented by typical craniofacial abnormalities as noticed in ZIKV embryopathies as a consequence of CB formation in the most susceptible NPCs in the developing embryo (Sharma et al., 2014a,b; Chabenne et al., 2014) and that CB formation occurs in the hippocampal CA-3 and dentate gyrus regions as a result of cerebral ischemia in vascular dementia (Jagtap et al., 2015). We emphasize that hippocampal CB formation is involved in major depressive disorders (MDDs). Hence any physiological and/or pharmacological interventions to prevent or inhibit free radical overproduction by antioxidants such as MTs and glutathione can provide neuroprotection (Sharma, 2014a,b). Moreover, any physicochemical

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injury and/or stress can induce hippocampal CB formation to cause major depression and posttraumatic stress disorder (Sharma, 2015a,b,c), whereas the MAO-B inhibitor selegiline provides neuroprotection by preventing brain regional disease-specific CB formation (Sharma, 2015a,b,c). In our publications, we reported ZIKV-induced CB formation in the most vulnerable developing NPCs to trigger charnolopathies involved in a diversified spectrum of developmental embryopathies (Sharma, 2016a,b), and recommended the therapeutic potential of a protein-rich, alcohol-free, well-nourished diet; B vitamins; folate; and antioxidants such as MTs, glutathione, and melatonin in the prevention of diversified charnolopathies, because these clinically beneficial antioxidants inhibit CB formation particularly in apoptosis, neurodegeneration, and early morbidity and mortality owing to impaired synaptic transmission and cognition.

Mosquito-Borne Diseases and Cancer It is recognized that mosquito-borne diseases have increased the global incidence of cancer. Hence it will be highly prudent to prevent their outbreak, spread, and transmission by successful, safe, and effective vector eradication program to reduce not only early morbidity and mortality but also multidrug-resistant malignancies, in addition to progressive neurodegenerative disorders of known and unknown etiopathogenesis as noticed in ZIKV disease. Nonspecific induction of CB formation causes gastrointestinal tract (GIT) symptoms, myelosuppression, alopecia, myocardial infarction, neurotoxicity, nephrotoxicity, hepatotoxicity, and pulmonary toxicity during chronic treatment of multidrug-resistant malignancies. Hence drugs may be developed to induce cancer stem cellespecific CB formation, inhibit charnolophagy, augment charnolophagosome destabilization, and augment CB sequestration in highly proliferative cancer stem cells to eradicate multidrug-resistant malignancies successfully with minimum or no adverse effects, and vice versa for the treatment of various cardiovascular disorders, neurodegenerative disorders, and infections such as mosquito (Aedes aegypti)-borne ZIKV disease.

Clinical Significance of Charnoly Body Theranostics We reported that the accumulation of CB at the junction of axon hillock can cause impaired axoplasmic transport of various ions, enzymes, hormones, neurotransmitters, neurotropic factors [brain-derived neurotropic factor (BDNF), nerve growth factor (NGF)-1, and insulin-like growth factor-1], and mitochondria at the synaptic terminals to cause impaired synaptic transmission and progressive degeneration, resulting in cognitive impairment accompanied by early morbidity and mortality (Sharma et al., 2014a,b). Basic knowledge acquired through scientific developments employing modern -omics biotechnology, next-generation sequencing, molecular genetics, epigenetics, and

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molecular imaging is remarkable and will certainly help prepare us to control any other virulent viral, bacterial, and/or fungal outbreak in the future. Although charnolostatic agents will have therapeutic potential during the acute phase of ZIKV disease, charnolocidal agents will be needed for immediate and complete remission of ZIKV disease. We need to be fully prepared far ahead of time so that individuals (particularly developing infants from economically poor countries) will not experience undesirable and deleterious consequences of ZIKV-linked charnolopathies involving microcephaly, embryopathies, Guillain-Barre´ syndrome (GBS), and reproductive anomalies. In addition to classical -omics (vaccinomics), next-generation sequencing and conventional molecular biological approaches will facilitate charnolopharmacotherapeutics not only of ZIKV disease but of several other progressive neurodegenerative and cardiovascular diseases and of cancer.

Clinical Significance of Charnoly Body Formation in Zika Virus and Other Diseases ZIKV has been proposed to induce organ- and disease-related MAO-Ae or MAOBespecific CB formation, accompanied by mitochondrial translocator protein (TSPO), Ca2þ, iron, and cholesterol delocalization in NPCs or any other highly vulnerable cell, including osteoblast stem cells, spermatogonia, spermatocytes, Sertoli cells, and oocytes, to cause diversified embryopathies and several craniofacial abnormalities. These pathophysiological events can cause selective loss of brain regional noradrenergic, serotonergic, and DA-ergic neurotransmission, respectively, owing to synaptic degeneration, which may lead to cognitive impairment and sensorimotor deficits in learning, intelligence, memory, and behavior, resulting in early morbidity and mortality. Significant impairment in sensorimotor performance is expected in individuals with ZIKV microcephaly in Brazil, Colombia, and several other countries including the United States and the Caribbean islands. Hence various potential prophylactic and therapeutic biomarkers as well as drug discovery targets have been proposed based on the systematic pathophysiological consequences of ZIKV-induced CB formation. These include but are not limited to ZIKV-induced downregulation of mitochondrial bioenergetics, CB formation triggering charnolophagy through lysosomal activation as a basic molecular mechanism of intracellular detoxification, charnolophagosome formation (a waste disposal container), CB sequestration, and apoptosis resulting in microcephaly and other diversified spectrum of embryopathies, infertility, and GBS.

Personalized Nanotheranostics It is envisaged that emerging nanotechnology will provide better, safe, effective, and economical personalized theranostic options for cancer and other drug-resistant chronic diseases including Alzheimer’s disease (AD), PD, chronic drug addiction, and MDDs, for

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which conventional medical treatment has limited prospectus. Hence future development in innovative NPs, nanomaterials, and nanodevices for targeted drug delivery using chronopharmacological approaches may provide better theranostics and reduce the cost of time-consuming, cumbersome, and potentially painful and futile therapeutic interventions with serious adverse effects. Personalized medicine can design time- and cost-effective theranostic protocols for each patient while taking into consideration genetic predisposition and individual variability to accomplish the best treatment with minimum or no adverse effects. For instance, treatment with patient-specific stem cells is a promising effort in this direction. Micellar NPs for the topical and transdermal drug delivery of pharmaceuticals and personal care products have been developed (Lee et al., 2010). In particular, specific CB and/or chronolophagy agonists and antagonists can be developed for the effective treatment of neurodegenerative diseases, cardiovascular diseases, and cancer. In addition to possible therapeutic strategies with small interfering RNA, microRNAs (miRNAs) are involved in posttranscriptional control and may be deregulated in chronic diseases and aging (Wang, 2009, Wang et al., 2011). Lactosyl gramicidinebased lipid NPs for the targeted delivery of anti-mir-155 to hepatocellular carcinoma were developed (Zhang et al., 2015). Further studies to explore the exact pathophysiological significance of dysregulated miRNAs in compromised mitochondrial bioenergetics causing CB formation in chronic illnesses will further expand our capability in nanotheranostics (Hamburg and Collins, 2010).

Fetal Alcohol Syndrome and Zika Virus Disease It is well established that alcohol consumption during any phase of pregnancy is deleterious. Alcohol induces deleterious effects during the first trimester of pregnancy that may be further complicated by intrauterine exposure to environmental toxins and/or microbial infections including ZIKV infection. Differential diagnosis of FAS is still a challenge. Hippocampal somatostatin and BDNF are reduced in FAS. In particular, hippocampal NPCs are highly susceptible to ethanol in addition to ZIKV virus. We reported CB as a biomarker as well as novel drug discovery target for the safe and effective prevention and treatment of ZIKV-induced embryopathies including microcephaly. ZIKV induces selective apoptosis of highly vulnerable NPCs derived from induced pluripotent stem cells (iPPs) through CB formation (Sharma, 2016a,b). CB appears to be caused by free radical overproduction as a result of compromised mitochondrial bioenergetics in a highly vulnerable cell (NPCs) in response to any physicochemical injury (including environmental toxins such as kainic acid and domoic acid, metal ions such as mercury and lead, and infections such as cytomegalovirus, rubella, and ZIKV), whereas “charnolophagy” is an energy-driven basic molecular mechanism of intracellular detoxification (Sharma and Ebadi, 2014a,b,c). We proposed that antiviral drugs preventing CB formation and/or augmenting “charnolophagy” will be highly beneficial for the prevention and/or effective treatment of ZIKV disease during the early phase of neuronal development, whereas “charnolophagosome”

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stabilizers, tissue-specific charnolophagy agonists, and “CB sequestration inhibitors” have promising therapeutic potential during the chronic phase of “ZIKV disease” (Sharma et al., 2016a,b,c).

Autophagy Versus Charnolophagy Christian de Duve, a Nobel Laureate from Belgium, discovered lysosomes and introduced the term “autophagy.” He was awarded the shared Nobel Prize for Physiology or Medicine in 1974, along with Albert Claude and George E. Palade, for describing the structure and function of organelles (lysosomes and peroxisomes) in biological cells. This year’s Nobel Prize in Physiology or Medicine was awarded to Prof. Yoshinori Ohsumi from Japan for discovering the basic molecular mechanisms of autophagy. As a doctoral student, the author discovered CB as a preapoptotic biomarker of compromised mitochondrial bioenergetics in the developing undernourished rat cerebellar Purkinje cells. He introduced the term “charnolophagy (CB autophagy)” by lysosomal activation because of free radical overproduction during nutritional stress, toxic insult, and/or infection, including ZIKV disease. CB is a universal biomarker of cell injury, whereas charnolophagy is a novel drug discovery target to evaluate intracellular detoxification. ZIKV compromises charnolophagy to cause microcephaly and several diversified embryopathies involving charnolopathy, as described in this chapter. The term “autophagy” can be used for eukaryotic as well as prokaryotic cells with no mitochondria and/or nuclei. However, “charnolophagy” is a more specific and restricted term that represents especially CB phagocytosis, and is formed as a consequence of compromised mitochondrial bioenergetics owing to free radical overproduction in a highly vulnerable cell in response to nutritional stress, environmental toxins, and/or microbial infection such as ZIKV infection. Thus charnolophagy can occur only in highly sensitive and specialized cells (particularly in eukaryotes) possessing structurally welldefined mitochondria as well as nuclei equipped with mitochondrial as well as nuclear genes involved in oxidative phosphorylation, growth, proliferation, differentiation, development, and apoptosis during nutritional stress, toxic exposure, and/or microbial infection (including ZIKV). Charnolophagy is one of the most significant molecular events that occurs during conception because of primarily nuclear DNA in the head and condensed mitochondria in the middle piece of a spermatocyte. The tail does not enter the oocyte during fertilization. Almost all mitochondrial energy is consumed to translocate male nuclear DNA near the female nuclear DNA during fertilization. The degenerated mitochondrial membranes of the middle piece are condensed to form CB, which is eliminated efficiently through energy-driven lysosomal activation in the oocytes by charnolophagy for the normal growth and development of the fetus. Hence charnolophagy is highly crucial for the normal and time-bound growth and development of the fetus. Inhibition of charnolophagy during fertilization can cause either zygotic death or diversified

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embryopathies, as noticed in severe undernutrition, toxin exposure, and FAS, and in viral infections including cytomegalovirus, rubella virus, and ZIKV. Hence the term “charnolophagy” should not be used indiscriminately and misinterpreted with general “autophagy.” Charnolophagy represents a particular energydriven CB autophagy, which involves autophagy of specific CBs in a highly vulnerable cell, as we discovered in developing undernourished Purkinje cells and in hippocampal neurons of kainic acide and domoic acideexposed cultured mice and in the CA-3 and dentate gyrus regions of mice exposed to intrauterine domoic acid during pregnancy (Sharma, 1985; Sharma et al., 1986, 1987, 1993a,b; Dakshinamurti et al., 1991, 1992, 1993; Sharma et al., 1994, 2013; Sharma and Ebadi, 2014a,b; Sharma, 2015a,b,c, 2016a,b; Sharma, 2017). Usually mitochondrial abundance in a cell depends on its metabolic activity and physiological energy (ATP) requirement. For instance, cardiomyocytes and smooth muscle cells have a maximum number of mitochondria compared with hepatocytes and neurons. In fact, 40% of the heart, 20% of the liver, and 15% of the CNS is composed of only mitochondria. Hence, the term “charnolophagy” will be more specific to these and other organs and diseases associated with these organs. Nevertheless, the natural abundance of mitochondria and the genetic susceptibility of the mitochondrial DNA (a neuron may have as many as 1000 mitochondria, which serve as powerhouses for ATP synthesis) render any metabolically active cell highly vulnerable to CB formation during oxidative and/or nitrative stress. For example, developing NPCs are highly susceptible to oxidative and nitrative stress of malnutrition, environmental toxins, drugs of abuse, and infections (including ZIKV). Free radicals (such as $OH and NO$) are generated as a by-product of mitochondrial oxidative phosphorylation. ATP requirements are significantly elevated during neurotropic ZIKV infection and free radical synthesis is augmented, which causes extensive mitochondrial degeneration with limited regenerative potential. Free radicals are highly reactive species and induce lipid peroxidation of mitochondrial membranes to cause structural and functional breakdown of polyunsaturated fatty acids. Owing to the natural abundance of free radicals in the mitochondria, particularly during oxidative and nitrative stress, inner mitochondrial membranes are destroyed first, as we discovered in severely undernourished developing rat cerebellar Purkinje neurons. Free radicals have a short half-life (1013 to 1014 s) and their overproduction during any physicochemical stress can induce mitochondrial destruction by augmenting CB formation, whereas charnolophagy is an efficient molecular mechanism of intracellular detoxification and is highly significant for normal health and well-being.

Phagolysosome Versus Charnolophagosome The term “phagolysosome” represents a significantly enlarged lysosome-containing phagocytosed subcellular components including plasma membranes [endoplasmic reticulum (ER), mitochondria, peroxisomes, Golgi body, nuclear membranes, and several

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other intracellular organelles], whereas charnolophagosomes represent lysosomes that have specifically phagocytosed CBs. Charnolophagosomes are usually localized in the vicinity of a CB; they are abnormally enlarged and electron-dense, and have a highly unstable structure. The incidence of charnolophagosomes is increased under nutritional stress, toxic exposure, and infection in a eukaryotic cell (particularly in the NPCs) in the developing embryo in response to ZIKV infection. The frequency of charnolophagosome formation is increased as a function of CB formation during nutritional stress. Thus charnolophagosomes represent a specific event that occurs exclusively after CB formation to enhance charnolophagy as a basic molecular mechanism of intracellular detoxification for normal cellular function (Sharma et al., 1986, 1987, 1993a,b). The frequency of CB formation, charnolophagy, and charnolophagosome formation was significantly increased during nutritional stress and was reduced by nutritional rehabilitation of 80 days in developing undernourished rat cerebellar Purkinje neurons. CB formation occurred particularly in dendrites and growth cones (possessing the maximum number of mitochondria) during severe nutritional stress. How many CBs a lysosome can phagocytose is yet to be established. However, the size of a charnolophagosome was w2.5 times greater than that of lysosomes in the developing undernourished rat cerebellar Purkinje neurons. Moreover, its electron density significantly increased, which facilitated their identification and quantitation. A positive correlation between CB formation and charnolophagosome formation with a correlation coefficient of 0.8 was noticed, as illustrated in Fig. 5.1. To estimate the mitochondrial bioenergetics quantitatively, we developed a kinetic equation: DE ¼ KDT, where DE is the difference in the 14C-labeled glucose use between normal well-fed and undernourished neurons; and DT is the difference in the duration of firing between normal and undernourished neurons. DE and DT approached 0 in 30 days’ undernourished developing rats that were subsequently nutritionally rehabilitated for 80 days.

Clinical Significance of Charnoly Body and Charnolophagy As described previously, charnolophagy occurs as consequence of free radical overproduction, which enhances lipid peroxidation to cause the structural and functional breakdown of polyunsaturated fatty acids in the plasma membranes [particularly mitochondrial membranes owing to their natural abundance, polyunsaturated fatty acids (linoleic acid, linolenic acid, and arachidonic acid), omega-3 fatty acids (ecosapentenoic acid, ecosahexanoic acid, and hexosapentanoic acid)], and highly susceptible mitochondrial DNA (mtDNA), which resides in a hostile environment of free radicals, generated as a by-product of oxidative phosphorylation during ATP synthesis in the electron transport chain. The incidence of lipid peroxidation is maximal in the brain compared with other peripheral tissues such as heart, liver, and kidney because more than 70% of the brain is composed only of lipids. Because of the natural abundance of physicochemically labile lipids and mitochondria, we notice CB formation more frequently in the developing malnourished or aging brain compared with other tissues. For instance, stroke occurs

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FIGURE 5.1 Charnoly body (CB) pathogenesis in undernourished rat cerebellar Purkinje neuron. (A) Purkinje neuron from 30-day protein malnourished rat cerebellar cortex demonstrating degenerating synaptic terminals. The degenerated synaptic terminals have become swollen and club-shaped with a cloudy appearance; whereas intact synaptic terminals appear rounded with a homogeneous appearance. (B) Accumulation of CBs in the axons causes axonal hypertrophy and blocks normal axoplasmic flow to cause degeneration of synaptic terminals. (C) Typical mature CB representing multilamellar, electron-dense stacks of degenerated mitochondrial membranes. (D) Charnolophagosome appears as abnormally enlarged, electron-dense lysosomes. The charnolophagosomes were localized in the vicinity of CBs. Open arrow: a typical charnolophagosome; star: degenerating mitochondrial membranes in the process of CB formation. (E) Regression analysis of CB formation and charnolophagosome formation. A positive correlation between CB formation and charnolophagosome (CPS) formation was observed with a correlation coefficient of 0.8. By definition, a charnolophagosome is a lysosome possessing phagocytosed CBs. It is 2.5 times larger than a lysosome and electron-dense and has a structurally highly labile plasma membrane. Magnification: (A and B) 1000; (C and D) 50,000.

more frequently in the aging brain, when the brain regional mitochondrial bioenergetics is significantly compromised as a function of time and lipid peroxidation, owing to cerebral ischemia. Brain is highly susceptible to ischemia and/or hypoglycemia. During cerebral ischemia (induced by hemorrhage or by acute ischemic stroke), millions of neurons are destroyed as a result of mitochondrial degeneration and CB formation. That is why stroke either kills or renders the patient impaired for rest of his or her life. Hence, charnolophagy becomes a highly significant event in such a situation: because it is one of the most crucial and efficient molecular mechanisms of intracellular detoxification during cerebral ischemia. Charnolophagy occurs more efficiently in the young adult brain compared with the aging brain with compromised mitochondrial bioenergetics that triggers CB formation involved in apoptosis and neuronal demise. That is why the prognosis of stroke is better in

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youth compared to old age: Charnolophagy and charnolophagosome stabilization are efficient in young healthy neurons compared with mitochondrial genome knockout (RhOmgko) aging neurons. Aging RhOmgko neurons are highly susceptible to CB formation, charnolophagy inhibition, CB sequestration, and charnolophagosome destabilization involved in progressive neurodegeneration, a leading cause of morbidity and mortality in aging and in ZIKV syndrome, as we have reported (Sharma, 2016a,b). In addition, various neuroinflammatory cytokines have a predominant role in CB pathogenesis. Proinflammatory cytokines IL-1b, tumor necrosis factor-a, and IL-6 inhibit charnolophagy, whereas antiinflammatory IL-4 and IL-10 induce charnolophagy to restore intracellular detoxification. Various antioxidants including glutathione, superoxide dismutase, and MTs provide neuroprotection by inhibiting CB formation as potent free radical scavengers and efficient charnolophagy agonists. Although antioxidants are effective, these are not highly potent; hence they have to be consumed in bulk quantities. Therefore, novel drugs inhibiting organ-specific CB formation, augmenting charnolophagy, stabilizing charnolophagosome, and preventing CB sequestration will be clinically significant against ZIKV disease and other related “charnolopathies” involved in a diversified spectrum of embryopathies of unknown etiopathogenesis. We have reported CB as a pleomorphic, electron-dense, multilamellar, preapoptotic biomarker of compromised mitochondrial bioenergetics. Nutritional stress and environmental toxins induce CB formation in highly vulnerable developing neurons whereas nutritional rehabilitation, physiological zinc supplementation, and MTs inhibit CB formation. Accumulation of CBs at the junction of the axon hillock impairs the axoplasmic transport of ions, neurotransmitters, neurotropic factors, and enzymes at the synaptic terminals to cause impaired neurotransmission, progressive neurodegenerations, cognitive impairment, and early morbidity and mortality. At the light microscopic level, degenerating synaptosomes become club-shaped and swollen, and have a cloudy appearance, as illustrated in Fig. 5.1. Therefore drugs may be developed to inhibit CB formation. In addition, nonspecific induction of CB formation in hyperproliferating cells results in adverse effects including alopecia, myelosuppression, GIT symptoms, cardiovascular toxicity, pulmonary toxicity, nephrotoxicity, neurotoxicity, and infertility in multidrug-resistant malignancies. Hence drugs may be developed to induce cancer stem cellespecific CB formation to cure multidrug-resistant malignancies and chronic infections. The natural abundance and genetic susceptibility of mitochondrial DNA qualify CB as an early, unique, and sensitive universal biomarker of clinical significance. The presence of calcium microcrystallization in widespread brain regions of ZIKV-infected developing brain with microcephaly further confirms the extensive degeneration of mitochondria as a consequence of free radicaleinduced CB formation in ZIKV embryopathies. Sequestration of lysosomal-resistant CB induces monoamine oxidase (MAO) release and cholesterol TSPO (18 kDa) delocalization to cause neurotransmitter (particularly

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serotonin, dopamine, and norepinephrine) and steroid (sex) hormone depletion, early morbidity, and mortality. Accumulation of CB at the junction of axon hillock causes impairment in the normal axoplasmic transport of various enzymes, ions, hormones, neurotransmitters, neurotropic factors (NGF and BDNF), and mitochondria to cause synaptic degeneration, resulting in impaired brain regional neurotransmission and leading to cognitive impairment in PD, AD, chronic drug addiction, and ZIKV disease. MTs and glutathione inhibit CB formation as free radical scavengers and provide zincmediated transcriptional regulation of genes involved in cell growth, proliferation, differentiation, and development (Sharma and Ebadi, 2013; Sharma and Ebadi, 2014a,b,c). In addition, MTs serve as CB antagonists to prevent obesity. The ubiquitous existence of mitochondria and genetic susceptibility of the single-stranded intron-less mtDNA qualifies CB as a universal biomarker of clinical significance, as described in this chapter and in several other of our research publications. It seems promising to develop mitochondrially targeted drugs for ZIKV disease and other clinical conditions beyond the scope of this book.

Metallothioneins Provide Mitochondrial Neuroprotection MTs are induced in severe nutritional stress and during clinical seizure discharge activity to prevent free radicalemediated CB formation and provide mitochondrial neuroprotection. To confirm the therapeutic potential of MTs further as CB antagonists, we discovered that although MT double-gene knockout (MTdko) mice exhibited no overt clinical symptoms of Parkinsonism, these genotypes are mildly obese and lethargic compared with MT transgenic (MTtrans) mice, which were lean, thin, and agile. Chronic administration of 1-methyl,4-phenyl,1,2,3,6-tetrahydropyridine induced severe body tremors, muscular rigidity, and complete immobilization in MTdko mice compared with MTtrans mice, which could still walk with stiff legs and an erect tail; this suggested the neuroprotective role of MTs in PD (Sharma et al., 2004). Various antipsychotic drugs such as chlorpromazine, chlorpramazine, risperidone, and domperidone are generally antidopaminergic because these are dopamine D2 receptor antagonists. These typical first-generation antipsychotic drugs alleviate the positive symptoms of schizophrenia compared with next-generation atypical antipsychotic drugs such as quetiapine, olanzapine, and clozapine, which alleviate the negative symptoms of schizophrenia and act on dopamine D3 and D4 receptors preferentially. Atypical antipsychotic drugs do not induce extrapyramidal symptoms as do typical antipsychotic drugs, but they can cause agranulocytosis; hence periodic blood analysis is required. These drugs can also induce hepatotoxicity, hypertension, hyperglycemia, obesity, and diabetes owing to the downregulation of brain regionespecific MTs and other antioxidants, CB induction, and charnolophagy inhibition in the mesolimbic dopaminergic system. Chronic use of antipsychotic drugs can cause parkinsonism, which is associated with extrapyramidal symptoms. Although synaptic changes and adverse effects may take only

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hours, these drugs require a minimum of 2e3 weeks to have a therapeutic effect. Chronic use of these drugs can induce reversible MAO-Bespecific CB formation to cause parkinsonism associated with extrapyramidal symptoms, whereas their discontinuation eliminates adverse symptoms owing to efficient MAO-Bespecific CB eradication through energy-driven charnolophagy, as a basic molecular mechanism of intracellular detoxification, as described in this chapter. About 70%e80% chronic drug addicts experience psychosis associated with schizophrenia because of excessive and uncontrolled DA-ergic neurotransmission. Hence anti-DA-ergic drugs alleviate the most distressing symptoms of schizophrenia to a certain extent, yet with adverse extrapyramidal symptoms. On the other hand, drugs that enhance DA-ergic neurotransmission and/or DA-ergic agonists are used to treat PD. Chronic use of these drugs induces hypersexuality and aggravates symptoms of schizophrenia in PD patients because of reversible MAO-Aespecific CB formation in the mesolimbic dopaminergic system. The symptoms of schizophrenia can be alleviated when these drugs are discontinued. Hence disease-specific CBs can be used as novel discovery targets for the future development of antipsychotic, anti-Alzheimer, antiparkinsonian, antiepileptic, antidepressant, antidiabetic, and antiobesity drugs with minimal or no adverse effects. Because individuals with ZIKV who have embryopathy may experience poor quality of life owing to sensorimotor deficits in later life, it will be highly prudent to develop CB-targeted drugs against ZIKV embryopathies, GBS, and reproductive disorders, collectively known as “ZIKV charnolopathies.”

Therapeutic Potential of Antioxidants In addition to MTs and glutathione, various other ROS-scavenging antioxidants such as resveratrol, polyphenols, lycopenes, catechin, sirtuins, rutins, and several lysine deacetylases derived from natural foods can easily pass through the bloodebrain barrier with no serious side effects and can modulate cellular epigenetic changes (histone acetylation and DNA methylation); hence they can be used to prevent and/or inhibit CB formation involved in compromised mitochondrial bioenergetics, MAOs downregulation, and TSPO delocalization. These therapeutic antioxidants have also antiapoptotic and antiinflammatory properties to inhibit progressive neurodegeneration in PD, AD, amyotrophic laterals sclerosis, Huntington’s disease, multiple sclerosis, chronic drug addiction, schizophrenia, diabetes, obesity, ZIKV disease, and several other diseases of unknown etiopathogenesis, through IL-10 induction and IL-6 inhibition. Although wine has resveratrol (250 mg/120 mL), it is insufficient to fulfill the daily requirement of 250 mg, which is 1000 times more compared with wine. Moreover, wine contains nitrosamines, which can cause cancer. To meet the daily requirement of resveratrol from wine alone, we would have to consume w300 bottles of 120 mL of wine,

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which would definitely cause hepatotoxicity and neurotoxicity much earlier than conferring the beneficial effects of resveratrol derived from the wine. Hence, resveratrol derived from natural foods or fresh grapes instead of wine is more beneficial than is generally publicized by the wine industry and is misinterpreted by consumers. This is highly significant for the normal growth and development of fetuses of pregnant women who reside or travel in ZIKV-prone areas. Because there is a limited scope of neuron replacement therapy, therapeutic interventions with these antioxidants seem practically feasible, because they can enter CNS freely without inducing deleterious adverse effects. However, their adequate target delivery and reduced potency remain a significant therapeutic challenge. Therefore, ROS-scavenging antioxidant-loaded NPs will have better CNS delivery and therapeutic potential in the effective clinical management of ZIKV-induced charnolopathies involved in diversified embryopathies. Because DJ is an extremely sensitive parameter for assessing mitochondrial bioenergetics, it can be considered the primary event during CB formation. We discovered that CB formation occurs primarily during nutritional and/or toxic insult to a very vulnerable cell (particularly hyperproliferating starving cells, because CB formation, charnolophagy, and/or charnolophagosome formation were not observed in normal, healthy developing neurons). In fact, nutritional rehabilitation for 80 days prevented or eliminated CB formation in developing undernourished Purkinje neurons. The elimination of CB through lysosomal activation (charnolophagy) is an efficient basic molecular mechanism of “intracellular detoxification,” particularly during conception of the oocyte. It is a highly significant event in “Mother Nature” that can decide the life and death of a zygote. Several toxins can inhibit charnolophagy to induce CB sequestration involved in craniofacial abnormalities, as noticed in FAS. Several drugs, including antiepileptic drugs, angiotensin-converting enzyme inhibitors, anesthetic agents, and ZIKV, also can induce congenital charnolopathies involved in diversified embryopathies, as we have reported (Sharma et al., 2014a,b; Sharma, 2016aee). During chronic conditions, when CB becomes lysosomal resistant, it cannot be phagocytosed efficiently. Lysosomalresistant CB formation can pose the serious problem of intracellular detoxification, which occurs in progressive neurodegenerative diseases, cardiovascular diseases, and cancer. Moreover, CB sequestration destroys neighboring cells during atherosclerotic plaque rupture in obesity, and in diabetes as a result of the release of iron, cytochrome C, caspase-3, Bax, and many other toxic substances. Nutritional rehabilitation, physiological zinc supplementation, and/or MTs can prevent CB formation, particularly during early neuronal development and aging, so that a healthy young life and aging can be enjoyed. By contrast, unhealthy lifestyle choices such as alcohol intake, cigarette smoking, a high-fat and salt-rich diet, or malnutrition, because of ignorance or poverty, may cause morbidity and early mortality owing to brain regionespecific CB formation. Antioxidants such as glutathione and MTs can be induced by zinc, a regular diet, and moderate exercise to circumvent free radical overproduction and prevent CB formation and progressive neurodegenerative disorders, including

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chronic drug addiction. Inhibition of charnolophagy may lead to progressive neurodegenerative diseases accompanied by neuronal inclusions. As antioxidants and antiapoptotic and antiinflammatory agents, MTs store, buffer, and release zinc ions even to dissolve amyloid plaques in AD. Hence drugs may be developed to inhibit CB formation to prevent or treat neurodegenerative diseases and cardiovascular diseases, and enhance cancer stem cellespecific CB formation for the clinical management of multidrugresistant malignancies.

Significance of Charnolopharmacotherapy There is a dire need for the safe and effective treatment of various neurodegenerative disorders; cardiovascular disorders; cancer; and drug-resistant bacterial, viral (including ZIKV), and fungal infections. Hence novel mitochondrial bioenergetics-based CB antagonists, charnolophagy agonists, charnolophagosome stabilizers, and CB sequestration-based drugs can be developed to prevent and/or cure chronic diseases. More specifically, drugs can be developed to enhance either mitochondrial bioenergetics and/or prevent disease-induced CB formation in a highly vulnerable cell, including iPPs, NPCs, endothelial progenitor cells, radial glial cells, hematopoietic stem cells, osteoblasts, placental Hofbauer cells, oocytes, Sertoli cells, spermatogonia, spermatocytes, Leydig cells, hair follicular cells, GIT cells, and numerous other highly vulnerable cells, to evaluate their prophylactic as well as therapeutic potential against chronic neurodegenerative diseases, cardiovascular diseases, cancer, and infections (including ZIKV). During the acute phase, the most appropriate strategy would be to develop drugs to inhibit the formation of CB and enhance charnolophagy as a basic molecular mechanism of intracellular detoxification (also called intracellular sanitation). Frequent abortions have been reported in ZIKV-infected pregnant women owing to zygotic death, as noticed in FAS, depending on the extent of alcohol consumed during pregnancy, because of the persistence of CB formation, the failure of charnolophagy, and CB sequestration. We have reported that zygotic death occurs as the result of the inefficient elimination of CB in the zygote soon after conception as a consequence of maternal alcohol abuse and/or ZIKV infection (Sharma et al., 2015; Sharma, 2016aee). Hence charnolophagy is a highly crucial biological event in the etiopathogenesis of diversified embryopathies in a newborn infant during ZIKV infection, particularly during first trimester (gastrulation period) of pregnancy. Thus it is clinically important to prevent charnolophagosome destabilization and CB sequestration involved in toxic cytochrome C, caspase-3, apoptosis inducing factor, and iron release resulting in apoptosis, progressive neurodegeneration, and diversified embryopathies (including microcephaly) in newborn infants and GBS in adults. It is also clinically critical to develop CB sequestration inhibitors during the chronic phase to prevent and/or cure ZIKV disease by developing an effective and safe vaccine and/or antiviral drugs. In addition, novel vaccines and/or antiviral drug(s) can be developed by targeting different phases of the viral lytic cycle, specific structural proteins,

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nonstructural protein-1, the enzyme helicase, and noncoding RNA against ZIKV disease, which are beyond the scope of this chapter. Ultimately we will have both preventive as well as therapeutic options available for the successful personalized theranostics of ZIKV disease to prevent its deleterious consequences. The clinical significance of CB formation, charnolophagy induction, charnolophagosome stabilization, and CB sequestration cannot be overemphasized, because these basic molecular events occur as a consequence of severe nutritional stress and toxic exposure, and in response to acute or chronic microbial ZIKV infection. These deleterious events induce free radical overproduction because the energy (ATP) requirements are significantly enhanced during cellular injury. A natural abundance of mitochondria with highly sensitive, single-stranded, intronless DNA in a metabolically active and functionally important cell renders vulnerable cells including but not limited to NPCs and myocytes highly susceptible to CB formation as the result of frequent epigenetic modifications. Hence charnolophagy has become the most viable option for the cell to get rid of toxic substances and remain healthy. Various flaviviruses have been shown to be connected with the ER in West Nile Virus (WNV)-infected HeLa cells. In one study, electron-dense virion and vesicle packets were noticed under transmission electron microscopy of WNV-infected HeLa cells (Saiz et al., 2016). Those investigators reported ER autophagy and cell-signaling pathways of the unfolded protein response (UPR), autophagy connections, and flaviviruses, including three arms of protein kinase Relike ER kinase (UPR, activating transcription factor-6, and inositol-requiring enzyme-1) of autophagy. Undoubtedly ER autophagy will occur in cells rich in ER, whereas charnolophagy is the earliest and predominant event that occurs primarily in highly vulnerable developing cells rich in mitochondria during physicochemical injury, including nutritional stress, toxic exposure, and/or microbial (ZIKV) infection. Hence ER autophagy can be distinguished from charnolophagy (CB autophagy) as described in this chapter. Other plasma membranes, including lysosomes, peroxisomes, ER, Golgi bodies, and/or nuclear membranes, are phagocytosed and eliminated by autophagy under severe cellular injury owing to extreme nutritional stress, toxic exposure, and/or microbial (ZIKV) infection. However, charnolophagy remains the primary and most frequent event in the proper maintenance of intracellular mitochondrial bioenergetics, epigenetic modulation, and detoxification because of the natural abundance of mitochondria and their pivotal function as intracellular powerhouses. The mitochondria are involved in ATP synthesis in the electron transport chain during oxidative phosphorylation. Free radicals (OH and NO$) are generated as by-products of oxidative phosphorylation in the electron transport chain during ATP synthesis. The energy requirement is significantly increased in a cell during severe nutritional stress, toxic exposure, and microbial (ZIKV) infection. Hence the mitochondria are readily destroyed as the result of free radical attack to the plasma membranes inducing lipid peroxidation to cause structural and functional degradation of polyunsaturated fatty acids such as linolenic acid, linoleic acid, and arachidonic acid. Charnolophagy may also occur

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rarely under normal physiological conditions as a basic molecular mechanism of intracellular detoxification. Nevertheless, it becomes a highly prevalent intracellular event in response to severe nutritional stress, toxic exposure, and/or microbial (ZIKV) infection. In addition, moderate exercise may enhance charnolophagy as an efficient basic molecular mechanism of intracellular detoxification.

Conclusion Nutritional stress, toxins exposure, and infections (including ZIKV) compromise mitochondrial bioenergetics to enhance free radical overproduction and lipid peroxidation. These deleterious events induce CB formation in highly vulnerable developing NPCs and induce apoptosis to cause neurodegeneration and microcephaly. Hence CB can be used as a universal biomarker, and charnolophagy (CB autophagy) as a novel drug discovery target against neurodegenerative disease, cardiovascular disease, cancer, and infection. Early therapeutic interventions to inhibit CB formation by nutritional rehabilitation, physiological zinc supplementation, antioxidants, healthy lifestyle choices (including personal and environmental hygiene), and moderate exercise will go a long way in preventing and/or treating microcephaly induced as the result of intrauterine alcohol abuse, toxins, and/or microbial (ZIKV) infections. Eventually the molecularly well-defined and genetically tailored practice of evidencebased physical therapy by developing novel NPs and nano-drug delivery devices/ systems will confer painless treatment with no adverse effects, which will improve the quality of our lives.

Acknowledgments The author expresses his sincere thanks and gratitude to Kallol Guha, president, Saint James School of Medicine, St. Vincent, for his moral support and encouragement.

References Bollimuntha, S., Singh, B., Shavali, S., Sharma, S., Ebadi, M., 2005. TRPC-1-Mediated inhibition of MPPþ toxicity in human SH-S-Y5Y neuroblastoma cells. J. Biol. Chem. 280, 2132e2140. Chabenne, A., Moon, C., Ojo, C., Khogali, A., Nepal, B., Sharma, S., 2014. Biomarkers in fetal alcohol syndrome (recent update). Biomarkers Genomic Med. 6, 12e22. Dakshinamurti, K., Sharma, S.K., Sundaram, M., 1991. Domoic acid induced seizure activity in normal rat. Neurosci. Lett. 127, 193e197. Dakshinamurti, K., Sharma, S.K., Watanabe, T., Sundaram, M., December 1992. Hippocampal changes in postnatal mice following intrauterine exposure to domoic acid. Epilepsia Vol. Suppl (Abstract). In: Proceedings of the American Epilepsy Society, Seattle, Washington, USA, p. 45. Dakshinamurti, K., Sharma, S.K., Sundaram, M., Watanabe, T., 1993. Hippocampal.changes in developing postnatal mice following Iintra-uterine exposure to domoic acid. J. Neurosci. 13, 4486e4495.

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Ebadi, M., Sharma, S.K., 2002. Mitochondrial a-synuclein-metallothionein interaction in Parkinson’s disease. FASEB J. 16, 697, 6. Ebadi, M., Govitrapong, P., Sharma, S., Muralikrishnan, D., Shavali, S., Pellet, L., Schaffer, R., Albano, C., Ekens, J., 2001. Ubiquinone (coenzyme Q10) and mitochondria in oxidative stress of Parkinson’s disease. Biol. Signals Receptors 10, 224e253. Ebadi, M., Sharma, S.K., Shavali, S., Rafaey, E.H., 2002. Neuroprotective action of selegiline. J. Neurosci. Res. 67, 285e289. Ebadi, M., Sharma, S., 2003. Peroxynitrite and mitochondrial dysfunction in the pathogenesis of Parkinson’s disease. Antioxid. Redox Signal. 5, 319e335. Ebadi, M., Wanpen, S., Shavali, S., Sharma, S., 2004a. Coenzyme Q10 stabilizes metallothionein in parkinson’s disease. In: Hiramatsu, M., Packer, L., Yoshikava, T. (Eds.), Molecular Interventions and Protection in Life Style-Related Diseases. Markel Dekker, Inc, New York. Ebadi, M., Sharma, S., Wanpen, S., Amornpan, A., 2004b. Coenzyme Q10 inhibits mitochondrial complex-1 downregulation and nuclear factor-kappa B activation. J. Cell. Mol. Med. 8, 213e222. Ebadi, M., Brown-Borg, H., Garrett, S., Singh, B., Shavali, S., Sharma, S., 2005a. Metallothionein-mediated neuroprotection in genetically-engineered mice models of Parkinson’s disease and aging. Mol. Brain Res. 134, 67e75. Ebadi, M., Sharma, S., Ghafourifar, P., Brwon-Borg, H., Refaey, H.E.I., 2005b. Peroxynitrite in the pathogenesis of Parkinson’s disease. Method Enzymol. 396, 276e298. Ebadi, M., Brown-Borg, H., Sharma, S., Shavali, S., El ReFaey, H, Carlson, E.C., 2006. Therapeutic efficacy of selegiline in neurodegenerative disorders and neurological diseases. Curr. Drug Targets 7, 1e17. Hamburg, M.A., Collins, F.C., 2010. The path to PM. N. Eng. J. Med. 363, 301e304. Jagtap, A., Gawande, S., Sharma, S., 2015. Biomarkers in vascular dementia (a recent update), 7, 43e56. Klongpanichapak, S., Govitropong, P., Sharma, S., Ebadi, M., 2006. Attenuation of cocaine and methamphetamine neurotoxicity by coenzyme Q10. Neurochem. Res. 31, 303e311. Kooncumchoo, P., Sharma, S., Porter, J., Govitrapong, P., Ebadi, M., 2006. Coenzyme Q10 provides neuroprotection in iron-induced apoptosis in dopaminergic neurons. J. Mol. Neurosci. 28, 125e141. Lee, R.W., Shenoy, D.B., Sheel, R., 2010. Micellar Nanoparticles: Applications for Topical and Passive Transdermal Drug Delivery. Chapter 2. In: Handbook of Non-Invasive Drug Delivery Systems. (NonInvasive and Minimally-Invasive Drug Delivery Systems for Pharmaceutical and Personal Care Products) A volume in Personal Care & Cosmetic Technology, pp. 37e58. ´ ngela Va´zquez-Calvo, A., Bla´zquez, A.B., 2016. ZIKV: the latest newcomer. Front. Microbiol. 7, Saiz, J.C., A 496. Sangchot, P., Sharma, S.K., Chetsawang, B., Govitropong, P., Ebadi, M., 2002. Deferoxamine attenuates iron-induced oxidative stress and prevents mitochondrial aggregation and a-synuclein translocation in SK-N-SH cells in culture. Develop. Neurosci. 24, 143e153. Sharma, S.K., 1985. Mossy fiber evoked unit activity in developing normal and undernourished rat Purkinje cells. In: Presented and Published in the Proceedings of the 13th World Congress of Neurology at Hamburg, Germany. September 1e6, vol. 232, p. 95. Sharma, S.K., 1988. Nutrition and brain development. In: Published in the Proceedings of the First World Congress of Clinical Nutrition. New Delhi, (India), pp. 5e8. Sharma, S., 2014a. Beyond Diet and Depression (Volume-2) Book. Nova Science Publishers, New York, U.S.A. Sharma, S., 2014b. Beyond Diet and Depression (Volume-1) Book. Nova Sciences Publishers, New York, U.S.A.

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Sharma, S., 2014c. Molecular Pharmacology of environmental neurotoxins. In: Kainic Acid: Neurotoxic Properties, Biological Sources, and Clinical Applications. Nova Science Publishers, New York, pp. 1e47. Sharma, S., 2014d. Charnoly body as a universal biomarker in nanomedicine. In: 2nd International Translational Nanomedicine Conference. Boston, July 25e27 (Invited Speaker). Sharma, S., 2014e. Mitochondrially-targeted nanomedicines. In: 5th World Gene Conference. Haikou, China Nov 13e15 (Invited Speaker and Chairperson). Sharma, S., 2014f. Charnoly body as a universal biomarker in drug discovery. In: 12th International Conference on Drug Discovery. Suzhou, China Nov 18e20 (Invited Speaker & Chair Person). Sharma, S., 2014g. Nanotheranostics in evidence based personalized medicine. Curr. Drug Targets 15, 915e930. Sharma, S., 2014h. Charnolopharmacotherapy in multi-drug resistant diseases. In: 5th International Conference in MediChem. Suzhou, China Nov 18e20 (Invited Speaker). Sharma, S., 2014i. Charnolopharmacotherapy of cancer and other diseases. In: 5th International Conference, Suzhou, China, Nov 18e20 (Invited Speaker). Sharma, S., Gawande, S., Jagtap,, A., Abeulela, R., Salman, Z., 2015. Fetal alcohol syndrome: prevention, diagnosis, & treatment. In: Alcohol Abuse: Prevalence, Risk Factors. Nova Science Publishers, New York, USA. Sharma, S., 2015a. Alleviating Stress of the Soldier & Civilian. Nova Science Publishers, New York. U.S.A. Sharma, S., 2015b. Monoamine Oxidase Inhibitors: Clinical Pharmacology, Benefits, & Adverse Effects. Nova Science Publishers, New York. U.S.A. Sharma, S., 2015c. Charnoly body as a universal biomarker in drug addiction. In: 3rd International Drug Addiction Conference. Oralando, Florida. USA. Aug, 2e5. Sharma, S., 2016a. Personalized Medicine (Beyond PET Biomarkers). Nova Science Publishers, New York. U.S.A. Sharma, S., 2016b. Progress in PET Radiopharmaceuticals. Nova Science Publishers, New York. U.S.A. Sharma, S., 2016c. Disease-specific charnoly body formation in neurodegenerative & other diseases. In: Drug Discovery & Therapy World Congress. Aug 22e25. Hynes International Convention Center. Boston Mass. U.S.A (Invited Speaker). Sharma, S., 2016d. Charnoly body as novel biomarker of nutritional stress in Alzheimer’s disease. In: 20 th International Coference of Funtional Foods in Health & Disease. Josheph P. Martin Memorial Convention Center, Harvard Medical School, Boston, Mass, U.S.A. Sep 22e23 (Invited Speaker). Sharma, S., 2016e. Charnoly body as a novel biomarker in ZIKV-induced microcephaly. In: Drug Discovery & Therapy World Congress (DDTWC-2016). Hynes Memorial Conventional Center, Boaton, Mass, U.S.A. Aug 21e25 (Invited Speaker). Sharma, S., 2017. Zika Virus Disease (Prevention and Cure). Nova Science Publishers, New York, USA. Sharma, S., Bolster, B., Dakshinamurti, K., 1994. Picrotoxin and pentylene tetrazole induced seizure activity in pyridoxine-deficient rats. J. Neurol. Sci. 121, 1e9. Sharma, S., Ebadi, M., 2003. Metallothionein attenuates 3-morpholinosydnonimone (SIN-1)-induced oxidative and nitrative stress in dopaminergic neurons. Antiox. Redox Signal. 5, 251e264. Sharma, S., Ebadi, M., 2008a. Coenzyme Q10 augments brain regional 18F-DOPA and 2-18F-Fluoro, 2-Deoxy, D-glucose uptake in metallothionein over-expressing weaver mouse. In: Proceedings of the World Congress of Molecular Imaging (WMIC 2008). Sep 10e13, 2008. Sharma, S., Ebadi, M., 2008b. Therapeutic potential of metallothioneins in Parkinson’s disease. In: Hahn, T.F., Werner, J. (Eds.), New Research on Parkinson’s Disease. Nova Science Publishers, New York, pp. 1e28.

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Sharma, S., Ebadi, M., 2011a. Metallothioneins as early & sensitive biomarkers of redox signaling in neurodegenerative disorders. J. Inst. Integr. Omics Appl. Biotechnol. (IIOAB J.) 2, 98e106. Sharma, S., Ebadi, M., 2011b. Therapeutic potential of metallothioneins as anti-inflammatory agents in polysubstance abuse. J. Inst. Integr. Omics Appl. Biotechnol. IIOAB J. 2, 50e61. Sharma, S., Ebadi, M., 2013. In-vivo molecular imaging in Parkinson’s disease. In: Pfeiffer, R.F., Wszolek, Z.K., Ebadi, M. (Eds.), Parkinson’s Disease, second ed. CRC Press Taylor & Francis Group, Boca Rotan, FL, USA, pp. 787e802. Chapter 58. Sharma, S., Ebadi, M., 2014a. Antioxidants as potential therapeutics in neurodegeneration. In: Laher, I. (Ed.), System Biology of Free Radicals and Antioxidants. Springer Verlag, Heidelberg, Germany, pp. 1e30. Chapter 85. Sharma, S., Ebadi, M., 2014b. Charnoly body as a universal biomarker of cell injury. Biomarkers Genomic Med. 6, 89e98. Sharma, S., Ebadi, M., 2014c. Significance of metallothioneins in aging brain. Neurochem. Int. 65, 40e48. Sharma, S.K., Nayar, U., Maheshwari, M.C., Gopinath, G., 1986. Ultrastructural studies of P-cell morphology in developing normal and undernourished rat cerebellar cortex. Electrophysiol. Corr. Neurol. India 34, 323e327. Sharma, S.K., Nayar, U., Maheshwari, M.C., Singh, B., 1987. Effect of undernutrition on developing rat cerebellum: some electrophysiological and neuromorphological correlates. J. Neurol. Sci. 78, 261e272. Sharma, S.K., Selvamurthy, W., Dakshinamurti, K., 1993a. Effect of environmental neurotoxins in the developing brain. Biometeorology 2, 447e455. Sharma, S.K., Nayar, U., Maheshwari, M.C., Singh, B., 1993b. Purkinje Cell evoked unit activity in developing undernourished rats. J. Neurol. Sci. 116, 212e219. Sharma, S., Carlson, E., Ebadi, M., 2003. The neuroprotective actions of selegiline in inhibiting 1-methyl, 4-phenyl, pyridinium ion (MPPþ)-induced apoptosis in dopaminergic neurons. J. Neurocytol. 32, 329e343. Sharma, S., Kheradpezhou, M., Shavali, S., EI Refaey, H., Eken, J., Hagen, C., Ebadi, M., 2004. Neuroprotective actions of coenzyme Q10 in Parkinson’s disease. Methods Enzymol. 382, 488e509. Sharma, S., Refaey, H.El, Ebadi, M., 2006. Complex-1 activity and 18F-DOPA uptake in genetically engineered mouse model of Parkinson’s disease and the neuroprotective role of coenzyme Q10. Brain Res. Bull. 70, 22e32. Sharma, S., Yang, B., Xi, X., Grotta, J., Aronowski, J., Savitz, S., 2011. IL-10 directly protects cortical neurons by activating PI-3 kinase and STAT-3 pathways. Brain Res. 1373, 189e194. Sharma, S., Rais, A., Sandhu, R., Nel, W., Ebadi, M., 2013. Clinical significance of metallothioneins in cell therapy and nanomedicine. Int. J. Nanomed. 8, 1477e1488. Sharma, S., Gawande, S., Jagtap, A., Abeulela, R., Salman, Z., 2014a. Fetal alcohol syndrome; prevention, diagnosis, & treatment. In: Alcohol Abuse: Prevalence, Risk Factors. Nova Science Publishers, New York, U.S.A. Sharma, S., Nepal, B., Moon, C.S., Chabenne, A., Khogali, A., Ojo, C., Hong, E., Goudet, R., SayedAhmad, A., Jacob, A., Murtaba, M., Firlit, M., 2014b. Psychology of craving. Open J. Med. Psychol. 3, 120e125. Sharma, S., Choga, J., Gupta, V., et al., 2016a. Charnoly body as a novel biomarker of nutritional stress in Alzheimer’s disease. Funct. Foods Health Dis. 6, 344e377. Sharma, S., Choga, J., Doghor, P., et al., 2016b. Charnoly body as a novel biomarker of nutritional stress in Alzheimer’s disease. In: 20th International Conference on Functional Foods in Health & Disease. Joseph P. Martin International Convention Center, Harvard Medical School, Boston. Mass., U.S.A. Sep 22e23.

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Sharma, S., Choga, J., Gupta, V., 2016c. Charnoly body as a novel biomarker of nutritional stress in Alzheimer’s Disease. Funct. Foods Health Dis. 6, 344e378. Wang, E., 2009. MicroRNA regulation and its biological significance in PM and aging. Curr. Genomics 10, 143. Wang, J.J., Zeng, Z.W., Xiao, R.Z., et al., 2011. Recent advances of chitosan nanoparticles as drug carriers. Int. J. Nanomed. 6, 765e774. Zhang, S., Lei, C., Liu, P., 2015. Association between variant amyloid deposits and motor deficits in FADassociated presenilin-1 mutations: a systematic review. Neurosci. Biobehavior. Rev. 56, 180e192.

Further Reading Ebadi, M., Sharma, S., 2006. Metallothioneins 1 and 2 attenuate peroxynitrite-induced oxidative stress in Parkinson’s disease. Exp. Biol. Med. 231, 1576e1583. Li, Z., Lin, Q., Ma, Q., Lu, C., Tzeng, C.M., 2014. Genetic predisposition to Parkinson’s disease and cancer. Curr. Cancer Drug Targets 14 (3), 310e321. Sharma, S., 2013. Charnoly body as a sensitive biomarker in Nanomedicine. In: International Translational Nonomedicine Conference. Boston, July 26e28 (Invited Speaker).

6

Protein, Carbohydrates, and Fats: Energy Metabolism Prabhakar Singh1, Rajesh K. Kesharwani2, Raj K. Keservani3 1

VEER BAHADUR SINGH PURVANCHA L UNIVERSITY, JAUNPUR, INDIA; 2 NIMS UNIVERSITY, J A NUP UR , I ND I A ; 3 RAJIV GANDHI PROUDYOGIKI V ISHWAVIDYALAYA, B HOPAL, INDIA

Introduction The major sources of energy in the diet for many people are carbohydrates and fats. Carbohydrates and fats were found to contribute nearly equally, as much as 46% and 42%, respectively, to the energy in the content of diets in the United States. Increasing Westernization, urbanization, and mechanization around the world are associated with changes in the dietary pattern toward one of high-fat, high energy-dense foods and a sedentary lifestyle (Popkin, 2001; W.H.O., 2000). Rather than being based on weight, equal energy must follow a comparison of carbohydrates and fat as energy sources in the diet. Carbohydrates, protein, and fat, the major micronutrients, are required to provide energy for maintenance, growth, and repair to the body. Energy driven by micronutrients is used in both physiological and psychological ways. Life expectancy around the world has increased as a result of advancements in diet and because of nutrition, hygiene, and control of infectious diseases. Various infectious and nutrient deficiency disease are being replaced in developing countries by new alarming threats to the health of populations, including obesity, cardiovascular disease, and diabetes (W.H.O., 2000). Traditional food habits are being replaced by fast foods, soft drinks, and increased meat consumption (Popkin, 2001). In the modern era the global diet has increased in energy density (Popkin, 2001), which is a major problem for countries that are at risk of micronutrient deficiencies and associated disorders (Pena and Bacallao, 2000). Metabolism of biomolecules involves the biochemical changes of that molecules to provide energy for work and growth. Nutrition is concerned with food contained in the form of carbohydrates, lipids, and proteins, and how the body is going to use it. All carbohydrates, lipids, and proteins must be ingested and digested before they are assimilated and used by the body. Carbohydrates, lipids, and proteins are major nutrients; however other micronutrients, such as vitamins, minerals, and trace elements, are also necessary to carbohydrate, lipid and protein metabolism and digestion but are required in much smaller quantities. Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00006-5 Copyright © 2017 Elsevier Inc. All rights reserved.

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Carbohydrates Carbohydrates are major energy-generating biomolecules that are widely distributed in plants and animals. Carbohydrates have the general formula (CH2O)n. The main dietary carbohydrates are monosaccharide and disaccharide sugars, e.g., fructose, glucose, lactose, and sucrose, and starch as polysaccharides. Currently, the World Health Organization (WHO) suggests that 55% of dietary energy should be carbohydrates. Most carbohydrates should be in the form of starch; however less than 10%e15% of energy intake should be in the form of sugars (W.H.O., 2000). Despite their role in energy, carbohydrates have several important structural and metabolic roles. Glucose is a major carbohydrate and the most important one. Several dietary carbohydrates are absorbed into the bloodstream as glucose after enzymatic hydrolysis of dietary starch and disaccharides (Swinburn et al., 2004).

Sugar in Foods Carbohydrates are major constituents of animal food and animal tissues. In the diet there is a reciprocal relationship between the percentage of carbohydrates and fat, because these two nutrients generally contribute over 80% of total energy (Swinburn et al., 2004). It has been demonstrated that people who have high total energy intakes tend to have a high total sugar intake (Williams, 2000; Naismith et al., 1995; Hill and Prentice, 1995; Gibson, 1993; Lewis et al., 1992), However in relative terms, a reciprocal relationship is also seen between the percentage of fat and sugar in the diet (Hill and Prentice, 1995). The study of sugar intake and BMI consistently evidences an inverse relation between sugar intake as a percentage of energy and BMI or the prevalence of obesity (Hill and Prentice, 1995). Carbohydrate malnutrition is associated with an energy imbalance (marasmus). Excess storage of carbohydrates as surplus energy causes obesity, a common disease in the current world that in turn results in many diseases, especially cardiovascular disease and type 2 diabetes mellitus (Swinburn et al., 2004). Sweetening foods increases their palatability, and it has been found that sweetness may lead to overconsumption (Drewnowski, 1992). However there appears to be a limit to the hedonistic response to sweetened foods (Rolls and Hetherington, 1989). Palatability of foods has been also found to increase with fat; hence processed foods contain both high sugar and high fat values, which may lead to weight gain (Anderson, 1995).

Sugar in Drinks Physiologically the energy density of fluids and foods may not have comparable effects on satiety and ad libitum food consumption (Mattes, 1996; Rolls et al., 1999a,b). The study of Tordoff and Alleva (1990) on the consumption of soda (1150 g/day for 3 weeks) sweetened with high-fructose corn syrup or aspartame demonstrated that high-fructose soda increased total energy intake by 335 kcal/day, which resulted in a significant mean weight gain of 0.66 kg. In comparison, for intake of aspartame soda, total energy intake decreased

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by 179 kcal/day and hence weight decreased nonsignificantly by 0.17 kg. Apart from this, it has been found that children who drink 9 oz/day or more of soda drink nearly 200 kcal/day more energy than do those of nondrinkers (Harnack et al., 1999). Similarly, Ludwig et al. (2001) found that high intake of sugar drinks over 19 months by 12-year-old children were more prone to develop obesity.

Specific Dynamic Action of Carbohydrates The phenomenon of the production of overheating by the body more than the calculated caloric value when a given food is metabolized is known as the specific dynamic action (SDA) of carbohydrates. The calculated SDA for carbohydrates is 5%, which explain why consumption of 100 cal of fat results in 113 cal, and 100 cal of carbohydrates results in 105 cal when they are metabolized in the body. SDA is also known as calorigenic action or thermogenic action, or the thermic action of food. A study explained that carbohydrates have lowest SDA, and then fat and protein (Swinburn et al., 2004).

Glycemic Index The glycemic index (GI) of food indicates the time course of postprandial glucose concentration that may be charted on a graph. Different carbohydrate diets induce blood glucose, and in turn serum insulin, to varying extents even when an equal amount of carbohydrates is eaten. It was reported that lowerGI diets have greater satiety value (Brand-Miller et al., 2002; Ludwig, 2000); this was thought to be due to increasing cholecystokinin and fullness after the meal (satiation) (Brand-Miller et al., 2002; Holt et al., 1992). Ludwig et al. found that rapid absorption of glucose from the food altered hormonal and metabolic functions, and it was found that ingestion of a high-GI meal promoted excessive food intake. Biochemical research explained that lower-GI diets induce fat oxidation instead of carbohydrate oxidation, however (Brand-Miller et al., 2002). A higher-GI diet induced insulin, which inhibits lipolysis and encourages fat storage (Brand-Miller et al., 2002; Ludwig et al., 1999), limiting available glucose and inducing overeating (Ludwig et al., 1999). Slabber et al. (1994) showed that a low-GI diet lowered insulin levels, which in turn increased weight loss more than did corresponding high-GI diets. Spieth et al. (2000) reported that low-GI diets were more effective than reduced-fat diets in treating childhood obesity.

Obesity and Carbohydrates The prevalence of obesity is increasing throughout the world’s population. This is particularly important for understanding the relationship between diet and obesity. Obese people tend to underreport intake more than do thin people and the underreporting is thought to be greatest for high-carbohydrate and high-fat foods (Heitmann and Lissner, 1995; Black et al., 1991). Despite feeding behavior, physical activity is also important in reference to the beginnings of weight gain and obesity (Di Pietro, 1995). The

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standard definitions of overweight (BMI  25 kg/m2) and obesity (30 kg/m2) were mainly derived from populations of European descent (W.H.O., 2000). The distribution of body fat, which is assessed using the waist circumference or the waistehip ratio, is an important self-regulating predictor of morbidity (Mc Keigue, 1996; Sjostrom, 1992). The susceptibility toward overweight and obesity is higher among children who watch more television (Lewis and Hill, 1998) as well as those with increased energy intakes (French et al., 2001; Taras et al., 1989). Higher carbohydrates, especially a diet with empty calories, tend to move the metabolic pathway from the oxidative phosphorylation of glucose to that of fatty acid synthesis. In addition, empty calories provide a lower satiety value. Carbohydrates are not the only causative agent for obesity, but fat and protein are also responsible. However major evidence suggests that a sedentary lifestyle and the lower basal metabolic rate (BMR) of people are largely responsible factors.

Protein Proteins are necessary macronutrients of food that supply essential as well as nonessential amino acids needed for the growth, repair, and maintenance of tissues. Protein is obtained from a variety of sources to supply amino acids for growth and repair. Humans are unable to synthesize nine amino acids out of the 20 found in protein. Nonessential amino acids are synthesized from essential amino acids in the body (Swinburn et al., 2004). The daily requirement of protein (about 65 and 50 g) in food by males and females, respectively, provides about 10%e15% of total energy in a balanced diet; however only about 5% of body energy comes from the catabolism of protein under normal circumstances (Gurr, 1991). It has been found that protein is the most satiating of macronutrients (Latner and Schwartz, 1999), mainly for people with a low habitual protein diet (Long et al., 2000) and it may influence body weight under ad libitum, reduced fat conditions (Astrup and Raben, 1995). It has been suggested that increasing protein intake may be helpful for controlling weight for some individuals, but the role of protein content in the diet is probably not an important determinant of the prevalence of obesity.

ProteineEnergy Malnutrition Proteineenergy malnutrition (PEM) is composed of a spectrum of biological disorders caused by the lack of food. Despite the name, it is not necessary for affected individuals to be experiencing a lack of protein, but rather a deficiency of total energy. Dietary proteins that would normally be used for tissue repair or growth are also used as fuel. PEM is rare in the developed world and is generally associated with children suffering from neglect or solitary malnourished elderly patients (Swinburn et al., 2004). The severity and clinical features of PEM indicate food deficiency in the forms of marasmus and kwashiorkor. PEM becomes life threatening when susceptibility to infectious diseases increases that would not normally be lethal (Swinburn et al., 2004).

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Lipids Lipids constitute about 15%e20% of the body weight in humans, in which triacylglycerols (TAG) comprise a maximum 85%e90% of body lipids. Most TAGs are neutral fat or depot fat stored in adipose tissue and serve as an energy reserve for the body. Lipids are a superior energy reserve compared with carbohydrates and protein. They also act as an insulating material to regulate body temperature. TAG is a highly concentrated form of energy that yields 9 kcal/g energy, compared with 4 kcal/g from carbohydrates and protein. For long-time stored energy, TAG is superior to glycogen because it is hydrophobic, and hence it is stored in anhydrous form; 1 g of glycogen combines with 2 g of water for storage (Satyanarayana and Chakrapani, 2008). An excess of body fat tissue may be related not only to energy expenditure and energy intake in humans but also to the type of diet, especially high-fat ones (Prentice, 1998; Flatt, 1995). A high-fat diet leads to several metabolic changes as reduced lipolytic activity in fat tissue, a reduction in leptin secretion and/or sensitivity, hypothalamic neuron apoptosis (Moraes et al., 2009), hyperphagia in humans (Westerterp et al., 2008), impairment of mitochondrial metabolism (Pomplun et al., 2007), insulin resistance, and obesity (Wajchenberg, 2000). Excess fat induced the production of the malonylcoenzyme A, which in turn negatively regulated glucose transporter type 4 efficiency. In such case, b-oxidation of fatty acids and the tricarboxylic acid cycle are temporarily uncoupled, which produces metabolites as by-products, and which then augment reactive oxygen species (ROS) production (Coelho et al., 2011). ROS induces oxidative stress, which actively oxidizes biomolecules’ carbohydrates, proteins, and fatty acids (Singh and Rizvi, 2012, 2013; Mehdi et al., 2012). In addition, the presence of oxidative stress and the modification of these biomolecules alter the activity of enzymes and the transport system activity of membranes and surround the plasma (Singh and Rizvi, 2015a,b). A comparative study showed that foods high in fat are less satiating than carbohydrates when equal calorically amounts of diet are eaten; a high satiety value of food is associated with a high volume of complex carbohydrate content (Blundell, 1996; Holt et al., 1995). Dietary lipids are fats and oils having concentrated sources of energy. In addition to producing energy, fats are also carriers for the fat-soluble vitamins A, D, E, and K. The WHO recommended that total fats not be more than 30% of the diet’s energy intake. Various studies related to manipulating energy density, fat, and carbohydrate content showed that at constant energy density and palatability, no energy intake occurs in a diet with varying fat and carbohydrate content (Stubbs et al., 1996); however, changes in energy density at constant fatecarbohydrate ratios influence total energy intake and weight (Rolls et al., 1999a,b).

Fat and Energy in the Diet and Obesity It has been found that weight changes occur when the concentration of energy changes. Clamping total energy produces similar weight changes irrespective of the

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macronutrient composition (Reaven, 1997; Golay et al., 1996; Garg et al., 1994). It was found that 16 ad libitum dietary trials showed that diets reduced in fat consistently reduced total energy intake and hence reduced weight (Astrup et al., 2000). In reference to energy, a reduction by 10% in the proportion of fat in the diet corresponded to a reduction of about 1 MJ of total energy per day (Astrup et al., 2000). Reducing the volume of fat in the diet consistently helped modestly reduce body weight, but beneficial effects would be more prominent if individuals simultaneously reduced other macronutrients in the diet or decreased the intake of certain high-volume foods such as staple carbohydrates. The composition of fat in diet can interfere in the progression of obesity because some fatty acids have specific roles in different metabolic activities, which in turn affect the oxidation and deposition rate of fat and hence regulate weight and/or composition. It has been reported that a high-fat diet generally induces hyperglycemia, but saturated fatty acids are more important for positive fat balance and accumulation in adipose tissue (Flatt, 1995; Flatt et al., 1985), in contrast to omega-3 and omega-6 polyunsaturated fatty acids (PUFA), which have been found to decrease energy intake and increase energy expenditure by modulating the activity of hormone-sensitive lipase, peroxisome proliferator-activated receptor a, and others (Wang et al., 2002). Table 6.1 lists food and nutritional information.

Dietary Fatty Acid and Lipid Metabolism The presence of unsaturated fatty acids in the diet has been reported to reduce total and high-density lipoprotein (HDL) and to increase biliary cholesterol secretion selectively (Morgado et al., 2005). Diets rich in omega-3 PUFA have been reported to reduce TAG, phospholipids, and cholesterol compared with diets rich in saturated fatty acids (Rokling-Andersen et al., 2009). In addition, replacement of saturated fatty acids by PUFA or monounsaturated fatty acids significantly reduced low-density lipoprotein (LDL) cholesterol and HDL cholesterol (Siri-Tarino et al., 2010).

Diet-Induced Thermogenesis/Specific Dynamic Action Daily energy expenditure consists of BMR, diet-induced thermogenesis, and physical activity. SDA is a phenomenon of the production of extra heat by the body over and above the calculated caloric value, when a given food is metabolized by the body. It is also known as calorigenic or thermogenic action, or the thermic action of food (Satyanarayana and Chakrapani, 2008). Diet-induced thermogenesis is an increase in energy expenditure above the BMR with the sequence: alcohol, protein, carbohydrate, and fat. A mixed-balance diet results in a diet that induces a 5%e15% of daily energy expenditure (Westerterp, 2004). The percent value of diet-induced thermogenesis is lower in high fat consumption and higher in relatively high protein and alcohol consumption; protein thermogenesis induced by food has an important effect on satiety.

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Table 6.1

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Food and Nutritional Formulation Energy Value (cal/g)

Food Carbohydrates sources: Sugar (99%), cereals (60%e80%), pulses (50%e60%), roots and tubers (20%e40%), and bread (5%e60%). RDA: 400 g/day (Adult man ¼ 70 kg)

In bomb calorimeter 4.1

In the Body 4.0

Formulation

Biological Importance

Digestible carbohydrates: e.g., glucose, starch, maltose, lactose, sucrose

Provide major source of energy Stored as reserve energy for quick needs Selective permeability and identity of membrane as sialic acids Components of genetic material as ribose and deoxyribose Improves glucose tolerance Prevents constipation Eliminates bacterial toxins Decreases gastrointestinal cancer Reduces plasma cholesterol level Satiety value Regarded as workhorses of cell: functions include enzyme Q hormones, blood-clotting factors, immunoglobulins, membrane receptors, storage proteins, genetic control, muscle contraction, respiration

Nondigestible carbohydrates: e.g., cellulose, inulin, chitin

Protein sources: Cereals 6%e12%, pulses 18%e22%, meat 18%e25%, eggs 10% e14%, milk 3%e4%, and leafy vegetables 1%e2% RDA: 0.8 g kg body weight/ day

5.4

4.0

Lipids Essential fatty acids RDA: 4 g/day Adult man ¼ 70 kg)

9.4

9

RDA, recommended dietary allowance.

Essential amino acids: arginine, valine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan Semiessential amino acids: arginine, histidine Nonessential amino acids: lycine, alanine, serine, cysteine, aspartate, asparagine, glutamate, glutamine, tyrosine, proline Essential fatty acids: linoleic acid, linolenic acid, and some amount of arachidonic acid Sources: vegetable oils and fish oils. Rich vegetable sources include sunflower oil, cotton seed oil, corn oil, soybean oil, etc. Nonessential fatty acids: triacylglycerols (fats and oils), saturated fatty acids

Development of healthy brain Structural components of biological membranes Participates in transport and use of cholesterol. Prevents fat accumulation in liver Requires prostaglandin 15%e50% of body energy requirement Major reserve source of energy

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Table 6.2 Calorific Values of Carbohydrates, Lipids, and Protein (Satyanarayana and Chakrapani, 2008) Energy Value (kcal/g) Macronutrient

In Bomb Calorimeter

In Body

Carbohydrates Fat Protein

4.1 9.1 5.4

4.0 9.0 4.0

SDA values for separate macronutrients are 0%e3% for fat, 5%e10% for carbohydrates, 20%e30% for protein (Acheson, 1993), and 10%e30% for alcohol (Westerterp et al., 1999). The main factor of SDA is the energy content of the food, followed by the amount of protein in the food. The thermic effect of alcohol is similar to that of protein. The higher value of SDA for protein shows that it is not a good source of energy. Fat is the best source of energy because of its lowering effect on SDA. The calorific values of carbohydrates, lipids, and protein are presented in Table 6.2.

Energy Metabolism and Obesity The balance of each macronutrient (carbohydrates, lipids, and protein) seems to involve rigorous control to regulate from intake to oxidation. An increase of carbohydrates and/ or protein is accompanied by an increased metabolism and oxidation rates of both nutrients (Stubbs et al., 1995). Obesity is major health problem in developed countries (Deitel, 2003). People with a BMI  40 kg/m2 or >35 with comorbidities are classified as morbidly obese. Lipid disorders such as dyslipidemia, hypercholesterolemia, and obesity are established as risk factors for the development of coronary artery disease and premature cardiovascular death (Uzun et al., 2004). In the current scenario, there is an increase in the occurrence of “metabolic syndrome,” a complex condition of obesity that has clinical features including insulin resistance, dyslipidemia, and hypertension (Campbel, 2006). Dyslipidemia, hypertension, and glucose dysmetabolism increase visceral obesity and insulin resistance in turn increases cardiovascular risk (Gaal et al., 2006). Lipid stored in adipose tissue represents excess energy consumption relative to energy expenditure. Adipose tissue serves as an integrator of various physiological pathways with increasing intensity as a result of the emergence of obesity. The role of adipocytes in calorie storage makes it well suited to regulating energy balance. In addition, fat metabolism adipocytes serve as regulators of glucose homeostasis. Hence adipocyte biology is necessary for understanding the pathophysiological basis of obesity and associated metabolic disorders (Rosen and Spiegelman, 2006). High-fat diets could lead to changes in adiposity, mitochondrial function, and insulin sensitivity. These changes have been evidenced as important in the etiology of obesity

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(Coelho et al., 2011). In addition, factors preventing obesity are generally considered to be a high intake of dietary nonstarch polysaccharides/fiber, lower total energy intake, regular physical activity, supportive home and school environments for children, and breastfeeding for babies (Swinburn et al., 2004). Factors inducing obesity are a high intake of energy-dense, micronutrient-poor foods, sedentary lifestyles, heavy marketing of energy-dense foods and fast-food outlets, sugar-sweetened soft drinks and fruit juices, and genetics (Swinburn et al., 2004). The main diets that promote obesity through passive overconsumption of total energy are as follows: energy-dense foods principally related to the fat content as well as the carbohydrate content, high-energy drinks, and large portion sizes of food. In a population the mean of total fat intake is an indicator of energy density, which should be less than 30% of energy. Similarly, the mean free sugar intake, which is less than 10% of energy, also reflects the low mean energy density of food and drink (WHO, 1996).

Conclusion Nutrition and energy disorders represent a serious threat to the health of the population of almost every country in the world. Carbohydrates, protein, and lipids are major macronutrients that provide energy, building blocks of the body, and reserve food for the body. In addition to these nutrients’ major function, all macronutrients participate in energy supply to the body with different energy values. There are rapidly developing discoveries about the energy metabolism of carbohydrates, lipids, and protein, and new insights are being revealed with each passing month that offer great hope for the prevention of various chronic diseases.

Abbreviations BMI Body mass index BMR Basal metabolic rate GI Glycemic index HDL High-density lipoprotein LDL Low-density lipoprotein PEM Proteineenergy malnutrition PUFA Polyunsaturated fatty acid ROS Reactive oxygen species SDA Specific dynamic action TAG Triacylglycerols WHO World Health Organization

Acknowledgments Dr. Prabhakar Singh acknowledges Prof. Dharani Dhar Dubey (Dean, Faculty of Sciences, Head, Department of Biotechnology, V.B.S. Purvanchal University) for his cooperative support and helpful nature, and for providing the research environment.

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References Acheson, K.J., 1993. Influence of autonomic nervous system on nutrient-induced thermogenesis in humans. Nutrition 9 (4), 373e380. Anderson, G.H., 1995. Sugars, sweetness, and food intake. Am. J. Clin. Nutr. 62 (Suppl.), 195se202s. Astrup, A., Raben, A., 1995. Carbohydrate and obesity. Int. J. Obes. Relat. Metab. Disord. 19 (Suppl. 5), S27eS37. Astrup, A., Grunwald, G.K., Melanson, E.L., Saris, W.H., Hill, J.O., 2000. The role of low-fat diets in body weight control: a metaanalysis of ad libitum dietary intervention studies. Int. J. Obes. Relat. Metab. Disord. 24 (12), 1545e1552. Black, A.E., Goldberg, G.R., Jebb, S.A., Livingstone, M.B.E., Cole, T., Prentice, A.M., 1991. Critical evaluation of energy intake data using fundamental principles of energy physiology: 2. Evaluating the results of published surveys. Eur. J. Clin. Nutr. 45, 583e599. Blundell, J.E., 1996. Food intake and body weight regulation. In: Bouchard, C., Bray, G.A. (Eds.), Regulation Body Weight: Biological and Behavioural Mechanisms. John Wiley & Sons, Chichester/ New York/Brisbane/Toronto/Singapore. Brand-Miller, J., Holt, S.H.A., Pawlak, D.B., McMillan, J., 2002. Glycemic index and obesity. Am. J. Clin. Nutr. 76, 281Se285S. Campbel, P., 2006. Obesity and diabetes. Nature 444 (7121). Coelho, D.F., Pereira-Lancha, L.O., Chaves, D.S., Diwan, D., Ferraz, R., Campos-Ferraz, P.L., Poortmans, J.R., Lancha Junior, A.H., 2011. Effect of high-fat diets on body composition, lipid metabolism and insulin sensitivity, and the role of exercise on these parameters. Braz. J. Med. Biol. Res. 44 (10), 966e972. Deitel, M., 2003. Overweight and obesity worldwide now estimated to involve 1.7 billion people. Obes. Surg. 13, 329e330. Di Pietro, L., 1995. Physical activity, body weight, and adiposity: an epidemiologic perspective. Exerc. Sport Sci. Rev. 23, 275e303. Drewnowski, A., Kurth, C., Holden-Wiltse, J., Saari, J., 1992. Food preferences in human obesity: carbohydrates versus fats. Appetite 18 (3), 207e221. Flatt, J.P., 1995. Use and storage of carbohydrate and fat. Am. J. Clin. Nutr. 61, 952Se959S. Flatt, J.P., Ravussin, E., Acheson, K.J., Jequier, E., 1985. Effects of dietary fat on postprandial substrate oxidation and on carbohydrate and fat balances. J. Clin. Invest. 76, 1019e1024. French, S.A., Story, M., Jeffery, R.W., 2001. Environmental influences on eating and physical activity. Annu. Rev. Public Health 22, 309e335. Gaal, L.F.V., Mertens, I.L., De Block, C.E., 2006. Mechanisms linking obesity with cardiovascular disease. Nature 444 (14), 875e880. Garg, A., Bantle, J.P., Henry, R.R., Coulston, A.M., Griver, K.A., Raatz, S.K., et al., 1994. Effects of varying carbohydrate content of diet in patients with non-insulin-dependent diabetes mellitus. J. Am. Med. Assoc. 271 (18), 1421e1428. Gibson, S.A., 1993. Consumption and sources of sugar in the diets of British school children. Are high sugar diets inferior? J. Hum. Nutr. Diet. 6, 355e371. Golay, A., Eigenheer, C., Morel, Y., Kujawski, P., Lehmann, T., de Tonnac, N., 1996. Weight-loss with low or high carbohydrate diet? Int. J. Obes. Relat. Metab. Disord. 20 (12), 1067e1072. Gurr, M.I., 1991. Diet, nutrition and the prevention of chronic diseases (WHO, 1990). Eur. J. Clin. Nutr. 45 (12), 619e623. Harnack, L., Stang, J., Story, M., 1999. Soft drink consumption among US children and adolescents: nutritional consequences. J. Am. Diet. Assoc. 99 (4), 436e441.

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Heitmann, B.L., Lissner, L., 1995. Dietary underreporting by obese individuals-is it specific or nonspecific? Br. Med. J. 311 (7011), 986e989. Hill, J.O., Prentice, A.M., 1995. Sugar and body weight regulation. Am. J. Clin. Nutr. 62 (Suppl. 1), 264Se273S. Discussion 273Se274S. Holt, S., Brand, J., Soveny, C., Hansky, J., 1992. Relationship of satiety to postprandial glycemic, insulin and cholecystokinin responses. Appetite 18 (2), 129e141. Holt, S., Miller, J.C., Petocz, P., Farmakalidis, E., 1995. A satiety index of common foods. Eur. J. Clin. Nutr. 49, 675e690. Latner, J.D., Schwartz, M., 1999. The effects of a high-carbohydrate, high-protein or balanced lunch upon later food intake and hunger ratings. Appetite 33 (1), 119e128. Lewis, M.K., Hill, A.J., 1998. Food advertising on British children’s television: a content analysis and experimental study with nine-year olds. Int. J. Obes. Relat. Metab. Disord. 22 (3), 206e214. Lewis, C.J., Park, Y.K., Dexter, P.B., Yetley, E.A., 1992. Nutrient intakes and body weights of persons consuming high and moderate levels of added sugars. J. Am. Diet. Assoc. 92 (6), 708e713. Long, S.J., Jeffcoat, A.R., Millward, D.J., 2000. Effect of habitual dietaryprotein intake on appetite and satiety. Appetite 35 (1), 79e88. Ludwig, D.S., 2000. Dietary glycemic index and obesity. J. Nutr. 130 (2S Suppl.), 280Se283S. Ludwig, D.S., Majzoub, J.A., Al-Zahrani, A., Dallal, G.E., Blanco, I., Roberts, S.B., 1999. High glycemic index foods, overeating, and obesity. Pediatrics 103 (3), E26. Ludwig, D.S., Peterson, K.E., Gortmaker, S.L., 2001. Relation between consumption of sugar-sweetened drinks and childhood obesity: a prospective, observational analysis. Lancet 357 (9255), 505e508. Mattes, R.D., 1996. Dietary compensation by humans for supplemental energy provided as ethanol or carbohydrate in fluids. Physiol. Behav. 59 (1), 179e187. Mc Keigue, P.M., 1996. Metabolic consequences of obesity and body fat pattern: lessons from migrant studies. In: Shetty, P.S., Mc Pherson, K. (Eds.), The Origins and Consequences of Obesity. John Wiley & Sons, Chichester. Mehdi, M.M., Singh, P., Rizvi, S.I., 2012. Erythrocyte sialic acid content during aging in humans: correlation with markers of oxidative stress. Dis. Markers 32 (3), 179e186. Moraes, J.C., Coope, A., Morari, J., Cintra, D.E., Roman, E.A., Pauli, J.R., et al., 2009. High-fat diet induces apoptosis of hypothalamic neurons. PLoS One 4, e5045. Morgado, N., Rigotti, A., Valenzuela, A., 2005. Comparative effect of fish oil feeding and other dietary fatty acids on plasma lipoproteins, biliary lipids, and hepatic expression of proteins involved in reverse cholesterol transport in the rat. Ann. Nutr. Metab. 49, 397e406. Naismith, D.J., Nelson, M., Burley, B., Gattenby, S., 1995. Does a highsugar diet promote overweight in children and lead to nutrient deficiencies? J. Hum. Nutr. Diet. 8, 249e254. Obesity: Preventing and Managing the Global Epidemic. Report of a WHO Consultation. WHO Technical Report Series No. 894, 2000. WHO, Geneva, Switzerland. Pena, M., Bacallao, J. (Eds.), 2000. Obesity and Poverty: A New Public Health Challenge. Pan American Health Organization (PAHO), Washington, DC. Pomplun, D., Voigt, A., Schulz, T.J., Thierbach, R., Pfeiffer, A.F., Ristow, M., 2007. Reduced expression of mitochondrial frataxin in mice exacerbates diet-induced obesity. Proc. Natl. Acad. Sci. USA 104, 6377e6381. Popkin, B.M., 2001. The nutrition transition and obesity in the developing world. J. Nutr. 131 (3), 871Se873S. Prentice, A.M., 1998. Manipulation of dietary fat and energy density and subsequent effects on substrate flux and food intake. Am. J. Clin. Nutr. 67, 535Se541S.

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Preparation and Use of Food-based Dietary Guidelines. Report of a Joint FAO/WHO Consultation. WHO Technical Report Series No. 880, 1996. World Health Organisation, Geneva, Switzerland. Reaven, G.M., 1997. Do high carbohydrate diets prevent the development or attenuate the manifestations (or both) of syndrome X? A viewpoint strongly against. Curr. Opin. Lipidol. 8 (1), 23e27. Rokling-Andersen, M.H., Rustan, A.C., Wensaas, A.J., Kaalhus, O., Wergedahl, H., Rost, T.H., et al., 2009. Marine n-3 fatty acids promote size reduction of visceral adipose depots, without altering body weight and composition, in male Wistar rats fed a high fat diet. Br. J. Nutr. 102, 995e1006. Rolls, B.J., Hetherington, M., 1989. The role of variety in eating and body weight regulation. In: Sheperd, R. (Ed.), Handbook of the Psychophysiology of Human Eating. John Wiley & Sons, Sussex, England. Rolls, B.J., Bell, E.A., Castellanos, V.H., Chow, M., Pelkman, C.L., Thorwart, M.L., 1999a. Energy density but not fat content of foods affected energy intake in lean and obese women. Am. J. Clin. Nutr. 69 (5), 863e871. Rolls, B.J., Bell, E.A., Thorwart, M.L., 1999b. Water incorporated into a food but not served with a food decreases energy intake in lean women. Am. J. Clin. Nutr. 70 (4), 448e455. Rosen, E.D., Spiegelman, B.M., 2006. Adipocytes as regulators of energy balance and glucose homeostasis. Nature 444, 847e853. Satyanarayana, U., Chakrapani, U., 2008. Biochemistry, fourth ed. Elsevier Inc. Singh, P., Rizvi, S.I., 2012. Anti-oxidative effect of curcumin against tert- butyl hydroperoxide induced oxidative stress in the human erythrocytes. Nat. Prod. J. 2, 69e73. Singh, P., Rizvi, S.I., 2013. Curcumin activates erythrocytes membrane acetylcholinesterase. Lett. Drug Des. Discov. 10 (6), 550e556. Singh, P., Rizvi, S.I., 2015a. Modulation effects of curcumin on erythrocyte ion-transporter activity. International Journal of Cell Biology 2015, 1e8. Singh, P., Rizvi, S.I., 2015b. Role of curcumin in modulating plasma PON1 arylesterase activity and susceptibility to LDL oxidation in oxidatively challenged Wistar rats. Lett. Drug Des. Discov. 12 (4), 319e323. Siri-Tarino, P.W., Sun, Q., Hu, F.B., Krauss, R.M., 2010. Saturated fat, carbohydrate, and cardiovascular disease. Am. J. Clin. Nutr. 91, 502e509. Sjostrom, L., 1992. Impacts of body weight, body composition, and adipose tissue distribution on morbidity and mortality. In: Stunkard, A.J., Wadden, T.A. (Eds.), Obesity: Theory and Therapy, second ed. Raven Press, New York. Slabber, M., Barnard, H.C., Kuyl, J.M., Dannhauser, A., Schall, R., 1994. Effects of a low-insulin-response, energy-restricted diet on weight loss and plasma insulin concentrations in hyperinsulinemic obese females. Am. J. Clin. Nutr. 60 (1), 48e53. Spieth, L.E., Harnish, J.D., Lenders, C.M., Raezer, L.B., Pereira, M.A., Hangen, S.J., et al., 2000. A low-glycemic index diet in the treatment of pediatric obesity. Arch. Pediatr. Adolesc. Med. 154 (9), 947e951. Stubbs, R.J., Harbron, C.G., Murgatroyd, P.R., Prentice, A.M., 1995. Covert manipulation of dietary fat and energy density: effect on substrate flux and food intake in men eating ad libitum. Am. J. Clin. Nutr. 62, 316e329. Stubbs, R.J., Harbron, C.G., Prentice, A.M., 1996. Covert manipulation of the dietary fat to carbohydrate ratio of isoenergetically dense diets: effect on food intake in feeding men ad libitum. Int. J. Obes. Relat. Metab. Disord. 20 (7), 651e660. Swinburn, B.A., Caterson, I., Seidell, J.C., James, W.P.T., 2004. Diet, nutrition and the prevention of excess weight gain and obesity. Public Health Nutr. 7 (1A), 123e146. Taras, H.L., Sallis, J.F., Patterson, T.L., Nader, P.R., Nelson, J.A., 1989. Television’s influence on children’s diet and physical activity. J. Dev. Behav. Pediatr. 10 (4), 176e180.

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Tordoff, M.G., Alleva, A.M., 1990. Effect of drinking soda sweetened with aspartame or high-fructose corn syrup on food intake and body weight. Am. J. Clin. Nutr. 51 (6), 963e969. Uzun, H., Zengin, K., Askin, M.T., Aydin, S., Simsek, G., Dariyerli, N., 2004. Changes in leptin, plasminogen activator factor and oxidative stress in morbidly obese patients following open and laparoscopic Swedish adjustable gastric banding. Obes. Surg. 14, 659e665. Wajchenberg, B.L., 2000. Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome. Endocr. Rev. 21, 697e738. Wang, H., Storlien, L.H., Huang, X.F., 2002. Effects of dietary fat types on body fatness, leptin, and ARC leptin receptor, NPY, and AgRP mRNA expression. Am. J. Physiol. Endocrinol. Metab. 282, E1352eE1359. Westerterp, K.R., 2004. Diet induced thermogenesis. Nutr. Metab. 1e5. http://dx.doi.org/10.1186/17437075-1-5. Westerterp, K.R., Wilson, S.A., Rolland, V., 1999. Diet induced thermogenesis measured over 24 h in a respiration chamber: effect of diet composition. Int. J. Obes. Relat. Metab. Disord. 23 (3), 287e292. Westerterp, K.R., Smeets, A., Lejeune, M.P., Wouters-Adriaens, M.P., Westerterp-Plantenga, M.S., 2008. Dietary fat oxidation as a function of body fat. Am. J. Clin. Nutr. 87, 132e135. Williams, P.T., 2000. Viewpoint. Sugar: is there a need for a dietary guideline in Australia? Aust. J. Nutr. Diet. 58, 26e31.

Further Reading Singh, P., Kesharwani, R.K., Misra, K., Rizvi, S.I., 2016. Modulation of erythrocyte plasma membrane redox system activity by curcumin. Biochem. Res. Int. 2016, 1e8.

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Role of Selected Medicinal Plants in Sports Nutrition and Energy Homeostasis Marijana Zovko Koncic UNIVERSITY OF Z AGREB, ZAGREB, CROATIA

Introduction The competition in modern sport is greater than ever, and the elite athletes’ desire to achieve better results has led to indiscriminate use of dietary supplements, including herbal ones. This has become a widespread and accepted practice in sport. Global supplement use in athletes is estimated to range from 40% to as high as 100%. Research conducted by the US Olympic Committee in 2004 found that about 90% of athletes use some form of dietary supplementation (Collins and Kalman, 2008; Williams et al., 2012). Most herbal supplements in sports can be classified either as adaptogens or ergogenic aids. While ergogenic aids are substances with performance-enhancing effects, the term adaptogen may suggest a plant that increases the adaptation to exercise-induced stress through nonspecific effects (Bleakney, 2008). Although elite athletes appear to use dietary supplements much more than their nonelite counterparts, an increasing number of amateur sportsmen are trying to improve their physical shape by the use of dietary supplements (Knapik et al., 2016). Although the marketing claims for some dietary substances include increase in energy, attenuation of pain, loss of excess weight, as well as enhancement of physical and cognitive performance and overall health status (Knapik et al., 2016), those claims are often based on little or no scientific evidence. Scientific scrutiny with controlled clinical trials has been used to critically examine such supplements only in the last two decades (Bucci, 2000). Furthermore, in addition to possible gains, these aids can be costly and potentially harmful. Since the possible performance-related benefits of most natural ingredients in the market are mild, they usually cannot meet the expectations of the users. Therefore, the producers often adulterate their products with drugs or new dietary ingredients not submitted to the FDA. Although such adulterants are illegal, their detection is not always timely, and they are often discovered only after an adverse event

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has already taken place. It is important to note that the sports performance/ bodybuilding supplements are among the three types of supplements most likely to cause medical problems due to the adulteration (Brown, 2016). The aim of this review is to evaluate the ergogenic and adaptogenic properties of selected medicinal plants: Citrus aurantium, Rhodiola rosea, Schisandra chinensis, Tribulus terrestris, Vitis vinifera, and Withania somnifera. Besides their composition and possible side effects, the available data on their activity, either alone or in combination with other herbs, will be reviewed.

Diverse Medicinal Plants Citrus aurantium Citrus  aurantium L., Rutaceae (bitter orange) is an evergreen tree with very fragrant flowers and orange, acidic-tasting fruit (Fleming, 2000). The fruit peel is rich in essential oil, mostly composed of terpenes, with limonene as the main constituent. The fruit also contains flavonoids (hesperidin, naringenin) and coumarins (umbelliferone, bergapten) (Khan and Ehab, 2010). However, in terms of its purported ergogenic and weight-loss properties, a protoalkaloid with adrenergic properties, p-synephrine, is by far the most interesting phytochemical in bitter orange (Preuss et al., 2002). The products containing p-synephrine and C. aurantium extract gained on popularity after products containing Ma huang or Ephedra species have been outlawed. Ephedra extracts contain ephedrine, a protoalkaloid with b-agonist properties. Ephedrine use may result in increased physical capabilities, thermogenesis, and appetite reduction. These effects are even more pronounced if ephedrine is combined with training and/or caffeine intake. However, ephedrine may cause serious adverse reactions, even with death outcomes. As a result, ephedrine supplements are banned in many countries (Bent et al., 2004). Numerous food supplement producers have tried to find appropriate substitute for Ephedra and ephedrine in their formulations. As an herbal product with similar protoalkaloid, psynephrine, C. aurantium has been perceived as such (Preuss et al., 2002; Westanmo, 2007). Trials on human subjects have shown that C. aurantium and p-synephrine may increase resting metabolic rate and energy expenditure, as well as decrease weight when given for 6e12 weeks (Gougeon et al., 2005; Preuss et al., 2002; Stohs et al., 2011). The flavonoids present in the C. aurantium extract also contribute to the observed effects (Stohs et al., 2011). Although p-synephrine has been included in the World Anti-Doping Agency monitoring program, scientific information about its effects on athletes’ performance is scarce, and the available evidence is somewhat contradictory. It seems that p-synephrine does not influence performance in sprint athletes. In a randomized and counterbalanced order, experienced sprinters (n ¼ 13) performed two experimental trials after the ingestion of 3 mg/kg p-synephrine or placebo. Forty-five minutes after the ingestion, the sprinters performed a squat jump, a countermovement jump, and 15-s repeated jump test, followed by 60-m and 100-m simulated sprint competitions. The jump heights and

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maximal running speed were the same for both treatments (Gutie´rrez-Hellı´n et al., 2016). The study performed by Haller et al., aimed primarily at studying cardiovascular and weight-loss properties of a combination of synephrine (21 mg) and caffeine (304 mg) in a moderately intense exercise (30 min on cycle ergometer at 75%e80% HRmax), reported that the treatment subjects perceived the exercise to be less difficult than those taking the placebo. Nevertheless, it is difficult to assess the influence of each ingredient on this effect (Haller et al., 2008). It seems that the p-synephrine does possess certain ergogenic properties, which can be enhanced with caffeine. Former athletes (n ¼ 12) who first performed a control resistance exercise protocol consisting of 6 sets of squats for up to 10 repetitions per set using 80% of their one repetition maximum were assigned to a treatment sequence consisting of use of three supplements: p-synephrine (100 mg), SC (100 mg of p-synephrine plus 100 mg of caffeine), or a placebo. The treatments were assigned in randomized, double-blind, balanced manner, and separated by 1 week. Each time, the subjects consumed the supplement for 3 days prior to exercise protocol and in the morning the protocol was performed. The treatments with p-synephrine produced a significantly greater number of repetitions and greater increase in volume load per protocol than control or placebo. In addition to that, the addition of caffeine to p-synephrine resulted in increased mean power and velocity for all six treatment sets. Blood lactate and perceived exertion were not changed (Ratamess et al., 2015). The structural likeness of p-synephrine and ephedrine has led to concern about the possible side effects of bitter orange. Several adverse cardiovascular effects have been possibly linked to the use of synephrine-containing supplements (Bouchard et al., 2005; Gange et al., 2006), including myocardial infarction (Nykamp, 2004). However, p-synephrine dose in many weight-loss products is almost 10-fold higher than p-synephrine content in the fruit (Westanmo, 2007). Some of the aforementioned adverse effects could be related to caffeine, a frequent constituent of weight-loss and sport supplements, including those with C. aurantium. Furthermore, a nonnatural synephrine isomer, m-synephrine, was found in some of the supplements, probably as a result of adulteration. m-synephrine is a stronger agonist of several a-receptor subtypes, which may result not only in better weight-loss properties, but also in the distinct cardiovascular effects. Even though the studies specifically designed to investigate cardiovascular of bitter orange and p-synephrine did not find significant adverse events such as alteration of electrocardiographic data or heart rate (HR) or blood pressure increase, caution on consumption of C. aurantiumecontaining products is to be advised (Rossato et al., 2011; Stohs, 2011).

Rhodiola rosea Rhodiola rosea L., Crassulaceae is widely distributed in colder regions of Europe and Asia. The common name of the plant, roseroot, originates from the scent of freshly cut underground organs, which reminds one of roses (Panossian et al., 2010). The main constituents of R. rosea root are free and glycosylated phenylpropanoids (tyrosol,

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salidroside), phenylpropenoids (rosin, rosavin), monoterpene derivatives (rosiridol, rosiridin), and about 0.05% of essential oil with a-pinene, limonene, and geraniol, the latter being responsible for the characteristic scent of the plant’s underground organs (Ali et al., 2008; Panossian et al., 2010). R. rosea is commonly used throughout Eastern Europe and Asia for its purported adaptogenic properties. Numerous older studies on the activity of R. rosea largely support such use (Panossian et al., 2010). Newer clinical trials have confirmed that R. rosea may improve mental performance, relieve fatigue, stress, and mild depression (Cropley et al., 2015; Darbinyan et al., 2000; Hung et al., 2011; Olsson et al., 2009). Commercially available R. rosea root extract, SHR-5, has displayed adaptogenic effects in several double-blind, randomized, placebo-controlled clinical trials. The observed effects included decreased fatigue in stress-related conditions as well as improved cognitive performance, mood, and attention. Even though the studies were performed for 2e 6 weeks, the effects could be perceived after a single dose in 1e2 h after the administration. Typically, the preparation was administrated orally in doses of 288e680 mg/ day (Darbinyan et al., 2000; Hung et al., 2011; Olsson et al., 2009; Panossian et al., 2010; Spasov et al., 2000). Another supplement containing R. rosea, ADAPT-232, has also been investigated for its adaptogenic properties. However, unlike SHR-5, which is a single active ingredient formulation, ADAPT-232 also contained Schisandra chinensis (Turcz.) Baill. and Eleutherococcus senticosus Maxim. In one double-blind, placebo-controlled, randomized study, ADAPT-232 improved attention, increased speed, and improved accuracy of female participants performing stressful cognitive tasks (Aslanyan et al., 2010). The observed adaptogenic properties have drawn the attention of researchers toward potential applications in sport. Noreen et al. investigated the effects of an acute oral dose of 3 mg/kg of R. rosea, testing it at submaximal exercise conditions in a double-blind, random crossover manner. The subjects (n ¼ 18) performed a standardized 10-min warm-up, followed by a 6-mile time trial on a bicycle ergometer 1 h upon the ingestion. The HR of the subjects using R. rosea was significantly lower during the warm-up than in those taking placebo. In addition, the time trial was significantly shorter in subjects taking R. rosea, while the perceived exertion was lower. Interestingly, mood and cognitive function did not differ between the treatments (Noreen et al., 2009). Both acute and 4-week effects of R. rosea supplementation were followed in a double-blind, placebo-controlled, randomized study. The participants (n ¼ 24) received 200 mg of R. rosea extract containing 3% rosavin and 1% salidroside. Acute R. rosea supplementation seemed to significantly increase time to exhaustion, peak oxygen uptake (VO2 peak), and peak carbon dioxide uptake. The other parameters (pulmonary ventilation, speed of limb movement in plate tapping test, the ability to sustain attention, aural and visual reaction time) were not changed. Interestingly, after 4-week R. rosea or placebo intake, treatment did not alter any of the observed variables (De Bock et al., 2004). Similarly, the performance was not altered in another two trials with subchronic R. rosea supplementation. Participants in the randomized, double-blinded study were experienced marathon runners (n ¼ 48) who received R. rosea (600 mg/day) or placebo

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supplementation starting 30 days before and ending 7 days after the marathon. The postmarathon decrease in muscle function, increases in muscle damage, delayed onset muscle soreness, extracellular HSP72, or plasma cytokines in experienced marathon runners did not differ between the treatment and the placebo group (Shanely et al., 2014). Furthermore, R. rosea supplementation (1500 mg/day, 4 days) did not cause significant differences between groups in phosphocreatine kinetics nor perceived exertion of resistance-trained men (n ¼ 12) who completed incremental forearmewrist flexion exercise to volitional fatigue (Walker et al., 2007). Intensive activity of skeletal muscles generates free radicals, and intense exercise can therefore result in oxidative damage to cellular constituents (Powers and Jackson, 2008). Therefore, R. rosea and other plant extracts with antioxidant polyphenols may affect biochemical antioxidant parameters during heavy physical activity. A randomized, doubleblind study evaluated the effects of 4-week R. rosea supplementation (200 mg/day) on the balance of oxidants and antioxidants in the serum and erythrocytes of competitive rowers. The participants (n ¼ 22) performed a test on a rowing ergometer at the beginning and at the end of the study. Total plasma antioxidant capacity in R. rosea group was significantly higher than in the placebo group. Furthermore, superoxide dismutase activity in erythrocytes was significantly lower in the athletes receiving R. rosea extracts. Conversely, supplementation with R. rosea had no effect on several other biochemical parameters, such as glutathione peroxidase activity, thiobarbituric acid reactive substances concentration in erythrocytes, creatine kinase (CK) activity in plasma, lactate levels in capillary blood samples, and uric acid concentrations in serum (Skarpanska-Stejnborn et al., 2009). Interestingly, a pilot study with participants taking 170 mg/day of R. rosea had contrasting results (Parisi et al., 2010). The crossover study was conducted on male athletes (n ¼ 14) who, following a chronic supplementation with R. rosea for 4 weeks, underwent a cardiopulmonary exhaustion test. Even though the treatment capsules also contained probiotics and antioxidants (of unspecified type and quantities), the authors failed to find any changes in antioxidant parameters (total antioxidant status, plasma malonyldialdheyde, and in vitro erythrocyte sensitivity to oxidative damage) caused by the treatment. Similar to the antioxidant parameters, the performance parameters, such as HRmax, perceived exertion, maximum rate of oxygen consumption (VO2 max), and duration of the test, were also unaffected. On the contrary, R. rosea intake reduced plasma-free fatty acids levels, blood lactate, and plasma CK, parameters that were unaffected by the previous trial (Skarpanska-Stejnborn et al., 2009). The possible sources of the discrepancies may be due to the interaction of the added probiotic and antioxidants, either with R. rosea or athletes’ organism. Other possible mechanisms of R. rosea action were also investigated. Even though Panossian et al. (2009) established that the mechanism of action of ADAPT-232 could be the modulation of expression of Hsp72, the study performed by Shanely et al. (2014) eliminates the possibility that R. rosea is responsible for that effect. It seems that the observed effects were not attained through the direct modulation of energy consumption either, as Walker et al. (2007) failed to find any influence of R. rosea supplementation on adenosine triphosphate turnover during or immediately after exercise.

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Besides the single-extract preparations, several combinations of R. rosea and Cordyceps sinensis were investigated for their effects on sport performance. A commercial extract (1000 mg of C. sinensis, 300 mg of R. rosea root extract, and 200 mg of chromium) was investigated for its ability to increase endurance and oxygen consumption. In a randomized, placebo-controlled, double-blind trial, the extract was administered to 17 amateur cyclists as recommended by the manufacturer: the loading phase (4 days, 6 caps/day) was followed by the maintenance phase (11 days, 3 caps/day). Each cyclist participated in two (pre- and postsupplementation) cycling tests. The authors observed no significant differences for investigated variables, including VO2 max, time to exhaustion, maximum power output, or HRmax (Earnest et al., 2004). Those results were confirmed in another study of the same supplement (Parcell et al., 2004), as well as by a study of another formula consisting of C. sinensis and R. rosea (Colson et al., 2005). Unlike the combination with R. rosea, C. sinensis may be potentially useful when combined with Rhodiola crenulata (Chen et al., 2014). A study of an R. crenulata (1400 mg/day) and C. sinensis (600 mg/day) combination on aerobic exercise capacity in high-altitude training was performed. It was found that the exhaustive run time was markedly longer in the treatment group, while the decline of parasympathetic activity was significantly prevented. R. rosea is generally considered safe. Only few minor side effects such as sleepiness, cold extremities (Aslanyan et al., 2010), dizziness, and dry mouth (Bystritsky et al., 2008) were reported in the studies.

Schisandra chinensis The fruit of Schisandra chinensis (Turcz.) Baill., Schisandraceae is spherical, bright red, and contains one to two reniform yellow seeds. In mandarin Chinese, it is called “wu-weizi,” which could be translated as “five-taste fruit.” The name is associated with different tastes of fruit parts: sour, sweet (skin and pulp), pungent, bitter, and salty (kernel). According to the traditional Chinese medicine, each taste affects different organs, rendering the fruit suitable for multiple applications. The fruit constituents include lignans such as schizandrin, gomisin A, and gomisin N (World Health Organisation, 2007). It is interesting to note that another species, Schisandra sphenanthera Rehder & E.H. Wilson, may also be used for similar purposes (Chen and Chen, 2004). Contemporary interest in S. chinensis is a result of a large number of pharmacological and clinical investigations carried out in the former USSR during the period of 1940e60. More than 30 studies pointed to the ability of S. chinensis to increase endurance and mental performance, stimulate central nervous system, as well as to increase accuracy of movement and physical working capacity. Unfortunately, the studies were not performed according to the current research standards. Although the studies on racing horses confirmed some of the ergogenic effects of S. chinensis (Ahumada et al., 1989; Hancke et al., 1994, 1996), modern clinical trials, which could confirm its sport-boosting properties, are still needed (Panossian and Wikman, 2008).

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In addition to the previously described study of ADAPT-232, the combined preparation with S. chinensis and other herbs (Aslanyan et al., 2010), a placebo-controlled, double-blind study investigated the influence of S. chinensis standardized extract on blood NO and cortisol concentration in several groups of trained athletes. The parameters were measured pre- and postexercise. In the beginning of the test, S. chinensis increased the concentration of NO and cortisol in blood plasma and saliva, similar to placebo-taking athletes after physical exercise. Furthermore, physical performance in athletes taking adaptogens increased versus athletes taking placebo. In contrast, after treatment with the adaptogen, heavy physical exercise did not increase salivary NO and cortisol in athletes, whereas in athletes treated with placebo, heavy physical exercise increased salivary NO. It was concluded that S. chinensis exhibits pro-stressor effects: it activates formation of both NO and cortisol in blood plasma and saliva. Those levels did not increase after physical exercise. It seems that the supplementation adapted the organism to the subsequent heavy physical loading (Panossian et al., 1999). Even though S. chinensis is widely used, no serious adverse effects have been reported. Overdose may cause restlessness, insomnia, or dyspnea (World Health Organization, 2007). One case of hemorrhagic stroke in a young, healthy male following use of a sports supplement containing schizandrol A was described. However, the product contained caffeine and other constituents, which may predispose to stroke and hemorrhage (Young et al., 2012).

Tribulus terrestris Tribulus terrestris L., Zygophyllaceae is an herb native to the Mediterranean region. Its fruit is used as an aphrodisiac, diuretic, galactagogue, and general and uterine tonic. The major constituents of the fruit are steroidal saponins, including gitonin, protodioscin, and tribulosaponins A and B. Other constituents include alkaloids (tribulusamides, harman, norharman) and flavonoids (kaempferol, quercetin, rutin) (World Health Organization, 2009b). T. terrestris fruit is among the most used herbal supplements with purported ergogenic and androgenic properties. It is usually combined with other herbs and chemicals, which are claimed to improve its efficiency and produce libido-, virility-, and vitality-enhancing effects. However, evidence emerging from the scientific literature is less convincing. For example, several studies investigated the ability of T. terrestris to influence androgen hormone status, and the obtained results were discouraging (Brown et al., 2000, 2001a,b; Saudan et al., 2008). Furthermore, two of the studies aimed at evaluating ergogenic potential of T. terrestris did not support its athletic-enhancing properties. The effects of T. terrestris supplementation (3.21 mg/kg day) in resistancetrained males (n ¼ 15), randomly assigned to a placebo or T. terrestris extract, were assessed. After 8 weeks, there were no significant between-groups differences in majority of the measured parameters. Maximal number of repetitions at 100e200% of body weight increased for the bench and leg press exercises in the placebo group, while the

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T. terrestris group experienced an increase in leg press strength only, but not for the bench press (Antonio et al., 2000). Those results were further confirmed in a study performed on elite male rugby league players (n ¼ 22) who were match-paired and randomly assigned in a double-blind manner to either a T. terrestris extract (450 mg/day) or placebo group. The subjects performed structured heavy resistance training as part of the club’s preseason preparations. After 5 weeks of training, strength and fat-free mass increased significantly, but without any between-group differences. No differences were noted in the urinary testosterone/epitestosterone ratio (Rogerson et al., 2007). In the study where the subjects using ANDRO-6 (300 mg of androstenedione, 150 mg DHEA, 750 mg T. terrestris, 625 mg chrysin, 300 mg indole-3-carbinol, and 540 mg saw palmetto) performed 3 days of resistance training per week for 8 weeks (n ¼ 20), the muscle strength of the subjects increased similarly from weeks 0e8 regardless if they had taken the supplement or a placebo (Brown et al., 2000). On the other hand, some of the performed studies did support some of the claims related to T. terrestris supplements. Athletes (n ¼ 32) were taking either commercial dietary supplement with T. terrestris (25 mg/kg day, 20 days) or no supplement at all. The group receiving T. terrestris had a statistically significant positive impact on anaerobic alactic muscular power and anaerobic alactic glycolytic power. During the experimental period, the percentage of granulocytes decreased and the percentage of leucocytes increased, which was a negative impact of the supplement on changes in athletes’ blood. CK has increased and the creatinine amount had a tendency to decline during the 20-day period of supplementation. The concentration of blood testosterone increased during the first half (10 days) of the experiment, but it remained the same during the remaining 10 days of the study (Milasius et al., 2009). The effects of T. terrestris extract (1250 mg/ day) were also investigated on trained male boxers (n ¼ 15). In an attempt to elucidate the mechanism of action, plasma androgens, insulin-like growth factor 1 (IGF-1), and IGF-1 binding protein-3 were assessed. The placebo and T. terrestris group undertook 3week high-intensity and 3-week high-volume trainings separated by a 4-week rest. The measured parameters were assessed before and at the end of the two trainings. The extract intake did not change muscle mass and plasma levels of androgenic hormones and IGF-1, but it significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers (Ma et al., 2015). One case of priapism was reported after consumption of an herbal supplement based on T. terrestris used for treatment of sexual dysfunction (Campanelli et al., 2016).

Vitis vinifera Vitis vinifera L., Vitaceae (grapevine) and other Vitis L. species are cultivated throughout the world. They are mostly used for wine production. Besides a high level of glucose, the fruit pulp contains organic acids and polyphenols such as flavonoids (quercetin), tannins, and stilbenes (resveratrol and viniferins), with whom many medicinal properties are associated (Fleming, 2000). Besides the pulp, another valuable grape product is grape

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seed extract (GSE), rich in proanthocyanidins, essential fatty acids, and tocopherols. Epidemiologic studies have shown that moderate wine, grapes, and GSE consumption may reduce the risk of cardiovascular diseases (Mason, 2007; Vaclavik and Christian, 2008). Presence of sugars and antioxidant phenolic compounds in grape pulp and seed makes it an excellent candidate for development of sport-related supplements. Several studies designed to examine potential differences in metabolism performance after consumption of raisins versus commercial products for short-term exercise boosts concluded that raisins are a cost-effective source of carbohydrate for preexercise feeding (Kern et al., 2007; Rietschier et al., 2011). Furthermore, addition of grape phenols to the diet of rodents improved run time to exhaustion, fitness, and skeletalemuscle mitochondrial function (Dolinsky et al., 2012). This has initiated several studies that investigated if such effects may occur in humans. However, their results were not always encouraging. Active young adults (n ¼ 40) were randomly assigned to a grape or placebo drink for 45 consecutive days. Treadmill running and high-intensity eccentric actions of the nondominant elbow flexors were performed before and after the supplementation. Statistical analysis did not find any grape consumptionerelated effect on treadmill running, VO2 max, work capacity, mood, perceived health status, inflammation, pain, or physical function responses to a mild injury induced by eccentric exercise (O’Connor et al., 2013). Furthermore, a randomized, placebo-controlled, double-blind trial (n ¼ 16) has shown that the resveratrol supplementation (4 weeks, 150 mg/day) did not increase aerobic or anaerobic capacity, exercise substrate utilization, or muscle fiberespecific adaptations of low-volume, high-intensity interval training (3 days/week). The comparison of VO2 peak, Wingate peak/average power, and training session performance suggested that resveratrol may actually be impairing/altering the adaptive response to training (Scribbans et al., 2014). However, a study aimed at evaluation of the effect of Vitrus labrusca organic grape juice intake (300 mL/day containing 5.32 mg/mL of polyphenols) on biochemical variables and microcirculatory parameters in triathlon athletes (n ¼ 10) has shown that organic grape juice intake improved glucose homeostasis (the peak levels of serum insulin increased, plasma glucose level decreased), antioxidant capacity (plasma uric acid increased while erythrocyte superoxide dismutase activity decreased), and microvascular function (the functional capillary density and red blood cell velocity increased) compared to baseline (Gonc¸alves et al., 2011). Positive, albeit moderate effects of grape constituents’ consumption were found in one study investigating the effects of combined resveratrol and quercetin combination (RQ). The subjects were taking 120 mg resveratrol and 225 mg quercetin for 6 days and 240 mg resveratrol and 450 mg quercetin on day 7 just prior to exercise. The study was a double-blind crossover design with 1-week washout between trials. Only the postexercise increase in F2-isoprostanes was significantly less with RQ than with placebo. However, the other measured parameters (protein carbonyls, antioxidant plasma parameters, Interleukin 8, and C-reactive protein) were not affected by treatment

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(McAnulty et al., 2013). Similarly moderate effects of resveratrol were found in a study on firefighters (n ¼ 60) supplemented or not with resveratrol (100 mg/day) for 90 days. The analyses were performed before and after a typical physical fitness test used to induce oxidative stress. Only antiinflammatory effects (reduced IL-6 and TNF-a level) were observed. In comparison with placebo, the supplementation did not alter hepatic enzymes’ (aspartate transaminase, alanine transaminase, and gamma-glutamyltransferase) plasma activities, glucose, lipid, and CK levels. The measured indicators of oxidative stress were also unaltered by supplementation (Macedo et al., 2015). The reason for apparent lack of activity may be either the low dose or low resveratrol absorption. The study investigating the effects of 500 mg of resveratrol plus 10 mg of piperine, a bioenhancer to increase bioavailibilty and efficacy of resveratrol versus placebo, demonstrated the treatment superiority. Participants (n ¼ 16) ingested the pills daily for 4 weeks and completed 3 sessions/week of submaximal endurance training of the wrist flexor muscles of the nondominant arm. In comparison with placebo group, the forearm skeletal muscle mitochondrial capacity in treatment group was significantly increased (Polley et al., 2016). The effects of GSE on heavy exercise-induced oxidative stress are well documented in animal studies (Belviranlı et al., 2012, 2013), but few studies were performed in human subjects. A combination of GSE (300 mg) and arginine (1.5 or 3.0 g) was able to increase physical working capacity at the fatigue threshold (PWCFT), estimated by an electromyographic procedure. The participants in the double-blind study were untrained college-age men (n ¼ 50), randomized into one of the three groups: placebo, 1.5 g arginine þ 300 mg GSE, or 3.0 g arginine þ 300 mg GSE. The subjects performed an incremental test to exhaustion on a cycle ergometer to determine their PWCFT before supplementation and after 4 weeks of supplementation. Significant mean increases of PWCFT were found in both supplement groups but not in the placebo group (Camic et al., 2010). Adverse effects of grape products (excluding wine) have not been reported. Fresh and dried fruits contain high levels of sugar and should therefore be cautiously used by diabetics (Ho et al., 2008).

Withania somnifera Withania somnifera (L.) Dunal, Solanaceae is a prominent adaptogen herb from traditional Indian medicine (Ayurveda). The part used for medicinal purposes is the long, tuberous root with characteristic horse-like odor to which the plant owes its name “ashwagandha” (Sanskrit for “smells like a horse”) (Department of Indian Systems of Medicine and Homeopathy, 1990). Interestingly, the root is considered to have both tonic and sedative properties, and it is therefore used for strengthening an exhausted nervous system to increase energy and improve overall health (Mishra et al., 2000; Pole, 2012). The principal bioactive compounds of W. somnifera are triterpene lactones known as withanolides (withaferin A, 27-deoxywithaferin A). Alkaloids (anaferine, anahygrine)

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and saponins (sitoindosides VIIeX) are also present (Ganzera et al., 2003; World Health Organization, 2009a). In animal studies, W. somnifera alone, or in a combination with other herbs, showed beneficial effect in exercise-induced oxidative stress (Misra et al., 2009) and led to behavior changes (Dhuley, 2000; Grandhi et al., 1994; Singh et al., 2000). However, despite its widespread use, large clinical trials are still lacking. A study investigating the effects of W. somnifera on mental and physical health of elderly subjects has confirmed some of its adaptogenic properties. The observed effects were mostly related to withanolide A, which was shown to prevent the formation of beta amyloid plaques (Singh et al., 2008). Even though W. somnifera is the ingredient of many supplements intended for use in sports, evidence of its activity is scarce. A randomized, prospective, double-blind, placebo-controlled clinical study followed healthy young male subjects (n ¼ 57) who received W. somnifera (600 mg/day, 8 weeks) or placebo. Following baseline measurements, both groups underwent resistance training for 8 weeks. The measurements were repeated at the end of week 8. Compared to the placebo, W. somnifera has increased muscle strength of the bench press exercise and the leg extension exercise, as well as the muscle size at the arms and chest. Furthermore, W. somnifera supplementation significantly decreased exercise-induced muscle damage as indicated by the stabilization of serum CK and led to a significantly greater increase in testosterone level and a significantly greater decrease in body fat percentage (Wankhede et al., 2015). In a study with elite cyclists, subjects (n ¼ 40) were chosen randomly and divided between W. somnifera (1000 mg/day, 8 weeks) and a placebo group. The baseline treadmill test for the cyclists was performed. The aerobic capacities in terms of VO2 max, metabolic equivalent, respiratory exchange ratio, and time to exhaustion were measured before and after the supplementation period. A significant improvement of all parameters was found in the treatment group, whereas the placebo group did not show any change from their baseline parameters (Shenoy et al., 2012). Another study (a single blind trial) investigated the effects of W. somnifera and Terminalia arjuna both individually and as a combination. The subjects (n ¼ 40) were young adults who received capsules with either W. somnifera, T. arjuna, their combination, or a placebo for 8 weeks. Subjects engaged in physical activities (sprint, vertical jumps, and balance board). Maximum velocity; average, absolute, and relative power; balance; VO2 max; and blood pressure were assessed before and after the 8-week period. The study showed that W. somnifera increased velocity, power, and VO2 max. When given in combination with T. arjuna, the improvement was seen in most of the parameters. However, the balance and diastolic blood pressure remained unchanged (Sandhu et al., 2010). At reasonable doses, W. somnifera is nontoxic. However, when taken in high doses, it may lead to nausea, vomiting, and diarrhea. In addition to its antiangiogenic and cytotoxic properties, W. somnifera has been used in traditional medicine to induce abortion. Therefore, its use during pregnancy or breastfeeding is contraindicated (World Health Organization, 2009a).

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Conclusions Numerous herbal supplements are used by elite and recreational athletes. However, much of the evidence concerning their efficacy is either anecdotal or based on earlier research, which suffers from methodological flaws. Newer studies indicate that while certain herbs do appear to have effects on performance and exercise-related recovery, the observed efficacy depends on numerous factors: product used, investigated sport, performed test, study setup, and others. The activity, if any, is rather supplement- and sport-specific than general. Therefore, more research is needed to unequivocally establish the activity of herbs, their combinations, and concentration of active principles in individual sports.

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Gange, C.A., Madias, C., Felix-Getzik, E.M., Weintraub, A.R., Estes, N.A.M., 2006. Variant angina associated with bitter orange in a dietary supplement. Mayo Clin. Proc. 81, 545e548. http://dx.doi.org/10. 4065/81.4.545. Ganzera, M., Choudhary, M.I., Khan, I.A., 2003. Quantitative HPLC analysis of withanolides in Withania somnifera. Fitoterapia 74, 68e76. Gonc¸alves, M.C., Bezerra, F.F., de Araujo Eleutherio, E.C., Bouskela, E., Koury, J., 2011. Organic grape juice intake improves functional capillary density and postocclusive reactive hyperemia in triathletes. Clinics 66, 1537e1541. http://dx.doi.org/10.1590/S1807-59322011000900005. Gougeon, R., Harrigan, K., Tremblay, J.-F., Hedrei, P., Lamarche, M., Morais, J.A., 2005. Increase in the thermic effect of food in women by adrenergic amines extracted from Citrus aurantium. Obes. Res. 13, 1187e1194. http://dx.doi.org/10.1038/oby.2005.141. Grandhi, A., Mujumdar, A.M., Patwardhan, B., 1994. A comparative pharmacological investigation of Ashwagandha and Ginseng. J. Ethnopharmacol 44, 131e135. Gutie´rrez-Hellı´n, J., Salinero, J.J., Abı´an-Vicen, J., Areces, F., Lara, B., Gallo, C., Puente, C., Del Coso, J., 2016. Acute consumption of p-synephrine does not enhance performance in sprint athletes. Appl. Physiol. Nutr. Metab. Physiol. Appl. Nutr. Metab. 41, 63e69. http://dx.doi.org/10.1139/apnm-20150299. Haller, C.A., Duan, M., Jacob, P., Benowitz, N., 2008. Human pharmacology of a performance-enhancing dietary supplement under resting and exercise conditions. Br. J. Clin. Pharmacol. 65, 833e840. http://dx.doi.org/10.1111/j.1365-2125.2008.03144.x. Hancke, J., Burgos, R., Ca´ceres, D., Brunetti, F., Durigon, A., Wikman, G., 1996. Reduction of serum hepatic transaminases and CPK in sport horses with poor performance treated with a standardized Schizandra chinensis fruit extract. Phytomedicine 3, 237e240. http://dx.doi.org/10.1016/S09447113(96)80059-6. Hancke, J., Burgos, R., Wikman, G., Ewertz, E., Ahumada, F., 1994. Schizandra chinensis, a potential phytodrug for recovery of sport horses. ResearchGate 65, 113e118. Ho, C.-T., Simon, J.E., Shahidi, F., Shao, Y. (Eds.), 2008. Dietary Supplements, first ed. American Chemical Society, Washington, DC. Hung, S.K., Perry, R., Ernst, E., 2011. The effectiveness and efficacy of Rhodiola rosea L.: a systematic review of randomized clinical trials. Phytomedicine 18, 235e244. http://dx.doi.org/10.1016/j. phymed.2010.08.014. Kern, M., Heslin, C.J., Rezende, R.S., 2007. Metabolic and performance effects of raisins versus sports gel as pre-exercise feedings in cyclists. J. Strength Cond. Res. 21, 1204e1207. http://dx.doi.org/10.1519/ R-21226.1. Khan, I.A., Ehab, A.A., 2010. Orange (bitter and sweet). In: Encyclopedia of Common Natural Ingredients: Used in Food, Drugs and Cosmetics. John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 477e482. Knapik, J.J., Steelman, R.A., Hoedebecke, S.S., Austin, K.G., Farina, E.K., Lieberman, H.R., 2016. Prevalence of dietary supplement use by athletes: systematic review and meta-analysis. Sports Med. 46, 103e123. http://dx.doi.org/10.1007/s40279-015-0387-7. Ma, Y., Guo, Z., Wang, X., 2015. Tribulus terrestris extracts alleviate muscle damage and promote anaerobic performance of trained male boxers and its mechanisms: roles of androgen, IGF-1, and IGF binding protein-3. J. Sport Health Sci.. http://dx.doi.org/10.1016/j. jshs.2015.12.003. Macedo, R.C.S., Vieira, A., Marin, D.P., Otton, R., 2015. Effects of chronic resveratrol supplementation in military firefighters undergo a physical fitness testea placebo-controlled, double blind study. Chem. Biol. Interact. 227, 89e95. http://dx.doi.org/10.1016/j.cbi.2014.12.033. Mason, P., 2007. Dietary-Supplements, third ed. Pharmaceutical Press, London.

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McAnulty, L.S., Miller, L.E., Hosick, P.A., Utter, A.C., Quindry, J.C., McAnulty, S.R., 2013. Effect of resveratrol and quercetin supplementation on redox status and inflammation after exercise. Appl. Physiol. Nutr. Metab. Physiol. Appl. Nutr. Metab. 38, 760e765. http://dx.doi.org/10.1139/apnm2012-0455. Milasius, K., Dadeliene, R., Skernevicius, J., 2009. The influence of the Tribulus terrestris extract on the parameters of the functional preparedness and athletes’ organism homeostasis. Fiziolohichnyi Zhurnal Kiev Ukr 1994 (55), 89e96. Mishra, L.C., Singh, B.B., Dagenais, S., 2000. Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern. Med. Rev. J. Clin. Ther. 5, 334e346. Misra, D.S., Maiti, R., Ghosh, D., 2009. Protection of swimming-induced oxidative stress in some vital organs by the treatment of composite extract of Withania somnifera, Ocimum sanctum and Zingiber officinalis in male rat. Afr. J. Tradit. Complement. Altern. Med. 6, 534e543. Noreen, E., Buckley, J., Lewis, S., 2009. The effects of an acute dose of Rhodiola rosea on exercise performance and cognitive function. J. Int. Soc. Sports Nutr. 6, 14. http://dx.doi.org/10.1186/15502783-6-S1-P14. Nykamp, D.L., 2004. Possible association of acute lateral-wall myocardial infarction and bitter orange supplement. Ann. Pharmacother. 38, 812e816. http://dx.doi.org/10.1345/aph.1D473. O’Connor, P.J., Caravalho, A.L., Freese, E.C., Cureton, K.J., 2013. Grape consumption’s effects on fitness, muscle injury, mood, and perceived health. Int. J. Sport Nutr. Exerc. Metab. 23, 57e64. Olsson, E., von Sche´ele, B., Panossian, A., 2009. A randomised, double-blind, placebo-controlled, parallel-group study of the standardised extract SHR-5 of the roots of Rhodiola rosea in the treatment of subjects with stress-related fatigue. Planta Med. 75, 105e112. http://dx.doi.org/10.1055/s-00281088346. Panossian, A., Wikman, G., 2008. Pharmacology of Schisandra chinensis Bail: an overview of Russian research and uses in medicine. J. Ethnopharmacol. 118, 183e212. http://dx.doi.org/10.1016/j.jep. 2008.04.020. Panossian, A., Wikman, G., Kaur, P., Asea, A., 2009. Adaptogens exert a stress-protective effect by modulation of expression of molecular chaperones. Phytomedicine 16, 617e622. http://dx.doi.org/ 10.1016/j.phymed.2008.12.003. Panossian, A., Wikman, G., Sarris, J., 2010. Rosenroot (Rhodiola rosea): traditional use, chemical composition, pharmacology and clinical efficacy. Phytomedicine 17, 481e493. http://dx.doi.org/10. 1016/j.phymed.2010.02.002. Panossian, A.G., Oganessian, A.S., Ambartsumian, M., Gabrielian, E.S., Wagner, H., Wikman, G., 1999. Effects of heavy physical exercise and adaptogens on nitric oxide content in human saliva. Phytomedicine Int. J. Phytother. Phytopharm. 6, 17e26. http://dx.doi.org/10.1016/S0944-7113(99) 80030-0. Parcell, A.C., Smith, J.M., Schulthies, S.S., Myrer, J.W., Fellingham, G., 2004. Cordyceps sinensis (CordyMax Cs-4) supplementation does not improve endurance exercise performance. Int. J. Sport Nutr. Exerc. Metab. 14, 236e242. Parisi, A., Tranchita, E., Duranti, G., Ciminelli, E., Quaranta, F., Ceci, R., Cerulli, C., Borrione, P., Sabatini, S., 2010. Effects of chronic Rhodiola rosea supplementation on sport performance and antioxidant capacity in trained male: preliminary results. J. Sports Med. Phys. Fitness 50, 57e63. Pole, S., 2012. In: Ayurvedic Medicine: The Principles of Traditional Practice, first ed. Singing Dragon, London. Polley, K.R., Jenkins, N., O’Connor, P., McCully, K., 2016. Influence of exercise training with resveratrol supplementation on skeletal muscle mitochondrial capacity. Appl. Physiol. Nutr. Metab. Physiol. Appl. Nutr. Metab. 41, 26e32. http://dx.doi.org/10.1139/apnm-2015-0370.

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Powers, S.K., Jackson, M.J., 2008. Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol. Rev. 88, 1243e1276. http://dx.doi.org/10.1152/physrev.00031. 2007. Preuss, H.G., DiFerdinando, D., Bagchi, M., Bagchi, D., 2002. Citrus aurantium as a thermogenic, weightreduction replacement for ephedra: an overview. J. Med. 33, 247e264. Ratamess, N.A., Bush, J.A., Kang, J., Kraemer, W.J., Stohs, S.J., Nocera, V.G., Leise, M.D., Diamond, K.B., Faigenbaum, A.D., 2015. The effects of supplementation with p-Synephrine alone and in combination with caffeine on resistance exercise performance. J. Int. Soc. Sports Nutr. 12, 35. http://dx.doi. org/10.1186/s12970-015-0096-5. Rietschier, H.L., Henagan, T.M., Earnest, C.P., Baker, B.L., Cortez, C.C., Stewart, L.K., 2011. Sun-dried raisins are a cost-effective alternative to Sports Jelly Beans in prolonged cycling. J. Strength Cond. Res. 25, 3150e3156. http://dx.doi.org/10.1519/JSC.0b013e31820f5089. Rogerson, S., Riches, C.J., Jennings, C., Weatherby, R.P., Meir, R.A., Marshall-Gradisnik, S.M., 2007. The effect of five weeks of Tribulus terrestris supplementation on muscle strength and body composition during preseason training in elite rugby league players. J. Strength Cond. Res. 21, 348e353. http://dx. doi.org/10.1519/R-18395.1. Rossato, L.G., Costa, V.M., Limberger, R.P., de Lourdes Bastos, M., Remia˜o, F., 2011. Synephrine: from trace concentrations to massive consumption in weight-loss. Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 49, 8e16. http://dx.doi.org/10.1016/j.fct.2010.11.007. Sandhu, J., Shah, B., Shenoy, S., Padhi, M., Chauhan, S., Lavekar, G., 2010. Effects of Withania somnifera (ashwagandha) and Terminalia arjuna (arjuna) on physical performance and cardiorespiratory endurance in healthy young adults. Int. J. Ayurveda Res. 1, 144. http://dx.doi.org/10.4103/0974-7788. 72485. Saudan, C., Baume, N., Emery, C., Strahm, E., Saugy, M., 2008. Short term impact of Tribulus terrestris intake on doping control analysis of endogenous steroids. Forensic Sci. Int. 178, e7e10. http://dx.doi. org/10.1016/j.forsciint.2008.01.003. Scribbans, T.D., Ma, J.K., Edgett, B.A., Vorobej, K.A., Mitchell, A.S., Zelt, J.G.E., Simpson, C.A., Quadrilatero, J., Gurd, B.J., 2014. Resveratrol supplementation does not augment performance adaptations or fibre-type-specific responses to high-intensity interval training in humans. Appl. Physiol. Nutr. Metab. Physiol. Appl. Nutr. Metab. 39, 1305e1313. http://dx.doi.org/10.1139/apnm2014-0070. Shanely, R.A., Nieman, D.C., Zwetsloot, K.A., Knab, A.M., Imagita, H., Luo, B., Davis, B., Zubeldia, J.M., 2014. Evaluation of Rhodiola rosea supplementation on skeletal muscle damage and inflammation in runners following a competitive marathon. Brain Behav. Immun. 39, 204e210. http://dx.doi.org/10. 1016/j.bbi.2013.09.005. Shenoy, S., Chaskar, U., Sandhu, J.S., Paadhi, M.M., 2012. Effects of eight-week supplementation of ashwagandha on cardiorespiratory endurance in elite Indian cyclists. J. Ayurveda Integr. Med. 3, 209e214. http://dx.doi.org/10.4103/0975-9476.104444. Singh, A., Saxena, E., Bhutani, K.K., 2000. Adrenocorticosterone alterations in male, albino mice treated with Trichopus zeylanicus, Withania somnifera and Panax ginseng preparations. Phytother. Res. 14, 122e125. Singh, R.H., Narsimhamurthy, K., Singh, G., 2008. Neuronutrient impact of Ayurvedic rasayana therapy in brain aging. Biogerontology 9, 369e374. http://dx.doi.org/10.1007/s10522-008-9185-z. Skarpanska-Stejnborn, A., Pilaczynska-Szczesniak, L., Basta, P., Deskur-Smielecka, E., et al., 2009. The influence of supplementation with Rhodiola rosea L. extract on selected redox parameters in professional rowers. Int. J. Sport Nutr. 19, 186.

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Spasov, A.A., Wikman, G.K., Mandrikov, V.B., Mironova, I.A., Neumoin, V.V., 2000. A double-blind, placebo-controlled pilot study of the stimulating and adaptogenic effect of Rhodiola rosea SHR-5 extract on the fatigue of students caused by stress during an examination period with a repeated low-dose regimen. Phytomedicine 7, 85e89. http://dx.doi.org/10.1016/S0944-7113(00)80078-1. Stohs, S.J., 2011. Synephrine: from trace concentrations to massive consumption in weight-loss. Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 49, 1472e1475. http://dx.doi.org/10.1016/j.fct. 2011.03.035. Stohs, S.J., Preuss, H.G., Keith, S.C., Keith, P.L., Miller, H., Kaats, G.R., 2011. Effects of p-synephrine alone and in combination with selected bioflavonoids on resting metabolism, blood pressure, heart rate and self-reported mood changes. Int. J. Med. Sci. 8, 295e301. Vaclavik, V., Christian, E., 2008. Essentials of Food Science. Springer, New York. Walker, T.B., Altobelli, S.A., Caprihan, A., Robergs, R.A., 2007. Failure of Rhodiola rosea to alter skeletal muscle phosphate kinetics in trained men. Metabolism 56, 1111e1117. http://dx.doi.org/10.1016/j. metabol.2007.04.004. Wankhede, S., Langade, D., Joshi, K., Sinha, S.R., Bhattacharyya, S., 2015. Examining the effect of Withania somnifera supplementation on muscle strength and recovery: a randomized controlled trial. J. Int. Soc. Sports Nutr. 12. http://dx.doi.org/10.1186/s12970-015-0104-9. Westanmo, A., 2007. Citrus aurantium. In: Tracy, T.S., Kingston, R.L. (Eds.), Herbal Products: Toxicology and Clinical Pharmacology. Humana Press, Totowa, pp. 233e244 (Chapter 15). Williams, M., Anderson, D., Rawson, E., 2012. In: Nutrition for Health, Fitness & Sport, tenth ed. McGraw-Hill Education, New York, NY. World Health Organisation, 2007. Fructus schisandrae. In: WHO Monographs on Selected Medicinal Plants. WHO, Geneva, pp. 296e313. World Health Organization, 2009a. Radix withaniae. In: WHO Monographs on Selected Medicinal Plants. WHO, Geneva, pp. 373e391. World Health Organization, 2009b. Fructus tribuli. In: WHO Monographs on Selected Medicinal Plants. WHO, Geneva, pp. 323e334. Young, C., Oladipo, O., Frasier, S., Putko, R., Chronister, S., Marovich, M., 2012. Hemorrhagic stroke in young healthy male following use of sports supplement Jack3d. Mil. Med. 177, 1450e1454. http://dx. doi.org/10.7205/MILMED-D-11-00342.

Further Reading Burke, J., Seda, G., Allen, D., Knee, T.S., 2007. A case of severe exercise-induced rhabdomyolysis associated with a weight-loss dietary supplement. Mil. Med. 172, 656e658. Gupta, G.L., Rana, A.C., 2007. Protective effect of Withania somnifera dunal root extract against protracted social isolation induced behavior in rats. Indian J. Physiol. Pharmacol. 51, 345e353. Shah, P.C., Trivedi, N.A., Bhatt, J.D., Hemavathi, K.G., 2006. Effect of Withania somnifera on forced swimming test induced immobility in mice and its interaction with various drugs. Indian J. Physiol. Pharmacol. 50, 409e415.

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Withania somnifera: Ethnobotany, Pharmacology, and Therapeutic Functions Muzamil Ahmad1, 2, Nawab J. Dar1, 2 2

1 INDIAN INSTITUT E OF INTEGRATIVE MEDICINE (CSIR) , SRINAGAR, INDIA; ACADEMY OF SCIENTIFIC AND INNOVATIVE RESEARCH, INDIAN INSTITUTE OF INT EGRATIVE MEDICINE (CSIR), JAM MU , IND IA

Introduction Withania somnifera (Linn.) Dunal is an evergreen, stout, woody shrub that grows to a height of 0.5e2.0 m above the ground. The plant is known as “Indian winter cherry” or “Indian ginseng” in English, “Ashwagandha” in Sanskrit, “Asgandh” or “Punir” in Hindi, and “Asgand” in Urdu. The plant belongs to the Solanaceae family of plant taxa and grows in hotter and drier parts of India and even in Himalayas up to an altitude of 5500 ft. The plant is commonly distributed, cultivated, or grows on its own in the drier parts of tropical and subtropical zones of the world. It grows from Canary Islands in the Mediterranean through tropical Africa to South Africa, Sri Lanka, Middle East, China, and India to warmer parts of Europe and to Australia. In India, it is cultivated as a medicinal crop and even grows wildly in waste lands. The plant has had enormous medicinal or therapeutic applications in Ayurvedic and Unani systems of medicine in India and has been used for more than 5000 years. Ashwagandha has been used as “Rasayana” in Ayurveda since its inception in 6000 B.C. It is an important herbal Rasayana and is known as “Sattvic Kapha Rasayana.” Rasayana is a herbal or metallic concoction that acts as a health promoter, rejuvenative agent, and tonic. Therapeutically and/or prophylactically, Ashwagandha is used as/ against adaptogenic, aphrodisiac, tonic, narcotic, diuretic, antihelminthic, astringent, depurative, thermogenic, and stimulant, antistress, antiinflammatory, anticarbuncle, antiulcer, debility from old age, rheumatism, vitiated conditions of Vata, leucoderma, constipation, insomnia, nervous breakdown, goiter, leucorrhoea, boils, pimples, flatulent colic, worms, piles, and oligospermia. Additionally, it is prescribed for snake venom and scorpion stings (Agarwal et al., 1999; Machiah et al., 2006; Machiah and Gowda, 2006). Moreover, it is used to clear white spots from the cornea, and there are reports that Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00008-9 Copyright © 2017 Elsevier Inc. All rights reserved.

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Ashwagandha is used in hysteria, anxiety, loss of memory, and syncope. Ashwagandha is used for emaciation of children (Sharma, 1999); however, when given with milk, it acts as tonic for children (Basu, 1935; Bhandari, 1970; Dar et al., 2015; Misra, 2004; Sharma et al., 1985). The history of Asgand being used in Unani system of medicine is mentioned in an old transcript: Kitab-al-Hashaish written by Dioscorides in 78 A.D. Asgand has been used against polyarthritis, rheumatoid arthritis, lumbago, painful swellings, spermatorrhoea, asthma, leucoderma, general debility, sexual debility, amnesia, anxiety neurosis, scabies, ulcers, marasmus, and leucorrhoea (Ali et al., 1997; Dar et al., 2015; Tiwari et al., 2014; Uddin et al., 2012). The plant has derived its names due to its certain characteristics. On account of its explicit antistress effects, the plant was ascribed its species name “somnifera,” which means “sleep-inducer” in Latin. The plant is called Ashwagandha because the roots of the plant exhibit the characteristic smell of a wet horse (“ashwa” means horse and “gandha” means smell). Additionally, it is called Indian ginseng because its pharmacologic effects and traditional uses are similar to that of Korean ginseng tea (Dar et al., 2015). Usually, roots of W. somnifera are manipulated for medicinal purposes. The plant retains its pharmacologic activity only for less than 2 years due to decomposition of its components. Owing to this drawback, fresh roots are harvested during JanuaryeMarch each year and shade-dried for good yield and better medicinal outcome. Leaves are used against fevers and painful swelling. Flowers have astringent, depurative, diuretic, and aphrodisiac properties. The seeds have some medicinal implications as being antihelminthic, removing white spots from the cornea, and increasing sperm count and testicular growth. The fruits are traditionally used topically against various skin diseases, carbuncles, and skin ulcers (Chopra et al., 2004; Kaur et al., 2004; Singh et al., 2011).

Chemical Composition Phytochemical characterization has revealed the presence of different chemical constituents in parts of the plant. To date, more than 12 alkaloids, 40 withanolides, and quite a few sitoindosides have been reported from the plant (Mirjalili et al., 2009). The main ingredients are shown in Table 8.1 and Fig. 8.1: Alkaloids: withanine, withaninine, withasomine, somniferine, tropeltigloate, somniferinine, somninine, and nicotine. Steroidal lactones: withaferin A, withanone, withanolide E, withanolide F, withanolide A, withanolide G, withanolide H, withanolide I, withanolide J, withanolide K, withanolide L, withanolide M. Steroids: cholesterol, b-sitosterol, stigmasterol, diosgenin, stigmastadien, sitoinosides VII, sitoinosides VIII, sitoinosides IX, sitoindosides. X Salts: cuscohygrine, anahygrine, tropine, pseudotropine, anaferine. Flavonoids: kaempferol, quercetin. Nitrogen-containing compounds: withanol, somnisol, and somnitol.

Toxicologic Studies of Withania somnifera Studies have revealed that extracts of W. somnifera are pretty safe for all age groups as well as both sexes and even during pregnancy. Hydroalcoholic root extract of

Table 8.1 Part of Plant Root Leaf

Fruit

Seed

Bioactive Compounds in the Different Parts of the Plant Bioactive Compounds Present Sitoindosides VII,VIII (acyl steryl glucoside), sitoindosides IX, X (glycowithanolide), withanine, withananine (alkaloids), withanolide A, viscosa lactone B, stigmasterol, and ashwagandhanolide Withaferin: withaferin A; withanone; withanolide D; withanolide E; withanolide B; 27-deoxywithaferin A; 2, 24-dienolide, trienolide (steroidal lactones); withanoside IV; withanolide Z, 7-hydroxywithanolide; 3a-methoxy-2, 3-dihydro; 4b, 17a-dihydroxy-1-1oxo; 5b, 6b-epoxy-22R-witha; 4b-dihydroxy-5b, 6b-epoxy; 1-oxo-22R-witha-2, 14e24, sitoindoside IX; 4-(1-hydroxy-2, 2-dimethylcyclo propanone; 2,3-dihydrowithaferin A; 24,25-dihydro-27 desoxywithaferin A, physagulin D; physagulin D (1–>6)-betaD-glucopyranosyl- (1–>4)-beta-D-glucopyranoside; 27-O-beta-D-glucopyranosylphysagulin D; 27-O-beta-D-lucopyranosylviscosalactone B; 4,16-dihydroxy-5beta, 6beta-epoxyphysagulin D, viscosalactone B; 5,20a (R)-dihydroxy-6a, 7a-epoxy-1-oxo- (5a) -witha-2, 24-dienolide (steroidal lactone) 2, 3-dihydrowithaferin-A-3beta-O-sulfate 5b, 6a, 14a, 17b,20b-pentahydroxy-1-oxo-20S,22R-witha-2, 2,4-dienolide, 6a,7a-epoxy-5a,14a,17a, 23 b- tetrahydroxy-1-oxo-22R-witha-2, 2,4-dienolide, 7a-hydroxy withanolide, withanolide glycosides, 17a- and 17b-withanolides, Withanone, 27-hydroxy withanolide A Withanolide eWS-2 (aliphatic ester), withanolide eWS-1 (aliphatic ketone)

FIGURE 8.1 Structure of main withanolides present in Withania somnifera.

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W. somnifera at 2000 mg/Kg body weight for acute and subacute oral toxicities in Wistar rats was found to be safe. For acute toxicity, the extract was administered at 2000 mg/Kg and observed for 14 days, and at 500, 1000, and 2000 mg/Kg and monitored for 28 days for subacute toxicity. No discernible changes were found in the body and organ weight and hematological parameters. In addition, W. somnifera was safe for developing rat fetuses and pregnant mothers. Acute toxicity studies involving Swiss albino mice revealed that a single intraperitoneal injection of 1100 mg/Kg did not exhibit any mortality within 24 h, but small increases above this dose led to deaths with an LD50 of 1260 mg/Kg of body weight (Dar et al., 2015; Sharada et al., 1993).

Pharmacokinetic Profile of Withania somnifera Upon oral administration of standardized W. somnifera aqueous extract in mice using multiple reaction monitoring, two major constituents, withaferin A and withanolide A, have been observed for pharmacokinetic properties. After a single dose of 1000 mg/Kg extract (equivalent to 0.4585 mg/Kg of withaferin A and 0.4785 mg/Kg of withanolide A), both of these withanolides exhibited a comparable pharmacokinetic profile. Mean plasma concentration (Cmax) of 16.69  4.02 ng/mL and 26.59  4.47 ng/mL was for withaferin A and withanolide A, with Tmax (time taken to reach Cmax) of 10 and 20 min, respectively, pointing to their rapid assimilation. There was T1/2 of 59.92  15.90 min and 45.22  9.95 min and clearance of 274.10  9.10 and 191.10  16.74 mL/min/Kg for withaferin A and withanolide A, respectively. On the whole, relative oral bioavailability of withaferin A was 1.44 times that of withanolide A. Another study demonstrated that in 7e8-week-old female Balb/c mice, a single dose of 4 mg/Kg withaferin A attains peak plasma concentration of up to 2 mM with a half-life of 1.36 h, with somewhat faster elimination of 0.151 ng/mL/min. In one more study involving six healthy buffalo calves, it has been established that after a single oral dose of 500 mg/Kg aqueous extract of W. somnifera, mean plasma concentration (248.16  16.12 mg/mL) hit the peak at 0.75 h. Additionally, mean plasma level of 6.55  0.12 mg/mL remained unchanged up to 3 h and mean therapeutic concentration (0.1 mg/mL) from 10 min to 3 h. Average half-life (t1/2) was observed to be 0.92  0.032 h, and total body clearance ranges from 2.26 to 3.09 L/Kg/h with an average of 2.78  0.12 L/Kg/h (Dar et al., 2015).

Neuroprotective Effects of Withania somnifera Immense precedent exists in the literature about the neuroprotective effects of W. somnifera. In glial cells as well as neuronal cells, leaf extract of W. somnifera and its component, withanone, mitigated scopolamine-induced toxicity. There was attenuation of neurofilament-H (NF-H), microtubule associated protein-2 (MAP-2), postsynaptic density protein-95 (PSD-95), and growth associated protein-43 (GAP-43) in neuronal cells and glial fibrillary acidic protein (GFAP) in glial cells, respectively. We have recently shown that withanone protected NMDA-induced neuro2a cells by decreasing intracellular Ca2þ, reactive oxygen species (ROS), mitochondrial membrane

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potential, and attenuation of MDA and Poly(ADP-Ribose) polymerase-1 (Parp-1) levels (Dar et al., 2016). W. somnifera was found to attenuate DNA damage and oxidative stress (Dar et al., 2016). Additionally, W. somnifera extract rescued glial cells from leadinduced toxicity by thwarting expression of GFAP and heat shock protein (HSP70), mortalin, and neural cell adhesion molecule. Glycowithanolides from W. somnifera exhibited significant antioxidant activity in the cortex and striatum of rat brain (Bhattacharya and Satyan, 1997). W. somnifera root powder salvaged the dying neurons in CA2 and CA3 subareas of the hippocampus of rats subjected to immobilization stress and prevented streptozotocin-induced oxidative stress in mice. Significant neurite outgrowth-promoting properties were exhibited by W. somnifera root extract or its constituents in human neuroblastoma cell lines. Withanolide A promoted both axonal and dendritic transformation and synaptic restoration in cortical neurons of rat treated with amyloid beta peptide (Ab). W. somnifera extract also reduced kainic acid toxicity by attenuating oxidative damage (Parihar and Hemnani, 2003). W. somnifera leaf extract abrogated glutamate-induced excitotoxicity in retinoic acid-differentiated C6 (glioma cell line from rat) and IMR-32 (human neuroblastoma cell line) cells.

Anti-Parkinson Effects of Withania somnifera Many studies have documented anti-Parkinson’s disease effects for W. somnifera. W. somnifera extracts have been demonstrated to protect Parkinson’s diseaseeinduced functional deficits and pathology induced by 6-hydroxydopamine (6-OHDA) in rat models. W. somnifera replenished the striatal dopamine and its metabolites levels by antioxidant mechanisms (Ahmad et al., 2005). W. somnifera root extract salvaged antioxidant balance, attenuated oxidant stress, stabilized catecholamine content, and improved functional deficits in midbrain of mice treated with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine. In addition, in rotenone-induced Parkinsonism in Drosophila melanogaster, standardized extract of W. somnifera clearly resolved oxidative stress, mitochondrial respiratory chain enzymes, and impaired cholinergic function, and it restored the levels of dopamine. Additionally, there was improvement in locomotor function and lethality (Manjunath and Muralidhara, 2015). Further on, W. somnifera root powder, owing to its antioxidant and antiinflammatory properties and ability to restore mitochondrial functions, attenuated rotenone-induced toxicity in cerebellum and striatum of mouse brain by restoring striatal dopamine levels (Manjunath and Muralidhara, 2013). Furthermore, in mouse model of Parkinson’s disease, Maneb and Paraquateinduced toxicities were attenuated by ethanolic root extract of W. somnifera. There was protection in tyrosine hydroxylase expression, normalization of nigral dopamine content, and improvement in locomotor through antiinflammatory, antiapoptotic, and antioxidant effects. W. somnifera masked the expression of inducible nitric oxide (NO) synthase, downregulated pro-apoptotic Bax, and upregulated antiapoptotic Bcl-2 protein expression that preceded decrease in the expression levels of GFAP (Prakash et al., 2014).

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Anti-Alzheimer Effects of Withania somnifera There is great hope and enthusiasm about the use of W. somnifera for drug development against Alzheimer’s disease. In healthy human subjects, standardized aqueous extract of W. somnifera led to better cognitive and psychomotor performance (Pingali et al., 2014). In a study involving transgenic mice overexpressing Ab, W. somnifera root extract corrected behavioral and pathological deficits besides Ab clearance by activating lipoprotein receptorerelated protein in liver (Sehgal et al., 2012). Molecular docking studies have shown that withanamides A and C uniquely bind to the active motif of Ab(25e35) that is suggestive of capability of withanamides to avoid the fibril formation and therefore protect cells from Ab toxicity (Jayaprakasam et al., 2010). Additionally, simulation studies have envisaged withanolide A-inhibiting human acetyl cholinesterase. Withanoside IV and its active metabolite “sominone” improved memory deficits in mice subjected to Ab(25e35) injections and salvaged degeneration of axons, dendrites, and synapses (Kuboyama et al., 2006). Moreover, W. somnifera attenuated acetylcholine esterase activity and cognitive decline induced by subchronic exposure to propoxur (carbamate insecticide) to rats (Yadav et al., 2010). Additionally, W. somnifera affords beneficial effects in streptozotocin-induced cognitive impairment by abrogating oxidative stress (Ahmed et al., 2013). In Abetreated SK-N-MC cells, the extract restored cell morphology and viability via the activation of peroxisome proliferatoreactivated receptor-g (Kurapati et al., 2013). W. somnifera salvaged SK-N-MC and differentiated PC12 cells from Ab(1e42), and hydrogen peroxide mediated toxicity by inhibiting acetyl cholinesterase activity.

Antiischemic and Antihypoxic Effects of Withania somnifera W. somnifera proved protective against transient middle cerebral artery occlusione induced injury in rats by reducing oxidative stress and lesion volume and restoring functional outcome (Chaudhary et al., 2003). Additionally, W. somnifera salvaged the infarct size in mice subjected to permanent distal middle cerebral artery occlusion by preventing expression of heme oxygenase-1 and blocking upregulation of the proapoptotic protein Parp-1 through apoptosis-inducing factor pathway. Also, W. somnifera reduced the semaphoring 3Aedependent inhibitory signals and thus stimulated repair mechanisms in dying neurons (Raghavan and Shah, 2014, 2015). W. somnifera root extract and withanolide A protected against hypobaric hypoxiae induced memory loss in rats and hippocampal neurodegeneration in vitro by provoking glutathione biosynthesis pathway and reduced glutathione (GSH) content in isolated hippocampal cells. These effects were through the Nrf-2 pathway and NO in a corticosterone-dependent manner (Baitharu et al., 2013, 2014).

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Cardioprotective Effects of Withania somnifera W. somnifera possesses hematopoietic, cardioprotective, and cardiotropic properties in humans and animals models. An Ayurvedic preparation having W. somnifera as one of the components demonstrated cardioprotection in animal models (Mohan et al., 2006; Thirunavukkarasu et al., 2006) via nuclear factor erythroid 2-related transcription factor (Nrf)-2 and by activating phase II detoxification enzymes and abrogating apoptosis (Reuland et al., 2013). W. somnifera distinctly normalized the oxidant status, attenuated apoptosis as measured by terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and prevented histopathologic changes of myocardium in rat model of coronary artery occlusion (Mohanty et al., 2008). Moreover, standardized extract of W. somnifera prevented doxorubicin-induced cardiotoxicity by attenuating biochemical changes of myocardium (Hamza et al., 2008).

Anticancer Effects of Withania somnifera W. somnifera or its chemical constituents have been used for prevention and treatment of various types of cancers in vivo or in vitro. Consistent with this, predictive analyses have pointed to withaferin A and withanone for cancer drug development (Vaishnavi et al., 2012). W. somnifera elicits its anticancer effects by several different pathways, including nuclear factor (NFk-b) and signal transducer and activator of transcription 3 (STAT3) signaling, PI3K (phosphoinositide 3-kinase)/AKT (a serine-threonine protein kinase) and mitogen-activated protein kinase (MAPK) signaling, angiogenesis inhibition, induction of oxidative stress, p53 signaling, granulocyteemacrophage colonystimulating factor signaling, death receptor signaling, apoptosis signaling, and by DNA damage regulation pathway (Widodo et al., 2008). Withaferin A killed melanoma cells by provoking ROS-mediated apoptosis. This process engaged mitochondrial pathway and entailed downregulation of Bcl-2, translocation of Bax to the mitochondrial membrane, release of cytochrome c into the cytosol, abolition of transmembrane potential, and concomitant activation of caspase 9 and 3, resulting in the downregulation of Parp-1 and resultant DNA fragmentation (Mayola et al., 2011). Withaferin A also stimulated the expression of tumor necrosis factor receptor (TNFR)-1 and annihilated Bid expression. Additionally, withaferin A hampered the binding of NFk-b to DNA and initiated nuclear cleavage of p65/Rel by activated caspase 3. Based on the foregoing studies, it was concluded that anticancer effects elicited by withaferin A are irrespective of the involvement of mitochondrial machinery (Malik et al., 2007). In human lymphoma U937 cells, withaferin A and radiation instigate apoptosis by increased generation of ROS, downregulation of Bcl-2, cleavage of Parp-1, activation of caspase 3, and stimulation of MAPK signaling (Yang et al., 2011a). Moreover, withaferin A exacerbated radiation-

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induced apoptosis in human renal cancer cells by increased production of ROS, by downregulation of Bcl-2 and dephosphorylation of AKT (Yang et al., 2011b), and by initiating endoplasmic reticulum (ER) stress (Choi et al., 2011). However, withaferin Aeinduced cytotoxicity is cell line specific and dependent on MAPK pathway (Hahm et al., 2014). In human breast cancer cells, withaferin A treatment prevented the formation of mammosphere by stimulating apoptosis and mitigation of complex III activity. Additionally, withaferin A activates Notch2 and Notch4, which inhibit migration in breast cancer cells (Lee et al., 2012). Moreover, withaferin A causes stoppage of phases G2 and M of cell cycle in human breast cancer cells (Stan et al., 2008). Also, withaferin A treatment blocked breast tumor development in xenograft and transgenic mouse models through activation of extracellular regulated kinases (ERK)/ribosomal s6 kinase (RSK) axis and upregulation of death receptor 5; additionally, these effects involved autophagy. However, a contradictory report predicted no role for autophagy in withaferin Aemediated killing of human breast cancer cells (Hahm and Singh, 2013). Additionally, withaferin A treatment checked the growth of experimental mammary tumors via inhibition of vimentin expression (Lee et al., 2015) by interfering with b-tubulin of cytoskeletal architecture (Antony et al., 2014). Similarly in a transgenic mouse, development and progression of mammary gland carcinoma was inhibited markedly by withaferin A treatment through decrease in tumor size area (Hahm et al., 2011, 2013; Kim and Singh, 2014). In human laryngeal carcinoma cells, W. somnifera root extract resulted in cytotoxicity by impeding cell cycle and angiogenesis (Mathur et al., 2006). Withaferin A hampers cell proliferation in human umbilical vein (HUVEC) endothelial cells by blocking expression of cyclin D1 and correcting the defects in proteasome (Mohan et al., 2004). Additionally, it restricts the growth of mesothelioma cells obtained from patients by apoptotic and proteasome inhibitory mechanisms (Yang et al., 2012). Withaferin A provoked apoptosis, ER stress, and PARP cleavage via inhibition of STAT3 pathway in a kidney cancer cell line (Choi et al., 2011; Um et al., 2012).

Antiinflammatory Effects of Withania somnifera Consistent with the indigenous medicinal uses of W. somnifera having a role in mitigating inflammation, W. somnifera has shown pronounced antiinflammation effects in various animal models. In trinitro benzyl sulfonic acid (TNBS)einduced inflammatory bowel disease, W. somnifera root extract showed muco-restorative capability by resolving inflammatory markers, including necrosis, edema, and neutrophil infiltration. In a murine model of nephritis, Withania root powder resolved the inflammation, and in a mouse model of lupus, it resolved inflammation by mitigating cytokines, tumor necrosis factor (TNF)-a, NO, and ROS. Withaferin A, one of the active constituents of W. somnifera, has shown to inhibit phorbol-12-myristate-13-acetate (PMA)einduced shedding of endothelial cell protein C receptor by inhibiting TNF-a and interleukin (IL)1b in human endothelial cells obtained from HUVEC. Additionally, it attenuated

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PMA-stimulated phosphorylation of p38, ERK 1/2, and c-Jun N-terminal kinase (JNK). Further in a mouse model, withaferin A attenuated cecal ligation and puncture-induced EPCR shedding by downregulating the expression and activity of TNF-aeconverting enzyme. Withaferin A protects vascular barrier integrity in HUVECs and in mice via inhibiting hyperpermeability induced by high-mobility group box 1 protein. Further on, withaferin A attenuated expression of cell adhesion molecules, adhesion and migration of leukocytes, production of IL-6 and TNF-a, and activation of NFk-b. Withaferin A blocks NFk-b translocation by preventing Ik-b phosphorylation and degradation in murine fibrosarcoma L929sA cells and human embryonic kidney 293T cells. Withaferin A also blocked TNF-a einduced expression of cell adhesion molecules by inactivation of AKT and NFk-b in human pulmonary epithelial cells. Additionally, withaferin A hampers NFk-b activation by inhibiting TNF-aeinduced expression of cell adhesion molecules by inactivation of AKT and targeting cysteine 179 located in catalytic site of inhibitor of nuclear factor kappa-B (IKKb). In cellular models of cystic fibrosis, withaferin A leads to inhibition of NFk-b and IL-8.

Antimicrobial Effects of Withania somnifera Traditionally, W. somnifera has been used against infections. The effectiveness of antimicrobial activity of W. somnifera varies from organism to organism and is mediated through gene silencing, immunopotentiation, cytotoxicity, etc. In agreement with traditional usage, extracts of W. somnifera have shown promising antibacterial and antifungal activity in laboratory settings. Methanolic leaf extract of W. somnifera has demonstrated strong zone inhibitory effect for gram-positive clinical isolates obtained from pus samples of methicillin-resistant Staphylococcus aureus and Enterococcus spp. (Bisht and Rawat, 2014). In addition, extracts of W. somnifera have shown antibacterial potential against some gram-negative bacteria such as Escherichia coli, Salmonella typhi, Proteus mirabilis, Citrobacter freundii, Pseudomonas aeruginosa, and Klebsiella pneumonia (Alam et al., 2012; Singh and Kumar, 2011). W. somnifera exhibited antibacterial activity against oral bacteria Streptococcus mutans and Streptococcus sobrinus sub-minimum inhibitory concentration (MIC) levels by restricting acid production, reducing acid tolerance, and hampering the formation of biofilm. W. somnifera has strong antiinhibitory activity against Salmonella typhimurium in culture. W. somnifera extracts enhanced the antibacterial effect of rifampicin and isoniazid against S. typhimurium and E. coli (Arora et al., 2004). In addition, in a mouse model of salmonellosis, W. somnifera has improved the survival rate and reduced bacterial load. In a mouse model of malaria, W. somnifera inhibited parasite load and attenuated packed cell volume drop effect; maximum activity was observed at 600 mg/lg. W. somnifera possesses anti-Leishmanial activity against freeliving promastigotes and intracellular amastigotes of Leishmania major with a maximum inhibitory effect of almost 50%. Withanolides, in dose as well as time-dependent manner, provoked death in Leishmania donovani in vitro in an apoptosis-like manner. This effect

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of withanolides was the result of DNA damage, cell cycle arrest, externalization of phosphatidylserine, and decrease in mitochondrial potential via the formation of ROS. The processes were probably due to inhibitory effect of W. somnifera on protein kinase C signaling pathway. W. somnifera enhanced the protective effects of cisplatin in L. donovanieinfected mice by augmenting the number of T cells (CD4þ, CD8þ) and natural killer celleassociated marker, while it had little effect on chloroquine-resistant Plasmodium berghei in mice. Flavonoids from W. somnifera have demonstrated marked fungicidal effects against Candida albicans with MIC of 0.039 and minimum fungicidal concentration (MFC) of 0.039 mg/mL, respectively. However, W. somnifera was ineffective against Aspergillus flavus and Aspergillus niger.

Antiarthritic Effects of Withania somnifera A great deal of literature suggests the role of W. somnifera in arthritis and helps collagen stabilization by inhibition of enzyme collagenase (Ganesan et al., 2011). Aqueous extracts of W. somnifera root powder produced cartilage-protective effects through inhibition of the gelatinase activity in vitro and by markedly attenuating NO release (Sumantran et al., 2008). Additionally, W. somnifera markedly reduced levels of proinflammatory cytokines TNF-a, IL-1b, and IL-12 p40 in peripheral blood mononuclear cells from human subjects and mononuclear cells derived from synovial fluid of rheumatoid arthritis patients challenged by lipopolysaccharide (LPS). These effects were due to blockade in translocation of the transcription factors NFk-b and activator protein1 (AP-1) and phosphorylation of Ik-b as evidenced from mouse cell line data. W. somnifera normalized LPS-induced NO release in mouse macrophage (RAW 264.7) cells and reduced cartilage loss in rat model of adjuvant-induced arthritis by restoring motor activity and radiological scores (Rasool and Varalakshmi, 2007). In addition, water extract of W. somnifera root prevented increased arthritic index, auto-antibodies, and C-reactive protein P in collagen-induced arthritis in rats (Khan et al., 2015; Gupta and Singh, 2014). Moreover, an Ayurvedic preparation of BV-9238 containing W. somnifera as one of constituents attenuated TNF-a and NO production without any cytotoxic effects in Freund’s complete adjuvanteinduced arthritis in rats and a mouse macrophage cell line (Dey et al., 2014). However, there are conflicting reports regarding withaferin A. It resulted in collagen degradation and inflammation through activation of microRNA 25 in rabbit articular chondrocytes. Moreover, it resulted in enhanced production of intracellular ROS followed by apoptosis and increased p53 expression through PI3K/AKT and JNK pathways in rabbit articular chondrocytes (Yu and Kim, 2013, 2014).

Antistress Effects of Withania somnifera W. somnifera is a well-known stress reliever being used in Ayurveda. In conformity with this, W. somnifera resulted in better stress endurance in animals as well as humans.

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W. somnifera root extracts normalized serum cortisol levels without causing any major side effects in human subjects. Glycowithanolides showed anxiolytic effects against pentylenetetrazole-induced anxiety in rats, and this effect was as good as that of known antidepressants. Additionally, it normalized rat brain levels of tribulin, an endocoid marker of clinical anxiety, and attenuated oxidative stresseinduced lipid peroxidation in the frontal cortex and striatum of rat after chronic footshock stress; EuMil, a polyherbal formulation, markedly restored cerebral monoamine levels that were enhanced by chronic electroshock stress. Further on, EuMil normalized chronic stresseinduced glucose intolerance and maintained male sexual behavior and frustration. In addition, there was recovery in cognitive dysfunction, immunosuppression, gastric ulceration, and plasma corticosterone levels. Another polyherbal formulation, Perment, exhibited antidepressant and anxiolytic activity in rats, which was partly due to activation of adrenergic and serotonergic systems (Ramanathan et al., 2011). The aqueous fraction of W. somnifera roots alleviated chronic stresseinduced reduction of T-cell population and upregulated Th1 cytokines in mice.

Antidiabetic Effects of Withania somnifera W. somnifera is used against diabetes in Indian systems of medicine. Dianix and Trasina polyherbal formulations prescribed in Ayurveda exhibited reasonable antidiabetic effects in human subjects. Additionally, W. somnifera root powder normalized blood glucose levels over a period of 30 days, and these effects were comparable to that of an oral hypoglycemic drug, Daonil. Aqueous extract of W. somnifera normalizes hyperglycemia in noninsulin-dependent diabetes mellitus in rats by improving insulin sensitivity (Anwer et al., 2008). Consistent with these studies, W. somnifera leaf and root extracts showed antidiabetic activity by normalizing glucose uptake in skeletal myotubes and adipocytes in a dose-dependent manner, the leaf extract being more effective than root extract in this activity (Gorelick et al., 2015). In alloxan-induced diabetes mellitus in rats, root and leaf extracts considerably attenuated levels of urine and blood glucose, glucose6-phosphatase, and tissue glycogen levels through nonenzymatic and enzymatic antioxidant mechanisms (Udayakumar et al., 2010). Interestingly, withaferin A hindered inflammatory response after cytokine-induced injury to islets in vitro and subsequent to transplantation (SoRelle et al., 2013), besides showing marked antiglycating actions (Babu et al., 2007).

Aphrodisiac Effects of Withania somnifera W. somnifera at 675 mg/day in 3 doses for 90 days given to oligospermic patients resulted in better sperm count, enhanced semen volume, and improvement in sperm motility from baseline, besides enhancing testosterone levels and serum leutinizing hormone content (Ambiye et al., 2013). In another study involving infertile men,

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W. somnifera normalized sperm count and motility in addition to attenuating serum levels of testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin (Ahmad et al., 2010). Reduced testosterone levels and low sperm count are indications of hampered spermatogenesis and more appropriately point to problems in functioning of Sertoli and Leydig cells (Sharpe, 1993). However, W. somnifera produced insignificant improvement in the management of psychogenic erectile dysfunction (Mamidi and Thakar, 2011). In laboratory setting, W. somnifera extracts reduced increase in testicular weight in addition to enhancing the diameters of seminiferous tubules and number of somniferous tubular cell layers in the testes of Wistar rats. Serum testosterone and follicle-stimulating hormone levels were lower, whereas interstitial cellestimulating hormone levels were higher in treated animals. Thus, W. somnifera provoked testicular development and spermatogenesis by influencing the somniferous tubules (AbdelMagied et al., 2001). In another study involving sexually sluggish mice, treatment with W. somnifera resulted in sperm production and serum testosterone levels. It also recovered pro-sexual behavior that includes chasing, nosing, and genital sniffing. Additionally, W. somnifera attenuated effects of cadmium toxicity on somniferous tubules and on motility and density of cauda epididymidal sperm (Mishra et al., 2012). In addition, W. somnifera treatment to 25-day-old rats led to significant changes in gonadotropin hormone levels with a parallel enhancement of ovarian weight and folliculogenesis (Al-Qarawi et al., 2000). However, a report has cautioned the use of W. somnifera against sexual incompetence, owing to its sedative effects or hyperprolactinemic or GABAergic or serotonergic activity. In this study, W. somnifera resulted in significant reduction in libido, sexual performance, sexual vigor, and penile erection, and these effects were moderately restored on termination of treatment. These antimasculine effects are not due to changes in testosterone levels or toxicity but may be attributed to hyperprolactinemic, GABAergic, serotonergic, or sedative activities of the extract (Ilayperuma et al., 2002).

Conclusion W. somnifera is a medicinal plant with well-known ethnopharmacological and pharmaceutical properties. It has been used since antiquity in folkloric medicine in India and has tremendous clinical applications in Indian systems of medicine (Fig. 8.2). In animal studies, W. somnifera and its chemical constituents exhibit diverse pharmacological properties such as antiinflammatory, antioxidant, inhibiting NFk-b transcription, activator of MAPK signaling pathways, antiapoptotic, angiogenic, and ER stressereducing effects. The fact that use of W. somnifera as a multipurpose medicinal agent against various clinical conditions and disease models is tremendously encouraging. However, additional scientific justification is warranted for its use in clinic.

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FIGURE 8.2 Withania somnifera exerts multiple pharmacologic actions such as neuroprotection, anticancer cardioprotection, antiinflammatory, antibacterial, antioxidant and antistress effects.

Acknowledgments Dr. Ahmad’s work was partly supported by the Ramalingaswami Fellowship/Re-entry Grant of Department of Biotechnology and financial assistance (MLP6009) as well as logistic support from the Council for Scientific and Industrial Research. Contents do not represent any governmental views.

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Agarwal, R., Diwanay, S., Patki, P., Patwardhan, B., 1999. Studies on immunomodulatory activity of Withania somnifera (Ashwagandha) extracts in experimental immune inflammation. J. Ethnopharmacol. 67, 27e35. Ahmad, M., Saleem, S., Ahmad, A.S., Ansari, M.A., Yousuf, S., Hoda, M.N., Islam, F., 2005. Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum. Exp. Toxicol. 24, 137e147. Ahmad, M.K., Mahdi, A.A., Shukla, K.K., Islam, N., Rajender, S., Madhukar, D., Shankhwar, S.N., Ahmad, S., 2010. Withania somnifera improves semen quality by regulating reproductive hormone levels and oxidative stress in seminal plasma of infertile males. Fertil. Steril. 94, 989e996. Ahmed, M.E., Javed, H., Khan, M.M., Vaibhav, K., Ahmad, A., Khan, A., Tabassum, R., Islam, F., Safhi, M. M., 2013. Attenuation of oxidative damage-associated cognitive decline by Withania somnifera in rat model of streptozotocin-induced cognitive impairment. Protoplasma 250, 1067e1078. Al-Qarawi, A., Abdel-Rahman, H., El-Badry, A., Harraz, F., Razig, N., Abdel-Magied, E., 2000. The effect of extracts of Cynomorium coccineum and Withania somnifera on gonadotrophins and ovarian follicles of immature Wistar rats. Phytother. Res. 14, 288e290. Alam, N., Hossain, M., Mottalib, M.A., Sulaiman, S.A., Gan, S.H., Khalil, M.I., 2012. Methanolic extracts of Withania somnifera leaves, fruits and roots possess antioxidant properties and antibacterial activities. BMC Complement. Altern. Med. 12, 175. Ali, M., Shuaib, M., Ansari, S.H., 1997. Withanolides from the stem bark of Withania somnifera. Phytochemistry 44, 1163e1168. Ambiye, V.R., Langade, D., Dongre, S., Aptikar, P., Kulkarni, M., Dongre, A., 2013. Clinical evaluation of the spermatogenic activity of the root extract of ashwagandha (Withania somnifera) in oligospermic males: a pilot study. Evidence-Based Complement. Altern. Med. 2013. Antony, M.L., Lee, J., Hahm, E.R., Kim, S.H., Marcus, A.I., Kumari, V., Ji, X., Yang, Z., Vowell, C.L., Wipf, P., Uechi, G.T., Yates, N.A., Romero, G., Sarkar, S.N., Singh, S.V., 2014. Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of beta-tubulin. J. Biol. Chem. 289, 1852e1865. Anwer, T., Sharma, M., Pillai, K.K., Iqbal, M., 2008. Effect of Withania somnifera on insulin sensitivity in non-insulin-dependent diabetes mellitus rats. Basic Clin. Pharmacol. Toxicol. 102, 498e503. Arora, S., Dhillon, S., Rani, G., Nagpal, A., 2004. The in vitro antibacterial/synergistic activities of Withania somnifera extracts. Fitoterapia 75, 385e388. Babu, P.V., Gokulakrishnan, A., Dhandayuthabani, R., Ameethkhan, D., Kumar, C.V., Ahamed, M.I., 2007. Protective effect of Withania somnifera (Solanaceae) on collagen glycation and cross-linking. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 147, 308e313. Baitharu, I., Jain, V., Deep, S.N., Hota, K.B., Hota, S.K., Prasad, D., Ilavazhagan, G., 2013. Withania somnifera root extract ameliorates hypobaric hypoxia induced memory impairment in rats. J. Ethnopharmacol. 145, 431e441. Baitharu, I., Jain, V., Deep, S.N., Shroff, S., Sahu, J.K., Naik, P.K., Ilavazhagan, G., 2014. Withanolide A prevents neurodegeneration by modulating hippocampal glutathione biosynthesis during hypoxia. PLoS One 9, e105311. Basu, K.A., 1935. In: Withania somnifera, Indian Medicinal Plants, second ed., vol. III. Lalit Mohan Basu, Allahabad, pp. 1774e1776. Bhandari, C., 1970. Ashwagandha (Withania somnifera) “Vanaushadhi Chandroday”. (An Encyclopedia of Indian Herbs), vol. 1. CS Series of Varanasi Vidyavilas Press, Varanasi, India, pp. 96e97. Bhattacharya, S.K., Satyan, K.S., 1997. Experimental methods for evaluation of psychotropic agents in rodents: IeAnti-anxiety agents. Indian J. Exp. Biol. 35, 565e575.

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Kim, S.H., Singh, S.V., 2014. Mammary cancer chemoprevention by withaferin A is accompanied by in vivo suppression of self-renewal of cancer stem cells. Cancer Prev. Res. (Phila.) 7, 738e747. Kuboyama, T., Tohda, C., Komatsu, K., 2006. Withanoside IV and its active metabolite, sominone, attenuate Abeta(25-35)-induced neurodegeneration. Eur. J. Neurosci. 23, 1417e1426. Kurapati, K.R., Atluri, V.S., Samikkannu, T., Nair, M.P., 2013. Ashwagandha (Withania somnifera) reverses beta-amyloid1-42 induced toxicity in human neuronal cells: implications in HIV-associated neurocognitive disorders (HAND). PLoS One 8, e77624. Lee, J., Sehrawat, A., Singh, S.V., 2012. Withaferin A causes activation of Notch2 and Notch4 in human breast cancer cells. Breast Cancer Res. Treat. 136, 45e56. Lee, J.H., Kim, J.E., Jang, Y.J., Lee, C.C., Lim, T.G., Jung, S.K., Lee, E., Lim, S.S., Heo, Y.S., Seo, S.G., Son, J. E., Kim, J.R., Lee, C.Y., Lee, H.J., Lee, K.W., 2015. Dehydroglyasperin C suppresses TPA-induced cell transformation through direct inhibition of MKK4 and PI3K. Mol. Carcinog. Machiah, D.K., Girish, K., Gowda, T.V., 2006. A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms. Compar. Biochem. Physiol. Part C Toxicol. Pharmacol. 143, 158e161. Machiah, D.K., Gowda, T.V., 2006. Purification of a post-synaptic neurotoxic phospholipase A 2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera. Biochimie 88, 701e710. Malik, F., Kumar, A., Bhushan, S., Khan, S., Bhatia, A., Suri, K.A., Qazi, G.N., Singh, J., 2007. Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine. Apoptosis 12, 2115e2133. Mamidi, P., Thakar, A., 2011. Efficacy of Ashwagandha (Withania somnifera Dunal. Linn.) in the management of psychogenic erectile dysfunction. AYU 32, 322. Manjunath, M.J., Muralidhara, 2013. Effect of Withania somnifera supplementation on rotenoneinduced oxidative damage in cerebellum and striatum of the male mice brain. Cent. Nerv. Syst. Agents Med. Chem. 13, 43e56. Manjunath, M.J., Muralidhara, 2015. Standardized extract of Withania somnifera (Ashwagandha) markedly offsets rotenone-induced locomotor deficits, oxidative impairments and neurotoxicity in Drosophila melanogaster. J. Food Sci. Technol. 52, 1971e1981. Mathur, R., Gupta, S.K., Singh, N., Mathur, S., Kochupillai, V., Velpandian, T., 2006. Evaluation of the effect of Withania somnifera root extracts on cell cycle and angiogenesis. J. Ethnopharmacol. 105, 336e341. Mayola, E., Gallerne, C., Esposti, D.D., Martel, C., Pervaiz, S., Larue, L., Debuire, B., Lemoine, A., Brenner, C., Lemaire, C., 2011. Withaferin A induces apoptosis in human melanoma cells through generation of reactive oxygen species and down-regulation of Bcl-2. Apoptosis 16, 1014e1027. Mirjalili, M.H., Moyano, E., Bonfill, M., Cusido, R.M., Palazon, J., 2009. Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 14, 2373e2393. Mishra, R.K., Verma, H.P., Singh, N., Singh, S.K., 2012. Male infertility: lifestyle and oriental remedies. J. Sci. Res. 56, 93e101. Misra, B., 2004. Ashwagandha e Bhavprakash Nigantu (Indian Materia Medica). Chaukhambha Bharti Academy, Varanasi, pp. 393e394. Mohan, I.K., Kumar, K.V., Naidu, M.U., Khan, M., Sundaram, C., 2006. Protective effect of CardiPro against doxorubicin-induced cardiotoxicity in mice. Phytomedicine 13, 222e229. Mohan, R., Hammers, H.J., Bargagna-Mohan, P., Zhan, X.H., Herbstritt, C.J., Ruiz, A., Zhang, L., Hanson, A.D., Conner, B.P., Rougas, J., Pribluda, V.S., 2004. Withaferin A is a potent inhibitor of angiogenesis. Angiogenesis 7, 115e122.

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Mohanty, I.R., Arya, D.S., Gupta, S.K., 2008. Withania somnifera provides cardioprotection and attenuates ischemia-reperfusion induced apoptosis. Clin. Nutr. 27, 635e642. Parihar, M.S., Hemnani, T., 2003. Phenolic antioxidants attenuate hippocampal neuronal cell damage against kainic acid induced excitotoxicity. J. Biosci. 28, 121e128. Pingali, U., Pilli, R., Fatima, N., 2014. Effect of standardized aqueous extract of Withania somnifera on tests of cognitive and psychomotor performance in healthy human participants. Pharmacognosy Res. 6, 12e18. Prakash, J., Chouhan, S., Yadav, S.K., Westfall, S., Rai, S.N., Singh, S.P., 2014. Withania somnifera alleviates parkinsonian phenotypes by inhibiting apoptotic pathways in dopaminergic neurons. Neurochem. Res. 39, 2527e2536. Raghavan, A., Shah, Z.A., 2014. Withania somnifera improves ischemic stroke outcomes by attenuating PARP1-AIF-mediated caspase-independent apoptosis. Mol. Neurobiol. Raghavan, A., Shah, Z.A., 2015. Withania somnifera: a pre-clinical study on neuroregenerative therapy for stroke. Neural Regen. Res. 10, 183e185. Ramanathan, M., Balaji, B., Justin, A., 2011. Behavioural and neurochemical evaluation of perment an herbal formulation in chronic unpredictable mild stress induced depressive model. Indian J. Exp. Biol. 49, 269e275. Rasool, M., Varalakshmi, P., 2007. Protective effect of Withania somnifera root powder in relation to lipid peroxidation, antioxidant status, glycoproteins and bone collagen on adjuvant-induced arthritis in rats. Fundam. Clin. Pharmacol. 21, 157e164. Reuland, D.J., Khademi, S., Castle, C.J., Irwin, D.C., McCord, J.M., Miller, B.F., Hamilton, K.L., 2013. Upregulation of phase II enzymes through phytochemical activation of Nrf2 protects cardiomyocytes against oxidant stress. Free Radic. Biol. Med. 56, 102e111. Sehgal, N., Gupta, A., Valli, R.K., Joshi, S.D., Mills, J.T., Hamel, E., Khanna, P., Jain, S.C., Thakur, S.S., Ravindranath, V., 2012. Withania somnifera reverses Alzheimer’s disease pathology by enhancing low-density lipoprotein receptor-related protein in liver. Proc. Natl. Acad. Sci. USA 109, 3510e3515. Sharada, A., Solomon, F.E., Devi, P.U., 1993. Toxicity of Withania somnifera root extract in rats and mice. Pharm. Biol. 31, 205e212. Sharma, P.V., 1999. Ashwagandha, Dravyaguna Vijana, Chaukhambha Viashwabharti. Varanasi, pp. 763e765. Sharma, S., Dahanukar, S.A., Karandikar, S.M., 1985. Effects of long-term administration of the roots of ashwagandha and shatavari in rats. Indian Drugs 29, 133e139. Sharpe, R., 1993. Declining sperm counts in meneis there an endocrine cause? J. Endocrinol. 136, 357e360. Singh, G., Kumar, P., 2011. Evaluation of antimicrobial efficacy of flavonoids of Withania somnifera L. Indian J. Pharm. Sci. 73, 473. Singh, N., Bhalla, M., de Jager, P., Gilca, M., 2011. An overview on ashwagandha: a Rasayana (rejuvenator) of Ayurveda. Afr. J. Tradit. Complement. Altern. Med. 8. SoRelle, J.A., Itoh, T., Peng, H., Kanak, M.A., Sugimoto, K., Matsumoto, S., Levy, M.F., Lawrence, M.C., Naziruddin, B., 2013. Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation. Diabetologia 56, 814e824. Stan, S.D., Zeng, Y., Singh, S.V., 2008. Ayurvedic medicine constituent withaferin a causes G2 and M phase cell cycle arrest in human breast cancer cells. Nutr. Cancer. 60 (Suppl. 1), 51e60. Sumantran, V.N., Chandwaskar, R., Joshi, A.K., Boddul, S., Patwardhan, B., Chopra, A., Wagh, U.V., 2008. The relationship between chondroprotective and antiinflammatory effects of Withania somnifera root and glucosamine sulphate on human osteoarthritic cartilage in vitro. Phytother. Res. 22, 1342e1348.

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Thirunavukkarasu, M., Penumathsa, S., Juhasz, B., Zhan, L., Bagchi, M., Yasmin, T., Shara, M.A., Thatte, H.S., Bagchi, D., Maulik, N., 2006. Enhanced cardiovascular function and energy level by a novel chromium (III)-supplement. Biofactors 27, 53e67. Tiwari, R., Chakraborty, S., Saminathan, M., Dhama, K., Singh, S.V., 2014. Ashwagandha (Withania somnifera): role in safeguarding health, immunomodulatory effects, combating infections and therapeutic applications: a review. J. Biol. Sci. 77e94. Udayakumar, R., Kasthurirengan, S., Vasudevan, A., Mariashibu, T.S., Rayan, J.J., Choi, C.W., Ganapathi, A., Kim, S.C., 2010. Antioxidant effect of dietary supplement Withania somnifera L. reduce blood glucose levels in alloxan-induced diabetic rats. Plant Foods Hum. Nutr. 65, 91e98. Uddin, Q., Samiulla, L., Singh, V., Jamil, S., 2012. Phytochemical and pharmacological profile of Withania somnifera Dunal: a review. J. Appl. Pharm. Sci. 2 (1), 170e175. Um, H.J., Min, K.J., Kim, D.E., Kwon, T.K., 2012. Withaferin A inhibits JAK/STAT3 signaling and induces apoptosis of human renal carcinoma Caki cells. Biochem. Biophys. Res. Commun. 427, 24e29. Vaishnavi, K., Saxena, N., Shah, N., Singh, R., Manjunath, K., Uthayakumar, M., Kanaujia, S.P., Kaul, S.C., Sekar, K., Wadhwa, R., 2012. Differential activities of the two closely related withanolides, Withaferin A and Withanone: bioinformatics and experimental evidences. PLoS One 7, e44419. Widodo, N., Takagi, Y., Shrestha, B.G., Ishii, T., Kaul, S.C., Wadhwa, R., 2008. Selective killing of cancer cells by leaf extract of Ashwagandha: components, activity and pathway analyses. Cancer Lett. 262, 37e47. Yadav, C.S., Kumar, V., Suke, S.G., Ahmed, R.S., Mediratta, P.K., Banerjee, B.D., 2010. Propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function: attenuation by Withania somnifera. Indian J. Biochem. Biophys. 47, 117e120. Yang, E.S., Choi, M.J., Kim, J.H., Choi, K.S., Kwon, T.K., 2011a. Combination of withaferin A and X-ray irradiation enhances apoptosis in U937 cells. Toxicol. In Vitro 25, 1803e1810. Yang, E.S., Choi, M.J., Kim, J.H., Choi, K.S., Kwon, T.K., 2011b. Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition. Chem. Biol. Interact. 190, 9e15. Yang, H., Wang, Y., Cheryan, V.T., Wu, W., Cui, C.Q., Polin, L.A., Pass, H.I., Dou, Q.P., Rishi, A.K., Wali, A., 2012. Withaferin A inhibits the proteasome activity in mesothelioma in vitro and in vivo. PLoS One 7, e41214. Yu, S.M., Kim, S.J., 2013. Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt, p38, and JNK pathways in rabbit articular chondrocytes. Exp. Cell Res. 319, 2822e2834. Yu, S.M., Kim, S.J., 2014. Withaferin A-caused production of intracellular reactive oxygen species modulates apoptosis via PI3K/Akt and JNKinase in rabbit articular chondrocytes. J. Korean Med. Sci. 29, 1042e1053.

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An Overview on Tribulus terrestris in Sports Nutrition and Energy Regulation  ski2, Andrzej Pokrywka1, 2, Barbara Morawin1, Jarosław Krzywan  -Lacny1 Agnieszka Zembron 1

UN IVERSITY OF ZIELONA GORA, ZIELONA GORA, POLAND; 2 NATIONAL CE NTRE FOR SPORT S MEDICINE, WARSAW, POLAND

Introduction Tribulus terrestris L. is an annual plant of the family Zygophyllaceae, which has been used for ages in traditional medicine in Greece, China, and India to energize, vitalize, and improve sexual function and physical performance in humans. The plant is a wellpatronized medicinal herb by Ayurvedic seers as well as by modern herbalists. It is used individually as a single therapeutic agent or as a prime or subordinate component of many compound formulations and food supplements. It is an annual shrub found in Mediterranean, subtropical, and desert climate regions around the world, including India, China, southern US, Mexico, Spain, and Bulgaria (Chhatre et al., 2014). Many different compounds with a variety of biological properties and chemical structures have been identified from T. terrestris, including steroidal saponins, phytosterols, tannins, terpenoids, amide derivatives, amino acids, and proteins. However, it is the steroidal molecular structure of the steroidal saponins, including protodioscin and protogracillin, that is thought to confer to T. terrestris unique biological activities (Neychev and Mitev, 2016). Nevertheless, the saponin content of this plant from different geographical regions is different (Kostova and Dinchev, 2005). The whole plant of T. terrestris has been explored exhaustively for its phytochemical and pharmacological activities, such as diuretic, aphrodisiac, antiurolithic, immunomodulatory, antihypertensive, antihyperlipidemic, antidiabetic, hepatoprotective, anticancer, anthelmintic, antibacterial, analgesic, and antiinflammatory (Chhatre et al., 2014; Hussain et al., 2009; Kim et al., 2011; Samani et al., 2016).

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Aphrodisiac Activity Analysis of phytochemical and pharmacological studies in humans and animals revealed an important role for T. terrestris in treating erectile dysfunction and sexual desire problems. Evaluation of hormonal effects of T. terrestris in primates, rabbit, and rat indicated that Tribulus may be useful in mild to moderate cases in the management of erectile dysfunction. It was possibly due to the presence of protodioscin in the extract (Gauthaman and Ganesan, 2008). Increased testosterone levels, along with increased dehydrotestosterone and dehydroepiandrosterone (DHEA) (due to conversion of protodioscin to DHEA), are suggestive of aphrodisiac activity (Gama et al., 2014). However, empirical evidence to support the hypothesis that these desirable effects are due to androgen-enhancing properties of Tribulus is inconclusive. While the mechanisms underlying T. terrestris aphrodisiac activity remain largely unknown, there is emerging compelling evidence from experimental studies in animals for possible endothelium and nitric oxideedependent mechanisms underlying T. terrestris aphrodisiac and pro-erectile activities (Neychev and Mitev, 2016). The therapeutic use of T. terrestris (112.5 mg of protodioscin per day for 84 days) was shown to have improved sperm quality in men with altered semen parameters and/or who are undergoing infertility treatment (Salgado et al., 2016). This plant is also likely to have a therapeutic effect on female reproduction at the molecular level (Abadjieva and Kistanova, 2016). The intake of 250 mg T. terrestris extract (one tablet thrice daily for 90 days) proved also safe and effective in the treatment of female sexual dysfunction (Gama et al., 2014). However, it should be kept in mind that the possibility of serious thrombotic events associated with therapy of T. terrestris was suggested (Liguori et al., 2015). Patients affected by premature ejaculation may also significantly benefit from oral therapy with T. terrestris with a combination of tryptophan, Satureja montana, and Phyllanthus emblica extracts (Sansalone et al., 2016). However, T. terrestris was ineffective in the treatment of idiopathic infertility; no statistically significant correlations were observed between semen parameters before and after a 3-month treatment (750 mg in three divided doses daily) (Roaiah et al., 2016). A limited number of animal studies displayed a significant increase in serum testosterone levels after T. terrestris administration, but this effect was only noted in humans when Tribulus was part of a combined supplement administration. However, most of the published data suggest that T. terrestris is ineffective in increasing testosterone levels in humans; thus marketing claims are unsubstantiated. Maybe the nitric oxide release effect of T. terrestris may offer a plausible explanation for the observed physiological responses to Tribulus supplementation, independent of the testosterone level (Qureshi et al., 2014). Nonetheless, T. terrestris is still widely used by athletes based on the belief, additionally fueled by claims in marketing information, that it can enhance testosterone or

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insulin-like growth factor 1 (IGF-1) concentrations, as well as being a legal and allowed alternative for anabolic steroids prohibited in sport by the World Anti-Doping Agency (WADA).

Supplementation in Sport Numerous articles on dietary supplementation in professional sport indicate that the use of supplements is highly prevalent among athletes (Fra˛czek et al., 2012; Mottram, 2015; Maughan et al., 2007; Tscholl et al., 2010). Athletes take supplements for many reasons, including potential performance benefits, prevention or treatment of a nutrient deficiency, convenience, or fear of “missing out” by not taking a particular supplement (Beck et al., 2015). Moreover, some athletes take several supplements simultaneously, even up to 26 products per day, although it seems redundant, particularly when their diet is well balanced (Corrigan and Kazlauskas, 2003; Shaw et al., 2016; Suzic Lazic et al., 2011; Tscholl et al., 2008). In addition, many studies do not confirm the ergogenic or anabolic effects of numerous products that are advertised by their manufacturers as performance enhancing. This also refers to supplements containing T. terrestris, which is a plant extract that has been suggested to stimulate leutinizing hormone, which stimulates the natural production of testosterone. Consequently, Tribulus has been marketed as a supplement that can increase testosterone and promote greater gains in strength and muscle mass during training (Kreider et al., 2010) as well as increasing red blood cell levels, thereby contributing to improvement in blood circulation and good oxygen transport (Arsyad, 1996). On the contrary, it may even increase the aromatization of testosterone to estradiol, which may be catastrophic for bodybuilders (Yavuz and ¨ zkum, 2014). There have also been reports on T. terrestris tonic activities, which result O from intensified protein synthesis and enhancement of the activity of enzymes associated with energy metabolism, an increase in iron absorption from small intestines, and inhibited lipid peroxidation during stress, which finally leads to more muscular strength and stamina (Joshi and Uniyal, 2008). However, several studies have indicated that Tribulus supplementation appears to have no effects on body composition, strength during training, or exercise performance (Antonio et al., 2000; Brown et al., 2001; Rogerson et al., 2007). In addition, a short-term treatment with T. terrestris showed no impact on the endogenous testosterone metabolism (Saudan et al., 2008) or on the urinary steroid profile (Van Eenoo et al., 2000), and 5 weeks of Tribulus supplementation did not cause any change in the testosterone-to-epitestosterone ratio (Rogerson et al., 2007). Likewise, after 3 months of daily use of T. terrestris (in three equally divided doses) by 30 randomized male patients, there was no change in serum-level testosterone (total and free) and luteinizing hormone (LH) (Roaiah et al., 2016). Also, in another study, the use of capsules containing 1250 mg T. terrestris extracts each day (during 3-week highintensity training and 3-week high-volume training) did not change muscle mass and plasma levels of testosterone, dihydrotestosterone (DHT), and IGF-1, but it significantly alleviated muscle damage and promoted anaerobic performance of trained male boxers,

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which may be related to the decrease of plasma IGF-1 binding protein-3 rather than androgen in plasma (Ma et al., 2015). After oral administration of T. terrestris extract for 84 days (1 pill orally every 8 h; each pill contained 250 mg of extract, including 37.5 mg of steroidal saponins), blood serum concentrations of DHT increased significantly. However, there were no significant differences in DHEA, free testosterone, follicle-stimulating hormone, LH, and prolactin concentrations. Protodioscin, the main phytochemical agent of the Tribulus genus, is known to convert testosterone into DHT (Salgado et al., 2016). Tribulus food supplement has a positive influence on athletes’ anaerobic alactic glycolytic power and aerobic capacity when energy is produced in the aerobic way (Milasius et al., 2010). One of the studies published in 2016 shows that treatment of rats with T. terrestris saponins significantly improved the performance of the overtrained rats, reflected by the extension of time to exhaustion, with a concomitant increase in body mass, relative mass, and protein levels of gastrocnemius. It might be attributed to the changes in androgen receptor axis and IGF-1 receptor signaling (Yin et al., 2016).

Contamination and/or Adulteration of Dietary Supplements The available data indicate that between 10% and 15% of supplements may contain prohibited substances. The analysis of press articles on doping cases dealt with by the Australian Sports Anti-Doping Authority, UK Anti-Doping, and the US Anti-Doping Agency in the period 2006e13 showed that 6.4e8.8% of the doping incidents were the result of supplement use (Outram and Stewart, 2015). In 2011e12, even 30% of doping cases in Poland involved the intake of supplements and sports foods whose doping contents resulted from contamination or intentional  ski, 2016). Moreover, such products may contain adulteration (Pokrywka and Krzywan substances that have not been medically analyzed. Some supplements contain excessive doses of potentially toxic ingredients, while others do not contain significant amounts of the ingredients listed on the label (Maughan, 2005). The presence of an undeclared substance in a supplement may also result from crosscontamination related to poor manufacturing practices and to the use of the same production line for several products. This is usually characterized by the presence of substances that are not necessarily related to the supplement claim (such as traces of steroids in vitamins and minerals). The majority of adulteration cases, however, are intentional and aimed at increasing the efficacy of the supplement (da Justa Neves and Caldas, 2015). A significant part of all reported cases of supplement contamination and/or adulteration concerns herbal aphrodisiacs (Gilard et al., 2015; Poplawska et al., 2013) and weight-loss products (De Carvalho et al., 2012; Pawar and Grundel, 2016), as  ski et al., 2014; well as supplements particularly recommended for athletes (Chołbin Geyer et al., 2008; Van Thuyne et al., 2006).

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Recommendations for Sport Nutrition With Supplements When planning their supplementation, athletes should take into account potentially adverse side effects of various components in dietary products and calculate the risk of antidoping rules violations. It is also advisable to refer to the available recommendations prior to a specific substance application. What may be of use here is the classification by the Australian Institute of Sport (AIS; http://www.ausport.gov.au/ais/nutrition), which is highly appreciated, not only by sports dietitians. In the years 2000e13, AIS has developed a Sports Supplements Program, which was designed for the specific needs of AIS and other Australian athletes. Among other aspects, the program was aimed to allow AIS athletes to focus on the use of supplements and specific sports diets as part of their nutrition plans and to minimize the risk of supplement use leading to an inadvertent doping offence (Pokrywka et al., 2014). The expertise and resources developed during the implementation of the program have been remodeled into the AIS Sports Supplement Framework. A key goal of the Framework is to minimize the risk of antidoping rules violations arising through the use of supplements and sports foods. The use of supplements and sports foods by Australian athletes involves a balance between potential benefits (for example, contribution to an evidencebased sports nutrition program) and potential risks (for example, waste of resources, distraction, poor role modeling, antidoping rules violations). For this reason, the special ABCD Classification system has been created, which ranks sports foods and supplement ingredients into four groups based on scientific evidence and other practical considerations that determine whether a product is safe, legal, and effective in improving sports performance. In each group, sports food and supplement ingredients are defined as follows:  Group A: supported for use in specific situations in sport using evidence-based protocols; provided or permitted for use by some athletes according to best practice protocols.  Group B: deserve further research and could be considered for provision to athletes under a research protocol or case-managed monitoring situation; provided to athletes within research or clinical monitoring situations.  Group C: have little meaningful proof of beneficial effects; not provided to athletes within supplement programs, may be permitted for individualized use by an athlete where there is specific approval from (or report to) a sports supplement panel.  Group D: banned or at high risk of contamination with substances that could lead to a positive drug test; should not be used by athletes. The classification is made via the consensus of an expert group and can evolve based on new knowledge and practical issues. T. terrestris and other testosterone boosters, as well as DHEA, androstenedione, 19-norandrostenione/ol, other prohormones, and maca

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root powder, are included in the subcategory of group D called “prohormones and hormone boosters.” Similarly, the International Society of Sports Nutrition grouped nutritional supplements under the following categories: 1. Apparently Effective (supplements that help people meet general caloric needs and/or the majority of research studies in relevant populations show is effective and safe). 2. Possibly Effective (supplements with initial studies supporting the theoretical rationale but requiring more research to determine how the supplement may affect training and/or performance). 3. Too Early To Tell (supplements with sensible theory but lacking sufficient research to support its current use). 4. Apparently Ineffective (supplements that lack a sound scientific rationale and/or research has clearly shown to be ineffective). T. terrestris is an example of a muscle-building supplement from group 4. The group also includes glutamine, smilax, isoflavones, sulfopolysaccharides (myostatin inhibitors), boron, chromium, conjugated linoleic acid, gamma oryzanol, prohormones, and vanadyl sulfate (Kreider et al., 2010). Relevant recommendations on sports supplementation have also been designed in Poland, for Polish sports associations. It was compiled by the National Centre for Sports Medicine and the Medical Commission of the Polish Olympic Committee in December 2012. The panel of experts analyzed the available data on dietary supplementation and functional food and made the assessment of these products with regard to current scientific evidence. The following groups were indicated as a result:  recommended products (group A)  products whose scientific analyses are equivocal but some valuable research suggests their applicability (group B)  products with no reliable proof of their functioning declared by the manufacturer, classified as not recommended (group C) Polish experts regarded products containing T. terrestris as inadvisable for athletes and classified it in group C. Group A included only caffeine, creatine, isotonic drinks,  ski, 2016). protein, and carbohydrates (Pokrywka and Krzywan

Risk of Violating Antidoping Rules The classification of T. terrestris as a not recommended sports nutritional component does not result from the fact that its natural ingredients are mentioned on the WADA prohibited list. The main reason is actually a high risk of contamination and/or adulteration of some products containing T. terrestris. The risk appears to have been warranted by a detection of anabolic steroids in a Tribulus leaf product designed for

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performance enhancement (Geyer et al., 2000). Traces of an anabolic steroid compound in a T. terrestris food supplement from Bulgaria were also reported in the Czech Republic (RASFF Annual Report, 2014). It needs to be noted that the WADA Prohibited List still has an open character. Apart from the examples of prohibited substances or methods in particular groups, some additional substances that are not located on the list but are characterized by “a similar chemical structure or similar biological effect(s)” can also be considered as doping. Such an attitude allows for triggering investigation procedures in case of applications of new pharmacological substances by athletes, including substances specially designed for doping purposes (Pokrywka et al., 2010). The manufacturers have already been found to replace prohormones with so-called designer steroids, i.e., substances with anabolic activity, most of which never made it to the market and were never tested on humans, and to create the supplements they dug up from old patents and scientific literature (Abushareeda et al., 2014; Cawley et al., 2016; Parr et al., 2011). To sum up, it is getting increasingly hard to prove “inadvertent doping” as the main cause of athletes’ doping infraction. According to many disciplinary committees that decide on the severity of punishment, the problem of dietary or/and nutritional supplements contamination or intentional adulteration has already been sufficiently publicized. Therefore, the athlete who takes products from such a market is aware of the risk of antidoping rules violations. As in any other case, it is the athlete who is first and foremost accountable for the doping control they undergo. Supplements and sports foods are used extensively, and although the use of some products may be ergogenic, the risk-to-benefit ratio needs to be carefully considered before embarking on the widespread use of supplements (Potgieter, 2013). It is worth quoting here the principles described in a practical guide to eating for health and performance prepared by the Nutrition Working Group of the International Olympic Committee based on an International Consensus Conference held at the IOC in Lausanne, Switzerland, in October 2010 (Maughan and Burke, 2012). Athletes must be aware of the strict liability principle that makes them responsible for everything they eat and drink. Ignorance is not an acceptable excuse for a positive doping result. Athletes should check all supplements with a medical officer or qualified sports nutrition professional. If there is any doubt at all, they should not use it. That does not only concern professional athletes. When deciding to use a sports food or supplement, each person who is proficient in any form of physical activity should take into consideration the following issues:     

Is it safe? Is it legal? Is there evidence that it works at the recommended dose? Am I aware of the correct protocols of how and when to take it? Can I afford it?

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To conclude, in the light of recent research results, the risk of T. terrestris being contaminated or adulterated with doping substances appears to outweigh the benefits of potentially improved sports performance induced by natural T. terrestris ingredients. Additionally, the previously mentioned recommendations clearly advise athletes against using T. terrestrisebased products.

References Abadjieva, D., Kistanova, E., 2016. Tribulus terrestris alters the expression of growth differentiation factor 9 and bone morphogenetic protein 15 in rabbit ovaries of mothers and F1 female offspring. PLoS One 11, e0150400. Abushareeda, W., Fragkaki, A., Vonaparti, A., Angelis, Y., Tsivou, M., Saad, K., Kraiem, S., Lyris, E., Alsayrafi, M., Georgakopoulos, C., 2014. Advances in the detection of designer steroids in antidoping. Bioanalysis 6, 881e896. Antonio, J., Uelmen, J., Rodriguez, R., Earnest, C., 2000. The effects of Tribulus terrestris on body composition and exercise performance in resistance-trained males. Int. J. Sport Nutr. Exerc. Metab. 10, 208e215. Arsyad, K.M., 1996. Effect of protodioscin on the quality and quantity of sperms from males with moderate idiopathic oligozoospermia. Medica 22, 614e618. Beck, K.L., Thomson, J.S., Swift, R.J., von Hurst, P.R., 2015. Role of nutrition in performance enhancement and postexercise recovery. Open Access J. Sports Med. 11, 259e267. Brown, G.A., Vukovich, M.D., Martini, E.R., Kohut, M.L., Franke, W.D., Jackson, D.A., King, D.S., 2001. Effects of androstenedione-herbal supplementation on serum sex hormone concentrations in 30- to 59-year-old men. Int. J. Vitamin Nutr. Res. 71, 293e301. Cawley, A., Blakey, K., Waller, C.C., McLeod, M.D., Boyd, S., Heather, A., McGrath, K.C., Handelsman, D.J. , Willis, A.C., 2016. Detection and metabolic investigations of a novel designer steroid: 3-chloro17a-methyl-5a-androstan-17b-ol. Drug Test. Anal. 8, 621e632. Chhatre, S., Nesari, T., Somani, G., Kanchan, D., Sathaye, S., 2014. Phytopharmacological overview of Tribulus terrestris. Pharmacognosy Rev. 8, 45e51.  ski, P., Wicka, M., Kowalczyk, K., Jarek, A., Kaliszewski, P., Pokrywka, A., Bulska, E., Chołbin Kwiatkowska, D., 2014. Detection of b-methylphenethylamine, a novel doping substance, by means of UPLC/MS/MS. Anal. Bioanal. Chem. 406, 3681e3688. Corrigan, B., Kazlauskas, R., 2003. Medication use in athletes selected for doping control at the Sydney Olympics (2000). Clin. J. Sport Med. 13, 33e40. da Justa Neves, D.B., Caldas, E.D., 2015. Dietary supplements: International legal framework and adulteration profiles, and characteristics of products on the Brazilian clandestine market. Regul. Toxicol. Pharmacol. 73, 93e104. De Carvalho, L.M., Cohen, P.A., Silva, C.V., Moreira, A.P., Falca˜o, T.M., Dal Molin, T.R., Zemolin, G., Martini, M., 2012. A new approach to determining pharmacologic adulteration of herbal weight loss products. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 29, 1661e1667. Fra˛czek, B., Gacek, M., Grzelak, A., 2012. Nutritional support of physical abilities in a professional athletes group. Problemy Higieny i Epidemiologii 93, 817e823. Gama, C.R., Lasmar, R., Gama, G.F., Abreu, C.S., Nunes, C.P., Geller, M., Oliveira, L., Santos, A., 2014. Clinical assessment of Tribulus terrestris extract in the treatment of female sexual dysfunction. Clin. Med. Insights Women. Health 7, 45e50.

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Gauthaman, K., Ganesan, A.P., 2008. The hormonal effects of Tribulus terrestris and its role in the management of male erectile dysfunctionean evaluation using primates, rabbit and rat. Phytomedicine 15, 44e54. Geyer, H., Mareck-Engelke, U., Reinhart, U., Thevis, M., Scha¨nzer, W., 2000. Positive doping cases with norandrosterone after application of contaminated nutritional supplements. Deutsche Zeitschrift fu¨r Sportmedizin 51, 378e382. Geyer, H., Parr, M.K., Koehler, K., Mareck, U., Scha¨nzer, W., Thevis, M., 2008. Nutritional supplements cross-contaminated and faked with doping substances. J. Mass Spectrom. 43, 892e902. Gilard, V., Balayssac, S., Tinaugus, A., Martins, N., Martino, R., Malet-Martino, M., 2015. Detection, identification and quantification by 1H NMR of adulterants in 150 herbal dietary supplements marketed for improving sexual performance. J. Pharm. Biomed. Anal. 102, 476e493. Hussain, A.A., Mohammed, A.A., Ibrahim, H.H., Abbas, A.H., 2009. Study the biological activities of Tribulus terrestris extracts. Int. J. Chem. Mol. Nuclear Mater. Metallur. Eng. 3, 510e512. Joshi, D.D., Uniyal, R.C., 2008. Different chemo types of Gokhru (Tribulus terrestris): a herb used for improving physique and physical performance. Int. J. Green Pharm. 2, 158e161. Kim, H.J., Kim, J.C., Min, J.S., Kim, M.J., Kim, J.A., Kor, M.H., Yoo, H.S., Ahn, J.K., 2011. Aqueous extract of Tribulus terrestris Linn induces cell growth arrest and apoptosis by down-regulating NF-kB signaling in liver cancer cells. J. Ethnopharmacol. 136, 197e203. Kostova, I., Dinchev, D., 2005. Saponins in Tribulus terrestris e chemistry and bioactivity. Phytochem. Rev. 4, 111e137. Kreider, R.B., Wilborn, C.D., Taylor, L., Campbell, B., Almada, A.L., Collins, R., Cooke, M., Earnest, C.P., Greenwood, M., Kalman, D.S., Kerksick, C.M., Kleiner, S.M., Leutholtz, B., Lopez, H., Lowery, L.M., Mendel, R., Smith, A., Spano, M., Wildman, R., Willoughby, D.S., Ziegenfuss, T.N., Antonio, J., 2010. ISSN exercise and sport nutrition review: research and recommendations. J. Int. Soc. Sports Nutr. 7, 7. Liguori, C., Placidi, F., Leonardis, F., Diomedi, M., Mercuri, N.B., Marciani, M.G., Stanzione, P., Sallustio, F., 2015. Development of collateral veins as a favorable prognostic factor for complete recovery in cerebral venous thrombosis due to Tribulus terrestris. Int. J. Stroke 10, E66eE67. Ma, Y., Guo, Z., Wang, X., 2015. Tribulus terrestris extracts alleviate muscle damage and promote anaerobic performance of trained male boxers and its mechanisms: roles of androgen, IGF-1, and IGF binding protein-3. J. Sport Health Sci. http://dx.doi.org/10.1016/j.jshs.2015.12.003. Maughan, R.J., 2005. Contamination of dietary supplements and positive drug tests in sport. J. Sports Sci. 23, 883e889. Maughan, R., Burke, L., 2012. Nutrition for athletes. In: A Practical Guide to Eating for Health and Performance Prepared by the Nutrition Working Group of the International Olympic Committee Based on an International Consensus Conference Held at the IOC in Lausanne in October 2010. IOC Medical Commission Working Group on Sports Nutrition, Lausanne. Maughan, R.J., Depiesse, F., Geyer, H., 2007. The use of dietary supplements by athletes. J. Sports Sci. 25, S103eS113. _ M., Ru  ta, D., Skernevi Milasius, K., Pe ciukoniene, cius, J., 2010. Efficacy of the Tribulus food supplement used by athletes. Acta Med. Lituanica 17, 65e70. Mottram, D., 2015. Inadvertent use of drugs in sport. Aspetar Sports Medicine Journal 4. Neychev, V., Mitev, V., 2016. Pro-sexual and androgen enhancing effects of Tribulus terrestris L.: fact or fiction. J. Ethnopharmacol. 179, 345e355. Outram, S., Stewart, B., 2015. Doping through supplement use: a review of the available empirical data. Int. J. Sport Nutr. Exerc. Metab. 25, 54e59.

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Parr, M.K., Pokrywka, A., Kwiatkowska, D., Scha¨nzer, W., 2011. Ingestion of designer supplements produced positive doping cases unexpected by the athletes. Biol. Sport 28, 153e157. Pawar, R.S., Grundel, E., 2016. Overview of regulation of dietary supplements in the USA and issues of adulteration with phenethylamines (PEAs). Drug Test. Anal. http://dx.doi.org/10.1002/dta.1980.  ski, J., 2016. Problem dopingu w sporcie. In: Braksator, W., Mamcarz, A. (Eds.), Pokrywka, A., Krzywan Kardiologia sportowa w praktyce klinicznej. Wydawnictwo Lekarskie PZWL, Warszawa, pp. 549e560. Pokrywka, A., Kwiatkowska, D., Kaliszewski, P., Grucza, R., 2010. Some aspects concerning modifications of the list of prohibited substances and methods in sport. Biol. Sport 27, 307e314.  ski, Z., Malczewska-Lenczowska, J., Fijałek, Z., Turek-Lepa, E., Grucza, R., 2014. Pokrywka, A., Obmin Insights into supplements with Tribulus terrestris used by athletes. J. Human Kinet. 41, 99e105. Poplawska, M., Blazewicz, A., Bukowinska, K., Fijalek, Z., 2013. Application of high-performance liquid chromatography with charged aerosol detection for universal quantitation of undeclared phosphodiesterase-5 inhibitors in herbal dietary supplements. J. Pharm. Biomed. Anal. 84, 232e243. Potgieter, S., 2013. Sport nutrition: a review of the latest guidelines for exercise and sport nutrition from the American College of Sport Nutrition, the International Olympic Committee and the International Society for Sports Nutrition. South Afr. J. Clin. Nutr. 26, 6e16. Qureshi, A., Naughton, D.P., Petroczi, A., 2014. A systematic review on the herbal extract Tribulus terrestris and the roots of its putative aphrodisiac and performance enhancing effect. J. Diet. Suppl. 11, 64e79. RASFF Annual Report, 2014. The rapid alert system for food and feed, 2015. Eur. Comm. Health Food Saf. Roaiah, M.F., Elkhayat, Y.I., Saleh, S.F., Abd El Salam, M.A., June 23, 2016. Prospective analysis on the effect of botanical medicine (Tribulus terrestris) on serum testosterone level and semen parameters in males with unexplained infertility. J. Diet. Suppl. 1e7. http://dx.doi.org/10.1080/19390211.2016. 1188193. Rogerson, S., Riches, C.J., Jennings, C., Weatherby, R.P., Meir, R.A., Marshall-Gradisnik, S.M., 2007. The effect of five weeks of Tribulus terrestris supplementation on muscle strength and body composition during preseason training in elite rugby league players. J. Strength Condition. Res. 21, 348e353. Salgado, R.M., Marques-Silva, M.H., Gonc¸alves, E., Mathias, A.C., Aguiar, J.G., Wolff, P., July 12, 2016. Effect of oral administration of Tribulus terrestris extract on semen quality and body fat index of infertile men. Andrologia. http://dx.doi.org/10.1111/and.12655. Samani, N.B., Jokar, A., Soveid, M., Heydari, M., Mosavat, S.H., 2016. Efficacy of the hydroalcoholic extract of Tribulus terrestris on the serum glucose and lipid profile of women with diabetes mellitus: a double-blind randomized placebo-controlled clinical trial. J. Evidence. Based Complement. Altern. Med. 21, NP91eNP97. Sansalone, S., Russo, G.I., Mondaini, N., Cantiello, F., Antonini, G., Cai, T., 2016. A combination of tryptophan, Satureja montana, Tribulus terrestris, Phyllanthus emblica extracts is able to improve sexual quality of life in patient with premature ejaculation. Archivio Italiano di Urologia e Andrologia 88, 171e176. Saudan, C., Baume, N., Emery, C., Strahm, E., Saugy, M., 2008. Short term impact of Tribulus terrestris intake on doping control analysis of endogenous steroids. Forensic Sci. Int. 178, e7e10. Shaw, G., Slater, G., Burke, L.M., 2016. Supplement use of elite Australian swimmers. Int. J. Sport Nutr. Exerc. Metab. 26, 249e258. Suzic Lazic, J., Dikic, N., Radivojevic, N., Mazic, S., Radovanovic, D., Mitrovic, N., Lazic, M., Zivanic, S., Suzic, S., 2011. Scand. J. Med. Sci. Sports 21, 260e267. Tscholl, P., Junge, A., Dvorak, J., 2008. The use of medication and nutritional supplements during FIFA World Cups 2002 and 2006. Br. J. Sports Med. 42, 725e730.

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Tscholl, P., Alonso, J.M., Dolle´, G., Junge, A., Dvorak, J., 2010. The use of drugs and nutritional supplements in top-level track and field athletes. Am. J. Sports Med. 38, 133e140. Van Eenoo, P., Delbeke, F.T., Desmet, N., De Backer, P., 2000. Excretion studies with Tribulus terrestris. In: Scha¨nzer, W., Geyer, H., Gotzmann, A., Mareck-Engelke, U. (Eds.), Recent Advances in Doping Analysis. Sport and Buch Strauß, Cologne, pp. 13e22. Van Thuyne, W., van Eenoo, P., Delbeke, F.T., 2006. Nutritional supplements: prevalence of use and contamination with doping agents. Nutr. Res. Rev. 19, 147e158. ¨ zkum, D., 2014. Herbs potentially enhancing sports performance. Curr. Top. Nutraceut. Yavuz, H.U., O Res. 12, 25e34. Yin, L., Wang, Q., Wang, X., Song, L.N., June 22, 2016. Effects of Tribulus terrestris saponins on exercise performance in overtraining rats and the underlying mechanisms. Can. J. Physiol. Pharmacol. 1e9. http://dx.doi.org/10.1139/cjpp-2016-0086.

10

The Use of Maca (Lepidium meyenii) for Health Care: An Overview of Systematic Reviews

Myeong Soo Lee1, Tae-Hun Kim2, Hye Won Lee1 1

KOREA INSTITUTE OF ORIENTAL ME DICI NE, DAEJEON, R EPUBLIC OF K OREA; 2 KY UNG HE E UNIVE RSITY, SEOUL, REPUBL IC OF KOREA

Introduction Maca is an Andean plant of the Brassica family that is widely grown in several South American countries (Gonzales, 2012; Gonzales et al., 2009). Two species are well known, Lepidium meyenii and Lepidium peruvianum (Hudson, 2009). They have been traditionally used as an adaptogenic plants to manage anemia, infertility, and female hormone balance, and they are used as food supplements throughout the world, including in China, Japan, Korea, Europe, and the US (Gonzales, 2012; Gonzales et al., 2009, 2014; Hudson, 2009). The total amount of exporting maca from Peru was about $6 million in 2010 (Gonzales, 2012). It has been claimed that maca has energy-boosting effects and it improves stamina and endurance in athletes (Grunewald and Bailey, 1993; Stone et al., 2009). Maca has also improved physical activity, immunity, and chronic fatigue. In the Inca Empire, the warriors ingested the maca in advance for battle to enhance their energy to assault their enemies (Quiro´s and Ca´rdenas, 1997; Wang et al., 2007). Maca has been regarded as a highly nutritious food and medicine (Gonzales, 2012; Lentz et al., 2006). A high number of nutrients, including carbohydrates, proteins, vitamins, and minerals, may make maca a powerful physical energizer as well as improve mental energy (Lentz et al., 2006). Many experimental studies reported that maca has beneficial effects on male reproduction, prostatic hyperplasia, sexual behavior, female fertility, osteoporosis, learning and memory, and protection against UV exposure (Gonzales, 2012; Gonzales et al., 2009, 2014). Several clinical studies have reported that maca is effective for increasing sperm quality (Gonzales et al., 2001) and managing menopausal symptoms, including depression and quality of life (Stojanovska et al., 2015). However, there is a large evidence gap between experimental and clinical studies. The number of clinical studies is small, and maca’s beneficial effects that have been found in experimental Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00010-7 Copyright © 2017 Elsevier Inc. All rights reserved.

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studies have not been confirmed yet. Therefore, this review is aimed at evaluating the current evidence of maca as caring for any type of health conditions by assessing the current systematic reviews (SRs).

Methods Data Sources Electronic literature searches were performed in December 2016 using Medline, Excerpta Medica dataBASE (EMBASE), The Allied and Complementary Medicine Database (AMED), the Cochrane Library, and 7 Korean Databases (DBs) (Korean Studies Information Service System, DBpia, the Korean Institute of Science and Technology Information, the Research Information Service System, KoreaMed, Korean National Assembly Library, and OASIS) and 1 Chinese DB (China National Knowledge Infrastructure) without restrictions of time or language. The search terms included “(maca OR Lepidium) AND (systematic OR systematic adj review OR meta-analysis OR review)” in Korean, Chinese, and English. We also have manual searches for our departmental files. The references in all located articles were searched manually for further relevant studies.

Study Selection Type of Study We included SRs or metaanalyses that had assessed the evidence specifically with the effectiveness of any type of maca from at least two controlled clinical trials. SRs assessing maca together with other complementary medicines without evaluating the two approaches separately were excluded. Reviews were defined as systematic if they included an explicit and repeatable methods section describing the search strategy and explicit inclusion/exclusion criteria. Reviews that did not use systematic methods were also excluded.

Type of Participants SRs or metaanalyses with patients with any type of conditions and healthy people were included.

Type of Intervention Any type of maca (Lepidium spp.) preparation, regardless of the origin of maca, was included. We also included the dietary supplement with maca.

Type of Control Intervention Any type of controls, including placebo and other active controls, were included.

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Type of Outcome Measures Any outcomes related to target conditions were included.

Data Extraction Abstracts of reviews located were thus inspected by two authors, and those appearing to meet the inclusion criteria were retrieved and read in full by both authors. Data from all identified SRs were extracted independently by two authors (Myeong Soo Lee and Tae-Hun Kim) using predefined criteria. Disagreements were resolved by discussion between the authors (Myeong Soo Lee, Tae-Hun Kim, and Hye Won Lee). Judgments about the quality of the primary studies were taken from the respective SRs.

Assessing the Methodological Quality of Systematic Reviews The assessing the methodological quality of systematic reviews (AMSTAR) checklist is a measurement tool for the assessment of SRs’ quality (Shea et al., 2007). It has 11 items that can be answered with one of the selections, including Yes, No, Can’t answer, and Not applicable. The AMSTAR score is the number of items checked with Yes, which suggests that the item was reported or conducted appropriately. In this review, we did overall quality assessment with this score for each study. We graded as highest quality if the score was 11, high quality if the score ranged from 8 to 10, medium quality if the score ranged from 4 to 7, and low quality if the score was below 3 points.

Results We located 271 eligible articles, of which 3 SRs were suitable for the inclusion criteria. The PRISMA flow diagram is presented in Fig. 10.1. Key data from all three SRs are listed in Table 10.1 (Lee et al., 2011, 2016; Shin et al., 2010). The first authors originated from the same group in Korea for all the included SRs. The SRs investigated reproduction-related or women’s related conditions, including menopausal symptoms (Lee et al., 2011), sexual function (Shin et al., 2010), and semen quality (Lee et al., 2016). The AMSTAR scores showed the high quality of the SRs in all of studies (Table 10.2). All reviews reported that the most of primary studies have poor or moderate methodological quality. For menopausal symptoms, four randomized clinical trials (RCTs) were included and reported positive conclusions (Lee et al., 2011). Two SRs are related with sexual function (Shin et al., 2010) and semen quality (Lee et al., 2016). Both SRs showed limited or suggestive evidence in a positive way. However, these reviews failed to avoid important limitations, such as a small number of included trials or poor quality of the primary studies.

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FIGURE 10.1 Flow diagram of literature search. SR, systematic review.

Table 10.1

Systematic Reviews of Maca for Health Care

First Author (Year), Country Maca Species Lee et al. (2011), Korea Lee et al. (2016), Korea Shin et al. (2010), Korea

Lepidium peruvianum and Lepidium meyenii L. meyenii

L. meyenii

Quality of Primary Studies

Quality of Review (AMSTARs)

Menopausal 4 RCTs symptoms

Moderate

8

Limited evidence for effectiveness of maca

Semen quality

Poor

8

Poor

7

Suggestive evidence for the effectiveness of maca Limited evidence for effectiveness of maca

Condition

Sexual function

No. of Primary Studies

5 studies (3RCTs, 2 UOSs) 4 RCTs

Conclusion (Quote)

AMSTAR, assessing the methodological quality of systematic reviews; RCT, randomized clinical trial; UOS, uncontrolled observational study.

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Table 10.2

171

Quality of Methodological Quality by AMSTAR Checklist Assessment

First Author (Year) [Ref], Country

1

2

3

4

5

6

7

8

9

10

Lee et al. (2011) Lee et al. (2016) Shin et al. (2010)

C C C

Y Y Y

Y Y Y

Y N Y

N N N

Y Y Y

Y Y Y

Y Y Y

Y Y Y

N N N

11

Total Score

Overall Quality Assessment

Y Y Y

8 7 8

High quality High quality High quality

The number in table headers mean the items of AMSTAR checklist and they means as followings. 1, was an “a priori” design provided?; 2, was there duplicate study selection and data extraction?; 3, was a comprehensive literature search performed?; 4, was the status of publication (i.e., gray literature) used as an inclusion criterion?; 5, was a list of studies (included and excluded) provided?; 6, were the characteristics of the included studies provided?; 7, was the scientific quality of the included studies assessed and documented?; 8, was the scientific quality of the included studies used appropriately in formulating conclusions?; 9, were the methods used to combine the findings of studies appropriate?; 10, was the likelihood of publication bias assessed?; 11, was the conflict of interest included?; AMSTAR, assessing the methodological quality of systematic reviews; AMSTAR score, the number of items checked with Y; C, can’t answer; N, no; NA, not applicable; Overall quality assessment, the highest quality (score ¼ 11), high quality (score ¼ 8e10), medium quality (score ¼ 4e7), and low quality (score ¼ 0e3); Y, yes.

Discussion Our overview found few rigorous SRs evaluating the efficacy of maca for health care. Most of the SRs had high-quality methods, but many included primary studies that had a high risk of bias. Common caveats included small sample sizes, lack of using random sequence generation, and allocation concealment, and these prevented them from having firm conclusions. It is meaningful to add two rigorous studies published in 2015 for updating the included SR. Two RCTs tested the effects of maca (L. meyenii) on menopausal symptoms (Stojanovska et al., 2015) and sexual function in women (Dording et al., 2015). Adding these two studies strengthens the evidence of maca to be managing menopausal symptoms measure with Greene Climacteric Scale (Stojanovska et al., 2015). Regarding efficacy of maca for sexual function, a new study builds up the beneficial evidence of maca for alleviating sexual dysfunction in women (Dording et al., 2015). Although we tried to search the literature comprehensively in international DBs in English, Chinese, and Korean regardless of publication languages, we cannot be totally confident that all articles were located. Our overview was aimed at evaluating the SRs rather than the individual primary studies, which means there is a risk of diluting the results of high-quality studies by including low-quality data. Furthermore, all of the included SRs were from our group, and this can be another bias, regardless of our efforts to have neutral decisions. Future studies on maca should be performed more rigorously and be well reported for establishing concrete evidence. We recommend that future investigators use the appropriate random sequence methods and report following CONSORT guidelines to avoid the transparency and any bias (Schulz et al., 2010). Our overview reported with the PRISMA reporting guidelines (Moher et al., 2009), and we urge that future SRs follow it for producing high-quality SRs.

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In conclusion, few SRs of maca have been published. Due to a number of caveats, the evidence is limited that maca is an effective therapy for menopause and sexual-related conditions.

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An Overview on Rhodiola rosea in Cardiovascular Health, Mood Alleviation, and Energy Metabolism Michael Duncan, Neil D. Clarke COVENTRY UNIVERS ITY, COVENTRY, U NITED KINGDOM

Introduction/Overview Rhodiola rosea (R. rosea), also known as Golden Root, Rose Root Rosavin, Rosenroot, Rhodiola Rhizome, Arctic Root, and Rhidola, is an herb that grows in high-altitude, highlatitude, typically mountainous regions of North America, Europe, and Asia. It is a flowering biennial and has been used in traditional folk medicine for centuries as a treatment for fatigue and mood disorders (Walker and Robergs, 2006). R. rosea has also been prescribed for cancer and tuberculosis in Mongolia (Khaidaev and Menshikova, 1978), to ingest for increased fertility in Russia (Saratikov and Krasnov, 1987), and also for use as a hair wash in Norway (Alm, 2004). The ingestion of R. rosea for physical effects is also historically rooted with data from Viking times indicating that it was used to boost endurance and physical strength (Magnusson, 1992). Although R. rosea has a long history of use in traditional and folk medicine, it has only more recently started to garner sustained scientific attention by virtue of its supposed therapeutic capacity as an adaptogen. Adaptogens are most commonly natural herbal products that are nontoxic in normal doses, produce a nonspecific response, and have a normalizing physiological influence (Brekhman and Dardymov, 1969). Consequently, there are now a growing number of scientific studies that have examined the effects of R. rosea ingestion or supplementation on a considerable range of outcomes, including depression (Amsterdam and Panossian, 2016), Alzheimer’s disease (Nabavi et al., 2015), cognitive function (Al-Kuraishy, 2016), inflammation (Shanley et al., 2014), anxiety and mood (Cropley et al., 2015), body composition (Chang et al., 2016), menopause (Gerbag and Brown, 2016), and athletic performance (DeBock et al., 2004). R. rosea has also been referred to as an ergogenic aid, i.e., a substance that enhances physical and/or mental performance, and consequently there has been increased recent interest in the utility of R. rosea ingestion on athletic performance. This is because the

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commonly reported effects of R. rosea ingestion align with enhanced performance in situations where physical and mental effort is required at the same time, particularly the reported fatigue-reducing effects of R. rosea. Scientists in the former Soviet Union studied R. rosea extensively, concluding that ingestion favorably influenced exercise performance (Brown et al., 2002). However, this prior work is largely not accessible in major international databases (Brown et al., 2002; Morgan and Bone, 2005), and those authors who have appraised this literature have concluded that it lacked experimental control and was methodologically limited (Abidov et al., 2003; Brown et al., 2002) to the extent that its conclusions cannot be relied upon. As a result, more recent studies have attempted to address these limitations and provide more robust evaluation of the potential efficacy of R. rosea ingestion for enhancing various aspects of human physiological and psychological performance. The current chapter provides an overview of the extant literature on the effect of R. rosea ingestion on cardiovascular health, mood alleviation, and energy metabolism in the context of exercise performance.

Active Constituents There are over 200 species of Rhodiola (Kelly, 2001), and each species has a distinctive pharmacology, although the phenylethanol salidroside is common throughout the genus and is considered the primary active constituent in R. rosea. R. rosea is distinguished from other species of Rhodiola by the presence of three water-soluble cinnamyl glycosides: rosavin, rosin, and rosarian. These three glycosides are commonly known as rosavins and are presumed to give R. rosea its adaptogenic characteristics (Abidov et al., 2003). It is, however, still unclear which specific compound(s) in R. rosea are active constituents. Most preparations of R. rosea are standardized to specific levels of marker compounds rosavin, salidroside, or both (Brown et al., 2002), and typically a ratio of 3:1 for rosavins and salidrosides has been used in animal-based experiments as reflective of the typical rosavin:salidroside ratios found in R. rosea (Mattioli and Perfumi, 2011). To date, R. rosea has been the species of the Rhodiola genus that has been the most intensively studied in humans, and it is the only Rhodiola species considered safe for human consumption (Brown et al., 2002). It is important to note that by far, the majority of early research on R. rosea’s pharmacological properties and active constituents was conducted and published in Russia and Bulgaria (Walker and Robergs, 2006). Much of this prior research has not been widely disseminated and suffers from considerable limitations. Only recently have scientists started to better evaluate the efficacy of R. rosea using more rigorous and robust designs. A full overview of the active constituents and pharmacology of R. rosea is beyond the scope of this chapter. Readers are referred to the review published by Brown et al. (2002) for a detailed history on the topic.

Rhodiola rosea and Exercise Performance Improving physical performance has been one of the main reasons why R. rosea has been either prescribed by sport and exercise scientists or medics, or has simply been

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ingested by athletes on the belief that it will result in improved performance. Taken collectively, the well-controlled studies that have examined the effect of R. rosea ingestion on various types of physical performance have largely been equivocal. Work in the 1960s and 1970s, primarily as part of Russian student dissertations, tended to report positive and performance-enhancing effects of R. rosea ingestion on exercise performance in trained athletes. This work was described by Brown et al. (2002), as it remains largely unpublished and untranslated. Consequently, there are questions over the methods and designs employed in these earlier studies. A summary of the exerciserelated studies examining the effect of R. rosea on performance and associated variables in humans is presented in Table 11.1. In 2006, Walker and Robergs provided what was then the most comprehensive review of the evidence on the ergogenic properties of R. rosea. This was then followed up by a systematic review on the effects of R. rosea on physical and mental fatigue by Ishaque et al. (2012). Both of these reviews report that the extant literature as to the effects of R. rosea ingestion are mixed. However, these reviews provided much needed information as, since the aforementioned Russian studies, research examining the effect of R. rosea on exercise performance remained poorly studied in humans until the 2000s. In one of these early 2000s studies, Spasov et al. (2000b) used a double-blind design to examine the effect of R. rosea ingestion (100 mg/day for 20 days) on performance of the PWC170 (physical work capacity) cycle ergometer test in Indian medical students. Spasov et al. (2000a) reported a 6.5% improvement in PWC170 performance in the R. rosea group (980  40 kgm/min) compared to placebo (920  52 kgm/min). Spasov et al. (2000a) also noted that there was a more rapid deceleration in heart rate postexercise in the R. rosea group compared to the placebo group. However, this last point was not well explored by Spasov et al. (2000a), nor could they identify any mechanism for their observations. It is also important to note that in the Spasov et al. (2000a) study, both the R. rosea and placebo groups produced significantly greater work capacity compared to baseline (810 kgm/min); thus a placebo effect cannot be ruled out here. DeBock et al. (2004) reported on both the acute and chronic effects of R. rosea ingestion on VO2 peak attained and time to exhaustion during cycle exercise and maximal isometric knee extension strength in a sample of 24 male and female students. They examined performance on the aforementioned variables following an acute (200mg ingestion before exercise) and a chronic (200 mg for 28 days) loading phase. Few studies have used this type of experimental design and uniquely in terms of the R. rosea literature to date. DeBock et al. (2004) reported a 5% improvement in time to exhaustion in the R. rosea trial compared to the placebo as a consequence of the acute loading phase. Conversely, following a 4-week period of loading with R. rosea, no differences in cardiovascular or performance variables were observed between R. rosea and placebo groups in the DeBock et al. (2004) study. The acute performance changes were attributed to the stimulation of b-endorphin activity in the body by DeBock et al. (2004). Subsequent data have suggested (Grossman and Sutton, 1985) that any change in b-endorphin activity is unlikely to explain the positive impact of acute R. rosea ingestion on cycling performance due to its inhibitory effect on the ventilator response to exercise.

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Table 11.1 Summary of Exercise-Related Studies Examining the Effect of R.Rosea on Performance and Associated Variables

Authors

Participants

Dose

Spasov et al. (2000a)

40 male students (17e19 years)

100 mg/day for 20 days

De Bock et al. (2004)

24 healthy male (21.8  0.3 years) and female (20.2  0.3 years) college students

20 mg before exercise

De Bock et al. (2004) Earnest et al. (2004)

24 healthy male (21.8  0.3 years) and female (20.2  0.3 years) college students 17 male competitive cyclists (31.6  2.8 years)

Colson et al. (2005)

8 males (18e34 years)

Noreen et al. (2013)

18 recreationally active females (22  3 years)

200 mg for 28 days

600 mg (mixed with Cordyceps sinensis) for 4 days, followed by 300 mg/day for 11 days 600 mg (mixed with C. sinensis) for 7 days, followed by 300 mg/day for 6 days 3 mg/kg body mass 60 min before exercise

Methods/ Variables Measured PWC170 (physical work capacity) cycle ergometry VO2 peak, cycle time to exhaustion

Muscle strength VO2 peak Muscle strength

Results 6.5% improvement compared to placebo

5% improvement over placebo Also noted lower heart rate 6 min into time to exhaustion test No effect No effect No effect

VO2 peak Peak power Lactate threshold VO2 peak Ventilatory threshold

No effect No effect No effect

6-mile cycle time trial time Average power during time trial Heart rate Blood lactate Salivary cortisol Salivary alpha amylase Rating of perceived exertion (RPE) Mood (Profile of Mood States; POMS)

1.6% improvement compared to placebo 3% improvement compared to placebo

No effect No effect

No effect No effect No effect Significantly higher than placebo Significantly lower than placebo Significantly lower scores for fatigue subscale post exercise than placebo. No effect for other POMS subscales

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Table 11.1 Summary of Exercise-Related Studies Examining the Effect of R.Rosea on Performance and Associated Variablesdcont’d

Authors

Participants

Dose

Duncan and Clarke (2014)

10 healthy males (26  6 years)

3-mg/kg body mass 60 min before exercise

Duncan et al. (2016)

12 males (24  6 years)

3-mg/kg body mass R. rosea 60 min before exercise 3-mg/kg body mass caffeine 60 min before exercise 3-mg/kg body mass R. rosea and 3-mg/kg caffeine 60 min before exercise

Methods/ Variables Measured 30 min cycling at 70% VO2 max Substrate utilization and energy expenditure Heart rate RPE

Mood States (Brunel Mood State Inventory (BRUMS)) Exercise Affect (Feeling State and Felt Arousal Scales) 5-km running time trial performance

Heart Rate RPE Exercise Affect (Feeling State and Felt Arousal Scales)

Results

No effect in energy expenditure, CHO or fat utilization No effect RPE significantly lower than placebo 30 min into exercise Significantly higher scores for vigor compared to placebo. No effect for other BRUMS subscales Scores for arousal and pleasure significantly higher than placebo 5% improvement in caffeine condition. No differences in R. rosea and R. rosea þ caffeine conditions No effect No effect No effect

This same study also showed no evidence of an ergogenic effect of R. rosea following 4 weeks loading. This is notable because the majority of past literature examining the efficacy of R. rosea ingestion on exercise performance has used a chronic loading model (Noreen et al.), and only more recently have human studies been conducted that examine the effect of acute R. rosea ingestion on performance. One previous study examining swimming to exhaustion in rats found that those treated with R. rosea (50 mg/kg) swam 24.6% longer than control rats (Abidov et al., 2003). Abidov et al. (2003) proposed that adenosine triphosphate (ATP) synthesis or resynthesis was the mechanism by which R. rosea enhanced exercise performance. This was based on observations of the

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ATP content in isolated rat muscle before and after 6 days of exhaustive swimming where ATP content decreased significantly less in rats that ingested R. rosea compared to controls. Few subsequent studies have considered ATP synthesis/resynthesis as a realistic mechanism by which R. rosea might positively influence exercise performance in humans. Alternative potential mechanisms have been proposed by recent research by Noreen et al. (2013) and Duncan and Clarke (2014), which will be explored later in this chapter. Work by Noreen et al. (2013) was one of the first studies to use a dose of R. rosea relative to body mass, and it examined the effect of 3-mg/kg body mass R. rosea on 6-mile cycle time trial performance in 18 females. The use of a relative dose of R. rosea is similar to procedures used in studies of other ergogenic aids, such as caffeine and sodium bicarbonate, and acknowledges that an absolute dose of R. rosea may have differential effects on participants who have relatively higher or lower body mass compared to each other. In their study, in addition to time trial time, rating of perceived exertion (RPE) was measured every 5 min during the time trial, as was heart rate. Blood lactate, salivary cortisol, and alpha amylase were assessed before a warm-up, after a warm-up, and then after the time trial. In addition, the Profile of Mood States questionnaire was completed pre- and postexercise by participants in both the R. rosea and placebo conditions. In their study, Noreen et al. (2013) reported that participants completed the 6-mile time trial significantly faster after ingestion of R. rosea compared to a carbohydrate placebo. There was no effect of R. rosea ingestion on blood lactate, salivary cortisol, and heart rate responses during the time trial. RPE values were also significantly lower in the R. rosea trial. Noreen et al. (2013) also presented data on the ratio of RPE relative to workload, which showed a more pronounced dampening of RPE in the R. rosea trial compared to the placebo. A similar approach to Noreen et al. (2013) was employed by Duncan and Clarke (2014) in terms of dose of R. rosea utilized. They asked 10 male recreational exercisers to undertake a 30-min cycle test at an intensity of 70% VO2 max on two occasions and once at 60 min, following ingestion of 3-mg/kg body mass R. rosea or a placebo (maltodextrin). The Duncan and Clarke (2014) study differed from those published previously in that a fixed-time, steady-state exercise trial was utilized rather than a more performance-oriented exercise trial, such as time to exhaustion or time trial as used by De Bock et al. (2004) and Noreen et al. (2013), respectively. In their study, Duncan et al. examined the effect of R. rosea ingestion on energy expenditure, carbohydrate, and fat metabolism. They also examined RPE and heart rate during the 30-min exercise trial. Mood state was assessed using the Brunel Mood State Inventory and exercise affect using the Feeling Scale and Felt Arousal Scale before and after the 30-min cycling trial. The rationale for the population sampled and experimental approach taken by Duncan and Clarke (2014) was that such data were more applicable to a typical gym-type scenario where active individuals would undertake a set bout of exercise. Duncan and Clarke (2014) reported that R. rosea ingestion did not significantly change energy expenditure, carbohydrate, or fat oxidation compared to placebo. There was also no significant difference in exercise heart rate between the R. rosea and placebo trials.

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However, while R. rosea ingestion did not appear to influence physiological responses to the 30-min cycle trial, there were differences in the psychological and perceptual variables examined by Duncan and Clarke (2014). RPE values were significantly lower in the R. rosea trial at the end of the 30-min exercise bout, as well as high scores for vigor on the Brunel Mood State Inventory (BRUMS) and significantly higher scores for pleasure on the Feeling Scale postexercise compared to placebo. Fig. 11.1 presents data from Duncan and Clarke (2014) for the feeling scale. These data potentially offer a viable explanation for any ergogenic effect of R. rosea ingestion during exercise. The Feeling Scale is used to quantify pleasure/displeasure on an 11-point Likert scale scored from 5 (Very Bad) to þ5 (Very Good). The data presented by Duncan and Clarke (2014) suggest that Feeling Scale scores were increased slightly following ingestion of R. rosea compared to placebo but before exercise. On exercise cessation, the Feeling Scales’ scores were maintained in the R. rosea trial. However, in the placebo trial, there was a distinct drop in feeling of pleasure at the same time point. This is an interesting observation, as no study on R. rosea had examined any potential impact on exercise affect. Duncan and Clarke (2014) suggested that hedonic theory may offer some explanations for the potential mechanism of action for R. rosea ingestion. This will be explored later in this chapter (Table 11.1).

Rhodiola rosea Combined With Other Substances A small number of studies have also examined the effect of R. rosea combined with other substances on various aspects of physical performance. Such studies do make it difficult

FIGURE 11.1 Mean  SE of perception of pleasure/displeasure before ingestion, after ingestion but before exercise and after exercise between Rhodiola rosea and placebo conditions (*P ¼ .0003). Taken from Duncan, M.J., Clarke, N.D., 2014. The effect of acute R. rosea ingestion on exercise heart rate, substrate utilisation, mood state, and perceptions of exertion, arousal, and pleasure/displeasure in active men. J. Sports Med. 2014, 563043.

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to attribute any effects reported to R. rosea alone but are worthy of mention, as they add to the understanding of any potential ergogenic effect of R. rosea. Studies by Earnest et al. (2004) and Colson et al. (2005) combined R. rosea with Cordyceps sinensis. Both used capsules of 1000 mg of C. sinensis and 300 mg R. rosea but in different total doses. In both cases, an initial loading dose of 6 capsules/day was employed for 4 days (Earnest et al., 2004) or 6 days (Colson et al., 2005), followed by maintenance doses of 3 capsules/ day for 11 days (Earnest et al., 2004) or 7 days (Colson et al., 2005). These were compared to placebo groups. In both these studies, no beneficial effect of substance ingestion was found on aerobic endurance performance and related parameters. The studies by Earnest et al. (2004) and Colson et al. (2005) are interesting in their use of combined substances that might have an ergogenic effect when combined. However, it is not clear why in these studies the addition of C. sinensis was thought to add to the ergocentricity of R. rosea, nor was the loading schedule of the substances clear in terms of an initial loading and then maintenance dose. It is thus not clear from the work of Earnest et al. (2004) and Colson et al. (2005) whether or not R. rosea can favorably influence exercise performance. More recently, Duncan et al. (2016) examined the effect of R. rosea (3 mg/kg), caffeine (3 mg/kg), combined caffeine, and R. rosea (3 mg/kg of each substance) on 5km running time trial compared to placebo in a sample of 12 males. In their study, acute caffeine ingestion significantly improved 5-km running time trial performance compared to placebo. However, ingestion of R. rosea or R. rosea combined with caffeine did not significantly improve time trial performance over that seen with placebo. In this study, there were also no differences in exercise heart rate, RPE, or measures of exercise affect across conditions. The design employed by Duncan et al. (2016) allows for the study of any effect of R. rosea and caffeine either alone or in combination of exercise performance, which was not possible in other studies where R. rosea combined with other substances has been examined. The authors of this study, however, concluded that although caffeine appears to offer ergogenic benefits for short-term running, R. rosea alone or when combined with caffeine did not offer any such benefit. In the Duncan et al. (2016) study, the choice to examine R. rosea in conjunction with caffeine was made based on the assumption that both caffeine and R. rosea had shown ergogenic effects on exercise performance in the literature. Duncan et al. (2016) argued that the combination of two ergogenic substances might be synergistic. While this does not appear to be the case in the aforementioned study, there is remit to further examine potential synergistic effects of R. rosea with other substances.

Mechanisms of Action Over the course of scientific study of the potential ergogenic effects of R. rosea ingestion on exercise performance and associated variables, a number of potential mechanisms of action have been proposed. To date, none of these have received any considerable

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empirical support, and the exact mechanism of action of R. rosea remains to be fully elucidated. Early research suggested R. rosea acted on inflammatory markers in a way that mimicked changes seen with long-term endurance training (Abidov et al., 2004). Blood levels of inflammatory markers such as C-reactive protein (CRP) and creatine kinase (CK) are normally elevated after exercise in proportion to the amount of exercise undertaken (Kasapis and Thompson, 2005; Lakka et al., 2005). However, in one study, participants who ingested 680 mg RHODAXTM (containing 60 mg R. Rosea) for 30 days prior to and 6 days following exhausting cycling exercise displayed lower levels of CRP and CK compared to those who ingested a placebo. The authors suggested that R. rosea ingestion may mimic adaptations that normally occur as a consequence of longer-term endurance training. Several other studies have supported the notion that R. rosea may influence inflammation and free radical formation. De Sanctis et al. (2004) introduced an oxidant (hypochlorous acid) to in vitro red blood cells that had been incubated with R. rosea. They reported that R. rosea protected, in a dose-dependent manner, human erythrocytes from the glutathione depletion, glyceraldehyde-3-phosphate dehydrogenase inactivation, and hemolysis induced by the oxidant. Likewise, in a study by Wing et al. (2003), participants were asked to ingest 1788 mg of R. rosea or a placebo for a 7day period while they also underwent repeated normobaric hypoxia exposure. Wing et al. reported that blood oxygen levels were not influenced in the R. rosea group, but lipid peroxides decreased. This was taken as an indication that R. rosea ingestion can decrease free radical formation caused by hypoxia. Thus, there is some tentative support for the notion that a mechanism by which R. rosea may positively influence exercise performance and associated physiological responses could be due to reduced inflammation and decreased free radical formation. However, the empirical data investigating this suggestion are sparse and certainly not sufficient to support these assertions to date. Other physiological-based mechanisms for the effect of R. rosea have also been suggested, such as R. rosea ingestion improving exercise performance via altered energy metabolism (Duncan and Clarke, 2014), activated by the synthesis or resynthesis of ATP in mitochondria and stimulated restorative energy processes after intense exercise (Abidov et al., 2003). Research has yet to support this assertion, with the most related exercise-type study to date showing no effect of R. rosea ingestion on exercise metabolism (Duncan and Clarke, 2014). The other purported mechanism by which R. rosea ingestion may positively influence exercise performance related to hedonic theory (Kahneman et al., 1993) and R. rosea’s potential influence on mood. Hedonic theory suggests that hedonic responses (pleasure or displeasure) following a behavior influence decisions regarding whether or not to repeat that behavior. As a consequence, if nutritional manipulation enhances the pleasure response to exercise, it is possible that this makes an individual more likely to exercise again and to exercise more vigorously. In relation to this suggestion, various authors have suggested that the performance enhancement reported as a consequence of R. rosea ingestion may result from this mechanism, subsequently impacting brain dopamine and attenuating perception of effort at a given workload (MacLeod, 1991).

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There is some evidence that RPE during exercise is higher when participants ingest the opioid receptor antagonist naloxone and that this is associated with a reduction in exercise performance (Hickey et al., 1992; Sgherza et al., 2002). Likewise, De Bock et al. (2004) attributed their observed increase in time to exhaustion following acute ingestion of R. rosea to an increase in opioid production. Disappointingly, De Bock et al. (2004) did not measure either endogenous opioids or RPE during exercise, which limits the extent to which they can attribute their responses to opioid production with R. rosea ingestion. Studies by Noreen et al. (2013) and Duncan and Clarke (2014) have added support to these suggestions via their reported reduced RPE values during exercise and changes in exercise affect/mood after ingestion of R. rosea. Taken with the postulations of hedonic theory and the reported changes in mood and exercise affect by Noreen et al. (2013) and Duncan and Clarke (2014), a mechanism for action of R. rosea through increased opioid production appears viable and attractive. However, no study to date has actually assessed endogenous opioid production as a consequence of R. rosea ingestion. Until this assessment takes place, the role of R. rosea in enhancing exercise performance via changes in exercise affect from increasing opioid production cannot be supported.

How to Take Supplementation of R. rosea tends to refer to either the SHR-5 extract in particular or an equivalent extract. R. rosea extracts used in most human clinical studies were standardized to a minimum of 3% rosavins and 0.8%e1% salidrosides because the naturally occurring ratio of these compounds in R. rosea root is approximately 3:1 (Khanum et al., 2005). The usage of R. rosea as a daily preventative against fatigue has been reported to be effective in doses as low as 50 mg. Acute usage of Rhodiola for fatigue and antistress has been noted to be taken in the 288e680 mg range. However, clinical studies report R. roseaeonly products ranging in dose from 50 mg to 660 mg per capsule, to a maximum of 1500 mg/day, suggesting a large margin of safety. However, as R. rosea has been shown to have a bell curve response, it is suggested that exceeding the aforementioned 680-mg dosage may be ineffective. Furthermore, studies reporting a positive effect of R. rosea on physical performance reported doses of 200 mg/day and 680 mg/ day, and those reporting a positive effect on mental fatigue reported doses between 100 and 576 mg/day. Finally, R. rosea is best absorbed when taken on an empty stomach at 30 min before a meal.

Side Effects R. rosea has demonstrated a very low occurrence of side effects demonstrating a low clinical toxicity. In rat toxicity studies, the lethal dose at which 50% of animals die was calculated to be 28.6 mL/kg, approximately 3360 mg/kg (Kurkin and Zapesochnaya, 1985). The equivalent dosage in a 70-kg man would be about 235 g or 235,000 mg.

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Because the usual clinical doses are 200e600 mg/d, there is a large margin of safety (Udintsev and Schakov, 1991). Overall, R. rosea appears to have very few side effects. In systematic review of 11 studies including 446 participants, only 5 adverse events were mentioned in 3 studies (Ishaque et al., 2012). Similarly, Hung et al. (2011) reported that eight random control trials (Darbinyan et al., 2000, 2007bib_Darbinyan_et_al_2007; De Bock et al., 2004; Olsson et al., 2009; Shevtsov et al., 2003; Spasov et al., 2000a,b; Wing et al., 2003) provided information on adverse events. Only three cases of adverse events, including headache (De Bock et al., 2004), hypersalivation (Shevtsov et al., 2003), and one unknown illness (Wing et al., 2003), were reported, and none of them were described as serious. The first two adverse events reported were both from the placebo groups. The unspecified adverse event, which caused the patient to drop out of the trial, was not specified in terms of group allocation. The authors concluded that no major risks have been associated with R. rosea. Therefore, it would appear that there are few side effects associated with R. rosea supplementation, and those identified are of a mild nature. Although no contraindications with other herbal or prescription medications have been identified, it is important to consider that R. rosea may have an additive effect with other substances exhibiting stimulant properties (Williams, 2005). Like many natural health products, the likelihood of adequate reporting of adverse events may be lower than conventional medications (Fugh-Berman and Ernst, 2001). However, as with any herbal supplements, patients should inform their primary health-care practitioner when taking R. rosea and experiment in training prior to important events.

Conclusions and Gap Analysis R. rosea appears a safe substance for human ingestion with few, if any, side effects. There is some evidence that is suggestive of a positive effect of R. rosea ingestion on exercise performance, potentially via changes in affect and dampened perception of exertion. This is a result of the purported effect of R. rosea as an opioid. However, of the research that has examined R. rosea, an equal body of studies suggest negligible or no effects of R. rosea ingestion on performance and associated variables. Consequently, there is a need for additional, well-controlled, double-blind laboratory studies on the effect of both acute and chronic ingestion of R. rosea on exercise performance and physiological and psychological responses during exercise. Without this, the suggested performance-enhancing benefits of ingesting R. rosea will remain, and research needs to comprehensively establish if R. rosea ingestion has any benefit over a placebo, or not, in relation to exercise performance. Future research then needs to ascertain the following:  if there is an optimum dose of R. rosea that might be ergogenic;  if the effects of R. rosea ingestion differ depending on the mode and duration of the exercise undertaken;  if training status influences the potential egocentricity of R. rosea; and

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 if chronic loading with R. rosea produces different effects to acute ingestion of R. rosea. As the literature stands, at the present time, the body of research that is currently available examines the effect of R. rosea ingestion, either acutely or chronically, is not sufficient to make robust conclusions as to the efficacy of R. rosea on exercise performance, metabolism, cardiovascular responses, or exercise affect.

References Abidov, M., Crendal, F., Grachev, S., Seifulla, R., Ziegenfuss, T., 2003. Effect of extracts from Rhodiola rosea and Rhodiola crenulata (Crassulaceae) roots on ATP content in mitochondria of skeletal muscles. Bull. Exp. Biol. Med. 136, 585e587. Abidov, M., Grachev, S., Seifulla, R.D., Ziegnenfuss, T., 2004. Extract of Rhodiola rosea radix reduces the level of C-reactive protein and creatinine kinase in the blood. Bull. Exp. Biol. Med. 138, 63e64. Al-Kuraishy, H.M., 2016. Central additive effect of Ginkgo biloba and Rhodiola rosea on psychomotor vigilance task and short term working memory accuracy. J. Intercult. Ethnopharmacol. 5, 7e13. Alm, T., 2004. Ethnobotany of Rhodiola rosea (Crassulaceae) in Norway. SIDA 21, 321e344. Amsterdam, J.D., Panossian, A.G., 2016. Rhodiola rosea L. as a putative botanical antidepressant. Phytomedicine 23, 770e783. Brekhman, I., Dardymov, I.V., 1969. New substances of plant origin which increase non-specific resistance. Annu. Rev. Pharmacol. 9, 419e430. Brown, R.P., Gerbarg, P.L., Ramazanov, Z., 2002. Rhodiola rosea: a phytomedicinal overview. HerbalGram 56, 40e52. Chang, J.C., Liao, Y.H., Chen, C.Y., Lin, C.H., 2016. Effect of Rhodiola mixture supplementation and exercise training on body composition and physical activity in rats. Med. Sci. Sports Exerc. 48, S246. Colson, S.N., Wyatt, F.B., Johnston, D.L., Autrey, L.D., FitzGerald, Y.L., Earnest, C.P., 2005. Cordyceps sinensis- and Rhodiola rosea-based supplementation in male cyclists and its effect on muscle tissue oxygen saturation. J. Strength Condition. Res. 19, 358e363. Cropley, M., Banks, A.P., Boyle, J., 2015. The effects of Rhodiola rosea L. extract on anxiety, stress, cognition and other mood symptoms. Phytother. Res. 29, 1934e1939. Darbinyan, V., Aslanyan, G., Amroyan, E., Gabrielyan, E., Malmstro¨m, C., Panossian, A., 2007. Clinical trial of Rhodiola rosea L. extract SHR-5 in the treatment of mild to moderate depression. Nord. J. Psychiatry 61, 343e348. Darbinyan, V., Kteyan, A., Panossian, A., Gabrielian, E., Wikman, G., Wagner, H., 2000. Rhodiola rosea in stress induced fatigueea double blind cross-over study of a standardized extract SHR-5 with a repeated low-dose regimen on the mental performance of healthy physicians during night duty. Phytomedicine 7 (5), 365e371. De Bock, K., Eijnde, B.O., Ramaekers, M., Hespel, P., 2004. Acute Rhodiola rosea intake can improve endurance exercise performance. Int. J. Sports Nutr. Exerc. Metab. 14, 298e307. DeSanctis, R., De Bellis, R., Scesa, C., Mancini, U., Cucchiarini, L., Dacha, M., 2004. In vitro protective effect of Rhodiola rosea extract against hypochlorous acid-induced oxidative damage in human erythrocytes. Biofactors 20, 147e159. Duncan, M.J., Clarke, N.D., 2014. The effect of acute Rhodiola rosea ingestion on exercise heart rate, substrate utilisation, mood state, and perceptions of exertion, arousal, and pleasure/displeasure in active men. J. Sports Med. 2014, 563043.

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Duncan, M.J., Tallis, J., Wilson, S., Clarke, N.D., 2016. The effect of caffeine and Rhodiola rosea, alone or in combination on 5 km running performance in men. J. Caffeine Res. 6, 40e48. Earnest, C.P., Morss, G.M., Wyatt, F., Jordan, A.N., Colson, S., Church, T.S., Fitzgerald, Y., Autrey, L., Jurca, R., Lucia, A., 2004. Effects of a commercial herbal-based formula on exercise performance in cyclists. Med. Sci. Sports Exerc. 36, 504e509. Fugh-Berman, A., Ernst, E., 2001. Herb-drug interactions: review and assessment of report reliability. Br. J. Clin. Pharmacol. 52, 587e595. Gerbag, P.L., Brown, R.P., 2016. Pause menopause with Rhodiola rosea, a natural selective estrogen receptor modulator. Phytomedicine 23, 763e769. Grossman, A., Sutton, J.R., 1985. Endorphins: what are they? How are they measured? What is their role in exercise? Med. Sci. Sports Exerc. 17, 74e81. Hickey, M.S., Franke, W.D., Herbert, W.G., Walberg-Rankin, J., Lee, J.C., 1992. Opioid antagonism, perceived exertion and tolerance to exercise-thermal stress. Int. J. Sports Med. 13, 326e331. Hung, S.K., Perry, R., Ernst, E., 2011. The effectiveness and efficacy of Rhodiola rosea L.: a systematic review of randomized clinical trials. Phytomedicine 18, 235e244. Ishaque, S., Shamseer, L., Bukutu, C., Vohra, S., 2012. Rhodiola rosea for physical and mental fatigue: a systematic review. BMC Complement. Altern. Med. 12, 70. Kahneman, D., Fredrickson, B.L., Schreiber, C.A., Redelmeier, D.A., 1993. When more pain is preferred to less: adding a better end. Psychol. Sci. 4, 401e405. Kasapis, C., Thompson, P.D., 2005. The effects of physical activity on serum C-reactive protein and inflammatory markers: a systematic review. J. Am. Coll. Cardiol. 45, 1563e1569. Kelly, G.S., 2001. Rhodiola rosea: a possible plant adaptogen. Altern. Med. Rev. 6, 293e302. Khaidaev, Z., Menshikova, T.A., 1978. Medicinal Plants in Mongolian Medicine. Mongolia: Ulan-Bator. Khanum, F., Bawa, A.S., Singh, B., 2005. Rhodiola rosea: a versatile adaptogen. Compr. Rev. Food Sci. Food Saf. 4, 55e62. Kurkin, V.A., Zapesochnaya, G.G., 1985. Chemical composition and pharmacological characteristics of Rhodiola rosea [review]. J. Med. Plants, Russ. Acad. Sci. 1231e1445. Lakka, T.A., Lakka, H.M., Rankinen, T., Leon, A.S., Rao, D.C., Skinner, J.S., Wilmore, J.H., Bouchard, C., 2005. Effect of exercise training on plasma levels of C-reactive protein in healthy adults: the HERITAGE Family Study. Eur. Heart J. 26, 2018e2025. MacLeod, C.M., 1991. Half a century of research on the Stroop effect: an integrative review. Psychol. Bull. 109, 163e203. Magnusson, B., 1992. Beauty: Herbs that Touch Us. Berndtssons, Ostersun, Sweden. Mattioli, L., Perfumi, M., 2011. Effects of a Rhodiola rosea L. extract on acquisition and expression of morphine tolerance and dependence in mice. J. Psychopharmacol. 25, 411e420. Morgan, M., Bone, K., 2005. Rhodiola: the arctic adaptogen. Townsend Lett. Doctors Patients 262, 26e28. Nabavi, S.M., Habtemariam, S., Daglia, M., Braidy, N., Loizzo, M.R., Tundis, R., et al., 2015. Neuroprotective effects of ginkgolide B against ischemic stroke: a review of current literature. Curr. Top. Med. Chem. 15, 2222e2232. Noreen, E.E., Buckley, J.G., Lewis, S.L., Brandauer, J., Stuempfle, K.J., 2013. The effects of an acute does of Rhodiola rosea on endurance exercise performance. J. Strength Condition. Res. 27, 839e847. Olsson, E.M., von Scheele, B., Panossian, A.G., 2009. A randomised, double-blind, placebo-controlled, parallel-group study of the standardised extract shr-5 of the roots of Rhodiola rosea in the treatment of subjects with stress-related fatigue. Planta Med. 75, 105e112. Saratikov, S.A., Krasnov, E.A., 1987. Rhodiola rosea Is a Valuable Medicinal Plant (Golden Root). Tomsk State University Press, Tomsk, Russia.

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Sgherza, A.L., Axen, K., Fain, R., Hoffman, R.S., Dunbar, C.C., Haas, F., 2002. Effect of naloxone on perceived exertion and exercise capacity during maximal cycle ergometry. J. Appl. Physiol. 93, 2023e2028. Shanley, R.A., Nieman, D.C., Zwetsloot, K.A., Knab, A.M., Imagita, H., Luo, B., Davis, B., Zubeldia, J.M., 2014. Evaluation of Rhodiola rosea supplementation on skeletal muscle damage and inflammation in runners following a competitive marathon. Brain Behav. Immun. 39, 24e39. Shevtsov, V.A., Zholus, B.I., Shervarly, V.I., Vol’skij, V.B., Korovin, Y.P., Khristich, M.P.N.A., Roslyakova, N. A., Wikman, G., 2003. A randomized trial of two different doses of a SHR-5 Rhodiola rosea extract versus placebo and control of capacity for mental work. Phytomedicine 10, 95e105. Spasov, A.A., Wikman, G.K., Mandrikov, V.B., Mironova, I.A., Neumoin, V.V., 2000a. A double-blind, placebo-controlled pilot study of the stimulating and adaptogenic effect of Rhodiola rosea SHR-5 extract on the fatigue of students caused by stress during an examination period with a repeated low-dose regimen. Phytomedicine 7, 85e89. Spasov, A.A., Mandrikov, V.B., Mironova, I.A., 2000b. The effect of the preparation rodakson on the psychophysiological and physical adaptation of students to an academic load. Eksperimentalnaia i Klinicheskaia Farmakologiia 63, 76e78. Udintsev, S.N., Schakhov, V.P., 1991. Decrease of cyclophosphamide haematotoxicity by Rhodiola rosea root extract in mice with Ehrlich and Lewis transplantable tumors. Eur. J. Cancer 27, 1182. Walker, T.B., Robergs, R.A., 2006. Does Rhodiola rosea possess ergogenic properties? Int. J. Sports Nutr. Exerc. Metab. 16, 305e315. Williamson, E.M., 2005. Interactions between herbal and conventional medicines. Expert Opin. Drug Saf. 4, 355e378. Wing, S.L., Askew, E.W., Luetkemeier, M.J., Ryujin, D.T., Kamimori, G.H., Grissom, C.K., 2003. Lack of effect of Rhodiola or oxygenated water supplementation on hypoxemia and oxidative stress. Wilderness Environ. Med. 14, 9e16.

Further Reading Lee, F.T., Kuo, T.Y., Liou, S.Y., Chien, C.T., 2009. Chronic Rhodiola rosea extract supplementation enforces exhaustive swimming tolerance. Am. J. Chin. Med. 37, 557e572.

Energy and Health Benefits of Shilajit

12

Sidney J. Stohs1, Kanhaiya Singh2, Amitava Das2, Sashwati Roy2, Chandan K. Sen2 1 CR EIGH TON UNIVERS ITY SCHOOL OF PHARMACY AND HEALTH PROFESSIONS, OMAHA, NE, U N I TE D ST AT ES ; 2 THE OHIO STATE UNIVERSITY WEXNER MEDI CAL CE NTER, COLUMBUS, OH, UNITED STATES

Introduction Shilajit is a resinous phytomineral exudate found in sedimentary rocks that has an extensive history of use in traditional folk medicine, including Ayurveda. It is a brown to black product that is extruded from layers of rocks in mountainous regions during the hottest months of the year. It is known by a variety of other names, including mumie, moomiyo, mummiyo, mumijo, silajatu, and salajeet. It is obtained from various mountainous regions of India, Tibet, China, Russia, Afghanistan, Nepal, and the former USSR (Caucasus, Ural, Altai, Sayan, Kazakhstan, Uzbekistan, Baykal, and Tajikistan) (Schepetkin et al., 2002; Agarwal et al., 2007; Stohs, 2014). Shilajit has had many applications in folk medicine with numerous anecdotal reports of therapeutic efficacy. Although there have been a limited number of well-designed, placebo-controlled human and animal studies establishing efficacy, various studies have confirmed the safety and efficacy of shilajit. Shilajit, as the proprietary product PrimaVie, received GRAS (generally recognized as safe) status in 2015. Shilajit has been used as an adaptogen and anabolic, and has been known for promoting both physical and mental energy (Acharya et al., 1988; Schepetkin et al., 2002; Ghosal, 2006; Agarwal et al., 2007; Wilson et al., 2011; Stohs, 2014). In the former USSR, it was used surreptitiously for many years to enhance performance of Olympic athletes and special military forces while reducing stress-related injuries and facilitating recovery (Bucci, 2000). Various studies indicate that it possesses antiinflammatory and antioxidant properties and functions as a chemoprotectant and immunomodulator. As a consequence of these broad properties, shilajit has historically been used to treat stomach disorders and ulcers, bone fractures, inflammatory joint conditions, impotence, nerve and cardiovascular disorders, diabetes, wounds, muscle and tendon strains, and urinary tract infections as well as being used to promote physical performance and Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00012-0 Copyright © 2017 Elsevier Inc. All rights reserved.

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energy. It is also used to promote general health and longevity. In the United States, it is used as a dietary supplement, either as a stand-alone product or in combination with other ingredients. This review will summarize the human and animal research supporting these health claims, with emphasis on well-controlled studies conducted and published in the past 10 years.

Chemistry The chemical composition of shilajit depends upon many environmental and geographical factors as well as whether the product has been processed and purified (Stohs, 2014; Raju, 2012; Agarwal et al., 2007; Ghosal, 2006; Schepetkin et al., 2003). The fulvic acid (Fig. 12.1) content has been shown to vary with the country and region of origin (Ghosal et al., 1991). Fulvic acid is a polyhydroxy polycarboxylic acid produced by the biodegradation of organic matter and has high chelating and complexing ability. High-quality products used in dietary supplements are standardized to contain at least 50% fulvic acids and equivalents (polymers and related structures) along with dibenzoa-pyrones (DBPs) and DBP chromoproteins. DBPs and DBP chromoproteins are usually present in greater than 10% (Raju, 2012; Stohs, 2014). More recently, five new diterpenoids, referred to as mumic acids AeE, have been isolated and structurally characterized using spectroscopic data and chemical derivatization (Kiren et al., 2014). High-quality products used in dietary supplements should have a water-soluble extraction value greater than 80%. As many as 40 or more total minerals have been reported in a polyphenolic complex in shilajit, with the majority of these in small or trace amounts (Frolova and Kiseleva, 1996). In processed shilajit, the sum of potassium, calcium, and magnesium generally make up over 90% of the total mineral content, with sulfur and sodium being the next most common minerals (Raju, 2012). Variations in color of shilajit are generally due to differences in the content of minerals such as iron, copper, and silver (Ghosal, 2006; Agarwal et al., 2007). OH OH

COOH

COOH

HOOC O OH HOOC COOH

OH

COOH O

FIGURE 12.1 Fulvic acid.

Chapter 12  Energy and Health Benefits of Shilajit 189

The physiological and pharmacological effects of shilajit are attributed to the DBPs, DBP chromoproteins (DBPs conjugated to proteins), fulvic acid, and various polymeric forms of fulvic acid (Ghosal, 2006; Sharma et al., 2003; Schepetkin et al., 2003; Raju, 2012). Some individuals believed that the primary effects of shilajit were due to the ability of fulvic acid constituents to chelate the minerals associated with the product and facilitate cellular penetration (Agarwal et al., 2007; Carrasco-Gallardo et al., 2012). The overall mineral content of shilajit is small. At the doses given, it is doubtful that significant amounts of minerals are absorbed and penetrate cells, since the vast majority of minerals that are present occur in exceedingly small amounts. For example, in a typical shilajit dose of 200 mg, the total mineral content will be 2e3 mg, with about 90% being potassium, calcium, and magnesium. To put this in perspective, the typical daily recommended intake for calcium is 1000e1200 mg, whereas the daily values for magnesium and potassium are 400 mg and 3000 mg, respectively. Because of the relative rarity of the material, its overall complex nature, the processing required to prepare the final product, and the difficulty in standardizing the finished product, counterfeiting and adulteration are major problems (Schepetkin et al., 2003; personal experience of author). As a consequence, consumers are cautioned to use products from known and reputable manufacturers and suppliers.

Safety Studies Various studies in animals and humans have demonstrated the safety of shilajit, which has led to its receiving a self-affirmed GRAS designation. The acute lethal dose at which 50% of animals die (LD50) of a purified shilajit in rats was found to be 1000 mg/kg when given intraperitoneally (IP) and was greater than 2000 mg/kg when shilajit was given orally (Acharya et al., 1988). No internal organ histological or morphological changes were observed in rabbits or mice given 100 and 500 mg/kg shilajit (mumie) orally in water for 30 days (Kelginbaev et al., 1973). Shilajit (mumie) was given to rats at doses of 200 and 1000 mg/kg for 90 days (subchronic toxicity study) and produced no adverse effects on liver, kidneys, heart, blood cells, or nervous and endocrine systems (Anisimov and Shakirzyanova, 1982). Furthermore, shilajit (mumie) did not cause any embryotoxic or teratogenic effects in pregnant rats (Anisimov and Shakirzyanova, 1982) or mice (Al-Hamaidi et al., 2003). The LD50 of fulvic acids, which had been isolated from shilajit, was 1268 mg/kg when given orally to rats (Ghosal, 2006), indicating low toxicity. In an unpublished toxicity study (Raju, 2012), “purified” (processed and standardized) shilajit administered to rats at doses of 200 mg/kg and 400 mg/kg orally for 90 days did not produce any hepatic, renal, hemopoietic, or behavioral effects, and at an oral dose of 2000 mg/kg was well tolerated. In addition, no significant changes in weights of vital organs were observed as compared to control animals. Furthermore, doses of 10, 30, and 100 mg/kg of processed shilajit given to mice did not produce any metaphase

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chromosomal aberrations in bone marrow (Raju, 2012), indicating that the shilajit was not genotoxic. In a placebo-controlled, double-blind, randomized study in arthritic dogs, the twice daily administration of 500 mg purified shilajit for 5 months resulted in no changes in physical parameters or serum biomarkers (Lawley et al., 2013). Biomarkers of liver [bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)], kidney (blood urea nitrogen and creatinine), heart, and muscle functions (creatine kinase) were assessed, while physical parameters measured included heart rate, body temperature, respiration rate, and body weight. Velmurugan et al. (2012) evaluated the safety of shilajit by administering groups of rats 500, 2500, or 5000 mg/kg daily for 90 days. The histology of all organs was normal except for what the authors referred to as “negligible changes” in liver and intestine at the highest dose. The weights of all organs were normal as compared to the control animals. Several studies have examined the human safety of shilajit. In a placebo-controlled study involving 20 healthy subjects given 2000 mg of processed shilajit daily for 45 days in capsule form, no significant changes in blood pressure, heart rate, or body weight were observed (Sharma et al., 2003). In addition, shilajit had no effect on blood glucose, urea, creatinine, uric acid, total protein, albumin, albumin/globulin ratio, alkaline phosphatase, ALT, or AST. Shilajit exhibited no evidence of systemic toxicity under these conditions. The administration of processed shilajit at a dose of 100 mg twice a day to 28 male subjects for 90 days had no significant effect on renal profile parameters, including urea, albumin, total protein, globulin, uric acid, bilirubin, alkaline phosphatase, ALT or AST (Biswas et al., 2009). Small but significant decreases in fasting blood glucose and creatinine levels were observed in shilajit-treated subjects. The results indicate that under these conditions, shilajit produced no evidence of systemic toxicity. In an unpublished safety study involving 43 healthy human volunteers (Raju, 2012), processed shilajit was given at a dose of 250 mg twice a day for 90 days. No changes in kidney or liver function tests were observed. Shilajit treatment decreased fasting blood sugar, uric acid, and erythrocyte sedimentation rate while increasing percent hemoglobin and platelet count. In a double-blind, placebo-controlled study, Sharma et al. (2003) administered 2000 mg of processed shilajit or placebo per day for 45 days to human subjects. Twenty subjects received the shilajit, whereas 10 subjects received the placebo. Significant decreases in serum cholesterol, low density lipoprotein, very low density lipoprotein, and triglycerides were observed in response to shilajit as compared to the placebo group. Improved antioxidant status in the form of increases in superoxide dismutase, vitamin C, and vitamin E was observed. High density lipoprotein also increased in shilajit-treated subjects. In summary, various studies with shilajit (mumie) in both animals and humans have demonstrated a very high degree of safety.

Chapter 12  Energy and Health Benefits of Shilajit 191

Research Studies For thousands of years, shilajit (moomiyo, mummiyo, and mumie) has been used in folk medicine in India and Northern Asia (Schepetkin et al., 2002; Agarwal et al., 2007; Wilson et al., 2011). It has also been used as a performance-enhancing agent in the former USSR for many years as well as the treatment of various human maladies. Most research regarding moomiyo (mumie, shilajit) involving sports performance in the former USSR has not been published. An ever-increasing number of studies have been conducted in animals that examine the physiological/pharmacological effects and mechanisms of action of shilajit. A growing number of studies in animals and in vitro systems using standardized materials have been conducted. However, the number of peer-reviewed scholarly publications in the scientific literature involving human subjects remains small. Much of the early literature have involved anecdotal reports, poorly controlled studies, studies involving products of unknown composition, and publication of results in obscure journals (Schepetkin et al., 2002; Goshal, 2006; Agarwal et al., 2007; Wilson et al., 2011). This review summarizes published human, animal, and in vitro research studies as well as a number of unpublished research reports involving well-designed studies.

Human Studies A number of human studies have examined the effects of shilajit on energy production, testosterone and spermatogenesis, and muscle adaptation. Biswas et al. (2009) evaluated the spermatogenic activity of shilajit. Thirty-five infertile (oligospermic) male subjects were given 100 mg processed shilajit in capsule form twice a day for 90 days. Significant increases in normal (18.9%) and total (61.4%) sperm count and sperm motility (12.4% e17.4%) were observed in the 28 subjects who completed the study. A significant decrease in semen malondialdehyde levels was also observed, indicating that shilajit exhibited antioxidant activity. Furthermore, in addition to the increase in sperm count, shilajit treatment significantly increased serum testosterone (23.5%) and folliclestimulating hormone (FSH) (9.4%) levels. In a randomized, placebo-controlled, double-blind study involving healthy male subjects (45e55 years), the effects of purified shilajit on serum testosterone levels were examined (Pandit et al., 2016). Seventy-five subjects (38 treated; 37 control) completed the study. Treated subjects received 250 mg shilajit twice daily for 90 days, which resulted in significant increases in serum total testosterone (31.0%), free testosterone (51.1%), and dehydroepiandrosterone (37.3%). No significant changes were observed in the gonadotropic hormones FSH and luteinizing hormone. These results supported and confirmed the beneficial androgenic effects of purified shilajit. An unpublished pilot study involving six healthy human volunteers examined energy production and physical activity (Raju, 2012). The subjects were given 200 mg processed shilajit once daily for 15 days. Treatment with shilajit significantly increased energy

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production and physical exercise (Harvard step test). In addition, the energy production was confirmed based on increases in adenosine triphosphate (ATP), ATP/adenosine diphosphate (ADP) ratio, coenzyme Q10 (CoQ10), total adenine nucleotides, adenylate energy charge, and uric acid levels in whole blood. This study would have benefited from an adequate number of subjects. Nutrition-dependent skeletal muscle adaptation involves the proper control of regulatory processes through an array of gene expression changes. Proper balance between protein synthesis and protein degradation is important in maintaining the skeletal muscle mass in mature individuals (Goodman et al., 2011). Preserving muscle mass during mechanical unloading during orthopedic and related injuries is an important clinical issue, and therapies aimed at increasing the rate of protein synthesis may serve as important interventions. Protein synthesis is the cumulative result of translational efficiency and translational capacity (McCarthy and Esser, 2010). Translational capacity is the amount of protein synthesized from one unit of RNA, while translational capacity is reflected by the amount of ribosomes present per unit of the tissue. A study was conducted to determine the effects of a purified and standardized shilajit in skeletal muscle adaptation in 16 overweight (average BMI of 28.9) individuals (Das et al., 2016). Subjects consumed shilajit at 250 mg orally twice daily after a baseline visit for 8 weeks followed by supplementation for 4 weeks with exercise. Global gene expression profile using Affymetrix GeneChip Human Transcriptome Array 2.0 identified a cluster of 17 extracellular matrixerelated probe sets that were significantly upregulated in muscles following 8 weeks of supplementation as compared to baseline. This cluster of genes included tenascin XB, decorin, myoferlin, collagen (types I, III, V, VI, and XIV), elastin, fibrillin I, and fibronectin I. The upregulation was confirmed by using real time polymerase chain reaction. Supplementation did not alter lipid profiles, blood glucose levels, or muscle damage markers, including creatine kinase and serum myoglobin (Das et al., 2016). The results indicated that supplementation with shilajit promoted skeletal muscle adaptation via upregulation of this set of related genes and therefore may be beneficial as a fitness and sports performance supplement. Data mining through Ingenuity Pathway Analysis tool (IPA) (Ingenuity Systems, www.ingenuity.com) identified novel biological pathways affected by administering shilajit orally in experimental animals. The analysis of generated major pathways suggested that the administration of shilajit improves both the translational efficiency and capacity of the tissue by targeting eukaryotic initiation factor 2 (eIF2) signaling (Correctedelog (P-value) ¼ 9.25), eIF4-p70S6K signaling (Correctedlog (P-value) ¼ 3.61), mechanistic target of rapamycin (mTOR) signaling (Correctedlog (P-value) ¼ 3.09), and regulating cellular junctional proteins (Correctedlog(P-value) ¼ 2.36) (Fig. 12.2, Table 12.1). mTOR signaling is one of the major protein synthesis pathways involved in increasing the translational efficiency of the cells (You et al., 2015). Previous studies have also shown that this increase in protein translation is through the phosphorylation of substrates such as eIF 4E binding protein 1 (4E-BP1) and p70 ribosomal protein S6 kinase (p70S6k), which then

0.15 5.0 0.10 2.5

0.05

RhoGDI Signaling

Breast Cancer Regulation by Stathmin1

Tight Junction Signaling

Remodeling of Epithelial Adherens Junctions

Protein Ubiquitination Pathway

Sertoli Cell-Sertoli Cell Junction Signaling

mTOR Signaling

phagosome maturation

Regulation of eIF4 and p70S6K Signaling

eIF2 Signaling

Threshold 0.0

Ratio

-log(B-H p-value)

0.20

7.5

0.00

FIGURE 12.2 The significant canonical pathways generated from the genes upregulated using Ingenuity Pathway Analysis tool (IPA) (Ingenuity Systems, www.ingenuity.com) having a elog(P value) of 1.3. BeH multiple testing correction. P-value was used as the scoring method for significant pathways sorting.

Table 12.1 Significant Canonical Pathways and Genes Which Were Upregulated After Supplementation With Purified and Standardized Shilajit Using Ingenuity Pathway Analysis Tool (IPA) Ingenuity Canonical Pathways

Llog(BeH P-value)

eIF2 signaling

9.25

Regulation of eIF4 and p70S6K signaling Phagosome maturation

3.61 3.46

mTOR signaling

3.09

Sertoli celleSertoli cell junction signaling Protein ubiquitination pathway

2.72

Remodeling of epithelial adherens junctions Tight junction signaling

2.36

Breast cancer regulation by Stathmin1 RhoGDI signaling

2.72

1.98 1.6 1.38

Molecules Involved RPL11, RAF1, EIF3C, RPLP1, RPS3A, RPS27, EIF4E, RPS4X, RPL27 A, EIF1, RPS20, RPS9, AKT3, RPS2, RPS3, RPL31, RPL34, RPL3, RPS10, EIF2S2, RPL10 A, RPL27, RPL26L1, RPS15 A, RPL13 A, RPSA, RPL38 RAF1, EIF3C, EIF1, RPS20, RPS3A, RPS27, RPS9, PPP2R5B, RPS10, AKT3, RPS15 A, RPS2, RPS3, RPS4X, EIF4E, EIF2S2, RPSA TUBA1B, CTSK, YKT6, HLA-A, PRDX1, TUBB4B, RAB7A, TUBB, ATP6AP1, GPAA1, TSG101, DYNLRB1, ATP6V0A1, TUBB6, CTSB MAPKAP1, EIF3C, RPS20, RPS3A, RPS27, STK11, RPS9, PPP2R5B, RAC1, RPS10, AKT3, RPS15 A, RPS2, RPS3, PRKD3, EIF4E, RPS4X, RPSA TUBA1B, RAF1, TJP2, TUBB4B, ACTB, RAC1, CTNNA1, JAM2, YBX3, NOS3, TUBB, TUBB6, PRKAR1B, PRKACA, AKT3, ACTG1 USP21, FZR1, CRYAB, HLA-A, HSPA9, HSPD1, HSPA8, TRAF6, PSMC1, PSMD10, HSP90AB1, CUL2, UBE2V1, PSMB1, PSMA4, ANAPC5, SMURF2, UBA1, UBC, PSMC3 TUBA1B, TUBB6, TUBB4B, ACTB, CTNNA1, RAB7A, TUBB, ACTG1, MAPRE3 TJP2, YKT6, MYL6, ACTB, PPP2R5B, RAC1, CTNNA1, JAM2, YBX3, GPAA1, PRKAR1B, PRKACA, AKT3, ACTG1 TUBA1B, RAF1, TUBB4B, PPP2R5B, RACK1, RAC1, TUBB, TSG101, GNAI2, CALM1 (includes others), PPP1R10, TUBB6, PRKAR1B, PRKACA, PRKD3 GNAI2, PAK6, CFL1, MYL6, WASF2, ACTB, RACK1, CD44, RAC1, RDX, ARHGDIA, ACTG1, MYL12A

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promote translation initiation by enhancing the formation of the elF4F complex and recruiting the 43S preinitiation complex to the 50 cap of almost all mRNAs (Haghighat et al., 1995; Hara et al., 1997). The rate of protein synthesis is also determined by the number of ribosomes available. Wnt/beta-catenin signaling pathway works independently from the mTOR pathway in the regulation of ribosomal biogenesis (Armstrong and Esser, 2005). The IPA guided network prediction of the genes upregulated in the shilajit group compared to placebo identified several genes involved in protein synthesis pathway (Fig. 12.3, Table 12.2). The upstream trigger (alpha catenin) activates an array of transporter proteins (SNX, S100A6, and TCOF1), translation regulators (YBX, LARP1), and enzymes (MYO19, LYZ, CSTB, and GART), which, in combination, help in the anabolism of structural proteins. In addition, ribosomal proteins (MRPS21, MRPL23, TCOF1, TBRG4, ASAP2, and AP3D1) were also upregulated in shilajit -administered mice compared to the placebo, supporting the fact that shilajit helps in maintaining the structural muscle mass by controlling both translation efficiency and translation capacity. A number of earlier studies that examined the health benefits of mumie (shilajit) in human subjects were published in Russian. These studies examined the effects of mumie on suppurative wounds (Muratova and Shakirov, 1968; Tazhimametov et al., 1987), peripheral nervous system diseases (Koziovskaia, 1968), bone fractures (Kelginbaev et al., 1973) and bone regeneration (Suleimanov, 1972), postoperative trepan cavities of the middle ear (Psakhis and Aizenberg, 1976), and benign prostatic hyperplasia (Andriukhova, 1997).

Animal Studies What may have been the first study to examine the effects of shilajit (mumie) on energy metabolism was conducted in rats (Shvetskii and Vorobeve, 1978). The study demonstrated that shilajit enhanced energy, protein, and nucleic acid metabolism. Bhattacharyya et al. (2009a,b) conducted a series of studies examining the effects of shilajit and its constituent DBPs on mitochondria and energy production. The treatment of mice IP with 20 mg of a mixture of the DBPs resulted in the detection of the DBPs and their redox products in hepatic mitochondria, the site of ATP and energy production (Bhattacharyya et al., 2009a). Furthermore, CoQ10 was augmented in plasma and organs relative to the control animals, and in vitro erythrocyte membrane lipid peroxidation was inhibited when rats were treated orally with 3,8-DBP. The results suggest a mechanism regarding how shilajit supports the energy-synthesizing ability of mitochondria, and provides at least a partial explanation for the physical performance and relief from fatigue reported in response to shilajit. In a subsequent study, Bhattacharyya et al. (2009b) examined the effects of shilajit on energy status in mice. When mice were forced to swim daily for 7 days and received either the placebo or 30 mg shilajit/kg orally per day for the last 4 days, the forced

Chapter 12  Energy and Health Benefits of Shilajit 195

MRPL23 TBRG4 ASAP2

MRPS21 TCOF1 FANCI

AP3D1 GART ANXA2

Cyclin D

FSCN1*

TNFAIP2 CTSB

MYO19

Gsk3

Collagen type I Wnt

LARP1 ILF3

TJP2

Arp2/3 CTNNA1

SHKBP1

SNX9

Alpha catenin

YBX3

LYZ

DCUN1D1

YBX2 ASB3/GPR75-ASB3

S100A6 TSNAX

FXR1

FAM3D DNPEP

FIGURE 12.3 Ingenuity Pathway Analysis tool (IPA)egenerated network of upregulated genes after shilajit supplementation suggesting the proteins involved in increasing translation efficiency and capacity.

swimming exercise resulted in an 82% decrease in muscle ATP levels. However, treatment with shilajit nearly doubled the ATP in muscle of mice forced to swim. Smaller effects of shilajit were observed with respect to ATP in blood and brain. CoQ10 administration resulted in a protection of muscle ATP similar to shilajit. In mice treated with a combination of shilajit and CoQ10, the muscle ATP levels were 2.44 times higher than untreated animals forced to swim. The primary biochemical function of CoQ10 is to aid in mitochondrial synthesis of ATP. The results support the contention that shilajit can increase energy, relieve fatigue, and support endurance. The effects of a processed and standardized shilajit (25, 50, and 100 mg/kg/day for 21 days) on various stress factors in rats forced to swim 15 min per day for 21 days were assessed (Surapaneni et al., 2012). The product contained 0.43% DBPs, 20.45%

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Table 12.2 Names and Cellular Localization of Proteins Involved in Increasing Translation Efficiency and Capacity After Shilajit Supplementation Symbol

Entrez Gene Name

Location

Family

Alpha catenin ANXA2 AP3D1

Alpha catenin Annexin A2 Adaptor-related protein complex 3 delta 1 subunit Actin-related protein complex ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 Ankyrin repeat and SOCS box containing 3 Collagen I Catenin alpha 1 Cathepsin B Cyclin D1 Defective in cullin neddylation 1 domain containing 1 Aspartyl aminopeptidase Family with sequence similarity 3 member D Fanconi anemia complementation group I Fascin actin-bundling protein 1 FMR1 autosomal homolog 1 Phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase Glycogen synthase kinase Interleukin enhancer binding factor 3

Cytoplasm Plasma membrane Cytoplasm

Group Other Transporter

Cytoplasm Nucleus

Complex Other

Cytoplasm Other Plasma Membrane Cytoplasm Nucleus Nucleus

Transcription regulator Complex Other Peptidase Group Other

Cytoplasm Extracellular space

Peptidase Cytokine

Nucleus Cytoplasm Cytoplasm Cytoplasm

Other Other Other Enzyme

Cytoplasm Nucleus Cytoplasm

Group Transcription regulator Translation regulator

Extracellular space Cytoplasm Cytoplasm Cytoplasm Cytoplasm Other Cytoplasm Cytoplasm

Enzyme Other Other Enzyme Transporter Other Transporter Other

Nucleus Plasma membrane Extracellular space Nucleus Extracellular space Cytoplasm Nucleus

Transporter Kinase Other Transporter Group Translation regulator Transcription regulator

Arp2/3 ASAP2 ASB3/GPR75-ASB3 Collagen type I CTNNA1 CTSB Cyclin D DCUN1D1 DNPEP FAM3D FANCI FSCN1 FXR1 GART

Gsk3 ILF3 LARP1 LYZ MRPL23 MRPS21 MYO19 S100A6 SHKBP1 SNX9 TBRG4 TCOF1 TJP2 TNFAIP2 TSNAX Wnt YBX2 YBX3

La ribonucleoprotein domain family, member 1 Lysozyme Mitochondrial ribosomal protein L23 Mitochondrial ribosomal protein S21 Myosin XIX S100 calcium binding protein A6 SH3KBP1 binding protein 1 Sorting nexin 9 Transforming growth factor beta regulator 4 Treacle ribosome biogenesis factor 1 Tight junction protein 2 TNF alpha-induced protein 2 Translin associated factor X Wnt Y-box binding protein 2 Y-box binding protein 3

Chapter 12  Energy and Health Benefits of Shilajit 197

DPB-chromoproteins, and 56.75% fulvic acids. Shilajit reversed the forced swimminge induced increase in immobility, the decrease in climbing behavior, the decrease in plasma corticosterone levels, and the decrease in adrenal gland weight. Shilajit treatment also prevented forced swimmingeinduced mitochondrial dysfunction as evidenced by stabilizing electron transport chain enzymes and mitochondrial membrane potential. In an early study by Visser (1987), fulvic acids were shown to stimulate respiration in rat liver mitochondria and also increased oxidative phosphorylation when present in concentrations between 40 and 360 mg/L. These results are consistent with the previously mentioned animal studies and provide mechanistic information regarding the increased energy and higher ATP levels. Several studies have examined the antiinflammatory effects of shilajit. In a welldesigned series of studies on the effects of shilajit performed in various animals species, Acharya et al. (1988) demonstrated that shilajit at a dose of 200 mg/kg IP exhibited significant analgesic activity as compared to controls using the rat tail flick method. Shilajit, at a dose of 50 mg/kg IP, also decreased carrageenan-induced inflammation in the rat paw by approximately 75%. Shilajit given orally at doses of 50e200 mg/kg twice a day to rats resulted in a significant, dose-dependent decrease in the gastric ulcer index, thereby demonstrating antiulcerogenic activity. Shilajit did not have significant activity with respect to the CNS, blood pressure, or skeletal muscle, and no antihistaminic activity based on studies in dogs, frogs, and guinea pigs. Shilajit exhibited antiulcerogenic and antiinflammatory activity when given to rats at a dose of 100 mg/kg orally twice a day (Goel et al., 1990). An increase in the mucosal barrier was believed to be produced by shilajit based on the decreased gastric ulcer index and increased carbohydrate/protein ratio. In addition, shilajit administration decreased carrageenan-induced acute pedal edema, granuloma pouch, and adjuvant-induced arthritis in rats, indicating significant antiinflammatory activity. Ghosal (2006) suggested that the antiulcerogenic effect of fulvic acids and the biphenyls present in shilajit were due to protection of the gastrointestinal mucosa with less loss of mucosal cells. Shilajit has also been shown to attenuate acetic acid and formalin-induced writhing in mice, thus demonstrating its antiinflammatory activity (Malekzadeh et al., 2015). A dose-dependent increase in the analgesic effects of shilajit was demonstrated at doses of 0.75, 7.5, and 75 mg/kg. No significant differences were observed between 75 and 750 mg/kg shilajit and up to 4 mg morphine or 30 mg sodium diclofenac, which were used as positive controls. The antiinflammatory and antiarthritic effects of shilajit have been studied in moderately arthritic dogs in a randomized, placebo-controlled, double-blind study (Lawley et al., 2013). Ten animals received either 500 mg shilajit twice daily or placebo for 5 months. The animals receiving shilajit showed significant pain reduction by day 60 with maximum reduction in pain by day 150. The authors concluded that shilajit markedly improved the daily life of the animals. The study suffers from the fact that there were only five treated and five control animals.

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Ghosal (2006) summarized a number of early studies on the immunomodulatory effects of processed shilajit, which suggest that shilajit enhances the lytic potential of polymorphonuclear leukocytes. The administration of a 200e600 mg/dose of shilajit to mice resulted in significant morphological and phagocytotic changes in peritoneal macrophages (Baumik et al., 1993; Ghosal et al., 1995), demonstrating its immunomodulatory capabilities. The antiinflammatory effects of shilajit may at least in part be explained by these effects. The effects of shilajit on spermatogenesis and ovogenesis in rats were investigated (Park et al., 2006). Shilajit administration daily for 6 weeks resulted in a significant increase in sperm count. Ovulation was induced in seven out of nine female rats in the shilajit group as opposed to three out of nine in the control group, indicating a questionable result in the female rats. The antioxidant effects of shilajit have been demonstrated in several animal studies. Shilajit was shown to prevent lead-induced oxidative stress in a 6-week feeding study in chicks (Kumar et al., 2010). Shilajit was included in the diet at 100 ppm. Antioxidant status was assessed based on glutathione peroxidase activity, glutathione reductase activity, catalase activity, glutathione content, and lipid peroxidation (thiobarbituric acid reactive substances). Shilajit treatment (25, 50, and 100 mg/kg/day for 21 days) also attenuated swimming-induced oxidative stress as evidenced by decreases in nitric oxide and lipid peroxidation and increases in catalase and superoxide dismutase activities (Surapaneni et al., 2012). The effects of shilajit on brain edema, intracranial pressure, and neurologic outcomes following traumatic brain injury have been studied in rats (Khaksari et al., 2013). Rats were treated IP with 0, 150, or 250 mg/kg shilajit 1, 24, 48, and 72 h after trauma. Intracranial pressure was significantly reduced at 24, 48, and 72 h after trauma in the shilajit-treated animals, while brain water and Evans blue dye uptake were also significantly decreased as compared to control. Neurological outcomes also significantly improved in the treated rats. In a study in mice, processed shilajit administration (0.1 and 1.0 mg/kg IP) resulted in significant inhibition of the development of tolerance to morphine (10 mg/kg IP twice daily) after 6 days of treatment (Tiwari et al., 2001). Shilajit per se did not exhibit any analgesic activity in the mice. No explanation was provided for the observed effect. Several studies have examined the antidiabetic effects of shilajit in animals. Bhattacharaya (1995) showed that the oral administration of 50 mg/kg and 100 mg/kg of a process and standardized shilajit attenuated streptozotocin-induced diabetes in rats. It also increased pancreatic islet superoxide dismutase, leading to a decrease in free radical production and accumulation. Kanikkannan et al. (1994) observed that a processed shilajit (1.0 mg/kg subcutaneously) prevented streptozotoxin-induced diabetes in rats. Furthermore, shilajit potentiated the hypoglycemic action of insulin. The cardioprotective effects of shilajit (mumie) have been studied in rats (Joukar et al., 2014). Rats received 250 or 500 mg shilajit per day orally for 7 days. Isoproterenol (85 mg/kg) was injected subcutaneously to induce myocardial damage. Shilajit pretreatment provided

Chapter 12  Energy and Health Benefits of Shilajit 199

significant protection against the cardiac damaging effects of the isoproterenol, including a reduction in the severity of cardiac lesions. The mechanisms involving protection were not clear to the authors. The ability of shilajit to protect against radiation-induced apoptosis in rat ovaries has been reported (Kececi et al., 2016). Rats were pretreated with shilajit or the vehicle and subject to irradiation. The animals were sacrificed 4 days after radiation exposure. Shilajit prevented the radiation-induced decreases in primordial, primary, preantral, and atretic follicles. Shilajit treatment also decreased the expression of p53, Bax, and caspase 3, thereby blocking the radiation-induced apoptotic pathway. Durg et al. (2015) examined the antiepileptic and antipsychotic activities of standardized shilajit in rats and mice. The animals were given 25 or 50 mg shilajit orally daily for 15 days. Seizures and psychotic behavior were then induced with isonicotinyl hydrazine (INH), pentylenetetrazole (PTZ), apomorphine, or electroshock. Shilajit pretreatment significantly decreased seizures induced by INH, PTZ, and electroshock, while shilajit significantly inhibited climbing and stereotypical behaviors induced by apomorphine. The authors suggested that the antiepileptic activity of shilajit may be due to enhancing the gamma aminobutyric acid (GABA) neurotransmitter (GABAergic) system, while the antipsychotic activity of shilajit may possibly be due to antidopaminergic and/or GABA-mimetic actions. The ability of shilajit to reduce alcohol withdrawal anxiety has been studied in mice (Bansal and Banerjee, 2016). Shilajit treatment significantly decreased ethanol intake and increased water consumption. The shilajit altered cortical-hypocampal dopamine in the mice but had no effect of GABA levels. In an early study, Schliebs et al. (1997) demonstrated that shilajit (40 mg/kg IP for 7 days) administration differentially affected cholinergic but not GABAergic or glutaminergic markers in rat brain as determined by brain slice histochemistry and autoradiography. The data suggested that shilajit preferentially affected cortical and basal forebrain cholinergic signal transduction cascade. An increase in the cholinergic signal transduction cascade could explain, at least in part, anecdotal reports of cognition and memory-enhancing effects of shilajit. Bhattarai et al. (2016) have examined the effects of shilajit on preoptic hypothalamic neurons in juvenile mice using a voltage clamp model. Shilajit induced a reproducible dose-dependent inward current, which persisted in the presence of tetrodotoxin, suggesting a postsynaptic action of shilajit, but was almost completely blocked by strychnine, a glycine receptor antagonist. The authors concluded that shilajit contains ingredients that influence hypothalamic neurophysiology through activation of strychnine-sensitive glycine receptoremediated responses postsynaptically. In a study involving rats, shilajit administration (400 mg/mL) exhibited an in vivo peripheral parasympathomimetic effect, which can provide at least a partial explanation for the reported effects on spermatogenesis, as well as the anecdotal reports on overall fertility and libido (Kaur et al., 2013). These conclusions were based on the administration of shilajit alone and in combination with acetylcholine, and they assessed changes in heart rate, blood pressure, respiratory rate, and neuromuscular transmission.

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In Vitro Studies Various in vitro studies have been conducted to obtain information regarding possible mechanisms of action of shilajit. The antioxidant activities of 3-hydroxy-DBPs and the 3,8-dihydroxy-DBPs, which are believed to be active constituents of shilajit, were demonstrated in vitro using five free radical scavenging assays (Battacharyya et al., 2009a). KU812 cells incubated with fulvic acid affected the expression of genes involved in signal transduction, cytokineecytokine receptor interaction, and immune response pathways, as well as cell adhesion molecule and IgE receptor b subunit responses (Motojima et al., 2011). These results demonstrate the wide range of potential physiological effects that can be modulated by fulvic acids and help explain the immunomodulatory responses observed as a result of shilajit ingestion. Mouse brainstem slices were incubated with shilajit in the presence of various receptor antagonists and channel blockers in order to assess their ability to exhibit glycine- and GABA-mimetic actions on the brainstem substantia gelatinosa neurons of the trigeminal subnucleus caudalis (Yin et al., 2011). Shilajit induced inward currents in a concentration-dependent manner. The results indicated that shilajit has CNS-sedating ingredients and may provide at least a partial explanation regarding reports of skeletomuscular pain relief as well as the antiepileptic and antipsychotic effects. Incubation of rat corpus cavernosum strips with shilajit (400 and 800 mg/mL) resulted in a concentration-dependent relaxation of the strips and enhanced acetylcholinemediated relaxation, suggesting an increased blood flow to the groin and therefore beneficial effects with regard to spermatogenesis and increased testosterone production (Kaur et al., 2013).

Discussion and Summary In recent years, a rapidly growing number of animal and human studies have been published regarding the physiological/pharmacological effects of shilajit (mumie, moomiyo) as well as its mechanisms of action. Studies in both animals and humans indicate that shilajit has a wide margin of safety and is free of adverse effects at the doses that are commonly used. Most human studies have focused on energy production and the androgenic effects of shilajit, including testosterone production and spermatogenesis. Shilajit was also shown to facilitate muscle adaptation. Published human and animal studies have shown that shilajit increases spermatogenesis in infertile males. An unpublished human study has also provided support for the beneficial effects with respect to both ATP and CoQ10 production. Animal studies have supported these observations, demonstrating that shilajit enhances energy (ATP) production, relieves fatigue, and promotes endurance. These effects may be mediated by the ability of shilajit to stabilize electron transport chain enzymes and mitochondrial membranes. Animal studies have also shown that

Chapter 12  Energy and Health Benefits of Shilajit 201

DBPs exhibited mitochondrial protective effects, which can further explain the beneficial effects with respect to physical performance and relief from fatigue. Extensive studies were reported to have been conducted within the USSR between 1960e1990 on the physical and mental revitalizing effects of shilajit. These studies were not published and were classified. Shilajit was purported to be widely used by USSR Olympic athletes, as well as military special forces, for its energy-producing and adaptogenic effects (Bucci, 2000; personal communication with Dr. N. Volkov). Various animal studies have shown that shilajit exhibits antioxidant, antiinflammatory and antiarthritic, immunomodulatory, and tissue-protective activities. In addition, antiepileptic and antipsychotic effects have been demonstrated in animals. This broad spectrum of effects has not been confirmed in human studies, in part because the studies have not been conducted. Animal studies have also examined the potential effects on various neurotransmitters and have demonstrated significant cholinergic and parasympathomimetic effects, which can explain the potential benefits with respect to cognitive function and enhanced fertility. Enhancement of the GABAergic system, binding to glycine receptors, and inhibition of dopaminergic actions have also been implicated in the antiepileptic and antipsychotic effects. Antiinflammatory and tissue protective effects may involve shilajitinduced decreased expression of signal transduction factors as p53, Bax, and caspase 3. Finally, a need exists for further studies, primarily in humans, with processed and standardized shilajit preparations. Consumers should keep in mind that products are marketed that do not conform to the chemical composition of “purified” shilajit and may be either adulterated or counterfeit. Therefore, caution must be taken with respect to the acquisition of shilajit, and only processed and standardized products should be consumed.

References Acharya, S.B., Frontan, M.H., Goel, R.K., Tripathi, S.K., Das, P.K., 1988. Pharmacology of shilajit. Ind. J. Expt. Biol. 26, 775e777. Agarwal, S.P., Khanna, R., Karmarkar, R., Anwer, M.K., Khar, R.K., 2007. Shilajit: a review. Phytother. Res. 21, 401e405. Al-Hamaidi, A., Saleh, R.A., Bedaiwy, M.A., 2003. Safe use of shilajit during pregnancy in female mice. Online J. Biol. Sci. 3, 681e684. Andriukhova, N.N., 1997. The treatment of benign prostatic hyperplasia using mumie vitas preparation. Lik. Sparava 6, 129e132. In Russian. Anisimov, V.E., Shakirzyanova, R.M., 1982. Application of mumie in therapeutic practice. Kazan Med. Zh 63, 65e68. In Russian. Armstrong, D.D., Esser, K.A., October 2005. Wnt/beta-catenin signaling activates growth-control genes during overload-induced skeletal muscle hypertrophy. Am. J. Physiol. Cell Physiol. 289 (4), C853eC859. Bansal, P., Banerjee, S., 2016. Effect of Withania somnifera and shilajit on alcohol addiction in mice. Pharmacogn. Mag. 12 (Suppl.), S121eS128.

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Baumik, S., Chattopadhyay, S., Ghosal, S., 1993. Effect of shilajit on mouse peritoneal macrophages. Phytother. Res. 7, 425e427. Bhattacharaya, S.K., 1995. Shilajit attenuates streptozotocin induced diabetes mellitus and decreases pancreatic islet superoxide dismutase activity in rats. Phytother. Res. 9, 41e44. Bhattacharayya, S., Pal, D., Banerjee, D., 2009a. Shilajit dibenzo-a-pyrones: mitochondria targeted antioxidants. Pharmacologyonline 2, 690e698. Bhattacharayya, S., Pal, D., Gupta, A.K., Ganguly, P., Majumder, U.K., Ghosal, S., 2009b. Beneficial effect of processed shilajit on swimming exercise induced impaired energy status of mice. Pharmacologyonline 1, 817e825. Bhattarai, J.P., Cho, D.H., Han, S.K., 2016. Activation of strychnine-sensitive glycine receptors by shilajit on preoptic hypothalamic neurons of juvenile mice. Chin. J. Physiol. 59, 39e45. Biswas, T.K., Pandit, S., Mondal, S., Biswas, S.K., Jana, U., Ghosh, T., Tripathi, P.C., Debnath, R.G., Auddy, R.G., Auddy, B., 2009. Clinical evaluation of spermatogenic activity of processed shilajit in oligospermia. Andrologia 42, 48e56. Bucci, L.R., 2000. Selected herbals and human exercise performance. Am. J. Clin. Nutr. 72 (2 Suppl.), 624Se636S. Carrasco-Gallardo, C., Guzman, L., Maccioni, R.B., 2012. Shilajit: a natural phytocomplex with potential precognitive activity. Int. J. Alzheimers Dis.. http://dx.doi.org/10.1155/2012/674142. Das, A., Datta, S., Rhea, B., Sinha, M., Veeraragavan, M., Gordillo, G., Roy, S., July 2016. The human skeletal transcriptome in response to oral shilajit supplementation. J. Med. Food 19 (7), 701e709. Durg, S., Veerapour, V.P., Thippeswamy, B.S., Ahamed, S.M., 2015. Antiepileptic and antipsychotic activities of standardized silajatu (shilajit) in experimental animals. Anc. Sci. Life 35, 110e117. Frolova, L.N., Kiseleva, T.L., 1996. Chemical composition of mumijo and methods for determining authenticity and quality. A review. Pharm. Chem. J. 8, 543e547. Ghosal, S., 2006. Shilajit in Perspective. Narosa Publishing House, New Delhi, India. Ghosal, S., Lal, J., Singh, S.K., Goel, R.K., Jaiswal, A.K., Bhattacharya, S.K., 1991. The need for formulation of shilajit by its isolated active constituents. Phytother. Res. 5, 211e216. Ghosal, S., Baumik, S., Chattopadhyay, S., 1995. Shilajit induced morphometric and functional changes in mouse peritoneal macrophages. Phytother. Res. 9, 194e198. Goel, R.K., Banerjee, R.S., Acharya, S.B., 1990. Antiulcerogenic and anti-inflammatory studies with shilajit. J. Ethnopharmacol. 29, 95e103. Goodman, C.A., Mayhew, D.L., Hornberger, T.A., 2011. Recent progress toward understanding the molecular mechanisms that regulate skeletal muscle mass. Cell Signal 23, 1896e1906. Haghighat, A., Mader, S., Pause, A., Sonenberg, N., 1995. Repression of cap-dependent translation by 4Ebinding protein 1: competition with p220 for binding to eukaryotic initiation factor-4E. EMBO J. 14, 5701e5709. Hara, K., Yonezawa, K., Kozlowski, M.T., Sugimoto, T., Andrabi, K., Weng, Q.P., Kasuga, M., Nishimoto, I., Avruch, J., 1997. Regulation of eIF-4E BP1 phosphorylation by mTOR. J. Biol. Chem. 272, 26457e26463. You, J.-S., Anderson, G.B., Matthew, S., Dooley, Hornberger, T.A., September 2015. The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice. Dis. Model Mech. 8 (9), 1059e1069. Joukar, S., Najafipour, H., Dabiri, S., Sheibani, M., Sharokhi, N., 2014. Cardioprotective effect of mumie (shilajit) on experimentally induced myocardial injury. Cardiovasc. Toxicol. 14, 214e221. Kanikkannan, N., Ramarao, P., Ghosal, S., 1994. Shilajit-induced potentiation of the hypoglycemic action of insulin and inhibition of streptozotocin induced diabetes in rats. Phytother. Res. 9, 478e481.

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Kaur, A., Kumar, P., Kumar, D., Kharya, M.D., Singh, N., 2013. Parasympathomimetic effect of shilajit accounts for relaxation of rat corpus cavernosum. Am. J. Mens Health 7, 119e127. Kececi, M., Akpolat, M., Gulle, K., Gencer, E., Sahbaz, A., 2016. Evaluation of preventive effect of shilajit on radiation-induced apoptosis on ovaries. Arch. Gynecol. Obstet. 293, 1255e1262. Kelginbaev, N.S., Sorokina, V.A., Stefanidu, A.G., Ismailova, V.N., 1973. Treatment of long tubular bone fractures with mumie assil preprations in experiments and clinical conditions. Eksp Khir Anesteziol. 18, 31e35. In Russian. Khaksari, M., Mahmmodi, R., Shahrokhi, N., Shabani, M., Joukar, S., Aqapour, M., 2013. The effects of shilajit on brain edema, intracranial pressure and neurologic outcomes following the traumatic brain injury in rats. Iran J. Basic Med. Sci. 16, 858e864. Kiren, Y, Nugroho, A.E., Hirasawa, Y., Shirota, O., Bekenova, M., Narbekovich, N.O., Shapilova, M., Maeno, H., Norita, H., 2014. Mumic acids A-E: new diterpenoids from mumiyo. J. Nat. Med. 68, 199e205. Koziovskaia, V., 1968. Treatment of peripheral nervous system diseases with Caucasian mumie. Vrach Deio 6, 88e92. In Russian. Kumar, M.R., Reddy, A.G., Anjaneyulu, Y., Reddy, G.D., 2010. Oxidative stress induced by lead and antioxidant potential of certain adaptogens in poultry. Toxicol. Int. 17, 45e48. Lawley, S., Gupta, R.C., Goad, J.T., Canerdy, T.D., Kalidindi, S.R., 2013. Anti-inflammatory and antiarthritic efficacy and safety of purified shilajit in moderately arthritic dogs. J. Vet. Sci. Anim. Husb. 1, 302e308. Malekzadeh, G., Cashti-Rhahmatabadi, M.H., Zanbagh, S., Akhavi Mirab-bashii, A., 2015. Mumijo attenuates chemically induced inflammatory pain in mice. Altern. Ther. Health Med. 21, 42e47. McCarthy, J.J., Esser, K.A., 2010. Anabolic and catabolic pathways regulating skeletal muscle mass. Curr. Opin. Clin. Nutr. Metab. Care 13 (3), 230e235. Motojima, H., Villareal, O., Han, J., Isoda, H., 2011. Microarray analysis of intermediate-type allergy in KU812 cells in response to fulvic acid. Cytotechnology 63, 181e190. Muratova, K.N., Shakirov, D.S., 1968. Clinical treatment of suppurative wounds with mumie. Khirurgiia (Mosk) 44, 122e124. In Russian. Pandit, S., Biswas, S., Jana, U., De, R.K., Mukhopadhyay, S.C., Biswas, T.K., 2016. Clinical evaluation of purified shilajit on testosterone levels in healthy volunteers. Andrologia 48, 570e575. Park, J.S., Kim, G.Y., Han, K., 2006. The spermatogenic and ovogenic effects of chronically administered shilajit to rats. J. Ethnopharmacol. 107, 349e353. Psakhis, B., Aizenberg, S.G., 1976. Use of mumie in the treatment of postoperative trepan cavities of the middle ear. Zh Ushn Nos Gori Bolezn 5, 57e61. In Russian. Raju, S., 2012. PrimaVieÒ. Technical Data Report. Natreon, Inc., 20 Janine Place, New Brunswick, NJ 08901. Schepetkin, I., Khebnikov, A., Kwon, B.S., 2002. Medical drugs from humus matter: focus on mumie. Drug Devel Res. 57, 140e159. Schepetkin, I.A., Khebnikov, A.I., Ah, S.Y., et al., 2003. Characterization and biological activities of humic substances from mumie. J. Agric. Food Chem. 51, 5245e5254. Schliebs, R., Liebmann, A., Bhattacharya, S.K., Kumar, A., Ghosal, S., Bigl, V., 1997. Systematic administration of defined extracts from with ania somnifera (Indian ginseng) and shilajit differentially affects cholinergic but not glutaminergic and GABAergic markers in rat brain. Neurochem. Int. 30, 181e190. Sharma, P., Jha, J., Shrinivas, V., et al., 2003. Shilajit: evaluation of its effects on blood chemistry of normal human subjects. Ancient Sci. Life 23, 114e119.

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Shvetskii, A.G., Vorobeva, L.M., 1978. Effect of the nonspecific biogenic stimulators pentoxyl and mumie on metabolic processes. Vopr Med. Khim 24, 102e108. In Russian. Stohs, S.J., 2014. Safety and efficacy of shilajit (mumie, moomiyo). Phytother. Res. 28, 475e479. Suleimanov, I., 1972. Effects of mumie on bone regeneration in patients subjected to surgery for osteoarticular tuberculosis. Ortop. Travmatol. Protez. 33, 64e68. In Russian. Surapaneni, D.K., Adapa, S.R., Preeti, K., Adapa, S.R.S.S., Teji, G.R., Veeraragavan, M., Krishnamurthy, S., 2012. Shilajit attenuates behavioral symptoms of chronic fatigue syndrome by modulating the hypothalamic-pituitary-adrenal axis and mitochondrial bioenergetics in rats. J. Ethnopharmacol. 143, 91e99. Tazhimametov, B.T., Usmanov, M.U., Dzhuraev, K.A., Sharipov, N.I., Zulfikarov, K., 1987. Effect of mumie on the healing of suppurative wounds. Klin Khir 1, 51e52. In Russian. Tiwari, P., Ramarao, P., Ghosal, S., 2001. Effects of shilajit on the development of tolerance to morphine in mice. Phytother. Res. 15, 177e179. Velmurugan, C., Vivek, B., Wilson, E., Bharathi, T., Sundaram, T., 2012. Evaluation of safety profile of black shilajit after 91 days repeated administration on rats. Asian Pac. J. Trop. Biomed. 2, 210e214. Visser, S.A., 1987. Effect of humic acid substances on mitochondrial respiration and oxidative phosphorylation. Sci. Total Environ. 62, 347e354. Wilson, E., Rajamanickam, G.V., Dubey, G.P., et al., 2011. Review on shilajit used in traditional Indian medicine. J. Ethnopharmacol. 136, 1e9. Yin, H., Yang, E.J., Park, S.J., Han, S.K., 2011. Glycine- and GABA-minetic actions of shilajit on the substantia gelatinosa neurons of the trigeminal subnucleus caudatis in mice. Korean J. Physiol. Pharmacol. 15, 285e289.

13

An Overview on Ginseng and Energy Metabolism

Haojun Zhang1, Dongliang Wang2, 3, Wenwen Ru2, 3, Yufeng Qin2, 3, Xiangshan Zhou2, 3 1 QILU UNIVERSITY OF TECHNOLOGY, JINAN, CHINA; 2 NATI O NAL ENGI NEERI NG TECHNOLOGY RESEARCH CE NTER OF GLUE OF TRADITIONAL MEDICINE, DONG ’E, CHINA; 3 SHANDONG DONG-E-E-JIAO CO., LTD., DONG’E, CHINA

Review The word “ginseng” has been applied to various herbs belonging to the genus Panax of the family Araliaceae, which has been used to treat everything in combination with other herbs over centuries. Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolius) are considered true ginseng, whereas Siberian ginseng or eleuthero (Eleutherococcus senticosus) is not. Twelve species and two infraspecific taxa are currently recognized as members of the genus Panax, as shown in Table 13.1 (Roskov et al., 2014; Shin et al., 2015). Table 13.1 Scientific and Common Names of Panax Plants (Roskov et al., 2014; Shin et al., 2015) Scientific Name

Rank

Panax Panax Panax Panax Panax Panax Panax Panax Panax Panax Panax Panax Panax Panax

Species Infraspecific taxon Infraspecific taxon Species Species Species Species Species Species Species Species Species Species Species

bipinnatifidus Seem. bipinnatifidus var. angustifolius bipinnatifidus var. bipinnatifidus ginseng C.A. Meyer japonicus (T. Nees) C.A. Meyer notoginseng (Burkill) F.H. Chen pseudoginseng Wall. quinquefolius L. sokpayensis Shiva K. Sharma & Pandit stipuleanatus H.T. Tsai & K.M. Feng trifolius L. vietnamensis Ha et Grushv wangianus S.C. Sun zingiberensis C.Y.Wu. & Feng

Common Name

Korean ginseng, ginseng Japanese ginseng Chinese ginseng, sanchi American ginseng

Vietnamese ginseng

Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00013-2 Copyright © 2017 Elsevier Inc. All rights reserved.

205

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Table 13.2 Chemical Constituents of Ginseng Root (Panax ginseng C.A. Meyer) (Ru et al., 2015; Xinbao et al., 2013) No Name

R1

R2

Source References

Protopanaxadiol ginsenosides 1

20S-Ginsengoside Ra1

-glc(2-1)glc

2

20S-Ginsengoside Ra2

-glc(2-1)glc

3

20S-Ginsengoside Ra3

-glc(2-1)glc

4

20S-Ginsengoside Ra4

-glc(2-1)glc(6) Bu

5

20S-Ginsengoside Ra5

-glc(2-1)glc(6) Ac

6 7 8 9 10

20S-Ginsengoside 20S-Ginsengoside 20S-Ginsengoside 20S-Ginsengoside 20S-Ginsengoside

-glc(2-1)glc(6) Bu -glc(2-1)glc(6) Bu -glc(2-1)glc(4) Bu -glc(2-1)glc(6)Bu -glc(2-1)glc

-glc(6-1) (4-1)xyl -glc(6-1) (2-1)xyl -glc(6-1) xyl -glc(6-1) (4-1)xyl -glc(6-1) (4-1)xyl -glc(6-1) -glc(6-1) -glc(6-1) -glc(6-1) -glc(6-1)

11 20S-Ginsengoside Rb2

glc(3-1) W,R

Besso et al. (1982) and Kasai et al. (1983) Besso et al. (1982) and Kasai et al. (1983) Matsuura et al. (1984)

ara(p)

W

Zhu et al. (2010)

ara(p)

W

Zhu et al. (2010)

glc ara(p) ara(f) ara(f) glc

W W W W W,R

-glc(2-1)glc

-glc(6-1) ara(p)

W,R

12 20SGginsengoside Rb3

-glc(2-1)glc

-glc(6-1) xyl

W,R

13 20S-Ginsengoside Rc

-glc(2-1)glc

-glc(6-1) ara(f)

W,R

14 20S-Ginsengoside Rd

-glc(2-1)glc

-glc

W,R

15 20S-Ginsengoside Rg3

-glc(2-1)glc

-H

W,R

16 17 18 19

20R-Ginsengoside Rg3 20R-Ginsengoside Rh2 20S-Ginsengoside Rh2 20S-Ginsengoside Rs1

-glc(2-1)glc -glc -glc -glc(2-1)glc(6) Ac

-H -H -H -glc(6-1) ara(p)

R Wa R W,R

20 20S-Ginsengoside Rs2

-glc(2-1)glc(6) Ac

-glc(6-1) ara(f)

W,R

21 20S-Ginsengoside Rs3 22 Malonyl-20S-ginsengoside Ra3 23 Malonyl-20S-ginsengoside Rb1

-glc(2-1)glc(6) Ac -glc(6-1) ara(f) R -glc(2-1)glc(6) mal -glc(6-1) ara(3-1) W xyl -glc(2-1)glc(6) mal -glc(6-1) glc W

Zhu et al. (2010) Zhu et al. (2010) Zhu et al. (2010) Zhu et al. (2010) Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Kasai et al. (1983) and Sanada et al. (1978) Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Kasai et al. (1983), Isao and Yoshiawa (1983) and Kim et al. (1995) Isao and Yoshiawa (1983) Zhong et al. (2008) Isao and Yoshiawa (1983) Kasai et al. (1983) and Zhu et al. (2010) Kasai et al. (1983) and Zhu et al. (2010) Baek et al. (1997) Ruan et al. (2010)

24 Malonyl-20S-ginsengoside Rb2 25 Malonyl-20S-ginsengoside Rc

-glc(2-1)glc(6) mal -glc(6-1) ara(p) -glc(2-1)glc(6) mal -glc(6-1) ara(f)

Ra6 Ra7 Ra8 Ra9 Rb1

ara(p)

W,R

ara(f)

W,R

W W

Zhu et al., 2010 and Kitagawa et al. (1983) Kitagawa et al. (1983) Kitagawa et al. (1983)

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207

Table 13.2 Chemical Constituents of Ginseng Root (Panax ginseng C.A. Meyer) (Ru et al., 2015; Xinbao et al., 2013)dcont’d No Name

R1

R2

Source References

26 Malonyl-20S-ginsengoside Rd 27 Malonyl-20S-notoginsengoside R4 28 20S-Gypenoside XVII 29 20S-Notoginsenoside Fe 30 20S-Notoginsenoside R4 31 20S-Pseudoginsenoside RC1 32 20S-Quinquenoside R1

-glc(2-1)glc(6) mal -glc -glc(2-1)glc(6) mal -glc(6-1) xyl -glc -glc(6-1) -glc -glc(6-1) -glc(2-1)glc -glc(6-1) xyl -glc(2-1)glc(6) Ac -glc -glc(2-1)glc(6) Ac -glc(6-1)

33 20S-Vinaginsenoside R16

-glc(2-1)xyl

-glc

W

34 20S-Ginsenoside Re

-glc(2-1)rha

-glc

W,R

35 36 37 38 39 40

-glc -glc(3-1)glc -glc -glc -glc -glc(2-1)glc

-glc(3-1) glc -glc -glc(4-1) glc -glc(6-1) ara(f) -glc(6) Bu -H

W W W W W W,R

41 20S-Ginsenoside Rg1

-glc

-glc

W,R

42 20S-Ginsenoside Rg2

-glc(2-1)rha

-H

W,R

43 20R-Ginsenoside Rg2

-glc(2-1)rha

-H

W,R

44 20-Gluco-20S-ginsenoside Rf

-glc(2-1)glc

-glc

W,R

45 20S-Ginsenoside Rh1

-glc

-H

R

46 20R-Ginsenoside Rh1 47 20S-Koryoginsenoside R1

-glc -glc(6-1)Bu

-H -glc

R W

48 20S-Notoginsenoside N 49 20S-Notoginsenoside R1

-glc(4-1)glc -glc(2-1)xyl

-glc -glc

W W,R

50 20S-Notoginsenoside R2

-glc(2-1)xyl

-H

W

51 20S-Yesanchinoside D

-glc(6)Ac

-glc

W

W glc(6-1) W

Kitagawa et al. (1983) Sun et al. (2007)

glc W ara(f) W glc(6-1) W,R

Zhu et al. (2010) Wang et al. (2013) Matsuura et al. (1984) and Dou et al. (2003) Zhu et al. (2010) Kasai et al. (1983) and Zhu et al. (2010) Zhu et al. (2010)

glc

W W,R

Protopanaxatriol ginsenosides

20S-Ginsenoside 20S-Ginsenoside 20S-Ginsenoside 20S-Ginsenoside 20S-Ginsenoside 20S-Ginsenoside

Re1 Re2 Re3 Re4 Re6 Rf

Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Yu et al. (2005) Yu et al. (2005) Yu et al. (2005) Yu et al. (2005) Yu et al. (2005) Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Kasai et al. (1983), (Isao and Yoshiawa, (1983) and Kim et al. (1995) and Nagai et al. (1971) Kasai et al. (1983), Sanada et al. (1974) and Isao and Yoshiawa (1983) Kasai et al. (1983), Isao and Yoshiawa (1983), Dou et al. (2003) Kasai et al. (1983) and Sanada et al. (1978) Isao and Yoshiawa (1983) and Kim et al. (1995) Isao and Yoshiawa (1983) Kim et al. (1995), Wang et al. (2013) and Yu et al. (2005) Yu et al. (2005) Kasai et al. (1983), Kim et al. (1995) and Yu et al. (2005) Dou et al. (2003) and Yu et al. (2005) Yu et al. (2005) (Continued )

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Table 13.2 Chemical Constituents of Ginseng Root (Panax ginseng C.A. Meyer) (Ru et al., 2015; Xinbao et al., 2013)dcont’d No Name

R1

R2

Source References

Protopanaxadiol and Protopanaxatriol ginsenosides with modified side chain 52 Ginsenjilinol 53 Ginsenoside F4

-glc(2-1)glc -glc(2-1)rha

-H e

W R

54 Ginsenoside Re5 55 Ginsenoside Rf2 56 Ginsenoside Rg5

-glc(2-1) glc -glc(2-1)rha -glc(2-1)glc

-H e e

W R R

57 Ginsenoside Rg6

-glc(2-1)rha

e

R

58 Ginsenoside Rh4

-glc

e

R

59 Ginsenoside Rk1

-glc(2-1)glc

-H

R

60 Ginsenoside Rk2 61 Ginsenoside Rk8

-glc -H

-H -Oglc

R R

62 Ginsenoside Rs4

-glc(2-1) glc(6)Ac

e

R

68 Ginsenoside Rs5

-glc(2-1) glc(6)Ac

-H

R

64 Ginsenoside Rs6 65 Ginsenoside Rs7 66 Koryoginsenoside R2

-glc(6)Ac -H -glc(2-1)glc

e -glc(6)Ac -glc(6-1)glc

R R W

67 Ginsenoside Ro

-glcUA(2-1)glc

-glc

W,R

68 Ginsenoside Ri 69 Ginsenoside Ro methyl ester

-H -ara(f) -(60 -Me)glcUA(2-1) -glc glc -(60 -PAE)glcUA(2-1) -glc glc

Wang et al. (2013) Ryu et al. (1996) and Zhang et al. (2012) Yu et al. (2005) Park et al. (1998) Zhang et al. (2012) and Baek et al. (1996a,b) and Kim et al. (1996) Zhang et al. (2012) and Baek et al. (1996a,b) Zhang et al. (2012), Baek et al. (1995a,b) and Park et al. (2002a,b) Zhang et al. (2012), Park et al. (2002a,b) Park et al. (2002) Zhang et al. (2012) and Park et al. (2002a,b) Zhang et al. (2012) and Park et al. (2002a,b) Zhang et al. (2012) and Park et al. (2002a,b) Park et al. (2002a,b) Fu et al. (1998) Kim et al. (1995)

Oleanane ginsenosides

70 Polyacetylene ginsenoside Ro

Wa W

Kasai et al. (1983) and Sanada et al. (1974) Fu et al. (1998) Zhang et al. (2002)

W

Zhang et al. (2002)

Alkaloids 71 N9-formylharman 72 Ethyl b-carboline 73 Perlolyrine 74 1-Carbobutoxy-b-carboline 75 1-Carbomethoxy-b-carboline

Han et al. (1986) and Han et al. (1987) Han et al. (1986) and Han et al. (1987) Han et al. (1986) and Han et al. (1987) Park et al. (1987) and 1988) Park et al. (1987) and 1988)

Chapter 13  An Overview on Ginseng and Energy Metabolism

209

Table 13.2 Chemical Constituents of Ginseng Root (Panax ginseng C.A. Meyer) (Ru et al., 2015; Xinbao et al., 2013)dcont’d No Name

R1

R2

Source References

Glucosides 76 Isomaltol-a-D-glucopyranoside 77 Ketopropyl-a-D-glucopyranoside 78 Adenosine

Matsuura et al. (1984) and Han et al. (1985) Matsuura et al. (1984) and Han et al. (1985) Matsuura et al. (1984) and Han et al. (1985)

Phenolic acids 79 Maltol (8-hydroxy-2-methyl-4pyrone) 80 Salicylic acid 81 Vanillic acid 82 p-Hydroxycinnamic acid

W. Wang et al. (2007) Han et al. (1981) Han et al. (1981) Han et al. (1981)

Others 83 Thiazole 84 Gomisin N 85 Gomisin A

Huh et al. (1990) Huh et al. (1990) Huh et al. (1990)

60 ’-Me, 60 0 -methyl ester; 60 ’-PAE, 60 0 -panaxytriol ester; Ac, acetyl; ara( f ), a-L-arabinofuranosyl; ara( p ), a-L-arabinopyranosyl; Bu, trans-but-2-enoyl; glc, b-D-glucopyranosyl; glcUA, b-D-glucopyranosiduronic acid; mal, malonyl; R, red ginseng;.W, white ginseng; Wa, woods grown ginseng; xyl, b-D-xylopyranosyl.

Ginseng is widely distributed in North America and in eastern Asia (mostly northeast China, the Korean peninsula, Russia, and Bhutan), typically in cooler climates. For more than 2000 years in China, ginseng (P. ginseng C.A. Mey.) has been considered an important component of Chinese traditional medicine that seeks to balance disharmonies in the body before they become full-flown diseases; it is also used in other East Asian countries such as Korea and Japan. Ginseng generally has a good safety profile and a low incidence of adverse effects (Coon and Ernst, 2002; Lee and Son, 2011). Hundreds of millions of Asian people consume P. ginseng not only for medicine and health supplements but also for agricultural products, food, dietary supplements, etc., depending on the consumer’s characteristics. Over the past decades, it has become increasingly popular in North America and Europe as well as other parts of the world (Brekhman and Dardymov, 1969; Hu, 1976; Cao et al., 1997). Along with the development of modern medicine and analytical methods, a large amount of the active chemical constituents and bioactivities of ginseng have been reported, such as polysaccharides, ginsenosides, peptides, polyacetylenic alcohol, and fatty acids. Ginseng’s pharmacological effects on the cardiovascular, immune, and central nervous system, and against cancer, diabetes mellitus, fatigue, and so on have been confirmed (Liu and Xio, 1992; Lu et al., 2009; Park et al., 2005). This chapter summarizes and discusses publications on ginseng and its effective components and biological activities to facilitate future scientific research on the plant.

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Introduction of Ginseng Ginseng refers specifically to the fleshy roots and rhizomes of the plant; however the leaf, berry, and other parts of ginseng are also medicinal sources (Xie et al., 2004, 2009; G.Y. Li et al., 2009). Ginseng products are divided into natural wild ginseng and farmed ginseng, which includes farmed ginseng, woods-grown ginseng, and wild-simulated ginseng. Wild ginseng is believed to have the best quality of all ginseng. According to different processing technologies, ginseng may also be divided into three categories: fresh, white, and red. Red ginseng is fresh ginseng that has been peeled, heated through steaming at a standard boiling temperature of 100 C (212 F), and then dried or sun-dried, whereas white ginseng is fresh ginseng that has been peeled and dried but not heated. The water content is reduced to 12% or less. The most common way to use dried ginseng is to marinate it in an herbal brew. The chemical constituents of ginseng are different in the three categories and result in different biological activities. Although the first use of ginseng was recorded around 2000 years ago, people in eastern Asia had already started to use ginseng 4500 years earlier. Because people know even more about traditional Chinese herbal medicine and demand better health, ginseng has become increasingly popular globally. Commercial ginseng is believed to be sold in over 35 countries. It has been reported that the world ginseng market is estimated to be worth $2084 million and total production was around 80,000 tons in 2013 (Baeg and So, 2013). Since the 1950s, when Petkov (1959) first reported the pharmacological properties of P. ginseng extract, more research regarding the traditional use, chemical constituents, and biological and pharmacological effects of ginseng has been published, especially over the past few years, owing to the rapid development of technology. Because ginseng contains complex constituents, research on the plant’s through pharmacognosy, phytochemistry, biosynthesis, and pharmacology has been reported in addition to traditional methods (Yun, 2001; Qi et al., 2010; Jia et al., 2009; Christensen, 2009; Angelova et al., 2008; Fuzzati, 2004). Normally ginseng displays restorative, tonic, and revitalizing properties owing to its active chemical constituents (Hu, 1977).

Active Chemical Constituents Several classes of compounds have been isolated from ginseng, including polysaccharides (Rittenbach et al., 2009), polyacetylene (Liu et al., 2007; Yang et al., 2010; Chan et al., 2010; Lee et al., 2004; Hirakura et al., 2000), phenolic components (Choi et al., 2010; Liu et al., 2009), amino acids (Naval et al., 2006), alkaloids (J.Y. Wang et al., 2006), and ginseng saponins. Ginseng saponins, also known as ginsenosides, panaxosides, triterpenoid saponins, and dammarane derivatives, have been found to be the predominant active constituents responsible for the pharmacological activities of ginseng (Kim and Park, 2011; Lee et al., 2013; Siddiqi et al., 2013; Kang et al., 2013; Lee et al., 2014; Lee and Kim, 2014). Hence ginseng is normally characterized by the presence of ginsenosides.

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Ginsenosides/Saponins Most ginseng saponins are a family of steroids with a four-trans-ring rigid steroid skeleton. It is believed that the first isolated ginseng saponin may be traced to 1854 (Park et al., 2005). The first reported isolation of six ginsenosides from P. ginseng occurred in the 1960s (Elyakov et al., 1964), and the chemical structures of several ginseng saponins were also characterized in the 1960s (Uvarova et al., 1965). After that, ginseng saponins were increasingly isolated and identified. Ginseng saponins can be isolated from various parts of the plant, most typically from the roots. The leaf, berry, and other parts of ginseng are also considered to be medicinal sources. To date, more than 200 saponins have been isolated from ginseng plants (Christensen, 2009). Studies have focused not only on ginseng root but also on the leaves and stems (Park et al., 2005; Jiang et al., 2008; Chen et al., 2009), flower buds (Nguyen et al., 2010a,b; Tung et al., 2010a,b; Nakamura et al., 2007), fruit (Wang et al., 2004; W. Wang et al., 2007), berries (C.Z. Wang et al., 2006a), and seeds (Sugimoto et al., 2009). As mentioned, the chemical constituents of ginseng are different owing to different processing technologies. Because the saponin profile of ginseng products can change because of steam or heat (C.Z. Wang et al., 2007; Sun et al., 2010), some ginseng saponins have also been isolated from processed roots (Liao et al., 2008; Lee et al., 2009), leaves (Nguyen et al., 2010a,b), flower buds (Tung et al., 2010a,b), and berries (C.Z. Wang et al., 2006b). From January 2000 to September 2010 alone, 123 new dammarane-type saponins were found from various parts of Panax plants (Qi et al., 2011). This chapter focuses on ginseng saponins found in three main kinds of ginseng root production over the past few decades: 2,3-oxidosqualene, believed to be the precursor of most ginseng saponins; and also b-sitosterol, a steroid commonly found in plants (Dou et al., 2008). Fig. 13.1 shows the proposed biosynthetic pathway of

HO

β-sitosterol

cycloartenol

cycloartenol synthase

HO 21

dammarenediol-II synthase

12

2,3-oxidosqualene

11 18 13 14 1 9 8 2 10 7 30 3 5 4 6 19

HO O

HO

29

dammarenediol-II

β-amyrin

HO

4

10 5

26

17

16 15

27

dammaranetype saponins

30

19 12 25 1

25

28

29

2 3

24 23

protopanaxadiol

β-amyrin synthase

HO

22

HO OH 20

HO

11 26 9 6

13 18 14

8 7

15

20

17 16

21 22 28

COOH

oleanane-type saponins

27

oleanolic acid

FIGURE 13.1 Biosynthetic pathways of ginseng saponins (Shin et al., 2015).

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

ginseng saponins and b-sitosterol. 2,3-Oxidosqualene may be cyclized into three different compounds (cycloartenol, dammarenediol-II, and b-amyrin); the latter two are the precursor of dammarane-type saponins and oleanane-type saponins, respectively (Shin et al., 2015). There are several ginseng saponin groups; two major ones are protopanaxadiol (PPD) and the protopanaxatriol (PPT), which are divided according to the position of sugar moieties at carbon. The PPD group has sugar moieties attached to C-3 and/or C-20 and the PPT group has sugar moieties at C-6 and/or C-20. Because PPD- and PPT-type chiral carbon C-20 position substitutes poor isobutyl, it is further divided into 20 (S) and 20 (R). Other groups include the ocotillol type with a five-membered epoxy ring at C-20, the oleanane type with a nonsteroidal structure, and the dammarane type with a modified C-20 side chain (Qi et al., 2010, 2011; Yoshikawa et al., 1998). As shown in Table 13.2 and Fig. 13.2, 70 ginseng saponins were isolated from the three main kinds of ginseng (P. ginseng C.A. Mey.) root production. Ginsenosides Rb1, Rb2, Re, and Rg1 are dammarane-type saponins biosynthesized from dammarenediol-II. By contrast, b-amyrin is the precursor of oleanane-type saponins. In P. ginseng, however, oleanane-type saponins are rare compared with ginsenoside Ro. The structures of these ginseng saponins are shown in Fig. 13.2. The major constituents of white and red ginseng are ginsenosides Rbl, Rb2, Rc, Rd, Rgl, Rg2, and Re, whereas ginsenosides Rg8, Rg5, and Rg6 are unique in red ginseng (Ryu et al., 1997). Normally heat and acidic conditions could denature ginsenosides, which is the reason for this difference. Once red ginseng is treated with heat, inner acidity is weaken by citric acid and other organic acids as a result of ginsenosides becoming denatured into several converted ginsenosides (Lee, 2014; Lee et al., 2015). The conversions from each ginsenoside are reported as follow: (Rg1/Rh1/Rh4, Rk3), (Re/Rg2/F4, Rg6), (Rf/Rg9, 20Z/Rg9, Rg10), and (Rb1, Rc, Rb2, Rd/Rg3/Rg5, Rk1, Rz1). The conversion mechanism of ginsenosides during red ginseng processing is shown in Fig. 13.3.

Polysaccharides The second most important phytochemical component of ginseng is polysaccharides and specific proteoglycans. Ginseng contains various saccharides, including monosaccharides, oligosaccharides, and polysaccharides. The polysaccharide content in ginseng has been reported at 40% (by weight). It was first isolated and documented in 1966 (Ovodov and Solov’eva, 1966). There are two types of polysaccharides: neutral and acidic. The higher biological activities are shown to occur in acid polysaccharides, known as ginsan, as pectin combined with glucouronic and galacturonic acid (Tomoda et al., 1993; Zhang et al., 2009; Baek et al., 2010). Several water-soluble ginseng oligosaccharides, a-Glcp-(1e6)-a-Glcp, a-Glcp-(1e6)-a-Glcp(1e4)-a-Glcp, a-Glcp-(1e6)-a-Glcp-(1e6)-a-Glcp-(1e4)-a-Glcp, and another six maltooligosaccharides (i.e., maltopentaose, maltohexaose, maltoheptaose, maltooctaose, maltononaose, and maltodecaose), were detected in 2012 (Wan et al., 2012;

Chapter 13  An Overview on Ginseng and Energy Metabolism

(A)

R2 O OH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

R2O OH

R2O OH

R 1O

R 1O

HO

20(S)-protopanaxadiol R1 R2 -glc(2-1)glc -glc(6-1) ara(p) (4-1) xyl -glc(2-1)glc -glc(6-1) ara(f) (2-1) xyl -glc(2-1)glc -glc(6-1) glc(3-1) xyl -glc(2-1)glc(6) Bu -glc(6-1) ara(p) (4-1) xyl -glc(2-1)glc(6) Ac -glc(6-1) ara(p) (4-1) xyl -glc(2-1)glc(6) Bu -glc(6-1) glc -glc(2-1)glc(6) Bu -glc(6-1) ara(p) -glc(2-1)glc(4) Bu -glc(6-1) ara(f) -glc(2-1)glc(6) Bu -glc(6-1) ara(f) -glc(2-1)glc -glc(6-1) glc -glc(2-1)glc -glc(6-1) ara(p) -glc(2-1)glc -glc(6-1) xyl -glc(2-1)glc -glc(6-1) ara(f) -glc(2-1)glc -glc -glc(2-1)glc -H -glc -H -glc(2-1)glc(6) Ac -glc(6-1) ara(p) -glc(2-1)glc(6) Ac -glc(6-1) ara(f) -glc(2-1)glc(6) Ac -glc(6-1) ara(f) -glc(2-1)glc(6) mal-glc(6-1) ara(3-1)xyl -glc(2-1)glc(6) mal-glc(6-1) glc -glc(2-1)glc(6) mal-glc(6-1) ara(p) -glc(2-1)glc(6) mal-glc(6-1) ara(f) -glc(2-1)glc(6) mal-glc -glc(2-1)glc(6) mal-glc(6-1) glc(6-1) xyl -glc -glc(6-1) glc -glc -glc(6-1) ara(f) -glc(2-1)glc -glc(6-1) glc(6-1) xyl -glc(2-1)glc(6) Ac -glc -glc(2-1)glc(6) Ac -glc(6-1) glc -glc(2-1)xyl -glc

OR1 20(S)-protopanaxatriol 34 35 36 37 38 39 40 41 42 44 45 47 48 49 50 51

213

R1 -glc(2-1) rha -glc -glc(3-1) glc -glc -glc -glc -glc(2-1) glc -glc -glc(2-1) rha -glc(2-1) glc -glc -glc(6-1) Bu -glc(4-1) glc -glc(2-1) xyl -glc(2-1) xyl -glc(6)Ac

20(R)-protopanaxadiol

R2 -glc -glc(3-1) glc -glc -glc(4-1) glc -glc(6-1) ara(f) -glc(6) Bu -H -glc -H -glc -H -glc -glc -glc -H -glc

R2O OH

R1 16 -glc(2-1)glc 17 -glc

R2O OH

HO OR1 20(R)-protopanaxatriol R1 43 -glc(2-1) rha 46 -glc

R2 -H -H

OH

OH

HO

R2 -H -H

HO

OR1 R1 R2 52 I-2-1 -glc(2-1) glc -H

OR1 R1 53 I-2-2 -glc(2-1) rha

OH R 2O OH

OH

OH

OH

OH

HO

HO OR1 R1 R2 54 I-2-3 -glc(2-1) glc -H

OR1 R1 57 I-2-5 -glc(2-1) rha

R1 R2 56 I-1-1 -glc(2-1) glc -H 62 I-1-4 -glc( 2-1) glc(6)Ac

R1 55 I-2-4 -glc(2-1) rha

OH

HO

R1O

OR1

OH

HO OR1 R1 58 I-2-6 -glc 64 I-2-8 -glc(6)Ac

OH

R1O 59 60 61 63 65

I-1-2 I-1-3 I-2-7 I-1-5 I-2-9

R2 R1 -glc(2-1) glc -glc -H -glc(2-1) glc(6)Ac -H

R2 -H -H -Oglc -H -glc(6)Ac

FIGURE 13.2 Structure of chemical constituents of ginseng root (Panax ginseng C.A. Meyer). (A) Ginseng saponins; (B) other chemical constituents (Ru et al., 2015; Xinbao et al., 2013).

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

(B)

R 2O OH

OH O

R1O

R 1O R1 -glc(2-1) glc

66 I-1-6

R1 II-1 -glcUA(2-1)glc II-1 -H II-1 -(6’-Me)glcUA(2-1)glc II-1 -(6’-PAE)glcUA(2-1)glc HO HO PAE=

R2 -glc(6-1)glc

N

N H

OR2

67 68 69 70

R2 -glc -ara(f) -glc -glc

R

71 R=H

74 R=COOCH3

72 R=COOC2H5

75 R=COOC4H9

73 R= O CH2OH

O

NH2 N O O

HO HO

N

O

HO HO

O 76

N

O

OH

OH

O

OH O

HO

OH O

77

O O

P

O

OH O

P

OH

O

O 79

OH

OH 78

HO

OH

OH

N

O O

OH

O

O

80

OH

H MeO

O

81

OH OH S O

R

MeO N

HO

83

Me

H

MeO

H Me

OMe 84 R:H gomisin N 85 R:OH gomisin A

82

FIGURE 13.2 Cont'd

Cai and Lei, 2016). Ginseng polysaccharides are clinically effective in preventing cancer and improving body immunity. The amount of acidic polysaccharides in red ginseng is three times higher than that in white ginseng. Although the chemical characteristics and content are not sufficiently defined through the acidic polysaccharides in red ginseng, it is clear that traditional red ginseng is more effective than white ginseng (Lee et al., 2015).

Polyacetylenes The roots of P. ginseng contain some nonwater-soluble substances, polyactylene compounds, from ginseng. The first identified polyacetylenic compound from P. ginseng was panaxynol (heptadeca-1,9-diene-4,6-diyne-3-ol); the second one was panaxydol (heptadeca-1-ene-9,10-epoxy-4,6-diyne-3-ol) (Takahashi and Yoshikura, 1966; Poplawski et al., 1980). To date, 20 kinds of polyacetylenic substances have been found (Okuda, 1992). Ginseng polyacetylenes have known toxicity and anticancer activities in vitro; however their anticancer activities are not clearly represented in vivo because of their chemical instability.

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(A) glc O OH

glc

HO OH

Rg2 (S, R) O glc – rha

HO

Re

- glc

- H2O

- glc

HO

O OH

OH

O glc – rha

HO

F4 O glc – rha

Rg1

HO

Rh1 (S. R)

HO

O glc

O glc

- H2 O

- H2O

- H2O

glc O OH

HO OH

OH

OH

OH

OH +

HO

20-gluco-Rf O glc – glc

Rg6 O glc – rha

HO

- glc HO OH

HO

20(E)-F4 O glc – rha

Rh4

HO

O glc

O glc +

OH

OH

OH

Rk3

HO

OH

- H2 O + Rf

HO

Rg9

HO

O glc – glc

+ 20(z)-Rg9

HO

HO

O glc – glc

O glc – glc

Rg10 O glc – glc

HO O glc

(B) glc

araf

glc

araf

O OH

O OH

- glc-araf

- mal. mal-Rc

R1 glc

R2

arap

glc

O OH

arap

mal-Rb2

glc

- glc-arap Rb2

R2

glc

glc

O OH

glc

- H2O Rk1

- mal.

- glc-glc

R2 glc

O OH

O OH

R2

R3

OH

R1: mal-glc-glc-OR2: glc-glc-O-

- mal. mal-Rd

R2

Rg3

Rb1

glc

R1

OH HO OH

O OH

mal-Rb1

R1

Rg5

R2

O OH - mal.

R1

OH

Rc

- glc

R3

Rz1

Rd

FIGURE 13.3 (A) Ginsenoside conversion mechanism in red ginseng processing. (B) Protopanaxatriol ginsenosides (Lee, 2014; Lee et al., 2015).

Alkaloids In 1986, three b-carboline alkaloids were isolated from ginseng roots by Han et al. (1986, 1987). In the next year, two other b-carboline alkaloids were reported by Jong et al. (Park et al., 1987, 1988). Their structures are shown in Fig. 13.2B.

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Glucosides Three glycosides (isomaltol-a-D-glucopyranoside, ketopropyl-a-D-glucopyranoside, and adenosine) were isolated from red ginseng based on spectral and chemical evidence. However, these compounds were not be found in white ginseng (Matsuura et al., 1984a,b; Han et al., 1985). Their structures are shown in Fig. 13.2B.

Phenolic Acid The first phenolic acid, maltol (3-hydroxy-2-methyl-4-pyrone), was isolated from ginseng roots in 1979 (Han et al., 1979). In 1981, another three identified phenolic acids, salicylic acid, vanillic acid, and p-hydroxycinnamic acid, were obtained from the ether-soluble acidic fraction of fresh ginseng (Han et al., 1981). Their structures are shown in Fig. 13.2B.

Others In addition to all of these constituents listed, in 1988 Jong et al (Park et al., 1988) isolated a thiazole (Kim et al., 1996). In 1990, two lingans, gomisin N and gomisin A, were isolated from Korean red ginseng (Huh et al., 1990). Their structures are shown in Fig. 13.2.

Pharmacological Effects Life depends on energy transformations; living organisms survive because of the exchange of energy within and without. The study of energy flow through living systems is called bioenergetics. The disorder of energy use and storage can lead to a series of problems such as metabolic syndrome (MetSyn), a cluster of at least three of the five (unfolding into a combination of nine) medical conditions: abdominal (central) obesity (cf. thin-outside-fat-inside), elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein levels, which are associated with a risk of developing cardiovascular disease and type 2 diabetes (Kaur, 2014; Felizola, 2015); furthermore disturbances in metabolic substrates, glucose, and fatty acids could produce nerve injury (Hinder et al., 2012). In 1956 Warburg (1956) found cancer cells to be able to elevate glycolysis in favor of mitochondrial respiration, which makes them better suited to cope with hypoxic conditions in solid tumors, and a study showed that elevated glycolysis can make cancer cells resistant to a therapeutically induced bioenergetic crisis. Many human diseases can cause a disorder of energy flow, or otherwise, whereas the constituents of ginseng are well known to have anticancer, antidiabetic, neuroregulatory, immune-enhancing, and blood floweimproving effects, as well as positive effects on memory enhancement and menopausal disorders (Liu and Xio, 1992; Lu et al., 2009; Park et al., 2005; Zhang et al., 1996; Yun et al., 2001; Joo et al., 2005; Jung et al., 2005).

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Anticancer Activity Saponin and nonsaponin compounds from ginseng roots have been shown to have powerful anticancer activity and were reported to show cytotoxic activities against various kinds of cancer cell lines in culture such as L1210, L5187Y, HeLa cells, sarcoma 180 cells, A549, SK-OV-3, SK-Mel-2, P388, and K562 (Baek et al., 1995a,b). Ginsenoside Rh2 was reported to inhibit human ovarian cancer growth in a nude mice model in 1991 by Kikuchi et al. (1991). After that, research on the anticancer potential of ginseng compounds was reported, such as Rb1, Rc, and Re acting as a weak phytoestrogen in MCF-7 human breast cancer cells, by binding and activating estrogen receptors at both the messenger RNA (mRNA) and protein levels (Y.J. Lee et al., 2003a,b); ginsenoside Rg3 and Rh2 inducing cell detachment and the inhibition of the proliferation of prostate cancer cells (H.S. Kim et al., 2004; G.Y. Li et al., 2009; W. Li et al., 2009); compound K, a ginsenoside metabolite, inhibiting the growth of human monocytic leukemia cell U937 by upregulating p21 and activating Jun N-terminal kinase in the G1 phase (Kang et al., 2005); Rg3 inhibiting tumor cell proliferation and inducing cell apoptosis in mice with induced liver cancer (Li et al., 2005); red ginseng as an adjuvant therapy in treating colorectal cancer via a synergistic action (Fishbein et al., 2009); and Rk1 inducing apoptosis in SK-MEL-2 human melanoma in vitro through upregulation of Fas, FasL, and Bax protein expression and downregulation of procaspase-8, procaspase-3, mutant p53, and bcl-2 protein expression (Kim et al., 2012), and so on.

Antidiabetic Activity In 1990, ginseng was reported to improve glucose homeostasis and insulin sensitivity (Sonnenborn and Proppert, 1991). Chung et al. (2001) reported that diabetic KKAy mice orally administered ginseng root for 4 weeks had reduced blood glucose levels similar to those that were insulin sensitized in 2001. Wild ginseng ethanol extract could prevent type 2 diabetes mellitus and possibly obesity in ICR mice by improving the insulin resistance index and decreasing the diameter of white and brown adipocytes (Yun et al., 2004). The most effective component of ginsenosides for streptozotocin-diabetic rats was found to be Rb2 (Yokozawa et al., 1985), which might inhibit palmitate-induced gluconeogenesis via adenosine monophosphateeactivated protein kinaseeinduced small heterodimer partner by relieving estrogen receptor (ER) stress (Lee et al., 2011; Jung and Chung, 2011).

Lipid-Regulating and Antithrombotic Activities Ginseng saponin has been found to have influence on lipid metabolism. Saponin could stimulate the absorption, metabolism, transport of lipids; decrease plasma cholesterol and triglyceride levels; and inhibit aortic atheroma formation in animals with hypercholesterolemia caused by long administration of high cholesterol or feeding on a diet-containing high cholesterol (Moon et al., 1984). Rg3 was identified as effective in

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

MetSyn in a comparison of the anti-MetSyn effect of vinegar-processed Radix Ginseng and unprocessed Radix Ginseng in a high-fat dieteinduced MetSyn ICR mouse model in 2006 (Yun et al., 2007). Red ginseng was reported to have a potent antithrombotic effect in vivo in 2006, and it may be beneficial to individuals with a high risk of thrombotic and cardiovascular diseases (Yu et al., 2006).

Immunoregulatory Activity In 1994, the aqueous extract of red ginseng was reported to have no antigenicity in guinea pigs but not to suppress immune reactions (Lee et al., 1994). Ginsan was reported to be a potent immunomodulator, producing several cytokines [tumor necrosis factor-a (TNF-a), interleukin (IL)-1b, IL-2, IL-6, IL-12, interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor] and stimulating lymphoid cells to proliferate (Song et al., 2003). Its mechanism of immunomodulator activity was studied in 2004; the results showed that ginsan at a dose of 100 mg/kg could cause marked elevation (1.7e2 fold) of heme oxygenase activity, decrease total hepatic cytochrome P-450 levels (by 20%e34%), and prolong zoxazolamine-induced paralysis time (by 65% e70%), with no evidence of causing hepatic injury, because serum aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase activities and levels of total bilirubin and albumin did not change (Song et al., 2004). It was also found to be able to improve g radiationeinduced immunosuppression and to have potential therapeutic effects on chronic fatigue syndrome (CFS) and synergistic immunostimulating activity against cyclophosphamide-induced immunosuppression with pidotimod (Han et al., 2005; Du et al., 2008; Wang et al., 2014).

Wound and Ulcer Healing Activity Several ginsenosides have been reported to have wound and ulcer healing activity: Rb2 enhancing epidermal cell proliferation by upregulating the expression of proliferationrelated factors; Rh3 from R5 improving chronic dermatitis or psoriasis by regulating IL-1b, TNF-a, and IFN-gamma produced by macrophage cells and Th cells; and Rb1 showing an antiulcer effect through increased mucus secretion (Choi, 2002; Shin et al., 2006; Jeong, 2002; Jeong et al., 2003).

Neuroregulation Activity In 1985, ginseng saponins were reported to be able to increase the amount of norepinephrine and dopamine in mouse brain. Ginseng total saponin (GTS) was reported to be able to modulate methamphetamine-induced striatal dopaminergic neuronal systems and modulate dopaminergic activity at both presynaptic and postsynaptic dopamine receptors (Oh et al., 1997; Kim et al., 1998). GTS might be useful in the prevention and therapy of behavioral side effects induced by psychotropic agents by attenuating the morphine-induced cyclic adenosine monophosphate signaling pathway (Kim et al., 2005). Ischemic brain injury was improved by ginsenoside Rh2 and compound K in 2004

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(Bae et al., 2004). Ginsenosides Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1 were found to regulate nociceptive processing induced by proinflammatory cytokines (TNF-a, IL-1b, and IFN-gamma) in 2008 (Seo et al., 2008). In 2009, red ginseng extract was reported to be able to modulate nerve growth factors (NGFs) expression in the steroid-induced polycystic ovary rat model by decreasing the ovarian concentration of NGF protein and NGF mRNA (Pak et al., 2009).

Health Effects In the philosophy of Chinese traditional medicine, ginseng is a tonic to remedy deficiencies in energy flow. Ginseng has been used as a panacea or to promote longevity; its active chemical constituents have tonic and revitalizing properties.

Antiaging Activity The importance of oxidative stress in the process of biological aging has been known for a long time (Muller et al., 2007). Excessive oxidative stress could lead to cell death and mitochondrial dysfunction (Bolli, 2007). Salicylic acid and vanillic acid have been reported to have potent antioxidant activity in the liver of ethanol-intoxicated mice, whereas p-hydroxycinnamic acid does not have this effect (Han et al., 1981). By comparing it with some antioxidant phenolic compounds, maltol was proved to be an antioxidant with little prooxidant activity in 1996 (Suh et al., 1996). Learning and memory in normal, aged, or brain-damaged animals could be improved using ginseng extract (Zhong et al., 2000; Kennedy and Scholey, 2003). It was reported that ginseng extract could enhance the age dependency of learning ability in the passive avoidance test in female rats (Jaenicke et al., 1991), and several ginsenosides have the function of ameliorating impaired memory function: Ginsenosides Rb1 and Rg1 accelerated memory acquisition of rats on a Y-maze task and enhanced the cognitive function of mice in a Morris water maze (Mook et al., 2001). Rg1 improved the scopolamine-induced impaired performance of rats in a radialarm maze; Rb1 and its metabolite M1 improved memory disorders, axonal atrophy, and synaptic loss in a mouse model of Alzheimer’s disease; and Rg3(S), Rg5, and Rk1 significantly reversed memory dysfunction (Tohda et al., 2004; Bao et al., 2005). Studies of ginseng extract and ginsenosides with regard to skin aging also showed many positive results. For example, ginsenoside Rb1 demonstrated antiaging activities in the skin resulting from an increase in type I collagen production and the suppression of UV-induced apoptosis (Cai et al., 2009; Kwok et al., 2012); compound K increased the amount of hyaluronan in the skin of hairless mice (S. Kim et al., 2004); and ginsenoside F1 protected human HaCaT keratinocytes against UVB-induced apoptosis (E.H. Lee et al., 2003).

Antifatigue Ginseng, particularly Asian ginseng, which is renowned as an herbal stimulant, unlike caffeinated substances, is both activating and restorative to central nervous system

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functions. It has been used in Chinese medicine to develop physical strength, especially in patients who experience severe fatigue (Saito et al., 1974). It was reported in 2010 that white ginseng polysaccharide possesses antifatigue activity and the acidic white ginseng polysaccharide (WGPA) showed higher potency to induce antifatigue activity compared with the neutral polysaccharide (Wang et al., 2010). Further study on the antifatigue activity of WGPA, its active component, and the mechanism that prevents CFS has been reported (Wang et al., 2014). In 2013, the first randomized, double-blind, controlled trial on the antifatigue effects of P. ginseng on subjects with idiopathic chronic fatigue showed that P. ginseng could be used to combat chronic fatigue, and that the mechanism may be related to its antioxidant properties (Kim et al., 2013). In 2015, a study on the effects of P. ginseng on fatigue induced by tumor growth showed that P. ginseng was safe and improved cancer-related fatigue as well as overall quality of life, appetite, and sleep at night (Yennurajalingam et al., 2015).

Others Many other studies related to the bioactivities of P. ginseng have been reported. For example, ginseng saponin could interact directly with Naþ-Kþ-adenosine triphosphatase before disruption of membrane barriers of sarcolemmal vesicles; GTS could modulate various cellular activities by inhibiting gap junction channel reconstitution; ginseng saponin could induce inositol triphosphateemediated Ca2þ release from ERs to activate the Ca2þ-activated Cl channel in Xenopus oocytes and it could be modulated by calmodulin; Rc could enhance IGABA in oocytes expressing human type A g-aminobutyric acidreceptor in Xenopus oocytes; and tissue culture root of wild P. ginseng could be a therapeutic agent for spermatogenic disorders (Lee et al., 1986; Hong et al., 1996; Choi et al., 2001a,b; Choi et al., 2001a,b, 2003; Lee et al., 2005; Park et al., 2006).

Conclusions Ginseng root (P. ginseng C.A. Mey.) has been used for dozens of centuries as a superior herb; it is listed as the top major tonic in the Chinese herbal classic, the Shennong Ben Cao Jing, and it is one of the most widely researched Chinese tonics. There is no doubt that strictly and traditionally defined ginseng and its extracts could improve human health because they have been used for thousands of years and have clinical efficacy proved by many Asian medical scientists. As modern science developed, more compounds were isolated from ginseng, especially the root, many of which have been confirmed to have certain pharmacological and/or health effects, especially ginsenosides, polysaccharides, and polyacetylenes. However more details about the relation between human health, disease, bioenergetics, and ginseng still need to be studied. In addition, further studies to isolate and define other constituents, new biological activities, and the mechanism of these effects need to be performed.

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Glycyrrhiza glabra (Licorice): Ethnobotany and Health Benefits Wang Xiaoying, Zhang Han, Wang Yu TIANJIN UNIVERSITY OF TRADITIONAL CHINESE M EDICINE, TIANJIN, C HINA

The licorice (Radix Glycyrrhizae or Liquiritiae radix), which is broadly used in medicine and commerce, is derived from the sweet root of various species of Glycyrrhiza (Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., or Glycyrrhiza inflata Bat., Leguminosae). Licorice is native to southern Europe and parts of Asia; it is one of the most commonly used herbal medicines and an important source of confectionery (Fiore et al., 2005). Therefore the health properties associated with licorice are well documented. According to the World Health Organization, licorice is employed as a demulcent in the treatment of sore throats and an expectorant for coughs and bronchial catarrh. Licorice also has a critical role in the prophylaxis and treatment of gastric and duodenal ulcers as well as dyspepsia. As an antiinflammatory agent, licorice reduces allergic reactions and prevents liver toxicity. In China, licorice is also documented to be effective for fatigue and debilitation (China, 2015). More than 20 triterpenoids and 300 flavonoids have been isolated from licorice to date (Yang et al., 2015). The major constituents are glycyrrhetic acid, flavonoids, isoflavonoids, hydroxycoumarins, and sterols, including b-sitosteroid, which may have glucocorticoid and mineralocorticoid activities (Seeff et al., 2001). Licorice roots are composed of approximately 3%e5% 18b-glycyrrhizin and 18b-glycyrrhizic acid (GA), which are considered to be the primary active components (Yu et al., 2012). When glycyrrhizin is taken orally, it is transformed into glycyrrhetic acid by intestinal bacteria and absorbed into the body (Akao et al., 1994). Active components of licorice are shown in Fig. 14.1. Many researchers have reported that these components could contribute to protecting the nervous, endocrine, respiratory, digestive, and cardiovascular systems. This chapter summarizes the active components, extractions, or preparations of licorice used medically that are supported by experimental or clinical data.

Effect on Respiratory System Licorice has a dramatic effect on inhibiting respiratory symptoms, especially airway injury. A randomized, double-blind comparison of licorice versus sugarewater gargle Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00014-4 Copyright © 2017 Elsevier Inc. All rights reserved.

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FIGURE 14.1 Chemical structures of active components in licorice.

proved the benefits of licorice in reducing the incidence of postoperative sore throat (POST) in patients who were intubated with double-lumen tubes (Ruetzler et al., 2013). Licorice gargle (0.5 g licorice in water) significantly attenuates the incidence and severity of POST (Agarwal et al., 2009). Another clinical trial reported that licorice lozenges reduced the distressing symptom of POST in the postoperative period among smokers (Gupta et al., 2013). The antiinflammation and transforming growth factor (TGF) signal pathway might be involved in licorice’s effect on the respiratory system. Moreover, liquiritin apioside decreased the cytotoxicity induced by cigarette extracts in a dosedependent manner and increased the expression of TGF-b and tumor necrosis factora (TNF-a) at the messenger RNA level in A549 cells as a protective agent against epithelial injury in chronic obstructive pulmonary disease (COPD) (Guan et al., 2012). In addition, glycyrrhetinic acid alleviates early-stage, radiation-induced lung injury by decreasing the expression of TGF-b1, Smad2, and Smad3 in lung tissue (Chen et al., 2016). Furthermore, licorice flavonoids (30 mg/kg) inhibit lipopolysaccharide (LPS)induced acute pulmonary inflammation by impairing the elevation of the content of water in the lung. Licorice is one of the most efficacious medicinal plants for the treatment of asthma (Javadi et al., 2016). According to the frequency of service prescribed for asthma among

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adults in Taiwan, 20,627 asthma patients (85.7%) used traditional Chinese medicines. Licorice was shown to be in the top three prescriptions, Ding-chuan-tang, Xiao-qinglong-tang, and Ma-xing-gan-shi-tang (the frequency was 23.1%, 15.1%, and 13.2%, respectively) (Wang et al., 2014). Licochalcone A (LA) is effective in treating inflammatory diseases such as asthma by suppressing the inhibitor of nuclear factor kB (NF-kB)/ thymic stromal lymphopoietin pathway in a dose- and time-dependent manner (Kim et al., 2015). LA (50 mg/kg) attenuates the allergic airway inflammation in a murine model of asthma by inhibiting the increase of T-helper type 2 cytokines such as interleukin (IL)-4, IL-5, and IL-13, and reducing serum levels of ovalbumin-specific immunoglobulin (Ig)E and IgG (Chu et al., 2013). Oral administration of glycyrrhizin (10 mg/ kg/day for 7 consecutive days) is benefit for lung histopathologic features in mice with chronic asthma (Hocaoglu et al., 2011). Other research indicates that the mechanism of glycyrrhizin on the airways might be work against b2-adrenergic receptor agonistinduced receptor internalization and cell apoptosis (Shi et al., 2011). Glycyrrhizin has a protective effect on acute lung injury caused by LPS, which induces sepsis and simultaneously reduces the alveolar capillary barrier (Zhao et al., 2016). G. uralensis flavonoids in antiasthma formula [antiasthma herbal medicine intervention (ASHMI)] reduce eosinophilic pulmonary inflammation and inhibit memory Th2 responses to antigen stimulation in culturing lung cells (Yang et al., 2013).

Hepatoprotective Effect Glycyrrhizin, GA, licorice flavonoid oil, and some preparations from licorice have strong hepatoprotective activity. Among them, glycyrrhizin has been developed as a hepatoprotective drug in China and Japan (Li et al., 2014). Glycyrrhizin preparation treats several liver diseases such as hepatitis B, hepatitis C, liver fibrosis, and cirrhosis. Stronger neominophagen C (SNMC), a Japanese preparation to treat chronic hepatitis that contains 0.2% glycyrrhizin, 0.1% cysteine, and 2% glycine, is marketed in Japan and India. Clinical trials on the hepatoprotective efficacy of licorice are listed in Table 14.1. Improving the activities of serum alanine aminotransferase (ALT) and aspartate transaminase (AST) is the principal indication for licorice on hepatoprotection. Licorice water extract reduces the elevation of serum transaminase induced by cadmium and significantly relieves liver cell swelling and necrosis (Lee et al., 2009a). Licorice extract (100 mg/kg) containing GA (15.77  0.34 mg/mg), liquiritin (14.55  0.42 mg/mg), and liquiritigenin (1.34  0.02 mg/mg) reverses the accumulation of hepatic lipid and enhances the activities of ALT and AST (Jung et al., 2016). GA (48 mg/kg/day) attenuates liver tissue inflammation, collagen deposition, and hydroxyproline levels in both bile duct ligation-induced rats and those with dimethylnitrosamine-stimulated hepatic fibrosis (Zhou et al., 2016). Glycyrrhizin and glycyrrhetinic acid inhibit experimental liver cirrhosis induced by carbon tetrachloride (Moro et al., 2008) and reduce the level of serum ALT, the content of serum globulin, and hepatic collagen proteins; they relieve interstitial inflammation and inhibit hyperplasia of hepatic fibrous tissue. Further

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Table 14.1 Components/ Preparations

Randomized Controlled Trials on Hepatoprotective Efficacy of Licorice Diseases

Patients

Alcohol consumption (40% ethanol) Chronic hepatitis B

12 (6 males and 6 females) 10

Healthy humans during light exercise Nonalcoholic fatty liver disease Subacute hepatic failure

34 females

SNMC

Time Period

Dose

Effect

References

12 d

0.1% e0.3%

Chigurupati et al. (2016)

12 month

Single dose

3 times/ week, short infusions 600 mg/d

Decrease alkaline phosphatase and plasma glutathione Regress biochemical disease activity

Mori et al. (2015)

66

2 months

2 g/d

Enhance fat oxidation; no change in lipid profile makers Decrease ALT and AST

18

30 d

Increase survival rate

Acharya et al. (1993)

Chronic hepatitis C

40

8 week

40 or 100 mL/d 100 mL/d

Kumada (2002)

SNMC

Chronic hepatitis C

84

8 week

Yo Jyo Hen Shi Ko

Nonalcoholic steatohepatitis

8

8 weeks

Improved liver histology and ALT levels Decrease cumulative hepatocellular carcinoma incidence Decrease ALT

Glycyrrhizin

Glycyrrhizic acid

Licorice flavonoid oil

Licorice root extract SNMC

100 mL/d 2e7 times/ week 500 mg three times/d

Eisenburg (1992)

Hajiaghamohammadi et al. (2012)

Arase et al. (1997)

Chande et al. (2006)

ALT, alanine aminotransferase; AST, aspartate transaminase; SNMC, stronger neominophagen C.

studies indicated that licorice saponins show significant hepatoprotective activities by lowering ALT and AST levels in primary rat hepatocytes injured by D-galactosamine (Zheng et al., 2015). Licorice flavonoids could also inhibit an increase in ALT activity in plasma by a model of T cellemediated fulminant hepatitis in mice (Feng et al., 2007). Preclinical animal trials showed that the hepatoprotective effect of licorice might be related to antioxidative stress, which is antiinflammatory and inhibits lipid peroxidation. Hepatoprotective effects of 18b-GA may be due to its ability to block the bioactivation of carbon tetrachloride and its free radical scavenging effects (Jeong et al., 2002). Licochalcones B and D strongly inhibited superoxide anion production and showed potent scavenging activity on diphenyl picryl hydrazinyl radical, which was related to

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inhibiting microsomal lipid peroxidation (Haraguchi et al., 1998). As a novel active ingredient, glycycoumarin ameliorates alcohol-induced hepatotoxicity via activation of Nrf2 and autophagy (Song et al., 2015). Glycyrrhizin inhibits the expression of type I and type III collagen in the liver tissue of rats with hepatic fibrosis, reduces hepatic necrosis, and promotes the regeneration of liver cells by antiinflammation, antilipid peroxidation, regulation of immunity, and lysosomal stability (Liang et al., 2015). Licorice flavonoids protect liver cells in rats with immunological liver injury induced by LPS (Xie et al., 2009).

Effect on Cardiovascular System Zhigancao decoction (roasted licorice decoction), which contains licorice, is a historical and typical prescription in Chinese medicine to treat almost any kind of arrhythmia. Zhigancao decoction appears to have beneficial effects on improving the total effective rate, relieving a number of ventricular premature beats in participants with premature ventricular contractions in the clinic (Liu et al., 2015). Extract of roasted licorice inhibits ventricular fibrillation, reduces the heart rate, and prolongs the QeT interval of electrocardiogram results (Liu and Jing, 2007). Roasted licorice injection could antagonize heart rhythm disorders induced by strophanthin G, aconitine, digoxin, and calcium chloride, which showed the effect of inhibiting calcium channel (Chen and Yuan, 1991). Active components of licorice such as flavones and triterpenes have also been investigated to protect the cardiovascular system and against endothelial dysfunction (Zhou et al., 2015; Feng et al., 2013). Hyperlipidemia is an important risk factor for cardiovascular disease. After patients with hypercholesterolemia and without significant stenosis consumed 0.2 g/d of ethanolic extract of licorice root for 12 months, mean carotid intima-media thickness, total cholesterol, low-density lipoprotein levels, and blood pressure decreased (Fogelman et al., 2016). Dietary licorice flavonoid oil significantly decreases hepatic cholesterol and plasma lipoprotein cholesterol levels by suppressing hydroxymethylglutaryl-CoA synthase activity (Honda et al., 2013). Glycyrrhizin (50 mg/kg) could decrease the content of cholesterol and triglyceride in the plasma of fructose-induced metabolic syndrome-X rats, which could reduce the activities of enzymatic antioxidants (superoxide dismutase and catalase) and elevate oxidative stress markers in metabolic syndrome to almost normal levels (Sil et al., 2013).

Immunity Regulation and Antiinflammation Effects The immune system is an incredibly intricate network of specialized cells that prevents infections and diseases by engulfing, modulating, and moderating malignant and foreign cells. The human immune system is composed of organs such as the spleen and thymus; in addition, lymph nodes and bone marrow contribute by producing and storing specific immune cells (Chaouat et al., 2007). Immune cells are of two major types: B cells and T cells. B cells are responsible for producing antibodies (immunoglobulins), proteins

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designed to recognize and mark specific antigens, whereas T cells are moieties charged with destroying antigens tagged with an antibody (Chaouat et al., 2007; Zhang et al., 2007). T cells have a critical role in controlling adaptive immune functions; their responses could be used to develop protective vaccines. They may induce tolerance to antigens that cause inappropriate immune responses, such as autoimmune diseases (Cooper and Alder, 2006; Li et al., 2007). In addition, phagocytes such as granulocytes, macrophages, and natural killer (NK) cells release pyrogens and interferon (IFN), which act as immunoregulatory moieties (Currier and Miller, 2002; Fauci et al., 2005). Cytokines are also effective in regulating immune responses. Other mediators such as TNF-a, IL, chemokines, and IFN also contribute to the proper functioning of the immune system (Timar et al., 2007). Licorice enhances the immune function and potentially increases mucosal immunity and antiinflammation effects in the peripheral tissues of pigs (Katayama et al., 2011). Meanwhile, licorice and roasted licorice reduce clinical arthritis scores, paw swelling, and histopathological changes on tissue plasminogen activatoreinduced acute inflammation and collageninduced arthritis in mice (Kim et al., 2010). Therefore, licorice holds immunoregulatory as well as antiinflammatory activities that are mainly attributed to its bioactive constituents, such as GA, glycyrol, and isoliquiritigenin (ILG). In this context, we focus on GA and its molecular mechanisms in regulating immunity as well as its antiinflammatory effects. GA and glycyrrhetinic acid are well-characterized components of licorice. GA generates glycyrrhetinic acid through metabolic processes in the human body. Therefore, the pharmacological effects of GA are essentially the same as those of glycyrrhetinic acid. GA, also called glycyrrhizin, is a triterpene glycoside from licorice root (G. glabra) and consists of one molecule of 18b-GA and two molecules of glucuronic acid (Matsui et al., 2004). Several immunomodulatory activities have been attributed to glycyrrhizin and GA. The reduction in cellular immunocompetence in gamma-irradiated mice can be recovered through treatment with glycyrrhizae and GA. These fractions are found to be effective in enhancing the leukocyte count and blastogenic responses of splenocytes to mitogens (Dorhoi et al., 2006). Glycyrrhizin selectively activates extrathymic T cells in the liver and in human T-cell lines (Kimura et al., 1992). GA is an inducer of type 2 antagonistic CD41 T cells in in vivo and in vitro studies (Kobayashi et al., 1993). In addition, it stimulates macrophage-derived NO production and upregulates inducible nitric oxide synthase expression through NF-kB transactivation in murine macrophages (Jeong and Kim, 2002). Both of them induce IFN activity and augment NK cell activity; glycyrrhizin is superior to GA in inducing IFN (Abe et al., 1982). On the contrary, GA induces the expression of Tolllike receptor 4 and its downstream signaling molecules, which have an important role in modulating innate immune responses against pathogens (Peng et al., 2011). Several mechanisms have been suggested for the antiinflammatory effects of GA. GA inhibits glucocorticoid metabolism and potentiates their effects. This potentiation is reported in skin and lung after coadministration with GA (Teelucksingh et al., 1990). Because GA is a potent inhibitor of 11b-hydroxysteroid hydroxygenase (11b-HSD) (Walker and Edwards, 1991), it causes an accumulation of glucocorticoids with antiinflammatory properties. Oral administration of GA or glycyrrhizin confirms this result.

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Glycyrrhizin inhibits reactive oxygen species generation by neutrophils, which are the potent mediator of tissue inflammation in the in vitro study. One of its antiinflammatory effects results from this inhibitory effect (Akamatsu et al., 1991; Wang and Nixon, 2001). By inhibiting Kv1.3 channels, GA has antiinflammatory effects in human Jurkat T cells (Wang et al., 2013a). Glycyrrhizin has been shown to increase the activity of dendritic cells, enhance the proliferation of allogenic T cells along with production of IFN-gamma and IL-10, and reduce IL-4 production (Bordbar et al., 2012). In contrast, GA impairs the capacity of dendritic cells to proliferate and initiate the T helper-1 response in LPSstimulated mature dendritic cells. GA also suppresses the expression of surface molecules CD80, CD86, and major histocompatibility complex classes I and II, and reduces levels of IL-12 production (Kim et al., 2013). G. glabra and glyderinine, a derivative of GA, also shows an antiinflammatory effect (Tokiwa et al., 2004). It also reduces myocardial inflammatory edema in experimental myocardial damage (Zakirov et al., 1999). GA does not inhibit either cyclooxygenase 1e or 2ecatalyzed prostaglandin biosynthesis with an IC50 value of 425 mM in an in vitro study. However, in another study, G. radix was involved in cyclooxygenase (COX-2) inhibition. Furthermore, G. radix increases corticosterone levels in rats. Also, glycyrrhizin and glycyrrhetinic acid are known to inhibit phospholipase A2 (Kase et al., 1998). Some derivatives of GA have shown inhibitory activity against IL-1beinduced prostaglandin E2 production in normal human dermal fibroblasts (Tsukahara et al., 2005). Some scientific evidence supports the hypothesis that the immunostimulating activity of licorice depends on the proper metabolism and functioning of various important mediators in adaptive or acquired immunity. The claims mentioned in the earlier section suggested that G. glabra could act as an immunostimulating agent (Brush et al., 2006). However, the mechanisms underlying the antiinflammatory activity of glycyrrhizin are still poorly understood. Use of licorice has been gaining wide popularity in the United States as well as other parts of the world, but the mechanism of action has not been subjected to thorough scientific investigation. Licorice holds therapeutic potential in clinical therapy to prevent or cure certain health risks with the additional benefit of reducing the costs of prevention. Indeed, findings suggested that licorice and its bioactive metabolites are effective in aiding the balance and proper function of the immune system through various modules of immune modification such as stimulation and suppression. Thus, the current scenario demands formal scientific research to explore the mode of action of licorice. Nutritionists, physicians, and other health professionals can use such information effectively to treat various ailments in vulnerable segments. Overall, licorice can be used as an additional tool for disease prevention and risk management.

Antitumor Activities The use of plants’ natural products in cancer treatment has received attention owing to their potentially wide safety margin and the capacity to complement conventional

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chemotherapeutic drugs. Plant-based products have demonstrated anticancer potential through different biological pathways. The extract as well as active ingredients of licorice have antitumor roles through diverse mechanisms. This part will deal with the pharmacological effects of licorice and its bioactive components in different tumor treatments, both in vivo and in vitro.

Breast Cancer Breast cancer continues to cause high cancer death rates among women worldwide. Despite early detection and apparently complete surgical resection, many patients die of metastatic cancer that remains undetected at diagnosis. Identification of dietary bioactive compounds lacking in toxicity and capable of blocking more than one tumor process could be a good strategy to treat breast cancer. Licorice root extract possesses cytostatic properties by inducing cell cycle arrest and suppressing the expression of the aryl hydrocarbon receptor in tumorigenic effects of endocrine-disrupting chemicalestimulated human breast cancer cells (Chu et al., 2014). The ethanol extract of licorice root induces apoptosis and G1 cell cycle arrest in MCF-7 human breast cancer cells (Jo et al., 2005). On the other hand, several derivatives of licorice components have the antitumor effects, including LA (Park et al., 2014), licochalcone E (Kwon et al., 2013), formononetin (Zhou et al., 2014), glycyrrhetinic acid (Wang et al., 2015), and GA. Notably, ILG has been most common reported among them. ILG, a flavonoid phytoestrogen from licorice, inhibits the migration and invasion of MDA-MB-231 cells by preventing anoikis resistance (Zheng et al., 2014). Meanwhile, ILG induces growth inhibition and apoptosis by downregulating the arachidonic acid metabolic network and deactivating phosphatidylinositol 3-kinase (PI3K)/Akt in human breast cancer. Remarkably, ILG induced growth inhibition and apoptosis of MDA-MB231 human breast cancer xenografts in nude mice, together with decreased intratumoral levels of eicosanoids and phospho-Akt [Thr(308)] (Chu et al., 2013). Interestingly, ILG inhibited the receptor activator of NK-kB ligand (RANKL)eosteoprotegerin ratio and COX-2 expression in human osteoblast hFOB1.19 cells stimulated with conditioned medium of metastatic breast cancer MDA-MB-231 cells. Thus, ILG can be a beneficial agent to inhibit and treat breast cancer celleassociated bone diseases by blocking the interaction between cancer cells and bone cells, by inhibiting osteoblastic RANKL expression (Lee et al., 2015). Licorice and its bioactive compounds could be promising multitarget agents to prevent breast cancer; future clinical trials are needed to examine the effectiveness of licorice in preventing human breast cancer.

Hepatocellular Carcinoma Hepatocellular carcinoma (HCC), one of the most common cancers in the world, causes nearly 600,000 deaths annually. Patients who have HCC are often diagnosed at a late stage and die within 7e8 months after diagnosis (Llovet et al., 2003). For years, standard chemotherapy and radiotherapy for HCC patients have remained disappointing

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(Arii et al., 2000). Efforts have been made, but no satisfactory drugs have been manufactured. Novel therapeutic agents for curing HCC are still highly in demand. Licorice and its derivatives have been reported to possess anti-HCC effects. Licorice flavonoid oil has a significant inhibitory effect on HCC (Nakagawa et al., 2010). Glycyrrhizae polysaccharide not only blocks the PI3K/Akt signal pathway (Chen et al., 2013), it reduces the proportion of T-regulatory cells and upregulated T-helper (Th) 1eTh2 cytokine ratio in HCC-bearing mice, which might partially cause the inhibition of tumor growth (He et al., 2011). GA, a pentacyclic triterpenoid from the roots of licorice plant, is widely used in HCCtargeted drug delivery systems (TDDS) owing to its highly expressed target binding sites on HCC cells, and induces the content of CYP enzymes significantly (Paolini et al., 1999). GA triggers a protective autophagy in HCC cells by activating extracellular signale regulated kinase (ERK), which might attenuate the anticancer effects of GA or chemotherapeutic drugs loaded with GA-modified TDDS (Tang et al., 2014). Meanwhile, GA significantly inhibits proliferation of the human hepatoma cell line by modulating inflammatory markers and inducing apoptosis without affecting the normal liver cell line (Hasan et al., 2016). GA can also inhibit the metabolic activation of hepatotoxin so as to protect against chemical-induced carcinogenicity (Chan et al., 2003). In a diethylnitrosamine-treated experimental animal study, as a chemopreventive agent of HCC, modulation of cell proliferation and apoptosis by GA may be associated with inhibition of HCC. Therefore, GA treatment may inhibit the occurrence of HCC (Shiota et al., 1999).

Prostate Cancer Prostate cancer is the most frequently diagnosed noncutaneous malignancy and the second leading cancer-related cause of death in men; it is responsible for nearly 30,000 deaths each year in the United States. Prostate cancer was estimated to be responsible for 28% (186,320) of all newly diagnosed cancers in 2010. Primary stages of the disease can be treated with surgery, androgen ablation, radiation therapy, or all of these. Patients undergoing hormonal therapy eventually develop aggressive hormone-unresponsive disease. Hence, the major focus in prostate cancer research is the discovery of better chemotherapeutic agents for the advanced hormone-resistant, metastatic form of this disease (Jemal et al., 2010). LA, isoangustone A (IAA), and ILG from licorice extract have been shown to possess inhibitory effects against prostate cancer in vitro and/or in vivo. LA, a novel estrogenic flavonoid isolated from PC-SPES composition herb licorice root, causes G2 and late-G1 arrest in androgen-independent PC-3 prostate cancer cells (Fu et al., 2004). However, LA induces caspase-dependent and autophagy-related cell death in androgen-dependent LNCaP prostate cancer cells by suppressing B-cell lymphoma 2 expression and the mechanistic target of rapamycin (mTOR) pathway (Yo et al., 2009). In the treatment of DU145 prostate cancer cells, licoricidin reduces cell migration and the secretion of matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1,

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urokinase-type plasminogen activator, and vascular endothelial growth factor, as well as the expression of adhesion molecules. These results indicate that licoricidin is a potent antimetastatic agent that can markedly inhibit the metastatic and invasive capacity of malignant prostate cancer cells (Park et al., 2010). IAA is an active compound from the hexaneeethanol extract of G. uralensis. The administration of IAA significantly attenuates the growth of prostate cancer cell cultures and xenograft tumors by inhibition CDK2 and mTOR (Lee et al., 2013a). In DU145 prostate cancer cells, IAA decreases DNA synthesis and induces G1 phase arrest, accompanied by a reduction of CDK2 and CDK4, as well as cyclin A and cyclin D1 (Seon et al., 2012). On the other hand, IAA increases apoptotic cells, the cleavage of poly(adenosine diphosphateribose) polymerase and caspases, and the levels of death receptor 4 and Mcl-1S (Seon et al., 2010). Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. ILG, a simple chalcone-type flavonoid derived from licorice, shallots, and bean sprouts, is a potent antioxidant with antiinflammatory and anticarcinogenic effects. ILG selectively inhibits the proliferation of prostate cancer C4-2 cells, which may be attributed in part to defective adenosine monophosphateeactivated protein kinase and ERK signaling pathways in C4-2 compared with IEC-6 cells (Zhang et al., 2010). In DU145 human prostate cancer cells and MAT-LyLu rat prostate cancer cells, ILG reduces cell proliferation by inducing apoptosis, which is mediated through mitochondrial events, and cell cycle arrest via the inhibition of ErbB3 signaling and the PI3K/Akt pathway (Jung et al., 2006; Kanazawa et al., 2003; Lee et al., 2009b), and it inhibits cancer cell invasion and migration by decreasing the JNK/activating protein-1 signaling pathway (Kwon et al., 2009). Altogether, both licorice and its bioactive derivatives suppress the proliferation of carcinoma by inducing apoptosis and cell cycle arrest, as well as inhibiting metastatic and invasive capacity. Hence, licorice and its bioactive compounds are potent chemopreventive agents that are potentially considered for clinical intervention in human cancer.

Effect on Gastrointestinal Tract The hydroalcoholic extract of G. glabra L. (50e200 mg/kg) exerted an antiulcergenic effect in an HCl/ethanol-induced ulcer that might be associated with an increase in gastric mucosal defensive factors (Jalilzadeh-Amin et al., 2015). A methanol extract of licorice root (Fm100) is the active antiulcer extract of licorice; it can completely inhibit the formation of gastric ulcer induced by the ligation of gastric ulcer in rats and decrease endogenous gastric acid secretion induced by acetylcholine and histamine (Ishii and Fujii, 1982). Secretin might work as a potential mediator of antiulcer actions of licorice because of its mucosal protective agents (Takeuchi et al., 1991). Licorice has antispasmodic effect on gastric smooth muscle. The total flavonoids have a significant spasmolytic effect on intestinal spasm induced by acetylcholine, histamine, and barium chloride. Six active constituents in licorice (GA, ILG, liquiritinapioside,

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liquiritigenin, isoliquiritin apioside, and glycycoumarin, 20 mmol/kg, intravenously) resulted in rapidly and significantly inhibited tetanic contractions (Lee et al., 2013b). Among these active ingredients from licorice, liquiritigenin proved to be strongest in antispasmodic effect (Nagai et al., 2006). Studies suggest that licorice may be a mild inhibitor of P-glycoprotein in intestinal mucosa (Yao et al., 2009).

Effect on Endocrine System Licorice and its active ingredients possess regulatory effects on the endocrine system, including potentiating the action of glucocorticoid cortisol, exerting estrogenic activity, and reducing testosterone synthesis. Among the compounds derived from licorice, glycyrrhizin, glycyrrhetinic acid, and carbenoxolone have inhibitory effects on 11b-HSD type 1 (11b-HSD1) and 2 (11b-HSD2), which catalyze the interconversion between active cortisol and insert cortisone. Glycyrrhetinic acid is approximately 200 times more potent than GA at inhibiting 11b-HSD1 (Makino, 2014). Carbenoxolone inhibits 11b-HSD1 activity in a concentration-dependent manner with an IC50 of 5 mM. Glycyrrhizin and glycyrrhizic acid also have potent inhibitory effects against 11b-HSD2 (dissociation constant of approximately 5e10 nM) to lower cortisol-induced mineralocorticoid activity and support hypothalamicepituitaryeadrenal axis function (Asl and Hosseinzadeh, 2008). This action of licorice provides therapeutic benefits for adrenal insufficiency, Addison disease, and postural hypotension treatment (Ferrari, 2010). However, inhibition of 11b-HSD2 activity by licorice overconsumption is generally detrimental to health owing to mineralocorticoid excess including hypokalemia, hypertension, and pseudohyperaldosteronism. In 2003, the Scientific Committee on Food recommended an upper limit of 100 mg/d for glycyrrhizin. Licorice and its extracts are widely available as dietary phytoestrogens for menopausal women as a natural alternative to hormone replacement therapy to relieve menopausal symptoms. Liquiritigenin, ILG, glabridin, calycoricone, methoxychalcone, vestitol, glyasperin C, glycycoumarin, and glicoricone have low binding affinity for estrogen receptors (ERs). Liquiritigenin and ILG have similar affinities to ER with IC50 values of 7.5 and 7.8 M, respectively, in competitive radiometric binding assays. ILG binds to ER with an IC50 value of 16 M, whereas liquiritigenin has weak affinity for ER (200 M) (Omar et al., 2012). However, a study reported that liquiritigenin, ILG, and 7 other components derived from licorice bind with approximately equal affinity to ERa and ERb, except for liquiritigenin and glyasperin C, which have more than 10 times preference for ERb. Liquiritigenin (10 6 M), ILG (10 6 M), methoxychalcone (3  10 6 M), and vestitol (3  10 6 M) stimulated MCF-7 cell proliferation. The highest level of proliferation induced by liquiritigenin and methoxychalcone was similar to that induced by E2 (10 10 M). Liquiritigenin, ILG, calycoricone, methoxychalcone, vestitol, and glycycoumarin also activated estrogen target gene expression including the progesterone receptor and GREB1 in cultured MCF-7 breast cancer cells. The stimulatory effect of licorice components is completely inhibited by cotreatment with antiestrogen reagent ICI182,78 (Hajirahimkhan et al., 2013).

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Licorice is used as a plant-derived antiandrogen to improve idiopathic hirsutism and polycystic ovary syndrome (Boonmuen et al., 2016). Licorice containing 0.5 g GA decreases serum testosterone and increases 17-hydroxyprogesterone by inhibiting 1720-lyase and 17-hydroxysteroid dehydrogenase activity after 4 and 7 days of administration in males (Faghihi et al., 2015). On the other hand, studies suggest that salivary testosterone and dehydroepiandrosterone increased after consuming licorice confectionery (3% licorice extract) for 7 days in male and female volunteers (Armanini et al., 1999). These findings lend support to the reasonable use of licorice as a promising strategy to treat hormone-dependent diseases.

Side Effects and Cautions In large amounts, licorice, which contains glycyrrhizin, might cause high blood pressure, salt and water retention, and low potassium levels, which could lead to heart problems. Thus people with heart disease or high blood pressure should be cautious about licorice. According to the Chinese Pharmacopeia, licorice is contraindicated in combination with Sargassum (Hai Zao), Herba Cirsii Japonici (Da Ji), Euphorbia kansui (Gan Sui), and Flos genkwa (Yuan Hua), which might attenuate liver function and cause cardiac toxicity (Wang et al., 2013b). It has been reported that licorice could alter enzyme activities of P450 isoforms and modulate drug transporter proteins such as P-glycoprotein, which leads to potential herbedrug interactions of licorice that are of concern. According to the US National Center for Complementary and Integrative Health (NCCIH), the recommended daily dose of licorice root is 5e15 g, equivalent to 200e600 mg of glycyrrhizin (https://nccih.nih.gov/health/liquoriceroot). In the Chinese Pharmacopeia (2015), the recommended dose of licorice is 2e10 g. Both the NCCIH and the European Medicines Agency report that the safety of using licorice as a dietary supplement for more than 4e6 weeks has not been thoroughly studied. Side effects from overdose or prolonged use (more than 4 weeks) might lead to symptoms such as water retention, hypokalemia, hypertension, or cardiac rhythm disorders.

Conclusion and Perspective Licorice is widely cultivated throughout Europe, the Middle East, and Asia. The major constituent of licorice, glycyrrhizin (also known as GA or glycyrrhizinic acid), is about 50 times sweeter than sucrose (common sugar). In Western countries, licorice is mainly used in nonmedicinal forms such as soft drinks, herbal teas, and tobacco products (Tobacco Documents Online http://tobaccodocuments.org/profiles/licorice.html). The medicinal potential of licorice needs further investigation and recognition in Western countries. Licorice has been extensively used by Chinese people to relieve and prevent cough, phlegm, dyspnea, spasms, and pain. It also relieves spasms of the smooth (involuntary) muscles and exhibits a cortisone-like action. In Ayurvedic medicine, licorice has a long

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history as a common remedy because of its expectorant, antiinflammatory, and laxative properties. In Chinese medicine, licorice is often combined with other herbs in many herbal formulas by harmonizing the characteristics of other herbs, alleviating the toxicity of herbs, and modulating the taste of herbs because of its sweet flavor. Licorice is also recorded as “National Venerable Master,” which has paradoxical roles, i.e., detoxifying/ strengthening efficacy and inducing/enhancing toxicity. When licorice is used in combination with some toxic herbs such as Radix aconiti lateralis praeparata (Fu zi), Rhizoma pinelliae (Ban xia), and Cinnabaris (Zhu sha), the toxicity of these herbs might be attenuated by licorice (Guo et al., 2014). Licorice roots, extracts, active ingredients such as glycyrrhetic acid, flavonoids and isoflavonoids, and also some prescriptions showed efficiency in regulating respiratory function, hepatoprotection, immunoregulation, antiinflammation, antineoplastic action, and gastroenteric protection. Based on this aspect, licorice could be considered the most important herb and the focus of research in herbal medicine. Further synergistic or antagonistic studies of licorice are needed for evaluation and validation based on both preclinical trials and clinical observation.

Abbreviations 11b-HSD 11b-Hydroxysteroid dehydrogenase ALT Alanine aminotransferase AST Aspartate transaminase COPD Chronic obstructive pulmonary disease ERs Estrogen receptors GA Glycyrrhizic acid HCC Hepatocellular carcinoma IAA Isoangustone A IFN Interferon IL Interleukin ILG Isoliquiritigenin LA Licochalcone A LPS Lipopolysaccharide NF-kB Nuclear factor kB POST Postoperative sore throat SNMC Stronger neominophagen TDDS Targeted drug delivery systems TGF Transforming growth factor TNF-a Tumor necrosis factor-a

References Abe, N., Ebina, T., Ishida, N., 1982. Interferon induction by glycyrrhizin and glycyrrhetinic acid in mice. Microbiol. Immunol. 26, 535e539. Acharya, S.K., Dasarathy, S., Tandon, A., Joshi, Y.K., Tandon, B.N., 1993. A preliminary open trial on interferon stimulator (SNMC) derived from Glycyrrhiza glabra in the treatment of subacute hepatic failure. Indian J. Med. Res. 98, 69e74.

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15

An Overview of Yohimbine in Sports Medicine

Nevio Cimolai UNIVE RSITY OF BRITISH COLUMB IA, V ANCOUVER, BC, CANADA

Introduction Whether from the root of Rauwolfia or the bark of the yohimbe tree, yohimbine has been used for various purposes both in the laboratory and for public consumption. Yohimbine is an indole alkaloid with a variety of physiological effects (Cimolai and Cimolai, 2011). The drug’s potential is sought in macerations of the tree bark or in crude or refined extractions. In sports medicine, yohimbine may be used alone or combined with an assortment of other physiologically active or inactive natural, purified, or semisynthetic products. Purified yohimbine has been available for prescription in some countries, but its use has diminished considerably as other novel pharmacological agents have been developed and have replaced yohimbine’s novelty. For nonprescription use, many preparations are commercially available for human consumption. Popular applications include additions to energy drinks, fat loss/slimming products, and dietary supplements. When obtained as a bark extract, the addition of a yohimbine product to an ingestible formulation lends the producer the opportunity to call the outcome a dietary supplement. Cohen et al. (2015) identified some interesting features of yohimbine that are present in dietary supplements in the United States. First, there is an incredible array of such commercial preparations, most of which consist of concoctions in which other ingredients coexist. A minority of these products formally list yohimbine as an active ingredient. Of those in which yohimbine is listed overtly as being present, some do not contain any of this drug. Quantities of yohimbine vary considerably from the recommended product dosing, and the amount present may not correlate with the stated quantity. In one product said to be indicated for fat loss, up to eight ingredients are named (Alkhatib et al., 2015). Venhuis et al. (2014) detailed serious adverse effects of one such cocktail of dietary supplements, termed Dexaprine. In this and other situations, additive or enhancing sympathomimetic overstimulation is achieved (Kearney et al., 2010). The attribution of toxicity or even death to one or more of these constituents becomes difficult in such a milieu of polypharmacy. Such mixtures would commonly include caffeine or other adrenergic agents (Rebiere et al., 2012; Venhuis Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00015-6 Copyright © 2017 Elsevier Inc. All rights reserved.

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et al., 2014). Combinations of yohimbine that may be touted for sports enhancement are analogous in their complexity to the use of yohimbine mixtures for sexual enhancement (Corazza et al., 2014). Analytical methods can be used to detect the presence, quantitation, and sourcing of yohimbine (Lucas et al., 2015). As a supplement, the use of tree bark powder that contains yohimbine is susceptible to considerable variation in dose. Older plants commonly contain a greater concentration of yohimbine. Available yohimbine products can be screened for yohimbine-associated alkaloids or other natural chemical derivations. Thus application of contemporary analytical methods can distinguish the presence of yohimbine from bark or the presence of purified yohimbine, i.e., adulterated supplements. Woolsey et al. (2014) conducted an interesting study of energy drink use in college students. They determined that the frequency of energy drink use among this educated cohort correlated with the likelihood that these students were more often to exploit the consumption of prescription stimulants. Correlating the latter to athletics, it is conceivable that yohimbine use might promote the use of other stimulant products. Some countries have banned yohimbine use in supplements and food (Cohen et al., 2015).

Mechanisms of Action Human and animal model responses to yohimbine are dose dependent, but subject-tosubject variability is evident for a fixed dose. In humans, doses as small as 1e5 mg may give measurable physiological responses. Commercial products often contain 5e30 mg, although higher doses have been used by individuals. Administration or consumption of yohimbine increases blood norepinephrine levels (Galitzky et al., 1990; Hedner et al., 1992). The mechanism of such an increase is yohimbine’s ability to create a2-adrenoreceptor antagonism. As norepinephrine rises either locally or systemically, various accentuated responses are achieved. In humans, an increase in heart rate is among the earlier responses. This can be followed variably by increases in blood pressure and by mood effects (Cimolai and Cimolai, 2011). Later physiological effects can be considered advances in autonomic reactions such as pupillary dilatation, sweating, agitation, tremor, nausea, penile erection, and headache. Toxicities of the central nervous system, kidney, or skin may occur from an excess of the drug. Yohimbine has a rapid distribution in normal volunteers (Hedner et al., 1992). Both intrahepatic and extrahepatic metabolism seem likely. The half-life is relatively short, so that an effect may be achieved within 1e2 h of ingestion but will resolve over the next 4e6 h. Increases in cardiac norepinephrine release have been observed (Wang et al., 2013). In animal models, brain norepinephrine is increased by yohimbine (Nirogi et al., 2012). Entrance into the human brain is rapid, and a concentration of binding sites exists especially in the cerebral cortex and the hippocampus (Nahimi et al., 2015). Among central or peripheral nerve effects, yohimbine may modify pain or inhibit noxious inhibitory controls (Bannister et al., 2015; Park et al., 2010).

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In theory, therefore, ingestion of yohimbine at appropriate timing could allow its effects clearly to last training or competition sessions in sports. On a first-level basis, one would anticipate yohimbine to act as a stimulant for any given athlete.

What Are the Needs for Sports Enhancement? If yohimbine were only a stimulant, it might attract attention for sports enhancement, but the effects of yohimbine continue to be expanded as basic and clinical research continue. Enhancement may conceivably take several paths, because the requirements in various sports can be diverse. Energization or stimulation is one of these facets. Other aspects of individual sports may benefit from combinations of physical enhancement (e.g., muscular bulking), weight loss, reduction of adipose tissue, impulsivity, concentration, pattern recognition, fear guarding, reduction in general fatigue, aerobic improvement, other metabolic optimization, and pain control or mitigation. The ability to excel in some niche sports, however, may also be impeded by changes in these factors. It is unlikely that any one pharmacological agent in itself will provide all of these benefits.

Theoretical Reflections Animal Model Research Yohimbine has consistently been used to initiate animal models for physiological stress (See and Waters, 2011). Central nervous system effects can be prolonged, and recurrent administration stimulates both recurrent stress and anxiety states (Figlewicz et al., 2014). Yohimbine can increase impulsivity (Sun et al., 2010). Similar effects in animals include a reinstatement of alcohol- or cocaine-seeking behaviors (Chen et al., 2015; Simms et al., 2011). In a general sense, yohimbine reinstates reward seeking (Chen et al., 2015). It can also bias animals toward increased risky behaviors when decision making (Montes et al., 2015). General decision making can be impaired, and inflexible perseverative behaviors may be fostered (Schwager et al., 2014; Wong and Marinelli, 2015). Fear extinction can be enhanced, and there may be an augmentation of extinction learning (Janak and Corbit, 2011; Singewald et al., 2015). The effect on fear extinction occurs in a dose-dependent fashion (Kaplan and Moore, 2011). The drug has been found to increase food-seeking behavior in general, and this can specifically include sucrose-seeking trends (Nair et al., 2011; Simms et al., 2011). Although yohimbine causes anorexia, an improvement in lipid profiles in a rat model of diet-induced obesity was identified (Dudek et al., 2015). The latter was accompanied by a reduction in intraperitoneal adipose tissue. Physical and emotional stressors are initially and normally accompanied by elevations in blood concentrations of glucose, but yohimbine impairs such a natural response (Sim et al., 2010). Exercise is associated with various physiological and feedback alterations that may ultimately increase pain thresholds. Yohimbine reversed the latter peripherally in animal studies (de Souza et al., 2013).

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Human Correlates As in animal models, yohimbine creates stress and anxiety responses in humans (See and Waters, 2011). It may also induce panic symptoms in a proportion of volunteers who are otherwise deemed normal (Kaplan et al., 2012). Startle and fear are affected (Soeter and Kindt, 2011). Yohimbine can strengthen fear memory and enhance fear extinction (Holmes and Quirk, 2010; Soeter and Kindt, 2012; Fitzgerald et al., 2014). However, one study ran contrary to the latter and could not show enhancement of fear extinction (Meyerbroeker et al., 2012). The drug may enhance extinction learning (Hofmann et al., 2011). Human impulsivity, as in animals, also increases with the ingestion of this pharmacological agent. An increased urge toward drug-seeking behavior was shown, but no effect was found on the urge to gamble (Elman et al., 2012; Greenwald et al., 2013). Nevertheless, in human social anxiety, yohimbine augments exposure therapy (Smits et al., 2014). Given these effects on stress, anxiety, impulsivity, and fear, one might anticipate precarious effects on human learning or performance. For example, yohimbine increased commission errors in normal volunteers (Moran-Santa Maria et al., 2016). There was no effect, however, on deliberative reasoning in healthy subjects (Margittai et al., 2016). In another setting, yohimbine enhanced memory consolidation but not memory retrieval or working memory (Wingenfeld et al., 2013). In sports, a fight-or-flight response, and for that matter even impulsivity, could be valuable for timely reactions. Such agitation could be detrimental in other sport contexts in which action and reaction are more measured and yet delayed.

Metabolic Effects in Humans There have been several controlled studies in humans of the metabolic effects of yohimbine but few have specifically included athletes. Most of these studies enrolled only 10e30 or more subjects. Doses administered have been variable but inclusive of the range of 15 to over 40 mg/day.

Obesity Berlin et al. (1986) conducted a randomized, placebo-controlled, double-blinded study over 8 weeks. Nineteen participants, mainly female, were administered 18 mg/day and a low-caloric diet consisting of 1000 kcal/day. There was no apparent effect on plasma lipids, either cholesterol or free fatty acids, and no effect on body weight. In that same year, however, Zahorska-Markiewicz et al. (1986) completed a nearly identical investigation. For 24 obese female participants, they gave 15 mg/day and included a placebo arm. However, the low-calorie diet was only 400 kcal/day. The investigators observed that plasma glycerol and free fatty acids were increased and that weight loss was achieved. Thereafter, Kucio et al. (1991) administered 20 mg/day to 20 obese females in a randomized, double-blinded, placebo-controlled study over 3 weeks. The latter was accompanied by a 1000-kcal/day diet. There was no evidence of lipolysis but yohimbine

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was associated with weight loss. Sax (1991) gave doses of 15e40 or more milligrams per day to obese adult men in a randomized, double-blind, placebo-controlled study over 6 months. An 1800-kcal diet was maintained. The outcomes did not show a net effect of yohimbine on plasma cholesterol. There was also no net effect on body weight, mass index, or fat. Berlan et al. (1991) gave 0.2 mg/kg per day to small groups of normal and obese females. The study was randomized and double-blinded, and a 15-day washout period was given between crossover. They found that plasma free fatty acids increased equally in normal and obese subjects.

Normal Males Galitzky et al. (1988, 1990) conducted two studies with doses ranging from 12 mg/day to 0.27 mg/kg per day. Patients served as their own controls. As in the study of Berlan et al. (1991), plasma free fatty acids were elevated by yohimbine. Herda et al. (2008) examined a dietary supplement that contained yohimbine. They studied the effect of a combination product on vertical jump height, isometric extensor leg strength, leg extension endurance, and forearm flexion endurance. A placebo control and washout period were used. There was no improvement in measured variables.

Athletes Ostojic (2006) studied 20 normal male athletes who were given 20 mg/day yohimbine. The study was randomized and placebo controlled, compared over 21 days. There was a decrease in body fat percentage with yohimbine but no change in body or muscle mass, or performance indicators. One other study found no differences for athletes and controls; yohimbine equally raised plasma cortisol, increased heart rate and blood pressure, and induced anxiety symptoms (Sommer et al., 2011). Overall, studies in athletes are limited regarding published data. Because yohimbine is a banned substance for amateur competitive athletics, it is difficult to imagine that much interest would be garnered for any other study relating to performance.

Proven and Potential Toxicities Toxicity is dose related, and most individuals who experience the inadvertent use of toxic doses will recover after a relatively short period of expectant restoration, which is measured in hours. Most side effects of excess include agitation, anxiety, palpitations, and high blood pressure (Cimolai and Cimolai, 2011). Even large overdoses will usually resolve with only supportive management. Anecdotal reports of excessive ingestion have detailed exposure to doses ranging from 200 to 5000 mg. Linden et al. (1985) described a female youth who ingested approximately 250 mg. She experienced anxiety, nausea, tachycardia, elevated blood pressure, headache, rash, chest pain, and various neurological symptoms. The latter included tremors, incoordination, paresthesia, and a dissociative mentation. She recovered after 11/2 days.

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Friesen et al. (1993) published a report of a male who ingested 200 mg. He manifested cardiovascular signs that would have been anticipated, but he also had pathological signs of the extremities. The toxic effects disappeared in less than 1 day. Varkey (1992) reported on a middle-aged man who ingested 350 mg. The individual experienced atrial fibrillation. His neurological presentation included confusion. Two citations documented the occurrence of seizures and loss of consciousness with much higher doses (3000 and 5000 mg for two patients) (Giampreti et al., 2009; Halcomb et al., 2006). Self-harm from street use of yohimbine have been witnessed. Deaths from yohimbine overdosing are uncommonly reported but nonetheless published (Anderson et al., 2013). Prolonged use of yohimbine has not been adequately studied and its potential toxicity cannot be extrapolated. Many excess ingestions of yohimbine occur in a setting where other toxic agents are coingested (Venhuis et al., 2014). Attribution of the toxicity of a specific agent is therefore difficult, and synergistic effects cannot be ruled out. Among human use of botanical products, yohimbine is among the most commonly identified in reported toxicities (Woolf et al., 2005). Kearney et al. (2010) stated that yohimbine reported as a toxin increased during the first decade of the new millennium. The latter also described, along with Sandler and Aronson (1993), anecdotes of both dermatological side effects and nephrotoxicity.

Would Yohimbine Be Unique in Its Activity for Sports? In general, science relies on the facts of yohimbine’s blocking actions, which consequently raise local and systemic norepinephrine levels. The net effect is that yohimbine effectively provides an adrenergic stimulus, however that should be manifest. If that were to be the entire effect of yohimbine, it would be one among many pharmacological agents that could theoretically fill the same role. Why then would yohimbine be preferred in any event? It is interesting that alternate receptors for yohimbine have not been defined or generally accepted. Some contemporary research on this basis could be called for. Individuals consuming yohimbine are likely to sense an effect on themselves given a sufficient dose. Although this effect will be greater than placebo for heart rate and agitation, it is unclear what role yohimbine may have in driving the thought process that a competitive advantage is at hand. That is, apart from the usual physiological effects, does the use of the drug promote the concept of enhancement for an athlete, and thereby derive its own benefit by promoting mind over matter or a placebo effect toward achievement?

Has Yohimbine Been Adequately Studied for Sports? Most studies in humans, as cited here, have had a relatively narrow view of human enhancement. These works have included few subjects and have been conducted over relatively short periods of time. Metaanalyses of the data provided to date would not be

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feasible. Ostojic’s study (2006) stands out as the only one addressing performance indicators while solely using yohimbine. Certainly a drug’s effect in sports performance must include a more complex and prolonged study period. Such studies, especially with regard to the degree of funding and work that would be required, are unlikely to be conducted in the near future. It is conceivable that yohimbine’s effect will continue to be measured only by its stimulant activity.

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Friesen, K., Palatnick, W., Tenenbein, M., 1993. Benign course after massive ingestion of yohimbine. J. Emerg. Med. 11, 287e288. Galitzky, J., Rivie`re, D., Tran, M.A., Montastruc, J.L., Berlan, M., 1990. Pharmacodynamic effects of chronic yohimbine treatment in healthy volunteers. Eur. J. Clin. Pharmacol. 39, 447e451. Galitzky, J., Taouis, M., Berlan, M., Rivie`re, D., Garrigues, M., Lafontan, M., 1988. a2-antagonist compounds and lipid mobilization: evidence for a lipid mobilizing effect of oral yohimbine in healthy male volunteers. Eur. J. Clin. Invest. 18, 587e594. Giampreti, A., Lonati, D., Locatelli, C., Rocchi, L., Campailla, M.T., 2009. Acute neurotoxicity after yohimbine ingestion by a body builder. Clin. Toxicol. 47, 827e829. Greenwald, M.K., Lundahl, L.H., Steinmiller, C.L., 2013. Yohimbine increases opioid-seeking behavior in heroin-dependent, buprenorphine-maintained individuals. Psychopharmacology 225, 811e824. Halcomb, H.S.E., Parab, S., Ravikumar, P.R., Hoffman, R.S., Nelson, L.S., 2006. Massive yohimbine overdose associated with sodium channel blockade. Clin. Toxicol. 44, 731. Hedner, T., Edgar, B., Edvinsson, L., Hedner, J., Persson, B., Pettersson, A., 1992. Yohimine pharmacokinetics and interaction with the sympathetic nervous system in normal volunteers. Eur. J. Clin. Pharmacol. 43, 651e656. Herda, T.J., Ryan, E.D., Stout, J.R., Cramer, J.T., 2008. Effects of a supplement designed to increase ATP levels on muscle strength, power output, and endurance. J. Int. Soc. Sports Nutr. 5, 3. Open Access. Hofmann, S.G., Smits, J.A., Asnaani, A., Gutner, C.A., Otto, M.W., 2011. Cognitive enhancers for anxiety disorders. Pharmacol. Biochem. Behav. 99, 275e284. Holmes, A., Quirk, G.J., 2010. Pharmacological facilitation of fear extinction and the search for adjunct treatments for anxiety disorders e the case of yohimbine. Trends Pharmacol. Sci. 31, 2e7. Janak, P.H., Corbit, L.H., 2011. Deepened extinction following compound stimulus presentation: noradrenergic modulation. Learn. Mem. 18, 1e10. Kaplan, G.B., Moore, K.A., 2011. The use of cognitive enhancers in animal models of fear extinction. Pharmacol. Biochem. Behav. 99, 217e228. Kaplan, J.S., Arnkoff, D.B., Glass, C.R., Tinsley, R., Geraci, M., Hernandez, E., Luckenbaugh, D., Drevets, W.C., Carlson, P.J., 2012. Avoidant coping in panic disorder: a yohimbine biological challenge study. Anxiety Stress Coping 25, 425e442. Kearney, T., Tu, N., Haller, C., 2010. Adverse drug events associated with yohimbine-containing products: a retrospective review of the California Poison Control System reported cases. Ann. Pharmacother. 44, 1022e1029. Kucio, C., Jonderko, K., Piskorska, D., 1991. Does yohimbine act as a slimming drug? Isr. J. Med. Sci. 27, 550e556. Linden, C.H., Vellman, W.P., Rumack, B., 1985. Yohimbine: a new street drug. Ann. Emerg. Med. 14, 1002e1004. Lucas, D., Neal-Kababick, J., Zweigenbaum, J., 2015. Characterization and quantitation of yohimbine and its analogs in botanicals and dietary supplements using LC/QTOF-MS and LC/QQQ-MS for determination of the presence of bark extract and yohimbine adulteration. J. AOAC Int. 98, 330e335. Margittai, Z., Nave, G., Strombach, T., van Wingerden, M., Schwabe, L., Kalenscher, T., 2016. Exogenous cortisol causes a shift from deliberative to intuitive thinking. Psychoneuroendocrinology 64, 131e135. Meyerbroeker, K., Powers, M.B., van Stegeren, A., Emmelkamp, P.M., 2012. Does yohimbine hydrochloride facilitate fear extinction in virtual reality treatment of fear of flying? A randomized placebo-controlled trial. Psychother. Psychosom. 81, 29e37. Montes, D.R., Stopper, C.M., Floresco, S.B., 2015. Noradrenergic modulation of risk/reward decision making. Psychopharmacology 232, 2681e2696.

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Moran-Santa Maria, M.M., Baker, N.L., McRae-Clark, A.L., Prisciandaro, J.J., Brady, K.T., 2016. Effects of yohimbine and drug cues on impulsivity and attention in cocaine-dependent men and women and sex-matched controls. Drugs. Alcohol Depend. 162, 56e63. Nahimi, A., Jakobsen, S., Munk, O.L., Vang, K., Phan, J.A., Rodell, A., Gjedde, A., 2015. Mapping a2 adrenoceptors of the human brain with 11C-yohimbine. J. Nucl. Med. 56, 392e398. Nair, S.G., Navarre, B.M., Cifani, C., Pickens, C.L., Bossert, J.M., Shaham, Y., 2011. Role of dorsal medial prefrontal cortex dopamine D1-family receptors in relapse to high-fat food seeking induced by the anxiogenic drug yohimbine. Neuropsychopharmacology 36, 497e510. Nirogi, R., Abraham, R., Jayarajan, P., Medapati, R.B., Shanmuganathan, D., Kandikere, V., Irappanavar, S., Saralaya, R., Benade, V., Bhyrapuneni, G., Muddana, N., 2012. Difference in the norepinephrine levels of experimental and non-experimental rats with age in the object recognition test. Brain Res. 1453, 40e45. Ostojic, S.M., 2006. Yohimbine: the effects on body composition and exercise performance in soccer players. Res. Sports Med. 14, 289e299. Park, C.H., Yong, A., Lee, S.H., 2010. Involvement of selective alpha-2 adrenoreceptor in sympathetically maintained pain. J. Korean Neurosurg. Soc. 47, 420e423. Rebiere, H., Guinot, P., Civade, C., Bonnet, P.A., Nicolas, A., 2012. Detection of hazardous weight-loss substances in adulterated slimming formulations using ultra-high-pressure liquid chromatography with diode-array detection. Food Additives & Contaminants Part A. Chem. Anal. Control Expo. Risk Assess. 29, 161e171. Sandler, B., Aronson, P., 1993. Yohimine-induced cutaneous drug eruption, progressive renal failure, and lupus-like syndrome. Urology 41, 343e345. Sax, L., 1991. Yohimbine does not affect fat distribution in men. Int. J. Obes. 15, 561e565. Schwager, A.L., Haack, A.K., Taha, S.A., 2014. Impaired flexibility in decision making in rats after administration of the pharmacological stressor yohimbine. Psychopharmacology 231, 3941e3952. See, R.E., Waters, R.P., 2011. Pharmacologically-induced stress: a cross-species probe for translational research in drug addiction and relapse. Am. J. Translat. Res. 3, 81e89. Sim, Y.B., Park, S.H., Kang, Y.J., Kim, S.M., Lee, J.K., Jung, J.S., Suh, H.W., 2010. The regulation of blood glucose level in physical and emotional stress models: possible involvement of adrenergic and glucocorticoid systems. Arch. Pharmacal. Res. 33, 1679e1683. Simms, J.A., Richards, J.K., Mill, D., Kanholm, I., Holgate, J.Y., Bartlett, S.E., 2011. Induction of multiple reinstatements of ethanol- and sucrose-seeking behaviour in Long-Evans rats by the a-2 adrenoreceptor antagonist yohimbine. Psychopharmacology 218, 101e110. Singewald, N., Schmuckermair, C., Whittle, N., Holmes, A., Ressler, K.J., 2015. Pharmacology of cognitive enhancers for exposure-based therapy of fear, anxiety and trauma-related disorders. Pharmacol. Ther. 149, 150e190. Smits, J.A., Rosenfield, D., Davis, M.L., Julian, K., Handelsman, P.R., Otto, M.W., Tuerk, P., Shiekh, M., Rosenfield, B., Hofmann, S.G., Powers, M.B., 2014. Yohimbine enhancement of exposure therapy for social anxiety disorder: a randomized controlled trial. Biol. Psychiat. 75, 840e846. Soeter, M., Kindt, M., 2011. Noradrenergic enhancement of associative fear memory in humans. Neurobiol. Learn. Mem. 96, 263e271. Soeter, M., Kindt, M., 2012. Stimulation of the noradrenergic system during memory formation impairs extinction learning but not the disruption of reconsolidation. Neuropsychopharmacology 37, 1204e1215. Sommer, M., Braumann, M., Althoff, T., Backhaus, J., Kordon, A., Junghanns, K., Ehrenthal, D., Bartmann, U., Hohagen, F., Broocks, A., 2011. Psychological and neurodendocrine responses to social stress and to the administration of the alpha-2-receptor antagonist, yohimbine, in highly trained endurance athletes in comparison to untrained healthy controls. Pharmacopsychiatry 44, 129e134.

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Sun, H., Green, T.A., Theobald, D.E., Birnbaum, S.G., Graham, D.L., Zeeb, F.D., Nestler, E.J., Winstanley, C.A., 2010. Yohimbine increases impulsivity through activation of cAMP response element binding in the orbitofrontal cortex. Biol. Psychiat. 67, 649e656. Varkey, S., 1992. Overdose of yohimbine. Br. Med. J. 304, 548. Venhuis, B., Keizers, P., van Riel, A., de Kaste, D., 2014. A cocktail of synthetic stimulants found in a dietary supplement associated with serious adverse events. Drug Test. Anal. http://dx.doi.org/10. 1002/dta.1664. Wang, Y., Yu, W., Wang, F., Wang, Y., Li, H., Lv, X., Lu, D., Wang, H., 2013. Yohimbine promotes cardiac NE release and prevents LPS-induced cardiac dysfunction via blockade of presynaptic a2Aadrenergic receptor. PLoS One 8 (5), e63622. Wingenfeld, K., Kuffel, A., Uhlmann, C., Terfehr, K., Schreiner, J., Kuehl, L.K., Otte, C., Lowe, B., Spitzer, C., 2013. Effects of noradrenergic stimulation on memory in patients with major depressive disorder. Stress 16, 191e201. Wong, W.C., Marinelli, M., 2015. Adolescent-onset of cocaine use is associated with heightened stressinduced reinstatement of cocaine seeking. Addict. Biol. http://dx.doi.org/10.1111/adb.12284. Woolf, A.D., Watson, W.A., Smolinske, S., Litovitz, T., 2005. The severity of toxic reactions to ephedra: comparisons to other botanical products and national trends from 1993e2002. Clin. Toxicol. 43, 347e355. Woolsey, C.L., Barnes, L.B., Jacobson, B.H., Kensinger, W.S., Barry, A.E., Beck, N.C., Resnik, A.G., Evans Jr., M.W., 2014. Frequency of energy drink use predicts illicit prescription stimulant use. Subst. Abuse 35, 96e103. Zahorska-Markiewicz, B., Kucio, C., Piskorska, D., 1986. Adrenergic control of lipolysis and metabolic responses in obesity. Horm. Metab. Res. 18, 693e697.

16

Black Ginger Extract Enhances Physical Fitness Performance and Muscular Endurance Kazuya Toda, Hiroshi Shimoda ORYZA OIL & FAT CHEMICAL CO. L TD., ICHINOMIYA, JAPAN

Introduction Black ginger, the rhizome of Kaempferia parviflora (Zingiberaceae), has traditionally been used as a food and folk medicine for more than 1000 years in Thailand. Dried rhizomes are generally pulverized and used as tea bags, whereas fresh rhizomes are utilized to brew wine. The wine preparation is increasingly used in Thailand as a tonic and aphrodisiac. As dietary supplements, rhizomes have been made into various preparations such as medicinal liquor, pills (powdered rhizome with honey), capsules, and tablets. In traditional Thai medicine, black ginger has been purported to cure allergies, asthma, impotence, gout, diarrhea, dysentery, peptic ulcers, and diabetes. Recent studies have demonstrated the biological activities of black ginger extract (K. parviflora extract; KPE), such as improved energy metabolism through the phosphorylation of 50 AMP-activated protein kinase (AMPK) (Toda et al., 2016a). KPE also enhances physical fitness and muscular endurance (Toda et al., 2016b; Promthep et al., 2015; Wattanathorn et al., 2012), and shows antiobesity (Akase et al., 2011; Shimada et al., 2011), gastric protective (Rujjanawate et al., 2005), antiglycation (Kusirisin et al., 2009), and aphrodisiac activities (Chaturapanich et al., 2012). As the constitutions, black ginger contains various polymethoxy flavonoids (PMF) such as 5-hydroxy-3,7-dimethoxyflavone (1), 5-hydroxy-7-methoxyflavone (2), 5-hydroxy-3,7,40 -trimethoxyflavone (3), 5-hydroxy-3,7,30 ,40 -tetramethoxyflavone (4), 3,5,7,30 ,40 -pentamethoxyflavone (5), 5,7,40 -trimethoxyflavone (6), 3,5,7,40 -tetramethoxyflavone (7), 5,7-dimethoxyflavone (8), 3,5,7-trimethoxyflavone (9), 5-hydroxy-7,40 dimethoxyflavone (10), 5,7,30 ,40 -tetramethoxyflavone (11), 5,30 -dihydroxy-3,7,

Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00016-8 Copyright © 2017 Elsevier Inc. All rights reserved.

261

262

SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Name

3

5

7

3′

4′

OCH3

OH

OCH3

H

H

H

OH

OCH3

H

H

5-hydroxy-3,7,4′-trimethoxyflavone (3)

OCH3

OH

OCH3

H

OCH3

5-hydroxy-3,7,3′,4′-tetramethoxyflavone (4)

OCH3

OH

OCH3

OCH3

OCH3

3,5,7,3′,4′-pentamethoxyflavone (5)

OCH3

OCH3

OCH3

OCH3

OCH3

H

OCH3

OCH3

H

OCH3

OCH3

OCH3

OCH3

H

OCH3

H

OCH3

OCH3

H

H

OCH3

OCH3

OCH3

H

H

H

OH

OCH3

H

OCH3

5-hydroxy-3,7-dimethoxyflavone (1) 5-hydroxy-7-methoxyflavone (2)

5,7,4′-trimethoxyflavone (6) 3,5,7,4′-tetramethoxyflavone (7) 5,7-dimethoxyflavone (8) 3,5,7-trimethoxyflavone (9) 5-hydroxy-7,4′-dimethoxyflavone (10) 5,7,3′,4′-tetramethoxyflavone (11)

H

OCH3

OCH3

OCH3

OCH3

OCH3

OH

OCH3

OH

OCH3

5,4′-dihydroxy-7-methoxyflavone (13)

H

OH

OCH3

H

OH

5-hydroxy-7,3′,4′-trimethoxyflavone (14)

H

OH

OCH3

OCH3

OCH3

4′-hydroxy-5,7-dimethoxyflavone (15)

H

OCH3

OCH3

H

OH

5,3′-dihydroxy-3,7,4′-trimethoxyflavone (12)

FIGURE 16.1 Chemical structures of polymethoxy flavonoids (PMF) in black ginger extract (Kaempferia parviflora extract; KPE) (Toda et al., 2016a).

40 -trimethoxyflavone (12), 5,40 -dihydroxy-7-methoxyflavone (13), 5-hydroxy-7,30 ,40 -trimethoxyflavone (14), and 40 -hydroxy-5,7-dimethoxyflavone (15) (Fig. 16.1). Regarding antiinflammatory effects of PMF, Horigome et al. (2014) previously reported that 4 and 8 more potently inhibited antigen-induced mast cell degranulation than nobiletin in citrus peel, a well-known antiinflammatory PMF. Sae-wong et al. (2011) demonstrated that 6, 8, and 11 markedly inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated RAW264.7 cells. Similarly, Tewtrakul and Subhadhirasakul (2008) showed that 4 (IC50 ¼ 16.1 mM) exhibited higher activity against NO release from RAW264.7 cells, followed by 10 (IC50 ¼ 24.5 mM) and 3 (IC50 ¼ 30.6 mM) than NG-nitro-L-arginine methyl ester, a positive control (NO synthase inhibitor, IC50 ¼ 61.8 mM). In addition, it was revealed that 4 exerted appreciable inhibitory effects against prostaglandin E2 release (IC50 ¼ 16.3 mM). Regarding lipid and glucose metabolism, Horikawa et al. (2012) found that 5 and 7 strongly induced the differentiation of 3T3-L1 preadipocytes to adipocytes and elevated mRNA levels and release of adiponectin. We also reported that 2, 3, and 8 enhanced the expression of glucose transporter type 4 (GLUT4) and peroxisome proliferatoreactivated receptor g coactivator-1a (PGC1a), while 8 enhanced the phosphorylation of AMPK (Toda et al., 2016a). Azuma et al. (2011) demonstrated the a-glucosidase inhibitory effects of 11 (IC50 ¼ 20.4 mM), 6 (54.3 mM), and 5 (64.3 mM). In addition, PMF has been shown to suppress prostate hyperplasia (Murata et al., 2013), tumors (Yenjai et al., 2009; Patanasethanont et al., 2007), and liver failure (Chaipech et al., 2012). Therefore, PMF in KPE have been suggested to work as active ingredients in dietary supplements or processed foods. In this section, we focus on the effects of KPE on physical fitness performance and muscular endurance.

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Mechanisms of Kaempferia parviflora Extract Underlying Increases in Physical Fitness by Kaempferia parviflora Extract KPE had been suggested to increase physical fitness based on its various effects on serum testosterone, muscular metabolism, muscular inflammation, and vasodilatation. Trisomboon et al. (2007) reported that black ginger significantly increased serum testosterone levels, but had no testosterone-like effect in male rats. Testosterone promotes the growth of skeletal muscle (anabolic effects) and exerts androgenic effects in males and females. Therefore, black ginger has been suggested to promote the hypertrophy of skeletal muscle by elevating serum testosterone levels. Muscular hypertrophy is involved in improvement of physical fitness, blood flow, hypothermia, and metabolic dysfunction. We previously investigated the effects of PMF in black ginger on muscular metabolism, particularly the phosphorylation of AMPK in C2C12 myoblasts (Toda et al., 2016a). These cells were treated with KPE (10 mg/mL) or 8 (10 mM) for 1 week. Cells were then collected to extract whole proteins. The phosphorylation of AMPK was evaluated by western blotting. The findings obtained revealed that KPE and 8 enhanced the expression of phosphorylated AMPK in C2C12 myoblasts (relative amounts of phosphorylated AMPK/total AMPK; control versus KPE versus 8; 1.0 versus 1.7 versus 2.3, respectively). AMPK is critically involved in the regulation of energy homeostasis (Kahn et al., 2005; Hardie et al., 2016; Astratenkova and Rogozkin, 2013), and its activation has been shown to enhance the metabolism of glucose and lipids (Nasri and Rafieian-Kopaei, 2014; Malin and Kashyap, 2014). Therefore, AMPK has been attracting attention for its application to antidiabetic or antiobesity treatments. Metformin, a typical AMPK activator, is known to improve high blood glucose levels in patients with type 2 diabetes. On the other hand, the phosphorylation of AMPK is linked to physical activity and muscular endurance. 5-Aminoimidazole-4-carboxyamide ribonucleotide, an agonist of AMPK, was previously reported to increase running endurance by up to 44% and decrease body fat in mice when orally administered for 4 weeks (Narkar et al., 2008). Consequently, the phosphorylation of AMPK has been suggested to improve physical fitness performance, muscular endurance, and fat metabolism. Hence, KPE and 8 may enhance the phosphorylation of AMPK, thereby promoting glucose and lipid metabolism. We also reported that 2, 3, and 8 significantly enhanced the expression of GLUT4 and PGC-1a in C2C12 myoblasts (Toda et al., 2016a). These genes play key roles in glucose and lipid metabolism. In addition, KPE also improved lactic acid metabolism, mitochondrial numbers, and the accumulation of muscular glycogens in C2C12 myoblasts. Therefore, the activation of muscular metabolism by several PMF in KPE is expected to improve not only physical fitness, but also obesity and hyperglycemia. Moreover, we also demonstrated that PMF, 1, 2, 3, 7, and 8 significantly suppressed LPS-induced increases in the mRNA expression of interleukin-6 and tumor necrosis factor-a (Toda et al., 2016a). Therefore, some PMF in black ginger are expected to suppress muscular inflammation.

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Wattanapitayakul et al. (2007) reported that KPE enhanced NO production in human umbilical vein endothelial cells and upregulated the mRNA and protein expression of endothelial NO synthase (eNOS). eNOS facilitates vascular function by generating NO in blood vessels and inhibits smooth muscle contraction and platelet aggregation. Since KPE enhances NO production, improvements in peripheral blood circulation are expected. Blood flow is one of the key factors for fitness performance. Muscles require large amounts of glucose, lipids, and oxygen during exercise, and these nutrients are transported in the blood. Therefore, KPE-induced improvements in peripheral blood circulation may contribute to fitness performance.

Effects of Kaempferia parviflora Extract on Physical Fitness and Muscular Endurance in Mice We evaluated the effects of KPE, which activates AMPK in C2C12 myoblasts, on improvements in physical fitness performance and muscular endurance in male ddY mice (Toda et al., 2016b). Mice were divided into two groups (n ¼ 15/group), and the KPE group was orally administered KPE (45 mg/kg/day) containing 1 (2.2%), 2 (1.7%), 3 (2.7%), 4 (1.0%), 5 (9.8%), 6 (10.5%), 7 (5.8%), and 8 (8.3%) for 4 weeks. The control group was orally administered the vehicle of KPE. The forced swimming test (ST), open-field test, inclined plane test, and wire hanging test were performed at 0, 1, 2, and 4 weeks. Muscular endurance in the KPE group was evaluated by the consecutive forced ST (CST) at 0, 1, 2, and 4 weeks. In the water pool, mice were bound by their tails to a weight that was approximately 10% of their body weights and forced to swim. In the CST, a second forced ST was performed 30 min after the first ST under the same conditions. ST was subsequently performed a total of seven times at 0, 0.5, 1, 1.5, 2, 2.5, and 3 h, as shown in Fig. 16.2, no significant differences were observed in the initial values obtained (0 h) at each week between the control and KPE groups. However, from the second and third measurements (0.5 or 1 h), decreases in the swimming time were smaller in the KPE group than in the control group (Fig. 16.2B and D). This suppression was confirmed from 1 week and became more apparent in a time-dependent manner (Fig. 16.2B and D). At 4 weeks, the duration of swimming for the last measurement (3 h) shortened to only 27% against the first measurement in the KPE group and to 78% in the control group (Fig. 16.2D). These findings indicate that KPE enhances muscular endurance or rapid recovery from swimming-induced fatigue. Physical fitness was also evaluated in the KPE group before and after ST based on fatigue using the physical fitness measurement tests (PT): ST, open-field test, inclined plane test, and wire hanging test, at 0, 1, 2, and 4 weeks. ST was performed under almost the same conditions as the CST. The other tests were performed twice, before and after ST, as described as follows. 1. The open-field test: A mouse was placed in a square field (30  30 cm) divided into nine areas. The number of times that the mouse moved to other areas was measured for 3 min.

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FIGURE 16.2 Kaempferia parviflora extract (KPE)eenhanced muscular endurance in the consecutive forced swimming test (CST) (Toda et al., 2016b). The CST was performed at 0 (A), 1 (B), 2 (C), and 4 (D) weeks. ST was repeated at 30-min intervals, and swimming times were measured for a total of seven times. Each point represents the mean with the S.E. (control; n ¼ 15, KPE; n ¼ 14). Open circle () for the control group and closed circle (C) for the KPE group. Asterisks denote significant differences from the initial value (0 week) at *: P < .05, **: P < .01, respectively. Daggers denote significant differences from the control at y: P < .05, yy: P < .01, respectively.

2. The inclined plate test: After the open-field test, the mouse was placed on a wooden board covered with a canvas, which was leaned gradually at a constant speed. The angle at which the mouse dropped from the board was measured. 3. The wire hanging test: After the inclined plane test, mice were bound by their tails to a weight that was 15% of their body weight. They were then placed on a square wire mesh (30  30 cm, mesh size 1 cm, and wire diameter f 1 mm). The wire mesh with the mouse on was turned upside down and placed at a height of 50 cm. The sides were shielded, and the time until the mouse dropped off was measured. In ST, which induced sufficient fatigue in mice, swimming times were slightly longer in the KPE group than in the control group from 1 week (Fig. 16.3A). In the

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FIGURE 16.3 Kaempferia parviflora extract (KPE)eenhanced physical fitness performance with or without fatigue loading (Toda et al., 2016b). Physical fitness measurement tests (PT), consisting of the forced swimming test (ST, A), open-field test (B and C), inclined plate test (D), and wire hanging test (E), were performed. Columns represented open column (,): control group and closed column (-): KPE group. The open-field test, inclined plate test, and wire hanging test were performed twice: before and after ST. Values in the open-field test before (B) and after (C) ST were indicated separately. Each column represents the mean with the S.E. (control; n ¼ 15, KPE; n ¼ 14). Asterisks denote significant differences from the initial result (0 week) at *: P < .05, **: P < .01, respectively. Daggers denote significant differences from the control at y: P < .05, yy: P < .01, respectively.

open-field test performed before ST (Fig. 16.3B), the number of movements in the control and KPE groups at 1, 2, and 4 weeks was significantly lower than that at 0 weeks. However, the number of movements after ST was significantly increased in the KPE group, but not in the control group at 1 and 2 weeks (Fig. 16.3C). Furthermore, the number of movements after ST was significantly increased in the KPE group at 4 weeks (Fig. 16.3C). In the inclined plate test, dropped angles were significantly greater in the KPE group than in the control group at 2 weeks (Fig. 16.3D). Dropped angles in the KPE group after ST were similar to those before ST at 2 and 4 weeks (Fig. 16.3D). In the wire hanging test, similar findings to those obtained in the inclined plate test were observed from 1 to 4 weeks. Based on the ratio calculated for the dropped time after and before ST, the ratio in the KPE group (96%) was almost twice that at 4 weeks (Fig. 16.3E), indicating enhanced grip strength in the KPE group. These findings suggest that KPE increases physical fitness performance (Fig. 16.3) and muscular endurance (Figs. 16.2 and 16.3).

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Clinical Trials In terms of clinical effects of KPE, three reports have been reported. Promthep et al. (2015) evaluated the effects of KPE on physical fitness in soccer players. Subjects (n ¼ 30/group) in both groups were administered either 180 mg of KPE in capsules or a placebo once daily for 12 weeks. In order to evaluate the effects of KPE on physical fitness and endurance, six physical performance tests were performed: a sit and reach test, a hand grip strength test, a back and leg strength test, a 40-yard technical test, a 50m sprint test, and a cardiorespiratory fitness test (VO2max). As shown in Table 16.1, righthand grip strength at 4, 8, and 12 weeks was significantly stronger in the KPE group than in the placebo group. Right- and left-hand grip strength significantly increased from the baseline in the KPE group. In addition, the 40-yard technical test and VO2max were slightly better after the ingestion of KPE than after the placebo. Consequently, KPE was suggested to enhance several aspects of physical fitness in soccer players. Table 16.1

The Results of Clinical Study in Athletes (Promthep et al., 2015) Predose Baseline Score

Items

Week 4

P-Value

Week 8

P-Value

Week 12

P-Value

Right-hand grip strength (kg/wt) Treatment (n ¼ 30) Placebo (n ¼ 30)

0.65  0.09 0.63  0.07

0.70  0.09* 0.034 0.66  0.07

0.68  0.10* 0.024 0.63  0.07

0.65  0.08* 0.038 0.62  0.07

0.65  0.10 0.469 0.62  0.07

0.64  0.08* 0.024 0.59  0.08

0.61  0.08 0.235 0.57  0.07

2.68  0.55 0.610 2.45  0.51

2.77  0.55 0.377 2.44  0.40

2.79  0.59 0.993 2.53  0.52

16.43  5.15 0.926 14.64  4.92

16.88  5.19 0.452 14.61  5.24

18.28  5.10 0.729 17.01  4.55

11.61  0.70 11.99  0.86

12.06  1.16 0.746 12.34  1.33

11.50  0.74 0.458 11.46  0.75

10.08  0.47 0.078 10.47  0.90

6.24  0.31 6.29  0.37

6.26  0.31 0.752 6.33  0.49

6.37  0.26 0.255 6.50  0.50

6.33  0.24 0.204 6.47  0.52

45.09  9.88 45.09  9.96

46.95  7.61 0.657 47.85  10.08

49.40  8.40 0.578 48.34  7.17

51.05  8.40 0.053 47.1  8.45

Left-hand grip strength (kg/wt) Treatment (n ¼ 30) Placebo (n ¼ 30)

0.62  0.08 0.60  0.08

Back and leg strength (kg/wt) Treatment (n ¼ 30) Placebo (n ¼ 30)

2.77  0.54 2.45  0.39

Sit and reach test (cm) Treatment (n ¼ 30) Placebo (n ¼ 30)

17.98  4.60 16.14  4.93

40-yard technical test (s) Treatment (n ¼ 30) Placebo (n ¼ 30) 50-m sprint (s) Treatment (n ¼ 30) Placebo (n ¼ 30) VO2

max

(mL/kg/min)

Treatment (n ¼ 30) Placebo (n ¼ 30)

Data were presented as the mean  SD (n ¼ 30/group). Significant differences from the placebo were indicated as *: P < .05.

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Wattanathorn et al. (2012) evaluated the effects of KPE on health-related quality physical fitness and oxidative status in healthy elderly subjects. Subjects (n ¼ 15/group) in three groups were administered either a placebo or KPE (25 or 90 mg) in capsules once daily for 8 weeks. In order to evaluate the effects of KPE on physical fitness tests, a 30-s chair stand test, hand grip test, 6-min walk test, and tandem test were performed. As shown in Table 16.2, the results of the 30-s chair stand test and 6-min walk test were superior in the KPE group at a dose of 90 mg/day than in the placebo group. Furthermore, KPE improved serum oxidative stress markers, including malondialdehyde levels and the activities of superoxide dismutase, catalase, and glutathione peroxidase. These findings suggest that the decreases induced in oxidative stress by KPE are one of the mechanisms underlying enhanced physical fitness in the elderly. We also evaluated the effects of KPE on health-related quality physical fitness in healthy volunteers aged between 20 and 62 years (Toda et al., 2016c). Healthy volunteers (n ¼ 24) were randomly divided into two groups: group A received KPE (30 mg/day) and

Table 16.2

The Results of Clinical Study in Elderly Subjects (Wattanathorn et al., 2012)

Measured Parameters

Group

Predose

1 month

2 months

Grip strength (Rt) (kg)

Placebo KP25 KP90 Placebo KP25 KP90 Placebo KP25 KP90 Placebo KP25 KP90 Placebo KP25 KP90 Placebo KP25 KP90 Placebo KP25 KP90 Placebo KP25 KP90

24.53  2.55 25.06  3.01 23.93  3.30 21.06  1.83 22.06  1.86 20.86  2.72 19.13  2.79 18.33  2.58 18.60  2.52 567.33  33.52 571.26  33.68 572.80  32.65 164.8  12.34 161.8  11.16 164.0  10.50 112.33  11.00 111.93  7.77 108.20  11.32 33.80  9.22 31.86  10.12 31.26  11.09 18.80  3.60 20.93  3.41 20.46  4.24

24.33  2.28 25.00  2.97 24.60  3.13 21.33  1.58 21.66  1.50 21.60  2.02 19.26  1.43 19.00  2.77 19.60  2.13 598.73  31.57 570.33  38.32 575.46  34.29 163.06  10.35 164.06  9.63 166.60  6.81 110.66  10.01 112.33  11.39 109.33  13.62 30.80  10.74 32.60  7.44 31.86  9.33 19.86  5.01 21.33  3.79 21.26  4.58

24.33  2.46 24.86  3.18 24.80  3.14 21.20  1.56 21.26  1.48 21.60  1.84 18.93  1.70 20.00  3.11 20.66  2.28# 571.26  32.05 575.53  36.04 601.26  33.70*# 156.06  9.80 162.26  8.93 168.46  6.90 109.0  10.20 111.8  10.16 110.46  13.31 31.66  10.41 32.73  7.67 33.40  8.94 21.20  4.57 21.26  3.19 22.06  3.93

Grip strength (Lt) (kg)

30-s chair stand test (s)

6-min walk test (m)

Tandem test (opened eye, right leg is in front) (s) Tandem test (opened eye, left leg is in front) (s) Tandem test (closed eye, right leg is in front) (s) Tandem test (closed eye, left leg is in front) (s)

Data were presented as the mean  SE (n ¼ 15/group). Significant differences from the placebo were indicated as *: P < .05, and those from the baseline were indicated as #: P < .05.

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then a placebo, while group B received a placebo and then KPE in a crossover trial. Subjects took one capsule containing KPE or the placebo once a day for 4 weeks. In order to evaluate the effects of KPE on physical fitness and endurance, a PT, consisting of a hand grip strength test, 30-s chair stand test, 5-m tandem walking test, and cycle ergometer test, was performed. The results obtained showed that grip strength after a 4week treatment with KPE significantly increased (right: þ2.2 kg, P < .05, left: þ2.8 kg, P < .01) from baseline. Significant improvements were also observed in the 30-s chair stand test (þ6.3 times, P < .01), 5-m tandem walking test (3.2 s, P < .05), and cycle ergometer test (þ8.6 kcal, P < .01). Grip strength in the right hand was significantly stronger after the ingestion of KPE (44.6 kg, P < .05) than after that of the placebo (43.0 kg). Net changes in grip strength in the left hand (þ2.8 vs. þ 0.0 kg, P < .05), the 30s chair stand test (þ6.3 vs. þ 1.7 times, P < .05), 5-m tandem walking test (3.2 vs. 0.9 s, P < .01), and cycle ergometer test (þ8.5 vs. þ 1.1 kcal, P < .01) were significantly greater after the intake of KPE than after ingestion of the placebo. These findings indicate that KPE enhances physical fitness, namely, grip strength, leg muscle strength, balance, endurance, and locomotor activity Table 16.3. On the other hand, KPE did not significantly change visual analogue scale fatigue scores in daily and postphysical fitness tests or chronic fatigue syndrome scores from those obtained with the placebo. However, in subjects without an exercise habit, KPE slightly improved these fatigue scores from the values in the placebo group following a 4week ingestion period. Muscular mass and metabolism may be reduced in the skeletal muscle of subjects without an exercise habit. KPE may improve these decreases. Therefore, we concluded that KPE may have more prominent effects on physical fitness and fatigue in the elderly and individuals lacking an exercise habit, among whom muscular metabolism has declined. Table 16.3

The Results of Clinical Study in Healthy Subjects (Toda et al., 2016c) Placebo

Measured Parameters

Before

After

KPE

Net Change (D) Before

After

Net Change (D)

All subjects Tiredness without exercise (%) Grip strength (R) (kg) Grip strength (L) (kg) 30-s chair stand test (s) 5-m tandem walking test (s) Cycle ergometer test (kcal) Tiredness after this test (%) Chronic fatigue syndrome score

34.6  4.3 32.0  3.5 2.57  4.5

34.5  4.0 29.4  4.3

42.8  2.5 40.1  2.5 25.3  2.1 12.2  0.8 47.4  4.5 56.7  4.5 16.1  1.2

42.4  2.4 38.9  2.2 21.4  1.7 13.8  1.3 44.3  4.0 52.7  5.4 15.7  1.3

43.0  2.5 0.21  0.7 40.1  2.5 0.03  0.8 27.0  1.8 1.71  0.9 11.4  0.9 0.87  0.5 48.6  4.3 1.13  1.4 51.0  4.9 5.08  4.3 13.7  1.1 2.38  1.1

5.14  4.2

44.6  2.6*,y 2.17  0.9 41.7  2.2yy 2.80  0.8* 27.6  1.7yy 6.27  1.7* 10.6  0.9y 3.17  1.3** 52.9  4.7yy 8.54  1.9** 46.8  3.9 5.87  4.3 13.0  1.2 2.75  1.2

Data were presented as the mean  SE (n ¼ 24). Significant differences from the placebo were indicated as *: P < .05, **: P < .01, and those from the baseline were indicated as y: P < .05, yy: P < .01 (t-test).

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Conclusion The findings described demonstrate the biological activities and clinical effects of KPE. In the last part, we summarized the positive effects of KPE on physical fitness performance and muscular endurance and described the underlying mechanisms. In the in vitro test using C2C12 myoblasts, several PMF, including 5,7-dimethoxyflavone (8), appeared to improve muscular metabolism by enhancing the expression of phosphorylated AMPK and significantly suppressed muscular inflammation (Toda et al., 2016a,b). Mice orally administered KPE showed improved physical fitness performance and muscular endurance in several tests (Toda et al., 2016b). Furthermore, KPE was shown to enhance physical fitness, namely, grip strength, leg muscle strength, balance, endurance, and locomotor activity in athletes (Promthep et al., 2015), the elderly (Wattanathorn et al., 2012), and healthy people (Toda et al., 2016c). Therefore, KPE may have health benefits for athletes and the elderly. KPE has the potential to improve locomotive dysfunctions in the elderly and enhance physical fitness and endurance in athletes.

References Akase, T., Shimada, T., Terabayashi, S., Ikeya, Y., Sanada, H., Aburada, M., 2011. Antiobesity effects of Kaempferia parviflora in spontaneously obese type II diabetic mice. J. Nat. Med. 65, 73e80. Astratenkova, I.V., Rogozkin, V.A., 2013. Participation AMPK in the regulation of skeletal muscles metabolism. Ross Fiziol Zh Im I M Sechenova 99, 657e673. Azuma, T., Kayano, S., Matsumura, Y., Konishi, Y., Tanaka, Y., Kikuzaki, H., 2011. Antimutagenic and a-glucosidase inhibitory effects of constituents from Kaempferia parviflora. Food Chem. 125, 471e475. Chaipech, S., Morikawa, T., Ninomiya, K., Yoshikawa, M., Pongpiriyadacha, Y., Hayakawa, T., Muraoka, O., 2012. Structures of two new phenolic glycosides, kaempferiaosides A and B, and hepatoprotective constituents from the rhizomes of Kaempferia parviflora. Chem. Pharm. Bull. 60, 62e69. Chaturapanich, G., Chaiyakul, S., Verawatnapakul, V., Yimlamai, T., Pholpramool, C., 2012. Enhancement of aphrodisiac activity in male rats by ethanol extract of Kaempferia parviflora and exercise training. Andrologia 44, 323e328. Hardie, D.G., Schaffer, B.E., Brunet, A., 2016. AMPK: an energy-sensing pathway with multiple inputs and outputs. Trends Cell Biol. 26, 190e201. Horigome, S., Yoshida, I., Tsuda, A., Harada, T., Yamaguchi, A., Yamazaki, K., Inohana, S., Isagawa, S., Kibune, N., Satoyama, T., Katsuda, S., Suzuki, S., Watai, M., Hirose, N., Mitsue, T., Shirakawa, H., Komai, M., 2014. Identification and evaluation of anti-inflammatory compounds from Kaempferia parviflora. Biosci. Biotechnol. Biochem. 78, 851e860. Horikawa, T., Shimada, T., Okabe, Y., Kinoshita, K., Koyama, K., Miyamoto, K., Ichinose, K., Takahashi, K. , Aburada, M., 2012. Polymethoxyflavonoids from Kaempferia parviflora induce adipogenesis on 3T3L1 preadipocytes by regulating transcription factors at an early stage of differentiation. Biol. Pharm. Bull. 35, 686e692. Kahn, B.B., Alquier, T., Carling, D., Hardie, D.G., 2005. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 1, 15e25.

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Kusirisin, W., Srichairatanakool, S., Lerttrakarnnon, P., Lailerd, N., Suttajit, M., Jaikang, C., Chaiyasut, C., 2009. Antioxidative activity, polyphenolic content and anti-glycation effect of some Thai medicinal plants traditionally used in diabetic patients. Med. Chem. 5, 139e147. Malin, S.K., Kashyap, S.R., 2014. Effects of metformin on weight loss: potential mechanisms. Curr. Opin. Endocrinol. Diabetes Obes. 21, 323e329. Murata, K., Hayashi, H., Matsumura, S., Matsuda, H., 2013. Suppression of benign prostate hyperplasia by Kaempferia parviflora rhizome. Pharmacognosy Res. 5, 309e314. Narkar, V.A., Downes, M., Yu, R.T., Embler, E., Wang, Y.X., Banayo, E., Mihaylova, M.M., Nelson, M.C., Zou, Y., Juguilon, H., Kang, H., Shaw, R.J., Evans, R.M., 2008. AMPK and PPARd agonists are exercise mimetics. Cell 134, 405e415. Nasri, H., Rafieian-Kopaei, M., 2014. Metformin: current knowledge. J. Res. Med. Sci. 19, 658e664. Patanasethanont, D., Nagai, J., Matsuura, C., Fukui, K., Sutthanut, K., Sripanidkulchai, B.O., Yumoto, R., Takano, M., 2007. Modulation of function of multidrug resistance associated-proteins by Kaempferia parviflora extracts and their components. Eur. J. Pharmacol. 566, 67e74. Promthep, K., Eungpinichpong, W., Sripanidkulchai, B., Chatchawan, U., 2015. Effect of Kaempferia parviflora extract on physical fitness of soccer players: a randomized double-blind placebocontrolled trial. Med. Sci. Monitor Basic Res. 21, 100e108. Rujjanawate, C., Kanjanapothi, D., Amornlerdpison, D., Pojanagaroon, S.J., 2005. Anti-gastric ulcer effect of Kaempferia parviflora. J. Ethnopharmacol. 102, 120e122. Sae-Wong, C., Matsuda, H., Tewtrakul, S., Tansakul, P., Nakamura, S., Nomura, Y., Yoshikawa, M., 2011. Suppressive effects of methoxyflavonoids isolated from Kaempferia parviflora on inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells. J. Ethnopharmacol. 136, 488e495. Shimada, T., Horikawa, T., Ikeya, Y., Matsuo, H., Kinoshita, K., Taguchi, T., Ichinose, K., Takahashi, K., Aburada, M., 2011. Preventive effect of Kaempferia parviflora ethyl acetate extract and its major components polymethoxyflavonoid on metabolic diseases. Fitoterapia 82, 1272e1278. Tewtrakul, S., Subhadhirasakul, S., 2008. Effects of compounds from Kaempferia parviflora on nitric oxide, prostaglandin E2 and tumor necrosis factor-alpha productions in RAW264.7 macrophage cells. J. Ethnopharmacol. 120, 81e84. Toda, K., Takeda, S., Hitoe, S., Nakamura, S., Matsuda, H., Shimoda, H., 2016a. Enhancement of energy production by black ginger extract containing polymethoxy flavonoids in myocytes through improving glucose, lactic acid and lipid metabolism. J. Nat. Med. 70, 163e172. Toda, K., Hitoe, S., Takeda, S., Shimoda, H., 2016b. Black ginger extract increases physical fitness performance and muscular endurance by improving inflammation and energy metabolism. Heliyon 2, e00115. Toda, K., Kohatsu, M., Takeda, S., Hitoe, S., Shimizu, N., Shimoda, H., 2016c. Enhancement of physical fitness by black ginger extract rich in polymethoxyflavones: a double-blind randomized crossover trial. Integr. Mol. Med. 3, 628e634. Trisomboon, H., Watanabe, G., Wetchasit, P., Taya, K., 2007. Effect of daily treatment with Thai herb, Kaempferia parviflora, in Hershberger assay using castrated immature rats. J. Reprod. Dev. 53, 351e356. Wattanapitayakul, S.K., Suwatronnakorn, M., Chularojmontri, L., Herunsalee, A., Niumsakul, S., Charuchongkolwongse, S., Chansuvanich, N., 2007. Kaempferia parviflora ethanolic extract promoted nitric oxide production in human umbilical vein endothelial cells. J. Ethnopharmacol. 110, 559e562. Wattanathorn, J., Muchimapura, S., Tong-Un, T., Saenghong, N., Thukhum-Mee, W., Sripanidkulchai, B., 2012. Positive modulation effect of 8-week consumption of Kaempferia parviflora on health-related physical fitness and oxidative status in healthy elderly volunteers. Evid. Based Complement. Altern. Med. 732816. Yenjai, C., Wanich, S., Pitchuanchom, S., Sripanidkulchai, B., 2009. Structural modification of 5,7-dimethoxyflavone from Kaempferia parviflora and biological activities. Arch. Pharmacal Res. 32, 1179e1184.

17

Role of Marine Nutraceuticals in Cardiovascular Health

Se-Kwon Kim1, Isuru Wijesekara2 1

PUKYONG NATIONAL UNIVERSITY, B USAN, REPUBL IC OF KOREA; 2 UNIVERSITY OF SRI JAYEWARDENEPURA, NUGEGODA, SRI LANKA

Introduction to Angiotensin-IeConverting Enzyme Inhibition Hypertension or high blood pressure is the most significant modifiable risk factor for vascular cognitive impairment and one of the most common reasons for mortality during pregnancy (Choi et al., 2015; Wang et al., 2016). The angiotensin-Ieconverting enzyme (ACE-I) in the renineangiotensin system has a crucial role in regulating blood pressure because it promotes the conversion of angiotensin-1 into the potent vasoconstrictor angiotensin-II and inactivates the catalytic function of bradykinin. Currently, inhibition of the activity of the enzyme ACE-I is considered to be a successful therapeutic approach to treating hypertension (He et al., 2013). Synthetic inhibitors such as captopril and enalapril have been used as drug candidates since the discovery of ACE-I inhibitory peptides in snake venom. However, these synthetic inhibitors of ACE-I have been shown to have several side effects such as cough, loss of taste, renal impairment, skin rashes, and angioneurotic edema. Therefore, researchers are interested in alternative ACE-I inhibitors from natural sources that have been reported to be in milk, muscle foods, plant foods, insects, and marine bioresources. Marine bioresources are a rich source of novel drug leads and are well-known potential ACE-I inhibitors (Fig. 17.1). The pharmaceutical value of marine-derived bioactives as potential antihypertensive candidates has been previously discussed. Among marine bioresources, bioactive peptides derived from fish muscles, invertebrates, and microalgae and macroalgae have frequently been discussed. In addition, phlorotannins from brown seaweeds and chitooligosaccharide derivatives from crustacean exoskeleton show potential ACE-I inhibitory effect (Murray and FitzGerald, September 2, 2016; Guang and Phillips, 2009; Wijesekara and Kim, 2010). This chapter briefly presents the ACE-I inhibitory effect of marine-derived bioactive agents.

Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00017-X Copyright © 2017 Elsevier Inc. All rights reserved.

273

274

SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

FIGURE 17.1 Marine-derived angiotensin-Ieconverting enzyme inhibitors.

Marine-Derived Angiotensin-IeConverting Enzyme Inhibitors Bioactive Peptides Generally, bioactive peptides are generated from the enzymatic hydrolysis or fermentation of native food proteins (Vercruysse et al., 2005). However, some ACE-I inhibitory peptides have been isolated directly from food materials without in vitro proteolytic hydrolysis. These food-derived peptides are considered to be milder and safer than synthetic drugs. Moreover, they usually have multifunctional properties and are easily absorbed. ACE-I inhibitory peptides contains generally 3e20 amino acids. Marinederived antihypertensive peptides have been isolated from fish muscles, marine invertebrates including mollusks, crustaceans, seaweeds, and microalgae (Table 17.1). The inhibition mode of ACE-I inhibitory peptides was evaluated by LineweavereBurk plots; most reported peptides act as competitive inhibitors. Some peptides show noncompetitive or uncompetitive inhibition. Table 17.1 Angiotensin-IeConverting Enzyme Inhibitory Peptides From Marine BioResources: Source, Amino Acid Sequence of the Peptide, and IC50 Value Source

Peptide

IC50 (mM)

References

Bigeye tuna

Trp-Pro-Glu-Ala-Ala-Glu-Leu-MetMet-Glu-Val-Asp-Pro Val-Ile-Tyr Met-Glu-Gly-Ala-Gln-Glu-Ala-Gln-Gly-Asp Tyr-Asn Tyr-Asn-Lys-Leu

21.6

Qian et al. (2007)

7.5 15.9 51 21

Fahmi et al. (2004) Zhao et al. (2009) Tsai et al. (2008) Suetsuna and Nakano (2000)

Sea bream Sea cucumber Hard clam Wakame

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275

These inhibitors can bind to the active site of ACE-I to block it, or to the inhibitor binding site that is remote from the active site to alter the enzyme conformation such that the substrate no longer binds to the active site. In addition, a noncompetitive mechanism has been observed in some peptides. Numerous in vivo studies of marinederived antihypertensive peptides in spontaneously hypertensive rats have shown potent ACE-I inhibitory activity.

Chitooligosaccharide Derivatives Chitin is the second most abundant biopolymer and is widely distributed in crustaceans and insects as the protective exoskeleton and cell walls of most fungi. Chitin is usually prepared from the shells of crabs and shrimp. Chitosan (Fig. 17.2), a partially deacetylated polymer of N-acetylglucosamine, has been yielded by alkaline deacetylation of chitin. Furthermore, chitooligosaccharides (COS) and chitosan oligosaccharides are chitin and chitosan derivatives, respectively, and can be generated by either enzymatic or chemical hydrolysis of chitin and chitosan. These chitin and chitosan derivatives have shown various bioactivities, which has made it possible to use them in the food, pharmaceuticals, cosmeceuticals, agriculture, and biomedicine fields. With results of the limited research to date, it is presumed that COS derivatives may have desirable properties to inhibit ACE activity. These findings suggested that the molecular weight and degree of deacetylation of COS derivatives are important factors for ACE-I inhibition. COS derivatives such as hetero-COS, aminoethyl COS, chitin derivatives (IC50 ¼ 0.06e0.1 mM), chitosan trimer oligomers (IC50 ¼ 0.9 mM), and carboxylated COS have been reported to be potent ACE inhibitors (Wijesekara and Kim, 2010; Hong et al., 1998). According to the LineweavereBurk plots, they are competitive inhibitors. Moreover, they show much lower IC50 values compared with all other marinederived ACE-I inhibitors.

Phlorotannins Phlorotannins are polymerized phenolic compounds consisting of phloroglucinol monomer units and are highly abundant in brown seaweeds. To date, several phlorotannins have been identified, including phloroglucinol (1), eckol (2), fucodiphloroethol G (3), phlorofucofuroeckol A (4), 7-phloroeckol (5), dieckol (6), and 6,6’-bieckol (7) (Fig. 17.3).

FIGURE 17.2 Chemical structure of chitosan.

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

HO

OH

HO

OH OH

OH OH

O O

HO

OH OH

OH HO

O

O HO

O HO

OH

OH

1

OH

2 HO

OH

HO

3 HO

OH

OH O

HO

OH

OH

5

4 HO

OH

OH

O HO

HO

O O

OH OH

O OH

OH HO OH

O OH

O

O

OH

OH

O O

O O

OH

O

HO

HO

OH

OH HO

OH

OH

O

HO

OH O

O O

O

O

OH

OH

O O

OH

OH

OH

OH HO

O

OH

OH

6

7

FIGURE 17.3 Phlorotannins from marine brown seaweeds; among them, eckol (2), phlorofucofuroeckol A (4), and dieckol (6) are potent angiotensin-Ieconverting enzyme inhibitors.

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277

Table 17.2 Angiotensin-IeConverting Enzyme Inhibitory Activity of Phlorotannins From Ecklonia stolonifera Phlorotannin

IC50 (mM)

References

Eckol Phlorofucofuroeckol A Dieckol

70.82  0.25 12.74  0.15 34.25  3.56

Jung et al. (2006) Jung et al. (2006) Jung et al. (2006)

Polyphenolic compounds inhibit ACE activity by sequestering the enzyme metal factor, Zn2þ ion (Liu et al., September 5, 2016). Therefore, it has been assumed that phlorotannins form a complex associated with proteins or glycoproteins to inhibit ACE activity. Phlorofucofuroeckol A from Ecklonia stolonifera (Phaeophyceae) have been shown more potent ACE-I inhibitory activity than eckol and dieckol (Table 17.2). Furthermore, Athukorala and Jeon (2005) reported that flavourzyme enzymatic digest of Ecklonia cava, which contains a high content of phlorotannins, is a potent ACE inhibitor, exhibited an IC50 of 0.3 mg/mL, and captopril, a commercial antihypertensive, exhibited an IC50 of 0.05 mg/mL. They hydrolyzed seven marine brown algal species (E. cava, Ishige okamurae, Sargassum fulvellum, Sargassum horneri, Sargassum coreanum, Sargassum thunbergii, and Scytosiphon lomentaria) and analyzed them for ACE inhibitory activities. Most of these algal species showed potent ACE inhibitory activities; however, E. cava was the most potent ACE inhibitor among them owing to its rich content of phlorotannins.

Fucoxanthin Fucoxanthin (Fig. 17.4) exists abundantly in brown seaweeds and contributes over 10% of the estimated total production of carotenoids in nature. There are a number of reported biological functions of fucoxanthin, including anticancer, antioxidant, antihypertensive, antiinflammatory, radio-protective, and antiobesity effects. Fucoxanthin from Undaria pinnatifida has been shown to reduce the development of hypertension and its related diseases in stroke-prone, spontaneously hypertensive rats (Ikeda et al., 2003). Furthermore, brown seaweeds Sargassum japonica and S. horneri extracts rich in fucoxanthin show potential ACE-I inhibition (Sivagnanam et al., 2015). However, the exact mechanism of how fucoxanthin inhibits ACE-I activity has not yet been completely explained. HO

CH3 CH3

CH3

CH3

O

O CH3

CH3

CH3

CH3

H3C H3C

FIGURE 17.4 Chemical structure of fucoxanthin.

OH

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Concluding Remarks Consumers pay much attention to natural bioactives as nutraceuticals, pharmaceuticals, and cosmeceuticals because they have few or no side effects as synthetic drugs. Natural ACE-I inhibitors derived from marine bioresources have been confirmed for their potential application as alternatives to hypertension. However, the activity of these ACE-I inhibitors have been observed only in vitro or in mouse model systems. Therefore, further research is needed to investigate their activity in human subjects. Collectively, it can be suggested that marine-derived ACE-I inhibitors such as bioactive peptides, COS, phlorotannins, and fucoxanthin are potential therapeutic candidates for preventing hypertension, and their involvement in future pharmaceuticals is promising.

References Athukorala, Y., Jeon, Y.-J., 2005. Screening for angiotensin 1-converting enzyme inhibitory activity of Ecklonia cava. J. Food Sci. Nutr. 10, 134e139. http://dx.doi.org/10.3746/jfn.2005.10.2.134. Choi, J.Y., Cui, Y., Kim, B.G., 2015. Interaction between hypertension and cerebral hypoperfusion in the development of cognitive dysfunction and white matter pathology in rats. Neuroscience 303, 115e125. http://dx.doi.org/10.1016/j.neuroscience.2015.06.056. Fahmi, A., Morimura, S., Guo, H.C., Shigematsu, T., Kida, K., Uemura, Y., 2004. Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. Process. Biochem. 39, 1195e1200. http://dx.doi.org/10.1016/S0032-9592(03)00223-1. Guang, C., Phillips, R.D., 2009. Plant food-derived angiotensin I converting enzyme inhibitory peptides. J. Agric. Food Chem. 57, 5113e5120. http://dx.doi.org/10.1021/jf900494d. He, H.-L., Liu, D., Ma, C.-B., 2013. Review on the angiotensin-I-converting enzyme (ACE) inhibitor peptides from marine proteins. Appl. Biochem. Biotechnol. 169, 738e749. http://dx.doi.org/10.1007/ s12010-012-0024-y. Hong, S.-P., Kim, M.-H., Oh, S.-W., Han, C.-K., Kim, Y.-H., 1998. ACE inhibitory and antihypertensive effect of chitosan oligosaccharides in SHR. Korean J. Food Sci. Technol. 30, 1476e1479. Ikeda, K., Kitamura, A., Machida, H., Watanabe, M., Negishi, H., Hiraoka, J., Nakano, T., 2003. Effect of Undaria pinnatifida (Wakame) on the development of cerebrovascular diseases in stroke-prone spontaneously hypertensive rats. Clin. Exp. Pharmacol. Physiol. 30, 44e48. http://dx.doi.org/10. 1046/j.1440-1681.2003.03786.x. Jung, H.A., Hyun, S.K., Kim, H.R., Choi, J.S., 2006. Angiotensin-converting enzyme I inhibitory activity of phlorotannins from Ecklonia stolonifera. Fish. Sci. 72, 1292e1299. http://dx.doi.org/10.1111/j.14442906.2006.01288.x. Liu, J.-C., Hsu, F.-L., Tsai, J.-C., Chan, P., Liu, J.Y.-H., Thomas, G.N., Tomlinson, B., Lo, M.-Y., Lin, J.-Y., 2003. Antihypertensive effects of tannins isolated from traditional Chinese herbs as non-specific inhibitors of angiontensin converting enzyme. Life Sci. 73, 1543e1555. http://www.ncbi.nlm.nih. gov/pubmed/12865094. Murray, B.A., FitzGerald, R.J., 2007. Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production. Curr. Pharm. Des. 13, 773e791. http://www. ncbi.nlm.nih.gov/pubmed/17430180. Qian, Z.J., Je, J.Y., Kim, S.K., 2007. Antihypertensive effect of angiotensin I converting enzyme-inhibitory peptide from hydrolysates of bigeye tuna dark muscle, Thunnus obesus. J. Agric. Food Chem. 55, 8398e8403. http://dx.doi.org/10.1021/jf0710635.

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Sivagnanam, S., Yin, S., Choi, J., Park, Y., Woo, H., Chun, B., 2015. Biological properties of fucoxanthin in oil recovered from two brown seaweeds using supercritical CO2 extraction. Mar. Drugs 13, 3422e3442. http://dx.doi.org/10.3390/md13063422. Suetsuna, K., Nakano, T., 2000. Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). J. Nutr. Biochem. 11, 450e454. http://dx.doi.org/10.1016/S09552863(00)00110-8. Tsai, J.S., Chen, J.L., Pan, B.S., 2008. ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria). Process. Biochem. 43, 743e747. http://dx.doi.org/10.1016/ j.procbio.2008.02.019. Vercruysse, L., Van Camp, J., Smagghe, G., 2005. ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein: a review. J. Agric. Food Chem. 53, 8106e8115. http://dx.doi.org/ 10.1021/jf0508908. Wang, T., Ding, J., Li, H., Xiang, J., Wen, P., Zhang, Q., Yin, L., Jiang, W., Shen, C., 2016. Antihypertensive activity of polysaccharide from Crassostrea gigas. Int. J. Biol. Macromol. 83, 195e197. http://dx.doi. org/10.1016/j.ijbiomac.2015.11.078. Wijesekara, I., Kim, S.-K., 2010. Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: prospects in the pharmaceutical industry. Mar. Drugs 8, 1080e1093. http://dx.doi.org/10. 3390/md8041080. Zhao, Y., Li, B., Dong, S., Liu, Z., Zhao, X., Wang, J., Zeng, M., 2009. A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate,. Peptides 30, 1028e1033. http://dx.doi.org/10. 1016/j.peptides.2009.03.002.

Royal Jelly in Medicinal to Functional Energy Drinks

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Hiroyoshi Moriyama1, Manashi Bagchi2, Debasis Bagchi3, 4 THE JAPANESE INSTITUTE FOR HE ALTH FOOD STANDARDS , TOKYO, JAPAN; DR. H ERBS, LLC, C ONCO RD , CA, UN ITED STATES; 3 UNIVERSITY OF HOUSTON COL LEGE OF PHARMACY, HOUSTON, TX, UNITED STATES; 4 CEPHAM RE SEARCH CENTER, PI SCATAWAY, NJ, UNITED STATES 1

2

Introduction The food we eat must be metabolically vital to sustain our daily lives and keep maintain our body healthy. Nutrients in the food include carbohydrates, fats, and proteins, which furnish energy (calories). In addition to such nutrients is fiber for bulk in the intestinal lumen. Vitamins are a group of compounds required in small amounts to support a wide variety of metabolic and physiologic functions; similarly, minerals are required in small quantities for both physiologic and biochemical functions, such as calcium, magnesium, and phosphorus supporting a structural function in the body (Murray et al., 2009). Although all these nutrients are required for humans to function properly and remain in good health and well-being, carbohydrates, fats, and protein, which constitute the bulk of the food eaten, are considered metabolic fuels. For example, when polysaccharides (carbohydrates) are digested, they undergo a series of hydrolysis to yield oligosaccharides, and then into free monosaccharides and disaccharides such as glucose and sucrose. Glucose that enters enzymatic degradation engages a process that produces acetyl-coenzyme A (CoA) via the tricarboxylic acid (TCA) cycle, the major catabolic pathway for acetyl-CoA in aerobic organisms including humans. At the end, adenosine triphosphate (ATP) is produced, whereas energy production takes place mainly in mitochondria in cells, which constitute various tissues in the human body. Such energy (calories) generated through complex biochemical reactions is indispensable for humans to be vital or even to sustain high labor- and stress-intensive workloads in everyday life. Similarly, fat, which is also an important source of energy as well as protein to yield acetyl-CoA, participates in the TCA cycle (Murray et al., 2009). Physiologic compounds are also present in essential nutrients in a wide variety of foods that we eat in daily life, although such compounds or bioactive (biological active) compounds exist in minute amounts in the foods, such as vegetables, whether cooked or uncooked, fruits, and many others including by-products of microorganisms and insects. Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00018-1 Copyright © 2017 Elsevier Inc. All rights reserved.

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In an attempt to search for such compounds from natural sources, a large number of bioactive compounds have been identified and isolated from the components of food in our diets: more specifically, wheat, rice, milk, eggs, and so on. Such bioactive compounds have been characterized according to their contribution to health benefits to humans, and they have often been consumed as dietary (food) supplements. Most of the bioactive compounds have been detected in botanicals. Botanicals and herbs are easily procurable for use and often they have a history of use as medicine as well as food, which validates them as safe to ingest. Much attention has been paid to bee products as part of botanical-derived products, such as bee pollen, honey, propolis, and royal jelly (RJ), and honey has been traditionally employed as valuable nourishment for many centuries. In ancient China RJ was used as a potent natural medicine as an aphrodisiac (Poplawsky, 2008), while the effect of RJ is anecdotal and has not been scientifically proven. However, the experience is that when RJ is fed to all of the larvae, queen bees are fed extra rations, which differentiates the queen bees from worker bees in their reproductive organs. Over the past decades, many bee scientists realized the significance of RJ in bee society and initiated in vitro and in vivo investigations to understand the mechanism of RJ and to determine whether its effects could be extended to humans. In this chapter, we describe the roles of RJ as a bee-derived functional product rich in nutrients with physiologic actions, formulated in medicinal and soft energy drinks that not only provide energy but also potentially enhance and modulate a variety of energyassociated functional activities. RJ is also used in a wide range of drinks from medicinal to soft energy drinks; the beverage formulas are prepared to fulfill food and drug regulatory requirements and to satisfy consumers’ expectations to become healthier.

Royal Jelly RJ is a secretory substance derived from the hypopharyngeal gland of young worker bees to feed young larva and has a pivotal role in rearing the queen bee. It is made from digested pollen and honey (Wilson-Rich, 2014). Throughout rearing, complex mechanisms within the beehive takes place, leading to the induction of young larva undergoing a series of hormonal and biochemical reactions. As a consequence, a queen bee comes into being. In the history of the search for bioactive compounds in RJ that induce the emergence of queen bees, it became clear that the main nutrients of queen and worker bees’ food are proteins, carbohydrates, and fats. Carbohydrates and fats are good and efficient sources of energy, but the supply of high-energy food alone is unlikely to cause a differentiation in bees. However there is no clear explanation or scientific evidence to reveal how RJ works to produce the queen bee. The main question that should be addressed is whether RJ has any such effect as seen in bees also in mammals including humans. To scrutinize the chemical composition of RJ, therefore, the following should be examined: identification of which bioactive compounds are involved, the underlying mechanistic action, and how RJ is beneficial to human health.

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Chemical Composition Data on the chemical composition of RJ are available from different sources (Lercker et al., 1981, 1982; Takenaka, 1982; Sabatini et al., 2009). Basically RJ is reported to be made of water (60%e70%); lipids (including fats) (3%e8%); 10-hydroxy-2-decenoic acid (10-HDA), which is a unique medium fatty acid whose chemical structure is shown in Fig. 18.1 (>1.4%); protein (9%e18%); fructose (3%e13%); glucose (3%e13%); and sucrose (0.5% e2.0%), as expressed in weight (g)/weight (100 g). Furthermore, data on freeze-dried or lyophilized RJ are available (Sabatini et al., 2009). The water content is >5%, whereas lipids, 10-HDA, and proteins are 8%e19%, >3.5%, and 27%e41%, respectively. Some of the constituents are present in trace amounts, such as vitamins, minerals, and adenosine, which is known to have various roles as bioactive compounds. Also, testosterone, which is a hormone that increases male power and endurance, has been found as a bioactive compound in RJ (Vittek and Slomiany, 1984). A wide spectrum of vitamins is contained in RJ, such as thiamine (B1), riboflavin (B2), thiamine (B1), niacin (B3), and folic acid; pyridoxine (B6), biotin (H), pantothenic acid, and inositol are also detected but with greater variations in their amounts (Vecchi et al., 1988). In the case of trace minerals, potassium (K), phosphorus (P), sulfur (S), sodium (Na), calcium (Ca), aluminum (Al), magnesium (Mg), Zn, Fe, Cu, and Mn are considered the highly essential minerals in RJ (Stocker et al., 2005). Apparently, the composition of postharvested RJ might differ to varied extents, depending on the storage temperature and time as they relate to the freshness, as measured by changes in the content of free amino acids. Such amino acids include proline, lysine, glutamate, a-alanine, phenylalanine, aspartate, and serine as RJ freshness biomarkers (Boselli et al., 2003). In connection to the RJ freshness biomarker, proteins were found not to be stable when stored at 40 C for a certain time, which potentially influences the effectiveness of the physiologic action or antifatigue (Kamakura et al., 2001). Furthermore, the contents of peptides and free amino acids in RJ are important as bioactive compounds, which may be well classified into three categories according to their physiologic functions: oxidationereduction, protein binding, and lipid transport (Ramadan and Al-Ghamdi, 2012). Regulations require the chemical composition of RJ to be identified as basic nutrients such as carbohydrates, protein, fats, calories, vitamins, minerals, and others so that consumers can understand the nutrition facts and energy in calories before its purchase and intake. Concomitantly, the presence and amounts of bioactive compounds such as

FIGURE 18.1 Structure of 10-hydroxy-2-decenoic acid, an important bioactive compound belonging to an important constituent in royal jelly.

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10-HDA (Ramadan and Al-Ghamdi, 2012) are expected to be stated on health food product labels to help consumers make better purchasing choices by comparing those with competitive products. In fact, labels of some foods and drinks state bioactive compounds such as polyphenols even though they are optional. However, if the contents of different bioactive compounds are high in health food products to motivate consumers to purchase them, it is obvious that the costs will rise accordingly, leading to less profit for sellers.

Standardization Fresh RJ is a viscous jelly substance that is whitish yellow in color. It has a sour odor and its taste is sour and sweet. Because of its vulnerable properties in preserving its intact chemical composition and its intrinsic bioactive properties after harvest and during storage, it is therefore necessary to process RJ to maintain its freshness so that it ca be stored for a longer period time to deliver the just-made quality of fresh RJ from production to store shelf. The degradation of RJ proteins is unavoidable during storage, which suggests that the temperature after harvest and during storage should be well managed (Kamakura et al., 2001), taking stability into consideration afterward to maintain the physiologic properties of fresh RJ. Generally, freeze-dried RJ (FD-RJ) demonstrates better stability in maintaining consistent amounts of nutrients, including bioactive compounds, in handling at storage and distribution, particularly degradable proteins and peptides in fresh RJ with susceptibility to microbial contaminations and biochemical interactions that may occur in the presence of water. An additional important advantage of the FD-RJ compared with fresh RJ is that it facilitates a longer shelf life and easier handling for the production of drinks as well as encapsulated forms, in part owing to the high solubility of FD-RJ. In many cases, FD-RJ is employed in experimental studies using animal model and human clinical trials because of its simplicity in the preparation of test samples and storage in laboratories, which may provide better results in examining the effects of RJ. Because of these advantages, many of the commercially available RJ formulated products in the marketplace are of FD-RJ powder (Fig. 18.2). Interestingly, no RJ international standards exist that encompass nutrition values as well as physicochemical properties. Some countries, including Japan, Brazil, Bulgaria, and Switzerland, have established standards. For example, Japanese quality standards are watched by The Japan Royal Jelly Fair Trade Council, whereas Japan Health and Nutrition Food Association establishes standards for RJ. As a biomarker, 10-HDA is a standardized constituent of both fresh RJ and FD-RJ, as identified in a previous study (Sabatini et al., 2009). As finished products, 500e3000 mg/day is recommended for intake. The standards include limitations for arsenic (As), which is to be kept >2 parts per million, and for pesticides such as benzene hexachloride and dichloro diphenyl trichloroethane, as well as microbial counts.

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(A)

Fresh RJ

Lyophilization

(B) FD-RJ

FIGURE 18.2 (A) Fresh royal jelly (RJ) and (B) freeze-dried royal jelly (FD-RJ). FD-RJ is the most commonly used form of RJ in drinks that are commercially available in the marketplace in Japan.

Safety RJ has a long history of use that goes back to the ancient China (Poplawsky, 2008). Supported by this history, it is deemed adequately safe to be ingested by humans. RJ is not toxic when injected into small experimental animals such as mice and rats at high doses of up to 3 g/kg body weight per day (Hashimoto et al., 1977). Another safety study in vitro that assessed the cytotoxic effects of RJ on lymphoblast cells showed no signs of toxicity (Spiridonov et al., 1989). On the other hand, an allergic response to RJ is a serious concern in both oral and dermal applications. Previous studies reported adverse effects of ingesting RJ, which included cases of asthma and anaphylaxis (Thien et al., 1996; Dutau and Rance, 2009). Topical applications of RJ were also reported that resulted in cases of skin contact dermatitis (Takahashi et al., 1983). However, in vitro and in vivo tests showed that alkaline proteaseetreated RJ did not evoke allergenic response and resulted in a hypoallergenic effect of the area treated with RJ (Moriyama et al., 2013). In fact, enzymetreated RJ has been commonly used in both oral and dermal applications to minimize or prevent allergic reactions. To prove further the safety of enzyme-treated RJ, additional safety studies have been performed, part of which were documented and submitted for the approval of an RJ Food with Specified Health Use (FOSHU) product with the health claim of “for those with high blood pressure,” as approved by the government in 2006. The safety requirements for obtaining the FOSHU approval are described, including overdose human clinical trial, which aims to confirm the presence of adverse effects of the RJ product with

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the oral dose usually three- to fivefold the established dose (Ohama et al., 2014). Other safety studies included a single-dose toxicity test (LD50), a 90-day toxicity test, a chromosomal aberration test, and a mutagenicity test, all of which concluded that the product was safe with no toxic effects.

Energy-Enhancing Actions Some of the biological effects of RJ were substantiated scientifically, but many of the studies were performed in vitro and in vivo. And consumers who purchase and take RJ expect to be healthy on the basis of the image created in the differentiation between the worker bee and the queen bee by the excessive feeding of RJ. Here mostly preclinical and a few clinical studies present potential actions indirectly or directly to support antifatigue and physical enhancing effects. Those physiologic actions of RJ, such as of sports performance and antifatigue, can be supported through supplementation.

Antifatigue Effect A methodology used in the antifatigue study was developed using mice by employing an apparatus with an adjustable-current swimming pool to determine the maximum swimming time (Matsumoto et al., 1966). In early 2000, the antifatigue effect of RJ was evaluated using the concept of the swimming apparatus in a small animal (Kamakura et al., 2001). Briefly, RJ was fresh and stored at 20 C after harvest. The experimental animal was male Std ddY mice. The study assessed the endurance level of mice, employing a water pool for swimming exercise in a set period of time after the administration of RJ; it also examined the extent to which exhaustion was relieved after exercise in the pool. The exhaustion and relief times under swimming were used as parameters. Other parameters measured were levels of accumulation of serum lactate and serum ammonia, and also muscle glycogen after swimming. Result of the study demonstrated that RJ possibly contributed to the amelioration of physical fatigue after exercise. Moreover, the antifatigue might be attributed to the content of 57-kDa protein detected in the fresh RJ, whereas RJ stored at 40 C for a week showed protein degradation and demonstrated no significant antifatigue effect. Other RJ content such as vitamins, 10HDA, and other fatty acids were unchanged even after 40 C storage. Although the underlying mechanism has not yet clarified, the presence of the 57-kDa protein in RJ was correlated with an improvement in the physical fatigue of swimming mice after the exercise administration of RJ (Kamakura et al., 2001). The effect of RJ on the restoration from fatigue using an animal model was also reported (Suzuki et al., 2009). The methodology was to monitor swimming mice, which includes the working gravitational load. It found that the strongest improvement in fatigue was observed at 1000 mg/kg RJ (Suzuki et al., 2009). The animal model studies repeatedly revealed that RJ exerted the effect of antifatigue. Again, the mechanistic action was not fully elucidated in light of how RJ would potentially be manifested in humans.

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A study using experimental animals administered RJ, which was designed to expose the animal to oxidative stress with fumonisin revealed the effect of antioxidative activity (El Nekeety et al., 2007). Proteins and polypeptides are known to show antioxidant activity (Elias et al., 2008). That study’s results using a small animal model (Kamakura et al., 2001) suggested that degraded proteins caused a loss of antifatigue activity. Furthermore, a number of experiments showed that proteins, peptides including dipeptides and tripeptides, and amino acids from various sources could provide antioxidative actions (Nagai et al., 2004, 2006; Jaminik et al., 2007; Zhuang et al., 2009). RJ, which is enzymatically hydrolyzed and extracted with water and alkaline solution, was examined for antioxidative activities (Nagai et al., 2004); the results of the study showed antioxidant activity in both extracts that depended on the amount. In studies of RJ, antioxidative peptides derived from RJ were reported to explain their structures and properties more specifically (Guo et al., 2007, 2009). It was demonstrated that the enzyme-treated hydrolysate of RJ protein had potent antioxidative activity on the peroxidation of linoleic acid and some of the dipeptides protected cultured human cells against oxidative stresseinduced cell death (Guo et al., 2007). In addition, the small peptides, dipeptides and tripeptides, derived from RJ protein were examined for antioxidative properties. Results revealed that the peptides contributed to potent hydroxyl radical scavenging activity, whereas the manner in which the antioxidative activity occurred differed among the peptides tested (Guo et al., 2009). A further study of the antioxidative action of RJ used baker’s yeast as cell model, and demonstrated that RJ lowered intracellular oxidation in a dose-dependent manner. On the basis of the protein profile analyzed, RJ in the cell does not act solely as a scavenger of reactive oxygen species, but also affects the expression of proteins such as antioxidant enzyme (Jaminik et al., 2007). As additional evidence, a few studies evaluated the relationship between antifatigue and peptides, although the peptides were of non-RJ origin (Wang et al., 2007, 2008; Yu et al., 2008; You et al., 2011). Evidence potentially supporting the effect of RJ peptide (Kamakura et al., 2001) led to the hypothesis of a possible mechanistic action of antifatigue in association with the antioxidation of peptides derived from the collagen hydrolysate of jellyfish (Ding et al., 2011). RJ has been widely used as a tonic in many countries; more clinical evidence needs to be accumulated regarding health claims such as the ameliorating effect of fatigue derived from physical exercise and training. It is challenging to establish optimal dose levels for a group of people for whom there are many differences in race, age, gender, dietary habits, etc. In addition, when human clinical trials are conducted to confirm physiologic effects on humans, RJ should be administered at optimal doses to subjects who need to receive appropriate prescreening under established inclusion and exclusion criteria, to obtain accurate and consistent results using standardized test samples of RJ. The number of subjects participating in the trial and the duration of the study are also crucial when any randomized controlled trial (RCT) is designed.

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Sustainability of Physical Performance Nutrition obviously has a remarkable role in enhancing physical performance, from children to aged individuals, particularly for those who require high contents of nutrition and oxygen or who are athletes or labor-intensive workers. For example, athletes continuously require a high content of energy in diet and oxygen, whereas nutrition not only provides calorie, it shapes muscles and bones, which should be well tolerated in sustaining activity during intensive exercise and training (Bagchi et al., 2013). One mechanism that delivers augmented oxygen supply to muscles during exercise involves red blood cells (RBCs). RBCs are well recognized for the transport of oxygen to the lungs in the respiratory system, whereas the diffusion of oxygen takes place in the lungs across the alveolar barrier with inspired air with oxygen into the blood. Oxygen then binds to hemoglobin in the vascular system, which is essential for oxidation to produce energy. Erythropoiesis is the process of producing RBCs undergoing a complicated metabolic pathway involving various hormones and others to produce RBCs. On the basis of 6 months of ingesting 100 mL liquid containing 3000 mg of RJ a day, the result of a randomized, placebo-controlled, double-blind trial speculated that stimulation of conversion from dehydroepiandrosterone sulfate (DHEA-S) to testosterone led to improvement of erythropoiesis, as shown in Fig. 18.3 (Morita et al., 2012). DHEA-S is the most abundant androgen in humans and is synthesized in the adrenal glands; it was significantly increased in subjects who ingested RJ (n ¼ 31) compared with the control group (n ¼ 30) (Mairba¨ur, November 12 2013). The study results also suggested that RJ did not promote iron metabolism or hemoglobin synthesis but eventually stimulated erythropoiesis or prolonged the life span of erythrocytes. This finding of a significant increase in RBCs supports the improvement in erythropoiesis. The effects of RBCs in exercise and training on oxygen supply were examined (Mairba¨ur, November 12 2013). It is known that the primary role of RBCs produced via erythropoiesis in exercise is to transport oxygen from the lungs to the cells and tissues and then to carry CO2 to the lungs for expiration. Furthermore, ATP and nitrogen oxide released from RBCs may contribute to vasodilation and an improvement of blood flow to muscles. As a result, the circulation of sufficient RBCs is required for this activity to function effectively. A study providing RJ as a supplement to young football players was reported (Joksimovic et al., 2011). The pilot study of football players who took RJ resulted in a statistically significant increase in body height, body mass, and muscle and bone components, and lower average values in fat components (Joksimovic et al., 2011). Despite the positive results, the dose used in the clinical experiment was not clearly mentioned, but the stated experimental duration was 2 months and the frequency of intake was four times per week. RJ was assessed in connection to its effects on muscle using in vivo and in vitro experiments regarding whether RJ could prevent the progression of sarcopenia in aged mice (Niu et al., 2011). Some of the results demonstrated that both RJ and protease-

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treated RJ enhanced muscle weight, grip strength, and the regenerating capacity of injured muscle. Sarcopenia is a term often used in global modern society; it is reported to be the age-related loss of muscle mass and strength. Consequences of sarcopenia are frailty and functional limitations in daily life, and thus deteriorating quality of life that further cause a higher mortality rate in elderly patients (Gronholdt-Klein et al., 2012). In addition, the population aged 60 years or older is growing at the rate of 2.6% per year (Nations United, 2009). In preclinical studies, RJ was found to prevent osteoporosis as a physiologic function (Narita et al., 2006; Hikada et al., 2006). It was also demonstrated in animal model experiment that as a bioactive substance, RJ potentially has an effect on sexual enhancement (Kohguchi et al., 2004). In the aged population, such an effect is beneficial to the quality of life associated with antiaging.

Formulations Japan is a leader in producing and consuming RJ in the world of medicinal and soft energy drinks. Medicinal energy drinks, which belong to both over the counter (OTC) drug and quasidrug and soft energy drinks, are available in the marketplace and are also accessible to consumers through various distribution channels including drugstores, most convenience stores, and supermarkets near the food products. In this section,

Dehydroepieandrosterone Sulfate

3β-hydroxysteroid hydorgenase type 2

Androstenedione 17β-hydroxysteroid dehyrogenase type 3

Testosterone SPECULATED

Improvement of erythropoeisis FIGURE 18.3 Proposed metabolic pathway for 6-month ingestion of royal jelly, which accelerates the conversion of dehydroepiandronsterone sulfate to testosterone, potentially resulting in the improvement of erythropoiesis. Modified Morita, H., Ikeda, T., Kajita, K., Fujioka, K., Mori, I., Okada, H., Uno, Y., Ishizuka, T.. 2012. Effect of royal jelly in ingestion for six months on healthy volunteers. Nutr. J. 11, 77. http://www.nutrtionj.com/content/11/1/77

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“designated” quasidrug drinks as medicinal energy drinks are elaborated upon according to the standpoint of formulating drinks with RJ.

Medicinal Energy Drinks Within commercially available medicinal energy drinks, drinks that belong to the classification of OTC drugs are prominent and mainly occupy the shelves of small- to largesize drugstores with toiletries and other items. According to the Pharmaceutical and Medical Device Regulation (Japan Pharmaceutical), three classes are assigned to OTC drugs: 1, 2, and 3. OTC drug drinks belong to classes 2 and 3, which are accompanied by some extent of health risks including possible drug interactions that have not yet been well investigated and adverse reactions, although they are accessible to consumers primarily in drug stores. Medicinal energy drinks here are referred to as “designated” quasidrugs. A quasidrug is defined as a unique classification of products with minimal to moderate pharmacologic activity restricted in use to specific indications; these include some energy drinks formulated with taurine and selected vitamin preparations for ingestion. Others belong to the class of hair tonics, bath preparations, skin whitening and acne products, antidandruff shampoos, and fluorinated toothpaste. “Designated” is added to quasidrug primarily for medicated drops and energy drinks to distinguish them from other quasidrug products (PMDR). Quasidrugs may be a category similar to, for example, a nonpharmacyebound drug in Germany (German Pharmacies, 2015). In this category, a significant number of energy drinks are available through outlets such as drug and convenient stores. Limited efficacies such as tonic actions are allowed to be described on their labels, and active ingredients are required to be formulated to make the product efficacious. Representative formulations with the label statements are shown in Tables 18.1 and 18.2. The active ingredients are mainly a combination of vitamin B complex and taurine, to allow drinks to have medicinal efficacy as indications. All ingredients of a quasidrug must have a monograph in the Japanese Pharmacopoeia (List of Quasi-Drug Ingredients) and comply with the given criteria. Indications for medicinal energy drinks are basically the same; however formulas vary to some extents to establish a better marketing image from other competitive medicinal energy drinks, and similar active ingredients are required, supporting their efficacy. In many cases, herbal extracts, in particular, are used to generate an efficacious image for consumers. For this reason, RJ continues to be the best ingredient choice to be incorporated into medicinal drinks for such an image. Despite solid clinical trials to establish efficacy allowed in “designated” quasidrug (medicinal) drinks, active ingredients are categorized as: (1) botanical- or animal-derived ingredients, (2) vitamins, (3) amino acids, and (4) others. Various combinations of the active ingredients classified in these categories provide differences in their marketing image and consumers’ perception of medicinal energy drinks. RJ could be part of Category 1 to support the physiologic actions of RBC enhancement and antiaging in the

Table 18.1

Formula and Other Statements on the Label of Medicinal Drink (50 mL) Contents

Inactive Ingredients

Fursultiamine hydrochloride (Vitamin B1₁ derivative) Riboflavin sodium phosphate (Vitamin B2₂ sodium phosphate) Pyridoxine hydrochloride (Vitamin B6₆) Nicotinamide Sodium L-aspartate Taurine (aminoethanesulfonic acid) Carnitine hydrochloride Anhydrous caffeine Royal jelly tincture

10 mg

Stevia extract Tartaric acid

15.25 mg

Citric acid hydrate Lactic acid

10 mg 60 mg 125 mg 1500 mg

Maltitol syrup Honey Sucrose Paraben Scents

100 mg

Ethanol

500 mg

Ethyl vanillin pH control chemicals

Precautions for Storage/Handling

Indication

Dosage and Direction

Precautions for Use

 Supply of nutrients in the following cases: physical fatigue, during or after illness, nutritional disorders, exhaustion with fever, during lactation  Nutrients and tonics  Weak constitution

Take one bottle (50 mL) 1 day (age 15 years) (Pay attention to overdose if other products containing vitamin or something are used concomitantly)

Stop taking this medicine  Store in a cool place away from immediately and consult a direct sunlight physician or pharmacist in  Keep out of the following case. Take this reach of children bottle with you  Do not take this  If the following sympmedicine after toms appear after taking the expiration this medicine: date skin rash, digestive organs: stomach discomfort  If your symptoms do not improve after taking this medicine for a certain period of time

(As royal jelly 500 mg) Note: The product sample was purchased at a drug store in Tokyo on September 1, 2016.

Stop taking this medicine and consult a physician or pharmacist if the following symptoms appear after taking this medicine and continue or worsen: diarrhea

Chapter 18  Royal Jelly in Medicinal to Functional Energy Drinks 291

Active Ingredients

292

Formula and Other Statements on Label of Medicinal Drink (50 mL)

Active Ingredients Vitamin B2₂ phosphate ester Vitamin B6₆ Vitamin B1₁ nitrate Royal jelly tincture (As royal jelly 500 mg) Taurine Glycine L-Arginine hydrochloride L-Lysine hydrochloride Eucommia ulmoides liquid extract (As leaves 60 mg) Nicotinamide Anhydrous caffeine

Contents

Inactive Ingredients

Indication

100 mg

 Supply of nutrients in Sodium the following cases: benzoate physical fatigue, Ethyl vanillin during or after illness, Sodium citrate nutritional disorders, Citric acid exhaustion with fever, Sucrose during lactation  Nutrients and tonics Glycerine  Weak constitution vanillin Paraben Propylene glycol Scents

0.06 mg

Acesulfame k

40 mg 50 mg

DL-Alanine Erythritol Sucralose

15 mg 10 mg 10 mg 300 mg

1000 mg 50 mg 100 mg

Propyl gallate DL-Malic acid Note: The product sample was purchased at a drug store in Tokyo on September 2, 2016.

Dosage and Direction Take one bottle (50 mL) per day (aged 15 years)

Precautions for Use

Precautions for Storage and Handling

Stop taking this medicine immediately and  Store a cool place away from direct consult a physician or pharmacist in the sunlight following case. Take this bottle with you  Keep out of the  If the following symptoms appear after reach of children taking this medicine: skin rash, digestive  Do not take this organs: stomach discomfort medicine after the  If your symptoms do not improve after expiration date taking this medicine for a certain period of time Stop taking this medicine and consult a physician or pharmacist if the following symptoms appear after taking this medicine and continue or worsen: diarrhea

SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Table 18.2

Chapter 18  Royal Jelly in Medicinal to Functional Energy Drinks 293

future. Vitamin B2 is to ameliorate fatigue. Taurine, which is in Category 3, amino acids, is for antifatigue and the enhancement of physical performance. RJ and other active ingredients adequately ensure the efficacy (indication) of medicinal energy drinks (Tables 18.1 and 18.2).

Soft Energy Drinks Soft energy drinks have become popular, particularly among younger generations because many of the drinks revitalize their strength and energize their physical condition and mental alertness. The effects are presumably attributable to high contents of caffeine. Medicinal energy drinks commonly contain 50 mg, whereas soft energy drinks sometimes contain as much as 50 mg or more per bottle, as labeled on some of the energy products available in the marketplace. Tables 18.3 and 18.4 each show a soft energy drink with RJ. The amount is not on the label, which implies that the content is small. In addition, RJ is considered an optional ingredient for stating its amount on the label, and the amounts of certain ingredients such as vitamins are required on the label. Nevertheless the formula in Table 18.4 states the content of RJ, although it is little. Soft energy drinks are classified as “food” and are often “so-called health foods” (SCHFs) (Ohama et al., 2014), which implies that neither health nor function claims are allowed in the domain of foods. The food regulatory system is clearly different from medicine, although a bundle of diversified food regulations are in existence regarding SCHF and foods with health claims (Ohama et al., 2014; Moriyama et al., 2016). For Table 18.3 Carbonated Soft Energy Drink Without Stated Amount of Royal Jelly Ingredients/Food Additive

Nutrition Facts/Ingredient Content (185 mL)

High-fructose corn syrup/sugar Royal jelly Acid flavor/flavor Vitamin C Niacin amide Caffeine Calcium pathothenate Water-soluble vitamin P Vitamin B₁ Vitamin B₆ Vitamin B₂ Threonine Sodium glutamate b-Carotene Vitamin B₁₂

Energy 54 kcal Protein and lipid 0 g Carbohydrate 13.5 g Sodium 5e12 mg Vitamin B₁ 0.67 mg Vitamin B₂ 0.75 mg Vitamin B₆ 1.0 mg Vitamin B₁₂ 1.4 mg Vitamin C 95 mg Niacin 8.6 mg Pantothenic acid 8.6 mg (Optional statement) Vitamin P 1.2 mg

Note:The product sample was purchased at a supermarket in Tokyo on September 2, 2016.

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

Table 18.4 Carbonated Soft Energy Drink With the Amount Statement of RJ Ingredients/Food Additive

Nutrition Facts/Ingredient Content (185 mL)

High-fructose corn syrup/sugar Korean ginseng extract Dextrin Royal jelly Acidifier Flavor Caffeine Vitamin C Arginine Niacin Vitamin E Vitamin B₂ Vitamin B₁ Vitamin A Vitamin D

Energy 99 kcal Protein and lipid 0 g Carbohydrate 26 g Sodium 9 mg Vitamin A 202 mg Vitamin B₁ 0.56 mg Vitamin B₂ 0.6 mg Niacin 5 mg Vitamin C 19 mg Vitamin D 1.5 mg Vitamin E 4 mg (Optional statement) Royal jelly 0.56 mg Arginine 19 mg Korean ginseng extract 9 mg Caffeine 37 mg

Note: The product sample was purchased at a supermarket in Tokyo on September 2, 2016.

sellers, labeling and promotional statements are strictly controlled by regulations under the supervision of the Consumer Affairs Agency in Japan, which is expected to protect consumers from being misled.

Marketing Challenges Soft energy drink is classified as a “food” or “functional food,” which is a fanciful term. Unlike a drug, a food is not able to make efficacy statements about prevention, cure, treatment, or other medical implications (Ohama et al., 2014). On the other hand, clinically proven claims are allowed in FOSHU products. Also, in 2015, a new regulatory framework was structured into the SCHF: namely, Food with Function Claims (FFCs) in Japan. Requirements for registering an SCHF as an FFC are detailed in a publication (Moriyama et al., 2016). Under the FFC regulatory system, however, it is highly possible that RJ is an active ingredient for functional energy drinks on the basis of results of the previous study (Gronholdt-Klein et al., 2012), which suggests that claims such as the enhancement of oxygen production and supply to the body are feasible with additional RCTs. Positioning RJ to make an ingredient with solid health or function claims should be supported by well-designed, rigorous clinical trials such as randomized, double-blind, placebo-controlled trials as the most recommended clinical standard. A clinical trial conducted to find the effects of RJ on swimmers that examined biochemical parameters seemed to have negative results presumably owing to the protocol’s design (Saritas¸ et al., 2011). According to the study, three different dose levels (500 mg, 1 g, and 2 g) were

Chapter 18  Royal Jelly in Medicinal to Functional Energy Drinks 295

administered to swimmers before and after supplementation. The study concluded that no significant difference was noted except for creatinine, whereas weight (kg) and body mass index (kg/m2) and body fat (%) showed no change compared with placebo. The dose levels used in the study could be one reason that led to positive effective results. For example, the study did not conclude that there was an antifatigue effect. However, according to the Japan Health and Nutrition Food Association, the recommended dose of RJ is as much as 3 g. The other possibility was the quality of the RJ sample as related to its bioavailability. Similarly, in the European Union (EU), there are no approved health claims for RJ provided in Regulation (EU) No. 432/2012. In the EU Register of Nutrition and Health Claims, 12 health claims are available for RJ, although they are not authorized. Claims related to tonic and structure/function enhancement are as follows. RJ: helps strengthen your body/strengthen the body (ID 1225) nourishes metabolism (ID 1226) helps to support the body’s vitality (ID 1231) is a reconstituent and tonic (ID 1703) stimulates blood circulation (ID 1227) could promote the protection of cells against certain harmful effects provoked by free radicals (ID 1229) nourishes the human body and supplies energy as stimulant. It supplies vitamins and minerals from natural sources. It has positive effects during menopause and for overall rejuvenation of the skin and human body (ID 3190) is associated with immune function or the immune system (ID 3191) Those health claims are not authorized on the basis of scientific evidence. Nevertheless those claims can potentially be made on the label if satisfactory scientific evidence is presented. Being able to make any of these claims is a remarkable benefit for stakeholders in the health food industry. The FFC of a Japanese new regulatory system seems to be beneficial, provided that systematic review of important active ingredients in an FFC product is suitably conducted on the basis of the assessment (review) of RCTs following the guidelines of the Consumer Affairs Agency (Guideline for Industry). For example, it is possible to introduce a “functional energy boosting drink” in the future. RJ certainly can be an active ingredient in an FFC product formulated with RJ that previously found it to “alleviate physical fatigue” or “support muscle function” if it is accompanied by sound RCTs that substantiate such claims.

Conclusion RJ demonstrated a wide range of biological properties such as antioxidant activity, as evidenced in many preclinical studies. Its applications in drinks are diverse, from medicinal drinks to soft energy drinks. RJ has been established as having had a good

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SUSTAINED ENERGY FOR ENHANCED HUMAN FUNCTIONS AND ACTIVITY

image as a food ingredient since ancient times because it is an extraordinary product of honey bees and has a unique role in the emergence of the queen bee. It is formulated in numerous types of SCHFs including functional drinks, by addressing some ambiguities in health benefits in many supplements in the marketplace. Therefore RJ requires more careful and rigorous assessment regarding its significance for human health benefits: for example, by conducting additional clinical trials, possibly using finished products with efficacious amounts of RJ. Many physiological properties of RJ are potentially related to antifatigue and muscle-enhancing activities. Such energy drinks are expected to become functional foods to provide a better quality of life for young and elderly people in modern society.

References Bagchi, D., Nair, S., Sen, C.K., 2013. Nutrition and Endurance Sports Performance Muscle Building, Endurance, and Strength, first ed. Academic Press, London. Boselli, E., Canoni, M.F., Sabatini, A.G., Marcazzan, G.L., Lercker, G., 2003. Determination and changes of free amino acids in royal jelly during storage. Apidologie 34, 129e137. Ding, J.-F., Li, Y.-Y., Xu, J.-J., Su, X.-R., Gao, X., Yue, F.-P., 2011. Study on effect of jelly fish collagen hydrolysate on anti-fatigue and anti-oxidantion. Food Hydrocolloids. http://dx.doi.org/10.1016/j/ foodhyd.2010.12.013. Dutau, G., Rance, F., 2009. Honey and honey-product allergies. Revue Francaise d’Allergologie 49, S16eSS22. El Nekeety, A.A., El-Khoy, W., Abbas, N.F., Ebaid, A., Amra, H.A., Bdel-Wahhab, M.A., 2007. Efficacy of royal jelly against the oxidative stress of fumonisin in rats. Toxicon 50, 256e269. Elias, J.R., Kellerby, S., Decker, A.E., 2008. Antioxidant activity of proteins and peptides. Crit. Rev. Food Sci. Nutr. 48, 430e441. EU regulation of nutrition and health claims make on foods. http://cc.europa.eu/nuhclaims/. German Pharmacies, Figures・Data・Facts. 2015. https://www.abda.de/fileadmin/assets/ZDF/ZDF_ 2015/ABDA_ZDF_2015_Brosch_english.pdf. Gronholdt-Klein, A.M., Wang, L., Ulfhake, B., 2012. Cellular degradation mechineries in age-related loss of muscle mass (Sarcopenia). In: Nagata, T. (Ed.), Senescence. Academia Edu., San Francisco, CA, pp. 269e286. Guideline for Industry. http://www.caa.go.jp/en/index.html. Guo, H., Ekusa, A., Iwai, K., Yonekura, M., Takahata, Y., Morimatsu, F., 2007. Royal jelly peptides inhibit lipid peroxidation in vitro and in vivo. J. Nutr. Sci. Vitaminol. 54, 191e195. Guo, H., Kouzuma, Y., Yonekura, M., 2009. Structures of properties of antioxidative peptides derived from royal jelly protein. Food Chem. 11, 238e245. Hashimoto, T., Takeuchi, K., Hara, M., Akatsuka, K., 1977. Pharmacological study on royal jelly (RJ). Acute and subacute toxicity tests on RJ in mice and rats. Bull. Meiji Coll. Pharm. 7, 1e13. Hikada, S., Okamoto, Y., Uchiyama, S., Nakatsuma, A., Hashimoto, K., Ohnishi, T., Yamaguchi, M., 2006. Royal Jelly prevents osteoporosis in rats: beneficial effects I ovariectomy model in bone tissue culture model. Evid. Based Compl. Altern. Med. 3, 339e348. Jaminik, P., Goranovi c, D., Raspo, P., 2007. Antioixdative action fo royal jelly in the yeast cell. Exp. Gerontol. http://dx.doi.org/10.1016/j.exger.2007.02.002. Japan Health, Nutrition Food Association, Royal jelly specification. http://www.jhnfa.org/health-02.html.

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Japan Pharmaceutical Manufacturer Association, Designated quasi-drug. http://www.jpma.or.jp/ english/parj/pdf/2015.pdf. Joksimovic, A., Stankovic, D., Joskimovic, I., Molnar, S., Joksimovic, S., 2011. Royal jelly as supplement for young football players. Sport Sci. 1, 62e67. Kamakura, M., Mitani, N., Fukuda, T., Fukushima, M., 2001. Antifatigue effect of fresh royal jell in mice. J. Nutr. Sci. Vitaminol. 47, 394e401. Kohguchi, M., Inoue, S., Ushio, S., Iwaki, K., Ikeda, M., Kurimoto, M., 2004. Effect of royal jelly diet on the testicular function of hamsters. Food Sci. Technol. Res. 10, 420e423. Lercker, G., Capella, P., Conte, L.S., Ruinji, F., Giordani, G., 1981. Components of royal jelly: I. Identification of organic acids. Lipids 16, 912e919. Lercker, G., Capella, P., Conte, L.S., Ruinji, F., Giordani, G., 1982. Components of royal jelly: II. The lipid fraction, hydrocarbons and sterols. J. Apicult. Res. 21, 178e184. List of Quasi-Drug Ingredients. http://jpdb.nihs.go.jp/kyokuhou/archives-e.htm. Mairba¨ur, H., November 12, 2013. Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells. Front. Physiol. 4. http://dx.doi.org/10.3389/phys.2013.00332. Article 332. Matsumoto, K., Ishihara, K., Tanaka, K., Inoue, K., Fushiki, T., 1966. An adjustable-current swimming for the evaluation of endurance capacity of mice. J. Appl. Physiol. 81, 1843e1849. Morita, H., Ikeda, T., Kajita, K., Fujioka, K., Mori, I., Okada, H., Uno, Y., Ishizuka, T., 2012. Effect of royal jelly in ingestion for six months on healthy volunteers. Nutr. J. 11, 77. http://www.nutrtionj.com/content/11/1/77. Moriyama, T., Yanagihara, M., Yano, E., Kimura, G., Seishima, M., Tani, H., Kanno, T., NakamuraHirota, T., Hashimoto, K., Tatefuji, T., Ogawa, T., kawamura, Y., 2013. Hypollaergenicity and immunological characterization of enzyme-treated royal jelly from Api mellifera. Biosci. Biotechnol. Biochem. 77, 789e795. Moriyama, H., Yoshinari, O., Bagchi, D., 2016. Chlorogenic acids in green coffee bean extract are the key ingredients in food with health and function claims. In: Bagchi, D., Moriyama, H., Swaroop, A. (Eds.), Green coffee Been Extract in Human Health. CRC Press, Boca Baton, pp. 19e30. Murray, R.K., Bender, D.A., Botham, K.M., Kennelly, P.J., Rodwell, V.W., Weil, P.A., 2009. Harper’s Illustrated Biochemistry, twenty eight ed. McGraw Hill, New York. Nagai, T., Inoue, R., Suzuki, N., Nagashima, T., 2004. Preparation and the functional properties of water and alkaline extract of royal jelly. Food Chem. 84, 181e186. Nagai, T., Inoue, R., Suzuki, N., Nagashima, T., 2006. Antioxidant properties of enzymatic hydrolysates from royal jelly. J. Med. Food 9, 363e367. Narita, Y., Nomura, J., Ohta, S., Inoh, Y., Suzuki, K., Araki, Y., Miyata, T., Isohama, Y., Abe, K., Miyata, T., Mishima, S., 2006. Royal jelly stimulates bone formation: physiologic and nutrigenomic studies with mice and cell lines. Biosci. Biotechnol. Biochem. 70, 2508e2514. Nations United, 2009. Department of Economic and Social Affairs Population Division: World Population Ageing 2009. United Nations, New York, p. 11. Niu, K., Guo, H., Ebihara, S., Asada, M., Ohrui, T., Furukawa, K., Ichinose, M., Yanai, K., Kudo, Y., Ari, H., Okazaki, T., Nagatomi, R., 2011. Royal jelly prevents the progression of sarcopenia in aged mice in aged mice in vivo and vitro. J. Gerontol. Ser. A Biol. Sci. Med. Sci. 68, 1482e1492. Ohama, H., Ikeda, H., Moriyama, H., 2014. Health foods and foods in health claims in Japan. In: Bagchi, D. (Ed.), Nutraceuticals and Functional Foods Regulations in the United States and Around the World, second ed. Academic Press, London, pp. 265e299. Pharmaceutical and Medical Device Regulation (PMDR), Drug classification. https://www.pmda.go.jp/ files/000152069.pdf. Poplawsky, A., 2008. Food for thought: royal jelly for the people. Cent. Sulcus 4, 3e4.

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Ramadan, F.A., Al-Ghamdi, A., 2012. Bioactive compounds and health-promting properties of royal jelly: a review. J. Funct. Food 4, 39e52. Regulation (EU) No. 432/2012 establishing a list of permitted health claims make on foods, other than those referring to the reduction of disease risk and to children’s development and health. http://eurlex.europa.eu/LexUriServ/LexUriServ.do?uri¼OJ:2012:136.0001:0040:en:PDF. Sabatini, A.G., Marcazzan, G., Caoni, M.F., Bogdanov, S., Almeida-Muradian, L.B., 2009. Quality and standardisation of royal jelly. J. Anal. At. Spectrom. 1, 1e6. Saritas¸, N., Yildiz, K., Bu¨yu¨kipkci, S., Cos¸kun, B., 2011. Effect of different levels of royal jelly on biomedical parameters of swimmers. Afr. J. Biotechnol. 10, 10718e10723. Spiridonov, N.A., Bakaneva, V.F., Narimanov, A., Arkhipov, V.V., 1989. Miotropic action and cytotoxicty of honey bee products. Farmatsiya 38, 62e63. Stocker, A., Schramel, P., Kettrup, A., Benegsch, E., 2005. Trace and mineral elements in royal jelly and homeostatic effects. J. Trace Elem. Med. Biol. 19, 183e189. Suzuki, I., Matsushima, Y., Hirai, H., Naito, Y., Hasegawa, M., Yuan, S., Hirosawa, K., Maehata, E., Tsurasaki, Y., Kurihara, M., Tomida, M., 2009. Study on the fatigue-restorative effect of royal jelly, propolis, and honey. Med. Biol. 153, 225e231. Takahashi, M., Matsuo, I., Ohkido, M., 1983. Contact dermatitis due to honeybee royal jelly. Contact Dermatitis 9, 452e455. Takenaka, T., 1982. Chemical composition of royal jelly. Honeybee Sci. 3, 69e74. The Japan Royal Jelly Fair Trade Council. Royal Standardization. http://www.rjkoutori.or.jp/mark/about. html. Thien, F.C.K., Leung, R., Baldo, B.A., Weiner, J.A., Plomley, R., Czarny, D., 1996. Ashma and anaphylaxis induced by royal jelly. Clin. Exp. Allergy 26, 216e222. Vecchi, M.A., Sabatini, A.G., Grazia, L., Tini, V., Zambonelli, C., 1988. II contento in vitamine come possible elelmento di caratterizzazione della gelatina reale. Apiccoltura 4, 139e146. Vittek, J., Slomiany, B., 1984. Testosterone in royal jelly. Cell Mol. Life Sci. 40, 104e106. Wang, H., Yin, H., Jin, H., 2007. The study of anti-fatigue effects of sea cucumber polyperide on mice. Food Mach. 23, 89e91. Wang, L., Zhang, H., Lu, R., 2008. The decapeptide CMS001 enhances swimming endurance in mice. Peptides 29, 1176e1182. Wilson-Rich, N., 2014. The Bee a Natural History. Princeton University Press. You, L., Zhao, M., Regenstein, J.M., 2011. In vitro antioxidant activity and in vivo anti-fatigue effect of loach (Misgurnus anguillicauadatus) peptides prepared by papain digestion. Food Chem. 124, 188e194. Yu, B., Lu, Z., Bie, X.-N., Lu, F.-X., Huang, X.-Q., 2008. Scavenging and anti-fatigue activity of fermented defatted soybean peptides. Eur. Food Res. Technol.y 226, 415e421. Zhuang, Y., Zhao, H., Li, B., 2009. Optimization of Antioxidant Activity by Response Surface Methodology in Hydrolysates of Jellyfish (Rhopilema Esculentum) Umbrella Collagen. Zhenjiang University Science B 10, pp. 572e579.

19

Role of Caffeine in Sports Nutrition Lucas Guimarães-Ferreira1, Eric T. Trexler2, Daniel A. Jaffe3, Jason M. Cholewa4 F E DE R AL UNI V ERSI TY OF E S PI RI TO SANTO , V I TO R I A, B R AZI L ; UNIVERSITY OF NORTH CAROLINA, CHAPEL HILL, NC, UNI TED STATES; 3 UNITED STATES MILITARY ACADEMY, WEST POINT, NY, UNI TED STATES; 4 CO AS TAL C AROLINA UNIVERSI TY, CONWAY, SC, UNITED STATES 1

2

Introduction Caffeine (1,3,7-trimethylxanthine) is a methylxanthine naturally present in a diversity of foods and beverages such as teas, cola soda, energy drinks, and chocolate (Barone and Roberts, 1996; Carrillo and Benitez, 2000). However, the main source of caffeine is through the consumption of coffee (Barone and Roberts, 1996) (Table 19.1). Coffee consumption is widespread across cultures in part due to the stimulating effects associated with it. In the US, 85% of adults consume caffeine regularly

Table 19.1

Caffeine Content on Food, Drinks, and Medication

Food or Drink

Serving Size

Caffeine Content (mg)

Instant coffee Brewed coffee Decaffeinated coffee Black tea Green tea Cola soda Energy drinks Hot chocolate Chocolate milk Chocolate bar/candy Baking chocolate OTC stimulant OTC pain reliever OTC cold medication

250 mL 250 mL 250 mL 250 mL 250 mL 355 mL 250 mL 250 mL 250 mL 28 g 28 g 1 capsule/tablet 1 capsule/tablet 1 capsule/tablet

10e170 40e110 2000 mg) can give rise to significant toxicities including nausea, vomiting, tachycardia, severe hypertension, arrhythmia, seizures, and even death; however, individuals sensitive to caffeine may exhibit adverse effects at lower doses (Cappelletti et al., 2015; Hoffman, 2011). The harmful aspects of caffeine overconsumption were first recognized in the early 19th century (Riksen et al., 2011), and up until 1980, reports of caffeine toxicity only occasionally appeared in the medical literature, often in the context of ingestions with other legal (e.g., amphetamine) or illegal (e.g., cocaine) stimulants (Hoffman, 2011). An interesting bellwether of the medical community’s growing anxiety over the caffeination of modern society comes from the number of articles published in the scientific literature since 1980 related to health concerns over this ubiquitous chemical and its myriad vehicles for ingestion (Gurley et al., 2015). Fig. 26.1 depicts the number of publications identified in the world scientific/medical literature from 1980 to 2013 that discusses adverse effects of caffeine or caffeine-containing products. During that time, the number of such publications has increased by a factor of eight, with the most

Number of Publications Identified

400 350 PubMed

WOS

300 250 200 150 100 50

2015

2014

2013

2012

2011

2010

2009

2008

2007

2006

2005

2004

2003

2002

2001

2000

0

Year FIGURE 26.1 Citation counts for articles about caffeine adverse effects in the PubMed and Web of Science (WOS) databases, by year.

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precipitous rise coming in the last 10 years. Contributors to this upsurge include caffeine-containing, multiingredient dietary supplements like Ephedra and Ephedra-free products as well as CEDS. If one dissects the timeline in Fig. 26.1, it is interesting to note that one of the most controversial categories of caffeine-containing products, those formulated with Ephedra (a natural source of ephedrine alkaloids) came into the US market in 1994 but were removed by the FDA in 2004, yet the greatest upswing in publications comes after 2004. (For a discussion of the Ephedra controversy, see Gurley et al., 2015.) Hidden within the escalating literature chronicling caffeine-associated health concerns is a cautionary subset linking CEDS consumption to adverse health effects. Beginning with Red Bull in the late 1990s, CEDS have become the fastest growing beverage category on the market in terms of number, variety of products, and sales revenue (Heckman et al., 2010). The boom in CEDS usage, especially among adolescents and young adults, has also sparked an upsurge in reports of caffeine-related adverse events among this population (Heckman et al., 2010; Worrall et al., 2005; Seifert et al., 2011; Trabulo et al., 2011; Gray et al., 2012; Wolk et al., 2012; Nordt et al., 2012; Benjo et al., 2012; Kaoukis et al., 2012; Rottlaender et al., 2012; Usman and Jawaid, 2012; Dikici et al., 2013; Pomeranz et al., 2013; Cotter et al., 2013; Seifert et al., 2013; Sepkowitz, 2013; Goldfarb et al., 2014; Huang et al., 2014; Greene et al., 2014; Samanta, 2015; Tofield, 2015; Sanchis-Gomar et al., 2015; Newton and Okuda, 2015; Solomin et al., 2015; Dikici et al., ¨ nal et al., 2015; Grant et al., 2016; Sattari et al., 2015; Ali et al., 2015; Saritas et al., 2015; U 2016; Mounir et al., 2015; Lippi et al., 2016; Gonzalez et al., 2015). Between 2000 and 2016, the number of publications appearing in the medical literature pertaining to CEDS has steadily increased (Fig. 26.2). Starting in 2008, the number of articles related to any aspect of CEDS intake has doubled every 2 years to a maximum of 359 in 2015 (Fig. 26.2). Many articles published during this period underscore safety concerns about CEDS. This remarkable surge in CEDS-related publications undoubtedly parallels the meteoric rise in CEDS consumption. CEDS differ from conventional caffeinated soft drinks in that they are not as highly carbonated (making them easier to drink quickly than conventional sodas), have higher caffeine content, and often contain vitamins, amino acids, L-carnitine, taurine, glucuronolactone, and botanical extracts like guarana, ginseng, Ginkgo biloba, and milk thistle, to name a few. The ambiguity of CEDS label claims for caffeine content may also contribute to the purported health risks linked to these beverages, although recent changes to accepted industry practices related to labeling have increased clarity related to existence and content of the ingredient mix in products. For a while, certain energy drinks were purposely formulated with alcohol, but safety concerns regarding caffeine/ alcohol combinations prompted the FDA to preclude sale of these products in 2011 (Arria and O’Brien, 2011; Attwood, 2012). Nevertheless, the concomitant consumption of energy drinks and alcoholic beverages remains a cause for alarm for both health-care professionals and law enforcement officials (Arria and O’Brien, 2011; Attwood, 2012; O’Brien et al., 2008; Howland and Rohsenow, 2013).

Chapter 26  Caffeine-Containing Energy Drinks/Shots 427

Number of publications identified

2500

2000

PubMed

WOS

1500

1000

500

1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

0

Year FIGURE 26.2 Citation counts for articles about caffeine-containing energy drinks/shots in the PubMed and Web of Science (WOS) databases, by year.

While much research has been conducted on the pharmacology of caffeine when administered either as a purified pharmaceutical ingredient or in the form of coffee or tea, fewer prospective studies have assessed the safety and efficacy of various formulations of CEDS. From the data currently available, it appears that the pharmacology/ toxicology of caffeine can be markedly influenced by a host of other physiological and environmental factors.

Pharmacology of Caffeine Caffeine’s stimulant effects on the cardiovascular system and CNS stem from four principal mechanisms: nonselective antagonism of G-coupled adenosine A1 and A2A receptors, inhibition of phosphodiesterases with the subsequent accumulation of cyclic AMP and an intensification of the effects of catecholamines, mobilization of intracellular calcium via activation of ryanodine receptor (RYR2) channels, and inhibition of gammaaminobutyric acid neurotransmission (Riksen et al., 2011; Benowitz, 1990). Only at higher serum concentrations (>25 mg/mL), which can be achieved with doses in excess of 1500 mg, do the latter three mechanisms appear to contribute significantly to caffeine pharmacodynamics (Cappelletti et al., 2015; Riksen et al., 2011). Caffeine’s dosedependent CNS stimulant effects (e.g., mood enhancement, wakefulness, insomnia, anxiety, tremors, and seizures) stem from antagonism of brain adenosine receptors,

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while antagonism of A1 and A2A receptors in the heart and vasculature account for its hemodynamic effects (e.g., increased heart rate, coronary and peripheral vasoconstriction, elevated blood pressure) (Riksen et al., 2011; Benowitz, 1990). As a result of adenosine antagonism, caffeine also stimulates the release of several neurotransmitters (e.g., dopamine, norepinephrine, serotonin), which also accounts for many of the drug’s indirect pharmacodynamic effects. (For an excellent review of the cardiovascular effects of caffeine, see Riksen et al., 2001.) Caffeine can also reduce cerebral, hepatic, and mesenteric blood flow as well as produce a diuretic effect via increased glomerular filtration and enhanced sodium and water excretion (Cappelletti et al., 2015). At higher doses, other pharmacodynamic effects include bronchodilation, lipolysis, hyperglycemia, and hypokalemia (Cappelletti et al., 2015; Benowitz, 1990). Caffeine-induced hypokalemia could contribute to ventricular arrhythmias and sudden death (Goldfarb et al., 2014). Chronic consumption of caffeine, however, can lead to pharmacological tolerance, which can occur within a few days (Riksen et al., 2011). Owing to the rapid development of tolerance, a person’s response to caffeine depends upon dose, dosing regularity, and their pharmacokinetic profile. Individual sensitivity to the effects of caffeine is well recognized (Nehlig et al., 1992). Such sensitivities may be attributable, in part, to an individual’s genetic makeup (Yang et al., 2010). Only recently has an appreciation developed for the effects that human receptor gene polymorphisms can have on the pharmacodynamics of caffeine. Adenosine A2A and a2-adrenergic receptor polymorphisms have been linked to caffeineinduced insomnia (Re´tey et al., 2007; Byrne et al., 2012), anxiety (Alsene et al., 2003; Childs et al., 2008), habitual coffee consumption (Cornelis et al., 2007), and blood pressure elevation (Renda et al., 2012), whereas animal studies hint that cardiac RYR2 mutations may increase caffeine’s arrhythmogenic potential (Jiang et al., 2010). Another gene polymorphism associated with caffeine adverse effects is the enzyme catechol-Omethyltransferase (COMT). In the case of functional COMT polymorphisms, the sympathomimetic effects of endogenous catecholamines (e.g., norepinephrine) are enhanced; following caffeine ingestion, such mutations have been linked to rapid heartbeat (Brathwaite et al., 2011), elevated blood pressure (Miller et al., 2012), and the incidence of acute coronary events (Happonen et al., 2006). Despite these emerging geneecaffeine relationships, more work is required before the functional variants involved in the caffeine response can be delineated. Caffeine’s physicochemical and pharmacokinetic properties set it apart from most phytochemicals. Caffeine is one of the few phytochemicals whose oral bioavailability is almost complete (Blanchard and Sawers, 1983). Peak blood concentrations of caffeine are usually achieved within an hour of ingestion (Arnaud, 2011; White et al., 2016). In the case of beverages, temperature and rate of consumption have little bearing on caffeine absorption kinetics (White et al., 2016). On account of its excellent aqueous solubility and small molecular weight, caffeine readily enters the intracellular space and is widely distributed; its volume of distribution mimics that of total body water (Arnaud, 2011). Accordingly, caffeine readily crosses the bloodebrain barrier and can be found in almost

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all body fluids and tissues (Arnaud, 2011). As a result of its disposition characteristics, the pharmacological consequences of caffeine doses commonly encountered in CEDS can be significantly exaggerated in children and lean or underweight adolescents when compared to adults. This may partially explain the increased incidence of adverse effects reported for CEDS in younger populations, as caffeine exposure in these populations (mg/kg) is likely much more enhanced (Substance Abuse and Mental Health Services Administration, 2013; Bashir et al., 2016). The pharmacokinetics of caffeine is dose dependent, which likely contributes to toxicities associated with many caffeine-containing dietary supplements (Denaro et al., 1990). Caffeine biotransformation is mediated primarily via hepatic cytochrome P450 1A2 (CYP1A2), and saturation of this pathway can occur at doses as low as 5 mg/kg (Arnaud, 2011). Caffeine’s principal CYP1A2-mediated metabolite in humans is paraxanthine, which exhibits pharmacological effects similar to its parent compound (Thorn et al., 2012; Benowitz et al., 1995), while minor metabolites include theophylline and theobromine (Thorn et al., 2012). Caffeine clearance is highly variable, and both genetic and environmental factors (e.g., diet, smoking, oral contraceptive use) are contributors to this variability (Arnaud, 2011; Carrillo and Benitez, 2000; Gunes and Dahl, 2008). Allelic variants in CYP1A2 can affect caffeine’s pharmacokinetics and pharmacological response. Among caffeine users, both “slow” and “rapid-metabolizer” phenotypes have been described, each corresponding to respective allelic variants that give rise to loss or gain of enzyme function. Habitual coffee use and higher consumption of coffee appear to correlate with rapid metabolizer phenotypes (homozygous CYP1A2*1A) (Josse et al., 2012), while slow metabolizer phenotypes (heterozygous CYP1A2*1F) have been linked to higher risks for hypertension (Palatini et al., 2009) and nonfatal myocardial infarction (Cornelis et al., 2006). In addition to possible pharmacogenetic factors, caffeine metabolism is also susceptible to a host of environmental influences. Smoking and diets rich in cruciferous vegetables induce CYP1A2 gene expression, presumably through activation of the aryl hydrocarbon nuclear receptor, resulting in enhanced caffeine clearance (Arnaud, 2011; Gunes and Dahl, 2008). Conversely, alcohol consumption, oral contraceptives, fluvoxamine, and quinolone antibiotics are known to inhibit CYP1A2 activity, lower caffeine clearance, and increase both area under the plasma concentration time curve and elimination of half-life (Carrillo and Benitez, 2000). When combined with other stimulants, whether legal (e.g., dextroamphetamine, methylphenidate, ephedrine) or illegal (e.g., cocaine, heroin, ecstasy), especially in the context of vigorous exercise, caffeine may increase the likelihood of serious adverse health effects like arrhythmia, heart attack, stroke, seizure, hypertensive crisis, and exertional heat illness (Morelli and Simola, 2011; Derlet et al., 1992; Vanattou-Saı¨foudine et al., 2012; Frau et al., 2013). Because many CEDS are marketed as athletic performance enhancers, it is worth noting that both exercise and obesity have exhibited equivocal effects on caffeine pharmacokinetics (Arnaud, 2011). Several studies suggest that caffeine disposition is not significantly altered during exercise (McLean and Graham, 2002; Haller et al., 2008), while others

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indicate that peak caffeine plasma levels may be enhanced (Collomp et al., 1991). Likewise, obesity’s effect on caffeine pharmacokinetics is also difficult to predict (Kamimori et al., 1987; Caraco et al., 1995; Skinner et al., 2014). Such ambiguities may contribute to the questionable safety and efficacy of CEDS. What is less ambiguous is that vigorous exercise may exacerbate the pharmacodynamic effects of caffeine (Daniels et al., 1998; Stebbins et al., 2001; Astorino et al., 2007; Woolf et al., 2008; Arciero and Ormsbee, 2009; Sung et al., 1995). Of particular concern is the recent finding that caffeine reduces myocardial blood flow during exercise (Higgins and Babu, 2013). Such consequences could have significant health repercussions, especially in caffeine-naı¨ve, untrained athletes (Sinclair and Geiger, 2000; Tunnicliffe et al., 2008). Collectively, these genetic and environmental influences can have a significant bearing on the safety of CEDS.

Safety of CEDS CEDS use has become widespread across a host of demographics within the United States. Surveys indicate that 30%e50% of adolescents and as many as 80% of college students regularly use CEDS (Seifert et al., 2011; Hoyte et al., 2013). CEDS usage is also common among US military personnel (Stephens et al., 2014). In general, CEDS are more popular among males than females (Ali et al., 2015). As for quantities consumed, a recent survey of emergency department patients found that 58% of young adults admitted to drinking an average of 3 CEDS per day over the last 30 days (Cotter et al., 2013). Whether this usage statistic translates to the general population, however, remains to be determined. Principal reasons for such high usage include increased alertness, improved mental and physical endurance, compensation for insufficient sleep, improved academic performance, athletic performance enhancement, or peer pressure (Bashir et al., 2016; Hoyte et al., 2013; Stephens et al., 2014; Taddeo et al., 2012). Aggressive marketing campaigns by manufacturers capitalize on these potential benefits, which drive energy drink sales. As the market increases, so increases the opportunity for adverse effects, fueling a growing anxiety within the medical community (Seifert et al., 2011; Ali et al., 2015; Saritas et al., 2015; Trabulo et al., 2011; Pomeranz et al., 2013). Many of the concerns expressed by health-care professionals center around the increasing number of anecdotal case reports, adverse event reports, calls to poison control centers, and emergency department visits linking suspected caffeine-related toxicities in young adults to energy drink consumption (Sepkowitz, 2013). According to the Drug Abuse Warning Network, the number of emergency department visits involving energy drinks doubled in the period 2007e2011, a statistic that likely mirrors the increase in the prevalence of energy drink consumption (Substance Abuse and Mental Health Services Administration, 2013). In fact, adverse health consequences linked to CEDS use or, more often, misuse, are the subjects of many of the publications constituting the recent upsurges in Figs. 26.1 and 26.2.

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The spike in reports of energy drinkerelated adverse events appears to stem from a combination of several factors. First, CEDS constitute the fastest growing segment of the US beverage market (Seifert et al., 2011) and are especially popular among young adults and adolescents (Trabulo et al., 2011). This popularity is bolstered by the marketing of CEDS on television channels (e.g., MTV2) frequently viewed by adolescents and young adults (Edmond et al., 2015). The increased consumption of CEDS and a corresponding heightened awareness of this increase has caused many health-care professionals to question potential benefits compared to risks with consumption of such products, whether safety concerns are supported by scientific data or not. Given the fact that health-care professionals are exposed to the same media attention regarding these products, it could also bias their perception of risk and increase reporting of suspected adverse effects involving CEDS products, whether related or not. Second, CEDSs, especially energy shots and beverage enhancers, contain higher quantities of caffeine than conventional soft drinks, tea, and coffee products. Table 26.1 Table 26.1

Caffeine-Containing Beverage Formulations and Their Caffeine Content

Beverage

Formulation

Caffeine Content (mg)

Serving Size (fl. oz.)

mg/fl. oz.

Coca-Cola Classic 5-h Energy decaf Tea (green) Mountain Dew Tea (black) Red Bull Full Throttle Monster NOS Rockstar Berzerk Zombie Blood Spike Shooter 5-h Energy Redline Extreme Shot AllDay Energy Nescafe Ice Java Redline Power Rush Ammo Energy Shot Stakk’d Caffeine Mixer Hijinks Energy Mixer ALRI HyperShot Energy Catalyst NRG Micro Shot Mio Energy DynaPep

Soda Energy shot Beverage Soda Beverage Energy drink Energy drink Energy drink Energy drink Energy drink Energy drink Energy shot Energy drink Energy shot Energy shot Energy shot Concentrate Energy shot Energy shot Concentrate Concentrate Energy shot Energy shot Energy shot Concentrate Energy shot

34 mg 6 mg 25 mg 54 mg 42 mg 80 mg 160 mg 160 mg 160 mg 160 mg 225 mg 80 mg 300 mg 200 mg 300 mg 210 mg 1880 mg 350 mg 171 mg 4590 mg 200 mg 500 mg 100 mg 130 mg 1080 mg 100 mg

12 oz. 2 oz. 8 oz. 12 oz. 8 oz. 8.5 oz. 16 oz. 16 oz. 16 oz. 16 oz. 16 oz. 3.4 oz. 8.4 oz. 2 oz. 3 oz. 2 oz. 16 oz. 2.5 oz. 1 oz. 25.5 oz. 1.1 oz. 2 oz. 0.23 oz. 0.2 oz. 1.62 oz. 0.14 oz.

2.8 3 3.1 4.5 5.2 9.4 10 10 10 10 14.1 23.5 35.7 100 100 105 118 140 171 180 182 250 435 650 667 714

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provides a comparison of caffeine content among conventional caffeinated beverages (e.g., coffee, tea, soda) to that of several brands of CEDS. Among the CEDS listed in Table 26.1, the caffeine content spans a wide range, with products containing as little as 3 mg per fluid ounce to as much as 700 mg per fluid ounce. From Table 26.1, one can easily see that popular energy drinks like Red Bull, Monster, and Rockstar have caffeine concentrations less than that found in a typical cup of regular brewed coffee. As such, adverse events reported for these brands are often the result of overconsumption, either with or without concurrent alcohol ingestion (Seifert et al., 2011; Trabulo et al., 2011; Gray et al., 2012; Wolk et al., 2012; Nordt et al., 2012; Benjo et al., 2012; Kaoukis et al., 2012; Rottlaender et al., 2012; Usman and Jawaid, 2012; Dikici et al., 2013; Pomeranz et al., 2013; Cotter et al., 2013; Seifert et al., 2013; Sepkowitz, 2013; Goldfarb et al., 2014; Huang et al., 2014; Greene et al., 2014; Samanta, 2015; Tofield, 2015; Sanchis-Gomar et al., 2015; Newton and Okuda, 2015; Solomin et al., 2015; Dikici et al., 2015; Ali et al., ¨ nal et al., 2015; Grant et al., 2016; Sattari et al., 2016; Mounir 2015; Saritas et al., 2015; U et al., 2015; Lippi et al., 2016; Gonzalez et al., 2015; Arria and O’Brien, 2011; Attwood, 2012; O’Brien et al., 2008; Howland and Rohsenow, 2013). A good illustration of how energy drink overconsumption can give rise to potential adverse health effects comes from a series of studies investigating the influence of energy drink ingestion on hemodynamic and cardiac physiology in healthy volunteers. Two prospective studies in healthy volunteers evaluated the effect of consuming 16 and 24 fl. oz. of Monster Energy Drink (Brothers et al., 2016) or 2 fl. oz. of 5-h energy shot (Shah et al., 2016) on blood pressure and electrocardiography. In both instances elevations, both systolic and diastolic blood pressure were noted, but no electrocardiographic anomalies were observed. The amount of caffeine ingested in either study did not exceed 240 mg. However, when the ingested volume of Monster Energy Drink was increased to 32 fl. oz. (320 mg caffeine) consumed within 1 h or less, significant elevations in systolic and diastolic blood pressure as well as prolongations in corrected QT intervals ( 500 milliseconds) were noted (Kozik et al., 2016; Shah et al., 2016). These findings suggest that a rapid consumption of 300 mg or greater of caffeine is sufficient to affect cardiac repolarization, which, in susceptible individuals, may be become clinically important. In the context of CEDS consumption, Chrysant and Chrysant (2015) corroborated this 300-mg caffeine threshold as being a practical limit where few cardiovascular complications are likely to occur. One additional cautionary finding recently uncovered in a series of prospective clinical trials involving Red Bull is the ability of the beverage to reduce cerebral blood flow and increase cerebrovascular resistance for up to 2 h after ingestion (Grasser et al., 2014, 2015). In these particular studies, no adverse effects were noted, so the clinical significance is unknown. Given caffeine’s ability to reduce coronary blood flow during exercise (Higgins and Babu, 2013), it remains to be determined whether this CEDSinduced cerebrovascular response is also augmented during vigorous exercise. The combination of CEDS and physical activity is common among adolescent athletes (Nowak and Jasionowski, 2016). Such unanticipated physiological responses lend credence to recommendations cautioning adolescents “to avoid CEDS consumption before or during sports practice” (Samanta, 2015).

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Based upon the previously described findings, energy shots and beverage enhancers would appear to be more problematic with regard to potential caffeine toxicity. Energy shots and microenergy shots are small-volume products, typically less than 3 fl. oz., with caffeine concentrations often in excess of 100 mg/fl oz. The potential for caffeine overdose is therefore greater for these products than that for larger-volume, less concentrated energy drinks. Beverage enhancers are relative newcomers to the energy drink arena. These products essentially serve as reservoirs for dispensing aliquots of caffeine into water or other beverages in order to enhance their stimulant properties. The accuracy of self-dispensed caffeine doses from these products is questionable. Consumers can readily “enhance” beverages with caffeine quantities that may be toxic. For example, an unsuspecting consumer could easily squeeze the entire contents of a 1.62-fl. oz. Mio Energy product into a beverage producing a drink with a caffeine content exceeding 1 g. The flavor profile of the product makes this less likely to occur due to the extreme bitter taste in a concentrated version of a self-made drink, but this quantity of caffeine could produce significant cardiovascular toxicity in many individuals. Given the ease with which enhancers and microshots can be misused, they pose a greater risk for accidental caffeine overdose. Until recently, caffeine content was not always indicated on CEDS product labels (Heckman et al., 2010). Today, however, most CEDS marketed as beverages instead of dietary supplements provide label claims for caffeine content, and the major manufacturers that control more than 95% of the market have agreed to accept industry practices to label accordingly. Nevertheless, the caffeine content for CEDS marketed as dietary supplements may not be discernable to consumers. Third, since energy drinks/shots are less carbonated, or in many instances noncarbonated, they may be more easily and quickly imbibed than hot coffee or typical carbonated soft drinks. Additionally, energy drinks are often formulated with a variety of botanical extracts. Besides purified caffeine, other typical components include guarana, yerba mate´, green tea, taurine (2-aminoethanesulfonic acid), glucuronolactone, Panax ginseng, yohimbe, and B vitamins (Heckman et al., 2010; Higgins et al., 2010). Many energy drink phytochemicals are present in lower quantities than those found in conventional botanical dietary supplements, while components like taurine and glucuronolactone are present at higher concentrations and may contribute to the purported benefits or suspected risks of energy drinks/shots (McLellan and Lieberman, 2012). For example, Red Bull, an energy drink containing taurine (1000 mg) and caffeine (80 mg), increased 24-h and daytime blood pressures over that of a compounded solution containing an equivalent dose of caffeine only (Franks et al., 2012). Consumption of a caffeine, taurine, and glucuronolactone formulation increased mean arterial blood pressure and platelet aggregation while decreasing endothelial function in healthy young adults (Worthley et al., 2010). Further evidence for taurine’s contribution to enhanced hemodynamic effects comes from an ex vivo preparation in which taurine exhibited both a positive inotropic effect and potentiated caffeine-induced cardiac muscle contraction (Steele et al., 1990). These additional elements aside, caffeine remains the principal psychoactive component of energy drinks and is responsible for the vast majority of reported toxicities (Cappelletti et al., 2015; Wolk et al., 2012).

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The final, and perhaps most controversial, contributor to the adversity surrounding CEDS is their consumption with alcohol (Wolk et al., 2012; O’Brien et al., 2008; Howland and Rohsenow, 2013). For a brief period between 2009 and 2011, caffeinated alcoholic beverages were commercially available in the United States before the FDA forced their removal from the market, deeming them adulterated under the Federal Food, Drug, and Cosmetic Act (Howland and Rohsenow, 2013). Nevertheless, this practice has continued with many consumers self-mixing energy drinks/shots with alcohol (Howland and Rohsenow, 2013). In the most comprehensive evaluation to date of scientific investigations into concurrent consumption of CEDS with alcohol (ACEDS), McKetin et al. provided the following assessment: (1) ACEDS consumers drink more alcohol and experience greater alcohol-related harm than other drinkers. It remains to be determined whether ACEDS use leads to increased alcohol consumption, but evidence supports that premise; (2) ACEDS increases mental stimulation and alertness, offsets fatigue from drinking, and increases the desire to continue imbibing; (3) ACEDS does not affect blood alcohol concentrations, nor does it mitigate perceived intoxication or perceived impairment; (4) ACEDS does not reverse alcohol-induced deficits in simple reaction time, focused attention, and basic psychomotor coordination, although the practice may attenuate alcohol-induced impairment on some complex psychomotor and cognitive tasks; (5) whether ACEDS directly increases alcohol consumption requires further investigation; and (6) researchers and policy makers need to be mindful of bias in industry-sponsored studies in this area of research (McKetin et al., 2015). Additionally, ACEDS has also been associated with greater risk-taking behaviors, such as driving under the influence of alcohol, binge drinking, or having unprotected sex (O’Brien et al., 2013; Berger et al., 2013). Such practices likely contribute to the rise in energy drinkerelated emergency department visits (Nordt et al., 2012; Cotter et al., 2013; Substance Abuse and Mental Health Services Administration, 2013). When consumed sensibly and in moderation, CEDS rarely produce significant adverse health effects. From the prospective clinical studies conducted to date, elevations in blood pressure and heart rate, as well as minor CNS effects (e.g., insomnia), are commonplace (Ali et al., 2015; Saritas et al., 2015). However, irresponsible overconsumption or coingestion of CEDS with alcohol or other stimulants certainly contributes to caffeine toxicity (Seifert et al., 2011; Sepkowitz, 2013; Substance Abuse and Mental Health Services Administration, 2013). Depending upon the specific product and the number of units imbibed, ingested caffeine doses can easily exceed 1000 mg. In healthy adults, a caffeine intake of 400 mg/day is considered safe; acute clinical toxicity begins at 1000 mg, and 5000e10,000 mg can be lethal (Cappelletti et al., 2015; Seifert et al., 2011). While often difficult to assign specific causation, reports linking CEDS to serious adverse health effects suggest that these products are vehicles for caffeine overdose. The growing number of caffeine-related overdoses among adolescents and young adults presenting to emergency departments corroborates this assessment. Of course, as discussed earlier, certain environmental, genetic, and medical circumstances

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Table 26.2

Recommendations Regarding Energy Drink Consumption in Adolescents

Do not consume more than one can/shot of CEDS (100 mg caffeine). Avoid CEDS consumption before participating in sports practice/events. Individuals with diagnosed cardiovascular anomalies consult physician before CEDS use. Do not combine CEDS with alcohol or prescription medications. Parents and coaches should educate themselves about CEDS adverse effects. Advise against overconsumption/abuse of CEDS Adapted from Sanchis-Gomar, F., Pareja-Galeano, H., Cervellin, G., Lippi, G., Earnest, C.P., 2015. Energy drink overconsumption in adolescents: implications for arrhythmias and other cardiovascular events. Can. J. Cardiol. 31, 572e575.

may predispose individuals to the effects of caffeine present in CEDS. Given the vulnerability of adolescents to CEDS, Table 26.2 provides a list of practical recommendations for this population (Sanchis-Gomar et al., 2015).

Efficacy of CEDS Improvements in mood and cognition are effects desired by just about every member of today’s fast-paced society. Reaction time, concentration, alertness, and subjective feelings of energy/vitality are important for athletes in many competitive sports. Strategies to enhance these attributes are often sought after by nonathletes and athletes alike. Over the last decade, several prospective studies have assessed the effects that energy drinks may have on these and other performance variables (Campbell et al., 2013). Like coffee, CEDS, when consumed sensibly, can improve and/or maintain mood and cognitive performance as determined by a variety of mood/cognition assessment instruments (Campbell et al., 2013; Smit et al., 2004; Scholey and Kennedy, 2004; Giles et al., 2012). Despite their varied ingredients, however, it appears that caffeine is the agent primarily responsible for the psychopharmacological effects of energy drinks (Smit et al., 2004; Scholey and Kennedy, 2004; Giles et al., 2012). The International Society of Sports Nutrition recently reviewed the ergogenic potential of energy drinks and came to the following conclusions: (1) the primary ergogenic nutrients in energy drinks appear to be caffeine and/or carbohydrate; (2) the ergogenic contribution of other nutrients besides caffeine is minor; (3) consuming an energy drink 60 min prior to exercise can improve mental focus and alertness, and may improve endurance performance in trained athletes; (4) ingestion of high-calorie energy drinks may promote weight gain; (5) children or adolescents should only consider using energy drinks with parental approval, and parents should be aware of potential adverse side effects; (6) indiscriminate use may lead to adverse events and harmful side effects; and (7) diabetics or individuals with underlying cardiovascular, hepatorenal, or neurologic disease who are taking prescription medications or other stimulants should avoid energy drinks unless approved by their physician (Campbell et al., 2013). These recommendations appear to represent a common sense approach to CEDS consumption across all populations likely to elect CEDS.

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Controversy In recent years, various publications have debated a potentially harmful association between the excessive intake of caffeine and significant adverse medical outcomes, including serious cardiovascular and neurologic dysfunction as discussed in previous sections. With the rising popularity of CEDS products, it’s not surprising that the debate has extended to these caffeine-containing products and an interest in quantifying their specific contribution to actual adverse effects and populations affected. Many of the publications citing CEDS adverse event risks up to this point have been limited to detailed questionnaires and surveys administered to various populations to canvass their past personal experience with CEDS use or anecdotal case reports involving individual patients reportedly consuming one or more CEDS. Epidemiological surveillance data from the United States National Poison Data System (NPDS), which tabulates poison control center data, has also been used. The NPDS database is largely representative of acute exposures and spontaneous reports of adverse effects associated with all types of substances and products, including caffeinated consumer products. The original publication linking pediatric toxicity with CEDS exposure using NPDS incident data was published in the journal Pediatrics in 2011 and has been routinely cited as supporting the premise that all CEDS products pose a significant risk to children, adolescents, and young adults (Seifert et al., 2011). Despite concluding there was a likely safety signal involving CEDS use and risk of serious adverse effects, further examination of the underlying primary data utilized in the publication indicates there were significant limitations related to the findings involving the populations cited. Although entitled “Health Effects of Energy Drinks on Children, Adolescents, and Young Adults,” the report relied on generic caffeinated product exposure data culled directly from published NPDS annual reports as a surrogate measure of pediatric toxicity associated with suspected CEDS exposure. For the years cited, all NPDS caffeine-containing products were culled and aggregated for inclusion in the analysis and included exposures to such products as OTC drugs, powdered caffeine, dietary supplements, and weight-loss aids. This prevented a distinction between CEDS and other caffeinated products in the NPDS annual reports. Furthermore, and as in many subsequent publications debating the risk of such products, the generic category of CEDS included products with variable caffeine doses and formulations such as tablets, capsules, liquids, powder, and concentrated shots as compared to mainstream beverage sources of caffeine like coffee, tea, soft drinks, or beverage versions of energy drinks. As a result of the methodology flaws and in the absence of a specific product breakdown, it is unknown how many CEDS exposures were actually reported to poison control centers in the years cited. The article abstract also cited the statistic that “of the 5448 US caffeine overdoses reported in 2007, 46% occurred in those younger than 19 years,” yet the article did not include any poison center data identifying either exposures or toxicity specific to CEDS or population subsets. At the time the 2011 Pediatrics article was published, the cited NPDS annual reports (Bronstein et al., 2007, 2009, 2008) did not have a generic category

Chapter 26  Caffeine-Containing Energy Drinks/Shots 437

for energy drink exposures, so determining if any serious CEDS exposures had been reported within the cited generic caffeinated product category was not possible. In essence, the 2011 Pediatrics article provided no numerator or denominator upon which to assess the relative safety or toxicity of these products. In 2013, Seifert et al. (2013) published a follow-up report entitled “An analysis of energy-drink toxicity in the National Poison Data System (NPDS).” In this report, exposures to then currently available nonalcoholic energy drinks (depicted as EDs) were tabulated by age and medical outcome for EDs containing either caffeine only or caffeine plus other additives. As in the first Seifert publication and contrary to NPDS nomenclature, all exposures were characterized as representing toxicity reported with product use. And, although a stated objective was to assess the incidence and outcomes of toxic exposures to caffeine-containing EDs, there was no denominator cited to perform adverse event incidence calculations based on servings/units sold during the period studied. Furthermore, all ED outcome data within the two classifications were aggregated for all products, yet caffeine content per ED serving varied widely, ranging from 80 mg to 505 mg, which prevented assessment of adverse effect characteristics potentially associated with specific products based on ED caffeine content. These and other identified inconsistencies were cited in a rebuttal by Barker and Seger, which included as follows: (1) depicting all exposures as “toxicity” (contrary to NPDS disclaimers); (2) distorting and exaggerating relative medical outcome percentages in various age groups by excluding 5 of the 10 NPDS medical outcome categories when performing calculations; and (3) excluding a breakdown of “amount ingested according to age and clinical course,” thereby limiting the validity of any conclusions about specific EDs and their safety or toxicity (Barker and Seger, 2014). Lastly, for the two publications involving NPDS incident data, neither publication included a review of individual case narratives for significant outcome or pediatric incidents to insure both report quality and accurate coding and inclusion of appropriate products and incident characteristics for the aggregate review. In fact, responding to a question regarding the accuracy of ED toxic exposures represented in the original article, Pediatrics recently issued a retraction and corrected the key table depicting ED toxicity to reflect that the cited numbers represented exposures (with or without associated adverse effects) rather than poisonings involving EDs (Seifert & et al., 2011). An ongoing and significant challenge in assessing the safety of products generically depicted as CEDS in various venues relates to how these products are defined in the marketplace as well as by the FDA. Recognizing this as an issue-creating confusion in the marketplace and application of regulatory labeling requirements, the FDA published guidance on the subject in the 2014 “Guidance for Industry” (Guidance for Industry). In this guidance, FDA distinguished between caffeinated dietary supplement products as opposed to caffeinated beverages, two product categories often mistakenly considered synonymous in adverse event analysis. Caffeinated products, such as “energy shots,” and various concentrated powdered or liquid products for reconstitution were distinguished from ready to consume beverage versions of products that include popular brands such

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as Red Bull, Monster, Rock Star, and others. Without this distinction, an objective evaluation of which products and formulations may be more likely to produce adverse effects under routine circumstances of use cannot be made. These distinctions are important. In recent years, many of the subsequent publications describing the potential for CEDS product adverse events cite the original Seifert paper published in 2011 where a presumption of CEDS involvement and resulting adverse effects was made. These data continue to be cited as evidence of CEDS-related injury and may bias objective investigation regarding which, if any, products pose a clinical risk of injury and under what circumstances or patterns of use. There are other challenges in assessing the safety or toxicity of products generically characterized as CEDS. In circumstances where evaluation of patients with adverse effects conducted in emergency departments reveals a history of energy drink consumption, rarely are the exposures confirmed by laboratory analysis of caffeine blood concentrations where exposure is confirmed and/or exposure quantity estimated. Not only is true exposure quantity unknown, but exact caffeine content of products involved can be significantly inaccurate. Recognizing the limitations of current CEDS, adverse event evaluations can lead to more robust investigations and a more thoughtful discussion related to both the safety and potential toxicity of caffeine exposure involving all classes of caffeinated products.

Conclusion Since their introduction in 1997, CEDS have become one of the most popular and widely consumed classes of caffeinated products in the United States. Although beverage and shot versions of these products are frequently lumped together in publication and media communications, the two versions can be significantly different in ingredient mix and caffeine content or concentration. Thus, not all products characterized as CEDS are created equal. What many consumers and health-care professionals often forget is that many of the beverage versions of CEDS are much like coffee, tea, or conventional sodas and often contain less caffeine than conventional coffees available at most coffee shops. Excluding specialty coffees brewed to contain high caffeine content, shot versions of CEDS often contain higher caffeine concentrations than conventional caffeinecontaining beverages. Despite intermittent case reports of adverse health effects linked to energy drinks as a class of product, current prospective evidence suggests that beverage versions of these products are safe when consumed sensibly. Furthermore, the American Beverage Association currently requires their member companies to adhere to a code of practices that require labeling of caffeine content from all sources, discourage rapid consumption, and prohibit marketing of their representative beverage versions of these products to children (ABA). Energy shots, microshots, and beverage enhancers, however, may pose a greater health risk than conventional beverage versions of energy drinks simply because they contain higher caffeine content in a smaller volume. Prospective studies that differentiate between the various versions of CEDS may help better define those products that may pose a high risk of adverse effects.

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Further Reading Cole, J., 1833. One the deleterious effects produced by drinking tea and coffee in excessive quantities. Lancet 2, 274e278. Ferreira, S.E., de Mello, M.T., Pompe´ia, S., de Souza-Formigoni, M.L.O., 2006. Effects of energy drink ingestion on alcohol intoxication. Alcohol. Clin. Exp. Res. 30, 598e605.

27

An Overview on the Constituents and Safety of Energy Beverages John P. Higgins1, 2, 3, 4, Karan Bhatti1 1

THE UNIVERSITY OF T EXAS HEALTH SCIENCE CENTER AT HOUSTON (UTHEALTH), HOUSTON, T X, UNIT ED STATE S ; 2 ME MORIAL HERM ANN IRONMAN SPORTS MEDICINE INSTITUTE, HOUSTON, TX, UNITED STATES; 3 LYNDON B . JOHNSON GENE RAL HOSPITAL, HOUSTON, TX, UNI TED STATES; 4 HEARTS ( HOUSTON EARL Y AGE RISK TESTING & SCREENING STUDY), HOUSTON, TX, UNITED STATES

Introduction Energy drinks were first introduced to the retail market in 1987 and have since grown into a multibillion-dollar industry (Higgins et al., 2010). They are frequently consumed by young adults, teenagers, office workers, those in the military, and athletes (Nowak and Jasionowski, 2016). Currently hundreds of different brands of energy drinks are marketed with caffeine content ranging from 50 to 505 mg per serving (Higgins et al., 2010). They are frequently marketed to improve stamina, concentration, athletic performance, endurance, and weight loss (Souza et al., 2016). However, well-conducted studies reveal conflicting results with respect to their abilities to improve physical stamina or academic performance (Champlin et al., 2016). Energy drinks contain a wide variety of active ingredients including vitamins, sugar, and herbal extracts, and contain elevated concentrations of caffeine (Higgins et al., 2010). Because of the lack of safety and efficacy trials required by the Food and Drug Administration (FDA), there has been no consistent scientific evidence to suggest that they boost endurance, stamina, and mental performance (Ibrahim and Iftikhar, 2014). However, multiple reports have shown that energy drinks have been associated with a variety of extracardiac and cardiac complications, particularly in susceptible individuals (Hampton, 2016; Higgins et al., 2015). Nonetheless, no industry standard currently or fully regulates energy drink contents, and many are not required to adhere to stringent guidelines by the FDA because they are classified as dietary supplements (Higgins and Babu, 2013).

Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00027-2 Copyright © 2017 Elsevier Inc. All rights reserved.

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Epidemiology The energy drink market was worth 30 billion United States dollars (USD) globally in 2011 and is estimated to reach 61 billion USD by 2021 (PRNewswire, 2015). Approximately 6% of the young adult and adolescent population consumes energy drinks on a daily basis, and an even greater number of adolescent athletes, 17%, consume them daily (Nowak and Jasionowski, 2016; Terry-McElrath et al., 2014). Furthermore, among those aged 11e35 years, 24e57% of the population reported that they drank an energy drink in the past few months (Ballard et al., 2010). Approximately 45% of the United States overseas troops consume energy drinks daily (Toblin et al., 2012; Sepkowitz, 2013). In addition, energy drinks have been, and continue to be, marketed to children and adolescents (From the American Academy of Pediatrics, 2011).

Constituents The most common ingredient currently found in energy drinks is caffeine (Higgins et al., 2010). Caffeine content varies among products, from large amounts of caffeine found in limited volume (energy shots) to similar amounts of caffeine in 8- or 16-fl oz cans or bottles (Higgins et al., 2010). The caffeine content in energy drinks frequently exceeds the recommended FDA-imposed limit of caffeine for soda by 6- to 12-fold (71 mg of caffeine per 12 fl oz or 0.2 mg of caffeine per milliliter (Attipoe et al., 2016; Seifert et al., 2011). Caffeine is usually found in two forms in energy drinks: from synthetic and natural sources (ACOG CommitteeOpinion No. 462, 2010). Caffeine is frequently added as a synthetic alkaloid rather than the naturally occurring constituent such as in tea or coffee (Higgins et al., 2010). Furthermore, many energy drinks fail to account for the natural caffeine contributed by “energy blend” ingredients (guarana, kola nut, or yerba mate) (Goldfarb et al., 2014; Heckman et al., 2010). Caffeine is a methylxanthine that is absorbed rapidly and completely by the body (Higgins et al., 2010). As an adenosine receptor antagonist it is a stimulant that can affect the activity of the central and peripheral nervous system. It generally reaches peak concentrations within 30e120 min of ingestion (Benowitz, 1990; Higgins and Babu, 2013). Caffeine is primarily metabolized in the liver by CYP1A2 to a number of physiologically active metabolites such as theobromine and theophylline (Schimpl et al., 2014). It has been shown that consuming more than 6 mg/kg caffeine saturates the hepatic caffeine metabolism (Benowitz, 1990). Adverse effects of caffeine typically occur at doses greater than 200e400 mg and include insomnia, nervousness, tachycardia, headache, nausea, and arrhythmia (Higgins and Babu, 2013). However significant individual variability exists in caffeine sensitivity (Benowitz, 1990). Other common ingredients found in energy drinks include niacin, taurine, pyridoxine, cyanocobalamin (vitamin B12), riboflavin (vitamin B2), pantothenic acid (vitamin B5),

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ginseng extract, ginkgo biloba extract, inositol, guarana, ephedra, yohimbine, kola nut, L-carnitine, theophylline, sugars, vitamins, and herbs (Higgins et al., 2015). The health effects of these ingredients individually or in combination have been poorly researched (Higgins et al., 2015). Caffeine and taurine are the two primary performance-enhancing ingredients found in energy drinks (Peveler et al., 2016). Interestingly, a metaanalysis evaluating the effects of energy drinks on muscle strength and endurance demonstrated a significant association between taurine dosage (mg) and performance (slope ¼ 0.0001; P ¼ .04), but not between caffeine dosage (mg) and performance (Souza et al., 2016). Furthermore, there is limited evidence to suggest that ingredients other than caffeine and taurine contribute to performance enhancement (McLellan and Lieberman, 2012). Importantly, a role for other active constituents, such as glucuronolactone, cannot be ruled out (Grasser et al., 2016). In addition, there is research that describes adverse events from these additives. Herbal additives in energy drinks have been associated with adverse effects, and many of the herbal compounds have been poorly investigated with regard to their safety profile (Higgins et al., 2010; McLellan and Lieberman, 2012). Finally, there has been concern about these additives and their interactions with caffeine, especially taurine and caffeine, which may potentially result in endothelial dysfunction, increased platelet aggregation, and vasospasm (Grasser et al., 2016; Higgins, 2013). A number of adverse effects are associated with energy drink consumption (Higgins et al., 2010; Higgins et al., 2015; Sanchis-Gomar et al., 2016; Wolk et al., 2012). Of note, most of these reports have been case reports and studies. We have divided these effects into extracardiac and cardiac effects (Table 27.1). Table 27.1

Summary of Adverse Effects Associated With Energy Drink Consumption

Noncardiac Neurologic Gastrointestinal Renal Endocrine Psychiatric

Epileptic seizures, stroke, subarachnoid hemorrhage, pontine myelinolysis, hallucinations, anxiety, agitation, headaches including migraine headaches Hepatitis, gastrointestinal upset Acute renal failure, rhabdomyolysis, metabolic acidosis Insulin resistance, obesity Acute psychosis, insomnia, high-risk behavior, aggressive behavior, caffeine withdrawal

Cardiac Vascular wall effects Hemodynamics Electrical effects Coronary disease Heart muscle

Endothelial dysfunction, aortic dissection Hypertension, tachycardia, postural orthostatic tachycardia syndrome Prolonged QT interval, supraventricular arrhythmia, ventricular tachycardia Coronary vasospasm, coronary artery thrombosis, coronary artery dissection, ST elevation, myocardial infarction, sudden cardiac death Takotsubo cardiomyopathy

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Extracardiac Effects Neurologic Many case reports have noted adverse neurologic effects in association with excessive energy drink consumption (Higgins et al., 2010; Wolk et al., 2012). These include epileptic seizures and generalized toniceclonic events (Iyadurai and Chung, 2007), reversible cerebral vasoconstriction, and intracerebral hemorrhage (Butragueno Laiseca et al., 2016; Samanta, 2015; Worrall et al., 2005).

Gastrointestinal Self-reported symptoms associated with energy drink consumption from emergency room visits indicate that about 6% of patients experience gastrointestinal upset with consumption, likely related to the emetic effects of caffeine (Nordt et al., 2012). Two cases have been reported in which patients developed elevated transaminases and jaundice after heavy energy drink consumption with suspected hepatitis; one of these patients had previously undergone orthotropic liver transplantation (Apestegui et al., 2011; Vivekanandarajah et al., 2011).

Renal Acute renal failure, rhabdomyolysis, and metabolic acidosis have been described in association with energy drink consumption (Greene et al., 2014; Wolk et al., 2012).

Endocrine Obesity is associated with energy drink consumption, because of the caloric content; a usual can of energy beverage contains 54e62 g carbohydrates, usually sucrose, high-fructose corn syrup, and/or glucose (BDJ, 2015; Higgins et al., 2010). In addition, the endocrine impacts of acute caffeine consumption include hyperinsulinemia and approximately a 30% decline in whole-body insulin sensitivity (Shearer, 2014; Shearer and Graham, 2014).

Psychiatric Acute psychosis has also been reported in the setting of energy drink use (Cerimele et al., 2010). Compared with caffeine users, adolescents and young adults who consumed energy drinks were more likely to report mind racing, restlessness or jitteriness, and trouble sleeping (Higgins et al., 2010). In addition, energy drink users were more likely to report indulging in risk-taking behaviors, including risky driving behaviors (e.g., fast driving and seat belt omission), sexual risk taking, tobacco use, marijuana use, psychedelic drug use, cocaine use, alcohol/binge drinking, other illegal drug use, mixing alcohol and energy drinks, and nonmedical use of prescription stimulants (Arria et al., 2014; Cofini et al., 2016; Miyake and Marmorstein, 2015). Energy drinks may serve as a

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gateway to other forms of drug dependence (Barrense-Dias et al., 2016; Reissig et al., 2009). Energy drinks are often combined with alcohol, and young adults who mix alcohol with energy drinks consume more alcohol and experience more related harm than do others who drink energy beverages (Holubcikova et al., 2016; McKetin et al., 2015).

Cardiac Effects Vascular Wall Effects Endothelial Dysfunction Endothelial cells form the inner lining of blood vessels; they have basal and inducible metabolic/synthetic functions that allow them to carry out multiple tasks (Deanfield et al., 2007). Endothelial cells have a key role in regulating vascular resistance, clotting blood, inducing inflammation, and providing a barrier function (Blanch et al., 2015). Endothelial function is seen as a barometer of vascular health, and abnormal endothelial cell function, termed “endothelial dysfunction,” is associated with vasoconstriction, poor vascular reactivity, prothrombosis, proadhesion, proinflammation, and growth promotion (Higgins et al., 2015; Veerasamy et al., 2015). Chronic endothelial dysfunction has been associated with coronary artery disease, cerebrovascular disease, and peripheral arterial disease (Veerasamy et al., 2015). In healthy individuals who consume caffeine and then exercise afterward, significant reductions in myocardial blood flow have been noted by indirect laboratory measures (Higgins and Babu, 2013). Caffeine has been shown to block adenosine receptors that modulate coronary vasomotor tone (Higgins and Babu, 2013). This effect is often more pronounced in caffeine-naive individuals or those who ingest higher doses of caffeine, such as are present in energy drinks. Most energy drinks also contain guarana, a rainforest vine with seeds that contain high levels of naturally occurring caffeine (Higgins et al., 2010). The immediate consumption of energy drinks has been shown to worsen reactive hyperemia, shown through the measurement of flow-mediated dilatation (Higgins and Ortiz, 2014; Higgins et al., 2014). A study of 11 healthy medical students [average age 24.5 years, average body mass index (BMI) 22.8 kg/m2], underwent baseline testing of endothelial function using the technique of endothelium-dependent flowmediated dilatation (Higgins et al., 2014). The subjects then consumed a 24-oz can of Monster energy drink and the protocol was repeated at 90 min after consumption. At 90 min of consumption of the energy drink, there was a significantly attenuated peak flow-mediated dilatation response (mean  standard deviation): baseline group 5.9%  4.6% versus energy drink group 1.9%  21%; P ¼ .03. Thus, acute exposure to an energy drink was shown to impair arterial endothelial dysfunction in heathy young individuals (Higgins et al., 2014). In addition, energy drink consumption has been shown to increase platelet aggregation acutely in healthy young adults (Pommerening et al., 2015). One study after

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consumption of 16 oz of a sugar-free energy drink (Monster Zero) or water in 32 healthy young doctors in training noted a significant increase in platelet aggregation activity 1 h after the energy drink (Pommerening et al., 2015). Another study of 34 young healthy males, mean age 22 years, noted a significant increase in platelet aggregation 1 h after they consumed 250 mL of a sugar-free energy drink (Worthley et al., 2010). A case report described an otherwise healthy 28-year-old motocross racing man who had a cardiac arrest after a day of consuming an excessive amount (8 cans) of an energy drink (Berger and Alford, 2009). He was noted to have severe coronary vasospasm, which suggested that excessive ingestion of caffeine and taurine contained in the energy drinks coupled with strenuous physical activity may produce myocardial ischemia by inducing coronary artery vasospasm (Berger and Alford, 2009). Reduced coronary blood flow may be a symptom of endothelial dysfunction that affects the ability of the endothelium to regulate vascular resistance (Veerasamy et al., 2015). This acute endothelial dysfunction can lead to ischemia resulting in serious arrhythmia, coronary artery vasospasm, and myocardial infarction (Higgins and Ortiz, 2014; Veerasamy et al., 2015).

Aortic Dissection The sympathetic surge that occurs in the body after consumption of energy drinks increases both pulse rate and blood pressure (Verster et al., 2016). This has been associated with precipitation of aortic dissection in individuals with an underlying predisposition (Higgins et al., 2010; Silverio et al., 2015). Patients with existing weakness of the aortic media (such as Marfan syndrome and bicuspid aortic valve) or other cardiovascular diseases have an increased likelihood of having an acute aortic dissection. These patients are susceptible to complications arising from acute rises in blood pressure and cardiac hemodynamics (Humphrey et al., 2015). Aortic dissection has been associated with consumption of a significant amount of energy drinks (400 mg caffeine and 5000 mg taurine), likely precipitated by increased hemodynamic stress resulting from elevations in heart rate, blood pressure, and cardiac contractility (Higgins et al., 2015).

Hemodynamics Elevated Blood Pressure Multiple studies have associated the consumption of energy drinks with acute hypertension (Grasser et al., 2015, 2016). This is consistent with known hemodynamic changes caused by caffeine consumption (Higgins and Babu, 2013; Higgins et al., 2010). Caffeine consumption can increase plasma renin and catecholamines (e.g., norepinephrine and dopamine) (Robertson et al., 1978; Svatikova et al., 2015). These substances stimulate the central nervous system, thereby increasing blood pressure and heart rate (Heckman et al., 2010). For example, norepinephrine individually increases alertness, enhances memory, focuses attention, and increases heart rate and blood pressure

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(Svatikova et al., 2015). Furthermore, synergistic effects between the various components in energy drinks affect blood pressure (Grasser et al., 2014). Studies have shown that the effects of caffeine on hemodynamics can last up to 5 h after ingestion (Donovan and DeVane, 2001). These effects might be amplified by strenuous activity after consumption (Baum and Weiss, 2001; Papaioannou et al., 2006). At 1e2 h after the consumption of energy drinks, healthy individuals typically have increases in blood pressure of 6e10 mmHg systolic and 3e6 mmHg diastolic (Grasser et al., 2014; Higgins et al., 2015; Shah et al., 2016). A study of 25 young, nonobese, healthy subjects (12 women, mean age 22.5 years, mean BMI 23.3 kg/m2) who had cardiovascular measurements performed before and 2 h after the ingestion of either 355 mL of the energy drink or 355 mL of tap water showed an overall negative hemodynamic profile in response to ingestion of Red Bull (Grasser et al., 2014). Findings included elevated blood pressure, increased double product (systolic blood pressure  heart rate), and lower cerebral blood flow velocity. Compared with baseline values, energy drink ingestion led to increases in both systolic blood pressure and diastolic blood pressure of 5.2 and 6.1 mmHg, respectively (Grasser et al., 2014). Taurine, a free-form amino acid commonly found in high quantities in energy drinks, may have a synergistic effect with caffeine on heart contractility. This was demonstrated in a double-blind crossover study: 13 conditioned athletes performed exhaustive endurance exercise at three different times (Baum and Weiss, 2001). Before exercise, they ingested Red Bull energy drink, a similar caffeine-only drink (without taurine), or placebo. Echocardiography was done before ingestion, before exercise, 40 min after ingestion, and in the recovery period. Stroke volume was significantly increased in only the Red Bull energy drink group in the recovery period, as a result of reduced end-systolic volume. This indicated that the consumption of Red Bull energy drink containing caffeine and taurine increased cardiac contractility significantly more than did the caffeine-only drink or placebo (Baum and Weiss, 2001). Furthermore energy drink consumption with caffeine and taurine has been shown by cardiac magnetic resonance imaging to increase peak systolic strain 1 h after consumption (Doerner et al., 2015). For healthy adults, it is unlikely that short-term exposure to minor fluctuations in blood pressure will cause significant side effects, and blood pressure usually returns to normal after cessation of energy drinks (Garcia et al., 2016; Usman and Jawaid, 2012). However in patients undergoing treatment for hypertension and who have complications owing to hypertension, elevations in blood pressure caused by energy drinks may have significant consequences (Higgins et al., 2015). It is recommended that in this patient group, individuals avoid consuming energy drinks (Higgins et al., 2010). Finally, there have been case reports of otherwise healthy children developing hypertension as a result of habitual energy drink consumption (Usman and Jawaid, 2012). More research is needed on the effects of chronic exposure to energy drinks (Garcia et al., 2016).

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Increased Heart Rate Acute consumption of energy drinks has been associated with small but significant increases in heart rate (Higgins et al., 2015). Studies have shown that heart rate in healthy individuals increased by 7.8% on day 1 of energy drink consumption and 11.8% by day 7 (Steinke et al., 2009). On average the heart rate in healthy individuals 1e2 h after consumption of energy drink increases by 3e7 beats per minute (Higgins et al., 2015).

Postural Orthostatic Tachycardia Syndrome Postural orthostatic tachycardia syndrome is a form of dysautonomia that is associated with the presence of excessive tachycardia upon standing (Dahan et al., 2016). This syndrome can be seen on rare occasions in normal healthy individuals who consume multiple energy drink cans a day (Terlizzi et al., 2008). Regarding this association, taurine and caffeine found in energy drinks can directly or indirectly affect cardiovascular function (Terlizzi et al., 2008). Taurine in particular is found in high concentrations in the brain; it has the ability to interfere with cardiovascular regulation in both experimental animals and humans, which might explain the adverse effect on autonomic function seen in these cases (Alford et al., 2001; Baum and Weiss, 2001; Bichler et al., 2006; Huxtable, 1992; Yang and Lin, 1983).

Electrical Effects Increased Corrected QT Interval An alarming concern regarding energy drink consumption is the increased QT-corrected (QTc) interval in young healthy individuals and those with preexisting genetic long QT syndrome (Higgins et al., 2015). The various ingredients in energy drinks, in addition to the high amounts of caffeine and taurine, work on multiple cardiac ion channels, and in certain circumstances they can be arrhythmogenic (Satoh, 2003). One study has shown that the QTc interval increased by 2.4% on day 1 and 5.0% on day 7 in healthy individuals who consumed energy drinks on a daily basis (Steinke et al., 2009). Another study in young healthy volunteers noted an increase in QTc interval of 3.4 ms 2 h after energy drink consumption (32-oz energy drink) compared with a decrease of 3.2 ms in the placebo group (P ¼ .03) (Shah et al., 2016). A case report described a young female who experienced an out-of-hospital cardiac arrest owing to torsade de pointes after consuming six cans of caffeinated energy drink within 4 h (Rottlaender et al., 2012). She was found to have type 1 long QT syndrome with KCNQ1 mutation (Rottlaender et al., 2012). QTc prolongation caused by energy drink consumption in case reports ranges from 25 to 107 ms (Higgins et al., 2015). A typical increase in QTc in normal healthy individuals 1e2 h after consumption of energy drinks is up to 22e25 ms (Higgins et al., 2015). This is of concern because the FDA (2005) suggests further testing when a change of greater than 10 ms is observed.

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Supraventricular Arrhythmia Caffeine consumption is associated with the precipitation and exacerbation of supraventricular arrhythmia (Artin et al., 2010). However the various other ingredients in energy drinks, including “energy blends” and herbs, are also potential triggers for arrhythmias (Sanchis-Gomar et al., 2015). Caffeine, taurine, and guarana all have arrhythmogenic properties (Ward et al., 2014). Atrial fibrillation, the most common arrhythmia seen in the elderly, is rare in the pediatric and young adult population (Rahman et al., 2016). When atrial fibrillation occurs in the pediatric population, it is commonly in association with underlying structural heart disease (Di Rocco et al., 2011). Nonetheless atrial fibrillation has been observed after the acute ingestion of energy drinks in young adults (Di Rocco et al., 2011; Izquierdo Fos et al., 2012). It is unknown whether these individuals have a genetic predisposition toward arrhythmia or whether ingredients in the energy drinks triggered these events (Turagam et al., 2015).

Ventricular Arrhythmia Ventricular arrhythmias such as ventricular fibrillation and ventricular tachycardia can lead to sudden cardiac arrest and death, and have been associated with energy drink consumption (Goldfarb et al., 2014). Caffeine is well known to increase circulating catecholamines in the body in a dose-dependent manner; it also causes hypokalemia and can suppress sodium channel conduction (Goldfarb et al., 2014). Furthermore, taurine in energy drinks may enhance the physiological effects of caffeine (Higgins et al., 2015). All of these factors may predispose an individual to ventricular arrhythmias. In addition, high doses of caffeine such as those found in energy drinks can exacerbate and worsen cardiac conditions, especially in individuals for whom stimulants are contraindicated (Seifert et al., 2011). This is most concerning in patients with channelopathies such as Brugada syndrome and hypertrophic cardiomyopathy (the most prevalent genetic cardiomyopathy in children and young adults). Patients with these conditions have increased hypertension, syncope, arrhythmias, and sudden cardiac death with energy drink consumption (Seifert et al., 2011). There have been published case reports in which young individuals are described to have experienced cardiac arrest owing to ventricular fibrillation and/or tachycardia after consuming large amounts of energy drinks (Higgins et al., 2015). In some of these individuals, a diagnosis of Brugada syndrome and hypertrophic cardiomyopathy was later uncovered (Goldfarb et al., 2014; Rutledge et al., 2012).

Coronary Disease Coronary Artery Spasm The high caffeine content in energy drinks is associated with competitive inhibition of the adenosine receptors, directly and indirectly resulting in a large amount of catecholamine release, causing a rapid efflux of calcium from the sarcoplasmic reticulum

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of the vascular smooth muscle cells, which can lead to coronary artery vasospasm (Holmgren et al., 2004; Scott et al., 2011). Taurine found in high quantities in energy drinks modulates calcium signaling, and toxic levels can effect both intracellular and extracellular calcium concentration (Heckman et al., 2010). Taurine combined with caffeine can lead to increased inotropy and can contribute to coronary artery vasospasm (Doerner et al., 2015; Scott et al., 2011). Cases have been reported in which patients have presented with significantly elevated troponins and ST segment elevations after consuming large amounts of energy drinks (Higgins et al., 2015). Workup for these patients revealed normal coronary angiograms, yet high levels of taurine and caffeine in the blood (Scott et al., 2011). Coronary artery vasospasm is typically associated with high levels of caffeine (400e800 mg) as well as taurine (2000e8000 mg) (Higgins et al., 2015).

Sudden Cardiac Death There are multiple case reports that described sudden cardiac death associated with or triggered by energy drink consumption in exercising individuals (Goldfarb et al., 2014). Sudden death can occur because caffeine reduces coronary artery flow reserve during exercise and increases myocardial oxygen demand (Higgins and Babu, 2013). Thus consuming energy drinks before exercise can decrease myocardial oxygen supply while exercise causes an increase in myocardial oxygen demand, resulting in classic supplyedemand mismatch (Higgins et al., 2015). Consequently this can lead to myocardial ischemia as a result of decreased oxygen supply to the heart muscle, resulting in ventricular arrhythmias and cardiovascular collapse (Berger and Alford, 2009; Higgins and Babu, 2013; Higgins and Ortiz, 2014; Higgins et al., 2014; Worthley et al., 2010). Although multiple confounding factors can lead to such events, individuals susceptible to the effects of energy drinks should avoid consumption until more safety data can be established (Higgins et al., 2010).

Myocardial Ischemia Myocardial ischemia has been associated with acute consumption of energy drinks. Possible mechanisms include increased myocardial oxygen demand (faster heart rate and raised blood pressure), reduced myocardial oxygen supply (endothelial dysfunction and vasospasm), sheer stress, and hypercoagulability (Lippi et al., 2016). Other cardiac complications related to energy drink consumptions have included spontaneous coronary artery dissection, ST-segment elevation myocardial infarction, and coronary artery thrombosis. Coronary artery thrombosis is likely related to hypercoagulability, endothelial dysfunction, and elevated norepinephrine levels (Benjo et al., 2012; Solomin et al., 2015).

Heart Muscle Disease Takotsubo (Stress) Cardiomyopathy Energy drinks contain multiple sympathomimetic substances such as caffeine (Higgins et al., 2015). Caffeine induces catecholamine release and causes a rise in myocyte

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intracellular calcium (Doerner et al., 2015; Higgins and Babu, 2013). Because energy drinks are consumed rapidly and sometimes in excessive amounts, this can predispose individuals to a large surge in catecholamines (Reissig et al., 2009). Furthermore caffeine is a competitive antagonist of adenosine receptors A1 and A2A in the central nervous system and myocardium, which alters neurotransmitter release and increases heart rate (Higgins and Babu, 2013). These factors can predispose individuals to Takotsubo cardiomyopathy, a well-recognized source of heart failure, lethal ventricular arrhythmias, and ventricular rupture (Higgins et al., 2015).

Effects in Specific Populations The health concerns regarding energy drinks are further highlighted in the adolescent population (Barrense-Dias et al., 2016; Hampton, 2016). Energy drink use is rapidly growing in this population (From the American Academy of Pediatrics, 2011; Terry-McElrath et al., 2014). Adverse effects can be exacerbated in this population because of the higher concentrations of caffeine in relation to body mass (Higgins and Babu, 2013). These concerns have led to the American Academy of Pediatrics recommending that energy drinks not be consumed by the adolescent population or used for hydration before, during, or after physical activity (NFHS and SMAC, 2014). In addition, because of their caffeine content as well as other herbal constituents that have not been fully evaluated, energy drinks should be avoided during pregnancy as well as by breastfeeding mothers (Thorlton et al., 2016).

Long-Term Effects There has been no significant research to establish the long-term effects of exposure to energy drinks and their possible complications (Higgins et al., 2015). However because of the multiple acute effects, one can postulate potential long-term effects. Chronic elevation of blood pressure over years can predispose individuals to coronary artery disease, cerebrovascular events, and renal dysfunction (Higgins et al., 2015). It is known that atherosclerosis occurs in the intima of medium-sized arteries at regions where blood flow is disturbed and triggered by an intricate interplay between endothelial dysfunction and subendothelial lipoprotein retention (Tabas et al., 2015). Chronic endothelial dysfunction can lead to increased risk for development of coronary artery disease and events, peripheral arterial disease, and increased risk for cerebrovascular accident (Higgins et al., 2015). In addition, consumption of energy drinks leads to hyperglycemia as well as an increase in total cholesterol, triglycerides, and low-density lipoprotein cholesterol, which are known to be cardiovascular risk factors (Lippi et al., 2016). Further research and data need to be obtained to establish the long-term safety of energy drink consumption.

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Conclusions Multiple noncardiovascular and cardiovascular adverse events are associated with energy drink consumption. These adverse events seem to be more common in certain vulnerable populations, including those aged under 18 years, pregnant or breastfeeding women, caffeine-naive or sensitive individuals, individuals taking stimulant or other caffeine-based medications, and those with certain cardiovascular or medical conditions and/or heavy consumption patterns (two or more energy drinks in one session). Energy drinks should not be consumed by children or adolescents and should not be used before, during, or after strenuous activity. Energy drink consumption should be avoided in individuals with existing medical conditions, especially cardiac conditions. More research on their safety and efficacy is required to determine what, if any, is a safe dose for consumption and who should avoid them. Finally, more randomized studies are required to determine whether energy drinks truly improve physical stamina and mental performance, as they purport in their advertisements.

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Benowitz, N.L., 1990. Clinical pharmacology of caffeine [Research Support, U.S. Gov’t, P.H.S. Review] Annu. Rev. Med. 41, 277e288. http://dx.doi.org/10.1146/annurev.me.41.020190.001425. Berger, A.J., Alford, K., 2009. Cardiac arrest in a young man following excess consumption of caffeinated “energy drinks” [Case Reports]. Med. J. Aust. 190 (1), 41e43. Bichler, A., Swenson, A., Harris, M.A., 2006. A combination of caffeine and taurine has no effect on short term memory but induces changes in heart rate and mean arterial blood pressure [Randomized Controlled Trial] Amino Acids 31 (4), 471e476. http://dx.doi.org/10.1007/s00726-005-0302-x. Blanch, N., Clifton, P.M., Keogh, J.B., 2015. A systematic review of vascular and endothelial function: effects of fruit, vegetable and potassium intake. Nutr. Metab. Cardiovasc. Dis. 25 (3), 253e266. http:// dx.doi.org/10.1016/j.numecd.2014.10.001. Butragueno Laiseca