Advances in Plant Phenolics: From Chemistry to Human Health 9780841232969, 0841232962, 9780841232952

Table of contents :
Content: Preface Isolation, HPLC Separation, and Antioxidant Activity1. Isolation and Analysis of Antioxidant Phytochemicals from Black Chokeberry, Maqui, and Goji Berry Dietary Supplements 2. Phenolic Compounds from the Brazilian Genus Lychnophora Mart. (Asteraceae) 3. Polyphenolic Profile of the Fruits Grown in Serbia 4. Analysis of C-Glycosyl Flavones and 3-Hydroxy-3-methylglutaryl-glycosyl Derivatives in Blood Oranges (Citrus sinensis (L.) Osbeck) Juices and Their Influence on Biological Activity 5. Determination of Ellagic Acid in the Wastes of Walnut, Chestnut, and Pomegranate Grown in Turkey Mass Spectrometry, NMR, and in Vitro Activities6. Extraction, Identification, and Potential Health Benefits of SpinachFlavonoids: A Review 7. Cryo-TOF-SIMS Visualization of Water-Soluble Compounds in Plants 8. Extraction and Identification of Health-Promoting Phytochemicals from Brussels Sprouts 9. Expanding Human Blood Metabolomics to the Analysis of Coenzymes and Antioxidants Using NMR Spectroscopy 10. Proanthocyanidins from Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Leaves: Structure Elucidation and Bioactive Functions11. Phenolic Compounds in Pomegranate (Punica granatum L.) and Potential Health Benefits 12. Chemical and Biological Properties of the Genus Abies Nanoencapsulation and Health Benefits of Various Phenolic Compounds13. Encapsulation of Polyphenols: An Effective Way To Enhance Their Bioavailability for Gut Health 14. Metabolic and Microbiome Innovations for Improving Phenolic Bioactives for Health 15. Xanthohumol, What a Delightful Problem Child! 16. Cooking Practice and the Matrix Effect on the Health Properties of Mediterranean Diet: A Study in Tomato Sauce 17. Garlic Grown from Air Bulbils and Its Potential Health Benefits18. Biological Activities of Phenolic Compounds from Fruit, Leaves, Heartwood, and Root of Artocarpus communis 19. Impact of Anthocyanins on Colorectal Cancer 20. Phenolic Compounds Accumulation in Wild and Domesticated Cladodes from Opuntia spp. and Its Benefits in Cardiovascular Diseases 21. Nanoencapsulation: An Advanced Nanotechnological Approach To Enhance the Biological Efficacy of Curcumin 22. Powering the Activity of Natural Phenol Compounds by Bioinspired Chemical Manipulation Editors' Biographies Author IndexSubject Index

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Advances in Plant Phenolics: From Chemistry to Human Health

ACS SYMPOSIUM SERIES 1286

Advances in Plant Phenolics: From Chemistry to Human Health Guddadarangavvanahally K. Jayaprakasha, Editor Texas A&M University College Station, Texas, United States

Bhimangouda S. Patil, Editor Texas A&M University College Station, Texas, United States

Giuseppe Gattuso, Editor University of Messina Messina, Italy

Sponsored by the ACS Division of Agricultural and Food Chemistry, Inc.

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

Library of Congress Cataloging-in-Publication Data Names: Jayaprakasha, Guddadarangavvanahally K., editor. Title: Advances in plant phenolics : from chemistry to human health / Guddadarangavvanahally K. Jayaprakasha, editor, Texas A&M University, College Station, Texas, United States, Bhimangouda S. Patil, editor, Texas A&M University, College Station, Texas, United States, Giuseppe Gattuso, editor, University of Messina, Messina, Italy ; sponsored by the ACS Division of Agricultural and Food Chemistry, Inc. Description: Washington, DC : American Chemical Society, [2018] | Series: ACS symposium series ; 1286 | Includes bibliographical references and index. Identifiers: LCCN 2018042468 (print) | LCCN 2018043650 (ebook) | ISBN 9780841232952 (ebook) | ISBN 9780841232969 (print) Subjects: LCSH: Phenols--Physiological effect. | Phytochemicals--Physiological effect. | Phenols in the body. Classification: LCC QP801.P4 (ebook) | LCC QP801.P4 A38 2018 (print) | DDC 547/.632--dc23 LC record available at https://lccn.loc.gov/2018042468

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2018 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

Contents Preface .............................................................................................................................. xi

Isolation, HPLC Separation, and Antioxidant Activity 1.

Isolation and Analysis of Antioxidant Phytochemicals from Black Chokeberry, Maqui, and Goji Berry Dietary Supplements ................................. 3 Jie Li, P. Annécie Benatrehina, Andrea L. Rague, Li Pan, A. Douglas Kinghorn, and C. Benjamin Naman

2.

Phenolic Compounds from the Brazilian Genus Lychnophora Mart. (Asteraceae) ............................................................................................................ 21 Daniel Petinatti Pavarini, Anelize Bauermeister, João Semir, Marcelo Monge, João Luís Callegari Lopes, and Norberto Peporine Lopes

3.

Polyphenolic Profile of the Fruits Grown in Serbia ............................................ 47 Živoslav Lj. Tešić, Uroš M. Gašić, and Dušanka M. Milojković-Opsenica

4.

Analysis of C-Glycosyl Flavones and 3-Hydroxy-3-methylglutaryl-glycosyl Derivatives in Blood Oranges (Citrus sinensis (L.) Osbeck) Juices and Their Influence on Biological Activity ............................................................................ 67 Davide Barreca, Ersilia Bellocco, Silvana Ficarra, Giuseppina Laganà, Antonio Galtieri, Ester Tellone, and Giuseppe Gattuso

5.

Determination of Ellagic Acid in the Wastes of Walnut, Chestnut, and Pomegranate Grown in Turkey ............................................................................ 81 G. Yalcin, C. Demirbag, I. Bahsi, L. Ozgul, D. Bilgic Alkaya, H. I. Onurlu, and S. Ayaz Seyhan

Mass Spectrometry, NMR, and in Vitro Activities 6.

Extraction, Identification, and Potential Health Benefits of Spinach Flavonoids: A Review .......................................................................................... 107 Jashbir Singh, G. K. Jayaprakasha, and Bhimanagouda S. Patil

7.

Cryo-TOF-SIMS Visualization of Water-Soluble Compounds in Plants ....... 137 D. Aoki, Y. Matsushita, and K. Fukushima

vii

8.

Extraction and Identification of Health-Promoting Phytochemicals from Brussels Sprouts ................................................................................................... 151 Haripriya Shanmugam, Guddadarangavvanahally K. Jayaprakasha, and Bhimanagouda S. Patil

9.

Expanding Human Blood Metabolomics to the Analysis of Coenzymes and Antioxidants Using NMR Spectroscopy ............................................................. 175 G. A. Nagana Gowda and Daniel Raftery

10. Proanthocyanidins from Chinese Bayberry (Myrica rubra Sieb. et Zucc.) Leaves: Structure Elucidation and Bioactive Functions .................................. 185 Yu Zhang, Shiguo Chen, Yu Fu, Haihua Yang, and Xingqian Ye 11. Phenolic Compounds in Pomegranate (Punica granatum L.) and Potential Health Benefits ..................................................................................................... 201 Muntha K. Reddy 12. Chemical and Biological Properties of the Genus Abies ................................... 225 Jinhee Kim and Eun-Jin Park

Nanoencapsulation and Health Benefits of Various Phenolic Compounds 13. Encapsulation of Polyphenols: An Effective Way To Enhance Their Bioavailability for Gut Health ............................................................................ 239 Deepak M. Kasote, G. K. Jayaprakasha, and Bhimanagouda S. Patil 14. Metabolic and Microbiome Innovations for Improving Phenolic Bioactives for Health .............................................................................................................. 261 Dipayan Sarkar and Kalidas Shetty 15. Xanthohumol, What a Delightful Problem Child! ............................................ 283 J. F. Stevens and J. S. Revel 16. Cooking Practice and the Matrix Effect on the Health Properties of Mediterranean Diet: A Study in Tomato Sauce ................................................ 305 José Fernando Rinaldi de Alvarenga, Julián Lozano-Castellón, Miriam Martínez-Huélamo, Anna Vallverdú-Queralt, and Rosa María Lamuela-Raventós 17. Garlic Grown from Air Bulbils and Its Potential Health Benefits .................. 315 Jerzy Zawistowski, Aneta Kopec, Elżbieta Jędrszczyk, Renata Francik, and Beata Bystrowska 18. Biological Activities of Phenolic Compounds from Fruit, Leaves, Heartwood, and Root of Artocarpus communis ................................................. 329 Jer-An Lin and Gow-Chin Yen 19. Impact of Anthocyanins on Colorectal Cancer ................................................. 339 Candice Mazewski and Elvira Gonzalez de Mejia

viii

20. Phenolic Compounds Accumulation in Wild and Domesticated Cladodes from Opuntia spp. and Its Benefits in Cardiovascular Diseases ...................... 371 Anne Negre-Salvayre, Françoise Guéraud, María del Socorro Santos-Díaz, and Ana Paulina Barba de la Rosa 21. Nanoencapsulation: An Advanced Nanotechnological Approach To Enhance the Biological Efficacy of Curcumin ................................................... 383 K. N. Chidambara Murthy, P. Monika, G. K. Jayaprakasha, and Bhimanagouda S. Patil 22. Powering the Activity of Natural Phenol Compounds by Bioinspired Chemical Manipulation ....................................................................................... 407 Lucia Panzella and Alessandra Napolitano Editors’ Biographies .................................................................................................... 427

Indexes Author Index ................................................................................................................ 431 Subject Index ................................................................................................................ 433

ix

Preface This ACS Symposium Series Book comes from the ACS symposium “Chemistry & Biological Activities of Phenolic Compounds from Fruits & Vegetables”, sponsored by the Division of Agricultural and Food Chemistry at the 253rd American Chemical Society National Meeting & Exposition in San Francisco, California, April 2–6, 2017. Diverse researchers in this field, including 42 scientists from 17 countries, came from all over the world to exchange ideas and results on food composition and analysis, HPLC separation, mass spectrometry, in vitro tests, in vivo studies, and human clinical trials. A world-class group of academic researchers and industrial scientists wrote the chapters published in this book to provide a state-of-the-art review and global perspective on this rapidly growing area of research. The growing number of scientific studies that have shown the ubiquitous occurrence of phenolics in plants, and their beneficial roles in human health, have driven increased public interest in the varied phenolic compounds present in fruits and vegetables since the '90s. More than 150,000 studies have appeared in the literature over this time span; this figure is doubtless an underestimate, due to the inherently heterogeneous nature of this topic, which intersects botany, chemistry, biology, pharmacology, and medicine. Epidemiological studies have shown that many phenolic derivatives possess strong antioxidant and radical scavenging activity, are associated with reduced risk for certain chronic diseases, and prevent some cardiovascular disorders and certain kinds of cancers. Moreover, polyphenols demonstrated for antiviral, antimicrobial, anti-inflammatory, antiulcer and anti-allergenic properties. The food industry, academia, and research institutions have invested considerable resources in this research. The interest in polyphenols has been also fueled by the current and growing awareness of healthy lifestyles and the beneficial effects that derive from the dietary intake of particular nutrients. As a result, the polyphenols containing nutraceutical and dietary supplements market is in high demand, with an estimated global value an about $750 million in 2015, and is expected reach up to $1.1 billion by end of 2022. The use of polyphenols as an ingredient for functional beverages in the health care, sports, and entertainment sector is one of the key factors for driving the growth of the polyphenols industry. Presently, the diverse researchers of the phenolics community invest their time and energy in a broad range of activities from the isolation and identification of new phenolic compounds from plant materials to the study of the effect and mechanism of action of known and novel phenolics in vitro and in vivo. Reflecting this diversity, the symposium covered widespread topics, with outstanding contributions ranging from isolation and identification of naturally occurring xi

bioactive compounds, to the understanding of their health benefits. This rich variety led us to organize the sessions, and the chapters of this book, in a thematic fashion following the ideal journey of a phenolic molecule from the plant to the human body: • • • • • •

Isolation, Food Composition, & Antioxidant Activity HPLC Separation, Mass Spectrometry & Antioxidant Activity Mass Spectrometry & In Vitro Biological Activities In Vitro Studies In Vivo Studies In Vivo & Human Clinical Trials

This book comprehensively describes the information presented at the ACS symposium “Chemistry & Biological Activities of Phenolic Compounds from Fruits & Vegetables”, providing a current review that will be useful for scientists, researchers, teachers, and engineers in general, and is particularly useful for chemists, biochemists, chemical engineers, biochemical engineers, and others in chemistry-related fields. The editors gratefully acknowledge all the authors for their patience, hard work, and timely contributions as well as rest of the speakers who gave oral presentations in the three-day symposium. Furthermore, we thank all the anonymous reviewers for their critical suggestions/comments that improved the chapters and made this book possible. Moreover, we thank Pratibha Acharya, Jashbir Singh, and Deepak Kasote, from VFIC, Texas A&M University, for their technical help. The editors also thank the ACS Books Division, Maryanne Rackl, Arlene Furman, Amanda Koenig, Jasmine Suarez, and Elizabeth Hernandez for their timely help in editorial editing and suggestions as well as individuals from the press, especially Mary Calvert and Pamela Kame for publishing this symposium series.

G. K. Jayaprakasha FRSC, FIC, FAGFD-ACS, FAFSTI Vegetable and Fruit Improvement Center Texas A&M University 1500 Research Parkway, A120 College Station, Texas 77845, United States

Bhimangouda S. Patil Vegetable and Fruit Improvement Center Texas A&M University 1500 Research Parkway, A120 College Station, Texas 77845, United States

xii

Giuseppe Gattuso Dipartimento di Scienze Chimiche Biologiche, Farmaceutiche ed Ambientali Università di Messina Viale F. Stagno d'Alcontres 31 98166 Messina, Italy

xiii

Isolation, HPLC Separation, and Antioxidant Activity

Chapter 1

Isolation and Analysis of Antioxidant Phytochemicals from Black Chokeberry, Maqui, and Goji Berry Dietary Supplements Jie Li,1 P. Annécie Benatrehina,1 Andrea L. Rague,1 Li Pan,1 A. Douglas Kinghorn,1 and C. Benjamin Naman*,2 1Division

of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States 2Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Ningbo University, Ningbo 315211, China *E-mail: [email protected].

The analysis of antioxidant and potentially cancer chemopreventive, or cytoprotective, phytochemicals that were isolated from berries including black chokeberry (Aronia melanocarpa), maqui (Aristotelia chilensis), and goji (Lyceum barbarum) has been reported recently. This chapter describes the research conducted, rationale, and draws connections from the summation of all three projects. For example, the re-isolation of some phytochemicals from multiple dietary supplement ingredients highlights the need for dereplication methods to be used in the course of research, yet understanding the distribution of these molecules in human dietary and supplementation ingredients remains important. The observation of low- and sub-micromolar ED50 (concentration yielding half efficacy) values for hydroxyl radical-scavenging and CD (concentration required to double activity) values for quinone-reductase inducing in vitro activities, summarized for the molecules isolated, highlights the potential health impact of phytochemicals from dietary supplement ingredients such as the three berries examined. Some of the phytochemicals described should eventually be evaluated in vivo for bioactivities, metabolism, and pharmaceutical properties, and can serve as marker compounds for chemical analysis.

© 2018 American Chemical Society

Introduction The phytochemical investigation of berries, such as black chokeberry, maqui, and goji berries, is analytical research that interfaces chemistry and biology. As such, many different research methods should be used in collaboration, and ideally with iterative feedback, to obtain optimal results. For example, the necessity of using previously identified and available standards for targeted chemical analysis of natural extracts demands continued discovery to enable such analytical procedures. In addition, the same targeted analysis can be used as a dereplication technique to preclude the inefficient “re-discovery” of known molecules, and thus improves new compound discovery. Furthermore, the discovery and analysis of small molecule natural products in foods and dietary supplements may lend evidence to anecdotal or reported health benefits of those source materials, after appropriate biological testing or assay. The same biological testing of crude and refined extracts can also efficiently guide the isolation of new or active molecules produced by the biological source, e.g., a berry. This type of interdisciplinary research has contributed for decades directly to the westernized drug repertoire and to the discovery pipeline in the form of lead molecules to be developed into drugs (1, 2). Accordingly, the detailed chemical investigation of many plants, fungi, and bacteria continues to take place in research laboratories worldwide, albeit primarily among academic institutions and small biotechnology companies since around the turn of the 21st century (3). One particularly interesting research area comprises understudied foodstuffs and plants with a history of dietary or traditional medicinal intake, for these may bear some inherent pharmacological properties and also other practical advantages for evaluation. Dietary Supplements In the United States, the use of dietary supplements for the overall well-being or improvement of health is quite popular (4). In total, the market for such products in the USA alone is estimated to be more than USD $40 billion per year (5). If one considers only the subset of products that is classified as comprising non-vitamin and non-mineral dietary supplements, this category can be termed “botanical dietary supplements”, or “herbal supplements”, and the U.S. market for retail sales of these exceeded USD $7 billion in 2016 (6). Popular botanical dietary supplements tend to include organic extracts generated from fruits and vegetables that may be well-known to the public consumer base, but many may also be considered by this population to be very exotic. For example, horehound (Marrubium vulgare) and cranberry (Vaccinium macropcarpon) are dietary supplement ingredients that can be found commonly on the shelves in retail pharmacies and health food stores, and are available online, and these two botanicals were ranked as the top-selling supplements in 2016 in the U.S. mainstream multi-outlet sales channel (6). Of course, the typical consumer is likely much more familiar with the dietary fruit, cranberry, than the medicinal plant, horehound. It is noteworthy that although, as is the case for horehound, some dietary supplement ingredients have a history of use in traditional medicines and current uses elsewhere in the world as “herbal remedies”, the U.S. Dietary 4

Supplements Health and Education Act of 1994 established regulations defining dietary supplements in America as being only for nutritional and preventive health purposes (7). However, a frequent consumer perception of general or specific health benefits from intake of antioxidants and other adaptogens has popularized many products, as have some disallowed health benefit claims made in “grass-roots” or direct advertising campaigns (6). There is growing evidence that some dietary supplements may be functional cancer chemopreventive agents, although much controversial debate surrounds this topic. Cancer Chemoprevention Cancer chemoprevention defines the prolonged use of chemical agents, in either pure form or in mixtures, to reduce the risk or diminish the occurrence of developing cancers (8). Mechanistically, this action can occur through a number of pathways, including the scavenging of free radicals or distribution of antioxidants in the system, the inhibition of phase I metabolic enzymes, or the induction of phase II metabolic enzymes, etc. (9) A common criticism of this research is the often overlooked or underappreciated aspect of bioavailability, pharmacokinetic properties, and metabolic fate of the chemicals being investigated. However, the clinical investigation and use of anti-inflammatory agents, antioxidants, and other chemopreventive agents have been reported (10, 11). Since such molecules need to have safe long-term use profiles, as well as a sustainable and affordable supply, many researchers have investigated phytochemicals from plants with a history of human dietary use, especially vegetables and fruits (12–14). A subgroup of these foodstuffs has emerged and attracted a considerable amount of research, leading to increased public attention, and this category comprises edible berries (15). Berries The crude extracts and purified phytochemicals of many berries have been studied chemically and biologically, for the associated health benefits, and have been broadly described by the international research community (15, 16). Due to the safety profile and typically high antioxidant potential of many berry extracts, and the important role that reactive oxygen species (ROS) play in many diseases including cancers, there have been many laboratory and clinical studies of berry extracts and phytochemicals related to cancer chemoprevention (17). Also, relevant animal models that involve the chemical induction of cancer in rodents have been developed to facilitate the more rudimentary investigation of cancer chemopreventive agents. In one such investigation, it was shown that while “control” rats treated with N-nitrosomethylbenzylamine developed esophageal cancer, “test” rats treated additionally with oral dietary intake of the extracts produced from each of several berries, including black raspberries, blueberries, goji berries, and noni, had significantly reduced tumorigenesis (18). However, the detailed phytochemical investigations of many important berries have yet to be completed, and the research conducted on three such samples of black chokeberry (Aronia melanocarpa), maqui (Aristotelia chilensis) berry, and goji (Lyceum barbarum) berry has been described recently and reviewed herein (19–22). In the 5

case of goji berry, several reports of phytochemical investigations do exist in the literature, and yet more and more meaningful results continue to emerge from the investigation of this berry (20, 23–25).

Bioassay-Guided Isolation of Berry Phytochemicals Bioassay-guided fractionation is a popular technique for the isolation and discovery of natural products. This method includes by design the repeated partitioning or chromatographic separation of crude natural product mixtures in connection with the testing of these fractions in one or more biological assay system. Fractions that are determined to be active return to the iterative cycle, whereas inactive fractions are discarded, or, preferably, set aside for study in alternative research projects (26–28). Thus, the detailed phytochemical investigation of plant extracts, berry fruits, and other natural product mixtures can be conducted in a manner that can be considered as being both “blind a priori” and also rational in experimental design and execution. Among the most significant mediating factors for the success of bioassay-guided fractionation research are the selection of one or more appropriate biological tests that are both relevant to the goal of the research program and are amenable to often complex mixtures of natural products, the use of effective but not disruptive (e.g. causing significant sample loss or degradation) chromatographic media as well as extraction and separation methods, and access to understudied or otherwise advantageous source or starting materials for investigation (26–29). Dietary supplement ingredients and berry products may be particularly interesting for study due to the extent of their human use and distribution, the large-scale commercial availability of many of these, and the striking fact that many such samples remain understudied by natural products researchers and other analytical scientists.

Sample Materials: Black Chokeberry, Maqui, and Goji Berries The literature shows that many berries have been previously examined with detailed analytical and phytochemical isolation experiments. Prior to the initiation of the described studies, three samples were selected for in-depth research because these had not been thoroughly investigated yet, they were used as direct dietary or supplemental ingredients, and some evidence existed of a history of their use in systems of traditional medicine. These were black chokeberry (Aronia melanocarpa), maqui (Aristotelia chilensis) berry, and African mango (Irvingia gabonensis), although the material procured for the lattermost project was eventually determined to be or to contain significant amounts of goji (Lyceum barbarum) berry (19–22). While the research manuscript conservatively reported “evidence of contamination, adulteration, and/or mislabeling” of the commercial sample investigated, which had been ordered and fulfilled as being African mango without any certification of authenticity being issued with the product, this sample is accordingly described throughout this chapter simply as being goji berry. 6

Selection of Bioassays The selection of an appropriate bioassay is paramount to leading subsequently to the isolation of molecules with the same activity being tested along the way. For the case of chemopreventive agents, there are numerous biological pathways and macromolecules that could be selected. However, it has been reported in the past that many phase II cellular detoxification enzymes are co-regulated (8). Furthermore, the enzyme quinone reductase, which initiates the conversion of quinones to semiquinones and hydroquinones, or quinols, has been reported as being a “reasonable biomarker for the potential chemoprotective effect of test agents against cancer initiation (8).” Accordingly, the quinone reductase induction bioassay was selected for sample testing in the work described here. This in vitro test was conducted using murine Hepa1c1c7 cells according to published protocols, and l-sulforaphane served as a positive control standard (30, 31). The quinone reductase induction assay was performed to test various concentrations of samples and calculate a CD value for each fraction and phytochemical, which corresponds to the concentration that would double the activity of quinone reductase in the experiment. In addition, due to the earlier-mentioned potential relevance of antioxidants for cancer chemoprevention, a convenient assay was also used to determine the hydroxyl radical-scavenging activity of fractions and purified phytochemicals (10, 11). This chemical assay measures the hydrogen peroxide-mediated radical oxidation of dichlorofluorescin (DCFH) to the fluorescent product dichlorofluorescein (DCF), has been well established and extensively used, and was conducted according to a method described previously (32, 33). Furthermore, quercetin was selected for use as a typical positive control standard in this assay (32, 33). The hydroxyl radical-scavenging assay was performed to test various concentrations of samples and calculate an ED50 value for each fraction and phytochemical, which corresponds to the concentration that would scavenge 50% of the hydroxyl radicals generated in the experiment. Extraction Methods A wide variety of methods exist today for the extraction of phytochemicals from berry fruits. Many of these methods have been described in a detailed set of review articles on natural product isolation (26, 27). For the extraction of berry phytochemicals in the presented work, a room temperature and atmospheric pressure methanolic solvent percolation was selected due to its affordability and convenience, as well as safety and phytochemical stability. Following solvent extraction and evaporation under vacuum, the crude extracts were suspended in a hydromethanolic mixture and further processed by sequential liquid-liquid extraction using organic solvents of increasing polarity starting with hexanes and proceeding to chloroform, ethyl acetate, and n-butanol to produce crude fractions from these partitions, and the remaining aqueous material. This procedure represents an augmentation of the “Wall-modified Kupchan partitioning scheme”, and generated extracts of crudely different composition, with clearly understandable polarity, or lipophilicity, by which to inform later chromatography experiments, as well as sequestering “nuisance” tannins into a 7

fraction to be tested separately, if desired (34). Each partition was then dried under vacuum and assayed for sample prioritization to take forward in other experiments. For example, the partitioning shown in Figure 1 demonstrates the sample processing that was conducted during the phytochemical investigation of black chokeberry (Aronia melanocarpa) by some of the coauthors of the present contribution (19, 21). It is quite noteworthy that, in this example and as for many other cases, although the crude extract was not observed to be bioactive at reasonable thresholds for the hydroxyl radical-scavenging and quinone reductase inducing assays (ED50 and CD >20 μg/mL), the crudely refined and significantly concentrated subfractions including CHCl3, EtOAc, and n-BuOH partitions were determined to be active. For the work presented on black chokeberry, only the EtOAc partition was prioritized for further study because this partition was shown to be active in both hydroxyl radical-scavenging and quinone reductase induction assays. However since they were also active in the hydroxyl radical-scavenging assay, one could envision meaningful outcomes from additionally investigating the CHCl3 and n-BuOH partitions (19, 21).

Figure 1. Solvent extraction and partitioning used for the initial processing and phytochemical prioritization of black chokeberry (Aronia melanocarpa) fruit.

8

Chromatography Optimal, or even feasible, methods for the purification of phytochemicals from natural product mixtures must be evaluated quite subjectively based on the source material, targeted molecular class(es) of interest, and sample quantity available, among other considerations. Many different methods have been developed and described, and nicely summarized in comprehensive review papers (26, 27). For the work presented on the isolation of investigation of black chokeberry phytochemicals, as shown in Figure 2, a series of different solid phase chromatographic media were used, including normal-phase silica gel, reversed-phase C18 bound silica, Sephadex LH-20 gel permeation resin, and densely packed reversed-phase C18 preparative HPLC columns (19, 21). Together with the iterative testing of bioactivity described above, these chromatography steps led to the purification of berry phytochemicals from active fractions. The specific methods used for similar studies on maqui and goji berry materials have been described elsewhere (20, 22).

Figure 2. Bioassay-guided chromatographic separations used for the detailed phytochemical investigation of black chokeberry (Aronia melanocarpa) fruit.

Structure Elucidation of Phytochemicals Most frequently, the elucidation of known and new structures for natural product isolates can be completed after the skilled scientist conducts careful interpretation of data collected using a traditional set of analytical tools, including NMR and IR spectroscopy, mass spectrometry (MS), UV spectrophotometry, as well as circular dichroism (CD) and optical rotation polarimetry. In other cases, X-ray crystallography, more advanced NMR techniques, MS/MS or 9

MSn fragmentation analysis, chemical degradation or total synthesis, and other techniques may be required (35–38). The consideration of what “the face of a molecule” looks like is largely subjective to the background and experimental preference of the scientist, as each individual may prefer to approach the same challenge from a different starting point (39). Structure determination for an unknown molecule is somewhat akin to the assembly of a jigsaw puzzle for which each contributing puzzle piece must first be constructed by the scientist, e.g., by the collection and interpretation of relevant analytical data. In the era of data analytics and informatics, a tremendous effort has been made to automate or semi-automate the structure elucidation of small molecules (37, 40). Computer-assisted structure elucidation can be imagined to not only improve the speed and efficiency of data interpretation, but also overcome significant intellectual challenges, remove any scientist biases, and ultimately serve as one additional application in the analytical chemistry toolkit. This computer-assisted structure elucidation software has become more readily available for use in both classroom and laboratory environments (41).

Isolated Compounds from Black Chokeberry, Maqui, and Goji Berry Dietary Supplement Ingredients Characterized Phytochemicals The vast majority of phytochemical isolates that were characterized by the present cohort of authors from goji, maqui, and black chokeberry were characterized structurally by traditional methods using CD, IR, MS, NMR and UV data (19–22). The observation of analogues, or molecules from the same structure class and subclass, e.g. 1–3 and 6–9, for example, makes the structure elucidation of each additional member of the class more straightforward to both the scientist, during the investigation, and the scientific community, after dissemination of the associated physical data. This was the case for 1–27, which were isolated from black chokeberry and characterized classically (19). Still, highly complex molecules may be present in the extract and impart important biological functions, as was the case for 28 and 29, a pair of fused pentacyclic flavonoid compounds that were also isolated from black chokeberry (21). The structures of these two molecules were ultimately determined only after computer-assisted structure elucidation in combination with significant efforts from trained scientists (21). The structures of all 29 molecules isolated from black chokeberry in these experiments are presented in Figure 3 (19, 21). In addition, the structures of 30–42 that were isolated from maqui berry, and 43–48 that were purified from goji berry are presented in Figures 4 and 5, respectively (20, 22).

10

Figure 3. Structures of the phytochemicals isolated and characterized from black chokeberry (Aronia melanocarpa) fruit (19, 21). Occurrence of Characterized Phytochemicals in Multiple Sources Although specialized phytochemicals are typically limited or even unique in distribution throughout Nature, it is also the case that many natural products are widely distributed. This more common occurrence is especially the case for those secondary metabolites that likely serve as intermediates and points of divergence in biosynthetic pathways. It may thus be unsurprising that several phytochemicals isolated and characterized in the research on black chokeberry, goji, and maqui berries were found to be equivalent. That is, compound 7 was isolated from both black chokeberry and goji berry, compounds 9, 14 and 24 from both black chokeberry and maqui berry, and compound 40 from both maqui and goji berries (19–22). This both follows the rational logic of bioactivity-guided isolation and highlights one of the criticisms of this practice, namely the re-isolation of molecules, since the same hydroxyl radical-scavenging 11

and quinone reductase-inducing bioassays were used to investigate all three of these materials. The discovery of several new phytochemicals from each sample, however, including 1, 28–30, and 43, shows that continued bioassay-guided isolation research still yields meaningful results.

Figure 4. Structures of the phytochemicals isolated and characterized from maqui (Aristotelia chilensis) berries (22).

Figure 5. Structures of the phytochemicals isolated and characterized from goji (Lyceum barbarum) berries (20). Adulteration, Contamination, and/or Mislabeling As noted earlier, the sample studied that turned out to be goji berry was procured with the intention to obtain African mango. In fact, it was only the isolation and characterization of the pyrrole compounds 43–46 from this material that led to the deeper investigation of the true identity of the source. The occurrence of some pyrrole alkaloids in goji berry extracts had been previously documented, whereas no class of alkaloids has been reported in African mango or other related species to date (20, 23–25, 42). Of course, a chemical fingerprinting and microscopic study of the sample material and authentic samples led to the observation, in both cases, of a confirmed match with goji berry and a distinction from African mango. This follows the report of a dramatic and striking trend of adulteration, contamination, and/or mislabeling of dietary supplements that are available for purchase on the open market (43–45). On November 7, 2017, the 12

US National Center for Complementary and Integrative Health (NCCIH, NIH) dispersed an e-mail to the public warning that, for dietary or herbal supplements, “what’s on the label may not be what’s in the bottle”. The resolution and reduction in the frequency of this problem is of urgent importance to the health and well-being of the many Americans and citizens elsewhere who choose to use these products. Biological Activity The biological study of the phytochemicals isolated from black chokeberry, maqui, and goji berries was conducted as during the respective bioassay-guided fractionation processes (19–22). Accordingly, the in vitro hydroxyl radicalscavenging and quinone reductase-inducing activities of 1–48, along with the associated IC50 values for cytotoxicity towards murine Hepa1c1c7 cells, are presented in Table 1. While the vast majority of phytochemicals tested were active at accepted thresholds for these bioassays, e.g., hydroxyl radical-scavenging ED50 < 20 μM or quinone reductase-inducing CD < 20 μM, others were not. Specifically, compounds 5–8, 10–13, 18, 41, 42 and 45–48 did not show significant activity in either test. It is common to identify isolated phytochemicals without inherent in vitro activities even after bioassay-guided fractionation, and this can occur for many reasons. For one simple example, several molecules may be purified from the same active parent fraction, and not every single one of them must display the desired activity. Another common reason for the isolation of inactive phytochemicals may be that synergistically acting agents are present in the parent mixture of purified molecules that enhanced the activity, or solubility, of one or more compounds, but rationally can no longer act together once separated. In this case, additional testing would be necessary to more thoroughly evaluate the biological function of the isolated phytochemicals, although these can be considered as laborious and often quite sample-demanding.

Table 1. In vitro hydroxyl radical-scavenging and quinone reductase-inducing activities of phytochemicals purified and characterized from black chokeberry, maqui, and goji berry samples by bioassay-guided isolationa compoundb

hydroxyl radicalscavenging

quinone reductase (QR)-induction QR cytotoxicity selectivity

EDED50c (μM)

CDd (μM)

IC50e (μM)

CIf

1

0.44 ± 0.04

>20

>100

ND

2

0.60 ± 0.07

>20

>100

ND

3

0.31 ± 0.04

>20

>100

ND

4

2.6 ± 0.32

>20

>100

ND

9

1.9 ± 0.16

4.3 ± 0.66

>100

>23.3

Continued on next page.

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Table 1. (Continued). In vitro hydroxyl radical-scavenging and quinone reductase-inducing activities of phytochemicals purified and characterized from black chokeberry, maqui, and goji berry samples by bioassay-guided isolationa compoundb

hydroxyl radicalscavenging

quinone reductase (QR)-induction QR cytotoxicity selectivity

EDED50c (μM)

CDd (μM)

IC50e (μM)

CIf

14

1.4 ± 0.16

>20

>100

ND

15

6.4 ± 0.66

>20

>100

ND

16

2.3 ± 0.33

>20

>100

ND

17

1.3 ± 0.12

>20

>100

ND

19

0.59 ± 0.08

>20

>100

ND

20

0.27 ± 0.02

>20

>100

ND

21

0.56 ± 0.07

>20

>100

ND

22

0.81 ± 0.11

6.7 ± 1.2

>100

>14.9

23

1.1 ± 0.13

3.1 ± 0.57

>100

>32.3

24

0.17 ± 0.03

19.2 ± 2.8

>100

>5.2

25

0.25 ± 0.04

>20

>100

ND

26

0.20 ± 0.03

>20

>100

ND

27

2.4 ± 0.34

>20

>100

ND

28

0.71 ± 0.09

7.4 ± 1.1

>100

>13.5

29

0.75 ± 0.09

8.8 ± 0.9

>100

>11.4

30

1.3 ± 0.12

>20

>100

ND

31

0.13 ± 0.02

>20

>100

ND

32

0.64 ± 0.06

>20

>100

ND

33

0.69 ± 0.08

>20

>100

ND

34

0.18 ± 0.02

>20

>100

ND

35

0.46 ± 0.05

>20

>100

ND

36

0.46 ± 0.04

>20

>100

ND

37

0.41 ± 0.05

>20

55.3 ± 7.8

ND

38

0.33 ± 0.04

14.1 ± 2.3

82.4 ± 11.9

5.8

39

1.8 ± 0.16

>20

>100

ND

40

>20

18.7 ± 2.9

>100

>5.1

Continued on next page.

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Table 1. (Continued). In vitro hydroxyl radical-scavenging and quinone reductase-inducing activities of phytochemicals purified and characterized from black chokeberry, maqui, and goji berry samples by bioassay-guided isolationa compoundb

hydroxyl radicalscavenging

quinone reductase (QR)-induction QR cytotoxicity selectivity

EDED50c (μM)

CDd (μM)

IC50e (μM)

CIf

43

16.7 ± 1.6

>20

>100

ND

44

11.9 ± 1.1

2.4 ± 0.38

>100

>42.6

Quercetin was used as a positive control standard for hydroxyl radical-scavenging assay, ED50 = 1.1 ± 0.11 μM. l-sulforaphane was used as a positive control standard for the quinone reductase-induction assay, CD = 0.53 ± 0.08 μM, IC50 = 13.2 ± 1.8 μM, CI = 24.9 ± 3.1 μM. b Compounds 5–8, 10–13, 18, 41, 42 and 45–48 did not show hydroxyl radicalscavenging (ED50 >20 μM) or quinone reductase-inducing (CD >20 μM) activity. c ED50, concentration scavenging hydroxyl radical by 50%. Data are presented as means ± SD (n = 3). Compounds with ED50