Pharmacological Activity-Based Quality Control of Chinese Herbs [1 ed.] 9781624176807, 9781604568233

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

PHARMACOLOGICAL ACTIVITY-BASED QUALITY CONTROL OF CHINESE HERBS

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Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

PHARMACOLOGICAL ACTIVITY-BASED QUALITY CONTROL OF CHINESE HERBS

SHAO-PING LI AND YI-TAO WANG

Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

EDITORS

Nova Science Publishers, Inc. New York

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Copyright © 2008 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Pharmacological activity based quality control of Chinese herbs / Shao-ping Li and Yi-Tao Wang (editors). p. ; cm. Includes bibliographical references and index. ISBN:  (eBook) 1. Medicine, Chinese. 2. Materia medica, Vegetable--Quality control. I. Li, Shao-ping. II. Wang, Yi-Tao. [DNLM: 1. Drugs, Chinese Herbal--pharmacology. 2. Drugs, Chinese Herbal--standards. 3. Medicine, Chinese Traditional. 4. Quality Control. QV 766 P536 2008] R601.P53 2008 616'.09--dc22 2008023339

Published by Nova Science Publishers, Inc.

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Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Contents

Preface

vii

I. Introduction Chapter 1

Strategies for Quality Control of Chinese Medicine Shao-ping Li

Chapter 2

Cell Materials Biospecific Extraction for Hypothesis of Active Components in Chinese Medicines Jing Wang, Jing Zhao, Li Yu and Shao-ping Li

11

Recent Development on Sample Preparation for Quality Control of Chinese Herbs Hua Yu, Feng-qing Yang, Yi-fan Han and Shao-ping Li

21

Recent Development on Analytical Techniques for Quality Control of Chinese Herbs Jia Guan, Xiao-jia Chen and Shao-ping Li

73

Chapter 3

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1

Chapter 4

Chapter 5

Applications of Capillary Electrophoresis - Mass Spectrometry Approach in Herbal Medicine Analyses Liya Ge, Jean Wan Hong Yong and Swee Ngin Tan

3

115

II. Pharmacological Activities and Quality Control of Selected Chinese Herbs

137

Chapter 6

Dongchongxiacao (冬虫夏草, Cordyceps sinensis) Shao-Ping Li and Feng-Qing Yang

139

Chapter 7

Rengongchongcao (人工虫草，Cultured Cordyceps sinensis) Kun Feng, Feng-qing Yang and Shao-ping Li

157

Chapter 8

Sanqi (三七，Panax notoginseng) Jian-bo Wan, Yi-tao Wang and Shao-ping Li

179

Chapter 9

Beimu (贝母，Fritillaria spp.) Hui-Jun Li, Yan Jiang and Ping Li

205

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vi

Contents

Chapter 10

Yinyanghuo (淫羊藿，Epimedium spp.) Xiao-jia Chen, Yi-tao Wang and Shao-ping Li

217

Chapter 11

Huangqi (黄芪，Astragalus spp.) Jun Li, Winnie Z.M. Li, Kui-jun Zhao, Wen Huang, Cathy W.C. Bi, Ran Duan, Tina T.X. Dong and Karl W.K. Tsim

235

Chapter 12

Lingzhi (灵芝，Ganoderma spp.) Jian-li Gao, Ying-bo Li and Shao-ping Li

251

Chapter 13

Chuanxiong (川芎，Ligusticum chuanxiong) Sunny Sun-kin Chan, Ru Yan and Ge Lin

273

Chapter 14

Ezhu (莪术, Rhizoma Curcumae) & Yujin (郁金, Radix curcumae) Feng-qing Yang and Shao-ping Li

291

Chapter 15

Guanghuoxiang (广藿香，Pogostemon cablin) Ying Zhang, Hui Cao, Michael Mengsu Yang and Peigen Xiao

311

Chapter 16

Wuweizi (五味子，Schisandra chinensis) Yan Lu, Jian-ping Gao and Dao-feng Chen

325

Chapter 17

Danshen (丹参，Salvia miltiorrhiza) Peng Li, Yi-tao Wang and Shao-ping Li

339

Chapter 18

Jinyinhua (金银花，Lonicera japonica) Jun Chen and Ping Li

365

Chapter 19

Juhua (菊花，Chrysanthemum morifolium) Jin-ao Duan, Yuping Tang and Dawei Qian

381

Chapter 20

Chishao (赤芍，Paeonia spp.) Quan-hong Zhu

391

Chapter 21

Chuipencao (垂盆草, Sedum sarmentosum) Li-fang Liu, Zhu-qing Wan, Le Xie and Xiao-li Ma

405

Chapter 22

Danggui (当归, Angelica sinensis) Sin-cheng Lao and Simon Ming-yuen Lee

417

Index

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

441

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Preface Natural products are the most successful source of drugs. Of the 877 small-molecule new chemical entities introduced between 1981 and 2002, roughly half were natural products, semi-synthetic natural product analogues or natural mimics. Chinese herbs (traditional Chinese medicines, TCMs) have been used for prevention and treatment of diseases for thousands years. It has been attracting intensive attention in the trends of back to nature. As herbal medicines, the conditions of growth, harvest time, process and storage etc. will undoubtedly affect the presence and concentration of the bioactive constituents, thus affecting their quality and efficacy. Up to date, there are few books focus on the relationship, which is the bridge between TCM and modern medical science, of traditional clinic uses, pharmacological activities and quality control of Chinese herbs. Unfortunately, bioactive compounds in Chinese herbs are usually not or only partially known. Actually, the active compounds considered in Chinese herbs may be different according to their clinical indication. In addition, it is considered that the curative effect of Chinese herbs is an integrative result of a number of bioactive compounds. Therefore, how to control the quality is a big problem. We would like to discuss the quality control based on their traditional clinical uses and pharmacological activities in this book, which is the fundamental step towards developing and modernizing such products into evidence based medicines. It will also help the peoples to understand TCMs in modern scientific angles. This book is designed primarily as a textbook for or adjunct to course in herbal remedies, food safety or quality control of herbs. It is also a professional reference whom is in the area of drug discovery, pharmaceutical analysis and food chemistry. I am grateful to Macao Science and Technology Development Fund and Research Committee of University of Macau for their support to our research projects. I would like to express my gratitude to Mr. Chen Yang, Yuan-jie Zhang, Miss Guo-xin Zhong, Bo Wang and Shuang Wang for their unstinting help in the search for literature, and the authors who kindly allowed reproduction of figures from their texts. Special thanks go to Miss Xiaojia Chen who patiently edited the manuscript prior to publication.

Shao-ping Li Spring 2008 in Macao Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

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I. Introduction

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In: Pharmacological Activity-Based Quality Control… ISBN 978-1-60456-823-3 Editors: Shao-ping Li and Yi-Tao Wang © 2008 Nova Science Publishers, Inc.

Chapter 1

Strategies for Quality Control of Chinese Medicine Shao-ping Li* Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China

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1. Introduction Traditional Chinese medicine (CM or TCM) has been attracting interest and acceptance in many countries. An estimated 1.5 billion people now use these preparations worldwide [1]. This may be primarily because of the general belief that herbal drugs are without any side effect besides being cheap and locally available [2]. However, as more people are using TCM products, there are increased reports on adverse reactions [3]. Therefore, quality control is crucial for ensuring the safety and effectiveness of CM. As we know, CM is usually used as whole plant and/or combination of several herbs, which contains more than tens to hundreds or even thousands of components. This greatly increases the difficulties of their quality control. Actually, it is considered that multiple constituents are usually responsible for the therapeutic effects of CM. These multiple constituents may work ‘synergistically’ and could hardly be separated into active parts. Moreover, the chemical constituents in CM may vary depending on their growth origins, harvest seasons, drying processes, store conditions, extraction approaches and other factors, while the quality of CM is strongly related to their therapeutic effects. Therefore, a rational and effective quality control method is the assurance of the safety and efficacy of CM. In this chapter, the strategies, related to the markers, reference compounds and approaches, for quality control of CM were reviewed and discussed.

*

E-mail address: [email protected]. Tel: +853-8397 4692. Fax: +853-2884 1358

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Shao-ping Li

2. Screening of Markers for Quality Control of CM As we know, the purpose of quality control is to ensure the safety and efficacy of CM. Therefore, the markers for quality control should be strongly correlated to the efficacy and safety of CM. Conventionally, separation followed with bioassay or bioassay guided separation is the method for discovering active compounds in CM. However, CM usually contains hundreds or even thousands of different compounds, which makes the separation and screen extremely difficult. HPLC is a powerful separation tool. Recently, a technique, HPLC coupled with bioassay detection, was developed to measure the activity of individual compounds on-line when they were eluted from an HPLC column [4, 5]. This technique makes it possible to directly identify active constituents in complex matrixes.

A Line:H PLC -D A D -M S A nalysis

H erbal extract

colum n

DAD

MS

B Line:A BTS Based A ssay A BTS·+

H PLC pum p

DAD

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reaction coil

Figure 1. Diagrammatic scheme of HPLC coupled with DAD-MS and ABTS-based assay for rapid screening and identification of antioxidants in herbal extract (adapted from Ref. [6] with permission).

A method using HPLC coupled with DAD-MS and ABTS-based assay was developed for identification of antioxidant components in essential oil of Angelica sinensis (AS oil) [6]. Fig. 1 shows the scheme of the instrumental setup. The sample mixture (herbal extract) is injected into HPLC system for separation, the eluent from DAD of Line A is split into two streams. The minor stream is introduced into MS for structure elucidation. The major one goes to Line B, which is used for ABTS assay, consists of a HPLC pump connects with a reaction coil through T-connector, and a detector. The continuous flow of free radical (e.g. ABTS•+) solution is introduced into the reaction coil and interacts with the eluent, and any bleaching of the initial color is detected as a negative peak. As shown in Fig. 2, compound (1) in AS oil has significant free radical scavenging activity. This antioxidant compound is identified as coniferyl ferulate (1) based on its UV spectra, MS data and previous reports [7, 8], which is further confirmed by comparing its retention time, UV spectra and MS data with those of pure coniferyl ferulate. Furthermore, bioassays show that the antioxidant capacity of AS oil is in strong accordance with the relative amount of coniferyl ferulate in the oil, which suggests that HPLC coupled with bioassay detection is a powerful approach for screening active

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Strategies for Quality Control of Chinese Medicine

5

components from complex matrix such as Chinese medicine. Indeed, this approach has attracted intensive attention [9-12]. Theoretically, modern pharmacological studies have shown that combining with some receptors or channels on cell membrane is the first step of drug action. Therefore, the ability of a drug to interact with cell membranes is very important for the behavior of the drug in the organism. In order to study the solute-binding component of cell membranes, a method called retardation chromatography was introduced by Bobinski and Stein [13]. However, the cell materials are less stable and the entrapment or immobilization procedure must be adapted to the kind of material that is to be analyzed and to the kind of gel matrix used [14]. In addition, interaction of components in CMs with biomembrane is rarely compatible to their separation on chromatography. Thus, cell materials biospecific extraction followed by HPLC analysis has been explored for screening bioactive compounds in CM, which was reviewed in Chapter 2. mAU

mAU 800

A

600 400

1

200

Intens x104 1.25 1.00 0.75 0.50 0.25 0.00

120

185.3

80 40

131.4 103.6 163.4 100

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0 200

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-40

B

O

O

-60 -80

OCH3

OCH3

-100 -120 -140 0

OH

HO

10

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min

Figure 2. Chromatograms of essential oil from Angelica sinensis (AS Oil) analyzed by HPLC coupled with (A) DAD-MS and (B) ABTS-based assay (from Ref. [6] with permission). Inserts are UV spectra, MS and structure of coniferyl ferulate (1)

3. Substitute of Standard for Quality Control of CM Generally, quality control of CM includes qualitative and quantitative analysis, while quantitative determination is not available without standard. However, reference compounds or chemical standards are the bottle neck for quality control of CMs because of their complexity. In the last decades, the great effort has been focused on this area during “Ninth Five-year” and “Tenth Five-year” programs in China. However, the supply of reference compounds is far from the requirement for quality control of CM. Up to date, there are only about 400 reference compounds supplied for TCM by National Institute for the Control of

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Shao-ping Li

Pharmaceutical and Biological Products. Especially, some pure chemical compounds are difficult to obtain because of their instability and/or trace, which hinders the development of quality control for CMs. Fortunately, gas chromatography-mass spectrometry (GC-MS) offers a powerful tool for identification of chemical components in essential oil. Using analogues as chemical standards, an alternative method, by which the content of identified components were determined or estimated, was developed to evaluate the quality of essential oils from CM without appropriate chemical standards [15, 16]. The results can be used for evaluating the quality of different samples or batches of essential oils from CM [15-18]. Recently, this strategy of resolving the shortage of reference compounds for quantitative analysis has attracted more attention [19, 20]. Danshen, the root and rhizome of Salvia miltiorrhiza Bunge, is one of the most important ancient Chinese herbal drugs. More than 20 phenolic acids of the water soluble constituents, which have antioxidant, anti-blood coagulation and cell protection activities, have been isolated from this plant since the 1980s [21]. Salvianolic acid B, one of the phenolic acids, has been used as the marker for the quality control of S. miltiorrhiza because it not only has the actions described above, but also is the characteristic constituent of S. miltiorrhiza [22]. However, the preparation and purification of salvianolic acid B is difficult because of its poor stability, and it is also expensive for daily control work. Therefore, methylparaben was used as a substitute reference substance of salvianolic acid B for determination of Radix Salviae miltiorrhizae and compound Danshen tablets [19]. The assay demonstrates that methylparaben instead of salvianolic acid B is feasible for quality control of S. miltiorrhiz and its products. It was also reported that the contents of arctiin, arctigenin, lappaol C and lappaol H was determined using arctiin as the reference compound with corrected factor method [20]. In addition, standard extract has also been proposed as reference standard for measuring each component in herbal products [23]. Silymarin, widely used for the treatment of toxic liver damage, hepatitis and cirrhosis, primarily consists of an isomeric mixture of active flavonolignans: silychristin (Sc), silydianin (Sd), and two groups of diastereoisomeric flavonolignans, silybin A (Sb A) and silybin B (Sb B), and isosilybin A (ISb A) and isosilybin B (ISb B) [24-26]. The different isomers of silymarin have been reported to have different biological activities [27-29]. Currently, all six individual purified standards are not available for the quantification of silymarin although there is a wealth of literature available. The lack of standards available for the quantification of Sb A, Sb B, ISb A and ISb B leads the search of alternative standard reference materials. Lee et al. [23] proposed using silymarin obtained from Sigma-Aldrich Co., with high purity of silymarin and the similar ratio profile of all six components in the commercial standard silymarin extracts, as the reference standard for the six individual constituents, which was used to evaluate each active constituent in seven commercial products from different brands. Especially, a new method for quantitative analyses performed without any standard using an evaporative light-scattering detector (ELSD) is also proposed [30]. However, the peak identification is a problem without matching reference compound. Mass spectrometry plays an important role for the identification of analytes [15-18, 31-33]. LC-MS was also applied for the identification of 23 flavonoids in the extract of Mexican oregano (Lippia graveolens H.B.K.), and ten flavonoids without matching standards among them were quantitatively estimated using eriodictyol 7-Oglucoside (for determination of three pentahydroxyflavanone monoglycosides), luteolin (for determination of 6-hydroxyluteolin), luteolin 7-O-glucoside (for determination of two 6hydroxyluteolin glycosides and scutallarein 7-O-hexoside), scutellarein (for determination of

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6-methoylscutallarein and 6,7-dimethoylscutallarein), galangin (for determination of methylgalangin), and phloridzin (for determination of 6-hydroxyphloretin 6’’-O-hexoside), respectively [34]. Indeed, using substitute and/or extract as standard are currently a practical and available strategy for resolving the shortage of standards for quality control of CM, the interest is how to improve the accuracy of quantification.

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4. Quality Control of CM Based on the Analysis of Chemical Markers In general, one or two markers or pharmacologically active components were currently employed for evaluating the quality and authenticity of CM. Among 195 CM raw materials with quantitative assay of pure compound recorded in Chinese Pharmacopoeia (2005), the quality of 154 herbs is evaluated using one marker, which is usually not strongly related to the safety and efficacy of CM. For example, Cordyceps sinensis, one of the valued CMs, is commonly used in hospitals in China and as a household remedy because of its multiple biological activities [35]. Adenosine has been used as a marker for quality control of natural Cordyceps sinensis in Chinese Pharmacopoeia [36]. However, fresh natural C. sinensis contains very little amount of adenosine, as compared to dry and processed one [37], and more interestingly cultured Cordyceps mycelium contains high level of adenosine [35]. In addition, the hypolipidemic activity of adenosine has never been reported. Moreover, inosine, the major biochemical metabolite of adenosine due to oxidative deamination, can stimulate axon growth in vitro and in the adult central nerve system [38]. It is interesting that natural Cordyceps contain much higher amount of inosine than the cultured ones, including C. sinensis and C. militaris [35]. Therefore, having adenosine as a marker for good quality of Cordyceps may not be indicative. Recently, multiple markers quantification is widely used for rationally and effectively evaluating the quality of CM [39-45]. But the markers and their contents should be determined based on their safety and efficacy. Otherwise, the quality of CM may not be controlled rationally. In China, raw ginseng and steam ginseng are usually used for the treatment of different diseases. But both qualities are controlled using ginsenosides Rg1, Re and Rb1 with the very similar content levels, i.e. the content of ginsenoside Rb1 should be more than 0.20% with sum of ginsenoside Rg1 and Re should be more than 0.30% and 0.25%, respectively [46]. It is obvious that multiple markers may also not accessible for quality control of CM rationally because of their complexity.

5. Quality Control of CM Based on the Fingerprint and Biofingerprint It is very difficult or impossible to evaluate the quality of CM using only a few chemical markers because of its complexity and the interaction of multiple chemical compounds may contribute to its therapeutic effects. In last decade, the construction of chromatographic fingerprints becomes one of the most powerful approaches for quality control of herbal medicines such as CM [47-53]. The Chinese State Food and Drug Administration (SFDA) would have begun to control the quality of Chinese medicine injections with the compulsory

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fingerprint analysis in 2004. To date, among 109 injections made from CM, the fingerprints of 72 injections have been developed though none has been compulsorily implemented to control their qualities [54]. Among the various experimental techniques, chromatographic methods are highly recommended for establishing the fingerprints of CM [55]. Chromatographic fingerprint analysis could be used for determining the identity, stability and consistency of CM as well as identification of adulterants [56]. However, an optimized chromatographic fingerprint should provide as detailed information regarding quality assessment based on the safety and efficacy as possible. Because of the complexity of ingredients in CM, more than one fingerprint may be needed for adequately assessing their quality. “Multiple chromatographic fingerprints” is an effective method [57], which has been used for quality control of the total alkaloids from Caulophyllum robustum [58]. Actually, the biologically active compounds are usually not or only partially known. In addition, there is little correlation between the chromatographic retention of the compounds and their bioactivities in conventional HPLC. Therefore, biological fingerprinting analysis (BFA), which is defined as the chromatograms and spectra of the complex small molecular system carrying with the information of their interaction with the biomolecules [59], was proposed and applied to biological fingerprinting analysis of TCMs [60-62]. In addition, cultured human cells were also used as a dynamic detector for the cellular responses and the DNA microarray was employed as a readout to fingerprint the global transcriptional response of a human liver cell line to the TCM decoction [63]. As the result, a set of nine signature genes were selected as reporters of the biological activities of the TCM product ISF-1 in human cells. Then, a cost-effective real time qRT-PCR technique could be routinely applied to evaluate the biological activities of individual production batches, which providing a sensitive and reliable biological quality control metric for botanical drug products.

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6. Conclusion To control the quality of CM, three aspects should be considered. They are quality marker, standard substance as well as advanced and standardized analytical techniques, including sample preparation and analysis. At present, the problems are quality marker has low correlation with the safety and efficacy of CM, shortage of standard substances and/or non-standardized analytical methods, which should be greatly improved. The strategies mentioned above are helpful to control the quality of CM.

References [1] Hosbach, I.; Neeb, G.; Hager, S.; Kirchhoff, S.; Kirschbaum, B. Anaesthesia 2003, 58, 282-283. [2] Gupta, L. M.; Raina, R., Curr. Sci. 1998, 75, 897-900. [3] Ko, R. J. J. Chin. Med. Assoc. 2004, 67, 109-116. [4] Koleva, I. I.; Niederländer, H. A. G.; van Beek, T. Anal. Chem. 2000, 72, 2323-2328. [5] Dapkevicius, A.; van Beek, T. A.; Niederländer, H. A. G. J. Chromatogr. A 2001, 912, 73-82. [6] Li, S. Y.; Yu, Y.; Li, S. P. J. Agric. Food Chem. 2007, 55, 3358-3362.

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[7] Lu, G. H.; Chan, K.; Liang, Y. Z.; Leung, K.; Chan, C. L.; Jiang, Z. H.; Zhao, Z. Z. J. Chromatogr. A 2005, 1073, 383-392. [8] Kong, L.; Yu, Z.; Bao, Y.; Su, X.; Zou, H.; Li, X. Anal. Bioanal. Chem. 2006, 386, 264-274. [9] Ingkaninan, K.; de Best, C. M.; van der Heijden, R.; Hofte, A. J. P.; Karabatak, B.; Irth, H.; Tjaden, U. R.; van der Greef, J.; Verpoorte, R. J. Chromatogr. A 2000, 872, 61-73. [10] van Bommel, M. R.; de Jong, A. P. J. M.; Tjadena, U. R.; Irth, H.; van der Greef, J. J. Chromatogr. A 2000, 886, 19-29. [11] Schenk, T.; Appels, N. M. G. M.; van Elswijk, D. A.; Irth, H.; Tjaden, U. R.; van der Greef, J. Anal. Biochem. 2003, 316, 118-126. [12] van Elswijk, D. A.; Diefenbach, O.; van der Berg, S.; Irth, H.; Tjaden, U. R.; van der Greef, J. J. Chromatogr. A 2003, 1020, 45-58. [13] Bobinski, H.; Stein, W. D. Nature 1966, 211, 1366-1368. [14] Gottschalk, I.; Lagerquist, C.; Zuo, S. S.; Lundqvist, A.; Lundahl, P., J. Chromatogr. B: Analyt Technol. Biomed. Life Sci. 2002, 768, 31-40. [15] Lao, S. C.; Li, S. P.; Kan, K. K. W.; Li, P.; Wan, J. B.; Wang, Y. T.; Dong, T. T. X.; Tsim, K. W. K. Anal. Chim. Acta 2004, 526, 131-137. [16] Yang, F. Q.; Li, S. P.; Chen, Y.; Lao, S. C.; Wang, Y. T.; Dong, T. T. X.; Tsim, K. W. K. J. Pharm. Biomed. Anal. 2005, 39, 552-558. [17] Qin, N. Y.; Yang, F. Q.; Wang, Y. T.; Li, S. P. J. Pharm. Biomed. Anal. 2007, 43, 486-492. [18] Tam, C. U.; Yang, F. Q.; Zhang, Q. W.; Guan, J.; Li, S. P. J. Pharm. Biomed. Anal. 2007, 44, 444-449. [19] Xie, Y.C.; Jin, S. H. Yao Wu Fen Xi Za Zhi 2007, 27, 497-502. [20] Huang, X.; Zheng, Y.; Zeng, Z. Y.; Gong, T. Chendu Zhong Yi Yao Da Xue Xue Bao 2005, 28, 42-44. [21] Wang, X.; Morris-Natschke, S. L.; Lee, K. H. Med. Res. Rev. 2007, 27, 133-148. [22] Pharmacopoeia Commission of PRC (Eds.), Pharmacopoeia of the People’s Republic of China; Chemical Industry Press: Beijing, China, 2005; Vol. I, pp 52. [23] Lee, J. I.; Narayan, M.; Barrett, J. S. J. Chromatogr. B 2007, 845, 95-103 [24] Kim, N. C.; Graf, T. N.; Sparacino, C. M.; Wani, M. C.; Wall, M. E. Org. Biomol. Chem. 2003, 1, 1684-1689. [25] Lee, D.Y.; Liu, Y. J. Nat. Prod. 2003, 66, 1171-1174. [26] Lee, J. I.; Hsu, B. H.; Wu, D.; Barrett, J. S. J. Chromatogr. A 2006, 1116, 57-68. [27] Křen, V.; Ulrichová, J.; Kosina, P.; Stevenson, D.; Sedmera, P.; Přikrylová, V.; Halada, P.; Šimánek, V. Drug Metab. Dispos. 2000, 28, 1513-1517. [28] Chlopčíková, Š.; Psotová, J.; Miketová, P.; Šimánek, V. Phytother. Res. 2004, 18, 107-110. [29] Škottová, N.; Krecman, V.; Šimánek, V. Phytother. Res. 1999, 13, 535-537. [30] Heron, S.; Maloumbi, M. G.; Dreux, M.; Verette, E.; Tchapla, A., J. Chromatogr. A 2007, 1161, 152-156. [31] Luque-Garcia, J. L.; Neubert, T. A. J. Chromatogr. A 2007, 1153, 259-276. [32] Villar-Garea, A.; Griese, M.; Imhof, A. J. Chromatogr. B 2007, 849, 105-114. [33] Wang, Y.; Karu, K.; Griffiths, W. J. Biochimie 2007, 89, 182-191. [34] Lin, L. Z.; Mukhopadhyay, S.; Robbins, R. J.; Harnly, J. M. J. Food Compost. Anal. 2007, 20, 361-369.

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[35] Li, S. P.; Yang, F. Q.; Tsim, K. W. K., J. Pharm. Biomed. Anal. 2006, 41, 1571-1584. [36] Pharmacopoeia Commission of PRC (Eds.), Pharmacopoeia of the People’s Republic of China; Chemical Industry Press: Beijing, China, 2005; Vol. I, pp 75. [37] Li, S. P.; Li, P.; Dong, T. T. X.; Tsim, K. W. K. Electrophoresis 2001, 22, 144-150. [38] Benowitz, L. I.; Goldberg, D. E.; Irwin, N. Prog. Brain Res. 2002, 137, 389-399. [39] Chen, J.; Song, Y.; Li, P. J. Chromatogr. A 2007, 1157, 217-226. [40] Wan, J. B.; Li, P.; Li, S.; Wang, Y.; Dong, T. T. X.; Tsim, K. W. K. J. Sep. Sci. 2006, 29, 2190-2196. [41] Murthy, P. B. S.; Raju, V. R.; Ramakrisana, T.; Chakravarthy, M. S.; Kumar, K. V.; Kannababu, S.; Subbaraju, G. V. Chem. Pharm. Bull. 2006, 54, 907-911. [42] Kim, S. N.; Ha, Y. W.; Shin, H.; Son, S. H.; Wu, S. J.; Kim, Y. S. J. Pharm. Biomed. Anal. 2007, 45, 164-170. [43] Yu, Q. T.; Qi, L. W.; Li, P.; Yi, L.; Zhao, J.; Bi, Z. J. Sep. Sci. 2007, 30, 1292-1299. [44] Yang, F. Q.; Wang, Y. T.; Li, S. P. J. Chromatogr. A 2006, 1134, 226-231. [45] Ha, Y. W.; Na, Y. C.; Seo, J. J.; Kim, S. N.; Linhardt, R. J.; Kim, Y. S. J. Chromatogr. A 2006, 1135, 27-35. [46] Pharmacopoeia Commission of PRC (Eds.), Pharmacopoeia of the People’s Republic of China; Chemical Industry Press: Beijing, China, 2005; Vol. I, pp 7, 105. [47] WHO. General Guidelines for Methodologies on Research and Evaluation of Traditional Medicine; World Health Organization: Geneva, 2000; Annex. I, pp 23. [48] FDA. General Regulatory Approaches. In: Guidance for Industry-Botanical Drug Products; US Food and Drug Administration: 2004; III, B, pp5. [49] EMEA. Final Proposals for Revision of the Note for Guidance on Quality of Herbal Remedies; European Medicines Evaluation Agency: 1999. [50] Calixto, J. B. Braz. J. Med. Biol. Res. 2000, 33, 179-189. [51] Philipsom, J. D. British Herbal Pharmacopoeia; British Herbal Medicine Association: 1996; Forward. [52] Indian Drug Manufacturers’ Association, Vedams Books International: 1998. [53] State Food and Drug Administration of China. Zhongguo Yao Pin Biao Zhun 2000, 1, 195-199. [54] http://www.39.net/focus/jkjd/174286.html (accessed 10/10/2007). [55] Liang, Y. Z.; Xie, P.; Chan, K. J. Chromatogr. B 2004, 812, 53-70. [56] Xie, P.; Chen, S.; Liang, Y. Z.; Wang, X.; Tian, R.; Upton, R. J. Chromatogr. A 2006, 1112, 171-180. [57] Fan, X. H.; Cheng, Y. Y.; Ye, Z. L.; Lin, R. C.; Qian, Z. Z. Anal. Chim. Acta 2006, 555, 217-224. [58] Li, Y.; Hu, Z.; He, L. J. Pharm. Biomed. Anal. 2007, 43, 1667-1672. [59] Su, X.; Kong, L.; Lei, X.; Hu, L.; Ye, M.; Zou, H. Mini Rev. Med. Chem. 2007, 7, 87-98. [60] Su, X.; Kong, L.; Li, X.; Chen, X.; Guo, M.; Zou, H. J. Chromatogr. A 2005, 1076, 118-126. [61] Su, X.; Kong, L.; Li, X.; Chen, X.; Guo, M.; Zou, H. J. Comb. Chem. 2006, 8, 544-550. [62] Su, X.; Hu, L.; Kong, L.; Lei, X.; Zou, H. J. Chromatogr. A 2007, 1154, 132-137. [63] Rong, J.; Tilton, R.; Shen, J.; Ng, K. M.; Liu, C.; Tam, P. K. H.; Lau, A. S. Y.; Cheng, Y. C. J. Ethnopharmacol. 2007, 113, 35-44.

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 2

Cell Materials Biospecific Extraction for Hypothesis of Active Components in Chinese Medicines Jing Wang1, Jing Zhao1,2, Li Yu1,3 and Shao-ping Li1,* 1 2

Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing, China 3 Nanjing University of Traditional Chinese Medicine, Nanjing, China

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1. Introduction Chemical substances derived from natural materials have been used to treat human disease since the dawn of medicine. Actually, natural products are the most successful source of drugs. Of the 877 small-molecule new chemical entities (NCEs) introduced between 1981 and 2002, roughly half (49%) were natural products, semi-synthetic natural product analogues or natural mimics [1]. Traditional Chinese medicines (CMs or TCMs) are natural therapeutic remedies used in China for thousands of years, which has attracted wide attention in drug discovery because of their excellent efficacy. However, CMs are complex mixtures consisting of hundreds or even thousands of different compounds. In most cases, biologically active compounds in CMs are not or only partially known. Therefore, the screening and analysis of bioactive components in CMs is very important not only for drug discovery but also for the quality control of crude drugs and elucidation of their therapeutic principles. A conventional procedure of discovering natural-product hits is that the compounds are extracted from the source, concentrated, fractionated and purified, yielding essentially a single biologically active compound. Though some active components such as berberine and artemisinin in Chinese medicine were discovered using this generic scheme, the way to increase the probability of success is still controversial, relegating most workers to trial-and-error *

E-mail address: [email protected]. Tel: +853-8397 4692. Fax: +853-2884 1358 (Corresponding author.)

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experiments. In addition, one must correlate the biological signal of interest with the effector compounds. It is usually difficult for CMs. Furthermore, it is the major bottleneck to yield sufficient quantities of the pure material for bioassay because often the target compounds represent much less than 1% by weight of the crude extract in CMs. Modern pharmacological studies have shown that combining with some receptors or channels on cell membrane is the first step of drug action. Therefore, the ability of a drug to interact with cell membranes is very important for the behavior of the drug in the organism. In order to study the solute-binding component of cell membranes, a method called retardation chromatography was introduced by Bobinski and Stein [2]. Biochromatography with immobilized human serum albumin (HSA) stationary phases has been applied to probe the interaction between the group of compounds in CMs and the protein [3-5]. However, the interaction of compounds and HSA is nonspecific. The use of immobilized membrane proteins as chromatographic stationary phases for the biospecific interaction between the proteins and solutes has been reviewed [6]. Recently, automated multiple ligand screening has also been developed so as to push frontal affinity chromatography (FAC) coupled to mass spectrometry detection more to the forefront as a moderate primary high-throughput screening system [7]. However, the affinity of the protein and solute may decrease upon protein purification [8]. On the other hand, a single protein is limited for elucidating the curative effect of CMs, which is an integrative result of a number of bioactive compounds. Therefore, immobilization of biomembrane or whole cells for chromatography has also attracted much attention in the recent years [9]. However, the cell systems were less stable and the entrapment or immobilization procedure must be adapted to the kind of material that is to be analyzed and to the kind of gel matrix used [10]. In addition, interaction of components in CMs with biomembrane is rarely compatible to their separation on chromatography. Thus, using biomembrane or whole cells chromatography, the separation and identification of compounds is a problem. A novel strategy for screening bioactive components in TCM namely cell materials biospecific extraction was proposed [11], which has attracted more attention recently [12-16]. Fig. 1 showed the schematic profile of screening bioactive components in CMs based on cell materials biospecific extraction. First, the extract of CMs is analyzed to obtain the chromatographic profile (chromatogram I). Then it is incubated together with cell materials, the potential bioactive compounds in the extract could be biospecifically adsorbed to cell materials, which decreased the concentrations of combined components in the extract (chromatogram II). Thus, comparing two chromatograms of the extract with the same concentration before and after interaction with cell materials, the bound compounds in the extract may be found. Furthermore, wash the cell materials to remove the non-specific adsorbed components. Then denature the cell materials to release the combined components which will be determined based on the chromatographic analysis (chromatogram III). Finally, the cell absorbed components in the extract also can be found when the cells are digested and analyzed (chromatogram IV). According to the procedure mentioned above, the compounds which can be specific interacted with cell materials in CMs extract will be discovered. All of these compounds are the bioactive candidates. This chapter summarized the current status and perspective of cell materials (cell biomacromolecule, cell membrane and whole cell) biospecific extraction for screening bioactive compounds in CMs.

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

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Figure 1. Flow scheme of cell materials biospecific extraction used for screening active components in traditional Chinese medicines.

2. Cell Macromolecules Biospecific Extraction Cell macromolecules include proteins (e.g. enzyme and receptor) and nucleic acids (i.e. DNA and RNA). They are first applied for screening the active components from CMs by Wang and his colleagues [17, 18]. It was reported that more than 400 extracts from 150 commonly used CMs were systematically screened using 11 receptors and enzymes binding assays. Immobilized polyclonal antibodies were also applied to mimic hepatitis C virus (HCV) NS3 protease in FAC-MS for screening active components in the extract of Phyllanthus urinaria, which resulted in brevifolin, brevifolin carboxylic acid, corilagin, ellagic acid, and phyllanthusiin U. These compounds showed high inhibitory activities to the virus [19]. FAC offers an effective tool for investigation of interaction between small molecules and macromolecules, such as enzyme [20-25], receptor [7, 26, 27], RNA [28] and DNA [29] in the complex systems, e.g. extracts of CMs.

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DNA, a carrier of genetic information, provides another group of targets for interaction with small molecules. Studies on the interaction of small molecules with DNA are important steps for elucidating the functional mechanisms of DNA, and may also provide a key to rational design of DNA-targeted drugs. Natural DNA has been immobilized on silica gel as stationary phase for screening and analysis of DNA binding active compounds in Rheum palmatum and Coptis chinensis. It was found that 14 compounds such as aloe-emodin, rhein, emodin, chrysophannol- 8-O-glucophranoside and physionl-8-O-glucophranoside in Rheum palmatum, 7 compounds, including berberine, palmatine and jatrorrhizine, in Coptis chinensis were active [30]. The latest applications of the general methodology, including chromatographic, electromigratic and mass spectrometric methods, used for elucidating the interactions between small molecules and biomacromolecules has been reviewed [31]. Cell macromolecules provide biospecific targets for screening the potential active compounds from CMs. But in most cases, the diseases are related to multi-target actions. Therefore, they can neither respond the authentic pharmacological action and nor embody the characteristic of multi-component and multi-target action of CMs screening based on the target of biomacromolecules. But using cell membrane or whole cell as the biospecific materials may predict the potential bioactivities of multiple compounds in CMs simultaneously.

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3. Cell Membrane Biospecific Extraction In order to analyze the interaction of active components and cell membrane proteins, rapid and gentle immobilization methods are usually applied. Red blood cell membranes were electrostatically adsorbed on ‘‘Celite’’ or DEAE-cellulose for chromatographic analysis of glucose binding component in 1966 [2, 32]. Cell membrane vesicle has also been immobilized on gel beads using a simple and mild freeze-thawing method [33-35]. This method was applied to determine biospecific interactions between membrane proteins and substrates and inhibitors [8, 36]. By incubating silica with the suspension of cell membranes, the whole surface of silica was covered by the cell membranes which could be immobilized [37]. This technique has been applied to screen vascular modulators in Angelica sinensis [38], Cladonia alpestris [39], Herba Epimedii [40], Cuscuta chinensis [41], Leontice robustum [42], Carthamus tinctorius L. [43] and Ligusticum chuanxiong [44] by using immobilized vascular cell membrane as stationary phase. Immobilized human endothelial cell ECV-304 membrane was also used for screening active compounds in Salvia miltiorrhiza [45] and Leontice robustum [46]. Similarly, Toll-like receptor 4 (TLR4) antagonists in Atractylodes macrocephala were identified using leucocytes membrane [47], and atractylenolide I with anti-inflammatory action was determined. In addition, immobilized red blood cell membrane was employed for determining the cytotoxic components in Cladonia fallax [48] and Libanotis buethorimensis [49]. The selectivity and specificity of the interaction has also been investigated using subtype receptors [50-53]. Actually, this technique suffers from the incompatibility between the solute-membrane interaction and the chromatographic separation of components in CMs. Cell membrane biospecific extraction is a novel alternative approach. By using free dog platelet biospecific extraction, active components in Radix Salviae miltiorrhizae [12] and Mailuoning Injection (a product of CMs) [13] have been studied under imitated physical environments. As a result, six specific active compounds in the water

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Cell Materials Biospecific Extraction for Hypothesis of Active Components…

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extracts of Radix Salviae Miltiorrhizae and eight characteristic active compounds in Mailuoning Injection were found.

C3

C1 C2

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4 3 2 1

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Figure 2. HPLC chromatograms of (1) aqueous extract of Angelica sinensis (WEAS), (2) desorption eluate of biomembrane interacted with WEAS, (3) final washing eluate of biomembrane interacted with WEAS and (4) desorption eluate of biomembrane interacted with phosphate buffer (from Ref. [11], with permission).

Angelica sinensis is a commonly used CM for tonifying blood and treating female irregular menstruation and amenorrhoea. It is also used for treatment of anemia, hypertension and cardiovascular diseases. Its active compounds were also hypothesized by using red cell membrane biospecific extraction [11]. Comparing HPLC chromatograms of water extract of Angelica sinensis (WEAS) and desorption eluate of biomembrane interacted with WEAS, four principal peaks, C1, C2, C3 and C4 were detected (Fig. 2). The components of peaks could be directly identified by MS, and peaks C1 and C4 were identified as ferulic acid and ligustilide, respectively. Peaks C2 and C3 were separated and purified by RP-HPLC according to their chromatograms and finally identified as senkyunolide-H and senkyunolideI. This technique is a feasible and flexible for screening active components in CMs.

4. Live Cell Biospecific Extraction Natural cellular receptors, mainly membrane-associated proteins, play their functions by binding specific ligands in a way that results in conformational change in the protein structure and then triggers a cellular response. According to their structures and functions in the cells, membrane receptors can be categorized into four different classes: ion-channel, G-proteinlinked, single transmembrane domain and enzyme-linked receptors [54]. Actually, live cells may be more suitable for the cell-compound interactions because cell membrane preparation may damage the structures and functions of proteins. Several methods have been reported to immobilize cells [55-59]. But the same problem of incompatibility between the solutemembrane interaction and the chromatographic separation still exists. Therefore, cell biospecific extraction followed by chromatographic analysis attracted more attention for the hypothesis of active components in CMs [14-16].

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

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Figure 3. HPLC chromatograms of (A) aqueous extract of Cordyceps sinensis (WECS) with RPMI 1640 solution, (B) supernatant of WECS after incubation with macrophage, (C) supernatant of WECS after incubation with heat denatured macrophage, and (D) supernatant of WECS after incubation with human hepatoma SMMC-7721 cell (from Ref. [16], with modification and permission). 1, uracil; 2, cytidine; 3, guanine; 4, uridine; 5, guanosine; 6, adenosine.

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Cell Materials Biospecific Extraction for Hypothesis of Active Components…

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Macrophage biospecific extraction and high performance liquid chromatography was developed for screening potential immunological active components in Cordyceps sinensis, a well-known and valued CM [16]. Comparing HPLC chromatograms of aqueous extract of C. sinensis (WECS) before and after biospecific extraction with macrophage, it was found that there were two peaks on chromatogram of WECS significantly decreased or even disappeared after the interaction (Fig. 3). This interaction was specific because the chromatograms of WECS were same before and after incubating with heat denatured macrophage or human hepatoma SMMC-7721 cell. These two compounds were identified as guanosine and adenosine with the help of UV spectra, MS data and reference standards. Their effects on mice macrophage were also investigated in vitro, which suggested that adenosine and guanosine had immune modulation activities. In some cases, the combined compounds may not be detected in desorption eluate of sample mixture after interacting with cells, which suggest that the compounds maybe enter into the cell instead of combination with cell membrane. If so, the compounds may be detected in cell digestion extract [15].

5. Conclusion

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Cell materials biospecific extraction, based on the modern pharmacological mechanism, offers a powerful tool for hypothesis of potential active components in CMs. It is flexible to chromatographic separation and MS detection. Commercial biospecific extraction cell materials such as immobilized proteins and nucleic acids may be developed in future. Especially, live cell biospecific extraction is effective and promising, which is well consistent with the characteristic of multi-component and multi-target action of CMs. Hyphened techniques such as GC/LC-MS and LC-NMR-MS will be intensively applied for the structural elucidation of the cell-combining components.

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

Newman, D. J.; Cragg, G. M.; Snader, K. M. J. Nat. Prod. 2003, 66, 1022-1037. Bobinski, H.; Stein, W. D. Nature 1966, 211, 1366-1368. Wang, H.; Zou, H. F.; Ni, J.; Guo, B. Chromatographia 2000, 52, 459-464. Wang, H.; Kong, L.; Zou, H. F.; Ni, J.; Zhang, Y. Chromatographia 1999, 50, 439-445. Wang, H.; Zou, H.; Ni, J.; Kong, L.; Gao, S.; Guo, B. J. Chromatogr. A 2000, 870, 501510. Lundqvist, A.; Lundahl, P. J. Biochem. Biophys. Methods 2001, 49, 507-521. Ng, W.; Dai, J. R.; Slon-Usakiewicz, J. J.; Redden, P. R.; Pasternak, A.; Reid, N., J. Biomol. Screen 2007, 12, 167-174. Haneskog, L.; Zeng, C. M.; Lundqvist, A.; Lundahl, P. Biochim. Biophys. Acta 1998, 1371, 1-4. Lundqvist, A.; Lundahl, P., J. Chromatogr. B: Biomed. Sci. Appl. 1997, 699, 209-220. Gottschalk, I.; Lagerquist, C.; Zuo, S. S.; Lundqvist, A.; Lundahl, P. J. Chromatogr. B: Analyt. Technol. Biomed. Life Sci. 2002, 768, 31-40. Dong, Z. B.; Li, S. P.; Hong, M.; Zhu, Q. J. Pharm. Biomed. Anal. 2005, 38, 664-669. Fan, H. W.; Yu, L.; Hong, M.; Zhu, Q. Zhongguo Yao Xue Za Zhi 2004, 39, 375-378.

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[13] Fan, H. W.; Zhu, Q.; Hong, M.; Yu, L. Zhongguo Yao Xue Za Zhi 2006, 41, 63-66. [14] Li, S. L.; Li, P.; Sheng, L. H.; Li, R. Y.; Qi, L. W.; Zhang, L. Y. J. Pharm. Biomed. Anal. 2006, 41, 576-581. [15] Zhang, H. Y.; Hu, C. X.; Liu, C. P.; Li, H. F.; Wang, J. S.; Yuan, K. L.; Tang, J. W.; Xu, G. W. J. Pharm. Biomed. Anal. 2007, 43, 151-157. [16] Yu, L.; Zhao, J.; Zhu, Q.; Li, S. P. J. Pharm. Biomed. Anal. 2007, 44, 439-443. [17] Wang, X.; Li, R. Z.; Ha, G. Q. Beijing Yi Ke Da Xue Xue Bao 1982, 14, 372-375. [18] Wang, X.; Han, G. Q.; Li, R. Z.; Pan, J. X.; Chen, Y. Y.; He, Y. Q.; Tu, F.; Wang, F.; Huang, L.; Lee, C.; Sandrino, M.; Chang, M. N.; Shen, T. Y. Beijing Yi Ke Da Xue Xue Bao 1986, 18, 31-38. [19] Luo, H.; Chen, L.; Li, Z.; Ding, Z.; Xu, X. Anal. Chem. 2003, 75, 3994-3998. [20] Schenk, T.; Appels, N. M. G. M.; van Elswijk, D. A.; Irth, H.; Tjaden, U. R.; van der Greef, J., Anal. Biochem. 2003, 316, 118-126. [21] Katayama, H.; Oda, Y. J. Chromatogr. B 2007, 855, 21-27. [22] Zhang, B.; Palcic, M. M.; Schriemer, D. C.; Alvarez-Manilla, G.; Pierce, M.; Hindsgaul, O. Anal. Biochem. 2001, 299, 173-182. [23] Chan, N. W. C.; Lewis, D. F.; Rosner, P. J.; Kelly, M. A.; Schriemer, D. C. Anal. Biochem. 2003, 319, 1-12. [24] Ng, E. S. M.; Yang, F.; Kameyama, A.; Palcic, M. M.; Hindsgaul, O.; Schriemer, D. C. Anal. Chem. 2005, 77, 6125-6133. [25] Hodgson, R. J.; Chen, Y.; Zhang, Z.; Tleugabulova, D.; Long, H.; Zhao, X. M.; Organ, M.; Brook, M. A.; Brennan, J. D. Anal. Chem. 2004, 76, 2780-2790. [26] Rich, R. L.; Hoth, L. R.; Geoghegan, K. F.; Brown, T. A.; LeMotte, P. K.; Simons, S. P.; Hensley, P.; Myszka, D. G. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 8562-8567. [27] Woodbury, C. P.; Venton, D. L. J. Chromatogr. B: Biomed. Sci. Appl. 1999, 725, 113-137. [28] Cummins, L. L.; Chen, S.; Blyn, L. B.; Sannes-Lowery, K. A.; Drader, J. J.; Griffey, R. H.; Hofstadler, S. A. J. Nat. Prod. 2003, 66, 1186-1190. [29] Su, X.; Qin, F.; Kong, L.; Ou, J.; Xie, C.; Zou, H., J. Chromatogr. B 2007, 845, 174-179. [30] Su, X.; Hu, L.; Kong, L.; Lei, X.; Zou, H. J. Chromatogr. A 2007, 1154, 132-137. [31] Tian, R.; Xu, S.; Lei, X.; Jin, W.; Ye, M.; Zou, H. Trends Analyt. Chem. 2005, 24, 810-825. [32] Bonsall, R. W.; Hunt, S. Nature 1966, 211, 1368-1370. [33] Brekkan, E.; Lundqvist, A.; Lundahl, P., Biochemistry 1996, 35, 12141-12145. [34] Lundqvist, A.; Lundahl, P. J. Chromatogr. A 1997, 776, 87-91. [35] Beigi, F.; Gottschalk, I.; Hägglund, C. L.; Haneskog, L.; Brekkan, E.; Zhang, Y. X.; Sterberg, T. Ö.; Lundahl, P. Int. J. Pharm. 1998, 164, 129-137. [36] Lundqvist, A.; Lundahl, P. J. Chromatogr. A 1999, 852, 93-96. [37] He, L. C.; Wang, S. C.; Geng, X. D. Chromatographia 2001, 54, 71-76. [38] Zhao, H. R.; Yang, G. D.; He, L. C.; Yang, Y. J. Zhongguo Yao Xue Za Zhi 2000, 35, 13-15. [39] Zhang, H. L.; Yang, G. D.; He, L. C.; Yang, Y. J. Zhongguo Yao Xue Za Zhi 2003, 38, 92-94. [40] Zhao, X.; Dang, G..; Yang, G.; He, L. Fen Xi Hua Xue 2002, 30, 195-197. [41] Wang, R. P.; Chen, Q.; He, L. C. Shaanxi Zhong Yi 2003, 24, 553-554.

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Gao, K.; Yang, G. D.; He, L. C. Zhongguo Yao Xue Za Zhi 2003, 38, 14-16. Chen, Q.; Wang, R. P.; He, L. C.; Xu, D. Q. Shaanxi Zhong Yi 2004, 25, 643-644. Liang, M. J.; He, L. C.; Yang, G. D. Life Sci. 2005, 78, 128-133. Lin, R.; Yang, G. D.; Wang, W. R.; Lin, J. T.; He, L. C.; Peng, N.; Han, C. J.; Liu, Y., Zhong Yao Cai 2006, 29, 1315-1317. Li, Y. P.; He, L. C. Ke Xue Tong Bao 2007, 52, 410-415. Li, C. Q.; He, L. C. Zhongguo Ke Xue C Sheng Ming Ke Xue 2005, 35, 545-550. Zhang, B.; He, L. C. Xian Dai Yi Yao Wei Sheng 2006, 22, 2303-2304. Zhang, Y. J.; He, L. C. Zhongguo Yao Xue Za Zhi 2005, 40, 463-465. Zhang, D.; Yuan, B.; Deng, X.; Yang, G.; He, L.; Zhang, Y.; Han, Q., Zhongguo Ke Xue (C) 2004,34, 173-177. Wang, Y.; Yuan, B.; Deng, X.; He, L.; Wang, S.; Zhang, Y.; Han, Q. Anal. Bioanal. Chem. 2006, 386, 2003-2011. Wang, Y.; Yuan, B.; Deng, X.; He, L.; Zhang, Y.; Han, Q. Anal. Biochem. 2005, 339, 198-205. Hou, J.; Yuan, B. X.; He, L. C.; Yang, G. D.; Mi, M. Zhongguo Yao Li Xue Yu Du Li Xue Za Zhi 2003, 17, 70-73. Subrahmanyam, S.; Piletsky, S. A.; Turner, A. P. Anal. Chem. 2002, 74, 3942-3951. Zeng, C. M.; Zhang, Y.; Lu, L.; Brekkan, E.; Lundqvist, A.; Lundahl, P. Biochim. Biophys. Acta 1997, 1325, 91-98. Jacobson, B. S.; Branton, D. Science 1977, 195, 302-304. Gottschalk, I.; Li, Y. M.; Lundahl, P. J. Chromatogr. B: Biomed. Sci. Appl. 2000, 739, 55-62. Gottschalk, I.; Gustavsson, P. E.; Ersson, B.; Lundahl, P. J. Chromatogr. B 2003, 784, 203-208. Gottschalk, I.; Lundqvist, A.; Zeng, C. M.; Hägglund, C. L.; Zuo, S. S.; Brekkan, E.; Eaker, D.; Lundahl, P. Eur. J. Biochem. 2000, 267, 6875-6882.

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 3

Recent Development on Sample Preparation for Quality Control of Chinese Herbs Hua Yu1,2, Feng-qing Yang1, Yi-fan Han3 and Shao-ping Li1,* 1

2

Institute of Chinese Medical Sciences, University of Macau Department of Biochemistry, The Hong Kong University of Science and Technology 3 Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University

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1. Introduction Pharmaceutical analysis plays an important role in the quality control of Chinese herbs, which can be designed to provide qualitative data and quantitative measurement. During the process, sample preparation is one of the key steps which greatly influences the repeatability and accuracy of the analysis. It is reported that 70-80% of analysis time is spent on sample preparation and more than 60% of analysis error derived from nonstandard sample pretreatment. Therefore, a proper sample preparation approach is very important for analysis. Most samples are not ready for direct introduction into instruments. For example, in the analysis of chemical compounds in Chinese herbs, it is not possible to analyze the herb directly. The compounds have to be extracted into a solution which can be analyzed by an instrument. Therefore, the principal objectives of sample preparation for quality analysis are: isolation of the analytes of interest from as many interfering compounds as possible, dissolution of the analytes in a suitable solvent and preconcentration. Sample extraction encompasses all those steps necessary to prepare the original sample for determination by the chosen method. Strategically, the extraction method chosen can influence the entire process, and will depend on: the physicochemical properties of the analyte(s), the nature of the matrix, the objectives of the analysis, and the limit of determination. Many extraction practices are *

E-mail address: [email protected]. Tel: 853-83974692. Fax: 853-28841358 (Corresponding author.)

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Hua Yu, Feng-qing Yang, Yi-fan Han et al.

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100

450

90

400

80

350

70

300

60

250

50

200

40

150

30 20

100

10

50

0

2001

2002

2003

2004

2005

2006

2007

Article numbers for SPE and SPME

Article numbers for PLE, SFE, MAE, LPME, ME and UE

based on classical methodologies of liquid/liquid or liquid/solid extraction. Sample extraction can be a time consuming, labor intensive process. Recent years, considerable efforts have been made to develop alternative and improved methods of extraction, and to make them commercially available. Some extraction methods are listed in Table 1. Fig. 1 showed their applications in sample preparation during last years. Their development for sample preparation of traditional Chinese medicine [1], food [2, 3], plant material [4] and nutraceuticals from plants [5] have been reviewed.

0

PLE (◆), SFE (◇), MAE (△), LPME (▲), ME (●), UE (○), SPE (■) and SPME (□) Source: The data collected from Web of Science.

Figure 1. The application trends of selected extraction methods for sample preparation viewed on publications growth during 2001-2007.

Table 1. Summary of extraction technologies for quality control of Chinese herbs Primary sample matrix Solid

Primary extraction phase Liquid

Classical Soxhlet

Liquid

Liquid

Solvent extraction

New technologies PLE MAE SFE UE SPE SPME LPME ME

PLE, Pressured liquid extraction; MAE, Microwave-assisted solvent extraction; SFE, Supercritical fluid extraction; UE, Ultrasonic extraction; SPE, Solid-phase extraction; SPME, Solid-phase microextraction; LPME, Liquid-phase microextraction; ME, Membrane extraction

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Recent Development on Sample Preparation for Quality Control of Chinese Herbs

23

2. Automated Soxhlet Extraction The Soxhlet extraction is a method used to extract solvent-soluble fractions from a solid medium. It was first introduced by the German chemist, Franz von Soxhlet, in 1879. Soxhlet has been a standard technique during more than one century, and at present, it is the main reference for evaluation of the performance of other leaching methods. There is a wide variety of official methods involving a sample preparation step based on Soxhlet extraction (Table 2). However, Soxhlet extraction has several significant drawbacks: 1) long time and large amount of solvent are required; 2) potential thermal decomposition of the target compounds cannot be ignored; 3) agitation cannot be provided for the acceleration of the process; 4) evaporation/concentration step after the extraction is mandatory due to the large amount of solvent used; 5) the technique is restricted to solvent selectivity and is not easily automated. Therefore, the conventional Soxhlet device, either the design or the operational procedure has been modified [6]. Table 2. Methods accepted as standards for the extraction of analytes Techniques

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Soxhlet extraction

Analytes

Standard Method

Semivolatile and nonvolatile organics

EPA 3540C

Fat in cacao products

AOAC 963.15

Polynuclear Aromatic Hydrocarbons

EPA 8100

Oilseeds

ISO 659-1988 (E)

Semivolatile and nonvolatile organics

EPA 3541

Semivolatile and nonvolatile organics

EPA 3545A

Semivolatile and nonvolatile organics

EPA 3546

Elements

ASTM D-5258

Total petroleum hydrocarbons

ASTM D-5765

Organic compounds

ASTM D-6010

Fat/Crude Fat

AOAC 985.15

Ultrasonic extraction (UE)

Organic

EPA 3550B

Nonvolatile and semivolatile organic

EPA 3550C

Supercritical fluid extraction (SFE)

Oil in oilseeds

AOAC 999.02

Semivolatile petroleum hydrocarbons

EPA 3560

Polynuclear Aromatic Hydrocarbons

EPA 3561

Polychlorinated Biphenyls and Organochlorine Pesticides Organic compounds Semivolatile and Nonvolatile Organics Glycoalkaloids (α-Solanine and α-Chacoine)

EPA 3562

Automated Soxhlet extraction Pressurized fluid extraction (PFE) Microwave-assisted extraction (MAE)

Solvent extraction Solid-phase extraction (SPE)

EPA 3511 EPA 3535 AOAC 997.13

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Hua Yu, Feng-qing Yang, Yi-fan Han et al.

④

②

①

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Figure 2. The five core elements of Büchi Extraction System B-811: (1) lower and (2) upper heater, (3) glass valve, (4) optical sensor, and (5) inert gas supply.

In order to develop liquid/liquid extractions, the conventional Soxhlet extractor has been adapted for the continuous extraction of a liquid with either a lighter or heavier solvent. Its performance is similar to a combination of functions of a separating funnel and a Soxhlet extractor, working in a manual or automated way. Fig. 2 shows an automated system (Büchi Extraction System B-811) which can be used to perform an extraction according to the original Soxhlet principle. Four different extraction methods are possible without making any changes to the unit: Soxhlet standard, Soxhlet warm, hot extraction and continuous extraction. The system has an inert gas supply to avoid oxidation during extraction and to accelerate the evaporation and drying process even with high boiling point solvents (up to 150°C). Whether it’s Soxhlet extraction or hot extraction, there is no need of assembly conversions. Every extraction mode can be set up with three main steps – extraction, flushing and drying – which are computer controlled guaranteeing reproducible conditions and reliable results [7]. In addition, combined with other technique, a novel microwave-assisted Soxhlet has been developed for extraction of fatty acid from acorn [8] and lipids from food products [9].

3. Pressurized Liquid Extraction (PLE) PLE is a new technique which was developed for sample pretreatment in recent years [10]. It was performed at elevated temperatures and high pressure which maintain the heated solvent in a liquid state during the extraction process. Therefore, PLE offers many advantages, such as short extraction time, little solvent consumption and high extraction efficiency. Benthin et al [11] were the first to conduct a comprehensive study on the feasibility of applying PLE to medicinal herbs. Generally, the major parameters which influence the extraction efficiency are type of solvent, particle size, temperature, static extraction time, flush volume and extraction cycle [12-14].

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Hubei

) cyperene, (

) β-selinene, (

D PL E SF E

Shandong

25

H

H D PL E SF E

Anhui

H

D PL E SF E

Percentage

100

H D PL E SF E

Recent Development on Sample Preparation for Quality Control of Chinese Herbs

80 60 40 20 0

(

) α-copaene, (

) β-cyperone and (

Zhejiang ) α-cyperone

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Figure 3. Comparison of 5 volatile compounds in different C. rotundus extracted by HD, SFE and PLE (from Ref. [22] with permission).

Optimization of PLE conditions has been reported using univariate [15-26] central composite [27-29], matrix [30] and orthogonal array [31] designs. It was reported that the extraction efficiency of strychnine in Strychnos nux-vomica increased about 4 folds when the temperature increased from 80 oC to 140 oC. The solvent required in PLE was approximately six times less and extraction time required was approximately 20-fold faster compared to Soxhlet extraction [32]. The amount of solvent and time required for PLE extraction of glycyrrhizin in Radix Glycyrrhizae was about half of those required by ultrasonic extraction [33]. Especially, the reproducibility of PLE is generally much better than that of conventional extraction methods [11,34,35]. Therefore, PLE is a good alternative method for sample preparation of Chinese herbs quality analysis (Table 3). It is noteworthy that different extraction methods have individual selectivity to the analytes. The investigation showed the contents of the five volatile components (α-copaene, cyperene, β-selinene, β-cyperone and αcyperone) in Cyperus rotundus extracted by hydrodistillation (HD), PLE and supercritical fluid extraction (SFE) are obviously different (Fig. 3). PLE had the highest extraction efficiency, while SFE had the best selectivity for extraction of β-cyperone and α-cyperone though its extraction yield was lower. HD may have more variation or be easily influenced by sample matrix because of its non-automated controlled parameters. Therefore, HD extraction efficiency was variant for individual compound in different raw materials [22]. Table 3. Application of PLE in sample preparation for the analysis of herbs Analytes Chloroacetanilide herbicides

Source Radix Pseudostellariae

Parameters Solvent: acetone-hexane (1:1, v/v) Particle size: not mentioned Temperature: 140 oC Static extraction time: 5 min Pressure: not mentioned Cycle: 1

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Ref. [18]

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Hua Yu, Feng-qing Yang, Yi-fan Han et al.

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Table 3. Continued Analytes Volatile components

Source Angelica roots

Parameters Solvent: n-hexane Particle size: not mentioned Temperature: 80 oC Static extraction time: 10 min Pressure: 1500 atm Cycle: 2

Ref. [23]

Essential oil

Pistacia Vera L.

Solvent: n-hexane Particle size: 40-60 mesh Temperature: 80 oC Static extraction time: 5 min Pressure: 10 bar Cycle: not mentioned

[56]

Z-ligustilide, Zbutylidenephthalid e and ferulic acid

Angelica sinensis

[27]

Non-polar compounds

Piper gaudichaudianum Kunth leaves

Solvent: MeOH Particle size: 0.125-0.2 mm Temperature: 110 oC Static extraction time: 25 min Pressure: 1500 psi Cycle: 1 Solvent: petroleum ether Particle size: ground leaves Temperature: 85 oC Static extraction time: 10 min Pressure: 10.34 Mpa Cycle: 1

Coumarins and furanocoumarins

Ammi majus L.

Solvent: chloroform followed by MeOH Particle size: not mentioned Temperature: 100 oC Static extraction time: 5min Pressure: 7MPa Cycle: 3

[50]

Sesquiterpenes

Curcuma rhizomes

Solvent: MeOH Particle size: 0.2-0.3 mm Temperature: 120 oC Static extraction time: 5 min Pressure: 1500 psi Cycle: 1 Solvent: MeOH Particle size: 0.2-0.3 mm Temperature: 100 oC Static extraction time: 5 min Pressure: 1000 psi Cycle: 1

[21]

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[28]

[37] [38]

Recent Development on Sample Preparation for Quality Control of Chinese Herbs Table 3. Continued

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Analytes Terpenes

Source Cyperus rotundus

Parameters Solvent: MeOH Particle size: 0.2-0.3 mm Temperature: 140 oC Static extraction time: 10 min Pressure: 1000 psi Cycle: 1

Ref. [22]

Rhizome (Jianghuang) and tuberous root (Yujin) of Curcuma longa

Solvent: MeOH Particle size: 0.15-0.20 mm Temperature: 140 oC Static extraction time: 5 min Pressure: 1000 psi Cycle: 1

[25]

Pogostemon cablin

Solvent: MeOH Particle size: 0.154 mm Temperature: 80 oC Static extraction time: 15 min Pressure: 1500 psi Cycle: 1

[49]

Triterpenes and sterols

Ganoderma lucidum, Ganoderma sinense

Solvent: MeOH Particle size: 0.2 mm Temperature: 100 oC Static extraction time: 5 min Pressure: 1500 psi Cycle: 1

[57]

Polycyclic aromatic hydrocarbons

Pine needles

Solvent: hexane-dichloromethane (1:1, v/v) Particle size: not mentioned Temperature: 150 oC Static extraction time: 10 min Pressure: 1500 psi Cycle: 2

[41]

Genipin-1-βgentiobioside, geniposide, gardenoside

Gardenia fruit

Solvent: MeOH-H2O (1:1, v/v) Particle size: not mentioned Temperature: 120 oC Static extraction time: 3 min Pressure: 12 Mpa Cycle: 3

[42]

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28

Hua Yu, Feng-qing Yang, Yi-fan Han et al. Table 3. Continued

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Analytes Saponins

Source Panax notoginseng

Parameters Solvent: MeOH Particle size: 0.3-0.45 mm Temperature: 150 oC Static extraction time: 15 min Pressure: 6.895×103 KPa Cycle: 1

Aesculus chinensis Bunge

Solvent: 70% (v/v) aqueous MeOH Particle size: 0.3mm Temperature: 120 oC Static extraction time: 7min Pressure: not mentioned Cycle: 2

[17]

Hyperoside, quercitrin, pseudohypericin, hyperforin and hypericin

Hypericum species

Solvent: MeOH Particle size: not mentioned Temperature: 40 oC Static extraction time: 5 min Pressure: 100 bar Cycle: 4

[51]

Saponins and fatty acids

Ziziphus jujuba (Suanzaoren)

Solvent: MeOH-ethyl acetate (95:5, v/v) Particle size: 40-60 mesh Temperature: 140 oC Static extraction time: 15 min Pressure: 1200 psi Cycle: 2

[26]

Rotenone

Derris elliptica and Derris malaccensis

[20]

Flavanoids

Lysimachia clethroide

Solvent: chloroform Particle size: 5 mm cubes Temperature: 50 oC Static extraction time: 6 min Pressure: 2000 psi Cycle: 1 Solvent: 50% aqueous ACN Particle size: not mentioned Temperature: 100 oC Static extraction time: 25 min Pressure: 1500 psi Cycle: 1 Solvent: 70% EtOH Particle size: 60-80 mesh Temperature: 120 oC Static extraction time: 10 min Pressure: 1500 psi Cycle: 1

Epimedium

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Ref. [34, 4346]

[29]

[16]

Recent Development on Sample Preparation for Quality Control of Chinese Herbs

29

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Table 3. Continued Analytes Naphthodianthron es and flavonoids

Source St. John’s wort (Hypericum perforatum L.)

Isoflavones

Radix Puerariae

Limonoid derivatives

Cortex Dictamni

Glycyrrhizin and ephedrine

Radix. glycyrrhizae and Herba Ephedrae

Alkaloids (Berberine and aristolochic acids)

Coptidis rhizoma and Radix aristolochiae

Intact glucosinolates

Isatis tinctoria leaves

Vitamin E

Lyophilized Corylus avellana L. nut

Parameters Solvent: MeOH, tetrahydrofuran, acetone for different compounds Particle size: 80 mesh Temperature: 150 oC Static extraction time: 5min Pressure: 2200 psi Cycle: 3 Solvent: 95% EtOH Particle size: 143.8 μm Temperature: 100 oC Static extraction time: 10 min Pressure: 1400 psi Cycle: 1 Solvent: MeOH Particle size: not mentioned Temperature: 150 oC Static extraction time: 5 min Pressure: 1500 psi Cycle: 1 Solvent: 0.4% of SDS and1.0% of Triton X-100 in 1 L of water Particle size: 0.5 mm Temperature: room temperature Static extraction time: 45-50 min Pressure: 10-20 bar Cycle: not mentioned Solvent: MeOH Particle size: 50%) those high molecular weight molecules of over 150 kDa, which were rather distinct as compared to the cultured products [65] There are several polysaccharides have been isolated from cultured Cordyceps, but few report on the polysaccharides from natural one (Table 3). 3.4. Amino Acid, Peptide and Protein Over 20% (w/w) of amino acid is found in Cordyceps, which could be responsible for the tonic and immuno-potentiating activity of Cordyceps [66]. But the content of total amino acids in cultured Cordyceps was lower than that in genuine natural Cordyceps [67]. Peptide,

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Kun Feng, Feng-qing Yang and Shao-ping Li

which consists of amino acids, usually shows biological activities. Several cyclo-diapeptides, with anti-tumor and immuno-enhancing activities, have been isolated from cultured Cordyceps [68]. The soluble proteins of natural and cultured Cordyceps were analyzed using polyacrylamide gel electrophoresis (PAGE), which indicated that the soluble protein zones of natural and cultured Cordyceps were obviously different [69]. The content of total water soluble protein in cultured Cordyceps mycelium was much higher than that in natural Cordyceps. SDS-PAGE analysis showed that there were 9 main sub-units of protein in natural Cordyceps, but 7 main sub-units in cultured one. The molecular weights of their common sub-units were 46971, 51087, 86681 and 95685, respectively [70]. Table 3 gave a summary of the major components in natural and cultured Cordyceps.

4. Pharmacological Activities of Cultured Cordyceps

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Up to date, there are many studies focus on the pharmacological activities and clinical therapeutic effects of C. sinensis have been reported. In 1998, Zhu et al [71, 72] systematically reviewed its preclinical in vitro and in vivo studies and clinical blinded or open-label trials. The beneficial effects of Cordyceps to the treatment of respiratory tract, kidney, cardiovascular system, liver, immune system, lipid and glucose metabolism diseases, and cancer could attribute to its multiple biological activities, including anti-asthma, improvement and protection of renal and liver function, antioxidation, antisenescence, improvement of blood circulation, anti-arrhythmia, immune potentiation or inhibition, antiinflammation, and antiatherosclerotic, hypolipidemic and hypoglycemic actions, etc. Since then, a few reviews [3, 73-78] and a great number of pharmacological/biological studies have been reported. According to the published articles, several major pharmacological effects of cultured Cordyceps are summarized as below. 4.1. Immunomodulatory and Anti-inflammatory Activity Cordyceps has bilateral effects on immune functions. The immune potentiaton [71, 7375, 77, 79-82] is achieved through: 1) enhancing phagocytosis of macrophage; 2) inducing increases of spleen weight, RNA, DNA and protein biosynthesis; 3) enhancing NK cell activity; 4) enhancing the production of interleukin-1 (IL-1), IL-2, IL-6, IL-10, IL-12, interferon (IFN), and tumor necrosis factor-α (TNF-α); 5) inducing transformation of lymphocytes, and increasing CD4+ and CD8+ T cells; 6) increasing the number of B cells and production of IgM, IgG, IgG1 and IgG2b; and 7) increasing the activity of acid phosphatase and arginase in alveolar macrophages. In contrast, the evidences for immune inhibition of cultured Cordyceps are [71, 73, 83-87]: 1) inhibiting proliferation of monocytes and lymphocyte; 2) inhibiting mixed lymphocyte reaction (MLR); 3) down-regulating the ratio of Th/Ts; 4) reducing production of IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-γ, cyclin E and tumour necrosis factor-α; 5) increasing the expression of soluble intercellular adhesion molecule-1 (sICAM-1); 6) arresting cell cycle at G1 phase in MLR and inhibiting the function of dentritic cells (DCs); 7) reducing the expression of AP-1 proteins, consisting of c-Fos and c-Jun, in activated T lymphocytes. C. sinensis mycelium extract also protected mouse against

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Group A streptococcus (GAS) infection by decreasing bacterial growth and dissemination, increasing IL-12 and IFN-a expression, and macrophage phagocytic activity [88]. The compounds related to immune modulation include nucleosides [89], polysaccharides [71, 73, 81, 90], sterols [86, 91] and peptides [92]. The different effects may derive from the variation of the contents and ratios of these compounds in different mycelia and/or extracts of Cordyceps (Table 2). Over-expression of the inflammatory mediators in macrophage is involved in many diseases, such as rheumatoid arthritis, atherosclerosis, chronic hepatitis and pulmonary fibrosis [93]. C. sinensis could bring about a down-regulation of inflammationrelated genes such as MCP-1 and TNF-α in the rat kidney following ischaemia/reperfusion [94]. It also reduced the increase of TNF-α and IL-8 in alveolus of chronic obstructive pulmonary diseases (CPOD) rats [95], and airway inflammation in sensitized rats [96]. The mechanisms of anti-inflammatory effects of Cordyceps may be related to its inhibition of nuclear transcription factor NF-κB activation [97] and inducible NO synthase expression [98].

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4.2. Effects on the Kidney Traditionally, Cordyceps sinensis is considered as a “kidney tonic” herb. It has been extensive used in the treatment of chronic renal diseases, including chronic nephritis, chronic pyelonephritis, chronic renal dysfunction or failure, and nephritic syndrome. These therapeutic efficacy can attribute to its kidney functions improvement and prevention against the damage caused by certain nephrotoxic chemicals [71, 73]. Ischemia/reperfusion (I/R) injury as a cause of delayed graft function is a major deterrent to long-term renal allograft survival [99]. C. sinensis extract significantly improved renal function and reduces the expression of inflammatory (MCP-1 and TNF-α) and apoptotic genes (Fas, FasL and caspase-3) in rat subjected to 60 minutes of ischemia and 3 days of reperfusion [94]. LDL may play a critical role in mediating mesangial cell hypertrophy or proliferation involved in the development of glomerulosclerosis. C. sinensis could inhibit the proliferation of cultured human glomerular mesangial cell induced by LDL [100]. It also decreased mortality ratio of chronic renal failure rats with 5/6 nephrectomy, declined level of serum creatinine (Scr) and blood urea nitrogen (BUN), delay the degree of glomerular sclerosis [101], down-regulated the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1) and TIMP-2 [102]. In patients with chronic renal failure (CRF), treatment with cultured Cordyceps (Cs-4, JinShuiBao) significantly improved renal functions, which showed reducing BNU and Scr, along with a very noticeable increasing total blood protein and calcium [103]. Furthermore, cultured Cordyceps polysaccharide (CP) could enhance the activity of superoxide dismutase (SOD) and decrease the content of malondialdehyde (MDA) in rat with CRF induced by freezing, which indicated that CP can ameliorate the renal injury by inhibiting lipid oxidation [104]. In addition, the protection effects of Cordyceps on kidney may closely related to the improvement of cell-mediated immune function and a delay in the deterioration of renal functions [105].

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4.3. Effects on the Cardiovascular System Cordyceps have broad effects on cardiovascular system, which includes: 1) dilation of arteries and improvement of nutritional blood supply to organs and extremities, 2) reducing the heart rate, 3) antiarrhythmic effects, 4) against acute myocardial ischemia and stressinduced myocardial infarction, and 5) anti-platelet aggregation, etc [71]. An aqueous extract of cultured C. sinensis mycelia, dialyzed overnight against buffer using a membrane cut off size of 3.5 kDa, showed hypotensive effect on anaesthetized rats and vaso-relaxant effect on isolated aorta [106]. Pre-treatment of petroleum extract of cultured Cordyceps by oral administration significantly antagonized arrhythmia in rats induced by aconitine and barium chloride, prolonged the onset time of arrhythmia, decreased the persistence duration and attenuated the degree of severity [107]. The extracts from cultured mycelia of C. sinensis have the protective effect on acute myocardial ischemia induced by pituitrin in rats [108], and myocardial injury induced by adriamycin in vitro [109]. The effects of C. sinensis on cardiovascular system may be correlated with its enhancing nitric oxide synthase (NOS) and superoside dismutase ( SOD) activity [110, 111], elevating intracellular calcium concentration [112] or against calcium overload [113], increasing delayed rectifier and transient outward K+ currents and decreasing inward rectifier K+ current [114], regulating cell immunity function and inducing production of INF-γ [115].

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4.4. Liver Protection Effects Cultured Cordyceps products have been used clinically for the treatment of chronic hepatitis and related disease conditions [116]. C. sinensis may decrease the damage to hepatocyte by CCl4, and inhibit hepatic fibrogenesis [117]. The mycelial extract of cultured Cordyceps sinensis could increase in vivo hepatic energy metabolism by increasing hepatic blood flow [118] and stimulating mitochondrial electron transport and ATP production [119]. The protective effect of Cordyceps on Bacillus Calmette Guerin (BCG)- and lipopolysaccharide-induced liver injury was demonstrated by markedly inhibited the activities of serum alanine and aspartate aminotransferases, decreased the liver- and spleen-enlarged index, reduced the level of lipid peroxide in serum and hepatic tissue, and reduced the level of serum TNF-α [120]. Cultured Cordyceps mycelia could also inhibit hepatic fibrogenesis derived from chronic liver injury, retard the development of cirrhosis, and notably ameliorate the liver function [121]. Its possible mechanism involves inhibiting transforming growth factor (TGF) β1 expression, and thereby, down regulating platelet-derived growth factor (PDGF) expression, preventing HSC activation and deposition of procollagen I and III. 4.5. Anti-tumor Activity C. sinensis has shown beneficial effects in cancer treatment because of its enhancement of cell-mediated immunity, oxygen free radical scavenging and augmentation of the cellular bioenergy system [71]. Besides, the extracts of cultured C. sinensis can inhibit proliferation of different type of cancer cell lines such as Jurkat, MCF-7, B16, HL-60 and HepG2 [98, 122].

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EtOAc extract of cultured C. sinensis also showed significant inhibiting effect on B16induced melanoma in C57BL/6 mice, causing about 60% decrease of tumor size over 27 days[122], while its water extract could inhibit spontaneous liver metastasis of Lewis lung carcinoma and B16 menaloma cells in syngeneic mice [123] . Apoptosis (programmed cell death) is an essential event in organism development and homeostasis. Inducing apoptosis has already proven an efficient method to treat cancer [124]. Supercritical carbon dioxide extract of cultured Cordyceps mycelia selectively inhibited the growth of colorectal carcinoma (HT-29 and HCT 116) and hepatocellular (Hep G2 and Hep 3B) cancer cells by the process of apoptosis [125]. Cultued Cordyceps mycelia water extract also showed apoptotic effects in SP2/0 cells [126, 127]. Recently, the effects of C. sinensis on apoptotic homeostasis have been reviewed [128]. The molecular mechanism of apoptotic effect of C. sinensis may attribute to its activating caspase-8-dependent and caspase-9independent pathways and down regulating NF-κB protein expression [129] and/or involving mitochondrion signal pathway with the loss of MTP (ΔΨm), cytochrome c release into the cytoplasm, the decrease in Bcl-2 protein level, and the translocation of Bax protein from cytoplasm into mitochondria [130]. Actually, inhibitory effect of C. sinensis on apoptosis is also investigated [128], though both water and alcohol extracts of cultured C. sinensis failed to inhibit apoptosis induced by hydrogen peroxide or CH-11, a Fas agonist antibody [131]. Cultured Cordyceps mycelia could alleviate apoptosis, in a non-alcoholic fatty liver disease (NFALD) model rat, by decreasing the level of active oxygen (by augmenting the amount of SOD), lowering the expression of Bax, increasing expression of Bcl-2 and activating NF-κB P65 [132].

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4.6. Anti-oxidation Activity Oxygen free radicals or reactive oxygen species (ROS) are well recognized to play an important role in biological systems and there are increasing evidences that a number of diseases, including cancer, atherosclerosis and cardiovascular diseases, neurodegenerative disorders and senescence are associated with accumulation of an excess of ROS, which results in oxidative damage to cell structures such as lipids and membranes, proteins and nucleic acids [133, 134]. Cordyceps has been applied to ameliorate conditions associated with aging and senescence in China for a long history [72]. The effects of Cordyceps are related to its antioxidant activity, or in part. The antioxidant activity of water extracts from different cultured Cordyceps mycelia was compared using the methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. They showed similar antioxidant activity in all assays. In addition, the antioxidant activities were increased to 1030 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from polysaccharides [63]. Furthermore, a polysaccharide, with molecular weight of ~210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. Treatment of the cells with the isolated polysaccharide at 100 μg/ml prior to H2O2 exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H2O2-induced production of malondialdehyde in cultured cells was markedly reduced, and glutathione peroxidase and superoxide dismutase activities were significantly attenuated by the polysaccharide treatment. These results suggest that the polysaccharide from Cordyceps can

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protect against the free radical-induced neuronal cell toxicity [135]. Another polysaccharide (PS), isolated from one of the anamorph strains of C. sinensis, significantly enhanced superoxide dismutase (SOD) activity of liver, brain and serum as well as glutathione peroxidase (GSH-Px) activity of liver and brain in H22 tumor-bearing mice. PS also significantly reduced the level of malondialdehyde (MDA) in liver and brain of tumor-bearing mice [136]. Hot-water extracts from cultured mycelia of C. sinensis showed the best effect on the inhibition of linoleic peroxidation among six in vitro assays, including inhibition of linoleic acid peroxidation; scavenging abilities on DPPH·, hydroxyl and superoxide anion radicals; the reducing power and the chelating ability on ferrous ions [51]. The extracts of cultured C. sinensis also inhibit accumulation of cholesteryl ester in macrophages via suppression of LDL oxidation [137]. In addition, the fraction R, obtained by supercritical carbon dioxide fluid extractive fractionation, showed strong scavenging free radicals ability [125].

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4.7. Hypoglycemic and Hypolipidemic Activities Studies have shown Cordyceps has obvious effects on metabolism of blood sugar and lipid, which can be applied to the treatment of diseases related with their metabolism disorders [71, 73]. Polysaccharide-enriched fractions from cultured C. sinensis orally administered produced hypoglycemic effects in rats induced by intraperitoneal injection of streptozotocin [138]. A polysaccharide from cultured C. sinensis, named CSP-1, significantly reduced the blood glucose level in normal and alloxan-induced diabetic mice, as well as in STZ-induced diabetic rats [44]. The serum insulin levels in diabetic animals were also increased by administration of CSP-1, which suggested that the hypoglycemic effect of CSP-1 may attribute to its stimulating insulin release from the residual pancreatic cells and/or reducing insulin metabolism in body. Actually, selective damage of islet cells in Type I diabetes could probably be due to low levels of antioxidant enzymes in the pancreas [139, 140]. An antioxidant can prevent the development of diabetes [141]. CSP-1 with antioxidant activity [135] may protect β-cells of islets of Langerhans in pancreas against oxidative damage result in serum insulin increase. Similar effects were also investigated [142]. Besides, the hypoglycemic mechanism may also be in association with the increase of glucose utilization in skeletal muscle [143] and cellular glucose uptake [144]. Hot-water extract of cultured C. sinensis mycelia, which contained 83.9% carbohydrate and 11.8% protein, decreased the total cholesterol, very low-density lipoprotein plus lowdensity lipoprotein (VLDL+LDL) cholesterol levels, and increased the high-density lipoprotein (HDL) cholesterol level in the serum of mice fed the cholesterol-enriched diet [145]. On the other hand, the water extracts of the fruiting bodies of cultured C. sinensis (WECS) could significantly suppress the increased serum lipid peroxide and aortic cholesteryl ester, a major lipid constituent in atherosclerotic lesions, levels in mice fed an atherogenic diet. The results suggest that WECS prevents cholesterol deposition in the aorta by inhibition of LDL oxidation mediated by free radicals rather than by reduction in serum lipid level [137]. In a word, cultured C. sinensis may exert beneficial effects on the formation of the atherosclerotic lesions.

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5. Conclusion As demand for Cordyceps sinensis products grows and the supply of wild material declines, mycelium of the asexual (anamorphic) stage grown under artificial culture conditions is increasingly used in medicinal products. However, there is no consensus strain and it is showed that certain populations contain different biologically active compounds, which may result in controversial effects in clinic. Therefore, definite species of fungus strain rather than cultured Cordyceps sinensis (mycelium) is necessary for clearly elucidation of its pharmacological effects and quality.

Acknowledgements The research was supported by grant from Macao Science and Technology Development Fund (077/2005/A and 028/2006/A2) to S. P. Li.

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[70] Shi, J. H.; Dang, H. N.; Wang, Y.; Bao, C. J., Yao Wu Sheng Wu Ji Shu 2003, 10, 303305. [71] Zhu, J. S.; Halpern, G. M.; Johns, K., J. Alt. Comp. Med. 1998, 4, 429-457. [72] Zhu, J. S.; Halpern, G. M.; Johns, K., J. Alt. Comp. Med. 1998, 4, 289-303. [73] Ng, T. B.; Wang, H. X., J. Pharm. Pharmacol. 2005, 57, 1509-1519. [74] Ma, D. Y.; Li, P.; Ji, H.; Hu, Z. Y., Zhong Yao Cai 2001, 24, 455-458. [75] Luo, Y. X., Zhongguo Shi Yong Jun 2003, 22, 39-42. [76] Hu, Z.; Li, H. P.; Ye, M. Q.; Yu, H. D.; Zou, G. L., An Ji Suan He Sheng Wu Zi Yuan 2003, 25, 20-22. [77] Zhao, K., R.; Wang, W. T.; Zhao, Z. Y., Guo Wai Yi Yao Zhi Wu Yao Fen Ce 2006, 21, 105-108. [78] Xu, D. C., Jun Wu Yan Jiu 2006, 4, 60-64. [79] Wei, T.; Tang, F. F.; Guo, Y.; Gong, X. J.; Zhang, P.; Wei, W. L.; Jin, Z. L., Shi Pin Ke Xue 2002, 23, 276-279. [80] Kuo, C. F.; Chen, C. C.; Lin.C.F.; Jan, M. S.; Huang, R. Y.; Luo, Y. H.; Chuang, W. J.; Sheu, C. C.; Lin, Y. S., Food Chem. Toxicol. 2007, 45, 278-285. [81] Wu, Y.; Sun, H.; Qin, F.; Pan, Y.; Sun, C., Phytother. Res. 2006, 20, 646-652. [82] Lee, S. K. W.; Wong, C. K.; Kong, S. K.; Leung, K. N.; Lam, C. W. K., Immunopharmacol. Immunotoxicol. 2006, 28, 341-360. [83] Wang, X. D.; Zhou, Y.; Zhang, L.; Wang, J. F.; Ge, D. Y.; Li, L., Beijing Zhong Yi Yao Da Xue Xue Bao 1998, 21, 34-36. [84] Wang, S. Y.; Meng, X. Q.; Chen, J. H.; Zhang, J. G.; He, Q., Zhongguo Zhong Xi Yi Jie He Za Zhi 2001, 21, 152-153. [85] Ma, L. L.; Yang, X. Y.; Gao, J. Z., Zhongguo Zhong Xi Yi Jie He Za Zhi 2007, 27, 905908. [86] Kuo, Y. C.; Weng, S. C.; Chou, C. J.; Chang, T. T.; Tsai, W. J., Brit. J. Pharmacol. 2003, 140, 895-906. [87] Kuo, Y. C.; Tsai, W. J.; Wang, J. Y.; Chang, S. C.; Lin, C. Y.; Shiao, M. S., Life Sci. 2001, 68, 1067-1082. [88] Kuo, C. F.; Chen, C. C.; Luo, Y. H.; Huang, R. Y.; Chuang, W. J.; Sheu, C. C.; Lin, Y. S., J. Med. Microbiol. 2005, 54, 795-802. [89] Yu, L.; Zhao, J.; Zhu, Q.; Li, S. P., J. Pharm. Biomed. Anal. 2007, 44, 439-443. [90] Cen, H. X.; Jia, X. B., Jiangsu Da Xue Xue Bao Yi Xue Ban 2005, 15, 75-78. [91] Yang, L. Y.; Chen, A.; Kuo, Y. C.; Lin, C. Y., J. Lab Clin. Med. 1999, 134, 492-500. [92] Liang, J.; He, L.; Fu, W. Z.; Su, A. R.; Ma, Y. Y.; Tan, H. Y.; Tan, Z. Y.; Li, F. W., Zhongguo Re Dai Yi Xue 2007, 7, 1104-1106. [93] Kim, H. G.; Shrestha, B.; Lim, S. Y.; Yoon, D. H.; Chang, W. C.; Shin, D. J.; Han, S. K.; Park, S. M.; Park, J. H.; Park, H. l.; Sung, J. M.; Jang, Y.; Chung, N.; Hwang, K. C.; Kim.T.W., Eur. J. Pharmacol. 2006, 545, 192-199. [94] Shahed, A. R.; Kim, S. I.; Shoskes, D. A., Transplant. Proc. 2001, 33, 2986-2987. [95] Guan, C. H.; Liu, J., Zhejiang Yi Xue 2007, 29, 186-188. [96] Lin, X. X.; Xie, Q. M.; Shen, W. H.; Chen, Y., Zhongguo Zhong Yao Za Zhi 2001, 26, 622-625. [97] Kim, K. M.; Kwon, Y. G.; Chung, H. T.; Yun, Y. G.; Pae, H. O.; Han, J. A.; Ha, K. S.; Kim, T. W.; Kim, Y. M., Toxicol. Appl. Pharmacol. 2003, 190, 1-8. [98] Rao, Y. K.; Fang, S. H.; Tzeng, Y. M., J. Ethnopharmacol. 2007, 114, 78-85.

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[129] Yang, H. Y.; Leu, S. F.; Wang, Y. K.; Wu, C. S.; Huang, B. M., Arch. Androl. 2006, 52, 103-110. [130] Zhang, Q. X.; Wu, J. Y., Exp. Biol. Med. 2007, 232, 52-57. [131] Buenz, E. J.; Weaver, J. G.; Bauer, B. A.; Chalpin, S. D.; Badley, A. D., J. Ethnopharmacol. 2004, 90, 57-62. [132] Zhang, X. X.; Shen, W., Chin. J. Gastroenterol. Hepatol. 2006, 15, 478-481. [133] Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J., Int. J. Biochem. Cell Biol. 2007, 39, 44-84. [134] Zhong, X. Q., Shaoguan Xue Yuan Xue Bao Zi Ran Ke Xue Ban 2006, 27, 87-90. [135] Li, S. P.; Zhao, K. J.; Ji, Z. N.; Song, Z. H.; Dong, T. T. X.; Lo, C. K.; Cheung, J. K. H.; Zhu, S. Q.; Tsim, K. W. K., Life Sci. 2003, 73, 2503-2513. [136] Chen, J.; Zhang, W.; Lu, T.; Li, J.; Zheng, Y.; Kong, L., Life Sci. 2006, 78, 2742-2748. [137] Yamaguchi, Y.; Kagota, S.; Nakamura, K.; Shinozuka, K.; Kunitomo, M., Phytother. Res. 2000, 14, 647-649. [138] Zhang, G. Q.; Huang, Y. D.; Bian, Y.; Wong, J. H.; Ng, T. B.; Wang, H. X., Appl. Microbiol. Biotechnol. 2006, 72, 1152-1156. [139] Kakkar, R.; Kalra, J.; Mantha, S. V.; Prasad, K., Mol. Cell. Biochem. 1995, 151, 113119. [140] Kakkar, R.; Mantha, S. V.; Radhi, J.; Prasad, K.; Kalra, J., Clin. Sci. 1998, 94, 623-632. [141] Prasad, K., Mol. Cell. Biochem. 2000, 209, 89-96. [142] Xiang, M.; Tang, J.; Chu, T.; Zhang, C. L.; Zou, X. L., Zhongguo Yi Yuan Yao Xue Za Zhi 2006, 26, 556-559. [143] Choi, S. B.; Park, C. H.; Choi, M. K.; Jun, D. W.; Park, S., Biosci. Biotechnol. Biochem. 2004, 68, 2257-2264. [144] Lo, H. C.; Tu, S. T.; Lin, K. C.; Lin, S. C., Life Sci. 2004, 74, 2897-2908. [145] Koh, J. H.; Kim, J. M.; Chang, U. J.; Suh, H. J., Biol. Pharm. Bull. 2003, 26, 84-87.

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 8

Sanqi (三七，Panax notoginseng) Jian-bo Wan, Yi-tao Wang and Shao-ping Li* Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China

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1. Introduction The root of Panax notoginseng (Burk.) F.H. Chen, called Sanqi in Chinese, is a highly valued and commonly used Chinese medicinal herb. It was first recorded in Ben Cao Gang Mu (Compendium of Materia Medica) by Li Shi-zhen in 1596 AD [1]. P. notoginseng has been cultivated for about 400 years in China. It was considered as one of the varieties from Panax pseudo-ginseng. Until 1948, Chen Feng-huai, a Chinese botanist, defined that Sanqi was an independent species of Panax genus, named officially as Panax notoginseng (Burk.) F.H. Chen [2]. At present, more than 85% of P. notoginseng (Fig. 1) in the market was produced from Wenshan, Yunnan Province, China [2]. According to Chinese pharmacological principles, P. notoginseng, characterized as warm nature, sweet and slightly bitter flavor, enters “liver and stomach channels”. It is well known for its efficacy in promoting blood circulation, removing blood stasis, inducing blood clotting, relieving swelling, and alleviating pain. P. notoginseng has been used as a creditable Chinese medicine for treatment of haemoptysis, haemostatic and haematoma, which is contained in several famous traditional Chinese medicinal products, such as Yunnan Bai Yao (a remedy for injury induced trauma and bleeding) and Pian Zai Huang (a remedy for relieving pain and detoxification). Modern pharmacological studies

*

E-mail: [email protected]. Tel: +853-8397 4692. Fax: +853-2884 1358 (Corresponding author.)

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A

B

C

D

900 800 Number of Papers

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Figure 1. The cultivation in Wenshan, Yunnan Province (A), original plant (B), fresh collected sample (C), and dried raw medicinal material (D) of Panax notoginseng.

700 600 500 400 300 200 100 0 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 Year

Figure 2. The numbers of literature published in last decade on Panax notoginseng. Source: SCI Finder scholar.

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have shown that P. notoginseng and its components were beneficial to human health [3, 4]. In the last decade, P. notoginseng has attracted more and more attention because of its powerful therapeutic effects (Fig. 2). Thus, it is very important to clearly understand the biological activities and quality control of P. notoginseng.

2. Pharmacological Activities of P. Notoginseng 2.1. Bilateral Effects on Blood Coagulation In ancient China, P. notoginseng was widely used for treatment of various types of wounds because it is a favorite medicine for both internal and external bleeding. On the other hand, P. notoginseng also showed the property of removing blood stasis and promoting blood circulation [2, 5]. Modern studies revealed that the bilateral effects of P. notoginseng, which depended on the dosage and form of medication, as well as pathologic status of organism [6], was attributed to its two groups of ingredients, dencichine and saponins.

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2.1.1. Hemostatic Activity P. notoginseng is commonly available in two forms, raw and steamed. Traditionally, raw P. notoginseng is used as a potential hemostatic agent. Modern studies showed that raw P. notoginseng and its preparation, Yunnan Bai Yao, provided appreciable hemostatic effects [7]. The alcohol extract of raw P. notoginseng resulted in the shortest bleeding time of rat, comparing with those treated with placebo and hexane extracts [8]. Dencichine (Fig. 3), a thermo-sensitive and non-protein amino acid, was found as the most effective hemostatic ingredient in P. notoginseng [9]. Indeed, dencichine can increase platelet counts, and promote the release of endogenic substances, such as arachidonic acid (AA), adenosine diphosphate (ADP), collagen, platelet factor III and Ca2+, to induce haemostasis [10, 11]. However, the steamed P. notoginseng, after heat treatment, contains less dencichine than the raw sample, which is the reason why raw P. notoginseng but not steamed one is usually used for stopping bleeding [12]. But it is noteworthy that dencichine has been considered as the main cause of a neurological disorder known as neurolathyrism [13, 14], though the neurotoxicity only occurred at high dose [10]. The content of dencichine in raw P. notoginseng is about 1.94 mg/g [12].

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R3 HO

O HO

O N H

O

R1

Saponin

R2

OH NH2

Dencichine

R1

R2

R3

Notoginsenoside A

-Oglc(2-1)glc

-H

(SCS1)a, -Oglc(6-1)glc

Notoginsenoside B

-Oglc(2-1)glc

-H

(SCS2)a, -Oglc(6-1)glc

Notoginsenoside C

-Oglc(2-1)glc

-H

(SCS3)a, -Oglc(6-1)glc

Notoginsenoside D

-Oglc(2-1)glc(2-1)xyl

-H

-Oglc(6-1)glc(6-1)xyl

Notoginsenoside E

-Oglc(2-1)glc

-H

(SCS4)a, -Oglc

Notoginsenoside Fa

-Oglc(2-1)glc(2-1)xyl

-H

-Oglc(6-1)glc

Notoginsenoside Fc

-Oglc(2-1)glc(2-1)xyl

-H

-Oglc(6-1)xyl

Notoginsenoside Fe

-Oglc

-H

-Oglc(6-1)araf

Notoginsenoside G

(SCS5)a, -Oglc(2-1)glc

-H

-Oglc

Notoginsenoside I

-Oglc(2-1)glc

-H

(SCS6)a, -Oglc(6-1)glc

Notoginsenoside K

-Oglc(2-1)glc

-H

(SCS4)a, -Oglc(6-1)glc

Notoginsenoside L

-Oglc(2-1)xyl

-H

-Oglc(6-1)glc

Saponin

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Protopanaxadiol Group

Notoginsenoside Q

-Oglc(2-1)glc(2-1)xyl

-H

-Oglc(6-1)xyl(4-1)xyl

Notoginsenoside R4

-Oglc(2-1)glc

-H

-Oglc(6-1)glc(6-1)xyl

Notoginsenoside S

-Oglc(2-1)glc(2-1)xy

-H

-Oglc(6-1)araf(5-1)xyl

Notoginsenoside T

-Oglc(2-1)glc(2-1)xyl

-H

-Oglc(6-1)glc(3-1)xyl

Ginsenoside Ra3

-Oglc(2-1)glc

-H

-Oglc(6-1)glc(3-1)xyl

Ginsenoside Rb1

-Oglc(2-1)glc

-H

-Oglc(6-1)glc

Ginsenoside Rb2

-Oglc(2-1)glc

-H

-Oglc(6-1)arap

Ginsenoside Rb3

-Oglc(2-1)glc

-H

-Oglc(6-1)xyl

Ginsenoside Rc

-Oglc(2-1)glc

-H

-Oglc(6-1)araf

Ginsenoside Rd

-Oglc(2-1)glc

-H

-Oglc

Ginsenoside Rg3

-Oglc(2-1)glc

-H

-OH

20(R)-Ginsenoside Rg3

-Oglc(2-1)glc

-H

-OH

Ginsenoside II

-Oglc(2-1)glc

-H

(SCS3)a, -Oglc

Ginsenoside F2

Oglc

-H

Oglc

Gypenoside IX

-Oglc

-H

-Oglc(6-1)xyl

Gypenoside XVII

-Oglc

-H

-Oglc(6-1)xyl

Saponin 1b

-Oglc(6-1)glc

-H

-Oglc

Saponin 2b

-Oglc

-H

-Oglc(2-1)arap

Figure 3. (Continued on next page.)

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Sanqi (三七，Panax notoginseng) Saponin

R1

R2

R3

Notoginsenoside H

-OH

-Oglc(2-1)xyl

(SCS1)a, -Oglc

Notoginsenoside J

-OH

-Oglc

(SCS7)a, -Oglc

Notoginsenoside M

-OH

-Oglc(6-1)glc

-Oglc

Ptotopanaxadiol Group

a b

Notoginsenoside N

-OH

-Oglc(4-1)glc

-Oglc

Notoginsenoside R1

-OH

-Oglc(2-1)xyl

-Oglc

Notoginsenoside R2

-OH

-Oglc(2-1)xyl

-OH

Notoginsenoside R3

-OH

-Oglc

-Oglc(6-1)glc

Notoginsenoside R6

-OH

-Oglc

-Oglc(6-1)glc*

Notoginsenoside R8

-OH

-Oglc

(SCS1)a,(20S)-OH

Notoginsenoside R9

-OH

-Oglc

(SCS1)a,(20R)-OH

Notoginsenoside T5

-OH

-Oglc(2-1)xyl

(SCS8)a

Ginsenoside Re

-OH

-Oglc(2-1)rha

-Oglc

Ginsenoside Rf

-OH

-Oglc(2-1)glc

-OH

20-O-glucoginsenoside Rf

-OH

-Oglc(2-1)glc

-Oglc

Ginsenoside Rg1

-OH

-Oglc

-Oglc

Ginsenoside Rg2

-OH

-Oglc(2-1)rha

-OH

Ginsenoside Rh1

-OH

-Oglc

-OH

Ginsenoside F1

-OH

-OH

-Oglc

Saponin 3b

-OH

-Oglc

(SCS1)a, -Oglc

SCS1-SCS8, side chain structure Not denominated

O Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

R3

R3

R3

OOH

R3 OOH

OH

SCS1

SCS2

SCS3

SCS4

OH

R3

R3 OH

OH

R1 SCS5

SCS6

SCS7

Glc, β-D-glucopyranosyl; Rha, α-L-rhamnopyranosyl; Arap, α-L-arabinopyranosyl; arabinofuranosyl; Xyl, β-D-xylopyranosyl; Glc*, α-D-glucopyranosyl.

Figure 3. Chemical structures of saponins and dencichine isolated from Panax notoginseng.

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SCS8 Araf,

α-L-

184

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2.1.2. Anti-platelet Aggregation

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P. notoginseng saponins (PNS) can reduce the platelet activation, and inhibit platelet aggregation and adhesiveness in vivo. PNS could also inhibit ADP-induced platelet aggregation in vitro in a concentration-related manner [15], which attributes to the increase content of cAMP and reduced production of thromboxane A2 in platelet [16]. PNS could also improve the fluidity and stability of platelet membranes [15]. CD62P, platelet surface selectin, is the marker of platelet activation, which mediates platelet-endothelial cell interactions. Several studies showed that PNS could reduce the expression of CD62P [17, 18]. Generally, 20(S)-protopanaxatriol type saponins (PTS), instead of 20(S)-protopanaxadiol ones (PDS), from P. notoginseng are considered as the main ingredients responsible for antiplatelet aggregation effect [16]. As we know, affinity method is unique among separation methods as it is the only technique that permits the purification of compounds based on biological functions rather than individual physical or chemical properties [19]. Compounds with affinities similar to a target receptor are also likely to share the same pharmacological properties. The methods for screening of potential active components in Chinese herbs using red cell membrane [20], macrophage [21] extraction and HPLC have been reported. Recently, we developed a method, human platelet extraction and HPLC-DAD-ESI-MS/MS, for screening potential anti-platelet aggregation agents in P. notoginseng. Two nucleosides, adenosine and guanosine, were identified. Bioassay showed that both adenosine and guanosine could inhibit rabbit platelet aggregation in vitro. The IC50 of adenosine on thrombin (THR)-induced rabbit platelet aggregation was 1.7×10-4 mg/ml (95% confidence interval of IC50:1.4×10-4-2.1×10-4 mg/ml), but it has no obvious dose-dependent effects on platelet aggregation induced by AA and ADP. Guanosine is an effective anti-platelet compound, which inhibits rabbit platelet aggregation induced by AA, ADP and THR with IC50 values (95% confidence interval) of 0.7 (0.6-0.8), 0.4 (0.3-0.5) and 0.9×10-1 (0.08-0.1) mg/ml, respectively. 2.1.3. Fibrinolytic Activity P. notoginseng appeared to activate fibrinolysis in vivo and in vitro. The pulverized root of P. notoginseng significantly reduced fibrinogenaemia in rats (p < 0.001), though doserelationship was not observed [22]. Notoginsenoside R1, one of the main ingredients of P. notoginseng, could increase the synthesis of tissue-type plasminogen activator and urine plasminogen activator, and decrease plasminogen activator inhibitor-1 (PAI-1) activity in cultured human endothelial cells from different vascular sources [23]. Notoginsenoside R1 also dose-dependently decreased tumor necrosis factor-α (TNF-α)-induced PAI-1 production at mRNA and protein levels and its secretion in human aortic smooth muscle cells by suppressing signal-related kinases and protein kinase B signaling pathways [24]. 2.2. Effects on Cardio-Cerebrovascular System 2.2.1. Anti-arrhythmic Action It was reported that PNS, PTS and PDS could protect against arrhythmia induced by ischemia and several chemicals such as strophanthin, barium chloride, aconitine, adrenaline Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

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and chloroform in vivo [25-29]. PDS could also reduce the spontaneous frequency of atrium dextrum and cardiac rate of rabbit, and prolong P-R and Q-T interval in the electrocardiogram, which suggested that PDS had the negative effects of autorhythmicity, frequency and conductibility [27]. On the other hand, PTS obviously opposed premature ventricualr contraction, ventricular fibrillation and duration of arrhythmia caused by ischemia of anterior descending coronary [28]. Actually, ginsenoside Rb1, one of the main ingredients of PNS, can inhibit Ca2+ entry on the myocardial cells of Cavia porcellus through voltagedependent and receptor-linked Ca2+ channels, which contribute to its anti-arrhythmic effect [30]. Therefore, the anti-arrhythmic effect of P. notoginseng may mainly attribute to the adjustment of ion channels and the direct inhibition of cardiac muscle, instead of blocking βadrenergic receptor and/or stimulating M-cholinergic receptor [25, 26].

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2.2.2. Protection on Myocardial Ischemia and Ischemia/Reperfusion Injury Ischemia is a condition in which blood flow (and thus oxygen) is restricted to a part of the body. Reperfusion injury (RI) refers to damage of tissue caused when blood supply returns to the tissue after a period of ischemia. Ischemia and reperfusion can cause serious brain damage in stroke or cardiac arrest [31-33]. A large number of studies have shown that P. notoginseng [34] and its ingredients, including PNS [35, 36], PTS [37], PDS [38], ginsenoside Rg1 [39] and Rb1 [40], possess the protective effect on cardiac and cerebral ischemia/RI. Among several mechanisms have been proposed to ischemia/RI, the formation of oxygen radicals and cellular calcium loading may be the most important factors in response to tissue damage [41]. P. notoginseng has been shown its free radicals scavenging ability and antioxidant activity [42, 43]. In an anti-aging rat model, PNS could significantly increase the serum levels and activities of superoxide dismutase, glutathion peroxidase and hydrogen peroxidase, as well as decrease the level of malondialdehyde [44]. During the ischemic period, increased Na+/Ca2+ exchange resulting in excessive Ca2+ entry into cells, which cause excitotoxicity and initiate the tissue ischemic damage [45, 46]. P. notoginseng and its saponins, considered as novel natural calcium antagonists, are able to inhibit not only the release of intracellular Ca2+, but also the inflow of extracellular Ca2+ [47, 48]. Actually, PNS selectively inhibits Ca2+ entry through receptor-operated Ca2+ channel [49]. PDS could block the potential-dependent and receptor-operated calcium channel in smooth muscle [47]. While ginsenoside-Rd remarkably inhibits Ca2+ entry through receptoroperated calcium channel and store-operated calcium channel without effects on voltagedependent inward Ca2+ current and Ca2+ release in vascular smooth muscle cells [50]. But ginsenoside Rg1 was not a Ca2+ channel antagonist [51, 52]. In addition, PNS, ginsenoside Rb1 and Rg1 could activate rat brain synaptosomal Na+-K+-ATPase for reducing the excessive intracellular Na+. Meanwhile, PNS and ginsenoside Rb1, not Rg1 could also inhibit Ca2+-Mg2+-ATPase [53]. Besides lowering intracellular Ca2+ level, the protective effects of P. notoginseng on ischemia/reperfusion injury was also related with its anti-inflammatory [54] and antiapoptotic activities [55]. Angiogenesis, the development of new blood vessels into area with limited blood flow, could offers promise as a novel treatment for myocardial ischemia disease [56, 57]. Recent studies have demonstrated that ginsenoside Rg1 and Re, the main ingredients from P. notoginseng, could promote angiogenesis in vitro and in vivo [58-60]. The pro-angiogenesis effect of ginsenoside Re on human umbilical vein endothelial cells (HUVECs) proliferation,

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migration, and tube formation were dose-dependent and reached a maximal level at a concentration of about 30 μg/ml [60]. Ginsenoside Rg1 can promote angiogenesis on HUVECs in multiple models, and this effect is partly due to the modulation of genes that are involved in the cytoskeletal dynamics, cell-cell adhesion and migration [58]. However, ginsenoside Rb1, another main ingredient of P. notoginseng, exerts an opposing effect and shows anti-angiogenic activity. Ginsenoside Rb1 could suppress the formation of endothelial tube-like structures through increasing the transcription, protein expression and secretion of pigment epithelium-derived factor, an anti-angiogenic protein [59, 61]. 2.2.3. Anti-atherosclerosis Atherosclerosis is a chronic disease caused by the deposition of fats, cholesterol, calcium, and other substances in the innermost layer of endothelium of the large and medium-sized arteries. Atherosclerosis is the underlying pathology of cardiovascular disease and causes more global death and disability than any other pathology [62] . It is increasingly appreciated as a complex and multifactorial disease that can be caused or promoted by elevated and modified LDL, vascular inflammation, endothelial dysfunction, free redicals, hypertension, activation of platelet and combinations of these or other factors [63, 64]. Several studies showed that P. notoginseng and PNS could interfere atherogenic formation, prevent its progression and visibly less the atherosclerotic lesion [65-67]. We also found that PNS could significantly decrease the extent of atherosclerotic lesion of apolipoprotein E deficient mice fed a western-type diet (21% milk fat, 0.15% cholesterol, w/w).

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2.2.3.1. Effects on Plasma Lipid Levels and Lipid Peroxidation Atherosclerosis has traditionally been attributed to disordered cholesterol metabolism with associated accumulation of lipid substrate in the arterial wall [68]. Therefore, intervention with agents that can decrease the plasma lipid level, elevate HDL concentration or prevent LDL oxidation should be helpful to restrict the development of atherosclerosis [69, 70]. P. notoginseng can obviously decrease the levels of plasma total cholesterol (TC) and triglycerides (TG) [71-73]. Its n-butanol extract dose-dependently reduced the concentration of serum TC, TG and LDL in rats and prevented the accumulation of abnormal lipid in hyperlipidemic rats via agitating liver receptors, LXR-alpha and FXR [74]. Furthermore, plasma HDL cholesterol concentration has been shown to be increased by P. notoginseng intake [75]. In addition, hot water extract of P. notoginseng could also inhibit ethanol-induced lipid peroxidation in mouse liver homogenate [76]. Those abilities of P. notoginseng may contribute to its anti-atherosclerotic property. 2.2.3.2. Anti-inflammation In the past decade, many lines of evidence indicate that atherosclerosis is a inflammatory response of the vascular wall, not merely the passive accumulation of lipids within artery walls [63, 77, 78]. Upon stimulation or injury, endothelial cells initiate a series of inflammatory responses by expressing of adhesion molecules and secreting inflammatory mediators such as cytokines (IL-1, IL-6 and TNF-α) and lipid mediators (prostaglandins, thromboxanes and platelet-activating factor) leading to leukocyte adhesion and emigration,

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which is central to inflammation [79]. Thus, anti-inflammation may help to reduce the risk of atherosclerosis. It is reported that ethanol extract of P. notoginseng, ginsenoside Rg1 and Rb1, with a concentration-dependent manner, inhibited the production of TNF-α and IL-6 of cultured macrophages (RAW264.7 cells) induced by lipopolysaccharide, as well as attenuated the expression of COX-2 and IL-1 beta mRNA [80]. PNS could inhibit the neutrophil function and phospholipase A2 activity, reduce NO and prostaglandin E2 production [81, 82], and leukocyte adhesion in venules under the inhibitory effect on the expression of adhesion molecules (CD11b and CD18) on neutrophils [83]. PNS could also inhibit NF-κB, a critical intracellular mediator of the inflammatory cascade of macrophages [84]. 2.2.3.3. Effects on Vascular Cells Atherosclerosis process is a multifactorial sequence of events involving vascular endothelial cells injury/dysfunction, smooth muscle cells migration and proliferation [85, 86]. Recent studies showed that P. notoginseng could protect endothelial cell against injury induced by endotoxin [87], balloon endothelial denudation [88] and hypoxia [89] in vivo. PNS was also shown to enhance endothelial cell function [90], promote the endothelial regeneration and reduce extracellular matrix thickening, which was related to downregulation of the expression of vascular endothelial growth factor and matrix metalloproteinase-2 [91]. In addition, PNS could inhibit the proliferation of aortic smooth muscle cells stimulated by hypercholesterolemic serum [92] and platelet derived growth factor [93].

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2.3. Effect on Central Nervous System P. notoginseng and its saponins could induce neuronal differentiation in neurosphere stem cells [94] and enhance axonal and dendritic formation activity [95]. PNS could also protect against posthypoxic cell damage of neurons in vitro via improving energy metabolism and preserving the structural integrity of neurons [96]. In addition, P. notoginseng, administered orally for 1 week, could improve the scopolamine-induced learning and memory deficit in rats [97]. Therefore, P. notoginseng may be utilized for treatment of neurodegenerative diseases. Several studies indicated that the saponins from the root [98], flower [99] and leaf [100] of P. notoginseng possessed quite good analgesic properties against the chemical and thermoinduced soreness, which were related to the agitation of opioid-like peptide receptor [101]. In addition, The P. notoginseng showed both inhibitory and stimulatory actions on the central nervous system, ascribing to the PTS and PDS, respectively [102]. 2.4. Miscellaneous Besides the effects mentioned above, P. notoginseng and its ingredients also possess extensive pharmacological activities, which are summarized in Table 1.

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Table 1. Some reported biological activities of Panax notoginseng and its components Bioactivity Immunomodulation Anti-carcinogenesis Hepato-protection Anti-fungus Renal protection Antihypertension Anti-HIV reverse transcriptase Enhancement of sperm motility Gastric mucosa protection Estrogen-like activity Hypoglycemic activity Reducing edema Antiradiation Anti-aging

Substance Polysaccharide, PNS, PDS Methanol extracts of P. notoginseng; ginsenosides Rb1, Rb3, Rc and Rg3; panaxydol Extract of P. notoginseng Protein PNS Saponins Xylanase (Protein); P. notoginseng Aqueous and n-butanol extracts of P. notoginseng; polysaccharide fraction; Ginsenoside Rb2 and Rc P. notoginseng Ginsenoside Rg1 Ginsenoside Rg1 PNS PNS PNS

Ref. [103-107] [108-110] [111-113] [114, 115] [116] [117] [118-119] [120-121] [122] [123] [124] [125] [126] [127]

3. Quality Control of P. Notoginseng

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As we know, the saponins in P. notoginseng have multiple pharmacologic activities, which are generally considered as the major bioactive components [3, 4]. To date, over 50 saponins (Fig. 3) have been isolated and elucidated from P. notoginseng [128]. In addition, dencichine is the main bioactive ingredient for hemostatic effect. So far, most of the published methods for quality control of P. notoginseng focused on these compounds. 3.1. Qualitative Analysis-Authentication of P. Notoginseng Several plants of the Panax genus, including P. ginseng C. A. Mey. (Ginseng), P. quinquefolius L. (American ginseng), and P. notoginseng (Burk.) F.H. Chen (Sanqi), are well-known Chinese medicines [129]. Although their morphological appearance are similar, and they all contain ginsenosides (saponins) which are usually considered to be their major active components [130], their traditional indications are significantly different. Therefore, the authentication of P. notoginseng from other medicinal plants such as ginseng and American ginseng of Panax genus is very important for ensuring the safety and efficacy of medication. Several methods, including unique compounds assay [131], chromatographic and spectral fingerprints [130, 132-136] and molecular biology techniques [137-142], have been employed for the authentication of P. notoginseng.

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1, notoginsenoside R1; 2-4, ginsenoside Rg1, Re, Rf; 5, pseudo-ginsenoside F11; 6-12, ginsenoside Rb1, Rg2, Rc, Rb2, Rb3, Rd and Rg3, respectively. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission from Ref. [130].

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Figure 4. Simulative mean chromatograms for Sanqi (SMC-NG), American ginseng (SMC-AG) and ginseng (SMC-G).

Notoginsenoside R1, the specific component in P. notoginseng, can be used as the chemical marker to distinguish P. notoginseng from the other medicinal plants of Panax genus [131]. The content ratios used for identification are usually ginsenosides Rg1/Rb1, Rg1/Re, malonylated (m)-Rb1/m-Rb2 and m-Rb1/m-Rc [130, 133, 143, 144]. Actually, the chemical characteristics among three species of Panax genus, including P. notoginseng, P. ginseng and P. quinquefolius, are obviously different (Fig. 4). Similarly, the three medicinal plants could also be identified based on their protein fingerprints determined by sodium dodecyl sulfate polyacrlyamide gel electrophoresis [136]. Molecular biology offers an approach for identification of Chinese medicinal materials based on DNA analysis because genetic composition is unique for each individual irrespective of the physical forms of samples, and is less affected by age, physiological conditions, environmental factors, harvest, storage and processing. Since random amplified polymorphic DNA assay (RAPD) was first used for authentication of P. notoginseng in 1990s [137], several other methods, such as restriction fragment length polymorphism (RFLP) [140], gradient PCR [141], multiplex amplification refractory mutation system (MARMS) [138] and enzyme-linked immunosorbent assay (ELISA) [142] have also been developed for the authentication of Panax species.

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3.2. Quantitative Analysis-Quality Evaluation of P. Notoginseng

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3.2.1. Sample Preparation Sample preparation is one of the most important and crucial steps in chemical analysis, and usually the most labour-intensive. The contents of desired chemical components in the herbs maybe considerable various when different sample preparation methods were employed [145]. Actually, ultrasonication [143, 146, 147], soxhlet extraction [148], hot reflux [149, 150] and immersion [151] have been employed for extraction of saponins from P. notoginseng using methanol, ethanol or their aqueous mixtures as solvents. Generally, an ideal sample preparation technique should be simple, efficient, cheap, selective and compatible with various analytical methods. It should also reduce the consumption of organic solvents. In last decade, one of the most promising sample preparation techniques is pressurized liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction) which has been applied in the sample preparation of P. notoginseng [128, 130, 134, 145, 152-154]. Comparing to conventional extraction methods, PLE technique has the advantages of short extraction time, less solvent consumption, high extraction efficiency and automatic operating capabilities [155, 156]. The different sample preparation methods for extraction of saponins from P. notoginseng, including PLE, ultrasonication, soxhlet extraction and immersion, were studied and compared [145]. The results showed that the extraction efficiency of PLE was the highest among the four extraction methods (Table 2). However, due to the thermo-sensitive property, Soxhlet, hot reflux and PLE are not suitable for the extraction of dencichine from P. notoginseng, which are usually instead with ultrasonication followed by centrifugation using water [148, 157], aqueous ethanol [158, 159] or methanol [160] as extractants. Immersion at the temperature of 40-50oC was also used for extraction of dencichine from P. notoginseng [161]. In addition, sample clean-up and/or analytes enrichment are very important and necessary for samples with complex matrices so as to avoid the contaminants disturb separation and detection, or to obtain highest sensitivity. Table 2. Comparison of different sample preparation methods for the saponins from Panax notoginseng. Methods PLE Ultrasonication Soxhlet Immersion

Solvent volume (ml) 20 30 60 30

Extraction Time (h) 0.25 2 6 48

Efficiency (%)a 7.36 5.77 6.99 6.00

RSD (%)b 1.84 4.59 5.15 10.33

Data are adapted from Ref. [145] and reproduced with permission of Elsevier Science. a

Total amount of nine saponins investigated, including notoginsenoside R1, ginsenoside Rg1, Re, Rf, Rb1, Rc, Rb2, Rb3 and Rd. b

Mean of RSD for nine saponins (n=3)

Solid-phase extraction (SPE) is a separation technique for extraction of compounds (called analytes) from a mixture of impurities. SPE has been successfully used to isolate saponins of P. notoginseng from a wide variety of matrices, including raw material [162], herbal product

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[163], rat tissues [164], serum [165], urine [166] and feces [167]. Similarly, macroporous adsorptive resin was also applied for sample preparation of P. notoginseng [168, 169]. 3.2.2. Analysis of Saponins 3.2.2.1. Thin Layer Chromatography Scanning (TLCs) Thin layer chromatography is a conventional technique for the analysis of herbs due to its easiness of use, low cost, versatility and simultaneous analysis of different samples. It is adopted by United States Pharmacopoeia and European Pharmacopoeia for the identification of plant derivatives [170]. Actually, TLC was a useful tool for qualitative analysis such as saponins [171-174]. The resolution, reproducibility and convenience of TLC have been greatly improved with high automation and decreased particle size. Simultaneous quantification of ginsenoside Rb1, Rb1, Rg1 and notoginsenoside R1 in P. notoginseng was performed by HPTLC equipped with automated sample applicator, development and densitometer [174]. The analytes were separated well using n-butanol-acetoacetate-water 3

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Figure 5. The HPTLC image (A) and scanning profiles of mixed standards (B) and the PLE extract of Panax notoginseng (C).

(1:1:2, v/v/v) as developing solvent (Fig. 5). Generally, TLC detection of ginsenosides can be achieved using chromogenic reagent such as p-anisaldehyde [173], 10% sulfuric acid-ethanol solution [175] and thionyl chloride vapor [172], which suffer unstable colorations and low

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sensitivity. Therefore, the quantitative determination of saponins from P. notoginseng is usually performed by HPLC.

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3.2.2.2. High Performance Liquid Chromatography HPLC is the most commonly used method for analysis of non-volatile compounds, and has been widely applied for the analysis of ginsenosides since 1970s [176]. Generally, chromatographic column, mobile phase, flow rate and eluent program are important factors of HPLC analysis affecting the separation result [177]. Actually, ocyadecyl columns (C-18 or ODS) are widely used for the separation of saponins from P. notoginseng [128, 130, 134, 145, 146, 153, 164-167] though C-8 [178] and NH2 [179] columns are casually applied. Acetonitrile-water system was usually utilized as the mobile phase because low wavelength of UV was used for detection. In some cases, additives such as KH2PO4 [148], acetic acid [180], phosphoric acid [181] and formic acid [182] were also added to the mobile phase for improving the separation results. Due to the similarity of the saponins, gradient elution was chosen in most cases for obtaining good separation. Especially, good separation between ginsenoside Rg1 and Re is usually difficult [151, 182, 183]. But a desired resolution can be obtained using appropriate gradient program [130, 145, 146, 184]. Meanwhile, flow rate is critical factor affecting the separation of ginsenoside Rg1 and Re [154]. A number of detection techniques, such as ultraviolet (UV), evaporative light scattering (ELSD), fluorescence and mass spectrometry (MS), can be used for HPLC analysis. Among them, UV detector is widely used [134, 145, 146, 179, 182, 184]. However, the saponins from P. notoginseng show poor UV absorptivity and low-wavelength UV (~200 nm) is required for the detection, which greatly increases the baseline noise and lowers the sensitivity of the detection. The choice of solvents and mobile-phase modifiers for improving separation are also restricted. In addition, refractive index (RI) detector is one of the least sensitive LC detectors and cannot be used for gradient elution, though it could be used as the detector [185]. Therefore, pre-column derivatization were developed for improving sensitivity of detection [186-188]. The double bond at the C24-C25 position of ginsenosides was converted into an aldehyde group by means of ozonolysis, then the aldehyde group reacts with FMOChydrazine forming the ginsenoside FMOC-hydrazone, and detected by fluorescence detector with excitation at 270 nm and emission at 310 nm. In this way, the limit of detection (LOD) of ginsenosides Rg1 and Rb1 was reported to be 2.0 ng (about 2.5 pmol) and 1.0 ng (about 0.9 pmol), respectively [186]. However, complex pretreatment and relatively low reproducibility restrict its application. ELSD is a universal, nonspecific detection method, in which signal intensity is related to the concentration of the solute in the effluent but not its optical characteristics. Therefore, ELSD can provide a stable baseline even with steep gradients [170], which has been also applied for quantitative determination of saponins in P. notoginseng [128, 130, 147, 154]. For obtaining best response, the two parameters, the flow rate of the nebulizer gas (nitrogen) and the drift tube temperature, should be optimized. Eleven saponins, including notoginsenoside R1, ginsenosides Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, Rd and Rg3, in the PLE extract of P. notoginseng were simultaneously determined using HPLC-ELSD with drift tube temperature of 60°C and nitrogen flow-rate of 1.4 L/min. LODs of the investigated saponins were less than 98 ng [154]. HPLC-ELSD was also used for investigation of the chemical characteristics

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of the different parts of P. notoginseng [128] and three medicinal plants of Panax genus, including ginseng, American ginseng and P. notoginseng [130]. m AU

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Figure 6. Chromatograms of the mixed standards of saponins from Panax notoginseng analyzed by (A) HPLC-DAD, (B) HPLC-ELSD, (C) HPLC-MS and (D) UPLC-PDA.

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However, good separation is necessary for quantification using UV or ELSD detection, which need a long time [146, 184, 189] for analysis of saponins from P. notoginseng (Fig. 6 A, B). The advantage of the mass spectrometer is that it may allow quantitative measurements to be made on unresolved peaks when their precursors or ions are different. A rapid method for qualitative and quantitative analysis of saponins in “XUESETONG” injection, made of PNS, was developed using HPLC-ESI-MS/MS in 20 min [190] (Fig. 6C). Actually, LC-MS has been widely applied for the qualitative and quantitative determination of saponins from P. ginseng [191-193] and P. notoginseng [180, 189, 190]. Besides electrospray ionization (ESI) interface [180, 189, 190, 194], fast-atom-bombardment (FAB) [195], fourier transform ion cyclotron resonance (FTICR) [196] and atmospheric pressure chemical ionization (APCI) [192, 197], were also used for ionization of ginsenosides. In general, negative ion mode was more sensitive, and provided straightforward structural information of saponins [170, 180, 191]. However, more structural information on ginsenosides was obtained in positive ion mode, which is helpful to confirm the quasimolecular ions [170, 189, 193]. Therefore, HPLC-MS is a powerful tool for analysis of saponins from P. notoginseng.

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3.2.2.3. Ultra Performance Liquid Chromatography The main limitation of HPLC for saponins of P. notoginseng was low efficient, the analytical time of most studies was more than 60 min for obtaining the good separation of ginsenoside Rg1 and Re [146, 184, 189, 190]. Ultra performance liquid chromatography (UPLC) , which utilizes silica particles 1.7μm, makes it possible to perform efficient separations in very short periods of time [198, 199]. It has the advantages of the fast analysis, high peak capacity, great resolution and good sensitivity [200]. But the small particles also create operating pressures that are very high (in the range of 6000-15,000 psi) [201]. Recently, Acquity UPLC was introduced as commercially available instrument, which has been applied for analysis of saponins from P. notoginseng [152, 199, 202]. Indeed, the separation and the determination of 11 saponins, named notoginsenoside R1, ginsenoside Rg1, Rg1, Re, Rf, Rb1, Rg2, Rc, Rb2, Rb3, Rd and Rg3 could be achieved on Acquity UPLC BEH C18 column (50mm×2.1mmi.d, 1.7μm) with water-acetonitrile gradient elution in 12 min, and the LOD of analytes were lower to 0.1-1.8 ng (Fig. 6D) [152]. In addition, UPLCMS has also been used for analysis of ginsenosides in rat urine [202], or saponins in raw and steamed P. notoginseng [199]. 3.2.2.4. Capillary Electrophoresis (CE) Capillary electrophoresis (CE) is a developing technique characterized by high efficiency, rapidity, low cost, small reagent requirement and easy turnover mode [203], which could overcome the several disadvantages of HPLC. Due to the absence of charge in dammarane triterpene saponins, capillary zone electrophoresis (CZE) was not suitable for determination of ginsenosides. Therefore, in micellar electrokinetic chromatography (MEKC), the low-molecular-mass surfactants are added to the buffer as pseudo-stationary phases, and the neutral analytes were able to be separated according to the differential partitioning of analytes between the micelle and the surrounding aqueous phase [204]. MEKC has been employed for analysis of several ginsenosides in P. ginseng [205], P. quinquefolius

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[206] and P. notoginseng [207]. However, the concentration sensitivity of CE is poor due to short optimal path and small volume of sample that can be injected. In order to improve the concentration sensitivity of MEKC, an on-line concentration technique, field-enhanced sample injection with reverse migrating micelles, was introduced to determine the ginsenoside Rd, Rc, Rb3, Rb1, Rh1, Rg2, Rf, Re and notoginsenoside R1 in P. notoginseng, which provide more than 100-fold increase in UV detector response with very high plate numbers [207]. 3.2.2.5. Other Methods Besides the methods mentioned above, gas chromatography (GC) [208, 209], NIR spectroscopy [181, 210] and ELISA [211-214], were also used for determination of saponins from P. notoginseng. Due to nonvolatile property of ginsenosides, the analytes should react with certain reagents to form volatile derivatives before GC analysis. Therefore, the pretreatment is complex with low reproducibility. NIR is a non-destructive technique which does not require sample preparation with the advantages of the rapidity and capability of use on-line assay, which has been employed to determine ginsenoside Rb1, Rb2, Rc, Rd, Re, Rf and Rg1 in P. notoginseng [181]. ELISA is a sensitive and selective method for assay of saponins from P. notoginseng. The LOD of ginsenoside Rg1 and Rb1 reached 20 ng/ml and 300 ng/ml, respectively [212]. Furthermore, no pretreatment of crude extracts is necessary and low quantities of specimen required [210, 212, 215]. However, the polyclonal or monoclonal antibody is required for ELISA. Their preparations are difficult and require special protocols because the molecular weight of second metabolites of plant is small [216]. Therefore, ELSIA application is limited on analysis of bioactive components from herbs.

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3.2.3. Analysis of Dencichine Dencichine, β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP), was first isolated from the seeds of Lathyrus sativus (grass pea seeds) [217]. It was identified as the main cause of a neurological disorder known as neurolathyrism [218]. In the 1980s, Japanese scientists found that dencichine was also present in P. notoginseng and was considered as the most effective compound for hemostatic activity of P. notoginseng [9]. Due to thermo-sensitivity and neurotoxicity of dencichine, several methods for its quantification have been developed. The colorimetric method which utilized the reaction of o-phthalaldehyde with α,βdiaminopropionic acid was first reported for determination of dencichine in 1970s [219]. In recent years, a few HPLC methods using direct assay [148], pre-column derivatization [157160, 220-222] and hydrophilic interaction [12] have been developed. The derivative reagents are para-nitrobenzyloxycarbonyl chloride [222], 9-fluorenylmethyl chloroformate [220], 6aminoquinolyl-N-hydroxysuccinim-idylcarbamate [158], phenylisothiocyanate [159] and 1fluoro-2,4-dinitroben-zene [221]. β-ODAP could be easily converted into its isomers αODAP at higher temperature or in acidic and basic solution [223], both forms could be determined using HPLC [222]. In addition, CZE [224], LC-MS [225], GC-MS with ethyl chloroformate derivatization [161] were also developed for the determination of dencichine.

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4. Conclusion P. notoginseng is a highly valued and important Chinese medicinal herb for the treatment of wide diseases related to hematological, immune, cardio-cerebral vascular, central nervous, endocrine and inflammatory system [3, 4]. Saponins are generally considered as its active compounds, which is the focus of quality control of P. notoginseng nowadays. With the development of molecular biological and pharmacological methodologies, the extensive bioactivity and its mechanism of P. notoginseng will be elucidated, and some other compounds such as polysaccharides and flavonoids may be concerned for quality control of P. notoginseng. On the other hand, the further study on dencichine should be performed to ensure the safety and efficacy of P. notoginseng.

Acknowledgment The research was supported by grants from Macao Science and Technology Development Fund (028/2006/A2 to S.P. Li and 049/2005/A-R1 to Y.T. Wang).

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[171] Zhou, Z. H.; Zhang, G. D.; Wang, J. F. Acta Pharm. Sin. 1981, 16, 535-540 [172] Vanhaelen-Fastre, R. J.; Faes, M. L.; Vanhaelen, M. H. J. Chromatogr. A 2000, 868, 269-276. [173] Corthout, J.; Naessens, T.; Apers, S.; Vlietinck, A. J. J. Pharm. Biomed. Anal. 1999, 21, 187-192. [174] Wan, J. B.; Li, S. P.; Jian, J. R.; Kong, L. Y.; Wang, Y. T. Zhongguo Tian Ran Yao Wu 2004, 2, 214-218. [175] Zhang, X.; Zhu, T. Q. Zhongguo Zhong Yao Za Zhi 2001, 26, 342. [176] Besso, H.; Saruwatari, Y.; Futamura, K.; Kunihiro, K.; Fuwa, T.; Tanaka, O. Planta Med 1979, 37, 226-233. [177] Dorsey, J. G.; Cooper, W. T.; Siles, B. A.; Foley, J. P.; Barth, H. G. Anal. Chem. 1998, 70, 591-644. [178] Su, J.; Zhu, W. Q. Yao Wu Feng Xi Za Zhi 2007, 25, 212-214. [179] Zhou, Y. C.; Zhao, H. Q.; Liang, N.; Qu, Y.; Lu, X. Y.; Zhang, F. M. Shenyang Yao Ke Da Xue Xue Bao 2003, 20, 27-31. [180] Li, L.; Tsao, R.; Dou, J.; Song, F.; Liu, Z.; Liu, S. Anal. Chim. Acta 2005, 536, 21-28. [181] Chen, Y.; Sorensen, L. K. Fresenius. J. Anal. Chem. 2000, 367, 491-496. [182] Li, L.; Zhang, J. L.; Sheng, Y. X.; Guo, D. A.; Wang, Q.; Guo, H. Z. J. Pharm. Biomed. Anal. 2005, 38, 45-51. [183] Kwon, S. W.; Han, S. B.; Park, I. H.; Kim, J. M.; Park, M. K.; Park, J. H. J. Chromatogr. A 2001, 921, 335-339. [184] Lau, A. J.; Seo, B. H.; Woo, S. O.; Koh, H. L. J. Chromatogr. A 2004, 1057, 141-149. [185] Di, F.; Shun, Y. K. Zhongguo Zhong Yao Za Zhi 1996, 21, 672-673. [186] Shangguan, D.; Han, H.; Zhao, R.; Zhao, Y.; Xiong, S.; Liu, G. J. Chromatogr. A 2001, 910, 367-372. [187] Park, M. K.; Kim, B. K.; Park, J. H.; Shin, Y. G. J. Liq. Chromatogr. 1995, 18, 20772088. [188] Park, M. K.; Kim, B. K.; Park, J. H.; Shin, Y. G.; Cho, K. H.; Do, Y. M. Arch. Pharm. Res. 1996, 19, 562-565. [189] Liu, J. H.; Wang, X.; Chai, S. Q.; Komatsu, K.; Namba, T. J. Chin. Pharm. Sci. 2004, 13, 225-236. [190] Lai, C. M.; Li, S. P.; Yu, H.; Wan, J. B.; Kan, K. W.; Wang, Y. T. J. Pharm. Biomed. Anal. 2006, 40, 669-678. [191] Fuzzati, N.; Gabetta, B.; Jayakar, K.; Pace, R.; Peterlongo, F. J. Chromatogr. A 1999, 69-79. [192] Ma, X. Q.; Liang, X. M. Phytochem. Anal. 2005, 16, 181-187. [193] Ji, Q. C.; Harkey, M. R.; Henderson, G. L.; Gershwin, M. E.; Stern, J. S.; Hackman, R. M. Phytochem. Anal. 2001, 12, 320-326. [194] Miao, X. S.; Metcalfe, C. D.; Hao, C.; March, R. E. J. Mass Spectrom. 2002, 37, 495-506. [195] Hattori, M.; Kawata, Y.; Kakiuchi, N.; Matsuura, K.; Namba, T. Shoyakugaku Zasshi 1988, 42, 228-235. [196] Tanaka, K.; Kubota, M.; Zhu, S.; Sankawa, U.; Komatsu, K. Nat. Prod. Commun. 2007, 2, 625-632. [197] Leung, K. S.; Chan, K.; Bensoussan, A.; Munroe, M. J. Phytochem. Anal. 2007, 18, 146-150.

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Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved. Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 9

Beimu (贝母，Fritillaria spp.) Hui-Jun Li a, Yan Jiang b and Ping Li a,* a

Key Laboratory of Modern Chinese Medicines (China Pharmaceutical University), Ministry of Education, Nanjing, 210009, China b Nanjing Forestry University, Nanjing, 210037, China

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1. Introduction Fritillaria L., one of the largest genera in the plant family of Liliaceae, comprises about 130 species of perennial herbs occurring in most temperate regions of the Northern Hemisphere, from North America, through Europe, the Mediterranean region and Central Asia, to China and Japan [1]. Fritillaria species are pharmacologically important because many species have traditionally been used as herbal remedies in Japanese, Turkish, Pakistani and Southeast Asian folk medicines [2,3]. In traditional Chinese medicine, “Beimu”, as an appellative name of dried bulbs of many Fritillaria species, is the most commonly used antitussive and expectorant herbal drug since ancient times. The earliest scientific description of Beimu was in “Shen-Nong Ben-Cao Jing” (the oldest herbological documents written in 2000 years ago), however, the herbological study [4] indicated that the early-used Beimu originated from Bolbostemma paniculatum (Maxim.) Franq. (Fam. Cucurbitaceae), and the genuine Beimu, derived from the bulbs of Fritillaria species, was described in “Ming-Yi Bie-Lu” (compiled by Hong-Jing Tao in A. D. 500) for the first time, since then it has been popularly prescribed for cough and fever by traditional Chinese doctors. In the 2005 edition of Chinese Pharmacopoeia [5], a total of 5 groups of Beimu (including 9 fritillaries) were officially recorded (Fig.1, Table 1). Besides, bulbs of some other Fritillaria species are often used as the substitutes in different local regions as the Chinese folk medicine.

*

E-mail: [email protected]. Tel: 86-25-85391244. Fax: 86-25-85322747. (Corresponding author)

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F.unibracteata

F.przewalskii

F.cirrhosa

F.delavayi

F.thungergii

F.hupehensis

F.pallidiflora

F.walujewii

F.ussuriensis Figure 1. The botanical origins of Beimu (in each set of image, the left refers to plants and the right to bulbs).

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Table 1. Comparison of various Beimu recorded in Chinese Pharmacopoeia Traditional action To remove heat, Sichuan, Tibet, Yunan moisten the lung, Sichuan, Qinghai Gansu, Qinghai, Sichuan resolve phlegm and relieve cough. Yunan, Sichuan, Qinghai, Tibet Hubei To remove heat and resolve phlegm, relieve cough and dispel the obstruction. Xinjiang Uygur To remove heat Autonomous Region from the lung, resolve phlegm, and reduce nodules.

Group

Species

Locations

ChuanBeimu

F. cirrhosa F. unibracteata F. przewalskii F. delavayi

HubeiBeimu

F. hupehensis

YiBeimu

F. walujewii F. pallidiflora

ZheBeimu

F. thunbergii

Zhejiang, Jiangsu

PingBeimu

F. ussuriensis

Heilongjiang, Jilin, Liaonin

To remove heat and reduce nodulation, resolve phlegm and relieve cough. To remove heat from the lung, relieve cough and resolve phlegm.

Indication Dry cough due to heat in the lung; cough with bloody sputum in consumptive diseases. Cough due to heatphlegm, subcutaneous nodule scrofula, swelling carbuncles and sore toxicity. Heat in the lung manifested by cough, stuffiness feeling in the chest and sticky sputum; scrofula and other inflammatory nodules. Attack of wind-heat on the lung; cough due to phlegm-heat; lung abscess; mastitis; sores, scrofula. Heat in the lung with cough, sticky and bloody sputum, and distress in the chest.

Fritillaria species have been the topic of research in many phytochemical and pharmacological laboratories owing to the structurally complex steroidal alkaloids and biologically fascinating effects. We have reviewed the developments in the field of Fritillaria steroidal alkaloids (FSAs) [6], and this review focuses on pharmacological activities and quality control of Beimu.

2. Pharmacological Activities of Beimu 2.1. Antitussive, Expectorant and Antiasthmatic Activities As the antitussive, expectorant and antiasthmatic effects of extracts and/or pure steroidal alkaloids from Fritillaria species correlate directly with the traditionally clinical usage of Beimu, a variety of related pharmacodynamic studies [7-18] have been extensively conducted in vitro and in vivo models (Table 2). It was found that both extracts and pure compounds showed positive results, which substantially verified the traditional curative effects of Beimu as a cough remedy. At the same time, the further investigations [19-22] on selective

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antagonism activity of FSAs at muscarinic receptors indicated that the mechanism of antiasthmatic action may be partly attributed to the selective antimuscarinic activity. Table 2. Antitussive, expectorant and antiasthmatic activities of Fritillaria spp. Active extracts and compounds Species F. thunbergii

F. anhuiensis F. przewalskii F. maximowiczii F. ebeiensis F. puqiensis F. unibracteata

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F. monantha F. wabuensis F. mellea F. taipaiensis var. ningxiaensis F. pallidiflora F. hupehensis

F. ussuriensis

Antiasthmatic activity

Ref.

Antitussive activity

Expectorant activity

Alcohol extract, total alkaloids, verticine, verticinone Alcohol extract, total alkaloids Alcohol extract, total alkaloids Alcohol extract, total alkaloids Total alkaloids, ebeiedine Puqietinone Alcohol extract, total alkaloids

Alcohol extract, total alkaloids

[7] [9]

Alcohol extract, total alkaloids Alcohol extract, total alkaloids Alcohol extract, total alkaloids Total alkaloids

[8]

Alcohol extract Alcohol extract Total alkaloids Total alkaloids Total alkaloids, delavine, hupeheninoside Alcohol extract

Alcohol extract Alcohol extract Alcohol extract Total alkaloids

Alcohol extract, total alkaloids Alcohol extract, total alkaloids Total alkaloids

Alcohol extract

[9] [9] [9] [11] [10] [12] [13] [14] [14] [15]

Total alkaloids Delavine

Total alkaloids Peimisine

[16] [17]

Alcohol extract

Alcohol extract

[18]

2.2. Antihypertensive Activity The antihypertensive effects of F. ussuriensis have been deeply tested. The aqueous extract showed a dose-dependent hypotensive effects in rats. The mechanism of hypotensive effects was demonstrated that F. ussuriensis extract not only showed a strongly angiotensin converting enzyme inhibitory activity but also direct NO/cGMP release from rat aortic rings [23]. The water extract could attenuate the increase of systolic blood pressure in the NG-nitroL-arginine methyester-induced hypertension via enhancing the generation of vascular NO and amelioration of renal functions [24]. Furthermore, verticinone, verticine and peimisine, three FSAs isolated from F. ussuriensis, inhibited angiotensin I converting enzyme activity in a dose-dependent manner [25].

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2.3. Anti-cholinesterase Activity Interestingly, on the contrast to the M-cholinolytic activity (mentioned above at Section 2.1), some FSAs, viz. ebeinone, eduardine, edpetilidine, verticinone, isoverticine, impericine, forticine, delavine, persicanidine A and imperialine isolated from F. imperialis [26,27], and N-demethylpuqietinone, hupeheninoside, ebeiedinone, yibeinoside A and chuanbeinone isolated from F. puqiensis, F. hupehensis, F. ebeiensis var. purpurea, showed acetylcholinesterase and butylcholinesterase inhibitory activity [28]. 2.4. Antitumour Activity The alkaloids ebeiedine [29] and puqietinone [30], isolated from F. ebeiensis and F. puqiensis, respectively, possessed antitumor activity against cervical cancer and Ehrlich ascites carcinoma in rats. The steroidal alkaloids N-dementylpuqietinone isolated from F. puqiensis, at a concentration of 10 μM, showed weak activity against the HL-60 human promyelocytic leukemia cell line (51% inhibition) [31]. 2.5. Miscellaneous Some other activities, such as antimicrobial activity [32], antiulcerogenic activity [33] and inhibition of platelet aggregation [34], were also ascribed to FSAs.

3. Quality control of Beimu Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

3.1. Qualitative analysis of Beimu It is quite common to find falsely labeled Beimu in herbal markets in China due to the complexity of botanical origin. Worse more, sometimes the adulterant Beimu is fraudulently sold for economic benefits, as the prices of various Beimu vary remarkably in markets. For instance, Chuan-Beimu is approximately 2–5 times more expensive than Yi-Beimu, 10–50 times than Ping-Beimu and 20–60 times than Zhe-Beimu. The tremendous financial incentives prompt dishonest merchants to adulterate Chuan-Beimu with immature ZheBeimu, which is morphologically similar to Chuan-Beimu. The adulteration adversely affects not only the commercial interest of consumers but also the safety and efficacy of the herbal drugs. The development of methods used to authenticate botanical origin of various Beimu is therefore of great value. Traditionally, the authentication of various Beimu has relied upon morphological and histological inspections [35-39]. In morphological analysis, the shape, size of the scale leaves were chosen as the differential characteristics; while in histological analysis, the starch granules, epidermal cells and stomata were selected. However, it was fairly difficult to discriminate unambiguously the specific species, due to lack of sufficiently distinctive characteristics within different Fritillaria bulbs. Hence, in many cases, such approaches are far from reliable.

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Recently, a large number of physical methods were devised by which one Fritillaria species could be distinguished from other similar ones. Wang et al [40] developed an X-ray diffraction method based on the differences of crystallininty of starch and X-ray diffraction spectra of Fritillaria powders, and five species including F. thungergii, F. ussurensis, F. pallidiflora, F. cirrhosa and F. hupehensis, could be identified. Chen et al [41] described a differential thermal analysis (DTA) to identify different species of Beimu and the adulterant Beimu (Bolbosemma panculatum and Tulipa edulis). Infrared (IR) spectroscopy, as a nondestructive and sensitive method, was also utilized in the qualitative analysis of Beimu [42-44]. Although these methods seem to be objective, the differential criteria are rather subtle and the primary data usually need mathematical analysis in order to extract the fine differences among samples. Developments in biological techniques and molecular genetic markers allow for the identification of plant species through the detection of species-specific alleles. More and more Chinese herbal drugs including some expensive and multi-origin herbs have been successfully authenticated by random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), etc. The molecular biological techniques were also used in service of species identification of Beimu [45-48]. Molecular methods carry apparent advantages over other methods: first, they are sufficiently sensitive for identification with a very small sample amount; second, they enable reliable and definitive species identification. However, there still remain some problems. The quality of DNA isolated from the processed samples tends to be unfavorable for analysis. On the other hands, they appear not simple and rapid methods, and would not be appropriate for routine use. Fingerprint analysis, especially chromatographic fingerprint analysis, has been playing more and more important role in quality control of traditional Chinese medicine. As a chromatographic profile is featured by the fundamental attributions of “integrity” and “fuzziness” or “sameness” and “difference” so as to chemically represent the samples investigated, the fingerprint provide sufficient qualitative information for quality control. An HPLC-ESI-MS fingerprint [49] was readily exploited for the purpose of identification of five Fritillaria species. Based on chemical chromatogram, fingerprint analysis would be a promising tool with regard to species identification. However, the application of chromatographic fingerprint is limited, as it requires firstly the chemical consistency of detected crude drugs, which unfortunately prone to be rather variant with different locations, ripeness stage as well as processed methods. In a word, although many qualitative analyses of Beimu are available, these methods have limitations, and the accurate identification of botanical origin is usually by the aid of combined methods. Extensive work is still needed to explore more reliable and reproducible methods to overcome the identification problem. 3.2. Quantitative Analysis of Beimu Although Beimu contains various types of metabolites including FSAs, triterpenoids, entkaurane-diterpenoids, nucleosides and sterols, the FSAs, responsible for the wide pharmacological spectra of Beimu, have been usually chosen as chemical markers for quality control in quantitative methods.

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3.2.1. Titration and Colorimetry The content of total alkaloids in Beimu, somewhat indicating the quality of crude drug, could be determined by titrametric analysis. Several titrametric models including acid-base titration [50], two-phase titration [51] and nonaqueous titration [52] were even employed for quantitative purpose. Because the real equivalent point was hard to catch accurately, the final results were usually deviated to some extent. In order to eliminate the accidental error caused by the deviation of equivalent point, the acidic dye colorimetry [53-57] was introduced. Under suitable pH condition, the Fritillaria alkaloids could quantitatively react with some reagents such as thymol blue or bromophenol green to form purple-green ion pairs, which could be extracted with chloroform and then determined using a spectrophotometer. Although the accidental error was eliminated, the analytical results were still strongly affected by the pH value and the resident water of the chloroform layer. 3.2.2. Thin-Layer Chromatography Scanning The individual Fritillaria alkaloids could be separated and determined by thin-layer chromatography scanning (TLCs) method. Those high-level FSAs such as verticine and verticinone in many Fritillaria species [58-65] and Beimu-containing compound formulas [66-69] had been quantified by various TLCs methods. Regardless of its broader application, TLCs suffers from the limitation of low reproducibility, which resulted from the loading, developing, visualizing, and scanning procedure. Furthermore, relatively low resolution and sensitivity also obscured the use of TLCs.

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3.2.3. Gas Chromatography Li et al [70,71] described two gas chromatography (GC) analytical methods, pre-column derivatization GC and direct GC, for the determination of the major active FSAs. When conducting the first method, all the hydroxyl groups in each molecular skeleton were required to react completely with derivative reagent (trimethylsilylimidazole) to ensure volatility of the products. Thus, although this method was applied for the assay of five FDAs (ebeiedine, ebeiedinone, verticine, verticinone and imperialine), it was not a rational method due to its tedious derivative procedure. Consequently, a direct GC method, using a commercially available capillary column (Supelco SAC-5 column, 30 m × 0.25 mm, i. d..), was exploited for the analysis of eight FSAs in Fritillaria species. Although verticine and verticinone coeluted, the established direct GC method, when compared with pre-column derivatization GC method, showed obvious advantages in simplicity, time-consumption, reproducibility, and accuracy. 3.2.4. High Performance Liquid Chromatography Nowadays, high performance liquid chromatography (HPLC) has become one of the most conventional analytical techniques for separation and quantification of targets in complicated matrices such as extract of traditional Chinese medicine, due to its high resolution, desirable reproducibility and powerful automation. Owing to major FSAs lacking

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conjugated unsaturation system and showing weak ultraviolet (UV) absorption, how to efficiently monitor the analytes eluted from column becomes a challenging task. In 2001 Lin et al [72] have well reviewed the application of HPLC for analysis of FSAs, and three detection strategies were summarized: (1) adding UV-absorbing chromophores via a precolumn derivatization, that is pre-column derivatization HPLC; (2) setting the detection wavelength at low wavelength (205 nm), that is HPLC-UV; (3) coupling with evaporative light scattering detection (ELSD), that is HPLC-ELSD. Among the three detection strategies, the pre-column derivatization HPLC method required time-consuming sample preparation and a complication of derivatizing reaction, and the HPLC-UV method inevitably caused high level of baseline noise and the relatively low sensitivity, whereas the HPLC-ELSD method seemed to becoming the routine technique for analysis of non-chromophoric FSAs in many laboratories, which was partly confirmed by the relevant publications on the quantitative analysis of Beimu since 2000 (Table 3). Table 3. Publicationsa) on HPLC analysis of Fritillaria steroidal alkaloids Species F. pallidiflora

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F. pallidiflora F. yuminensis F. maximowiczii

F. wabuensis

F. ussuriensis

F. monantha

F. thunbergii

Analytes Column Imperialine, Kromasil C18 imperialine-3β- (200mm × glucoside 4.6mm, i. d.), 5 μm

Mobile phase Acetonitrilewatertriethylamine (71 : 29 : 0.03)

Detection ELSD (Alltech Associates, USA), drift tube temperature of 88°C, nitrogen flow-rate of 2.38L/min Peimissine, ELSD (Alltech Associates, Waters Nova- Gradient elution with imperialine, USA), drift tube Pak C18 acetonitrile and temperature of 90°C, imperialine-3β- (150mm × glucoside, nitrogen flow-rate of 2.0 3.9mm, i. d.), 5 water (containing yibeinoside A, μm L/min 0.1% sinpeinine A diethylamine) AcetonitrileImperialine Shim-pack ELSD (Sedex 75LT, waterODS C18 France), drift tube diethylamine temperature of 40°C, (150mm × nitrogen pressure of 1.6 bar 4.6mm, i. d.), 5 (60 : 40 : 0.3) μm AcetonitrileELSD (Alltech Associates, Verticine Reliasil C18 0.02% USA), drift tube (250mm × temperature of 87°C, 4.6mm, i. d.), 5 triethylamine (57 : 43) nitrogen flow-rate of 2.3 μm L/min ELSD (Alltech Associates, Verticine, Hypersil ODS2 AcetonitrileUSA), drift tube verticinone C18 (200mm × 0.03% temperature of 83.5°C, 4.6mm, i. d.), 5 diethylamine (65 : 35) nitrogen flow-rate of μm 2.2L/min Gradient ELSD (Waters 2420 ELSD, Verticine, X Terra -C18 elution with USA), drift tube verticinone (150mm × 3.9mm, i. d.), 5 acetonitrile and temperature of 85 °C, 10 mmol/L nitrogen pressure of 0.2 μm NH4HCO3 MPa water (pH=10.10)

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

Ref. [73]

[74]

[75]

[76]

[77]

[78]

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Table 3. Continued Species F. thunbergii

Analytes Verticine, verticinone

Column Hypersil BDSC18 (250mm × 4.0mm, i. d.), 5 μm

Verticine

Kromasil ODS C18 (250mm × 4.6mm, i. d.), 5 μm

Mobile phase Acetonitrilewaterdiethylamine (70 : 30 : 0.03)

Detection ELSD (Alltech Associates, USA), drift tube temperature of 85 °C, nitrogen flow-rate of 2.2 L/min Detection at UV 208nm

Methanolwater (containing 7.5 mmol/L SDS, pH = 4.5 ± 0.1) (69: 31) Verticinone Hypersil Pre-column derivatization AcetonitrileSampleb) ODS2-C18 with 2, 40.1 mol/L CH3COONH4 dinitrophenylhydrazone and (150mm × detection at UV 375 nm. 4.6mm, i. d.), 5 water (pH = 5.0) μm (39 : 61) Imperialine Supelco C8 Gradient Samplec) ELSD (Alltech Associates, elution with USA), drift tube (150mm × temperature of 72°C, 4.6mm, i. d.), 3 wateracetonitrilenitrogen flow-rate of 2.2 μm methanol L/min. containing 0.6% triethylamine a The cited references were defined after the year of 2000. b Plasma colleted from mice after intravenous administration of verticinone. c Plasma colleted from rat after intravenous or oral administration of imperialine. Copyright © 2008. Nova Science Publishers, Incorporated. All rights reserved.

F. ussuriensis

Ref. [79]

[80]

[81]

[82]

Actually, apart from the weak UV absorption of FSAs, other challenges for chemical analysts still remain as encountered with Beimu: (1) how to fully resolve chromatographically the isomeric or stereoisomeric FSAs present in extract of Fritillaria species; and (2) how to quantify the minor or trace FSAs. Recently, HPLC coupled with mass spectrometry (MS) is increasing employed as a powerful tool combining the high resolution, high sensitivity and high selectivity for the analysis of medicinal plants. Inspired by this hyphenated approach, an HPLC couple with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) method [83] was newly developed by our research team for quantification of 26 naturally occurring steroidal alkaloids in Fritillaria species. Compared with previously published LC or GC methods, this HPLC/TOF-MS method offered improvements not only to the peak identification with an unequivocal assignment under the help of accurate mass measurements within 4 ppm error, but also to sensitivity with limits of detection remarkably down to 0.0014-0.0335 μg/ml.

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4. Conclusion As a valued traditional Chinese medicine, Beimu possesses a variety of pharmacological activities, such as antitussive, expectorant, antiasthmatic, antihypertensive, anti-cholinesterase activities. For quality control and standardization of herbal Beimu, species identification is an important issue because of its complicated botanical origins. Unfortunately, the present qualitative methods have limitations, extensive work is still needed to explore more reliable and reproducible methods to resolve the identification problem. With respect to the quantification of FSAs in Beimu, the most recently developed HPLC-TOF MS assay is likely to be the most suitable and readily adoptable quantitative method for the simultaneous determination of the major and minor bioactive ingredients in plant matrices.

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References [1] Wang, F. Z.; Tang, J. Liliaceae. In Flora Republicae Popularis Sinicae. Delectis Florae Republicae Popularis Sinicae Agenda Academiae Sinicae Edita, Ed. Science Press, Beijing, China, 1980; Vol.14, pp.101-116. [2] Qian, Z. Z.; Nohara T. Phytochemistry 1995, 40, 979-981. [3] Akhtar, M. N.; Rahman, A.; Choudhary, M. I.; Sener, B.; Erdogan, I.; Tsuda, Y. Phytochemistry 2003, 63, 115-122. [4] Xie, H. M.; Wang, M. C.; Lu R. X. J. Chin. Med. Mater. 2000, 23, 423-427. [5] The State Pharmacopoeia of the People’s Republic of China. Pharmacopoeia of The People’s Republic of China, Vol. I; Chemical Industry Press: Beijing, China, 2005. [6] Li, H. J.; Jiang, Y.; Li, P. Nat. Prod. Rep. 2006, 23, 735-752. [7] Qian, B. C.; Xu, H. J. Acta Pharm. Sin. 1985, 20, 306-308. [8] Wang, L. Y.; Han, C. H.; Wang, P.; Ni, G. Y.; Xu, S. Y. Chin. Pharmacol. Bull. 1988, 4, 375-377. [9] Li, P.; Ji, H.; Xu, G. J.; Xu, L. S. J. Chin. Pharm. Univ. 1993, 24, 360-362. [10] Ji, H.; Li, P.; Yao, L.; Zhou, S.; Xu, G. J. J. Chin. Pharm. Univ. 1993, 24, 95-97. [11] Li, P.; Ji, H.; Zhou, S.; Yao, L.; Xu, G. J.; Xu, L. S. Chin. Tradit. Herb. Drugs 1993, 24, 475-477. [12] Yao, L. N.; Sun, H. Q.; Jiang, Z.; Chen, Y. D.; Lu, C. B. J. Tongji Med. Univ. 1993, 22, 47-49. [13] Zhang, Z. Z.; Fan, C. S. Jiangxi J. Tradit. Chin. Med. 1993, 24, 179-180. [14] Mo, Z. J.; Tang, X. Y.; Sun, Z.; Li W. Chin. J. Chin. Mater. Med. 1998, 23, 51-52. [15] Yan, L.; Li, H. Q.; Dai, G. D. J. Ningxia Med. Coll. 1999, 21, 164-165. [16] Wang, L. H.; Ji, H.; Wang, C. L.; He, Q.; Li P. J. Chin. Pharm. Univ. 2003, 34, 172-176. [17] Zhang, Y. H.; Ruan, H. L.; Pi, H. F.; Cai, J. Y.; Zeng, F. B.; Zhao, W.; Wu, J. Z. Chin. Tradit. Herb. Drugs 2005, 36, 1205-1207. [18] Du, S. F. Trad. Chin. Drug Res. Clin. Pharmacol. 1996, 7, 45-46. [19] Gilani, A. H.; Shaheen, F.; Christopoulos, A.; Mitchelson, F. Life Sci. 1997, 60, 535-544. [20] Zhou, Y.; Ji, H.; Li, P.; Jiang, Y. J. Chin. Pharm. Univ. 2003, 34, 58-60. [21] Ji, H.; Wang, L. H.; Li, P.; Jiang, Y. Chin. J. Nat. Med. 2005, 3, 116-120.

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In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-Tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 10

Yinyanghuo (淫羊藿，Epimedium spp.) Xiao-jia Chen, Yi-tao Wang and Shao-ping Li* Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China

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1. Introduction The plants of Epimedium genus (Family Berberidaceae) are widespread in North Africa, north of Italy, Black Sea, West Himalayan, China, North Korea, Japan and Northeast Asia. There are about 50 species of Epimedium found throughout the world. China, where there are about 40 species, is the geographical distribution center of Epimedium [1]. Based on the distribution character, resource quantity and growing with other species mingled, four main production regions in China were suggested [2]: Northeastern China where only E. koreanum grows, northwestern and northern China are main distribution of E. brevicornu, E. sagittatum mainly grows in eastern and southern China, and a lot of other species of Epimedium distribute in southwestern China (Fig. 1). For example, besides E. pubescens, there are still 13 species of Epimedium grow in Sichuan, while E. wushanense and other 9 species of Epimedium can be found in Guizhou Province. According to the Chinese Pharmacopoeia, the dried aerial part of E. brevicornu Maxim., E. sagittatum (Sieb. et Zucc.) Maxim., E. pubescens Maxim., E. wushanense T.S.Ying and E. koreanum Nakai are used as Yinyanghuo (Fig. 2), a well-known Chinese herbal medicine [3]. It is first recorded in Shen Nong Ben Cao Jing (Han Dynasty of China, 2000 years ago), the oldest book of Materia Medica in China, and it has been used to treat diseases for about 2000 years. The Chinese name, Yinyanghuo (horny goat weed), derived from the phenomenon that goats have excessive sexual intercourse after they graze on the herbs. Traditionally, Yinyanghuo has the effects of reinforcing the kidney yang, strengthening the tendons and bones, and relieving rheumatic conditions, which can be used for treatment of impotence,

*

E-mail address: [email protected]. Tel: 853-83974692. Fax: 853-28841358 (Corresponding author)

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● E. brevicornu; ▲ E. sagittatum; ○ E. pubescens; ◆ E. wushanense; ■ E. koreanum

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Figure 1. The distribution of major species of Epimedium in China.

A

B

C

D

Figure 2. The plants and raw materials of (A, B) E. pubescens and (C, D) E. koreanum.

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seminal emission, weakness of the limbs, rheumatoid arthralgia with numbness and muscle contracture, and climacteric hypertension [3]. Actually, many other species of Epimedium, not used as Yinyanghuo, are also used in folk medicines. Thus, quality control of Yinyanghuo, which is very important for the safety and efficacy of medication, was reviewed based on its pharmacological activities and bioactive components.

2. Pharmacological Activities

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2.1. Estrogen-Like Activity Estrogens are steroid hormones which exhibit a broad range of physiological activities. The sudden drop in estrogen levels during menopause will cause bone loss and increase the risk of heart disease. Estrogen replacement therapy is the main treatment for menopause symptoms. The mechanism of estrogen action is binding with the estrogen receptors (ERs) in target cells. There are two ERs, ERα and ERβ, mediate the effects of the hormone. Their structure, chromosomal location and tissue distribution are different. The biological effect of an ER ligand in a specific tissue is determined by the expression of ERα and ERβ in that tissue. The activated steroid-receptor complex interacts with nuclear chromatin to initiate hormone-specific RNA synthesis. The attachment of two estrogen-linked receptors to the genome is required for a response, which results in the synthesis of specific proteins that mediate a number of physiologic functions [4]. ERs are widely distributed in human body, such as uterus, ovary, bone, vascular endothelium, smooth muscle and the central nervous system, etc. So estrogens have effects on related systems. However, estrogens have unacceptable adverse effects such as breast and endometrial cancer, thromboembolism and myocardial infarction, etc. Thus, search for selective estrogen receptor modulators, which have beneficial effects for menopausal symptoms and bone health without increasing the risk of breast cancer and cardiovascular disease, are the objectives of modern pharmaceutical research. Phytoestrogens are plant-derived non-steroidal compounds with estrogen-like biological activities. They include the subclasses of isoflavonoids, flavonoids, stilbenes and lignans, whose structures are very similar to the estradiol, and exhibit selective estrogen receptor modulating activities [5]. Actually, extract of Epimedium with rich flavonoids was found to be a potent and specific estrogenic mimic. Its activities on ERs have been investigated using estrogen-responsive bioassays, recombinant yeast bioassay and the Ishikawa Var-I assay [6], stably transfected ERα and ERβ-responsive cell lines [7], or breast cancer (MCF-7) cells [8, 9]. The studies also showed that Epimedium had a broad range of therapeutic applications in skeletal, cardiovascular, reproductive, and nervous system, etc. 2.1.1. Prevention of Osteoporosis One of the most important indications of estrogens is anti-osteoporosis. Effects of Epimedium on the skeletal system have been demonstrated both in vitro and in vivo. Rat models with osteoporosis induced by ovariectomy [10-15], retinoic acid [16-19] or glucocorticoid [20, 21] were used in vivo studies. It is found that the flavonoids of several species of Epimedium, such as E. sagittatum, E. pubescens and E. brevicornu, were able to increase the bone mineral density, the content of calcium and phosphorus in serum and bone,

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decrease the concentration of calcium in urine, and inhibit the activity of alkaline phosphatase. Histological results showed that the flavonoids can increase the bone mass, thickness and number of trabecular, reduce the trabecular separation [10-21]. In addition, combination of diethylstilbestrol and the flavonoids will have a synergetic effect on osteoporosis without adverse effects on uterus [12]. The effects of Epimedium were also investigated by using osteoblast or osteoclast as models in vitro. Osteoblast models usually use osteoblast-like cells (OS-732 [22] or UMR 106 [23, 24]) or osteoblasts obtained from new born rat calvaria by digestive enzymes [25-27] or by inducing human marrow mesenchymal stem cells [28]. It is shown that the total flavonoids [22, 25, 27] and some single flavonoids (icariin, icariside I, icariside II, epimedin B and epimedin C) [23, 24, 26, 28] of Epimedium can stimulate the proliferation, differentiation and maturation of the osteoblasts, increase the expression of osteoporogeterin mRNA [27], and production of bone morphogenetic protein 2 [28]. The effects of icariin on the osteoclasts are investigated by culturing osteoclasts with bone slices and seeing its influence on the bone resorption pits. The results showed that icariin could dose-dependently inhibit the osteoclasts formation and bone resorption [29-31].

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2.1.2. Effects on Cardiovascular Function It is known that phytoestrogens have protection effects on cardiovascular system. Effects of icariin and water extract from E. grandiflorum on myocardial activities of rabbits were studied. The myocardial and circulatory indices indicated that both icariin and the water extract decreased the peripheral resistance, which suggested their usefulness in treatment of hypertension-complicated coronary disease [32]. The vasodilating mechanism of icariin might attribute to its blocking calcium channels [33]. Icariin also promotes the differentiation of embryonic stem cells into cardiomyocytes in vitro. The effect was related to its increasing and accelerating gene expression of α-cardiac major histocompatibility complex and myosin light chain 2v, regulating the cell cycles and inducing apoptosis, and elevation of the cAMP/cGMP ratio in embryonic stem cells, as well as upregulation of endogenous NO generation during the early stages of cardiac development [34, 35]. The effect of total flavonoids of Epimedium (TFE) on experimental acute myocardial infarction was also investigated. TFE could significantly attenuate the degree and extent of myocardial infarction with the decreased activities of serum creatine phosphokinase, lactate dehydrogenase and the level of malondialdehyde, as well as increased serum superoxide dismutase activity [36, 37]. Several experiments proved that Epimedium has a favorable effect on hemorheology and hemodynamics. Flavonoids of E. brevicornu could inhibit thrombosis by reducing blood mucosity and inhibiting agglutination of red cell [38]. In addition, the effect of water extract from E. koreanum on erythrocytes was investigated by atomic force microscopy (AFM). The images of the surface structures showed clear concave and progressive increase of surface roughness of erythrocyte after incubation with the extract at concentration of 0.2 or 0.05 g/L, far below its critical hemolytic levels. The AFM results also indicated that the granules of the fine surface structure increased, which caused by aggregation of membrane protein. Further study showed that the change in surface topography of erythrocyte membrane might be connected with the increase of intracellular free Ca2+ induced by the extract [39].

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2.1.3. Effects on Breast Cancer Estrogens drive the growth of ER positive breast tumors, and selective estrogen receptor modulators, such as tamoxifen, are used to reduce tumor recurrence. It is found that ethanol extract from leaves of E. brevicornu has potent (EC50: 1.3μg/mL) and specific ER-stimulatory activity. It increased estrogen-responsive human breast cancer cell proliferation at low doses, but paradoxically caused profound inhibition of growth at higher doses. Using bioassayguided fractionation, a prenylflavone, breviflavone B, was isolated. It exerted bilateral effects, stimulation and inhibition, on breast cancer cell proliferation, mimicking the effects of E. brevicornu. In contrast to estradiol and genistein, high doses (>2 μM) of breviflavone B almost eliminated ERα protein; a process that may be mediated through increased proteasome degradation [40].

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2.1.4. Neuroprotective Activities Estrogens are proved to be beneficial to central nervous system. Epimedium also have potential neuroprotective activities with fewer side effects [41-44]. The polysaccharide and flavonoids complex of E. wushanense can raise the hypothalamic monoamine neurotransmitter levels in aging male rats and improve the learning and memory in aging rats and mice. It can also inhibit the activities of acetylcholine esterase both in brain tissues and whole blood in mice [41]. The study in vitro showed that methanol extract of E. sagittatum had significant stimulation activity on neurite outgrowth of PC12h cell, an established cultured cell of rat pheochromocytoma. Bioassay guided fractionation discovered that six prenylated flavonol glycosides i.e. ikarisoside A, icarisid II, epimedoside A, icariin, epimedin B and epimedokoreanoside-I were the active ingredients [42]. Icaritin, an active natural ingredient from E. sagittatum, was investigated to assess its neuroprotective effect against the toxicity induced by Aβ25-35 in primary cultured rat cortical neuronal cells. Aβ25-35 induced neuronal toxicity, characterized by decreased cell viability, lactate dehydrogenase release, and neuronal DNA condensation, is associated with both the loss of membrane potential and the decrease of the expression of Bcl-2 family proteins. The phenotype alternation induced by Aβ25-35 could be reversed by icaritin. The neuroprotective effects of icaritin mentioned above were estrogen receptor dependent due to the blocking action could be induced by estrogen receptor antagonist, ICI 182780. PD98059, a specific inhibitor of MEK [mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) kinase], could attenuate the protective effects, which implied MAPK/ERK pathway may also be involved in and partly contributed to the neuroprotective effects of icaritin [43]. In addition, antidepressant-like effect of icariin was also investigated in behavioral despair models of Kunming strain of male mice. It can significantly shorten immobility time in the forced swimming test (FST) and the tail suspension test. Moreover, it was observed that the stress of FST exposure induced increases of monoamine oxidase A and B activities, and decreases of monoamine neurotransmitter levels in brain, as well as the increases of corticotropin-releasing factor levels in serum, which could be reversed by icariin. These results suggested that icariin possessed potent antidepressant-like properties that were mediated via neurochemical and neuroendocrine systems [44].

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2.2. Promotion of Male Sexual Function In China, there is a long history of using Epimedium to treat sexual dysfunction. Modern pharmacological studies show that Epimedium has stimulation on reproductive system [4549]. Flavonoids extracted from E. brevicornu and icariin can increase the weights of pituitary gland, epididymis and seminal vesicle, secretion of stimulate testosterone and luteinizing hormone in immature rats. The increase of testosterone was also observed in vitro [45, 46]. On the other hand, water extract of E. brevicornu can relax the corpus cavernosum smooth muscle, and both the extract and icariin can also improve penile erectile function. These results suggest that Epimedium may have a therapeutic effect on erectile dysfunction, which is correlated with NO-cGMP-PDE 5 pathway [47-49].

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2.3. Immunomodulation Activity There are many reports for the effects of Epimedium, enhancement and/or suppression, on immune system. Therefore, it is assumed that Epimedium is a bilateral modulator to immune system. It is found that both Epimedium polysaccharise (EPS) and icariin have immunostimulant effect on thymus. EPS can enhance the proliferation and interleukin-2 (IL-2) production of thymocytes, decrease the L3T4+ and Lyt2+ cell numbers, and decrease responses to concanavalin A (Con A) stimulation of the thymus, which possibly caused by the migration of thymocytes from the thymus to peripheral organs promoted by EPS. Icariin has no influence on L3T4+ and Lyt2+ cell numbers, so it increase response to Con A stimulation to thymus cell [50]. It is also found that icariin could induce a weak and delayed proliferation of peripheral blood mononuclear cells from healthy donors in vitro. Both T- (TCR αβ+) and B cells are its target cells, where icariin increased the ratio of CD4-8+ and CD4+8- cells. Icariin in certain concentrations could also increase lymphokine-activated killer (LAK) cell activity in both tumor patients and healthy donors, and natural killer (NK) cell activity in tumor patients. Moreover, icariin also could stimulate the production of tumor necrosis factor-α in monocytes from healthy donors [51]. These findings suggest that icariin may be applied to adoptive immunotherapy, which may be superior to IL-2 alone because the former can induce generation of LAK cells at appropriate concentration [51]. In addition, the immune function of immunodepressant mice can be enhanced by Epimedium. It is found that n-butanol fraction from aerial parts of E. hunanense and epimedin C could significantly enhance the response of spleen antibody-forming cells, lymphocyte proliferation and IL-2 production in mice treated with hydrocortisone acetate [52]. Total flavonoids from E. wushanese had antagonistic effect on the decrease of IL-2 and NK cell activities induced by N-hydroxyurea [53]. It is also shown that the aqueous extract of E. koreanum could significantly block the suppression of total serum and antigen-specific immunoglobulin G2a antibodies, phagocytic activity, delayed-type hypersensitivity response and interferon-γ production by oral ovalbumin [54]. Actually, Epimedium polysaccharides could increase the spleen index, thymus index, hemolysin level, hemolytic ability of spleen plaque-forming cells, and the number of white blood cells in cyclophosphamide treated mice and tumor-bearing mice [55].

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223

In contrast to immune potentiation, Epimedium was also shown its suppression on immunity function. Baohuoside-1, a flavonoid isolated from E. davidii, had selectively immunosuppressive effects on T-cell and B-cell activation in vitro, which could effectively prevent rat heart allograft rejection in vivo [56]. It is also found that the methanolic extract of root and rhizome of E. alpinum (MEEA) exerted stimulatory or inhibitory effects on immune response depending on its concentration. At low concentration, it could significantly enhance proliferation of splenocytes and thymocytes triggered by Con A, whereas at high concentration, it would suppress lymphocytes proliferation. These effects are due to the stimulation or inhibition of IL-2 production and the up or down-regulation of IL-2 receptor α expression. In addition, higher concentrations of MEEA may induce apoptosis [57]. 2.4. Miscellaneous Besides the pharmacological activities mentioned above, Epimedium also have cytotoxic effects on the cells of leukemia, nasopharyngeal carcinoma, pulmonary carcinoma, gastric carcinoma and hepatoma [58-61]. In addition, Epimedium also has antihepatotoxic [62, 63], anti-inflammatory [64], antiviral [65], antioxidant [66, 67] and anti-aging [68-70] activities. Up to date, most activities of Epimedium mainly attribute to its flavonoids [11-19, 21-53, 5667, 69].

3. Quality Control

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3.1. Identification There are about 50 species of Epimedium, which are very similar in their appearance but significantly different with chemical components and biological activities, in the world. Thus, the exact identity is assurance of safety and efficacy of medication. Conventionally, morphological and microscopic characteristics are used for identification of herbs, which need rich professional experience. Recently, genetic characteristics have been utilized for the identification of plant species. Molecular markers, such as random amplified polymorphic DNA (RAPD) [71] and restriction fragment length polymorphism (RFLP) [7, 71] are good candidates for the identification and authentication of Epimedium species. Different species were easily distinguished by its typical amplified band pattern. Moreover, internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA [72] and 5S rRNA gene spacer sequences [72, 73] were also used to identify the medicine. Among the five species of Epimedium listed in Chinese Pharmacopoeia, the diversity of E. koreanum is obvious [7, 72, 73]. 3.2. Quantitative Analysis At present, flavonoids, mainly icariin, are believed to be the major active components in Epimedium. And among more than 130 compounds identified in different species of Epimedium, most are the flavonoids [74]. Thus, quantitative analysis mostly focused on the flavonoids in Epimedium. Up to date, a series of methods, including UV-Vis

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spectrophotometry [75, 76], thin layer chromatography [77-82], high performance liquid chromatography [83-96], ultra-performance liquid chromatography (UPLC) [97, 98], capillary electrophoresis (CE) [99-104] and capillary electrochromatography (CEC) [105], have been reported to quantify the contents of flavonoids in Epimedium. 3.2.1. HPLC HPLC was the most frequently used method for quality control of Epimedium. In the early years, researchers usually determined icariin only [83-85]. However, it is well known that interaction of multiple chemical compounds contributes to the therapeutic effect of Chinese medicine. Therefore, the analysis of multiple components is necessary and helpful to control the quality of Chinese medicine. HPLC methods for simultaneous determination of multiple flavonoids were summarized in Table 1. Mass spectrometry (MS) was also utilized to help identify the flavonoids [91, 93, 95, 96, 106-110], but there was few reports for the quantification of HPLC-MS [95, 96]. Table 1. Summary of simultaneous determination of multiple flavonoids in Epimedium using HPLC

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Analytes

Column

Mobile phase

Ref

Epimedin A, epimedin B, epimedin C, icariin Ikarisoside C, epimedoside A, epimedin B, epimedin C, icariin, ikarisoside F, baohuoside II, sagittatoside B, baohuoside I Icariin, epimedin A, epimedin B, epimedin C Epimedin C, icariin

ODS (4mm×150mm, 5μm) DISK-Cphenyl (3.9mm×300mm, 10μm)

water-acetonitrile (73:27) acetonitrile-1.4% aqueous acetic acid with gradient elution

[86]

Shim-pack CLC-ODS (6.0mm×150mm) Hypersil BDS-C18 (250mm×4mm, 5μm

[88]

Rouhuoside, icariin

Epimedin A, epimedin B, epimedin C, icariin

BECKMAN COULTER TM-C18 (250mm×4.5mm, 5μm) Zorbax ODS (4.6mm×250mm, 5μm)

water-acetonitrile (70:30) acetonitrile-0.05% phosphoric acid with gradient elution acetonitrile-water (25:75)

[91]

Epimedin A, epimedin B, epimedin C, icariin

Zorbax SB-C8 (4.6mm×250mm, 5μm)

Hexandraside E, kaempferol-3-Orhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2’’-Orhamnosyl icariside II, baohuoside I

Zorbax SB-C18 (4.6mm×250mm, 5μm)

acetonitrile-0.5% aqueous acetic acid (30:70) acetonitrile-1% aqueous acetic acid with gradient elution acetonitrile-water with gradient elution

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

[87]

[89]

[90]

[92]

[93]

225

Yinyanghuo (淫羊藿，Epimedium spp.) Table 1. Continued Analytes Icariin, epimedin C, sagittatoside B Icariin, epimedin A, epimedin B, epimedin C, hyperin

Mobile phase

Ref

Venusil ASB-C18 (4.6mm×250mm, 5μm) Capcell Pak C18 (2.0mm×150mm, 5μm)

acetonitrile-water with gradient elution 5mM ammonium formate (pH 4.0)-90% acetonitrile containing 5nM ammonium formate (pH 4.0) with gradient elution

[94] [96]

R4

R1 R3O

Column

O OR5

Glc=β-D-glucose Rha=α-L-rhamnose

OR2

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OH

Xyl=β-D-xylose

O

M.W.=Molecular Weight

Compounds

R1

R2

R3

R4

R5

M.W

2拻 -O-rhamnosyl icariside II baohuoside I

-CH2CH=C(CH3)2

-rha(2-1)rha

-H

-H

-CH3

660

-CH2CH=C(CH3)2

-rha

-H

-H

-CH3

514

baohuoside II

-CH2CH=C(CH3)2

-rha

-H

-H

-H

500

baohuoside VII

-CH2CH=C(CH3)2

-rha(4-1)glc

-H

-H

-CH3

676

caohuoside C

-CH2CH=C(CH3)2

-rha

-H

-OH

-CH3

530

epimedin A

-CH2CH=C(CH3)2

-rha(2-1)glc

-glc

-H

-CH3

838

epimedin B

-CH2CH=C(CH3)2

-rha(2-1)xyl

-glc

-H

-CH3

808

epimedin C

-CH2CH=C(CH3)2

-rha(2-1)rha

-glc

-H

-CH3

822

epimedoside C

-CH2CH=C(CH3)2

-H

-glc

-H

-H

516

hexandraside E

-CH2CH=C(CH3)2

-glc

-glc

-H

-H

678

hexandraside F

-CH2CH=C(CH3)2

-rha(3-1)glc

-glc

-H

-CH3

838

icariin

-CH2CH=C(CH3)2

-rha

-glc

-H

-CH3

676

kaempferol-3-O-rhamnoside

-H

-rha

-H

-H

-H

432

sagittatoside A

-CH2CH=C(CH3)2

-rha(2-1)glc

-H

-H

-CH3

676

sagittatoside B

-CH2CH=C(CH3)2

-rha(2-1)xyl

-H

-H

-CH3

646

Figure 3. Chemical structures of 15 investigated flavonoids.

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In our study [93], 15 flavonoids (Fig. 3), including hexandraside E, kaempferol-3-Orhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2’’-Orhamnosyl icariside II and baohuoside I, were simultaneously quantified in different species

A

m AU 175 150 125 100 75 50 25 0 0

AU 0.30

7

1 5

2 10

0.10

4

9 11+121314

15

20

25

30

35

40

15

0.05

45

m in

7

20 1

2

C

10

5 4 3 15

m AU 120

8 20

25

11+121314 910

6.00

1314 1112

8.00

10.00

15 12.00 m in

6 7

30

35

40

5 4

0.02 45

m in

6

0.00 0.00

2 2.00

14 13 1112

3 9 4.00

6.00

8.00

10.00

15

12.00 m in

6

0.25

7

0.20

7

0.15

20

1

0 0

5

2 10

4 3

175 150 125 100 75 50 25 0 0

5 8

15

m AU

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4.00

AU

100

40

20

25

14 11+1213 9 30

35

40

0.10

15

0.05

2

3

45

m in

0.00

2.00

6

4.00

5

10

15

8 20

12.00 m in

25

15

0.10

911+12 13 30

35

3 5 4

0.00 40

45

m in

0.00

2.00

14 1112 13

9

4.00

6.00

8.00

10.00

15 12.00 m in

AU 7

0.12 7

80

0.10 0.08

40 20 0 5

10

15

m AU

5

0.06

5 4 6 3

1

9 11+121314

20

25

30

35

40

4

0.04

15

0.02

1

45

m in

0.00

2.00

AU

6

6

3

0.00

80

10 4.00

6.00

1112 1314

8.00

10.00

15 12.00 m in

6

0.16 7

60

0.12

40 20

1

2

0 0

10.00

7

0.20 3 5 4

8.00

0.30

14 2

6.00

15

6

0.40 7

1

14 12 13

9

AU

m AU 100

0

5 4

0.00

60

F

2.00

0.04

60

E

0.00 AU

0.06

80

D

910

0.08

0 5

4 3

0.10 15

40

1 2

0.00

6

60

5

0.15

5 3

80

0

6

0.20 6

m AU

B

7

0.25

5

10

14

45 3 15

0.04

11+1213 910 20

25

30

35

0.00 40

7

0.08 15

45

m in

0.00

2 2.00

45 3 4.00

14 8 6.00

910 8.00

12 13 10.00

Figure 4. (Continued on next page.)

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15 12.00 m in

227

Yinyanghuo (淫羊藿，Epimedium spp.) m AU

G

0

H

67

60 50 40 30 20 10 0

3

0.12 0.10 0.08 0.06 0.04 0.02 0.00

1 5

45

2 10

11+12

15

8 20

m AU 120

25

13

9 10 30

35

40

45

m in

1

20

2

0 5

10

15

6 45 15

14

3

2.00

13

910 4.00 3

15

11

45 2 6.00

8.00

10.00

12.00m in

7

0.12

60 40

7

0.16

3

80

0.00

6

AU 0.20

7

100

0

AU

15 14

8

20

25

30

HPLC

35

40

6 45

0.08

11+12 9 1314 10

0.04

8

0.00 45

m in

0.00

2.00

4.00

6.00

9 10 8.00

11

1314 10.00

15 12.00 m in

UPLC

1, hexandraside E; 2, kaempferol-3-O-rhamnoside; 3, hexandraside F; 4, epimedin A; 5, epimedin B; 6, epimedin C; 7, icariin; 8, epimedoside C; 9, baohuoside II; 10, caohuoside C; 11, baohuoside VII; 12, sagittatoside A; 13, sagittatoside B; 14, 2’’-O-rhamnosyl icariside II; 15, baohuoside I.

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Figure 4. Typical HPLC (left) and UPLC (right) chromatograms of extracts of (A) E. brevicornu; (B) E. sagittatum; (C) E. pubescens; (D) E. wushanense; (E) E. koreanum; (F) E. acuminatum; (G) E. davidii (Adapted from Ref. 93 and 98, with permission.)

of Epimedium using HPLC. The typical HPLC chromatograms of some species of Epimedium were shown in Fig. 4 (left). The results showed that the contents of the 15 investigated flavonoids were greatly variant in different species or locations of Epimedium. The content of icariin, which is considered as the major active component and the quality marker of Yinyanghuo, is not always the highest flavonoid in the samples. The flavonoids, such as epimedin A, B, C and baohuoside I, whose pharmacological activities have been verified [26, 56, 61], are also abundant in the species of Epimedium. Thus the analysis of multiple components is more reasonable for quality control of Yinyanghuo. In order to evaluate the variation of Epimedium, hierarchical clustering analysis was performed based on characteristics of 15 investigated compounds peaks in HPLC profiles. The results showed that 26 samples were divided into three main clusters, which are in accordance with their flavonoids contents. Especially, samples in the cluster with low content of flavonoids are mainly the species of Epimedium not used as Yinyanghuo according to Chinese pharmacopoeia. In addition, it is found that E. acuminatum, E. myrianthum and E. davidii also have high content of flavonoids, which may have similar therapeutic efficacy with Yinyanghuo though further investigation is need. As a result, epimedin A, B, C and icariin are suggested as the reasonable markers for quality control of the species of Epimedium used as Yinyanghuo [93]. 3.2.2. UPLC Although HPLC is stable and reliable, it suffers from relative long analysis time and high solvent consumption. UPLC retains the practicality and principles of HPLC while creating a step-function improvement in chromatographic performance. It utilizes sub-2 µm particles,

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mobile phases at high linear velocities, and instrumentation that operates at higher pressures than those used in HPLC, so as to obtain dramatic increases in resolution, sensitivity and speed of analysis. Nowadays, UPLC is also used to determine flavonoids in Epimedium [97, 98]. 15 flavonoids in 37 samples from 17 species of Epimedium were analyzed using UPLC in a short time (Fig. 4, right). It is shown that the results were consistent with those determined by HPLC, but UPLC method has higher sensitivity, lower solvent consumption and much shorter analysis time (Table 2). Fingerprint was introduced to evaluate the homogeneity of Epimedium. However, the similarity of the 37 tested samples was pretty low because of the great chemical variation among different species of Epimedium. Therefore, simulative mean chromatogram was generated using the samples (one used for validation) of the cluster with high content of flavonoids (high content cluster) after hierarchical clustering analysis. The similarity calculation was carried out after standardization of UPLC profiles. The correlation coefficient (entire chromatogram) of the sample for validation to mutual mode of high content cluster was 0.96, while the value for samples of middle and low content clusters were 0.70±0.15 and 0.18±0.03, respectively [98]. Table 2. Comparison of HPLC and UPLC Parameters Column Solvent consumption Analysis time Linearity

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Sensitivity Precision Repeatability Recovery

HPLC SB-C18 (250×4.6mm, 5 µm) ~50 mL 50 min R2 > 0.9997

UPLC BEH-C18 (50×2.1mm, 1.7 µm) ~3 mL 13 min R2 > 0.9997

LOD25

>35

37.5

25

>50

36.3

>17.5

>25

35

37.5

25

>50

>36.3

>17.5

>25

35

37.5

>25

50

36.3

>17.5

25

4.375

4.688

3.125

12.5

9.075

17.5

25

4.375

4.688

6.25

12.5

18.15

17.5

>25

35

18.75

25

25

36.3

>17.5

>25

>35

37.5

25

>50

>36.3

>17.5

>25

17.5

18.75

25

50

36.3

>17.5

>25

35

37.5

25

50

>36.3

>17.5

2.3. Antivirus Activity Flavonoids have been regarded as potential anti-HIV agents [4]. Acacetin-7-O- β-Dgalactopyranoside from C. morifolium was showed as an active anti-HIV principle [5]. Lee et al reported that apigenin-7-O-β-D-(4″-caffeoyl) glucuronide from C. morifolium showed

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Jin-ao Duan, Yuping Tang and Dawei Qian

strong HIV-1 integrase inhibitory activity (IC50 = 7.2 ± 3.4 μg/mL) and anti-HIV activity in a cell culture assay (EC50 = 41.86 ± 1.43 mg/mL) using HIV-I (IIIB) infected MT-4 cells [6]. 2.4. Antitumor Activity Fifteen pentacyclic triterpene diols and triols from the non-saponifiable lipid fraction of the edible flower extract of C. morifolium were evaluated for their inhibitory effects on Epstein–Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-Otetradecanoylphorbol-13-acetate, in Raji cells as a primary screening test for antitumorpromoters. All of the compounds tested showed inhibitory effects against EBV-EA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Evaluation of the cytotoxic activity of six compounds against human cancer cell lines revealed that arnidiol possesses a wide range of cytotoxicity, with GI50 values (concentration that yields 50% growth) of mostly less than 6 μM [4,7]. Ellagic acid from C. morifolium have been shown to be a potent inhibitor towards lens aldose reductase [8]

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2.5. Antimutagenic Activity The methanol extract from the flower heads of C. morifolium showed a suppressive effect on umu gene expression of the Son of sevenless (SOS) response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3- (5-nitro-2-furyl) acrylamide (furylfuramide). Suppressive compounds were isolated and identified as the flavonoids acacetin, apigenin, luteolin, and quercetin. They suppressed the furylfuramide-induced SOS response in the umu test. And they suppressed 60.2, 75.7, 90.0, and 66.6% of the SOSinducing activity at a concentration of 0.70 μmol/mL. The ID50 values of acacetin, apigenin, luteolin, and quercetin were 0.62, 0.55, 0.44, and 0.59 μmol/mL. These compounds had the suppressive effects on umu gene expression of the SOS response against other mutagens, 4nitroquinolin 1-oxide and N-methyl-N'-nitro-N- nitrosoguanidine, which do not require livermetabolizing enzymes. These compounds also showed the suppression of SOS-inducing activity against the other mutagens aflatoxin B1 and 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole (Trp-P-1), which require liver- metabolizing enzymes, and UV irradiation. In addition to the antimutagenic activities of these compounds against furylfuramide, Trp-P-1 and activated Trp-P-1 were also assayed by the Ames test using S. typhimurium TA100 [9]. 2.6. Anti-inflammatoy Activity Akihisa et al revealed the anti-inflammatory activity of the flower extract of C. morifolium [10]. Fourteen triterpene diols and triols from the edible flower extract of C. morifolium were evaluated with respect to their anti-inflammatory activity against 12-Otetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice. All of the triterpenes showed marked inhibitory activity, with a 50% inhibitory dose (ID50) of 0.03-1.0 mg/ear, which was more inhibitive than quercetin (ID50 = 1.6 mg/ear), a known inhibitor of TPAinduced inflammation in mice [11].

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2.7. Antioxidant Activity Kim et al isolated two new dicaffeoylquinic acids, 3,5-dicaffeoyl-epi-quinic acid and 1,3dicaffeoyl-epi-quinic acid from C. morifolium together with six known dicaffeoylquinic acid derivatives and three flavonoids. These compounds were assessed for antioxidant activities in the DPPH radical and superoxide anion radical scavenging systems. Most of the isolates showed strong antioxidant activities in these assay systems. Two new compounds showed potent superoxide anion radical scavenging activity (IC50 = 2.9 ± 0.1 for 3,5-dicaffeoyl-epiquinic acid and 2.6 ± 0.4 mg/mL for 1,3-dicaffeoyl-epi-quinic acid, respectively) in the xanthine/xanthine oxidase system as compared to quercetin and also showed potent DPPH radical scavenging activity (IC50 = 5.6 ± 0.1 for 3,5-dicaffeoyl-epi-quinic acid and 5.8 ± 0.2 microg/mL for 1,3-dicaffeoyl-epi-quinic acid, respectively) [12].

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2.8. Antitubercular Activity Akihisa et al obtained twenty-eight 3-hydroxy triterpenoids from the non-saponifiable lipid fraction of the flower extract of C. morifolium and one lupane-type 3α-hydroxy triterpenoid, which were tested for their antitubercular activity against Mycobacterium tuberculosis strain H37Rv using the Microplate Alamar Blue Assay (MABA). Fifteen compounds showed a minimum inhibitory concentration (MIC) in the range of 4-64 mg/mL, among which maniladiol (MIC 4 mg/mL), 3-epilupeol (4 mg/mL), and 4,5α-epoxyhelianol (6 mg/mL) exhibited the highest activity. Cytotoxicity of 3-epilupeol against Vero cells gave an IC50 value of over 62.5 mg/mL, suggesting some degree of selectivity for M. tuberculosis [13]. Zheng et al [14] obtained two new acidic polysaccharides, F4 and F5 from the flowers of C. morifolium, biological tests revealed that F4 and F5 could simulate the mitogen induced T and B lymphocyte proliferation in vitro.

3. Quality Control The chemical analysis and quality control of Chrysanthemum flower and extracts is reviewed here. Important constituents present in the medicinally used flower are the flavonoids and glycosides, caffeoylquinic acids, pentacyclic triterpene diols and triols, and essential oils etc. In Chinese Pharmacopeia, chlorogenic acid is assayed for the part of the quality control. Use octadecylsilane bonded silica gel as the stationary phase and a mixture of 0.1 mol/L sodium dihydrogen phosphate buffer and methanol (70:30) as the mobile phase. As detector a spectrophotometer set at 328 nm. The number of theoretical plates of the column is not less than 2500, calculated with reference to the peak of chlorogenic acid. It contains not less than 0.20 percent of chlorogenic acid, calculated with reference to the dried drug [15].

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3.1. Flavonoids and Their Glycosides Of all the compound classes present in Chrysanthemum flower, the flavonoids and glycosides have received by far the most attention. This is due to that Chrysanthemum species have also been demonstrated to produce a wide variety of flavonoids and phenols [6,16,17]. For qualitative identification of flavonoids, HPLC is a common method [18-20]. And liquid chromatography/mass spectrometry (LC/MS) was considered as the most powerful approach [21-23]. LC/MS has been widely used to identify all kinds of flavonoids in different plant samples such as cocoa, fresh herbs, cauliflower, mango and kale [23-27]. It is ascribed that LC/MS not only provides the molecular mass of the different constituents but also is able to identify unstable compounds in solution, such as acylated flavonoids [21]. Moreover, LC/MSn could also be used to differentiate O-glycosides, C-glycosides and O,C-glycosides [20,21,27] as well as the position of some functional groups such as hydroxyl and carboxylic groups in flavonoids [29-31]. Recently, the chromatographic fingerprints have become a pivotal tool in the quality control of complex herbal plants and vegetables [32]. In the study of Lai et al [33], a liquid chromatography/diode array detector-atmospheric pressure chemical ionization/mass spectrometry (LC/DAD-APCI/MS) was successfully developed to identify and characterize the main flavonoids and caffeoylquinic acids of three common Compositae plants (C. morifolium, Artemisia annua, and C. coronarium). Identifications were performed by comparing the retention time, UV and mass spectra of samples with standards or/and earlier publications. The crude methanolic extracts of these plants were assayed directly using LC/MS without any further pretreatment. The proposed method is rapid and reproducible and is useful for characterization and evaluation of different plant flavonoids and caffeoylquinic acids. A total of 41 different flavonoids and 6 caffeoylquinic acids were identified and confirmed by APCI-MS. The main components of three Compositae plants were also compared. Although there exist some similarities in the flavonoidic content of the leaf and flower of C. morifolium, significant variations in their varities and concentrations were observed. Artemisia annua processes substantial amount of alkylated derivatives of flavones and C. coronarium contains only caffeoylquinic acids. These findings suggest that although all the plants studied are from the same Compositae family, their flavonoids and phenolic compositions are markedly different. The proposed method is useful for further chromatographic fingerprint of plant flavonoids. 3.2. Triterpene Diols and Triols Recently, many bio-active triterpene derivatives were discovered from Chrysanthemum flower. In Toshihiro’s group [7], fifteen pentacyclic triterpene diols and triols, consisting of six taraxastanes, faradiol, heliantriol B, heliantriol C, 22α-methoxyfaradiol, arnidiol, and faradiol α-epoxide; five oleananes, maniladiol, erythrodiol, longispinogenin, coflodiol, and heliantriol A1; two ursanes, brein and uvaol; and two lupanes, calenduladiol and heliantriol B2, isolated from the non-saponifiable lipid fraction of the edible flower extract of Chrysanthemum flower. All of the compounds tested showed inhibitory effects against EBVEA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Hu et al [34,35] also obtained five triterpenes in

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Chrysanthemum flower from Hangzhou, China. However, the analyses of these triterpenes have not received by far any attention. 3.3. Essential Oils Many species of Chrysanthemum genus produce essential oils and several representatives are important herbs and spices used in some part of the world. To date, Chrysanthemum genus have been chemically investigated and revealed by GC-MS about 50 volatiles components in total and wide essential oil from Chrysanthemum flower. The monoterpenes, sesquiterpene and their oxides, including chrysanthenon, chrysanthenol, borneol, monobornyl phthalate, bornyl acetate, rank highest in importance [36-38].

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4. Conclusion Juhua, as a folk medicine, used for treatment of the common cold, headache and dizziness in Asia for centuries. They have been found to possess various biological activities such as antibacterial, antimutagenic, antitubercular and anti-inflammatoy activities etc. And some important bio-active constituents were discovered, such as caffeoylquinic acids with potent superoxide anion and DPPH radical scavenging activites, flavonoids with antimutagenic activity, terpenoids with antitubercular, anti-inflammatoy and anti-tumor activities. In the chemical analysis and quality control of Chrysanthemum flower and extracts, important constituents present in the medicinally used flowers are caffeoylquinic acids, i.e., chlorogenic acid, and some flavonoids, such as apigenin, luteolin, and luteolin 7-O-β-Dglucoside. Many publications only deal with the analysis of the flavonoids. Separation and detection can be routinely carried out by HPLC with DAD or MS. TLC is another possibility. No analysis procedure for terpenoids and chromatographic fingerprint has been published so far. Some further development in this area can be foreseen.

Acknowledgments This work was supported by 2006 “Qinglan Project” Scientific and technological innovation team training program of Jiangsu College and University, and 2006 the Returned Student Initial Funding from Nanjing University of Traditional Chinese Medicine. For other helpful assistance, we are pleased to thank Prof. Shihui Qian, Drs. Jianming Guo, Erxin Shang, Jian Zhang, and Shulan Su.

References [1] Zhang, J.; Li, Y.; Qian, D.; Qian, S.; Duan, J.; Ding, A. Shizhen Guoyi Guoyao 2006, 17, 1941-1942. [2] Jiang, H.; Xia, Q.; Xu, W.; Zheng, M. Pharmazie 2004, 59, 565-567.

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[3] Sun, X.; Duan, J. Study on the active constituents and their activities in the flower of Chrysanthemum morifolium Ramat. jiangsu Natural Science Foundation Project Materials. 2005, pp31-37. [4] Wang H. K.; Xia, Y.; Yang, Z. Y.; Natschke, S. L.; Lee, K. H. Adv. Exp. Med. Biol., 1998, 439, 191-225. [5] Hu, C. Q.; Chen, K.; Shi, Q.; Kilkuskie, R. E.; Cheng, Y. C.; Lee, K. H. J. Nat. Prod. 1994, 57, 42-51. [6] Lee, J. S.; Kim, H. J.; Lee, Y. S. Planta Med. 2003, 69, 859-861. [7] Ukiya, M.; Akihisa, T.; Tokuda, H.; Suzuki, H.; Mukainaka, T.; Ichiishi, E.; Yasukawa, K.; Kasahara, Y., Nishino, H. Cancer Lett. 2002, 177, 7-12. [8] Terashima, S.; Shimizu, M.; Horie, S.; Morita, N. Chem. Pharm. Bull. 1991, 39, 33463347. [9] Miyazawa, M.; Hisama, M. Biosci. Biotechnol. Biochem. 2003, 67, 2091-2099. [10] Akihisa, T.; Yasukawa, K.; Oinuma, H.; Kasahara, Y.; Yamanouchi, S.; Takido, M.; Kumaki, K.; Tamura, T. Phytochemistry 1996, 43, 1255-1260. [11] Ukiya, M.; Akihisa, T.; Yasukawa, K.; Kasahara, Y.; Kimura, Y.; Koike, K.; Nikaido, T.; Takido, M. J. Agric. Food Chem. 2001, 49, 3187-3197. [12] Kim, H. J.; Lee, Y. S. Planta Med. 2005, 71, 871-876. [13] Akihisa, T.; Franzblau, S. G.; Ukiya, M.; Okuda, H.; Zhang, F.; Yasukawa, K.; Suzuki, T.; Kimura, Y. Biol. Pharm. Bull. 2005, 28, 158-160. [14] Zheng, Y.; Wang, X. S.; Fang, J. J. Asian Nat. Prod. Res. 2006, 8, 217-222. [15] Chinese Pharmacopoeia Commision. Pharmacopoeia of the People’s Republic of China. People’s Medical Publishing House: Beijing, 2005; Vol I, pp57-58. [16] Matsuda, H.; Morikawa, T.; Toguchida, I.; Harima, S.; Yoshikawa, M. Chem. Pharm. Bull. 2002, 50, 972-975. [17] Hu, C. Q.; Chen, K.; Shi, Q.; Kilkuskie, R. E.; Cheng, Y. C.; Lee, K. H. J. Nat. Prod. 1994, 57, 42-51. [18] Guo, Q.; Qian, D.; He, X.; Liu, L.; Ju, J.; Zhu, L., Duan, J.; Cai, Y. Zhongguo Zhongyao Zazhi 2002, 27, 896-898. [19] Qian, D.; Zhu, L.; Peng, Y.; Shen, H. Xiandai Zhongyao Yanjiu yu Shijian 2005, 19, 1416. [20] zhu, L.; Duan, J.; Shen, H.; Qian, D.; Zhang, J. zhongchengyao 2007, 29, 1-3. [21] Cuyckens, F.; Claeys, M. J. Mass Spectrometr. 2004, 39, 1-15. [22] Li, Q. M.; van den Heuvel, H.; Delorenzo, O.; Corthout, J.; Pieters, L. A. C.; Vlietinck, A. J.; Claeys, M. J. Chromatogr. 1991, 562, 435-446. [23] Sánchez-Rabaneda, F.; Jáuregui, O.; Casals, I.; Andrés-Lacueva, C.; Izquierdo-Pulido, M.; Lamuela-Raventós, R. M. J. Mass Spectrometr. 2003, 38, 35-42. [24] Justesen, U. J. Chromatogr. A 2000, 902, 369-379. [25] Llorach, R.; Gil-Izquierdo, A.; Ferreres, F.; Tomás-Barberán, F.A. J. Agric. Food Chem. 2003, 51, 3895-3899. [26] Schieber, A.; Berardini, N.; Carle, R. J. Agric. Food Chem. 2003, 51, 5006-5011. [27] Zhang, J. M.; Satterfield, M. B.; Brodbelt, J. S.; Britz, S. J.; Clevidence, B.; Novotny, J. A. Anal. Chem. 2003, 75, 6401-6407. [28] Cuyckens, F.; Rozenberg, R.; de Hoffman, E.; Claeys, M. J. Mass Spectrometr. 2001, 36, 1203-1210.

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[29] Miketova, P.; Schram, K. H.; Whitney, J.; Kearns, E. H.; Timmermann, B. N. J. Mass Spectrometr. 1999, 34, 1240-1252. [30] Schram, K.; Miketova, P.; Slanina, J.; Humpa, O.; Taborska, E. J. Mass Spectrometr. 2004, 34, 384-395. [31] Clifford, M. N.; Knight, S.; Kuhnert, N. J. Agric. Food Chem. 2005, 53, 3821-3832. [32] Gong, F.; Liang, Y. Z.; Xie, P. S.; Chau, F. T. J. Chromatogr. A 2003, 1002, 25-40. [33] Lai, J. P.; Lim, Y. H., Su, J.; S., J.; S.; H. M.; Ong, C. N. J. Chromatogr. B 2007, 848, 215-225. [34] Hu, L.; Chen, Z. Acta Botan. Sin. 1997, 39, 85-90. [35] Hu, L.; Chen, Z. Phytochemistry 1997, 44, 1287-1290. [36] Liu, W.; Guo, T. Henan Zhongyi Xuekan 1995, 10, 12-13. [37] Huang, B.; Liu, J. Zhongyi Yanjiu 1997, 10, 14-16. [38] Bao, Z.; Qin, Z.; Xu, R.; Xu, J.; Ji, Q. Shipin Kexue 2003, 24, 120-121.

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In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-Tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 20

Chishao (赤芍，Paeonia spp.) Quan-hong Zhu* School of Traditional Chinese Medicine, Southern Medical University

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1. Introduction Chishao (Radix Paeoniae rubra）is the root of Paeonia lactiflora Pall. or Paeonia veitchii Lynch (Fig. 1) and it is a crude drug used in many traditional prescriptions in China. It is commonly used for activating blood circulation, regulating menstruation, alleviating pain and treating liver disease and cancer. This crude drug is produced mainly in Inner Mongolia, Heilongjiang, Jilin, Liaoning and Sichuan Provinces and the quality of the medicinal root produced in Duolun, Inner Mongolia is better than that of in other areas of China. The root is dug in spring and autumn and then dried by the air after removing the stem, fibrous and sediment.

2. Pharmacological Activities According to the theory of Traditional Chinese Medicine (TCM), Chishao is bitter in flavor, slightly cold in nature and acts on the liver channel. It has the functions of clearing heat, cooling blood, dissipating stasis and relieving pains. In clinic, it has been traditionally indicated for the syndromes caused by blood-heat, blood stasis and liver-fire, such as macula, apostaxis, amenorrhea, algomenorrhea, menoxenia, trauma swelling, superficial suppurative infections and conjunctival congestion. At present, it is often used for the treatment of gynaecological problems, liver diseases and vascular disorders. *

E-mail address: [email protected]. Tel: +86 20 6164 8261. Fax: +86 20 6164 8261 (Corresponding author)

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A

B

C

Figure 1. The plant of (A) and the raw material (B and C) of Paeonia veitchii Lynch. Fig. 1A and 1B were adopted from: Pharmacopoeia Commission of the Ministry of Public Health, A Colour Atlas of the Chinese Materia Medica Specified in Pharmacopoeia of the People’s Republic of China (1995 edition), Guangdong Science & Technology Publishing House, Guangzhou, China. 1996.

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2.1. Effects on Hematological System In view of the theory of TCM, the main functions of Chishao are promoting blood circulation and removing blood stasis. Therefore, the pharmacological actions of the crude drug are closely related with hematological system. 2.1.1. Antithrombotic and Antiplatelet Aggregation Activities Chishao decoction and Chishao injection administrated intragastrically to rats and rabbits respectively were noted to prolong thrombus formation time (TFT), prothrombin time (PT), kaolin partial thromboplastin time (KPTT), thrombin time, and to reduce the length and mass of thrombus [1,2]. The effect of Chishao on enzyme activity was studied in another study in which the aqueous extract of Chishao significantly prolonged fibrin coagulation time (CT), activated plasminogen, and inhibited the activation of urokinase to plasminogen. This indicated that the effects of Chishao on inhibiting thrombase and activating plasminogen were the basis of its antithrombotic action [3]. Clinical studies to evaluate its effect on platelet function showed that Chishao had significant inhibitory effects on platelet aggregation induced by adnephrin, adenosine diphosphate (ADP) and snake venom. The increase of cyclic adenosine monophosphate (cAMP) level in platelet may be contributing to the antiplatelet

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aggregation effect of Chishao [4]. The antithrombotic and antiplatelet aggregation activities of Chishao were further confirmed by the extracts of Chishao [5]. Total Paeony glycoside (TPG), the major active constituents extracted from Chishao, remarkably prolonged PT, KPTT, CT and artery TFT, and inhibited not only ADP-induced pulmonary thrombosis but also thrombosis formation caused by electro-irritation [6-8]. In addition, TPG inhibited ADPinduced platelet aggregation and caused the increase in plasma 6-keto-prostaglandin F1α (PGF1α), decrease in plasma thromboxane B2 (TXB2), and regulation in serum nitric oxide (NO)/endothelin [9]. Recently the same group reported that administration of TPG reduced the activities of coagulation factor II and V, and increased antithrombin-III activity. This may possibly explain the anticoagulation mechanism of Chishao [10,11].

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2.1.2. Effects on Erythrocyte The effect of Chishao on erythrocyte was studied in mesentery of rats subjected to IIIº empyrosis by boiling water. It was observed that Chishao extract administrated intragastrically to rats could improve the microartery constriction, significantly reduce the leucocytic adhere in microvein, alleviate the erythrocyte aggregation, and thus improved the disorder of mesentery microcirculation [12]. The administration of Chishao injection to hyperlipidemia rabbits caused decrease in platelet cytosolic free Ca, and increase in basal and calmodulin-stimulated activity of erythrocyte membrane Ca2+-Mg2+-ATPase. This may be contributing to the beneficial effect of Chishao on hemorheology [13]. In another study it was found that erythrocyte permeability was remarkably improved in hepatitis B virus (HBV) patient serum after treated with aqueous extract of Chishao and the membrane structure of erythrocyte was stabilized [14]. The effect of Chishao on erythrocyte was studied in later experiments where Chishao inhibited markedly erythrocyte aggregation and had no obvious influence on red blood cell (RBC) [15]. The results were validated by scanner in which the gray scale was correlated with the degree of erythrocyte aggregation [16]. The hemorheology effect of TPG was also reported [6,17,18]. TPG could markedly decrease whole blood viscosity, plasma relative viscosity, flbrinogen level and RBC aggregation index in bloodstasis rats. It suggested that the antithrombosis mechanism of Chishao may be related with the improvement of hemorheology. 2.2. Cardiovascular Effects 2.2.1. Anti-artherosclerosis (AS) Activity Hyperlipemia is one of the main reasons for AS and reduction in blood fat will be beneficial to AS therapy. Administration of Chishao extract to hyperlipemia rabbits could regulate TXA2/PGI2 change and decrease lipid peroxidation (LPO) in plasma, membrane lipid, Ca, phospholipids and plaque areas in aorta [19]. The decrease in serum total cholesterol (TCh), low density lipoprotein-cholesterol (LDL-Ch), very low density lipoprotein-cholesterol (VLDL-Ch) and triglyeride (TG), and increase in high density lipoprotein-cholesterol (HDL-Ch), HDL2-Ch were also observed. Regulating TXA2/PGI2 balance and LPO through Ca2+ metabolism may be one of anti-AS mechanisms of Chishao [20]. Later on these findings were confirmed in a clinical study where Chishao injection

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administrated to stroke patients by acupoints significantly decreased TCh, apolipoprotein B (apoB), increased apoA1 and HDL [21]. Recently the effect of Chishao on stability of AS plaque in apoE-/- mice was addressed. Chishao exerted action on regulating blood lipid, increasing collogen area and had no effect on inflammatory reaction [22]. It was reported in another study that Chishao decoction caused a significant regression of AS lesion in rabbits injured with immunoreaction and fed with a high-cholesterol diet [23]. Migration of smooth muscle cell (SMC) from arterol media to intima and proliferation of intimal SMC are also one of the main causes of AS. Chishao extract added into the medium of cultured rabbit SMCs caused the inhibition of SMCs proliferation, decrease in malondialdehyde (MDA) level and LDL-Ch oxidation [24,25]. Later on the same group reported that AS rabbits administrated Chishao extract caused the inhibition of oncogene C-myc, C-fos and V-sis expression in addition to regression of coronary arteries and aortic AS lesion [26]. The preventive effect of Chishao on restenosis after carotid balloon injury (CBJ) in rabbits was confirmed in another experiment where Chishao extract remarkably decreased the hyperplastic intima area and proliferating cell nuclear antigen positive expression, inhibited collagen type I proliferation, and alleviated new intima generation. The inhibition of Chishao on neointimal hyperplasis may be partly attributed to its inhibition to renin-angiotensin system activation [27,28]. It suggested that Chishao had preventive effect on vascular remodeling and restenosis in AS rabbits. The physiological and pathological changes of the cardiovascular diseases, especially hyperlipemia and AS were commonly reflected on the disorder of endothelial cell. Chishao extract relaxed PGF2α-precontracted aortic ring preparations of isolated rat aorta that contained endothelium, but failed to relax aortic rings without endothelium. It was then further observed that Chishao extract significantly increased not only endothelium-dependent relaxation but also superoxide dismutase (SOD) activity. These findings indicated that Chishao had protective effects on endothelial cells and their function [29,30].

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2.2.2. Cardioprotective and Antioxidant Activities Initial experiment demonstrated that Chishao extract had protective effect on early myocardial function in rats subjected to empyrosis caused by boiling water [31]. The antioxidative effect of TPG was studied on cardiomyoctyes injured by isoprenaline (ISO). It was observed that TPG could improve the levels of SOD, MDA, NO, the beating frequency of cardiomyocytes, enzyme activities of glutamic oxaloacetic aminotransferase, lactic acid dehydrogenase (LDH), creatine kinase and survival rate of cardiomyocytes [32,33]. These observations suggested that TPG had protective action on injured cardiomyocytes induced by ISO in dose-dependent manner. The beneficial effect of TPG was further evidenced in acute myocardial ischemia mongrel canines. TPG significantly prevented the increase in serum level of free fatty acid (FFA) and decreased LPO through increasing the activities of SOD, glutathione peroxidase. The increase in myocardial flow, decrease in coronary artery resistance, myocardial consumption of oxygen, oxygen consumption index and oxygen uptake rate was also observed [34,35]. Furthermore, the effects of Chishao on portal hypertension, pulmonary artery hypertension and adult respiratory distress syndrome were also reported [36-38]. These findings indicated that TPG had obvious improvement on haemodynamics and myocardial function by improving epicardial electrocardiogram, increasing myocardial blood supply, antagonizing LPO reaction, stabilizing cell membrane and protecting the activity of endogenous antioxidase. The mechanism of the effect may

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relate to the enhancement of antioxidation in cells, the reduction of membrane damage caused by free radical and lipid peroxide, the improvement of myocardial ischemia and hypoxia, the maintain of normal energy metabolism and systolic function, and the inhibition of cell apoptosis.

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2.3. Effects on Cerebral Ischemic Injury The physiological and pathological mechanism of ischemic brain was closely related with neuropeptide. The aqueous extract of Chishao prevented the increase in leu-enkephalin and βendorphin level after ischemia by ligation. This may possibly explain the beneficial effect of Chishao on ischemic cerebrovascular diseases [39]. The protective effects of TPG on ischemic brain injury have been studied systematically by another group. TPG showed a significant protection against ischemia injury in cultured primary cortex neurons and PC12 cells stimulated by hypoxia, hypoglucose, free radicle injury, caffeine injury, NO injury and neurotoxicity and excitatory amino acid injury, and the survival number of neurons in injury models was also increased obviously [40,41]. TPG reduced notably LDH level, Ca2+ concentration in cytoplasm and had protective effect on Ca2+ overloading injury induced respectively by caffeine, potassium chloride and N-methyl-D-aspartate in cultured primary cortex neurons and PC12 cells [42,43]. The same group subsequently reported the protective effect of TPG in vivo. TPG administrated intragastrically to cerebral ischemia-reperfusion mice prevented the increase of MDA and NO level, increased SOD level, inhibited the decrease of LDH, and improved the learning and memory capacity of model mice significantly [44]. TPG remarkably prolonged the gasp time of decapatative mice, lessened the cerebral water content and decreased the permeability of cerebral capillary [45]. The protective effect of TPG against focal cerebral ischemia-reperfusion injury was also evidenced in rats. TPG caused the alleviation of infarction zone, increase of anti-oxidase activity, inhibition of LPO reaction, improvement of energy metabolism after infarction and thus protected brain tissue from injury induced by focal cerebral ischemia-reperfusion. The pathological slices also proved its protective effect on neuron [46,47]. These observations got further supported by another study conducted in global cerebral ischemia-reperfusion impairment gerbils [48]. In addition to protection of cerebral ischemic injury, Chishao also exhibited the protective effect on renal ischemia-reperfusion injury and the acute lung injury induced by intestinal ischemia-reperfusion [49,50]. 2.4. Effects on Liver 2.4.1. Hepatoprotective Activities Early studies revealed that Chishao in a range of proper concentrations could promote the synthesis of hepatic cell DNA in vitro [51]. Later on the hepatoprotective effects of Chishao have been studied in experimental liver damage model in rats and mice by several authors. Rats subjected to D-galactocamine-induced acute liver injury were administrated Chishao injection by vena caudalis and then caused a marked increase in plasma fibronectin level. The

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englobement function of reticuloendothelial system to prevent liver from immunologic injury was improved and thus hepatocytes were protected. Those observations were corroborated by pathological studies [52,53]. The hepatoprotective effect of TPG got further supported by another study. TPG could significantly lower the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and MDA, but enhanced SOD activity in the liver tissue and markedly ameliorated the liver damage induced by D-galactosamine in mice. The mechanism of which maybe related to its antiperoxidation of lipid [54].

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2.4.2. Anti-hepatic Fibrosis Effects Liver fibrosis represents a major medical problem with significant morbidity. The model of liver fibrosis is mainly induced by drug in the experimental studies, such as carbon tetrachloride (CCl4). Chishao decoction given orally before and after CCl4 administration rats significantly prevented the increase in the levels of hyaluronic acid (HA) and hydroxyproline (HYP) in liver tissue and the increase in serum ALT, HA, collagen type IV, precollagen type III and laminine (LN). It was thus concluded that Chishao repressed collagen hyperplasy as well as base hyperplasy [55,56]. In another experiment it was observed that the aqueous extract of Chishao caused a significant increase in serum transaminase activities, ALT, AST, NO, HA and LN levels, and contents of HYP and MDA in CCl4-induced rat livers [57]. Liver fibrosis caused by the abnormal accumulation of basilaris substantia other than liver cell was commonly called adiposis hepatica in China. Rats were fed with a high fat diet and intraperitoneally administrated tetracycline to induce adiposis hepatica and then administrated with Chishao extract. It was observed that the levels of ALT, AST, MDA, FFA, TG were decreased. This suggested that the anti-adiposis hepatica effect of Chishao may be attributed to the lipid metabolism promotion and anti-LPO [58,59]. Hepatic stellate cell (HSC) plays an important role in the formation of liver fibrosis. To inhibit the proliferation and induce the apoptosis of HSC may be an ideal way to prevent against the liver fibrosis. This got supported by the experiment conducted in dogs. The serum before and after Chishao administration was taken and interacted with HSC-T6 established by acetaldehyde. The promotion of HSC-T6 apoptosis was observed and the apoptosis rate increased significantly. The proportion of HSC-T6 in G0/G1 phase rose obviously, while reduced in S phase and there were no any changes in G2/M phase. After treated with aqueous extract of Chishao, the increase in protein level of bcl-2 was decreased and the decrease in bax and caspase-3 was increased significantly. These findings may possibly explain the mechanism of Chishao on inhibiting HSC-T6 proliferation, promoting HSC-T6 apoptosis, and the beneficial effect of Chishao against liver fibrosis [60,61]. 2.4.3. Anti-HBV Activities The therapeutic use of Chishao for hepatitis was based on empirical observations and has been verified repeatedly in clinic, especially in treatment for severe hepatitis, icterohepatitis, viral hepatitis, and etc [62]. However, scientific investigations into the pharmacological actions and critical evaluation of its anti-hepatitis effects were only done in recent years. Chishao decoction was intragastrically administrated to rats and then the serum of rats was isolated. The inhibition of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) was observed [63]. The aqueous extract of Chishao from Yunnan Province showed

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obvious inhibition of HBsAg and HBeAg levels in the extracellular medium of HepG 2.2.15 cells in a dose-dependent manner. Those results demonstrated anti-HBV activity of Chishao. However in view of anti-HBV activity of Chishao, this needs to be further explored in vivo [64].

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2.5 Anti-endotoxin Activities Of late several publications regarding the potential anti-endotoxin activities of Chishao have been reported. The anti-endotoxin components of Chishao were isolated, and then combined with the equal volume of lipopolysaccharide (LPS). After added in cultured mice RAW 264.7 cell line, it was found that the effective components had high binding capability with LPS and inhibited significantly the release of TNF-α from LPS-stimulated cells in vitro in a dose-dependent manner [65,66]. The pathological changes caused by LPS accorded with the noxious heat blood stasis syndrome (NH-BS) in view of TCM. Therefore the effects of Chishao on serum proteome in rats suffering from NH-BS were studied. The changes in volume of serum protein (xPr) were observed by group comparison. The results suggested that the molecular basis of therapeutic effect of Chishao decoction on NH-BS might be through the regulation of xPr l, 2, 3, 4, 9 and 16 [67]. In another experiment it was observed that Chishao decoction exhibited obvious neutralization with endotoxin in vitro as well as TPG, and decreased significantly the death rate in mice attacked intravenously by bacterial endotoxin. It clearly indicated that Chishao decoction had anti-endotoxin activities and TPG was the main effective fraction, while volatile oil and polysaccharide in Chishao showed no evident actions [68]. These observations got further supported by another study where the protective effect of Chishao in LPS-induced acute lung injury rats was observed. It was concluded that not only the inhibition of the abnormal increase of inducible nitric oxide synthase expression and the increase of endothelial nitric oxide synthase expression in lung tissue, but also the inducement of heine oxygenase expression was contributing to the beneficial effects of Chishao [69,70]. 2.6. Antitumor Effects Clinical studies have revealed that Chishao is effective in tumor therapy. The antitumor effect of Chishao extract, aqueous extract (Extract A), 70% alcoholic extract (Extract C) and n-butanolic extract (Extract D) was studied respectively. It was found that Extract D showed inhibitory effect on bearing S180 sarcoma mice. Although Extract A or Extract C showed no obvious antitumor effects, they showed obvious inhibitory effect on S180 sarcoma and extended significantly the live time of leukemiac 615 mice when combined respectively with cyclophosphamide and methotrexate. These findings indicated Chishao had synergic antitumor effects. Extract A, C and D could increase the level of cAMP in S180 sarcoma, S180 ascites carcinoma and Lewis lung carcinoma. This may be contributing to the antitumor effects of Chishao [71]. In another experiment Chishao preparation showed significant increase in serum level of vascular endothelial growth factor (VEGF) and microvessel density in tumor tissue of rats innoculated Walker 256 cell and Lewis lung carcinoma respectively. These observations implied that Chishao were able to promote invasion and metastasis of tumor by enhancing the expression of VEGF and angiogenesis in experimental rats [72,73].

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The molecular mechanism of antitumor effect of Chishao was studied on human hepatoma cell lines. The aqueous extract of Chishao had inhibitory effect and cytotoxicity on both HepG2 and Hep3B cell lines [74,75]. A dose- and time- dependent anti-proliferative effect was observed on HepG2 cell. The cytotoxicity was through the activation of the cell apoptosis which was independent of p53 pathway as Hep3B cell was p53-deficient. Furthermore, the expression of Bax and p53 mRNA was up-regulated while Bcl-2 mRNA was down-regulated dose-dependently on HepG2 cell treated Chishao. Thus it induced the tumor cell apoptosis and may possibly explain the mechanism underlying the antitumor effects of Chishao. These findings got further confirmed by TPG [76]. Besides hepatoma cells, Chishao had apoptosisinducing activity on HL-60 cells which occurred via the mitochondrial route and that the apoptosis-conducting mechanism acted through a cascade involving caspase-3 [77]. The beneficial effect of Chishao on immune system may explain some of its antitumor activity. TPG administrated to tumor-bearing mice showed increase in interleukin (IL)-2 and TNF-α secretion, CD8 expression, recovery in CD4+/CD8+ balance, and up-regulation in decreased IL-4 [78,79]. These observations suggested that the immune-enhancing effect may be one of its antitumor mechanisms of Chishao [80].

3. Quality Control

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Chishao used in clinic is a crude drug which is not cultivated as many other raw materials but grows in wild. Thus the quality of the root is influenced by natural and artificial conditions, such as species, habitat, biogeocoenosis, process, storage and season for collecting. The quality control of the drug is necessary to guarantee the safety, efficacy, constancy and controllability of Chishao. 3.1. Controlled by Effective Component—Paeoniflorin In addition to the origin, character and micro identification, the qualitative and quantitative analysis for the quality control of Chishao using paeoniflorin as the reference substance are also recorded in Chinese Pharmacopiea (Ch. P) (2005). The crude drug was identified by thin layer chromatography (TLC) in which the same blue-purple dot was emerged in the corresponding place as paeoniflorin in the chromatogram of sample. The content of paeoniflorin was determined by high performance liquid chromatography (HPLC) and not less than 1.8%. This standard has been accepted and followed naturally in several research aspects of Chishao until now, for instance in processing the medicinal materials [81], optimizing the extraction technology [82-90], especially in controlling the quality of Chishao [91-95], its extract [96-98] and preparations [99-101]. Although the quality control is stable and feasible via monitoring the content of paeoniflorin in Chishao, and to some extent paeoniflorin showed the similar pharmacological activities to Chishao in hepatorpotective activity [102], antihyperlipidemic effect [103], neuroprotective action [104-106], antithrombosis [107], ameliorating learning and memory effect [108,109], and so on, it does not represented the whole pharmacological effects and will be insufficient for the quality control of Chishao.

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The therapic integrity effects of the crude drug itself can not be elucidated by anyone of the constituents in Chishao according to the theory of TCM. Furthermore, paeoniflorin is the quality control component of not only Chishao but also Baishao (Radix Paeoniae alba), and the content of paeoniflorin should be not lower than 1.8% and 1.6% respectively in Ch. P (2005). Although Chishao and Baishao all are the roots of Paeonia lactiflora of the family Ranunculaceae, the former is a heat-clearing drug for clearing heat, cooling blood, dissipating stasis and relieving pains, and the latter is a nourishing blood drug for suppressing hyperactivity of liver, relieving pain, replenishing blood, regulating menstruation and astringing sweating, That means the two drugs with different effects and clinical uses are controlled by nearly the same quality standard and paeoniflorin is not the characteristic component existed only in Chishao. A comparative study on content of major constituents between Chishao and Baishao by mecellar electrokinetic chromatography have revealed that the contents of paeoniflorin and D-catechin in Chishao were more than 6% and 0.05% respectively，while the paeoniflorin in Baishao was less than 6.0%, and D-catechin was not detected. It indicated that the content of paeoniflorin had better raise in order to distinguish from Baishao and D-catechin, another component of promoting blood flow to remove stasis, also can be used as a marker in the quality control of Chishao [110].

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3.2. Controlled by Fingerprint The fingerprint has been adopted by several countries for controlling production process of phytomedicine. Since the effects of Chinese herbs as well as phytomedicines are the complex action of multi-components, the fingerprints of Chinese herbs have been studied in China recently. HPLC fingerprint was established for quality assessment of Chishao. It was demonstrated that the ratio of peak area of different habitat samples were different. Nine peaks were designated and the chemical components were obviously different between Baishao and Chishao [111,112]. Hence the method was suitable to differentiate Chishao from different sources conveniently and provided new evidence for the two drugs’ quality control in good reproducibility, precision and stability. The infrared spectrum fingerprint was also an effective method of quality control. The samples of Chishao were compared using Foruier transform infrared (FTIR) absorption spectrometry. It was found that the frequency, intensity and shape of infrared absorption spectra were obviously different between wild and cultivated groups, and infrared absorption peaks of Chishao in Duolun were of distinct characteristics. The samples clustered by FTIR fingerprint agreed with their geographical origins. This suggested that FTIR technique was an operable method in the quality control and discrimination of Chishao in the place of the genuine [113,114]. These findings were confirmed in another experiment where the samples of Chishao were determined by horizontal attenuated total reflectance FTIR. The differences of wave number-absorbance between official and unofficial samples were obvious by principal component analysis [115].

4. Conclusion Chishao has been used in clinic for a long history as an effective drug for activating blood circulation and dissipating blood stasis. It has been widely used in the treatment of

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gynaecological problems, liver diseases and vascular disorders, and the benefit effects of Chishao have been proved in practice. The modern pharmacological studies have manifested the extensively biological activities of Chishao in the level of molecule, cell, tissue and global animal. However the mechanisms of its pharmacological actions and the relationship with clinical diseases have not been clearly stated. Furthermore the traditional pharmacological activities which accord with TCM theory have not been verified completely. Although the quality control of Chishao has progressed a lot with the development of modern analytical technologies, the quality control and assessment system of Chishao was not consummate. Chishao contains glycosides (most notably paeoniflorin), flavonoids, proanthocyanidins, tannins, terpenoids, triterpenoids, and complex polysaccharides that may all contribute to its medicinal effects. The quality control should reflect the integrity and complex action of multicomponents of Chishao and the parameters must be kept pace with the biological effects to ensure consistent quality of Chishao. TPG, the major active fraction extracted from Chishao, has been demonstrated that it has the similar pharmacological activities to Chishao in many ways (stated above) and thus TPG used as a marker in the quality control of Chishao may be more reasonable Furthermore, the establishment of fingerprint which reflect the characteristics of Chishao will be an ideal way for the quality control and of great benefit to connection with world market. However, it is necessary and significant to formulate a proper quality assessment standard, to clarify the pharmacodynamics foundation and thus to elucidate the mechanism of action and clinical indications of Chishao.

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[103] Yang, H. O.; Ko, W. K.; Kim, J. Y.; Ro, H. S. Fitoterapia. 2004, 75, 45-49. [104] Liu, D. Z.; Xie, K. Q.; Ji, X. Q.; Ye, Y.; Jiang, C. J.; Zhu, X. Z. Br. J. Pharmacol. 2005, 146, 604-611. [105] Tsai, T. Y.; Wu, S. N.; Liu, Y. C.; Wu, A. Z.; Tsai, Y. C. Eur. J. Pharmacol. 2005, 523, 16-24. [106] Zhang, G. Q.; Hao, X. M.; Chen, S. Z.; Zhou, P. A.; Cheng, H. P.; Wu, C. H. Acta. Pharmacol. Sin. 2003, 24, 1248-1252. [107] Ye, J.; Duan, H. Yang, X. Yan, W.; Zheng, X. Planta Med. 2001, 67, 766-767. [108] Tabata, K. Matsumoto, K.; Murakami, Y.; Watanabe, H. Biol. Pharm. Bull. 2001, 24, 496-500. [109] Abdel-Hafez, A. A.; Meselhy, M. R.; Nakamura, N.; Hattori, M.; Watanabe, H.; Murakami, Y.; El-Gendy, M. A.; Mahfouz, N. M.; Mohamed, T. A. Biol. Pharm. Bull. 1998, 21, 1174-1179. [110] Zhou, H. T.; Luo, Y. Q.; Hu, S. L.; Li, R. K.; Liu, H. W.; Feng, X. F. Zhongguo Yao Xue Za Zhi. 2003, 38, 654-657. [111] Zhang, K. R.; Bi, K. S. Zhong Cao Yao. 2003, 34, 1048-1051. [112] Wang, Q.; Lu, R. X.; Yu, H. L.; Liu, P.; Liu, Z. T.; Bi, K. S.; Guo, D. A. Zhongguo Yao Xue Za Zhi. 2007, 42, 581-584. [113] Zhou, H. T.; Hu, S. L.; Feng, X. F.; Yang, J. Y.; Sun, S. Q. Zhong Cao Yao. 2002, 33, 834-837. [114] Xu, Y. Q.; Huang, H.; Zhou, Q.; Zhou, H. T.; Hu, S. L.; Sun, S. Q. Fen Xi Hua Xue. 2003, 31, 5-9. [115] Chen, C. G.; Li, D. T.; Chen, J. H.; Kong, L. C.; Cheng, Z. F. Zhongguo Yao Xue Za Zhi. 2006, 41, 580-582.

Pharmacological Activity-Based Quality Control of Chinese Herbs, edited by Shao-Ping Li, Nova Science Publishers, Incorporated, 2008. ProQuest

In: Pharmacological Activity-Based Quality Control… Editors: Shao-ping Li and Yi-Tao Wang

ISBN: 978-1-60456-823-3 © 2008 Nova Science Publishers, Inc.

Chapter 21

Chuipencao (垂盆草，Sedum sarmentosum) Li-fang Liu*, Zhu-qing Wan, Le Xie and Xiao-li Ma The Key Laboratory of Modern Chinese Medicines (Ministry of Education) and Department of Pharmacognosy, China Pharmaceutical University, Nanjing 210038, China

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1. Introduction The traditional Chinese medicine Herba Sedi (Chuipencao) is one of the commonly used drugs included in Chinese Pharmacopeia, which derived from fresh or dried herb of Sedum sarmentosum Bunge (Fig. 1), a plant distributed in the whole country of China. Herba Sedi, with the actions of removing damp-heat and counteracting toxicity, is clinically used to treat the jaundice with oliguria caused by damp-heat, acute and chronic hepatitis, carbuncles and sores. The main chemical constituents contained in Herba Sedi are cyanophoric glycosides, flavonoids, triterpenes, amino acids and carbohydrates. Many studies have been done on the chemical composition and pharmacologic action of Herba Sedi since 1940’s. This part attempts to condense the essentials of the achievements on chemical composition, pharmacological activities and quality control of Herba Sedi.

*

E-mail address: [email protected]. Tel: 86-25-85391253. Fax: 86-25-85301528 (Corresponding author)

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A

B Figure 1. The plant (A) and raw material (B) of Sedum sarmentosum Bunge.

2. Chemical Compositions Numerous studies focused on the chemical components of Sedum sarmentosum Bunge during the past decades. Many compounds were isolated from its ethanol extract [1-3]. The main components from this herb are reviewed as below. 2.1. Cyanophoric Glycosides A special water-soluble cyanophoric glycoside, named as sarmentosin, was found in 1979 by Shengding Fang [4] and its chemical structure was elucidated as 2-cyano-4-O-β-Dcounter-butylene glycose-2-alcoh. In 1982, the author further reported the chemical property of sarmentosin in detail and revealed its transformation process to isosarmentosine as well

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[5]. Experiments have been done to demonstrate that sarmentosin has the activity to decrease the level of serum glutamate-pyruvate transaminase (GPT), while the isosarmentosine does not show this activity. 2.2. Flavonoids Thirteen flavonoids have been isolated from S. sarmentosum Bunge by Aimin He, et al. [6], i.e. tricin, tricin-7-glucoside, luteolin, luteolin-7-glucoside, liquiritigenin, liquiritin, isoliquiritin, isoliquiritigenin, isorhamnetin-7-glucoside, isorhamnetin-3,7- diglucoside, limocitrin, limocitrin-3-glucoside, and limocitrin-3,7- diglucoside. In 2000, Jinhuo Pan [7] isolated two new flavanoids from n-butanol and water fractions of S. sarmentosum Bunge. Their chemical structures were elucidated as 5, 4’-dioxy-8, 3’dimethoxyl-3, 7-dicho-O-glucoside and quercetin-3-L-rhamnoside. According to Hyuncheol [8], five flavanoids, which have the inhibitory action to angiotonin ferase were isolated from S. sarmentosum Bunge. Among that, one new flavanoid (quercetin-3-O-α-(6’’’-glyco-caffeic acid)-β-1,2-rhamnoside) and four known flavanoids (quercetin-3-O-α-(6’’’-coumadin glucosyl)-β-1,2-rhamnoside, isothamnetin-3-β-glucoside, quercetin-3-β-glucoside and trifolitin-3-α-arabinoside) were contained.

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2.3. Triterpenoids and Steroids Four triterpenoids, δ-amyrone, δ-amyrin, 3-epi-δ-amyrin, and 18-β-hydroperoxy-olean12-en-3-one were isolated in 1998 [9]. All of them showed significant hepatic protection effect and there was reciprocal transformation among δ-amyrone, δ-amyrin and 3-epi-δamyrin. On the other hand, four steroids were also isolated and identified in 1997 [10], which are β-sitosterol, daucosterol, 3β,6β-stigmast-4-en-3,6-diol and a new compound, 3β, 4α, 14α, 20R, 24R-4,14 -dimethylergost-9(11)-en-3-ol, which was named as sarentosterol. 2.4. Alkaloids Some alkaloids, including methylisopelletierine, dihydroisopelletierine, 3-formyl-1,4dihydroxy-dihydropyran and N-methy-2β-hydroxypropylpiperidine, were isolated from this plant in 1949 [11]. 2.5. Others Analysis study was done through an automatic amino acid analyzer and an atomic absorption spectrophotometer [12]. The results showed that there are many kinds of amino acids in S. sarmentosum Bunge, some of them have higher contents than others, such as glutamic acid, methionine, isoleucine, leucine, phenylalanine, lysine, histidine, and alanine. Moreover, sedoheptulose, sucrose, fluctose, glucose and β-d-galactose- (3-1)-β-D-glucose(4’-1)-O-β-D-allose have also been found in S. sarmentosum Bunge. Taimin Wei [13] reported that dioctadecyl sulfide and palmic acid were obtained when they isolated ethyl

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acetate soluble portion of 95% ethanol extract from S. sarmentosum Bunge. In addition, syringic acid, 3,5-dicho-methoxyl-4-ortho- hydroxybenzoic acid, tannins, glues and phlegmatic temperament were also found in this herb [14,15].

3. Pharmacological Activities Both pharmacological experiments and clinical applications have demonstrated that Herba Sedi had a good therapeutic effect to treat acute and chronic virus hepatitis. Meanwhile, it also shows its potential use in immune suppression [16], hepatocellular protection, GPT reduction and antiviral action. Recently, many research focuses on its inhibition of angiotensin-converting enzyme (ACE) [8] and estrogenic actions [17]. These showed that the Herba Sedi would possibly be used to treat hypertensive disease and to improve the quality of life for female in climacteric period. 3.1. Hepatoprotective Effect The action of hepatoprotective of Herba Sedi was mostly emphasized during the past years. Many experiments concluded as follow were designed to reveal the possible action pathway for this effect.

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3.1.1. Promoting Substance and Energy Metabolism in Hepatic Cells The hepatoprotective effect of the aqueous extract of Herba Sedi and sarmentosin through hepatic injury model of rats induced by galactosamine or carbon tetrachloride was observed [18,19]. The results showed sarmentosin from this herb had an obviously protection activity to hepatic injury of mice induced by carbon tetrachloride at the dose of 0.5~1.0 mg/20 g through increasing the contents of glycogen, glycose-6-phosphatase and lactate dehydrogenase in hepatic cells, which is helpful for the glycol-metabolism. Moreover, it might promote substance and energy metabolism in hepatic cells through increasing the activities of succinate dehydrogenase and adenosine triphosphase. This is beneficial for the energy supply during the recovery of hepatic injury. 3.1.2. Againsting the Oxidation Damage Other experiments investigated the effects of aqueous extract from Herba Sedi in the oxygen-derived free radicals and lipid peroxidation damages of mice’s liver induced by acute alcoholism [20]. Results showed that the exhaustion of tathion of mice and the produce of one main kind of lipid peroxidized products-malonaldehyde could be prevented after orally taking the Herba Sedi (10 g/kg) one or three times a day. The results further indicated this herb could protect the hepatic cells against the damage from lipid peroxidation.

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3.1.3. Inhibiting the Delayed Type Hypersensitivity The further study reported that 100 or 500 mg/kg aqueous extract from Herba Sedi could decrease the clearance rate of carbon particle and reduce the intake of carbon particle in liver and spleen of mice [21]. However, it had no significant effect on the capability of peritoneal macrophage in swallow chicken red blood-cell. 100mg/kg of Herba Sedi would obviously inhibit the delayed type hypersensitivity (DTH) induced by 2,4,6-trinitrochlo-robbenzene and the DTH induced by sheep red blood-cell at the administration in induction phase, while the Herba Sedi had no significant influence to the above two types of DTH at the administration in effective phase. This demonstrated that this herb had no treatment effect on the effected DTH. The possible mechanism of this effect might be in the inhibition of mononuclear macrophage system. 3.1.4. Inhibiting the Proliferation of Viral Hepatoma Carcinoma Cell The human and murine hepatoma cells were cultivated in alkaloid fractions from Herba Sedi (50~150 μg/mL) for 24~48 hs. Although no gangrene phenomenon was observed in the alkaloids solution, while the number of cancer cells were decreased obviously. It revealed the alkaloids from Herba Sedi had a significant dose-dependent activity on anti-proliferation of Hepatoma G2 in its growing period of G1 in murine and human. Therefore, it might extend the life span of persons with liver cancer through inhibiting the proliferation of viral hepatoma carcinoma cell [22].

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3.1.5. Inhibiting the Effusions of Inflammation Factors From the experiments of the effect of Herba Sedi on duck hepatovirus by natural infection model of duck hepatitis B virus (DHBV) [23], Herba Sedi was not a simple inhibitor of alanine aminotransferase (ALT). The reason was it didn’t show inhibitory action in DHBV and adipose degeneration of hepatic cells. These results elucidated that Herba Sedi decreased the value of ALT through inhibiting the effusions of inflammation factors to reduce the hepatocellular damage. Moreover, the active fractions of this drug in hepatoprotective were also investigated. Besides aqueous extracts, n-butanol extracts from Herba Sedi could also significantly reduce the levels of ALT and aspartate aminotranferase (P