Chemical and pharmacological evaluations of panax notoginseng and swietenia macrophylla

CHEMICAL AND PHARMACOLOGICAL EVALUATIONS OF PANAX NOTOGINSENG AND SWIETENIA MACROPHYLLA TOH DING FUNG (B.Sc. (Pharm.) (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to extend my sincere gratitude to my thesis supervisor, Prof Koh Hwee Ling, for her patient guidance, suggestions and advice throughout the whole course of this project and thesis write-up. I would also like to thank Dr Eric Chan Chun-Yong and Miss New Lee Sun, for their helpful guidance and advice in the metabolomics work on P. notoginseng. I would like to thank Dr Neo Soek-Ying and Dr Alvin Teo for their advice and guidance in the cancer cell work. I would like to express my heartfelt gratitude Mr Li Lin for his help in LC-MS and NMR and Mr Johannes Murti Jaya for his help in preliminary work on isolation of limonoids. I am also grateful to the financial support of a research scholarship from the National University of Singapore research scholarship. The technical assistance from the laboratory officers in the Department of Pharmacy, especially Ms Ng Sek Eng is greatly appreciated. I also wish to thank everyone in the department who has helped me in one way or another, especially my laboratory mates (namely, Li Lin, Jianhong, Dhaval and Peiling) for their help and enjoyable times in the laboratory. Special thanks also go to all my fellow friends in the department for their moral support, helpful discussions, and for sharing all the woes and wonderful times together during my postgraduate years. Last but not least, I would like to thank my family for their understanding and continuous support. ii LIST OF PUBLICATIONS AND CONFERENCE PRESENTATIONS Publications 1. Toh DF, New LS, Koh HL, Chan EC. Ultra-high performance liquid chromatography/timeof-flight mass spectrometry (UHPLC/TOFMS) for time-dependent profiling of raw and steamed Panax notoginseng. J Pharm Biomed Anal 2010; 52: 43-50. 2. Toh DF, Patel DN, Chan EC, Teo A, Neo S-Y, Koh HL. Antiproliferative effects of raw and steamed extracts of Panax notoginseng and its ginsenoside constituents on human liver cancer cells. Chinese medicine 2011, In press. 3. Lau AJ, Toh DF, Chua TK, Pang YK, Woo SO, Koh HL. Antiplatelet and anticoagulant effects of Panax notoginseng: comparison of raw and steamed Panax notoginseng with Panax ginseng and Panax quinquefolium. J Ethnopharmacol 2009; 125: 380-386. 4. Chan EC, Yap SL, Lau AJ, Leow PC, Toh DF, Koh HL. Ultra-performance liquid chromatography/time-of-flight mass spectrometry based metabolomics of raw and steamed Panax notoginseng. Rapid Commun Mass Spectrom 2007; 21: 519-528. 5. Toh DF, New LS, Chan EC, Koh HL. Ultra-performance Liquid Chromatography - Time of flight Mass Spectrometry (UPLC-TOFMS) metabolite profiling of raw and steamed Panax notoginseng: Application on commercial products. (In preparation) Conference presentations 1. Toh DF, Koh HL. A review of the traditional uses, phytochemical constituents and biological activities of Swietenia macrophylla, 3rd Medicinal Chemistry Symposium, 28 July 2008, National University Singapore, Singapore. 2. Toh DF, Koh HL. Medicinal plants in Drug Discovery: A focus on Swietenia macrophylla, Medicinal Chemistry Symposium, 23 January 2008, National University Singapore, Singapore. 3. Toh DF, Koh HL. A literature review of the traditional uses, phytochemical constituents and biological activities of the medicinal plant Swietenia macrophylla, The Inaugural Singapore-Taiwan-Hong Kong (CU) Meeting of Pharmacologists, 28-29 May 2007, National University Singapore, Singapore. 4. Ching JH, Toh DF, Tan CH, Koh HL. Antiplatelet activities of Ardisia elliptica and Swietenia macrophylla, Congress of the Asian-Pacific Society on Thrombosis and Haemostasis, 18-20 September 2008, Grand Copthorne Waterfront Hotel. 5. Low WL, Toh DF, Liesbet Tan, Belinda Tan, Low MY, Chan CL, Koh HL. Safety of botanical health supplements and other complementary medicine, Research Awareness Symposium 2009, April 2009, National University Singapore, Singapore. iii 6. Tan CJ, Ching JH, Toh DF, Neo SY, Koh HL. Effects of Ardisia elliptica and Strobilanthes crispus on hepatocellular carcinoma cell proliferation. Research Awareness Symposium 2009, April 2009, National University Singapore, Singapore. 7. Patel DN, Toh DF, New LS, Chan EC, Koh HL. Quality control of Panax notoginseng using metabolomics Approach, 4th PharmSci@Asia 2009 Symposium, 27 May 2009, China Pharmaceutical University, China. 8. Patel DN, Toh DF, New LS, Chan EC, Koh HL. Effects of steaming on the types and concentrations of ginsenosides in Panax notoginseng, Conference on Recent Development in Chinese Herbal Medicine jointly with the Consortium for Globalization of Chinese Medicine (CGCM), 25-26 January 2010, Nanyang Technological University, Singapore. iv TABLE OF CONTENTS Title page i Acknowledgements ii List of publications and conference presentations iii Table of contents v Summary ix List of tables xi List of figures xiii List of abbreviations xvi Chapter 1. 1.1 1.2 1.3 1.4 Introduction Herbal medicine 1.1.1 Importance of herbal medicine 1.1.2 Quality control of herbal medicine 1.1.2.1 Importance of quality control 1.1.2.2 Traditional methods of quality control of herbal medicine 1.1.2.3 Modern methods of quality control of herbal medicine 1.1.2.4 Metabolomics Medicinal plants and herbal medicine as potential sources of novel therapeutics 1.2.1 Cancer 1.2.2 Cardiovascular diseases 1.2.3 Medicinal plants in drug discovery 1.2.3.1 Cancer 1.2.3.2 Cardiovascular diseases Panax notoginseng 1.3.1 Introduction 1.3.2 Processing of P. notoginseng 1.3.3 Chemical constituents of P. notoginseng 1.3.4 Quality control of P. notoginseng 1.3.5 Pharmacological studies of P. notoginseng Swietenia macrophylla 1.4.1 Introduction 1.4.2 Botanical description 1.4.3 Identification of Swietenia species 1.4.4 Traditional uses and biological activities 1.4.5 Phytochemical constituents Chapter 2. Hypothesis and Objectives Chapter 3. Ultra-high pressure liquid chromatography / time-of-flight mass spectrometry (UHPLC/TOFMS) analysis of raw and steamed Panax notoginseng and application on commercial products 3.1 Development of metabolomic platform for time-dependent profiling 3.1.1 Introduction 3.1.2 Objective 3.1.3 Experimental 13 23 25 26 27 31 36 37 39 41 48 50 51 51 53 61 66 69 75 v 3.2 3.3 3.1.3.1 Materials and reagents 3.1.3.2 Steaming of raw P. notoginseng herb 3.1.3.3 Extraction and sample preparation 3.1.3.4 Instrumentation 3.1.3.5 Method validation 3.1.3.6 Chemometric/Multivariate data analysis 3.1.3.7 Statistical analysis 3.1.4 Results and Discussion 3.1.4.1 Ultra-high performance liquid chromatography time-of-flight mass spectrometry (UHPLC/TOFMS) 3.1.4.2 Method validation 3.1.4.3 PCA & PLS-DA analysis 3.1.4.4 Loadings plot 3.1.4.5 Univariate data analyses of therapeutically important ginsenosides 3.1.4.6 Differences of raw and steamed P. notoginseng in claimed therapeutic effects and scientific evidence Application of method developed on commercial products 3.2.1 Introduction 3.2.2 Objective 3.2.3 Experimental 3.2.3.1 Materials and reagents 3.2.3.2 Sample preparation 3.2.3.3 UHPLC/TOFMS method 3.2.3.4 Chemometric/Multivariate data analysis 3.2.4 Results and Discussion 3.2.4.1 Optimization and processing of raw, 2h and 4h steamed samples 3.2.4.2 Application on four pairs of raw and steamed commercial products (1R,S to 4R,S) 3.2.4.3 Application on thirteen commercial products labeled as “raw” (5R to 17R) Conclusion Chapter 4. 4.1 4.2 4.3 4.4 75 75 76 76 77 78 79 79 83 84 89 97 97 101 101 101 101 104 104 104 108 116 126 Chemical analysis of Panax notoginseng and Investigation of its antiproliferative activities against liver cancer cells Introduction 4.1.1 Liver cancer 4.1.2 P. notoginseng as a potential source of anticancer therapeutics Objective Experimental 4.3.1 LC-LTQ Orbitrap FTMS Analysis 4.3.1.1 Sample preparation 4.3.1.2 Standards preparation 4.3.1.3 LC-LTQ Orbitrap FTMS method 4.3.1.4 Method validation 4.3.1.5 Data analysis 4.3.2 Cell culture 4.3.3 Cell proliferation analysis using WST-1 assay – Cell proliferation screening 4.3.4 Statistical analysis of results Results and discussion 4.4.1 UHPLC/TOFMS 127 130 133 134 134 134 135 136 136 137 137 138 vi 4.4.1.1 4.5 UHPLC/TOFMS metabolite profiles of raw and steamed extracts of P. notoginseng 4.4.1.2 Use of Chemometrics in the Identification of important ginsenosides responsible for the differences between raw and steamed P. notoginseng 4.4.2 LC-LTQ Orbitrap FTMS 4.4.2.1 Method validation 4.4.2.2 Effects of steaming on the ginsenoside compositions of P. notoginseng 4.4.3 Effects of raw and steamed P. notoginseng extracts on human liver cancer cells 4.4.4 Effects of ginsenosides on the proliferation of human liver cancer cells 4.4.5 Changes of the important ginsenosides upon steaming 4.4.6 Dose-response effects of selected ginsenosides on SNU449 cell line Conclusion Chapter 5. 5.1 5.2 5.3 139 141 144 147 150 154 160 164 166 Chemical and pharmacological evaluations of Swietenia macrophylla Isolation and identification of chemical components 5.1.1 Introduction 5.1.2 Objective 5.1.3 Experimental 5.1.3.1 Materials and reagents 5.1.3.2 Extraction 5.1.3.3 Isolation and purification of compounds from kernel extract 5.1.3.4 Isolation and purification of compounds from seed coat extract 5.1.3.5 Identification 5.1.4 Results and Discussion 5.1.5 Conclusion Screening for antiproliferative activities against liver cancer cells 5.2.1 Introduction 5.2.2 Objective 5.2.3 Experimental 5.2.3.1 Materials and reagents 5.2.3.2 Cell culture 5.2.3.3 Sample preparation 5.2.3.4 WST-1 Assay: extracts, fractions, compounds 5.2.4 Results and Discussion 5.2.4.1 Effects of Swietenia macrophylla extracts on human liver cancer cells in vitro 5.2.4.2 Effects of compounds isolated from ether extract of seed coat against human liver cancer cells in vitro 5.2.4.3 Effects of compounds isolated from ether extract of kernel against human liver cancer cells in vitro 5.2.4.4 Dose-response effects of compounds on SNU449, SNU182 and HepG2 cell lines 5.2.5 Conclusion Screening for antiplatelet and anticoagulant activities 5.3.1 Introduction 5.3.2 Objective 5.3.3 Experimental 5.3.3.1 Materials and reagents 5.3.3.2 Sample preparation 5.3.3.3 In vitro platelet aggregation assay 5.3.3.4 In vitro plasma coagulation assay 167 168 168 168 169 170 171 172 191 191 193 193 193 193 194 194 198 201 204 207 208 209 209 210 210 212 vii 5.3.4 5.3.5 5.4 5.3.3.5 Statistical analysis Results and Discussion 5.3.4.1 Effects of Swietenia macrophylla extracts aggregation 5.3.4.2 Effects of compounds isolated from ether extract macrophylla on platelet aggregation 5.3.4.3 Effects of Swietenia macrophylla extracts coagulation 5.3.4.4 Effects of compounds isolated from ether extract macrophylla on plasma coagulation Conclusion 213 on platelet 214 of Swietenia 216 on plasma 220 of Swietenia 222 Conclusion Chapter 6. 226 226 Conclusions and future prospects 227 234 Bibliography Appendices Appendix I Typical chromatograms of standard mixtures of ginsenosides (A and B) and P. notoginseng (raw and 15h steamed) samples 261 Appendix II Schematic equations for the chemical conversions of the ginsenosides 263 Appendix III 13 264 C and 1H NMR spectra of compounds isolated from Swietenia macrophylla viii SUMMARY The overall objectives of this work are to chemically and pharmacologically evaluate medicinal plants, namely, to develop a metabolomic platform for the quality control of raw and steamed P. notoginseng and study the effects of steaming of raw herb in terms of chemical and biological activity and to investigate the chemical components and biological activity of the seeds of S. macrophylla. Metabolomic platform using ultra-high pressure liquid chromatography / time-of-flight mass spectrometry (UHPLC/TOFMS) and chemometrics has been successfully developed and validated for the time-dependent profiling of raw and differentially steamed P. notoginseng. It has also been successfully applied to commercial products of raw and steamed P. notoginseng. Important ginsenosides of the raw and steamed form were identified. Two commercial products were found to have been subjected to heat treatment despite being labeled as the “raw” form. The raw and steamed P. notoginseng were also evaluated for their antiproliferative activities against three liver cancer cell lines, namely, SNU449, SNU182 and HepG2. The antiproliferative activities of P. notoginseng increased with progressive steaming up to 24h as this process enriched the bioactive components such as ginsenosides Rh2, Rk1, Rk3 and 20SRg3. These antiproliferative ginsenosides were identified in the complex multi-ingredient mixture in P. notoginseng using the metabolomics method developed by correlating the changes of antiproliferative activity of P. notoginseng with different steaming durations. The platform facilitates drug discovery from P. notoginseng. Ginsenoside Rh2 was found to be the most potent antiproliferative component with an IC50 of 56 µM as compared to the well-known anticancer agent 20S-Rg3 of 199 µM against the same cell line SNU449. This is the first report of the antiproliferative activity of ginsenoside Rk3. Besides Chinese herbs, locally grown medicinal plants are also important resources. In this work, the seeds of S. macrophylla are investigated for their chemical components and pharmacological activities, namely, antiproliferative and antithrombotic (antiplatelet and ix anticoagulant) activities. Two pure compounds and five partially purified compounds were isolated from the seed coat while five partially purified compounds were isolated from the kernel. The two pure compounds were determined to be the limonoids swietenolide and swietenine. The compounds isolated are potentially bioactive compounds responsible for the various pharmacological activities in S. macrophylla. Some extracts and partially purified compounds showed promising antiproliferative and antiplatelet for the first time. Some of the partially purified compounds had IC50 values comparable to that of aspirin in antiplatelet activity. Hence, they could be potential leads for antiplatelet drugs. In conclusion, a metabolomics platform has been successfully developed for the quality control of P. notoginseng and its products, and facilitates drug discovery. The chemical and pharmacological investigations on S. macrophylla show that it is a useful medicinal plant with promising anticancer and antiplatelet activity. 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(A) Standard mixture A RT: 0.00 - 31.00 SM: 7G NL: 1.18E7 TIC MS mixed-stda1-10ppmdf-pos21apr2010 -1 26.12 100 29.32 95 90 15.87 20S-Rg3 85 80 Rh2 10.21 75 70 65 Relative Abundance Rk1 Re 60 55 28.94 Rc 50 Rk3 45 40 23.50 35 30 26.54 25 20 15 10 16.86 0.16 1.70 3.11 3.65 5.05 5.64 7.05 7.66 8.49 11.92 12.37 9.81 13.24 14.24 15.25 17.29 22.75 20.14 22.36 19.99 21.05 24.14 27.20 27.80 25.83 30.94 0 10 12 14 (B) Standard mixture B RT: 0.00 - 31.00 16 Time (min) 18 20 22 24 26 28 30 20R-Rh1 SM: 7G NL: 1.44E7 9.64 100 20S-Rh1 Rg1 95 16.43 TIC MS mixed-stdb1-10ppmdf-pos21apr2010 -1 18.11 90 15.87 15.11 85 10.17 80 75 70 65 Relative Abundance 60 Rb1 R1 55 50 Rd 45 40 35 30 25 20 15 10 9.04 10.69 0.19 1.72 2.42 3.75 4.25 5.68 6.57 6.90 7.73 19.41 14.26 16.73 11.08 12.70 19.79 21.12 21.35 23.39 25.73 25.98 26.59 28.77 29.10 29.26 0 10 12 14 16 Time (min) 18 20 22 24 26 28 30 261 (C) Raw P. notoginseng RT: 0.00 - 31.00 SM: 7G NL: 1.01E7 14.78 100 TIC MS Notogins e ng-Raw1200x-Pos29Apr2010 -1 Rg1 + Re 95 90 Rb1 10.07 85 80 75 70 65 Relative Abundance 60 55 50 45 Rd 40 35 30 17.77 29.09 28.88 25 9.60 20 R1 1.66 15 10 13.30 1.95 2.90 3.74 1.07 4.67 4.99 6.21 6.35 8.54 9.37 10.52 13.95 15.59 12.57 19.03 16.98 17.98 20.16 21.24 22.36 23.08 23.35 25.71 30.13 27.58 0 10 12 14 16 Time (min) 18 20 22 24 26 28 30 (D) 15h steamed P. notoginseng RT: 0.00 - 31.00 SM: 7G NL: 2.71E6 TIC MS notoginsen g-s15h1200x-pos03may201 0-1 29.02 100 28.85 95 20S-Rg3 20S-Rh1 90 85 25.71 15.71 Rg5 80 75 Rh4 Rk1 70 65 Rk3 Relative Abundance 60 55 23.86 50 45 40 14.88 35 Rb1 30 1.62 20R-Rh1 Rh2 23.12 26.14 16.24 25 20 22.69 10.11 10 1.47 2.60 3.74 4.14 4.97 5.64 7.10 7.41 6.96 12.81 10.65 9.05 12.54 17.05 14.07 18.59 11.23 29.24 22.00 17.86 15 19.37 28.09 28.29 26.40 24.09 21.29 20.77 29.65 0 10 12 14 16 Time (min) 18 20 22 24 26 28 30 262 Appendix II Schematic equations for the chemical conversions of the ginsenosides 263 Appendix III C and 1H NMR spectra of compounds (dissolved in CDCl3) isolated from Swietenia macrophylla. 13 Compound: sce1 H NMR of sce1 13 C NMR of sce1 264 Compound: sce2 H NMR of sce2 13 C NMR of sce2 265 Compound: sce3 H NMR of sce3 13 C NMR of sce3 266 Compound: sce4 H NMR of sce4 13 C NMR of sce4 267 Compound: sce5 H NMR of sce5 13 C NMR of sce5 268 Compound: sce6 H NMR of sce6 13 C NMR of sce6 269 Compound: eks1 H NMR of eks1 13 C NMR of eks1 270 Compound: eks6 H NMR of eks6 13 C NMR of eks6 271 [...]... isolated from the ether extract of seed coat of S macrophylla on platelet aggregation 216 Figure 5.10 Effects of compounds isolated from the ether extract of kernel of S macrophylla on platelet aggregation 218 Figure 5.11 Effects of extracts of seed coat and kernel of S macrophylla on prothrombim time (PT) 221 Figure 5.12 Effects of extracts of seed coat and kernel of S macrophylla on activated partial... loadings plot of PLS-DA model of raw and steamed P notoginseng 93 Figure 3.8 Chemical structures of some saponins present in raw and steamed P notoginseng 96 Figure 3.9 Plots of integrated peak area of the [M+HCOO]- ions of (A) 20S-Rg3 and (B) Rh2 against the duration of steaming process (hours) for the batch 1 samples 98 Figure 3.10 Typical total ion chromatograms (TICs) of raw and steamed P notoginseng. .. plot of raw and steamed P notoginseng samples (2, 6, 9, 15 and 24 h) 142 Figure 4.3 Loadings plot of PLS-DA model of raw and steamed P notoginseng samples (2, 6, 9, 12 and 15 h) 143 Figure 4.4 The saponins content in the (A) raw and differentially (B) steamed P notoginseng samples 149 Figure 4.5 In vitro antiproliferative effects of raw and steamed Panax notoginseng extracts in SNU449, SNU182 and HepG2... form of whole root (coated and uncoated) and slices 37 Figure 1.2 Chinese proprietary medicines (CPMs) of P notoginseng available in raw and steamed form 40 Figure 1.3 Chemical structures of some saponins in P notoginseng 42 Figure 1.4 The fruits and seeds of S macrophylla 52 Figure 1.5 Chemical structures of some limonoids in S macrophylla 64 Figure 3.1 Representative total ion chromatograms (TICs) of. .. concentrations of the partially purified compounds isolated from the ether extract of the seed coat of the seeds of S macrophylla on proliferation of SNU449, SNU182 and HepG2 human liver cancer cells (n = 3) 205 Table 5.9 Inhibitory concentrations of the ether extracts and partially purified compounds of S macrophylla and aspirin (n = 3) 219 xii LIST OF FIGURES Page no Figure 1.1 The root of P notoginseng. .. (2h and 4h) obtained from UHPLC/TOFMS 105 Figure 3.11 PCA score plot of raw and steamed (2hr and 4hr steamed) P .notoginseng 107 Figure 3.12 PLS-DA score plot of raw and steamed (2hr and 4hr steamed) P .notoginseng 109 xiii Figure 3.13 PCA score plot of raw and steamed (2hr and 4hr steamed) P .notoginseng in comparison to the commercial products (pairs 1R/1S to 4R/4S) 110 Figure 3.14 PLS-DA score plot of. .. Table 5.5 1 188 H-NMR chemical shifts (δ, ppm) of samples eks1 and eks6, 54 xi obtained in CDCl3 at 300 MHz Table 5.6 13 C-NMR chemical shifts (δ, ppm) of samples eks1 and eks6, obtained in CDCl3 at 300 MHz 189 Table 5.7 Inhibitory concentrations of the ether, methanol and water extracts of the seed coat and kernel of the seeds of S macrophylla on proliferation of SNU449, SNU182 and HepG2 human liver... extract of seed coat of S macrophylla in SNU449, SNU182 and HepG2 human liver cancer cells 199 Figure 5.7 In vitro antiproliferative effects of isolated compounds from ether extract of kernel of S macrophylla in SNU449, SNU182 and HepG2 human liver cancer cells 202 Figure 5.8 Effects of extracts of (A) seed coat and (B) kernel of S macrophylla on platelet aggregation 215 Figure 5.9 Effects of compounds... 12 and 15h steamed samples 88 Figure 3.4 Plot of PLS-DA cross-validated predicted Y scores of raw and steamed P notoginseng samples (2, 6, 9, 12 and 15h) belonging to the batch 1 model and batch 2 prediction set 90 Figure 3.5 Two dimensional PLS-DA score plot of raw and steamed P notoginseng 91 Figure 3.6 Loadings plot of PLS-DA model of raw and steamed P notoginseng 92 Figure 3.7 An enlarged view of. .. (TICs) of raw and steamed P notoginseng extracted samples (2h and 15h) obtained from UHPLC/TOFMS 80 Figure 3.2 PCA score plot of raw and steamed P notoginseng samples (1, 2, 4, 6, 9, 12, 15 and 24 h) 86 Figure 3.3 (A) Three dimensional PLS-DA score plot of batch 1 raw and steamed P notoginseng samples (1, 2, 4, 6, 9, 12, 15 and 24h); (B) Three dimensional PLS-DA score plot of batch 1 model set and batch . study the effects of steaming of raw herb in terms of chemical and biological activity and to investigate the chemical components and biological activity of the seeds of S. macrophylla. Metabolomic. Koh HL. Antiplatelet and anticoagulant effects of Panax notoginseng: comparison of raw and steamed Panax notoginseng with Panax ginseng and Panax quinquefolium. J Ethnopharmacol 2009; 125:. 164 4.5 Conclusion 166 Chapter 5. Chemical and pharmacological evaluations of Swietenia macrophylla 5.1 Isolation and identification of chemical components 5.1.1 Introduction