Phenolic Acid Rich Fraction of Gynura Procumbens As Potential Antihyperlipidemic and Antioxidant Agents

PHENOLIC ACID RICH FRACTION OF GYNURA PROCUMBENS AS POTENTIAL ANTIHYPERLIPIDEMIC AND ANTIOXIDANT AGENTS

KISANTINI A/P MURUGESU

UNIVERSITI SAINS MALAYSIA

2018 PHENOLIC ACID RICH FRACTION OF GYNURA PROCUMBENS AS POTENTIAL ANTIHYPERLIPIDEMIC AND ANTIOXIDANT AGENTS

KISANTINI A/P MURUGESU

Thesis submitted in fulfillment of the requirements for the degree of Master of Science

June 2018 ACKNOWLEDGEMENT

Foremost, I am grateful to the Almighty Superior God for the good health and well being that were crucial for the completion of this thesis.

I am extremely indebted to my advisor, Prof. Dr. Amirin Sadikun for many insightful conversations, encouragement and sincere advice that have been extended to me from the beginning of study until his retirement day. He was always available for me whenever I had questions about my research and consistently shared his expertise whenever he thought I needed it. I could not have imagined of having a better advisor for my research.

Besides my advisor, I would like to thank my main supervisor, Assoc. Prof. Dr.

Vikneswaran Murugaiyah and co-supervisor, Prof. Dr. Mohd Zaini Bin Asmawi for their valuable guidance, immense knowledge and useful comments throughout my Masters study period. Their patience and clarity when giving information have helped me a lot during my research and thesis writing. To my advisor and supervisors, thank you for steering me in the right direction.

Next, I wish to thank my fellow colleagues, Dr. Sultan, Dr. Mohammad Ali, Ida,

Hidayah, Farhana, Syafinaz, Shahrul, Chung Wan Jie, Liew Wai Lam, Saiful and

Mathew for their sincere assistance and generosity in sharing knowledge during the study. In particular, I am grateful to Dr. Sultan for his valuable contribution, advice and enlightenment to the success of this research.

I take this opportunity to thank the department staffs, science officers and technicians for their patience in entertaining our complaints, continuous guidance in handling equipment and for giving access to the research facilities. Without their support and cooperation it would not be possible to conduct this research.

Last but not least, a million thanks to my parents, siblings and my partner for unfailing support and enduring love throughout my years of study. Thank you for being there for me during my hard times, I will be thankful forever for your love. Without you all, I wouldn’t have achieved this position in my life. I wish you all the happiness in the world.

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TABLE OF CONTENTS

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iv

LIST OF TABLES xi

LIST OF FIGURES xiv

LIST OF APPENDICES xviii

LIST OF ABBREVIATIONS xxi

LIST OF SYMBOLS xxv

ABSTRAK xxvi

ABSTRACT xxviii

CHAPTER 1 - INTRODUCTION

1.1 Background 1

1.2 Problem statement 4

1.3 Hypothesis 5

1.4 Research objectives 5

CHAPTER 2 - LITERATURE REVIEW

2.1 The genus Gynura 8

2.2 Gynura procumbens (Lour.) Merr. 8

2.2.1 Description of Gynura procumbens (Lour.) Merr. 8

2.2.2 Chemical constituents of Gynura procumbens (Lour.) Merr. 11

2.2.3 Pharmacological activities of Gynura procumbens (Lour.) Merr. 21

2.2.3(a) Wound healing activity 21

2.2.3(b) Anticancer activity 21

2.2.3(c) Antiulcerogenic activity 21

2.2.3(d) Cardiovascular activity 22

2.2.3(e) Ultraviolet (UV) protective activity 22

2.2.3(f) Immunomodulatory activity 22

2.2.3(g) Antihypertensive activity 23

2.2.3(h) Anti-inflammatory activity 23

2.2.3(i) Antiherpetic activity 24

2.2.3(j) Antidiabetic activity 24

2.2.3(k) Antioxidant activity 25

2.2.3(l) Antiplasmodial activity 25

2.2.3(m) Toxicity 25

2.3 Caffeoylquinic acids 26

2.4 Standardization 28

2.4.1 Preliminary screening and chemical group quantification 31

2.4.2 Phytochemical fingerprint profiling 32

2.5 Hyperlipidemia 33

2.5.1 Lipids 33

2.5.2 Classifications of hyperlipidemia 35

2.5.3 Risk factors and clinical symptoms of hyperlipidemia 37

2.5.4 Biosynthesis of lipids 38

2.5.5 Management of hyperlipidemia 41

2.5.6 Laboratory animal models of acute hyperlipidemia 45

2.5.7 High fat diet-induced chronic hyperlipidemic model 46

2.5.8 Natural products in the treatment of hyperlipidemia 47

2.6 Reactive oxygen species and antioxidants 49

CHAPTER 3 - MATERIALS AND METHODS

3.1 Materials and equipment 55

SECTION I - CHEMISTRY

3.2 Preparation and characterization of extract and fractions of Gynura procumbens 59

3.2.1 Extraction and fractionation of Gynura procumbens leaves 59

3.2.2 Qualitative analysis of ethanolic extract and fractions of Gynura procumbens 61

3.2.2(a) Thin layer chromatography 61

3.2.2(b) Ultraviolet-visible spectroscopy 61

3.2.2(c) Fourier transform infra-red spectroscopy 61

3.2.3 Sub-fractionation of 50 % methanolic fraction 62

3.3 Isolation of chlorogenic acid 63

3.3.1 Spectroscopic & physical characterization of chlorogenic acid 65

3.3.1(a) Nuclear magnetic resonance spectroscopy 65

3.3.1(b) Liquid chromatography–mass spectrometry 65

3.3.1(c) Melting point 66

3.4 Determination of phenolic and flavonoid contents of ethanolic extract and fractions

of Gynura procumbens 66

3.4.1 Total phenolic content 66

3.4.2 Total flavonoid content 67

3.5 Evaluation of antioxidant activities of ethanolic extract and fractions of Gynura

procumbens and chlorogenic acid 68

3.5.1 Ferric reducing antioxidant power assay 68

3.5.2 DPPH free radical scavenging assay 69

3.5.3 ABTS radical scavenging assay 70

3.6 Development and validation of a HPLC method for quantification of phenolic acids

and standardization of Gynura procumbens plant samples 72

3.6.1 Method development 72

3.6.1(a) Chromatographic conditions 72

3.6.2 Sample preparation 72

3.6.2(a) Preparation of standard solutions of phenolic acids 72

3.6.2(b) Preparation of 50 % methanolic fraction of Gynura procumbens

ethanolic extract 73

3.6.3 Parameters investigated during method development 73

3.6.4 Peak purity 74

3.6.5 Method validation 74

3.6.5(a) Linearity, limit of detection (LOD) and limit of quantification (LOQ) 74

3.6.5(b) Precision, accuracy and recovery 75

3.6.5(c) Quantification of samples 76

SECTION II - PHARMACOLOGY

3.7 Antihyperlipidemic evaluation of Gynura procumbens 77

3.7.1 Experimental animals 77

3.7.2 Antihyperlipidemic evaluation of standardized ethanolic extract, fractions and

chlorogenic acid of Gynura procumbens in poloxamer-407-induced

hyperlipidemic rats 77

3.7.2(a) Induction of hyperlipidemia 77

3.7.2(b) Experimental design 78

3.7.2(c) Collection of blood sample 79

3.7.2(d) Analysis of lipid parameters 80

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3.7.3 Antihyperlipidemic evaluation of standardized 50 % methanolic fraction of

Gynura procumbens in high fat diet-induced hyperlipidemic rats 81

3.7.3(a) Induction of hyperlipidemia 81

3.7.3(b) Experimental design 82

3.7.3(c) Collection of biological samples 82

3.7.4 Statistical analysis 83

3.8 Evaluation of mechanism of antihyperlipidemic activity of standardized 50 %

methanolic fraction and chlorogenic acid of Gynura procumbens 84

3.8.1 Evaluation of inhibitory activity of standardized 50 % methanolic fraction

and chlorogenic acid of Gynura procumbens on HMG-CoA reductase and

pancreatic lipase enzymes 84

3.8.1(a) Preparation of standardized 50 % methanolic fraction and chlorogenic

acid 84

3.8.1(b) HMG-CoA reductase inhibitory activity 84

3.8.1(c) Pancreatic lipase inhibitory activity 85

3.8.2 Evaluation of effect of standardized 50 % methanolic fraction of Gynura

procumbens on lipids and bile acids excretions 86

3.8.2(a) Liver lipids estimation 86

3.8.2(b) Fecal lipids estimation 87

3.8.2(c) Fecal bile acids estimation 87

3.8.3 Statistical analysis 88

3.9 Toxicological evaluation of standardized 50 % methanolic fraction and chlorogenic

acid of Gynura procumbens 88

3.9.1 In vitro cytotoxicity evaluation of standardized 50 % methanolic fraction and

chlorogenic acid of Gynura procumbens 88

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3.9.1(a) Cell culture 88

3.9.1(b) MTT assay 89

3.9.2 Acute toxicity evaluation of standardized 50 % methanolic fraction of Gynura

procumbens 90

3.9.2(a) Animals 90

3.9.2(b) Treatment dose 91

3.9.2(c) Observations 91

CHAPTER 4 - RESULTS & DISCUSSION

SECTION I - CHEMISTRY

4.1 Extraction and fractionation of Gynura procumbens leaves 92

4.2 Isolation of chlorogenic acid 96

4.3 Polyphenolics content of ethanolic extract and fractions of Gynura procumbens 110

4.4 Antioxidant activities of ethanolic extract, fractions and chlorogenic acid of Gynura

procumbens 111

4.5 Development and validation of a HPLC method for standardization of Gynura

procumbens extracts and fractions 118

SECTION II - PHARMACOLOGY

4.6 Antihyperlipidemic effect of standardized ethanolic extract, fractions and

chlorogenic acid of Gynura procumbens in poloxamer-407 induced hyperlipidemic

rats 141

4.7 Antihyperlipidemic effect of standardized 50 % methanolic fraction of Gynura

procumbens in high fat diet-induced hyperlipidemic rats 156

4.8 Mechanism of antihyperlipidemic activity of standardized 50 % methanolic fraction

and chlorogenic acid of Gynura procumbens 178

4.8.1 Inhibitory activity of standardized 50 % methanolic fraction and chlorogenic

acid of Gynura procumbens on HMG-CoA reductase and pancreatic lipase

enzymes 178

4.8.2 Effect of standardized 50 % methanolic fraction of Gynura procumbens on

lipids and bile acids secretion 185

4.9 Toxicity of standardized 50 % methanolic fraction and chlorogenic acid of Gynura

procumbens 190

4.9.1 Cytotoxicity 190

4.9.2 Acute toxicity 193

4.10 Summary of results 196

CHAPTER 5 - CONCLUSION

5.1 Conclusion 198

5.2 Limitations 201

5.3 Suggestions for future studies 201

REFERENCES 202

APPENDICES

LIST OF TABLES

Page

Table 2.1 Taxonomic classification of Gynura procumbens (Lour.) Merr 9

Table 2.2 Properties and functions of plasma lipoproteins 34

Table 2.3 Blood lipid levels guidelines 35

Table 2.4 The Fredrickson classification 36

Table 2.5 Summary of uses, side effects and mechanisms of lipid 43 lowering drugs

Table 2.5-1 Continued 44

Table 2.6 Free radicals and non radicals 50

Table 2.7 List of in vitro antioxidant assays 54

Table 2.8 List of in vivo antioxidant assays 54

Table 3.1 List of chemicals and solvents 55

Table 3.1-1 Continued 56

Table 3.2 List of standards and drugs 57

Table 3.3 List of kits 57

Table 3.4 List of cell lines 57

Table 3.5 List of consumables and equipment 57

Table 3.5-1 Continued 58

Table 3.6 List of instruments 58

Table 3.6-1 Continued 59

Table 3.7 Solvent ratio used for elution of 50 % methanolic fraction 63 using dry-flash column chromatography

Table 3.8 Solvent ratio used for elution of chlorogenic acid using 65 Sephadex LH-20 column

Table 3.9 Parameters investigated during method development 73

Table 3.10 Preparation of samples for measurement of HMG-CoA 85 reductase inhibitory activity

Table 3.11 Preparation of samples for measurement of pancreatic lipase 86 inhibitory activity

Table 4.1 Percentage yield of ethanolic extract and different fractions of 92 Gynura procumbens

Table 4.2 Comparison of 13C NMR and 1H NMR data of compound A 103 with the literature values

Table 4.3 Polyphenolic contents and antioxidant activities of ethanolic 113 extract, fractions and chlorogenic acid of Gynura procumbens

Table 4.4 Retention times and occurrence of marker compounds eluted 122 from phenyl-hexyl column using mobile system ACN:0.25 % AAW (21.5:78.5, v/v)

Table 4.5 Calibration results, LOD and LOQ of marker compounds 127

Table 4.6 Recovery, within-day and between-day precision and accuracy 128 values of marker compounds

Table 4.7 Content of caffeoylquinic acids in ethanolic extract, various 130 fractions and plant samples from different states

Table 4.8 Relative organ weights of high fat diet-induced chronic 170 hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Table 4.9 Hematological parameters of high fat diet-induced chronic 171 hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Table 4.10 Biochemical parameters of high fat diet-induced chronic 172 hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

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Table 4.11 Percentage inhibition of cell growth by standardized 50 % 191 methanolic fraction and chlorogenic acid of Gynura procumbens

Table 4.12 Body weights (g) of female rats given different doses on the 194 0th, 7th and 14th day

Table 4.13 Summary of findings 196

Table 4.13-1 Continued 197

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LIST OF FIGURES

Page

Figure 1.1 Flow chart of the study 7

Figure 2.1 Gynura procumbens plant 9

Figure 2.2 Biosynthesis of lipids 40

Figure 4.1 Structure of compound A 97

Figure 4.2 1H NMR spectrum of compound A 99

Figure 4.3 Expansion of 1H NMR spectrum of compound A 100

Figure 4.4 Expansion of 1H NMR spectrum of compound A 101

Figure 4.5 13C NMR spectrum of compound A 102

Figure 4.6 LCMS spectrum of compound A 105

Figure 4.7 Fragmentation pattern of chlorogenic acid 106

Figure 4.7-1 Continued 107

Figure 4.8 UV-vis spectrum of compound A 107

Figure 4.9 FTIR spectrum of compound A 109

Figure 4.10 Typical chromatogram of mixed standards (1) CA; (2) 3,4DC; 123 (3) 3,5DC; (4) 4,5DC; (5) 1,3DC; (6) 1,5DC at 1000 µg/mL

Figure 4.11 Typical peak purity spectra of 50 % methanolic fraction of 126 Gynura procumbens (A) CA; (B) 3,4DC; (C) 3,5DC; (D) 4,5DC at 1000 µg/mL at 218, 244, 262, 280 and 340 nm

Figure 4.12 HPLC chromatogram of ethanolic extract and fractions of 131 Gynura procumbens (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC at 1000 µg/mL

Figure 4.13 HPLC chromatogram of ethanolic extracts of Gynura 137 procumbens leaves collected from different geographical locations (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC at 1000 µg/mL

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Figure 4.14 HPLC chromatogram of ethanolic extracts of Gynura 138 procumbens stems collected from different geographical locations (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC at 1000 µg/mL

Figure 4.15 Total cholesterol levels for all groups at zero hour and after 6 144 hours from induction using poloxamer-407

Figure 4.16 Total cholesterol levels of poloxamer-407 induced 145 hyperlipidemic rats after treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens at different time points

Figure 4.17 Triglycerides levels of poloxamer-407 induced hyperlipidemic 146 rats after treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens at different time points

Figure 4.18 Low-density lipoprotein-cholesterol levels of poloxamer-407 147 induced hyperlipidemic rats after 58 hours treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens

Figure 4.19 Very low-density lipoprotein-cholesterol levels of poloxamer- 147 407 induced hyperlipidemic rats after 58 hours treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens

Figure 4.20 High-density lipoprotein-cholesterol levels of poloxamer-407 148 induced hyperlipidemic rats after 58 hours treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens

Figure 4.21 Atherogenic index levels of poloxamer-407 induced 148 hyperlipidemic rats after 58 hours treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens

Figure 4.22 Coronary risk index levels of poloxamer-407 induced 149 hyperlipidemic rats after 58 hours treatment with standardized ethanolic extract, fractions and chlorogenic acid of Gynura procumbens

Figure 4.23 Total cholesterol levels for all groups after 2 weeks 159 optimization

Figure 4.24 Total cholesterol levels of high fat diet-induced chronic 160 hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.25 Triglycerides levels of high fat diet induced-chronic 161 hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.26 Low-density lipoprotein-cholesterol levels of high fat diet- 162 induced chronic hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.27 Very low-density lipoprotein-cholesterol levels of high fat 162 diet-induced chronic hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.28 High-density lipoprotein-cholesterol levels of high fat diet- 163 induced chronic hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.29 Atherogenic index levels of high fat diet-induced chronic 163 hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.30 Coronary risk index levels of high fat diet-induced chronic 164 hyperlipidemic rats after treatment with different doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.31 Food consumption of high fat diet-induced chronic 166 hyperlipidemic rats during treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.32 Average body weights for all groups after 2 weeks 166 optimization

Figure 4.33 Average body weights for all groups after 2 weeks 167 optimization period and during treatment period with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

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Figure 4.34 Effects of different doses of standardized 50 % methanolic 173 fraction of Gynura procumbens on rat liver histology of high fat diet-induced hyperlipidemic rats as assessed by H&E staining, (A) Normal control; (B) Hyperlipidemic control; (C) Atorvastatin 20 mg/kg; (D) S(F2) 125 mg/kg; (E) S(F2) 250 mg/kg; (F) S(F2) 500 mg/kg (Magnification × 100)

Figure 4.35 HMG-CoA reductase inhibitory activity of standardized 50 % 179 methanolic fraction and chlorogenic acid of Gynura procumbens

Figure 4.36 Pancreatic lipase inhibitory activity of standardized 50 % 179 methanolic fraction of Gynura procumbens

Figure 4.37 Pancreatic lipase inhibitory activity of chlorogenic acid of 180 Gynura procumbens

Figure 4.38 Liver total cholesterol levels of high fat diet-induced chronic 186 hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.39 Liver triglycerides levels of high fat diet-induced chronic 186 hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.40 Fecal total cholesterol levels of high fat diet-induced chronic 187 hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

Figure 4.41 Fecal bile acids levels of high fat diet-induced chronic 187 hyperlipidemic rats after treatment with various doses of standardized 50 % methanolic fraction of Gynura procumbens for 5 weeks

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LIST OF APPENDICES

Appendix A

Figure I TLC profile of ethanolic extract and fractions of Gynura procumbens eluted from Amberlite XAD-2 column, spraying reagent: NP/PEG, wavelength: 365 nm

Figure II UV-vis spectrum of ethanolic extract of Gynura procumbens

Figure III UV-vis spectrum of water fraction of Gynura procumbens

Figure IV UV-vis spectrum of 50 % methanolic fraction of Gynura procumbens

Figure V UV-vis spectrum of acetone fraction of Gynura procumbens

Appendix B

Table I Marker compounds used in the method development

Table II Retention times of marker compounds at mobile phase composition of 83 % AAW:17 % ACN using LiChroCART RP-18

Table III Retention times of marker compounds at mobile phase composition of 86 % AAW:14 % ACN using Chromolith High Resolution RP- 18

Table IV Retention times of marker compounds at mobile phase composition of 88 % AAW:12 % ACN using SeQuant ZIC-HILIC

Table V Retention times and occurrence of marker compounds eluted from ZORBAX Eclipse Plus phenyl-hexyl column using mobile system ACN:0.25 % AAW (21.5:78.5, v/v)

Figure VI Chromatogram obtained by varying the mobile system using LiChroCART RP-18 column (A) 78 % AAW:22 % ACN; (B) 80 % AAW:20 % ACN; (C) 83 % AAW:17 % ACN; (D) 85 % AAW:15 % ACN at 1000 µg/mL

Figure VII Chromatogram of marker compounds (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC; (5) 1,3DC; (6) 1,5DC at 1000 µg/mL, column: LiChroCART RP-18, mobile system: 83 % AAW:17 % ACN

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Figure VIII Chromatogram obtained by varying the mobile system using Chromolith High Resolution RP-18 column (A) 80 % AAW:20 % ACN; (B) 84 % AAW:16 % ACN; (C) 86 % AAW:14 % ACN; (D) 90 % AAW:10 % ACN at 1000 µg/mL

Figure IX Chromatogram of marker compounds (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC; (5) 1,3DC; (6) 1,5DC at 1000 µg/mL, column: Chromolith High Resolution RP-18, mobile system: 86 % AAW:14 % ACN

Figure X Chromatogram obtained by varying the mobile system using SeQuant ZIC-HILIC column (A) 80 % AAW:20 % ACN; (B) 88 % AAW:12 % ACN; (C) 90 % AAW:10 % ACN; (D) 92 % AAW:8 % ACN; (E) 94 % AAW:6 % ACN at 1000 µg/mL

Figure XI Chromatogram of marker compounds (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC; (5) 1,3DC; (6) 1,5DC at 1000 µg/mL, column: SeQuant ZIC-HILIC, mobile system: 88 % AAW:12 % ACN

Figure XII Chromatogram obtained by varying the flow rate using SeQuant ZIC-HILIC column (A) 90 % AAW:10 % ACN, 0.8 mL/min; (B) 90 % AAW:10 % ACN, 1.0 mL/min; (C) 92 % AAW:8 % ACN, 0.8 mL/min; (D) 92 % AAW:8 % ACN, 1.0 mL/min at 1000 µg/mL

Figure XIII Chromatogram obtained by varying the modifier using SeQuant ZIC-HILIC column (A) 88.5 % AAW:11.5 % ACN; (B) 88.5 % FAW:11.5 % ACN; (C) 89.5 % AAW:10.5 % ACN; (D) 89.5 % FAW:10.5 % ACN at 1000 µg/mL

Figure XIV Chromatogram obtained by varying the mobile system using ZORBAX Eclipse Plus phenyl-hexyl column (A) 78 % AAW:22 % ACN; (B) 78.5 % AAW:21.5 % ACN; (C) 79.5 % AAW:20.5 % ACN; (D) 81 % AAW:19 % ACN at 1000 µg/mL

Figure XV Chromatogram of marker compounds (1) CA; (2) 3,4DC; (3) 3,5DC; (4) 4,5DC; (5) 1,3DC; (6) 1,5DC at 1000 µg/mL, column: ZORBAX Eclipse Plus phenyl-hexyl, mobile system: 78.5 % AAW:21.5 % ACN

Figure XVI Chromatogram obtained using ZORBAX Eclipse Plus phenyl- hexyl column with mobile system ACN:0.25 % AAW (21.5:78.5, v/v) (A) F2 without spiking (B) F2 spiked with compound 5 and 6 (at 1000 µg/mL)

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Appendix C

Figure XVII Effect of standardized 50 % methanolic fraction of Gynura procumbens on colon cancer cell line, HCT-116

Figure XVIII Effect of standardized 50 % methanolic fraction of Gynura procumbens on cervical cancer cell line, Hela

Figure XIX Effect of standardized 50 % methanolic fraction of Gynura procumbens on breast cancer cell line, MCF-7

Figure XX Effect of standardized 50 % methanolic fraction of Gynura procumbens on endothelial cell line, Eahy

Figure XXI Effect of chlorogenic acid of Gynura procumbens on colon cancer cell line, HCT-116

Figure XXII Effect of chlorogenic acid of Gynura procumbens on cervical cancer cell line, Hela

Figure XXIII Effect of chlorogenic acid of Gynura procumbens on breast cancer cell line, MCF-7

Figure XXIV Effect of chlorogenic acid of Gynura procumbens on endothelial cell line, Eahy

LIST OF ABBREVIATIONS

1,3DC 1,3-dicaffeoylquinic acid 1,5DC 1,5-dicaffeoylquinic acid 3,4DC 3,4-dicaffeoylquinic acid 3,5DC 3,5-dicaffeoylquinic acid 4,5DC 4,5-dicaffeoylquinic acid 13C Carbon-13 1H Proton AAW Acetic acid in water ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) ACN Acetonitrile AI Atherogenic index ALT Alanine aminotransferase ANOVA Analysis of variance Apo Apolipoprotein ARASC Animal Research and Service Centre AST Aspartate aminotransferase ATP Adenosine triphosphate CA Chlorogenic acid CE Catechin equivalent CETP Cholesterol ester transfer protein cm Centimeter cm-1 Unit for wavenumber CMC Carboxymethylcellulose CO2 Carbon dioxide CoA Coenzyme A CRI Coronary risk index CYP51 Lanosterol 14α-demethylase CYP7A1 Cholesterol 7α-hydroxylase d Doublet DAD Diode array detector dd Doublet of doublets DHCR Dehydrocholesterol reductase dL Deciliter DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 2,2-diphenyl-1-picrylhydrazyl Eahy Human endothelial cells ELISA Enzyme-linked immunosorbent assay Ethanolic extract 95 % ethanolic extract F1 Water fraction F2 50 % methanolic fraction F3 Acetone fraction FAS Fatty acid synthase

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FAW Formic acid in water FeSO4 Ferrous sulphate FPP Farnesyl pyrophosphate FRAP Ferric reducing antioxidant power g Gram GAE Gallic acid equivalent GC Gas chromatography GPP Geranyl pyrophosphate h Hour H&E Hematoxylin and Eosin HaCat Human keratinocyte HCl Hydrochloric acid HCT-116 Human colon cancer cells HDL-C High-density lipoprotein-cholesterol HeLa Human cervical cancer cells HepG2 cells Human liver carcinoma cells HFD High fat diet HILIC Hydrophilic Interaction Liquid Chromatography HMG-CoA 3-hydroxy-3-methylglutaryl-coenzyme A HPLC High-performance liquid chromatography HSV Herpes simplex virus Hz Hertz i.p. Intraperitoneal i.v Intravenous IC50 Half maximal inhibitory concentration ICH International Council for Harmonization IDL-C Intermediate-density lipoprotein-cholesterol IPP Isopentenyl-diphosphate delta IR Infrared spectroscopy IU International unit J Coupling constant in Hertz kg Kilogram L Liter LCAT Lecithin–cholesterol acyltransferase LD50 Lethal dose LDL-C Low-density lipoprotein-cholesterol LOD Limit of detection LOQ Limit of quantification LPL Lipoprotein lipase M Molar m Multiplet m/z Mass-to-charge ratio MCF-7 Human breast cancer cells MeOH Methanol mg Milligram min Minutes mL Milliliter

xxii mm Millimeter mM Millimolar mmol Millimoles mol Moles MS Mass spectrometry MTT 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4- sulfophenyl)-2H tetrazolium salt MW Molecular weight NADH Nicotinamide adenine dinucleotide NADP+ Oxidized nicotinamide adenine dinucleotide phosphate NADPH Nicotinamide adenine dinucleotide phosphate NF-ĸb Nuclear Factor Kappa B nm Nanometer NMR Nuclear magnetic resonance NP-PEG Natural product-polyethylene glycol O.D. Optical density OECD Organization for Economic Cooperation and Development p Probability value p-407 Poloxamer-407 pH Potential of hydrogen PPARα Peroxisome proliferator-activated receptor alpha ppm Parts per million PTFE Polytetrafluoroethylene QE Quercetin equivalent ROS Reactive oxygen species RP Reverse phase rpm Revolutions per minute RPMI Roswell Park Memorial Institute RSD Relative standard deviation S(ethanolic extract) Standardized ethanolic extract S(F1) Standardized F1 S(F2) Standardized F2 S(F3) Standardized F3 SD Sprague Dawley sec Seconds SEM Standard error of mean s-ICAM-1 Soluble-intercellular adhesion molecule 1 SPE Solid phase extraction SPSS Statistical Package for the Social Sciences s-VCAM-1 Soluble-vascular cell adhesion molecule 1 T cells T helper cell TC Total cholesterol td Triplet of doublets TEAC Trolox equivalent antioxidant capacity TFC Total flavonoid content TG Triglycerides TLC Thin layer chromatography

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TPC Total phenolic content TPTZ 2,4,6-Tris(2-pyridyl)-s-triazine U Unit UV-vis Ultraviolet–visible v/v Volume over volume VLDL-C Very low-density lipoprotein-cholesterol w/v Weight over volume w/w Weight over weight WHO World Health Organization

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LIST OF SYMBOLS

α Alpha

Å Angström

β Beta

0C Degree Celsius

°E East

γ Gamma

λ Lambda

°N North

π Pi

μ Micro

< Less than

> More than

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FRAKSI GYNURA PROCUMBENS YANG DIPERKAYA DENGAN ASID FENOLIK SEBAGAI AGEN ANTIHIPERLIPIDEMIK DAN ANTIOKSIDA BERPOTENSI

ABSTRAK

G. procumbens boleh didapati di kebanyakan negara Asia Tenggara dan daunnya digunakan sebagai ubat untuk merawat pelbagai penyakit termasuk hiperlipidemia.

Objektif kajian ini termasuk pemencilan sebatian penanda, pemiawaian dan analisis fitokimia sampel tumbuhan G. procumbens dan penyelidikan kesan antioksida dan antihiperlipidemik mereka dalam model tikus akut dan kronik, penentuan tindakan mekanisma dan profil toksikologi mereka. Sampel daun G. procumbens telah diekstrak dengan 95 % etanol dan difraksinasi kepada tiga fraksi: F1 (air), F2 (50 % metanol) dan

F3 (aseton). Pemurnian selanjutnya terhadap F2 telah menghasilkan asid fenolik, asid klorogenik (CA). Analisis antioksida mendedahkan bahawa F2 mempunyai kandungan fenolik tertinggi dan aktiviti antioksida yang kuat. CA juga menunjukkan aktiviti antioksida yang kuat setanding dengan piawaian rujukan. Sampel-sampel G. procumbens, ekstrak dan fraksi-fraksi dipiawaikan menggunakan asid fenolik (asid kafioyilkuinik: CA, 3,4DC, 3,5DC dan 4,5DC) sebagai sebatian penanda dengan kaedah

RP-HPLC yang disahkan. Analisis fitokimia mendedahkan bahawa, F2 diperkaya dengan asid kafioyilkuinik berbanding dengan fraksi-fraksi lain. Kajian antihiperlipidemik akut ekstrak, fraksi-fraksi dan CA G. procumbens yang telah dipiawaikan menyatakan bahawa S(F2) dan CA mempunyai aktiviti antihiperlipidemik yang paling kuat untuk merendahkan tahap TC dan TG dalam tikus hiperlipidemik akut diinduksi dengan p-407 (500 mg/kg; ip). Di samping itu, S(F2) juga mengurangkan

xxvi tahap LDL-C, VLDL-C, AI dan CRI secara ketara selepas rawatan selama 58 jam diikuti oleh CA. Oleh itu, S(F2) telah dipilih untuk penyiasatan lanjut menggunakan model hiperlipidemik kronik. Rawatan selama lima minggu dengan pelbagai dos S(F2) menghasilkan kesan antihiperlipidemik bersandarkan dos, di mana kesan yang paling kuat diberikan oleh 500 mg/kg S(F2). Fraksi tersebut didapati merendahkan tahap TC,

TG, LDL-C, VLDL-C, CRI dan AI pada masa yang sama meningkatkan paras HDL-C tikus hiperlipidemik secara ketara. Kedua-dua S(F2) dan CA telah menunjukkan aktiviti perencatan pada enzim HMG-CoA reduktase sementara, hanya CA mempamerkan aktiviti perencatan sederhana terhadap enzim lipase pankreas. Di samping itu, S(F2) menunjukkan keberkesanan bersandarkan dos dimana dos 500 mg/kg menurunkan tahap sintesis lipid hati (TC dan TG) dan meningkatkan perkumuhan lipid (TC) dan asid hempedu melalui tinja. CA menunjukkan kesan sitotoksik sederhana terhadap MCF-7 dan Hela, manakala ia adalah selamat pada sel-sel HCT-116 dan Eahy. Sebaliknya,

S(F2) tidak menunjukkan kesan sitotoksik pada semua sel kanser dan normal. Kajian toksisiti akut pada S(F2) menunjukkan bahawa fraksi tersebut adalah selamat dan LD50 dianggarkan lebih daripada 5000 mg/kg. Kesimpulannya, fraksi bioaktif S(F2) berpotensi sebagai agen merendahkan lipid yang boleh menghalang hiperlipidemia dan penyakit kardiovaskular dengan mengurangkan tahap lipid serum.

xxvii

PHENOLIC ACID RICH FRACTION OF GYNURA PROCUMBENS AS

POTENTIAL ANTIHYPERLIPIDEMIC AND ANTIOXIDANT AGENTS

ABSTRACT

G. procumbens is found in most of the Southeast Asian countries and the leaves are used as a folk medicine to treat various illnesses including hyperlipidemia. The objectives of the present study include isolation of marker compound(s), standardization and phytochemical analysis of G. procumbens plant samples and investigation of their antioxidant and antihyperlipidemic effects in acute and chronic rat models, determination of their mechanisms of action and toxicological profiles. The leaves of G. procumbens were macerated with 95 % ethanol and fractionated into three fractions: F1

(water), F2 (50 % MeOH) and F3 (acetone). Further purification of F2 yielded a phenolic acid, chlorogenic acid (CA). Antioxidant analyses revealed that, F2 possessed highest phenolics content and strong antioxidant activities. Likewise, CA exhibited potent antioxidant activities that were comparable to reference standards. The G. procumbens plant samples, extract and fractions were standardized using phenolic acids

(caffeoylquinic acids: CA, 3,4DC, 3,5DC and 4,5DC) as marker compounds by a validated RP-HPLC method. Phytochemical analysis revealed that, F2 was enriched with caffeoylquinic acids compared to other fractions. Acute antihyperlipidemic study of standardized extract, fractions and CA of G. procumbens indicated that S(F2) and CA had most potent antihyperlipidemic activity on lowering TC and TG levels in p-407 (500 mg/kg; i.p.) induced acute hyperlipidemic rats. In addition, S(F2) also significantly reduced levels of LDL-C, VLDL-C, AI and CRI after 58 h treatment followed by CA.

xxviii

Hence S(F2) was chosen for further investigations in chronic hyperlipidemic model.

Five weeks of treatment with various doses of S(F2) resulted in dose dependent antihyperlipidemic effects, whereby the most potent effect was exerted by 500 mg/kg of

S(F2). The fraction significantly decreased TC, TG, LDL-C, VLDL-C, CRI and AI levels while increased HDL-C level of hyperlipidemic rats. S(F2) and CA showed inhibitory effect on HMG-CoA reductase enzyme while, only CA had moderate inhibitory activity on pancreatic lipase enzyme. In addition, S(F2) (500 mg/kg) dose dependently reduced liver lipids synthesis (TC and TG) and increased the excretion of lipids (TC) and bile acids via feces. CA showed mild cytotoxic effects against MCF-7 and Hela, while it was safe on HCT-116 and Eahy cells. In contrast, S(F2) exhibited no cytotoxic effects on all cancer and normal cell lines. Acute toxicity on S(F2) indicated that the fraction was safe and LD50 was greater than 5000 mg/kg. In conclusion, S(F2) bioactive fraction is a potential lipid-lowering agent which may prevent hyperlipidemia and cardiovascular diseases by lowering serum lipid levels.

xxix

CHAPTER 1

INTRODUCTION

1.1 Background

Around the world, non-communicable diseases such as cardiovascular diseases, cancer, diabetes and chronic respiratory diseases kill 40 million people each year (WHO, 2017).

Health data gathered from countries around the world revealed that cardiovascular diseases remain the leading cause of death with ≈17.7 million deaths annually (WHO,

2017). In Malaysia, 73 % of total deaths are contributed by non-communicable diseases with cardiovascular diseases being the main contributor (NHMS, 2015). There are a number of risk factors associated with the development of cardiovascular diseases such as hyperlipidemia, obesity, hypertension and diabetes which are caused by unhealthy lifestyle factors which include smoking, unhealthy diet and lack of physical exercises

(Buttar et al., 2005). Among the risk factors, hyperlipidemia plays a significant role in inducing atherosclerosis which leads to heart attack, stroke, angina and various types of heart and blood vessel disorders, collectively belong to cardiovascular diseases (WHO,

2017; Kaur et al., 2013).

Hyperlipidemia is defined as elevated levels of blood total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and declining of high- density lipoprotein-cholesterol (HDL-C). WHO (2017) revealed that hyperlipidemia associated diseases cause approximately 2.6 million deaths annually worldwide and the prevalence is the highest in Europe with 54 % for both sexes. While in Malaysia, NHMS

(2015) revealed that 47.7 % of total population aged above 18 is suffering from this condition, which has doubled since 2006 (20.7 %). The incidence of hyperlipidemia is known to be one of the main contributors for cardiovascular diseases in Malaysia

(NHMS, 2015). Despite the availability of various antihyperlipidemic drugs (statins, niacin, fibrates, bile acid sequestrants) in the market, many still opt for herbal medicines as an alternative. The demand and popularity for herbal medicines in both developed and developing countries are due to the presumed minor side effects, good efficacy in management of human diseases and their availability at affordable prices (Gunjan et al.,

2015).

Herbs or medicinal plants usually have broad pharmacological activities mainly due to their bioactive phytochemicals such as the flavonoids, alkaloids, terpenoids, glycosides and lignans. In addition to their medicinal properties, medicinal plants are also well known for their antioxidant potential to fight against reactive oxygen species

(ROS) which is the main contributor to the development of many diseases (Chodak et al., 2011). Of interest, a particular attention has been focused on phenolic acids especially mono- and di-caffeoylquinic acids due to their extended range of pharmacological activities besides their known antioxidant and radical scavenging activities (Križman et al., 2007).

Numerous studies on various plants have revealed that consumption of polyphenols/phenolic acids rich plants may reduce the risk of cardiovascular diseases caused by hyperlipidemia via lipid lowering mechanism (Križman et al., 2007; Inbaraj et al., 2010). Moreover, literature have also confirmed that medicinal plants having both

2 antihyperlipidemic and antioxidant properties are better in managing disorders associated with hyperlipidemia which are usually accompanied by increased oxidative stress (Patil et al., 2014). Studies also have revealed that caffeic acid derivatives such as

1,3-dicaffeoylquinic acid (1,3DC), 1,5-dicaffeoylquinic acid (1,5DC), 3,4- dicaffeoylquinic acid (3,4DC), 3,5-dicaffeoylquinic acid (3,5DC) and 4,5- dicaffeoylquinic acid (4,5DC) possessed better antioxidant potential and displayed protective role against cardiovascular diseases nearly tenfold higher than normal mono- caffeoylquinic acids (Chen et al., 2013; Wang et al., 2009; Inbaraj et al., 2010).

Considering all the factors mentioned above, it is rationale to investigate a plant that has been in existence in the traditional medicinal system for a long time and is used for management of various human diseases.

Gynura procumbens (Lour.) Merr from Asteraceae family is locally known as

‘Sambung nyawa’ or ‘Bai bing cha’ by the Malay and Chinese communities respectively

(Shwter et al., 2014). This decumbent perennial herbaceous shrub is abundantly distributed in South East Asian countries such as Malaysia, Thailand, Indonesia,

Philippines, China and Borneo (Kaewseejan & Siriamornpun, 2015). Traditionally, the leaves of this plant are used to treat ailments such as fever, migraine, inflammation, kidney problems, rheumatism, diabetes, high blood pressure, rashes and constipation

(Kaewseejan & Siriamornpun, 2015; Kaewseejan et al., 2015; Wu et al., 2011). The plant is non-toxic and the leaves are still being used as food source by the natives where it is generally consumed raw as salad. Several studies have reported that the leaves of G. procumbens possessed a vast range of pharmacological activities such as antioxidant, antifungal, antiherpes simplex virus, antihyperglycemic, antibacterial, anti-

3 inflammatory, antiulcer, anticancer and antihypertensive (Wu et al., 2011; Abrika et al.,

2013; Iskander et al., 2002; Kaewseejan et al., 2012; Mahmood et al., 2010; Mustafa et al., 2010; Rahman & Asad, 2013). Bioactive chemical constituents isolated from

G. procumbens leaves include flavonoids, phenolic acids (gallic acid, p-coumaric and ferulic acids), tannins, terpenoids, saponins, alkaloids, coumarins, anthocyanins and stigmasterol (Kaewseejan & Siriamornpun, 2015; Rahman & Asad, 2013).

1.2 Problem statement

Previous studies reported that 95 % ethanolic extract (ethanolic extract) of

G. procumbens leaves contained high contents of phenolics and flavonoids (Kaewseejan

& Siriamornpun, 2015). However, not many studies were carried out to determine the classes/individual compounds or fractions of G. procumbens extracts that are responsible for its pharmacological activities. Zhang and Tan (2000), first reported on the lipid lowering effect of ethanolic extract of G. procumbens in streptozotocin-induced diabetic rats. Preliminary findings by Meng (2011) and Saeed (2013) revealed that the ethanolic extract of G. procumbens had promising antihyperlipidemic effect by reducing blood lipid levels of the acute and chronic hyperlipidemic rat models. However, to date, a question still remains on which class(es) of chemical constituent(s) or compound(s) is/are responsible for the antihyperlipidemic activity observed by the earlier researchers.

Meng (2011) has also reported on the isolation of two major phenolic acids from the ethanolic extract of G. procumbens, namely chlorogenic acid (CA) and 3,5DC. This has created an interest to work on the bioactivity of the fractions of ethanolic extract of G. procumbens to evaluate their antihyperlipidemic effects and further investigate the possible mechanisms of action and toxicity.

1.3 Hypothesis

Phenolic acid rich fraction is expected to be fractionated from the ethanolic extract of G. procumbens using resin column technology. Phenolic acid(s) will then be isolated from the bioactive fraction and the isolated compound(s) will be used as marker compounds to standardize G. procumbens plant samples. The phenolic acid rich bioactive fraction is expected to possess potent antioxidant and antihyperlipidemic activities. Hence, the fraction and the isolated compound(s) should be able to cure hyperlipidemia via multiple mechanisms such as inhibition of lipid related enzymes, increased secretion of fecal bile acids and excretion of lipids. The bioactive fraction and isolated compound(s) are also expected to be safe for ingestion.

1.4 Research objectives

The main objective of the present study is to evaluate antihyperlipidemic activity of standardized phenolic acid rich fraction of G. procumbens using animal models and to investigate the mechanisms of its antihyperlipidemic action.

The sub-objectives of present study include:

1. To fractionate the ethanolic extract of G. procumbens using resin column

technology.

2. To isolate and characterize phenolic acid(s) from ethanolic extract of G.

procumbens.

3. To evaluate antioxidant activities of ethanolic extract of G. procumbens and its

fractions and phenolic acid(s).

4. To standardize ethanolic extract of G. procumbens and its fractions using

phenolic acid(s) isolated as marker compounds.

5. To evaluate antihyperlipidemic activities of standardized ethanolic extract of G.

procumbens and its fractions and phenolic acid(s) in chemically-induced

hyperlipidemic rats.

6. To evaluate antihyperlipidemic activity of standardized bioactive fraction of G.

procumbens in high fat diet (HFD)-induced hyperlipidemic rats.

7. To investigate the mechanisms of antihyperlipidemic activity of standardized

bioactive fraction of G. procumbens and its phenolic acid(s) on:

a) inhibition of selected enzymes involved in lipids biosynthesis and

metabolism

b) removal of excess lipids through bile acids and feces

8. To investigate the potential cytotoxicity and acute toxicity of the standardized

bioactive fraction of G. procumbens and its phenolic acid(s).

The research flow is summarized in Figure 1.1.

Ethanolic extract

Fractionation & isolation of marker compound(s)

Antioxidant study Phytochemical analysis Antihyperlipidemic study Toxicological study

In vitro HPLC P-407-induced HFD-induced Mechanisms of Cytotoxicity Acute study

antioxidant for standardization acute chronic anti- study for for extract, fractions using phenolic hyperlipidemic hyperlipidemic hyperlipidemic standardized standardized & phenolic acid(s) acid(s) & model for model for activity bioactive bioactive standards standardized standardized fraction & fraction extract, bioactive phenolic fractions & fraction acid(s) TPC Quantification of phenolic TFC G. procumbens acid(s) FRAP samples from 5 Enzymes MCF-7 Effects on DPPH different inhibitory Hela lipids & bile ABTS locations activities of HCT-116 acids standardized Eahy excretion bioactive fraction & HMG-CoA phenolic reductase & acid(s) Figure 1.1: Flow chart of the study pancreatic lipase

CHAPTER 2

LITERATURE REVIEW

2.1 The genus Gynura

The genus Gynura was first described by Cassini in 1825 and so far forty four species have been identified globally (Vanijajiva & Kadereit, 2011). Gynura is a perennial herbaceous shrub that belongs to Asteraceae-Senecioneae family which is widely distributed in tropical countries such as Africa, Southeast Asia, Southern Japan,

Southern China, Northern Australia and New Guinea (Kaewseejan & Siriamornpun,

2015). About hundreds of research papers have been published on the phytochemical and pharmacological studies of the genus, Gynura. Among the species that have been studied are G. angulosa, G. aurantiaca, G. bicolor, G. calciphila, G. crepidioides, G. cusimbua, G. divaricata, G. elliptica, G. formosana, G. japonica, G. medica, G. nepalensis, G. pseudochina, G. scandens and G. segetum (Vanijajiva & Kadereit, 2011).

2.2 Gynura procumbens (Lour.) Merr.

2.2.1 Description of Gynura procumbens (Lour.) Merr.

G. procumbens (Figure 2.1) is an evergreen plant belonging to the Astereceae

(Compositae) family indigenous to Southeast Asia especially Malaysia, Indonesia and

Thailand (Bhore et al., 2010). In Malaysia, this plant species can be found growing wild or cultivated and has limited distribution at the western part of peninsular Malaysia

(Keng et al., 2009). The plant has several scientific synonyms such as Cacalia

procumbens Lour., Calacia procumbens Lour., G. sarmentosa DC. and Cacalia sarmentosa Blume (Mustaffa et al., 2011). Taxonomic classification of G. procumbens is shown in Table 2.1.

Figure 2.1: Gynura procumbens plant

Table 2.1: Taxonomic classification of Gynura procumbens (Lour.) Merr.

Domain Eukaryota Kingdom Plantae Subkingdom Viridiplantae Class Spermatophyta Order Asterales Family Asteraceae Subfamily Asteroideae Genus Gynura Species procumbens Scientific name Gynura procumbens

G. procumbens is commonly known as sambung nyawa (prolongation of life), daun dewa, akar sebiak or kecam akar by the Malays while ‘bai bing cha’ (100 ailments) by the Chinese in Malaysia (Tan et al., 2016). The reason why the plant is called sambung nyawa and ‘bai bing cha’ is due to the vast range of uses in traditional medicine. Preparations of G. procumbens have been utilized traditionally for the treatment of skin conditions, kidney problems, rheumatism, hypertension, diabetes, viral infection, ringworm infection, constipation, inflammation, eruptive fever, rashes, migraine and cancer (Bhore et al., 2010; Mustaffa et al., 2011; Tan et al., 2016).

Even the leaves of this plant have been used extensively in different ways as a part of food in different cultures. In Malaysia, the fresh leaves are edible as salad known as ‘ulam’, eaten with rice and used as flavoring for food. Meanwhile in Thailand, the leaves are either eaten raw or boiled to be used as a garnish in food preparations such as curries, salads, soups and entrees. Scientific investigations on the leaves of this plant have shown that it’s safe for consumption (Rosidah et al., 2009).

G. procumbens is a fast growing decumbent shrub that can grow up to 10-25 cm tall (Keng et al., 2009). The plant is highly branched with hairy leaves arranged alternately on hairy stem (Rahman & Asad, 2013; Saiman et al., 2012; Keng et al., 2009;

Mustaffa et al., 2011). The leaves of this species normally are green in color, succulent, hairy on both surfaces, differ in shape either ovate, elliptic or lanceolate, 3.5-8.0 cm long, 0.8–3.5 cm wide, with cuneate or rounded base and an acute or obtuse attenuated apex (Rahman & Asad, 2013; Li et al., 2015; Keng et al., 2009; Mustaffa et al., 2011).

The stem of the plant is fleshy and has purple tint. The flowering heads are 1–1.5 cm

long, yellow in color, narrow and panicled (Rahman & Asad, 2013). The plant produces purple flowers that are tubular and bisexual (Sunarwidhi et al., 2014; Keng et al., 2009).

2.2.2 Chemical constituents of Gynura procumbens (Lour.) Merr.

In 1996, Sadikun and the co-workers isolated a mixture of sterol and sterol glycosides from the leaves of G. procumbens. Flash column chromatography of petroleum ether extract, followed by further purification using preparative thin layer chromatography

(TLC) yielded mixture of β-sitosterol [1] and stigmasterol [2]. In addition, the chloroform extract subjected to silica gel column chromatography afforded two more compounds 3-O-β-D-glucopyranosyl β-sitosterol [3] and 3-O-β-D-glucopyranosyl stigmasterol [4]. The group also isolated a nucleic acid, adenosine [5] and two flavanol glycosides, kaempferol-3-O-α-L-rhamnosyl-(1→2)-O-β-D-glucopyranoside [6] and kaempferol-3-O-β-D-glucopyranoside [7] (Sadikun et al., 1996).

Two types of di-caffeoylquinic acids, 3,5DC [8] and 4,5DC [9] have been identified by Jiratchariyakul et al. (2000) from G. procumbens which was found to inhibit the replication of herpes causing viruses. Later, Akowuah et al. (2002) isolated four compounds from the n-butanol fraction of petroleum ether extract of G. procumbens leaves, namely quercetin-3-O-rhamnosyl (1→6) glucoside [10], kaempferol-3-O-rhamnosyl (1→6) glucoside [11], kaempferol-3-O-glucoside [7] and quercetin-3-O-rhamnosyl (1→2) galactoside [12].

High-performance TLC (HPTLC) analysis of G. procumbens methanolic extract and its fractions resulted in identification of two phenolics namely, kaempferol-3-O-

rutinoside [11] and astragalin [7] (Rosidah et al., 2008). Nine types of phenolic acids were identified using reverse phase-high-performance liquid chromatography (RP-

HPLC) analysis in the ethanolic extract, ethyl acetate fraction and its sub-fractions fractionated using Sephadex LH-20 column chromatography. The phenolic acids identified were, hydroxybenzoic acids: p-hydroxybenzoic acid [13], gallic acid [14], protocatechuic acid [15], vanillic acid [16], syringic acid [17] and hydroxycinnamic acids: caffeic acid [18], p-coumaric acid [19], ferulic acid [20] and sinapic acid [21]

(Kaewseejan et al., 2015).

The aerial parts of G. procumbens were extracted according to polarity using petroleum ether, dichloromethane and ethanol. The ethanolic extract which showed virucidal and antireplicative actions against herpes simplex virus-1 (HSV-1) and HSV-2 upon further purification afforded kaempferyl-3-O-훼-L-rhamnosyl(1→6)-훽-D- glucopyranoside [11], 3,5DC [8], kaempferyl glucopyranoside [7], 4,5DC [9], kaempferyl-3-O-훼-L-rhamnosyl(1→6)-훽-D-galactopyranoside [22], CA [23], quercetin-

3-O-훽-D-glucopyranoside [24], kaempferol [25], 훽-sitosterol [1], stigmasterol [2], 훽- sitosteryl glucoside [3], stigmasteryl glucoside [4] and 1,2-bis-dodecanoyl-3-훼-D- glucopyranosyl-sn-glycerol [26] (Jarikasem et al., 2013).

Phytochemical analysis of the antihyperglycemic active fraction using HPLC revealed the presence of kaempferol-3,7-di-O-β-D-glucoside [27], which was shown to be responsible for antihyperglycemic activity of ethanolic extract of G. procumbens

(June et al., 2012). Zhang and co-workers investigated methanol (MeOH) extract of the

leaves of G. procumbens which afforded a new sesquiterpenoid, muurol-4-ene-1β, 3β,

10β-triol [28], two sesquiterpene glycosides, muurol-4-ene-1β,3β,10β-triol 3-O-β-D- glucopyranoside [29] and muurol-4-ene-1β,3β,15-triol 3-O-β-D-glucopyranoside [30] and three known sesquiterpenoids, schensianol A [31], negunfurol [32], and 4 β,10α- aromadendranediol [33] (Zhang et al., 2014b).

[1] β-sitosterol [2] Stigmasterol

[3] 3-O-β-D-glucopyranosyl β-sitosterol

[4] 3-O-β-D-glucopyranosyl stigmasterol [5] Adenosine

[6] Kaempferol-3-O-α-L-rhamnosyl-(1→2)-O-β-D-glucopyranoside

[7] Kaempferol-3-O-β-D-glucopyranoside

[8] 3,5DC

[9] 4,5DC

[10] Quercetin-3-O-rhamnosyl (1→6) glucoside [11] Kaempferol-3-O-rhamnosyl (1→6) glucoside

[1 2] Quercetin-3-O-rhamnosyl (1→2) galactoside

[1 3] p-hydroxybenzoic acid [14] Gallic acid

[15] Protocatechuic acid [16] Vanillic acid

[17] Syringic acid [18] Caffeic acid [19] p-coumaric acid

[20] Ferulic acid [21] Sinapic acid

[23] CA

[22] K aempferyl-3-O-훼-L-rhamnosyl(1→6)-훽-D-galactopyranoside

[25] Kaempferol

[24] Quercetin-3-O-훽-D-glucopyranoside

[26] 1,2-bis-dodecanoyl-3-훼-D-glucopyranosyl-sn-glycerol

[27] Kaempferol-3,7-di-O-β-D-glucoside

[28] Muurol-4-ene-1β, 3β, 10β-triol

[29] Muurol-4-ene-1β,3β,10β-triol 3-O-β-D-glucopyranoside

[30] Muurol-4-ene-1β,3β,15-triol 3-O-β-D-glucopyranoside

[31] Schensianol A

[32] Negunfurol

[33] 4β,10α-aromadendranediol

2.2.3 Pharmacological activities of Gynura procumbens (Lour.) Merr.

2.2.3(a) Wound healing activity

Zahra et al. (2011) reported that ethanolic extract of G. procumbens leaves significantly healed the wound (2 cm in diameter) created on the dorsal neck of rats. After 14 days, wounds treated by ethanol extract and intrasite gel showed signs of wound healing with less scars and healed earlier than those treated with vehicle (gum acacia) (Zahra et al.,

2011).

2.2.3(b) Anticancer activity

In 2012, Nisa and the team conducted anticancer study of G. procumbens ethanolic extract in rats model of liver cancer induced using 7,12-dimethylbenz(a) antracene.

Histopathology results revealed that G. procumbens ethanolic extract (300 mg/kg) significantly decreased proliferation of liver cells in cancer induced rats compared to untreated group, thus G. procumbens can be used as chemopreventive agent to inhibit carcinogenesis (Nisa et al., 2012).

2.2.3(c) Antiulcerogenic activity

G. procumbens ethanolic leaf extract was investigated for its antiulcerogenic activity at dose range of 50-400 mg/kg in absolute ethanol induced gastric lesions in rats. The animals treated with plant extracts exhibited significant ulcer protection evidenced by reduction in ulcer area and edema compared to untreated ulcer control group (Mahmood et al., 2010).

2.2.3(d) Cardiovascular activity

Kaur et al. (2012) reported cardiovascular activity of ethanolic (95 %, 75 %, 50 %, 25

%, v/v) and aqueous extracts of G. procumbens using rat aorta rings procedure. Aqueous extract was found to exhibit effective dose dependent vasorelaxation and negative chronotropic and ionotropic effects. Data suggested that cardiovascular activity possessed by aqueous extract can possibly be attributed to the high content of polyphenolic compounds (Kaur et al., 2012).

2.2.3(e) Ultraviolet (UV) protective activity

Protective activity of G. procumbens on photoaging skin caused by UV radiation has been evaluated by Kim and the team in year 2011 using human dermal fibroblasts. The result showed that 20 μg/mL of ethanolic extract of G. procumbens inhibited matrix metalloproteinase-1 (MMP-1) and MMP-9 expressions up to 70 % and 73 %, respectively. The extract effectively reduced ROS production and showed marked inhibitory effect on pro-inflammatory cytokine mediators, interleukin-6 (IL-6) and IL-8 in human HaCat keratinocyte (Kim et al., 2011).

2.2.3(f) Immunomodulatory activity

Effects of G. procumbens leaves ethanolic extract on immunocompetent T cells (CD4+

T cells, CD4+CD25+ T cells, and B220+ cells) were investigated by Dwijayanti and

Rifa'I to study its immunomodulatory activity in splenic cells. The result indicated that

G. procumbens ethanolic extract increased the production of T cells compared to control. The study concluded that G. procumbens possessed immunomodulatory activity

and only a very minimal dose (0.1 μg/mL and 1.0 μg/mL) was required to promote T cell activation (Dwijayanti & Rifa'i, 2015).

2.2.3(g) Antihypertensive activity

Water extract (300 and 600 mg/kg) of G. procumbens, orally fed via gastric gavage to spontaneously hypertensive rats for four weeks showed significant lowering of mean atrial pressure and heart rate in dose dependent manner. Significant increase in urine flow rate was also observed in treated spontaneously hypertensive rats, hence removed excess sodium and water. G. procumbens water extract also showed antihypertensive activity by inhibiting pressor responses induced by acetylcholine, phenylephrine, methoxamine, angiotensin II, and isoprenaline. Overall, the extract was able to lower blood pressure through non selective pathway by stimulating vasodilation, heart stabilization and diuretic effect (Kaur et al., 2013).

2.2.3(h) Anti-inflammatory activity

Water and ethyl acetate fractions of the ethanolic extract of G. procumbens were subjected to anti-inflammatory evaluation against ear inflammation induced by croton oil. The water fraction did not show any anti-inflammatory effect, while ethyl acetate fraction showed good anti-inflammatory activity by inhibiting increase in ear thickness due to inflammation. Among its sub-fractions, hexane (inhibition 44.6 %) and toluene

(inhibition 34.8 %) sub-fractions showed comparable activity to reference drug, hydrocortisone (inhibition 35.0 %) (Iskander et al., 2002).

2.2.3(i) Antiherpetic activity

Jarikasem and team worked on antiherpetic effects of G. procumbens against HSV-1 and

HSV-2. The ethanolic extract of the plant showed virucidal and antireplicative activity thus it was further fractionated to afford four fractions: F1 (water), F2 (50 % MeOH), F3

(MeOH) and F4 (ethyl acetate). All fractions except F1 showed antiherpetic activity against HSV-1 and HSV-2. The active fractions contained a mixture of di-caffeoylquinic acids, mixture of 훽-sitosterol and stigmasterol, mixture of 훽-sitosteryl and stigmasteryl glucosides and 1,2-bis-dodecanoyl-3-훼-D-glucopyranosyl-sn-glycerol, all showed potent antiherpatic activity against HSV-1 and HSV-2 (Jarikasem et al., 2013).

2.2.3(j) Antidiabetic activity

The leaves of G. procumbens were subjected to sequential extraction in aqueous ethanol of various percentages (95 %, 75 %, 50 %, 25 %) and tested for antidiabetic activity in streptozotocin-induced diabetic rats. In acute study, the extracts lowered fasting blood glucose level significantly in diabetic rats; however, the lowering effect was the greatest in 25 % ethanolic extract. Furthermore, in sub-chronic study, the 25 % ethanolic extract exerted highest fasting blood glucose lowering effect by 49.38 % and 65.43 % on day 7 and 14 respectively, similar to effect of metformin (7th day: 46.26 % and 14th day: 65.42

%). In addition, 25 % ethanolic and aqueous extracts suppressed peak fasting blood glucose in subcutaneous glucose tolerance test for 90 min, which was comparable to the effect exerted by metformin (Algariri et al., 2013).