QUALITY CONTROL ANALYSIS, STANDARDIZATION AND STABILITY STUDIES OF LONGIFOLIA AND EFFECT OF ITS SAPONIN CONTENT ON SOLUBILITY AND TOXICITY OF EURYCOMANONE

NURSYAZURA BINTI KHARI

UNIVERSITI SAINS MALAYSIA 2014

QUALITY CONTROL ANALYSIS, STANDARDIZATION AND STABILITY STUDIES OF AND EFFECT OF ITS SAPONIN CONTENT ON SOLUBILITY AND TOXICITY OF EURYCOMANONE

By

NURSYAZURA BINTI KHARI

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

AUGUST 2014 1 ACKNOWLEDGEMENTS

Alhamdulillah, all praises to Allah the almighty who gave me the knowledge, strength and the determination to complete this study and finish my thesis successfully. I would like to express my sincere gratitude to my supervisor, Prof.

Zhari Ismail for his support, guidance, encouragement and supervision throughout my research. This study would not have been completed without his help. I would like to convey my sincere words of appreciation to my colleague Dr. Abdalrahim

F.A. Aisha for his invaluable assistance, support and guidance which made the work possible. Special thanks to Dr. Amin Malik Shah Bin Abdul Majid, for allowed me to work in EMAN lab and to all the students at the EMAN lab for their support and assistance, particularly Miss. Suzana Hashim helped in MTT assay. My sincere thanks to both Ms. Nor Dyana Binti Zakaria from INFORMM, USM and Mr. Masrul

Mansor from School of Biological Sciences, USM whom has helped me in performing particle size and Transmission Electron Microscope (TEM) analysis. I would like to thank Universiti Sains Malaysia for giving me the opportunity and providing me with all the necessary facilities that made my study possible and to the

Malaysian Ministry of Higher Education for awarding me the Budget Mini scholarship for two years, which has supported me to pay the fees. Special thanks to my colleagues and my entire friend at Pharmaceutical Chemistry research laboratory especially Dr. Beh Hooi Kheng, Nurul Najwa, Noor Hafizoh, Gheniya, Munira,

Shahrul and Amirul for their encouragement and support. Last but not least, I wish to express my deepest love and gratitude to my parents, Khari Bin Yaakob and Sabehah

Binti Shuib, my sister Nurfarina Binti Khari and beloved husband Mohd Baijuri Bin

Azmi for their support, understanding and endless love, through the duration of my studies. ii

2 TABLE OF CONTENTS

1 ACKNOWLEDGEMENTS ...... ii

2 TABLE OF CONTENTS ...... iii

3 LIST OF TABLES...... viii

4 LIST OF FIGURES...... x

5 LIST OF ABBREVIATION...... xvi

6 ABSTRAK...... xix

7 ABSTRACT ...... xix

1 CHAPTER ‎1 INTRODUCTION ...... 1 1.1 Significance of Medicinal ...... 1 1.2 Efficacy, Safety and Quality Control in the Standardization of Herbal Medicine ...... 2 1.3 Problem Statements and Justification of the Research ...... 5 1.4 Objectives of the Study ...... 6

2 CHAPTER ‎2 LITERATURE REVIEW ...... 7 2.1 Eurycoma longifolia Jack ...... 7 2.1.1 ...... 7 2.1.2 Vernacular Names ...... 7 2.1.3 Description ...... 8 2.1.4 Review of Chemical Constituents of Eurycoma longifolia ...... 10 2.1.5 Review of the Pharmacological Activities of Eurycoma longifolia...... 14 2.2 Stability Studies ...... 18 2.2.1 Physical Factors Effecting Stability ...... 19 2.2.1.1 Temperature ...... 19 2.2.1.2 Moisture ...... 19 2.2.1.3 Light...... 19 2.2.2 Chemicals Factors Effecting Stability ...... 20

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2.2.2.1 Hydrolysis ...... 20 2.2.2.2 Oxidation ...... 20 2.2.2.3 Isomerisation and Polymerization ...... 20 2.2.3 Accelerated Stability ...... 21 2.3 Saponins ...... 22 2.3.1 Definition and Chemical Compositions ...... 22 2.3.2 A Review of the Pharmacological Activities of Saponins ...... 23

3 CHAPTER ‎3 MATERIALS AND METHODS ...... 28 3.1 Quality Control Analysis of Eurycoma longifolia Raw Material ...... 28 3.1.1 Preparation of Plant Materials ...... 28 3.1.2 Moisture and Ash Content ...... 29 3.1.3 Acid Insoluble Ash Content ...... 29 3.1.4 Alcohol Soluble Extractives ...... 29 3.1.5 Water Soluble Extractives ...... 30 3.1.6 Determination of Heavy Metals ...... 31 3.1.7 Optimization of Extraction Time and Solvent Ratio ...... 31 3.1.7.1 Plant Material and Small Scale Extraction ...... 31 3.1.7.2 Chemical Profiling of Extracts from Different Extraction Time and Solvent Ratio ...... 32 3.2 Analytical Studies of Eurycoma longifolia Extracts and Commercial Products Using Spectroscopic and Chromatographic Techniques ...... 33 3.2.1 Material and Methods...... 33 3.2.1.1 Chemicals and Solvents...... 33 3.2.1.2 Instrumentations...... 33 3.2.1.3 Marker...... 34 3.2.2 Preparation of Extracts from Raw Materials...... 34 3.2.3 Preparation of Commercial Products...... 35 3.2.4 Fourier Transform Infrared (FTIR) Spectroscopy and PCA ...... 36 3.2.5 High Performance Thin-Layer Chromatography (HPTLC)...... 36 3.2.6 High Performance Liquid Chromatography (HPLC) ...... 37 3.2.6.1 Preparation of Marker Compound ...... 37 3.2.6.2 Preparation of Samples ...... 38 3.2.6.3 Optimization and Validation of HPLC Method ...... 38

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3.2.6.4 Quantification of Eurycomanone in Eurycoma longifolia Extracts and Commercial Products ...... 40 3.2.7 Analysis of Primary and Secondary Metabolites in Eurycoma longifolia Extracts and Commercial Products...... 40 3.2.7.1 Material and Methods ...... 40 3.2.7.2 Instruments...... 40 3.2.7.3 Determination of Total Phenols ...... 41 3.2.7.4 Determination of Glycosaponins ...... 42 3.2.7.5 Determination of Total Polysaccharides ...... 42 3.2.7.6 Determination of Total Protein Using Lowry’s Method ...... 43 3.3 Stability Studies on Eurycoma longifolia Root, Stem and Water Extracts ...... 44 3.3.1 Stability Study Protocol ...... 44 3.3.2 Quantification of Eurycomanone by HPLC ...... 45 3.3.3 Calculation of Chemical Kinetics ...... 45 3.3.3.1 Determination of the Order of Degradation Reaction ...... 45 3.3.3.2 Determination of the Rate Constant (K) ...... 46 3.3.3.3 Determination of Activation Energy (Ea) and Pre- exponential Factor (A) ...... 46 3.3.3.4 Determination of Shelf Life of the Reaction ...... 47 3.4 Studies of Saponins on the Solubility and Cytotoxicity of Eurycomanone...... 48 3.4.1 Preparation of Saponins and Eurycomanone-rich Extracts ...... 48 3.4.2 Analysis of Saponins and Eurycomanone-rich Extracts...... 49 3.4.2.1 Identification Using HPLC Analysis ...... 49 3.4.2.2 Phytochemical Screening of Saponins ...... 49 3.4.3 Effect of Saponins on the Solubility of Eurycomanone-rich Extract 50 3.4.4 Determination of Critical Micellar Concentration (CMC) ...... 50 3.4.5 Measurement of Particle Size ...... 51 3.4.6 Transmission Electron Microscope (TEM) ...... 51 3.4.7 Effect of Saponins on the Cytotoxicity of Eurycomanone-rich Extract ...... 51 3.4.7.1 Cell Line and Culture Condition ...... 51

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3.4.7.2 Harvesting and Counting of Cells ...... 52 3.4.7.3 Cytotoxicity ...... 52 3.4.7.4 Preparation of Cells ...... 52 3.4.7.5 Treatment of Cells ...... 53 3.4.7.6 Viability Testing by MTT Test ...... 53 3.4.7.7 Analysis of Results ...... 54

4 CHAPTER ‎4 RESULTS AND DISCUSSION ...... 55 4.1 Quality Control Analysis of Eurycoma longifolia Raw Material...... 55 4.1.1 Moisture and Ash Content ...... 55 4.1.2 Acid Insoluble Ash Content ...... 58 4.1.3 Alcohol and Water Soluble Extractives...... 59 4.1.4 Heavy Metals Content of Eurycoma longifolia Raw Material ...... 62 4.1.5 Optimization of Extraction Time and Solvent Ratio ...... 64 4.1.5.1 Extraction Parameters and Percentage Yield ...... 64 4.1.5.2 Chemical Profiling of Extracts from Different Extraction Time and Solvent Ratio ...... 65 4.2 Chemical Profiling and Standardization of Eurycoma longifolia ...... 71 4.2.1 Determination of Percentage Yield ...... 71 4.2.2 ATR-FTIR Spectra Profiles of Eurycoma longifolia Extracts from Different Localities and Eurycoma longifolia Commercial Products from Different Brands ...... 72 4.2.3 Principal Compenant Analysis (PCA) ...... 79 4.2.4 HPTLC Profiles of Eurycoma longifolia Extracts from Different Localities and Eurycoma longifolia Commercial Products from Different Brands ...... 84 4.2.5 Optimization of HPLC Method for Quantitative Analysis of Eurycoma longifolia ...... 91 4.2.5.1 Validation of HPLC Method ...... 92 4.2.6 HPLC Analysis of Eurycoma longifolia Extracts from Different Localities and Eurycoma longifolia Commercial Products from Different Brands ...... 95 4.2.7 Analysis of Primary and Secondary Metabolites in Eurycoma Longifolia Extracts and Commercial Products ...... 103

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4.2.7.1 Determination of Total Phenolics ...... 103 4.2.7.2 Determination of Glycosaponins and Total Polysaccharides...... 105 4.2.7.3 Comparison of Total Proteins Contents Using Lowry's Method...... 109 4.3 Stability Studies of Eurycoma longifolia Extracts ...... 111 4.3.1 Percentage Remaining of the Marker Compound ...... 113 4.3.2 Determination of the Order of the Degradation Reaction ...... 116 4.3.3 Determination of the Rate Constant (K) ...... 119 4.3.4 Estimation of Activation Energy (Ea) and Pre-exponential Factor (A) ...... 123 4.3.5 Estimation of Shelf Life (t90) ...... 124 4.4 Studies of Saponins-rich Extract on Eurycomanone-rich Extract ...... 128 4.4.1 HPLC Profiles of Saponins and Eurycomanone-rich Extracts ...... 128 4.4.2 Determination and Identification of Saponins in Eurycoma longifolia Root...... 130 4.4.3 Effect of Saponins on the Solubility of Eurycomanone-rich Extract ...... 132 4.4.4 Critical Micellar Concentration ...... 135 4.4.5 Particle Size ...... 139 4.4.6 Transmission Electron Microscope (TEM) ...... 140 4.4.7 Effect of Saponins on the Cytotoxicity of Eurycomanone-rich Extract ...... 141

5 CHAPTER 5‎ CONCLUSION ...... 145 5.1 Conclusion ...... 145 5.2 Suggestions for Further Studies ...... 150

6 REFERENCES ...... 157

7 APPENDICES ...... 161

8 LIST OF PUBLICATIONS ...... 168

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LIST OF TABLES Pages

Table 2.1 Chemical constituents of Eurycoma longifolia 10

Table 3.1 List of locations, label and suppliers of Eurycoma 28 longifolia root, stem and leaf raw material

Table 3.2 Microwave digestion setting 31

Table 3.3 List of Eurycoma longifolia products 35

Table 3.4 The gradient mobile phase applied 37

Table 3.5 Storage conditions and relative humidity (RH) in the 45 stability studies

Table 3.6 Changes of colour for steroidal and triterpenoidal 49 saponins

Table 4.1 Heavy metal content of Eurycoma longifolia root, stem 63 and leaf raw material and their limit (WHO, 1998)

Table 4.2 Precision analysis of retention time and peak area in 93 terms of %RSD. Retention (RT) time and peak area (PA) are presented as average ±SD (n=5)

Table 4.3 LOD and LOQ value using HPLC method. Results are 94 displayed as average ±SD (n=5)

Table 4.4 Pre-exponential factor of eurycomanone in Eurycoma 124 longifolia root, stem and leaf water extracts

Table 4.5 Absorbance of Liebermann-Burchard reagent, reference 131 and mixture solution from 390 – 450 nm

Table 4.6 Fluorescence intensity of pyrene added to various 136 concentration of crude root water extract (TA)

Table 4.7 Fluorescence intensity of pyrene added to various 137 concentration of the saponins-rich extract

Table 4.8 Fluorescence intensity of pyrene added to various 138 concentration of the eurycomanone-rich extract

Table 4.9 Particle size of root water extract. The results are 139 presented as average ± SD (n=3)

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Table 4.10 Summary of the cytotoxic effect of eurycomanone (25 144 µg/mL), saponins and their combinations on HCT 116 cells. The table presents the percentage inhibition of individual treatments and their combinations, and the fold change in the cytotoxicity of combinations compared to their additive cytotoxicity. The results are displayed as average ± SD (n = 3)

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LIST OF FIGURES Pages

Figure 2.1 Pictures of Eurycoma longifolia (a) - (b) whole plant, (c) 9 and (d) root Figure 2.2 Chemical structure of Eurycomanone 14

Figure 2.3 Aglycone skeletons of (a) steroidal spirostane and (b) 23 triterpenoid saponins. R = sugar moiety

Figure 3.1 Flow chart of preparation of saponins and 48 eurycomanone-rich extracts from root water extract

Figure 4.1 Total moisture contents of Eurycoma longifolia root 58 stem and leaf raw material. Results are expressed as the mean ± SD (n=3)

Figure 4.2 Total ash contents of Eurycoma longifolia root, stem 57 and leaf raw material. Results are expressed as the mean ± SD (n=3)

Figure 4.3 Acid insoluble ash contents of Eurycoma longifolia root, 58 stem and leaf raw material. Results are expressed as the mean ± SD (n=3)

Figure 4.4 Extractive values of Eurycoma longifolia root raw 61 material from five different localities

Figure 4.5 Extractive values of Eurycoma longifolia stem raw 61 material from five different localities

Figure 4.6 Extractive values of Eurycoma longifolia leaf raw 61 material from five different localities

Figure 4.7 Parameters and percentage yield for different extraction 64 method of Eurycoma longifolia root from Perak

Figure 4.8 HPTLC fingerprints of Eurycoma longifolia root water 66 extracts (Perak) from different extraction time and solvent ratio at 254 and 366 nm; in image (a) Eurycomanone, (b) 4 h ratio 1:10, (c) 6 h ratio 1:10, (d) 8 h ratio 1:10, (e) 4 h ratio 1:15, (f) 4 h ratio 1:20

Figure 4.9 UV-Vis spectrum of eurycomanone standard, λmax was 67 between 248 and 255 nm

Figure 4.10 2D HPTLC densitogram of the eurycomanone standard 67 and Eurycoma longifolia root water extracts from different extraction time and solvent ratio at 254 nm

x

Figure 4.11 HPTLC chromatogram of eurycomanone standard and 68 Eurycoma longifolia root water extracts from different extraction time and solvent ratio obtained at 254 nm; in image (a) Eurycomanone, (b) 4 h ratio 1:10, (c) 6 h ratio 1:10, (d) 8 h ratio 1:10, (e) 4 h ratio 1:15, (f) 4 h ratio 1:20

Figure 4.12 Concentration of eurycomanone in Eurycoma longifolia 70 root water extracts from different extraction time and solvent ratio

Figure 4.13 Percentage yield of water extracts of different parts of 71 Eurycoma longifolia from five different localities

Figure 4.14 ATR-FTIR profile of the Eurycoma longifolia root 74 water extracts from five different localities

Figure 4.15 ATR-FTIR profile of the Eurycoma longifolia stem 75 water extracts from five different localities

Figure 4.16 ATR-FT-IR profile of the Eurycoma longifolia leaf 76 water extracts from five different localities

Figure 4.17 ATR-FT-IR profile of the Eurycoma longifolia 77 commercial products form six different brands

Figure 4.18 Score plot (A) of the two principal components analysis 81 of ATR-FTIR spectra of Eurycoma longifolia root water extracts from five different localities at 4000-650 cm-1

Figure 4.19 Score plot (A) of the two principal components analysis 82 of ATR-FTIR spectra of Eurycoma longifolia stem water extracts from five different localities at 4000-650 cm-1 Figure 4.20 Score plot (A) of the two principal components analysis 82 of ATR-FTIR spectra of Eurycoma longifolia leaf water extracts from five different localities at 4000-650 cm-1

Figure 4.21 Score plot (A) of the two principal components analysis 83 of ATR-FTIR spectra of Eurycoma longifolia commercial products from six different brands at 4000- 650 cm-1

Figure 4.22 Score plot (A) of the two principal components analysis 83 of ATR-FTIR spectra of Eurycoma longifolia root water extracts from five different localities and commercial products from six different brands at 4000-650 cm-1

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Figure 4.23 HPTLC profile of Eurycoma longifolia root, stem and 85 leaf water extracts from five different localities observed under UV 254 nm

Figure 4.24 HPTLC profile of Eurycoma longifolia root, stem and 85 leaf water extracts from five different localities observed under UV 366 nm

Figure 4.25 3D densitograms of eurycomanone and Eurycoma 86 longifolia root, stem and leaf water extracts from five different localities at 254 nm

Figure 4.26 HPTLC profile of eurycomanone and Eurycoma 88 longifolia commercial products from six different brands observed under UV 254 nm

Figure 4.27 HPTLC profile of eurycomanone and Eurycoma 88 longifolia commercial products from six different brands observed under UV 366 nm

Figure 4.28 3D densitograms of HPTLC of the eurycomanone and 89 Eurycoma longifolia commercial products from six different brands at 254 nm

Figure 4.29 Chromatogram of eurycomanone and Eurycoma 90 longifolia commercial products from six different brands obtained by HPTLC method at 254 nm

Figures 4.30 HPLC chromatogram of eurycomanone and Eurycoma 98 longifolia root water extracts from five different localities

Figures 4.31 HPLC chromatogram of eurycomanone and Eurycoma 99 longifolia stem water extracts from five different localities

Figures 4.32 HPLC chromatogram of eurycomanone and Eurycoma 100 longifolia leaf water extracts from five different localities

Figure 4.33 HPLC chromatogram of eurycomanone and Eurycoma 101 longifolia commercial products from six different brands

Figure 4.34 Concentration of eurycomanone in Eurycoma longifolia 102 root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)

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Figure 4.35 Concentration of eurycomanone in Eurycoma longifolia 102 commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)

Figure 4.36 Total phenolics contents in Eurycoma longifolia root, 104 stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)

Figure 4.37 Total phenolics contents in Eurycoma longifolia 104 commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)

Figure 4.38 Total glycosaponins contents in Eurycoma longifolia 107 root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)

Figure 4.39 Total polysaccharides contents in Eurycoma longifolia 107 root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)

Figure 4.40 Total glycosaponins contents of Eurycoma longifolia 108 commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)

Figure 4.41 Total polysaccharides contents of Eurycoma longifolia 108 commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)

Figure 4.42 Comparison of total proteins contents of Eurycoma 110 longifolia root, stem and leaf water extracts by using Lowry method and Bradford method. Results are shown as %wt/wt ±SD (n=3)

Figure 4.43 Comparison of total proteins contents of Eurycoma 110 longifolia commercial products by using Lowry method and Bradford method. Results are shown as %wt/wt ±SD (n=3)

Figure 4.44 Remaining percentage concentration of eurycomnone in 114 root (BR) stored at different storage conditions over 6 months period

Figure 4.45 Remaining percentage concentration of eurycomnone in 115 stem (BS) stored at different storage conditions over 6 months period

Figure 4.46 Remaining percentage concentration of eurycomnone in 115 leaf (BL) stored at different storage conditions over 6 months period

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Figure 4.47 Plot of (ln C%) against time for eurycomanone in root 117 (BR) at different storage conditions

Figure 4.48 Plot of (ln C%) against time for eurycomanone in stem 117 (BS) at different storage conditions

Figure 4.49 Plot of (ln C%) against time for eurycomanone in leaf 118 (BL) at different storage conditions

Figure 4.50 Rate constant (K) of degradation reaction of 120 eurycomanone in Eurycoma longifolia root, stem and leaf water extracts

Figure 4.51 Arrhenius plot for eurycomanone in Eurycoma 121 longifolia root water extract (BR)

Figure 4.52 Arrhenius plot for eurycomanone in Eurycoma 121 longifolia stem water extract (BS)

Figure 4.53 Arrhenius plot for eurycomanone in Eurycoma 122 longifolia leaf water extract (BL)

Figure 4.54 Activation energy of eurycomanone in Eurycoma 123 longifolia root, stem and leaf water extracts

Figure 4.55 Calculated shelf life of eurycomanone in Eurycoma 127 longifolia root, stem and leaf water extracts

Figure 4.56 Chromatogram of a) saponins-rich extract, b) 129 eurycomanone-rich extract and c) eurycomanone standard

Figure 4.57 UV spectra for Liebermann-Burchard reagent, reference 131 and mixture solution from 700 – 200 nm

Figure 4.58 Retention time (RT) and area of eurycomanone and 134 unknown compounds from solubility analysis by HPLC

Figure 4.59 Chromatogram of eurycomanone-rich extract alone and 134 combination of various saponins-rich extract ratios with eurycomanone-rich extract

Figure 4.60 Critical micellar concentration of crude root water 136 extract (TA)

Figure 4.61 Critical micellar concentration of saponins-rich extract 137

Figure 4.62 Critical micellar concentration of eurycomanone-rich 138 extract

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Figure 4.63 Size distribution by intensity of root water extract (TA) 139

Figure 4.64 TEM of reconstituted crude root water extract (TA). The 140 sample was analyzed at a magnification of 53K, 120kV

Figure 4.65 The dose response curves of eurycomanone-rich extract 141 on HCT 116 cells

Figure 4.66 Effect of saponins-rich extract and eurycomanone-rich 142 extract on HCT 116. a) Untreated cell (negative control), b) Treated with eurycomanone-rich extract at 100µg/mL, and c) Treated with saponins-rich extract at 100µg/mL

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LIST OF ABBREVIATION

µg Microgram

µg/mL Microgram per milliliter

µL Microliter

µM Micro molar

µm Micrometer

AAS Atomic Absorption Spectroscopy

As Arsenic

ATR-FTIR Attenuated Total Reflectance- Fourier Transform Infrared Spectroscopy

BSA Bovine Serum Albumin

Cd Cadmium cm Centimetre

CO2 Carbon Dioxide

DMSO Dimethyl sulfoxide

ED50 Effective Dose for 50% g Gram g/L Gram per liter

GC Gas chromatography h Hour

H2O Water

HCl Hydrochloric Acid

Hg Mercury

HNO3 Nitric Acid

HPLC High Performance Liquid Chromatography

HPTLC High Performance Thin-Layer Chromatography

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IC50 Inhibitory Concentration 50

ICH International Conference on Harmonisation kJ/mol Kilo Joule per Mole

LC50 Lethal Concentration

LD50 Lethal Dose 50

LD95 Lethal Dose 95

LOD Limit of Detection

LOQ Limit of Quantification mg Milligram mg/kg Milligram per Kilogram

MIC Minimum Inhibitory Concentration min Minutes mL Milliliter mL/min Milliliter per Minutes mm Millimeter mM Millimolar

N Normal nm Nanometer

ºC Degree Celsius

Pb Lead

PCA Principal Component Analysis

PCS Photon Correlation Spectroscopy ppm Part per Million

PSI Pounds per Square Inch

RH Relative Humidity rpm Round per Minutes

RSD Relative Standard Deviation

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RT Room Temperature

SD Standard Deviation t1/2 Half Life

TEM Transmission Electron Microscopy

TGA Thermo gravimetric Analysers

UV-Vis Ultra Violet and Visible v/v Volume per Volume

W Watt w/v Weight per Volume wt/wt Weight per Weight

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ANALISIS KAWALAN KUALITI, PEMIAWAIAN DAN KAJIAN

KESTABILAN EURYCOMA LONGIFOLIA DAN KESAN KANDUNGAN

SAPONINNYA TERHADAP KELARUTAN DAN KETOKSIKAN

EURIKOMANON

ABSTRAK

Ujian kawalan kualiti dilakukan pada bahan mentah akar, batang dan daun E. longifolia daripada lima lokasi yang berbeza untuk menentukan julat nilai kawalan kualiti. Kandungan kelembapan, abu dan abu tidak larut asid bagi akar berada di dalam julat yang ditetapkan oleh Standard Perubatan Herba Asean dan Monograf

Herba Malaysia Jilid 1. Julat nilai bagi kawalan kualiti bahan mentah batang dan daun telah ditetapkan. Peratusan ekstrak larut air adalah lebih tinggi berbanding ekstrak larut alkohol untuk semua bahagian tumbuhan. Peratusan ekstrak yang diperolehi dengan kaedah panas adalah lebih tinggi berbanding dengan kaedah sejuk untuk kedua-dua ekstrak larut air dan alkohol. Kandungan Pb, Cd, As dan Hg dalam bahan mentah E. longifolia didapati berada dalam julat yang diterima Pertubuhan

Kesihatan Dunia. Kaedah pengekstrakan telah dioptimumkan dengan menggunakan nisbah serbuk akar: pelarut dan masa pengekstrakan yang berbeza. Keputusan menunjukkan pengeluaran ekstrak yang terbaik ialah pada nisbah serbuk akar: air

1:15 selama 4 jam setiap kitaran dan diulang sehingga 3 kitaran refluk. Peratusan hasil dan profil kimia yang melibatkan analisis kualitatif (HPTLC, UV dan HPLC) dan analisis kuantitatif (HPLC) telah dijalankan untuk semua ekstrak.

Kaedah analisis spektroskopi dan kromatografi digunakan untuk pemprofilan kimia dan pemiawaian ektrak air bagi akar, batang, daun termasuk enam sampel

xix komersial E. Longifolia berdaftar. Kaedah HPLC memberi peratusan eurycomanone bagi akar, batang, daun dan sampel komersial di dalam julat 0.89 ± 0.01% sehingga

3.28 ± 0.01% , 0.23 ± 0.00% sehingga 1.10 ± 0.00%, 0.10 ± 0.01% sehingga 0.34 ±

0.02% dan 0.07 ± 0.00% sehingga 0.16 ± 0.00%. Peratusan jumlah fenol, glycosaponins, polisakarida dan protein bagi akar adalah masing-masing dari julat

5.94 ± 0.23% sehingga 10.10 ± 0.08%, 40.12 ± 1.05% sehingga 44.45 ± 0.54%,

25.30 ± 0.15% sehingga 51.30 ± 2.56% dan 21.42 ± 0.6% sehingga 44.92 ± 2.5%.

Peratusan jumlah fenol, glycosaponins, polisakarida dan protein bagi batang adalah masing-masing dari julat 4.2 ± 0.13% sehingga 8.76 ± 0.3%, 24.09 ± 1.06% sehingga

47.24 ± 1.07%, 20.53 ± 0.48% sehingga 68.18 ± 0.22% dan 12.17 ± 0.6% sehingga

48.20 ± 2.0%. Peratusan jumlah fenol, glycosaponins, polisakarida dan protein bagi daun adalah masing-masing dari julat 15.88 ± 0.1% sehingga 22.33 ± 0.28%, 52.26 ±

0.31% sehingga 61.58 ± 0.44%, 7.59 ± 0.04% sehingga 12.59 ± 0.11% dan 58.90 ±

1.6% sehingga 81.58 ± 2.3%. Peratusan jumlah fenol, glycosaponins, polisakarida dan protein bagi sampel komersial adalah masing-masing dari julat 2.53 ± 0.00% sehingga 3.34 ± 0.01%, 3.31 ± 0.31% sehingga 16.44 ± 0.21%, 5.98 ± 0.54% sehingga 66.17 ± 3.96% dan 6.4 ± 0.2% sehingga 7.8 ± 0.3 %.

Dalam kajian kestabilan dipercepatkan, ekstrak (BR, BS dan BL) adalah lebih stabil pada 30ºC berbanding suhu lain yang dikaji dan anggaran hayat (t90) BR, BS dan BL adalah 0.31, 0.23 dan 0.36 bulan, masing-masing, pada 30ºC. Eurycomanone mempunyai jangka hayat yang lebih panjang dalam ektrak daun (BL) berbanding ekstrak akar (BR) dan batang (BS). Penanda sebatian kimia mengikuti degradasi tertib pertama dan degradasinya menjadi lebih cepat pada suhu dan kelembapan yang lebih tinggi. Oleh itu, untuk jangka hayat yang lebih panjang, adalah disyorkan untuk

xx menyimpan E. longifolia ekstrak pada suhu lebih rendah dari 30ºC di dalam bekas yang tertutup rapat.

Saponins meningkatkan kebolehlarutan eurycomanone melalui pembentukan miseles seperti yang ditunjukkan oleh kehadiran zarah sub-mikron. Kepekatan Misel

Kritikal (CMC) dianggarkan bernilai 267 μg/mL untuk ekstrak air akar mentah (TA),

57 μg/mL untuk ekstrak kaya saponin dan 847 μg/mL untuk ekstrak kaya eurycomanone. Keputusan ini menunjukkan kehadiran miseles dan menunjukkan peranan saponin dalam pembentukan miseles, seperti yang ditunjukkan oleh nilai

CMC rendah dalam ekstrak kaya saponin berbanding dengan nilai CMC ekstrak kaya eurycomanone. Kesan sitotoksik ekstrak kaya eurycomanone terhadap HCT 116 sel karsinoma kolorektal menunjukkan kepekatan perencatan median (IC50 ) 22.14 ± 1.0

μg/mL, manakala ekstrak kaya saponins pada julat kepekatan yang sama tidak menunjukkan kesan sitotoksik terhadap sel HCT 116. Kesan sitotoksik gabungan ekstrak kaya saponin dan ekstrak kaya eurycomanone terhadap sel HCT 116 telah meningkatkan peratusan perencatan, bergantung kepada dos. Didapati bahawa dengan penambahan ekstrak kaya saponin dengan ekstrak kaya eurycomanone, kesan sitotoksik dapat dikurangkan terutamanya pada ekstrak kaya saponins yang berkepekatan rendah (25, 50 dan 75 μg/mL), dan tidak mempunyai kesan pada kepekatan tinggi (100 μg / mL). Ini menunjukkan kehadiran saponins di dalam ekstrak E. longifolia dapat melindungi kesan sitotoksik eurycomanone. Tambahan pula, penemuan yang diperolehi menunjukkan bahawa penggunaan ekstrak mentah

E. longifolia lebih selamat berbanding penggunaan sebatian tulen atau ekstrak eurycomanone pekat.

xxi

3 QUALITY CONTROL ANALYSIS, STANDARDIZATION AND

STABILITY STUDIES OF EURYCOMA LONGIFOLIA AND EFFECT OF

ITS SAPONIN CONTENT ON SOLUBILITY AND TOXICITY OF

EURYCOMANONE

4 ABSTRACT

Quality control test was done on E. longifolia root, stem and leaf from five different localities in order to determine the range of quality control values.

Moisture, ash and acid insoluble ash content of all the root raw material were found to be within acceptable limit set by Standard of Asean Herbal Medicine and

Malaysian Herbal Monograph Volume 1. The range of quality control values of the stem and leaf raw material have been set. The percentages of water soluble extracts were higher as compared to alcohol soluble extracts in all parts of the plant.

Percentage extractive values by the hot method were higher as compared to the cold method for both water and alcohol soluble extracts. The amounts of Pb, Cd, As and

Hg in E. longifolia raw material were found within acceptable limit according to

World Health Organization. Extraction method was optimized by different root powder: solvent ratio and extraction time. Results suggest the best extraction conditions at the root powder: water ratio of 1:15 and at 3 reflux cycles each for 4 h.

The percentage yield and chemical profile involving qualitative analysis (HPTLC,

UV and HPLC) and quantitative analysis (HPLC) were carried out for all the extracts.

Analytical spectroscopy and chromatographic methods were used for chemical profiling and standardization of E. longifolia root, stem, leaf water extracts

xxii and six registered commercial products. HPLC validated methods give the percentage of eurycomanone for root, stem, leaf and commercial products in a ranges

0.89 ± 0.01% to 3.28 ± 0.01%, 0.23 ± 0.10% to 1.10 ± 0.00%, 0.10 ± 0.00% to 0.34

± 0.01% and 0.07 ± 0.00% to 0.16 ± 0.00%, respectively. The percentage of total phenolics, glycosaponins, polysaccharides and protein for root ranging from 5.94 ±

0.23% to 10.10 ± 0.08%, 40.12 ± 1.05% to 44.45 ± 0.54%, 25.30 ± 0.15% to 51.30 ±

2.56% and 21.42 ± 0.6% to 44.92 ± 2.5%, respectively. The percentage of total phenolics, glycosaponins, polysaccharides and protein for stem ranging from 4.20 ±

0.13% to 8.76 ± 0.30%, 24.09 ± 1.06% to 47.24 ± 1.07%, 20.53 ± 0.48% to 68.18 ±

0.22% and 12.17 ± 0.6% to 48.20 ± 2.0%, respectively. The percentage of total phenolics, glycosaponins, polysaccharides and protein for leaf ranging from 15.88 ±

0.10% to 22.33 ± 0.28%, 52.26 ± 0.31% to 61.58 ± 0.44%, 7.59 ± 0.04% to 12.59 ±

0.11% and 58.90 ± 1.6% to 81.58 ± 2.3%, respectively. The percentage of total phenolics, glycosaponins, polysaccharides and protein for commercial products ranging from 2.53 ± 0.00% to 3.34 ± 0.01%, 3.31 ± 0.31% to 16.44 ± 0.21%, 5.98 ±

0.54% to 66.17 ± 3.96% and 6.4 ± 0.2% to 7.8 ± 0.3 %, respectively.

In accelerated stability study, extracts (BR, BS and BL) were more stable at

30ºC compared to other temperatures studied, and the estimated shelf life (t90) of BR,

BS and BL were 0.31, 0.23 and 0.36 months, respectively, at 30ºC. Eurycomanone has higher shelf life in the leaf (BL) as compared to root (BR) and stem (BS). The marker compound followed the first order degradation and its degradation rate was increased by increasing storage temperature and humidity. Therefore, in order to guarantee a longer shelf life of the products, it is recommended to store the products containing E. Longifolia extract at below 30ºC in a tightly closed container.

xxiii

Saponins increase the solubility of eurycomanone through the formation of micelles as indicated by the presence of sub-micron particles. The Critical Micellar

Concentration (CMC), was estimated to be 267 µg/mL for the crude root water extract (TA), 57 µg/mL for saponins-rich extract and 847 µg/mL for eurycomanone- rich extract. These results indicate the presence of micelles and show the role of saponins in the formation of these micelles as indicated by the lower CMC value in saponins-rich extract compared to the CMC value of eurycomanone-rich extract.

Cytotoxicity effect of eurycomanone-rich extract on HCT 116 colorectal carcinoma cells showed dose dependent growth inhibitory effect with median inhibitory concentration (IC50) of 22.14 ± 1.0 µg/mL, whereas the saponins-rich extract at the same concentration range did not show any cytotoxic effect on the same cells.

Cytotoxicity effect of combination saponins and eurycomanone-rich extract on HCT

116 cells was increased in a dose dependent manner. It was found that adding the saponins-rich extract to the eurycomanone-rich extract reduced the cytotoxic effect of the later particularly at low concentration of the saponins-rich extract (25, 50 and

75 μg/mL), and has no effect at higher concentration (100 μg/mL). These findings indicate that the presence of saponins in the E. longifolia extracts protect against the cytotoxic effect of the eurycomanone. Furthermore, these findings indicate that using the crude extracts of E. longifolia could be safer than using pure compounds or concentrated fractions of eurycomanone.

xxiv

1 CHAPTER 1 INTRODUCTION

1.1 Significance of Medicinal Plants

Since historically, medicinal plants have played an important role in human life. Plants have been used extensively as food and medicine for the treatment of a range of diseases. The sale and use of herbal medicines have increased significantly over the past 20 years. The use of herbal medicines in the US increased from 2.5% in

1990 to 12.1% in 1997, with a total of $5 billion being spent (Eisenberg et al., 1998).

Within the UK, over 30% of a random sample of 5010 adults were taking, or had been taken, herbal remedies (Thomas et al., 2001). Furthermore, 80% of the world's population in developing countries depend on the use of herbal and other traditional medicines for their primary health care (Khalid et al., 2009).

Medicinal plants are available from a variety of plant material which is from leaf, stem, root and bark. They contain many biologically active compounds and can be used to treat mild or chronic diseases. Herbs are easy to be obtained, prepared and consumed in many ways, using either fresh raw materials or dried ingredients.

Herbal remedies are also available in the form of capsules, tablets or powder.

1

1.2 Efficacy, Safety and Quality Control in the Standardization of Herbal

Medicine

Determination of quality aspects of herbal products can be a problem because of the variation in the content and concentrations of constituents of plant material, different range of extraction techniques and processing steps used by different manufacturers. Many of the herbal products are prepared from a single plant or a combination of two or more different types of medicinal plants. It is unusual to have different herbal ingredients in one product but the problem is to determine which components contribute to biological activities. Pharmacological activities and consistency in herbal products are based on chemical constituent’s changes which possess the activity of the herbs. To overcome this problem, consistent products can be produced through standardization.

Standardization of herbal materials is carried out to ensure the consistency and repeatability of a particular extract in ensuring guaranteed potency through acceptable levels of active compounds. This process can be performed by chemical

(HPTLC, HPLC/GC and FTIR) or biological (antioxidant) analysis. Standardization is an important step where the active constituents are known. However, for many herbs the active constituents are not known. In these cases, products may be standardized on the content of certain ‘marker’ compound (chemical characteristic of the herb or present in large quantities). Although there are many benefits proven, natural products are not getting wider acceptance in the mainstream of pharmaceuticals due to lack of standardization. In order to bring these remedies into the mainstream pharmaceutical market, strong scientific evidence is required to support the efficacy claims of these products (Barnes, 2003).

2

Statement that herbal drugs are very safe and free from side effects is not absolutely true. The plants contain hundreds of constituents and some are highly toxic such as the most cytotoxic anti-cancer plant-derived drugs, digitalis, the pyrrolizidine alkaloids, ephedrine, phorbol esters, etc. There are two kinds of side effects reported. The first, considered to be intrinsic to herbal drugs themselves, for example related to predictable toxicity, over-dosage and interaction with conventional drugs, as reported for modern medicines. The most known side effects reported for herbal drugs are extrinsic to the preparation for example because of several manufacturing problems; misidentification of plants, lack of standardization, failure of good manufacturing practice, contamination, substitution and adulteration of plants and incorrect preparation or dosage (Calixto, 2000).

3

Due to increasingly widespread use of herbs, it is important to ensure the safety of the herbal products. Therefore, the quality control and standardization of raw material and the herbal preparation should be permanently conducted.

Standardization and quality control of herbals is the process involved in the evaluation of crude drugs physicochemical aspects such as selection and handling of crude material, safety, efficacy and stability assessment of finished products, documentation, safety and risk based on experience, provision of product information to consumer and product promotion. However, more attention is given to the following quality index such as macro and microscopic examination, foreign organic matter, ash values, moisture content, extractive values, crude fibre, qualitative chemical evaluation, chromatography’s examination, quantitative chemical evaluation and toxicological studies (WHO, 1996a; WHO, 1996b; WHO,

1992).

Several factors can also affect the quality control and consequently the therapeutic value of herbal medicines such as the use of fresh plants, light, temperature, nutrients, water availability, duration and time collection, methods of collecting, drying, packaging, storage, transportation of raw material, age, part of the collected plant and other factors. Method of extraction, contamination with microorganism, heavy metals, pesticides and etc, can also affect the quality, safety and efficacy of herbal drugs (Calixto, 2000).

4

1.3 Problem Statements and Justification of the Research

In Malaysia, dietary supplements are widely consumed for general health and well-being and it is easily available in community pharmacies, health food stores, night markets, grocery stores and also obtained via the internet. Currently, 183 products manufactured from E. longifolia are available and registered with the

National Pharmaceutical Control Bureau (Appendix A). However, there is still a lack of information on its chemical profile in the commercial products. All parts (root, stem and leaf) of E. longifolia are useful and contributed to different biological activities, thus indicating the need to perform quality control tests. The toxic potential of eurycomanone as a major marker compound makes it necessary to standardize the plant extracts in commercial products to comply with the set standards in order to protect consumers from possible toxic effects. Stability studies must be performed in order to estimate the shelf life, suitable temperature storage and stability of eurycomanone compound in all parts of the plants. The stability of E. longifolia extracts need to comply to ICH standard guidelines. The solubility and toxicity of eurycomanone needs improvements. Since saponins are the major primary metabolites in this plant, their effect on the solubility and toxicity of eurycomanone was investigated.

5

1.4 Objectives of the Study

The objectives of this study are as the following:

1. To perform quality control tests on E. longifolia root, stem and leaf raw

material from five different locations and to optimize the extraction method

for the root.

2. To develop and validate reliable analytical methods for chemical profiling

and standardization of E. longifolia root, stem and leaf water extracts and

several selected commercial products.

3. To perform and study the accelerated stability profile of E. longifolia water

extract of the root, stem and leaf using eurycomanone as a marker.

4. To study the effect of saponins on the solubility and cytotoxicity of

eurycomanone-rich extract.

6

2 CHAPTER 2 LITERATURE REVIEW

2.1 Eurycoma longifolia Jack

2.1.1 Taxonomy

Taxonomically, E. longifolia Jack is classified as the following scheme:

Kingdom: Plantae

Division: Magnoliophyta

Class: Magnoliopsida

Order:

Family:

Genus: Eurycoma

(Comprehensive information can be found at http://en.wikipedia.org/wiki/Eurycoma_longifolia)

2.1.2 Vernacular Names

Some of the common and popular names of this plant includes Long Jack,

Malaysian Ginseng, Local Ginseng, Natural Viagra, Pasak Bumi, Payung Ali,

Penawar Pahit, Setunjang Bumi, Bedara Pahit, Tongkat Baginda, Pokok Syurga,

Tongkat Ali Hitam, Pokok Jelas, Cay ba binh, Ian- don, and Jelaih (Bhat and Karim,

2010).

7

2.1.3 Plant Description

E. longifolia Jack is a slender, evergreen flowering belongs to the family

Simaroubaceae, is occurring naturally in jungles of Malaysia and Indonesia. The female and male reach a height of up to 15 meters (Wernsdorfer et al., 2009) and bearing fruits after nearly 2–3 years of cultivation. However, it is generally believed that for complete maturation of the plant, it might take up to 25 years. But, for commercial uses, most of the times, the roots are harvested after 4 years of cultivation. The fruits are green in color, 2–3 cm long and turn to dark red after ripening. The leaves are pinnate, spirally arranged, long (10–15 inches) with 10–30 leaflets. The are produced in large panicles and the plant is dioecious with male and female flowers borne on different trees. The plant grows profusely in sandy, well-drained soil in the presence of partial shade and with adequate quantity of water. This plant is widely sought ‘singly’ or as an ‘essential component’ in herbal remedies for a variety of illnesses and as health supplements. Almost all the parts of

Tongkat Ali have been used traditionally for therapeutic purposes (Bhat and Karim,

2010). There are four different species of Tongkat Ali, namely Eurycoma longifolia,

Eurycoma apiculata, Polyathia bullata and Goniothalamus sp. (Aziz et al., 2003).

Among the four, Eurycoma longifolia is the most commonly used species for the extract production (Athimulam et al., 2006).

8

(a) (b)

(c) (d)

Figure 2.1: Pictures of Eurycoma longifolia (a) - (b) whole plant, (c) leaves and (d) root

9

2.1.4 Review of Chemical Constituents of Eurycoma longifolia

Various chemical constituents have been identified from the different parts of

E. longifolia. Reviews on the identified chemicals of E. longifolia are summarized in

Table 2.1.

Table 2.1: Chemical constituents of Eurycoma longifolia

Plant Extract Chemical constituents References part Root Aqueous quassinoids (e.g. eurycolactones A-E, (Chua et al., 2011; eurycomalides A-B, eurycomalactone, Chua et al., 2012; 6α-hydroxyeurycomalactone, 7α- Yusmazura et al., hydroxyeurycomalactone, eurycomanone, 2009) 13α(21)-epoxyeurycomanone, 12,15- diacetyl-13α(21)-epoxy- eurycomanone,12-acetyl-13,21- dihydroeurycomanone, 15-acetyl- 13α(21)-epoxyeurycomanone, 3,4ε- dihydroeurycomanone, 13,21- dihydroeurycomanone, eurycomanol, 13β,18-dihydroeurycomanol, 13β, 21- dihydroxyeurycomanol, eurycomanol-2- O-β-D-glycopyranoside, 11- dehydroklaineanone, 15β- hydroxyklaineanone, 14,15β- dihydroxyklaineanone, 5α,14β,15β- trihydroxyklaineanone,15β-O-acetyl-14- hydroxyklaineanone, 6α-acetoxy-14,15β- dihydroxyklaineanone, 6α-acetoxy-15β- hydroxyklaineanone, laurycolactones A- B, longilactone, dehydroxylongilactone, 2,3-dehydro-4α-hydroxylongilactone, ailanthone, (α/β-epoxide) ailanthone, chaparrinone (α-methyl), 3,4ε- dihydrochaparrinone, picrasinoside B, klaineanolide B, iandonoside B, eurycomaoside, 16-α-O- methylneoquassin, samaderin B and glaucarubolone), canthin-6-one alkaloids (e.g. canthin-6-one, 9-methoxycanthin-6- one, 5,9-dimethoxycanthin-6-one, 9,10- dimethoxycanthin-6-one, 11-

10

hydroxycanthin-6-one, 1-hydroxy-11- methoxycanthin-6-one, 10-hydroxy-9- methoxycanthin-6-one, 11-hydroxy-10- methoxycanthin-6-one, 11-O-β-D- glucopyranosylcanthin-6-one, canthin-6- one-3N-oxide, 9-methoxycanthin-6-one- 3N-oxide and 9-methoxy-3- methylcanthin-5,6-dione), β-carboline alkaloids (e.g. β-carboline-1-propionic acid, 7-hydroxy-β-carboline-1-propionic acid, 7-methoxy-β-carboline-1-propionic acid, and 1-methoxymethyl-β-carboline), squalene-type triterpene (e.g. eurylene and 11/14-deacetyl eurylene), biphenylneolignans (e.g. 2,2’-dimethoxy- 4-(3-hydroxy-1-propenyl)-4’-(1,2,3- trihydroxypropyl) diphenyl ethers (isomer), 2-hydroxy-3,2’,6’-trimethoxy- 4’-(2,3-epoxy-1-hydroxypropyl)-5-(3- hydroxy-1-propenyl)-biphenyl and 2- hydroxy-3,2’-dimethoxy-4’-(2,3-epoxy- 1-hydroxypropyl)-5-(3-hydroxy-1- propenyl)-biphenyl) and others (e.g. isoleucine, calcium, magnesium and potassium)

Methanol quassinoids (e.g. eurycolactones A-F, (Kardono et al., eurycomalides A-B, eurycomalactone, 1991; 6α-hydroxyeurycomalactone, 6-hydroxy- Chan et al., 1991; 5,6-dehydroeurycomalactone, 5,6- Chan et al., 1992; dehydroeurycomalactone, eurycomanone, Ang et al., 2000; 13β,21-dihydroxyeurycomanol, 14,15β- Ang et al., 2002; dihydroxyklaineanone, 5α,14β,15β- Kuo et al., 2003b; trihydroxyklaineanone, laurycolactones Teh et al., 2010; A-B, longilactone, 6- Kuo et al., 2003a) dehydroxylongilactone, 2,3-dehydro-4α- hydroxylongilactone and pasakbumin B- C), canthin-6-one alkaloids (e.g. canthin- 6-one, 1-hydroxycanthin-6-one, 9- hydroxycanthin-6-one, 5- hydroxymethylcanthin-6-one, 5- methoxycanthin-6-one, 9- methoxycanthin-6-one, 10- methoxycanthin-6-one, 1-hydroxy-9-

11

methoxycanthin-6-one, 4-hydroxy-5- methoxycanthin-6-one, 8-hydroxy-9- methoxycanthin-6-one, 5- hydroxymethyl-9-methoxycanthin-6-one, 4,5-dimethoxycanthin-6-one, 9,10- dimethoxycanthin-6-one, canthin-6-one 9-O-β-glucopyranoside, canthin-6-one- 3N-oxide, 9-hydroxycanthin-6-one-3N- oxide and 9-methoxycanthin-6-one-3N- oxide), β-carboline alkaloids (e.g. β- carboline-1-propionic acid, 7-methoxy-β- carboline-1-propionic acid, methyl β- carboline-1-carboxylate, n-pentyl β- carboline-1-propionate and picrasidine L), triterpenes (e.g. eurylene, mixture of β-sitosterol and stigmasterol, and β- sitosteryl glucoside) and others (e.g. scopoletin, fraxidin, scopolin, ρ- hydroxybenzaldehyde, syringic aldehyde, 2,4’-dihydroxy-3’-methoxyacetophenone, 2,3-dihydroxy-1-(4’-hydroxy-3’- methoxyphenyl)-propan-1-one, 3- hydroxy-1-(4’-hydroxy-3’- methoxyphenyl)propan-1-one, threo-1,2- bis-(4-hydroxy-3- methoxyphenyl)propane-1,3-diol, vanillic acid, protocatechuic acid, nicotinic acid, syringic acid, sodium syringate, sodium ρ-hydroxybenzoate, lariciresinol, erythro- 1-C-syringylglycerol, threo-1-C- syringylglycerol, erythro- guaiacylglycerol, threo-guaiacylglycerol, iandonone, adenosine, guanosine, thymidine, alanine, proline, arginine, serine, glucose and fructose).

Ethanol quassinoids (e.g. eurycomanone, 13,21- (Chan et al., 1989; (50%) dihydroeurycomanone, 13α(21)- Chan and Choo, epoxyeurycomanone, longilactone, 2002; eurycomalactone, 14,15β- Chan et al., 2004) dihydroxyklaineanone, eurycomanol, eurycomanol-2-O-β-glucopyranoside) and a canthin-6-one alkaloid (e.g. 9- methoxycanthin-6-one).]

12

Aqueous Volatile components were also detected (Shafiqul et al., and in the aqueous and methanol root 2006) Methanol extracts, e.g. 3-methylbutanal, 1-butanol, 1-pentanol, 2-hexadecanol, acetol, nonanal, acetic acid, 2-methylhexanol, benzaldehyde, -2,3-butanediol, butyrolactone, 2-furanmethanol, 3- methylbutanoic acid, 2(5H)-furanone, curcumene, hexanoic acid, butylated hydroxytoluene, 1-(1H-pyrrol-2-yl)- ethanone, (R)-(−)-massoilactone, 1H- pyrrole-2-carboxaldehyde, 3-phenoxy-1- propanol, octanoic acid, [1R,2S,5R]-1’- [butyn-3-one-1-yl]-menthol, 2- phenoxyethanol, ethyl p-ethoxybenzoate, nonanoic acid, 4-ethynyl-4-hydroxy- 3,5,5-trimethyl-2-cyclohex-1-enone, 2,4- bis(1,1-dimethylehtyl)phenol, diethyl phthalate, benzoic acid, 2,3,6,7- tetrahydro 4a,8a-butano-[1,4]dioxino[2,3- b]-1,4dioxin.

Leaves Ethanol Quassinoids (lonilactone, 6-dehydro (Jiwajinda et al., lonilactone,11-dehydroklaineanone, 12- 2001) epi dehydroklaineanone, 15β- hydroxyklaineanone, 14,15 β- dihydroxyklaineanone, and 15-β-O- acetyl-14-hydroxyklaineanone)

13

OH OH CH OH 2 O CH OH O 3 OH H

O O H H CH3 Figure 2.2: Chemical structure of Eurycomanone

2.1.5 Review of Pharmacological Activities of Eurycoma longifolia

E. longifolia Jack (Tongkat Ali) is one of the most popular herbs in Malaysia.

This plant is a symbol of man’s ego and strength because it has been claimed by

Malaysians to improve strength and power during sexual activities; it increases male virility and sexual prowess and is usually taken as a decoction of the roots in water

(Gimlette and Thomson, 1977). The root has also been traditionally used for fever, afterbirth medication, boils, wounds, ulcer, syphilis, bleeding gums, ache, dysentery, glandular swelling, oedema and tonic (Gimlette and Burkill, 1930; Burkill and

Haniff, 1930). The leaves of the plant have been used by traditional healers to cure malaria, ulcers, prevent gum diseases and for the treatment of sexually transmitted diseases such as syphilis and gonorrhoea (Bhat and Karim, 2010).

E. longifolia is reported to have many pharmacological activities.

Antimalarial activity of E. longifolia has been investigated by several studies.

Eurycomanone, pasakbumi B and 7-methoxy--carboline-1-propionic acid isolated from E. longifolia root exhibited potent anti-plasmodial activity against

P. falciparum (Chan et al., 1986). In another study, it was shown that eurycomanone,

13,21 dihydroeurycomanone, 13α(21)-epoxyeurycomanone, eurycomalactone, and 9- methoxycanthin-6-one isolated from 50% aqueous ethanol root extracts displayed 14 anti-plasmodial activity against chloroquine-resistant Gombak A isolate of

P. falciparum with IC50 in the range of 0.23-1.56 µg/mL (Chan et al., 2004). A mixture of semi-purified root extract (13β,18-dihydroeurycomanol, eurycomanol-2-

О-β-D-glucopyranoside, eurycomanol and eurycomanone) of 0.07-5.00 µg/mL demonstrated more than 50% inhibition of P. falciparum strain (Kuo et al., 2004;

Ang et al., 1995). The standardized methanol extract of E. longifolia root at the concentration of 10, 30 and 60 mg/kg BW in combination with artemisinin given orally to mice for 3 days showed suppression of 63, 67 and 80% of Plasmodium yoelii infection in mice, respectively, whilst 80% suppression of P. yoelii infection was observed with subcutaneous treatment of E. longifolia (10 mg/kg BW) combined with artemisinin (Mohd et al., 2007).

Cytotoxicity of E. longifolia has been reported in many studies.

Eurycomanone isolated from E. longifolia roots exhibited potent cytotoxicity with

IC50 values of 45 ± 0.15 μg/mL of hepatoma (HepG2) cells through apoptosis mechanism (Yusmazura et al., 2009). In another study, Eurycomalactone, 6- dehydroxylongilactone, 9-methoxycanthin-6-one, canthin-6-one, longilactone,

14,15β-dihydroxyklaineanone, pasakbumin C and canthin-6-one 9-O-β- glucopyranoside isolated from E. longifolia roots exhibited potent cytotoxicity towards human lung cancer (A549) cell lines with ED50 in the range of <2.5 to 4.6

µg/mL, while eurycomalactone, 6-dehydroxylongilactone, 9-methoxycanthin-6-one,

14,15β-dihydroxyklaineanone, eurycomanone, pasakbumin B and pasakbumin C exhibited potent cytotoxicity towards human breast cancer MCF-7 cells (ED50 <2.5-

4.5 µg/mL) (Kuo et al., 2004). 9-methoxycanthin-6-one, 9-methoxycanthin-6-one-N- oxide, 9-hydroxycanthin-6-one and 9-hydroxycanthin-6-one-N-oxide isolated from

15

E. longifolia roots exhibited cytotoxic effects against human cancer cell types including breast, colon, fibrosarcoma, lung, melanoma, KB and murine lymphocytic leukemia (P-388) with ED50 in the range of 1.4 to 11.7 µg/mL. Eurycomanone was active against all cells above including vincristine-resistant KB cells (ED50 0.2-11.3

µg/mL), except P-388 cells (Kardono et al., 1991). A different study reported that eurycomanone, 21-dihydroeurycomanone and 14,15β-dihydroxyklaineanone from

E. longifolia roots showed a potent cytotoxic effect against KB cells with IC50 values of 0.98, 0.81 and 0.96 µM, respectively (Chan et al., 2004).

Many studies reported the effect of aphrodisiac activity in E. longifolia.

Aqueous extract of E. longifolia root (30, 60, 90, 150 mg/kg) given via oral gavage to Sprague Dawley male rats for 28 days enhanced sexual activities and sperm quality (sperm count, motility, morphology and viability) of the treated rats. In another study, chloroform, methanol, butanol and water fractions of E. longifolia root given orally to 3-4 month old male Sprague Dawley albino rats for 10 days showed a dose-dependent increase in mounting frequency from 5.3 (400 mg/kg) to 5.4 (800 mg/kg), 4.9 to 5.4, 4.8 to 5.2 and 5.2 to 5.3, respectively (Ang and Sim, 1997). These fractions (500 mg/kg) contributed towards sexual motivation activity in adult, middle-aged male albino mice and in retired breeders (Ang et al., 2003a), as well as enhanced sexual qualities of middle-aged male rats (Ang et al., 2003b). Different studies reported that methanol, chloroform, water and butanol fractions of E. longifolia root (800 mg/kg) given via oral gavage to 9-month old Sprague Dawley rats for 10 days showed changes in sexual behaviour such as increased orientation activities towards the receptive female rats (anogenital sniffing, licking and mounting), increased genital grooming towards themselves and restricted movements

16 to a particular area of the cage (Ang and Lee, 2002). The fractions (800 mg/kg) were also found to promote sexual arousal in sexually sluggish old male rats (Ang et al.,

2004). Additionally, these fractions (800 mg/kg) given orally for 12 weeks to testosterone-stimulated castrated intact male rats revealed pro-androgenic property as evidenced by the enhanced growth of the laevator ani muscle (Ang and Cheang,

2001).

Anti-diabetic property of E.longifolia was reported. In this study, antihyperglycaemic activity of aqueous extract of E. longifolia root (150 mg/kg body weight) was observed in streptozotocin-induced hyperglycaemic Sprague Dawley rats after 10 days of treatment (Husen et al., 2004).

Anxiolytic activity effect of E. longifolia was investigated. The result showed that chloroform, methanol, butanol and water fractions of E. longifolia root (0.3 g/kg) was administered by oral gavage to inbred adult albino mice (35-40 g) exerted anxiolytic effect (Ang and Cheang, 1999).

The effect of E.longifolia on anti-osteoporosis was investigated. In this study, supplementation of E. longifolia root aqueous extract given to 12-month old orchidectomised Sprague Dawley rats for 6 weeks showed maintenance of bone calcium level (Shuid et al., 2011).

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2.2 Stability Studies

Stability is defined as the time during which a drug to maintains its chemical integrity and labelled potency within the certain limits. The stability of a pharmaceutical preparation is its degree of resistance to chemical and physical changes. Products should be consistent in its effectiveness and claimed potency or may change only within the limits set by the legal provisions until the expiration date

(Racz, 1989). Stability is one of the most important factors that determine whether a compound or mixture of compounds can be developed into a pharmaceutical product.

Stability studies provide evidences on how the quality of a drug substance varies with the passage of time under the influence of environmental factors (WHO, 1996).

Stability studies before developing a dosage form are the first quantitative assessment of chemical stability of a new product and its possible formulation.

Stability studies are used to recommend storage conditions and predict shelf life of medicinal products.

The stability studies are important for three main reasons. Firstly, it is for the patient safety by making sure that the patient receives a uniform dose of drug throughout the whole shelf life of the product. Even though a drug may have been shown to be safe for use, this is not necessarily true for the decomposition products.

Secondly, consideration must be given to the relevant legal requirements concerned on the identity, strength, purity and quality of the drug. Finally, stability study is important to prevent the economic impact of marketing an unstable product. The stability of a product is influenced by a number of factors, which may be classified into two main categories, physical and chemical factors.

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2.2.1 Physical Factors Effecting Stability

2.2.1.1 Temperature

Temperature increases the rate of degradation of active ingredients due to the increase in kinetic energy, which increases the number of colliding molecules. The decomposition normally increases by two to three times for every 10°C temperature rises. Therefore, it is important to be aware of it while keeping raw material and in production of a product especially when heating is required. It is important especially in the case of thermo labile and volatile constituents.

2.2.1.2 Moisture

Moisture content increases the rate of decomposition and makes the product susceptible to hydrolysis. For raw herbal powders or extract, it allows the growth of microbes that not only deteriorate the constituents but can produce toxic substances.

2.2.1.3 Light

Sunlight as an energy form can initiate and accelerates decomposition. Photo labile constituents of extracts deteriorate on expose to sunlight (Rawlins, 1977; Pugh,

2002).

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2.2.2 Chemicals Factors Effecting Stability

The constituents of extracts also undergo chemical degradation over the time.

The chemical degradation is further classified as follows:

2.2.2.1 Hydrolysis

Hydrolysis, reaction of the substance with water is the most common cause of drug degradation (Waterman et al., 2002). It is considered as the main cause of deterioration of drugs, especially for aqueous-based preparations.

2.2.2.2 Oxidation

Oxidation is defined as the removal of an electropositive atom, radical or electron, or addition of an electronegative atom, radical or electron. Decomposition of pharmaceutical preparation due to oxidation is nearly as probable as that of hydrolysis. The rate of oxidation is depends on the temperature. For example, peroxidation of fatty acids, break down of fatty acids into aldehydes and ketones, accelerates as the temperatures exceeds 50°C (Rawlins, 1977).

2.2.2.3 Isomerisation and Polymerization

Polymerization is defined as the addition of similar molecules whereas isomerisation is the change in isomeric forms. These phenomena change the biological activity of compounds.

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2.2.3 Accelerated Stability

The stability study of pharmaceuticals performed at exaggerated conditions is called accelerated stability. The accelerated stability study is performed for fast prediction of stability. Degradation is usually slow at room temperature and shelf life may go up to several years. Stability testing for this type will be time consuming and expensive since the period can be as long as two years. Therefore, accelerated stability testing is devised for rapid prediction of long term stability of a product. The stability of drugs at room temperature is the main purpose of this study but it would take a long time to conduct. Therefore, stability studies are conducted at elevated temperatures and the data is evaluated by applying Arrhenius equation

(Sungthongjeen, 2004).

An estimation of shelf life (t90), a time in which a product retains 90% of its original potency, may be made by accelerating the decomposition process and extrapolating the results to normal storage conditions. Therefore, stability tests are often conducted under elevated temperatures to accelerate the degradation process in order to get a fast stability prediction. Application of the principles of chemical kinetics on the results of accelerated stability tests enables us to make prediction and estimate the shelf life of a product at normal temperature.

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2.3 Saponins

2.3.1 Definition and Chemical Compositions

'Saponin' name is derived from the Latin word sapo, which means 'soap', because saponin molecules form a soap- like foams when shaken with water

(Vincken et al., 2007). Saponins are found in great number of plant species and usually found in roots, tubers, leaves, blooms or seeds. Saponins can be classified into two groups based on the nature of their aglycone skeleton. The first group consists of the steroidal saponins and the second group consists of the triterpenoid saponins. Steroidal saponins consist of a steroidal aglycone, a C27 spirostane skeleton, generally comprising of a six-ring structure (Figure 1.1 (a)). Triterpenoid saponins consist of a triterpenoid aglycone, which consists of a C30 skeleton, comprising of a pentacyclic structure (Figure 1.1 (b)) (Sparg et al., 2004).

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CH3 a 3CH O CH3

O CH3

RO

3CH CH3

b CH CH 3 3 CH3

CH3 RO

CH3 3CH

Figure 2.3: Aglycone skeletons of (a) steroidal spirostane and (b) triterpenoid saponins. R = sugar moiety

2.3.2 A Review of the Pharmacological Activities of Saponins

Pharmacological activities of saponins have been reported in several studies.

Haemolytic activity in a mixture of saponins isolated from Maesa lanceolata Forssk.

(Myrsinaceae) was reported. In this study, the maesasaponin mixture showed very high haemolytic activity, haemolysing 50% of the human erythrocytes (1% suspension in phosphate buffer saline) at a concentration of 1.6 µg/mL (Sindambiwe et al., 1998).

The Molluscicidal activity of saponins has been reported in many studies. A six-oleanane-type triterpenoid saponin mixture (maesasaponin mixture, isolated from

Maesa lanceolata) was tested for molluscicidal activity against Biomphalaria glabrata snails. The saponin mixture showed high toxicity, with LD95 and LD50 values of 4.1 and 2.3 µg/mL, respectively (Sindambiwe et al., 1998). In another

23 study, the biodegradability of molluscicidal water-extracted saponins from the berries of Phytolacca dodecandra was reported. From the study, the saponins in an aqueous extract of Phytolacca dodecandra readily biodegraded (t1/2 = 15.8 h) when consumed within 10 days in aquatic environments under aerobic conditions. As a result, the use of Phytolacca dodecandra berries for snail control in schistosomiasis infested water bodies are environmentally acceptable (Mølgaard et al., 2000). One of the ten isolated saponins from the leaves of Maesa lanceolata showed molluscicidal activity against Biomphalaria glabrata snails. Maesasaponin VI2 is responsible for a large part of the activity of the mixture. This saponin had a LC50 value of 0.5 mg/mL in its isolated form (Apers et al., 2001).

The effect of anti-inflammatory activity of saponins has been reported by several studies. Fruticesaponin B, a bidesmosidic saponin with an unbranched saccharide moiety isolated from Bupleurum fruticescens L. (Apiaceae), was shown to have the highest anti inflammatory activity of the all the saponins tested in the mouse oedema assays (Just et al., 1998). Saponins isolated from Bupleurum rotundifolium

L. (Apiaceae) were reported to have anti-inflammatory activity against both 12-O- tetradecanoylphorbol-13-acetate (TPA) induced ear oedema and chronic skin inflammation (Navarro et al., 2001) Kalopanaxsaponin A and pictoside A, both two isolated triterpenoid saponins from the stem bark of Kalopanax pictus Nakai

(Araliaceae) showed significant anti-inflammatory activity at the oral dose of 50 mg/mL (Li et al., 2002).

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