quality control analysis, standardization and stability studies of ...
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QUALITY CONTROL ANALYSIS, STANDARDIZATION AND STABILITY STUDIES OF EURYCOMA LONGIFOLIA AND EFFECT OF ITS SAPONIN CONTENT ON SOLUBILITY AND TOXICITY OF EURYCOMANONE NURSYAZURA BINTI KHARI UNIVERSITI SAINS MALAYSIA 2014
Transcript of quality control analysis, standardization and stability studies of ...
QUALITY CONTROL ANALYSIS,
STANDARDIZATION AND STABILITY STUDIES OF
EURYCOMA 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 EURYCOMA LONGIFOLIA 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
ii
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.
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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 Plants ................................................................. 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 Taxonomy ....................................................................................... .7
2.1.2 Vernacular Names .......................................................................... .7
2.1.3 Plant 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 Leaf 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 longifolia root, stem and leaf raw material
28
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 stability studies
45
Table 3.6 Changes of colour for steroidal and triterpenoidal saponins
49
Table 4.1 Heavy metal content of Eurycoma longifolia root, stem and leaf raw material and their limit (WHO, 1998)
63
Table 4.2 Precision analysis of retention time and peak area in terms of %RSD. Retention (RT) time and peak area (PA) are presented as average ±SD (n=5)
93
Table 4.3 LOD and LOQ value using HPLC method. Results are displayed as average ±SD (n=5)
94
Table 4.4 Pre-exponential factor of eurycomanone in Eurycoma longifolia root, stem and leaf water extracts
124
Table 4.5 Absorbance of Liebermann-Burchard reagent, reference and mixture solution from 390 – 450 nm
131
Table 4.6 Fluorescence intensity of pyrene added to various concentration of crude root water extract (TA)
136
Table 4.7 Fluorescence intensity of pyrene added to various concentration of the saponins-rich extract
137
Table 4.8 Fluorescence intensity of pyrene added to various concentration of the eurycomanone-rich extract
138
Table 4.9 Particle size of root water extract. The results are presented as average ± SD (n=3)
139
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Table 4.10 Summary of the cytotoxic effect of eurycomanone (25 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)
144
x
LIST OF FIGURES
Pages
Figure 2.1 Pictures of Eurycoma longifolia (a) - (b) whole plant, (c) leaves and (d) root
9
Figure 2.2 Chemical structure of Eurycomanone 14
Figure 2.3 Aglycone skeletons of (a) steroidal spirostane and (b) triterpenoid saponins. R = sugar moiety
23
Figure 3.1 Flow chart of preparation of saponins and eurycomanone-rich extracts from root water extract
48
Figure 4.1 Total moisture contents of Eurycoma longifolia root stem and leaf raw material. Results are expressed as the mean ± SD (n=3)
58
Figure 4.2 Total ash contents of Eurycoma longifolia root, stem and leaf raw material. Results are expressed as the mean ± SD (n=3)
57
Figure 4.3 Acid insoluble ash contents of Eurycoma longifolia root, stem and leaf raw material. Results are expressed as the mean ± SD (n=3)
58
Figure 4.4 Extractive values of Eurycoma longifolia root raw material from five different localities
61
Figure 4.5 Extractive values of Eurycoma longifolia stem raw material from five different localities
61
Figure 4.6 Extractive values of Eurycoma longifolia leaf raw material from five different localities
61
Figure 4.7 Parameters and percentage yield for different extraction method of Eurycoma longifolia root from Perak
64
Figure 4.8 HPTLC fingerprints of Eurycoma longifolia root water 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
66
Figure 4.9 UV-Vis spectrum of eurycomanone standard, λmax was between 248 and 255 nm
67
Figure 4.10 2D HPTLC densitogram of the eurycomanone standard and Eurycoma longifolia root water extracts from different extraction time and solvent ratio at 254 nm
67
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Figure 4.11 HPTLC chromatogram of eurycomanone standard and 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
68
Figure 4.12 Concentration of eurycomanone in Eurycoma longifolia root water extracts from different extraction time and solvent ratio
70
Figure 4.13 Percentage yield of water extracts of different parts of Eurycoma longifolia from five different localities
71
Figure 4.14 ATR-FTIR profile of the Eurycoma longifolia root water extracts from five different localities
74
Figure 4.15 ATR-FTIR profile of the Eurycoma longifolia stem water extracts from five different localities
75
Figure 4.16 ATR-FT-IR profile of the Eurycoma longifolia leaf water extracts from five different localities
76
Figure 4.17 ATR-FT-IR profile of the Eurycoma longifolia commercial products form six different brands
77
Figure 4.18 Score plot (A) of the two principal components analysis of ATR-FTIR spectra of Eurycoma longifolia root water extracts from five different localities at 4000-650 cm-1
81
Figure 4.19 Score plot (A) of the two principal components analysis of ATR-FTIR spectra of Eurycoma longifolia stem water extracts from five different localities at 4000-650 cm-1
82
Figure 4.20 Score plot (A) of the two principal components analysis of ATR-FTIR spectra of Eurycoma longifolia leaf water extracts from five different localities at 4000-650 cm-1
82
Figure 4.21 Score plot (A) of the two principal components analysis of ATR-FTIR spectra of Eurycoma longifolia commercial products from six different brands at 4000-650 cm-1
83
Figure 4.22 Score plot (A) of the two principal components analysis 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
83
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Figure 4.23 HPTLC profile of Eurycoma longifolia root, stem and leaf water extracts from five different localities observed under UV 254 nm
85
Figure 4.24 HPTLC profile of Eurycoma longifolia root, stem and leaf water extracts from five different localities observed under UV 366 nm
85
Figure 4.25 3D densitograms of eurycomanone and Eurycoma longifolia root, stem and leaf water extracts from five different localities at 254 nm
86
Figure 4.26 HPTLC profile of eurycomanone and Eurycoma longifolia commercial products from six different brands observed under UV 254 nm
88
Figure 4.27 HPTLC profile of eurycomanone and Eurycoma longifolia commercial products from six different brands observed under UV 366 nm
88
Figure 4.28 3D densitograms of HPTLC of the eurycomanone and Eurycoma longifolia commercial products from six different brands at 254 nm
89
Figure 4.29 Chromatogram of eurycomanone and Eurycoma longifolia commercial products from six different brands obtained by HPTLC method at 254 nm
90
Figures 4.30 HPLC chromatogram of eurycomanone and Eurycoma longifolia root water extracts from five different localities
98
Figures 4.31 HPLC chromatogram of eurycomanone and Eurycoma longifolia stem water extracts from five different localities
99
Figures 4.32 HPLC chromatogram of eurycomanone and Eurycoma longifolia leaf water extracts from five different localities
100
Figure 4.33 HPLC chromatogram of eurycomanone and Eurycoma longifolia commercial products from six different brands
101
Figure 4.34 Concentration of eurycomanone in Eurycoma longifolia root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)
102
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Figure 4.35 Concentration of eurycomanone in Eurycoma longifolia commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)
102
Figure 4.36 Total phenolics contents in Eurycoma longifolia root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)
104
Figure 4.37 Total phenolics contents in Eurycoma longifolia commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)
104
Figure 4.38 Total glycosaponins contents in Eurycoma longifolia root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)
107
Figure 4.39 Total polysaccharides contents in Eurycoma longifolia root, stem and leaf water extracts from five different localities. Results are shown as %wt/wt ±SD (n=3)
107
Figure 4.40 Total glycosaponins contents of Eurycoma longifolia commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)
108
Figure 4.41 Total polysaccharides contents of Eurycoma longifolia commercial products from six different brands. Results are shown as %wt/wt ±SD (n=3)
108
Figure 4.42 Comparison of total proteins contents of Eurycoma longifolia root, stem and leaf water extracts by using Lowry method and Bradford method. Results are shown as %wt/wt ±SD (n=3)
110
Figure 4.43 Comparison of total proteins contents of Eurycoma longifolia commercial products by using Lowry method and Bradford method. Results are shown as %wt/wt ±SD (n=3)
110
Figure 4.44 Remaining percentage concentration of eurycomnone in root (BR) stored at different storage conditions over 6 months period
114
Figure 4.45 Remaining percentage concentration of eurycomnone in stem (BS) stored at different storage conditions over 6 months period
115
Figure 4.46 Remaining percentage concentration of eurycomnone in leaf (BL) stored at different storage conditions over 6 months period
115
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Figure 4.47 Plot of (ln C%) against time for eurycomanone in root (BR) at different storage conditions
117
Figure 4.48 Plot of (ln C%) against time for eurycomanone in stem (BS) at different storage conditions
117
Figure 4.49 Plot of (ln C%) against time for eurycomanone in leaf (BL) at different storage conditions
118
Figure 4.50 Rate constant (K) of degradation reaction of eurycomanone in Eurycoma longifolia root, stem and leaf water extracts
120
Figure 4.51 Arrhenius plot for eurycomanone in Eurycoma longifolia root water extract (BR)
121
Figure 4.52 Arrhenius plot for eurycomanone in Eurycoma longifolia stem water extract (BS)
121
Figure 4.53 Arrhenius plot for eurycomanone in Eurycoma longifolia leaf water extract (BL)
122
Figure 4.54 Activation energy of eurycomanone in Eurycoma longifolia root, stem and leaf water extracts
123
Figure 4.55 Calculated shelf life of eurycomanone in Eurycoma longifolia root, stem and leaf water extracts
127
Figure 4.56 Chromatogram of a) saponins-rich extract, b) eurycomanone-rich extract and c) eurycomanone standard
129
Figure 4.57 UV spectra for Liebermann-Burchard reagent, reference and mixture solution from 700 – 200 nm
131
Figure 4.58
Retention time (RT) and area of eurycomanone and unknown compounds from solubility analysis by HPLC
134
Figure 4.59 Chromatogram of eurycomanone-rich extract alone and combination of various saponins-rich extract ratios with eurycomanone-rich extract
134
Figure 4.60 Critical micellar concentration of crude root water extract (TA)
136
Figure 4.61 Critical micellar concentration of saponins-rich extract 137
Figure 4.62 Critical micellar concentration of eurycomanone-rich extract
138
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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 sample was analyzed at a magnification of 53K, 120kV
140
Figure 4.65 The dose response curves of eurycomanone-rich extract on HCT 116 cells
141
Figure 4.66 Effect of saponins-rich extract and eurycomanone-rich 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
142
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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
kJ/mol
International Conference on Harmonisation
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
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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
xxi
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.
xxii
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
xxiii
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.
xxiv
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.
1
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.
2
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).
3
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).
4
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).
5
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.
6
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.
7
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: Sapindales
Family: Simaroubaceae
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).
8
2.1.3 Plant Description
E. longifolia Jack is a slender, evergreen flowering tree belongs to the family
Simaroubaceae, is occurring naturally in jungles of Malaysia and Indonesia. The
female and male trees 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 flowers 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).
9
(a)
(b)
(c)
(d)
Figure 2.1: Pictures of Eurycoma longifolia (a) - (b) whole plant, (c) leaves and (d) root
10
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 part
Extract Chemical constituents References
Root Aqueous quassinoids (e.g. eurycolactones A-E, eurycomalides A-B, eurycomalactone, 6α-hydroxyeurycomalactone, 7α-hydroxyeurycomalactone, eurycomanone, 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-
(Chua et al., 2011; Chua et al., 2012; Yusmazura et al., 2009)
11
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, eurycomalides A-B, eurycomalactone, 6α-hydroxyeurycomalactone, 6-hydroxy-5,6-dehydroeurycomalactone, 5,6-dehydroeurycomalactone, eurycomanone, 13β,21-dihydroxyeurycomanol, 14,15β-dihydroxyklaineanone, 5α,14β,15β-trihydroxyklaineanone, laurycolactones A-B, longilactone, 6-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-
(Kardono et al., 1991; Chan et al., 1991; Chan et al., 1992; Ang et al., 2000; Ang et al., 2002; Kuo et al., 2003b; Teh et al., 2010; Kuo et al., 2003a)
12
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 (50%)
quassinoids (e.g. eurycomanone, 13,21-dihydroeurycomanone, 13α(21)-epoxyeurycomanone, longilactone, eurycomalactone, 14,15β-dihydroxyklaineanone, eurycomanol, eurycomanol-2-O-β-glucopyranoside) and a canthin-6-one alkaloid (e.g. 9-methoxycanthin-6-one).]
(Chan et al., 1989; Chan and Choo, 2002; Chan et al., 2004)
13
Aqueous
and Methanol
Volatile components were also detected in the aqueous and methanol root 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.
(Shafiqul et al., 2006)
Leaves Ethanol Quassinoids (lonilactone, 6-dehydro lonilactone,11-dehydroklaineanone, 12-epi dehydroklaineanone, 15β-hydroxyklaineanone, 14,15 β-dihydroxyklaineanone, and 15-β-O-acetyl-14-hydroxyklaineanone)
(Jiwajinda et al., 2001)
14
CH3
O
OHCH3
O O
OHOH
CH2
OHOH
O
H H
H
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
15
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
16
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
17
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.
19
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).
20
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).
23
O
RO
CH3
CH3
CH3O
CH3
RO
CH3CH3
CH3 CH3
CH3
CH3
CH3 CH3
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
a
b
24
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).
