ANTIOXIDANT EFFECTS OF RHODOMYRTUS TOMENTOSA (KEMUNTING) EXTRACT ON THE DEVELOPMENT OF
EXPERIMENTAL ATHEROSCLEROTIC-INDUCED NEW ZEALAND WHITE RABBIT
MUHAMAD FAHRIN BIN MASKAM
DISSERTATION SUBMITTED IN FULLFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE
INSTITUTE OF BIOLOGICAL SCIENCES FACULTY OF SCIENCE
UNIVERSITY OF MALAYA KUALA LUMPUR
2011
UNIVERSITI MALAYA
Nama: Muhamad Fahrin Bin Maskam (No. K.P/Pasport: 790616-14-5443) No. Pendaftaran/Matrik: SGR 070098
Nama Ijazah:Master Of Science
Tajuk Kertas Projek/Laporan Penyelidikan/Disertasi/Tesis (“Hasil Kerja ini”):
Antioxidant Effects of Rhodomyrtus tomentosa (Kemunting) Extract On The Development Of Experimental Atherosclerotic-Induced New Zealand White Rabbit
Bidang Penyelidikan: Physiology
Saya dengan sesungguhnya dan sebenarnya mengaku bahawa:
(1) Saya adalah satu-satunya pengarang/penulis Hasil Kerja ini;
(2) Hasil Kerja ini adalah asli;
(3) Apa-apa penggunaan mana-mana hasil kerja yang mengandungi hakcipta telah dilakukan secara urusan yang wajar dan bagi maksud yang dibenarkan dan apa-
apa petikan, ekstrak, rujukan atau pengeluaran semula daripada atau kepada mana-mana hasil kerja yang mengandungi hakcipta telah dinyatakan dengan sejelasnya dan secukupnya dan satu pengiktirafan tajuk hasil kerja tersebut dan pengarang/penulisnya telah dilakukan di dalam Hasil Kerja ini;
(4) Saya tidak mempunyai apa-apa pengetahuan sebenar atau patut
semunasabahnya tahu bahawa penghasilan Hasil Kerja ini melanggar suatu hakcipta hasil kerja yang lain;
(5) Saya dengan ini menyerahkan kesemua dan tiap-tiap hak yang terkandung di dalam hakcipta Hasil Kerja ini kepada Universiti Malaya (“UM”) yang seterusnya mula dari sekarang adalah tuan punya kepada hakcipta di dalam Hasil Kerja ini dan apa-apa pengeluaran semula atau penggunaan dalam apa jua bentuk atau dengan apa juga cara sekalipun adalah dilarang tanpa terlebih dahulu mendapat kebenaran bertulis dari UM;
(6) Saya sedar sepenuhnya sekiranya dalam masa penghasilan Hasil Kerja ini saya telah melanggar suatu hakcipta hasil kerja yang lain sama ada dengan niat atau sebaliknya, saya boleh dikenakan tindakan undang-undang atau apa-apa tindakan lain sebagaimana yang diputuskan oleh UM.
Tandatangan Calon Tarikh
Diperbuat dan sesungguhnya diakui di hadapan ,
Tandatangan Saksi Tarikh
Nama:
Jawatan:
ABSTRACT
The in vitro study was conducted to determine the antioxidant activity of R. tomentosa in water, methanol, chloroform and petroleum ether extract. In DPPH assay, methanol extract had the highest antioxidant capacity with IC50 of 107µg/ml. Antioxidant activity of R. tomentosa measured using FRAP assay also showed that methanol extract had the highest antioxidant capacity 0.162nm at concentration of 500µg/ml. While in Metal Chelating assay again showed methanol extract had the most chelating ability of all extracts with 36% at concentration of 100µg/ml. The chemical compounds found in R.
tomentosa were quercetin, gallic acid and tannic acid from HPLC analyses; malic acid, caffeic acid, dihydrocaffeic acid, quinic acid, brevifolin carboxylic acid, octadecenoic acid and galloyl glucose from GCMS analyses. Total phenolic content (TPC) of the extracts estimated as gallic acid equivalent was highest in water extract at 66.515mg/g dry mass. Total flavonoid content (TFC) estimation was highest in water extract measured at 1.828mg/ml. Toxicity of the extracts was assessed in vitro by Artemia salina lethality. The result showed, water extract of R. tomentosa was the most non- toxic with LC50 at 616.083µg/ml. Group of rabbits treated with tomentosa extract had significantly lower (p<0.05) in LDL and total cholesterol (TC) against cholesterol group. Meanwhile high density lipoprotein (HDL) did show significant increase (p<0.05) compared to cholesterol group. The study also showed a significant reduction of lipid peroxidation index indicated by low TBARs-MDA level (p<0.05) in tomentosa group compared to cholesterol group. Histopathology analysis of aorta presented with thick foam cell formation in cholesterol group, however there were less foam cell formation in tomentosa and simvastatin group. Kidney and liver showed less infiltration of inflammatory cell. In conclusion, the supplementation of 50mg/kg/day of R.
tomentosa extract was able to prevent and reduce the progression of atherosclerotic
plaque development induced by dietary cholesterol of 1%. The reduced progression was due to the chemical compounds rich in antioxidant and other bioactive compounds.
ABSTRAK
Kajian in vitro telah dijalankan untuk menguji aktiviti antioksidan R. tomentosa dalam ekstrak air, methanol, chlorofom dan petroleum ether. Dalam ujian DPPH, ekstrak methanol menunjukkan kadar antioksidan yang tertinggi dengan IC50 pada 107µg/ml.
Aktiviti antioksidan dalam R. tomentosa yang diuji menggunakan teknik FRAP juga menunjukkan methanol ekstrak sebagai yang tertinggi dengan kapasiti 0.162nm pada kepekatan 500µg/ml. Teknik Metal Chelating juga menunjukkan ekstrak methanol sebagai ekstrak yang paling tinggi keupayaan pada 36% untuk kepekatan 100µg/ml.
Bahan kimia yang telah dikenalpasti ialah quercetin, asid galik dan asid tanik melalui analisis HPLC; asid malik, kafeik asid, dihidrokafeik asid, kuinik asid, brevifolin karboksilik asid, oktadekenoik asid dan galoil glukosa melalui analisa GCMS.
Kandungan keseluruhan phenolic (TPC) adalah yang tertinggi dalam ekstrak air pada 66.515mg/g berat kering. Kandungan keseluruhan flavanoid (TFC) juga paling tinggi dalam ekstrak air pada 1.828mg/ml. Kesan toksik pada ekstrak dianalisa dengan ujian in vitro Artemia salina.hasil ujian ini menunjukkan ekstrak air R. tomentosa adalah yang paling selamat dengan nilai LC50 616.083µg/ml. Kumpulan arnab yang telah diberikan ekstrak tomentosa telah menunjukkan pengurangan (p<0.05) dalam lipoprotein berketumpatan rendah (LDL) dan kolesterol total (TC) berbanding dengan kumpulan kolesterol. Manakala kolesterol berketumpatan tinggi (HDL) menunjukkan peningkatan signifikasi (p<0.05) berbanding dengan kumpulan kolesterol. Kajian ini juga menunjukkan pengurangan signifikasi (p<0.05) pada indeks peroksidasi lipid yang diindikasi oleh TBARs-MDApada kumpulan tomentosa berbanding dengan kumpulan kolesterol. Analisis histopatologi pada aorta menunjukkan pembentukan tebal sel foam pada kumpulan kolesterol, walaubagaimanapun terdapat pengurangan sel busa dalam kumpulan tomentosa dan kumpulan simvastatin. Organ seperti ginjal dan hati telah terdapat pengurangan daripada segi infiltrasi sel-sel inflamasi. Kesimpulan daripada
kajian ini adalah ekstrak R. tomentosa sebanyak 50mg/kg/sehari telah berjaya mengurangkan pembentukan ateroskerosis yang diaruhkan oleh diet kolesterol sebanyak 1%. Pengurangan proses pembentukan adalah kerana terdapat bahan kimia yang kaya dengan antioksidan dan beberapa bahan bioaktif lain.
ACKNOWLEDGEMENTS
I would like to express my heartfelt gratitude to Dr Jamaludin Bin Mohamad, lecturer of the Department of Biohealth, Faculty of Science, University Malaya, for his advice throughout the duration of this thesis, continuous guidance and his patience have provided a good basis for the present thesis.
I wish also to thank to Associate Professor Dr Mahmood Ameen Abdulla from Department of Molecular Medicine, Faculty of Medicine, University of Malaya for assisting in animal and histology research and Associate Professor Dr Zulkhairi Amom from Department of Human Anatomy, Faculty Of Medicine, University Putra Malaysia for their support, both in scientific matter, valuable comments and suggestions.
My master research would not be completed without very helpful staff members from Biohealth Department. I like to thanks Mr Mohd Rozlan Md Tam, Assistant Officer of Science, Elias Megat Ahmad, Assistant Officer of Science Syed Mohd Aliff Syed Alias, Laboratory Assistant and Ruzaimah Sadimin, General Office Assistant for providing working facilities and positive attitudes towards my studies and greatly acknowledged for their helpfulness.
I also wish to thank Nor Syuhada Ahmad Bashah, tutor from department of Molecular Medicine for hers guidance in statistical analysis. Throughout this master research, I have greatly indebted to my colleagues Farah Liyana Jamaludin, Faezah Mohd, Apiah Mohd Amin, and Che Wan Imanina who have helped me with my work research. Their kind supports and guidance that have been great value in this study.
And lastly I would like to thank for the financial support of research grant provided by University Malaya.
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CONTENTS
ABSTRACT i
ABSTRAK iii
ACKOWLEDGEMENTS v
CONTENTS vi
LIST OF TABLES x
LIST OF FIGURES xii
LIST OF SYMBOLS AND ABBREVIATIONS xvi
CHAPTER 1.0 INTRODUCTION 1
CHAPTER 2.0 LITERATURE REVIEW 6
2.1 Studied plant - Rhodomyrtus tomentosa 6
2.2 Description 7
2.3 Chemical Constituent of R. tomentosa 8
2.4 Medicinal uses 12
2.5 Antioxidants 14
2.6 Role of antioxidants in prevention of atherosclerosis 17
2.7 Lipid Peroxidation 20
2.8 Role of lipid peroxidation 24
2.9 Cholesterol 27
2.10 Plasma Lipids 29
2.11 Lipoprotein Transport in the Blood 30
2.12 Cholesterol Homeostasis in Physiological System 32
2.13 Low Density Lipoprotein (LDL) 35
2.14 High Density Lipoprotein (HDL) 36
2.15 Triglycerides (TG) 38
2.16 Hypercholesterolemia 39
2.17 Lipid Lowering Drugs as Treatment of Hypercholesterolemia 42
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2.18 Malondialdehyde (MDA) 46
CHAPTER 3.0: MATERIALS AND METHOD 48
3.1 Plant materials 48
3.2 Extraction of Plant compound 48
3.3 Separation of Chemical Compound 49
3.3.1 Identification of chemical compounds in R. tomentosa 49
3.4 HPLC and GCMS analysis 52
3.4.1 HPLC analysis 52
3.4.1.1 Sample preparation for HPLC 52
3.4.1.2 Instruments for HPLC analyses 52
3.4.2 GCMS analysis 53
3.4.2.1 Sample preparation for GCMS 53
3.4.2.2 Instruments for GCMS analyses 53
3.5 Antioxidant Assay 54
3.5.1 DPPH assay 54
3.5.2 Ferric Reducing Power Assay 55
3.5.3 Metal Chelating Assay 56
3.6 Total Phenolic Content (TPC) 59
3.7 Total Flavonoid Content (TFC) 60
3.8 Brine Shrimp Lethality Assay (BSLA) 61
3.9 Animal Study 62
3.9.1 Maximum Tolerated Dose (MTD) 62
3.9.2 Experimental Animals 63
3.9.2.1 Dietary composition to Experimental Animals 63
3.9.2.2 Preparation of simvastatin drug treatment 64
3.9.2.3 Preparation of Plant extract treatments 64
3.9.2.4 Preparation of Blood samplings 65
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3.10 Lipid Profile analysis 66
3.10.1 Total Cholesterol (TC) 66
3.10.2 High Density Lipoprotein (HDL) 68
3.10.3 Triacylglycerides (TG) 70
3.10.4 Low Density Lipoprotein (LDL) 71
3.11 Lipid Peroxidation index, TBARs-Melondialdehyde (MDA) analysis 72
3.12 Histology study 74
3.12.1 Aorta preparation 74
3.12.2 Sudan IV staining 75
3.12.3 Haematoxyline and Eosin Staining (H and E) 76
3.12.4 Tissue sectioning and staining 76
3.13 Statistical analysis 77
CHAPTER 4.0 RESULTS 78
4.1 Phytochemical Detection by (TLC) on Rhodomyrtus tomentosa extracts 78
4.2 HPLC and GCMS analyses 83
4.3 Antioxidant assays 98
4.4 Total Phenolic Content (TPC) 102
4.5 Total Flavonoid Content (TFC) 104
4.6 Brine Shrimp Lethality Assay (BSLA) 106
4.7 Maximum Tolerated Dose (MTD) 109
4.8 Organ Weight and Parameter 111
4.9 Lipid Profile Test 116
4.10 TBARS-Malondialdehyde (MDA) 126
4.11 Histology examination of Hematoxyline and Eosin (H and E) 129
4.11.1 Histology examination of aorta 129
4.11.2 Histology examination of kidney 133
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4.11.3 Histology examination of liver 137
4.12 Evaluation of Atherosclerotic Area Macroscopically 141
CHAPTER 5.0 DISCUSSION 146
CONCLUSION 190
REFERENCES 192
APPENDIX 217
BIODATA OF AUTHOR 276
LIST OF TABLES
Table Page
2.1 Scientific Classification of Rhodomyrtus tomentosa 6
2.3 Chemical compounds isolated from Rhodomyrtus tomentosa 9
2.17 Frederickson/WHO Classification of hyperlipoproteinaemia 42
3.5.1 Reaction of mixtures containing ascorbic acid, DPPH, and methanol 54
as positive standard references 3.5.3 Reaction mixtures of deionized water, EDTA, FeCl2 2mM 57
and ferrozine 5mM 3.9.2.1 Dietary composition and treatment for each group of animal 63
3.12.3 Tissue dehydration in a tissue processor machine 76
3.12.4 Colourisation with Hematoxyline and Eosin (H&E) 77
4.1.1 TLC analysis of crude water extract from the fruits of Rhodomyrtus 79
tomentosa 4.1.2 TLC analysis of crude methanol extract from the fruits of Rhodomyrtus 80
tomentosa 4.1.3 TLC analysis of crude chlorofoam extract from the fruits of Rhodomyrtus 81 tomentosa 4.1.4 TLC analysis of crude petroleum ether extract from the fruits of 82
Rhodomyrtus tomentosa 4.2.1 Retention time of standard during HPLC analysis 83
4.2.2 Retention time of R. tomentosa extract during HPLC analysis 83
4.2.3 Identification of phytochemicals in water extracts from R. tomentosa 96
fruit extract 4.4.1 Total phenolic content of different Rhodomyrtus tomentosa extracts 102
4.5.1 Total flavonoid content of different Rhodomyrtus tomentosa extracts 104
4.6.1 LC50 of crude water extract of Rhodomyrtus tomentosa 106
4.6.2 LC50 of crude methanol extract of Rhodomyrtus tomentosa 107
4.6.3 LC50 of crude chlorofoam extract of Rhodomyrtus tomentosa 107
4.6.4 LC50 ofcrude petroleum ether extract of Rhodomyrtus tomentosa 108
4.7.1 Maximum tolerated dose of water extract of Rhodomyrtus tomentosa 109 based on body weight effect of male rabbit
4.7.2 Maximum tolerated dose of water extract of Rhodomyrtus tomentosa 109 based on body weight effect of female rabbit
4.7.3 Maximum tolerated dose of methanol extract of 110 Rhodomyrtus tomentosa based on body weight effect of male rabbit
4.7.4 Maximum tolerated dose of methanol extract of 110 Rhodomyrtus tomentosa based on body weight effect of female rabbit
LIST OF FIGURES
Figure Page
2.2.1 Rhodomyrtus tomentosa fruits 7
2.2.2 Rhodomyrtus tomentosa flowers 7
2.6 Role of antioxidants in atherogenic process 19
2.8 Role of oxidatively-modified low-density lipoprotein (LDL) in atherogenesis 26
2.11 Schematic diagram of cholesterol transport 31
2.12 Transcription process by SREPs in endoplasmic reticulum 34
2.14 HDLs protection against atherosclerosis by several mechanisms 36
2.16.1 Type of aortic lesion 40
2.16.2 Pathways in evolution and progression of human atherosclerotic lesions 41
2.16 Structure of simvastatin 43
3.3 Diagram of determination of Retention factor (Rf) value on TLC plate 50
3.10.1 Pathways of chemical reactions in total cholesterol (TC) measurement 67
3.10.2 Pathways of chemical reaction in high density lipoprotein (HDL) 68
Measurement 3.10.3 Pathways of chemical reactions in triglycerides (TG) measurement 70
3.10.4 Pathways of chemical reactions in low density lipoprotein (LDL) 71
measurement 4.2.1 HPLC chromatogram of standard gallic acid at wavelength 280nm 4nm. 84
4.2.2 HPLC chromatogram of standard gallic acid at wavelength 360nm 4nm 84
4.2.3 HPLC chromatogram of R. tomentosa at wavelength 280nm 4nm 85
4.2.4 HPLC chromatogram of R. tomentosa at wavelength 360nm 4nm 85
4.2.5 HPLC chromatogram of standard quercetin acid at wavelength 280nm 4nm 86 4.2.6 HPLC chromatogram of standard quercetin acid at wavelength 360nm 4nm 86
4.2.7 HPLC chromatogram of R. tomentosa at wavelength 280nm 4nm 87
4.2.8 HPLC chromatogram of R. tomentosa at wavelength 360nm 4nm 87
4.2.9 HPLC chromatogram of standard tannic acid at wavelength 280nm 4nm 88
4.2.10 HPLC chromatogram of standard tannic acid at wavelength 360nm 4nm 88
4.2.11 HPLC chromatogram of R. tomentosa at wavelength 280nm 4nm 89
4.2.12 HPLC chromatogram of R. tomentosa at wavelength 360nm 4nm 89
4.2.13 ESI-MS at scan mode of water extract of R. tomentosa fruit 90
TOF-MS 100-750 4.2.14 ESI-MS at scan mode of water extract of R. tomentosa fruit 91
TOF-MS 100-165 4.2.15 ESI-MS at scan mode of water extract of R. tomentosa fruit 92
TOF-MS 165-184 4.2.16 ESI-MS at scan mode of water extract of R. tomentosa fruit 93
TOF-MS 184-215 4.2.17 ESI-MS at scan mode of water extract of R. tomentosa fruit 94
TOF-MS 255-325 4.2.18 ESI-MS at scan mode of water extract of R. tomentosa fruit 95
TOF-MS 334-365 4.3.1 Free-radical scavenging activity of crude extract from fruits of 99
R. tomentosa measured by DPPH assay. 4.3.2 Ferric Reducing Antioxidant Power of crude extract from fruits of 100
R. tomentosa. 4.3.3 Metal chelating ability of the crude extract from the fruits of R. tomentosa 101
4.4.1 Graph of standard gallic acid 103
4.5.1 Graph of Standard quecertin 105
4.8.1 Dissected heart organs 111
4.8.2 Dissected liver organs 112
4.8.3 Dissected kidney organs 113
4.8.4 Heart, Liver and Kidney weight of experimental rabbits 114
4.9.1 The effect of R. tomentosa water extract on body weight at different week 117
4.9.2 The effect of R. tomentosa water extract on Triacylglycerides (TG) 119
at different week 4.9.3 The effect of R. tomentosa water extract on Total Cholesterol (TC) 121
at different week 4.9.4 The effect of R. tomentosa water extract on Low Density Lipoprotein 123 (LDL) at different week
4.9.5: The effect of R. tomentosa water extract on High Density Lipoprotein 125 (HDL) at different week
4.10.1 The effect of R .tomentosa water extract on lipid peroxidation index, 128 indicated by malondialdehyde (MDA) at different time
4.11.1 Cross section of aorta tissue specimen from normal group stained with 129 H & E at LM X 10
4.11.2 Cross section of aorta tissue specimen from normal group stained with 129 H&E at LM X 20
4.11.3 Cross section of aorta tissue specimen from cholesterol group stained with 130 H&E at LM X 4
4.11.4 Cross section of aorta tissue specimen from cholesterol group stained with 130 H&E at LM X 20
4.11.5 Cross section of aorta tissue specimen from R. tomentosa group stained 131 with H&E at LM X 10
4.11.6 Cross section of aorta tissue specimen from R. tomentosa group stained 131 with H&E at LM X 20
4.11.7 Cross section of aorta tissue specimen from simvastatin group stained 132 with H&E at LM X 10
4.11.8 Cross section of aorta tissue specimen from simvastatin group stained 132 with H&E at LM X 20
4.11.9 Cross section of kidney tissue specimen from normal group with 133 H&E at LM X 10
4.11.10 Cross section of kidney tissue specimen from normal group with 133 H&E at LM X 20
4.11.11 Cross section of kidney tissue specimen from cholesterol group with 134 H&E at LM X 10
4.11.12 Cross section of kidney tissue specimen from cholesterol group with 134 H&E at LM X 20
4.11.13 Cross section of kidney tissue specimen from R. tomentosa group 135 with H&E at LM X 10
4.11.14 Cross section of kidney tissue specimen from R. tomentosa group 135 with H&E at LM X 20
4.11.15 Cross section of kidney tissue specimen from simvastatin group 136 with H&E at LM X 10
4.11.16 Cross section of kidney tissue specimen from simvastatin group 136 with H&E at LM X 10
4.11.17Cross section of liver tissue specimen from normal group 137 with H&E at LM X 10
4.11.18 Cross section of liver tissue specimen from normal group 137 with H&E at LM X 20
4.11.19 Cross section of liver tissue specimen from cholesterol group 138 with H&E at LM X 10
4.11.20 Cross section of liver tissue specimen from cholesterol group 138 with H&E at LM X 20
4.11.21 Cross section of liver tissue specimen from R. tomentosa group 139 with H&E at LM X 10
4.11.22 Cross section of liver tissue specimen from R. tomentosa group 139 with H&E at LM X 10
4.11.23 Cross section of liver tissue specimen from simvastatin group 140 with H&E at LM X 10
4.11.24 Cross section of liver tissue specimen from simvastatin group 140 with H&E at LM X 20
4.12.1 Histomorphoric analysis of intimal lesion area in different group 141 4.12.2 Aortas of Rabbit in Normal group which stained with Sudan IV without 142 showing any atherosclerotic area
4.12.3 Aortas of Rabbit in Cholesterol group which stained with Sudan IV 143 showing marked atherosclerotic area
4.12.4 Aortas of Rabbit in Tomentosa group which stained with Sudan IV 144 showing atherosclerotic area
4.12.5 Aortas of Rabbit in Simvastatin group which stained with Sudan IV 145 showing atherosclerotic area
5.1 TBA-MDA complex 176
LIST OF SYMBOLS AND ABBREVIATIONS
Abs = absorbance
BHA = butylated hydroxyanisole
EDTA = ethylene-diamine-tetraacetic acid BSLA = brine shrimp lethality assay TLC = thin layer chromatography
CC = column chromatography
CH3CN = acetonitrile
EDTA = ethylenediaminetetraacetic acid EtOAc = ethyl acetate
EtOH = ethanol
FeCl2 = ferrus chloride
GAE = gallic acid equivalents
HPLC = high performance liquid chromatography H2O = distilled water
H2SO4 = sulphuric acid HOAc = acetic acid
HCl = hydrochloric
KI = kalium iodide
Na2HPO4 = sodium phosphate monobasic OD = optical density
PUFA = polyunsaturated fatty acid
UV = ultraviolet
Rf = relative mobility
ºC = degree celsius
cm = centimeter
g = gram
µg = microgram
mg = milligram
m = meter
M = molarity mM = milimol
ml = milliliter
mm = millimeter
µl = microliter
L = liter
nm = nanometer
rpm = revolutions per minute
min = minute
w/v = weight/volume
LM = longitudinal magnification
% = percentage
IC50 = half maximal inhibitory activity
≤ = less than or equal to
≥ = more than or equal to + = add
- = minus / = divide
BIODATA OF THE AUTHOR
Born on 16th June 1979, Kuala Lumpur, Muhamad Fahrin Bin Maskam took an early interest in science when in early childhood. He went to Sekolah Rendah Kebangsaan Jalan Gurney, Kuala Lumpur, Sekolah Menengah Seksyen 18, Shah Alam, and Maktab Rendah Sains Mara, Jasin for his primary and secondary education. Graduated from Universiti Putra Malaysia in Biomedical Sciences, he further developed his fascination in medical and research especially in physiology. Graduating from biomedical sciences, he had many opportunities working in medical and research line through various organization and company. In addition to his working experience, he has gained invaluable skills such as first response life savers that were thought in hospitals for medical personnel. With the awareness of cardiovascular disease as the main cause of mortality in Malaysia, he took this challenge for his contribution in prevention of this disease. Throughout his study in master’s degree, he had been to various conferences and seminars to share his findings regarding this research and to gain more knowledge in scientific research. He hoped that throughout all his effort, he managed to contribute towards the development of natural products in providing another alternative treatment of cardiovascular diseases.
