FACTORS REGULATING NITRIC OXIDE
PRODUCTION IN SPONTANEOUSLY HYPERTENSIVE RATS TREATED WITH Piper sarmentosum
BY
TAHER F T ELSHAMI
A dissertation in fulfillment of the requirement for the degree of Master of Medical Sciences
Kulliyyah of Medicine
International Islamic University Malaysia
APRIL 2018
ii
ABSTRACT
Introduction: Hypertension is a major risk factor for cardiovascular diseases making it one of the leading causes of death worldwide. Piper sarmentosum (PS) leaves have been widely used in traditional medicine for the treatment of diseases including hypertension. Phenolic compounds are the major active constituents in PS. The dose of Piper sarmentosum aqueous extract (PSAE) which reduces the blood pressure (BP) effectively has been patented as Kadukmy™. Objectives: This study aims to evaluate the antihypertensive potential of Kadukmy™ and to investigate the factors modulating nitric oxide (NO) production through its antioxidant activities. Method: The PS leaves were extracted with distilled water and freeze-dried. The PSAE was examined to quantify their antioxidant activities through the DPPH test, FRAP test and then screened for the total phenolic content (TPC) and total flavonoids content (TFC). In spontaneously hypertensive rats, the arterial blood pressure and heart rate were recorded using the non-invasive tail-cuff technique. The antihypertensive effect of Kadukmy™ was evaluated using four different groups (n=6); C group (negative control), K group (Kadukmy™), P group (3 mg/kg perindopril), M group (Kadukmy™ + 1.5 mg/kg perindopril) in spontaneously hypertensive rats (SHR) for four weeks. The arterial blood pressure and heart rate were weekly recorded using the tail-cuff technique for four weeks. Then the animals were sacrificed, and their blood was collected for the determination of the serum NO level using Griess assay.
Asymmetric dimethylarginine (ADMA) and arginine level were determined using HPLC. Results: The PSAE showed good in-vitro antioxidant activities. Kadukmy™
group showed a significant lowering effect of blood pressure compared to the control group. Kadukmy™ antihypertensive activity was associated with an increase in serum NO level and a decrease in ADMA level. Meanwhile, the arginine level does not change significantly. Conclusion: PSAE showed an antihypertensive effect in Kadukmy™ treated SHR. Its high antioxidant activity enhances the clearance of ADMA which leads to the increase in the NO level. Keywords: Piper sarmentosum, Kadukmy™, antioxidant activity, spontaneously hypertensive rats, blood pressure, heart rate, hypertension, nitric oxide, asymmetric dimethylarginine, non-invasive blood pressure tail cuff.
iii
ثحبلا ةصلاخ
الم دحأ و ،ةيومدلا ةيعولأاو بلقلا ضارملأ يسيئر رطخ لماع وه مدلا طغض عافترا :ةمدق لأا
في ةافولل ةيسيئرلا بابس
.لماعلا ءانحأ عيجم ضارملأا جلاعل ليدبلا بطلا في عساو قاطن ىلع موسوتنمراس بريابلل ءارضلخا قاروا تمدختسا دقو
ليجست تم دقو .موسوتنمراس برياب في ةيسيئرلا ةطشنلا تانوكلما يه ةيلونيفلا تابكرلما .مدلا طغض عافترا كلذ في ابم يلع قلطا موسوتنمراس بريابلل يئالما صلختسلما نم عاترخا ةءارب طغض عافترا نم ةيجلاع ةعرجك )يمكوداك( مسا ه
ةساردلا هذه نم فدلها .مدلا وه
مييقت ةردق يمكوداك ظفخ ىلع
لاو مدلا طغض عافترا حت
قق نم ةيساسلأا تايللآا
للاخ نم لمعلل دوجو
داضم تا ا ةدسكلأ ا قاروا في موسوتنمراس بريابل
ةقيرط . ثحبلا جارختسا تم : صلختسم
قاروأ
بريابل رطقلما ءالماب موسوتنمراس ديمجتتلاب فيفجتلا للاخ نم قوحسم ليا اهليوتح ثم نم
يئالما صلختسلما صحف تم .
رابتخا للاخ نم ةدسكلأل ةداضلما هتطشنلأ يمكلا سايقملل موسوتنمراس بريابلل DPPH
رابتخاو FRAP
( يلكلا ليونيفلا ىوتحملل هصحفو TPC
( يلكلا ديونوفلافلا ىوتمحو ) TFC
.) عافتراب ةضيرم نارئفلا مادختسا تم
ثحبلا اذه في يثارو مدلا طغض تم .
سايق مادختساب بلقلا تابرض لدعمو نيايرشلا مدلا طغض نم سيقي زاهج
ليذلا للاخ يرثأت مييقت تم .
لا ةردق يمكوداك مدلا طغض ظفخ ىلع
مادختساب 4
( ةفلتمخ تاعوممج n = 6
ةعوملمجا )
C ةعوملمجا . K
؛)يمكوداك(
ةعوملمجا P ( 3 ةعوملمجا ؛)ليربودنيرب غك / غلم M
+ يمكوداك(
1.5 غك / غلم
)ليربودنيرب حترقلما جلاعلا ءاطعا تم
لكشب رمتسم تابرض لدعمو نيايرشلا مدلا طغض ليجست تم .عيباسأ ةعبرأ ةدلم
نم بلقلا نارئفلا ليذ للاخ
لبق جلاعلا ءاطعا تم ثم .عيباسأ ةعبرأ ةدلم ايعوبسأ
حبذ نارئفلا ديدحتل مدلا عجمو ،
( كيترينلا ديسكأ ىوتسم NO
سيرغ صحف مادختساب ) Griess reaction
يننيجرأ ليثيم يئانث ليلتح تم . لا
يرغ
( لثامتم ADMA مادختساب يننيجرأ ىوتسمو )
HPLC رهظأ :جئاتنلا .
صلختسلما لما
يئا موسوتنمراس بريابلل
جئاتن بتخا في ةديج ا ر
تا مدلا طغض في يربك ضافنخا يرثأت يمكوداك ةعوممج ترهظأ .ةدسكلأا تاداضم ةطشنأ
ةعوممج عم ةنراقم C
طاشنلا طبترا . ظفالخا
طغضل ا نم مدلا ىوتسم في ةدايز عم موسوتنمراس بريابل
كيترينلا ديسكأ
( NO ىوتسم في ضافنخاو ) يننيجرأ ليثيم يئانث
لا ( لثامتم يرغ ADMA
).
لم امنيب يرغتي لكشب يننيجرأ ىوتسم
لخا .يربك ةصلا يرثأت ةساردلا هذه دكؤت : ا
موسوتنمراس بريابل ضرم ىلع
.مدلا طغض عافترا للاخ نم كلذو
ةدايز في
ىوتسم كيترينلا ديسكأ (
NO ىوتسم في ضافنخاو ) يننيجرأ ليثيم يئانث
لا ( لثامتم يرغ ADMA
).
لا لما تاملك ةيحاتف
،يمكوداك ،موسوتنمراس برياب : مدلا طغض ،ةدسكلأا تاداضم
، بلقلا تابرض لدعم
، عافترا
مدلا طغض ،
،كيترينلا ديسكأ يننيجرأ ليثيم يئانث
لا
.لثامتم يرغ
iv
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences.
………..
Maizura Mohd. Zainudin Supervisor
………..
Abul Bashar Mohammed Helaluddin
Co-Supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Medical Sciences.
………..
Rosazra BT. Roslan Internal Examiner
This dissertation was submitted to the Department of Basic Medical Sciences and is accepted as a fulfillment of the requirement for the degree of Master of Medical Sciences.
………..
Mohammed Imad A. Mustafa Mahmud
Head, Department of Basic Medical Sciences
This dissertation was submitted to the Kulliyyah of Medicine and is accepted as a fulfillment of the requirement for the degree of Master of Medical Sciences.
………..
Azmi Md Nor
Dean, Kulliyyah of Medicine
v
DECLARATION
I hereby declare that this dissertation is the result of my own investigation, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.
Taher F T Elshami
Signature………….…………. Date …...
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF
FAIR USE OF UNPUBLISHED RESEARCH
FACTORS REGULATING NITRIC OXIDE PRODUCTION IN SPONTANEOUSLY HYPERTENSIVE RATS TREATED WITH
Piper sarmentosum
I declare that the copyright holder of this dissertation is jointly owned by the student and IIUM.
Copyright ©2018 by Taher F T Elshami and International Islamic University Malaysia. All rights reserved.
No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.
1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgment.
2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.
By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.
Affirmed by Taher F T Elshami
……..……..……… ……….……..
Signature Date
vii
ACKNOWLEDGEMENTS
This work would never have been possible or completed without the support and guidance of several people in my life. First, I would like to express my very great appreciation to my supervisor, Assistant Professor Dr. Maizura Mohd. Zainudin, for her exemplary guidance, monitoring, and constant support throughout the period of the study. I also take this opportunity to express a deep sense of gratitude to my co- supervisor Assistant Professor Abdul Bashar Helaludin.
This study was carries out at the physiology laboratory at International Islamic University Malasia and was supported by fundamental research grant scheme (FRGS) institution, under grant number (FRGS/1/2016/WAB11/UIAM/03/1).
I would also like to extend my thanks to the technicians of the laboratory of the Basic Medical Sciences Department, Faculty of Medicine for their help in offering me the resources in running the program.
Many thanks to my postgraduate friends, who supported and always shared me valuable advice and experiences. In particular, thank you to Mohamed Alaama, who helped me in the HPLC analysis.
Finally, this thesis is dedicated to the memory of my father, and to my mother and family for their invaluable support throughout my life.
viii
TABLE OF CONTENTS
Abstract ... ii
asbnrtAncbtcartsbA ... iii
Approval Page ... iv
Declaration ... v
Copyright ... vi
Acknowledgements ... vii
Table of Contents ... viii
List of Tables ... xi
List of Figures ... xii
List of Abbreviation/ Symbols ... xiv
CHAPTER ONE: INTRODUCTION ... 1
1.1 Research Background ... 1
1.2 Problem Statement ... 3
1.3 Significant of The Research... 3
1.4 Objectives ... 3
1.4.1 General Objective... 3
1.4.2 Specific Objectives... 3
1.5 Research Hypothesis ... 4
CHAPTER TWO: LITERATURE REVIEW ... 5
2.1 Piper Sarmentosum ... 5
2.1.1 Overview of Piper sarmentosum (PS) ... 5
2.1.2 Uses of Piper sarmentosum (PS) ... 6
2.1.3 Pharmacokinetic of Piper sarmentosum ... 9
2.1.4 Chemical Constituents in Piper sarmentosum ... 10
2.1.5 Safety and Toxicology of Piper sarmentosum ... 12
2.1.6 Kadukmy™………...13
2.2 Experimental Model of Hypertension ... 13
2.2.1 Spontaneously Hypertensive Rats as the Model of Primary Hypertension ... 15
2.3 Hypertension ... 16
2.3.1 Epidemiology of Hypertension ... 16
2.3.2 Definition of Hypertension ... 17
2.3.3 Risk Factors for Hypertension ... 18
2.4 Types of the Hypertension ... 19
2.4.1 Primary (Essential) Hypertension ... 19
2.4.2 Secondary Hypertension ... 20
2.5 Regulation of Blood Pressure ... 21
2.5.1 Haemodynamic Basic of Blood Pressure ... 21
2.5.2 Autonomic Nervous System Mediates the Short-Term Regulation of The Mean Arterial Blood Pressure ... 22
2.5.3 The Renal-Body Fluid Feedback Mechanism for Long-Term BP Regulation ... 23
2.6 Pathophysiology of Essential Hypertension ... 24
ix
2.6.1 Sympathetic Hyperactivity and Essential Hypertension ... 25
2.6.2 Renal Mechanism of Hypertension ... 26
2.6.3 Endothelial Dysfunction and Hypertension ... 26
2.6.1.1 Nitric Oxide ... 29
2.6.1.2 Asymmetric Dimethylarginine (ADMA) ... 31
CHAPTER THREE: METHODOLOGY ... 34
3.1 Materials ... 34
3.1.1 Piper Sarmentosum Aqueous Extract (PSAE) ... 34
3.1.2 Experimental Animal ... 34
3.1.3 Chemicals and Reagents ... 35
3.1.4 Anesthetic Agent ... 34
3.1.5 Apparatus ... 36
3.1.5.1 Blood Pressure Instrument ... 36
3.1.5.2 HPLC Instruments ... 36
3.1.5.3 HPLC Column ... 36
3.1.5.4 Nitrogen Evaporators ... 36
3.1.5.5 Weighing Balances ... 36
3.1.1.6 pH Meter ... 37
3.1.5.7 Hot Plate ... 37
3.1.5.8 Lab-Dryer ... 37
3.1.5.9 Centrifuge ... 37
3.1.5.10 Vortex ... 37
3.1.5.11 Ultrasonic Bath ... 37
3.1.5.12 Freeze Dryer Machine ... 38
3.1.6 Glassware ... 38
3.2 Preparation Piper Sarmentosum Aqueous Extract (PSAE) of Leaves ... 38
3.3 Antioxidant Activity of Piper Sarmentosum Aqueous Extracts (PSAE) ... 41
3.3.1 Determination of DPPH Activity of PSAE ... 41
3.3.2 Determination of Ferric-Reducing Antioxidant Power Activity (FRAP) of PSAE ... 42
3.3.3 Determination of Total Phenolic Content Assay of PSAE ... 43
3.3.4 Determination of Total Flavonoid Content Assay Of PSAE ... 43
3.4 Sample Size Calculation ... 44
3.5 Study Design ... 44
3.6 Animal Experiment ... 46
3.6.1 Measurement Body Weight... 46
3.6.2 Dose Preparation ... 46
3.6.3 Administration of Kadukmy™ and Perindopril into the Animals. .. 46
3.6.4 Measurement of Blood Pressure ... 46
3.6.5 Administration of The Anaesthetic Agent ... 47
3.6.6 Blood Collection ... 49
3.7 Biochemical Parameters ... 50
3.7.1 Determination of Serum Nitric Oxide ... 50
3.7.1.1 Deproteinization Methods of Serum ... 50
3.7.1.2 Total Nitrite and Nitrate Determination ... 50
3.7.2 Determination of the Endogenous Nitric Oxide Synthase Inhibitor Asymmetric Dimethylarginine (ADMA) in Plasma Sample By HPLC ... 49
x
3.7.2.1 HPLC Buffers ... 49
3.7.2.2 Preparation of Standards ... 50
3.7.2.3 Sample Preparation For HPLC ... 50
3.7.2.4 Derivatization Reagents ... 52
3.7.2.5 HPLC Analysis ... 53
3.8 Statistical Analysis... 54
CHAPTER FOUR: RESULTS ... 55
4.1 In-Vitro Antioxidant Activities ... 55
4.1.1 DPPH Radical Scavenging Activity ... 55
4.1.2 Ferric Reducing Antioxidant Power (FRAP) ... 55
4.1.3 Total Phenol and Total Flavonoid Contents (TPC & TFC) ... 56
4.2 Body Weight ... 58
4.3 Antihypertensive Evaluation ... 59
4.3.1 Systolic Blood Pressure ... 59
4.3.2 Diastolic Blood Pressure ... 60
4.3.3 Mean Arterial Pressure ... 59
4.4 Heart Rate ... 62
4.5 Serum Nitric Oxide (NO) Level ... 63
4.5.1 Linearity ... 63
4.5.2 Serum Nitric Oxide Level ... 64
4.6 Plasma Asymmetric Dimethylarginine (ADMA) and Arginine Level ... 65
4.6.1 Optimized Run Time and Chromatogram ... 65
4.6.2 Linearity ... 65
4.6.3 System Suitability ... 68
4.6.4 Plasma ADMA Level ... 69
4.6.5 Plasma Arginine Level ... 70
CHAPTER FIVE: DISCUSSION ... 69
5.1 In-Vitro Antioxidant Activity of Piper Sarmentosum ... 69
5.2 Antihypertensive Activity of Piper Sarmentosum (Kadukmy™) in Spontaneously Hypertensive Rats ... 72
5.3 Piper Sarmentosum Role Through the Regulation Factors of Nitric Oxide Production ... 73
5.4 Summary of the Research………... 72
5.5 Limitation and Recommendation For Future Studies ... 75
CHAPTER SIX: CONCLUSION ... 76
REFERENCES ... 77
APPENDIX I HERBARIUM VOUCHER SPECIMEN INFORMATION ... 83
APPENDIX II: ETHICAL APPROVAL LETTER FROM IACUC-IIUM……..86
xi
LIST OF TABLES
Table 2.1 The most common animal models for hypertension experimental study 14 Table 2.2 Classification of BP according to the JNC7 guideline, the JNC8 panel
member report and the 2017 ACC/AHA guideline. 18
Table 2.3 List of Secondary C68auses of Hypertension 20
Table 3.1 HPLC Gradient conditions of mobile phase 52
Table 4.1 DPPH and FRAP activates on extracts 55
Table 4.2 TPC and TFC activates on extract 56
Table 4.3 Table 4.3 The effect of Kadukmy™ and perindopril administration on
body weight in antihypertensive study of PSAE on . 58
Table 4.4 System suitability parameters for ADMA and arginine 66
xii
LIST OF FIGURES
Figure 2.1 Parts of Piper sarmentosum Roxb 6
Figure 2.2 Pharmacokinetic model of sarmentosine, sarmentine and pellitorine after administering ethanol extract of fruit of PS in rat 10 Figure 2.3 Structure of the three phenylpropanoyl amides compounds 11 Figure 2.4 HPLC analysis profiles of methanolic PS leaves extracts 12 Figure 2.5 Prevalence of hypertension around the world between gender and
different level of income 17
Figure 2.6 Factors regulating mean arterial pressure. 22
Figure 2.7 Autonomic nervous system and control of blood pressure 23 Figure 2.8 Regulatory functions of the endothelium. Normal or antiatherogenic
vs. dysfunction or atherogenic properties 27
Figure 2.9 Association of endothelial dysfunction in the pathophysiology of
hypertension 29
Figure 2.10 Basic synthesis of nitric oxide 29
Figure 2.11 Role of nitric oxide and cGMP in smooth muscle vasorelaxation 31 Figure 2.12 Structure of arginine and asymmetric dimethylarginine (ADMA) 32 Figure 2.13 Endogenous inhibitors of L-arginine (asymmetric (ADMA) and
symmetric dimethylarginine (SDMA)) 33
Figure 3.1 PS leaves harvesting 38
Figure 3.2 PSAE concentration process 38
Figure 3.3 PSAE Storage at -80 °C for 24 hours before moved to freeze drier 39 Figure3.4 PSAE freeze drying process 39 Figure 3.5 Reaction of DPPH test 40 Figure 3.6 Reaction of FRAP test 41
xiii
Figure 3.7 Reaction of TPC and TFC test 43
Figure 3.8 Animal handling and experimental design 44
Figure 3.9 Administration of PSAE aqueous extract to SHR via oral gavage 46
Figure 3.10 Blood pressure measured by using the tail-cuff technique by CODA non-invasive blood pressure system. 47
Figure 3.11 Intravenous injection of ketamine and xylazine to the lateral vein of the rat tail 47
Figure 4.1 Standard curve for FRAP calculation. 55
Figure 4.2 Standard curve for TPC calculation 55
Figure 4.3 Standard curve for TFC calculation 55
Figure 4.4 Systolic blood pressure of each group of experiment over 4 weeks of treatment. 57
Figure 4.5 Diastolic blood pressure of each group of experiment over 4 weeks of treatment. 58
Figure 4.6 Diastolic blood pressure of each group of experiment over 4 weeks of treatment. 59
Figure 4.7 Heart rate of each group of experiment over 4 weeks of treatment 60
Figure 4.8 Typical linear curve of NO with R2 of 0.9994 61
Figure 4.9 Serum nitric oxide (NO) levels in the experimental groups 62
Figure 4.10 HPLC chromatogram for blank (A); arginine, IS and ADMA (B) 64
Figure 4.11 Standard curve for ADMA (A) and arginine (B) 65
Figure 4.12 Plasma asymmetric dimethylarginine (ADMA) levels in the experimental groups 67
Figure 4.13 Plasma arginine levels in the experimental groups 68
Figure 5.1 Mechanism of PSAE to reduce blood pressure 73
xiv
LIST OF ABBREVIATION/ SYMBOLS
% Percentage
ºC Degree Celsius
+ -
Plus Minus
= Equal to
< Less than
> More than
± ( ) , . :
; ABP ACE ADMA Ang II ANP BP Ca2+
CAT1
Plus minus Left parenthesis Right parenthesis Comma
Full stop Colon Semicolon
Arterial blood pressure
Angiotensin-converting enzyme Asymmetric dimethylarginine Angiotensin ii
Atrial natriuretic peptide Blood pressure
Calcium
Cationic amino acid transporter 1
xv cGMP
CHD CNS CO CVD DBP DDAH ED eNOS ET-1 FRAP g GC GFR H HCl HUVECs IIUM K+ kg KOP LDL MAP MDA
Cyclic guanosine monophosphate Coronary heart disease
Central nervous system Cardiac output
Cardiovascular disease Diastolic blood pressure Dimethylaminohydrolase Endothelial dysfunction
Endothelial nitric oxide synthase Endothelin-1
Ferric-reducing antioxidant power activity Gram
Guanylate cyclase Glomerular filtration rate Hours
Hydrochloric acid
Human umbilical vein endothelial cells International Islamic University Malaysia Potassium
Kilogram
Kulliyyah of Pharmacy Low-density lipoprotein Mean arterial blood pressure Malondialdehyde
xvi MI
MIC ml mM µl MMA MPA NADPH NE NGF NO OPA PS PSAE RAAS SBP SDMA SHR SNS
TM
TPC TPR WHO WKY
Myocardial infarction
Minor inhibition concentration Milliliter
Millimole Microliter
Monomethylarginine 3-mercaptopropionic acid
Nicotinamide adenine dinucleotide phosphate Norepinephrine
Nerve growth factor Nitric oxide
O-phthalaldehyde Piper sarmentosum
Piper sarmentosum aqueous extract Renin-angiotensin-aldosterone system Systolic blood pressure
Symmetric dimethylarginine Spontaneously hypertensive rats Sympathetic nervous system Trade mark sign
Total phenolic content Total peripheral resistance World Health Organization Wistar Kyoto rats
1
CHAPTER ONE INTRODUCTION
1.1 RESEARCH BACKGROUND
Hypertension is the most prevalent chronic cardiovascular diseases (CVD). Globally, almost 7.6 million deaths due to hypertension were annually recorded. More than one billion patients around the world including over 5.8 million Malaysians have high blood pressure (BP) (Kew et al., 2015; Hall et al., 2012). Hypertension is a precursor to stroke, myocardial infarction (MI), coronary heart disease (CHD) and renal failure.
For decades, hypertension has been extensively investigated in clinical and pre- clinical studies to understand the mechanisms of the disease as well as to test new potential drugs. Several animal models of hypertension have been developed. One of these models is the spontaneously hypertensive rats (SHR), which is widely used as an essential hypertension model to study the antihypertensive prospective of some natural products.
Herbal products have gained increasing popularity in the last decade, and are now used by approximately 20% of the population (Bent, 2008). In the 21st Century, 11% of the 252 drugs approved by the World Health Organization (WHO) have originated from plants (Veeresham, 2012). Many research on plant extracts has exhibited that natural products contain natural active compounds to treat diseases including hypertension. The importance of investigating the mechanism of action of natural products will provide an idea of how these compounds work in our body.
2
There is a strong evidence that endothelial dysfunction (ED) plays an important role in the pathogenesis of hypertension. Endothelial cells produce nitric oxide(NO) as a response to the increase of BP leads to vasodilatation. NO is produced by a healthy endothelial cell as a signalling molecule to dilate blood vessel, ensure optimum blood supply and reduce BP. ED occur as a result of imbalance between antioxidant availability and free radicals which causes oxidative stress. Reduce NO availability due to oxidative stress is a hallmark of ED.
Several researchers found that many plants and herbs including Piper sarmentosum (PS) has high antioxidant activity (Thent, Seong Lin, Das, & Zakaria, 2012). They also suggested that additional antioxidant from plants may help in ameliorating blood pressure and able to prevent the development of hypertension. There are various methods used to measure the antioxidant activity of a plant including DPPH test, FRAP test and the screening of total phenolic content (TPC) and total flavonoids content (TFC).
PS is a wild herbal plant. It is called Kaduk in Malaysia. It is one of the natural products that show a potential ability to cure diseases including hypertension. Piper sarmentosum aqueous extract (PSAE) has been shown to increase NO bioavailability with decrease malondialdehyde (MDA) level (Mohd Zainudin et al., 2015). Lack of research to investigate the mechanism of PS motivates this research which aims to evaluate the activity of PSAE in reducing blood pressure and examining possible mediated effects of NO production.
3 1.2 PROBLEM STATEMENT
Previous studies with PSAE (Kadukmy™) have shown antihypertensive effects, with decreased oxidative stress and increased NO production with improvement in ED.
However, the mechanism of production of NO is as yet unclear.
1.3 SIGNIFICANT OF THE RESEARCH
This research explores the factors that regulate NO production in PSAE treated SHR.
1.4 OBJECTIVES 1.4.1 General Objective
This research aims to study the PSAE antioxidant activity, blood pressure lowering effects on SHR, and the mechanism responsible.
1.4.2 Specific Objectives
1. To investigate the antioxidant activity and compound through DPPH and FRAP tests as well as examine the total phenolic content (TPC) and total flavonoids content (TFC) of PSAE in vitro.
2. To determine blood pressure indices in Kadukmy™ treated SHR.
3. To determine the serum NO level in Kadukmy™ treated SHR.
4. To determine the plasma ADMA level in Kadukmy™ treated SHR.
5. To determine the plasma arginine level in Kadukmy™ treated SHR.
6. To compare the antihypertensive effect of Kadukmy™ treatment with Perindopril.
4 1.5 RESEARCH HYPOTHESIS
1. PSAE has high antioxidant activity, TPC and TFC in vitro.
2. PSAE decreases the systolic blood pressure, diastolic blood pressure and mean arterial pressure.
3. PSAE decreases the oxidative stress that leads to an increased clearance of ADMA causing increased serum NO.
5
CHAPTER TWO LITERATURE REVIEW
2.1 PIPER SARMENTOSUM
2.1.1 Overview of Piper sarmentosum (PS)
The Piperaceae family is one of the largest plant family herbs found in Southeast Asia.
Piperaceae is also called ‘pepper family’ which includes herbs such as ‘Piper caninum, Piper sarmentosum, piper betle’. The pepper family comprises more than 300 species in Southeast Asia. PS, on the other hand, is a creeping terrestrial herb which is found in many countries like Malaysia, Philippines, Myanmar, China, Thailand, Borneo, Cambodia, Laos, Java. (Hamil, Memon, Majid, & Ismail, 2016).
Kaduk is the local name of Piper sarmentosum Roxb in Malaysia whilst in Thailand it is known as Cha-plu (Sharifah Farhana Syed Ab Rahman, 2016). PS is a stoloniferous sapling because it grows as a ground sprawl herbaceous creeper, and propagates in clusters up to 60 cm in height. The plant is usually found in uncultivated land and forests (Wiart, 2006). The leaves are thin and bright green, in a heart shape with 5-7 obvious veins which originate from the base, and the leaves. The flowers are bar-shaped and white in color, and the fruits are obovoid berries (Figure 2.1) (Chan &
Wong, 2014).
6
Figure 2.1 Parts of Piper sarmentosum Roxb (A) Leaves, (B) Flowers, (C) Fruits
2.1.2 Uses of Piper sarmentosum (PS)
One of the major uses of PS leaves and fruits is for medicinal purposes as part of the traditional medicinal practice. It has been used by the elderly to improve their memory.
There is a documented evidence that PS has an acetylcholine inhibitory activity using the Ellman’s method with the Flow Injection Analysis technique (Khan, Elhussein, Khan, & Khan, 2012). Laboratory studies have exhibited that PS has high antioxidant efficacy as it shows 87.6% superoxide free radical scavenging activity, 96% radical scavenging activity, and 98% lipid peroxidation inhibitory activity (Thent, Seong Lin, Das, & Zakaria, 2012).
Various clinical and animal studies conducted using PS have identified numerous potential health benefits of PS leaves consumption. It has been traditionally used to treat a number of minor ailments such as headache, chest pain, cough,
7
expectorants, muscle pain, backache, toothache, waist pain, menstrual pain and joint pain (Syed Ab Rahman, 2016; Peungvicha et al., 1998; Ridtitid et al., 1998;
Pongmarutai, 1989; Li, 1980; Han et al., 1992; Mathew et al., 2004; Hussain, Ismail, Sadikun, & Ibrahim, 2011; Yong et al., 2013; Thent, Seong Lin, Das, & Zakaria, 2012;
Daud, Azahar, Zainal Abidin, & Tawang, 2015; Amran et al., 2010; Mohd Zainudin, Zakaria, & Megat Mohd Nordin, 2015).
It has been well documented that PS leaves are able to lower the blood glucose level in normal and diabetic rat models. However, the methanolic extract of PS was more potent than the water extract (P Peungvicha, 1998). More recent studies have shown that PS could reduce ultra-structural degenerative changes in proximal aorta and cardiac tissues in diabetic cardiovascular tissues. PS has been recognised to contain antioxidant compounds like quercetin (flavonoid compounds). Quercetin has a definite role in reducing blood glucose levels, enhancing the regeneration of the pancreatic islets, and promoting insulin release in diabetic rats (Thent et al., 2012).
It has also been reported that PS has anti-bacterial activity against Bacillus subtilis, Escherichia coli and A.actinomycetemcomitans. The same study also identified that the four phenylpropanoids isolated from PS leaves ( 1 - allyl - 2, 6-dimethoxy - 3, 4-methylenedioxybenzene) have antibacterial effects. (Taweechaisupapong, Singhara, Lertsatitthanakorn, & Khunkitti, 2010). The methanol extract of the PS leaves demonstrated antibacterial activity against both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus and methicillin-resistant S.
aureus (MRSA) (Rain, 2005).
The anti-tuberculosis potential of PS was also investigated against Mycobacterium tuberculosis using a colorimetric microplate-based assay. A positive activity was observed in the PS with a minor inhibition concentration (MIC) of 800
8
μg/ml. The amides isolated from the fruits of PS showed anti-tuberculosis activities against Mycobacterium tuberculosis. Seven compounds extracted from the roots of PS displayed anti-mycobacterial activities (Suriyati Mohamad, 2011). In addition to that, the chloroform extract from the roots and fruits of PS also displayed anti-malarial activities. According to Suriyati Mohamad (2011), the amide compounds (Sarmentine and 1-piperettyl pyrrolidine) isolated from the fruits had revealed anti-plasmodial activities.
PS also demonstrated anti-nociceptive properties. The Piper sarmentosum aqueous extract (PSAE) exhibited a painkiller effect at the central and peripheral levels.
The anti-nociceptive activity was examined by means of abdominal constriction using hot-plate tests. The inhibition of Cyclooxygenase (COX) and prostaglandins were suggested at the peripheral level. The aqueous extract of PS showed significant anti- nociceptive actions and anti-inflammatory activities using the carrageenan-induced paw oedema test, which could be due to its effect on the COX or prostaglandins (Mathew et al., 2004).
PS had also been shown to display cytotoxic activity; the chloroform leaf extract of PS was assessed for anti-cancer activity using the MTT cell-viability test. This study showed that PS extract inhibited HepG2 and HUVEC human cancer cells with IC50
values of 76 and 64 μg/ml respectively (Atiax, E., Ahmad, F., Sirat, H. M., & Arbain 2011). However, in a subsequent study conducted by Yong et al., (2013), dichamanetin that was isolated from PS significantly decreased the cell viability of various types of human cancer cells (MDA-MB-231 breast cancer and HT-29 colon, DU145 prostate).
A potential therapeutic role of PS in the treatment of hypertension had also been suggested recently (Mohd Zainudin et al., 2015). It had been shown that the PSAE could reduce oxidative stress injury on blood vessels in vivo. Their findings suggested that
