COPYRIGHTpsasir.upm.edu.my/id/eprint/66043/1/FPSK (p) 2016 6 IR.pdf · aktiviti antinosiseptif, penjelasan mekanisma tindakan dan mengenal pasti komponen bioaktif dari ekstrak daun

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UNIVERSITI PUTRA MALAYSIA

ANTINOCICEPTIVE ACTIVITY, ELUCIDATION OF MECHANISM OF ACTION AND IDENTIFICATION OF BIOACTIVE COMPONENTS OF

Muntingia calabura L. LEAF EXTRACTS

MOHD HIJAZ MOHD SANI

FPSK(p) 2016 6

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ANTINOCICEPTIVE ACTIVITY, ELUCIDATION OF MECHANISM OF

ACTION AND IDENTIFICATION OF BIOACTIVE COMPONENTS OF

Muntingia calabura L. LEAF EXTRACTS

By

MOHD.HIJAZ MOHD SANI

Thesis submitted to the School of Graduate Studies, University Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Doctor of Philosophy

June 2016

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Copyright © Universiti Putra Malaysia

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Abstract of thesis presented to the senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Doctor of Philosophy

ANTINOCICEPTIVE ACTIVITY, ELUCIDATION OF MECHANISM OF

ACTION AND IDENTIFICATION OF

BIOACTIVE COMPONENTS OF MUNTINGIA CALABURA L. LEAF

EXTRACTS

By


June 2016

Chairman: Associate Professor Zainul Amiruddin Zakaria, PhD

Faculty: Medicine and Health Sciences

Research into plant as an alternative treatment for pain has been widely explored due to

its potentially low adverse effect with great therapeutic activity. Muntingia calabura L.

(Tiliaceae) has recently gained a medicinal status as well as attention from throughout

the world. The present study examined the potential antinociceptive activity of

methanol extract of Muntingia calabura (MEMC) leaves using the acetic acid-induced

abdominal constriction, formalin and hot plate test following the determination of its

safety using acute toxicity test. Then the possible involvement of MEMC-induced

antinociception through capsaicin, glutamate, bradykinin, opioidergic, dopaminergic serotonergic, noradrenergic, adenosisnergic, protein kinase C (PKC), nitric oxide

(NO)/cyclic guanosine monophosphate (cGMP) and potassium channel pathway was

evaluated. Experimental animals (n=6) were pretreated orally with 10% dimethyl

sulfoxide (DMSO; negative control), 5 mg/kg morphine or 100 mg/kg aspirin (positive

control) or 100, 250, and 500 mg/kg of MEMC, followed by the administration of

receptor antagonists and/or induction of nociception. The crude MEMC extract was

further partitioned into three fractions: petroleum ether extract (PEMC), ethyl acetate

extract (EAMC) and aqueous extract (AEMC). The antinociceptive profiling

demonstrates PEMC produced a significantly better activity than EAMC and AEMC.

Possible mechanism of PEMC action was studied using the same assays. Result on the

acute toxicity study shows no mortality and significant behavioural and physiological changes detected. Oral administration of MEMC and PEMC shows a dose-dependent

inhibition in acetic acid-induced abdominal constriction test, formalin-, capsaicin-.

glutamate-, bradykinin- and PMA-induced paw licking test, while only the highest dose

produced significant pain inhibition in hot plate test. Furthermore, the antinociception

caused by MEMC and PEMC in acetic acid-induced abdominal constriction test was

significantly attenuated by intraperitoneal treatment of naloxone (non-specific opioid

receptor antagonist; 5 mg/kg), naltrindole (δ opioid receptor antagonist; 1 mg/kg) nor-

binaltorphimine (κ opioid receptor antagonist; 1 mg/kg), β-funaltraxamine (μ opioid

antagonist; 10 mg/kg), pindolol (5-HT1A receptor antagonist; 1 mg/kg), caffeine (non-

selective adenosine receptor antagonist; 3 mg/kg) and yohimbine (α2 adrenoceptor

antagonist; 0.15 mg/kg). While both extracts did not act on dopaminergic receptor system, PEMC, but not MEMC, was significantly reversed by i.p. injection of atropine

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(non-selective cholinergic antagonist; 10 mg/kg). At the same time, MEMC and PEMC

were found to inhibit pain through NO/cGMP/PKC as well as potassium channel

pathways. The potential antinociceptive activity seen was probably due to synergistic

effect of flavonoids, tannins, polyphenolic compounds, triterpene and steroid present in

the extracts based on their phytochemical screening. High performance liquid

chromatography analysis shows several peaks, detected at different wavelengths of the

chromatogram, which were suggested to be flavonoid-based compounds. In conclusion,

the synergistic effects of various bioactive components suggested to be responsible in

the antinociceptive activity of MEMC as well as the semi-purified PEMC extract which

involved in central and peripheral pain pathways. The activation of potassium pathway

as well as opioid, adenosinergic, noradrenergic and serotonergic receptors while inhibits NO/cGMP/PKC pathways, TRPV1, glutamate and bradykinin receptors might

be the possible mode of action of both the extracts in exerting their analgesic effect.

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Abstrak tesis yang dikemukakan kepada senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

AKTIVITI ANTINOSISEPTIF, PENJELASAN MEKANISMA TINDAKAN

DAN MENGENAL PASTI KOMPONEN BIOAKTIF DARI EKSTRAK DAUN

MUNTINGIA CALABURA L.

Oleh


Jun 2016

Pengerusi: Profesor Madya Zainul Amiruddin Zakaria, PhD

Fakulti: Perubatan dan Sains Kesihatan

Penyelidikan ke atas tumbuhan sebagai perubatan alternatif untuk kesakitan telah luas

diteroka kerana potensi kesan sampingan yang rendah disamping memberi aktiviti

terapeutik yang kuat. Muntingia calabura L. (Tiliaceae) baru-baru ini telah mendapat

status tumbuhan perubatan dan juga perhatian penyelidik dari serata dunia.

Penyelidikan ini mengkaji potensi aktiviti antinosiseptif ekstrak metanol dari daun

Muntingia calabura (MEMC) menggunakan ujian penggeliatan perut mencit, ujian

penjilatan tapak kaki tikus dan ujian plat panas selepas ujian keselamatan toksik akut.

Selepas itu, mekanisma tindakan antinosiseptif MEMC dikaji menggunakan model

eksperimen capsaicin, glutamate, bradykinin, opioidergik, dopaminergic, serotonergic, noradrenergic, adenosinergic, protin kinas C (PKC), saluran nitrik oksida (NO)/kitaran

guanosin monofosfat (cGMP) dan saluran kalium. Haiwan ujikaji (n=6) dirawat secara

oral dengan 10% dimetil sulfoksida (DMSO; kawalan negatif), 5 mg/kg morfin atau

100 mg/kg aspirin (kawalan positif), atau 100, 250 dan 500 mg/kg MEMC, diikuti

dengan pemberian reseptor antagonis dan/atau perangsang kesakitan. Ekstrak kasar

MEMC kemudiannya dibahagikan kepada tiga pecahan: ekstrak petroleum eter

(PEMC), ekstrak etil asetat (EAMC) dan ekstrak akues (AEMC). Mekanisma tindakan

yang mungkin untuk PEMC dikaji menggunakan model eksperimen yang sama.

Keputusan toksik akut menunjukkan tiada kematian serta perubahan signifikan

terhadap tingkah laku dan fisiologi. Pemberian MEMC dan PEMC secara oral

menunjukkan perencatan secara signifikan dalam ujian penggeliatan perut mencit serta ujian penjilatan tapak kaki mencit oleh formalin, capsaicin, glutamate, bradykinin dan

PMA, manakala hanya dos paling tinggi menunjukkan penurunan kesakitan yang

signifikan dalam ujian plat panas. Lebih dari itu, kesan antinosiseptif oleh MEMC dan

PEMC dalam ujian pengeliatan perut mencit dilemahkan secara signifikan selepas

dicabar dengan naloxone (reseptor antagonis opioid tidak spesifik; 5 mg/kg),

naltrindole (reseptor antagonis opioid δ; 1 mg/kg) nor-binaltorphimine (reseptor

antagonis opioid κ; 1 mg/kg), β-funaltraxamine (reseptor antagonis opioid μ; 10

mg/kg), pindolol (reseptor antagonis 5-HT1A; 1 mg/kg), kafein (reseptor antagonis

adenosin tidak spesifik; 3 mg/kg) and yohimbine (reseptor antagonis α2; 0.15 mg/kg).

Walaupun kedua-dua ekstrak tidak bertindak ke atas reseptor dopaminergik, aktiviti

PEMC, tetapi tidak MEMC, secara signifikannya direncat oleh atropin (reseptor antagonis kolinergik tidak spesifik; 10 mg/kg). Pada masa yang sama, MEMC dan

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PEMC dibuktikan dapat merencat kesakitan melalui saluran NO/cGMP/PKC dan juga

saluran kalium. Potensi aktiviti antinosiseptif yang ditunjukkan berkemungkinan

disebabkan oleh kesan sinergistik flavonoid, tannin, kompaun polyphenolic, triterpene

dan steroid yang ada di dalam ekstrak berdasarkan dari saringan fitokimia yang dibuat.

Analisa kromatografi cecair berprestasi tinggi (HPLC) menunjukkan beberapa puncak,

dikesan dari gelombang berbeza, yang disyorkan sebagai kompaun dari kategori

flavonoid. Kesimpulannya, kesan sinergistik oleh komponen bioaktif yang berbeza

mungkin bertanggungjawab ke atas aktiviti antinosiseptif MEMC dan juga separa-tulen

PEMC yang terlibat dalam saluran kesakitan periferal dan pusat. Pengaktifan saluran

kalium serta reseptor opioid, adenosinergik, noradrenergik and serotonergik disamping

merencat saluran NO/cGMP/PKC serta reseptor TRPV1, glutamate dan bradykinin berkemungkinan ialah mekanisma tindakan yang diambil oleh kedua-dua ekstrak dalam

kesan antinosiseptif yang ditunjukkan.

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ACKNOWLEDGEMENT

Alhamdulillah, all praise and thanks is for Allah subhanahu wa ta’ala for helping me

overcome all the challenges that I had gone through in the study.

I wish to extend my deepest appreciation to my supervisor, Associate Professor Dr.

Zainul Amiruddin Zakaria for his supervision, guidance, valuable advice, patience and

continuous support throughout the course of this project. I truly thank him for giving

me the opportunity to be his postgraduate student. I would like to express my gratitude

and appreciation to my co-supervisors, Associate Professor Dr. Arifah Abdul Kadir and

Dr. Manraj Singh Cheema for their guidance, invaluable advice and support.

Special thanks to my colleague, Tavamani Balan, for her continuous assistance

throughout the study. I would like to thank all my fellow pharmacology lab mates, Kushairi Ahmad, Farhana Yahya, Fauzi Fahmi, Salahuddin and Siti Syariah Mamat for

their companion, cooperation and care towards me. My sincere appreciation dedicates

to the Faculty of Medicine and Heatlth Sciences, Universiti Putra Malaysia for giving

me the opportunity to carry out this project.

Last but not least, my extreme gratitude to my wife, Dr Siti Sarah binti Darussalam for

her patience, support, encouragement and motivation which really help me

accomplished this study. I also owe a depth gratitude to my mother and the family of

my wife for their patience and continuous support.

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This thesis was submitted to the senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The

members of the supervisory committee were as follows:

Zainul Amiruddin Zakaria, PhD

Associate Professor

Faculty of Medicine and Health Sciences Universiti Putra Malaysia

(Chairman)

Arifah Abdul Kadir, PhD

Associate Professor

Faculty of Veterinary Medicine

Universiti Putra Malaysia

(Member)

Manraj Singh Cheema, PhD

Senior Lecturer

Faculty of Medicine and Health Sciences Universiti Putra Malaysia

(Member)

_______________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean School of Graduate Studies

Universiti Putra Malaysia

Date:

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Declaration by graduate student

I hereby confirm that:

This thesis is my original work;

Quotations, illustrations and citations have been duly referenced;

This thesis has not been submitted previously or concurrently for any other degree

at any other institutions;

Intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

Written permission must be obtained from supervisor and the office of Deputy Vice-Chancellor (Research and Innovation) before thesis is published (in the form

of written, printed or in electronic form) including books, journals, modules,

proceedings, popular writings, seminar papers, manuscripts, posters, reports,

lecture notes, learning modules, or any other materials as stated in the Universiti

Putra Malaysia (Research) 2012;

There is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: _____________________ Date: __________________

Name and Matric No.: Mohd.Hijaz Mohd Sani GS28051

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Declaration of The Members of Supervisory Committee

This is to confirm that:

The research conducted and the writing of this thesis was under our supervision;

Supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (revision 2012-2013) are adhered to.

Signature:

Name of Chairman of Supervisory

Committee: Zainul Amiruddin Zakaria, PhD

Signature:

Name of Member of

Supervisory

Committee: Arifah Abdul Kadir, PhD

Signature:

Name of Member of

Supervisory

Committee: Manraj Singh Cheema, PhD

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVATIONS xvii

CHAPTER

1 INTRODUCTION

1.1 Introduction 1

1.2 Hypothesis 2

1.3 Objectives 2

2 LITERATURE REVIEW

2.1 Pain and nociception 3

2.2 Types of pain 4 2.3 Pain theory 6

2.4 Pain processing and sensitization 8

2.5 Pain receptor 9

2.5.1 TRPV1 receptor 10

2.5.2 Glutamate receptor 11

2.5.3 Bradykinin receptor 11

2.5.4 Involvement of protein kinase C 11

2.5.5 Involvement of Nitric Oxide and cGMP pathway 12

2.5.6 Involvement of potassium channels 13

2.5.7 Adenosinergic receptor 13

2.5.8 Serotonergic receptor 14

2.5.9 Cholinergic receptor 14 2.5.10 Dopaminergic receptor 15

2.5.11 Adrenergic receptor 16

2.6 Non-steroidal anti-inflammatory drugs 16

2.7 Opiate and opioid 17

2.7.1 Opioid receptors 18

2.7.2 Endogenous opioid 18

2.7.3 Exogenous opioid 19

2.7.4 Opioid antagonist 20

2.8 Natural product as an alternative analgesic agent 20

2.9 Muntingia calabura L. 21

2.9.1 Traditional uses 22 2.9.2 Scientific findings 24

2.10 Antinociceptive assays 31

2.10.1 Acetic acid-induced abdominal constriction test 31

2.10.2 Hot plate test 32

2.10.3 Formalin test 33

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3 PREPARATION OF METHANOL EXTRACT OF MUNTINGIA

CALABURA LEAVES (MEMC) AND ITS PARTITIONS: ACUTE

TOXICITY STUDY

3.1 Introduction 34

3.2 Materials and methods

3.2.1 Chemicals 34

3.2.2 Plant collection 34

3.2.3 Methanol extraction process 35

3.2.4 Preparation of petroleum ether, ethyl acetate and

aqueous partition

35

3.2.5 Acute toxicity study 35 3.3 Statistical analysis 36

3.4 Result

3.4.1 Preparation of extracts 36

3.4.2 Acute toxicity study 36

3.5 Discussion 41

4 DETERMINATION OF THE ANTINOCICEPTIVE ACTIVITY

OF MEMC

4.1 Introduction 42


4.2.1 Preparation of extract 42 4.2.2 Preparation of drugs 43

4.2.3 Experimental animals 43

4.2.4 Acetic acid-induced abdominal writhing test 43



4.3 Statistical analysis 44

4.4 Result

4.4.1 Acetic acid-induced abdominal writhing test 44



4.5 Discussion 49

5 POSSIBLE MECHANISM OF ACTION THAT MODULATED

THE ANTINOCICEPTION OF MEMC

5.1 Introduction 52


5.2.1 Preparation of extract 52

5.2.2 Preparation of drugs 52


5.2.4 Capsaicin-induced paw licking test 53

5.2.5 Glutamate-induced paw licking test 53

5.2.6 Phorbol 12-myristate 13-acetate (PMA)-induced

nociception

53

5.2.7 Bradykinin-induced nociception 54

5.2.8 Involvement of Nitric Oxide/cGMP pathway 54


5.2.10 Involvement of various non-opioid receptors 54

5.2.11 Involvement of opioid receptor and its subtype 55


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5.4 Result




nociception

55






5.5 Discussion 67

6 ANTINOCICEPTIVE PROFILE OF VARIOUS PARTITIONS

OBTAINED FROM MEMC: DETERMINATION OF THE MOST

EFFECTIVE PARTITION

6.1 Introduction 73

6.2 Materials and methods 73




6.2.4 Determination of the most effective partition using

acetic acid-induced abdominal constriction test

74




6.4 Result

6.4.1 Determination of the most effective partition using

acetic acid-induced abdominal constriction test

74



6.5 Discussion 79

7 POSSIBLE MECHANISMS OF ACTION THAT MODULATE

ANTINOCICEPTION EXERTED BY PETROLEUM ETHER

PARTITION (PEMC) OF MEMC

7.1 Introduction 81









nociception

82






7.4 Result

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nociception

84





7.5 Discussion 95

8 PHYTOCHEMICAL SCREENING AND HIGH

PERFORMANCE LIQUID CHROMATOGRAPHY ANALYSIS

OF MEMC AND PEMC

8.1 Introduction 97

8.2 Method

8.2.1 Phytochemical screening of dried leaves, methanol

extract and petroleum ether partition of M. calabura

97

8.2.2 HPLC profile of MEMC and PEMC at various

wavelengths

98

8.3 Results 99

8.4 Discussion 106

9 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATION

107

BIBLIOGRAPHY 111

APPENDICES 135

BIODATA OF STUDENT 137

LIST OF PUBLICATIONS 138

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LIST OF TABLES

Table

Page

1 Ethnomedicinal uses of M. calabura

23

2 Pharmacological activities of M. calabura

25

3 Bioactive compounds of M. calabura

26

4 Recorded body weight of control group and rats treated with high dose extract

37

5 Comparison of relative organ weight (in gram) between control

and MEMC-pretreated group obtained at the end of study

38

6 Comparison of complete blood counts between control and

MEMC-pretreated group following the toxicity study

39

7 The comparison of serum biochemistry level between control and

MEMC-pretreated group from the acute toxicity study

40

8 Effect of MEMC on the hot plate test on mice.

47

9 Effect of PEMC in formalin-induced paw licking test.

77

10 Antinociceptive profile of PEMC assessed using the hot-plate test

in mice.

78

11 Comparison on the phytochemical constituents between the leaves

of M. calabura and MEMC.

100

12 Comparison on the phytochemical constituents between the leaves

of M. calabura and PEMC

101

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LIST OF FIGURES

Figure

Page

1 Descartes' drawing of a pathway-oriented pain mechanism

7

2 Leaves and fruits of Muntingia calabura L.

22

3 Effect of MEMC in acetic acid-induced abdominal

constriction test in mice.

46

4 Effect of MEMC in formalin-induced paw licking test.

48

5 Effect of MEMC on capsaicin-induced paw licking test in rats.

58

6 Effect of MEMC on glutamate-induced paw licking test in

rats.

59

7 Effect of MEMC on PMA-induced paw licking in rats.

60

8 The antinociceptive effect of MEMC against bradykinin-

induced paw licking.

61

9A Effect of L-arginine, L-NAME and their combination on

MEMC antinociception as assessed by acetic acid-induced

abdominal constriction test.

62

9B Effects of L-arginine, methylene blue and their combination

on MEMC antinociception as assessed by acetic acid-induced


63

10 Effect of MEMC on nociception in the acetic acid-induced

abdominal constriction test in mice following pre-treatment

with potassium channels inhibitors.

64

11 The involvement of several non-opioid receptor antagonists in

MEMC-induced antinociception against acetic acid-induced

abdominal writhing test in mice.

65

12 Analysis of opioid receptor subtypes involvement in MEMC-

induced antinociception against acetic acid-induced writhing

test in mice.

66

13 Comparison between the numbers of abdominal constriction in

mice during the acetic acid-induced abdominal constriction test.

76

14 Effect of PEMC on capsaicin-induced paw licking test in rats.

86

15 Effect of PEMC on glutamate-induced paw licking test in rats.

85

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16 The antinociceptive effect of PEMC against bradykinin-

induced paw licking.

93

17 Effect of PEMC on nociception induced by PMA on rats

94

18A Effects of L-arginine, L-NAME and their combination on

PEMC antinociception as assessed by acetic acid-induced


95

18B Effects of L-arginine, methylene blue and their combination

on PEMC antinociception as assessed by acetic acid-induced abdominal constriction test.

96

19 Effect of PEMC on nociception in the acetic acid-induced

abdominal constriction test in mice following pre-treatment

with potassium channels inhibitors.

97

20 The involvement of several non-opioid receptor antagonists in

PEMC-induced antinociception against acetic acid-induced

abdominal writhing test in mice.

98

21 Analysis of opioid receptor subtypes involvement in PEMC-induced antinociception against acetic acid-induced writhing

test in mice.

99

22 The UV spectra analysis of MEMC

102

23 Chromatogram of MEMC at 254 nm showing the presence of

flavonoids type compounds, namely rutin, quercitrin and

fisetin based on the comparison of their respective UV spectra

analysis

103

24 The HPLC profile of PEMC

104

25 Comparison of the HPLC profile of PEMC against several

pure flavonoids at the wavelength of 300 and 366 nm.

105

26 Graphical abstract summarizing the possible mechanism of

action of MEMC and PEMC.

109

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LIST OF ABBREVIATIONS

5-HT Indole 5-hydroxytrptamine/Serotonin

AA Arachidonic acid

ACh Acetylcholine

AEMC Aqueous extract of Muntingia calabura

AMPA α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid

ANOVA Analysis of variance

AR Noradrenergic receptor

ASA Acetylsalicylic acid ATP Adenosine triphosphate

Ca2+ Calcium ion

cAMP Cyclic adenosine monophosphate

cGMP Cyclic guanosine monophosphate

Cl- Chloride ion

CNS Central nervous system

COX Cyclooxygenase

DAG Diacylglycerol

dH2O Distilled water

DMSO Dimethyl sulfoxide

DRG Dorsal root ganglion EAMC Ethyl acetate extract of Muntingia calabura

ED50 Median effective dose

FDA Food and drug association

HPLC High performance liquid chromatography

i.p. Intraperitoneal

i.pl. intraplantar

IASP International association for the study of pain

K+ Potassium ion

L-NAME NG-nitro-L-arginine methyl esters

LOX Lypooxygenase

mACh Muscarinic acetylcholine

MB Methylene blue MEMC Methanol extract of Muntingia calabura

Na+ Sodium ion

nACh Nicotinic acetylcholine

NMDA N-methyl-D-aspartate

NO Nitric oxide

NOS Nitric oxide synthase

NSAIDs Non-steroidal anti-inflammatory drugs

p.o. Orally

PAG periaqueductal grey

PEMC Petroleum ether extract of Muntingia calabura

PGs Prostaglandins PKC Protein kinase C

PLC Phospholipase C

PMA Phorbol 12-myristate 13-acetate

PKA Protein kinase A

PNS Peripheral nervous system

S.E.M. Standard error of mean

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sGC Soluble guanylyl cyclase

SNAT Sodium-coupled neural amino acid transporter

TRPV1 Transient receptor potential vanilloid subtype 1

VR1 Vanilloid receptor subtype 1

WHO World health organization

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CHAPTER 1

INTRODUCTION

1.1 Introduction

Pain is a universal experience that can span an enormous spectrum of intensity from

mild discomfort to excruciating agony. In other words, pain is a fundamental and

inevitable form of human suffering, the experience of which is unique to each

individual (Hawthorn and Redmond, 1998). The vocabulary of pain always suggests a

negative event which often relates to an unpleasant experience. Contrary, in basic

biological terms, pain is indeed a protective mechanism. Pain is indispensable for life

and survival that serves a protective function by signalling the presence of noxious,

tissue-damaging condition. From a medical standpoint, the subjective description and

indication of the location of pain may help to pinpoint the underlying cause of disease

(Tortora and Grabowski, 2003).

Human being has been pondering and trying to understand why they feel pain and how

to reduce it. Through the years, understanding and treatment of pain have changed and

developed in accordance with science. Initially viewed religiously as a consequence to

human wrong doing or due to evil spirit entered the body, it has changed to a more

scientific concept. In the first century, the emphasis in medicine shifted from plants to

pills. The usage of natural herbs as analgesia has declined as synthetic drugs with

specific actions were embraced by pharmaceutical industry (Gillis, 1998). However,

these contemporary analgesics, such as opiates and non-steroidal anti-inflammatory

drugs, are often not suitable in all patients and cases, particularly chronic pain on

account of their limitation such as potency, side effects and propensity to lead to

tolerance. As a result, the continuing search for other alternatives is necessary.

Medicinal plants are known to be an important source of new chemical substances with

potential therapeutic effects. The huge discovery of cinchona, morphine, as well as

aspirin which were derived from plant has laid the foundation for further research in

natural plants (Raskin and Ripoll, 2004). The research into plants which are employed

as pain-relievers in traditional ethno-medicine is therefore one of the productive and

logical strategies in the search for new analgesic drugs (Vongtau et al., 2004). Natural

medicine has a net worth of around US$40 billion in global market. The herbal

remedies not only can still be found in ethnic and health food stores, but are also

available in pharmacies and grocery stores (Rasooli, 2011). Data from World Health

Organization (WHO) shows that more than a half of world populations make use of

herbal medicine to relief painful or unpleasant symptoms, with at least 30% are

prescribed (WHO, 1978).

Focusing on natural products as an alternative to many medications, it has been a major

interest not only among scientists but also attract interest from both pharmaceutical

companies as well as financial support from the government. Natural products as

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referred to Holt and Chandra (2002) are herbs, herbal concoctions, dietary

supplements, traditional Chinese medicines or alternative medicines. Natural products

research is guided by ethno-pharmacological knowledge and has brought substantial

contributions to drug innovation by providing novel chemical structures and/or

mechanism of actions (Rates, 2001).

1.2 Hypothesis

MEMC possess significant antinociceptive activity due to the presence of

flavonoids-based compounds.

1.3 Objectives

General:

a) To evaluate the antinociceptive activity of crude and the most effective

partition of methanol extract of Muntingia calabura (MEMC).

Specific:

a) To determine the safety of MEMC using single high-dose acute toxicity model

b) To determine antinociceptive profiles of MEMC using various animal models

c) To elucidate the possible mechanisms of action that take part in the

antinociception of MEMC

d) To determine the most effective partition of MEMC using acetic acid-induced


e) To elucidate the possible mechanisms of action that take part in the

antinociception of PEMC

f) To elucidate and identify the possible bioactive compounds responsible for the

MEMC and PEMC triggered antinociception using the phytochemical

screening test and HPLC analysis

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BIODATA OF STUDENT

Mohd.Hijaz Mohd Sani was born in Kota Kinabalu, Sabah, Malaysia on April 14th

,

1987. He received his early primary education at Sekolah Kebangsaan Malawa,

Telipok (1994-1999). He then continued his secondary education at Sekolah

Menengah St. Peter, Telipok from 200-2004 before being accepted into Labuan

Matriculation College (2005-2006). In July 2006, his application to further his study in

bachelor degree was accepted by Universiti Putra Malaysia. He completed his degree in

Biomedical Sciences in four years. During the study period, his passion and interest

towards research particularly in medicine field was developed, which encouraged him

to further his study. He pursues his study in Master of Science (Pharmacology) at the

same university. After a year, he managed to complete reasonable amount of his

research which allowed him to publish his first scientific journal. Following this, his

determination prompts him to apply for conversion from master to doctor of

philosophy which was successful.

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LIST OF PUBLICATIONS

Journal:

M.H. Mohd. Sani, Z.A. Zakaria, T. Balan, L.K. Teh and M.Z. Salleh (2012).

Antinociceptive Activity of Methanol Extract of Muntingia calabura Leaves and

the Mechanisms of Action Involved. Evidence-based Complementary and

Alternative Medicine. Article ID 890361. DOI:10.1155/2012/890361

Z.A. Zakaria, M.H.M. Sani, M.N.H. Abdullah, A. Zuraini, A. Abdul Kader, L.K. Teh

and M.Z. Salleh (2014). Antinociceptive Activity of Methanolic Extract of

Muntingia calabura Leaves: Further Elucidation of the Possible Mechanisms.

BMC Complementary and Alternative Medicine. 14: 63.

Z.A. Zakaria M.H.M Sani, A.A. Kadir, L.K. Teh, M.Z. Salleh, (2016). Antinociceptive

effect of semi-purified petroleum ether partition of Muntinia calabura leaves.

Sociedade Brasileira de Farmacognosia, doi: 10.1016/j.bjp.2015.12.007

Zainul Amiruddin Zakaria, Mohammed Hijaz Sani, Mohammed Fazli Mohammar,

Nurul Shulehaf Mansor, Zurina Shaameri, Teh Ley Kek et al. 2013.

Antinociceptive activity of a synthetic oxopyrrolidine-based compound,

ASH21374, and determination of its possible mechanisms. Can. J. Pharmacol.

91: 1143-1153

Mohd. Hijaz Mohd Sani, Muhammad Taher, Deny Susanti, Teh Ley Kek, Mohd. Zaki

Salleh, Zainul Amiruddin Zakaria. 2014. Mechanisms of α-Mangostin-induced

antinociception in a rodent model. Biological Research for Nursing. 17(1): 68-

77

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