UNIVERSITI PUTRA MALAYSIA

ANTINOCICEPTIVE ACTIVITY OF HARUAN (Channa striatus) TRADITIONAL EXTRACT

ZULEEN DELINA FASYA BINTI ABDUL GHANI

FPSK(m) 2010 10

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ANTINOCICEPTIVE ACTIVITY OF HARUAN (Channa striatus)

TRADITIONAL EXTRACT

By

ZULEEN DELINA FASYA BINTI ABDUL GHANI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the

Degree of Master of Science.

January 2010

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DEDICATION

This thesis is dedicated to all of the following people who have inspired

me in a very special way of my life:

My self – To try the best I can do.

Mak and Ayah – For your eternal love and supports.

Along and Adik – For reminding me not to give up.

My family and friends – For being there.

My supervisor and co-supervisor – For trusting me.

“Patience, persistence and perspiration make an unbeatable

combination for success” – Napoleon Hill (1883-1970)

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science.

ANTINOCICEPTIVE ACTIVITY OF HARUAN (Channa striatus) TRADITIONAL EXTRACT

By

ZULEEN DELINA FASYA BINTI ABDUL GHANI

January 2010

Chair: Professor Dr. Abdul Manan Mat Jais

Faculty: Medicine and Health Sciences

The objectives of this study were to determine the amino acid and fatty acid

composition of Haruan Traditional Extract (HTE), to elucidate the mechanism

responsible for its antinociceptive activity and to clarify the relationship

between the presence of amino acids and fatty acids with the expected

activity. The HTE was obtained by steaming fresh fillet of Channa striatus

with pressure cooker at 100oC, for 3 hrs after 1 hr of seasoning process. For

the antinociceptive activities of HTE at different concentration, the extracts

were purified, filtered and prepared in concentrations of 100 (stock solution),

50 and 10% dilution in distilled water and subjected to the antinociceptive

studies. The antinociceptive activities of HTE was done on different

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molecular weight, where some of the extract was subjected to the

centrifugation-filtration process with Ultraspeed Centrifuge for 7k G in 3 hr

using Vivaspin Polythersulfone Membrane with different pore size (3k, 5k,

10k and 30k NMWL) while part of it would be kept in non-filtered conditions /

stock solution prior to the antinociceptive assays. Lastly, for the elucidation of

the involvement of opioid receptor in HTE antinociceptive activities, the

antinociceptive action of the extract with the highest antinociceptive activity

resulted from the prior study were tested against naloxone in all

antinociceptive assays in a bid to further elucidate probable mechanisms of

antinociception. In all of the above-mentioned studies were involved the use

of 0.6% acetic acid-induced abdominal constriction, 5% formalin-induced

paw-licking, and 50oC hot plate test in rodent to evaluate the HTE

antinociceptive activities. For the determination of amino acid and fatty acid

the stock solution HTE was run into the High Performance Liquid

Chromatography (HPLC) and Gas Chromatography (GC), respectively. All

data obtained were analyzed using the One-Way Analysis of Variance

(ANOVA) followed by Dunnet test with P<0.05 as the limit of significance.

From the data obtained, the HTE was found to contain major amino acid

cysteine, histidine, glutamic acid, glycine and aspartic acid. In addition, the

HTE was found to have a high myristic acid followed by palmitic, arachidonic,

linolenic and oleic acid. The HTE exhibited concentration/dosage-dependent

manner significant antinociceptive activity (P<0.05). On the other hand, at

different molecular, HTE showed the size-dependent in abdominal

constriction, hot plate and first phase of formalin test. HTE also were showed

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to have an involvement of central antinociceptive mechanism when tested

against the naloxone.

As a conclusion, the HTE contains all the important amino acids and fatty

acids and possesses the antinociceptive activities. Thus, proved the

traditional claimed for reducing pain, inflammation and promote wound

healing properties.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk Ijazah Sarjana Sains.

AKTIVITI ANTINOSISEPTIF TERHADAP EKSTRAK TRADITIONAL HARUAN (Channa striatus)

Oleh

ZULEEN DELINA FASYA BINTI ABDUL GHANI

Januari 2010

Pengerusi: Professor Dr. Abdul Manan Mat Jais

Fakulti: Perubatan dan Sains Kesihatan

Objektif penyelidikan ini adalah untuk menentukan komposisi asid amino

dan asid lemak Ekstrak Tradisional Haruan (HTE) bagi mengenalpasti

mekanisma yang melibatkan aktiviti antinosiseptif serta menjelaskan

hubungkait di antara kehadiran asid amino dan asid lemak dengan aktiviti

antinosiseptive yang dijangkakan. HTE diperoleh melalui proses pengukusan

isi ikan Channa striatus (haruan) segar dengan menggunakan periuk tekanan

pada suhu 100oC selama 3 jam, selepas proses perapan selama 1 jam.

Ketika aktiviti antinosiseptif HTE dalam berlainan kepekatan, ekstrak perlu

dibersihkan, disaring dan disediakan dalam kepekatan 100 (larutan stok), 50,

10% dengan dilarutkan menggunakan air suling sebagai bahan untuk kajian

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antinosiseptif. Untuk aktiviti antinosiseptif HTE dalam berlainan berat

molekul, sebahagian daripada ekstrak HTE akan melalui proses emparan –

saringan dengan menggunakan pengempar berkelajuan Ultra (7k G) selama

3 jam bersama-sama membran Vivaspin Polythersulfone yang mempunyai

pelbagai saiz liang (3k, 5k, 10k dan 30k NMWL). Manakala sebahagian lagi

akan dibiarkan dalam keadaan asal iaitu tanpa saringan / larutan tulen untuk

analisa antinosiseptif. Akhir sekali, untuk mengenalpasti kehadiran reseptor

opioid dalam aktiviti antinosiseptif HTE, kesan ekstrak antinosiseptif dengan

keputusan yang tertinggi daripada kajian terdahulu akan diuji bersama-sama

naloxone dalam semua ujian antinosiseptif bagi menentukan kebarangkalian

mekanisma antinosiseptif. Dalam semua ujian yang disebutkan di atas,

sebanyak 0.6% asid asetik digunakan untuk merangsang pencerutan

abdomen, 5% formalin bagi merangsang haiwan untuk menjilat tapak kaki

dan ujian 50oC kepanasan piring terhadap tikus digunakan bagi menentukan

aktiviti antinosiseptif HTE. Untuk penentuan asid amino dan asid lemak,

larutan stok / 100% HTE akan dianalisa dengan menggunakan High

Performance Liquid Chromatography (HPLC) dan Gas Chromatography

(GC). Kesemua data yang diperolehi akan dianalisis dengan menggunakan

kaedah One-Way ANOVA diikuti ujian Dunnet dengan limit signifikasi

P<0.05.

Daripada data yang diperolehi, HTE telah dikenalpasti mengandungi asid

amino dengan komponen majornya adalah terdiri daripada asid sistin,

histidin, glutamik, glisin dan asid aspartik. Selain itu, HTE juga dikenalpasti

mengandungi kandungan asid myristic yang tinggi, diikuti asid palmitik,

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arakidonik, linolenik, dan oleik. HTE menunjukkan pergantungan dos aktiviti

dengan signifikasi (P<0.05). Ketika kajian aktiviti antinosiseptif HTE dalam

berlainan saiz tapisan, keputusan menunjukkan bahawa saiz dipengaruhi

oleh pencerutan abdomen, piring panas serta fasa pertama ujian Formalin.

HTE juga menunjukkan penglibatan mekanisma pusat antisiseptif apabila

diuji dengan naloxone.

Kesimpulannya, HTE mengandungi semua asid amino serta sebahagian asid

lemak yang penting dan mempunyai aktiviti antinosiseptif. Oleh itu,

dibuktikan bahawa secara tradisional untuk mengurangkan kesakitan,

keradangan serta pemulihan kecederaan.

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ACKNOWLEDGEMENTS

“In the name of Allah S.W.T., the most benevolent and Merciful.

All gratification is referred to ALLAH S.W.T.”

Firstly I would like to take this opportunity to thank the chairman of my

supervisory committee, Professor Dr. Abdul Manan B. Mat Jais, whose

expert guidance and support has helped me to complete this research. His

kindness, affection, encouragement and moral support gave me the courage

and ability to overcome all the problems I have faced from time to time during

the course of my work. I would like to extend my appreciation to him for his

invaluable advice and continuous comment and support, which brighten my

future through the experiences that I have gained from him.

I am also very grateful to Professor Dr. Roslan B. Sulaiman, whose expert

guidance and support has helped me to overcome most of the problems that

I have encountered during the course of my study.

I am also indebted to and wish express my appreciation to Professor Dr.

Mustaffa Ali B. Mohamed for his interest and invaluable suggestion that had

enabled me to carry on with my project successfully.

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I would like to thank the Ministry of Sciences, Technology and Environmental

for their belief in me and for providing me a scholarship (National Science

Fellowship), which has enable me to continue my research project as well as

my life as a student and to UPEN Pahang (BSP©/BTK/006; Tabung Amanah-

6362300-14001) for trusting and supporting grant of this research project.

The list of thanks also goes to all of my friends and the people who have

known me for the joy and laughter and memories that we have shared

together during my study at Universiti Putra Malaysia.

Lastly, I would like to express my most sincere and warmest gratitude to my

mother (Rohana Bt. Katan), my father (Abdul Ghani B. Bakar) and all my

family members for their prayers, love, generosity and moral supports that

they have provided during the course of my study.

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I certify that a Thesis Examination Committee has met on 6 January 2010 to conduct the final examination of Zuleen Delina Fasya Binti Abdul Ghani on her thesis entitled “Antinociceptive Activity of Haruan (Channa striatus) Traditional Extract” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the degree of Master of Science. Members of the Thesis Examination Committee were as follows: Sabrina Sukardi, PhD Associate Professor Department of Biomedical Sciences Faculty Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Zuraini Ahmad, PhD Associate Professor Department of Biomedical Sciences Faculty Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Roslida Abd. Hamid @ Abd. Razak, PhD Department of Biomedical Sciences Faculty Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Ahmad Rohi Ghazali, PhD Associate Professor Department of Biomedical Science Faculty of Allied Health Sciences Universiti Kebangsaan Malaysia (External Examiner)

BUJANG BIN KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date: 12 February 2010

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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 Master of Science. The members of the Supervisory Committee were as follows: Abdul Manan Mat Jais, PhD Professor, Department of Biomedical Sciences, Faculty Medicine and Health Sciences, Universiti Putra Malaysia. (Chairman) Mohd Roslan Sulaiman, PhD Professor, Department of Biomedical Sciences, Faculty Medicine and Health Sciences, Universiti Putra Malaysia. (Member) Mustaffa Ali Mohammed, PhD Professor, Department of Pharmacology, Faculty of Medicine, Universiti Malaya. (Member)

HASANAH MOHD GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 17 March 2010

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DECLARATION

I decline that the thesis is my original work except for quotations and citations

which have been duly acknowledged. I also declare that has not been

previously, and is not concurrently, submitted for any other degree at

Universiti Putra Malaysia or at any other institution.

ZULEEN DELINA FASYA BINTI ABDUL GHANI

Date: 16 March 2010

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TABLE OF CONTENTS

Page

DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvii LIST OF FIGURES xviii LIST OF ABBREVIATIONS xx

CHAPTER

1 INTRODUCTION

1.1 Marine Natural Product 2

1.2 Channa striatus 3

1.3 Justification of the Study 4

1.4 Objectives of the Study 4

2 LITERATURE REVIEW

2.1 Natural Product 6

2.2 Fish Natural Product 7

2.3 Channa striatus 9

2.4 Pain 10

2.4.1 Mechanism of Pain 12

2.5 Nociception 16

2.5.1 Nocicepton Neurons 16

2.6 Analgesic Drugs 17

2.6.1 Non-Opioid Analgesic Compounds 18

2.6.2 Opioid Analgesic Compounds 19

2.7 Aspirin – A NSAID’s With Analgesic Properties 20

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2.8 Bioactive Compounds – Amino Acid and Fatty

Acid

22

3 MATERIALS AND METHODS

3.1 Preparation of Fresh Fillet C. striatus 24

3.2 Preparation of Haruan Traditional Extract (HTE) 25

3.3 Preparation of Different Concentration of HTE 25

3.4 Preparation of Different Filter Sizes of HTE 25

3.5 Determination of Amino Acid and Fatty Acid

Composition of THE

26

3.5.1 Determination of Protein Composition of

THE

26

3.5.2 Determination of Amino Acid Composition

of THE

27

3.5.3 Determination of Fatty Acid Composition

of THE

29

3.6 Antinociceptive Studies 32

3.6.1 Preparation of the Dosage Drugs 32

3.6.2 Experimental Animals 32

3.6.3 Establishment of the Antinociceptive

Profile of THE

33

3.6.4 Abdominal Constriction Test 34

3.6.5 Formalin Test 35

3.6.6 Hotplate Test 35

3.6.7 Antinociceptive Test Pre-Treated With

Naloxone

36

3.7 Statistical Analysis 36

4 RESULTS

4.1 Amino Acid and Fatty Acid Composition of HTE 37

4.2 Antinociceptive Activities of THE at Different

Concentration

47

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4.3 Antinociceptive Activities of THE from Different

Filter Sizes

52

4.4 Elucidation of the Involvement of Opioid

Receptor in HTE Antinociceptive Activities

57

5 DISCUSSION 62 6 SUMMARY, CONCLUSION AND

RECOMMENDATION FOR FUTURE STUDIES 72

REFERENCES 75 APPENDICES 85 BIODATA OF STUDENT 96 LIST OF PUBLICATIONS 98