APHRODISIAC ACTIVITY and SCREENING for ACUTE TOXICITY of ASIAN SWAMP EEL, Monopterus Albus EXTRACTS on MALE MICE

APHRODISIAC ACTIVITY AND SCREENING FOR ACUTE TOXICITY OF ASIAN SWAMP EEL, Monopterus albus EXTRACTS ON MALE MICE

NOR INTAN FAZLEEN BINTI ZULKEFLI

A thesis submitted in fulfilment of the requirement for the degree of Master of Health Sciences

Kulliyyah of Allied Health Sciences International Islamic University Malaysia

FEBRUARY 2017

ABSTRACT

Monopterus albus is one of the terrestrial animal sources that is widely consumed by the local people, who believe in the medicinal properties offered by the species. However, there is not many research has been done on the species to prove the claims. Therefore, this study was conducted to evaluate the potential of M. albus extracts as an aphrodisiac and to characterize the components of the extracts. This study comprises of three phases of study: aphrodisiac, fertility and acute toxicity test. Two types of extraction were used; aqueous and lipid. Prior to treatment, the extracts were analysed for the mineral contents and fatty acid analysis using inductively coupled plasma optical emission spectrometry (ICP-OES) and Gas Chromatography Mass Spectrometry (GC-MS), respectively. Then, the sexual behaviour was observed in eight groups of ICR male mice with four animals in each group (n = 4). The doses used for both extracts were 50, 100 and 200 mg/kg. Negative control group received 0.9% saline, meanwhile positive control group received 5 mg/kg Sildenafil citrate or known as Viagra®. After an hour administration (i.p.) of the respective doses, the observation of sexual behaviour was conducted. The male mouse was paired with a female mouse for three hours to determine the effect of extracts on sexual behaviour. In addition, mating assessment also was carried out at similar dose level. On the sixth day, the copulatory plug was observed on the female mice used to confirm the presence of ejaculation. Afterwards, the male mice were slaughtered to evaluate the sperm count and motility as well as testicular histology. Another group of male mice were used for the analysis of testosterone levels. After that, toxicity test was conducted to determine the effect of a single dose of lipid extract in rats. The rats administered orally with the doses of 5, 50, 300 and 2000 mg/kg of M. albus lipid extract and control group received 0.9% saline. The survival and toxicity signs were observed for 14 days. Besides that, the blood analysis and histopathological examination of liver and kidney tissues also were investigated. From the analysis of free fatty acid, it shows that lipid extracts of M. albus contained higher level of arachidonic acid (AA), the essential fatty acid which influences the reproduction. The results obtained shows that all doses of lipid extract has significantly increased (p ≤ 0.05) the mounting frequency (MF) and sperm count as compared to negative control. Meanwhile, in aqueous extract, only the dose of 200 mg/kg shows significant difference of MF but no improvement in sperm count. In the analysis of testosterone levels, only the dose of 200 mg/kg lipid extract and positive control group showed tendency to increase the hormone levels in treated mice. Whereas, in the toxicity study, the haematological, biochemical and histological analyses showed no significant difference (p ≥ 0.05) as compared to the control. The treated rats showed no acute signs of toxicity and no mortality was observed. Therefore, the M. albus was safe to be consumed by the people and has great potential as an aphrodisiac agent. Thus, the study suggests that M. albus lipid extract could be used as an alternative source for sexual activity in men.

خالصة ﺍﻟﺐﺤﺚ

إْ حُىاْ ِٕىبخىط أٌبىط )Monopterus albus( ٔىم ِٓ األمسبن اٌخخٕفظ يف اذلىاء وحغخهٍه هًٍ ٔـبق واعن ِٓ لبً اٌغىبْ احملٍُني، اٌزَٓ َؤِٕىْ ببخلظبئض اٌـبُت ذلزا إٌىام. وِن رٌه فٍُظ ٕ٘بٌه أحببد ِوخربة أصشَج هًٍ ٘زا إٌىم إلرببث حٍه ادلضاهُ. وٌزٌه، فئْ ٘زٖ اٌذساعت هتذف إىل حمُُُ ِغخخٍظبث M. albus وّٕشؾ صٕغٍ وٌىطف ِىىٔبث ادلغخخٍظبث. وحخأٌف ٘زٖ اٌذساعت ِٓ رالد ِشاحً: أوذلب، اخخببس اربسة اٌشهىة اجلٕغُت واخلظىبت واخخببس اٌغُّت احلبدة. مت اعخخذاَ ٔىهني ِٓ ادلغخخٍظبث ادلبئٍ واٌذ٘ين لبً اٌوالس. ولذ مت حتًٍُ ادلغخخٍظبث هٓ احملخىَبث ادلوذُٔت وحتًٍُ األمحبع اٌذُٕ٘ت ببعخخذاَ احلذ ببإلػبفت إىل اٌبالصِب اٌبظشَت ِـُبفُت االٔبوبد (ICP-OES) واٌغبص اٌٍىين اٌـُفٍ اٌىخٍٍ (GC-MS) ، هًٍ اٌخىايل. ٌىحق أْ اٌغٍىن اجلٕغٍ يف مثبين رلّىهبث ِٓ روىس اٌفئشاْ ِن أسبوت حُىأبث يف وً رلّىهت )ْ = 4(. ووبٔج اجلشهبث ادلغخخذِت ٌىال ِٓ ادلغخخٍظبث 25 و 055 و 055 ٍِغُ / وغُ. حٍمج رلّىهت ادلشالبت اٌغٍبُت 0.0٪ ِٓ احملٍىي ادلٍحٍ، حٍمج رلّىهت ادلشالبت اإلجيببُت يف ٘زٖ األرٕبء 5 ٍِغ / وغ فُبغشا عُخشاث أو ادلوشوفت ببعُ .®Viagra بوذ اداسة عبهت (.i.p)ِٓ حٕبوي اجلشهبث، أصشَج ِشالبت اٌغٍىن اجلٕغٍ. ووبٔج حمخشْ روىس اٌفئشاْ ِن إٔبد اٌفئشاْ دلذة رالد عبهبث ٌخحذَذ أرش ادلغخخٍظبث هًٍ اٌغٍىن اجلٕغٍ. وببإلػبفت إىل رٌه، مت إصشاء حمُُُ اٌخضاوس هًٍ ِغخىي صشهت ممبرٍت. يف اٌُىَ اٌغبدط، ولذ ٌىحق حىىْ copulatory plug يف إٔبد اٌفئشاْ واٌيت حغخخذَ ٌخأوُذ وصىد اٌمزف. بوذ رٌه، مت ربح روىس اٌفئشاْ ٌخمُُُ هذد احلُىأبث ادلٕىَت وحشوخهب ووزٌه األٔغضت اخلظُت. واعخخذِج رلّىهت أخشي ِٓ روىس اٌفئشاْ ٌخحًٍُ ِغخىَبث ٘شِىْ اٌخغخىعخريوْ. بوذ رٌه، مت إصشاء اخخببس اٌغُّت ٌخحذَذ حأرري صشهت واحذة ِٓ ادلغخخٍض اٌذ٘ين يف اٌفئشاْ. مت اهـبء اٌفئشاْ هٓ ؿشَك اٌفُ صشهت ِٓ 2، 25، 055 و 0555 ٍِغ / وغ ِٓ ادلغخخٍض اٌذ٘ين ِٓ M. albus وحٍمج اجملّىهت اؼٌببـت احملٍىي ادلٍحٍ 5.0٪. ولذ ٌىحلج هالِبث بمبء ومسُت دلذة 01 َىِب. وببالػبفت اىل رٌه، مت اٌخحمُك يف حتًٍُ اٌذَ وفحض أٔغضت اٌىبذ وأٔغضت اٌىًٍ. ِٓ خالي حتًٍُ األمحبع اٌذُٕ٘ت احلشة ربج أْ ادلغخخٍض اٌذ٘ين اٌىاسد ِٓ M. albus ٌٗ ِغخىي ِفوىي أهًٍ ِٓ محغ األساوُذؤُه (AA) ، واألمحبع اٌذُٕ٘ت األعبعُت اٌيت حؤرش هًٍ اإلجنبة. ِٓ إٌخبئش ادلخحظً هٍُهب حبني أْ وً صشهت ِٓ ادلغخخٍض اٌذ٘ين اصداد بشىً ٍِحىف )ص ≥ 5.52( هًٍ وحرية ِخظبهذة وهذد احلُىأبث ادلٕىَت ببدلمبسٔت ِن اجملّىهت اؼٌببـت اٌغٍبُت. ويف اٌىلج ٔفغٗ، يف ادلغخخٍض ادلبئٍ، صشهت 055 ٍِغ / وغ حلهش اخخالفب وبريا يف MF وٌىٓ ٌُظ ٕ٘بن أٌ حتغٓ يف هذد احلُىأبث ادلٕىَت. يف حتًٍُ ِغخىَبث ٘شِىْ اٌخغخىعخريوْ، يف صشهت 055 ٍِغُ / وغُ ِٓ ادلغخخٍض اٌذ٘ين واجملّىهت اؼٌببـت اإلجيببُت أكهشحب اجتب٘بٌضَبدة ِغخىَبث اذلشِىْ يف اٌفئشاْ اٌيت هىجلج. يف حني، يف دساعت اٌغُّت، وأِشاع اٌذَ، أكهشث اٌخحبًٌُ اٌبُىوُُّبئُت وإٌغُضُت أٔٗ ال َىصذ فشق وبري )p ≤ 5.52( ببدلمبسٔت ِن اجملّىهت اؼٌببـت. مل ٔلهش اٌفئشاْ أٌ هالِبث حبدة اٌغُّت ومل َالحق أٌ وفُبث. وٌزٌه، وبْ M. albus آِٓ إىل أْ بغخهٍه ِٓ لبً اٌشوىة وٌذَٗ إِىبٔبث وبرية وّزري ٌٍشهىة اجلٕغُت. و٘ىزا، حشي اٌذساعت أْ ادلغخخٍض اٌذ٘ين ي M. albus ميىٓ أْ َغخخذَ وّظذس بذًَ ٌٍٕشبؽ اجلٕغٍ ٌذي اٌشصبي.

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APPROVAL PAGE

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 thesis for the degree of Master of Health Sciences.

...... Ridzwan Hashim Supervisor

...... Mohd Shukri Baba Internal Examiner

...... Wan Omar Abdullah External Examiner

This thesis was submitted to the Department of Biomedical Science and is accepted as a fulfilment of the requirement for the degree of Master of Health Sciences.

...... Ibrahim Adham Taib Head, Department of Biomedical Science

This thesis was submitted to the Kulliyyah of Allied Health Sciences and is accepted as a fulfilment of the requirement for the degree of Master of Health Sciences.

...... Wan Azdie Mohd Abu Bakar Dean, Kulliyyah of Allied Health Sciences

iv DECLARATION

I hereby declare that this thesis is the result of my own investigations, 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.

Nor Intan Fazleen Binti Zulkefli

Signature …………………………………… Date ……………………..

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

APHRODISIAC ACTIVITY AND SCREENING FOR ACUTE TOXICITY OF ASIAN SWAMP EEL, Monopterus albus EXTRACTS ON MALE MICE

I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM.

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 acknowledgement.

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 Nor Intan Fazleen Binti Zulkefli

………..………… ….….………….. Signature Date

To my beloved family, may they always be blessed by Allah S.W.T.

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ACKNOWLEDGEMENTS

In the name of Allah the Most Beneficient and the Most Merciful

Alhamdulillah, I sincerely would like to state my gratitude to Most Beneficient and Most Merciful creator, Allah S.W.T for permitting me to complete this thesis, as well as blessing me the joy of accomplishment after the years spent in sweat and tears to finish this thesis to the best of my efforts.

Fisrt and foremost, a special thanks to my supervisor, Prof Dato‟ Paduka Dr. Hj. Ridzwan Hashim. I truly appreciated the advice and guidance that he has given me from the beginning until the end of this study. His continuous encouragement pushed me to continue my research until the last page of this thesis.

I am also would like to forward my special thanks to all science officers, assistant science officer, medical lab technologist and to all who have contributed their efforts and works, whether directly or indirectly, for this study at Kulliyyah of Allied Health Sciences. Your kindness has not gone unnoticed.

My sincere gratitude also goes to my beloved parents, Zulkefli Ibrahim and Sharifah Salleh, my siblings for their constant moral and physical supports, understanding, motivation as well as their endless love and care which kept me strong in facing the hardship throughout the completion of this study. Not to forget to all my postgraduate colleagues who had assisted from the beginning of this study until the thesis is submitted. May Allah S.W.T. showered his blessing to all of us till the end of time.

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TABLE OF CONTENTS

Abstract ...... ii Abstract in Arabic ...... iii Approval Page ...... iii Declaration ...... v Copyright Page ...... vi Dedication ...... vii Acknowledgements ...... viii Table of Contents ...... ix List of Tables ...... xii List of Figures ...... xiv List of Abbreviations ...... xvi List of Symbols ...... xvi

CHAPTER ONE: INTRODUCTION ...... 1 1.1 Research Background ...... 1 1.2 Significance of Study ...... 5 1.3 Objective ...... 6 1.3.1 General Objective ...... 6 1.3.2 Specific Objectives ...... 6

CHAPTER TWO: LITERATURE REVIEW ...... 7 2.1 Asian Swamp Eel; Monopterus albus ...... 7 2.1.1Taxonomy and Distribution of Asian Swamp Eel ...... 7 2.1.2 Habitat of Asian Swamp Eel ...... 8 2.1.3 Morphology of Asian Swamp Eel ...... 9 2.1.4 Life Cycle of Asian Swamp Eel ...... 10 2.2 Nutritional Value of Monopterus albus ...... 12 2.3 Traditional Medicinal Value and Beliefs ...... 13 2.4 Scientific Research on the Asian Swamp Eel ...... 13 2.4.1 Antimicrobial and Antifungal Activity ...... 13 2.4.2 Antiproliferative Activity ...... 14 2.4.3 Alternative Skin Gelatin ...... 15 2.4.4 Wound Healing ...... 16 2.5 Aphrodisiac ...... 17 2.5.1 Plant Resources as Aphrodisiac ...... 17 2.5.2 Marine Resources as Aphrodisiacs ...... 19 2.6 Toxicity Study ...... 20 2.6.1 Heavy Metals Uptake by Monopterus albus ...... 22 2.6.2 Acute Toxicity Study of Monopterus albus Aqueous Extract ...... 23

CHAPTER THREE: METHODOLOGY ...... 24 3.1 Materials and Equipments ...... 24 3.1.1 Animals ...... 23 3.1.2 Chemicals/Reagents/ Solutions/ Drugs ...... 24 3.1.3 Consumable/Disposable Items ...... 27 3.1.4 Equipment ...... 28 3.2 Samples Collection and Eel Fillet Preparation ...... 29 3.3 Eel Fillet Extraction ...... 30 3.3.1 Aqueous Extraction ...... 30 3.3.2 Lipid Extraction ...... 30 3.4 Mineral Content Analysis ...... 31 3.5 Free Fatty Acid Analysis ...... 32 3.6 Experimental Animals ...... 33 3.6.1 Aphrodisiac and Male Fertility Study ...... 33 3.6.2 Toxicity Study ...... 33 3.7 Aphrodisiac Study ...... 34 3.7.1 Preparation of Treatment Solutions ...... 34 3.7.2 Experimental Design ...... 36 3.7.3 Sexual Behaviour Analysis ...... 37 3.7.4 Mating Assessment ...... 39 3.7.4.1 Vaginal smear ...... 39 3.8 Male Fertility Study ...... 40 3.8.1 Sperm Count ...... 40 3.8.2 Sperm Motility ...... 41 3.8.3 Histopathological Observation of Testis ...... 41 3.9 Serum Testoterone Assay ...... 42 3.10 Acute Toxicity Study ...... 43 3.10.1 Preparation of Treatment Solutions ...... 43 3.10.2 Experimental Design ...... 43 3.10.3 Physical Observation, Body Weight and Mortality ...... 43 3.10.4 Haematological and Biochemical Analyses ...... 44 3.10.5 Histopathological Analysis ...... 44 3.11 Statistical Analysis ...... 45

CHAPTER FOUR: RESULTS ...... 46 4.1 Mineral Content Analysis ...... 46 4.2 Free Fatty Acid Analysis ...... 47 4.2.1 Total Lipid Content ...... 47 4.2.2 Free Fatty Acid Profiles ...... 47 4.3 Aphrodisiac Study ...... 49 4.3.1 Analysis of Sexual Behaviour ...... 49 4.3.1.1 Behaviour observation ...... 49 4.3.1.2 Effect of M. albus aqueous extract on sexual behaviour ...... 51 4.3.1.3 Effect of M. albus lipid extract on sexual behaviour ...... 53 4.3.1.4 Effect of M. albus extracts on mounting frequency ...... 55 4.3.2 Analysis of Mating Assessment ...... 56 4.4 Male Fertility Study ...... 57 4.4.1 Sperm Count and Motility ...... 57

4.4.2 Histopathology of Testis ...... 58 4.5 Analysis of Testosterone Serum Levels ...... 60 4.6 Acute Toxicity Study ...... 61 4.6.1 Mortality and Physical Observation ...... 61 4.6.2 Body Weight Observation ...... 62 4.6.3 Haematological and Biochemical Analysis ...... 62 4.6.4 Gross Organ Observation ...... 66 4.6.5 Histopathological Observation ...... 66

CHAPTER FIVE: DISCUSSION ...... 72 5.1 Effects of M. albus Extracts on Aphrodisiac and Fertility Properties ...... 72 5.2 Effects of M. albus Extracts on Acute Toxicity Study ...... 82 5.3 Aphrodisiac Research from Islamic Perspectives ...... 86

CHAPTER SIX: CONCLUSION ...... 89 6.1 Conclusion ...... 89 6.1.1 Limitation ...... 90 6.1.2 Future Work ...... 91

REFERENCES ...... 92

APPENDIX A: FLOW CHART ...... 104 APPENDIX B: ORIGINAL DATA ...... 105 APPENDIX C: PUBLISHED ARTICLES/PAPERS/BOOK CHAPTERS ...... 106 APPENDIX D: CONFERENCES/SEMINARS ...... 108 APPENDIX E: IACUC ETHICS COMMITTEE APPROVAL ...... 109

LIST OF TABLES

Table No. Page No.

2.1 Summarization of nutritional elements in Asian swamp eel, Monopterus albus as being analysed by Razak et al. (2001), Tok et al. (2009), and Rahman et al. (2012). 12 3.1 Animals used for aphrodisiac and toxicity studies.. 24 3.2 List of chemicals/reagents/solutions used in lipid extraction, mineral content analysis and free fatty acid analysis. 25 3.3 List of chemicals/ reagents/solutions/drugs used in the aphrodisiac and fertility study. 25 3.4 List of chemicals/reagents/solutions used in histopathology. 26 3.5 The consumable/disposable items used throughout the study. 27

3.6 The equipment used throughout the study. 28 4.1 Mineral content of Monopterus albus. 46 4.2 Fatty acid composition in lipid extracts of Monopterus albus fillet. 47 4.3 Summary of behavioral observation of ICR male mice in all treated groups (n=4) over 3 hrs of observation. The volume administered (i.p.) for all the mice were 0.2 mL 50 4.4 Effects of different doses of M. albus fillet aqueous extract on sexual behaviour of ICR male mice after 1 hr administration (i.p.) of treatment (MF = mounting frequency, IF = intromission frequency, ML = mounting latency, IL = intromission latency, EJ = ejaculation). 51 4.5 Effects of different doses of M. albus fillet lipid extract on sexual behaviour in ICR male mice after 1 hr administration (i.p.) of treatment (MF = mounting frequency, IF = intromission frequency, ML = mounting latency, IL = intromission latency, EJ = ejaculation). 53 4.6 Effect of aqueous and lipid extracts of Monopterus albus on the number of mounting in ICR male mice for 3 hrs. 55 4.7 Effects of treatment of Monopterus albus extracts on epididymal sperm count and sperm motility of ICR male mice 57

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4.8 Effect of different doses of M. albus fillet extracts on the levels of serum testosterone in ICR male mice after 6 days treatment (- = not detected). 60 4.9 Mortality and physical observation of control and treated rats for 14 days test period. All the rats received the same volume (2 mL) orally of either saline or lipid extraction of Monopterus albus fillet. 61 4.10 Body weight of female Sprague-Dawley rats before and after treatment with Monopterus albus fillet lipid extract during the acute toxicity study. All the rats received the same volume (2 mL) orally of either saline or lipid extraction of M. albus fillet. 62 4.11 Effects of different doses of Monopterus albus lipid extract given orally on haematology parameters of Sprague-Dawley rats after 14 days. All the rats received the same volume (2 mL) of either saline or lipid extraction of M. albus fillet. 63 4.12 Effects of different doses of Monopterus albus lipid extract given orally on blood chemistry parameters of Sprague- Dawley rats after 14 days. All the rats received the same volume (2 mL) of either saline or lipid extraction of M. albus fillet. 65

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LIST OF FIGURES

Figure No. Page No. 2.1 (a) The cylindrical and tapering shape of Asian swamp eel, (b) ventral side of Asian swamp eel with lighter in colour. 9

2.2 Life cycle of Asian swamp eel, Monopterus albus. 11

3.1 (a) Fresh samples and (b) fillet of Monopterus albus. 29 3.2 (a) Aqueous extraction and (b) lipid extraction of Monopterus albus fillet. 35

3.3 Flow chart of the overall procedure for aphrodisiac study. 36 3.4 ICR female mouse with opening of vagina shows maximum receptivity towards male mouse. 37

3.5 ICR Male mouse engaged in mounting a female mouse. 38 4.1 Vaginal smears of female mouse in Sildenafil citrate group which showed reproductive stage of (a) estrus and (b) metestrus. Magn: 400X 56 4.2 Copulatory plug in a female mouse after overnight mating with a male mouse. 56 4.3 Section of testicular tissue from (a) Saline group and (b) Sildenafil citrate group of ICR male mice. Normal Lumen (L), spermatogonia (Sg), primary spermatocyte (Sp), spermatozoa (Sp), Sertoli cell (SC). Magn: 400X. 58 4.4 Section of testicular tissue from groups of ICR male mice treated with (a) 50 (b) 100, (c) 200 mg/kg lipid extract and (d) 200 mg/kg aqueous extract of Monopterus albus, respectively. Normal Lumen (L), spermatogonia (Sg), primary spematocyte (Sp), spermatozoa (Sz), Sertoli cell (Sc). Magn: 400X. 59 4.5 Liver tissues from control group of female Sprague-Dawley rats. Normal hepatocytes (H), central vein (CV), nuclei (N), sinusoid (Si) and Kupfer cell (K). (a) Magn: 200X (b) Magn: 400X. 67 4.6 Kidney tissues from control group of Sprague-Dawley rats. Normal glomeruli (G), Bowman‟s space (BS), proximal convulated tubule (PT) and distal convulated tubule (DT). (a) Magn: 200X (b) Magn: 400X. 67

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4.7 Liver tissues from female Sprague-Dawley rats treated with 5 mg/kg M. albus fillet lipid extracts. Normal hepatocytes (H), central vein (CV), nuclei (N), sinusoid (Si), part of portal triads (PT) and Kupfer cell (K). (a) Magn: 200X (b) Magn: 400X. 68 4.8 Kidney tissues from female Sprague-Dawley rats treated with 5 mg/kg M. albus lipid extracts. Normal glomeruli (G), Bowman‟s space (BS), proximal convulated tubule (PT) and distal convulated tubule (DT). (a)Magn: 200X (b)Magn: 400X. 68 4.9 Liver tissues from female Sprague-Dawley rats treated with 50 mg/kg M. albus lipid extracts. Normal hepatocytes (H), central vein (CV), nuclei (N), sinusoid (Si) and part of portal triads (PT). (a) Magn: 200X (b) Magn: 400X 69 4.10 Kidney tissues from female Sprague-Dawley rats treated with 50 mg/kg M. albus lipid extracts. Normal glomeruli (G), Bowman‟s space (BS), collecting tubule (CT), proximal convulated tubule (PT) and distal convulated tubule (DT). (a) Magn: 200X (b) Magn: 400X. 69 4.11 Liver tissues from female Sprague-Dawley rats treated with 300 mg/kg M. albus lipid extracts. Hepatocytes (H) with degenerative changes detected around the central vein (CV). Normal nuclei (N), sinusoid (Si) and Kupfer cell (K). (a)Magn: 200X (b) Magn: 400X. 70 4.12 Kidney tissues from female Sprague-Dawley rats treated with 300 mg/kg M. albus lipid extracts. Normal glomeruli (G), Bowman‟s space (BS), collecting tubule (CT), proximal convulated tubule (PT) and distal convulated tubule (DT). (a) Magn: 200X (b) Magn: 400X. 70 4.13 Liver tissues from female Sprague-Dawley rats treated with 2000 mg/kg M. albus fillet lipid extracts showed hepatocytes (H) with very mild insignificant changes detected. (a) Magn: 200X (b) Magn: 400X. 71 4.14 Kidney tissues from female Sprague-Dawley rats treated with 2000 mg/kg M. albus fillet lipid extracts. Normal glomeruli (G), Bowman‟s space (BS), collecting tubule (CT), proximal convulated tubule (PT) and distal convulated tubule (DT). (a) Magn: 200X (b) Magn: 400X. 71

LIST OF ABBREVIATIONS

ANOVA Analysis of Variance AA Arachidonic acid ADH Andiuretic hormone AP Alkaline phosphatase ALT Alanine transaminase AST Aspartate aminotransferase BF3-CH3OH Boron trifluoride methanolic sodium hydroxide BHT Butylated hyroxytoluene CHCl3 Chloroform CH3OH Methanol C15H24O Butylated hydroxytoluene CK Creatine kinase CO2 Carbon dioxide DHA Docosahexaenoic acid EDTA Ethylenediaminetetraacetic acid EPA Eicosapentaenoic acid GCMS Gas chromatography mass spectrophometer GGT/ɣ-GT Gamma-glutamyl transferase HDL High-density lipoprotein HB Haemoglobin HCT Haematocrit HCl Hydrochloric acid IF Intromission frequency IL Intromission latency LDH Lactate dehydrogenase LDL Low-density lipoprotein MCH Mean corpuscular hemoglobin MCHC Mean corpuscular hemoglobin concentration MCV Mean corpuscular volume MF Mounting frequency ML Mounting latency MUFA Monounsaturated fatty acid NA Not applicable NaCl Sodium chloride Na2SO4 Sodium sulphate NaOH Sodium hydroxide (NH4)2SO4 Ammonium sulphate PCV Packed cell volume PUFA Polyunsaturated fatty acid RBC Red blood cell S.E.M Standard Error Mean SFA Saturated fatty acid UV Ultraviolet WBC White blood cell

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LIST OF SYMBOLS

% percentage °C degree celcius b.w. body weight cm centimetre fL fluid ounce ɣ gamma g gram hr hour i.p. intraperitoneal kJ kilojoules kg kilogram L litre µg microgram µL microliter µmol miromolar mbar millibars mg milligram mL mililitre min minute mmol milimolar ω omega p probability rpm revolutions per minute sec seconds w/w weight/weight

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CHAPTER ONE

INTRODUCTION

1.1 RESEARCH BACKGROUND

Sexual activity is vital component of a normal and healthy lifestyle. The sex is the most intimate, imperative and integral part of every individual and can be a source of pleasure and fulfillment. The basic and fundamental purpose of sex and sexuality is the continuation of progeny. However, the problems in sexuality may disturbed interpersonal relationship and even can cause divorce in married couples. The main sexual problems are related to sexual desire and sexual dysfunction (Gonzales,

Cordova, Vega, Chung, Villena and Gonez, 2003). It was estimated approximately

30% of couples infertility and sexual dysfunction are due to male factors (Isidori,

Pozza, Gianfrilli and Isidori, 2006). According to Schwartz and Kloner (2011), sexual dysfunction is a serious medical and social symptom that occurs in 10% - 52% of men and 25% - 63% of women. Male sexual dysfunction, which includes erectile dysfunction (ED), affects 34.8% of men aged between 40 - 70 years (Schwartz and

Kloner, 2011). Epidemiological studies have demonstrated a high prevalence and incidence of ED in developed countries (Hatzimouraditis, Amar, Eardley, Giuliano,

Hatzichristou and Montorsi, 2010), and therefore it is consider being an important health problem.

Erectile dysfunction defined as the persistent or recurrent inability to achieve or maintain a penile erection of sufficient for sexual performance (Bella, Lee, Carrier,

Benard and Brock, 2015). The major causes of ED include obesity, diabetes mellitus, hypertension, cardiovascular disease, increasing age and non-healthy lifestyle such as

smoking, drug abuse, stress and unhealthy food habits. There are many alternative ways for the treatment of ED including psychotherapy, surgery, mechanical devices, drugs and oral phosphodiesterase type 5 inhibitors (PDE 5). Since 1998, Viagra®

(Sildenafil citrate), was the first and the most favorable PDE 5 inhibitor that have been demonstrated to be effective in treating ED (Jackson, Gillies and Osterloh, 2005).

Viagra® will increase the level of cyclic Guanosine Mono-Phosphate (cGMP) in the corpus cavernosal smooth muscle cells, leading to facilitation and prolongation of penile erection (Meinhardt, Kropman and Vermeji., 1999). However, in some men, the oral prescription drug is not compatible and the satisfaction is below the expectation. Nevertheless, the intake of Viagra ® caused certain side effects to the patients such as headache, flushing, dyspepsia, nasal congestion, dizziness and sometimes can cause blurred vision (Schiff and Mulhall, 2005). According to

Hatzimouraditis et al. (2010), the intake of prescription drug should be considered with timing or frequency of intercourse and interactions with food or alcohol, for effective results in sexual activity.

Therefore, there is a demand to look for traditional medicine as opposed to conventional medicine which offers least invasive and successful treatment in sexual dysfunction. Since ancient times, a large number of plants have been tested throughout the world for the possible aphrodisiac to relieve sexual dysfunction and enhance fertility properties (Kabbashi, Dafaalla, Hassan, Idris, Abdoun and Modawe,

2016). The medicinal plants are extensively used as aphrodisiac that provide nutritional value, thereby, improving sexual performance and libido (Yakubu, Akanji and Oladiji, 2007). Aphrodisiac is defined as a substance in a form of food or drug that is capable to enhance sexual activity and help in fertility. It can be categorized

according to their mode of action into three types: those that increase libido (sexual desire), potency (effectiveness of erection) and sexual pleasure (Sandroni, 2001).

Plant materials such as Myristica fragrans, Tribulus terrestris, Asparagus racemosus, Ginkgo biloba and Eurycoma longifolia have been used as a male aphrodisiac remedy (Patel, Kumar, Prasad and Hemalatha, 2011). Those plants have been tested for their aphrodisiac activity in vivo, in vitro and human clinical trials to support the claims. Besides these plants, some marine resources are also known to have aphrodisiac value. This includes sea cucumber, Holothuria scabra (Nurjanah,

Gumbira-Sa‟id, Suprihatin and Riani, 2008), oyster, Crassostrea iredalei (Ridzwan,

Hanani, Siti Norshuhada, Farah Hanis and Aileen, 2013) and sea slug, Aplysia dactylomela (Ridzwan, Noor Atika Elliyana, Farah Hanis and Jamaluddin, 2014).

According to Wang and Croll (2006), several types of marine resources such as octopus, sea hare, soft-shell clam and oyster contains high level of steroid.

Previous studies have indicated that sex steroids involved in the male hormone testosterone secretion. It is able to stimulate hypoactive sexual desire in men

(Matsumoto, 1994; Seidman, 2000). Therefore, many people prefer to use natural sources of therapy because they believe the possible bioactive aphrodisiacs such as steroid were derived from the animals. Steroids hormone plays an important role in sexual behaviour. For example, steroid sandfish extract (Holothuria scabra) able to enrich testosterone and cholesterol concentration in blood serum and fastening secondary reproduction characteristics in male chick (Riani, Gumbira-Said, Syamsu,

Kustiariyah, Kaseno and Cordova, 2013). Thus, sandfish steroid has potential to be used as an aphrodisiac for men.

Currently, one of terrestrial resources that aroused interest in Asian countries is rice field eel, Monopterus albus or locally known as „belut sawah‟. Ancient herbal

and traditional medical records show that eel has been traditionally used to treat bronchitis, tuberculosis, anxiety and for life longevity. Studies on this species is limited to its fatty acid composition (Razak, Basri, Dzulkefly, Razak and Salleh,

2001), heavy metals uptake by M. albus (Sow, Ismail and Zulkifli, 2012), antibacterial, antifungal and antiproliferative of M. albus (Nik Mohd Ikhram and

Ridzwan, 2013; Atif, Zahri, Esa, Zilfalil, Rao and Nordin, 2015) and eel skin gelatin as alternative to bovine gelatin (Nurul and Sarbon, 2015). However, there is no study on aphrodisiac properties of this eel, except, Chan and Philips (1969), reported gonad of M. albus produced sex steroid which is commonly found in most other vertebrate animals. Nowadays, not only by the Chinese, Japanese and Koreans but also by the

Malays who recognize eel as an aphrodisiac. But, to this day, the claims of benefit values from M. albus were not yet widely discovered. Thus, this study was conducted to assess the potential of M. albus extract in inducing sexual activity and fertility in male mice.

1.2 SIGNIFICANCE OF STUDY

There are a number of conventional medical treatments which act as sex stimulant and enhancing the sexual desire and activity in men and women. Although the use of medicine has shown the significant improvement in treating sexual dysfunction, it also caused severe unwanted side effects. The use of synthetic aphrodisiac will dilate blood vessel in other parts of body and causing headache and fainting in patient.

Therefore, people nowadays intended to look for safe and effective natural resources as opposed to synthetic medicine. Interestingly, people from many countries like to eat eel, but it has been a long period before it was discovered where eels came from.

Traditionally, eel has always been viewed as a health enhancing food and even

Malays currently consume eel especially, ricefield eel as an aphrodisiac.

Medical research had found that oily fish like eel contains high amounts of

Omega-3 and Omega-6 polyunsaturated fatty acids (PUFA) which comprises mainly of eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) (Razak et al., 2001;

Suseno, Saraswati, Hayati and Izaki, 2014). Dietary intake of these PUFA is necessary since human cannot synthesis these fatty acids because lack of fatty acid desaturase enzymes. The PUFA, therefore, need to be provided in the diet as these

PUFA are essential for numerous processes include promoting reproduction.

Therefore, the present study will determine the nutritional value presence in the lipid extraction of M. albus and reveal the association between the extraction and its effect on sexual behaviour in male mice. In addition, the toxicity effect of the extraction also was conducted in this study. It is hoped that the findings from the study may provide scientific evident for the role of M. albus in aphrodisiac as acclaimed by the locals.

1.3 OBJECTIVE

1.3.1 General Objective

The overall objectives of this research project is to evaluate aphrodisiac activity as well as fertility potential on male animals, to characterize the M. albus extracts and its toxicity level.

1.3.2 Specific Objectives

1. To evaluate the effects of M. albus aqueous and lipid extracts on

aphrodisiac activity of male mice.

2. To evaluate the effective extract on the male mice fertility.

3. To identify the components of the most effective extract.

4. To identify the toxicity level of the effective extract.

CHAPTER TWO

LITERATURE REVIEW

2.1 ASIAN SWAMP EEL; Monopterus albus

2.1.1 Taxonomy and Distribution of Asian swamp eel

Asian swamp eel, Monopterus albus (Zuiew, 1793) belongs to the family synbranchidae of the order synbranchiformes (Rosen and Greenwood, 1976). It is commonly known as rice eel, rice field eel, rice paddy eel or „belut sawah‟. A brief taxonomy classifying rice field eel are as follows:

Kingdom : Animalia

Phylum : Chordata

Class : Actinopterygii

Order : Synbranchiformes

Family : Synbranchidae

Genus : Monopterus

Species : M. albus

The Asian swamp eel is native to the tropical and subtropical areas of northern

India and Burma to China, Thailand, Philipines, Malaysia, Indonesia, and possibly north eastern Australia (Collins, Trexler, Nico, and Rawlings, 2002). In the early

1990s, it was found in several freshwater in North America including Hawaii, Florida and Georgia due to an aquarium release or a fish farm escaped (Fuller, Nico and

Williams, 1999).