UNIVERSITI PUTRA MALAYSIA

FARAH IDAYU BINTI NASIR

FPSK(m) 2013 37

ANTIDEPRESSANT PROPERTIES OF MITRAGYNINE, AN ALKALOID ISOLATED FROM MITRAGYNA SPECIOSA

KORTH, IN MICE MODEL OF DEPRESSION

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ANTIDEPRESSANT PROPERTIES OF MITRAGYNINE, AN ALKALOID ISOLATED FROM MITRAGYNA SPECIOSA KORTH, IN

MICE MODEL OF DEPRESSION

FARAH IDAYU BINTI NASIR

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2013

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ANTIDEPRESSANT PROPERTIES OF MITRAGYNINE, AN ALKALOID ISOLATED FROM MITRAGYNA SPECIOSA KORTH, IN MICE MODEL OF

DEPRESSION

By

FARAH IDAYU BINTI NASIR

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, In Fulfilment of the Requirements for the Degree of Master of

Science

April 2013

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COPYRIGHT

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icons, photographs and all other artwork, is copyright material of Universiti

Putra Malaysia unless otherwise stated. Use may be made of any material

contained within the thesis for non-commercial purposes from the copyright

holder. Commercial use of material may only be made with the express,

prior, written permission of Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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Abstract Submitted to Senate of Universiti Putra Malaysia, In Fulfilment of the Requirements for Degree of Master of Science

ANTIDEPRESSANT PROPERTIES OF MITRAGYNINE, AN ALKALOID ISOLATED FROM MITRAGYNA SPECIOSA KORTH, IN MICE MODEL OF

DEPRESSION

By

FARAH IDAYU BINTI NASIR

April 2013

Chairman : Mohamad Taufik Hidayat Bin Baharuldin, PhD

Faculty : Medicine and Health Sciences

Major depressions are among the most prevalent disease of the central nervous

system with a high morbidity and mortality. Available antidepressants that used

as pharmacotherapy for depression produce a lot of adverse effects towards

depressed patient. Therefore, safer treatments for treating mental illness like

depression are still needed. On the other hand, drugs obtained from natural

sources are perceived to have at least risk and low side- effects profiles, while

having the ability to cure mental disorder. Mitragynine (MG) is the major alkaloid

identified in Mitragyna speciosa Korth which has been used in traditional

medicine. The antinociceptive action of MG is due to its role on opioid system to

stimulate the release of endogenous noradrenaline and serotonin from nerve

terminal. However, none has been reported on the mechanism action of MG via

spectrum of antidepressant studies. Based on the principle that MG has a

significant role in producing antinociceptive action, it might as well beneficial as

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antidepressant. Hence, the present investigation evaluated the antidepressant

effect of MG in the mouse forced swim test (FST) and tail suspension test (TST)

together with its effects on hypothalamic-pituitary-adrenal (HPA) axis by

measuring the corticosterone concentration of mice exposed to FST and TST.

An open-field test (OFT) was used to study any association of immobility in the

FST and TST with psychomotor stimulant effect of MG. Male ICR mice were

randomly assigned to six treatment groups (n=8): Group I (vehicle control

group), Group II received reference drug 20 mg/kg, fluoxetine (selective

serotonin reuptake inhibitor, SSRI), Group III received tricyclic antidepressant

drug, amitriptyline hydrochloride 10 mg/kg and Group IV, V and VI received 5,

10 and 30 mg/kg of MG. MG at doses of 10 mg/kg and 30 mg/kg significantly

reduced the immobility time of mice in both FST and TST without any significant

effect on locomotor (crossing) activity in OFT. Moreover, MG significantly

reduced the released of corticosterone in mice exposed to FST and TST at dose

of 10 mg/kg and 30 mg/kg.In order to investigate the involvement of MG on

cannabinoid system, a group of animals were randomly assigned into four

experimental groups (8 mice per group). The groups were consist of group I,

that served as control treatment; group II was given MG (10 mg/kg i.p.); group III

was given cannabinoid receptor (CB1) antagonist drug, AM 251 (0.5 mg/kg i.p.)

and finally group IV was given pre-treatment of AM 251 (0.5 mg/kg i.p.) followed

by treatment of MG (10 mg/kg i.p.). The results showed that pre- treatment of

mice with AM 251 produced significant reduction in immobility time as compared

with treatment of MG alone and treatment of AM 251 alone. In terms of

corticosterone level, pre- treatment of mice with AM 251 significantly increased

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the level of corticosterone concentrations as compared with treatment of MG

alone and treatment of AM 251 alone after exposed to FST and TST. These

data suggest antidepressant effect produced by MG is not likely through its

action on cannabinoid receptor system.

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

KESAN ANTI-KEMURUNGAN MITRAGYNINE, ALKALOID DARI

MITRAGYNA SPECIOSA KORTH, DALAM MODEL KEMURUNGAN MENCIT

Oleh

FARAH IDAYU BINTI NASIR

April 2013

Pengerusi : Mohamad Taufik Hidayat Bin Baharuldin, PhD

Fakulti : Perubatan dan Sains Kesihatan

Kemurungan adalah salah satu masalah sistem saraf pusat yang sering dihidapi

dengan perkadaran yang tinggi dari segi motiliti dan morbiditi. Dadah anti-

kemurungan yang digunakan bagi terapi farmakologi untuk kemurungan

menghasilkan banyak kesan sampingan terhadap pesakit yang menghidap

kemurungan. Oleh itu, rawatan yang lebih selamat untuk merawat penyakit

mental seperti kemurungan masih diperlukan. Ubat-ubatan yang diperolehi

daripada sumber semula jadi dilihat mempunyai kurang risiko berbahaya dan

kesan sampingan, di samping mempunyai keupayaan untuk menyembuhkan

penyakit mental.Mitragynin (MG) adalah alkaloid utama yang dikenalpasti dari

tumbuhan Mitragyna speciosa Korth di mana ia telah digunakan sebagai ubat

tradisional. Kebanyakan penyelidikan lebih tertumpu pada tindakan kesan

tahan sakit MG kerana peranannya pada sistem opioid untuk merangsang

pembebasan noradrenalin dalaman dan serotonin dari terminal saraf.

Berdasarkan pada prinsip bahawa MG mempunyai peranan penting dalam

menghasilkan tindakan anti sakit, ia mungkin juga bermanfaat sebagai ubat anti-

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kemurungan.Malah banyak kajian telah dilaporkan mengenai kesan dadah anti-

kemurungan juga mampu memberikan kesan anti-sakit dan digunakan secara

meluas dalam rawatan sakit kronik. Oleh itu, kajian terkini ini menilai kesan anti-

kemurungan mitragynin ke atas dua model mencit iaitu ujian paksa-renang

(FST) dan ujian penggantungan ekor (TST) juga kesan mitragynin ke atas

sistem neuroendokrin iaitu sistem paksi- hipotalamik-pituitari dengan mengukur

aras kortikosteron mencit yang terdedah kepada ujian paksa-renang (FST) dan

ujian penggantungan ekor (TST). Mencit jantan ICR ditentukan secara rawak

kepada enam kumpulan setara (n=8): Kumpulan I (kumpulan kontrol menerima

pelarut), Kumpulan II menerima ubat anti-kemurungan yang digunakan sebagai

rujukan iaitu 20mg/kg (i.p.) fluoxetine (SSRI), Kumpulan III menerima ubat anti-

kemurungan trisiklik ,amitriptyline hidroklorik, 10 mg/kg (i.p.), Kumpulan IV, V,VI

menerima mitragynin dos 5, 10 dan 30 mg/kg.Dalam kajian ini, MG pada dos 10

mg/ kg dan 30 mg/kg mengurangkan masa tempoh pegun mencit dalam kedua-

dua FST dan TST tanpa kesan signifikan ke atas aktiviti motor dalam ujian

lapangan motor (OFT). Selain itu, MG juga merendahkan kadar perembesan

kortikosteron mencit yang terdedah kepada FST dan TST pada dos 10mg/kg

dan 30mg/kg. Dalam usaha untuk menyiasat penglibatan MG pada sistem

cannabinoid, mencit-mencit telah diasingkan secara rawak kepada empat

kumpulan eksperimen (n=8). Kumpulan-kumpulan ini terdiri daripada kumpulan I

yang berkhidmat sebagai rawatan kawalan; kumpulan II telah diberikan MG (10

mg/ kg i.p.); kumpulan III telah diberi dadah antagonist reseptor cannabinoid

(CB1) AM 251 (0.5 mg / kg i.p.) dan akhirnya kumpulan (IV) telah diberikan pra-

rawatan AM 251 (0.5 mg/kg i.p.) diikuti rawatan MG (10 mg/kg). Menurut

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keputusan yang terhasil, pra-rawatan tikus dengan AM 251 diikuti MG

menghasilkan pengurangan ketara dalam masa pegun mencit berbanding

dengan rawatan MG bersendirian dan rawatan AM 251 bersendirian. Dari segi

tahap kortikosteron, pra-rawatan tikus dengan AM 251 diikuti MG meningkatkan

tahap kortikosteron berbanding dengan rawatan MG bersendirian dan rawatan

AM 251 bersendirian selepas terdedah kepada FST dan TST. Data-data ini

mencadangkan kesan anti-kemurungan yang dihasilkan oleh MG tidak mungkin

melalui tindakan pada sistem reseptor cannabinoid.

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ACKNOWLEDGEMENTS

Alhamdulillah, in the name of ALLAH S.W.T, my gratitude to the Almighty

ALLAH for blessing me with the strength, courage and patience as I finished the

journey of my master research and thesis.

First and foremost, I wish to take this opportunity to express my heartfelt thanks

and appreciation to Dr. Mohamad Taufik Hidayat Bin Baharuldin as the

chairman of my supervisory committee for his encouragement, invaluable

advice, inspiring guidance, constructive comment and support and most of all for

his understanding and patience throughout the duration of my research project.

Special appreciation and thanks to members of the supervisory committee,

Assoc. Prof. DaDr. Sharida Fakurazi and Dr. Mohamad Aris Mohd Moklas for

their advice and constructive criticisms. It was a great experience for me to have

the opportunity to do the research under their supervision.

Thousand thanks to every staff in Department of Human Anatomy UPM, for their

cooperation and support which enabled me to perform the animal study

successfully. Not forgetting staff in Animal House, FPSK, UPM who were always

being very helpful and also Science Officer from Chemical Pathology Unit for

providing the technical assistance.

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Precious thanks also to all my research group friends, Evhy Apryani, Nurul

Raudzah and Nur Shamima for their immeasurable support, assistance and

encouragement. The knowledge you guys shared is always great,

Last but not least, I express my deepest appreciation and million thanks to my

family especially my mum, Norlily Ismail, my abah, Nasir Ibrahim, my younger

sister, Farah Azieka Nasir and my other half, Azaman Ali for their sustained

support, love and continuous encouragement throughout the accomplishment of

this thesis. The prayers and support they gave will never melt till the end of my

life. Thanks for your kindness and endless encouragement. Finally, I really hope

that this particular thesis will be a beneficial source of knowledge to others.

Thank you.

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I certify that a Thesis Examination Committee has met on 22 April 2013 to conduct the final examination of Farah Idayu Binti Nasir on her Master of Science thesis entitled “Antidepressant Properties of Mitragynine, An Alkaloid Isolated From Mitragyna speciosa Korth, in Mice Model of Depression 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 Master of Science (Physiology). Members of the Examination Committee were as follows: Dr. Rusliza Binti Basir, PhD

Senior Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Chairman) Prof. Dr. Mohd Roslan Bin Sulaiman, PhD

Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner 1) Prof. Madya Dr. Mohamad Aziz Bin Dollah, PhD

Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner 2) Prof. Dr. Nasaruddin Abdul Aziz, PhD

Professor Faculty of Medicine Cyberjaya University College of Medical Sciences (External Examiner)

NORITAH OMAR, PhD

Assoc. Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 2 August 2013

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The 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: Mohamad Taufik Hidayat Bin Baharuldin, PhD

Senior Lecturer Faculty of Medicine and Health Science Universiti Putra Malaysia (Chairman)

Sharida Binti Fakurazi, PhD Associate Professor Faculty of Medicine and Health Science Universiti Putra Malaysia (Member) Mohamad Aris Bin Mohamad Moklas, PhD

Senior Lecturer Faculty of Medicine and Health Science 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

I declare that this Master thesis is based on my original work except for quotations and citations which have been dully acknowledged. I also declare that it has not been previously, and concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

FARAH IDAYU BINTI NASIR

Date: 22 April 2013

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TABLE OF CONTENTS

Page ABSTRACT ii ABSTRAK v ACKNOWLEDGEMENTS viii APPROVAL x DECLARATION xii LIST O TABLES xvi LIST OF FIGURES xviii LIST OF ABBREVIATIONS xxi CHAPTER

1 INTRODUCTION 1 1.1 Research objectives 7

1.1.1 General objectives 7 1.1.2 Specific objectives 7 1.2 Hypothesis of study 8

2 LlTERATURE REVIEW 9 2.1 Mitragyna speciosa Korth 9 2.1.1 Mitragyna genus 9 2.1.2 Distribution of Mitragyna speciosa 10 2.1.3 Biogeography and ecology of

Mitragyna speciosa 11

2.1.4 Ethno botanical use of Mitragyna speciosa

13

2.1.5 Alkaloid of Mitragyna speciosa Korth 16 2.1.6 Side effects of Mitragyna speciosa leaves 19 2.1.7 Pharmacological effect of Mitragyna

speciosa Korth Extract (reported studies) 20

2.1.8 Pharmacological effect of mitragynine (reported studies)

23

2.2 Depression 28 2.2.1 Prevalence of Depression 28 2.2.2 Diagnostic criteria for Depression 32 2.2.3 Subtypes of Depression 34 2.3 Hypothalamic-pituitary-adrenal Axis (HPA axis) 36 2.3.1 HPA axis in Depression 39 2.4 Biogenic amine (monoamine neurotransmitters) 40 2.4.1 Serotonin (5-HT) 41 2.4.2 Dopamine (DA) 43 2.4.3 Noradrenaline (NA) 45 2.5 Antidepressant 46

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2.5.1 Development of Antidepressant 47 2.5.2 Selective serotonin reuptake inhibitor (SSRI) 49 2.5.3 Tricyclic antidepressant (TCA) 51 2.5.4 Monoamine oxidase Inhibitor (MAOI) 51 2.6 Cannabinoid system 53 2.6.1 Cannabinoid receptor system 53 2.6.2 Cannabiniod endogenous ligand

(Endocannabinoids) 55

2.6.3 CB1 antagonist 56 2.6.4 Cannabinoid system in HPA axis regulation 57

3 METHODOLOGY 59 3.1 Preparation of mitragynine from Mitragyna

speciosa leaves 59

3.1.1 Collection of plant material 59 3.1.2 Crude methanol extraction of Mitragyna

speciosa leaves 59

3.1.3 Acid and Base process of crude methanol extract

60

3.1.4 Process of separation of non-polar compounds

61

3.1.5 Fractionation of alkaloid extract using column chromatography

62

3.1.6 Phytochemical screening by using Thin Layer Chromatography (TLC)

63

3.1.7 Nuclear Magnetic Resonance Spectra Analysis (NMR)

66

3.2 Experimental Animals Procedure 68 3.2.1 Pharmacological Treatments 68 3.2.2 Animals 68 3.2.3 Drug Administration 69 3.2.4 Forced swim test (FST) 71 3.2.5 Tail suspension test (TST) 72 3.2.6 Open-field test (OFT) 73 3.3 Blood Analysis 75 3.3.1 Blood sample collection 75 3.3.2 Blood Centrifugation and Storage 75 3.3.3 Assay of corticosterone levels 75 3.4 Statistical analysis 76

4 RESULTS 77 4.1 Effect of mitragynine (MG) on immobility time in

forced swim test (FST) 77

4.2 Effect of mitragynine (MG) on immobility time in mouse tail suspension test (TST)

79

4.3 Effect of mitragynine (MG) on locomotor activity in the mice open-field test (OFT)

81

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4.4 Effect of mitragynine (MG) on serum corticosterone levels in mice exposed to forced swim test (FST)

83

4.5 Effect of mitragynine (MG) on serum corticosterone levels in mice exposed to tail suspension test (TST)

85

4.6 Effect mitragynine (MG) in mice pre-treated with AM 251 on immobility periods in mouse forced swim test (FST)

87

4.7 Effect of mitragynine (MG) in mice pre-treated with AM 251 on immobility periods in the mouse tail suspension test (TST)

89

4.8 Effect of mitragynine (MG) in mice pre-treated with AM 251 on locomotor activity in the mice open-field test (OFT)

91

4.9 Effect of mitragynine (MG) in mice pre-treated with AM 251 on serum corticosterone levels in mice exposed to forced swim test (FST)

93

4.10 Effect of mitragynine (MG) in mice pre-treated with AM 251 serum corticosterone levels in mice exposed to tail suspension test (TST)

95

5 DISCUSSION 97 6 CONCLUSION AND RECOMMENDATION 110

REFERENCES 112 APPENDICES 134 BIODATA OF STUDENT 147 LIST OF PUBLICATIONS 148