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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