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UNIVERSITI PUTRA MALAYSIA

EFFECTS OF MITRAGYNINE FROM MITRAGYNA SPECIOSA Korth. LEAVES ON WORKING MEMORY IN MICE

EVHY APRYANI

FPSK(m) 2011 8

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EFFECTS OF MITRAGYNINE FROM MITRAGYNA SPECIOSA Korth. LEAVES ON WORKING MEMORY IN MICE

EVHY APRYANI

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2010

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EFFECTS OF MITRAGYNINE FROM MITRAGYNA SPECIOSA Korth. LEAVES ON WORKING MEMORY IN MICE

EVHY APRYANI

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

Science

March 2011

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

EFFECTS OF MITRAGYNINE FROM MITRAGYNA SPECIOSA Korth. LEAVES ON WORKING MEMORY IN MICE

By

EVHY APRYANI

MARCH 2011

Chairman : Mohamad Taufik Hidayat Baharuldin, PhD

Faculty : Medicine and Health of Sciences

Repeated exposure to opiates can lead to deficits in cognitive function. A

study in 2000 reported that opioid users have the risk for the brain damage.

In the present study, we examined the working memory effects of

mitragynine extracted from Mitragyna speciosa mature leaves using object

location task and the motor activity in open field test. Mitragynine of the dose

5, 10 and 15 mg/kg were administered intraperitoneally (IP) to mice for 28

consecutive days and evaluated on day 28 after the last dose treatment.

Scopolamine was used as the positive control drug. After the completion of

behavioral study, mice were sacrificed and the brain was used to evaluate

any changes in hippocampal morphology and in the expression of N-Methyl-

D-aspartic Acid Receptors-1 (NMDAR1) by using immunohistochemical

method.

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It was observed, that the administration of mitragynine significantly reduced

the locomotor acitivity in open-field test compared with vehicle ( p < 0.05).

Some studies have been reported an association between exploratory

behavior to the level of cognitive function. in object location task, mitragynine

treated mice did not show any significance discrimination between the object

that had changed position than the object that had remain in a constant

position (p > 0.05). The analysis of the results obtained indicated that mice

with cognitive impairment will spend more time exploring the familiar object

than the novel one. An absence of any difference in the exploration of the two

objects during the second phase can be interpreted as a memory deficit.

Histological study did not show any morphological changes but

immunohistochemical study showed down-regulation in the expression of

NMDAR1. Our results suggested that chronic administration of mitragynine

may alter the cognitive behavioral function in mice, while preserving the brain

morphology.

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

EFFECTS OF MITRAGYNINE FROM MITRAGYNA SPECIOSA Korth. LEAVES ON WORKING MEMORY IN MICE

Oleh

EVHY APRYANI

MAC, 2011

Pengerusi : Mohammad Taufik Hidayat Baharuldin, PhD

Fakulti : Perubatan dan Sains Kesihatan

Paparan berulang opiat boleh menyebabkan defisit dalam fungsi kognitif.

Sebuah kajian pada tahun 2000 melaporkan bahawa pengguna opioid

mempunyai risiko kerosakan otak. Penelitian ini bertujuan untuk mengetahui

kesan mitragynine yang diekstrak daripada daun Mitragyna speciosa matang

terhadap memori kerja mencit. Kesan kognitif dipelajari dengan

menggunakan lokasi objek tugas dan aktiviti motor di uji lapangan terbuka.

Mitragynine 5, 10 dan 15 mg / kg dan dikendalikan oleh intraperitoneal (IP)

selama 28 hari berturut-turut dan diuji pada hari ke-28 selepas rawatan dos

terkini. Skopolamin digunakan sebagai kawalan positif dadah. Setelah

selesai mengkaji perilaku, mencit dikorbankan dan otak digunakan untuk

menilai perubahan histologis dan ekspresi NMDAR1 menggunakan kajian

imunohistokimia.

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Dalam kajian ini terdapat pengaruh yang menonjol pada aktiviti lokomotor

mencit. Mitragynine secara signifikan mengurangkan aktiviti lokomotor dalam

uji lapangan terbuka berbanding dengan control (p < 0.05). Di lokasi objek

tugas, mitragynine tidak menunjukkan adanya diskriminasi signifikansi antara

objek yang berubah kedudukan dari objek yang tetap dalam posisi yang

sama (p > 0.05). Pengajian histologi tidak menunjukkan perubahan morfologi

tetapi kajian imunohistokimia menunjukkan perubahan dalam ekspresi

NMDAR1 di hipokampus.Keputusan kajian kami menunjukkan bahawa

rawatan kronik mitragynine boleh menukar perilaku fungsi kognitif pada

mencit.

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ACKNOWLEDGEMENTS

In the name of Allah, The Most gracious and Most merciful I would like to say

Alhamdulillah because of with His blessing I have completed the project and

the preparation of this thesis.

Firstly, I would like to express my gratitude and respect to Mohamad Taufik

Hidayat Baharuldin, PhD as the chairman of my supervisory committee, who

constantly motivated me with his knowledge and insight. I am also grateful to

Associate Professor Sharida Fakurazi, PhD and Mohamad Aris Mohd

Moklas, PhD as the member of my supervisory committee for sharing their

experience and expertise.

And to science officers and all staff also the members of the Human Anatomy

department in UPM, who have immensely contributed to my personal and

professional time at UPM. The group has been a source of friendship as well

as good advice and collaboration.

My time in Malaysia was made enjoyable in large part due to the many

friends and groups that became a part of my life. I am grateful for time spent

with housemates and friends, and also for my backpacking buddies for our

memorable trips and for many other people and memories.

Lastly, I would like to thank my family for all their love and encouragement.

For my parents who raised me with love of science and supported me in all

my pursuits. For the supports of my brother, sister and Adrianus Andi Prijatno

during the final stage of this MSc is so appreciated. Thank you.

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I certify that an Examination Committee has met on date/month/year to conduct the final examination of Evhy Apryani on her Master of Science thesis entitle “The effects of mitragynine from Mitragyna speciosa Korth. leaves on working and memory function in mice” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Master of Science.

Members of the Examination Committee were as follows:

Professor Dr. Fauziah Othman Jabatan Anatomi Manusia Fakulti Perubatan dan Sains Kesehatan University Putra Malaysia (Chairman) Professor Dr. Khatiza Haida Ali Fakulti Perubatan dan Sains Kesehatan University Putra Malaysia (Internal Examiner) DR Roslida binti Abd Hamid Fakulti Perubatan dan Sains Kesehatan University Putra Malaysia (Internal Examiner) Professor Dr. Mohd Hamim Rajikin Fakulti Perubatan dan Sains Kesehatan Universiti Teknologi Mara Malaysia (External Examiner)

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

Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee were as follows:

Mohamad Taufik Hidayat Baharuldin, PhD Senior lecturer Faculty of Medicine and Health of Sciences Universiti Putra Malaysia (Chairman)

Mohamad Aris Mohd Moklas, PhD Senior lecturer Faculty of Medicine and Health of Sciences Universiti Putra Malaysia (Member)

Sharida Fakurazi, PhD Associate Professor Faculty of Medicine and Health of Sciences Universiti Putra Malaysia (Member)

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

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DECLARATION

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

EVHY APRYANI

Date: 2 March 2011

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TABLE OF CONTENTS

ABSTRACT ABSTRAK

Page iv vi

ACKNOWLEDGEMENT viii DECLARATION xi LIST OF TABLES xv LIST OF FIGURES xvi LIST OF ABBREVIATIONS xvii

CHAPTER

1 INTRODUCTION

1

1.1.Research Background 1

1.2.Objectives 6

1.2.1 General Objective 6

1.2.2 Specific Objectives 6

2 LITERATURE REVIEW 7

2.1 Introduction to Mitragyna speciosa Korth 7

2.1.2 Chemical constituent of Mitragyna speciosa Korth

10

2.2 Overview of Mitragynine 11

2.2.1 Pharmacokinetics of Mitragynine 13

2.2.2 Pharmacodynamic activity of Mitragynine 13

2.3 Neurochemical systems involved in learning and memory

14

2.3.1 Cholinergic neurons and its role in memory process

15

2.3.2 Role of serotonin in memory 16

2.3.3 Dopaminergic function in prefrontal cortex is critical for memory and learning process

17

2.3.4 Involvement of GABAergic system in memory and learning

18

2.3.5 Glutamate and its role in learning and memory function

19

2.4 Molecular Basis of Learning and Memory 20

2.4.1 Short-term potentiation 21

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2.4.2 Long term potentiation 22

3 THE EXTRACTION OF MITRAGYNINE FROM Mitragyna speciosa Korth. LEAVES

26

3.1 Introduction 26

3.2 Materials and Methodology 29

3.2.1 Materials 29

3.2.2 Sample Collection 29

3.3 Results 34

3.4 Discussion 39

4 THE EFFECTS OF MITRAGYNINE FROM Mitragyna speciosa Korth LEAVES ON WORKING MEMORY IN MICE

42

4.1 Introduction 42

4.2 Materials and Methodology 44

4.2.1 Preparation of Mitragynine 44

4.2.2. Experimental Animals 44

4.2.3 Object Location Task 45

4.2.4 Open Field Test 46

4.2.5 Statistical Analysis 47

4.3 Result 48

4.4 Discussion 51

5 EFFECTS ON THE EXPRESSION ON NMDAR1 RELATED TO WORKING AND MEMORY FUNCTION AFTER CHRONIC INTAKE OF MITRAGYNINE

54

5.1 Introduction 54

5.2 Materials 56

5.2.1 Chemicals and reagents 56

5.2.2 Instruments and Equipments 56

5.2.3 Experimental Animal 57

5.2.4 Statistical analysis 63

5.3 Results 64

5.4 Discussion 68

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6 DISCUSSION, CONCLUSION AND THE FUTURE RECOMMENDATION RESEARCH

72

REFERENCES 75

APPENDIX 1 88

BIODATA OF STUDENT 89