dysoxylum grande hiern as a potential source of

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Dysoxylum grande HIERN AS A POTENTIAL SOURCE OF BIOLOGICALLY ACTIVE CONSTITUENTS

LOW KOK WAH

FS 2012 100

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By

LOW KOK WAH

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

In Fulfilment of the Requirement for the Degree of Master of Science

June 2012

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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


By

LOW KOK WAH

June 2012

Chairman : Intan Safinar Ismail, PhD

Faculty : Science

Dysoxylum grande was chosen to be studied due to the lack of reported scientific

investigations on the plant and interesting phytochemical findings on another plant of

the same genus, Dysoxylum acutangulum. Two biological activities; anti-

acetylcholinesterase and ichthyotoxicity were used as the bioassay in guiding the

fractionation step. As a result, nine compounds were isolated from the ichthyotoxic

EtOAc leaves fraction. Among them, eight were identified as new compounds named

as 20S,24R-epoxy-7�,25-dihydroxy-3,4-secodammar-4(28)-en-3-oic acid (CP1),

grandol A (CP2), grandol B (CP3), grandol C (CP4), grandol D (CP5), grandol E

(CP8), grandol F (CP9) and grandol G (CP10) and the known compound was

identified as �-sitosteryl-�-D-glucopyranoside (CP11). All new compounds were

identified based on thorough analysis of spectroscopic data including 1D and 2D

NMR. The structure for the known compound was established by spectral data

analyses and by comparison with reported investigations. In ichthyotoxic assay

carried out on Zebra fish (Danio rerio), EtOAc fraction (leaves) was found to

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possess significant ichthyotoxicity compared to other fractions. However, the

isolated pure compounds did not provide any IC50 values for the ichtyotoxicity test

due to their limited amounts. All compounds were tested for anti-acetylcholinesterase

activity using Thin Layer Chromatography (TLC)-bioautography with fast blue B

salt. Only grandol A (CP2) and grandol B (CP3) showed positive results. However,

the obtained IC50 for both of these compounds, grandol A (CP2) and B (CP3), using

Ellman’s method was not significant (>200 µg/mL).

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

Dysoxylum grande HIERN SEBAGAI SUMBER BERPOTENSI BAGI KANDUNGAN SEBATIAN AKTIF BIOLOGI

Oleh

LOW KOK WAH

Jun 2012

Pengerusi : Intan Safinar Ismail, PhD

Fakulty : Sains

Dysoxylum grande telah dipilih untuk kajian penyelidikan memandangkan

kurangnya penyelidikan yang dilaporkan secara saintifik ke atas tumbuhan ini dan

penemuan fitokimia yang menarik ke atas tumbuhan lain dalam genus yang sama,

iaitu Dysoxylum acutangulum. Dua bioaktiviti; ‘anti-acetylcholinesterase’ dan

‘ichthytoxicity’ telah digunakan sebagai bioassai dalam membantu langkah

pemeringkatan. Hasilnya, sembilan sebatian telah diasingkan daripada fraksi daun

EtOAc yang ‘ichthyotoxic’.Antaranya, lapan sebatian baru telah dikenapasti yang

dinamakan sebagai asid 20S,24R-epoxy-7�,25-dihydroxy-3,4-secodammar-4(28)-en-

3-oic (CP1), grandol A (CP2), grandol B (CP3), grandol C (CP4), grandol D (CP5),

grandol E (CP8), grandol F (CP9) dan grandol G (CP10) dan satu sebatian yang

telah diketahui iaitu �-sitosteril-�-D-glukopiranosida (CP11). Semua sebatian baru

telah dikenalpasti melalui analisis ke atas data spektroskopi termasuk 1D dan 2D

NMR. Struktur bagi sebatian yang telah diketahui telah dikenalpasti oleh analisis

data spektrum dan perbandingan dengan penyelidikan yang dilaporkan sebelum ini.

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Dalam assai ‘ichthyotoxic’ yang dijalankan ke atas ikan Zebra (Danio rerio), fraksi

EtOAc (daun) didapati mempunyai aktiviti yang ketara berbanding dengan fraksi

yang lain. Namun demikian, sebatian tulen yang dipisahkan tidak dapat memperolehi

nilai IC50 dalam ‘ichtyotoxicity’ disebabkan oleh kuantiti sebatian yang tidak

mencukupi. Semua sebatian juga telah diuji dengan ‘anti-acetylcholinesterase’

menggunakan kromatografi lapisan nipis (TLC)-bioautografi dengan ‘fast blue salt

B’. Hanya grandol A (CP2) dan grandol B (CP3) menunjukkan hasil yang positif.

Namun demikian, nilai IC50 yang diperolehi bagi kedua-dua sebatian dengan

mengguanakan kaedah Ellman didapati tidak ketara (>200 µg/mL).

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ACKNOWLEDGEMENTS

I would like to express my deep and sincere gratitude to my supervisor, Dr. Intan

Safinar Ismail, whose encouragement, guidance and support from the initial to the

final level enabled me to develop an understanding of the project. Her wide

knowledge and her logical way of thinking have been of great value for me. Without

her guidance, this project would not be accomplished successfully.

Acknowledgement also goes to my co-supervisor, Associate Professor Dr. Faridah

Abas who was abundantly helpful and offered invaluable assistance, support and

guidance.

I am also very grateful and would like to express thousands of thanks to Science

Officers, Mr. Salahudin, Mdm Mazina and Mdm Zurina in acquiring spectroscopic

data and also special thanks to Mr. Shamsul and Mr. Tajuddin in obtaining plant

samples.

During this research I have collaborated with many colleagues for whom I have great

regards, and I wish to extend my warmest thanks to all those who have helped me

with my work at the Laboratory of Natural Products, Institute Bioscience, Universiti

Putra Malaysia.

Lastly, I wish to thank my entire extended family for providing a loving environment

for me and supporting me spiritually throughout my life.

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I certify that a Thesis Examination Committee has met on 26 June 2012 to conduct the final examination of Low Kok Wah on his thesis entitled “Dysoxylum grade Hiern as a Potential Source of Biologically Active Constituents” in accordance with the Universities and University College 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.

Members of the Thesis Examination Committee were as follows:

Mohd Aspollah bin Hj Md Sukari, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman) Gwendoline Ee Cheng Lian, PhD Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Siti Mariam binti Mohd Nor, PhD Doctor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Hasnah Mohd Sirat, PhD Professor Faculty of Science Universiti Teknologi Malaysia (External Examiner) �

____________________ SEOW HENG FONG, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 27 Septermber 2012

��

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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:

Intan Safinar Ismail, PhD Lecturer Institute of Bioscience Universiti Putra Malaysia (Chairman) Faridah Abas, PhD Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member)

_____________________ BUJANG BIN KIM HUAT 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.

__________________________ LOW KOK WAH

Date: 26 June 2012

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TABLE OF CONTENTS

Page

ABSTRACT i ABSTRAK iii ACKNOLEDGEMENTS v APPROVAL vi DECLARATION viii LIST OF TABLES xi LIST OF CHART xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xvii

CHAPTER

1 Introduction 1.1 Natural Products 1 1.2 Meliaceae 2 1.3 Ichthyotoxic Assay 4 1.4 Acetylcholinesterase (AChE) Assay 4 1.5 Problems Statement 5 1.6 Research Objectives 6

2 Literature Review 2.1 Genus – Dysoxylum 7 2.2 Medicinal Uses of Genus Dysoxylum 7 2.3 Bioactivities Studies on the Genus Dysoxylum 8 2.4 Dysoxylum grande 13 2.4.1 Properties 13 3 Experimental 3.1 General Instrumentations 14 3.2 Chromatographic Techniques 14 3.3 Experimental Methods 17 3.3.1 Plant Material 17 3.3.2 Extraction 17 3.3.3 Isolation of Compounds from Dysoxylum 19

grande Leaves EtOAc Fraction

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3.4 Spectral Data of Isolated Compounds 27 3.5 Dysoxylum grande bark 37 3.6 Bioassay Procedures 37 3.6.1 Anti-acetylcholinesterase Activity 37 3.6.2 TLC Bioautographic Acetylcholinesterase 37

(ACh) Assay 3.6.3 96-wells Microplate Method 39

(Ellman’s Method) – Ellman et al., 1976 3.6.4 Ichthyotoxicity Assay 40 4 Results and Discussion 4.1 Chemical Constituents of Dysoxylum grade (Leaves) 41 4.2 Ichthyotoxic Test 42 4.3 Anti-acetylcholinesterase 46 4.4 Structural Elucidation of the Chemical Constituents 49

4.4.1 20S,24R-epoxy-7�,25-dihydroxy- 49 3,4-secodammar-4(28)-en-3-oic acid (CP1) 4.4.2 Grandol A (CP2) 59 4.4.3 Grandol B (CP3) 76 4.4.4 Grandol C (CP4) 88 4.4.5 Grandol D (CP5, identical with CP6, CP7) 101 4.4.6 Grandol E (CP8) 111 4.4.7 Grandol F (CP9) 121 4.4.8 Grandol G (CP10) 131 4.4.9 �-Sitosteryl-�-D-glucopyranoside (CP11) 141 5 Conclusion and Recommendation 146 5.1 Conclusion 146

REFERENCES 148 APPENDICES 151 BIODATA OF STUDENT 155

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LIST OF TABLES

Table Page

1 HPLC Elution Gradients Used in Analysis of EtOAc Fraction 16

2 Ichthyotoxicity of the Bark and Leaves of Dysoxylum grande 42

3 Ichthyotoxicity of the HM Fractions 43

4 Ichthyotoxicity of the CM Fractions 44

5 Ichthyotoxicity of the HA Fractions 45

6 1H NMR, 13C NMR, HMBC and Selected NOESY of CP1 56 in CDCl3 (400 MHz) 7 13C NMR data for CP1, 20S,24S-epoxy-7�,25-dihydroxy- 57

3,4-secodammar-4(28)-en-3-oic acid (19) and 20S,24R-epoxy- 25-hydroxy-3,4-secodammar-4(28)-en-3-oic acid (20)

8 1H NMR, 13C NMR, HMBC and Selected NOESY of CP2 74 in CDCl3 (500 MHz)

9 1H NMR, 13C NMR, HMBC and Selected NOESY of CP3 86 in CDCl3 (500 MHz) 10 1H NMR, 13C NMR, HMBC and Selected NOESY of CP4 99 in CDCl3 (500 MHz) 11 1H NMR, 13C NMR, HMBC and Selected NOESY of CP5 109 in CDCl3 (500 MHz) 12 1H NMR, 13C NMR, HMBC and Selected NOESY of CP8 119 in CDCl3 (500 MHz) 13 1H NMR, 13C NMR and HMBC of CP9 in CDCl3 (500 MHz) 129

14 1H NMR, 13C NMR and HMBC of CP10 in CDCl3 (500 MHz) 139

15 1H NMR (500 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) 142

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LIST OF CHARTS

Chart Page

1 Summary of Isolated Compounds Based On Ichthyotoxicity 48

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LIST OF FIGURES

Figure Page

1 Dysoxylum grande Plant Parts 13

3.1 Extraction of Leaves or Bark of Dysoxylum grande 18

3.2 Fractionation of Dysoxylum grande Leaves EtOAc Fraction 19

3.3 Fractionation of HM 28 Fraction (EtOAc Extract) 20

3.4 Purification of CM 9802 Fraction 21

3.5 Fractionation of CM 9604 Fraction 22

3.6 Fractionation of CE 55:1-4 23

3.7 Purification of CM 9406 Fraction 24

3.8 Fractionation of HM 37 Fraction 25

3.9 Purification of HE2: 46 and HE2: 37 Fractions 26

3.10 Danio rerio (Zebra Fish) 40

4.1 Profile TLC (Sprayed with 10% H2SO4 in Ethanol) 46 and AChE Profile TLC of HM Fractions

4.2 AChE Profile TLC of CP2 and CP3 47

4.3 HRESIMS of CP1 50

4.4 Structure of 20S,24R-epoxy-7�,25-dihydroxy-3,4- 50 secodammar-4(28)-en-3-oic acid (CP1) 4.5 1H NMR Spectrum of CP1 in CDCl3 (400 MHz) 51

4.6 13C NMR Spectrum of CP1 in CDCl3 (400 MHz) 52

4.7 13C NMR Spectrum of CP1 in Pyridine (500 MHz) 53

4.8 HMBC NMR Spectrum of CP1 54

4.9 NOESY 1H NMR Spectrum of CP1 55

4.10 Comparison Structure for CP1 with 19 and 20 58


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4.12 Infrared (IR) Spectrum of CP2 60

4.13 Structures of CP2 and 4α-methyl-5α-cholest-8-en-3β-ol (21) 61

4.14 1H NMR Spectrum of CP2 in CDCl3 (500 MHz) 62


4.16 DEPT NMR Spectrum of CP2 64

4.17 HMBC Correlations in Partial Structures of CP2 65

4.18 COSY (1H-1H) NMR Spectrum of CP2 67

4.19 HMQC NMR Spectrum of CP2 68

4.20 HMQC (Expand) NMR Spectrum of CP2 69


4.22 HMBC (Expand) NMR Spectrum of CP2 71




4.26 Structure of CP3 and Partial HMBC Correlations in Ring A and B 76

4.27 1H NMR Spectrum of CP3 in CDCl3 (500 MHz) 77







4.34 Selected NOESY 1H NMR Spectrum of CP3 85



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4.37 Structure of CP4 and Partial HMBC Correlations in Ring A, B, C 88 and D 4.38 1H NMR Spectrum of CP4 in CDCl3 (500MHz) 89

4.39 13C NMR Spectrum of CP4 in CDCl3 (500MHz) 90


4.41 HMQC (Expand) NMR Spectrum of CP4 92





4.46 Selected NOESY 1H NMR Spectrum of CP4 98



4.49 Structure of CP5 and Partial HMBC Correlations in Ring A and B 101

4.50 1H NMR Spectrum of CP5 in CDCl3 (500MHz) 102



4.53 HSQC NMR Spectrum of CP5 105






4.59 Structure of CP8 111


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4.75 Structure of CP9 (Above) and CP3 (Below) 128









4.84 Structure of CP10 (Above) and CP5 (Below) 138

4.85 Mass Spectra of �-sitosteryl-�-D-glucopyranoside 140

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4.86 1H-NMR Spectrum of �-sitosteryl-�-D-glucopyranoside (CP11) 143 in Pyridine-d5 (500MHz) 4.87 NMR Spectrum of �-sitosteryl-�-D-glucopyranoside (CP11) in 144 Pyridine-d5 (500 MHz)

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LIST OF ABBREVIATIONS

� Chemical shift

bp Boiling point

br Broad

COSY Correlation spectroscopy

d Doublet

dd Doublet of doublets

dt Doublet of triplets

EIMS Electron Impact Mass Spectrum

HMBC Heteronuclear Multiple Bond Correlation

HRMS High Resolution Mass Spectrometry

HSQC Heteronuclear Single Quantum Correlation

Hz Hertz

J Coupling constant

m Multiplet

m/z Mass per charge

MHz MegaHertz

mp Melting Point

nm Nanometer

NOESY Nuclear Overhauser Effect Spectroscopy

q Quartet

Rf Retention factor

s Singlet

t Triplet

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CHAPTER 1

Introduction

1.1 Natural Products

Natural products have been an overwhelming success as a rich source of therapeutically

effective medicines. Approximately 60% of the world’s population is fully relies on

plants for medication. Among 520 new drugs approved between 1983 and 1994, 60 –

80% of antibacterial and anticancer drugs are derived from natural products and 39% are

natural products or derived from natural products. Natural products in the present are

commonly used to cure diseases which include anticholesteremic agents (e.g. levostatin

and derivatives), antibiotics (e.g. erythromycin and derivatives, such as clarithromycin),

antitumor agents (e.g. paclitaxel and camptothecin derivatives such as topotecan and

irinotecan) and immunosuppressive agents (e.g. cyclosporine A and FK 506) (Bindseil

et al., 2001).

Current commercial evidence supports the use of natural products. A study in 1999 has

shown twenty best-selling non-protein drugs were either derived from or developed as

the result of substances generated by natural products such as simvastatin, levostatin,

enalapril, pravastatin, atorvastatin, augmentin, ciprofloxacin, clarithromycin and

cyclosporine. These drugs have combined annual sales of more than US$16 billion.

Current developments based on natural products include the antimalarial drug

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artemisinin and the anticancer agents such as taxol, docetaxel and camptothecin.

Therefore, the use of natural products has been the most successful strategy for the

discovery of new medicines (Harvey, 2000).

1.2 Meliaceae

The Meliaceae family is characterized by the presence of limonoid triterpenes, many of

which are biologically active against insects. Two limonoids have been commercialized

which are azadirachtin in the U.S. and toosendanin in China from the Asian species

Azadirachta indica and Melia azedarach. Extracts of several species from the genera

Cedrela, Trichilia, Swietenia and Guarea from the Neotropics have demonstrated

moderate to good growth reducing activity to European corn borer (ECB), Ostrinia

nubilalis Hubner. Consequently, some of these species have been the subject of

chemical and biological investigations in order to isolate new insecticidal agents

(Jimenez et al., 1998).

Limonoids are an important group of metabolically altered-triterpenes, which are limited

in their distribution. In recent years a large number of pharmacological studies have

been carried out to indicate their beneficial effects. The medicinal properties reported

include anti-cancer, anti-malarial, antimicrobial, anti-HIV, anti-viral and several others.

Post-study health evaluation has established no ill effects among study subjects

consuming high doses of limonin glucoside. Some of them have also shown allelopathic

potential. The citrus limonoids exhibit promising health benefits (anti-cancer, cardio

protective, anti-oxidant etc.) but are the major causes of concern due to their extreme

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bitterness. There is a need to develop an acceptable and versatile debittering method that

can substantially remove or mitigate the bitterness of fruits and juice (Amit and

Shailendra, 2005).

Folk medicines from the Meliaceae plants have been traditionally used to cure malarial

disease in many tropical and sub-tropical countries. The elevated incidence of malaria in

recent years has been attributed to the development of resistance of the malarial protozoa

(Plasmodium falciparum) to chloroquine and to the development of resistance of the

vector mosquitoes to insecticides. In Africa, all species of Khaya used in the German

timber and furniture industry contribute to malarial diseases. These plants usually

contain a variety of limonoids and they possess antimalarial activities. Limonoids are the

most representative class of secondary metabolites in the order Rutales, which includes

the Rutaceae, Meliaceae, and Simaroubaceae families. They are tetranortriterpenoids

with 4,4,8-trimethyl-17-furanylsteroidal skeleton, with several oxygenated functions.

Several limonoids of angolensates, khivorins, mexicanolides, and fissinolides have been

isolated from different parts of Khaya anthotheca. The limonoids have several beneficial

properties such as low mammalian toxicity, lack of neurotoxic mode of action, low

persistence in the environment and biodegradability and are reported to be new sources

of biologically active natural products (Lee et al., 2007).

In our study group, two plants from the Meliaceae family had been investigated from

which two novel triterpenes, acutaxylines A, B and three-type limonoids, sandrapins A,

B and C have been successfully isolated from the Dysoxylum acutangulum and

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Sandoricum koetjape respectively. The methanol extract from the Dysoxylum

acutangulum leaves showed cytotoxicity against human cancer cells (Ismail et al., 2009).

1.3 Ichthyotoxic Assay

Ichthyotoxic assay is a quick preliminary test carried out to determine the possible

potential biological activities that may be possessed by the plant under study. Ichthyo

itself means fish and this test is performed by using fish to exhibit the significant

toxicity. Based on the previous findings, the ichthyotoxic compounds often exhibited

various biological activities such as insecticidal, anti-tumor and antibacterial (Ismail et

al., 2003). Therefore, this assay is a suitable method to preliminarily identify the active

fraction/compound. Several researchers had performed ichthyotoxic assay as a

preliminary screening for biologically active substance. Ismail et al. (2003) had used this

assay as a preliminary test to determine the potential biological activities in Sandoricum

koetjape bark against killie-fish. In this study, it was shown that the ichthyotoxic

compounds also exhibited cytotoxicity.

1.4 Acetylcholinesterase (AChE) Assay

Studies have shown that the inhibition of brain acetylcholinesterase (AChE) plays an

important role for the treatment of many degenerative diseases such as shoulder

tendonitis, heart disease, inflammatory bowel disease, rheumatoid arthritis, asthma and

gouty arthritis which are always associated with inflammatory process (Fawole et al.,

2010). Inhibition of brain acetylcholinesterase (AChE) has been the major therapeutic

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target of cholinesterase inhibitor (ChEI) treatment strategies for Alzheimer's disease

(AD), Parkinson's disease, myasthenia gravis, dementia, ataxia, and senile. However,

existing anticholinesterase drugs such as tacrine, physostigmine and

heptylphysostigmine, donepezil and galantamine were reported to have several side

effect and dangerous (Sancheti et al., 2010). Due to this, more new anticholinesterase

drugs are needed to replace them. In this study, two acetylcholinesterase (AChE) assay

methods were used to screen the fraction/compounds, which are Thin Layer

Chromatography (TLC)-bioautography with fast blue B salt and Ellman’s method.

1.5 Problems Statement

The world’s population relies on plants for medication since ancient. However, there are

still diseases which are unable to be cured or the existing drugs are having several side

and/or adverse effects which could be dangerous when used. Therefore, there is need to

increase the volume of research to find out more potential drugs from natural products.

Tapping on plant is one of the many ways to discover the potential bioactive compounds

natural resources. As Dysoxylum grande is one of the least explored plants of Malaysia

but being traditional claimed as having bioactivities, we decided to explore the

possibilities of discovering the potential properties of it through some set objectives.

1.6 Research Objectives

This study focuses on the isolation and characterization of the chemical constituents

from Dysoxylum grande leaves and the bioactivities of the isolated compounds. All the

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compounds obtained were characterized using thorough spectroscopic data obtained

including 1H, 13C Nuclear Magnetic Resonance (NMR), 2D-NMR, mass spectroscopy

(MS) and infrared (IR) spectroscopy.

Due to the lack of scientific report on this Meliaceae plant species and interesting

characteristics of compounds with various biological activities, D. grande was chosen to

be studied based on the objectives of:

1. To discover pure compounds from the ichthyotoxic plant part.

2. To determine the possible bioactivity (ies) of the isolated compounds.

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REFERENCES

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Aladesanmi, A. J., & Hoffmann, J. J. (1994). Additional alkaloids from the stem of Dysoxylum lenticellare. Phytochemistry, 35(5), 1361-1362.

Aladesanmi, A. J., Adewunmi, C. O., Kelley, C. J., Leary, J. D., Bischoff, T. A., Zhang, X., et al. (1988). Lenticellarine, a molluscicidal alkaloid from Dysoxylum lenticellare. Phytochemistry, 27(12), 3789-3792.

Alan, H. (2000). Strategies for discovering drugs from previously unexplored natural products. Drug Discovery Today, 5(7), 294-300.

Amit, R. & Shailendra, S. (2005). Limonoids: Overview of Significant Bioactive Triterpenes Distributed in Plants Kingdom. Biological and Pharmaceutical Bulletin, 29(2), 191-201.

Barry W, W., & Robert, K. (2010). Chapter 68 - Cholinesterases Hayes' Handbook of Pesticide Toxicology. New York: Academic Press. 3,1457-1478.

Beastall, G. H., Tyndall, A. M., Rees, H. H. & Goodwin, T. W. (1974). Sterols in Porphyridium species. 4-Methyl-5-cholesta-8,22-dien-3-ol and 4,24-dimethyl-5-cholesta-8,22-dien-3-ol: two novel sterols from Porphyridium cruentum. European Journal of Biochemistry. 41, 301-309.

Bindseil, K. U., Jakupovic, J., Wolf, D., Lavayre, J., Leboul, J., & van der Pyl, D. (2001). Pure compound libraries; a new perspective for natural product based drug discovery. Drug Discovery Today, 6(16), 840-847.

Cronquist, A. (1988). The Evolution and Classification of Flowering Plants. The New York Botanical Garden, New York.

Fawole, O. A., Amoo, S. O., Ndhlala, A. R., Light, M. E., Finnie, J. F., & Van Staden, J. (2010). Anti-inflammatory, anticholinesterase, antioxidant and phytochemical properties of medicinal plants used for pain-related ailments in South Africa. Journal of Ethnopharmacology, 127(2), 235-241.

Fujioka, T., Yamamoto, M., Kashiwada, Y., Fujii, H., Mihashi, K., Ikeshiro, Y.,...Lee, K. H. (1998). Novel cytotoxic diterpenes from the stem of Dysoxylum Kuskusense. Bioorganic and Medicinal Chemistry Letters, 8(24), 3479-3482.

Fujita, T. & Nakayama, M. (1993). Monoterpene glycosides and other constituents from Perilla frutescens. Phytochemistry. 34(6), 1545-1548.

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He, X.-F., Wang, X.-N., Yin, S., Dong, L., & Yue, J.-M. (2011). Ring A-seco triterpenoids with antibacterial activity from Dysoxylum hainanense. Bioorganic & Medicinal Chemistry Letters, 21(1), 125-129.

Hooker, J. D., & Brid, F. I. (1875). India. 1, 547.

Ismail, S. I., Ito, H., Hatano, T., Taniguchi, S., & Yoshida, T. (2003). Modified limonoids from the leaves of Sandoricum koetjape. Phytochemistry, 64(8), 1345-1349.

Ismail, S. I., Ito, H., Mukainaka, T., Higashihara, H., Enjo, F. (2003). Ichthyotoxic and Anticarcinogenic Effects of Triterpenoids from Sandoricum koetjape Bark. Biological and Pharmaceutical Bulletin, 26(9), 1351-1353.

Ismail, S. I., Nagakura, Y., Hirasawa, Y., Hosoya, T., Lazim, M. I. M., Lajis, N. H., Morita, H. (2009). Acutaxylines A and B, two novel triterpenes from Dysoxylum acutangulum. Tetrahedron Letters, 50(34), 4830-4832.

Jimenez, A., Villarreal, C., Toscano, R. A., Cook, M., Arnason, J. T., Bye, R., & Mata R. (1998). Limonoids from Swietenia humilis and Guarea grandiflora (Meliaceae) Taken in part from the PhD and MS theses of C. Villarreal and M. A. JimÃ©nez, respectively. Phytochemistry, 49(7), 1981-1988.

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BIODATA OF STUDENT

Low Kok Wah was born in Taiping, Perak, Malaysia on the 25th March 1984. He had

his early education at Sekolah Jenis Kebangsaan Hua Lian, Taiping and secondary

education at Sekolah Menengah Jenis Kebangsaan Hua Lian. He completed his Sijil

Pelajaran Malaysia (SPM) and Sijil Tinggi Pelajaran Malaysia (STPM) at the same

school. After that, he was offered to study in Universiti Putra Malaysia in Bachelor

of Science (Honours) degree in Industrial Chemistry. Then he worked as a Quality

Assurance Executive at Lion Dri Sdn Bhd for two years. He resigned to further study

at Universiti Putra Malaysia for Master in Science (Natural Product). He was also

offered as a Grand Research Fellow holder during his MSc study.

dysoxylum grande hiern as a potential source of

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