IN VITRO AND IN SILICO STUDIES ON

SELECTED MALAYSIA CULINARY

PLANTS AGAINST DENGUE

NS2B-NS3 PROTEASE

YONG KAI SING

UNIVERSITI SAINS MALAYSIA

2016

IN VITRO AND IN SILICO STUDIES ON

SELECTED MALAYSIA CULINARY

PLANTS AGAINST DENGUE

NS2B-NS3 PROTEASE

by

YONG KAI SING

Thesis submitted in fulfillment of the requirements

for the degree of Master of Science

April 2016

ii

ACKNOWLEDGMENT

During the Master research project, bridging theory on books and practical in lab is

tough for me as a fresh degree graduate at the initial stage. Therefore, I learned to seek

technical advice from experts and set achievable objectives for completing the research work.

Hereby, I claim all the work in this thesis are done by me and willing to take the

responsibility for it.

First, I would like to express gratitude to my supervisor, Professor Dr. Habibah A.

Wahab for her advice and encouragement throughout this project. Her constructive

recommendation and willingness to give time for planning and development of this research

work is highly appreciated. In addition, a thank you to the Dean of School of Pharmaceutical

Sciences, USM and Director General of Malaysian Institute of Pharmaceuticals and

Nutraceuticals (IPHARM) in providing the resources and facilities for this research project.

This project would have been impossible without the financial support from Universiti Sains

Malaysia Fellowship, Agilent Bio-analytical Industry Development Program and grants from

the Ministry of Science and Technology, Malaysia.

I wish to acknowledge the help provided by Dr. Maywan Hariono for his brilliance

and inspiration in the lab. I would like to extend my thanks to Dr. Choi Sy Bing for

providing me her guidance and consultation in computational work. Many people, especially

all the members from Pharmaceutical Design and Simulation Lab (PhDS) have made

valuable suggestions to improve this project. I wish to thanks Mr Fizal, Mr. Zainuddin and

Mr. Anwar from School of Pharmaceutical Sciences for their help in handling the

instruments. I express my warm thanks to my family members and friends who have been

giving their endless love and support for completing this project. Last but not least, this

thesis is dedicated to my partner, Chun Siang Teo, for his love and support during the past

three years for finalizing this thesis.

iii

TABLE OF CONTENTS

Page

ACKNOWLEGMENT .......................................................................................................... ii

TABLE OF CONTENTS ..................................................................................................... iii

LIST OF TABLES ................................................................................................................. vi

LIST OF FIGURES ............................................................................................................ viii

LIST OF ABBREVIATIONS AND SYMBOLS ................................................................. xi

ABSTRAK ........................................................................................................................... xiii

ABSTRACT .......................................................................................................................... xiv

CHAPTER ONE: INTRODUCTION ................................................................................... 1

1.1 Statement of Problem................................................................................................... 1

1.2 Objectives and Scopes of Research ............................................................................. 3

CHAPTER TWO: LITERATURE REVIEW ...................................................................... 5

2.1 Dengue ......................................................................................................................... 5

2.2 Dengue Drug Discovery .............................................................................................. 8

2.2.1 Dengue Virus .................................................................................................. 8

2.2.2 Dengue Drug Targets .................................................................................... 10

2.2.3 NS2B-NS3 Protease ...................................................................................... 11

2.2.4 Potential Dengue NS2B-NS3 Inhibitor......................................................... 13

2.3 Natural Product in Drug Discovery ........................................................................... 21

2.4 Malaysia Culinary Plants ........................................................................................... 23

2.4.1 Curcuma longa ............................................................................................. 24

2.4.2 Cymbopogon citratus .................................................................................... 26

2.4.3 Murraya koenigii .......................................................................................... 27

2.4.4 Ocimum basilicum ........................................................................................ 29

2.4.5 Pandanus amaryllifolius ............................................................................... 31

2.4.6 Persicaria odorata ........................................................................................ 32

2.4.7 Zingiber officinale ........................................................................................ 33

2.5 Bioassay Guided Isolation ......................................................................................... 35

2.6 Computation in Drug Discovery ................................................................................ 36

2.7 Molecular Docking Simulation .................................................................................. 39

iv

CHAPTER THREE: MATERIALS AND METHODOLOGY ........................................ 42

3.1 In vitro Study ............................................................................................................ 42

3.1.1 Materials ...................................................................................................... 42

3.1.2 Preparation of Buffer .................................................................................... 43

3.1.3 Enzymatic Bioassay for Screening Fractions ............................................... 43

3.2 Extraction and Separation ......................................................................................... 44

3.2.1 Materials ....................................................................................................... 44

3.2.2 Sample Preparation ....................................................................................... 44

3.2.3 Crude Extraction ........................................................................................... 44

3.2.4 Liquid-liquid Extraction ............................................................................... 45

3.2.5 Separation and Isolation ............................................................................... 45

3.3 Structure Elucidation ................................................................................................ 46

3.3.1 Materials ...................................................................................................... 46

3.3.2 High Performance Liquid Chromatography ................................................ 46

3.3.3 Nuclear Magnetic Resonance Spectroscopy ................................................ 46

3.3.4 Fourier Transform Infrared Spectrometer .................................................... 47

3.3.5 Liquid Chromatography Mass Spectrometry ............................................... 47

3.4 IC50 Study .................................................................................................................. 47

3.4.1 Enzymatic Bioassay for the Determination of IC50 ...................................... 47

3.4.2 Detergent-based Enzymatic Bioassay Protocol ............................................ 47

3.5 In silico Study ........................................................................................................... 47

3.5.1 Materials ...................................................................................................... 47

3.5.2 Preparation of Protein Structure ................................................................... 48

3.5.3 Preparation of Ligand Structure ................................................................... 48

3.5.4 Docking Parameter ....................................................................................... 48

CHAPTER FOUR: RESULTS AND DISCUSSION ......................................................... 50

4.1 In vitro Study ............................................................................................................ 50

4.1.1 Optimization of Enzyme Concentration ....................................................... 50

4.1.2 Bioactivity of Selected Culinary Plants ........................................................ 51

4.1.3 Bioactivity of Fractions from Persicaria odorata ........................................ 52

4.1.4 Bioactivity of Fractions from Murraya koenigii ........................................... 53

4.2 Extraction and Separation ......................................................................................... 55

4.2.1 Persicaria odorata ....................................................................................... 55

4.2.2 Murraya koenigii ......................................................................................... 57

4.3 Structure Elucidation ................................................................................................ 60

v

4.3.1 Chemical Constituent from Persicaria odorata ........................................... 60

4.3.2 Chemical Constituent from Murraya koenigii .............................................. 70

4.4 IC50 Study ................................................................................................................. 78

4.4.1 Enzymatic Assay of Panduratin A, F2_1 and Bismahanine ........................ 78

4.4.2 Detergent-based Enzymatic Assay of F2_1 and Bismahanine ..................... 80

4.5 In silico Study ........................................................................................................... 81

4.5.1 Control Docking ........................................................................................... 81

4.5.2 Docking of Panduratin A .............................................................................. 83

4.5.3 Docking of Compound F2_1 ........................................................................ 84

4.5.4 Docking of Bismahanine .............................................................................. 87

CHAPTER FIVE: CONCLUSION ..................................................................................... 93

5.1 Accomplishment of the Objectives ............................................................................ 93

5.2 Future Work ............................................................................................................... 94

5.3 Concluding Remarks ................................................................................................. 95

REFERENCES...................................................................................................................... 96

LIST OF APPENDICES

APPENDICES

LIST OF PUBLICATIONS

vi

LIST OF TABLES

Page

Table 2.1 Compounds with reported Dengue NS2B-NS3pro inhibitory

activities

16

Table 2.2 The Selected Malaysia Culinary Plants and Their Scientific Names

24

Table 2.3 Taxonomy of Curcuma longa

24

Table 2.4 Taxonomy of Cymbopogon citratus

26

Table 2.5 Taxonomy of Murraya koenigii

28

Table 2.6 Taxonomy of Ocimum basilicum 30

Table 2.7 Taxonomy of Pandanus amaryllifolius 31

Table 2.8 Taxonomy of Persicaria odorata

32

Table 2.9 Taxonomy of Zingiber officinale 34

Table 2.10 Success drug candidates develop from in silico study

38

Table 4.1 Percentage Inhibition of Selected Malaysia Culinary Plants against

NS2B-NS3 Protease Bioassay at Concentration 200 μg/mL

52

Table 4.2 Percentage Inhibition of Partitions from P. odorata against NS2B-

NS3 Protease Bioassay at Concentration 200 μg/mL

52

Table 4.3 Percentage Inhibition of Ethyl Acetate and Hexane Fractions of P.

odorata

53

Table 4.4 Percentage Inhibition of Partitions from M. koenigii against NS2B-

NS3 Protease Bioassay at Concentration 200 μg/mL

53

Table 4.5 The percentage Inhibition of Hexane Fractions and Sub-fractions

from M. koenigii against NS2B-NS3 Protease Bioassay at

Concentration 200 μg/mL

54

Table 4.6 Percentage Inhibition of Ethyl Acetate Fractions and Sub-fractions

from M. koenigii against NS2B-NS3 Protease Bioassay at

Concentration 200 μg/mL

55

Table 4.7 Chemical Shift of 13

C-NMR for Compound F2_1 (MeOD, 125

MHz)

64

Table 4.8 Chemical Shift of 1H-NMR for Compound F2_1 (MeOD, 500

MHz)

66

Table 4.9 The Correlation from 2D-NMR of Compound F2_1 (MeOD, 500

MHz)

67

Table 4.10 Chemical Shift of 13

C-NMR for Compound E22E4f (CDCl3, 125 73

vii

MHz)

Table 4.11 Chemical Shift of 1H-NMR for compound E22E4f (CDCl3, 500

MHz)

75

Table 4.12 The Correlation from 2D-NMR of Compound E22E4f (CDCl3, 500

MHz)

76

Table 4.13 Summary of Important Interactions between Control Docked

Tetrapeptide and Amino Acid Residue of 3U1I

82

Table 4.14 Summary of Important Interactions between Panduratin A and

Amino Acid Residue of 3U1I

83

Table 4.15 Summary of Important Interactions between Compound F2_1 and

3U1I

85

Table 4.16 Summary of Important Interactions between Bismahanine and

Amino Acid Residues of 3U1I

88

Table 4.17 Free Energy of Binding for Panduratin A, Compound F2_1 and

Bismahanine Docked with 3U1I

90

Table 4.18 Summary of Important Amino Acid Residues of 3U1I between

Control Docked Tetrapeptide, Panduratin A, Compound F2_1 and

Bismahanine

91

viii

LIST OF FIGURES

Page

Figure 2.1 Dengue Infection Cases in Malaysia from Year 1998 to 2014 6

Figure 2.2 Dengue Death Cases in Malaysia from Year 1998 to 2014 7

Figure 2.3 Flavivirus Life Cycle 9

Figure 2.4 Flaviviral Polyprotein 12

Figure 2.5 (a) NS2B (yellow ribbon) Surround NS3 Protease form PDB ID

3U1I (b) Important Residues Interact with Tetrapeptide (stick)

13

Figure 2.6 Structure of Small Molecule Inhibitors (a) Panduratin A, (b)

Arylcyanoacrylamide and (c) Ribavarin

15

Figure 2.7 Chemical structures of (a) curcumin, (b) ar-turmerone and (c)

zingerone

25

Figure 2.8 Photos of (left) whole plant and (right) rhizome of C. longa 26

Figure 2.9 Chemical structures of (a) limonene, (b) citronelal and (c) geraniol 27

Figure 2.10 Photos of whole plant of C. citratus 27

Figure 2.11 Carbazole Alkaloids (a) Mahanine, (b) pyrayafoline-D and (c)

Murrayafoline

28

Figure 2.12 Photo of (left) Whole Plant, (up right) Fruit and (down right)

Leaves of M. koenigii

29

Figure 2.13 Chemical structures of (a) linalool, (b) eugenol and (c) rosmaric

acid

30

Figure 2.14 Photo of whole plant of O. basilicum 31

Figure 2.15 Structure of (a) pandanamine and (b) 2-acetyl-1-pyrroline 31

Figure 2.16 Photo of whole plant of P. amaryllifolius 32

Figure 2.17 Chemical structures of (a) polygodial, (b) caryophyllene and (c)

ocimene

33

Figure 2.18 Photo of whole plant of P. odorata 33

Figure 2.19 Chemical structures of chemical constituents (a) 6-gingerol, (b)

zingerone and (c) shogaols

34

Figure 2.20 Photos of (left) whole plant and (right) rhizome of Z. officinale 35

Figure 2.21 Modern Drug Discovery Research Process include CAMM

Activities (yellow), Experimental ADME-Tox and Compound

Profiling Activities (blue)

37

ix

Figure 3.1 Flow Diagram of Methodology 42

Figure 4.1 (a) Michaelis-Menten Graph and (b) Lineweaver-Burk Graph 50

Figure 4.2 Maceration Extraction and Liquid-liquid Extraction for P. odorata 56

Figure 4.3 Result of Fractionation of Ethyl Acetate Partitions for P. odorata 56

Figure 4.4 Result of Fractionation of Hexane Partitions for P. odorata 57

Figure 4.5 Maceration Extraction and Liquid-liquid Extraction for M. koenigii

58

Figure 4.6 The Separation and Isolation of Hexane Fractions for M. koenigii

59

Figure 4.7 Result of Separation and Isolation of Ethyl Acetate Fractions for

M. koenigii

59

Figure 4.8 Thin Layer Chromatography of Fraction E2 (a) was Separated into

10 Sub-fractions from E9 to E18 (b) in Solvent System

Chloroform:Acetone (9.5:0.5)

60

Figure 4.9 Result of Recycling Isolation of Ethyl Acetate Fraction for P.

odorata

61

Figure 4.10 Result of Fraction F2_1 for P. odorata in analytical HPLC 61

Figure 4.11 UV Spectrum for Compound F2_1 62

Figure 4.12 Numbering for Compound F2_1 62

Figure 4.13 13

C-NMR Spectra of F2_1 63

Figure 4.14 1H-NMR Spectra of F2_1 65

Figure 4.15 1H-

1H COSY (-) and

1H-

13C HMBC (→) Correlations of F2_1

68

Figure 4.16 ESI-MS of Compound F2_1 69

Figure 4.17 Infrared Spectrum of Compound F2_1 70

Figure 4.18 HPLC Chromatogram of Compound E22E4f 70

Figure 4.19 UV Spectrum of Compound E22E4f 71

Figure 4.20 Numbering for Compound E22E4f 71

Figure 4.21 13

C-NMR Spectra of E22E4f 72

Figure 4.22 1H-NMR Spectra of E22E4f 74

Figure 4.23 1H-

1H COSY (-) and

1H-

13C HMBC (→) Correlations of

Compound E22E4f

77

x

Figure 4.24 LCMS-QTof of Compound E22E4f 77

Figure 4.25 Infrared Spectrum of Compound E22E4f 78

Figure 4.26 Inhibition Activity of (a) Panduratin A, (b) F2_1 and (c) E22E4f in

Dengue Assay

79

Figure 4.27 Optimization of Detergent in Dengue Assay 80

Figure 4.28 Inhibition Activity of (a) F2_1 and (b) E22E4f in Dengue

Detergent Based Assay

81

Figure 4.29 Tetrapeptide in S1 Pocket (yellow), S2 Pocket (orange), S3 Pocket

(cyan) and S4 Pocket (purple) of NS2B-NS3 Protease. RMSD of

Control Docked Pose (purple color) of Tetrapeptide Inhibitor with

Crystal Structure (green color) of Tetrapeptide is 1.3 Å.

83

Figure 4.30 Interaction of Docked Conformation of Panduratin A (green) with

Important Residues of 3U1I. Dotted line is hydrogen bond

interactions.

84

Figure 4.31 Interaction of Docked Conformation of Compound F2_1 (stick

representation) with Important Residues of 3U1I. Orange line is pi

interactions while dotted line is hydrogen bond interactions.

86

Figure 4.32 Visualization of Molecular Interactions of Compound F2_1 with

NS2B-NS3 Protease. Dotted line is hydrogen bond interactions

while straight line is pi interactions.

87

Figure 4.33 Interaction of Docked Conformation of Bismahanine (green) with

Important Residues of 3U1I. Dotted line is hydrogen bond

interactions while orange line is pi interactions.

89

Figure 4.34 Visualization of Molecular Interactions of Bismahanine with

NS2B-NS3 Protease

90

xi

LIST OF ABBREVIATIONS AND SYMBOLS

Ac Acetone

AMC 7-Amino-4-methyl-coumarin

Arg Arginine

Asn Asparagine

Asp Aspartic acid

Boc tert-Butyloxycarbonyl

br Broad

BuOH n-Butanol

CDCl3 Chloroform-D

CHCl3 Chloroform

Conc. Concentration

COSY Correlation Spectroscopy

d Doublet

DENV2 Dengue Virus Type 2

DMSO Dimethyl sulfoxide

ESI ElectroSpray Ionisation

EtOAc Ethyl acetate

FEB Free energy of binding

RFU Relative fluorescence unit

g Gram

Gly Glycine

Hex n-Hexane

His Histidine

HMBC Heteronuclear Multiple Bond Correlation

HPLC High Performance Liquid Chromatography

HSQC Heteronuclear Single Quantum Correlation

Hz Hertz

H2O Water

IC50 Half maximal inhibitory concentration

IR Infrared J Coupling constant kcal/mol Kilocalories per mol

L Litre

Lys Lysine

m Multiplet

MCA Methyl Cumaryl Amide

MeOD Methanol-D

MeOH Methanol

Met Methionine

MHz Mega Hertz

mL Milli litre

MS Mass Spectrometry m/z Mass to charge ratio

nm Nanometer

NPCC Normal Phase Column Chromatography

Phe Phenylalanine

PLC Preparative Thin Layer Chromatography

ppm Part per million

xii

Pro Proline Q-Tof Quadrupole time-of-flight Rf Retention factor

RP-TLC Reverse Phase Thin Layer Chromatography

RMSD Root mean square deviation r

2 Correlation coefficient

s Singlet

Ser Serine

SO4 Sulfate

t Triplet

Thr Threonine

TLC Thin Layer Chromatography

Tyr Tyrosine

UV Ultraviolet

Val Valine 1H-NMR Proton Nuclear Magnetic Resonance

13C-NMR Carbon Nuclear Magnetic Resonance

δ Chemical Shift

2D Two dimension

% Percentage μM Micro Molar Å Angstrom

xiii

KAJIAN IN VITRO DAN IN SILICO KEATAS TUMBUHAN MASAKAN

MALAYSIA TERPILIH TERHADAP PROTEASE DENGGI NS2B-NS3

ABSTRAK

Di Malaysia, statistik jangkitan denggi setakat tahun 2015 sudah melebihi 100,000

kes. Walaubagaimanapun sehingga kini tidak ada calon vaksin dan ubat untuk denggi yang

boleh didapati di pasaran. Oleh itu, penemuan ubat untuk denggi adalah penting untuk

mencari calon ubat yang sesuai. Kajian ini melibatkan pencirian kimia juzuk, eksperimen in

vitro dan simulasi in silico untuk tumbuh-tumbuhan yang digunakan dalam masakan harian

di Malaysia. Dalam kajian ini, kunyit, serai wangi, daun kari, daun selasih, daun pandan,

daun kesum dan halia dipilih dalam saringan bioaktiviti terhadap NS2B-NS3 protease.

Tumbuh-tumbuhan tempatan ini dipilih sebab mereka memaparkan keputusan yang menarik

dalam penyaringan maya pada kajian yang lepas. Antara tujuh tumbuhan, daun kesum dan

daun kari didapati sangat aktif dalam merencat denggi NS2B-NS3 protease dengan 93% dan

88% perencatan masing-masing. F2_1 (IC50 17.4 μM) diasingkan dari daun kesum

menyerupai Vanicoside A mempunyai perencatan yang lebih baik daripada rujukan

Panduratin A (IC50 94.60 μM) manakala Bismahanine (IC50 93.1 μM) diasingkan dari daun

kari mempunyai perencatan yang serupa dengan Panduratin A. Dari hasil pendokan, sebatian

F2_1 mempunyai interaksi polar dengan amino asid pada struktur krystal 3U1I manakala

Bismahanine mempunyai interaksi ikatan hydrogen dengan triad pemangkin (His51, Asp75,

Ser135) yang lebih baik daripada enzim protease. Walau bagaimanapun, F2_1 dan

Bismahanine cenderung untuk beragregat pada kepekatan mikro molar dan IC50 masing-

masing 319.4 μM dan 327.3 μM dalam asai berasaskan detergen. Kajian masa depan boleh

dilakukan pada pengubahsuaian kimia pada struktur Bismahanine bagi merekabentuk

perencat denggi NS2B-NS3 yang lebih baik.

xiv

IN VITRO AND IN SILICO STUDIES ON SELECTED MALAYSIA CULINARY

PLANTS AGAINST DENGUE NS2B-NS3 PROTEASE

ABSTRACT

In Malaysia, dengue infection was reported more than 100,000 cases in year 2015.

To date there is no marketed drug candidate available for Dengue disease. Therefore, drug

discovery for dengue is important to find a suitable drug candidate. This study involves

characterization of chemical constituent, in vitro experiment and in silico simulation for anti-

dengue from culinary plants in Malaysia. In this study, turmeric, lemon grass, curry leaves,

sweet basil, screw pine, Vietnamese mint and ginger were screened for bioactivity against

dengue NS2B-NS3 protease. The rational of choosing these native culinary plants because

they show interesting result in virtual screening in previous study. Out of seven plants,

Vietnamese mint and curry leaves were found highly active in inhibiting dengue NS2B-NS3

protease with 93% and 88% inhibition, respectively. Compound F2_1 (IC50 of 17.4 μM)

isolated from Vietnamese mint which is similar to be Vanicoside A showed better inhibition

than reference Panduratin A (IC50 of 94.60μM) while Bismahanine (IC50 of 93.1 μM)

isolated form curry leaves has similar inhibition activity with Panduratin A. From docking

result, compound F2_1 formed polar interaction with amino acid residues of protease crystal

structure, 3U1I while Bismahanine has hydrogen bond interactions with the catalytic triad

(His51, Asp75, Ser135) of protease which is more preferable. However, F2_1 and

Bismahanine tend to aggregate at micromolar concentration and the IC50 increased to 319.4

μM and 327.3 μM respectively in the detergent-based assay. Future studies especially on the

chemical modification of the structure of Bismahanine could be carried out in order to

discover more potent dengue protease inhibitors.

1

CHAPTER ONE

INTRODUCTION

1.1 Statement of Problem

Dengue disease, an arthropod-borne viral infection, has affected more than 100

countries worldwide. Every year, nearly 390 million cases of this infection are reported and

over 3.9 billion people are at risk (Bhatt et al., 2013). There is an increasing trend of dengue

cases due to climate change, flourishing of tourism industry (Freedman et al., 2006),

unorganized urbanization and poor water management system. In Malaysia, it is an endemic

and epidemic disease which increases yearly. In the year 2013, 43,346 cases reported and

this number increased drastically to 103,610 cases in 2014 (WHO, 2014) and 107,079 cases

in the year 2015 (WHO, 2015).

To date, there is no efficient marketed drug to treat dengue. Lack of suitable animal

model (Zompi & Harris, 2012) for in vivo study slow down the dengue drug discovery

compounded with the fact that part of the mechanisms of dengue virus infection is still

unclear (Thullier et al., 2001).

Only few peptides (Yin et al., 2006), non-peptide (Ganesh et al., 2005), small

molecules (Deng et al., 2012) and natural compounds (de Sousa et al., 2015) have been

reported to have inhibition activity towards dengue NS2B-NS3 protease. For instance,

aprotinin inhibits dengue virus activity at submicromolar concentrations. However it cannot

be developed as drug due to its large unstable peptide structure that would envelops enzyme

and blocks substrate from approaching the active site and form non-competitive inhibition

(Leung et al., 2001). Ribavirin, a RNA virus inhibitor, suppresses virus replication in vitro

but shows protective effect in animal study (Leyssen et al., 2008). Kalata B1 cyclotide, one

type of plant protein, which is designed and synthesized from natural cyclotide has potent

inhibition from dengue NS2B-NS3 protease (Gao et al., 2010). The findings of cyclohexenyl

2

chalcone derivatives isolated from Bosenbergia rotunda as competitive inhibitors to DEN2

NS2B-NS3 protease (Kiat et al., 2006) have encouraged researchers to find more potential

drugs from natural product.

Natural product has been established as excellent lead for therapeutic field, for

example, taxol from Taxus brevifolia that treats cancer and artemisinin from Artemisia

annua is widely used in treating malaria (Phillipson, 2001). Vinblastine from Catharanthus

roseus (L.) is a vinca alkaloid and chemical analogue of vincristine. Both compounds are

chemotherapy medication used to treat acute lymphocytic leukemia, acute myeloid leukemia,

Hodgkin's disease, neuroblastoma, and small cell lung cancer (Lahlou, 2013).

Interestingly, the endemic cases of dengue usually occur in developing or

underdeveloped countries with inadequate facilities where most patients could not afford

expensive treatment. Drug discovery and development of dengue disease should not

contribute to high-priced medicines that burden the patients. Therefore exploration of plant

compounds is suitable as they are natural and cheap sources for remedy. Malaysia is rich in

flora and fauna with over 16,000 flowering plants (Napis et al., 2001) and 3000 species of

medicinal plants, providing broad environment and good opportunity to drug discovery and

development for dengue disease.

In addition, computational method has an apparent important role in drug discovery

and development from the stage of target identification, lead discovery, and lead

optimization, preclinical to clinical trials since 1980s (Ou Yang et al., 2012). In silico

methods decrease the amount of resources required for trial experiment, simulate the

working environment and predict chemical interaction, and therefore aid in improving

efficacy and efficiency of drug discovery (Kapetanovic, 2008).

3

Some commercial drugs begin their research with computer aided drug design

method. Captopril is the first success drug candidate using structure based drug design. It is

an angiotensin-converting enzyme (ACE) inhibitor used for hypertension and treat

congestive heart failure (Cushman et al., 1977). Dorzolamide is carbonic anhydrase inhibitor

use in the treatment of the ocular disease, glaucoma (Greer et al., 1994). Nelfinavir is HIV

protease inhibitor (Wlodawer & Vondrasek, 1998). Zanamivir demonstrate the power of

rational, structure-based and computer assisted drug design. It is neuraminidase inhibitor

received US FDA approval in 1999 for the treatment of the influenza A and B viruses. (von

Itzstein et al., 1993). Imatinib is tyrosine kinase inhibitor for bcr-abl fusion protein

(Philadelphia chromosome-positive leukemias) with the help of rational drug design

approach (Nagar et al., 2002).

In this study, the main aim was to utilise local natural product as the sources of

candidate for dengue inhibitors confirmed by the in vitro enzymatic assay. The mechanism

of action of inhibitor will then be investigated thoroughly using in silico molecular docking

study.

1.2 Objectives and Scopes of Research

This study involved the screening of the inhibitory activity of selected Malaysian

culinary plants using in vitro dengue enzyme bioassay. Seven plants were selected for

extraction, separation and characterisation of the natural products. Each extract’s fraction

was evaluated in dengue enzymatic bioassay system and the active fraction was further sub-

fractionated or isolated. Structure elucidation of chemical constituent was carried out with

High Performance Liquid Chromatography (HPLC), Fourier Transform Infrared

Spectroscopy (FTIR), Quadrupole Time-of-flight Mass Spectrometry (QTof-MS) and

Nuclear Magnetic Resonance (NMR). Bioactivity of chemical constituent was evaluated

using dengue enzymatic bioassay. Molecular interaction between chemical constituent and

protein structure was studied with in silico method using software Autodock 4.2.

4

Specifically, the objectives of this study are:

1. To determine the inhibition activity of fractions from Malaysian culinary plants

towards dengue protease NS2B-NS3 using in vitro method.

2. To characterise chemical constituents from Malaysian culinary plants with potential

anti dengue property.

3. To study the interaction between chemical constituent and dengue protease using in

silico docking method.

21

2.3 Natural Product in Drug Discovery

Plants are used as medicine by folks since ancient time (Weldegerima, 2009).

Traditionally, plants with fewer side effects are the major driver in the study of

phytochemical of natural products (Phillipson, 2001). Sources of natural products however

are not only limited to higher plant species but also include terrestrial plants, microorganisms,

marine organisms, vertebrates and invertebrates. The traditional way of drug discovery

involving natural product is a high cost and time consuming. Thus, in modern days, different

kinds of strategies such as genetic engineering, high speed dereplication and advanced

methods in separation are used to expand the usage of natural products in drug discovery

(Harvey, 2000). Among the families of secondary metabolites, nitrogen containing alkaloids

are the largest group of drug (Raskin et al., 2002) while terpenoids make important

contribution as well (Cragg, 1998).

Drug discovery nowadays encompasses multidisciplinary approach from natural

product research, combinatorial synthetic chemistry to biosynthetic pathways research and

proteomics or genomics research. Drug discovery has shifted from natural product to

microorganism field when the first antibiotic penicillin was derived from fungi at 1920’s.

Natural products derived from microbes are the outcome of genes or non-ribosomal peptide

synthetases or pathways (Baker et al., 2007). Antibiotics such as erythromycin,

chloramphenicol and streptomycin isolated from various microbes are still being used as

drugs today. Over sixty percent of drug in the market today have history related to natural

product or natural product inspired. Among the 175 small molecules discovered in cancer

research between 1940s and 2010, 48.6% are natural products or their derivatives. This

proves the effectiveness of natural product in treating diseases and nature is shown to have

important role in providing new scaffold for diseases (Newman & Cragg, 2012).

Big pharmaceutical companies emphasize the research focusing on synthetic

chemistry, combinatorial chemistry and genomics over the past decades. Synthetic chemistry

22

and combinatorial chemistry have been at the forefront of the development of drug discovery

for decades but there are limitations. Synthetic drug discovery output lacked new lead

compounds from its library as the time passed by (Lee & Schneider, 2001). Combinatorial

chemistry research is the combination of all possible chemical building blocks but the

outcome of the huge library of compounds brings unfruitful effect. Sorafenib from Bayer as

anti-tumor compound is the only new chemical entity as the product of combinatorial

chemistry research (Newman & Cragg, 2007). Bioassay-guided extraction, separation and

isolation with various types of chromatography technology are widely used nowadays

(Sticher, 2008). A successful example for bioassay guided isolation method is the discovery

of novel HIV inhibitor betulinic acid and derivatives (Itokawa et al., 2008).

Discovery of new drug scaffold and pharmacophore from natural product offer new

opportunity in the isolation of new bioactive compounds for lead discovery. So far, plants

are still the majority sources for licensed drugs. For example, immunosuppressive agent such

as cyclosporine A, antitumor agents such as paclitaxel and topotecan, anti-cholesterolemic

agents such as lovastatin are derived from natural products (Bindseil et al., 2001). Aspirin,

morphine, quinine, paclitaxel and artemisinin are also the examples of drug developed from

natural products (Cragg et al., 1997).

On the other hand, the disadvantage of natural product is the difficulties for

resupplying the same species of plant. The isolated compounds might be in small quantities

or are not enough for study of lead optimization, lead development and clinical trials.

However, many plant derived drugs cannot be fully synthesized due to its complexity and

high cost such as atropine and reserpine. Thus, natural product drug discovery need

collaboration from fields of natural product chemistry, pharmacognosy, pharmacology,

ethnobotany and life sciences to get a novel marketed drug for future enhancement.

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2.4 Malaysia Culinary Plant

Malaysia, located in the tropical area has rainforests which consist of 15,000 species

of higher plants. According to literature review, Cymbopogon citratus (Cavalcanti et al.,

2004), Curcuma longa (Kalaivani et al., 2012), Ocimum basilicum (Murugan et al., 2007),

Pandanus amaryllifolius (Pratama et al., 2009), Zingiber officinale (Kalaivani et al., 2012)

and Murraya koenigii (Kovendan et al., 2012) have potential inhibition activity towards

larvicidal activity against Aedes aegypti. Previously, in a virtual screening study in our lab,

some of these edible plants were found to have good inhibitions towards the dengue protease

(PhDs, unpublished result).

Scientists have discovered that some Malaysian culinary plants were able to give

promising vector control activity as shown in Table 2.2. Leave extracts of Pandanus

amaryllifolius killed 100% of mosquito larvae of Aedes aegypti at 0.9% concentration

(Pratama et al., 2009). Leaf extracts of Murraya koenigii had larvicidal property against

house-resting mosquitoes in the tropical country (Kovendan et al., 2012). LC50 value of

Ocimum basilicum was 3.7 for larvae first instar, 4.1 for second instar, 4.6 for third instar

and 5.1 for fourth instar suggesting that the plant can be used to control larvicidal activity for

A. aegypti. Essential oil of C. citratus is also a promising larvicide against mosquitoes

(Cavalcanti et al., 2004), while the essential oil of Z. officinale and C. longa caused larval

mortality within 24 hour at concentrations of 50.78 ppm and 192 ppm respectively

(Kalaivani et al., 2012). Only P. odorata did not have any literature review regarding its

larvicidal activity. Thus, it would be interesting for these plants to be further investigated for

anti dengue properties in enzymatic level.

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Table 2.2 The Selected Malaysia Culinary Plants and Their Scientific Names

Scientific Name Common

Name

Malay

Name

Extract LC50

(ppm)

Reference

Curcuma longa Turmeric Kunyit Oil 115.6 (Kalaivani et

al., 2012)

Cymbopogon

citratus

Lemon grass Serai Oil 69 (Cavalcanti et

al., 2004)

Murraya koenigii Curry leaves Daun kari Hex 963.53 (Kovendan et

al., 2012)

Ocimum basilicum Sweet basil Daun selasih Oil 148.5 (Kalaivani et

al., 2012)

Pandanus

amaryllifolius

Screw pine Daun pandan CHCl3 221.67 (Mardiyah &

Satoto, 2014)

Persicaria odorata Vietnamese

mint

Daun kesum - - -

Zingiber officinale Ginger Halia Oil 40.5 (Kalaivani et

al., 2012)

2.4.1 Curcuma longa

In Chinese and Indian traditional medicines, Curcuma longa is used for the

treatment of abdominal pains, sprains and swelling as well as stomach tonic and blood

purifier. The common name for C. longa is turmeric and it is widely used as spice in food

preparation. Table 2.3 shows the taxonomy of this plant.

Table 2.3 Taxonomy of Curcuma longa

Rank Scientific Name Common Name

Kingdom Plantae Plant

Subkingdom Tracheobionta Vascular plant

Superdivision Spermatophyta Seed plant

Division Magnoliophyta Flowering plant

Class Liliopsida Monocotyledons

Order Zingiberales -

Family Zingiberaceae Ginger family

Genus Curcuma L. -

Species Curcuma longa L. Turmeric

The yellow pigment curcumin (diferuloylmethane) shows in Figure 2.7a is the major

chemical constituent extracted from the rhizome of the plant. Essential oil extracted from

rhizome contains borneol (0.5%), sabiene (0.6%), α-phellanderene (1%), cineol (1%),

zingiberene (25%) and sesquiterpines (53%) (Jayaprakasha et al., 2005). Ar-turmerone