ashraf ahmed ali abdusalsalam

52 Pages·2017·1.44 MB·English

IN-VITRO AND MOLECULAR DOCKING
STUDIES OF SEVEN SELECTED
MYRTACEAE PLANTS FOR
NEURAMINIDASE ACTIVITY

by

ASHRAF AHMED ALI ABDUSALSALAM

Thesis submitted in fulfillment of the requirements
for the degree of
Doctor of Philosophy

September 2016
IN-VITRO AND MOLECULAR DOCKING
STUDIES OF SEVEN SELECTED
MYRTACEAE PLANTS FOR
NEURAMINIDASE ACTIVITY

ASHRAF AHMED ALI ABDUSALSALAM

UNIVERSITI SAINS MALAYSIA
2016
ACKNOWLEDGEMENT

First and foremost, I would like to give my warmest and greatest thanks to the
Almighty Allah, who gave me the courage and strength to completing this project.
Second I would like to extend my thanks and appreciation to my supervisor associate
Professor Dr. Nornisah Binti Mohamed for her full cooperation and encouragement
motivation and enormous knowledge for helping me to make this work come true.
Her understanding, kindness, expertise in guiding students, helped me greatly in
overcoming the difficulties encountered during the course of my study and in
completing the thesis. Her wonderful personality, will continue to influence and shape
my behavior throughout my life.
My gratitude also goes to my co-supervisors Professor Dr: Hasnah Binti Osman and
Dr. Ezatul Ezleen Kamarulzaman for their guidance, advice and support.
I would like to acknowledge the School of Pharmaceutical Sciences (Dean, academic
and technical staff ) for allowing me to utilize all the necessary facilities to enable to
complete this study.
I appreciate the help from the all group members of Pharmaceutical Design and
Simulation laboratory (PhDs) and to my fellow lab mates in School of Pharmaceutical
Science Universiti Sains Malaysia, especially Muhammad Fakhrul.

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Last but not least, I would like to express my appreciation to my dear parents, my
brother and sisters who always give me a prayer and blessing to finish my study.
Finally, my deep and special thanks are addressed to my lovely wife Dr. Gazala Ben-
Hander and Estabrak and Ahmad my daughter and son.
)نيملاعلا بر لله دمحلا(
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TABLE OF CONTENTS

Acknowledgment ………………………………………………………………….. ii
Table of Contents ………………………………………………………………… iv
List of Tables ……………………………………………………………………… xiii
List of Figures …………………………………………………………….............. xvi
List of Symbols and Abbreviations ……………………………………………… xx
Abstrak ……………………………………………………………………………. xxiii
Abstract …………………………………………………………………………… xxvi
CHAPTER ONE: INTRODUCTION …………………………………............... 1
1.1 The influenza A viruses ………………………………............................... 1
1.2 Influenza pandemics ………………………………………………………. 2
1.3 Swine influenza virus (SIV) ………………………………………………. 3
1.4 Life cycle of influenza A virus…………………………………………… 4
1.5 Sialic acid …………………………………................................................. 6
1.6 Neuraminidase NA …………………………………................................... 7
1.7 Neuraminidase inhibitors (NAIs)……………………………...................... 8
1.8 Neuraminidase active Site………………………………………………… 10
1.9 Natural products…………………………………………………………… 12
1.9.1 Family of Myrtaceae ……………………………………………..... 14
1.9.1(a) Psidium guajava…………………………………............ 15
1.9.1(a)(i) General introduct…………………………. 15
1.9.1(a)(ii) Traditional medicinal uses of Psidium 16
guajava……………………………............
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1.9.1(a)(iii) Chemical constituents of Psidium guajava 18
1.9.1(a)(iv) Essential oils of Psidium guajava………… 24
1.9.1(a)(v) Biological activities of Psidium guajava…. 29
1.9.1(b) Syzygium cumini………………………………………… 30
1.9.1(b)(i) General introduction……………………… 30
1.9.1(b)(ii) Traditional medicinal uses of Syzygium 31
cumini……………………………………

1.9.1(b)(iii) Chemical constituents of Syzygium cumini.. 31
1.9.1(b)(iv) Essential oils of Syzygium cumini………… 31
1.9.1(b)(v) Biological activities of Syzygium cumini…. 32
1.9.1(c) Syzygium grande…………………………………………. 33
1.9.1(c)(i) General introduction……………………… 33
1.9.1(c)(ii) Traditional medicinal uses of Syzygium 34
grande……………………………………..

1.9.1(c)(iii) Chemical constituents of Syzygium grande 34
1.9.1(c)(iv) Essential oils of Syzygium grande……… 34
1.9.1(c)(v) Biological activities of Syzygium grande… 34
1.9.1(d) Syzygium jambos………………………………………. 36
1.9.1(d)(i) General introduction……………………… 36
1.9.1(d)(ii) Traditional medicinal uses of Syzygium 36
jambos……………………………………..
1.9.1(d)(iii) Chemical constituents of Syzygium 36
jambos……………………………………..
1.9.1(d)(iv) Essential oils of Syzygium jambos………... 37
1.9.1(d)(v) Biological activities of Syzygium jambos… 37
1.9.1(e) Syzygium samarangense……………………………….. 38
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1.9.1(e)(i) General introduction……………………… 38
1.9.1(e)(ii) Traditional medicinal uses of Syzygium 39
samarangense……………………………
1.9.1(e)(iii) Chemical constituents of Syzygium 39
samarangense…………………………….

1.9.1(e)(iv) Essential oils of Syzygium samarangense… 39
1.9.1(e)(v) Biological activities of Syzygium 40
samarangense……………………………..

1.9.1(f) Syzygium malaccense cultivated jambu bol……………... 41
1.9.1(f)(i) General introduction……………………… 41
1.9.1(f)(ii) Traditional medicinal uses of Syzygium 42
malaccense cultivated jambu bol…………

1.9.1(f)(iii) Chemical constituents of Syzygium 42
malaccense cultivated jambu bol………….

1.9.1(f)(iv) Essential oils of Syzygium malaccense 42
cultivated jambu bol………………………
1.9.1(f)(v) Biological activities of Syzygium 43
malaccense cultivated jambu bol………….

1.9.1(g) Syzygium malaccense cultivated jambu susu……………. 44
1.9.1(g)(i) General introduction……………………… 44
1.9.1(g)(ii) Essential oil of Syzygium malaccense 45
cultivated jambu susu……………………...

1.9.1(g)(iii) Biological activities of Syzygium 45
malaccense cultivated jambu susu………...

1.10 Separation Techniques …………………………………………..…............ 46

1.10.1 Thin Layer Chromatography ( TLC)…….......................................... 46

1.10.2 Column Chromatography ( CC)………………………..................... 47

1.11 Essential oil ( CC)…………………………………………………………… 47

1.11.1 Methods for analyzing essential oil …………………………….. 48
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1.11.1(a) Gas chromatography………………….………………. 48
1.11.1(b) Gas chromatography-mass spectrometry (GC-MS)……. 48
1.12 Molecular docking………………………………………….……….……… 49
1.13 Problem statements…………………………………………………………. 52
1.14 Objectives of the research…………………………………………............... 53
CHAPTER TWO: ISOLATION, CHARACTERIZATION AND BIOASSAY 54
OF THE FRACTIONS AND ISOLATED COMPOUND 5

2.1 Introduction …………………………………................................................ 55
2.2 Materials and methods ………………………………................................... 57
2.2.1 Plant material …………………………………………………….. 58
2.2.2 Instrumentations...………………………………………………… 59
2.2.2(a) X-ray Crystallographic analysis…………………………. 59
2.2.2(b) Turner biosystems Modulus Microplate reader and 59
GLOMAX multi detection system (Promega) ………….
2.2.2(c) Nuclear magnetic resonance (NMR) spectroscopy……… 60
2.2.2(d) Liquid Chromatography- Mass-Spectroscopy. ………….. 60
2.2.2(e) High Performance Liquid Chromatography……………... 60
2.2.2(f) Infrared (IR) Spectroscopy………………………………. 60
2.2.2(g) Gas Chromatography-Mass Spectroscopy………............. 61
2.2.3 Extraction and fractionations of four fruits and seven leaves of 61
plants……………………………………………………………......

2.2.3(a) Extraction of fruits ………………………………………. 61
2.2.3(b) Fractionation of fruits ….……………………………….. 61
2.2.3(c) Extraction of leaves.……………………………………... 62
2.2.3(d) Fractionations of leaves………………..………………… 62
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2.2.3(e) Extraction of fruit for semi-prep HPLC…………............. 62
2.2.4 Isolation and purification of active compounds from fruit and leaves 63
of Psidium guajava…………………………………………..

2.2.4(a) Isolation of compounds from of Psidium guajava fuit 63
using column chromatography………………………….

2.2.4(b) Isolation of compounds from of Psidium guajava leaves 65
using column chromatography…..……………………….

2.2.5 Studies of neuraminidase Inhibitory Assay (MUNANA Assay)…… 66
2.2.5(a) Preparation of stock solution…………………...………. 66
2.2.5(a)(i) 325 mM MES …………………………...... 66
2.2.5(a)(ii) 100 mM CaCl ……………………………. 67
2
2.2.5(a)(iii) 1M Glycine ……………………………... 67

2.2.5(b) Preparation of working solution………………...……….. 67
2.2.5(b)(i) 32.5 mM MES Buffer …………………… 67
2.2.5(b)(ii) Stop solution……………………………… 67
2.2.5(c) Preparation of substrate, enzyme and inhibitors………… 68
2.2.5(c)(i) MUNANA substrate……………………… 68
2.2.5(c)(ii) Neuraminidase enzymes………………….. 68
2.2.5(c)(iii) Preparation of inhibitors (plant extracts, 68
fractions and compounds)…………………

2.2.5(d) Neuraminidase inhibition assay protocol……………… 69
2.3 Results and discussion………………………………………………........... 70
2.3.1 Yied of extracts and fractions………………………………………. 70
2.3.1(a) Fruit MeOH extracts…………………………………….. 70
2.3.1(b) Fractions of fruit MeOH extract…………………………. 71
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2.3.1(c) Leaves MeOH extracts…………………………………... 72
2.3.1(d) Fractions of leaves MeOH extract……………………….. 73
2.3.2 Process of the choose the active fraction for isolation ……………... 74
2.3.3 NA inhibition for methanolic crude extracts……………………….. 75
2.3.3(a) Fruit MeOH extract……………………………………… 75
2.3.3(b) Leave MeOH extract…………………………………….. 76
2.3.4 NA inhibition for the fractions of seven varieties………………….. 77
2.3.4(a) Bacterial NA inhibition assay ………….......................... 77
2.3.4(b) H1N1 NA % inhibition assay……………………………. 81
2.3.5 Charactrization of isolated compounds from ethyl acetate fraction of 91
Psidium guajava fruit…………………………………………...

2.3.5(a) Compound ETO1 ……………………………………….. 91
2.3.5(b) Compound ETO2 …………………………….................. 98
2.3.6 Characterization of isolated compounds from Psidium guajava fruit 101
using semi-prep HPLC………………………………………………

2.3.6(a) Compound PG1 …………………………………………. 102
2.3.6(b) Compound PG2 …………………………………………. 106
2.3.7 Characterization of isolated compounds from ethyl acetate fraction 111
of Psidium guajava leave……………………………………………

2.3.7(a) Compound BF2 …………………………………………. 111
2.3.7(b) Compound BF6 ……………………….………………… 117
2.3.8 NA inhibition assay for isolated compounds (bacteria and H1N1 123
virus)……………………………………………………………….

2.4 Conclusion…………………………………………………………………. 126

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