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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
ii
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.
iii
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.
)الحمد هلل رب العالمين(
iv
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
guajava……………………………............
16
v
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
cumini……………………………………
31
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
grande……………………………………..
34
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
jambos……………………………………..
36
1.9.1(d)(iii) Chemical constituents of Syzygium
jambos……………………………………..
36
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
vi
1.9.1(e)(i) General introduction……………………… 38
1.9.1(e)(ii) Traditional medicinal uses of Syzygium
samarangense……………………………
39
1.9.1(e)(iii) Chemical constituents of Syzygium
samarangense…………………………….
39
1.9.1(e)(iv) Essential oils of Syzygium samarangense… 39
1.9.1(e)(v) Biological activities of Syzygium
samarangense……………………………..
40
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
malaccense cultivated jambu bol…………
42
1.9.1(f)(iii) Chemical constituents of Syzygium
malaccense cultivated jambu bol………….
42
1.9.1(f)(iv) Essential oils of Syzygium malaccense
cultivated jambu bol………………………
42
1.9.1(f)(v) Biological activities of Syzygium
malaccense cultivated jambu bol………….
43
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
cultivated jambu susu……………………...
45
1.9.1(g)(iii) Biological activities of Syzygium
malaccense cultivated jambu susu………...
45
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
vii
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
OF THE FRACTIONS AND ISOLATED COMPOUND
54
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
GLOMAX multi detection system (Promega) ………….
59
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
plants……………………………………………………………......
61
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
viii
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
of Psidium guajava…………………………………………..
63
2.2.4(a) Isolation of compounds from of Psidium guajava fuit
using column chromatography………………………….
63
2.2.4(b) Isolation of compounds from of Psidium guajava leaves
using column chromatography…..……………………….
65
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 CaCl2……………………………. 67
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,
fractions and compounds)…………………
68
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
ix
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
Psidium guajava fruit…………………………………………...
91
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
using semi-prep HPLC………………………………………………
101
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
of Psidium guajava leave……………………………………………
111
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
virus)……………………………………………………………….
123
2.4 Conclusion………………………………………………………………….
126
x
CHAPTER THREE: ESSENTIAL OIL ANALYSIS AND
NEURAMINIDASE INHIBITION ACTIVITY
128
3.1 Introduction …………………………………................................................. 128
3.2 Materials and methods…………………………….......................................... 129
3.2.1 Collection of plant material.............................................................. 129
3.2.2 Extraction of essential oil………………………………................. 129
3.2.3 GC-MS and head-space condition………………………………… 130
3.2.4 Retention index calculation……………………………………….. 131
3.2.5 Identification of essential oil component………………................. 132
3.3 Results and discussion ..................................................................................... 132
3.3.1 Percentage yield of the essential oils................................................ 132
3.3.2 Essential oil analysis........................................................................ 133
3.3.2(a) Identification of chemical constituents of Psidium guajava
.....................................................................
133
3.3.2(b) Identification of chemical constituents of Syzygium
cumini................................................................................
137
3.3.2(c) Identification of chemical constituents of Syzygium
grande ...............................................................................
141
3.3.2(d) Identification of chemical constituents of Syzygium
malaccense cultivated jambo bol…………………...........
143
3.3.2(e) Identification of chemical constituents of Syzygium
jambos……………………………………………………
144
3.3.2(f) Identification of chemical constituents of Syzygium
samarangense……………………………………...
146
3.3.2(g) Identification of chemical constituents of Syzygium
malaccense cultivated jambo susu……………………….
149
3.3.3 Evaluation of neuraminidase inhibition (MUNANA Assay)……......
152
3.3.3(a) Inhibition against Clostridium perfringens Neuraminidase
Enzyme………………..............................
152
xi
3.4 Conclusions ..................................................................................................... 156
CHAPTER FOUR: IN SILICO MOLECULAR DOCKING ANALYSIS
OFCOMPOUNDS FROM ESSENTIAL OIL OF
Syzygium malaccense cultivated jambu bol, Syzygium
jambos, Syzygium malaccense cultivated jambu susu AND
ISOLATED COMPOUNDS FROM Psidium guajava AS
POSSIBLE NEURAMINIDASE H1N1 INHIBITOR….
157
4.1 Introduction ..................................................................................................... 157
4.2 Materials and Methods..................................................................................... 158
4.2.1 Material........................................................................................... 158
4.2.2 Preparation of ligands ..................................................................... 158
4.2.3 Protein preparation........................................................................... 159
4.2.4 Determination of active site............................................................. 159
4.2.5 Molecular Docking Simulation………………………………….. 159
4.3 Results and discussion ..................................................................................... 161
4.3.1 Control docking of the conservative binding pocket using
oseltamivir…………………………………………………………..
161
4.3.2 Control docking of the adjacent binding pocket (150-cavity) reported
compound………………………………………………….
162
4.3.3 Oseltamivir…………………………………………………………. 162
4.3.4 Compound NSC164640…………………………………………….. 163
4.3.5 Molecular docking of the compounds isolated from Psidium
guajava………………………………………………………………
165
4.3.5(a) Molecular docking of the isolated coFmpounds interact
with the conservative active site………………………...
166
4.3.5(b) Molecular docking of the isolated compounds interact
with adjacent active site (150-cavity) …………………..
171
4.3.6 Molecular docking of compounds identified from active essential
oils…………………………………………………………………...
174
xii
4.3.7 Correlation between the bioassay and molecular docking of isolated
and identified compounds…………………………………………...
175
4.4 Conclusion ....................................................................................................... 185
CHAPTER FIVE: CONCLUDING REMARKS AND
RECOMMENDATIONS FOR FUTURE STUDIES
186
5.1 Concluding remarks …………………………………………..................... 188
REFERENCES …………………………………………………………………... 190
APPENDICES ………………………………………………………….................
213
LIST OF PUBLICATIONS AND PRESENTATIONS AT CONFERENCE
xiii
LIST OF TABLES
Page
Table 1.1 Ethnomedical uses of Psidium guajava.
17
Table 2.1 Solvent, chemicals and reagent used.
57
Table 2.2 Voucher number of the seven selected plants.
59
Table 2.3 Yields of MeOH extracts of fruits.
71
Table 2.4 List of fraction from fruit, solvent used and amount
obtained.
72
Table 2.5 List of leaves MeOH extract.
73
Table 2.6 Yield of fractions from MeOH extracts of leaves.
74
Table 2.7 IC50 of MeOH extract of one fruit and seven leaves.
87
Table 2.8 IC50 of fractions of one fruit and seven leaves.
87
Table 2.9 Percentage of inhibition of MeOH extract of one fruit and
seven leaves at 250 μg mL-1.
88
Table 2.10 Percentage of inhibition for fractions of one fruit and seven
leaves.
90
Table 2.11 Comparison of the 13C-NMR spectral data (CD3OD 500 and
125MHz) of compound ETO1 with some available data in
the literature.
95
Table 2.12 Crystal data and structure refinement details for ETO1
96
Table 2.13 Geometry parameter, bond lengths (Å) and angle (o) of
ETO1
97
Table 2.14 Crystallographic date of compound ETO2
99
Table 2.15 Geometry parameter, bond lengths (Ǻ) and angles (o) of
ETO2
100
Table 2.16 Comparison of the 13C-NMR spectral data (CD3OD 500 and
125 MHz) of compound PG1 with some available data in
the literature
106
xiv
Table 2.17 Comparison of the 13C-NMR spectral data (C5D5N 500 and
125 MHz) of compound PG2 with some available data in
the literature
111
Table 2.18 Comparison of the 13C-NMR spectral data (C5D5N 500 and
125MHz) of compound BF2with some available data in the
literature
113
Table 2.19 Comparison of the 13C-NMR spectral data (C5D5N 500 and
125 MHz) of compound BF6 with some available data in
the literature
122
Table 2.20 The inhibition and IC50 for the isolated compounds.
124
Table 3.1 Essential oil yield of seven species.
133
Table 3.2 Composition of the volatile oil from fruits of Psidium
guajava L. (components listed in order of elution on the HP-
5 column).
134
Table 3.3 Comparsion the obtained terenoild with previously reported
of psidium guajava.
136
Table 3.4 Chemical composition of volatile oil from leaves of
Syzygium cumini. (Components listed in order of elution on
the HP-5 column).
138
Table 3.5 .Comparsion the obtained terenoild with previously reported
of Syzygium cumini.
140
Table 3.6 Chemical composition of volatile oil from leaves of
Syzygium grande. (Components listed in order of elution on
the HP-5 column).
141
Table 3.7 Chemical composition of volatile oil from leaves of
Syzygium malaccense cultivated - Jambu bol (components
listed in order of elution on the HP-5 column).
143
Table 3.8 Chemical composition of volatile oil from leaves of
Syzygium jambos (components listed in order of elution on
the HP-5 column).
145
Table 3.9 Chemical composition of volatile oil from leaves of
Syzygium samarangens (components listed in order of
elution on the HP-5 column).
147
xv
Table 3.10 Chemical composition of volatile oil from leaves of
Syzygium malacanse cultivated jambo susu (components
listed in order of elution on the HP-5 column).
150
Table 3.11 IC50 valuse for crude oil extract against NA from H1N1.
155
Table 4.1 Summary of hydrogen bonds between oseltamivir and
important interacting amino acid residue of 3TI6.
163
Table 4.2 Summary of the interaction between compound
NSC164640 and the amino acid residue at the 150-cavity in
the 3TI6.
165
Table 4.3 The binding energy and types of interaction of the isolated
compounds.
170
Table 4.4 The binding energy and types of interaction of the isolated
compounds.
172
Table 4.5 Binding energies and interaction of ten compounds from
essential oil with the protein. 184
xvi
LIST OF FIGURES
Page
Figure 1.1 Schematic diagram of influenza A viruses (Horimoto and
Kawaoka, 2005).
2
Figure 1.2 Schematic diagram of the influenza viral life cycle (Das et al.,
2010).
4
Figure 1.3 Structure of N-acetyl and N-glycolylneuraminic acids. These
SA differ at position 5 of the pyranose ring. N-
Acetylneuraminic acid is the precursor of N-glycolylneuraminic
acid; enzymatic hydroxylation of the former results in the latter.
7
Figure 1.4 Structures of (a) peramivir, (b) laninamivir, (c) Oseltamivir, (d)
Zanamivir, (e) DANA.
10
Figure 1.5 The neuraminidase active site The conserved NA residues
(R118, D151, R152, R224, E227, E276, R292, and R371).
12
Figure 1.6 Psidium guajava plants (a) flower, (b) leaves (c) fruit and (d)
tree.
16
Figure 1.7 Syzygium cumini plants (a) flower, (b) fruit, (c) leaves and (d)
tree.
33
Figure 1.8 Syzygium grande plants (a) flower, (b) fruit, (c) leaves and (d)
tree.
35
Figure 1.9 Syzygium jambos plants (a) flower, (b) fruit, (c) leaves and (d)
tree.
38
Figure 1.10 Syzygium samarangense plants (a) flower, (b) fruit, (c) leaves
and (d) tree.
41
Figure 1.11 Syzygium malaccense cultivated jamb bol (a) flower, (b) fruit,
(c) leaves and (d) tree.
44
Figure 1.12 Syzygium malaccense cultivated jamb susu plants (a) flower, (b)
fruit, (c) leaves and (d) tree.
46
Figure 2.1 Flow chart of research activities.
56
Figure 2.2 Isolation scheme of ETO1 and ETO2 compounds.
64
Figure 2.3 Isolation scheme of BF2 and BF6 compounds. 66
xvii
Figure 2.4 Illistration of 96-well Microplate for NA Assay. Well with red
colors represent serial dilution of NA, while well with green
green color represent negative control (without NA) and yellow
colors represent blank control.
70
Figure 2.5 Bacterial NA % inhibition activity of fruits MeOH extract.
75
Figure 2.6 Bacterial NA % inhibition activity of leave MeOH extract.
76
Figure 2.7 Bacterial NA % inhibition activity of P. guajava fruit MeOH
extract and fractions.
78
Figure 2.8 Bacterial NA % inhibition activity of (a) P. guajava (b) S.
cumini (c) S. grande (d) S. jambos (e) S. samarangense (f)
S. malaccense cultivated jambu bol (g) S. malaccense
cultivated jambu susu leaves MeOH extracts and fractions.
78
Figure 2.9 H1N1 NA % inhibition activity of P. guajava fruit MeOH
extracts.
82
Figure 2.10 H1N1 NA % inhibition activity of (a) P. guajava (b) S.
cumini (c) S. grande (d) S. jambos (e) S. samarangense (f) S.
malaccense cultivated jambu bol (g) S. malaccense cultivated
jambu susu leaves MeOH extracts and fractions.
83
Figure 2.11 13C-NMR spectrum (CD3OD 125 MHz) of compound ETO1.
93
Figure 2.12 13C-NMR spectrum (CD3OD125 MHz) of compound ETO1.
94
Figure 2.13 X-ray crystal structure of Kojic acid.
98
Figure 2.14 X-ray crystal structure of 1,3-benzothiazole-oxalic acid (2/1).
101
Figure 2.15 The HPLC chromatogram separation of MeOH extract from
Psidium guajava.
102
Figure 2.16 1H-NMR spectrum (CD3OD 500 MHz) of compound PG1.
104
Figure 2.17 13C-NMR spectrum (CD3OD 125 MHz) of compound PG1. 105
Figure 2.18 1H-NMR spectrum (C5D5N 500 MHz) of compound PG2.
108
Figure 2.19 13C-NMR spectrum (C5D5N 125 MHz) of compound PG2.
109
Figure 2.20 1H-NMR spectrum (C5D5N 500 MHz) of compound BF2.
114
xviii
Figure 2.21 13C-NMR spectrum (C5D5N 125 MHz) of compound BF2.
115
Figure 2.22 1H-NMR spectrum (C5D5N 500 MHz) of compound BF6.
119
Figure 2.23 13C-NMR spectrum (C5D5N 125 MHz) of compound BF6.
120
Figure 2.24 Bacterial NA % inhibition activity of isolated compounds from
P. guajava.
153
Figure 2.25 Virus H1N1 % inhibition activity of isolated compounds from
P. guajava.
124
Figure 3.1 Hydrodistillation apparatus used for extraction of essential oil.
130
Figure 3.2 Percentage of inhibition between Neuraminidase against
essential oil extracts.
150
Figure 3.3 Percentage of inhibition between Neuraminidase from H1N1
against essential oil extracts.
154
Figure 3.4 Inhibition activity compounds identified from essential oil
against of NA.
153
Figure 4.1 The grid box of covering the active site of 3TI6.
160
Figure 4.2 Superimposition between the receptor-ligand complex of
docked conformation A interaction (yellow) and the crystal
structure (Red) of NA, H1N1 B.
161
Figure 4.3 the docked conformation of oseltamivir and 3TI6 suggested by
molecular docking. Green dashed lines represented H-bonds
between oseltamivir and important amino acids residues of
3TI6.
163
Figure 4.4a 3D representation of the compound NSC164640 attached to the
150-cavity in the 3TI6.
164
Figure 4.4b 2D representation of the compound NSC164640 attached to the
150-cavity in the 3TI6.
165
Figure 4.5 Superimposition of the oseltamivir red with the three isolated
compounds in the active site with DANA (blue) ursolic acid
(yellow) ursolic aldehyde (light blue) 1,3-benzothiazole-oxalic
acid (purple).
168
xix
Figure 4.6 2D representation of the three isolated compounds and dana in
the 3TI6 active site (a) DANA, (b) 1,3-benzothiazole-oxalic
acid, (c) ursolic acid and (d) ursolic aldehyde.
168
Figure 4.7 Superimposition of the three isolated compounds that attached
to the 150-cavity kojic acid (yellow) gallic acid (pink) quercetin
(purple) and the OTV (red) attached to the conservative active
site.
171
Figure 4.8 2D representation of the three isolated compounds and in the
3TI6 active site (a) quercetin, (b) gallic acid and (c) kojic acid.
173
Figure 4.9 2D representation of the ten identified compounds and in the
3TI6 active site (a) γ-terpinene, (b) humulene oxide (c) borneol
(d) carvacrol (e) furfural (f) Isopulegol (g) methyl salicylate (h)
Safranal (i) tau-cadinol and (j) Curcumene.
178
xx
LIST OF SYMBOLS AND ABBREVIATIONS
µg Microgram
µM Micromolar
13C NMR Carbon Neuclear magnetic Resonance
1D NMR One Dimentional Neuclear magnetic Resonance
1H NMR Proton Nuclear magnetic Resonance
1H-13C HMBC Heteronuclear Multiple-Bond Correlation
1H-13C HSQC Heteronuclear Single-Quantum Correlation
1H-1H COSY Homonuclear Correlation Spectroscopy
2D NMR Two Dimentional Neuclear magnetic Resonance
Ala Alanine
Arg Arginine
Asn Asparagine
Asp Aspartic acid
BuoH Butanol
CADD Computer aided drug design
CC Column chromatography
CHCl3 Chloroform
d Doublet
dd Doublets of doublet
ddd Doublets of doublet of doublet
DEPT NMR Distortionless Enhancement by Polarization Transfer
EtoAc Ethyl acetate
FT-IR Fourier Transform Infrared
Glu Glutamic acid
Gly Glycine
HA Hemagglutinin.
HS-GC/MS HeadSpace Gas Chromatography Mass Spectrometry
Hz Hertz
xxi
IC50 Half-maximal inhibitory concentration
Lys Lysine
m Multiplet
m/z Mass/charge
M1 Matrex
M2 Ion chanel
MES 2-(N-Morpholino)EthaneSulfonic Acid
Methanol-d5 Deuterated Methanol
mg Milligram
MHz Mega Hertz
MS Mass spectrscopy
MUNANA 2′-(4-methylumbelliferyl)-α-d- N-acetylneuraminic acid
NA Neuraminidase
NAI Neuraminidase inhibitor
Neu5AC N-acetylneuraminic acid
NP nucleoprotein
PA Viral RNA polymerase
PB1 Viral RNA polymerase
PB2 Viral RNA polymerase
PDB Protein Data Bank
PTLC Preparative Thin Layer Chromatography
Pyridine- d5 Deuterated Pyridine
s Singlet
SEMI-PREP
HPLC
Semi Preparative High Performance Liquid Chromatography
Ser Serine
SPE Solid phase microextraction
t Triplet
TLC Thin Layer Chromatography
Tyr Tyrosine
v/v Volume/volume
xxii
w/v Weight/volume
δ Chemical shift
xxiii
KAJIAN IN- VITRO DAN PENDOKAN MOLEKUL TUJUH TUMBUHAN
MYRTACEAE TERPILIH UNTUK AKTIVITI NEURAMINIDASE
ABSTRAK
Virus influenza A menyebabkan masalah kesihatan yang teruk kepada
manusia dan beberapa spesies haiwan yang boleh membawa kepada morbiditi dan
kematian dalam kalangan pesakit yang dijangkiti. Oseltamivir (Tamiflu) dan
zanamavir (Relenza) adalah dua drug perencat NA terkini yang telah diluluskan oleh
Pentadbiran Makanan dan Drug (FDA, USA). Walaubagaimana pun, oleh kerana
kemunculan rintangan virus terhadap kedua-dua drug ini, terdapat keperluan
mendesak untuk menemui suatu perencat neuraminidase alternatif. Kajian ini telah
dijalankan untuk menyiasat aktiviti perencatan neuraminidase tujuh tumbuhan terpilih
daripada keluarga Myrtaceae: P. guajava, S. cumini, S. grande, S. jambos, S.
malaccense jambu bol, S. samaranges, dan S. malaccense jambu susu. Tumbuh-
tumbuhan telah diekstrak menggunakan metanol dan difraksi menggunakan n-
heksana, kloroform, etil asetat dan butanol. Minyak pati daripada daun tujuh terpilih
ini telah diasingkan menggunakan hidropenyulingan dan dianalisis dengan
kromatografi-spektrometri jisim. Ekstrak mentah, fraksi, sebatian dan minyak pati
telah diuji terhadap assai enzim neuraminidase Clostridium perfringens dan H1N1.
Saringan maya menggunakan Autodock Vina terhadap tapak aktif protein
neuraminidase H1N1 (kod PDB: 3TI6) telah digunakan untuk menyaring sebatian-
sebatian yang dikenal pasti daripada minyak pati, dan kemudian sebatian-sebatian
yang dikenal pasti diubahsuai dengan menambahkan kumpulan fungsi yang ada pada
oseltamivir iaitu drug piawai. Pemencilan telah dilakukan dari fraksi etil asetat (daun
xxiv
dan buah-buahan) daripada P. guajava untuk menghasilkan satu sebatian baru, 1,3-
benzotiazol-asid oksalik dan lima sebatian yang telah dikenali, asid kojik, asid galik,
kuersetin, asid ursolik dan ursolik aldehid. Kebanyakan daripada ekstrak
mempamerkan perencatan yang bagus, buah P. guajava 89% dan tujuh daun yang
menpunyai kadar perencatan antara 70 hingga 89% bagi C. perfringens. Bagi fraksi
yang lain, perencatan antara 12.2 hingga 97% terhadap NA daripada C. perfringens
sementara 12.6 hingga 97.9% terhadap NA daripada H1N1. Tiga pati minyak
menunjukkan perencatan tinggi sehingga 57.8, 88.7 dan 91.2% dalam kalangan tujuh
pati minyak. IC50 bagi dua sebatian terpencil, kuersetin adalah 229 µM, asid galik
adalah 187 µM dan DANA 4.3 µM untuk NA bakteria, dan kuersetin 127 µM, asid
galik 258 µM dan DANA 3.9 µM untuk NA virus H1N1. Pelbagai sebatian utama
telah dikenalpasti daripada setiap varieti adalah 20.7% α-pinena daripada P. guajava,
25.1% Z-β-osimena daripada S. cumini, 33.1% δ-terpinena daripada S. samarangense,
(Z)-3-heksanol 23.5% daripada S. malaccense jambu susu dan 62.5%, 20.5% dan
23.0% β-kariofilena masing-masing daripada S. grande, S.malaccense jambu bol dan
S. Jambos. Pendokan molekul in silico sebatian terpencil daripada P. guajava dan
sebatian yang dikenal pasti daripada minyak pati aktif telah dikaitkan dengan
keputusan eksperimen. Semua sebatian mempamerkan interaksi dengan protein
sasaran pada dua tapak mengikat berbeza, iaitu tapak aktif konservatif dan poket
bersebelahan (kaviti 150). Hubungan antara in silico dan keputusan eksperimen
menunjukkan bahawa aktiviti yang diperolehi bukan disebabkan oleh sebatian tunggal
tetapi dari kesan sinergi, yang mana semua sebatian menyumbang untuk merencat
neuraminidase. Kesimpulannya ekstrak mentah, fraksi, tiga daripada tujuh minyak
xxv
pati tumbuh-tumbuhan terpilih menunjukkan perencatan neuraminidase yang baik dan
boleh menjadi sumber perencat neuraminidase.
xxvi
IN-VITRO AND MOLECULAR DOCKING STUDIES OF SEVEN
SELECTED MYRTACEAE PLANTS FOR NEURAMINIDASE ACTIVITY
ABSTRACT
Influenza A virus cause severe health problems to humans and several animal
species which could result in morbidity and mortality in infected patients. Oseltamivir
(Tamiflu) and zanamivir (Relenza) are two current drug used NA inhibitors that have
been approved by the Food and Drug Administration (FAD, USA). However, due to
the emergence of virus resistance to these drugs, there is an urgent need to discover
an alternative neuraminidase inhibitors. The present study was undertaken to
investigate the neuraminidase inhibition activity of seven selected plants that belong
to the family of Myrtaceae; Psidium guajava, Syzygium cumini, Syzygium grande,
Syzygium jambos, Syzygium malaccense cultivated jambu bol, Syzygium samaranges,
and Syzygium malaccense cultivated jambu susu. The plants were extracted using
methanol and fractionated using n-hexane, chloroform, ethyl acetate and butanol.
Essential oils from leaves of seven selected plants were isolated by hydrodistillation
and analyzed by head-space chromatography–mass spectrometry. The crude extract,
fractions, compounds and essential oil were subjected to neuraminidase inhibition
assay against Clostridium perfringens and H1N1 neuraminidase enzyme. Molecular
docking using Autodocktools 4.2 were applied against the active site of neuraminidase
H1N1 protein (PDB code: 3TI6) for the isolated and identified compounds from
essential oil. The isolation was done from the ethyl acetate fraction of (leave and fruit)
of P. guajava to yield one new compound 1,3-benzothiazole-oxalic acid and five
xxvii
known compounds kojic acid, gallic acid, quercetin, ursolic acid and ursolic aldehyde.
Good inhibition exhibited by most of the extracts, P. guajava fruit 89% and seven
leaves ranging from 70 to 89% against C. perfringens. For the other fractions the
inhibition ranged from 12.2 to 97% against NA from C. perfringens while 12.6-97.9%
against NA from H1N1. Three essential oils showed high inhibition up to 57.8, 88.7
and 91.2% among the seven oils. The IC50 of two isolated compounds quercetin was
229 µM, gallic acid was 187 µM and DANA 4.3 µM for the bacterial NA, and
quercetin was 127 µM, gallic acid was 258 µM and DANA 3.9 µM for the NA H1N1
virus. Different major compounds identified in essential oil from each variety were
20.7% α-pinene in P. guajava, 25.1% Z-β-ocimene in S. cumini, 33.1% δ-terpinene in
S. samarangense, (Z)-3-hexanol (23.5%) in S. malaccense cultivated jambu susu and
62.5%, 20.5% and 23.0% β-caryophyllene in S. grande, S. malaccense cultivated
jambu bol and S. Jambos, respectively. The in silico molecular docking of isolated
compounds from P. guajava and identified compounds from active essential oil were
correlated with the experimental results. All the compounds exhibited interactions
with the target protein at two different binding sites, namely, conservative active site
and adjacent pocket (150-cavity). The correlation between the in silico and
experimental results revealed that the obtained activity was not due to a single
compound but from a synergistic effect, where all the compounds contributed to
inhibit neuraminidase. In conclusion crude extracts, fractions, three out of seven
essential oils of selected plants showed good neuraminidase inhibition and could be
source of neuraminidase inhibitors.
1
CHAPTER ONE
INTRODUCTION
1.1 The influenza A viruses
The human-human transmission rate of the influenza virus A (H1N1) virus
and their effects on human health attracted global attention in 2009. Influenza A
viruses are isolated from numerous host species, such as marine mammals, horses,
pigs, domestic birds, humans, and dogs (Webster et al., 1992). Influenza A viruses
belong to the Orthomyxoviridae family and cause infrequent epidemics that usually
result in increased death rates compared with seasonal influenza epidemics. These
viruses have an RNA genome consisting of eight gene segments (Knipe et al., 2007;
Wright et al., 2007) with two neuraminidase (NA) and a hemagglutinin (HA) as
surface glycoproteins in the envelope (Lamb et al., 2001; Wright et al., 2001).
Influenza A viruses are sorted into antigenic HA and NA subtypes. A total of 16
antigenic HA (H1–H16) and 9 NA (N1–N9) subtypes have been confirmed. H1N1,
also called swine flu, consists of an H1 subtype HA and an N1 subtype NA (Schwahn
et al., 2010). H3N2, H2N2, and H1N1 are considered the most important influenza A
subtypes that infect humans during seasonal epidemics (Taubenberger et al., 2005).
Influenza A viruses contain a genome consisting of eight segments involving a
negative-sense, single-stranded RNA that encodes one or two proteins Figure 1.1.
2
Fig. 1.1 Schematic diagram of influenza A viruses (Horimoto and Kawaoka, 2005)
1.2 Influenza pandemics
Influenza viruses cause global pandemics as well as repeated epidemics. In the
20th century, three major worldwide pandemics of influenza occurred at different
interval in 1918, 1957, and 1968 (Taubenberger and Morens, 2006; Kilbourne, 2006).
Spanish flu in 1918 was the most devastating pandemic that caused the death of 50
million people (Johnson and Mueller, 2002). The subsequent epidemics in 1957
occurred in Asia and Hong Kong flu pandemic of 1968; these two epidemics were
milder, together caused a total of approximately two million deaths (Kilbourne, 2006).
An important number of infected people would be detected resulting from the recently
emerged H1N1 influenza virus and spread in many worldwide locations. In the
3
southwestern United States and Mexico 2009 new influenza A (H1N1) has been
reported (Pan et al., 2010). During the epidemic 2009 H1N1 the new strain of swine
flu (hosted by pigs) attracted the attentions of most of the organizations, because this
epidemic gain the ability of transfer from human to human.
1.3 Swine influenza virus (SIV)
A global influenza preparedness has been developed by World Health
Organization (WHO), this plan used to define the pandemic stages which can be used
as national measures before and during the pandemic. Concerning the swine influenza
(H1N1) pandemic, the WHO raised alert levels according to the different H1N1
phases, namely phases 4, 5, and 6, on 27 April 2009, 29 April 2009, and 11 June 2009
respectively, these grouped into six-phases, phase 1-3 correlate with preparedness
these including the capacity development and the response planning activities, while
or phases 4-6 gave clear signal the need for serious response and more mitigation
efforts.. Afterward, the WHO declared a full-blown influenza pandemic. Laboratories
showed genuine cases of H1N1 reported from more than 214 countries in June 2010,
with the casualties reaching 18,156 (WHO, 2009).
The pandemic H1N1 virus that spread in 2009 comprised various re-assorted
viral genes from different sources. The virus was introduced to populations in 1998
from the avian virus with a North American lineage; this H1N1 virus consisted of two
polymerase genes, eight segmented genomic RNAs, PA, and PB2 (Krause µµ., 2010).
Two different surface glycoproteins, NA and HA (Lamb and Krug, 2001; Wright and
Webster, 2001), have important protective immunity within a host (Garten et al.,
4
2009). This unique genetic combination may contribute to the improved fitness of the
H1N1 in humans and its human-to-human transmissibility, No molecular functions
have been shown before to confer the increased human-to-human transmissibility of
the 2009 H1N1.
1.4 Life cycle of influenza A virus
The HA protein is critical for binding cellular receptors and fusing viral and
endosomal membranes (Figure 1.2) (Das et al., 2010). The life cycle of the influenza
A virus is divided into different stages: (1) viral entry to the host cell, (2) entry of viral
ribonucleoprotein complex (vRNP) into the nucleus, (3) transcription and replication
of the viral genome, (4) vRNP exportation from the nucleus (5) assembly, and then
(6) budding at the plasma membrane of the host cell (Samji, 2009).
Fig. 1.2 Schematic diagram of the influenza viral life cycle (Das et al., 2010).
5
Influenza is spread via tiny droplets expelled through coughing or sneezing,
thus can be easily inhaled thereby transmitting the disease. Once the virus has entered
the host within the respiratory tract, particularly in the nose, mouth and throat, it will
fuse to the plasma membrane of permissive cells lining in the respiratory tract. The
projecting spikes on the viral lipid membrane known as hemagglutinin (HA) bind to
sialic acid (SA) of the host cell's membrane. Two major linkages between SA and the
carbohydrates which are important for the HA specificity is the α (2, 3) and α (2, 6)
found in different species (Samji, 2009). Humans influenza recognize α (2, 6)
linkages, avian influenza; α (2, 3) linkages while swine influenza recognize both
linkages (Skehel and Wiley, 2000). This explains the importance of swine being a
good mixing vessel for avian and human influenza viruses, hence producing
dangerous pathogenic viruses.
After the attachment, receptor mediated endocytosis occurs and the virus is
engulfed by the cell in an endosome at low pH of around 5 to 6. The acidic
environment of the endosome induces conformational change in HA, bringing both
the viral and endosomal membranes into contact with each other (Skehel and Wiley,
2000; Huang et al., 2003) and also opens up the M2 ion channel that acts as a proton
selective ion channel, thus acidifies the viral core (Pinto and Lamb, 2006; Holsinger
et al., 1994).
The viral constituents (vRNP) will then be released into the cytoplasm and
migrate through the cytoplasm to the cell's nucleus. Viral transcription and replication
occurs in the nucleus. The influenza viral genome is made up of negative sense strands
of RNA and converted into a positive sense RNA for the genome to be transcribed.
6
The virus replicates its genome and forms new gene particles (Samji, 2009). New
genes and proteins then migrate from the nucleus through the cytoplasm and to the
cell membrane. The newly fanned viral begin to bud through the host cell's plasma
membrane to form viral particles and eventually released into the extracellular
environment to invade new cells (Scheiffele et al., 1999). The new viral particles are
coated with sialic (neuraminic) acid (SA), the same substance that bound them to the
exterior of the cell they had invaded (Samji, 2009; Scheiffele et al., 1999). The
nascent viruses would stick to the membrane of the cell in which they had been
formed, thus, unable to separate and travel to other cells in the respiratory tract.
The HA spike of one virus could stick to the SA coating of another, thereby
causing viral aggregation and impeding the capability of the viruses to spread to, and
invade, new cells. However, NA molecules, which are also part of the wrapping on
the newly formed viral particles, cleave viral SA, thereby freeing the viruses from the
host cell and from each other (Samji, 2009; Von Itzstein and Thomson, 2009). Drug
that blocked the action of viral NA would thus prevent the escape of newly formed
viruses from the host cells and thus preclude viral spread to other cells. This is the
main reason as to why NA is taken as a drug target for developing agents in influenza
virus drug discovery.
1.5 Sialic acid
Sialic acid (SA) is a common term for nine-carbon acidic amino sugars (5-
amino-3,5-dideoxy-D-glycero-D-galacto-nonulosonic acid). Two new compounds,
namely, N-glycolylneuraminic acid and N-acetylneuraminic acid (Neu5Ac), are
obtained by substituting the N-glycolyl group and N-acetyl with the amino group in
7
SA (Figure 1.3). The hydroxyl group can be substituted with phosphate, methyl,
sulfate, or lactoyl residues (Suzuki et al., 2000). Human influenza viruses preferably
bind to the sialic acid-α2,6-galactose SAα2,6Gal linkage, whereas equine and avian
influenza viruses preferably bind to the (SAa2,3Gal) linkage (Connor et al., 1994).
Fig. 1.3 Structure of (a) N-acetyl and (b) N-glycolylneuraminic acids. These SA differ
at position 5 of the pyranose ring. N-Acetylneuraminic acid is the precursor of N-
glycolylneuraminic acid; enzymatic hydroxylation of the former results in the latter.
1.6 Neuraminidase NA
Influenza A viruses carry two surface glycoproteins, namely, HA and NA.
Both proteins recognize the same host cell molecule, that is, SA and Neu5Ac, which
are found as the terminal carbohydrate unit of the upper respiratory tract and lung-
associated glycoconjugates (Von Itzstein and Thomson, 2009). NA encodes six RNA
segments and is known as the second major surface antigen of virions. NA acts as a
pair of biological scissors clipping Neu5Ac residues, thus facilitating movement and
viral release (Von Itzstein and Thomson, 2009). The NA morphology can be divided
into three domains: the cytoplasmic domain of six amino acids, the N-terminal
hydrophobic domain, and the actual stalk domain (Colman, 1994). The cytoplasmic
domain contains six amino acids completely conserved in the NA of influenza A. This
8
condition is essential for NA inclusion into virions and for the assembly of the
influenza A virus.
Scientists have found nine different NAs (N9). However, only N1 and N2 are
important in human infection, whereas the other NAs have only been found in seven
animals. Neuraminidase is the major surface protein of the influenza virus that
extracellularly acts by cleaving the terminal neuraminic acid from cellular receptors
recognized by the HA. According to phylogenetic studies, the neuraminidase of the
influenza virus is divided into two groups. Group one consist of N1, N4, N5, and N8,
whereas the other group consist of N2, N3, N6, N7, and N9 (Gong et al., 2007).
The neuraminidase plays an extremely important role in the pathogenesis of
viral infection. That is, it facilitates the release of newly formed virions from the host
cell surface to the neighbor cells that facilitated the spread of the virus. NA acts as a
receptor-destroying enzyme in the influenza virus by removing the terminal SA of a
viral receptor (De Clercq, 2006).
1.7 Neuraminidase inhibitors (NAIs)
Inhibitors block the active site of the neuraminidase and then prevent the
release and spread of new virions. After infection, the patient should take an antiviral
drug during the early stage if possible to achieve an effective treatment. Antiviral
drugs are important role in the management of infection caused by the seasonal
influenza virus. Adamantanes (M2 ion channel blockers) and NA inhibitors (NAIs)
are two classes of antiviral drugs licensed to prevent and treat infections caused by
the influenza A virus (Moscona, 2008). The first inhibitor analog of SA is 2,3-
9
didehydro-2-deoxy-N-acetylneuraminic acid or Neu5Ac2en (DANA) Figure 1.4
(Meindl et al., 1974).
The only two drugs approved by the Food and Drug Administration,
oseltamivir and zanamivir currently effective for the treatment of influenza infections,
the seasonal influenza virus, and the pandemic H1N1 virus. Recently, peramivir has
been developed as a new neuraminidase inhibitor administered intravenously (Babu
et al., 2000) Figure 1.4. It is currently approved for emergency use in Japan and Korea.
In other countries, this drug is still undergoing clinical trials. Peramivir is based on
DANA, but it contains a cyclopentane ring and has the features of both oseltamivir
and zanamivir Figure 1.4. Yamashita et al. (2010) found a new potent NA inhibitor,
laninamivir considered as a second-generation acts as a long-acting NA inhibito.
Administered as single inhaled dose, laninamivir is found to be effective against the
wild-type strain of the influenza A and B viruses and the oseltamivir-resistant H274Y
mutations of seasonal H1N1 viruses, namely, H5N1 and H1N1 (Sugaya and Ohashi,
2010; Nguyen et al., 2010; Yamashita, 2010).
10
Fig. 1.4 Structures of (a) peramivir, (b) laninamivir, (c) oseltamivir, (d) zanamivir, (e)
DANA.
1.8 Neuraminidase Active Site
The common feature among all influenza NAs is the active site Figure 1.5;
with well formed large and rigid pockets form, can be considered as a common feature
11
among influenza NAs. SA and neuraminidase with conserved amino acids interact in
a similar fashion with both substrate and inhibitor molecules (Xu et al., 2008; Kim et
al., 1999). Therefore, antiviral drugs affecting the spread of viral replication have been
developed.
The presence of four hydrophobic residues and ten Arg, Asp, and Glu residues
make the neuraminidase active site highly polar (Von Itzstein and Thomson, 2009;
Zhang and Xu, 2006). The active site in NA is divided into five regions from S1 to
S5. S1 includes Arg 118, Arg 292, and Arg 371, which all bind to the carboxylate of
substrate. S2 consists of Glu 119 and Glu 227, which are acidic residues responsible
for forming the negative-charge region of the active site (Zhang and Xu, 2006). The
hydrophobic region present at S3 forms at the side chains of Ile 222 and Trp 178,
which are adjacent to the polar region of the Arg 152 side chain, and interacts with
water molecules (Zhang and Xu, 2006; Russell et al., 2006). S4 is primarily a
hydrophobic region derived from the side chains of Ile 222, Ala 246, and Arg 224. S5
has a mixed polarity and comprises of Ala 246 methyl and Glu 276 carboxylate that
forms a hydrogen bond with the substrate hydroxyl group (Zhang and Xu, 2006).
The joining pockets of eight highly conserved amino acid residues are linked
together, thereby causing the NA active site of these amino acids to directly come into
contact with Neu5Ac and its derivatives (Von Itzstein, 2007). The active site is
divided into three major pockets according to the crystal structure. Pocket 1 is formed
by Glu 276, Glu 277, and Asn 294, which interact with the glycerol moiety of SA
(Zhang and Xu, 2006). Pocket 2 forms a hydrophobic pocket that is not utilized by
sialic acid for binding (Kim et al., 1997) and comprises the Ala 246, Ile 222, and Arg
224 side chains. Interactions with potential inhibitors are provided by these residues
12
because they are highly conserved. Pocket 3 is the largest pocket consisting of
numerous amino acids, such as Glu 119, Asp 151, Arg 152, Trp 178, Ser 179, Th 222,
and Glu 227, all of which interact with the N-acetyl and C-4 hydroxyl groups of sialic
acid (Kim et al., 1997).
Fig. 1.5 The neuraminidase active site, conserved NA residues (R118, D151, R152,
R224, E227, E276, R292, and R371) (Yen et al., 2006)
1.9 Natural products
Natural products have many sources, such as microbes, plants, animals, or
prebiotic origins (Vuorelaa et al., 2004). Many classes of compounds can be found in
natural products. Natural products serve as natural sources of most drugs. Most of the
useful therapeutic agents are derived from higher plants. In today’s clinical settings,
13
over 50% of all drugs are based on natural products and their analogs with 25% of
such drugs containing higher plant-derived natural products (Balandrin et al., 1993).
According to WHO, around 80% of people in developing countries depend on
traditional medicine for their daily healthcare (WHO, 2009). Approximately 85% of
these medicines are plant extracts; whereby, around 3.5 to 4 billion people worldwide
rely on natural plants as drug sources (Farnsworth, 1988). Great efforts have been
exerted to discover new antiviral drugs. Many reviews have published discoveries of
new natural-based drugs for treating viral infections (Khan et al., 2005; Naithani et
al., 2008). Different reviews have discussed the significant role of traditional
medicine in the development and use of antiviral compounds. Traditional medicines
such as Chakma medicines and Ayurvedic are interestingly good sources of potential
antiviral drugs (Khan et al., 2005). Many active compounds from natural sources are
used as therapeutics against different functionally diverse and genetic viruses;
examples of these compounds include lignans, alkaloids, thiophenes, chlorophyllins,
saponins, furyl compounds, limonoids, peptides, and proteins (Chattopadhyay and
Bhattacharya, 2008; Naithani et al., 2008). The mechanism of anti-viral active
compounds can be explained in terms of their scavenging capacities, DNA inhibition,
antioxidant activities, RNA synthesis, and inhibition of viral reproduction
(Chattopadhyay and Naik, 2007).
According to several pharmacological and chemical studies, some bioactive
compounds used to treat influenza are obtained from medicinal plants. Varieties of
alkaloids, polyphenols, saponins, and flavonoids isolated from medicinal plants have
been extensively tested for anti-influenza activity, which is reflected in the ability of
14
bioactive compounds to block adherence, duplication, penetration, and maturation
during viral propagation (You et al., 2013).
Malaysia is home to over 15,000 species of flowering plants and more than
1,100 species of fern allies and ferns, whereby most of the plants are known as
endemic plants (Napis et al., 2001). A series of chemical structures can be observed
in Malaysian flora, especially in secondary flora metabolites. Malaysian plants used
as medicine by locals are well documented in books and papers on ethnobotany and
ethnopharmacology, with about 1,300 plant species used for medicinal purposes
(Napis et al., 2001). The traditional medicines available in the markets are mostly in
crude form and are sold by traditional medical practitioners. Only 100 species from
high-species density available have been systematically evaluated for medicinal
properties (Napis et al., 2001).
1.9.1 Family of Myrtaceae
The Myrtaceae family known as a large family of more than 5,650 species is
organized in 130 to 150 genera. This family is considered as the eighth largest
flowering plant family. It has centers of diversity in the wet tropics, particularly South
America, Australia, and tropical Asia with occurrences in Africa and Europe
(Grattapaglia et al., 2010).
The main genera are Eugenia, Syzygium, Psidium, Pimenta, Myrtus,
Pseuocaryophyllus, Plinia, Leptospermum, Melaleuca, and Eucalyptus. Numerous
members of this family are used in folk medicine for their antirheumatic, anti-
inflammatory, antidiarrheal, antioxidant, and antimicrobial properties (Govaerts et al.,
15
2008). Syzygium gaertner is considered as the largest genus of the Myrtaceae family,
with approximately 1,200 species distributed in subtropical and tropical regions
(Biffin et al., 2010). Some promising fruits belonging to the Syzygium genus found in
Malaysia are under-utilized but are recognized to be useful for medical and nutritional
purposes. A number of species from the Syzygium genus have been cultivated for their
edible, colorful flesh (Rabeta et al., 2013).
1.9.1(a) Psidium guajava
1.9.1(a)(i) General introduction
P. guajava originated in Mexico (Rios et al., 1977) and extends throughout
the South America, Europe, Africa, and Asia. Archaeological evidence shows that
during pre-Columbian times, this plant was widely used and known in Peru. This plant
can adapt to various climate conditions and is found in tropical and subtropical areas
worldwide (Stone, 1970). P. guajava Linn. (Myrtaceae family) is commonly called
guava, goyavier in French, goiabeiro in Portugal, banjiro in Japanese, and guaiaba in
Brazil (Killion, 2000). P. guajava is known in Malaysian communities as jambu batu
and is considered as an important food crop with medicinal use. P. guajava is a small,
thin tree with a height of 10 m and a smooth peeling bark. It has a short petiolate
leaves that are 5-15 cm long. Its flowers have whitish petals measuring up to 20 cm
long as well as numerous stamens. The fruit is pear-shaped, small, 3-6 cm long and
reddish-yellow when ripe (Stone, 1970). The P. guajava plants are shown in Figure
1.6.
16
Fig. 1.6 Psidium guajava plants (a) flower, (b) fruit, (c) leaves and (d) tree (Finca
Tropical 2011)
1.9.1(a)(ii) Traditional medicinal uses of Psidium guajava
P. guajava was traditionally used as an anti-diarrheal agent and to treat
dysentery. Its use as antioxidant, lipid lowering agent, and anti-bacterial agent and its
hypoglycemic activities have also been reported (Rahmat et al., 2004). P. guajava is
used in many parts of the world to treat different diseases and conditions, such as
fever, pain, wounds, caries, inflammation, and diabetes, as reported in many ethno-
pharmacological studies (Table 1.1).
a
d c
b
17
Table 1.1: Ethnomedical uses of Psidium guajava.
Place, country Part(s)
used
Ethno medical uses Preparation(s) Reference
Malaysia Leaves Vomiting, diarrhea and stomach ache Maceration Alsarhan et al.,
2010
Panama, Cuba, Costa Rica,
M´exico,Nicaragua, Panam´a,
Per´u, Venezuela, Mozambique,
Guatemala, Argentina
Leaves anti-inflammatory Externally applied hot
on inflammations
M´endez, 1990;
Valdiz´an and
Maldonado,
1972
Kinshasa, Congo
Leaves,
bark
Diarrhoea, antiamoebic Infusion or decoction
tisane
Tona et al. 1999
Latin America,
Mozambique
Leaves Diarrhoea, stomach ache Infusion or decoction Pontikis, 1996
South Africa Leaves Diabetes mellitus, hypertension Infusion or decoction Oh et al., 2005;
Ojewole, 2005
China Leaves Diarrhoea, antiseptic, Diabetes mellitus Infusion or decoction Teixeira et al.,
2003
Brazil Ripe
fruit,
flowers
,
and
leaves
Anorexia, cholera, diarrhoea, digestive
problems, dysentery, gastric insufficiency,
inflamed mucous membranes, laryngitis,
mouth
(swelling), skin problems, sore throat, ulcers,
vaginal discharge
Mashed, Decoction Holetz et al.,
2002; Cybele et
al., 1995
18
1.9.1(a)(iii) Chemical constituents of Psidium guajava
P. guajava contains many phytochemicals including essential oils,
polysaccharides, vitamins (Macleod and Troconis, 1975; Smith and Siwatibau, 1975),
triterpenoid acid and sesquiterpenoid alcohols (Begum et al., 2002; Wilson and Shaw,
1978), saponins, flavonoids , steroids, tannins, glycosides, alkaloids, (Geidam et al.,
2007; Narayana et al., 2001; Cho et al., 2003). P. guajava is very rich in vitamins and
antioxidants and also has a high content of lycopene, lutein and zeaxanthine (Hobert
and Tietze, 1998; Tee et al., 1997). The P. guajava fruit has high water content and
low amounts of proteins, fats, and carbohydrates. This fruit also contains thiamine,
iron, niacin, manganese, and vitamins A and C. Ascorbic acid is the major constituent
of its skin (Gutierrez et al., 2008).
Different compounds were isolated from fruits and leave of P. guajava such
as gallic acid (1), protocatechuic acid (2), (Okuda et al., 1984) caffeic acid (Liang et
al., 2005) (3), ferulic acid (Zhu et al., 1997) (4), chlorogenic acid (Liang et al., 2005)
(5), ellagic acid (Misra and Seshadri, 1968) (6), quercetin (Liang et al., 2005) (7),
leucocyanidin (Nadkarni and Nadkarni, 1999) (8), kaempferol (Liang et al., 2005)
(9), quercetin 3-β-galactoside (Liang et al., 2005) (10), kaempferol 3-β-glucoside
(Liang et al., 2005) (11), guaijaverin (Prabu et al., 2006) (12), morin-3-O-α-L-
arabinopyranoside (Arima and Danno, 2002) (13), mecocyanin (Liang et al., 2005)
(14), quercitrin (Liang et al., 2005) (15), zeatin riboside (Nagar and Rao, 1981) (16),
oeanolic acid (Siddiqui et al., 2002) (17), ursolic acid (Begum et al., 2002) (18), β-
sitosterol (Begum et al., 2002) (19), uvaol (Begum et al., 2004) (20), goreishic acid I
(Begum et al., 2002) (21), 2-α-hydroxyursolic acid (Begum et al., 2002) (22),
19
guavanoic acid (Begum et al., 2004) (23) , guavacoumaric acid (Begum et al., 2002)
(24), guajavolide (Begum et al., 2004) (25), 2α-hydroxy-3β-P-E-coumaroyloxyurs-
12,18-dien-28-oic acid (Begum et al., 2002) (26), α-sitosterol-3-O-β-D-
glucopyranoside (Begum et al., 2002) (27).
OH
OH
OH
O
HO
OH
OH
O
HO
(1) (2)
HO
HO
OH
OH
O
(3) (4)
O
HO
HO
OH
O
O
(5) (6)
20
O
OH
HO
OH
O
OH
OH
(7) (8)
OHO
OH
OH
OH
O
(9)
O
OH
HO
O
O
O
OH
OH
OH
HO
OH
OH
O
O
HO
OH
OH
O
O
OH
OH
OH
HO
(10) (11)
21
O
OH
HO
O
O
O
OH
OH
OH
HO OH
O
OH
HO
O
O
O
OH
OH
HO OH
OH
(12) (13)
O
O
HO
OH
OH
O
O
OH
OH
HO
OH
O
OH
OH
OH
HO
O
OH
HO
O
O
O
OH
OH
OH
HO CH3
OH
(14) (15)
N
NH
N
N
HN
CH3
OH
(16) (17)
22
(18) (19)
(20) (21)
(22) (23)
23
(24) (25)
(26)
(27)
24
1.9.1(a)(iv) Essential oil of Psidium guajava
Analysis of essential oils from Psidium guajava (fruit and leaves) using GC
and GC-MS enabled the identification different constituents varied from
Monoterpenes and Sesquiterpenes. Essential oils are extracted from the fruit, leaves,
and seeds of P. guajava. The yield and composition of the obtained oils vary from one
part to another. Monoterpene hydrocarbon E-β-Ocimene, (Li et al., 1999) (28), Z-β-
Ocimene (Li et al., 1999) (29),, Myrcene (Li et al., 1999) (30), p-cymene (Kenneth et
al., 1970) (31),, α-pinene (Kenneth et al., 1970) (32), β-pinene (Jordan et al., 2003)
(33), camphene (Jordan et al., 2003) (34), γ-terpinene (Jordan et al., 2003) (35)
identified from leaves and fruits with different composition. Oxygenated
monoterpenes identified from seed and leaves including 1,8-cineole (Kenneth et al.,
1970) (36), citronellol (Kenneth et al., 1970) (37) , linalool (Kenneth et al., 1970)
(38), limonene (Kenneth et al., 1970) (39) . Sesquiterpenes hydrocarbon from bark,
leaves and fruits include α-copaene (Oliver-Bever, 1986) (40), β-copaene (Adam et
al., 2011) (41), 1-epi-cubenol (Ruther, 2000) (42), α-cubebene (Porat et al., 2001)
(43), α-selinene (Ruther, 2000) (44), α-Humulene (Paniandy et al., 2000) (45) β-
caryophyllene (Adam et al., 2011) (46), γ-cadinene (Ruther, 2000) (47), germacrene
D (Oliver-Bever, 1986) (48). Another important constituent oxygenated
sesquiterpenes identified with GC-MS from leaves and fruits showed different
composition include spathulenol (Porat et al., 2001) (59), ar-turmerone (Paniandy et
al., 2000) (50), δ-cadinol (Ruther, 2000) (51), α-cadinol (Oliver-Bever, 1986) (52),
caryophyllene oxide (Ruther, 2000) (53), humulene epoxide II (Paniandy et al., 2000)
(54).
