Studies on the antiviral effect of some plant derived compounds

20 Pages·2015·1.1 MB·English

Cairo University
Faculty of Veterinary Medicine
Department of Virology
Studies on the antiviral effect of some
plant derived compounds
A Thesis presented by
Omar Mohamed Ibrahim Yousef
B.V.Sc. (1997), M.V.Sc. (2003), Faculty of Veterinary Medicine, Cairo University

For the degree of
Doctor of Philosophy (Ph. D.)
(Virology)

Under the supervision of
Prof. MOHAMED ABD EL-HAMEED SHALABY

Prof. of Virology,
Faculty of Veterinary Medicine,
Cairo University
Prof. AHMED ABD EL-GHANI EL SANOUSI
Prof. of Virology,
Faculty of Veterinary Medicine,
Cairo University
2012

ABSTRACT

AIM: To investigate the in vitro antiviral potential of Quercus ilex (wood) extract
against two RNA viruses that cause important diseases in veterinary and human medicine
in comparison to extracts of green tea and pomegranate which are previously reported to
have antiviral properties.
METHODS: The crude aqueous methanol extracts (CAMEs) of Q. ilex
(wood), Punica granatum (rind), and Camellia sinensis (leaves), in addition to crude
ethanolic extract (CEE) of P. granatum (arils) were firstly evaluated for cytotoxicity on
two different cell lines, MDCK and MDBK. Afterward, evaluated for their in vitro
antiviral activities against Influenza A (H9N2) virus and Bovine viral diarrhea virus
(BVDV) by means of (MTT/ XTT) based colorimetric assays.
RESULTS: On MDCK cells, The Q. ilex (wood) CAME exhibited a considerable
Influenza A virus inhibition percent (IP) > 97 %, which was equivalent to C. sinensis
(leaves) CAME, while being less cytotoxic to these cells, and at the same time, showed
more potent antiviral activity than pomegranate extracts and Amantadine on the same cell
line.
On the other hand, The Q. ilex (wood) CAME anti-BVDV activity on MDBK cell
line was found to be more pronounced than the other tested plant extracts on MDBK cells
with (IP > 97 %) at virus dose of 100 TCID .
50
CONCLUSION: Q. ilex (wood) extract shown antiviral activity, is suggesting
this plant extract as a candidate for further activity-monitored fractionation to identify its
active constituents.

Key words: Antiviral, Quercus ilex, Punica granatum, Camellia sinensis, BVDV, H9N2.
ACKNOWLEDGEMENT
Everlasting gratitude to “Allah” whom I attribute
any success in achieving anything in my life

Prof. Mohamed Abd El-Hameed
My sincere thanks are due to
Shalaby
, Professor of virology, Faculty of veterinary medicine, Cairo
University, for his skillful supervision of this thesis and for kindly offering his
experience, time and constructive criticism throughout this work.
Prof. Ahmed Abd El-Ghani El-Sanousi
My thanks also extend to ,
Professor of virology, Faculty of veterinary medicine, Cairo University, for his
knowledgeable supervision, valuable direction and encouragement and for his
generous attitude.
Prof. Mahmoud Nawar and Dr.
I express my sincere gratitude to
Mohamed El-Raiy
, Department of Phytochemistry, National research
center, Cairo (Egypt), for theirs great help and cooperation in extraction of
plant materials.
Prof. Hussain Ghaly
I would like to express my appreciation to , Head of
render pest department, veterinary serum & vaccine research institute,
Abbasia, Cairo (Egypt) for his kind providing of the BVD virus.
Dr. Layla Bassiony,
I am particularly thankful to Principle Instructor
Dr. Iman El Maamon
of WHO polio eradication program and , head of
WHO eastern mediterranean regional reference laboratory for diagnosis of
Polio virus and enteroviruses, holding company for biological products &
vaccines (VACSERA), Agouza, Cairo, Egypt, for the facilities and support
they offered to me during the work.
all staff members
Thanks should be given to of WHO eastern
mediterranean regional reference laboratory for diagnosis of Polio virus and
Dr. Samia
enteroviruses, (VACSERA), Agouza, Cairo, Egypt, specially
Abdel-Rahman Dr. Tamer Hassan
and for their precious help and
support.
Dr. Ali Fahmy
My heartfelt thanks are due to , head of Rabies research
dept., (VACSERA), Agouza, Cairo, Egypt, for his generous offering of advice
and experience.
all staff members
Great Thanks are due to of virology department,
Faculty of veterinary medicine, Cairo University, for their friendly cooperation
and advice.
I would like to dedicate the greatest thank and express all my deep gratitude to
mother wife sons daughters
my , , and for their prayers, patience and
endless support.
~
LISTS

CONTENTS
PAGE
1. Introduction …………………………………. 1
2. Review of Literature
2.1. THE CURRENT CHALLENGE OF VIRAL RESISTANCE …………………………………. 5
2.2. BOVINE VIRAL DIARRHEA VIRUS BIOLOGY …………………………………. 8
2.2.1. BVDV VETERINARY IMPORTANCE …………………………………. 10
2.2.2. BVDV AS SARROGATE MODEL FOR HCV …………………………………. 12
2.2.3. PLANTS TESTED AGAINST BVDV ( AND/OR HCV) …………………………………. 13
2.3. INFLUENZA A VIRUS (IAV) …………………………………. 15
2.3.1. AIV H9N2 VETERINARY IMPORTANCE …………………………………. 17
2.3. 2. AIV AS ZOONOTIC DANGER …………………………………. 18
2.3.3. DRUGS TESTED AGAINST AIV …………………………………. 20
2.3.4. AIV DRUG RESISTANCE …………………………………. 21
2.3.5. PLANTS TESTED AGAINST AIV …………………………………. 22
2.4. ANTIVIRAL CHEMOTHERAPEUTIC AGENTS …………………………………. 22
2. 5. PLANT ORIGIN ANTIVIRALS
2.5.1. GENERAL KNOWLEDGE …………………………………. 24
2.5.2. PLANT POLYPHENOLIC COMPOUNDS …………………………………. 29
2.5.3. EXTRACTION OF PLANT FOR ANTIVIRAL
…………………………………. 30
TESTING
2.6. GREEN TEA
2.6.1. GENERAL KNOWLEDGE …………………………………. 32
LISTS

2.6.2. G. TEA PROVEN ANTIVIRAL ACTIVITY …………………………………. 33
2.7. POMEGRANATE
2.7.1. GENERAL KNOWLEDGE …………………………………. 34
2.7.2. POMEGRANATE PROVEN ANTIVIRAL
…………………………………. 35
ACTIVITY
2.8. Quercus ilex
2.8.1 GENERAL KNOWLEDGE …………………………………. 36
2.8.2. Q. ilex ANTIMICROBIAL ACTIVITY …………………………………. 38
2.8.3. Q. ilex ANTIVIRAL ACTIVITY …………………………………. 38
2.9. FUTURE GUIDELINES IN ANTIVIRAL ASSESSMENT
…………………………………. 38
OF MEDICINAL PLANTS
3-Materials and Methods
3.1.Materials …………………………………. 42
3.2.Methods …………………………………. 48
4-Results …………………………………. 59
5-Discussion …………………………………. 102
6-Summary …………………………………. 114
7-References …………………………………. 117
8-Arabic Summary
LISTS

List of tables

TABLE TITLE PAGE

1 Antiviral agents’ potential targets in virus life cycle. ……… 23
2 The 4 plant extracts used for antiviral testing. ……… 44
Microplate reader absorbance results of cytotoxicity assay of
3 ……… 60
Q. ilex on MDBK cells (MTT)
Microplate reader absorbance results of cytotoxicity assay of
4 ……… 61
C. sinensis on MDBK cells (MTT)
Microplate reader absorbance results of cytotoxicity assay of
5 ……… 62
P. granatum (rinds) on MDBK cells (MTT)
Microplate reader absorbance results of cytotoxicity assay of
6 ……… 63
P. granatum (arils) on MDBK cells (XTT)
Results of virus infectivity titration assay of BVDV on
7 ……… 65
MDBK cells (CPE)
Microplate reader mean absorbance results of PILOT
8 antiviral assay of Q. ilex on MDBK cells against BVDV ……… 67
(MTT).
Microplate reader mean absorbance results of PILOT
9 ……… 69
antiviral assay of C. sinensis on MDBK cells against BVDV
(MTT).
Microplate reader mean absorbance results of PILOT
10 ……… 71
antiviral assay of P. rinds on MDBK cells against BVDV
(XTT).
LISTS

Results of virus inhibition percentages (IP %) of pilot
11 ……… 72
antiviral assessment of different plant extracts on MDBK
cells against BVDV
Microplate reader mean absorbance results of virus titration
12 ……… 74
assay of BVDV on MDBK cells (MTT)

Microplate reader mean absorbance results of antiviral assay
13 ……… 76
of Q. ilex on MDBK cells against BVDV (XTT).
Microplate reader mean absorbance results of antiviral assay
14 ……… 78
of C. sinensis on MDBK cells against BVDV (XTT).
Microplate reader mean absorbance results of antiviral assay
15 ……… 80
of P. rinds on MDBK cells against BVDV (XTT)
.
Microplate reader mean absorbance results of antiviral assay
16 ……… 82
of P. arils on MDBK cells against BVDV (XTT).
Results of virus inhibition percentages (IP %) of main
17 ……… 83
antiviral assay of different plant extracts on MDBK cells
against BVDV.
Microplate reader mean absorbance results of cytotoxicity
18 ……… 85
assay of Q. ilex on MDCK cells (MTT)
Microplate reader mean absorbance results of cytotoxicity
19 ……… 86
assay of C. sinensis on MDCK cells (MTT)
Microplate reader mean absorbance results of cytotoxicity
20 ……… 87
assay of P. granatum (rinds) on MDCK cells (MTT)
Microplate reader mean absorbance results of cytotoxicity
21 ……… 88
assay of P. granatum (arils) on MDCK cells (MTT)
LISTS

Microplate reader mean absorbance results of virus titration
22 ……… 90
assay of H9N2 on MDCK cells (MTT)

Cell culture HA results of virus titration assay of H9N2 on
23 ……… 92
MDCK cells (1% RBCs solution)

Microplate reader mean absorbance results of antiviral assay
24 ……… 94
of Q. ilex on MDCK cells against H9N2 (MTT).

Microplate reader mean absorbance results of antiviral assay
25 ……… 96
of C. sinensis on MDCK cells against H9N2 (MTT).
Microplate reader mean absorbance results of antiviral assay
26 ……… 98
of P. rinds on MDCK cells against H9N2 (MTT).

Microplate reader mean absorbance results of antiviral assay
27 ……… 100
of P. arils on MDCK cells against H9N2 (MTT).

Results of virus inhibition percentages (IP %) of main
28 ……… 101
antiviral assay of different plant extracts on MDCK cells
against H9N2 virus.