hepatoprotective, immunomodulatory and antibacterial effects of selected malaysian medicinal ...

216 Pages·2013·6.96 MB·English

HEPATOPROTECTIVE, IMMUNOMODULATORY AND
ANTIBACTERIAL EFFECTS OF SELECTED MALAYSIAN
MEDICINAL PLANT EXTRACTS

MOHAMMED ABDULLAH MAHDI ALSHAWSH

THESIS SUBMITTED IN FULFILMENT OF THE
REQUIREMENTS FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY

FACULTY OF MEDICINE
UNIVERSITY OF MALAYA
Kuala Lumpur

2013
UNIVERSITI MALAYA
ORIGINAL LITERARY WORK DECLARATION
Name of Candidate: Mohammed Abdullah Mahdi Alshawsh
(Passport No: 04160181)
Registration/Matric No: MHA090017
Name of Degree: PhD
Title of Thesis (“this Work”): Hepatoprotective, Immunomodulatory and Antibacterial
Effects of Selected Malaysian Medicinal Plant Extracts
Field of Study: Immunology
I do solemnly and sincerely declare that:
(1) I am the sole author/writer of this Work;
(2) This Work is original;
(3) Any use of any work in which copyright exists was done by way of fair dealing and
for permitted purposes and any excerpt or extract from, or reference to or reproduction
of any copyright work has been disclosed expressly and sufficiently and the title of the
Work and its authorship have been acknowledged in this work;
(4) I do not have any actual knowledge nor do I ought reasonably to know that the
making of this work constitutes an infringement of any copyright work;
(5) I hereby assign all and every rights in the copyright to this work to the University of
Malaya (“UM”), who henceforth shall be owner of the copyright in this work and that
any reproduction or use in any form or by any means whatsoever is prohibited without
the written consent of UM having been first had and obtained;
(6) I am fully aware that if in the course of making this work I have infringed any
copyright whether intentionally or otherwise, I may be subject to legal action or any
other action as may be determined by UM.

Candidate’s Signature Date
Subscribed and solemnly declared before,

Witness’s Signature Date
Name:
Designation:
ABSTRACT

Orthosiphon stamineus Benth and Morinda citrifolia L. are considered an important
traditional folk medicine and commonly used in Malaysia for treating many diseases. In
this study, the ethanol extracts of O. stamineus and M. citrifolia were evaluated for their
hepatoprotective and antioxidant activities; in vivo against thioacetamide-induced liver
cirrhosis in rats and in vitro against H O -induced hepatotoxicity in WRL-68 liver cell
2 2
line, as well as to investigate their immunomodulatory and antibacterial effects.
Orthosiphon stamineus and Morinda citrifolia were evaluated for their antioxidant
activities using DPPH, ABTS and FRAP. In addition the phenolic and flavonoids
contents were determined. Seven groups of adult SD rats were used in the
hepatoprotective experiment. Group 1 as normal control group, while groups 2 to 7
were injected intraperitoneally with 200 mg/kg of thioacetamide (TAA) thrice weekly
for two months and orally administered respectively with 10 % Tween 20, 50 mg/kg
silymarin, 200 mg/kg and 100 mg/kg ethanol extract of O. stamineus and M. citrifolia
daily for two months. The hepatoprotective activity was evaluated using the following
parameters; body and liver weight, serum liver biochemical markers, liver gross
morphology and histopathology, as well as endogenous antioxidant markers.
Furthermore, the liver fibrosis related genes namely; TGFβ1, MMP2, TIMP1 and Coll α
were estimated for the change in gene expression levels using RT-PCR. In addition, the
ethanol crude extracts of O. stamineus and M. citrifolia and their isolated fractions were
investigated against H O -induced hepatotoxicity in WRL-68 liver cell line and the
2 2
percentage of cell viability using MTT assay and the antioxidant level markers were
assessed. The immunomodulatory potential of the extracts were investigated by MTT
assay against human peripheral blood mononuclear cells (PBMCs), while the
antibacterial activity was investigated by disc diffusion and determination of minimum
ii
inhibitory concentration (MIC) against four Gram-positive and Gram-negative bacterial
strains. Finally, LC-MS was used for identification of the active constituents of the
fractions that proved to have hepatoprotective activity.
Orthosiphon stamineus exhibited significant free radical scavenging activity with DPPH
(IC 21.4 µg/ml), at the same time, showed high total phenolic and flavonoidal
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contents. In animal experiments, the hepatotoxic group showed a coarse granulation on
the liver surface when compared to the smooth aspect observed on the liver surface of
normal and treatment groups. Histopathological study confirmed the result. Moreover,
there was a significant (P < 0.05) increase in serum liver biochemical parameters (ALT,
AST, ALP and bilirubin) and the level of liver lipid peroxidation index
malondialdehyde (MDA), accompanied by a significant decrease in the level of total
protein, albumin, catalase, superoxide dismutase and glutathione peroxidase in the TAA
control group comparing with normal group. The 200 mg/kg treatment groups of both
plants significantly restored the elevated liver function enzymes and antioxidant
parameters near to normal and significantly down-regulated the expression of the liver
fibrosis genes. The oxidative stress by H O resulted in a decrease of cell viability to
2 2
41.9 %, while pre-treatment with crude extracts of O. stamineus and M. citrifolia, as
well as with fraction 3 of O. stamineus and fraction 2 of M. citrifolia were found
significantly (P < 0.01) increase the cell viability to 81.1 %, 76.4 %, 95.1 % and 86.1 %
respectively at concentration 100 µg/ml. The hepatoprotective and antioxidant activity
could be claimed to the following flavonoids; ponkanetin, eupatorin, TMF and
salvigenin that were identified in O. stamineus F3 and scopoletin and P- coumaric acid,
which were identified in M. citrifolia F2. In conclusion, this study showed that O.
stamineus and M. citrifolia exhibit potent antioxidant properties, immunomodulatory
activity and could be an effective herbal and efficient remedy for chemical-induced
hepatic cirrhosis, thus may be a highly promising candidate drugs.
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ABSTRAK
Orthosiphon stamineus dan Morinda citrifolia dikenali sebagai ubatan tradisional yang
penting dan kerap digunakan untuk mengubati pelbagai penyakit di Malaysia. Bagi
kajian ini, ekstrak etanol O. stamineus dan M. citrifolia diuji untuk menilai aktiviti
pelindung hati dan antioksidan masing-masing; secara in vivo di dalam tikus yang
mengalami sirosis hati akibat thioacetamide dan secara in vitro di dalam sel-sel hati
WRL-68 yang mengalami ketoksikan akibat H O , serta menyiasat kesan modulasi
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sistem imun dan antimikrobial.
O. stamineus dan M. citrifolia dinilai untuk aktiviti antioksidan dengan menggunakan
DPPH, ABTS, FRAP, jumlah fenol dan asai kandungan flavanoid. Tujuh kumpulan
tikus SD dewasa digunakan untuk eksperimen melindungi hati. Kumpulan 1 adalah
kumpulan kawalan normal, manakala kumpulan-kumpulan 2 ke 7 disuntik secara
intraperitoneal dengan 200 mg/kg thioacetamide (TAA) tiga kali seminggu selama dua
bulan dan diberi 10 % Tween 20, 50 mg/kg silymarin, 200 mg/kg atau 100 mg/kg
ekstrak etanol O. stamineus and M. citrifolia secara oral selama dua bulan. Aktiviti
perlindungan hati dinilai melalui parameter-parameter berikut; berat badan dan hati,
petanda biokimia hati di dalam serum, morfologi nyata dan histopatologi hati serta
petanda antioksidan tisu hati. Di samping itu, gen yang berkait dengan fibrosis hati,
yakni; TGFβ1, MMP2, TIMP1 dan Coll α diuji secara RT-PCR untuk mengesan
perubahan di dalam ekspresi gen. Ekstrak etanol kasar daripada O. stamineus dan M.
citrifolia serta pecahan kromatografi masing-masing pula diperiksa secara in vitro di
dalam sel hati WRL-68 yang mengalami ketoksikan akibat H O dan penilaian
2 2
peratusan viabiliti sel menggunakan asai MTT dan petanda paras antioksidan. Potensi
untuk memodulasi sistem imun oleh ekstrak-ekstrak ini dikaji melalui asai MTT ke atas
sel-sel mononuklear darah periferi manusia (PBMC). Keberkesanan antibakteria dikaji
dengan menggunakan cakera sebar dan kepekatan minimum untuk mencapai perencatan
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(MIC) ke atas empat jenis bakteria Gram-positif dan Gram-negatif. LC-MS digunakan
untuk mengenal-pasti juzuk aktif yang terbukti mempunyai aktiviti yang mampu
melindungi hati.
O. stamineus menampakkan aktiviti memerangkap radikal bebas yang signifikan
dengan DPPH IC 21.4 µg/ml, dan juga menunjukkan kandungan jumlah fenol dan
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flavanoid yang tinggi. Eksperimen tikus menunjukkan kumpulan ketoksikan hati
mempunyai granulasi kasar di permukaan hati berbanding dengan permukaan licin yang
dilihat pada hati-hati daripada kumpulan normal dan kumpulan yang dirawat. Kajian
histopatologi mengesahkan keputusan ini; di samping itu terdapat penambahan
signifikan (P < 0.05) bagi parameter biokimia hati di dalam serum (ALT, AST, ALP
dan Bilirubin) dan paras indeks malondialdehyde (MDA) bagi pengoksidan lipid hati,
berserta pengurangan signifikan paras jumlah protein, Albumin,
katalase, superoxide dismutase danglutathione peroksida bagi kumpulan kawalan TAA
berbanding kumpulan normal. Kumpulan-kumpulan yang dirawat dengan dos 200
mg/kg memulihkan paras tinggi enzim fungsi hati dan parameter antioksidan hampir ke
paras normal dan menurunkan dengan signifikan regulasi ekspresi gen fibrosis hati.
Tekanan oksidatif H O daripada mengakibatkan viabiliti sel merosot ke 41.9 %,
2 2
manakala prarawatan dengan ekstrak O. stamineus, M. citrifolia, pecahan 3 O.
stamineus dan pecahan 2 M. citrifolia berkepekatan 100 100 µg/ml menyebabkan
penambahan signifikan (P < 0.01) bagi viabiliti sel ke 81.1 %, 76.4 %, 95.1 % dan 86.1
%. Kesimpulan kajian ini ialah O. stamineus dan M. citrifolia mempunyai kesan
antioksidan yang kuat, aktiviti modulasi sistem imun dan berkesan sebagai rawatan
herba yang efisien untuk sirosis hati yang diakibatkan oleh bahan kimia, dan
mempunyai potensi yang tinggi untuk dijadikan ubat.
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ACKNOWLEDGEMENT

All praises to the Almighty Allah, the most Gracious and Merciful, Who is omnipotent
and all giving, for affording me the strength and determination to complete this study. I
would like to express my gratitude to my supervisor Prof. Dr. Mahmood Ameen
Abdulla for his guidance, continued support and encouragement throughout this work.
I am particularly grateful for my co-supervisor Dr. Salmah Ismail, who provided me
needed support, good comments and valuable suggestions.
I wish to express my thanks to Ministry of Higher Education- Yemen for the
scholarship and I would like to thank University of Malaya, Malaysia for supporting
this work by research grants No. (PS182/2009C) and (PV047/2011B).
I wish to mention and thank: Animal house unit staff, CDL-UM Hospital, CARIF,
Biotechnology Department members and for all staff in Molecular Medicine
Department, faculty of medicine, University of Malaya.

Acknowledgments are gratefully made to the following: Zahra A. Amin, Mustafa
Kassim, Pouya, Miss Kim, Lisa, Khadijeh Gholami and Iman Mustafa, as well as to Dr.
Riyadh Alhammadi for the contribution in varying degrees of this work. I am greatly
indebted to the immunology Lab mates and all Molecular Medicine Department
students.
I wish to express my thanks for my Mother and father who always pray for me, for my
wife who support and encourage me, for my kids who make me laugh. Finally, I wish to
acknowledge with thanks all those who, cooperated with me during this work, in all lab
work and who read, reviewed and offered numerous helpful suggestions and
corrections.
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TABLE OF CONTENTS

ABSTRACT ................................................................................................................. ii
ABSTRAK .................................................................................................................. iv
ACKNOWLEDGEMENT .......................................................................................... ivi
TABLE OF CONTENTS ............................................................................................ vii
LIST OF FIGURE S .................................................................................................. xiii
LIST OF TABLES ..................................................................................................... xvi
LIST OF SYMBOLS AND ABBREVIATIONS ...................................................... xviii
CHAPTER I ................................................................................................................. 1
INTRODUCTION ........................................................................................................ 1
1.1 Introduction ......................................................................................................... 1
1.2 Objectives ........................................................................................................... 6
1.2.1 General ......................................................................................................... 6
1.2.2 Specific ........................................................................................................ 6
CHAPTER II ................................................................................................................ 7
LITERATURE REVIEW .............................................................................................. 7
2.1 Liver cirrhosis ..................................................................................................... 7
2.1.1 Causes of liver cirrhosis ................................................................................ 9
2.1.2 Pathology ................................................................................................... 10
2.1.3 Immunology (molecular and cellular aspects of liver cirrhosis) ................... 13
2.1.4 Symptoms and complication ....................................................................... 17
2.1.5 Diagnosis.................................................................................................... 18
2.1.6 Prevention .................................................................................................. 19
2.1.7 Treatment ................................................................................................... 19
2.1.8 Liver cirrhosis and medicinal plants............................................................ 20
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2.2 Approaches to study hepatoprotective activity and screening models ................. 22
2.2.1 In vivo models ............................................................................................ 22
2.2.1.1 Thioacetamide induced hepatotoxicity model ....................................... 23
2.2.1.2 Carbon tetrachloride induced hepatotoxicity model .............................. 24
2.2.1.3 Paracetamol induced hepatotoxicity model .......................................... 24
2.2.2 In vitro methods ......................................................................................... 25
2.2.3 Markers for hepatoprotective activity evaluation......................................... 26
2.2.4 Silymarin .................................................................................................... 27
2.3 Antioxidant activity ........................................................................................... 28
2.3.1 Free radicals and reactive oxygen species ................................................... 28
2.3.2 Oxidative stress in liver cirrhosis ................................................................ 29
2.3.3 Antioxidant and liver cirrhosis .................................................................... 30
2.3.4 Evaluation of antioxidant activity ............................................................... 31
2.4 Immunomodulatory activity .............................................................................. 32
2.5 Antibacterial activity ......................................................................................... 33
2.6 Investigated medicinal plants............................................................................. 35
2.6.1 Orthosiphon stamineus ............................................................................... 35
2.6.1.1 Description .......................................................................................... 35
2.6.1.2 Uses, pharmacological properties and safety ........................................ 36
2.6.1.3 Phytochemistry .................................................................................... 37
2.6.2 Morinda citrifolia ....................................................................................... 38
2.6.2.1 Description .......................................................................................... 39
2.6.2.2 Uses, pharmacological properties and safety ........................................ 40
2.6.2.3 Phytochemistry .................................................................................... 41
CHAPTER III ............................................................................................................. 43
METHODOLOGY ..................................................................................................... 43
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3.1 Plant materials and chemicals ............................................................................ 43
3.2 Experiment design ............................................................................................. 44
3.3 Antioxidant activity ........................................................................................... 45
3.3.1 Scavenging activity of DPPH ..................................................................... 45
3.3.2 Ferric reducing antioxidant power (FRAP) assay ........................................ 45
3.3.3 ABTS assay ................................................................................................ 46
3.3.4 Total phenolic content (TPC) and Flavonoids determination ....................... 47
3.4 Acute toxicity .................................................................................................... 47
3.5 In vivo hepatoprotective activity of plant extracts .............................................. 47
3.5.1 Animals ...................................................................................................... 47
3.5.2 In vivo hepatoprotective activity experimental design ................................. 48
3.5.3 Biochemical and histopathological examination.......................................... 49
3.6 In vivo antioxidant activity in liver tissue ........................................................... 50
3.7 Gene expression assay using real time PCR (RT-PCR) ...................................... 50
3.7.1 RNA isolation and purification ................................................................... 51
3.7.2 Reverse transcription and cDNA synthesis.................................................. 52
3.7.3 Real time PCR amplification ...................................................................... 54
3.8 Immunomodulatory activity .............................................................................. 57
3.8.1 Peripheral blood mononuclear cell (PBMCs) isolation and cell culture ....... 57
3.8.2 MTT cell viability assay ............................................................................. 58
3.9 Profiling and Fractionation of crude extracts ..................................................... 58
3.10 In vitro hepatoprotective activity of plant crude extracts and isolated fractions 59
3.10.1 Cell line and culture conditions ................................................................. 59
3.10.2 Hydrogen peroxide treatment evaluation ................................................... 60
3.10.3 In vitro hepatoprotective activity and cell viability test ............................. 60
3.11 In vitro antioxidant for cell line experiment ..................................................... 62
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