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UNIVERSITI PUTRA MALAYSIA
ANTIMICROBIAL, IMMUNOMODULATORY AND TUMOR
CELLS- SELECTIVE CYTOTOXIC ACTIVITIES OF BRASSICA
OLERACEA L. AND VIGNA RADIATA L.
RAND R. HAFIDH
FSTM 2011 27
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ANTIMICROBIAL, IMMUNOMODULATORY AND TUMOR CELLS-
SELECTIVE CYTOTOXIC ACTIVITIES OF BRASSICA OLERACEA L. AND
VIGNA RADIATA L.
By
RAND R. HAFIDH
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,
in Fulfillment of the Requirements for the Degree of Doctor of Philosophy
May 2011
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DEDICATION
I dedicate the results of this work and my degree to the people
who without them i wouldn’t be able to reach what i have
reached, my beloved husband, my caring mother, my father, my
grandmother Adilaa and my aunt Lamiaa.
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment
of the requirement of the degree of doctor of philosophy
ANTIMICROBIAL, IMMUNOMODULATORY AND TUMOR CELLS-
SELECTIVE CYTOTOXIC ACTIVITIES OF BRASSICA OLERACEA L. AND
VIGNA RADIATA L.
By
RAND R. HAFIDH
May 2011
Chair: Professor Dr. Fatimah Abu Bakar, PhD
Faculty: Food Science and Technology
The world today faces a great challenge in the emergence of drug-resistant microbes
and dangerous cancers against most of the commonly known antimicrobial and
anticancer drugs, respectively. Screening for effective, new, and cheap remedies
from natural resources has become the goal of many scientists all around the world.
The plants’ methanol extracts used in this study were red cabbage (RC) and mung
bean sprout (MBS) which both showed remarkable antiviral activities against herpes
simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV). The mode of
action of RC extract was shown to be prophylactic against RSV (the concentration
that inhibit 50% of virus cytopathic effect (IC50) = 13.9 mg/ml and selectivity index
(SI) = 17.67). Against HSV-1, RC extract showed a remarkable viricidal activity
(IC50 = 11.51 mg/ml and SI = 34.52) with less prophylactic activity (IC50 = 32.97
mg/ml and SI = 11.71). MBS extract had viricidal effect (IC50 = 15.62 mg/ml and SI
= 14.18), (IC50 = 7.62 mg/ml and SI = 18.23) and to a lesser extent prophylactic
(IC50 = 17.23 mg/ml and SI = 12.82), (IC50 = 12.72 mg/ml and SI = 10.9) against
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RSV and HSV-1 viruses, respectively. One of the good explaination on the effective
prophylactic activities of MBS and RC extracts was their excuisite ability to induce
production of high levels of IFNβ, TNFα, IL-1, and IL-6 cytokines (300-900%
higher levels when compared to untreated cells; P<0.0001) in virally infected cells.
These antiviral cytokines proved to play critical and synergistic role in the antiviral
resistance of human cells. For the cytotoxic effects of the tested extracts, RC extract
showed selective cytotoxic effect against human cervix adenocarcinoma cells
(HeLa), (SI = 10.88) while it showed less selectivity against human hepatocellular
carcinoma cells (HepG2), (SI = 8.93). On the contrary, MBS extract showed a
selective cytotoxic effect against both HeLa and HepG2 cells with SI values of 12.44
and 11.94, respectively. In an attempt to disclose part of the underlying mechansims
of cytotoxic effect of both extracts using concentrations lower than IC50, RC
induced the production of the anticancer cytokine TNFα (up to 600% increase;
P<0.0001) whereas MBS induced the production of both anticancer cytokines TNFα
(up to 700% increase; P<0.0001) and IFNβ (up to 300% increase; P<0.001). The
increase in the anticancer cytokine levels was dose-dependent. In addition, the IC50
of RC extract induced cell cycle arrest in G0/G1 phase in cancer cells increasing the
percentage of cells from 66.87 to 76.96% and from 57.83 to 70.41% in the treated
HeLa and HepG2 cells, respectively. On the other hand, the IC50 of MBS extract
induced cell cycle arrest in G0/G1 phase only in HeLa cells (62.87 to 80.48%). Both
extracts induced remarkably apoptosis in the treated human cancer cells in a dose-
and time-dependent manner. The induction of apoptosis in cancerous cells 12-16h
after exposure to RC and MBS extracts was significant via caspase-dependent
pathways including the extrinsic pathway by upregulating caspase 8 (about 8-32
folds) and the intrinsic pathway by upregulating both caspase 9 (about 32-256 folds)
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and Bax genes (about 8-32 folds). Furthermore, both extracts upregulated highly
caspase 7 (about 32-512 folds) which could induce apoptosis via caspase-
independent pathway. More to the point, Cdk-inhibitor proteins were upregulated
(p21: about 8-512 folds, p27: about 4-16 folds, and p53: about 16-32 folds) 12-16h
after exposure to both extracts and their upregulation might be the main mechanism
used by both extracts to exert G1 cell cycle arrest and ultimately the final fate of
cells, apoptosis. For the immunomodulatory effect of tested extracts, RC extract
showed potent anti-inflammatory activity by inhibiting the production of the pro-
inflammatory cytokine, IFNγ (about 80% decrease; P<0.01) in peripheral blood
mononuclear cells (PBMC). On the other hand, MBS extract was effective
immunomodulator agent as TNFα and IFNβ cytokines are also potent
immunomodulators. In addition, MBS extract showed an immunopolarizing effect by
inducing IFNγ (about 300% increase; P<0.01) and inhibiting IL-4 production (about
75% decrease; P<0.01) in PBMC. The immunopolarization effect of MBS extract
was dose-dependent. For the antibacterial and antifungal potential of the tested
extracts, antibacterial and antifungal activities against multiple drug resistant (MDR),
non-MDR bacteria, and fungi were shown to be significant for both extracts. The
antibacterial and antifungal activities were dose-dependent. Remarkably,
antibacterial activity of RC extract was evident particularly against highly infectious
microorganisms such as Methicillin-resistant Staphylococcus aureus (MRSA),
Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae,
Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as
against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. MBS
extract revealed potential antibacterial and antifungal activities against MRSA,
Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumonaie,
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Staphylococcus aureus, and Salmonella Typhimurium as well as against human
fungal pathogens, Trichophyton rubrum and Trichoderma harzianum. The findings
of the electron microscopy together with phenotype microarray (PM) supported these
results for the antibacterial activity. PM screening was a valuable tool in the search
for compounds that can inhibit bacterial growth by affecting certain metabolic
pathways like peptidoglycan synthesis pathway. This pathway appeared to be
targeted by both RC and MBS extracts at metabolic points differ from that of other
available drugs. These points could be targets for discovering new therapeutic agents
for the currently studied microbes. The considerable results of this study could be
explained by the powerful antioxidant activity of RC and MBS which is most likely
due to the phenolic and antioxidant compounds present in these plants which act
individually or synergistically to bring to light the importance of these plants as
candidates for new therapeutic agents.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai
memenuhi keperluan ijazah dari doktor falsafah
AKTIVITI ANTIMIKROB, IMUNOMODULATOR DAN SEL-SELEKTIF
TUMOR SITOTOKSIK DARIPADA BRASSICA OLERACEA L. DAN VIGNA
RADIATA L.
Oleh
RAND R. HAFIDH
Mei 2011
Pengerusi: Profesor Dr. Fatimah Abu Bakar, PhD
Fakulti: Sains dan Technologi Makanan
Dunia saat ini menghadapi cabaran besar dengan munculnya mikrob resistan
terhadap ubat dan kanser berbahaya terhadap sebahagian besar antimikrob umumnya
dikenali dan ubat antikanser, masing-masing. Penyaringan untuk Penemuan remedi
yang berkesan, baru, dan murah daripada sumber alam semulajadi menjadi matlamat
ramai saintis di seluruh dunia. Ekstrak metanol yang digunakan dalam penelitian ini
adalah kubis merah (RC) dan kacang hijau tunas (MBS) yang keduanya
menunjukkan aktiviti antivirus yang luar biasa terhadap jenis virus herpes simpleks 1
(HSV-1) dan virus RSV (RSV). Mod tindakan RC ekstrak terbukti profilaksis
terhadap RSV (kepekatan yang menghalang 50% daripada kesan virus sitofatik
(IC50) = 13.9 mg / ml dan indeks selektivitas (SI) = 17.67). Terhadap HSV-1, RC
ekstrak menunjukkan aktiviti virisidal luar biasa (IC50 = 11.51 mg / ml dan SI =
34.52) dengan aktiviti profilaktik kurang (IC50 = 32.97 mg / ml dan SI = 11.71).
Kesan virisidal ekstrak MBS (IC50 = 15.62 mg / ml dan SI = 14.18), (IC50 = 7.62
mg / ml dan SI = 18.23) dan ksan profilaksis tahap yang lebih rendah (IC50 = 17.23
mg / ml dan SI = 12.82), (IC50 = 12.72 mg / ml dan SI = 10.9) terhadap RSV dan
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HSV-1 virus, masing-masing. Salah satu alasan yang baik menjelaskan kegiatan
profilaksis berkesan ekstrak MBS dan RC adalah kemampuan mereka untuk
menginduksi pengeluaran peringkat tinggi IFNβ, TNFa, IL-1, dan IL-6 sitokin (tahap
300-900% lebih tinggi bila dibandingkan dengan yang tidak dirawat sel; P <0.0001)
pada sel yang dijangkiti virus. Sitokin ini terbukti memainkan peranan kritikal dan
sinergis dalam kesan rintangan sel antivirus manusia. Untuk antikanser ekstrak, RC
ekstrak menunjukkan aktiviti anti-kanser selektif terhadap sel adenokarsinoma
serviks manusia (Hela), (SI = 10.88), sementara kurang selektiviti terhadap sel
karsinoma hepatoseluler manusia (HepG2), (SI = 8.93). MBS ekstrak masing-masing
menunjukkan aktiviti anti-kanser selektif terhadap kedua HeLa dan sel-sel HepG2
dengan nilai SI 12.44, dan 11.94. Dalam usaha untuk mendedahkan sebahagian
daripada mekanisma yang mendasari aktiviti anti-kanser daripada kedua-dua ekstrak
dengan kepekatan yang lebih rendah daripada IC50, RC mengaruh pengeluaran
sitokin TNFa antikanser (meningkat hingga 600%, P <0.0001) sedangkan MBS
mengaruh pengeluaran antikanser sitokin TNFa (sehingga 700% meningkat; L
<0,0001) dan IFNβ (hingga mencapai 300%, P <0,001). Peningkatan kadar sitokin
antikanser adalah dos-bergantung. Selain itu, IC50 dari ekstrak RC mengaruh sel
penangkapan kitaran sel pada fasa G0/G1 dalam sel kanser dengan meningkatkan
peratusan sel 66.87-76.96% dan 57.83-70.41% dalam sel HeLa dan HepG2, masing-
masing. Selain itu IC50 dari ekstrak MBS mengaruh penangkapan kitaran sel pada
fasa G0/G1 hanya dalam sel Hela (62.87-80.48%). Kedua-dua ekstrak mengaruh
apoptosis dalam sel kanser manusia dengan cara-dos dan masa-dependen. Induksi
apoptosis dalam sel kanser selepas pendedahan 12-16h untuk ekstrak RC dan MBS
yang signifikan melalui laluan caspase-dependent termasuk laluan ekstrinsik
menggunakan upregulating caspase 8 (sekitar 8-32 lipatan) dan laluan intrinsik
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dengan upregulating kedua-dua caspase 9 (tentang 32-256 lipatan) dan gen Bax
(sekitar 8-32 lipatan). Seterusnya, kedua-dua ekstrak terselaras tinggi terhadap
caspase 7 (sekitar 32-512 lipatan) yang boleh mengaruh apoptosis melalui laluan
bebas-caspase. Lebih penting lagi, protein Cdk-inhibitor terselaras (p21: sekitar 8-
512 lipatan, p27: sekitar 4-16 lipatan, dan p53: sekitar 16-32 lipatan) 12-16h setelah
terdedak kepada untuk kedua-dua ekstrak dan upregulation mungkin merupakan
mekanisme utama yang digunakan oleh kedua-dua ekstrak untuk mengerahkan
penangkapan kitaran sel G1 dan akhirnya, apoptosis. Untuk kesan imunomodulator
dari ekstrak diuji, RC ekstrak menunjukkan aktiviti anti-inflamasi tinggi dengan
menghalang pengeluaran sitokin pro-inflamasi, IFNγ (sekitar 80% menurun, P
<0.01) pada sel mononuklear darah periferi (PBMC). MBS ekstrak sebagai agen
imunomodulator berkesan sitokin TNFα dan IFNβ. Selain itu, MBS ekstrak
menunjukkan kesan immunopolarizing dengan mengaruh IFNγ (sekitar 300%
meningkat; P <0.01) menghalas dan pengeluaran IL-4 (penurunan sekitar 75%, P
<0.01) pada PBMC. Kesan immunopolarization ekstrak MBS adalah dos-
bergantung. Untuk potensi antibakteria dan antikulat dari ekstrak diuji, aktiviti
antibakteria dan antikulat terhadap beberapa dadah rintang bakteria (MDR), non-
MDR, dan kulat menunjukkan kesan signifikan bagi kedua-dua ekstrak. Aktiviti
antibakteria dan antikulat adalah bergantung dos. Hebatnya, aktiviti antibakteria
ekstrak RC adalah jelas terutama terhadap mikroorganisme yang sangat berjangkit
seperti Staphylococcus aureus resistan methicillin-(MRSA), Escherichia coli O157:
H7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, dan
Salmonella enterica serovar Typhimurium serta terhadap kulat patogen manusia,
Trichophyton rubrum dan Aspergillus terreus. Ekstrak MBS menunjukkan aktiviti
antibakteria dan antikulat berpotensi terhadap MRSA, Escherichia coli O157: H7,
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Pseudomonas aeruginosa, Klebsiella pneumonaie, Staphylococcus aureus, dan
Salmonella Typhimurium serta kulat patogen manusia, Trichophyton rubrum dan
Trichoderma harzianum. Penemuan menggunakan mikroskop elektron bersama-
sama dengan microarray fenotipe (PM) menyokong keputusan untuk aktiviti
antibakteria. Penyaringan PM adalah alat yang berharga dalam mencari bahan yang
boleh menghalang pertumbuhan bakteria dengan mempengaruhi pusat metabolisme
tertentu seperti pusat sintesis peptidoglikan. Laluan ini ampaknya menjadi sasaran
oleh kedua-dua ekstrak RC dan MBS pada titik metabolisme yang berbeza dari ubat
lain yang sedia ada. Titik-titik ini boleh menjadi sasaran untuk mencari agen terapi
baharu bagi mikrob yang dihaji. Keputusan kajian ini menunjukkan aktiviti
antioksidan yang kuat dari RC dan MBS berkemungkinan besar disebabkan oleh
sebatian antioksidan fenolik yang wujud dalam tanaman yang bertindak secara
individu atau secara sinergis tik untuk menjadikan pentingnya tanaman ini sebagai
sumber baru agen terapi.
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ACKNOWLEDGEMENTS
This work was highly supported by the superior guidance and encouragment of Dr.
Fatimah Abu Bakar, the supervisor committee. The finanical support from Univeristi
Putra Malaysia was highly helpfull to full all the requirments of this study. I want to
appreciate and thank Dr. Farid Azizi Jalilian for his kind advices and teaching which
he has provided to push this work forward.
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I certify that an Examination Committee has met on 3rd
May 2011 to conduct the
final examination of Rand R. Hafidh on her PhD thesis entitled “Antimicrobial,
Immunomodulatory and Tumor Cells-Selective Cytotoxic Activities of Brassica
oleracea L. and Vigna radiata L.” in accordance with Universiti Pertanian Malaysia
(Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree)
Regulations 1981. The Committee recommends that the student be awarded Doctor
of Philosophy degree.
Members of the Examination Committee were as follows:
Mariana Nor Shamsudin, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Chairman)
Son Radu, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Internal Examiner)
Alfi Khatib, PhD
Lecturer
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Internal Examiner)
Peter John Houghton, PhD
Professor
Pharmaceutical Science Research Division
King’s College London
United Kingdom
(External Examiner)
___________________________________
SHAMSUDDIN SULAIMAN, PhD
Professor and Deputy Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 21th
June 2011
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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been
accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.
The members of the Supervisory Committee were as follows:
Fatimah Abu Bakar, PhD
Professor
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Chairman)
Faridah Abas, PhD
Lecturer
Faculty of Food Science and Technology
Universiti Putra Malaysia
(Member)
Zamberi Sekawi, PhD
Associate Professor
Faculty of Medicine and Health Sciences
Universiti Putra Malaysia
(Member)
Fatemeh Jahanshiri, PhD
Lecturer
Faculty of Biotechnology and Biomolecular Sciences
Universiti Putra Malaysia
(Member)
___________________________________
HASANAH MOHD. GHAZALI, PhD
Professor and Dean
School of Graduate Studies
Universiti Putra Malaysia
Date: 14th
July 2011
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DECLARATION
I declare that the thesis is my original work except for quotations and citations which
have been duly acknowledged. I also declare that it has not been previously, and is
not concurrently, submitted for any other degree at Universiti Putra Malaysia or at
any other institution.
____________________________
RAND R. HAFIDH
Date: 2nd
June 2011
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TABLE OF CONTENTS
Page
DEDICATION ii
ABSTRACT iii
ABSTRAK vii
AKNOWLEDGMENTS xi
APPROVAL xii
DECLARATION xiv
LIST OF TABLES xix
LIST OF FIGURES xxiv
LIST OF ABBREVIATIONS xxx
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 7
2.1 The Natural Products and Phytochemicals 7
2.2 Red Cabbage (Brassica oleracea L. var capitata f.
rubra)
10
2.3 Mung Bean Sprout (Vigna radiata (L.) R. Wilczek) 13
2.4 The Current Drugs and their Problems 15
2.4.1 Antiviral Drugs 15
2.4.2 Antibacterial and Antifungal Drugs 17
2.4.3 Anticancer Drugs 21
2.4.4 The Immunomodulatory Drugs 24
2.5 Overcome the Problems of the Current Available
Drugs by Using Natural Products
27
2.6 The Notorious Microbes Addressed by the Current
Research
30
2.6.1 Respiratory Syncytial Virus (RSV) 30
2.6.2 Herpes Simplex Virus Type 1 (HSV-1) 31
2.6.3 Methicilline-Resistant Staphylococcus
aureus (MRSA)
33
2.6.4 Enterobacteriaceae 35
2.6.5 Pseudomonas aeruginosa 40
2.6.6 The Dermatophytes 41
2.6.7 Trichoderma harzianum 43
2.6.8 Aspergillus terrus 44
2.7
The Cancer Models Addressed by the Current
Research
45
2.7.1 Hepatocellular Carcinoma (HCC) 45
2.7.2 Cervical Cancer 46
3 ANTIVIRAL PROPERTIES OF RED CABBAGE
(BRASSICA OLERACEA L.) AND MUNG BEAN
SPROUT (VIGNA RADIATA L.)
3.1 Introduction 48
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3.2 Materials and Methods 49
3.2.1 Preparation of the Extracts 49
3.2.2 Preparation of Stock Extracts 50
3.2.3
Specific Cells Propagation and
Cryopreservation
50
3.2.4 Virus Propagation (RSV and HSV-1) 51
3.2.5 Virus Titration 51
3.2.6 Cytotoxicity Assay 52
3.2.7 Virus Yield Reduction Assay 56
3.2.8 Virucidal Activity Assay 59
3.2.9 Prophylactic Activity Assay 63
3.2.10
Cytokine Production by Human Embryonic
Fibroblast Cell Lines (MRC-5) in Response
to HSV-1 Infection and Extracts’ Treatment
65
3.2.11 Data Analysis 69
3.3 Results 69
3.3.1
Estimating the Antiviral Activity by Virus
Yield Reduction Assay
70
3.3.2
The Direct Virus Inactivation (DVI) and the
Inhibition of Virus Replication (IVC) by RC
Extract
71
3.3.3
The Direct Virus Inactivation (DVI) and the
Inhibition of Virus Replication (IVC) by
MBS Extract
76
3.3.4
Comparison of the Virucidal and the
Prophylactic Properties of RC and MBS
Extracts Against RSV together, and with
Ribavirin
84
3.3.5 Comparison of the Virucidal and the
Prophylactic Properties of RC and MBS
Extracts against HSV-1 together, and with
Acyclovir
88
3.3.6
The Antiviral Cytokines Produced by the
Treated-Viral Infected Cells
92
3.4 Discussion 97
3.5 Conclusion 115
4 THE CYTOTOXIC AND IMMUNMODULATORY
PROPERTIES OF RED CABBAGE (BRASSICA
OLERACEA L.) AND MUNG BEAN SPROUT (VIGNA
RADIATA L.)
4.1 Introduction 117
4.2 Materials and Methods 119
4.2.1
Cancer Cells Propagation and
Cryopreservation
119
4.2.2
Isolation of Human Peripheral Blood
Mononuclear Cells (PBMC)
120
4.2.3 MTS Tetrazolium Assay 120
4.2.4 Cytokine Production by PBMC 123
4.2.5 Sandwich Enzyme-Linked Immunosorbent 124
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Assay (ELISA)
4.2.6 Cytokine Production by Human Cancer Cell
Lines
127
4.2.7 Analysis of Apoptosis 127
4.2.8 Data Analysis 134
4.3 Results 135
4.3.1 Cytotoxicity on Human Cancer Cells 135
4.3.2 Non-Specific Immune Response by PBMC 141
4.3.3
Specific Immune Response by Human
Cancer Cells
150
4.3.4
RC and MBS Extract Induced Apoptosis and
Cell Cycle Arrest in Human Cancer Cells
160
4.3.5
The Apoptosis- and Cell Cycle- Related
Genes in Human Cancer Cells Treated with
RC and MBS Extracts
174
4.4 Discussion 185
4.4.1
The Cytotoxic Effects of RC and MBS
Extracts
185
4.4.2
Immunomodulatory Activities of RC and
MBS Extracts
202
4.5 Conclusion 207
5 ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES
OF OF RED CABBAGE (BRASSICA OLERACEA L.)
AND MUNG BEAN SPROUT (VIGNA RADIATA L.)
5.1 Introduction 210
5.2 Materials and Methods 211
5.2.1 Preparation of Stock Extracts 211
5.2.2 Microorganisms and Media 212
5.2.3
Selection of Multidrug Resistant (MDR)
Bacteria
213
5.2.4 Antimicrobial Sensitivity Tests 214
5.2.5 Scanning Electron Microscope (SEM) and
Transmission Electron Microscope (TEM)
217
5.2.6 Biolog Phenotype Microarray Technology 219
5.2.7 Data Analysis 222
5.3 Results 223
5.3.1 The MDR Bacteria 223
5.3.2 Disc Diffusion Assay for Bacteria 225
5.3.3 Microdilution Assay for Bacteria 228
5.3.4 Antimicrobial Assays for Fungi 235
5.3.5 Mode of the Antimicrobial Action 240
5.3.6
SEM and TEM Electron Microscope
Observations
241
5.3.7
Phenotype Microarray for E. coli and S.
aureus Bacteria Treated with RC and MBS
Extracts
244
5.4 Discussion 263
5.4.1
The Antimicrobial Activity of the Extracts
in the Current Research
263
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5.4.2 Antimicrobial Activity of RC Extract 265
5.4.3 Antimicrobial Activity of MBS Extract 274
5.5 Conclusion 282
6 SUMMARY, GENERAL CONCLUSION AND
RECOMMENDATIONS FOR FUTURE RESEARCH
284
REFERENCES/BIBLIOGRAPHY 293
APPENDIX 339
BIODATA OF STUDENT 343
LIST OF PUBLICATIONS 349