UNIVERSITI PUTRA MALAYSIA

IN VITRO STUDY OF AQUEOUS AND METHANOL EXTRACTS OF

TINOSPORA CRISPA (L.) HOOK. F. & THOMSON IN EARLY STAGE

ATHEROGENESIS

IHSAN SAFWAN BIN KAMARAZAMAN

FPSK(m) 2012 24

© COPYRIG

HT UPM

IN VITRO STUDY OF AQUEOUS AND METHANOL EXTRACTS OF TINOSPORA

CRISPA (L.) HOOK. F. & THOMSON IN EARLY STAGE ATHEROGENESIS

By

IHSAN SAFWAN BIN KAMARAZAMAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

April 2012

© COPYRIG

HT UPM

ii

DEDICATION

I dedicate this work for all of my family, especially to my mother, Sabariah Bt.

Ismail, my father, Kamarazaman B. Abd. Rahman and my wife, Solehatun Bt.

Mhd. Bani, for giving me inspiration to pursue my study in this field. Special

appreciation to my supervisor, Professor Dr. Zulkhairi Hj. Amom who contributed

greatly for my career development and personal growth. Last but not least, I

would like to express my appreciation to my research group members, Daryl,

Kamal, Amalina, Fazali, Sakinah, Khairunnur Fairuz, Nawal and Jalil and all

colleagues who were involved in this project.

© COPYRIG

HT UPM

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment of the requirement for the degree of Master Science

IN VITRO STUDY OF AQUEOUS AND METHANOL EXTRACTS OF

TINOSPORA CRISPA (L.) HOOK. F. & THOMSON IN EARLY STAGE

ATHEROGENESIS.

By

IHSAN SAFWAN BIN KAMARAZAMAN

April 2012

Chairman : Zulkhairi bin Haji Amom, PhD

Faculty : Medicine and Health Sciences

Tinospora crispa, locally known as ‘Patawali’ in Malaysia, is a plant belonging to

the family of Menispermaceae. It is a climber that can be found in primary

rainforest widely distributed in Malaysia, Indonesia, Thailand and Vietnam. T.

crispa has been traditionally used to treat diabetes, hypertension and lumbago

and reported to have antidiabetic, hypotensive and anti-inflammatory activity.

The aimed of this study was to investigate the antioxidant properties of this plant

as well as its ability to attenuate the release of oxidant and inflammatory

markers in induced oxidation and inflammation in human umbilical vein cells

(HUVECs).

© COPYRIG

HT UPM

iv

In vitro studies have been conducted to evaluate antioxidant properties of T.

crispa. The radical scavenging activity was tested by 1,1-diphenyl-2-

picrylhydraxyl (DPPH) assay. The result showed DPPH scavenging activity of T.

crispa aqueous (TCAE) and methanol extract (TCME) were 82 ± 1.78 and 73 ±

1.01%, respectively. The ability of T. crispa extracts to reduce Fe3+ to Fe2+ were

tested by FRAP assay and the result showed FRAP value of TCAE and TCME

were 1.04 ± 0.27 and 1.64 ± 0.06 mmol/L, respectively. Total flavonoids content

(TFC) and total phenolics content (TPC) were also measured. The result

showed TFC value of TCAE and TCME were 205.58 ± 3.5 and 223 ± 10.49 mg

QE/g sample, respectively while TPC value of TCAE and TCME were 32.58 ±

0.68 and 41.64 ± 0.97 mg GAE/mg sample, respectively.

The antioxidant enzymes activities and the level of anti-inflammatory markers in

HUVECs treated with TCAE and TCME to counter the oxidative effect by

hydrogen peroxide (H2O2) or inflammatory effect by tumor necrosis factor- α

(TNF-α) were also measured. HUVECs were seeded at 1 x 106 cell/well in 6-well

plate and the treatments were divided into 3 groups; normal control, negative

control, and treated groups. In the negative control (NC) group, HUVECs were

exposed to either 250 µM H2O2 or 10 ng/mL TNF-α alone, whereas in the

treated groups HUVECs were pretreated with various concentrations of TCAE

and TCME (100, 200, 400 and 600 µg/mL) for 30 minutes prior to exposure to

H2O2 (250 µM) or TNF-α (10 ng/mL). In the normal control groups, HUVECs

were incubated with culture medium only. The cells were incubated for 24 hours

at 37 oC with 5% CO2 supply for further analysis. Assays that were performed in

© COPYRIG

HT UPM

v

present study were antioxidant enzyme activities such as catalase (CAT),

superoxide dismutase (SOD) and glutathione peroxidase (GPx), lipid

peroxidation level by malondealdehyde (MDA) assay, and inflammatory markers

such as nitric oxide (NO), intercellular cell adhesion molecule (ICAM-1), vascular

cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1)

and macrophage colony stimulating factor (M-CSF).

Results of antioxidant enzymes activity assays (CAT, SOD and GPx) showed

TCAE and TCME at a concentration ranges from 100-600 µg/ml significantly

increased (p<0.05) the level of those antioxidant enzymes compared to NC.

Results of MDA assay showed significant reduction (p<0.05) of MDA level in

HUVECs treated with TCAE and TCME compared to NC. Concomitantly, the

level of NO expression in HUVECs treated with TCAE and TCME was

significantly elevated (p<0.05) compared to NC. In addition, inflammatory

markers assays showed TCAE and TCME have significantly reduced (p<0.05,

0.01) the secretion of ICAM-1, VCAM-1 and M-CSF as compared to NC.

However, secretion of MCP-1 was not reduced by the treatment of TCAE and

TCME. Taken together, this study suggest that TCAE and TCME can effectively

prevent oxidative stress by H2O2 and inflammation by TNF-α on HUVECs, which

might be importance in the treatment of atherosclerosis.

© COPYRIG

HT UPM

vi

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah master Sains

KAJIAN IN VITRO EKSTRAK AKUES DAN METANOL TINOSPORA CRISPA

(L.) HOOK. F. & THOMSON PADA PERINGKAT AWAL ATEROGENESIS

Oleh

IHSAN SAFWAN BIN KAMARAZAMAN

April 2012

Pengerusi : Zulkhairi bin Haji Amom, PhD

Fakulti : Perubatan & Sains Kesihatan

Tinospora crispa, dikenali sebagai ‘Patawali’ di Malaysia, adalah sejenis

tumbuhan tergolong dari famili Menispermaceae. Ia adalah pokok pemanjat

yang boleh ditemui di hutan primer di Malaysia, Indonesia, Thailand dan

Vietnam. T. crispa telah digunakan secara tradisi untuk merawat diabetis, darah

tinggi dan sakit belakang. T. crispa sebelum ini dilaporkan mempunyai kesan

antidiabetik, hipotensif dan anti-inflamasi. Tujuan kajian ini dijalankan adalah

untuk mengkaji keupayaan antioksidan tumbuhan ini dan juga keupayaan

tumbuhan ini merencat isyarat pengoksidaan dan inflamasi pada sel vena

endothelial umbilikal manusia (HUVECs) teraruh.

Kajian in vitro telah dijalankan untuk menilai kesan antioksidan oleh T. crispa.

Kesan perambatan radikal dilakukan menggunakan asai DPPH dan keputusan

© COPYRIG

HT UPM

vii

ujian ini menunjukkan kesan perambatan radikal DPPH oleh ekstrak akues T.

crispa (TCAE) adalah 82 ± 1.78% dan 73 ± 1.01% bagi ekstrak metanol

(TCME). Keupayaan ekstrak T. crispa untuk menurunkan Fe3+ kepada Fe2+

diuji dengan asai FRAP dan keputusannya menunjukkan nilai FRAP bagi TCAE

adalah 1.04 ± 0.27 mmol/L manakala nilai FRAP bagi TCME adalah 1.64 ± 0.06

mmol/L. Kandungan flavonoid total (TFC) dan kandungan fenolik total (TPC)

juga dikaji. Keputusan kajian ini menunjukkan nilai TFC bagi TCAE adalah

sebanyak 205.58 ± 3.5 QE/g sampel manakala nilai TPC bagi TCME adalah

sebanyak 223 ± 10.49 mg QE/g sampel. Nilai TPC bagi TCAE adalah sebanyak

32.58 ± 0.68 mg GAE/mg sampel manakala nilai TPC bagi TCME adalah

sebanyak 41.64 ± 0.97 mg GAE/mg sampel.

Kajian ini juga menilai akitiviti enzim antioksidan dan aras penghasilan isyarat

anti-inflamasi pada sel HUVEC yang dirawat TCAE dan TCME terhadap kesan

pengoksidaan oleh hidrogen peroksida (H2O2) dan inflamasi oleh faktor nekrosis

tumor-α (TNF-α). HUVEC sebanyak 1 x 106 dikultur pada plat 96 telaga dan

rawatan dibahagikan kepada 3 kumpulan iaitu kumpulan kawalan normal,

kumpulan kawalan negatif (NC) dan juga kumpulan rawatan. Bagi kumpulan

kawalan negatif, HUVEC hanya dirawat samada H2O2 dengan kepekatan 250

µM atau TNF-α dengan kepekatan 10 ng/ml manakala bagi kumpulan rawatan,

HUVEC dirawat dengan TCAE atau TCME pada kepekatan 100, 200, 400 dan

600 µg/ml selama 30 minit sebelum oleh samada kepada H2O2 (250 µM) atau

TNF-α (10 ng/ml). Bagi kumpulan normal pula, hanya mempunyai HUVEC. Sel

kemudian dieram dalam inkubator selama 24 jam pada 37 oC dengan

© COPYRIG

HT UPM

viii

kehadiran 5% CO2 bagi analisis seterusnya. Asai yang dilakukan dalam kajian

ini ialah asai enzim antoksidan seperti catalase (CAT), superoxide dismutase

(SOD) dan glutathione peroxide (GPx), asai peroksidaan lipid iaitu asai

malondealdehyde (MDA) dan juga asai inflamasi seperti nitrik oksida (NO),

intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion

molecule-1 (V-CAM-1), monocyte chemotactic protein-1 (MCP-1) dan

macrophage cell stimulating factor (M-CSF).

Bagi asai aktiviti enzim antioksidan (CAT, SOD dan GPx), keputusan ujian ini

menunjukkan TCAE dan TCME pada kepekatan antara 100-600 µg/ml telah

menyebabkan peningkatan yang ketara (p<0.05) bagi aras enzim antioksida

tersebut. Bagi asai MDA pula, keputusan ujian ini menunjukkan penurunan

ketara (p<0.05, 0.01) bagi aras MDA dalam HUVEC yang dirawat oleh TCAE

dan TCME berbanding NC. Pada masa yang sama, penghasilan NO dalam

HUVEC yang dirawat oleh TCAE dan TCME meningkat secara ketara (p<0.05)

berbanding NC. Selain itu, isyarat inflamasi menunjukkan TCAE dan TCME

telah menyebabkan penurunan signifikan (p<0.05, 0.01) pada aras penghasilan

ICAM-1, VCAM-1 dan M-CSF berbanding NC. Walaubagaimanapun,

penghasilan MCP-1 tidak berkurangan dengan rawatan TCAE dan TCME.

Secara keseluruhannya, kajian ini menunjukkan bahawa TCAE dan TCME

boleh melindungi sel HUVEC daripada kesan pengoksidaan oleh H2O2 dan juga

kesan inflamasi oleh TNF-α, menunjukkan kegunaannya dalam rawatan

aterosklerosis.

© COPYRIG

HT UPM

ix

ACKNOWLEDGEMENT

First and foremost, I am grateful to Allah with His blessing and compassion. I

wish to express my gratitude all the people that help and support me in finishing

this study. A sincere gratitude to my supervisor, Associate Professor Dr.

Zulkhairi Hj. Amom for his support, advice and lesson that he gave to me. He is

always patient to me in teaching me about the proper way to perform research

and guide me throughout my study and always gives brilliant suggestions and

solutions for me. I also would like to gratitude my co-supervisors, Dr. Abdah

Mohd Akim and Dr Rasadah Mat Ali for their kind advises and supports to me. I

want to acknowledge them for their scientific advise, critical reading of the thesis

and for their valuable comment and suggestion. I’m also would like to show my

gratitude to all the staffs of Human Anatomy Lab, Faculty of Medicine and

Human Health, UPM for helping me in preparing all the equipment to be ready in

the laboratory. I would like to thanks to all my fellow postgraduate students,

Daryl, Kamal, Amalina, Khairunnur Fairuz, Fazali, Nawal, Jalil, Faiz, Saifuddin,

Afiq, Khairulashraf and all the people that helped me during my period of study.

Last but not least, my special gratitude for my mother, father, brothers, sister

and my wife for their loving support.

© COPYRIG

HT UPM

x

I certify that an Examination Committee has met on 5 April 2012 to conduct the final examination of Ihsan Safwan Bin Kamarazaman on his Master thesis entitle In Vitro Study of Aqueous and Methanol Extracts of Tinospora Crispa (L.) Hook. F. & Thomson in Early Stage Atherogenesis in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulation 1981. The Committee recommends that the student be awarded the Master of Science.

Members of the Examination Committee were as follows Dr. Mohamad Taufik Hidayat Baharuldin Senior Lecturer Department of Human Anatomy Faculty of Medicine and Health Sciences Universiti Putra Malaysia Prof. Asmah Rahmat Professor Department of Nutrition and Dietetics Faculty of Medicine and Health Sciences Universiti Putra Malaysia Dr. Zainul Amiruddin Zakaria Associate Professor Department of Biomedical Science Faculty of Medicine and Health Sciences Universiti Putra Malaysia Dr. Nor Fadilah Rajab Associate Professor Biomedical Science Programme School of Diagnostic and Applied Health Sciences Faculty of Health Sciences Universiti Kebangsaan Malaysia Jalan Raja Muda Abdul Aziz 50300 Kuala Lumpur

HASANAH MOHD. GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date

© COPYRIG

HT UPM

xi

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the Master of Science. The members of the Supervisory Committee were as follows:

Zulkhairi Hj. Amom, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia. (Chairman) Abdah Md Akim, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia. (member) Rasadah Mat Ali, PhD Senior Research Officer Natural Product Division Forest Research Institute Malaysia. (member) BUJANG BIN KIM HUAT

Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

© COPYRIG

HT UPM

xii

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 any other institutions.

IHSAN SAFWAN BIN KAMARAZAMAN

Date: 5 April 2012

© COPYRIG

HT UPM

xiii

TABLE OF CONTENT

Page ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENT ix APPROVAL x DECLARATION xii LIST OF FIGURES xvii LIST OF APPENDICES xviii LIST OF TABLES xix LIST OF ABBREVIATION xx CHAPTER 1 INTRODUCTION 1.1 Background 1 1.2 Objectives of the Study 5 1.2.1 General Objective 5 1.2.2 Specific Objectives 6 1.3 Hypotheses 6 2 LITERATURE REVIEW 8 2.1 Atherosclerosis 8 2.1.1 Atherogenesis 9 2.2 Free Radical 12 2.3 Reactive Oxygen Species (ROS) 12 2.3.1 ROS and Atherosclerosis 13 2.4 Antioxidants 14 2.4.1 Dietery Antioxidants and Atherosclerosis 14 2.4.2 Polyphenols 15 2.4. 2.4.21 Polyphenols and Atherosclerosis 15 2.4.3 Flavonoids 16 2.4.3.1 Flavonoids and Atherosclerosis 17 2.5 Antioxidant Enzymes 18 2.6 Endothelial Cell 20 2.6.1 Endothelial Cell Injury 21 2.6.2 Human Umbillical Vein Endothelial Cells (HUVECs) 24 2.7 Nitric Oxide 26

2.7.1 Nitric Oxide and Atherosclerosis 27 2.8 Adhesion Molecule 28 2.8.1 Intercellular Cell Adhesion Molecule (ICAM-1) 29 2.8.2 Vascular Cell Adhesion Molecule (VCAM-1) 29 2.8.3 Soluble Adhesion Molecule and Atherosclerosis 30 2.9 Chemokines 31 2.9.1 Monocyte Chemotactic Protein-1 (MCP-1) 32 2.9.3 Macrophage Colony Stimulating Factor (M-CSF) 34

© COPYRIG

HT UPM

xiv

2.10 Lipoprotein 37 2.10.1 Oxidized Low Density Lipoprotein (ox-LDL) and

Atherosclerosis 40

2.11 Tinospora crispa 41 2.11.1 Taxonomy of Tinospora crispa 41 2.11.2 Chemical Content of Tinospora crispa 41 2.11.3 Traditional Usage of T. crispa 42 2.11.4 Medicinal Properties of T. crispa 42 3 AQUEOUS AND METHANOL EXTRACTS OF TINOSPORA

CRISPA POSESS INHIBITORY PROPERTIES ON H2O2-INDUCED LIPID PEROXIDATION AND MODULATE ANTIOXIDANT ENZYME ACTIVITY

45

3.1 Introduction 45 3.2 Materials And Methods 47 Material 3.2.1 Plant and Experimental HUVECs 47 3.2.2 Chemicals and Reagents 47 3.2.2.1 Antioxidant Activity, Phenolics and Flavonoids

Content of Tinospora crispa 47

3.2.2.2 In Vitro Study of TCAE and TCME on HUVEC Signaling Molecule and Antioxidant Enzyme

48

3.2.3 Apparatus 48 Methods 49 3.2.4 Preparation of TCAE and TCME 49 3.2.5 Antioxidant Activity of TCAE and TCME 50 3.2.5.1 1,1-diphenyl-2-picrylhydrazil (DPPH) Test 50 3.2.5.2 Ferric Reducing Antioxidant Power (FRAP) 50 3.2.6 Determination of Total Phenolics Content (TPC) 51 3.2.7 Determination of Total Flavonoids Content (TFC) 52 3.2.8 Cell Culutures Procedures 52 3.2.8.1 Preparation of Cell Culture Medium 52 3.2.8.2 Thawing the Cells 52 3.2.8.3 Subculturing the Cells 53 3.2.8.4 Cryopreservation of Cells 54 3.2.9 MTT assay 54 3.2.9.1 Determination of Huvecs Viability Against

TCAE and TCME 55

3.2.9.2 Determination o The Ability of TCAE and TCME to Attenuate the Cytotoxic Effect of H2O2

55

3.2.10 Preparation of Cells 56 3.2.11 Preparation of Samples 57 3.2.12 Determination of Lipid Peroxidation Index 57 3.2.12.1 MDA Assay 57 3.2.12.2 Protein Assay 58 3.2.13 Antioxidant Enzyme Assay 58

© COPYRIG

HT UPM

xv

3.2.13.1 CAT Activity Assay 58 3.2.13.2 SOD Activity Assay 60 3.2.13.3 GPx Activity Assay 62 3.2.14 Statistical Analysis 64 3.3 Result 65 3.3.1 Antioxidant Activity, Total Flavonoids Content and

Total Phenolics Content of TCAE and TCME 65

3.3.2 Determination of Median Inhibitory Concentration (IC50) of TCAE and TCME on HUVECs

67

3.3.3 Effective Concentration of TCAE and TCME to Prevent HUVECs from Oxidation by H2O2

70

3.3.4 Determination of Lipid Peroxidation Index 73 3.3.5 CAT Activity Assay 75 3.3.6 SOD Activity Assay 77 3.3.7 GPx Activity Assay 79 3.4 DISCUSSION 81 3.5 CONCLUSION 85 4

AQUEOUS AND METHANOL EXTRACTS OF TINOSPORA CRISPA POSSESS INHIBITORY PROPERTIES ON TNF-α-INDUCED INFLAMMATION ON HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS (HUVECS)

86

4.1 Introduction 86 4.2 Materials And Methods 88 Materials 88 4.2.1 Chemical and Reagent 88 4.2.2 Apparatus 88 Methods 89 4.2.3 Preparation of Cells 89 4.2.4 Total NO Assay 90 4.2.5 Determination of Adhesion Molecule Expression 91 4.2.5.1 Intercellular Adhesion Molecule-1 (ICAM-1)

Assay 91

4.2.5.2 Vascular Cell Adhesion Molecule-1 (VCAM-1) Assay

92

4.2.6 Determination of Inflammatory Marker 94 4.2.6.1 Monocyte Chemotactic Protein-1 (MCP-1)

Assay 94

4.2.6.2 Macrophage Colony Stimulating Factor (M-CSF) Assay

95

4.2.7 Statistical Analysis 97 4.3 Results 97 4.3.1 Effect of TCAE and TCME on NO Expression 97 4.3.2 Effect of TCAE and TCME on ICAM-1 Expression 100 4.3.3 Effect of TCAE and TCME on VCAM-1 Expression 102 4.3.4 Effect of TCAE and TCME on MCP-1 Expression 104 4.3.5 Effect of TCAE and TCME on M-CSF Expression 106

© COPYRIG

HT UPM

xvi

4.4 Discussion 108 4.5 Conclusion 112 5 DISCUSSION 113 5.1 General Discussion 113 5.2 Conclusion 118 5.3 Recommendations 119 REFERENCES 120 BIODATA OF STUDENT 150 LIST OF PUBLICATIONS 152 APPENDICES 153