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UNIVERSITI PUTRA MALAYSIA

EFFECTS OF COLA NUT [COLA NITIDA (VENT.) SCHOTT & ENDL.] AQUEOUS EXTRACT ON RAT LIVER DURING

HEPATOCARCINOGENESIS

MOHAMMAD KADIVAR

FPSK(m) 2010 31

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EFFECTS OF COLA NUT [COLA NITIDA (VENT.)

SCHOTT & ENDL.] AQUEOUS EXTRACT ON RAT

LIVER DURING HEPATOCARCINOGENESIS

MOHAMMAD KADIVAR

MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2010

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EFFECTS OF COLA NUT [COLA NITIDA (VENT.) SCHOTT & ENDL.]

AQUEOUS EXTRACT ON RAT LIVER DURING HEPATOCARCINOGENESIS

By

MOHAMMAD KADIVAR

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

Fulfilment of the Requirements for the Degree of Master of Science.

November 2010

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This thesis is dedicated to my lovely parents, Maryam Nowroozalizadeh Shirazi and

Mohammad Rahim Kadivar, who taught me the value of education. I am deeply

indebted to them for their love, endless support and unwavering faith in me.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Master of Science

EFFECTS OF COLA NUT [COLA NITIDA (VENT.) SCHOTT & ENDL.]

AQUEOUS EXTRACT ON RAT LIVER DURING HEPATOCARCINOGENESIS

By

MOHAMMAD KADIVAR

November 2010

Chair: Professor Fauziah Othman, PhD

Faculty: Medicine and Health Sciences

The effect of Cola nitida aqueous extract in hepatocarcinogenesis induced male Sprague

Dawley rat livers, and elemental analysis of the cola nut was studied to investigate the

possible anticancer activity. The unprocessed cola nuts were observed for their surface

morphological structure under the scanning electron microscope (SEM). Together with

the imaging, samples were then elementally analyzed using energy dispersive x-ray

microanalysis attached to variable pressure scanning electron microscopy (EDX-

VPSEM). SEM study of cola nut illustrates numerous crystals packed in clusters within

the cell wall. The elemental analysis results revealed that the cola nut contained high

amount of oxygen and carbon, in addition to potassium, phosphorus and magnesium.

Potassium, magnesium and phosphorous have been well reported as co-factors of

antioxidant enzymes to protect the body from oxygen free radicals. Additionally, these

elements play important roles in metabolic mechanisms in the body.

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Hepatocarcinogenesis was induced in rat livers according to the modified Solt and

Farber method. Diethylnitrosamine (DEN) was injected into the rats at 200 mg/kg body

weight to initiate hepatocarcinogenesis and after two weeks this was followed by

feeding 0.02% 2-Acetylaminofluorene (AAF) to promote the hepatocarcinogenesis. The

DEN/AAF induced rats were treated with 1, 2.5, and 5% (w/v) concentrations of cola

nut extract or 0.001, 0.0025, and 0.005% w/v dilutions of glycyrrhizin as a drug control.

There were normal and cancer controls; in addition, three groups of normal rats were

treated with three concentrations of cola nut to observe the side effect of the cola nut on

normal livers.

The body and liver weight profile results of this study showed no significant difference

between treated groups compared to normal and cancer controls. The similarity in body

weight gain and relative liver weight results might occur because of the short length of

the in vivo experiment (eleven weeks).

The supplementation of cola nut extract decreased the level of plasma and microsomal

GGT and GST tumor marker enzymes significantly in DEN/AAF induced liver tissues

even better than glycyrrhizin. Additionally, it was revealed that cola nut extract has no

effect on the level of GST and GGT enzymes in normal cells.

The histological and ultrastructural examination as well as the lesions scoring results

demonstrated that the cola nut extract reduced neoplastic stage of the

hepatocarcinogenic liver cells more than glycyrrhizin based on their abnormal

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morphology, inflammation, necrosis, and fibrosis. Moreover, rat’s normal hepatocytes

treated with cola nut extract illustrated normal features.

TUNEL assay results showed the significant increase in the number of apoptotic cells in

hepatocarcinogenic liver tissues treated with cola nut extract and glycyrrhizin. These

results showed that cola nut did not induce the apoptosis in normal liver cells.

Quantitative real-time RT-PCR results revealed that although the level of alpha-

fetoprotein (AFP) mRNA increased in DEN/AAF induced liver cells, but the

supplementation of the cola nut and glycyrrhizin decreased it predominantly in

hepatocarcinogenic liver cells treated with cola nut extract.

These findings suggest that cola nut might act as a promising anticancer against

hepatocarcinogenesis with even higher efficacy compared to glycyrrhizin, without any

side effects in normal liver cells.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

KESAN EKSTRAK AKUEUS COLA NUT [COLA NITIDA (VENT.) SCHOTT &

ENDL.] TERHADAP HATI TIKUS SEMASA HEPATOCARCINOGENESIS

Oleh

MOHAMMAD KADIVAR

November 2010

Pengerusi: Profesor Fauziah Othman, PhD

Fakulti: Perubatan dan Sains Kesihatan

Kesan ekstrak akueus Cola nitida dalam hati tikus Sprague Dawley yang diaruh

hepatokarsinogenesis, dan analisis elemen terhadap cola nut dijalankan untuk mengkaji

kemunglanan aktiviti antikanser yang dimiliki. Cola nut yang belum diproses

diperhatikan untuk struktur morfologi permukaan di bawah SEM. Bersama dengan

pengimejan, analasis elemen sampel dijalankan menggunakan EDX-VPSEM. Kajian

SEM terhadap cola nut menunjukkan pelbagai hablur-hablur bersusun dalam kumpulan

di dinding sel. Hasil analisis unsur elemen menunjukkan cola nut mengandungi jumlah

oksigen dan karbon, yang tinggi di samping kalium, fosforus dan magnesium. Kalium,

magnesium dan fosforus telah diketahui umum sebagai ko-faktor bagi enzim

antioksidan untuk melindungi tubuh dari radikal bebas oksigen. Tambahan pula, unsur-

unsur ini memainkan peranan yang penting dalan mekanisme-mekanisme metabolik di

dalam tubuh.

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Aruhan hepatokarsinogenesis berdasarkan teknik Solt dan Farber yang diubah suai.

Sebanyak 200 mg/kg DEN disuntik ke dalam tikus untuk memulakan

hepatokarsinogenesis dan selepas 2 minggu diikuti dengan memberi 0.02% AAF dalam

makanan untuk penggalakan. Tikus yang diaruh DEN/AAF dirawat dengan 1, 2.5, dan

5% (w/v) kepekatan ekstrak cola nut atau 0.001, 0.0025, dan 0.005% w/v glycyrrhizin

sebagai dadah kawalan. Terdapat normal dan kawalan kanser, dan 3 kumpulan tikus

normal dirawat dengan tiga kepekatan cola nut yang berbeza untuk melihat kesan

sampingan cola nut pada hati normal.

Profil berat badan dan hati menunjukkan tiada perbezaan yang ketara antara kumpulan

yang dirawat berbanding dengan normal dan kawalan kanser. Persamaan dalam

penambahan berat badan dan hasil berat relatif hati kemungkinannya berlaku kerana

jangka masa eksperimen in vivo yang singkat (11 minggu).

Penambahan ekstrak cola nut mengurangkan paras plasma dan enzim penanda barah

mikrosom GGT dan GST dalam tisu hati yang diaruh DEN/AAF malah lebih baik dari

glycyrrhizin. Malah, ekstrak cola nut juga telah terbukti tiada kesan terhadap paras

enzim GST dan GGTs dalam sel normal.

Pemeriksaan histologi dan ultrastruktur beserta hasil skor lesi menunjukkan ekstrak cola

nut mengurangkan paras neoplastik hati sel hepatocarcinogenik lebih baik daripada

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glycyrrhizin. Tambahan lagi, hepatosit normal yang dirawat dengan ekstrak cola nut

menggambarkan ciri-ciri normal.

Asai TUNEL menunjukkan peningkatan yang signifikan dalam bilangan sel apoptotik

tisu hati hepatokarsinogenik dirawat dengan ekstrak cola nut dan glycyrrhizin. Hasil ini

menunjukkan bahawa cola nut tidak mengaruh apoptosis dalam sel hati normal.

Hasil kuantitatif real-time RT-PCR menunjukkan bahawa walaupun paras AFP mRNA

meningkat pada sel hati yang diaruh dengan DEN/AAF, penambahan cola nut dan

glycyrrhizin mengurangkan “predominantly” dalam sel hati tikus yang diaruh

hepatokarsinogenesis yang dirawat dengan ekstrak cola nut.

Hasil kajian ini menunjukkan bahawa cola nut berkemungkinan bertindak sebagai anti

kanser dan agen kemopreventif yang lebih baik berbanding dengan glycyrrhizn, tanpa

sebarang kesan sampingan dalam sel hati normal.

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ACKNOWLEDGMENT

First and foremost, I have to thank the GOD, without whose gracious help it would have

been impossible to accomplish this work.

I would like to express my sincere thanks to Professor Dr. Fauziah Othman for giving

me an opportunity to pursue the Master of Science program and for her support and

guidance throughout this project. I would also like to thank Professor Dr. Asmah

Rahmat for serving as a member of my MSc program committee.

I am thanking all the staff of Faculty of Medicine and Health Sciences as well as

Institute of Bioscience for their steady assistance and friendship. Additionally, I would

like to thank my friends for their encouragement in my way.

My special gratitude to my hero, my father, and my beloved mother, whose love and

affection is the source of inspiration and encouragement for my studies.

Finally yet importantly, I am eternally grateful for my dear wife, Zeynab

Adabimohazab, for her love, patience, sacrifices, unlimited support throughout the

years, and for being the sunshine of my life.

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I certify that a Thesis Examination Committee has met on 16 November 2010 to

conduct the final examination of Mohammad Kadivar on his thesis entitled “Effects of

Cola Nut [Cola Nitida (Vent.) Schott & Endl.] Aqueous Extract on Rat Liver During

Hepatocarcinogenesis” in accordance with the Universities and University College Act

1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A)106] 15 March

1998. The committee recommends that the student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Saidi bin Moin, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Patimah binti Ismail, PhD

Professor



(Internal Examiner)

Chong Pei Pei, PhD

Associate Professor



(Internal Examiner)

Nor Fadilah Rajab, PhD

Associate Professor

Universiti Kebangsaan Malaysia

(External Examiner)

_________________________

SHAMSUDDIN SULAIMAN, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 18 January 2011

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This thesis was submitted to the senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Fauziah Othman, PhD

Professor



(Chairman)

Asmah Rahmat, PhD

Professor



(Member)

_______________________________

HASANAH MOHD GHAZALI, PhD

Professor and Dean

School of Graduate Studies


Date:

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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.

__________________________

MOHAMMAD KADIVAR

Date: 16 November 2010

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TABLE OF CONTENTS

Page

DEDICATION

ABSTRACT

ABSTRAK

ACKNOWLEDGEMENTS

APPROVAL

DECLARATION

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATION

CHAPTER

1 INTRODUCTION

2 LITERATURE REVIEW

2.1 Cola nitida

2.1.1 Cola Nut

2.1.2 Elemental Analysis of Cola Nut

2.2 Energy Dispersive X-ray Microanalysis (EDX) Attached to

Variable Pressure Scanning Electron Microscopy (VPSEM)

2.3 Cancer

2.3.1 Liver Cancer

2.3.2 Carcinogenesis

2.3.3 Hepatocarcinogenesis

2.4 Liver

2.5 Anticancer Drugs and Glycyrrhizin

2.6 Chemical Carcinogens

2.6.1 Diethylnitrosamine (DEN)

2.6.2 2-Acetylaminofluorene (AAF)

2.7 Enzyme Tumor Markers Activities

2.7.1 Gamma-glutamyl Transpeptidase (GGT)

2.7.2 Glutathione S-transferase (GST)

2.8 Histology

2.8.1 Light Microscopy

2.8.2 Lesion Scoring

2.8.3 Transmission Electron Microscopy

2.9 Apoptosis

2.10 TUNEL Assay

2.11 Alpha-fetoprotein

2.11.1 Alpha-fetoprotein Gene Expression and

Hepatocellular Carcinoma

2.11.2 Quantitative Real-Time RT-PCR

2.11.3 Primer Design

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3 MATERIALS AND METHODS

3.1 Cola Nut

3.1.1 Elemental Analysis Using EDX-VPSEM

3.1.2 Preparation of Cola Nut Aqueous Extract

3.2 Diethylnitrosamine Preparation

3.3 2-Acetylaminofluorene Preparation

3.4 In vivo Study

3.4.1 Pre-treatment

3.4.2 Treatment

3.4.3 Post Treatment

3.5 Enzyme Tumor Markers Activities

3.5.1 Preparation of Cytosolic and Microsomal Fractions

3.5.2 Gamma-glutamyl transpeptidase Assay

3.5.3 Glutathione S-transferase Assay

3.6 Histology




3.7 TUNEL Assay

3.8 Real-time Quantitative Reverse Transcriptase Polymerase

Chain Reaction

3.8.1 RNA Extraction

3.8.2 Primer Design

3.8.3 Real-time Quantitative RT-PCR

3.8.4 Real-time RT-PCR Results Analysis

3.9 Data Analysis

4 RESULTS

4.1 Elemental Analysis

4.2 In Vivo Study

4.2.1 Body Weight Profile

4.2.2 Liver Weight and Relative Liver Weight

4.3 Histology




4.4 TUNEL Assay

4.5 Enzyme Tumor Markers Activity

4.5.1 Gamma-glutamyl Transpeptidase

4.5.2 Glutathione S-transferase

4.6 Expression Level of Alpha-Fetoprotein Gene

(Real-Time RT-PCR)

4.6.1 Primer Design

4.6.2 Quantitative Real-Time RT-PCR

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5 DISCUSSION

5.1 Elemental Analysis of Cola Nut

5.2 Body Weight profile, Liver Weight and Relative Liver Weight

5.3 Tumor Markers Enzymes Activity

5.3.1 Gamma-glutamyl Transpeptidase (GGT)

5.3.2 Glutathione S-transferase (GST)

5.4 Histology




5.5 Detection of Apoptosis by TUNEL Assay

5.6 Alfa-Fetoprotein Gene Expression

6 CONCLUSION

BIBLIOGRAPHY

APPENDICES

BIODATA OF STUDENT

LIST OF PUBLICATIONS

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