UNIVERSITI PUTRA MALAYSIA

HYPOGLYCEMIC AND ANTIOXIDATIVE EFFECTS OF EUGENIA AROMATICA AND ARCHIDENDRONE JIRINGA ON DIABETIC RATS

RADHIAH BINTI SHUKRI

FSTM 2006 20

HYPOGLYCEMIC AND ANTIOXIDATIVE EFFECTS OF EUGENIA AROMATICA AND ARCHIDENDRONE JIRINGA ON DIABETIC RATS

RADHIAH BINTI SHUKRI

MASTER OF SCIENCE UNIVERSITI PUTRA MALAYSIA

2006

HYPOGLYCEMIC AND ANTIOXIDATIVE EFFECTS OF EUGENIA AROMATICA AND ARCHIDENDRONE JIRINGA ON DIABETIC RATS

By

RADHIAH BINTI SHUKRI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of Science

June 2006

To my husband and family…….

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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

HYPOGLYCEMIC AND ANTIOXIDATIVE EFFECTS OF EUGENIA

AROMATICA AND ARCHIDENDRONE JIRINGA ON DIABETIC RATS

By

RADHIAH BINTI SHUKRI

June 2006

Chairman : Professor Suhaila binti Mohamed, PhD

Faculty : Food Science and Technology

The study conducted for 15 weeks involved 56 Sprague Dawley male rats aged three

weeks that were divided into seven groups. Two control groups were normal rats and

induced-diabetic rats given a basal diet, four other groups were 2 normal rat groups and

2 induced-diabetic rat groups either supplemented with a basal diet containing 5% of

cloves (Eugenia aromatica) or jering (Archidendrone jiringa) respectively. The basal

diet that contained Glibenclamide (3mg/kg body weight) was supplemented to the

remaining one group of diabetic rats. Body weight and feed consumption were

monitored weekly and daily respectively. During a 3 weeks interval, blood samples were

drawn via cardiac puncture for the purpose of biochemical analysis that consisted of

glutathione peroxidase (GSH-Px), catalase (CAT) and superoxide dismutase (SOD)

activities; malondialdehyde (MDA) level and levels of urea, creatinine, alanine

aminotransferase (AST) and aspartate aminotransferase (AST). Somatic index and

histological changes of liver, heart, lung, eye, brain, kidney and pancreas of the

experimental rats were also evaluated.

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The results observed showed a slight lowering of blood glucose level of 6.1±0.4 and

6.2±0.5 mmol/l for cloves and jering supplemented STZ-diabetic rats respectively. The

body weight of the diabetic rats supplemented with the herbs was also improved with R2

value of 0.9924 and 0.9068 for jering and cloves, respectively. Weak anti-oxidative

property in blood and organs was revealed with the supplementation of the herbs but

was more effective in cloves. While evidence of toxicity of jering was shown mainly in

the liver, kidney and heart of normal and diabetic rats, cloves was seen to be toxic to the

pancreas of diabetic rats through histology. Jering-supplemented normal and diabetic

groups had high cardiosomatic index with 0.51±0.11 (NJ) and 0.49±0.04 (DJ), necrotic

hepatocytes and Kupffer cells of 50.5±5.0 (NJ) and 71.2±5.2 (DJ), respectively.

However, toxicity effect of these herbs towards certain organs at the 5% dose given

suggests that a dose-response effect need to be further studied.

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

KESAN HIPOGLISEMIK DAN CIRI-CIRI ANTIOKSIDAN OLEH EUGENIA AROMATICA DAN ARCHIDENDRONE JIRINGA TERHADAP TIKUS

KENCING MANIS

Oleh

RADHIAH BINTI SHUKRI

Jun 2006

Pengerusi : Profesor Suhaila binti Mohamed, PhD

Fakulti : Sains dan Teknologi Makanan

Kajian yang telah dijalankan selama 15 minggu melibatkan 56 ekor tikus Sprague

Dawley jantan yang berumur 3 minggu yang dibahagikan kepada 7 kumpulan.

Sementara 2 kumpulan kawalan tikus normal dan tikus kencing manis yang diaruh

hanya diberikan diet normal, 4 lagi kumpulan yang terdiri daripada 2 kumpulan tikus

normal dan 2 kumpulan tikus kencing manis diberikan sama ada diet yang mengandungi

5% cengkih (Eugenia aromatica) atau 5% jering (Archidendrone jiringa). Diet yang

mengandungi Glibenclamide (3mg/kg berat badan) diberikan kepada satu lagi kumpulan

tikus kencing manis. Berat badan diselia seminggu sekali manakala jumlah makanan

diselia setiap hari. Setiap 3 minggu, sampel darah diambil melalui jantung untuk

dianalisa secara biokimia iaitu aktiviti glutation peroksidase (GSH-Px), katalase (CAT)

and superoksid dismutase (SOD); paras malondialdehid (MDA) dan paras urea,

kreatinin, alanin aminotransferase (AST) dan aspartat aminotransferase (AST). Indeks

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somatik dan perubahan histologi bagi hati, jantung, paru-paru, mata, otak, buah

pinggang dan pankreas tikus eksperimen turut dinilai.

Keputusan yang diperolehi menunjukkan penurunan yang tidak ketara paras glukos

dalam darah sebanyak 6.1±0.4 dan 6.2±0.5 mmol/l masing-masing bagi tikus kencing

manis STZ yang diberikan cengkih dan jering. Berat badan tikus-tikus kencing manis

yang diberikan herba juga menunjukkan pencapaian yang baik dengan nilai R2 0.9924

dan 0.9068 for masing-masing bagi jering dan cengkih. Penstabilan ciri-ciri tekanan

oksidatif yang tidak ketara dalam darah dan organ-organ juga ditunjukkan dengan

pemberian herba tetapi cengkih lebih menunjukkan kesan yang baik. Ketoksikan jering

dapat dikesan terutamanya dalam hati, buah pinggang dan jantung tikus normal dan tikus

kencing manis sementara cengkih menunjukkan kesan toksik pada pankreas tikus

kencing manis melalui histologi. Kumpulan tikus normal dan kencing manis yang

diberikan jering mempunyai kardiosomatik indeks yang tinggi dengan nilai 0.51±0.11

(NJ) dan 0.49±0.04 (DJ) serta hepatosit nekrotik dan sel Kupffer yang tinggi dengan

nilai 50.5±5.0 (NJ) dan 71.2±5.2 (DJ).

Kesan toksik 5% herba ini terhadap organ-organ tertentu menunjukkan bahawa kajian

lanjut berkenaan dengan respon kepada dos yang berbeza perlu dijalankan.

ACKNOWLEDGEMENTS

Alhamdulillah, first of all I would like to express my thanks and gratitude to Almighty

Allah S.W.T. who has given me the capability to complete this research and my selawat

and salam to His righteous messenger, prophet Mohammad S.A.W.

I would like to take this opportunity to express my deepest gratitude to my supervisor,

Prof. Suhaila binti Mohamed for her valuable suggestions, constructive criticisms,

guidance and patience throughout the research. Special appreciation and gratitude is

extended to Assoc. Prof. Noordin bin Mohamed Mustapha and Dr. Nazimah binti Sheikh

Abdul Hamid for the constant guidance, tremendous encouragement and constructive

comments.

Special thanks to all the staff of Faculty of Food Science and Technology and Faculty of

Veterinary Medicine especially staffs of Animal House, Post Mortem Laboratory,

Histology Laboratory and Biochemistry Laboratory to have contributed in one way or

another towards the success of the research.

My heartful gratitude goes to my husband and family for their encouragement, patience,

understanding, support and unwavering love throughout the years of my study.

To Patricia, Siti Anarita, Imilia, Azlina, Nora, Ashraf, Ainul Salhani, and Sanaz whose

help, suggestions, comments and moral supports have helped in the improvement and

completion of this thesis- a million thanks to all of you. Last but not least, thanks to

everybody that was involved that contributes to the success of this research.

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I certify that an Examination Committee has met on 30 Jun 2006 to conduct the final examination of Radhiah binti Shukri on her Master of Science thesis entitled “Hypoglycemic and Antioxidative Effects of Eugenia aromatica and Archidendrone jiringa on Diabetic Rats” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Azizah binti Abdul Hamid, PhD Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Asmah binti Rahmat, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Maznah binti Ismail, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Jamaluddin bin Mohamed, PhD Professor Faculty of Health Sciences Universiti Kebangsaan Malaysia (External Examiner)

_________________________________ HASANAH MOHD. GHAZALI, PhD

Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:

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This thesis 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 are as follows: Suhaila binti Mohamed, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Noordin bin Mohamed Mustapha, PhD Associate Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Member) Nazimah binti Sheikh Abdul Hamid, PhD Senior Lecturer Faculty of Food Science and Technology Universiti Putra Malaysia (Member)

_______________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia

Date: 14 DECEMBER 2006

DECLARATION I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.

________________________

RADHIAH BINTI SHUKRI

Date:

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TABLE OF CONTENTS DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF ABBREVIATIONS

Page 2 3 5 7 8 10 14 17 18 20

CHAPTER

1 2

INTRODUCTION LITERATURE REVIEW 2.1 Classification of Diabetes Mellitus 2.1.1 Type 1 Diabetes Mellitus 2.1.2 Type 2 Diabetes Mellitus 2.2 Oxidative Stress 2.2.1 Definition 2.2.2 Reactive Oxygen and Free Radicals 2.2.3 Oxidative Stress in Diabetes Mellitus 2.2.4 Antioxidant Enzymes 2.2.5 Lipid Peroxidation 2.3 Complications in Diabetes Mellitus 2.3.1 Retinopathy 2.3.2 Neuropathy 2.3.3 Nephropathy 2.3.4 Hepatoxicity 2.3.5 Cardiovascular Disorders 2.4 Glycation and Advanced Glycation Endproducts 2.4.1 Harmful Effects of Glycation and AGEs 2.5 Common Drugs for Diabetes Mellitus 2.6 Plants as Potential Anti-diabetes 2.7 Eugenia aromatica 2.7.1 Mycology of Eugenia aromatica 2.7.2 Therapeutic Uses 2.7.3 General Composition and Properties 2.8 Archidendrone jiringa 2.8.1 Mycology of Archidendrone jiringa 2.8.2 Therapeutic Uses

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25 25 25 26 27 27 28 31 33 37 38 39 40 42 44 47 48 50 52 53 61 61 62 63 67 67 68

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2.8.3 General Composition and Properties MATERIALS AND METHODS 3.1 Materials 3.2 Methods 3.2.1 Plant material 3.2.2 Specimens 3.2.3 Induction of Experimental Diabetes 3.2.4 Experimental Design 3.2.5 Feed Preparation 3.2.6 Sampling 3.2.7 Plasma and Red Blood Cells Preparation 3.2.8 Biochemical Analysis 3.2.9 Statistical Analysis EFFICACY OF ARCHIDENDRONE JIRINGA AND EUGENIA AROMATICA ON STREPTOZOTOCIN-INDUCED DIABETIC RATS 4.1 Introduction 4.2 Materials and Methods 4.2.1 Feed Consumption, Body Weight Blood and Glucose

Level Monitoring 4.2.2 Biochemical Analysis 4.3 Results and Discussion 4.3.1 Feed Consumption 4.3.2 Body Weight 4.3.3 Blood Glucose Level 4.3.4 Lipid Peroxidation 4.3.5 Glutathione Peroxidase Activity 4.3.6 Superoxide Dismutase Activity 4.3.7 Catalase Activity TISSUE BIOCHEMICAL AND MORPHOLOGIC CHANGES IN STREPTOZOTOCIN-INDUCED DIABETIC RATS 5.1 Introduction 5.2 Materials and Methods 5.2.1 Organ Extract Preparation 5.2.2 Biochemical Analysis 5.2.3 Organ Biomarkers 5.2.4 Histology 5.3 Results and Discussion 5.3.1 Heart 5.3.2 Kidney 5.3.3 Liver 5.3.4 Brain 5.3.5 Pancreas

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71 71 72 72 73 73 73 74 74 75 75 79

80 80 80

80 81 81 81 83 86 91 95 97 100

102 102 102 103 103 103 103 104 104 115 131 149 154

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5.3.6 Lungs 5.3.7 Lens GENERAL CONCLUSION

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REFERENCES APPENDICES BIODATA OF THE AUTHOR

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LIST OF TABLES

Table

Page

2.1

2.2

2.3

2.4

2.5

2.6

2.7

4.1

4.2

4.3

4.4

4.5

4.6

4.7

5.1

Some factors determining the status of oxidative stress in biological systems.

Common drugs used for diabetes mellitus.

Anti-diabetes plants and their effects.

Anti-diabetes plants and active compounds responsible for the hypoglycaemic effects.

Constituents identified in clove bud oil.

Proximate composition of cloves (100g edible portion).

Proximate composition of jering (100g edible portion).

The feed consumption (g/3 weeks) of rats during the experimental period (Mean± SD).

The body weight (g/3 weeks) of rats during the experimental period (Mean± SD). The blood glucose level (mmol/l) of rats during the experimental period (Mean± SD). The MDA level in plasma (nmol/ml) of rats during the experimental period (Mean± SD). The GSH-Px activity in red blood cells (U/ml) of rats during the experimental period (Mean± SD). The SOD activity in red blood cells (U/min) of rats during the experimental period (Mean± SD). The CAT activity in red blood cells (U/min) of rats during the experimental period (Mean± SD). The weight of heart and cardiosomatic index of rats at the end of the experimental period (Mean ± SD).

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52

55

59

64

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69

82

84

87

92

96

98

101

105

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5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

5.10

5.11

5.12

5.13

5.14

5.15

5.16

The cardiac oxidative and anti-oxidative status of rats at the end of the experimental period (Mean ± SD). Quantitative histology scores of lesion in the heart of experimental groups (Mean ± SD). The weight of the kidney and nephrosomatic index of rats at the end of the experimental period (Mean ± SD). The renal oxidative and anti-oxidative status of rats at the end of the experimental period (Mean ± SD). Urea in plasma (mmol/l) of experimental rats during the experimental period (Mean± SD). Creatinine (umol/l) in plasma of experimental rats during the experimental period (Mean± SD). Quantitative histology in the kidney of experimental groups (Means ± SD). The weight of the liver and hepatosomatic index of rats at the end of the experimental period (Mean± SD). The liver oxidative and anti-oxidative status of rats at the end of the experimental period (Mean± SD). ALT (U/l) level in plasma of experimental rats during the experimental period (Mean± SD). AST (U/l) level in plasma of experimental rats during the experimental period (Mean± SD). Quantitative histology in the liver of experimental groups. The weight of the brain and encephalosomatic index of rats at the end of the experimental period (Mean± SD). The brain oxidative and anti-oxidative status of rats at the end of the experimental period (Mean± SD). Quantitative histology in the brain of experimental groups.

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116

118

122

124

127

132

134

137

139

141

149

150

153

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5.17

5.18

5.19

5.20

5.21

The weight of the pancreas and pancreasomatic index of rats at the end of the experimental period (Mean±SD). The pancreas oxidative and anti-oxidative status of rats at the end of the experimental period (Mean±SD). Quantitative histology in the pancreas of experimental groups. The weight of the lungs and pneuosomatic index of rats at the end of the experimental period (Mean±SD).

The lungs oxidative and anti-oxidative status of rats at the end of the experimental period (Mean± SD).

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LIST OF FIGURES

Figure Page

2.1

2.2

2.3

2.4

Products of free radicals. Glycation of a protein by glucose and the subsequent formation of AGEs. The cloves of Eugenia aromatica The seeds of Archidendrone jiringa

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LIST OF PLATES

Plate

Page

5.16

5.17

5.18

5.19

5.20

5.21

5.22

5.23

5.24

5.25

5.26

5.27

5.28

5.29

5.30

5.31

5.32

5.33

5.34

5.35

5.36

5.37

Photomicrograph, heart of rat from N group at necropsy. Photomicrograph, heart of rat from D group at necropsy. Photomicrograph, heart of rat from DC group at necropsy. Photomicrograph, heart of rat from DJ group at necropsy. Photomicrograph, heart of rat from DG group. Photomicrograph, heart of rat from DG group at necropsy. Photomicrograph, heart of rat from D group at necropsy. Photomicrograph, kidney of rat from N group at necropsy. Photomicrograph, kidney of rat from D group at necropsy. Photomicrograph, kidney of rat from N group at necropsy. Photomicrograph, kidney of rat from D group at necropsy. Photomicrograph, kidney of rat from D group at necropsy. Photomicrograph, kidney of rat from NJ group at necropsy Photomicrograph, kidney of rat from NJ group at necropsy. Photomicrograph, liver of rat from N group at necropsy. Photomicrograph, liver of rat from D group at necropsy. Photomicrograph, liver of rat from NJ group at necropsy. Photomicrograph, liver of rat from NJ group at necropsy. Photomicrograph, liver of rat from DJ group at necropsy. Photomicrograph, liver of rat from N group at necropsy. Photomicrograph, liver of rat from D group at necropsy. Photomicrograph, liver of rat from NJ group at necropsy.

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114

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115

128

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129

129

130

130

131

143

144

144

145

145

146

146

147

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5.38

5.39

5.40

5.41

5.42

5.43

5.44

5.45

5.46

5.47

5.48

Photomicrograph, liver of rat from NJ group at necropsy. Photomicrograph, liver of rat from DJ group at necropsy. Photomicrograph, brain of rat from N group at necropsy. Photomicrograph, pancreas of rat from N group at necropsy. Photomicrograph, pancreas of rat from D group at necropsy. Photomicrograph, pancreas of rat from NJ group at necropsy. Photomicrograph, pancreas of rat from DC group at necropsy. Photomicrograph, pancreas of rat from DC group at necropsy. Photomicrograph, pancreas of rat from NJ group at necropsy. Photomicrograph, lens of rat from DG group at necropsy. Photomicrograph, lens of rat from D group at necropsy.

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154

162

162

163

163

164

164

170

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LIST OF ABBREVIATIONS

3-DG

AGEs

ALT

AST

C

CAT

CML

D

DC

DJ

DG

DM

DR

EDTA

g

G

GBM

GFR

GSH

GSH-Px

GSSG

H2O2

3-deoxyglucosones

Advanced glycation end product

Alanine aminotransferase

Aspartate aminotransferase

Cloves

Catalase

N-carboxymethyl-lysine

Diabetic control

Diabetic rats treated with cloves

Diabetic rats treated with jering

Diabetic rats treated with Glibenclamide

Diabetes mellitus

Diabetic retinopathy

Ethylene diamine tetra-acetic acid

Gram

Glibenclamide

Glomerular basement membrane

Glomerular filtration rate

Glutathione

Glutathione peroxidase

Oxidized glutathione

Hydrogen peroxides

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HCL

IDDM

IU

J

KCl

MDA

mg

MGO

Ml

N

NC

NJ

NASH

NIDDM

NO

O2

PKC

RBC

ROS

SD

SOD

STZ

TBA

Hydrochloric acid

Insulin-dependent diabetes mellitus

International Unit

Jering

Potassium chloride

Malondialdehyde

Miligram

Methylglyoxal

Mililiter

Normal control

Normal rats supplemented with cloves

Normal rats supplemented with jering

Non-alcoholic steatohepatitis

Nitric oxide

Non-insulin-dependent diabetes mellitus

Oxygen

Protein kinase C

Red blood cells

Reactive oxygen species

Standard deviation

Superoxide dismutase

Streptozotocin

Thiobarbituric acid

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TBARS

TGF- β

Thiobarbituric acid reactive substances

Growth factor β

CHAPTER 1

INTRODUCTION

For the most part of this century, health concerns in the field of human nutrition that

have been centered around deficiency disorders of macro and micronutrients with

emphasis on the role of essential nutrients in health and disease. In recent years, various

dietary constituents have been found to provide protections against any disease. Any

significant role by dietary intervention is encouraging and emerging as an acceptable

approach for controlling the diabetes mellitus incidence worldwide (Dasgupta et al.,

2004). Currently there are over 150 million diabetics worldwide and this number is

likely to increase to 300 million or more by the year 2025 due to increase in sedentary

lifestyle, consumption of energy rich diet, and obesity (King et al., 1998). Prevalence of

diabetes mellitus among Malaysians was 10.5% in 1996 and is dangerously increasing to

15% in 2003 (Mafauzy, 2005).

Despite remarkable progress in the management of diabetes mellitus by synthetic drugs,

there has been a renewed interest in indigenous anti-diabetic agents, especially the

medicinal plants attributed with therapeutic virtues (Grover et al., 2003). High

prevalence and long-term complications of diabetes mellitus (Palumbo, 2001) have

prompted a search for new oral hypoglycemic agents from such antidiabetic plants

(Grover et al., 2003). The ethno-botanical information reports about 800 plants that may

possess anti-diabetic potential. Many plant extracts and plant products have been shown

to have significant antioxidant activity as well as having hypoglycemic properties (Sekar

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