assessment of hypoglycemic and antioxidative...
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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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3 4 5
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
65
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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110
116
118
122
124
127
132
134
137
139
141
149
150
153
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16
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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160
165
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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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49
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70
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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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112
113
113
114
114
115
128
128
129
129
130
130
131
143
144
144
145
145
146
146
147
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19
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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148
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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