UNIVERSITI PUTRA MALAYSIA INSULINOTROPIC PROPERTIES OF SEVERAL MALAYSIAN HERBS AND GANODERMA SPECIES MOHD SAUFI BASTAMI. FBSB 2006 2 INSULINOTROPIC PROPERTIES OF SEVERAL MALAYSIAN HERBS AND GANODERMA SPECIES BY MOHD SAUFI BASTAMI Thesis submitted to School of Graduate Studies, Universiti Putra Malaysia, in fulfilment of the Requirements for the Degree of Master of Science January 2006 of Abstract of thesis presented to the Senate of Universiti Putra Malaysia in f~dfilment the requirement for the degree of Master of Science INSULINOTROPIC PROPERTIES OF SEVERAL MALAYSIAN HERBS AND GANODERMA SPECIES BY MOHD SAUFI BASTAMI January 2006 Chairman : Muhajir Hamid, PhD Faculty : Biotechnology and Biomolecular Sciences Diabetes Mellitus incident is currently one of the major and common diseases in Malaysia and increasing every year. To date, no herbs or drugs are able to completely cure the disease. In this study, insulinotropic properties of Malaysian herbs were investigated using pancreatic P cells BRIN-BD1 1. Three out of 13 herbs samples studied were belongs to fungi (Ganoderma sp). The studied showed seven samples are able to induce insulin secretion over 200 % by BRIN-BD11 cell lines. Based on traditional practitioner reports, Gynuraprocumbens was choosed for further investigation on its anti- hyperglycemic properties. Glucose tolerance test were carried on normal and streptozotocin-induced diabetic rats showed at 1000 mglkgl b.w Gynura procumbens methanolic extract was able to reduce blood glucose level in rats. Toxicity towards BRIN-BD11 cells was analyzed using MTT assay and alkaline comet assay. Toxicity levels at ICS0was 300 uglml using MTT assay. Furthermore, Alkaline Comet Assay has proved this toxicity is not due to DNA damage in BRIN-BDl 1 cells. Acute toxicity was carried on ICR strain mice for 14 days. Gynuraprocumbens did not show any toxicity at concentration 7 glkg b.w after administered orally. These results suggest Gynura procumbens and other herbs contain active constituents and have potentials for diabetes treatment. Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains CIRI-CIIU INSULINOTROPIK BEBERAPA HERBA MALAYSIA DAN SPESIES GANODERMA Oleh MOHD SAUFI BASTAMI Januari 2006 Pengerusi : Muhajir Hamid, PhD Fakulti : Bioteknologi dan Sains Biomolekul Kes penyakit Diabetis Mellitus atau Kencing Manis kini menjadi penyakit utama dihadapi oleh kebanyakkan pesakit manakala bilangannya semakin meningkat setiap tahun di Malaysia. Sehingga kini masih belum ada ubat atau herba yang dapat memulihkan daripada penyakit ini sepenuhnya. Dalam kajian ini, ciri-ciri insulinotropik dari tumbuhan herba Malaysia di jalankan bagi melihat kebolehan tumbuhan herba merangsang penghasilan insulin oleh sel selanjar model sel P pankreas BRIN-BDll. Sejumlah tiga dari 13 sampel yang di kaji terdiri dari golongan kulat (Ganoderma sp). Kajian mendapati tujuh daripada sample kajian berupaya merangsang penghasilan insulin lebih dari 200 % oleh sel BRIN-BD1 1. Gynura procumbens dipilih berdasarkan laporan pengamal perubatann tradisional untuk ujian lanjut ciri-ciri anti hyperglycemia. Seterusnya, kajian toleransi glukosa ekstrak methanol Gynura procumbens terhadap tikus normal dan tikus diabetik yang di aruh oleh streptozotocin mendapati 1000 mglkg b.w ekstrak berupaya menurunkan paras glukosa darah. Kajian toksisiti terhadap sel BRIN- BDl l mengunakan asai MTT dan Asai Komet Beralkali dijalankan. Asai MTT memberikan keputusan paras toksisiti Gynura procumbens pada ICsoadalah 300 uglml. Walaubagaimanapun, Asai Komet Beralkali membuktikan toksisiti ini berlaku bukan disebabkan oleh pemusnahan DNA di dalam sel BRIN-BD11. Tahap toksisiti akut dijalankan kepada mencit dari strain ICR untuk tempoh 14 hari. Gynura procumbens didapati tidak memberi kesan toksik pada kepekatan 7 glkg b.w selepas ekstrak di masukkan secara oral. Keputusan ini menunjukkan Gynura procumbens dan tumbuhan herba yang lain mengandungi komponen aktif dan berpotensi dalam rawatan kencing manis. ACKNOWLEDGEMENTS I would like to express my sincere thanks and appreciation to my supervisor Dr. Muhajir Hamid for his advice, support, encouragement and his patience. I am especially grateful to him for his intellectual guidance and constructive comments throughout the course of this study and during the preparation of the manuscript and for being so freely available at all stages for discussion. Sincere appreciations are extended to my supervisory committee, Prof Dr. Abd. Manaf Ali and Associate Prof Dr. Khozirah Shaari for their advice and support throughout the past years and for imparting valuable knowledge. Without their help, this study could not be completed successfully. Many thanks are also due to my lab mates, Pn. Nazrien Kaman, Ms. Siti Pauliena Bohari and Mr. Tajul Anuar which giving support and sharing their time during the previous studies which help on numerous improvements. An acknowledgement is due to En. Hussain Jiragon on helping me on providing materials needed and not forgotten to Ms. Hairiah Yunus for all the guidance and support on completing this thesis. My special thanks also goes to Head Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Associate Prof. Dr Raja Noor Zaliha and to all staff at the department. To the Graduate School Office of Universiti Putra Malaysia, grateful acknowledged for providing me with a Master Science studentship to perform this research. Finally, sincere thanks are fondly expressed to my family and my beloved wife Noazira Idris for their love, patience and support throughout this work. You never know how much I appreciate it - a big thank you. I certify that an Examination Committee met on 13'" Febuary 2006 to conduct the final examination of Mohd Saufi Bastami on his Master of Science thesis entitled "Insulinotropic Properties of Several Malaysian Herbs and Ganoderma Species" in accordance with Universiti Putra Malaysia (Higher Degree) Act 1980 and Universiti Putra Malaysia (Higher Degree) Regulations 1981. The committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows: Johari Ramli, PhD Associate Professor Faculty of Biotechnology and Biomolecules Science Universiti Putra Malaysia (Chairman) Mohd Noor Abd. Wahab, PhD Associate Professor Faculty of Biotechnology and Biomolecules Science Universiti Putra Malaysia (Internal Examiner) Amin Ismail, PhD Lecturer Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Internal Examiner) Jamaludin Hj Mohamed, PhD Professor, Faculty of Allied Health Sciences Universiti Kebangsaan Malaysia (External Examiner) School of Graduate Studies Universiti Putra Malaysia Date : vii This thesis submitted to Senate of Universiti Putra Malaysia has been accepted as fulfillment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows: MUHAJIR HAMID, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman) ABDUL MANAF ALI, PhD Professor, Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) KHOZIRAH SHAARI, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Member) AINI IDERIS, PhD ProfessorIDean School of Graduate Studies Universiti Putra Malaysia ... Vlll 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 is has not been previously or concurrently submitted for any other degree at UPM or other institutions. Date: TABLE OF CONTENTS Page DEDICATION 1 .. ABSTRACT 11 ... ABSTRAK 111 ACKNOWLEDGEMENTS iv APPROVAL SHEETS vii DECLARATION 1X TABLES OF CONTENTS X LIST OF FIGURES xii LIST OF TABLES xv ABBREVIATION xvi CHAPTER 1 INTRODUCTION LITERATURE REVIEW 5 2.1 Diabetes Mellitus 5 2.1.1 Types of Diabetes 6 2.1.2 Type 1 : Insulin -Dependent Diabetes Mellitus 6 2.1.3 Type 2 : Non-Insulin Dependent Diabetes Mellitus 7 2.1.4 Other Types of Diabetes 2.1.5 Complications of Diabetes 2.1.6 Diabetes and Its Impact Insulin 2.2.1 Insulin Synthesis and Characteristic 2.2.2 Insulin Mechanism and Action 2.2.3 Other Usage of Insulin Treatment Strategies For Diabetes 2.3.1 Insulin Injection 1 Replacement 2.3.2 Pancreas or Islet Transplantation in Diabetes 2.3.3 Diet Control on Diabetic Patient 2.3.4 Commonly Used Drugs on Diabetes Treatment Medicinal Plants As Alternative Disease Treatments Insulin Secreting Cell Lines MATERIALS AND METHODS 3.1 Plant Samples 3.2 Animals 3.3 Plants Sample Extraction 3.4 BIUN-BD 11 Cells Culture Passage Cytotoxicity Assay Towards Plants And Ganoderma Samples 28 Insulin Release Activity From Insulin Secreting Cell Lines 30 Rat Insulin Elisa Assay 31 Streptozotocin Induce Diabetic Rats 31 Oral Glucose Tolerance Test By Gynura Procumbens Methanol Extract 32 The SCGE / Comet Assay 33 BRIN-BDI 1 Cells Morphological Examination By AOPI Assay 34 Acute Toxicity Study Of Gynura Procumbens Methanol Extract Towards ICR Mice 35 Statistical Analysis 35 RESULTS AND DISCUSSION Cytotoxicity Effects of Plants And Ganoderma Samples Towards BRIN-BD 1 1 Cells 36 Insulin Secretion Responses on Malaysian Traditional Plants. 39 Insulin Secretion Response by Ganoderma Species. 68 Insulinotropic Properties of Malaysian Herbs As Alternative In Diabetes Treatment. 74 Comparison Between Glybenclamide, Tolbutamide And Plants Sample On Percentage Insulin Release. 80 Oral Glucose Tolerance Test On Gynura Procumbens. 84 In Vitro Cytotoxicity Study (MTT Assay) Of BRIN-BD1 1 Cells Treated By Gynura Procumbens 89 Single Cells Electrophoresis Assay (Comet Assay) of Gynura Procumbens Extract On BRIN-BD 1 1 Cells 91 Morphological Examination of BRIN-BD 1 1 Cells By AOPI Assay 93 Acute Toxicity Study of Gynura Procumbens Extract On ICR Mice 97 CONCLUSION REFERENCES APPENDIX BIODATA OF THE AUTHOR LIST OF FIGURES Figure Page 2.1 Human insulin amino acids sequences on chain A and chain B 11 3.1 Morphology of BRIN-BD1 1 cells as assessed by phase contrast 29 microscopy (X 200 magnification.) 4.1 The effect of Glybenclamide drugs on insulin secretion by BRIN- BDl 1 cell lines after 30 minutes incubation. 4.2 Percentage insulin release by BRIN-BDll cell lines compared to control after 30 minutes treatment with Glybenclamide. 4.3 The effect of Tolbutamide drugs on insulin secretion by BRIN-BDl 1 cell lines after 30 minutes incubation. 4.4 Percentage insulin release by BRIN-BDl1 cell lines compared to control after 30 minutes treatment with Tolbutamide. 4.5 The effect of A.paniculata methanol extract on insulin secretion by BRIN-BD1 1 cell lines after 30 minutes incubation. 4.6 Percentage insulin release by BRIN-BD11 cell lines compared to control after 30 minutes treatment with A.paniculata. 4.7 The effect of different A.paniculata fractions on insulin secretion by BRIN-BD1 1 cell lines after 30 minutes incubation. 4.8 The effect of F.deltodia methanol extract on insulin secretion by BRIN-BD 11 cell lines after 30 minutes incubation. 4.9 Percentage insulin release by BRIN-BDl1 cell lines compared to control after 30 minutes treatment with F. deltodia methanol extract. 4.10 The effect of Gynura procumbens methanol extract on insulin secretion by BRIN-BD 11 cell lines after 30 minutes incubation. 4.1 1 Percentage insulin release by BRIN-BDll cell lines compared to control after 30 minutes treatment with G.procumbens methanol extract. 4.12 The effect of Labisia pumila methanol extract on insulin secretion by BRIN-BDl 1 cell lines after 30 minutes incubation. xii 4.13 Percentage insulin release by BRIN-BD1 1 cell lines compared to control after 30 minutes treatment with L.pumila methanol extract. 4.14 The effect of Momordica charantia methanol extract on insulin secretion by BRIN-BD1 1 cell lines after 30 minutes incubation. 4.15 Percentage insulin release by BRIN-BD11 cell lines compared to control after 30 minutes treatment with Momordica charantia methanol extract. 4.16 The effect of Morinda citrifolia methanol extract on insulin secretion by BRIN-BD1 1 cell lines after 30 minutes incubation. 4.17 Percentage insulin release by BRIN-BDl 1 cell lines compared to control after 30 minutes treatment with Morinda citrifolia methanol extract. 4.18 The effect of Orthosiphon spicatus methanol extract on insulin secretion by BRIN-BD1 1 cell lines after 30 minutes incubation. 4.19 Percentage insulin release by BRIN-BD11 cell lines compared to control after 30 minutes treatment with Orthosiphon spicatus methanol extract. 4.20 The effect of Tinospora cripsa methanol extract on insulin secretion by BRlN-BD1 1 cell lines after 30 minutes incubation. 4.21 Percentage insulin release by BRIN-BD11 cell lines compared to control after 30 minutes treatment with Tinospora crispa methanol extract. 4.22 The effect of Zingiber officinale methanol extract on insulin secretion by BRIN-BDl I cell lines after 30 minutes incubation. 4.23 Percentage insulin release by BRIN-BDll cell lines compared to control after 30 minutes treatment with Zingiber ofJicinale methanol extract. 4.24 The effect of Phylantus nirruri methanol extract on insulin secretion by BRIN-BD1 1 cell lines after 30 minutes incubation. 4.25 The effect of G.lucidum extract on insulin secretion by BRIN-BD11 cell lines after 30 minutes incubation. ... Xlll 4.26 The effect of G.tropicum extract on insulin secretion by BRIN-BDl 1 cell lines after 30 minutes incubation. 4.27 The effect of G.tsugae extract on insulin secretion by BRIN-BD11 cell lines after 30 minutes incubation. 4.28 Percentage insulin release by BRIN-BDI 1 cell lines compared to control after 30 minutes treatment with various species of Ganodenna extract. 4.29 The effect of G.procumbens methanol extract leaves on oral glucose tolerance test (OGTT) in normal rats 4.30 The effect of G.procumbens methanol extract leaves on oral glucose tolerance test (OGTT) in diabetic rats 4.31 The effect of Gynura procumbens methanol extract on percentage relative viability of BRIN-BD1 1 cells line. 4.32 SCGE of BRIN-BDI 1 after treatment with Gynura procumbens methanol extract. 4.33 BRIN-BDI 1 cells processed in the comet assay. Image of target cells DNA after micro electrophoresis below fluorescent microscope. xiv LIST OF TABLES Table Page 2.1 An insulin amino acids sequence in vertebrates. 13 3.1 List of plants used in the experiment, source, part used and their local 25 name. 4.1 Inhibition concentration 50 % (ICSo) of Malaysians herbs and Ganoderma sp. on BRIN-BDl 1 cells by cytotoxicity assay compared to tolbutamide drugs. 4.2 Comparison percentages of insulin released between glybenclamide and tolbutamide. 4.3 Potentiating activity of insulin-secretory activity by methanol extract and drugs 4.4 Morphological examination of BRIN-BD1 1 cells by AOPI assay 4.5 Acute toxicity study of Gynuraprocumbens methanol extract on ICR mice LIST OF ABBREVIATIONS Body weight Carbon dioxide cmL Centimeter square DMSO Dimethyl sulfoxide DNA Deoxyribonucleic Acid "C Degree Celsius EDTA Ethylenediamine tetraacetic acid ELISA Enzyme-Linked Immunosorbent Assay EtBr Ethidium Bromide FBS Feotal bovine serum g Gram IC50 Concentration of 50% inhibitory KRBB Krebs Ringer bicarbonate buffer LMA Low melting agarose M Molar mA mili Ampere ml Milliliter mM milimolar MTT 3-[4,5-dimetiltiazol-2-yl]-2,5-dimetiltetrazolium bromide N Normality NaCl Sodium Chloride NMA Normal melting agarose nrn Nanometer OGTT Oral glucose tolerance test PBS Phosphate buffer saline RPMI 1640 Roswell Park Memorial Institute 1640 Medium W Watt xvi v/v Volume /volume ul Microliter mg Miligram w/v Weight / volume mmol Milimole xvii CHAPTER 1 INTRODUCTION Herbal and natural products have been used for centuries throughout the world in every culture especially on disease treatments by traditional method. Traditional medicines are medicine is made from natural resources whether plant, fauna or mineral. It can be found throughout the world either in the water or land. Malaysia is known for its diversity which are claimed to possess medicinal value. The Malaysians also practice traditional and herbal remedies as an alternative choice for disease treatment. Traditional medicines are still a source of disease treatment of the world population. It has been shown by Phillipson and Wright that 80 % of the world populations depends on traditional medicine (Rahrnan et al., 1999). In two separate surveys recently conducted in Australia and United States respectively that almost 48.5 % and 34 % of respondent have used at least one form of unconventional therapy including herbal medicine (Grover et al., 2001). Furthermore, many of the current commercial drugs are derived from traditional plants based. In the United States, approximately 25 % of prescription contains active ingredients derived from plants. However the proportion in others countries are varies, such as in Germany, 40 % of drugs are based on plant materials (Lee, 1999). Well known drug for headache, aspirin, was originally created from two herbs, - white willow bark and meadowsweet (Lee, 1999). Traditional herbs were also generating an economic value to some countries in the world on medicinal aspect. About 30 % of the worldwide sales of drugs are based on natural product or herbs. Developing new potential drugs might cost millions dollars. Pharmaceutical companies have to spend IJS$ 350 million dollars to develop new drugs (Grabley & Thiericke, 1999). Malaysian herbal market is valued at about US$ 20 billion and growing at a rate of 20 % annually. Much of it is imported either from Indonesia, China or India (Kadir & Lope Pihie, 2001). Malaysian herbal research and development are focusing into few herbs discoveries such as anti-malarial, cytotoxicity, anticancer agents from Goniothalamus and male aphrodisiacs agent from the root extract of Eurycoma lingofolia (Kadir & Shaari, 2000). There are many more potential herbs which were used by traditional practitioner which are undiscovered. Noor and Ashcroft (1989) described that over the past years; scientific and medical knowledge on the role of plant-derived product in the treatment of diabetes mellitus has advanced and created an exciting new area of research which could provide valuable information for the development of alternative drugs. With rich of natural resources, almost 6800 species of seed plants and 600 seedless plants, Malaysia may able to develop drugs towards many diseases. Most studies on remedies usage are not really understood. Scientific studies have been carried out on several plants which believed to possess anti-diabetic properties. Andrographis paniculata, the king of bitter is used since immemorial times as Chinese and Ayuverdic medicine mainly for liver disease and dysentery was combined with Orthosiphon spicatus on diabetes treatment (Akowuah et al., 2004). In India, hypoglycaemic activity of Momordica charantia or bitter gourd, known as 'peria katak' in Malaysia, was studied since 1981 by Virdi et al., (2003). Meanwhile, Gynura procumbens, found in various part of Southeast Asia, has been used for the treatment of eruptive fevers, rash and kidney disease (Zhang & Tan, 2000). Other potential herbs reported to have anti hyperglycaemic activity are Alium cepa, Allium sativum, Murraya koeinigii, Viscum album, Sumbucus nigra, Tinospora crispa and Morinda citrifolia (Grover et al., 2002). Most of these plants are easily found in Malaysia. Development for potential drugs on diabetes treatments are not limited to plants, but also able to be produced from Ganodenna. Ganoderma lucidum which known as 'Ling Zhi ' in China or 'reishi' in Japan was studied by Tomoda et al., (1986), is reported to have hypoglycaemic activity. It is also deemed as an elixir of life that could augment good health and well-being. However, hypoglycaemic activity and medicinal value of Ganoderma tropicum and Ganoderma tsugae are unknown. About 1200 species of Malaysian plants have been reported to have medicinal value. Phytochemical screening, which aimed at scoring the presence of different types of chemical compounds such as alkaloids, flavonoids, saponins, terpenoids, eugenols, polyphenols, sterols, tannins, aloin, plysacharides, diterpenes and glycosides have been carried out (Kadir and Shaari, 2000; Grover et al., 2002). These phytochemical compounds have been reported to have anti-tumor, anti-oxidant also male and women aprodiasiac properties (Kadir and Shaari, 2000). It is also believed to have potential on enhancing insulin secretion from pancreatic cells since some Malaysian herbs were used by traditional practioner on diabetes treatment. The main objectives of the experiment are to study insulinothropic properties of some Malaysian Herbs including some species of Ganoderma. Plants that have potential on diabetes treatment as reported by traditional practioner will be used for insulinotrophic acting using rat pancreatic p cell lines. Measurement was carried on insulin secreting cell lines (BRIN-BDII cell lines) using insulin ELISA methods. Various insulin concentrations are released by BRIN- BDl 1 cell lines affect from its stimulator. Plants and Ganoderma samples was extracted and tested towards BRIN-BDI 1 cells on their ability in enhancing insulin secretion and determine the cytotoxicity level and value by [3-(4, 5-dimethylthiazol-2-y1)-2,5-dimethyltetrazolium bromide (MTT) assay. The second objective is to study the anti-hyperglycemic properties of Gynura procumbens. A further study of Gynura procumbens methanolic extract on hypoglycaemic activities and its toxicity was carried as Gynura procumbens was reported by traditional practitioner to be safe taken as edible and useful in controlling diabetes. Study were carried to determine the maximum insulin levels secreted by BRIN-BDI 1 cell lines and in vivo study of OGTT (oral glucose tolerance test) on normal and induced diabetic rats by Gynura procumbens methanolic extract. Toxicity level of Gynura procumbens methanolic extract towards BRIN-BDl I cell lines and ICR strain mice will be evaluated. The genotoxicity towards BRIN-BDl 1 also were studied by Comet Assay and AOPI assay. CHAPTER 2 LITERATURE REVIEW 2.1 Diabetes Mellitus Diabetes mellitus (DM) is a complex disorder of carbohydrate, fat and protein metabolism. It is primarily a result of a defect in secretion or action of insulin, the hormone that facilitates and control the use of glucose in the cells. Due to the deficiency of insulin, diabetic patients have an impaired tolerance to glucose that leads to numbers of short term and long term complications. Early diagnosis of diabetes is determined by blood glucose level. Glucose levels before meal are generally run between 4-7 mmolll or 60-100 mgldl in normal people (Williams & Porte, 1974; Dagget, 1981). Diabetes not only limited to human being but also occurred on canine especially on type 1 diabetes, insulin dependent diabetis mellitus (IDDM) (Davison et al., 2002). Diabetes Mellitus is the commonest endocrine disorder that affects more than 100 million people worldwide (6 % of the population) and projected to grow over 220 millions within 40-50 years (Proietto et al., 1999). In next 10 years, diabetes may affect about five times more people than it does now (Bailey, 2000; Grover et al., 2002). Diabetes derived fiom Greek for siphon, which refers to the copious excretion of water that characterizes the disease, meanwhile mellitus came fiom Latin word for honey, characterized by the high sugar content in urine. In Malaysia, diabetes mellitus is increasing towards serious level. The Ministry of Health has recorded 657,988 people in Malaysia suffering from diabetes in 2002 increasing from 525,858 in year 2001 (Abdullah, 2003). The number of diabetics estimated to be increased to 0.8 million cases in 2025. From the reports of admissions to government hospitals in Peninsular Malaysia, diabetes mellitus had increased from 5024 cases in 1979 to 17808 cases in year 1990, and in year 2020 about 144 600 Malaysians will suffering diabetes (Amos et al., 1997). 2.1.1 Types of Diabetes Diabetes can be divided into three distinct types, in which subtypes have been identified. 2.1.2 Type 1: Insulin -Dependent Diabetes Mellitus (IDDM) Type 1 or insulin-dependant diabetes mellitus (IDDM) occurs in approximately 10 % of all diabetics' patients in western world. The symptoms are hyperglycaemia due to secondary insulin deficiency, occurring as a result of autoimmune destruction of pancreatic endocrine p cells (Perfetti & Ahmad, 2000). Type 1 commonly occurs on childhood (Docherty, 2001). This type of diabetes accounts for 3 % of all diabetes worldwide in 1997, meanwhile type 2 is by far the most common type (Proietto et al., 1999). 2.1.3 Type 2: Non-Insulin Dependent Diabetes Mellitus (NIDDM) Non-insulin dependent diabetes mellitus occurs when insulin is no longer reacting as metabolic hormone in reducing blood sugar level (insulin resistant) or insufficient ability to secrete insulin (Proietto et al., 1999; Perfetti & Ahrnad, 2000). It is reported that type 2 diabetes has strong genetic tendency (Proietto et al., 1999). Combination of insulin resistant and insufficient insulin released has accounting 90-95% of diabetes in developed countries (Husen et al., 2004). About 90 % of diabetics in western world are categorized by type 2 rather than type 1. Type 2 diabetic shows direct contact with obesity. Hartz et al., 1983 and Kissebah et al., 1984, reports that a close relationship between upper body obesity with type 2 diabetes by increased free fatty acid oxidation could impair glucose oxidation thus leading to insulin resistance (Proietto et al., 1999). The risk of diabetes seems to be increased ten times in women with both severe obesity and high waist to hips ratio. 2.1.4 Other Types of Diabetes Other types of diabetes including entities secondary to or associated with certain other conditions or syndromes. Diabetes may be secondary to pancreatic disease removal of pancreatic tissues, endocrine disease such as acromegaly, Cushing's syndrome, glucagonoma, somatostatinoma, or the administration of certain drugs, hormones or chemicals that causing hyperglycaemia (Hamid, 1999).