KESAN PENURUNAN PARAS GLUKOSA-DARAH OLEH EKSTRAK BIJI SWIETENIA MACROPHYLLA DALAM TIKUS NORMAL DAN TIKUS DIABETIK TERARUH -STREPTOZOTOCIN oleh MOHD AKMAL BIN HASHIM Tesis yang diserahkan untuk memenuhi keperluan bagi Ijazah Sarjana Sains UNIVERSITI SAINS MALAYSIA Januari 2012 BLOOD-GLUCOSE LOWERING EFFECT OF SWIETENIA MACROPHYLLA SEED EXTRACTS IN NORMAL AND STREPTOZOTOCIN-INDUCED DIABETIC RATS by MOHD AKMAL BIN HASHIM Thesis submitted in fulfillment of the requirements for the degree of Master of Science UNIVERSITI SAINS MALAYSIA January 2012 In the Name of ALLAH The Most Loving and the Most Merciful This thesis is dedicated to my parents and family for doing their best to educate and support me and to all my friends for their kindness in helping me to complete this study ii ACKNOWLEDGEMENTS First and foremost, I would like to express all praises to Almighty Allah who made all things possible. This research project would not have been possible without the support of many people. I would like to acknowledge and extend my heartfelt gratitude to the following persons who have made the completion of this study possible; my main supervisor Prof. Mohd Zaini Asmawi, for showing me some example that related to the topic of my project, Assoc. Prof. Amirin Sadikun for his advice, guide and assistance throughout the chemical aspect of this work, not to forget Prof. Zhari for his kindness providing the permission for using facilities at Centre for Herbal Standardization (CHEST). I also wish to express my sincere gratitude to the Dean of School of Pharmaceutical Sciences, Prof Syed Azhar Syed Sulaiman for approving me to pursue my study in this school. Further thanks to Institute of Postgraduate Studies and Unversiti Sains Malaysia for providing me with a good environment and facilities to complete this project Needless to mention that Mr. Abdul Razak, Science Officer from Centre of Drug Research (CDR), who had been a source of technical assistance and for his timely guidance in the conduct of part of the studies. I would also like to thank Mr. Roseli Hassan, Puan Zurina, Mr. Yam, Mr. Selva, Madam Yong, Mr. Hassan, Mr. Farid and staff from Animal Research & Wellness Unit (ARWU) for all their valuable assistance in the project work. Words are inadequate in offering my thanks to all of them, for their encouragement and cooperation in carrying out the project work. iii Finally, yet importantly, I would like to express my heartfelt thanks to my beloved parents for their blessings, my friends/labmates, Lim Chung Pin, Najihah, Thaiga, Elsnoussi, Sook Yee, Norazimah and others for their help and wishes for the successful completion of this project. They shared their knowledge, their ideas, and numerous tips all of which culminated in the completion of this study. Thanks guys. As for the closure, I would also like to convey thanks to the Ministry of Higher Education (MOHE) and School of Pharmacy, USM for providing the financial means and laboratory facilities. iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iv LIST OF TABLES xi LIST OF FIGURES xii LIST OF ABBREVIATIONS xviii ABSTRAK xix ABSTRACT xxi CHAPTER ONE : INTRODUCTION 1 1.0 Pharmacognosy 1 1.1 Herbs as an alternative medicine 2 1.2 Compounds from herbs with anti-diabetic activity 4 1.3 Approaches to anti-diabetic screening of Swietenia macrophylla (SM) seeds 7 extract. 1.3.1 Serial extraction of S. macrophylla seeds. 7 1.3.2 In-vivo and in-vitro study of S. macrophylla seeds extracts 8 1.3.2.1 In-vivo test 8 1.3.2.2 In-vitro test 9 1.3.3 Single dose and repeated doses (14 days) studies 10 1.3.4 Phytochemical screening 11 v 1.4 S. macrophylla King 13 1.4.1 The morphology of fruit and seeds of S. macrophylla 15 1.4.2 Phytochemistry of S. macrophylla 16 1.4.3 Medicinal value of S. macrophylla 19 1.5 Diabetes mellitus, disease of the millennium 20 1.5.1 What causes diabetes? 21 1.5.2 Pathophysiology of diabetes mellitus 23 1.5.3 Treatment for diabetes mellitus : 24 Oral medications a) Insulin Secretagogues. 25 b) Insulin Sensitizers 25 c) α -glucosidase inhibitors 26 d) Biguanide 26 Parenteral medication e) Insulin 26 1.6 Objectives of study 29 vi CHAPTER TWO : LITERATURE REVIEW 30 2.0 Introduction 30 2.1 Diabetes mellitus 30 2.2 S. macrophylla 33 CHAPTER THREE : MATERIALS AND METHODS 36 3.1 Materials : 36 3.1.1 Chemicals and solvents 36 3.1.2 Drugs 37 3.1.3 Instruments 37 3.2 Methods 39 3.2.1 Collection, identification and preparation of the plant material 39 3.2.2 Solvents and apparatus for extraction 39 3.2.3 Extraction procedure 39 3.2.4 Fractionation of the aqueous extract of S. macrophylla seeds 41 using liquid-liquid separation technique. 3.3 Pharmacological studies 43 3.3.1 In- vivo studies : 43 3.3.1.a Animals 43 3.3.1.b Chemicals and apparatus 43 3.3.1.c Effect of different anti-diabetic drugs and Insulin on 43 different experimental models 3.3.1.d Vehicle test for the extracts 44 vii 3.3.1.e Measurement of blood glucose level by tail-prick 44 method 3.3.1.f Induction of diabetic rats 44 3.3.1.g Intra-peritoneal glucose tolerance test (IPGTT) 45 3.3.1.h Hypoglycaemic test in normal rats 45 3.3.1.i Anti-hyperglycaemic test in diabetic rats 46 3.3.1.j The effect of n-butanol fraction in antihyperglycaemic 46 test of diabetic rats on plasma insulin concentration 3.3.1.k 14 days treatment 47 3.3.2 In- vitro studies 47 3.3.2.a Study of inhibition of glucose absorption by everted 47 intestine sacs 3.3.2.b Study of glucose uptake by isolated rat abdominal muscle 48 3.4 Chemical analysis 49 3.4 (a) Gas chromatography-mass spectrometry (GC-MS) analysis on 49 petroleum-ether extract of S. macrophylla seeds. 3.4 (b) Thin layer chromatography (TLC) analysis on n-butanol fraction of 49 aqueous extract of S. macrophylla seed extract 3.4 ( c) Liquid chromatography-mass spectrometry (LC-MS) analysis on 50 n-butanol fraction of aqueous extract of S. macrophylla seed extract viii 3.5 Phytochemical screening of n-butanol fractions 51 3.5.a Detection of alkaloids 51 3.5.b Total phenolic Assay (TPA) 51 3.5.c Total flavonoid Assay (TFA) 52 3.5.d Detection of saponins (Frothing test) 52 3.6 Statistical Analysis 52 CHAPTER FOUR : RESULTS 53 4. Extraction of S. macrophylla seeds 53 4.1 Effect of anti-diabetic drugs (glibenclamide, metformin) and 54 insulin in hypoglycaemic, antihyperglycaemic and intraperitoneal glucose tolerance tests. 4.2 Hypoglycaemic test in normal rats 58 4.3 Intraperitoneal glucose tolerance test (I.P.G.T.T) in normal rats 59 4.4 Antihyperglycaemic test in diabetic rats 60 4.5 Fractionation of aqueous extract 67 4.6 Antihyperglycaemic test on different fractions of aqueous extract 68 4.7 Dose-response study of the n-butanol fractions 70 4.8 The effect of n-butanol fraction and glibenclamide on the 72 plasma insulin levels of STZ-induced diabetic rats ix 4.9 The effect of daily oral administration of Petroleum ether 74 and aqueous extracts on body weight and blood glucose level of STZ-induced diabetic rats 4.10 In-vitro study of n- butanol fraction of S. macrophylla seeds 77 aqueous extract a) Effect on glucose absorption by everted sac preparation 78 b) Effect on glucose uptake by isolated abdominal muscle 79 4.11 GC-MS analysis on petroleum-ether extract of S. macrophylla seed 80 4.12 Phytochemical investigation and thin layer chromatography 85 (TLC) analysis on n-butanol fraction of aqueous extract of S. macrophylla seed extract 4.12.a Detection of alkaloids 85 4.12.b Detection of saponin 85 4.12.c TLC analysis of the n-butanol fraction 85 4.13 LC-MS analysis on aqueous extract and n-butanol fraction of 88 S. macrophylla seed 4.14 Total phenolic and total flavonoid assay of the n-butanol fraction 96 x CHAPTER FIVE : DISCUSSION AND CONCLUSIONS 97 5.1 Preliminary and acute studies 97 5.2 Fourteen days treatment 107 5.3 Antihyperglycaemic activity guided fractionation of the 108 aqueous extracts 5.4 Possible antihyperglycaemic mechanism of the n-butanol fraction 110 5.5 Phytochemical investigations and chemistry analysis of the n-butanol 114 fraction and GC-MS analysis of the petroleum ether extract 5.5.1 GC-MS analysis of the petroleum ether extract 114 5.5.2 Phytochemical investigations and chemistry analysis of the 115 n-butanol fraction REFERENCES 119 APPENDICES 133 xi LIST OF TABLES Page Table 1.1: Some of the herbs commonly used in Malaysia for treating diabetes. 3 Sources : (http://jamu-herbal.com, accessed on 2 July 2009) Table 1.2: Some of the secondary metabolites that have been reported to 4-6 have anti diabetic properties. Table 1.3 : Causes of diabetes mellitus (San, 1991). 22 Table 1.4 : Types of insulin 28 Table 4.1 : The amount of extracts obtained from the seeds of S. macrophylla 53 King (Meliaceae) Table 4.2 : The main compounds identified in petroleum ether extract of 82 Swietenia macrophylla seeds Table 4.3: The Rf values in chromatogram of n-butanol fraction, aqueous 85-86 extract, rutin and isoquercitrin observed under UV 365 and 240 nm. Table 4.4: Compounds detected by LC-MS 88 Table 4.5 : Total phenolic and total flavonoid contents of n-butanol 96 fraction of S. macrophylla seeds extracts. xii LIST OF FIGURES Page Figure 1.1 Flow chart for the study of plants used in traditional medicine 12 (Lue Pieters and Arnold., 2005) Figure 1.2 : a) The tree 14 b)The leaves c) The fruit d) The seeds Figure 1.3 The fruit of S. macrophylla 15 Figure 1.4 : The seeds of S. macrophylla 16 Figure 1.5 : Swietenine 17 Figure 1.6 : Swietenolide diacetate 18 Figure 1.7 : Swietenolide monohydrate 18 Figure 1.8 : Andirobin 18 Figure 1.9 : Swietemahonin A 18 Figure 1.10 : Febrifugin 18 Figure 1.11 : Overview of the pathogenesis of type 2 diabetes mellitus 24 adopted from (http://www.pharmainfo.net) Figure 1.12 : Pancreatic human insulin structure. 27 Adopted from abpischools.org.uk (12.20p.m, 25 Mac2011) Figure 2.1 : Prevalence of diabetes worldwide 31 (www.exsulin.com, accessed on 17 December 2010). Figure 3.1 Maceration ( cold method) 40 Figure 3.2 Soxhlet extraction ( hot method) 41 Figure 3.3 : Liquid-liquid separation procedure using separatory funnel 42 xiii Figure 4.1 : The effect of oral administration of normal saline (10ml/kg), 55 glibenclamide (10mg/kg) and metformin (500mg/kg), on normal rats. Each value represents the mean ± s.e.m of six rats (n=6). * indicates statistically significant difference (p<0.05) compared to the control group. Figure 4.2 : The effect of oral administration of normal saline, 56 glibenclamide (10mg/kg) and metformin (500mg/kg), on normal rats loaded with 500mg/kg of glucose. Each value represents the mean ± s.e.m of six rats (n=6). * indicates statistically significant difference (p<0.05) compared to the control group. Figure 4.3 : The effect of oral administration of normal saline 10ml/kg, 57 glibenclamide (10mg/kg), metformin (500mg/kg) and subcutaneous administration of insulin (5 I.U/kg), on STZ-induced diabetic rats. Each value represents the mean ± s.e.m of six rats (n=6). * indicates statistically significant difference (p<0.05) compared to the control group. Figure 4.4 : The effect of oral administration of S.macrophylla extracts 61 obtained by maceration method (1g/kg) on normal rats. Each value represents the mean ± s.e.m of six rats (n=6). * indicates statistically significant difference (p<0.05) compared to the control group. Figure 4.5 : The effect of oral administration of S. macrophylla extracts 62 obtained by Soxhlet’s method (1g/kg ) on normal rats. Each value represents the mean ± s.e.m of six rats (n=6). * and ** indicates statistically significant difference (p<0.05) and (p<0.001) compared to the control group respectively. xiv Figure 4.6 : The effect of oral administration of S.macrophylla extracts 63 obtained by maceration method (1g/kg) and metformin (500mg/kg) , on normal rats loaded with 500mg/kg of glucose. Each value represents the mean ± s.e.m of six rats (n=6). * and ** indicates statistically significant difference (p<0.05) and (p<0.005) compared to the control group respectively. Figure 4.7 : The effect of oral administration of S.macrophylla extracts 64 obtained by Soxhlet’s extraction method (1g/kg) and metformin (500mg/kg) on normal rats loaded with 500mg/kg of glucose. Each value represents the mean ± s.e.m of six rats (n=6). *, ** and *** indicate statistically significant difference (p<0.05), (p<0.01) and (p<0.001) compared to the control group respectively. Figure 4.8 : The effect of oral administration of S.macrophylla extracts 65 obtained by maceration method (1g/kg) and subcutaneous administration of insulin (5 I.U/kg) on STZ-induced diabetic rats. Each value represents the mean ± s.e.m of six rats (n=6). * and *** indicates statistically significant difference (p<0.05) and (p<0.001) compared to the control group respectively. Figure 4.9 : The effect of oral administration of S.macrophylla extracts 66 obtained by Soxhlet’s extraction method (1g/kg) and subcutaneous administration of insulin (5 I.U/kg) on STZ-induced diabetic rats. Each value represents the mean ± s.e.m of six rats (n=6). *, ** and *** indicate statistically significant difference (p<0.05), (p<0.01) and (p<0.001) compared to the control group respectively. Figure 4.10 : Amount and percentage of fractions obtained from 67 liquid-liquid separation of aqueous extract of S.macrophylla Figure 4.11 : The effect of oral administration of chloroform, ethyl 69 acetate, n-butanol and aqueous fractions of S.macrophylla (1g/kg) and insulin (5 I.U/kg) subcutaneously on STZ-induced diabetic rats. Each value represents the mean ± s.e.m of six rats (n=6). *, **, *** and indicates statistically significant difference (p<0.05), (p<0.01), (p<0.005) and (p<0.001) compared to the control group respectively. xv Figure 4.12 : The effect of oral administration of n- butanol fractions 71 of S. macrophylla extracts (500mg/kg, 1g/kg, 2g/kg) and insulin (5I.U/kg) subcutaneously on STZ-induced diabetic rats. Each value represents the mean ± s.e.m of six rats (n=6). *, **, *** and indicates statistically significant difference (p<0.05), (p<0.01), (p<0.005) and (p<0.001) compared to the control group respectively. Figure 4.13 : The effect of oral administration of n- butanol fractions of 73 S.macrophylla extracts (500mg/kg, 1g/kg, 2g/kg) and glibenclamide (10mg/kg), on insulin levels of STZ-induced diabetic rats. Each value represents the mean ± s.e.m of six rats (n=6). ** and *** indicates statistically significant difference (p<0.01) and (p<0.005) compared to the control group respectively. Figure 4.14 : The effect of daily oral administration of 4% tween 80 75 (control, 10ml/kg), metformin (500mg/kg), petroleum ether (Pet-Ether) and aqueous extracts for 14 days on body weight of STZ-induced diabetic rats. Each value represents the mean ± s.e.m of five rats (n=5). ** indicates statistically significant difference (P<0.01) compared to day 0 (before treatment). Figure 4.15 : The effect of daily oral administration of 4% tween 80 76 (control, 10ml/kg), metformin (500mg/kg), petroleum ether (Pet-Ether) and aqueous extracts for 14 days on blood glucose level of STZ-induced diabetic rats. Each value represents the mean ± s.e.m of five rats (n=5). * and ** indicates statistically significant difference of (p<0.05) and (p<0.005) respectively compared to day 0 (before treatment). Figure 4.16 : Effect of n-butanol fraction of S. macrophylla extract and 78 acarbose on glucose absorption by everted intestine. Each value represents the mean ± S.E.M (n=8) ; ** indicate significant differences (p<0.01) between treated groups and control group. xvi Figure 4.17: Effect of n-butanol fraction of S. macrophylla extract 79 and metformin on glucose uptake in isolated rat abdominal muscle (in the present or absence of insulin). Each value represents the mean ± S.E.M (n=8) ; * indicate significant different between treated groups with control (without insulin); p< 0.05, *** indicate significant different between treated group and control (with insulin); p<0.05, ** indicate significant different in glucose uptake by the muscle in the condition without insulin and in the presence of insulin; p<0.05. Figure 4.18 : The compounds identified in petroleum-ether extract 81 of S. macrophylla Figure 4.19 (a) : Chromatogram of petroleum –ether extract by GC-MS 83 analysis Figure 4.19 (b) : Chromatogram of petroleum –ether extract by GC-MS 84 analysis, focus on fucosterol and β-sitosterol Figure 4.20 : TLC profile of n-butanol fraction (n-But), aqueous 87 extract (H20), rutin and isoquercitrin after being developed with ethyl acetate : formic acid : glacial acetic acid : water (100;11;11:26) as mobile phase , under (a) 340 nm, (b) 254 nm Figure 4.21 : Total ion chromatogram for rutin 89 Figure 4.22 : Total ion chromatogram for isoquercitrin 90 xvii Figure 4.23 : Total ion chromatogram for aqueous extract of 91 S. macrophylla seeds Figure 4.24 : Total ion chromatogram for aqueous extract of 92 S. macrophylla seeds (combined analysis) Figure 4.25 : Total ion chromatogram for n-butanol fraction of 93 aqueous extract of S. macrophylla seeds Figure 4.26 : Total ion chromatogram for n-butanol fraction of aqueous 94 extract of S. macrophylla seeds (combined analysis) Figure 4.27 : Rutin 95 Figure 4.28 : Iso-quercitrin 95 xviii LIST OF ABBREVIATIONS STZ Streptozotocin FBG Fasting blood glucose SAR Structure activity relationship DM Diabetes mellitus idDM Insulin dependant diabetes mellitus nidDM Non-insulin dependant diabetes mellitus IPGTT Intraperitoneal glucose tolerance test TZD Thiazolidinedione GIT Gastrointestinal tract GC-MS Gas chromatography-mass spectrometer LC-MS Liquid chromatography-mass spectrometer HPTLC High performance Thin layer chromatography TFA Total flavonoid assay OHA Oral hypoglycaemic agent TPA Total phenolic assay GAE Gallic acid equivalent QE Quercetin equivalent MW Molecular weight xix KESAN PENURUNAN PARAS GLUKOSA-DARAH OLEH EKSTRAK BIJI SWIETENIA MACROPHYLLA DALAM TIKUS NORMAL DAN TIKUS DIABETIK TERARUH -STREPTOZOTOCIN ABSTRAK Biji S.macrophylla telah digunapakai oleh orang yang terdahulu untuk merawat pelbagai jenis penyakit termasuk diabetes. Matlamat kajian ini adalah untuk menyiasat aktiviti antidiabetik ekstrak tumbuhan ini dan menentukan sebatian aktif yang mungkin menyumbang kepada aktiviti tersebut. Biji S.macrophylla yang telah hancur dan kering, diekstrak secara bersiri dengan Petroleum-eter, kloroform, metanol dan juga air melalui dua kaedah berbeza; pengekstrakan Soxhlet dan juga kaedah maserasi. Ekstrak-ekstrak yang diperolehi , diuji bagi aktiviti hipoglisemik ke atas tikus normal, perencatan kenaikan paras glukosa darah dalam ujian toleransi glukosa secara intraperitoneal (IPGTT) dan juga aktiviti antihiperglisemia ke atas tikus diabetik teraruh- streptozotocin. Semua ekstrak daripada kaedah pengekstrakan Soxhlet didapati tidak mempunyai aktiviti antidiabetik. Manakala bagi ekstrak yang diperolehi melalui kaedah maserasi, didapati bahawa tiada ekstrak yang dapat menurunkan paras glukosa darah dalam ujian hipoglisemik ke atas tikus normal. Walaubagaimanapun, ekstrak petroleum-eter dan juga air yang diperolehi melalui kaedah maserasi, merencat dengan signifikan kenaikan paras glukosa darah dalam ujian IPGTT. Bagi tikus diabetik teraruh- streptozotocin, hanya ekstrak air berupaya menurunkan paras glukosa darah selepas 3, 5 dan 7 jam pemberian. Pemberian ekstrak petroleum-eter dan juga air secara oral sebanyak dua kali sehari, ke atas tikus diabetik menunjukkan hanya ekstrak air (1000mg/kg/sehari) dengan signifikan menurunkan paras glukosa darah puasa selepas xx empat belas hari rawatan. Ekstrak air biji S.macrophylla kemudiannya difraksikan dengan kloroform, etil-asetat dan juga n-butanol. Ujian selanjutnya menunjukkan bahawa fraksi n-butanol adalah yang paling poten dalam menurunkan paras glukosa darah puasa tikus diabetik teraruh- streptozotocin. Fraksi n-butanol didapati meningkatkan dengan signifikan penggunaan glukosa oleh sediaan otot abdomen terasing dan juga merencat penyerapan glukosa oleh kantung usus terbalik terasing. Ini mencadangkan bahawa kesan penurunan paras glukosa darah oleh fraksi n-butanol, setidak-tidaknya adalah melalui perencatan penyerapan glukosa pada saluran gastrousus dan juga meningkatkan penggunaan glukosa oleh otot. Analisa ekstrak petroleum-eter dengan menggunakan spektrometer jirim-kromatografi gas (GCMS) menunjukkan kehadiran fukosterol, β-sitosterol, asid linoleik dan juga γ-tokoferol yang mungkin bertanggungjawab ke atas aktiviti anti-hiperglisemik. Analisa ke atas fraksi n-butanol dengan menggunakan ujian saringan fitokimia dan juga spektrometer jirim-kromatografi cecair (LCMS) masing-masing telah menunjukkan fraksi tersebut mengandungi flavonoid, alkaloid dan juga saponin, dan flavonoid yang telah dikenalpasti adalah rutin dan isoquercitrin. Kesimpulannya, kesan penurunan glukosa mungkin disumbangkan oleh sebatian tak berkutub yang merencat kenaikan paras glukosa darah ke atas tikus yang diberikan glukosa dan juga sebatian berkutub yang menurunkan paras glukosa darah ke atas tikus diabetik teraruh-streptozotocin. Penemuan-penemuan ini menyokong penggunaan tradisional Swietenia macrophylla untuk merawat diabetes xxi BLOOD-GLUCOSE LOWERING EFFECT OF SWIETENIA MACROPHYLLA SEED EXTRACTS IN NORMAL AND STREPTOZOTOCIN-INDUCED DIABETIC RATS ABSTRACT Swietenia macrophylla (meliaceae) seeds have been used in folklore for treating various diseases including diabetes. The aim of the present work was to investigate the antidiabetic activity of the plant extracts and determine the active compound(s) that might contribute to the activity. Pulverized, dried powder of S. macrophylla seeds was extracted serially with petroleum ether, chloroform, methanol and water by two different methods; Soxhlet extraction and maceration methods. The extracts obtained were examined for hypoglycaemic activity in normal rats, inhibition of the rise of blood glucose level in intraperitoneal glucose tolerance test (IPGTT) and antihyperglycaemic activity in streptozotocin-induced diabetic rats. It was found that all the extracts obtained from Soxhlet method did not possess antidiabetic activity. As for the extracts obtained from the maceration method, none of the extracts lowered the blood glucose levels in hypoglycaemic test in normal rats. However, petroleum-ether and aqueous extracts obtained by maceration method significantly inhibited the rise of blood glucose levels in IPGTT. In streptozotocin-induced diabetic rats, only aqueous extract at 3,5 and 7 hours after administration reduced the blood glucose level. Twice daily oral administration of aqueous and petroleum-ether extracts to diabetic rats showed that only aqueous extract (1000mg/kg/daily) significantly decreased the fasting blood glucose levels after 14 days xxii treatment. The aqueous extract of S. macrophylla seeds was then fractionated with chloroform, ethyl-acetate and n-butanol. Further test showed that n-butanol fraction was the most potent in lowering the fasting blood glucose levels in streptozotocin-induced diabetic rats. The n-butanol fraction significantly increase the utilization of glucose by isolated abdominal muscles preparation and inhibited glucose absorption by isolated everted intestine. It may suggests that the blood glucose lowering effect of n-butanol fraction is by inhibition of glucose absorption in gastrointestinal tract and increasing utilization of glucose by muscles. Analysis of petroleum-ether extract by Gas-chromatography-mass spectrometer (GC-MS) showed the presence of fucosterol, β-sitosterol, linoleic acid and γ-tocopherol that might be responsible for the antihyperglycaemic activity. Analysis of n-butanol fraction, using phytochemical screening and LC-MS respectively indicated that the fraction contained flavonoids, alkaloids and saponins and the flavonoids that have been identified were rutin and isoquercitrin. In conclusion, the glucose lowering effect might be contributed by the non polar compound(s) which inhibit the rise in blood glucose level in glucose loaded rats and polar compound(s) which reduced the blood glucose level in streptozotocin-induced diabetic rats. These findings support the traditional use of Swietenia macrophylla for treatment of diabetes. 1 CHAPTER 1 INTRODUCTION 1.0 Pharmacognosy Pharmacognosy is the study of drugs of natural origin. It is one of the five major divisions of the pharmaceutical curriculum. In other words, pharmacognosy also means a knowledge of drugs or pharmaceuticals, that has been a part of healing arts and sciences, since mankind first began to treat illnesses (Varro et al., 1988). The ancient people, gathered herbs, animals, plants, and minerals and concocted them into plenty of ill-flavoured pungent mixtures, in order to treat diseases. The way of creating medication have progressed from an era of empiricism to the present age of specific therapeutic agent. Recently, as a result of the intense concern with all aspects of ecology, there has been a renewed interest in so-called “natural” foods and drugs. The availability of an extremely wide variety of these products, ranging from the mint tea to ginseng chewing gum, has stimulated the scientific community to learn more about them. Nowadays, some natural compounds of drugs constituents have been partially replaced in commerce by synthetic compounds of identical chemical structure and therapeutic properties. For instance, natural camphor is obtained from the camphor tree by steam distillation, but in contrast, synthetic camphor was manufactured by either of two methods: by total synthesis from vinyl chloride and cyclopentadiene (a complete synthetic process) or semi synthesis from pine stumps (involve chemical modification of a natural product) illnesses ( Varro et al., 1988). 2 In future, the achievements of science and technology will be so great that, when brought to bear upon the mysteries of nature that have long puzzled us, those mysteries will yield their secrets amazingly. The plant and animal kingdoms will continue to serve mankind in the 21st century just as they have done since the dawn of history. Significant new drugs of natural origin and new methods of producing them will continue to be important parts for sustainability of human kinds. 1.1 Herbs as an alternative medicine In recent years, medicinal herbs and their preparations have been increasingly considered in the treatment of illness, and at the the same time, Malaysia is a country that treasures its natural forests. These forests are the largest source of natural products which can be used for medicinal purpose. Since the early years, the Malays have been using plant products such as the leaves, seeds, roots, stems, flowers and fruit as ingredients in the preparation of traditional medicine. In the old days, plants were used primarily for treatment rather than cure, due to the tendency of practitioners of traditional medicine diagnosed diseases in a holistic manner. However, traditional medicine remains a popular method of treatment since it has been used in immemorial time until today. Malaysia is a country with plenty of natural resources, rich with medicinal values. There are over 6000 species of tropical plants all over the country. Only in Peninsular Malaysia , there are 550 genera containing 1,300 species and most of these are medicinal plants good for the human body (Muhammad & Mustafa.,1994). Most drugs currently used as medicine today, are products of synthetic chemicals derived from natural chemicals. There are many types of traditional medicines that are widely used in 3 Malaysia for treating diabetes. It is a mixture of various plant parts. Some of the plants commonly used in Malaysia for treating diabetes are listed in Table 1. The composition of the herbs mixture varies in different places. Local name Scientific name Family Part Used Mengkudu Morinda citrifolia L Rubiaceae Leaves Lidah buaya Aloe vera L. Liliaceae Leaves Kunyit Curcuma Domestica Val. Zingiberceae Rhizome Kacang soya Glycine max [L.] Merr. Papilionaceae (Leguminoceae). Seeds Jambu batu Psidium guava L Myrtaceae Fruit Bawang merah Allium cepa L. Liliaceae The entire parts Table 1.1 : Some of the herbs commonly used in Malaysia for treating diabetes. Sources : (http://jamu-herbal.com, accessed on 2 July 2009) 4 1.2 Compounds from herbs with antidiabetic activity Anti-diabetic secondary metabolites Diabetes mellitus is a debilitating and often life-threatening disorder with increasing incidence throughout the world (WHO, 1985). Literature surveys show that more than 400 plant species were reported to have anti-diabetic activity, and most of the anti-diabetic natural products were, so far, isolated from plants (Mukherjee, 1981; Rai, 1995). SECONDARY METABOLITES Description Flavonoids Originate from a large group of polyphenolic compounds, commonly in plants.There are the pigments responsible for the shades of yellow, orange and red in flowering plants. Many are endowed with biological activities, such as anti-diabetic, anti-inflammatory,antiallergic, antischemic, antiplatelet and anti-tumoral activities. (Catherine and Lester, 2003) Alkaloids An organic nitrogenous compounds with a limited distribution in nature. May occur in various parts of the plants: in seeds, fruits, leaves, in underground stems, roots, rhizomes and barks. The pharmacologic actions varies widely : from anti-diabetic to analgesics and narcotics whereas others are central stimulants. (Varro et al.,1988). 5 Diterpenes Naturally occurring organic compounds that comprise two monoterpene molecules; found in some essential oils; reported to have antibacterial, anti-diabetic, antiviral, antifungal, anti-inflammatory and expectorant properties ( Jonas., 2005) Triterpenoids Terpenoid derivatives of triterpene molecules. They may have useful activities including immunostimulation, anti-tumour-promoting activity, antiinflammatory and anti-cancer properties . ( Joseph et al., 2002) Sesquiterpene lactones It is another member of the terpene group; comprises three units of isoprene. Molecular formula is C15H24O6. Reported to be useful as an antiseptic, antibacterial, or anti-inflammatory, anti-diabetic and as a calming agent by aromatherapists. ( Jonas., 2005). Saponins A group of glycosides that is widely distributed in the higher plants. It form colloidal solutions in water that foam upon shaking. They have a bitter and acrid taste . Saponins have been reported to beneficial to control blood cholesterol levels, bone health, cancer, diabetic and building up of the immune system. (Varro et al., 1988). 6 Anthocyanins Water-soluble, terrestrial plant pigments that can be classified chemically as both flavonoid and phenolic. Act as a colouring agents for fruit juices and wine. These pigments are linked to an amazingly broad range of health benefits such as treating diabetes, hypertension, vision disorders, microbial infections and also diarrhea. ( Jonas., 2005) Lignans A type of polyphenolic compound that occurs naturally in some plants. Sources that are particularly rich in lignin, in order of highest to lowest concentration are, flax, sesame, sunflower, and pumpkin seeds. This substance has an excellent anti-oxidant properties and belief to help in preventing the development of diabetes and arteriosclerosis. (Iqbal ahmad et al., 2006) Tannins A naturally occurring polyphenolic compound with antioxidant and anticarcinogenic effects. In vitro/animal studies indicate it may have health benefits in treating diabetes (Liu X et al., 2005) Table 1.2 : Some of the secondary metabolites that have been reported to have anti-diabetic properties. 7 1.3 Approaches to anti-diabetic screening of S. macrophylla seeds extract. 1.3.1 Extraction of S. macrophylla seeds. Extraction is a process of transfering a solute from one phase to another. In other words, this is the separation of a substance from a mixture by dissolving that substance in a suitable solvent. Extraction is one of the important parts in bioactivity guided isolation study. Figure 1.1 shows the flow chart for the study of plants used in traditional medicine (Luc Pieters & Arnold., 2005). A successful extraction, should begin with careful selection of solvents and preparation of the plant samples. The extraction method chosen should try to minimize interference from compounds that may be co-extracted with the target compounds, and contamination of the extract obtained. The extraction method used if possible can prevent decomposition of important metabolites or avoid artifact formation as a result of extraction conditions or solvent impurities. In this study,’ serial extraction’ has been chosen as the method of extraction . Serial extraction refers to the use of increasing polarity of solvents in order to remove different polarity of secondary metabolites from the plants. The solvents used in this study were petroleum ether, chloroform, methanol and water. 8 1.3.2 In-vivo and in-vitro studies of S. macrophylla seeds extracts 1.3.2.1 In-vivo experiment. In- vivo experiment defines as experiment conducted in a living body. In vivo testing is often employed over in vitro because it provided overall effects of an experiment on a living subject (Wikipedia.org, accessed on 10 Dec. 2010). This is due to, some conditions in the in-vitro are unrealistic or unsimilar to the actual environment within the living organism. For instance, if the active entity of a substance is its metabolite, the substance might not show any activity if being tested using in-vitro method. In this study, we have chosen three different in-vivo experiments using the actual animal models , which includes both normal and diabetic rats. The experiments are hypoglycemic and intraperitoneal glucose tolerance tests in normal rats, and also antihyperglycaemic test in diabetic rats. For the animal models of diabetes mellitus, the condition can be induced through pharmacological, surgical or genetic manipulations in several animal species. Most experiments in diabetes are carried out on rodents, although some studies are performed in larger animals. The classical model employed by Banting and Best was pancreatectomy in dogs (Bliss, 2000). However, majority of studies published in the field of ethnopharmacology between 1996 and 2006 employed models, where diabetes was induced through administration of chemicals such as streptozotocin (STZ, 69%) and alloxan (31%). This model has been useful for the study of multiple aspects of the disease. Both drugs exert their diabetogenic action when they are administered parenterally: intravenously, intraperitoneally or subcutaneously. The dose of these agents required for inducing diabetes depends on the animal species, route of 9 administration and nutritional status. According to the administered dose of these agents, syndromes similar to either type 1, type 2 diabetes mellitus or glucose intolerance can be induced (Lenzen et al., 1996; Mythili et al., 2004). Methods are available, but the pH and type of buffer employed as well as the preparation of the solution of either alloxan or streptozotocin in the day of the experiments are still arguable (Yu et al., 2000; Lei et al., 2005;; Miranda et al., 2006). In this study, the diabetic rats are being induced by a single intraperitoneal (i.p) injection of 60mg/kg streptozotocin(STZ) dissolved immediately in normal saline (0.9% NaCl, pH 4.5) before used, in overnight (16 hours) fasted rats (Dominguez et al,. 2000). Hyperglycemia (diabetes) was confirmed by the elevated blood glucose level determined at 72 hours after STZ injection and the symptom of polyurea. The rats found with a fasting blood glucose (FBG) level above 15mmol/L were considered to be diabetic and used in the experiment. 1.3.2.2 In-vitro experiment In vitro is the opposite of in vivo, which means a biological process or experiment that is performed not in a living organism but in a controlled environment, such as in a test tube or Petri dish (Kail et al., 2006). In-vitro test involved the use of organs, tissues, cells, cellular components, proteins, and/or biomolecules. It is a better test for deducing biological mechanisms of action compare to the in-vivo approach. However, the controlled conditions present in the in vitro system differ significantly from those in vivo, and may give misleading results due to the fact that living organisms are extremely complex functional systems that are made up of genes, protein molecules, RNA molecules, small organic compounds, inorganic ions and complexes in 10 an environment that is spatially organized by membranes and, in the case of multicellular organisms, organ systems (Alberts and Bruce., 2008). Therefore, in vitro studies are usually followed by in vivo studies. In this study, we used the isolated tissue preparation in order to determine or elucidate the mechanism of actions of the extract. Two different experiments will be carried out to determine whether the antihyperglycaemic properties of these extracts act through extrapancreatic mechanism of action. The first experiment involve the study of glucose uptake by muscles using isolated abdominal muscle preparation of Gray and Flatt, (1998) with some modification. Another experiment is to study the effect of the extracts on glucose absorption in the gastrointestinal tract by using the modified averted sac technique of Wilson and Wiseman, (1954). Increase of glucose uptake by muscles and inhibition of glucose absorption in the gastrointestinal tract are among the extrapancreatic mechanisms known to cause the lowering of blood glucose levels. 1.3.3 Single dose and repeated doses (14 days) studies Acute studies (single dose) are short-term studies with exposure periods usually less than 24 hours or maybe last for 48 to 96 hours. This study is usually implemented for the purpose of screening for various kinds of bio-activities in living organism. Opposite to the acute study, is the chronic study (repeated doses). In certain conditions, a single dose study might not give positive result or exert any potential bio-activity when being tested. However, this does not conclude that the bio-activities are not there or in other words the plant or compound do not possess the bio-activity. Chronic study (repeated dose) in animals are usually necessary to evaluate toxic characteristics of a chemical arise from repeated exposure over a period of time. 11 Repeated dose studies, mostly have been carried out after initial information on toxicity has been obtained from acute toxicity test. Duration of exposure normally depends on nature of chemical and circumstances. In my study for the fourteen days treatment, the extracts were administered orally, twice daily (9.00 a.m and 9.00 p.m) for fourteen days . The animals were observed individually after dosing, daily for fourteen days. Each animal was observed for toxic sign and recorded. Body weight and fasting blood sugar level were measured once a week. The baseline (day 0, before the treatment start) and final (day 15,after two weeks treatment) weight and fasting blood sugar level were examined and recorded. 1.3.4 Phytochemical screening Phytochemistry, or easily defined as the chemistry of plants, is one of the early subdivisions of organic chemistry. This division is importance in the identification of plant substances of medicinal value. It deals with the the study of chemicals derived from plants (phytochemicals). Phytochemicals are chemical compounds such as β-carotene that occur naturally in plants. The term is generally used to refer to those chemicals that may affect health, but are not yet established as essential nutrients (Satyanand Tyagi., 2010) However, the use of phytochemicals comes with a caution sign. These compounds were not always beneficial under all circumstances especially in high doses. Certain biochemicals and vitamins, have been found to encourage the growth of cancer cells and their use is being discouraged in patients undergoing cancer treatments. Although it has many benefits in certain conditions, high doses of beta-carotene 12 supplements are associated with an increased risk of lung cancer in male smokers. (Russel R et al., 1999) With the development of new phytochemical methods, new information has become available for use in conjunction with plant taxonomy; which contributed to the modern field of chemotaxonomy, or biochemical (Britannica.com., accessed on 23 Jan 2011). *SAR studies (Structure-activity-relationship studies) Figure 1.1 Flow chart for the study of plants used in traditional medicine (Luc Pieters and Arnold., 2005) 13 1.4 Swietenia macrophylla (SM) King Swietenia macrophylla King, from family Meliaceae is also known as big-leaf mahogany or Honduras mahogany. It is a tropical timber tree that can grow to a height of 40-60 m. This tree produces a fruit commonly called “sky fruit” because the fruit seems to hang upwards from the tree. In Malaysia, the locals usually called it as ‘Tunjuk Langit’. This plant is naturally distributed from the Atlantic regions of south-east Mexico, through Central America, northern South America and across the southern Amazon Basin, in Bolivia and Brazil, but nowadays this plant is cultivated in South East Asia region for various purposes (Lugo et al,2003). ‘Tunjuk Langit’ has been processed commercially to a wide range of health foods and healthcare products.  Scientific classification : Kingdom: Plantae Order: Sapindales Family: Meliaceae Genus: Swietenia Species : S. macrophylla 14 Figure 1.2 : a) Tree b) Leaves c) Fruit d) Seeds 15 This plant is an umbrella-shaped crown. fast-growing perennial tree with tall straight, cylindrical bole clear of branches for 12-18m, often with high buttresses. It has pinnate leaves arranged alternately and clustered at the ends of branchlets. Each leaf consists of 3- 6 pairs of opposite or occasionally subopposite leaflets that are typically 9-14 x 3-5 cm, usually oblong to oblong-lanceolate or ovate-lanceolate (Lugo et al.,2003). 1.4.1 The morphology of fruit and seeds of S. macrophylla Figure 1.3 The fruit of S. macrophylla The fruits are large (12-15 x 6-8 cm), woody, erect, capsules, oblong to slightly sub-globulus. The outer valves are thick and becoming woody with a coriaceous surface when mature. When dry, the 4 or 5 valved fruits split open from the base, or from the base and the apex simultaneously. The centre of the fruit is a thick, woody 5 angled columella extending to the apex from which the seeds hang pendulous by their wing, leaving conspicuous seed scars after their release. 16 Figure 1.4 : The seeds of S. macrophylla The seeds are chestnut colored and 7.5-12 cm in length with wings. There are arranged in irregular forms inside the fruit. There are usually about 35-45 winged seeds per fruit and it takes around 13,000 to 20,000 seeds to reach a kilogram weight. This seeds ripen from the end of January to beginning of March and varies according to location. 1.4.2 Phytochemistry of Swietenia macrophylla Phytochemical study of Swietenia macrophylla King has revealed various compound exist in this plant. The first phytochemistry study on S. macrophylla was reported in 1951 by Guha Sircar et al where they successfully isolate two tetranotriterpenoids known as swietenine (figure 1.5) and swietenolide. Later, Mootoo et al in 1999, reported that, upto fifteen limonoids have been isolated from S. macrophylla, which are 7-deacetoxy-7-oxogedunin, andirobin (figure 1.8), and thirteen bicyclononanolides. In 1999, they successfully discovered another two new compounds which are augustineolide and 3β,6-dihydroxydihydrocarapin from the same plant . In addition, they also mentioned another nine known compounds that have been identified 17 from S. macrophylla which are; proceranolide, 6-O acetylswietenolide, 3,6-O,O- diacetylswietenolide, Khayasin T, Swietemahonins E-G, 2-hydroxyswietenine and 6-deoxyswietenine also known as febrifugin (figure 1.10). Further phytochemical investigations have revealed the presence of limonoids such as 8, 30-epoxyswietenine acetate, swietenine acetate, swietenolide diacetate (figure 1.6), and swietenolide tiglate, in the seeds of S. macrophylla (Chakravarty & Chatterjee, 1955; Chakravarty, Chatterjee, & Krishnagar, 1957; Chan, Tang, & Toh, 1976; Connolly, Henderson, McCrindle, Overton, & Bhacca, 1964; Connolly & Labbe, 1980; Ghosh, Chakrabartty, & Chatterjee, 1960). Other parts of the plant, which is the leaves of S. macrophylla, have been reported to yield essential oils which contain himachalene, germacrene D, germacrene A, cadina-1,4-diene, hexadecanoic acid, and ethyl hexadecanoate (Marisi et al., 2003). Seok-Keik Tan et al in 2009, also mentioned four new phragmalin-type limonoids, named swietephragmin H-J and swietemacrophine have been isolated from the leaves of S. macrophylla King. The barks of S. macrophylla have been reported to possess triterpenoids, limonoids, flavonoids and tannins according to Guha Sircar et al in 1951 and Mootoo et al in 1999. Figure 1.5 18 Figure 1.6 Swietenolide diacetate Figure 1.7 Swietenolide monohydrate Figure 1.8 Andirobin Figure 1.9 Swietemahonin A Figure 1.10 Febrifugin 19 1.4.3 Medicinal value of S. macrophylla Herbal medicines have been used since the dawn of civilization to maintain health and to treat disease. There is a tremendous historical legacy in folklore uses of plant preparations in medicines. Scientific studies on plants used in ethnomedicine led to the discovery of many valuable drugs (Shylesh BS and Padikkale J, 2000). The seeds of S. macrophylla have been used for Leishmaniasis and abortion medicine by an Amazonian Bolivian ethnic group (Bourdy et al.,2000) and for treatment of hypertension, diabetes and malaria as folk medicines in Indonesia (Kadota et al., 1990a). Murningsih et al in 2005, reported anti-malarial activity of water extract of S. macrophylla seeds against Plasmodium falciparum . The seeds also have antibabesial (Murningsih et al.,2005) and anti-diarrhoeal activities (Maiti et al.,2007). The seeds also have been reported to possess antiinflammatory, antimutagenicity and antitumor activity (Maiti et al., 2007). In Malaysia, the seeds of S. macrophylla, are used as a folk medicine for treating diabetes and high blood pressure (Chan et al, 1976). A decoction of the crushed seeds of this tree is also used to treat skin ailments and wounds (Munoz et al, 2000). The seeds are also used for diarrhea and a survey of literature showed that the organic and aqueous extracts of S. macrophylla seeds possess a wide array of biological properties including anti-diabetic (Maiti et al, 2007a) . In the same year, Maiti et al also discovered that S. macrophylla also possess anti-diarrhoeal (Maiti et al,2007b) and antibabesial activities (Maiti et al,2007c). Anti-microbial and anti-malarial activities have also been reported with this plant (Soediro et al,1990, Jean et al, 2000). 20 Guevera AP et al in 1996, mention that the seed of S.macrophylla had been reported to have anti-inflammatory, antimutagenicity, and anti-tumor activities. S.macrophylla also have been shown to exhibit insect antifeedant activities (Nsiama et al., 2008), anti-fungal (Govindachari et al., 1999) and in Malaysia, the seeds are used as a folk medicine for treatment of hypertension (Chan, Tang, & Toh, 1976). Munoz et al (2000) reported that this plant also has anti-inflammatory activity. The barks of S. macrophylla also reported to possess anti-HIV, antimicrobial, antimalarial, and antitumor activities (Munoz et al, 2000) . 1.5 Diabetes mellitus, (DM) disease of the millennium Diabetes is one of the oldest diseases known to mankind. It was first mentioned in the Ebers Payrus (Egypt 1500BC) and also called as ‘honey urine’ by Sushrutha in India in 400 BC (Larry A. Distiller.,1980). The Latin word ‘mel’ which means honey was used, and the disease came to be known as diabetes mellitus that is, the passing of honeyed urine. This is still the full name of the disease. Diabetes occur when the quantity of insulin produced is too little to allow for glucose to be used or to be stored. In other words, this disease will appear when a person’s body does not make enough insulin or cannot use insulin properly. Therefore, the hallmark of diabetes, which is due to insulin deficiency, is a rapid increase in the level of glucose in the blood to more than normal levels. Diabetes can be divided into two groups : Type 1 which is known as insulin dependant diabetes mellitus (idDM) and also Type 2 ; non-insulin dependant diabetes mellitus (nidDM). 21 According to World Health Organization (WHO) data, based on a survey conducted in 1996 in Malaysia , it was estimated that around 700,000 to 900,000 adults aged 30 and above were having diabetes or there are 8 diabetic patients in every 100 adults. 1.5.1 What causes diabetes? The causes of diabetes mellitus are rather ill understood even now. However, as mentioned previously, the most important cause of diabetes mellitus is the failure of beta cells in the pancreas to secrete the insulin hormone sufficiently to the body needs. Causes of type 1 diabetes differs from type 2 in many aspects . Type 1 diabetes is mainly due to the destruction of insulin-producing beta cells of the pancreas with resulting loss of insulin production. The actual process causing the damage of the beta cells is still unknown although there are many theories. As for type 2 diabetes, which usually develops gradually, differs greatly from the type 1. The cause is unknown. One type of type 2 is associated with obesity with marked insulin resistance. Many factors are blamed for the cause of this type of diabetes but recently more interest has been shown in the possibility of faulty neural mechanisms of metabolic control. Unlike type 1 diabetes, islet cell antibodies are not found in type 2 diabetes. Table 3 shows some of the causes of diabetes mellitus. 22 Table 1.3 : Causes of diabetes mellitus (Lee Y.S., 1991). CAUSES OF DIABETES MELLITUS TYPE 1 : a) Viral Infection ?mumps ?Coxsackie B4 virus ? Rubella b) Hereditary c) Auto- immunity d) Toxin to Islet cells. TYPE 2 : a) Hereditary + i ? diet ii ? exercise b) Obesity 23 1.5.2 Pathophysiology of diabetes mellitus (DM) Glucose is a simple sugar found in food. Glucose is an essential nutrient that provides energy for the proper functioning of the body cells. Carbohydrates are broken down to glucose in the small intestine, and the glucose is then absorbed by the intestinal cells into the bloodstream, and is carried by the bloodstream to all the cells in the body where it is utilized. However, glucose cannot enter the cells alone and needs insulin to aid in its transport into the cells. Without insulin, the cells become starved of glucose energy despite the presence of abundant glucose in the blood stream. The abundant, unutilized glucose is wastefully excreted in the urine. Insulin is a hormone that is produced by specialized cells (beta cells) of the pancreas. (The pancreas is a deep-seated organ in the abdomen located behind the stomach). In addition to helping glucose enter the cells, insulin is also important in tightly regulating the level of glucose in the blood. After a meal, the blood glucose level rises. In response to the increased glucose level, the pancreas normally releases more insulin into the bloodstream to help glucose enter the cells and lower blood glucose levels after a meal. When the blood glucose levels are lowered, the insulin release from the pancreas is turned down. As outlined above, in patients with diabetes, the insulin is either absent, relatively insufficient for the body's needs, or not used properly by the body. All of these factors cause elevated levels of blood glucose (hyperglycemia). Over a period of time, high glucose level in the bloodstream can lead to severe complications, such as eye disorders, cardiovascular diseases, kidney damage and nerve problems. 24 Figure 1.11: Overview of the pathogenesis of type 2 diabetes mellitus ( adopted from (http://www.pharmainfo.net ) 1.5.3 Treatment for diabetes mellitus (DM) When diet and exercise unable to lower the raised blood sugar level to an acceptable value, the use of drugs became a must. Many Type 2 diabetics will require the use of hypoglycaemic drugs to lower the blood sugar. However, almost all Type 1 diabetics will not respond to oral hypoglycaemic drugs, hence the only way to overcome this type of diabetes is through administration of Insulin. Generally, there are four main groups of oral hypoglycaemic agents (OHA) available for the treatment of diabetes, which are classified according to their mode of actions. They are Insulin secretagogues, insulin sensitizers, α-glucosidase inhibitors and biguanide (Lebovitz 2001). * FFA = free fatty acid 25 1.5.3.a Insulin Secretagogues. Insulin secretagogues can be divided into 2 subclasses: sulfonylureas and non-sulfonylureas. The basic action of sulphonylureas is normalizing the release of insulin by some healthy β-cells of the pancreas (Lee Y.S.,1991). It also reduces the production of glucose in the liver and the absorption of glucose from the gut. Some sulphonylureas are said to enhance insulin sensitivity in the peripheral tissues. Sulfonylureas that are currently available are glipizide, gliclazide, glibenclamide, glimerpide and glyburide. As for the non-sulphonylureas, the mechanism of action of these drugs is similar to that of the sulfonylureas , except they bind to the sulfonylurea receptor at a different site and with different kinetics than the sulfonylureas. Nateglinide and repaglinide are some of the drugs belong to this group. 1.5.3.b Insulin Sensitizers Thiazolidinediones (TZD) are the main insulin sensitizers currently available. Examples of TZDs are rosiglitazone and pioglitazone. TZDs are selective agonists for the peroxisome proliferator-activated receptor gamma (PPAR γ), which is most highly expressed in adipocytes. These nuclear receptors, which are ligand-activated transcription factors, play an integral part in the regulation of the expression of a variety of genes involved in carbohydrate and lipid metabolism (Lebovitz.,2001). TZDs require insulin to be present for their activity, where they increase the insulin sensitivity, particularly in the peripheral tissues.

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