MIR MOHAMMAD REZA SEYED HASSANI
ANTIHYPERGLYCEMIC ACTIVITY OF TINOSPORA CRISPAEXTRACTS AND FRACTIONS IN RATMIR MOHAMMAD REZA SEYED HASSANIUNIVERSITI SAINS MALAYSIA2015
ANTIHYPERGLYCEMIC ACTIVITY OF TINOSPORACRISPA EXTRACTS AND FRACTIONS IN RATByMIR MOHAMMAD REZA SEYED HASSANIThesis submitted in fulfillment of the requirements for the degree ofMaster of ScienceAugust 2015
ACKNOWLEDGEMENTSAll praises to Allah the Almighty, without whom will everything would ceaseto be, who gave me the strength, inspiration and patience to continue this research.First of all Special thanks to my parents Mir Jafar, Mahin and my brotherMir Morteza for their supports all this years. I would like to express my deepgratitude and sincere appreciation to my supervisor Prof. Dr. Mohd Zaini Asmawiwho provided me the germ of the idea for this research. Over the months of researchand analysis, his deep and broad knowledge, constructive suggestions, stimulatingdiscussion and comments on the preparation of this thesis as well as his wisdom andwit helped that germ to sprout/germinate and grow. He had always open door for meto see him for whatever duration or reason although he was busy with his otherstudents. I also wish to thank my co-supervisor Dr. Roziahanim Binti Mahmud forher guidance and assistance throughout the chemical aspect of this work. I wouldlike to express my gratitude to the Dean of School of Pharmaceutical SciencesDr. Munavvar Zubaid Abdul Sattar, for giving me the chance to pursue my highereducation in this school. Also, my thanks go to the Institute of Postgraduate Studiesand Universiti Sains Malaysia for their kind cooperation and assistance rendered inthe preparation of this thesis and a warm thanks to director of International office,Assoc. Prof. Dr. Anees Janee Ali for his helps, kindness and guides. A number ofother academic and non-academic staff at the Universiti Sains Malaysia also gave metheir support and assistance, including Mr. Rosli, the laboratory assistant in thepharmacology research laboratory and Mr. jasmie for his helps. Also, I am verygrateful to all my colleagues who were always my real friends Abdulmenem, Adlin,Bassel, Faramarz, Idris, Item, Michael, Nasiba, Raghdaa and Sook yee for theirhelps, kindness and supports.ii
TABLE OF CONTENTSPageACKNOWLEDGMENTiiTABLE OF CONTENTSiiiLIST OF TABLESixLIST OF FIGURESxLIST OF ABBREVIATIONSxiiiLIST OF PUBLICATION ASSOCIATED WITH THIS THESISxivABSTRAKxvABSTRACTxviiCHAPTER ONE: INTRODUCTION1.1 Introduction11.2 Diabetes Mellitus31.3 Classification of Diabetes Mellitus51.4 sm91.5 Antidiabetic pha-glucosidase inhibitors111.5.4Thiazolidinedione12iii
1.5.5Incretin mimetics / GLP-1 analogues131.5.6Meglitinide131.5.7Dipeptidyl peptidase-4 inhibitor / Gliptins131.5.8Insulin151.5.8.1 Rapid action insulin151.5.8.2 Short action insulin161.5.8.3 Intermediate action insulin161.5.8.4 Long action insulin161.6 The plant, Tinospora crispa181.6.1Botanical aspects181.6.2Background201.7 Antihyperglycemic drug methodologies221.7.1in vitro techniques221.7.2in vivo techniques231.8 Hypoglycemia studies241.8.1Mechanisms of action of hypoglycemic agents241.8.2Glucose loading test241.9 Phytochemical screening of water and methanol extractsof Tinospora crispa251.1026Objectives of the present studyCHAPTER TWO: MATERIALS AND METHODS2.0 Introduction materials and methods272.1 Materials and their sources272.2 Instrument used and their sources292.3 Methods30iv
2.3.1Experimental animals302.3.2Plant material collection302.3.3Preparation of extracts322.3.4Antidiabetic evaluation of Tinospora crispa extracts332.3.4.1 Hypoglycemic activity in normal rats332.3.4.2 Intraperitoneal glucose tolerance test (IPGTT) in normal rats332.3.4.3 Diabetes induction342.3.4.4 Antihyperglycemic effect of Glibenclamide and Metformin on 35diabetic rats2.3.4.5 Antihyperglycemic activity of crude extracts of Tinospora 35crispa in STZ induced diabetic rats2.3.5Water extract fractionation using solvent-solvent extraction method362.3.5.1 Intraperitoneal glucose tolerance activity (IPGTT) in normal 38rats2.3.5.2 Antihyperglycemic activity of fractions of crude water extract, 38n-butanol and aqueous fraction in STZ induced diabetic rats2.3.5.3 Dose-response study of the most active aqueous fraction of 39Tinospora crispa water extract2.4 In vitro antidiabetic mechanism study of the most effective fraction2.4.139Effect of the fractions on glucose uptake in isolated rat abdominal 39muscle2.4.1.1 Buffer solution preparation392.4.1.2 Experimental setup402.4.2Effect of the fractions on glucose absorption in isolated rat intestine 402.4.2.1 Experimental setup402.5 Determination of lipid level in blood sample of treated rat with petroleum 42ether, chloroform, methanol and water extracts of Tinospora crispav
2.6 Phytochemical screening of the water and the methanolic extractsof Tinospora crispa stems2.6.1Detection of Alkaloids43432.6.1.1 Preparation of Dragendorffs reagent432.6.1.2 Alkaloid study432.6.2Detection of flavonoids (H2SO4 Test)442.6.3Detection of cardiac glycosides442.6.4Detection of steroids442.6.5Detection of tannins452.6.6Detection of terpenoids452.6.7Detection of saponins452.7 Gas Chromatography Mass Spectrophotometry (GC-MS) analysis ofTinospora crispa water fraction462.7.1Sample Preparation and Derivatization462.7.2GC-MS Chromatographic Condition462.8 Development and validation of High Performance Liquid Chromatography 47(HPLC) methods2.8.1Preparation of standards and water fractions of Tinospora crispa for 47HPLC analysis2.8.2Tinospora crispa fraction Samples472.8.3Chromatographic conditions for Analysis of SalsolinolHydrobromide482.8.4Chromatographic conditions for Analysis of Gallic Acid48502.9 Data AnalysisCHAPTER THREE: RESULTS513.1 Extraction Process3.1.1Hypoglycemic activity in normal ratsvi51
3.1.2Intraperitoneal glucose tolerance test (IPGTT) in normal rats523.1.3Effect of glibenclamide and metformin in subcutaneous glucosetolerance tests523.1.4Antihyperglycemic effect of Glibenclamide and Metformin indiabetic rats523.1.5Antihyperglycemic activity in Streptozotocin-induced diabetic rats533.1.5.1 Effect of daily oral administration of Tinospora crispa extracts on the 53body weight of diabetic rats.3.2 Fractionation of the water extract of Tinospora crispa603.2.1Intraperitoneal glucose tolerance test (IPGTT) in normal rats3.2.2Antihyperglycemic activity of fractions of the water extract on 61diabetic rats Intraperitoneal3.2.2.1 Dose response study of the most active fraction (aqueous fraction)3.2.36061Effect of daily oral administration of water fraction of water extract 62of Tinospora crispa on body weight.3.3 In vitro studies673.3.1Effect of water and n-butanol fractions on glucose uptake of isolated 67rat abdominal muscles3.3.2Effect of water and n-butanol fractions on glucose absorption of 67isolated rat intestine3.4 The effect of petroleum ether, chloroform, methanol and water extracts of 71Tinospora crispa on lipid profiles3.5 Phytochemical screening of water and methanol extracts of Tinospora crispa 713.6 Gas chromatography–mass spectrometry (GC-MS) analysis773.7 Development and validation of High Performance Liquid Chromatography 82(HPLC) methodsCHAPTER FOUR: DISCUSSION AND CONCLUSION874.1 Antidiabetic study914.2 In vitro and in vivovii
4.3 Phytochemical screening934.4 Conclusion954.5 Recommendation for further studies96REFERENCES97APPENDICES107viii
LIST OF TABLESPageTable 1.3Chemical structures of important alpha-glucosidase inhibitor drugs.12Table 2.1Chemicals and reagents.27Table 2.2Instrument used in this study29Table 2.3Elements combined for preparing 2 L of Kreb’s ringer BicarbonateBuffer.39Table 2.4Gradient elution program used in separation of Gallic acid in waterfractions of Tinospora crispa.49Table 3.1The amount of extracts obtained from the stems of Tinospora crispa51Table 3.2The amount of fractions obtained from the water extract ofTinospora crispa stems.60Table 3.3.Results of phytochemical screening of water extract and methanolextract of Tinospora crispa72Table 3.4Volatile chemical compounds in water fraction of water extract ofTinospora crispa identified by GCMS.77Table 3.5Intra-day and inter-day precision of salsolinol hydrobromide andgallic acid (based on HPLC method, n 6).85Table 3.6Accuracy of the HPLC methods in water fractions of Tinosporacrispa.85ix
LIST OF FIGURESPage1.1Pancreas system71.2Chemical structure of metformin101.3Chemical structures of important alpha-glucosidase inhibitor drugs121.4Mechanism of DPP-4 inhibitors and GLP-1141.5Antidiabetic Drugs action on different organs171.6The stem of Tinospora crispa plant.181.7The Tinospora crispa plant192.1(i) Leaves and stems of Tinospora crispa, (ii) Documentcertification authentication of plant specimen (voucher no. 11509)312.2The Tinospora crispa plant dried in the oven322.3Schematic diagram of Tinospora crispa water extract fractionationusing solvent-solvent extraction method372.4GC-MS Function and its configuration473.1Effect of oral administration of the petroleum ether, chloroform,methanol and water extracts of Tinospora crispa stems at a dose of 1g/kg on the blood glucose levels of normal rats. Values areexpressed as the mean SEM of six animals. * P 0.05 comparedwith the diabetic control.543.2Effect of oral administration of petroleum ether, chloroform,methanol and water extracts of Tinospora crispa and followed onehour later with glucose 1.0 g/kg intraperitoneally on the bloodglucose levels of normal rats. Values are expressed as the mean SEM of six animals. * P 0.05 compared to the control group.3.3Effect of oral administration of glibenclamide (10 mg/kg) andmetformin (500 mg/kg) and followed one hour later with glucose1.0g/kg intraperitoneally on the blood glucose level of normal rat.Values are expressed as the mean SEM of six animals. * P 0.05compared to the diabetic control563.4Effect of daily oral administration of glibenclamide (10 mg/kg) andmetformin (500 mg/kg) for 12 days on streptozotocin-induceddiabetic rats. Values are expressed as mean SEM of six animals.* P 0.05 and ** P 0.01 compared to the diabetic control group.57x55
3.5Effect of daily oral administration of Tinospora crispa petroleumether, chloroform, methanol and water extracts on the blood glucoselevels of STZ- induced, diabetic rats. Values are expressed as themean SEM; of six animals. * P 0.05 and ** P 0.01 comparedto the diabetic control group.583.6Effect of daily oral administration of 1 g/kg of the petroleum ether,chloroform, methanol, and water extracts of Tinospora crispa stemson the body weights of STZ-induced diabetic rats. Values areexpressed as the mean SEM of six animals.* P 0.05 compared tothe diabetic control group593.7Effect of oral administration of n-butanol and water fractions ofTinospora crispa water extract on the blood glucose levels ofnormal rats intraperitoneally loaded with 1 g/kg glucose. Values areexpressed as the mean SEM of six animals.* P 0.05 compared tocontrol group633.8Effect of oral administration of n-butanol and water fractions of thewater extract of Tinospora crispa on the blood glucose levels ofSTZ-induced diabetic rats. Values are expressed as the mean SEMof six animals.*P 0.05 and ** P 0.01 compared to the diabeticcontrol group643.9Effect of daily oral administration of different doses of waterfraction (500, 250 and 100 mg/kg) on the blood glucose levels ofSTZ-induced diabetic rats. Values are expressed as the mean SEMof six animals. ** P 0.01 compared to diabetic control group.653.10Effect of daily oral administration of different doses of waterfraction (500, 250 and 100 mg/kg) on the body weight of STZinduced diabetic rats. Values are expressed as the mean SEM ofsix animals.663.11Effect of water and n-butanol fractions on glucose uptake of isolatedrat abdominal muscle incubated in Tyrode’s solution. Values are themean of 6 determinations SEM.683.12Effect of water and n-butanol fractions on glucose uptake byisolated rat abdominal muscle when incubated in the Tyrode’ssolution containing 1 IU/ml insulin. Values are expressed as themean of 6 determinations SEM. *P 0.05 vs. the control.693.13Effect of acarbose, water fraction and n-butanol fraction on glucoseabsorption by everted rat jejunum.* P 0.05; ** P 0.01 vs. thecontrol.70xi
3.14Effect of daily oral administration of petroleum ether, chloroform,methanol and water extracts of Tinospora crispa stems 1 g/kg for 9days on the triglycerides levels of diabetic rats. Values are expressedas the mean SEM of six animals.733.15Effect of oral administration of the petroleum ether, chloroform,methanol and water extracts of Tinospora crispa stems 1 g/kg for 9days on the LDL levels of diabetic rats. Values are expressed as themean SEM of six animals. * P 0.05 compared with the diabeticcontrol.743.16Effect of a daily oral administration of the petroleum ether,chloroform, methanol and water extracts of Tinospora crispa stems1 g/kg for 9 days on the HDL levels of diabetic rats. Values areexpressed as the mean SEM of six animals.* P 0.05 comparedwith the diabetic control.753.17Effect of a daily oral administration of petroleum ether, chloroform,methanol and water extracts of Tinospora crispa stems 1 g/kg for 9days on total cholesterol levels of diabetic rats. Values are expressedas the mean SEM of six animals.* P 0.05 and ** P 0.01compared with the diabetic control.763.18GC-MS analysis of water fraction ethanolic solution of Tinosporacrispa783.19AGC-MS analysis: Detection of dodecanol in water fraction ofTinospora crispa793.19BGC-MS analysis: Detection of dodecyl acrylate in water fraction ofTinospora crispa793.19CGC-MS analysis: Detection of pentadecanoic acid in water fractionof Tinospora crispa803.19DGC-MS analysis: Detection of propanoic acid in water fraction ofTinospora crispa803.19EGC-MS analysis: Detection of decanedioic acid in water fraction ofTinospora.crispa813.20HPLC chromatograms of water fractions of Tinospora crispa andGallic acid. Note: Gallic acid standard (A), water fractions ofTinospora crisp (B)833.21HPLC chromatograms of water fractions of Tinospora crispa andSalsolinol hydrobromide. Note: Salsolinol hydrobromide standard(A), water fractions of Tinospora crisp (B)84xii
LIST OF ABBREVIATIONS0CDegree Celsius%PercentAFAqueous fractionANOVAAnalysis of varianceBFn-butanol fractionb.w.Body weightet al.And othersgGramGC-MSGas chromatography–mass spectrometryHFn-hexane fractionHPLCHigh-performance liquid chromatographyIDDMInsulin dependent diabetes mellitusi.p.IntraperitonealIPGTTIntraperitoneal glucose tolerance testIUInternational unitsKgKilogramKRBKrebs-Ringer bicarbonateOGTTOral glucose tolerance testSTZStreptozotocinWFWater fractionxiii
LIST OF PUBLICATION ASSOCIATED WITH THIS THESISJornal PablicationAcceptedMir Mohammad Reza Seyed Hassani, Ashfaq Ahmad, Mohd. Zaini normoglycemic,anti-hyperglycemic and dyslipidemic activities of different extracts of Tinosporacrispa on diabetic rat (2015).On ReviewMir Mohammad Reza Seyed Hassani, Item Justin Atangwho and Mohd. ZainAsmawi. Antihyperglycemic activity of extract and fractions of Tinosporacrispa in diabetic rats and intestine (2015).Poster PresentationMir Mohammad Reza Seyed Hasani,Pros Dr. Mohd. Zaini Asmawi, ParastooZarghami Moghaddam, Helen Hemati. Evaluation of anti-hyperglycemic effectof Tinospora crispa in traditional medicine in in vivo model. University ofMedical Sciences, Bojnurd, Iran (2013).xiv
AKTIVITI ANTIHIPERGLISEMIK EKSTRAK DAN FRAKSI TINOSPORACRISPA PADA TIKUSABSTRAKTinospora crispa (Menispermaceae) yang nama tempatanya dikenali sebagai"akar patawali", telah lama digunakan dalam perubatan tradisional Malaysia untukrawatan kencing manis. Tujuan kajian ini adalah untuk mengesahkan kesanhipoglisemik dan antihiperglisemik ekstrak Tinospora crispa yang berbeza bezadalam tikus normal dan tikus diabetik aruhan streptozotocin (STZ). Serbuk batangTinospora crispa kering diekstraksi secara berturut-turut scara maserasi dengan eterpetroleum, kloroform, metanol dan air untuk menghasilkan empat ekstrak. Tidak satupun daripada keempat-empat ekstrak (1.0 g / kg) yang diberikan secara oral dapatmenurunkan paras glukosa darah tikus normal yang menunjukkan kesanhipoglisemik.Keempat-empat ekstrak juga tidak menghalang kenaikan paras glukosa darah tikusyang dimuatkan dengan glukosa secara intraperitoneal. Pemberian ekstrak secaraoral 1 g / kg setiap hari selama 9 hari pada tikus diabetik aruhan STZ mendapatihanya ekstrak air dapat mengurangkan paras glukosa darah secara signifikan(P 0.05) berbanding dengan kumpulan kawalan yang menunjukkan bahawa ekstrakair mempunyai kesan antihyperglisemik. Ekstrak air kemudiannya difraksikan untukmendapatkan fraksi n-butanol dan fraksi air. Walau bagaimanapun, rawatan setiaphari untuk 12 hari mendapati hanya fraksi air (0.5 g / kg) menurunkan paras glukosadarah tikus diabetic aruhan-STZ. Penyaringan fitokimia ekstrak air Tinospora crispamenunjukkan kehadiran alkaloid, saponin dan glikosida. GC-MS fraksi airmenunjukkan kehadiran dodesil akrilat, asid pentadekanoik dan asid propanoikxv
manakala analisis HPLC menunjukkan kehadiran asid galik dan salsolinol. Dodesilakrilat, asid pentadekanoik, asid propanoik dan asid galik telah dilaporkan sebelumini memiliki kesan antidiabetik. Secara keseluruhan, hasil kajian ini menunjukkanTinospora crispa mempunyai kesan antidiabetik yang mungkin menyokongpenggunaannya di kalangan pesakit kencing manis. Beberapa sebatian yangdikenalpasti diatas mungkin telah menyumbang secara individu atau dalam gabungankepada aktiviti antidiabetik tersebut.xvi
ANTIHYPERGLCEMIC ACTIVITY OF TINOSPORA CRISPAEXTRACTS AND FRACTIONS IN RATS.ABSTRACTTinospora crispa (Menispermaceae), locally known as “akar patawali”, haslong been used in Malaysian traditional medicine for treatment of diabetes. The aimof this study was to verify the hypoglycemic and antihyperglycemic effects ofdifferent Tinospora crispa extracts in normal and streptozotocin (STZ)-induceddiabetic rats. Pulverized Tinospora crispa dried stems were extracted successively bymaceration with petroleum ether, chloroform, methanol and water to yield the fourextracts. None of the four extracts (1.0 g/kg) administered orally lowered the bloodglucose levels of normal rats which suggest that they have no hypoglycemic effect.The four extracts also did not inhibit the rise of blood glucose level of glucoseintraperitoneally loaded rats. Daily oral administration of the extract 1 g/kg for9 days in STZ-induced diabetic rats found that only the water extract managed tolower the blood glucose levels significantly (P 0.05), as compared with the controlgroup which suggests that it has antihyperglycaemic effect. The water extract wasthen fractionated to obtain n-butanol and aqueous fractions. However, dailytreatment for 12 days found that only water fraction (0.5 g/kg) lowered the bloodglucose level of STZ-induced diabetic rats. Phytochemical screening of the waterextracts of Tinospora crispa indicated the presence of alkaloids, glycosides andsaponins. GC-MS of water fraction showed the presence of dodecyl acrylate,pentadecanoic acid and propanoic acid whereas HPLC analysis showed the presenceof gallic acid and salsolinol. Dodecyl acrylate, pentadecanoic acid, propanoic acidand gallic acid have been previously reported to possess antidiabetic effect. Overall,xvii
the findings of this study showed Tinospora crispa possesses antidiabetic effectwhich may justified its use in diabetic patients. Some of the identified compoundsabove might have contributed individually or in combination to its antidiabeticactivity.xviii
CHAPTER ONE : INTRODUCTION1.1 IntroductionThe use of herbal remedies, in different cultures and traditional medicines, ha
MIR MOHAMMAD REZA SEYED HASSANI UNIVERSITI SAINS MALAYSIA 2015. ANTIHYPERGLYCEMIC ACTIVITY OF TINOSPORA CRISPA EXTRACTS AND FRACTIONS IN RAT By MIR MOHAMMAD REZA SEYED HASSANI Thesis submitted in fulfillment of the requirements for the degree of Master of Science August 2015 . ii
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