1. PRESENTED BY SUMAIYA NAHID M.PHARM USTC Histopathological changes of alloxan induced diabetic rat after administration of methanolic extracts of Syzigium cumini (L) Skeels seeds and isolation & characterization of compounds from ethyl acetate fraction.
2. Introduction Historically, plants have provided a source of inspiration for novel drug compounds. Plant derived medicines have made large contributions to human health and well being. Cutler, Stephen J.; Cutler, Horace G. (2000)Used as anticancer agent
4. Literature Review Significant blood glucose lowering activity was observed in fasted, fed, glucose loaded and diabetic rats at a single oral dose of 250mg/kg body weight. (Rahul Gupta and A. M. Saxena, 2011) Syzygium cumini seed kernel (aqueous suspension) was screened for its antidiabetic activity at the dose levels of 1g, 2g, 4g and 6 g/kg body weight.(R.Bhaskaran Nair and G.Santhakumari, 1986) The antibacterial and DPPH radical scavenging activities of the leaf extracts and leaf essential oil of Syzygium cumini Linn were investigated ( L.joji reddy, Beena jose, 1986) Isolation and identification of the putative antidiabetic compound, Mycaminose from the S. cumini seeds. ( A. Kumar, R. Ilavarasan, T. Jayachandran et al, 2008) 7-hydroxycalamenene , methyl--orsellinate , -sitosterol and oleanolic acid were isolated from the pet-ether and carbon tetrachloride soluble fractions of seeds of Syzygium cumini. (Md. Al Amin Sikder, Mohammad A. Kaisar et al 2012)
5. To evaluate the effectiveness of Syzygium cumini (L.) Skeels seeds in the management of Type 2 diabetes by following experiments- 1. Determination of phytochemical constituents of the crude extract 2.Evaluation of hypoglycemic activity 3. Estimation of lipid profile contents 4. Histopathological examination of Pancreas, Kidney, Liver, Heart, and Spleen. 5. Isolation and Characterization of compounds from Syzigium cumini (L.) Skeels seeds Aim of the Research
6. Phytochemical Screening Fehling Test Flavonoid test Shake Test Phenolic Test Alkaloid Test PolyPhenol test Steroid Test Glycoside Test Tannin Test Chemical constituent Name of Test Positive Results Alkaloids Wagner`s test + Glycosides General test ++ PolyPhenol Ferric Chloride test +++ Steroids Salkowski`s test ++ Tannins Acetic anhydride Test + Flavonoids Conc. HCl & alcoholic test ++ Saponins Shake test (aq. Solution) + Reducing sugar Fehling`s test ++
7. Determination of Total Phenolic Content (Danijela Bursac, kivacavic et al.2009) ) Materials & Method: Folin-Ciocalteu Reagent Sodium carbonate Methanol Test tubes Volumetric flask pipette Incubator UV spectrophotometer Standard- Gallic acid y = 0.0055x + 0.1474 R = 0.9503 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 50 100 150 200 250 Absorbanceat750nm Concentrationg/ml Calibration curve of Gallic acid Addition of FCR Addition of Sodium carbonate Absorbance measurement Fig: Total Phenolic Content of different extracts Danijela Bursac kivacavic et al.2009 0 20 40 60 80 100 120 140 n-hex DCM EtoAc n-Bu Aqu Totalphenoliccontent(g/ml) 82 g/ml 30 g/ml 122 g/ml 42 g/ml 120 g/ml
8. Total Flavonoid Content Determination Materials : Aluminum chloride NaNO2 NaOH Vortex and Incubator UV spectrophotometer Standard- Quercetine Aluminum chloride Method Extracts solutions prepared for absorbance measurement after the addition of NaNO2, AlCl3 & NaOH respectively y = 0.0411x + 0.0246 R = 0.9837 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 2 4 6 8 10 12 absorbance concentration (g/ml) absorbance Fig: Total flavonoid content of different extracts Danijela Bursac kivacavic et al.2009 Calibration curve for Quercetine -0.5 0 0.5 1 1.5 2 2.5 3 3.5 n_Hex DCM EtoAc n-But Aqu Totalflavinoidcontent(g/ml) 0.04 g/ml 1.2 g/ml 3 g/ml 0.9 g/ml 0.9 g/ml
9. Modified Kupchan partitioning of Methanolic crude extracts Ethyl Acetate Extract Powdered Plant Material N-Hexane Extract Methanolic Extract (5 gm) Aqueous Methanolic Solution Marc (Reject) DCM Extract Aqueous Methanolic Solution + 12.5ml water Aqueous Methanolic Solution + 16ml Water Aqueous Methanolic Solution+ 16ml Water 90ml Methanol+ 10ml Water Extraction with hexane(1003ml) Extraction with DCM(1003ml) Extraction with Butanol (1003ml) Butanol extract Aqueous extract Extraction with EA (1003ml) N-Hexane fraction DCM fraction EA fraction Butanol fraction Fractions for Evaporation Fig: Modified Kupchan partitioning of Methanolic crude extracts ( Hu, X. 1999).
10. Evaluation of Hypoglycemic Activity a) Evaluation of In vitro hypoglycemic activity - -amylase inhibition activity 1. Iodine-starch method 2. DNSA method b) Evaluation of In vivo hypoglycemic activity 1. Alloxan induced method
11. Addition of PPA Fig- comparative -amylase inhibition activity study of Ethyl acetate extract with standard Discussion: The ethyl acetate extract showed an increasing in vitro -amylase inhibition activity with the concentration in comparison with the Acarbose as standard and is an indication of its hypoglycemic action. * Xiao Z, Storms R, 2006 -20 0 20 40 60 80 100 120 0.1 0.3 0.5 0.7 1.1 1.3 1.5 %inhibition concentration (g/ml) Acarbose (standard) Ethyl acetate extract * Mean SEM, n = 3 Iodine-Starch Method * Addition of Iodine solution Blue-Violet complex Absorbance at 620nm Preparation of extracts solution Addition of starch sol * P < 0.07, ** p