Givotia Moluccana Analysis

Givotia Moluccana AnalysisMATERIALS AND METHODS4.1. PLANT MATERIAL4.1.1. COLLECTION OF PLANTThe plant aeriform parts of Givotia moluccanawas amass and Authentified by Dr. K. Madhava Chetty, Department of Botany, Sri Venkateswara University, Tirupathi (AP).4.1.2. PREPARATION OF THE EXTRACTThe dried leaves of G. moluccana was collected, cleaned, dried and powdered in a grinder - mixer to obtain a coarse powder and then passed through 40 mesh sieve. About coulomb0 gm of powdered drug was get outed with aqueous ethanol by soxhlet apparatus. The stock was carried out until the drug becomes exhausted. The solvent was recovered from their extract by distillation under reduced pressure. The dried extract thus obtained was kept in a desicator and was used for further experiments.4.2. EXPERIMENTAL ANIMALSHealthy adult male wistar rats weighing between 150-200gm were used for the present study. The animals were housed in groups of six and maintained under amount conditions (272C, relative humidity 44 56% and light and dark cycles of 10 and 14 hours respectively) and fed with type rat diet and purified drinking pissing ad libitum for 1 week before and during the experiments.All experiments and protocols described in present study were approved by the Institutional Animal Ethical direction (IAEC) of P.Rami Reddy Memorial Collage of Pharmacy (1423/PO/a/11/CPCSEA/102/2014).All the experiments were per organize in the morning fit to current guidelines for the care of laboratory animals and the ethical guidelines for the probe of experimental pain in conscious animals (Zimmerman, 1983).4.3. DRUGS AND CHEMICALSEpinephrine, DTNB, Triphenyl tetrazolium chloride and isoproterenol were obtained from Sigma-Aldrich, Bangalore. Thiobarbituric window glass (TBA), trichloro acetic astringent, hydrogen peroxide were obtained from SD fine chemicals Ltd Mumbai. Sodium dihydrogen phosphate, potassium dihydrogen phosphate, tris buffer and all separate reagents used were of anal ytical grade. CK-MB, LDH, SGOT, SGPT, ALP, Total cholesterol, HDL, and triglyceride estimation kits were obtained from Erba diagnostic Ltd. India.4.4. INSTRUMENTSAnalytical Auto analyzer (MaxLyzer NB-201), UV-Visible spectrophotometer (Shimadzu, Model no 2203), Electronic balance (Shimadzu, Model no DS-852 J), create from raw stuff homogeniger (Ever shine, Model no 607), Remi centrifuge (Remi, Model no KKLO-9013).4.5 ACUTE ORAL TOXICITY STUDYThe acute oral toxicity study was done according to OECD 423 guidelines. Wistar albino rats of either sex were selected randomly and divided into six groups (n = 6). The animals were fasted overnight and extract in doses of cytosine, 250, 500, 1000, 2000 and 5000 mg/kg body weight, were ad momentistered orally to II VI groups. mathematical group I which received vehicle (CMC) served as control. The animals were observed continuously for 2 hr, and then intermittently for 6 hr and at the end of 24 hours, the pattern of deaths was noted to d etermine LD50 of the extract (Annie et al., 2004).4.6. EXPERIMENTAL DESIGN4.6.1. NEPHROPROTECTIVE ACTIVITYThe experimental animals were randomly divided in to 5 groups (n= 6) and treated for duration of 21 long time as per the treatment schedule given in table no 3. Nephrotoxicity was induced by administration of Gentamycin (80 mg/kg I.P) daily for 7 days. Ethanolic extract of G. moluccanawas freshly suspended in CMC and administered to animals by oral feeding needle.Table no 3 Treatment schedule Evaluation of nephroprotective activity of EEGM against gentamycin induced nephrotoxicity in Wistar Rats.I.P = Intra peritoneal, P.O = Per oral.4.6.2. COLLECTION OF BLOOD AND URINE SAMPLESThe line of reasoning samples were collected from the retrorbital venous plexus of rats without any coagulant for the separation of serum, at the regular intervals of the treatment. After collecting the blood in effindraf tubes they were kept for 1 h at room temperature and serum was separated by centrif ugation at 2000 rpm for 15 min and stored until analyzed for miscellaneous biochemical parameters. Urine was collected over 24 hours on the 21st day by keeping the test animals in metabolic cages. The volume of collected urine samples was measured followed by estimation of biochemical parameters, namely urine Creatinine, urine uric stinging and urine urea.4.7. PARAMETERS MONITERED4.7.1. BIOCHEMICAL ESTMATIONSi. Estimation of Urea (Berthelot system) commandmentThe reaction sequence employed in the assay is as followsUrea + water supply Urease 2NH3 + carbon dioxideNH3 + Salicylate +Hypochlorite Nitropruside 2-2-Dicarboxy IndophenolUrease catalyses the conversion of Urea to Ammonia and Carbondioxide. The ammonia released reacts with a compartmentalisation of Slicylate. Hypochlorite and Nitropruside to yield a blue-green colored compound (Indophenol). The intensity of color produced is proportional to the concentration of urea in the sample and is measured photometrically at 578 n m or with yellow filter.Reagent preparationTransfer the entire Enzyme Concentrate (1A) into Urease Reagent (1) with the dropper (or) microtip provided.Assay resultPipette into test tubes labeled Blank (B), touchstone(S), Test(T) as follows. cock and Read absorbance of Standard (S) and Test (T) against Blank (B) at 578 nm (570-620 nm) or with yellow filter.The final color is stable for 30 min. at R.T.Calculations declension urea nitrogen in mg/dl = a X 0.467Urine Urea in gm/24 hours = a X 24 hrs urine volume in litres.ii. Estimation of BUN (GLDH-Urease Method)Methodology Talke and Schubert, Tiffany et al.PrincipleThe estimation of Urea in serum involves the following enzyme catalyzed reactionsUrea + H2O Urease 2NH3 + CO2 NH3 + -KG + NADH GLDH Glutamate + NAD-KG -KetoglutarateGLDH Glutamate dehydrogenaseThe rate of decrease in absorbance is monitored at 340 nm and is directly proportional to urea concentration in the sample. mathematical function cockle intimately, and aspirate standard followed by samples.CalculationDetermine absorbance change (A) for the standard and unknown samples by using the formula.A = A1 A2Urea = A of Test Concentration of(mg/dl) A OF Standard Standard (mg/dl)iii. Estimation of uric acid (Uricase/POD)PrincipleUric acid is oxidized to Allontoin and hydrogenperoxide by the enzyme uricase. In presence of peroxidase, released hydrogen peroxide is coupled with phenylamine derivative and 4-amino antipyrine (4-aap) to form colored chromogen complex. Absorbence of colored dye is measured at 550 nm and is proportional to Uric acid concentration in the sample (Schultz, 1984 Teivedi et al., 1978). Uric acid + 2H2O Uricase Allontoin + CO2 + H2O2H2O2 + Aniline derivative + 4-AAP POD Chromogen complex + H2O2ProcedureMix well. Incubate at 37C for 5 minutes.Programme the analyzer as per assay parameters.Blank the analyzer with reagent blank.Measure absorbance of standard followed by the test.Calculate results as per given calculation formula.Ca lculationsSerum/plasma/uric acid = Absorbance of Test 6(mg/dl) Absorbance of StandardUrine uric acid = Dilution 24 hours urine volume in dl. factor (mg/day)Conversion component partUric acid concentration in mmol/L = Uric acid in mg/dL 0.059iv. Estimation of Creatinine (Mod. Jaffes Kinetic Method)PrinciplePicric acid in an alkaline spiritualist reacts with creatinine to form an orange coloured complex with the alkaline picrate. Intensity of the colour formed during the fixed time is directly proportional to the amount of creatinine present in the sample.Creatinine + Alkaline Picrate Orange Coloured ComplexProcedurePipette into clean dry test tubes labeled as Standard (S) or Test (T)Mix well and read the initial absorbance A for the Standard and Test 1 after exactly 30 seconds. Read another absorbance A of the Standard 2 and Test exactly 60 seconds later. Calculate the change in absorbance A for both the Standard and Test.For Standard AS = A2 S A1 SFor Test AT = A2 T A1 TCa lculationsCreatinine in mg/dl = 2.0Urine Creatinine in g/L = x 1.0Urine Creatinine g/24 Hrs. = Urine Creatinine in g/L x Vol. of urine in 24 Hrs.v. Estimation of Total Protein (Biuret Method )MethodologyThe peptide bonds of protein react copper ions in alkaline resolving power to form blue-violet complex, (biuret reaction). Each copper ion complexing with 5 or 6 peptide bonds. Tartarate is added as a stabilizer whilst iodide is used to prevent auto-reduction of the alkaline copper complex. The color formed is proportional to the protein concentration and is measured at 546nm (520-560nm).ProcedureIncubate for 10 minutes at 37 C. Read absorbance of the standard and each test at 546 nm( 520-560 nm) against reagent blank.CalculationsCalculate the results as followsTotal Protein = Absorbance of Test Concentration of(g/dl) Absorbance of Standard Standard (g/dl)vi. Estimation of albumin (Bromocresol Green)PrincipleAt pH 3.68, Albumin acts as a cation and binds to the anionic dye Bromo cresol Green (BCG),forming a green colored complex. The color intensity of the complex is proportional to Albumin concentration in the sample (Gendler Proteins, 1984 Gustsfsson, 1978).Albumin + BCG Ph 3.68 Green colored complex.ProcedureMix well. Incubate at Room Temperature (15-30C) for 1 minute.Programme the analyzer as per assay parameters.Blank the analyzer with reagent blank.Measure absorbance of standard followed by the test.Calculate results as per given calculation formula.Calculations Albumin (g/dL) = Absorbance of Test 4Absorbance of StandardGlobulin = Total Protein AlbuminConversion factorAlbumin concentration in g/L = Albumin concentration in g/dL 10vii. Estimation of Cholestrol (CHOD-PAP Method)Methodology Modified Roeschlau,s MethodPrincipleThe estimation of cholesterol involves the following enzyme catalyzed reactions.Cholestrol ester CE Ckolestrol + Fatty acid Cholestrol + O2 CHOD Cholest-4-en-3-one + H2O22H2O2 + 4AAP + Phenol POD 4H2O + QuinoneimineCE Cholest rol esteraseCHOD Cholestrol Oxidase4AAP 4-AminoantipyrineProcedureMix well and incubate at 370C for 10 minutes. Aspirate Blank followed by Standard and Tests. Read the absorbance of standard and each test tube against blank at 505 nm or 505/670 nm on bichromic analyzer.CalculationsCholestrol (mg/dL) = Absorbance of Test Concentration of Standard (mg/dl)Absorbance of Standardviii. Estimation of Glucose (GOD POP Method)Methodology Trinder, s Method.PrincipleGucose + O2 + H2O Glucose oxidase Gluconic acid + H2O2 H2O2 + 4HBA + 4AAP Peroxidase Quinonemine Dye + 2 H2O4AAP 4-Aminoantipyrine4HBA 4-Hydroxy benzoic acid.The intensity of the pink color formed is proportional to the glucose concentration and can be measured photometrically between 500 to 540 nm.ProcedureMix well and incubate for 10 minutes at 370 C. Read the absorbance of standard and each test tube against reagent blank at 505 nm (500-540nm) or 505/670 nm on bichromic analyzer.CalculationsGlucose = Absorbance of Test X C oncentration of Standard (mg/dl)(mg/dL) Absorbance of Standardix. Estimation of Bilirubin (BIT BID)Methodology Diazo Method of Pearlman LeePrincipleBilirubin reacts with diazotized sulphanilic acid in acidic medium to form pink colored azobilirubin with absorbance directly proportional to Bilirubin concentration. Direct Bilirubin, being water soluble directly reacts in acidic medium. However Indirect or unconjugated Bilirubin is solubilised using a surfactant and then it reacts similar to Direct Bilirubin.Reagent preparationProcedureMix well and incubate for 5 minutes at 370 C for Total Bilirubin and Direct Bilirubin. Read Absorbance at 546/630 nm against Reagent Blank.Calculations with FactorsTotal Bilirubin (mg/dl) = Abs. of Test Factor (23).4.7.2. IN VIVO ANTIOXIDANT PARAMETERSPreparation of homogenateThe homogenate of heart was prepared as follows for the remaining animals.Reagents0.25 M sucrose origin 85.87 g of sucrose was dissolved in 1000 ml of distilled water10 mM tris buffer solution 1.2 g of tris was dissolved in 900 ml of distilled water. pH was adjusted to 7.4 with 1M HCl and diluted up to 1000 ml.ProcedureKidneys were excised and chopped with surgical scalp into fine slices and were chilled in the cold 0.25 M sucrose, quickly blotted with filter paper. The tissue was minced and homogenized in ice cold 10 mM tris HCl buffer (to pH 7.4) at a concentration of 10% (w/v) with 25 stokes of tight teflon pestle of glass homogenizer at a secureness of 2500 rpm. The prolonged homogenization under hypotonic condition was designed to disrupt as far as possible the ventricular structure of cells so as to release soluble protein and leave only membrane and non-vascular matter in a sedimentable form. It was then centrifuged at 5000 rpm at 20o C temperature and clear supported was separated and used to estimate reduced glutathione (GSH), catalase (CAT) and lipidperoxidation (LPO).a). Catalase (CAT)Catalase was estimated by the method of Hugo E. Aebi method hydrogen peroxide hydrogen-peroxidoreductase.PrincipleIn UV range H2O2 can be followed directly by the decrease in absorbence (O.D 240) per unit time is measure of catalase activity.H2O2 H2 + O2RDOH H2O + ROH + ADecomposition of H2O2 = Decrease in absorbance at 240 nmReagentsphosphate buffer (50 mM, pH 7.0)Dissolve 6.81 g KH2PO4 in distilled water and make up to 1000 ml.Dissolve 8.9 g NaH2PO4. 2H2O in distilled water and make up to 1000 ml.Mix the solution A and B in proportion 115 (v/v)Hydrogen peroxide (30 mM/I) Dilute 0.34 ml of 30% Hydrogen peroxide with phosphate buffer up to 100 ml.ProcedureDilute homogenate 20 times with Phosphate buffer pH 7.0Calculation enter (A / B) 2297.3Where,A Initial absorbanceB final absorbance (after 30 second)Units = moles of H2O2 consumed/min/mgb). Reduced glutathione (GSH)Reduced glutathione was determined by the method of Moran et al., 1979.ReagentsTCA (10% w/v) solution accurately weighed 10 g of TCA was dissolved in 100 ml of distilled water .Phosphate buffer (0.2 M, pH 8)DTNB reagent (0.6 M) 60 mg of 5,5- dithio bis (2-nitro benzoic acid) was dissolved in 100 ml of 0.2 M sodium phosphate (pH 8).Standard glutathione Prepared by dissolving 10 mg of reduced glutathione in 100 ml of distilled water.ProcedureTo 1 ml of sample, 1 ml of 10% TCA was added. The precipitated fraction was centrifuged and to 0.5 ml supernatant, 2 ml DTNB was added. The final volume was made up to 3 ml with phosphate buffer. The colour developed was read at 412 nm. The amount of glutathione was expressed as g of GSH/mg protein, reduced glutathione was used as standard (100 g/ml).Y Absorbance of test samplec). Lipid peroxidationLipid peroxidation was determined by the method of Slater and Sawsyer et al., 1971ReagentsThiobarbituric acid 0.67% w/v in 1M tris hydrochloride pH -7, 0.67 g of thiobarbituric acid was dissolved in 100 ml of distilled water.Trichloroacetic acid (20% w/v) 20 g of TCA was dissolved in 100 ml of distilled water.Standard malond ialdehyde (0-25 n.mol)A stock solution containing 50 mm/ml of 1, 1,3,3-tetra ethoxy propane in tris hydrochloride buffer in pH -7, 10 ml of stock solution was diluted to 100 ml to get a working standard 50 nm malondialdehyde/ml. This was used for preparation of calibration curves.Procedure2 ml of sample was manifold with 2 ml of 20% TCA and kept in ice for 15 min. The precipitate was separated by centrifugation and 2 ml of samples of clear supernatant solution were mixed with 2 ml aq. 0.67% TBA solution. This mixture was heated on a boiling water bath for 10 min. It was cooled in ice for 5 min and absorbance was read at 535 nm. The values were expressed as nm of MDA formed/mg of protein values are normalized to protein content of tissues.Y Absorbance differences of final (after 3 min) and initial reading of test sample.