Identification of Plant Material: Corallocarpus Epigaeus L

Identification of Plant squ atomic number 18 Corallocarpus Epigaeus L6. MATERIALS AND METHODS6.1 CollectionThe jell material was sedate from the Tirupati, Andhra pradesh, India in the calendar month of February 2014. The plant taxonomical au thusticated by Prof Dr. K. Madhava chetty, Department of Botany, SV University, Tirupati. The voucher specimen of Corallocarpus epigaeus L has been preserved in our laboratory for further prayer and reference.ChemicalsVarious reagents like Mayers reagent, Wagners reagent, Hagers reagent, Dragendr off-keys reagent, napthol etymon, Fehlings solution A B, Barfoeds reagent, Millons reagent, Ninhydrins solution, cuso4, ethanol 95%, potassium hydroxide, concentrated HNO3, pyridine, atomic number 11 nitroprus status, sodium picrate, concentrated HNO3, pyridine, sodium nitroprusside, sodium picrate, concentrated H2SO4, Glacial acetic acid, ferric chloride, Ammonium hydroxide solution, Potassium dichromate solution, Thionyl chloride solution, P henolpthalein, Chloroform, etc. were received from threadbare suppliers to Dept of Pharmacology, SIPS-Proddatur.6.1.1 Preparation of Whole Plant ethanolic press out of Corallocarpus epigaeus LThe fresh leaves of Corallocarpus epigaeus L. The sieved pulverise was stored in airtight container and kept at room temperature for further study. The dried powdered material (250gm) was extracted with 95% ethanol using soxhlet apparatus for somewhat 72hours.Figure no 12SOXHLET APPARATUS DISTILLATION APPARATUS aft(preno bital) extraction with solvent, the marc was dried in hot air oven below 50o c and was concentrated by distilling off the solvent and e desiccationa raiseg to dryness. The dried extract was subjected to anterior phytochemical screening for detection of several(a) phytoconstituents.6.1.2 Qualitative Phytochemical Analysis74The ethanolic extract Corallocarpus epigaeus L was subjected to motley analytical studys in order to identify various phytoconstituents. streamlet f or AlkaloidsMayers testTo 1 ml of the extract, a lay or cardinal drop of Mayers reagent was added by the side of test tube. Appearance of a white or creamy come down indicates aim of alkaloids.Wagners turn upTo 1 ml of the extract, a hardly a(prenominal)(prenominal) drops of Wagners reagent was added. Development of reddish brownished people of glossiness indicates the nominal head of alkaloids.Hagers showTo the 1 ml of the extract, fewerer drops of Hagers reagent was added. A prominent chickenhearted emblazon indicates the test as positive.Dragendroffs testingTo the 1 ml of the extract, few drop of Dragendroffs reagent was added. A prominent yellow colour indicates the test as positive. tribulation for CarbohydratesBenedicts shewTo 5 ml of Benedicts reagent, 1 ml of the extract solution was added and boiled for 2 heartbeat and cooled. geological formation of red come down shows the strawman of carbohydrates.Molischs TestTo 2 ml of extract, two drops of alcoho lic solution of -naphthol was added and shaken well. posterior 1 ml of concentrated sulphuric acid was added slowly along the side of the test tubes and allowed to stand. A violet ring indicates the presence of carbohydrates.Fehlings TestTo 1 ml of the extract, add equal quantity of Fehling solution A and B were added. Appearance of red precipitate indicates the presence of sugars.Barfoeds TestTo 2 ml of the extract, 2 ml of Barfoed reagent was added and mixed well. It was modify for 1-2 moment in boiling peeing bath and cooled. initializeion of red precipitateIndicates the presence of sugars.Test for Protein and Amino AcidsMillions TestTo 2 ml of the extract, few drops of Million reagent was added. A white precipitateIndicates the presence of proteins.Ninhydrin TestTo the 2 ml of the extract, two drops of Ninhydrin solution was added. A property purple color indicates the presence of amino acids, proteins and peptides.Biurett TestTo 1 ml of the extract, one or two drop of 1% copper sulfate solution was added and tothis 1 ml of ethanol (95%) was added, followed by excess of potassium hydroxide pellets. The pink layer in ethanolic layer indicates the presence of proteins.Xanthoprotein TestTo 1 ml of the extract, add 1 ml of concentrated azotic acid was added resulting in the formation of a white precipitate which is then boiled and cooled. Then 20% sodium hydroxide in ammonium hydroxide was added. Orange colour indicates the presence of aromatic amino acids.Test for Glycosides legitimates Test2 ml of extract was dissolved in the solution of pyridine. Then sodium nitroprusside was added, to make it alkaline. The change in the colour from yellow to orange was not observed, which indicates the presence of glycosides.Baljets TestTo 1 ml of the extract, 1 ml of sodium picrate solution was added. The colour from yellow to orange reveals the presence of glycosides.Borntragers TestTo 1 ml of extract, few ml of sulphuric acid was added, boiled, filtered and extr acted with chloroform. The chloroform layer was than inured with few ml of ammonia. The formation of red colour indicates the presence shows the presence of anthraquinone glycosides.Keller Killani TestThe extract was dissolved in acetic acid containing traces of ferric chloride and was transferred to a test tube containing sulphuric acid. At the junction, the formation of reddish brown colour, which gradually turns to dark, confirms the presence of glycosides.Test for FlavonoidsShinoda TestTo 1 ml of extract, milligram turnings was added and 1-2 drops of concentrated hydrochloric acid was added drop wise. Formation of pink to crimson colour indicates the presence of flavonoids.Alkaline reagent TestThe aqueous solution of the extract was hard-boiled with 10% ammonium hydroxide solution. Yellow fluorescence indicates the presence of flavonoids.Tests for Tannins and Phenolic compounds ferric chloride TestTo 1 ml of the extract, add few drops of neutral 5% ferric chloride solution. Formation of dark greenish colour shows the presence of phenolic compounds. To the extract add potassium dichromate solution, formation of a precipitate shows the presence of tannins and phenolic compounds.Test for TriterpenoidsTwo or three granules of tin metal were added to thionyl chloride solution present in a test tube. Later 1 ml of extract solution was added. The formation of pink colour indicates the presence of triterpenoids.Test for SaponinsThe 1 ml of the extract was diluted with distilled water and the volume was made up to 20 ml. The abeyance was shaken in a graduated cylinder for 15 minutes. Appearance of foam indicates the presence of saponins.Tests for Fixed OilSpot testA teentsy quantity of the extract was pressed between two filter papers. Appearance of sebaceous stain on the presence of fixed oils.Saponification TestA few drops of 0.5 N alcoholic potassium hydroxide solution was added to a small quantity of extract along with a drop of phenolphthalein. The mi xture was heated on water bath for 2 hrs. Formation of the soap or partial neutralization of alkali indicates the presence of fixed oil.Tests for SteroidsLibermann Buchard Test1ml of the extract was dissolved in 2 ml of chloroform in a dry test tube. 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid was added to it. The solution turns to red, then gamey and finally bluish green, indicating the presence of steroids.Salkowski TestThe extract was dissolved in chloroform the extract in chloroform and equal volume of concentrated sulphuric acid was added. Formation of bluish red to cherry red colour in chloroform layer and green fluorescence observed acid indicates the presence of steroids.6.2 INVITRO ANTICANCER bodily functionCell bloodsCOLO 320 kiosk lineswere obtained from sugen Life Sciences Pvt. Ltd., Tirupati and cultured in RPMI 1640 medium (Difco, invitrogen corp, Canada). commonplace information74Organism Homo sapiens, humanTissue ColonProduct Format Fr ozenMorphology Cells are rounded and refractileCulture Properties loosely adherent, multi stall aggregatesBiosafety Level 1Disease DukestypeC,colorectaladenocarcinomasAge 55 years sexual practice FemaleEthnicity CaucasianStorage Condition Liquid nitrogen vapor phaseGenesExpressed Serotonin,norepinephrine,epinephrine,Adreno Cortico Tropic Hormone (ACTH), parathyroid hormone.Tumorigenic outcomesYes, in nude miceCommentsCells are weakly positive for keratinsCulture MethodThe base medium for this cell line is conventionalismted RPMI-1640 Medium.6.2.1 Tryphan Blue dye Exclusion Assay Method75 tabularise 5 List of instrumentsTable 6 List of ChemicalsExperimental externaliseThe designed study consists of three hosts viz Negitive control, Control, Test. In the Negative control assembly the cell lines were incubated with the medium for a period of 24 hours. This group was designed to rule out the possibility of any growth inhibitory offspring of certain compounds of medium.The contro l group was designed to rule out the effect of any residual or traces of solvent with which the extract was prepared on the growth inhibition of cell lines. Here the solvent employed was ethanol and hence it is added at the denseness of 0.1% (v/v) in distilled water.In test group different concentrations of test extract i.e, 10, 25, 50, 75 and 100g/ml are incubated with colo 320 cell lines for a period of 24 hours. This group was utilise to study the effect on cell line viability.Table7 Experimental radiation diagram to Study the Effect of Ethanolic Extract of Corallocarpus epigaeus L on colo 320 Cell Line Viability by Tryphan Blue Assay purpose1. An aliquot of cell suspension being tested for viability was centrifuged for 5 min andsupernatant was discarded. The size of the aliquot depends on the approximate number of cells present. The aliquot is taken such that it contained a well-off number of cells to count in a haemocytometer when suspended in 1 ml PBS and then diluted agai n by flux with 0.4% Tryphan blue (e.g., 5 105 cells/ml).2. The cell pellet was resuspended in 1 ml PBS or serum free complete medium. blood serum proteins stain with Tryphan blue and can produce misleading results. Hence determinations must be made in serum-free solution.3. Mix 1 part of 0.4% Tryphan blue and 1 part cell suspension (dilution of cells) cells were mixed and allowed to incubate at room temperature for approximately 3 minutes, Cells were counted within 3 to 5 min of mixing with Tryphan blue, as longer incubation periods will lead to cell death and reduced viability counts. Mixing was performed in a well of a microtiter plate or a small plastic tube using 10 to20 l each of cell suspension and Tryphan blue.4. A drop of the Tryphan blue/cell mixture was applied on to haemocytometer. The haemocytometer on the present of a binocular microscope and cells were focused clearly.5. The no. of unstained (viable) and stained (nonviable) cells were counted apiece in the haemocy tometer.Seeding of CellsCOLO 320 cells were cultured to reach the 80-90% confluency using RPMI 1640 medium. After reaching the coveted confluency, culture was collected and centrifuged at 3000 rpm for 10 minutes to hold up cell pellet. The pellet was resuspended in 1ml of fresh culture media. Cell concentration was determined by Tryphan blue assay was performed by mixing 50 litres of culture and 50 litres of 0.4% tryphan blue dye. Finally cells seeded in 24 well plates at the concentration 10000 cells/ml and incubated at 5% co2 incubator at 370 c for 24 hours.Drug intercessionCells were maintained in 24 well plates in triplicate for every(prenominal) concentration, and treated with different concentrations of (10, 25, 50, 75, 100gm). Corallocarpus epigaeus L, test compound and control groups were treated with medium and ethanol. The treated cells were incubated for 24 hours in 5% co2 incubator at 370c.Invitro Cytotoxic AssayAfter 24 hours incubation the cells were collected from each well in eppendroffs and centrifuged at 3000rpm for 10 min to bilk cell pellet, to the pellet 50lit of each medium and tryphan blue was added and mixed well to suspend the pellet. Cytotoxicity was screened by performing tryphan blue assay. region of growth inhibition was calculated by using the following formula6.2.2 Micro Culture Tetrazolium Assay76Plant Material Used Whole plant ethanolic extract of Corallocarpus epigaeus L.PrincipleThis assay is based on the capacity of mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow coloured watersolublesubstrate3(4,5dimethylthiazolyl)2,5diphenyltetrazoliumbromideintoaninsolublepurplecolouredformazanproductwhosecolouredismeasuredbymeansofELISAreaderat540nm.Onlyviablecellswithactivemitochondria reducesignificant amountsof MTT, since reduction of MTT can solely occur in metabolically active cells.Figure 13 lessening of MTT to a formazan compound by mitochondrial EnzymesCell LinesHuman colorectal adenocarc inoma- colo 320were obtained by sugen Life Sciences Pvt. Ltd., Tirupati from an authenticated supplier. Stock culture of these cell lines were cultured in RPMI -1640 with 10% inactivated newborn bovine serum, Penicillin (100 IU/ml), Streptomycin (100g/ml)) under humidified. The cells were dissociated in 0.2% trypsin and 0.02% EDTA in phosphate buffered saline solution. The stock culture was grown in 25cm 2 tissue culture flasks and cytotoxicity experiments were carried out in 96 well microtiter plates.ProcedureCell lines in the exponential growth phase were selected, washed, trypsinized and suspended in complete culture media i.e, RPMI 1640.The microtiter plates and incubated for 24hrs during which a partial monolayer was formed.They were then exposed to various concentrations of the extract (1-100g/ml). Control wells received only the nutrition medium.The plates were incubated at 37C and 48 hrs and cells were periodically checked for granularity, shoplifting and swelling. After 48 hrs, the sample solution in wells was flicked off and 50l of MTT dye was added to each well.The plates were gently shaken and incubated for 4 hrs at 370C in 5%CO2 incubator. The supernatant was removed and 50 l of DMSO was added.The plates were gently shaken to solubilise the formed formazan. The absorbance was measured at 540nm.The percentageage of growth inhibition was calculated using the following formula,Values of absorbance were reborn into percentage of residual viability. Usually the Inhibition concentration 50% (IC50) is elect as the best biological marker of cytotoxicity. The IC50 value represents the concentration of the test extracts that reduced 50% of cell inhibition.Statistical analysisStatistical rating of data was done by one-way analysis of variance (ANOVA) followed by Tukeys multiple comparison test on viability using computing machine based fitting program (Prism graph pad prism variance 6.03) statistical significance was set at p,0.05.IC50 was calculated b y additive interpolation method using the formulaIC50 = (D-C)+CWhere A = The root point on the curve, expressed in percent inhibition, that is less than 50%B = The first point on the curve, expressed in percent inhibition, that is greater than or equal to 50%C = The concentration of inhibitor that gives A% inhibitionD = The concentration of inhibitor that gives B % inhibition