Essay Example on It was double diluted with RPMI tissue culture media








It was double diluted with RPMI tissue culture media layered on 3ml Ficoll hypaque Sigma USA and centrifuged at 1500rpm for 15 minutes The lymphocyte ring obtained was collected in a separate NUNC sterile tube containing 13 ml RPMI The contents of the tube were mixed well and centrifuged at 1000 rpm for 5min The supernatant was discarded and the pellet was dissolved in 1ml RPMIC RPMI containing 20 FCS The cell viability was checked using Trypan blue method Phytohaemagglutinin PHA was added at a concentration of 1µl per 1million cells 500 µl of the culture was added to each well of a 24 well NUNC plate The culture was incubated at 37 0 C in a 5 CO2 incubator for 24 hrs Addition of plant extract After 24hrs 1µl of IL 2 1 100 in RPMIC was added to each well along with 1µl of different dilutions 2 3 4 5 mg ml of the aqueous extract of C collinus One of the wells in which the standard drug AZT was added was used as the positive control The plates were incubated at 37 0 C in a 5 CO2 incubator for 72 hrs Different dilutions of the extracts were also added to wells with healthy lymphocytes and incubated at 37 0 C in a 5 CO2 incubator for 72 hrs Cytotoxicity assay was carried out using Trypan blue dye Mishell and Shiigi 1980 Measurement of p24 antigen expression

The effect of the crude extracts of C collinus on HIV 1 replication in vitro was tested by viral core protein p 24 expression using a commercial kit Vironistika HIV 1 Antigen Micro Elisa system Germany as described previously by Gary et al 2003 HIV infected cell cultures containing the HIV 1 antigen was incubated in Micro Elisa strips whose wells were coated with antibodies murine monoclonal to HIV 1 p24 core antigen Following incubation the specimen was aspirated and the wells were washed with phosphate buffer Subsequently anti HIV 1 conjugate Antibody to HIV 1 human coupled to horse radish peroxidase was added The labeled antibodies bind to the previously formed solid phase antibody antigen complexes Following wash and incubation with TMB tetramethylbenzidine substrate a blue colour was developed which turned yellow and the reaction was stopped with sulfuric acid Optical density OD was measured using the ELISA reader Percentage of p24 antigen inhibition was calculated by comparing OD of the treated wells with the control well The results were presented as the average and standard error of triplicate experiments The statistical significance was checked at P 0 5 Antibacterial activity 

A modification of the well diffusion assay protocol given by Juliani et al 2002 was used to screen the extract for antimicrobial activity against Salmonella typhi MTCC 734 Salmonella paratyphi A MTCC 3220 Vibrio cholerae MTCC 3904 and Klebsiella pneumoniae MTCC 109 Nutrient agar plates were swabbed with the respective broth culture of the organisms and kept for 15 minutes for absorption to take place Wells were made in agar plates using the broad end of a sterile Pasteur pipette 5 mm diameter Then 10 µl of the crude extract in methanol ethyl acetate chloroform and hot water at concentrations 2 3 4 and 5 mg ml were added to each well A mixture of penicillin and streptomycin 1 1 was used as positive control The various solvents in which dilutions were made were used as negative controls Plates were incubated at 37ºC for 24 hours and the diameters of the inhibition zones were measured in millimeter after the incubation period Phytochemical analysis by HPLC Standards and chemicals HPLC grade methanol other chemicals acetone ethanol benzene petroleum ether and Authentic polyphenol standards were purchased from Himedia Laboratories 

Mumbai India Sample preparation and HPLC Analysis Powdered leaf was used for HPLC analysis The samples were then heated under reflux for 1 hour with 6 ml of 25 hydrochloric acid and 20 ml MeOH the hydrolysate was diluted with methanol to 100ml and filtered PTFE Syringe filter Whatman UK 1 ml of this solution was injected for HPLC analysis Analysis was performed after three separate extraction of each sample and each extract was diluted and injected in triplicate Polyphenols in the samples was identified by comparison of their retention times with the standard compounds Chromatographic equipment and condition The chromatographic analyses were performed on a 250 mm 4 6 mm i d C18 ODS Shimadzu Japan with 0 5 aqueous solution of Orthophosphoric acid and Methanol HPLC Grade as mobile phase at a flow rate of 1 mL min 1 The HPLC equipment comprised Hewlett Packard HP 1050 ChemStation Software an HP model35900 interface unit an HP 9000 Series 300 computer and an HP DeskJet 500 Printer A Waters 486 tunable absorbance detector was operated at 254 nm detector sensitivity was 0 05 AUFS and the column oven temperature was 30 C Determinations were performed after three separate extractions of each sample and each extract was injected in triplicate

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