LlectionThe rhizomes of Alpinia pahangensis were collected from Pahang, Malaysia. This species was authenticated by Professor Dr Halijah Ibrahim, from Faculty of Science, University of Malaya and also a voucher specimen (No. KLU 46177) deposited within the university herbarium.Phang et al. BMC Complementary and Alternative Medicine 2013, 13:243 http://www.biomedcentral/1472-6882/13/Page 3 ofReagents and chemicalsButylated hydroxyanisole (BHA), ascorbic acid, FolinCiocalteu’s phenol reagent, -carotene, linoleic acid, Tween 80, gallic acid and two,2-diphenyl-1-picrylhydrazyl (DPPH), potassium ferricyanide had been acquired from Sigma-Aldrich. Methanol, hexane, ethyl acetate and trichloroacetic acid were obtained from Merck. All solvents had been bought in analytical grade.Human cell line and culture medium[33]. The crude methanolic extract, hexane fraction, ethyl acetate fraction and positive control (BHA and ascorbic acid) had been dissolved in methanol while water fraction was dissolved in distilled water. The total phenolic content material (mg/ g of plant extract) within the crude aqueous methanol extract and its fractions expressed in gallic acid equivalents (GAE). Imply values have been calculated from three measurement.DPPH radical scavenging assayThe cell lines had been purchased in the American Tissue Culture Collection (ATCC, USA). The human cell lines applied have been nasopharyngeal epidermoid carcinoma cell line (KB), cervical carcinoma cell line (Ca Ski), colon adenocarcinoma cell line (HT-29), colon carcinoma cell line (HCT 116), lung adenocarcinoma epithelial cell line (A549), hormone-dependent breast carcinoma cell line (MCF7) and non-cancer human fibroblast cell line (MRC5). The cells were propagated making use of the following growth media: RPMI (Sigma) for MCF7, Ca Ski, HT-29 cell lines, McCOY’s (Sigma) for HCT 116 cell line, and EMEM (Sigma) for MRC5 and KB cell lines, supplemented with ten foetal bovine serum (PAA Lab, Austria), one hundred g/ml penicillin/streptomycin (PAA Lab, Austria) and 50 g/ml of fungizone (PAA Lab, Austria). The cells were offered new media every 2 to 3 days until 90 confluency.Prostaglandin D2 medchemexpress The viability on the cells was checked ahead of and soon after therapy by the tryphan blue exclusion dye system.MSNBA Protocol Frozen cell stocks were stored in liquid nitrogen (-196 ) before use.PMID:23847952 Extraction and fractionationThe DPPH radical scavenging activity was determined working with the approach as described by Phang et al. [33]. An aliquot of extract of different concentrations have been mixed with 0.8 of DPPH resolution (0.02 mL) in methanol. Reaction mixtures have been mixed nicely and incubated at room temperature for 30 minutes. Absorbance was read at 520 nm applying spectrophotometer (UV-2450 Shimadzu). Methanol was used as blank and DPPH answer with out addition of extract was utilized as handle. BHA and ascorbic acid were used as standards. The percentage inhibition activity was calculated as [(A0 – A1)/A0] 100, exactly where A0 was the absorbance with the handle, and A1 was the absorbance of your extract/standard. The IC50 value was determined by interpolation from non-linear regression of plot of percentage of inhibiton against the concentration of extracts, which can be defined as the amount of extract necessary to scavenge 50 of DPPH radicals.-carotene bleaching assayThe dried, ground rhizomes of Alpinia pahangensis (200 g) have been soaked in 80 aqueous methanol (three L) for 3 days at space temperature. The solvent-containing extract was then filtered along with the filtrate obtained was evaporated applying a rotary evaporator at 40 beneath va.
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