Total flavonoid content The TFC of the crude extracts and polyphenolic rich fractions was determined using the aluminium tri chloride sellckchem assay as described by Dewanto et al. A standard curve was prepared using rutin Inhibitors,Modulators,Libraries hydrate. Into a 96 well plate was pipetted 20 ul rutin hydrate stand ard, crude extract, or polyphenolic rich fraction, as well as 20 ul sodium ni trate solution, 20 ul aluminum trichloride solu tion and 100 ul sodium hydroxide solution. Absorbance was measured at 570 nm. Results are expressed as rutin equivalents which were calculated using the following equation where, c concentration calculated from standard curve, v volume obtained from initial ex traction of plant material, DF dilution factor of sample. and m total weight of extract.

Inhibitors,Modulators,Libraries Determination of cell free antioxidant activity Trolox equivalence antioxidant capacity assay The 2,2 azino bis radical ABTS scavenging activity of crude extracts and polyphenolic rich fractions were determined using the TEAC assay as described by Re et al. Aqueous ABTS was prepared in distilled water and ox idized using 2. 5 mM potassium peroxidisulfate at 4 C for Inhibitors,Modulators,Libraries 16 h. ABTS was diluted with distilled water to an ab sorbance of 0. 70 0. 02 absorbance units at 734 nm. A standard curve was prepared using Trolox and samples were tested at four differ ent concentrations. Into a cuvette was pipetted 20 ul Trolox standard, crude extract, polyphenolic rich fraction or ascorbic acid as well as 2 ml ABTS. Absorbance was measured at 734 nm after 1 min incubation.

Results are expressed as Trolox equivalents which were calculated using the following equation where, slope slope of Trolox standards curve. slope slope of sample curve. 1,1 diphenyl 2 picrylhydrazyl radical assay The DPPH radical scavenging activity of crude extracts and polyphenolic rich fractions were determined using the DPPH assay as described by Gyamfi et al. A standard curve was Inhibitors,Modulators,Libraries prepared using Trolox and samples were tested at four differ ent concentrations. Into a 96 well plate was pipetted 15 ul Trolox standard, Inhibitors,Modulators,Libraries crude extract, polyphenolic rich fraction or ascorbic acid as well as 185 ul DPPH solution. Absorbance was mea sured after 15 min at 570 nm. Results are expressed as TE using the equation in Section Trolox equivalence antioxidant capacity assay.

Cytotoxicity Culture, maintenance and seeding of cells Normal human dermal fibroblasts were pur chased from Southern Medical, while 3T3 L1 murine pre adipocyte and C2C12 murine myoblast cell lines were pur chased from the American Type Culture Collection. Adherent NHDF, 3T3 L1 and C2C12 cells were cultured in 10% foetal calf serum supplemented Dulbeccos selleck Gefitinib Modi fied Eagle Medium with penicillin and streptomycin at 37?C and 5% CO2. Once cells became confluent, flasks were rinsed with PBS and cells enzymatically detached with Trypsin Versene so lution for 5 to 10 min.