Saponin was used as positive control. Efficacy to protect against AAPH induced ROS generation from this source The ability of crude extracts and polyphenolic rich fractions to attenuate Inhibitors,Modulators,Libraries AAPH induced ROS generation was mea sured using the 2, 7 dichlorodihydrofluorescein diacetate method as described by Jakubowski and Bartosz. Into pre seeded U937 white plates was pipetted a 20 ul medium, crude extract, polyphenolic rich fraction or 1 mM Trolox and 5 uM DCFHDA, which was incubated for 1 h at 37?C and 5% CO2. Plates were washed with PBS and treated with 1. 5 mM AAPH. Fluorescence was mea sured over a period of 3 h at ex 485 nm and em 520 nm. Percentage inhibition was determined using the following equation where, AUC average area under curve of AAPH exposed cells. AUC average area under curve of sample treated, AAPH exposed cells.

Efficacy to protect against AAPH induced apoptosis The ability of crude extracts and polyphenolic rich frac tions to attenuate AAPH induced apoptosis was mea sured using the caspase 3 activity assay as described by Banjerdpongchai et al. Staurosporine was employed Inhibitors,Modulators,Libraries as positive control. Pre seeded U937 AAPH exposed plates were centrifuged, medium replaced with 25 ul cold Inhibitors,Modulators,Libraries lysis buffer and incubated for 15 min on ice. Thereafter, a 100 ul caspase 3 substrate buffer containing Ac DEVD AMC was added and plates were incubated for 3 h at 37?C. Fluorescence was mea sured at ex 355 nm and em 460 nm. Efficacy to protect against AAPH induced lipid peroxidation The ability of crude extracts and polyphenolic rich frac tions to attenuate AAPH induced lipid peroxidation was measured using the thiobarbituric acid assay as described by Stern et al.

Hydrogen peroxide was used as positive control. From pre seeded U937 AAPH exposed plates were Inhibitors,Modulators,Libraries taken aliquots of supernatant, which were mixed with 200 ul trichloroacetic acid and 400 ul TBA and in cubated at 95?C for 20 min. 3 Methyl butan 1 ol was added to the mixture, vortex mixed and the organic layer was left to separate from the aqueous layer. Into a white 96 well plate was transferred 100 ul of the organic layer and the fluorescence measured at ex 544 nm and em 590 nm. Efficacy to protect against AAPH induced GSH depletion The ability of crude extracts and polyphenolic rich frac tions to attenuate AAPH induced GSH depletion was measured using the monochlorobimane assay as de scribed by Fernandez Checa and Kaplowitz.

H2O2 was used as positive control. Into pre seeded U937 AAPH exposed plates was pipetted Inhibitors,Modulators,Libraries 50 uM monochlorobimane and plates were incubated for 1 h. Plates were the full report washed twice with PBS after which the fluorescence was measured at ex 355 nm and em 460 nm. Statistical analyses All experiments were performed in triplicate on three separate days and results expressed as mean SEM using GraphPad Prism 4.