Ltd. and Sigma Aldrich, Mumbai. Human Liver cancer HEP G2 (Hepatoma) cell lines were obtained from National Centre for Cell Sciences, Pune. Silver
nanoparticles were synthesized from the aqueous leaf extract of M. pubescens by reducing silver nitrate. M. pubescens leaves extract (333 mg/ml) was used to reduce 10 ml of 1 mM silver nitrate. 13 The recovered nanoparticle sample was used for antioxidant and anticancer studies. Different concentrations of 20–200 μg/ml of silver nanoparticles were added, in equal volume, to 0.1 mM ethanolic DPPH solution. The mixture was shaken vigorously and allowed to stand for 20 min in the dark at room temperature MK-2206 mouse and the absorbance was monitored at 517 nm. DPPH solution without silver nanoparticles served as the control. α tocopherol was used as the standard for the concentration range as considered
for the sample. DPPH radical scavenging activity % was calculated for the sample and the standard using the following formula: %scavengingactivity=Absorbancecontrol−AbsorbancesampleAbsorbancecontrol×100 The non-enzymatic Phenazine methosulfate/Nicotinamide adenine dinucleotide (PMS/NADH) system generated superoxide radicals, which reduced Nitro blue tetrazolium (NBT) Panobinostat manufacturer to a purple formazan. NBT solution of about 1 ml (156 μM NBT in 100 mM phosphate buffer, pH 8) was mixed with 1 ml of NADH solution (468 μM in 100 mM phosphate buffer, pH 8). To this 0.1 ml of sample solution (1 mg/mL) was added. The reaction was started by adding 100 μl of PMS solution (60 μM PMS in 10 mM Phosphate buffer, pH 8). The mixture was incubated at 25 °C for 5 min. A control was performed without the sample. Absorbance was measured at 560 nm. α tocopherol with concentration 100 μg/ml was used as the standard and the inhibition percentage of superoxide
anion generated was calculated as in DPPH assay. The sample of 100 μg/ml concentration all was added to 1.0 ml of iron-EDTA solution (0.13% Ferrous ammonium sulfate and 0.26% Ethylenediaminetetraacetic acid), 0.5 ml of EDTA solution (0.018%), and 1.0 ml of Dimethyl sulfoxide (DMSO) (0.85% v/v in 0.1 M phosphate buffer, pH 7.4). The reaction was initiated by adding 0.5 ml of ascorbic acid (0.22%) and incubated at 80–90 °C for 15 min in a water bath. After incubation the reaction was terminated by the addition of 1.0 ml of ice-cold Trichloroacetic acid (17.5% w/v). Nash reagent of about 3 ml (75.0 g of ammonium acetate, 3.0 ml of glacial acetic acid and 2 ml of acetyl acetone were mixed and raised to 1 L with distilled water) was added and left at room temperature for 15 min. The reaction mixture without sample was used as the control. The intensity of the color formed was measured at 412 nm. α tocopherol with concentration 100 μg/ml was used as the standard. The percentage hydroxyl radical scavenging activity was calculated as in DPPH assay. To 250 μl of sample (100 μg), 0.05 ml of 2 mM ferrous chloride was added. The reaction was initiated by the addition of 0.