The dried extracts have been stored at twenty C. For use in cell culture therapy, the dried extracts had been dissolved in DMSO and diluted in ultrapure water to get stock solutions which have been sterile filtered by 0. 2 um syringe filters. Stock remedies have been di luted in ultrapure water to make appropriate extract con centrations for testing. The ultimate concentration of DMSO during the cell culture reaction mixture was lower than 1%. Ex tracts have been kept at 4 C. Determination of complete phenolic content material Complete phenolic material of C. sativum extracts was established utilizing the Folin Ciocalteau system with some modifications. Briefly, 500 ul of one,ten Folin Ciocalteau phenol reagent was added to ten ul of sample, regular or favourable control. The mixture was allowed to stand for 5 min just before the addition of 350 ul of 10% sodium carbonate.
The resulting response mixture was incubated in the selleck chemicalCC-292 dark at room temperature for a more two h. Ab sorbance was then measured at 765 nm working with a spectro photometer. Gallic acid was employed because the standard. Rutin and quercetin were employed as constructive controls. Final results were expressed in milli grams of gallic acid equivalents per gram dried extract. All experiments have been carried out in triplicate. Ferric cutting down antioxidant electrical power assay The antioxidant activity based upon the ferric cutting down abil ity of C. sativum extracts was estimated based on the assay by Benzie Strain with some modifications. A operating reagent was ready fresh by mixing ten ml of 300 mM acetate buffer with 1 ml of ten mM 2,four,6 tripyri dyl s triazine in forty mM of hydrochloric acid and 1 ml of 20 mM FeCl3.
6H2O. The freshly pre pared FRAP reagent was pre warmed at 37 C for 5 min just after which a blank reading was taken at 595 nm using a plate reader. Subsequently, three ul of sample, regular or optimistic control and 9 selleck chemical ul of water was added to 90 ul with the FRAP reagent. Soak up ance readings were measured instantaneously on addition from the FRAP reagent and once again at 4 min following the start from the reaction. The change in absorbance during the 4 min reaction was calculated by comparison on the absorbance modifications of FeSO4. 7H2O towards a common curve examined in parallel. Rutin and quercetin have been used as posi tive controls. Results had been expressed as mmol ferric redu cing action with the extracts per gram of dried extract. All experiments were carried out in triplicate. DPPH radical scavenging exercise Radical scavenging actions of C.
sativum sequential ex tracts have been determined by 1,one diphenyl two picrylhydrazyl radical scavenging assay with some modifi cations. The extract was extra to 120 ul of 0. 04 mg ml DPPH remedy in methanol. The extracts tested ranged from 0 5000 ug ml. The mixtures were mixed nicely and incubated inside the dark for 30 min. The reduction of DPPH absorption was measured at 515 nm applying a plate reader.