sativum ethyl acetate extract was subjected to acid hydrolysis to release cost-free polyphenols from their glycosides according towards the technique of Nuutila, Kammio virta, Oksman Caldentey, with slight modifications. Briefly, twenty mg of dried extract in 0. four ml of six N HCl and 1. six ml of HPLC grade methanol with twenty mM butylated hydroxytoluene as antioxidant was heated at 90 C for two h. The mixture was centrifuged at ten,000 rpm for 5 min as well as the supernatant was filtered as a result of a 0. two um syringe filter and stored at four C for HPLC analysis. HPLC analysis was carried out working with a SPD 20A HPLC program. Reverse phase separation was per formed at forty C applying a Purospher STAR RP 18 endcapped column. The mobile phase con sisted of trifluoroacetic acid in ultrapure water at pH 2. six and acetonitrile.
The gradient pro gram consisted of, 0% to twelve. 5% B for two. five min, twelve. 5% to 100% B for 17. 5 min and 100% B for 10 min. The flow rate was kept at 1 ml min and injection volume was ten ul. The chromatogram peaks had been detected at 254 nm. Information ac quisition and processing was carried out using LCsolution software program. The buy SB 431542 compounds were identi fied by evaluating the retention occasions of peaks with stan dards. The extract was then spiked with all the specifications to confirm their presence. Unidentified peaks had been collected manually as well as mobile phase was air dried. The dried fraction was stored at four C for evaluation with GC MS. Fuel chromatography mass spectrometry analysis Prior to GC MS examination, chemical derivatisation was carried out to reduce the polarity of practical groups by reconstituting 400 ug of the dried fraction S1 with 500 ul of HPLC grade ethyl acetate and twenty ul of N,O bis trifluoroacetamide, and heated at 70 C for 40 min.
The GC MS analyses had been carried out in the GCMS QP2010 program fitted that has a ZB five capillary column. The carrier fuel was helium by using a flow charge of one. 08 ml min. The column temperature was set at 100 C for five min, 100 275 C at ten C min, selelck kinase inhibitor and last but not least held for twenty min in 275 C. Sample volume injected was one ul with a split ratio of two,one. The injector temperature was 250 C and the detector temperature was 290 C. The MS oper ating parameters were, ionisation likely, 70 eV, ion supply temperature, 200 C, solvent delay, 3. 0 min, scan pace, 2500 amu s, scan range, forty 500 amu and de tector voltage, one. five kV. Compound identification was verified based upon mass spectral information by personal computer match ing with Wiley 229, NIST 107, NIST 21 and PMW tox2 libraries. Statistical evaluation Information are presented as mean common deviation. Statistical analyses had been carried out by a single way examination of variance with Tukeys a number of compari sons and the College students t test. A P value of 0.