Extract/fraction and 10 ng/ml of TNF- at a final concentration
Extract/fraction and 10 ng/ml of TNF- at a final concentration in the fresh medium. The cells after incubation for 6 h at 37 were washed with PBS. Cells obtained were lysed in 50 l of 1X reporter lysis buffer with one freeze/thaw cycle (-80 / 37 ). By using the Luciferase Assay System from Promega (Madison, WI, USA) the inhibition of NFkB was recorded in a luminometer according to the manufacturer’s instructions. In this assay cells not instigated with TNF- were used as negative control whereas the cells not treated with DMSO and TNF- were considered as positive control. The experiment was performed in three replications. After determination of percentage inhibition of NFkB the IC50 values were computed [44]. The data were then plotted graphically as dose response curve after changing the concentration of extract/fractions to the log scale.Inhibition of LPS-induced nitric oxide synthesis (nitrite assay)Sprague-Dawley rats of either sex weighing 150?00 g were used in this experiment to perform the analgesic test [43]. Before the animals were used to determine the analgesic effect of various extract/fractions; animals were pre-tested on hot plate analgesimeter (Havard apparatus Ltd., UK) which was maintained at 55 ?0.1 to exclude the rats exhibiting >15 s of latency period. Animals were divided randomly into 15 groups with six rats in each group. In this study the animals of negative control were injected with normal saline 2 ml, i.p. whereas the animals of the positive groups received 10 mg/kg, p.o. of the diclofenac sodium and morphine respectively. Animals of the other groups PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27689333 were administered with doses of 100 mg/kg, p.o. and 200 mg/kg, p.o. of the FXM and its derived fractions; FXH, FXC, FXE, FXB and FXA. Latency period (time for which rat remains PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28499442 on the hot plate without licking or flicking of hind limb or jumping) in second was recorded for each animal of the group at 0 and after 30, 60 and 120 min of dose administration.To examine the anti-inflammatory Rocaglamide A site effects of the crude methanol extract and fractions from leaves of F. xanthoxyloides, LPS-stimulated macrophage model was used [45]. RAW 264.7 cells (ATCC-TIB-71) were seeded at a density of 10 ?104 cells per well in 96-well plate on 10 FBS containing DMEM and incubated for 24 h at 37 . Extract/ fractions in 10 DMSO solution at a final concentration of 0.5, 10, 15, 20, 25, 30, 40 g/ml in 1 FBS-containing phenol red free DMEM were added and incubated further for 15 min at 37 . Cells were stimulated by the addition of 1 g/ml of LPS and incubated further for 20 h at 37 . Cells not stimulated with LPS were considered as a negative control whereas cells treated with LPS and DMSO used as positive control. To determine the anti-inflammatory potential of extract/fractions, an aliquot of 100 l of the incubation media was transferred to 96-well plate and allowedYounis et al. BMC Complementary and Alternative Medicine (2016) 16:Page 5 ofto react with Griess reagent (90 l of 1 sulfanilamide in 5 phosphoric acid, and 90 l of N-(1-naphthyl) ethylenediamine). Absorbance of the reaction mixture was recorded at 540 nm. The experiment was performed in three replications. After determination of percentage inhibition of nitric oxide IC50 values were computed. The data were then plotted graphically as dose response curve after changing the concentration of extract/fractions to the log scale.Cytotoxicity studies for dose response100 mg/kg and 200 mg/kg, p.o. of FXM and its derived frac.