Purification Of N-BuOH Extract Smilax China L. And Its Effect On RAW264.7 Macrophages Inflammatory

This experiment through the establishment of RAW264.7 macrophages inflammation model induced by lipopolysaccharide.Smilax China L.n-Bu OH extract and its purification parts together with lipopolysaccharide intervention cells,study of Smilax China L.anti-inflammatory activity in vitro,and further discuss the mechanism of anti-inflammatory.Crude Smilax China L.was extracted by ultrasound-assisted method,after extracted by petroleum ether,ethyl acetate and n-butanol,gain the n-butanol extraction,D101 resin purified saponins with strong adsorption capacity of saponins were selected,optimize the purification process to get D5(D101 50% alcohol eluent).Selected to S-8 resin purified other substances with weak adsorption capacity of saponins and obtain S3,S5,S7(S-8 30%,50%,70% alcohol eluent).MTT assay was used to detect the relative vitality of cells,determine the optimum concentration of sample.Lipopolysaccharide stimulates RAW264.7 macrophages to build inflammatory model in vitro.The secretion levels of TNF-α,IL-1β,IL-6 were determined by Elisa,screening the active site of Smilax China L..The secretion level of nitric oxide(NO)were determined by Griess,and RT-q PCR was used to measure the m RNA expression levels of TNF-α,IL-1β,IL-6 and Caspase-3 in macrophages.Western blot(WB)were used to determine the protein expression levels of Caspase-3,Caspase-9,Bcl-2,Bcl-XL,TNF-R1,p-IκBα,p-IKKβ and p-p65,and the apoptotic cells were detected by nuclear dye Hochest33342.Finaly,the chemical composition and cleavage behavior of anti-inflammatory active site S5 and D5 were deduced by UPLC-DAD-ESI-MS.Results showed as follow:(1)D101 and S-8 macropore resins were selected to purify the saponins and phenolics.After purification,D101 resins purify the total saponins content was up to 78.47%(D5),and S-8 resins purify the phenolics content was up to 80.26%(S5).(2)The extraction of Smilax China L.(n-Bu OH extracton,S5,D5)different degree of decreased the secretion and m RNA expression levels of TNF-α,IL-1β,IL-6.Simultaneously decreased the secretion of NO in cell supernatant in inflammatory RAW264.7 macrophages.(3)The anti-inflammatory components of Smilax China L.different degree of down-regulation protein expression levels of TNF R1,p-IκBα、p-IKKβ and p-p65 by inhibiting the activation of the NF-κB pathway in macrophages,thereby inhibition of inflammation,and most significant inhibitory effect of D5.(4)The anti-inflammatory components of Smilax China L.different degree of down-regulation protein expression levels of Caspase-3 and Caspase-9,inhibiting the transcription of Caspase-3,and significantly up-regulation protein expression of Bcl-XL and Bcl-2 in macrophages,and the most significant regulation effect of S5.(5)Cell fluorescent dye test results show that S5 and D5 significantly relieve cell apoptosis by lipopolysaccharide,and S5 anti-apoptotic effect is most obvious,corresponding with the results of WB.(6)Anti-inflammatory active site of Smilax China L.,the main contents of D5 are pseduoprotodioscin,protodioscin and gracillin;the main contents of S5 are 4-Hydroxy 3-methoxybenzoic acid 4-β-D-glucoside,neochlorogenic acid,astilbin,quercetin,Kaempferol-3-O-glucorhamnoside and aesculin.Conclusion: n-Bu OH extracton anti-inflammatory components of Smilax China L.,may inhibited the activation of the NF-κB pathway,thus inhibiting cytokines secretion and transcription,and regulate the expression of apoptosis related proteins,inhibiting cell apoptosis caused by inflammation,finally achieve anti-inflammatory effect.