| Background and objective:Smilax china L.is a traditional Chinese herbal medicine,which possessed damp-inhibiting and wind-dispersing efficacy.Its chinese medicine prepatations contains of Kumquat capsule,Carborundum tablets,et al.It is mainly used to treat inflammation of department of gynaecology disease,especially has curative effect in treating pelvic inflammatory disease in clinic.Our group isolated the ethyl acetate fraction from the total extract of Smilax china L.,which was superior to general extract of Smilax china L..To further study the effective compounds of Smilax china L.,an extraction and separation was made to seperate several monomer compounds from the ethyl acetate fraction which contained Ampelopsin F(AMPF).Our first screening of these monomers' effects found that AMPF have significant anti-inflammation effect in human endometrial stromal cells(hESC),which suggested that it may have anti-inflammatory effect.To further explore the anti-inflammation active of monomer compound AMPF in Chinese traditional medicine Smilax china L.,with a view to find out potential effective monomers from traditional Chinese medicine.Methods:The extraction,separation and active identification of AMPF.With the methods of silica gel,MCI,ODS,LH-20 column chromatography,we seperated several monomer compounds from active ethyl acetate fraction of Smilax china L.,and characterized the monomer compounds' structure by using MS and NMR spectroscopy' methods to identify these monomer compounds.Screening the anti-inflammation by observing the inhibition of expression of IL-1? in hESC.We observed cytotoxic effect of AMPF on cells in vitro experiment,to explore the best dosage levels.Using CCK 8 cell activity kits to detect the influence of the cell proliferation activity of RAW 264.7 cells and microglial BV-2 cells after treated with AMPF(5,10,20,40 and 80 mg/L).Selected two cell lines included RAW 264.7 cells and microglial BV-2,constructing cell inflammation model in vitro by LPS,to detect the inhibition of AMPF in releasing of inflammatory markers in inflammational response cells.The experiment set LPS model group,the control group and different dose groups(10,20,40,and 100 mg/L)of AMPF.In addition,to the control group,the rest of the group were treated with LPS on 1 mg/L final concentration in order to stimulate cells inflammation model after 1 h treated with AMPF in cell culture system;After 24 h of dosing process,enzyme-linked immuno assay(ELISA)to detect interleukin 1?(IL-1?),interleukin 6(IL-6)and tumor necrosis factor a(TNF-?)concentration in culture supernatant of each group;Using the NO kit to detect the nitric oxide(NO)content in the cell culture supernatant.The inhibition activity on proteins of inflammation related pathway of AMPF.The experiment set LPS model group,the control group and AMPF different dose groups(10,20,40,and 80 mg/L).In addition to the control group,the rest of the group were treated with LPS on 1 mg/L final concentration in order to stimulate cells inflammation model after 1 h treated with AMPF in cell culture system;After 24 h of dosing process,Western Blot was conducted to detect the expression of Cyclooxygenase-2(COX-2)and Nitric oxide synthases(iNOS)proteins in each RAW 264.7 cells and microglial BV-2 cells.Results:AMPF was seperated from the ethyl acetate fraction of Smilax china L.and can significantly inhibite the expression of inflammation factor IL-1? in LPS-induced inflammatory hESC in diferent mass concentrations(10,20 and 40 mg/L)of AMPF(P<0.05).After 24 h treated with different mass concentrations(0,5,10,20,40 and 80 mg/L)of AMPF on RAW 264.7 cells,the survival rate of RAW 264.7 cells was higher than 90%,and AMPF didn't have significant influence on activity of RAW 264.7 cells.The results show that after 24 h treated with LPS,expression of inflammatory markers significantly improved(P<0.05).10,20 and 40 mg/L of AMPF could inhibit the expression of IL-1?(P<0.05),and treated with 40 mg/L of AMPF could significantly inhibite the expression of IL-6 and TNF-a(P<0.05).20 and 40 mg/L of AMPF could significantly inhibite the expression of protein COX-2 in LPS-induced inflammatory RAW 264.7 cells.10,20 and 40 mg/L of AMPF inhibite the expression of protein iNOS in LPS-induced inflammatory RAW 264.7 cells.40 mg/L of AMPF could significantly inhibite the expression of NO in LPS-induced inflammatory RAW 264.7 cells(P<0.05).After 24 h treated with different mass concentrations(0,10,20,40 and 80 mg/L)of AMPF on microglial BV-2 cells,the survival rate of microglial BV-2 cells was higher than 90%,and AMPF didn't have significant influence on activity of microglial BV-2 cells.The results show that after 24 h treated with LPS,expression of inflammatory markers significantly improved(P<0.05).20,40 and 80 mg/L of AMPF significantly inhibit the expression of IL-1? in LPS-induced inflammatory microglial BV-2 cells(P<0.05),and 80 mg/L of AMPF significantly inhibite the expression of IL-6 and TNF-a in LPS-induced inflammatory microglial BV-2 cells(P<0.05).Treated with 10,20,40 and 100 mg/L of AMPF could significantly inhibite the expression of protein COX-2 in LPS-induced inflammatory microglial BV-2 cells.10,20,40 and 80 mg/L of AMPF significantly inhibite the expression of NO in the supernant of LPS-induced inflammatory microglial BV-2 cells(P<0.05).Conclusions:Monomer compound AMPF extracted from the ethyl acetate parts of Smilax china L.could significantly inhibit the expression of IL-1? in the supernat of LPS-induced inflammatory hESC,which firstly validate the anti-inflammation activity of AMPF.Moreover,further study of anti-inflammation effects of AMPF was conducted.Selected two cell lines included RAW 264.7 cells and microglial BV-2,we finally constructed cell inflammation model in vitro by LPS.AMPF could inhibite LPS-induced inflammatory of RAW 264.7 and microglial BV-2 cells,which anti-inflammatory mechanism may be related to inhibite the expression of IL-1?,IL 6 and TNF-?,regulate the expression of COX-2 and iNOS proteins,and inhibit the release of NO. |
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