Study On Anti-inflammatory Effects And The Chemical Basis Of Fuke Qianjin Capsules

Objective:To investigate the anti-inflammatory effects of Fuke Qianjin capsules both in vivo and in vitro.To fractionate Fuke Qianjin capsules total extract into different fractions to compare the difference in anti-inflammatory effects.To clarify the mechanisms of the anti-inflammatory effects of Fuke Qianjin capsules.To characterize Fuke Qianjin capsules chemical constituents and the absorbed compounds in rats after oral administration of FKA,and to provide a pattern for Chinese medicinal formula research and lay a foundation for the secondary development of Fuke Qianjin capsules.Method:（1）To fractionate Fuke Qianjin capsules total extract into volatile oil fraction（FKV）,polysaccharide fraction（FKP）,n-butanol fraction and chloroform fraction（FKT）by using solvent extraction method,ethanol subsiding method and supercritical CO₂ extraction method.（2）Anti-inflammatory experiment in vitro was performed on mouse mononuclear macrophage RAW264.7 cells.The cytotoxicity of the drug and the appropriate experimental concentration of the drug adnimistration was determined by MTT assay.RAW264.7 cell inflammation model was established by using lipopolysaccharide（LPS）stimulation.ELISA and Griess methods were used to detect the effects of different extracts of Fuke Qianjin capsules on the levels of inflammatory factors（TNF-α,IL-6,NO）.Simultaneously,Western blot and confocal laser scanning were used to observe the expression of related proteins（IκBα,NF-κB p65 and iNOS）.（3）In estrum-treated SD rats,pelvic inflammation model were established by the infection of the mixture of Staphylococcus aureus,Escherichia coli and Ureaplasma urealyticum.HE staining was used to observe the infiltration of endometrial neutrophils,the degree of ovarian tubal adhesions,and the degree of pelvic effusion to evaluate the drug efficacy against pelvic inflammation.（4）The chemical composition of the active fraction FKV was characterized by GC-MS method and by matching with the database;UPLC-MS and UNIFY software were used to determine the composition of FKA;Rat plasma sample after oral administration of FKA were collected.The UPLC-MS data of FKA administrated rat plasma sample were performed a binary comparison with the UPLC-MS data of FKA and blank rat plasma sample respectively to certify the obsorbed compounds of FKA.Result:（1）FKA,FKP,FKF,FKV reduced the release of TNF-α,IL-6,and NO in different degrees.Results of western blot and confocal laser scanning showed the possible mechanism that they inhibited NF-κB p65 nuclear translocation by suppressing degradation of IκBα,which down-regulated the release of LPS-stimulated inflammatory factors including TNF-α,IL-6 in RAW264.7.In addition,the LPS-induced iNOS expression declined,and further extracellular NO level reduced.（2）The results in vivo showed that in control group,endometrium was smooth,only a small amount of neutrophils infiltrated,and there was no obvious tissue edema in the fallopian tubes.In pelvic inflammation model group,there was obvious neutrophil infiltration in the endometrium compared to the normal group.Severe tissue edema,visible cilia were observed in the fallopian tubes,which indicated that the pelvic inflammation model was successful.In the drug administration groups,the positive drug group improved occlusion of the fallopian tubes to a certain extent.In the FKA and FKP groups,cilia adhesion was reduced,but effusion was seen;adhesion and effusion were significantly improved in FKV and FKF groups.（3）GC-MS analysis of FKV matched 26 compounds,among which,alkenoic acids were the main components.UPLC-MS analysis of FKA matched 65 compounds,among which 17 obsorbed compounds were detected in FKA administrated rat plasma.Conclusion:Anti-inflammatory activity screening experiment in vitro indicates FKA,FKP,FKV,FKF all have a certain anti-inflammatory activity but in different degrees.Experiment in vivo further verifies that Fuke Qianjin capsules have a great efficacy in pelvic inflammatory disease.Given the experiment results both in vivo and in vitro,FKV is superior to other groups in anti-inflammatory activity.The chemical composition analysis of effective fraction,the chemical composition analysis of FKA,and the analysis of obsorbed compounds provide theoretical support for the quality standardization of Fuke Qianjin capsules.