Study On Chemical Constituents And Effect Of Anti-Gout Of Fissistigma Retusum Merr.

Fissistigma.retusum Merr.belongs to the family Annonaceae.In China it distributed in Guangxi,Guangdong,Fujian and Yunnan,et al.The genus are mostly used as traditional folk herbs,which have biological activities of activating blood and reducing stasis,dispelling wind and dehumidification,and anti-inflammatory.In recent years,reports on the genus have gradually increased,and the main directions of reports are chemical composition and pharmacological activity.However,reviewing the relevant literature,it was found that no research group has yet conducted research on the chemical composition and pharmacological activity of F.retusum.Herein,we takes F.retusum as the research object,and researches and discusses its chemical composition,pharmacological effects,and content determination.The specific research content and results are as follows:(1)To separate and identify the chemical composition of the dichloromethane site of F.retusum by Silica gel column chromatography,Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography were used.A total of 20 compounds were isolated and their structures were identified by NMR spectroscopic techniques and references,these 20 compounds were identified as : goniopedaline(1),aristololactam AⅢa(2),piperolactam A(3),piperolactam B(4),aristololactam AⅠa(5),cepharanone B(6),aristololactam AⅡ(7),O-methylmoscatoline(8),lysicamine(9),Ethyl p-hydroxybenzoate(10),Ethyl 3,4-dihydroxybenzoate(11),telisatins A(12),telisatins B(13),5,6,7,8-tetramethoxyflavone(14),7-Hydroxy-5,6,8-trimethoxyflavone(15),2-hydroxy-3,4,5,6-tetramethoxydihydrochalcone(16),(+)-syringaresinol(17),(+)-isolariciresinol(18),2,6-dimethoxy-p-benzoquinone(19),(+)-bosciali(20),These include 11 alkaloids,3 flavonoids,2 lignins and 4 others.(2)The anti-gout effects and mechanisms of F.retusum were investigated by network pharmacology and molecular docking.By reviewing the literature,18 compounds in F.retusum were selected as the research objects of network pharmacology.Through the collection of related targets of 18 compounds,gout target collection,common target screening,GO function analysis and KEGG pathway enrichment analysis,164 potential related targets of F.retusum for the treatment of gout diseases were obtained,and 17 core targets were selected,mainly GAPDH,AKT1,EGFR,VEGFA,ALB,STAT3,SRC,HSP90AA1.GO analysis screened out 287 cellular biological processes,30 cell composition and 48 molecular functions;KEGG pathway enrichment results involved a total of 146 signaling pathways,including cancer pathways,PI3 KAkt signaling pathways,lipid and atherosclerosis signaling pathways,EGFR tyrosine kinaserelated signaling pathways,etc.The core components were selected through Cytoscape software,and molecular docking was performed with related proteins on the PI3K-Akt signaling pathway,and the compounds with good docking results were selected for in vitro experimental verification.The results of cell experiments show that the compounds 5,6,7,8-tetramethoxyflavone,lysicamine,aristololactam AⅡ and piperolactam B all have obvious inhibitory effects on the excessive secretion of TNF-α and IL-1β,and they also have obvious inhibitory effects on PI3 K p-PI3 K and p-AKT protein expression.(3)HPLC technology was used to establish a method for the determination of the content of alkaloids of aristololactam AⅡ,gohiopedaline,piperolactam B,aristololactam BⅡ and flavonoid of 5,6,7,8-tetramethoxyflavone in F.retusum of extract.At the same time,the total alkaloid content in F.retusum was determined by acid dye colorimetric method.The results show that the content of the five core ingredients in F.retusum of extract is 8.711、3.921、3.612、1.312、35.650 mg/g,the total alkaloid content is 18.8 mg/g.(4)Through the combined HS-GC-MS technology,the volatile components of four F.were measured and analyzed,including F.retusum,F.uonicum,F.acuminatissimum and F.oldhamii var.longistipitatum.A total of 47 kinds of compounds were identified,including hydrocarbons,alcohols,and a small number of esters and oxides.The combined HS-GC-MS technology was first used to analyze the four Fissistigma,and the results showed that there were certain differences in the volatile components and content.