Extraction And Separation Of Active Components From Medicinal Fungi Phellinus Igniarius And Inonotus Obliquus And Its Anti-gout Activity

Phellinus igniarius?P.igniarius?and Inonotus obliquus?I.obliquus?are precious and rare large-scale medicinal fungi,both of them possess extensive biological activity.At present,the research focuses on the antitumor functions of polysaccharides,and relatively few studies on their small molecular compounds.It has been reported that P.igniarius and I.obliquus showed certain characteristics in terms of health care,disease treatment,and rehabilitation after illness.The gout,as a highly-occurring metabolic chronic disease,has seriously affected people's quality of life.The studies have reported that the extracts of medicinal fungis had anti-gout effect,but the related research on the active ingredients and mechanism has not been reported.Therefore,the mechanism of action and activity compounds of P.igniarius and I.obliquus about gout-related inflammation needs to be further studied.In this thesis,the activity compounds of P.igniarius and I.obliquus were analyzed by chromatography and mass spectrometry.Then,xanthine oxidase?XOD?was used as the target to quickly screen potential enzyme inhibitors from crude extracts.Based on the activity,high-performance counter-current chromatography?HPCCC?was used to isolate and purify the lead compounds of P.igniarius and I.obliquus,and further evaluated the activity and mechanism of the isolated compounds from the aspects of enzymatic reaction kinetics and cell biology.The specific content is as follows:Phenol-sulfuric acid,nitrite-aluminum nitrate,vanillin-glacial acetic acid,and BCA methods were used to compare the contents of polysaccharides,flavonoids,triterpenoids,and total proteins in 10 different sources of P.igniarius.LC-MS technology was applied to preliminary identify 15 chemical constituents of P.igniarius extract,and three potential XOD inhibitors were screened from crude extract by ultrafiltration technology.It was separated by HPCCC technology and identified by mass spectrometry.Davallialactone 28.30 mg,HypholomineB 72.15 mg,InoscavinA46.50 mg were isolated from 10.00 g crude P.igniarius extract.The purity was98.75%,96.43%and 90.46%by HPLC.A RP-HPLC method was developed for the quantitative analysis of Osmundacetone in the extracts of I.obliquus.The method was used to evaluate content of Osmundacetone of different solvents and methods extracts.The LC-MS technology was used to identify the five chemical constituents in the extracts of I.obliquus.The ultrafiltration technology was developed to screen 2 potential XOD inhibitors from the crude extracts.Then,they were separated by HPCCC technology and identified by mass spectrometry.Protocatechuic aldehyde 85.60 mg and Osmundacetone 36.35 mg were isolated from 10.00 g crude I.obliquus extract.The purity of both compounds was above 95.00%by HPLC.The enzymatic reaction kinetics method was used to evaluate the XOD inhibitory of five compounds isolated from P.igniarius and I.obliquus,and the mechanism was judged by the Lineweaver-Burk method.The results demonstrated that Davallialactone,HypholomineB,InoscavinA,protocatechuic aldehyde and osmundacetone showed reversible XOD inhibitory effects.Specifically,davallialactone was a competitive inhibitor,hypholomineB was a competitive inhibitor,inoscavinA was an un-competitive inhibitor,protocatechuic aldehyde and osmundacetone were competitive inhibitors with inhibition constants of K_i=71.63,29.72,68.97,57.85 and 35.40?g/mL,respectively.The results showed that the five compounds had better XOD inhibition ability,with IC₅₀of 56.04,81.35,17.83,38.80 and 23.00?g/mL,respectively.Furthermore,molecular biology method was applied to verify the anti-gout activity of 5 compounds.Acute gout cell model was established with MSU-induced RAW264.7cells,which was developed to evaluate the anti-gout activity the extracts and 5compounds with allopurinol as a positive drug.The ELISA method was used to determine the contents of IL-1?and ICAM-1 in model cells in different administration groups.The results showed that the addition of two extracts and five compounds could inhibit the release of inflammatory factors IL-1?and ICAM-1 in the gout inflammation cells.The RT-PCR method was used to evaluate the effect of different administration groups on the expression of IL-1?and ICAM-1 mRNA genes in model cells.After testing,it was found that the expression of related genes in RAW264.7cells induced by MSU significantly increased,and the expression of related genes in the drug group?5 monomer compounds?significantly decreased.The results demonstrated that the drug group could reduce the expression levels of inflammatory genes of IL-1?and ICAM-1,and protect the acute gout cells.Cell experiments and evaluation of the enzymatic reaction both showed that the five monomer compounds isolated from P.igniarius and I.obliquus revealed a good anti-gout activity.In this research,analysis and identification of the chemical compounds,activity screening,guided isolation,activity evaluation,and mechanism of anti-gout activity of P.igniarius and I.obliquus were systematically studied.It provides a theoretical basis for the development of characteristic resources of P.igniarius and I.obliquus.