Study On The Anti Gout Active Components From Poria Cum And Polyporus Umbellatus

Gout is a heterogeneous disease caused by a chronic abnormality in purine metabolism in the body,resulting in increased blood uric acid levels or reduced uric acid excretion causing tissue damage.In recent years,the incidence of gout has been increasing year by year,with a trend towards younger age groups,and has become a common and incurable disease worldwide.Poria cum and Polyporus Umbellatus are medicinal fungi of the family Polyporaceae,with a wide range of biological activities.Modern studies had shown that Poria cum and Polyporus Umbellatus have certain pharmacological effects in anti-gout and other aspects.At present,the research on the active ingredients of Poria cum and Polyporus Umbellatus is mostly focused on polysaccharide substances,but the research on small molecule compounds is less reported.The anti-gout effects of Poria cum and Polyporus Umbellatus as extracts have been reported in the literature,but studies on their active ingredients and mechanisms of action have not been reported.Therefore,in this thesis,the extraction,isolation and in vitro activity of the antigout active ingredients in Poria cum and Polyporus Umbellatus were investigated by using mathematical modeling,mass spectrometry and chromatography to establish a rapid screening method for the antigout active ingredients in Poria cum and Polyporus Umbellatus.It provides experimental basis and data support for the development and utilization of Poria cum and Polyporus Umbellatus.The specific research work is as follows:1.The results showed that the best extraction process for the triterpenoids in Poria cum was 75%ethanol by volume,16 m L/g liquid to material ratio,1.0 h extraction time,3 times extraction,and 2.30%total triterpene yield.The best extraction process of ergosterol in Polyporus Umbellatus was 80%ethanol concentration,16 m L/g liquid to material ratio,1.4 h extraction time,3 times extraction,and the yield of ergosterol was2.31%.2.Four potential antigout active ingredients were screened from Poria cum,namely,3-O-acetyl-16α-Dehydrometenolic acid,Dehydropachymic acid,Pachymic acid and Dehydrotrametenolic acid,with binding intensities of 10.91%,19.68%,37.49%,24.64%and docking binding energies of-6.32 kcal/mol,-6.70 kcal/mol,-7.34 kcal/mol,-7.32 kcal/mol,respectively,using ultrafiltration experiments.Four potential antigout active ingredients were screened from Polyporus Umbellatus,namely polyporusterone B,polyporusterone A,ergosta-4,6,8（14）,22-tetraen-3-one and ergosta-7,22-dien-3-one,with binding intensities of 40.77%,50.29%,17.53%and 15.17%.The docking binding energies were-8.6 kcal/mol,-8.7 kcal/mol,-8.1 kcal/mol,and-7.8 kcal/mol,respectively.The main force for the compounds to bind to xanthine oxidase was hydrogen bonding,and the molecular docking results were consistent with the results of ultrafiltration experiments.3.Using high-speed counter-current chromatography combined with semi-preparative high performance liquid chromatography,four anti-gout active ingredients were isolated from each of Poria cum and Polyporus Umbellatus,identified by UPLC-Q-Exacive and NMR techniques were 3-O-acetyl-16α-Dehydrometenolic acid,Dehydropachymic acid,Pachymic acid and Dehydrotrametenolic acid and polyporusterone B,polyporusterone A,ergosta-4,6,8（14）,22-tetraen-3-one and ergosta-7,22-dien-3-one,all with purity of more than 90%.4.An enzymatic reaction kinetic method was used to evaluate the inhibitory ability of the active ingredients in Poria cum and Polyporus Umbellatus on xanthine oxidase,and the mechanism of action was determined using the Lineweaver-Burk double inverse graphing method.The results showed that the eight compounds showed good inhibitory effect on XOD in the suitable concentration range,and the inhibition rate of XOD gradually became larger as the concentration of the compounds increased.The IC₅₀of3-O-acetyl-16α-Dehydrometenolic acid,Dehydropachymic acid,Pachymic acid and Dehydrotrametenolic acid on XOD were 46.05μg/m L,35.17μg/m L,23.70μg/m L and32.89μg/m L,respectively.The IC₅₀of polyporusterone B,polyporusterone A,ergosta-4,6,8（14）,22-tetraen-3-one and ergosta-7,22-dien-3-one were 20.07μg/m L,16.00μg/m L,27.70μg/m L and 31.95μg/m L,respectively.5.The results of the network pharmacology study showed that there were 158targets associated with gout for the four active ingredients in Poria cum,and 832 targets associated with gout were retrieved through the Gene Cards database,and 36 shared targets were obtained after Wayne analysis of the genes of potentially related targets for both.From the results of the pathway enrichment study,it is clear that the main metabolic pathways involve lipid and atherosclerosis,cancer,IL-17 signaling pathways among other pathways.There were 197 targets related to gout for the four active ingredients of Polyporus Umbellatus,and 832 targets related to gout were retrieved from Gene Cards database,and 38 common targets were obtained after Wayne analysis of the genes of the two potentially related targets.From the results of the pathway enrichment study,it is clear that the main metabolic pathways involve lipid and atherosclerosis,cancer and other pathways.It is hypothesized that the potential xanthine oxidase inhibitors in Poria cum and Polyporus Umbellatus may have a therapeutic effect on gout by affecting the above metabolic pathways.In summary,a rapid screening method for xanthine oxidase inhibitors in Poria cum and Polyporus Umbellatus was developed using a combination of mathematical modelling,mass spectrometry and chromatography techniques.In this study,we conducted analytical identification,activity screening,guided isolation and activity evaluation of antigout active ingredients in Poria cum and Polyporus Umbellatus of the family Polyporaceae to provide a theoretical basis for the development of the characteristic resources Poria cum and Polyporus Umbellatus,the results provide a theoretical basis for further research on the anti-gout and other pharmacological effects of Poria cocos and Polyporus Umbellatus.