Studies On The Active Constituents And Anti-gout Mechanism Of Sanghuangporus Vaninii

In recent years,gout and hyperuricemia have become diseases that cannot be ignored.With the change of modern lifestyle,the number of gout patients has increased substantially,and the age of onset is gradually becoming younger.Therefore,it is urgent to develop medicines and health products with independent intellectual property rights.Medicinal fungi can produce active secondary metabolites,which can effectively prevent and treat gout related diseases.The occurrence of gout is directly related to the high content of uric acid in the body,and Sanghuangporus vaninii(hereinafter referred to as S.vaninii)has activities such as inhibiting uric acid.Compared with the toxic and side effects of synthetic drugs,natural drug-feeding fungi are more in line with modern health concepts.Therefore,it is of practical significance to further strengthen the basic research on the drug efficacy of medicinal fungi,and to explore the mechanism of anti-gout of S.vaninii from the molecular level.Firstly,taking the same S.vaninii strain as the research object,the contents of total polysaccharide(water extract)and total polyphenol(alcohol extract)in the extracts of wild S.vaninii and in different growth years were evaluated using the phenol sulfuric acid and folinphenol methods.The main active components of alcohol extract of different growth years S.vaninii were analyzed and compared by liquid chromatograph-Mass spectrometer(LC-MS).The results showed that the content of total polysaccharide was higher in biennial S.vaninii,while that of total polyphenols was higher in quadrennial S.vaninii.The results of LC-MS analysis showed that there were fewer active components in the mycelium of S.vaninii,which was consistent with the results of total polyphenol content determination.The main components in the extracts of the artificial and wild S.vaninii were similar,and there were more components in the wild S.vaninii,but some components in the artificial were much higher than those in the wild.Six compounds were identified by LC-MS,namely phelinin B,phelligridimer A,davallialactone,phellinstatin,hypholomine B,and inoscavin A.Secondly,the inhibition rate of Xanthine oxidase(XO)was used as the index to evaluate the inhibition ability of the S.vaninii extract with different methods.Ethanol(95%)was used as the extraction solvent to prepare the extract by ultrasonic,reflux and cold leaching,and the content of total polyphenols and the inhibition rate of XO were determined.A rapid in vitro pharmacodynamic screening method was established with XO as the target,and the potential XO inhibitors were screened by ultrafiltration mass spectrometry.The compounds were separated by high performance countercurrent chromatography(HPCCC),and their chemical structures were identified by chromatography-mass spectrometry.The results showed that the100 μg/m L S.vaninii extraction had the highest inhibition rate of XO(88.21%),and the cold extract had the lowest inhibition rate(75.88%).Seven potential XO inhibitors were screened from the extracts by ultrafiltration mass spectrometry.Preliminary identification was osmuncacetone、hypholomine B、inoscavin A、phelligridin D、phelligridin C、3-[4,5-Dihydroxy-2-(6-methyl-4-oxo-4H-pyran-2-yl)Phenyl]-6-[(E)-2-(3,4-dihydroxyphenyl)vinyl]-4-hydroxy-2H-pyran-2-one 、 phelligrins A.Two compounds were isolated by HPCCC technique and identified as phellinin B and davallialactone,respectively,with purity of more than 90%.Thirdly,RAW264.7 cells were induced by monosodium urate(MSU)to establish an acute gout cell injury model.The effects of S.vaninii extract on the contents of TNF-α,IL-1β,SOD,intracellular reactive oxygen species(ROS)and nitric oxide(NO)in the model cells were studied,and the protective effect of the extract on acute gout cells was evaluated.Western Blot was used to investigate the expression of NLRP3 inflammasome and TLR4/NF-κB Inflammation signaling pathway.The results showed that compared with model group,the contents of ⅠL-1β of 5.00 μg/m L extract group was significantly decreased(p< 0.05),and the contents of ⅠL-1β and TNF-α were extremely significantly decreased in both 10.00 and 20.00 μg/m L extract groups(p< 0.01).The ROS contents of 5.00,10.00 and 20.00 μg/m L extract groups were extremely significantly decreased(p< 0.05).The NO contents of 10.00 and20.00 μg/m L extract groups were significantly and extremely significantly decreased(p< 0.05,p< 0.01).The SOD contents of 5.00,10.00 and 20.00 μg/m L extract groups was significantly increased(p< 0.05).The results of Western Blot further confirmed that the extract could down-regulate the expression levels of TLR4,My D88,NF-κB,NLRP3,ⅠL-1β,and inhibit the NLRP3 inflammasome and the signal transduction of TLR4/NF-κB pathway,so as to play an anti-gout effect.Finally,davallialactone,a monomer compound with good XO inhibition activity,was selected,and the mechanism of interaction between inhibitor and XO was analyzed by multi-spectral method and molecular docking simulation technology.The results showed that the collision between davallialactone and XO led to fluorescence quenching and conformational changes in XO,which were mainly driven by hydrophobicity and hydrogen bonding.In addition,the results of circular dichroism spectroscopy show that davallialactone can change the secondary structure of XO,in which the contents of α-helix,β-turning and irregular curling are decreased,while the contents of β-folding are increased.Davallialactone can induce the compact and loose structure of XO,and reduce the catalytic capacity of XO.Molecular simulations further showed that davallialactone was located at the entrance of the hydrophobicity of XO,interacting with amino acid residues Arg912,Gln1040,Ser1080,Gln1194,Ser1256,and Glu1261,and regulating the entry of xanthine or davallialactone into the molybdopterin center,leading to the inhibition of XO.We also observed face-to-faceπ-π interactions between the aryl ring of davallialactone and Phe798.Based on multidisciplinary theories and methods,the pharmacodynamic substance base and anti-gout activity of local characteristics and unconventional edible and medicinal fungi of S.vaninii were studied in this paper,which is of great significance for the protection of edible and medicinal fungi resources in China and further development and utilization.