Optimization Of Culture Conditions For Phellinus Igniarius And A Study On Its Hypouricemic Activity

Gout is a common metabolic disease resulting from hyperuricemia,and the majority of gout patients experience recurrent acute attacks,joint swelling and joint pain.Currently,the available drugs for gout are predominantly allopurinol and febuxostat tablets,which exert their effects mainly through the reduction of uric acid production.However,there are severe toxic side effects of these drugs during treatment.Therefore,a search for low-toxicity,safe and effective drugs for the treatment of gout in the form of natural compounds has become an intense focus of research.Phellinus igniarius,also known as“Sang Chen","Hu Sun Yan"and"Sang Huang Gu"in China,is increasingly favored by researchers for its broad spectrum of biological activity.In this research,we focused on Phellinus igniarius and optimized its culture conditions.Then,we extracted its bioactive components and investigated the hypouricemic activity.Firstly,the medium was optimized based on the potato dextrose medium.With increasing concentrations of lignin in the glucose potato medium,the colony diameters?solid-culture?and mycelium yields?liquid-culture?of Phellinus igniarius in different groups continued to increase,reaching the maximum when the lignin concentration was0.06 g/L.After 7 days of fermentation with the optimized medium,the productions were harvested and subjected to centrifuging and ultrasonic treatment to obtain the extracellular extract?EEP?and the intracellular extract?IEP?.Subsequently,the toxic effects of EEP and IEP on human normal liver cell line L02 were determined by MTT assay and FACS.The results showed that there were little toxic effects on cells when the concentration of IEP is lower than 2.34 g/L and the concentration of EEP is lower than 3.15 g/L.This was consistent with the phenomenons observed by the fluorescence microscope.To further explore the hypouricemic activity of Phellinus igniarius,the hyperuricemia cell model and the hyperuricemia animal model were established.Hypoxanthine was used to establish the hyperuricemia cell model.When the concentration of hypoxanthine was 67.5 mg/L,XO and HGPRT which play key roles in the uric acid production were significantly induced to overexpress,indicating the hyperuricemia cell model was successfully constructed.Then,the cell model was treated with different concentrations of EEP and IEP.The results showed that EEP and IEP could significantly induce the up-regulation of HGPRT and down-regulation of XO when the concentration of EEP was 2.34 g/L and the concentration of IEP was 3.15 g/L.Yeast extract and sodium urate were used to construct the hyperuricemia mouse model and the hyperuricemia rat model,respectively.With the gavage administration of EEP and IEP,the incidence of joint swelling of rats and serum uric acid of mice both decreased significantly compared with the control groups.These results indicated that Phellinus igniarius could effectively inhibit the production of uric acid and had good therapeutic efficacy in the treatment of gout.The metabolism of uric acid is accompanied by the generation of reactive oxygen species?ROS?and uric acid is considered to be an important marker of oxidative stress.So in this research,the antioxidant effect of Phellinus igniarius and its protective effects against oxidative stress in cells were evaluated.The results showed that EEP and IEP exhibited strong scavenging ability for the DPPH radical and hydroxyl radical.At the indicated concentrations of EEP and IEP,the DPPH scavenging rates were 65.0%?25.0g/L EEP?and 80.0%?4.96 g/L IEP?,respectively,while the hydroxyl radical scavenging rates were 90.0%?6.30 g/L EEP?and 76.0%?4.96 g/L IEP?.EEP and IEP also had strong reducing capacities.When the concentration of EEP was 25.0 g/L and the concentration of IEP was 18.75 g/L,the value of OD₇₀₀ each was 1 and 1.3.However,Compared with the scavenging ability for the above radicals,the scavenging ability of EEP and IEP for superoxide anions was relatively weak.When the concentration of EEP was 25.0 g/L and the concentration of IEP was 18.75 g/L,the scavenging rates only reached 18%and 42%,respectively.By FACS and fluorescence microscopy analysis,the juglone-evoked oxidative stress in L02 cells was significantly inhibited by EEP and IEP.These suggested that the extracts of Phellinus igniarius had strong antioxidant activity and could effectively attenuate the effect of oxidative stress generated during the uric acid production.In summary,Phellinus igniarius has good hypouricemic activity,and the mechanism of the hypouricemic activity is related to the regulation of the expression of XO and HGPRT in cells.Its antioxidant activity will further improve the therapeutic effect of Phellinus igniarius on gout.At the same time,the growth-promoting effect of lignin on Phellinus igniarius was also studied in this research.We believe that this research will provide a foundation for the development and utilization of Phellinus igniarius.