| Polygonum cuspidatum(P.cuspidatum)is a perennial herb whose rhizome extracts have various active functions,including anti-inflammatory,antioxidant,antibacterial,antitumor,and uric acid-lowering effects.In recent years,with the rapid pace of life leading to increased social pressure,changes in dietary habits,and the continuous emergence of aging problems,hyperuricemia(HUA)in China has shown a trend of high incidence and younger age.Literature studies have demonstrated the significant therapeutic effect of P.cuspidatum on HUA,but its pharmacological basis is unclear.Researches of P.cuspidatum mainly focus on the qualitative and quantitative analysis of its crude extracts or certain chemical components,while the studies of its in vivo anti-HUA functional activity have been relatively limited.Therefore,this paper focuses on clarifying the main functional components with anti-HUA activity in P.cuspidatum and preliminarily exploring its related mechanism of action,with specific research content as follows:1.Screening and evaluation of anti-HUA functional components of P.cuspidatum based on mouse model.The P.cuspidatum was extracted with ethanol and treated with alcoholic precipitation by water extraction to obtain the alcoholic precipitation phase(A).Mixed extracts of alcoholic soluble phase and alcoholic extracts were separated by macroporous resin to obtain four fractions(B、C、D、E).The results of the cell activity assay showed that the crude extract of P.cuspidatum was low toxic and fractions D and E significantly reduced the viability of mouse leukemia cells of monocyte macrophage(RAW264.7)(P<0.001).The anti-HUA functional activity of crude extracts were investigated in HUA mice induced by potassium oxonate and hypoxanthine,and it was found that crude extract D significantly reduced the serum uric acid(UA)level in mice(P<0.001).The crude extract D was separated by MCI column to obtain five subfractions(D-1、D-2、D-3、D-4、D-5),and the subfractions were studied again using animal models,concluding that the most significant decreases in UA level(P<0.001)and creatinine level(P<0.01)were observed in the D-4 group.There was a significant upward trend in the serum albumin to globulin ratio in mice(P<0.05)and both tumor necrosis factor-α and interleukin 6 levels were significantly improved(P<0.001).Western blot analysis showed a significant decrease in protein expression of nuclear transcription factor-κB p65(NF-B p65)in the kidneys of D-4 group mice.The results of oxidative stress evaluation indicated that intervention with D-4 fraction enhanced superoxide dismutase,catalase and glutathione S-transferase activity and reduced malondialdehyde levels in the liver of HUA mice,and the effect was superior to that of allopurinol.2.Structural identification and process optimization of monomeric compounds based on in vitro enzyme activity.On the basis of subdividing components,further separation of D-4 component was performed using ODS column,and five monomer compounds were obtained after purification.In enzymology studies in vitro,both compound 4 and compound 5effectively inhibited xanthine oxidase(XOD)activity,with compound 4 showing a stronger inhibitory effect,and a half maximal inhibitory concentration of 14.35 μg/m L for the enzyme.Compound 4 inhibited XOD in a reversible competitive manner with a kinetic inhibition constant of 15.29 μg/m L.In addition,compound 4 exhibited a more pronounced inhibitory effect on the proliferation of RAW264.7 cells in a dose-dependent manner.Mass spectrometry and nuclear magnetic resonance technology was used to analyze the structure of the compounds.Finally,two monomeric compounds 4 and 5 with strong anti-HUA activity were preliminarily identified,which are 1-(4-hydroxy-2-methoxyphenyl)-2-(4-hydroxy-3,5-dimethylphenyl)-butane-1,2,3-triol and 1-(4-hydroxy-2-methoxyphenyl)-2-(4-hydroxy-3,5-dimethylphenyl)-3-methylbutane-1,2-diol,both of which were isolated from P.cuspidatum for the first time.The extraction process of anti-HUA functional components from P.cuspidatum was optimized using the extraction rate of compound 4 as an evaluation indicator.Combining external factors such as cost and energy consumption,the optimized extraction process was as follows: the optimal material-to-liquid ratio of 1:20 g/m L,extraction time of 3.2 h,extraction temperature of 60°C,ethanol concentration of 70%,extraction times of 2 times.Under these conditions,the extraction rate of compound 4 reached(2.41±0.03)%.3.Research on the action mechanism of anti-HUA functional components based on untargeted metabolomics technology.Using gas chromatography-time of flight mass spectrometry,the impact of D-4 fraction treatment of P.cuspidatum on relevant metabolic pathways in mice was assessed.Serum metabolomic analysis revealed that the anti-HUA functional activity of D-4 fraction was closely related to galactose metabolism,taurine and hypotaurine metabolism,purine metabolism,and energy metabolism.Intervention with P.cuspidatum also induced alterations in the hepatic metabolome and lipidome of HUA mice.The differential metabolites in the metabolome mainly involved carbohydrates,organic acids,purines,and amino acids,which were primarily enriched in amino acid metabolism,suger metabolism,and lipid metabolism pathways,with significant upregulation of expression in the pathways of alanine,aspartate,and glutamate metabolism(P=5.57E-4,Impact=0.291).The lipidome mainly screened out glycerides,glycerophospholipids,and sphingolipids,identifying35 significantly different lipids,among which four major phospholipids and sphingomyelin(SM)lipids in sphingolipids showed a significant increase in overall content.The most significant upregulation of SM 39:5;O2 level was observed(P=0.005,FC=4.665),while the overall level of ceramide(CER)lipids decreased.This suggested that P.cuspidatum may exert a positive impact on cells by downregulating the SM-CER pathway,activating pro-proliferation and anti-apoptotic signals,thereby alleviating liver metabolic abnormalities and further maintaining uric acid homeostasis. |
PDF Full Text Request