Study On Uric Acid-lowering Effect And Molecular Mechanism Of Polyphenols In Prunus Salicina Lindl.cv."Furong"

The prevalence of hyperuricemia(HUA)in China continues to increase and tends to be younger.At present,although the commonly used drugs for the prevention and treatment of hyperuricemia can significantly reduce the level of serum uric acid,there are also common side effects of western medicine,such as liver and kidney damage and allergic reactions.It is of great scientific significance and application value to study and find new safe and efficient active ingredients for reducing uric acid to prevent and improve hyperuricemia.Many studies have shown that plant polyphenols have the effect of lowering uric acid.Furong plum(Prunus salicina Lindl.cv."Furong")is a red-flesh fruit with Fujian characteristics and is rich in polyphenols.In this paper,the components and ultrasonic-assisted extraction conditions of polyphenols from Prunus salicina Lindl.cv."Furong"(PPSF)were investigated.The uric acid-lowering effect of PPSF on xanthine oxidase activity in vitro was analyzed.The anti-inflammatory and oxidative stress effects of PPSF on sodium urate-induced cellular inflammation were evaluated by in vitro cell experiments.Finally,the effect of PPSF on hyperuricemia was explored from the perspective of in vivo animal experiments,and its mechanism of action was explored.This research played certain roles in offering the theory support and practice guidance to promote extra value of Furong plum products,preventing and treating hyperuricemia,and developing new functional foods.The main research contents and results of this paper are as follows:(1)Component analysis and ultrasonic-assisted extraction of PPSFThe contents of phenolic compounds,flavonoids,and the phenolic components in Furong plum at 60%,80%,and full maturity were determined,respectively,and the ultrasonic-assisted extraction parameters of PPSF were optimized.This can screen the maturity of Furong plum suitable for extracting polyphenols and provide a theoretical basis for the development and consumption guidance of the functional components of PPSF.The results indicated that the contents of polyphenols,total flavonoids,and anthocyanins in the fruits of Furong plum showed an increasing trend at the mature stage.The total phenolic content was between 214～320 mg GAE/100 g,and the free polyphenol content accounted for more than 90%of the total phenols.Polyphenols mainly existed in the form of flavan-3-ols(catechins,epicatechins,proanthocyanidins),phenolic acids(chlorogenic acid,ferulic acid,protocatechuic acid),and flavonols(myricetin,quercetin).Epicatechin was the highest monomeric phenolic compound in Furong plum,with a content of up to 55.96～56.85 mg/100 g.Based on the results of the study,a fully mature Furong plum was selected as the raw material for extracting polyphenols.Through single factor and response surface analysis,the optimal conditions for ultrasonic-assisted extraction of PPSF were as follows:extraction temperature 60℃,extraction time 47 min,ethanol concentration 61%,ultrasonic power 420 W,material liquid ratio 1:20.Under these conditions,the yield of polyphenols was 43.85 ± 0.74 mg GAE/g.(2)Uric acid-reducing and antioxidant activity of PPSF in vitroXanthine oxidase(XOD)is an important enzyme that catalyzes the formation of uric acid in the human body.The method of screening uric acid-lowering substances in vitro with xanthine oxidase(XOD)as a target is the fastest and cost-saving method.In addition,antioxidants can inhibit the oxidation of hypoxanthine and xanthine,reducing uric acid production.In this paper,the XOD inhibition and antioxidant system in vitro were established to investigate the inhibition and inhibition kinetics of PPSF on XOD and to evaluate its scavenging ability on hydroxyl radical and DPPH radical.The results depicted that PPSF had a good inhibitory effect on XOD,and the IC50(concentration of 50%inhibition rate)was 77.64 μg/mL.The inhibitory effect exhibited a certain concentration dependence.The inhibition of XOD by polyphenols was a reversible process.With the increase of its concentration,the Michaelis constant(Km)decreased gradually,and the maximum reaction rate(Vmax)increased gradually.PPSF can inhibit XOD in a mixed way.Polyphenols can not only compete with xanthine for the active site of XOD but also bind to other parts of XOD to inhibit the formation of the enzyme-substrate complex.The inhibition constant(Ki)was 16.53 mmol/L,and the inhibition coefficient(α)was 0.26.At the same time,PPSF had a strong antioxidant capacity in vitro.The free and bound polyphenols of fully mature Furong plum had the strongest DPPH scavenging activity,with IC50 values of 38.57 μg/mL and 22.69μg/mL,respectively.PPSF had a strong ability to scavenge hydroxyl radicals,and the IC50 of bound polyphenols was 116.78～126.65μg/mL.(3)Anti-inflammatory effect of PPSF on RAW 264.7 macrophages induced by sodium urateThe body is at a high uric acid level,and urate crystals accumulate in joints and surrounding tissues,leading to acute gouty arthritis,and inducing inflammatory cells such as macrophages to secrete inflammatory factors,triggering an inflammatory response.At the same time,it will stimulate oxidative stress and aggravate oxidative damage.RAW 264.7 macrophages were induced by monosodium urate(MSU)to establish an inflammatory model,and the effects of PPSF on inflammation and oxidative stress of macrophages were investigated.Studies have indicated that PPSF has significant anti-inflammatory activity and antioxidant activity at the cellular level.It could inhibit the expression of inflammatory factors such as TNF-α,IL-1β,and IL-18 in RAW 264.7 macrophages and activate cell SOD activity,effectively reducing the levels of ROS and MDA in cells.It can effectively reduce the levels of ROS and MDA in cells and play a role in alleviating cellular inflammation.Transcriptome RNA-seq analysis exhibited that 15377 genes were differentially expressed in the MSU group compared with the blank control group,including 9699 upregulated genes and 5678 downregulated genes.Compared with the MSU group,8585 genes were differentially expressed in the polyphenol group,including 4421 up-regulated genes and 4164 down-regulated genes.Compared with the blank control group,the polyphenol group had 11907 differentially expressed genes,including 7012 up-regulated genes and 4895 down-regulated genes.And the related functional analysis of these differential genes was carried out.Among them,GO analysis depicted that the most enriched items were related to material metabolism and chemical reaction.KEGG analysis exhibited that PPSF regulated MSU-induced RAW 264.7 macrophage inflammation through the HIF-1 signaling pathway,FoxO signaling pathway,and IL-17 signaling pathway.The expression of inflammatory factor receptors IL-17RA,IL-17RB,and IL-17RE in RAW 264.7 macrophages was down-regulated by PPSF,the expression of IL-17RC was up-regulated,and the expression of transcription activator AP-1 was inhibited,while the expression of nuclear transcription factor NF-κB was promoted.In summary,PPSF had anti-inflammatory activity and metabolic regulation on RAW 264.7 macrophages induced by sodium urate and could effectively antagonize oxidative stress.(4)Effect and mechanism of PPSF on reducing uric acid in vivoThe hyperuricemia(HUA)mice model was used to investigate the effects of PPSF on biochemical indexes,serum metabolites,and metabolic phenotypes of mice.The mechanism of reducing uric acid of polyphenols extract was explored by GC-MS metabolomics strategy.This could provide a research basis for the application of plum polyphenol extract in the prevention and treatment of hyperuricemia.The results depicted that PPSF could effectively improve the body weight of mice and significantly reduce the organ index of hyperuricemic mice.Secondly,the contents of uric acid,creatinine,and urea nitrogen in the serum were obviously decreased,and XOD and ADA in the liver were significantly inhibited.PPSF could significantly alleviate hyperuricemia.Using GC/MS metabolomic analysis,PPSF regulated 28 potential biomarkers in hyperuricemic mice.After the intervention of PPSF,the contents of 25 biomarkers,including 2-keto adipic acid,cyclohexenol β-epoxide,pantothenic acid,uric acid,D-glyceric acid,hexadecanol,D-galacturonic acid,sorbitol,creatine,ribose,sucrose,malonamide,pyrogallic acid,vanillin,tagatose,threitol,oxamide,galacturonic acid,orotic acid,oxalic acid,glycerol,2-monopalmitin glyceride,D-glycerophosphate,citric acid and cannabinoid in the serum of hyperuricemia mice were down-regulated,and the contents of 3 biomarkers including 2,6-diaminopimelic acid,aminomalonic acid,and glycine in the serum were up-regulated.The changing trend of these potential biomarkers reflected that PPSF could regulate the purine metabolism,glycolipid metabolism,pentose phosphate pathway,and galactose metabolism in hyperuricemic mice.The potential mechanism of reducing uric acid by PPSF was to reduce the generation of uric acid from the source.Under the intervention of PPSF,the content of 5’-phosphate ribose in pentose phosphate pathway was down regulated,thus reducing the content of hypoxanthine nucleotide,adenine nucleotide and guanine nucleotide.The production of uric acid precursors was reduced,which indirectly regulated the level of serum uric acid.On the other hand,in the purine metabolism pathway,PPSF could effectively inhibit the activities of key enzymes such as XOD and ADA in the uric acid production pathway.ADA mainly mediated the formation of excessive uric acid precursors,while XOD mainly mediated the formation of final uric acid.The activity of ADA enzyme was weakened,thus the process of adenine nucleoside deamination and conversion to hypoxanthine was inhibited.The activity of XOD enzyme was weakened,so the oxidation of hypoxanthine and xanthine to uric acid was inhibited.Finally,the increase of serum uric acid level was inhibited to achieve the effect of reducing uric acid.