The Antihyperuricemic Activity Of Soy Sauce, The Isolation And Identification Of Substances From Soy Sauce With XOD Inhibitory Activities,and Their Action Mechanisms With XOD

Soy sauce is a kind of traditional fermented condiment with various active components and many physiological functions.The antihyperuricemic activity of soy sauce was evaluated in vivo in this paper.The xanthine oxidase（XOD）inhibitory activity-guided purification and identification of active compounds were performed.The XOD inhibitory activities of the active compounds were evaluated,and the action mechanisms of active compounds with XOD and the metabolic absorption characteristics were discussed.The antioxidants in soy sauce were enriched by macroporous resins,and the contents and antioxidant activities of typical soy isoflavones,furanones,pyranones,and phenolic acids in soy sauce were determined.The key small molecule antioxidant compounds in soy sauce were identified by antioxidants omission experiments.The effect of fermentation process on the XOD inhibitory activity and antioxidant activity of soy sauce was determined by statistical analysis.The results of this paper were as follows:（1）It was demonstrated by animal experiments that soy sauce could reduce the serum uric acid levels and xanthine oxidase（XOD）activities of hyperuricemic rats.Soy sauce was extracted with ethyl acetate and partitioned sequentially into ethyl acetate and water fractions.The XOD inhibitory activity of the ethyl acetate fraction(IC₅₀ value was 1.56±0.06 mg/mL)was stronger than that of water fraction.On the basis of XOD inhibitory activity-guided purification,ten compounds including 3,4-dihydroxy ethyl cinnamate,diisobutyl terephthalate,harman,daizein,flazin,catechol,thymine,genistein,uracil and cyclo（L-Pro-L-Ala）were obtained.It was the first time that 3,4-dihydroxy ethyl cinnamate,diisobutyl terephthalate and cyclo（L-Pro-L-Ala）had been identified from soy sauce.（2）The XOD inhibitory activities and antioxidant activities of the ten isolated compounds from soy sauce.Flazin,catechol and genistein were detected to be not only potent XOD inhibitors(IC₅₀ values were 0.51 mM,2.01 mM and 2.17 mM,respectively),but also strong antioxidants（ORAC values were 1.37 mol TE/mol,3.65 mol TE/mol and 10.56 mol TE/mol,respectively）.According to fluorescence quenching,3,4-dihydroxy ethyl cinnamate,flazin,catechol and genistein could bind to XOD,and there was only one binding site for them.According to the Linweaver-Burk curves,3,4-dihydroxycinnamate,flazin,catechol and genistein were the hybrid inhibitors of XOD dominated by non-competitive inhibition mechanism.Molecular docking experiments showed that flazin could enter into the catalytic center of XOD to interact with Glu1261,Arg912,Gly796 and Gln1194 by hydrophobic forces,and four hydrogen bonds were formed by flazin with Lys1045,Gln1194 and Arg912.According to ADME（absorption,distribution,metabolism and excretion）simulation,flazin had good intestinal absorption and low blood brain penetration,so flazin had good oral bioavailability in vivo.（3）The adsorption and desorption characteristics of seven macroporous resins on the antioxidants in soy sauce were investigated.SP-207 and SP-825 resins possessed good adsorption and desorption capacities,and could effectively and rapidly enrich the antioxidants in soy sauce.The adsorption kinetics and thermodynamics of the antioxidants in soy sauce onto SP-207 and SP-825 resins were studied.The pseudo-second-order kinetics and Langmuir isotherm models were demonstrated to be appropriate to describe the whole exothermic and physical adsorption processes of antioxidants onto resins,which were influenced by boundary layer diffusion and intraparticle diffusion stages.The 60%ethanol eluted fraction from soy sauce purified by SP-825 resin column possessed the strongest antioxidant activity.The antioxidant activities and contents of typical soy isoflavones,furanones,pyranones,and phenolic acids in the SP-60%fraction were determined.These compounds contributed to50.02%of the total antioxidant activity of the SP-60%fraction.The key small molecule antioxidant compounds in soy sauce were identified as 4-ethylguaiacol,catechol,daidzein,and 4-ethylphenol by the antioxidants omission experiments.（4）The XOD inhibitory activities and antioxidant activities of 35 commercial soy sauces（including dark and light soy sauces）produced by three fermentation processes were compared,and it was found that the average XOD inhibitory activities were ranked as follows:low-salt solid-state fermentation（LSF）soy sauce>Japanese style fermented（JSF）soy sauce>high-salt liquid fermentation（HLF）soy sauce,but there were no significant differences;Dark soy sauce>light soy sauce,but there were no significant differences.The average DPPH radical scavenging activities and ORAC were ranked as follows:HLF soy sauce>LSF soy sauce>JSF soy sauce（p<0.05）;Dark soy sauce>light soy sauce（p<0.05）.The average superoxide anion radical scavenging activities were ranked as follows:JSF soy sauce>HLF soy sauce（p<0.05）;HLF soy sauce>LSF soy sauce（p>0.05）;Light soy sauce>dark soy sauce（p<0.05）.DPPH free radical scavenging activities and ORAC could effectively reflect the antioxidant activities of maillard reaction products and maillard reaction intermediate products in soy sauce.The changes in the browning indexes and antioxidant activities of soy sauce after macroporous resin enrichment were examined.It was found that the macromolecular maillard reaction products and maillard reaction intermediate products played an important role in the antioxidant activity of soy sauce.It was speculated that in addition to maillard reaction products and maillard reaction intermediate products,small molecule active substances were also important for the antioxidant activity of soy sauce.