The Mechanism And Active Components Of TongFengTangSan Against Hyperuricemia Based On Spectrum-Effect Relationship And Metabonomics

Hyperuricemia is a metabolic disease caused by the disorder of purine metabolism in the human body,and the excretion of uric acid is lower than the rate of uric acid production.Long-term hyperuricemia can easily lead to the deposition of sodium urate crystals in the joints and cause gout.First-line drugs such as allopurinol and benzbromarone,which are currently clinically used to treat hyperuricemia,usually have serious adverse reactions,such as skin damage,digestive system damage,and hematopoietic system damage.Therefore,there is an urgent need to develop safe and effective alternative drugs.As a traditional prescription in Tibetan areas,Tong Feng Tang San is composed of Myrobalan,Lezhe and Zha Tam Gao.It has a good anti-gout effect and is often used by people in Tibetan areas to treat gout.However,the mechanism of its anti-hyperuricemia and its active ingredients have not been studied yet.Therefore,this subject will integrate spectral efficacy,metabolomics technology and molecular docking methods to study the potential uric acid-lowering pharmacological components and mechanism of Tong Feng Tang San.The main contents are as follows:1.Anti-hyperuricemia efficacy of Tong Feng Tang San and different fractions were studied,so as to provide the basis for the follow-up spectrum-effect relationship research.The 85% alcohol extract of Tong Feng Tang San(CF)was adsorbed with AB-8 macroporous resin and eluted using water,30%,60%,and 90% ethanol to procure the corresponding fractions(SX,CF30,CF60,CF90).Serum uric acid(s UA),serum creatinine(CRE),serum urea nitrogen(BUN),serum xanthine oxidase activity(s XOD),liver xanthine oxidase activity(l XOD),serum interleukin-1β(IL-1β)and serum interleukin-6(IL-6)as efficacy evaluation indicators,and it was found that85% alcohol extract of Tong Feng Tang San(CF)could significantly reduce the level of s UA,CRE,s XOD,l XOD and IL-1β(P < 0.05)SX can significantly reduce CRE,l XOD,IL-1β and s XOD(P < 0.05);CF30 can significantly reduce l XOD,IL-1β and s XOD(P < 0.05);CF60 can significantly reduce s UA,CRE,s XOD,l XOD,IL-1β and IL-6(P < 0.05);CF90 can significantly reduce CRE,l XOD,IL-1β and s XOD(P <0.05).Among the four fractions of TFTS,CF60 has the strongest uric acid-lowering effect,followed by SX,CF30,and CF90;CF60 has the strongest anti-inflammatory effect,followed by CF90,SX,and CF30;SX has the strongest XOD inhibition effect,followed by CF90,CF60,and CF30;SX has the better renal regualtion effect,followed by CF60,CF90,and CF30;CF90 has a better inhibiiton effect on IL-1β,followed by CF60,SX,CF30;among four fractions,only CF60 had a significant inhibitory effect on IL-6.In a words,the result shows that CF60 is the main fraction of CF.2.UPLC-Q-TOF/MS technology was used to qualitatively analyze the components of Tong Feng Tang San,and a total of 98 compounds were identified.Combined with the s UA level-lowering effect of four fractions of Tong Feng Tang San,the spectrum-effect relationship was analyzed by orthogonal partial least squares method,Spearman correlation analysis,and gray correlation analysis method,and 32 potential anti-hyperuricemia components were screened.3.Metabolites in plasma,kidney tissue and liver tissue of hyperuricemia rats were investigated by UPLC-Q-TOF/MS-based non-targeted metabonomics.The differential metabolites and their pathways in hyperuricemia were screened out based on VIP value and P value.The results of plasma metabolomics showed that 25 metabolites and 5 metabolic pathways were significantly changed.After CF intervention,6 differential metabolites in plasma were reversed and involved in purine metabolism and riboflavin metabolism;after SX intervention,4 differential metabolites in plasma were reversed and involved in 2 pathways;after CF30 intervention,the 4 differential metabolites in plasma were reversed and involved in two pathways;after CF60 intervention,8 differential metabolites in plasma were reversed and involved in two pathways;after CF90 intervention,4 differential metabolites in plasma were reversed and involved in one pathways.Kidney differential metabolites involved 2 pathways;8 differential metabolites were recalled after CF60 intervention,involving 2 pathways;4 differential metabolites were recalled after CF90 intervention,involving 1 metabolic pathway.The results of renal metabolomics showed that 18 metabolites changed significantly in rats with hyperuricemia.CF reversed 13 metabolites and involved in purine metabolism and βalanine metabolism;SX reversed 6 differential metabolites and no obvious pathway was reversed;13 differential metabolites were reversed after CF30 intervention,involving in one metabolic pathway;13 differential metabolites were reversed after CF60 intervention,involving 1 metabolic pathway;3 differential metabolites were reversed after CF90 intervention,and no pathway was reversed.The results of liver metabolomics showed that 11 metabolites changed significantly in hyperuricemia rats,6 metabolites were reversed after CF intervention,3 differential metabolites were reversed after SX intervention,and 7 differential metabolites were reversed after CF30 intervention;after CF60 intervention,9 differential metabolites were reversed;after CF90 intervention,5 differential metabolites were reversed;in the liver,the metabolism of alanine,aspartic acid and glutamic acid in hyperuricemia rats changed significantly,CF improved alanine,aspartate,and glutamate metabolic pathways and Ascorbate and aldarate metabolism.Among the different fractions of Tong Feng Tang San,SX significantly regulated Alanine,aspartate and glutamate metabolism and Pentose and glucuronate interconversions.CF30,CF60 and CF90 all significantly regulated ascorbic acid and aldonic acid metabolic pathways.The above results show that the regulation effect of Tong Feng Tang San on hyperuricemia rats involve purine metabolism,riboflavin metabolism pathway,β-alanine metabolism,ascorbic acid and aldonic acid metabolism,and alanine metabolism.Among the five metabolic pathways,purine metabolism is the common pathway of plasma and kidney metabolism,so it is speculated that purine metabolism may be the main way for Tong Feng Tang San against hyperuricemia.In addition,the correlation analysis between metabolites regulated by four fractions of Tong Feng Tang San and the anti-hyperuricemia efficacy revealed that riboflavin,uric acid,indoxyl glucuronide,uridine and citrulline are the key biomarkers of Tong Feng Tang San against hyperuricemia.4.Affinity of the active ingredients of Tong Feng Tang San and the targets of purine metabolic pathways was evaluated by molecular docking technology.It was found that,except for PNP,1-O-Galloyl-6-O-Cinnamoylglucose,Feruloyl dopamine,Cibotiumbaroside A,Urolithin B 3-O-glucuronide,Apigenin-7-O-glucoside and Murrayamine I had strong affinity with 5’-NT,AMPD,ADA,GDA and XOD.It is speculated that these six compounds are closely related to the purine metabolism of Tong Feng Tang San.In summary,Tong Feng Tang San has a significant anti-hyperuricemia effect,and its mechanism may be mainly related to the regulation of purine metabolism.CF60 may be the main active site of CF.