Molecular Mechanism Of Anthocyanins From Purple Sweet Potato(Ipomoea Batatas L.)in Attenuating Abnormal Uric Acid Metabolism

Abnormal uric acid metabolism generally refers to a condition in which the level of serum uric acid is too high,and which in turn deposits in the body and eventually causes gout.Its typical representative disease,hyperuricemia(HUA),has developed into the “fourth hyper-disease” after “hyperglycemia,hypertension and hyperlipidemia”,showing a trend of “high prevalence and youthfulness” in China.Uric acid belongs to the terminal product of human's purine metabolic pathway and is regulated by various bioactive enzymes in the pathway during its production.When the public frequently and intensively consumes high purine food,it will lead to a large amount of uric acid being produced,which exceeds the body's own metabolic capacity and causes abnormal uric acid metabolism disease.At present,there is no effective method for completely curing abnormal uric acid in clinic.Drugs such as xanthine oxidase(XO)inhibitor allopurinol not only need to be in the long-term medication,but also have strong toxic and side effects,which brings heavy burden to patients.Therefore,natural activity compounds with high efficiency and low toxicity,especially flavonoids with special structure,have a huge potential market for nutritional intervention of abnormal uric acid metabolism,which are also hot research topics in recent years.The purpose of this study was to investigate the substance basis and molecular mechanism of anthocyanins from purple sweet potato in attenuating abnormal uric acid metabolism.Anthocyanin-rich Purple Sweet Potato Extract(APSPE)was prepared by acid-alcohol extraction,ethyl acetate extraction and AB-8 macroporous resin.Its XO inhibitory activity was compared with 9 commercial anthocyanin enrichments from different sources.Different secondary purified fractions from APSPE were separated by ODS reverse-prepared packed column.Characterization and quantification of anthocyanin fractions were carried out by high performance liquid chromatography(HPLC).Anthocyanin fraction with best inhibitory effect was screened by XO inhibition experiment in vitro.On this basis,the interaction betweenanthocyanin fractions and XO was further compared using fluorescence spectroscopy.The interaction mechanism between the best performing fraction Highly-Acylated Anthocyanins from Purple Sweet Potato(HAA-PSP)and XO was further investigated by fluorescence quenching spectroscopy and circular dichroism chromatography.The interaction mode between typical anthocyanin molecules in fractions and XO was studied by computer simulation molecular docking method,and the binding mode and key groups of interaction between anthocyanins and XO active sites were discussed theoretically.The biological activity of HAA-PSP in reducing uric acid in vivo,its regulation on key enzymes of purine metabolic pathway and the molecular mechanism of its intervention on abnormal uric acid metabolism were systematically evaluated by using HUA mice model.Finally,the combination of HAA-PSP and allopurinol at a certain concentration was used to study the individual and combined effects of allopurinol and HAA-PSP on improving uric acid level,renal injury,oxidative stress in HUA mice,and to investigate whether HAA-PSP can alleviate renal inflammation and functional damage in patients with hyperuricemia by reducing the dosage of allopurinol on the premise of guaranteeing the therapeutic effect of reducing uric acid.The main results are as follows:(1)The crude extract of anthocyanins from purple sweet potato was prepare by acid-alcohol extraction and ethyl acetate extraction.Anthocyanin-rich Purple Sweet Potato Extract(APSPE)was purified by AB-8 macroporous resin.Compared with 9commercial anthocyanin enrichments from different sources,APSPE has obvious advantages in XO inhibition,showing obvious "dose-effect relationship".Four secondary purified fractions from APSPE were separated by ODS reverse-prepared packed column.The results of classic pH differential analysis and HPLC showed that the main component of Fraction 1 was anthocyanin copigment,Fraction 2 was mono-acylated anthocyanin with molecular weight of 900,Fraction 3 was di-acylated anthocyanins with molecular weight of 1100,and Fraction 4 has an extremely lowanthocyanin content,which is presumed to be a residual impurity component.In the inhibition of XO activity in vitro,Fraction 3 had the strongest inhibitory effect on XO activity,followed by Fraction 2.The results indicated that the main compounds inhibiting XO in purple sweet potato was anthocyanins rather than copigment,and the inhibition of highly-acylated anthocyanins with large molecular weight in purple sweet potato is much stronger than that of the low-acylated anthocyanins with small molecular weight.(2)Fluorescence quenching spectra and circular dichroism analysis of Fraction 1,2and 3 showed that with the increase of anthocyanins content,the intrinsic fluorescence intensity of XO decreased,suggesting that suggesting that there was interaction between anthocyanins fractions and XO.Among them,Fraction 3,the Highly-Acylated Anthocyanins from Purple Sweet Potato,a.k.a.HAA-PSP,has the best fluorescence quenching effect on XO.Further analysis of quenching data shows that the fluorescence quenching mechanism of HAA-PSP on XO is static quenching,and there is only a single binding site on the active cavity of XO.The main force in the interaction is hydrophobic interaction,and the binding process may be an endothermic spontaneous reaction driven by entropy.From the results of circular dichroism,it is known that with the increase of the molar ratio of HAA-PSP to XO,the ellipticity of XO circular dichroism peaks gradually decreases,which indicates that HAA-PSP can affect the secondary structure of XO.Its binding with XO can rearrange the conformation of the enzyme,influence the structure of its hydrogen bond network,and lead to the denser secondary structure of XO,which limits the binding process of substrate to XO,and ultimately lead to the reduction of the catalytic activity of XO,resulting in the inhibition effect.(3)The results of molecular docking showed that the binding ability of di-acylated anthocyanins to XO was higher than that of mono-acylated anthocyanins and anthocyanins without acyl groups.Acyl groups played an important role in the recognition and binding of macromolecular anthocyanins to XO active sites.Amongthem,acyl groups carried by sophoroside in anthocyanins from purple sweet potato can effectively insert into the active cavity of XO and interact with the active amino acid residues such as Lys 771,Glu 802,Glu 879,Arg 880,Val 1011,Pro1012,etc.Other structures except acyl groups can interact with the amino acid residues near the active channel of XO and cover the active channel,which will hinder the process of substrate entering the active cavity and binding with molybdenum pterin from two aspects.Hydrophobic interaction is the main driving force for the interaction between highly-acylated anthocyanins and XO,with hydrogen bond being the auxiliary interaction force.Different types of acyl groups have some influence on the inhibitory effect of enzyme activity caused by interaction with XO.Compared with cyanidin anthocyanins,anthocyanins with peonidin structure are more susceptible to different acyl groups.Caffeoyl group is a dominant group compared to other groups such as feruloyl,which assists the binding of anthocyanins to XO enzymes.(4)The results of in vivo experiments in HUA mice showed that HAA-PSP could effectively regulate the serum uric acid level in HUA mice,and the effect was significantly stronger than APSPE.During administration,HAA-PSP did not cause significant toxic damage to the organs of mice and could significantly regulate the levels of serum urea nitrogen and creatinine and weaken the renal damage in HUA mice.At the same time,HAA-PSP can effectively inhibit the activities of xanthine oxidase(XO),adenosine deaminase(ADA)and 5'-nucleotidase(5'-NT),which are key biological enzymes in purine metabolic pathway.It can inhibit the production of uric acid and its precursors in mice with hyperuricemia from the source,so as to reduce the level of uric acid.(5)HAA-PSP was mixed with allopurinol at a certain concentration to carry out animal experiments in HUA mice.The results showed that when HAA-PSP was combined with allopurinol,its uric acid-lowering activity in HUA mice was more significant than that of HAA-PSP used alone.It was speculated that there might be some synergistic effect between HAA-PSP and allopurinol,that is,it could reduce theintake of allopurinol while maintaining the uric acid-lowering activity and attenuating abnormal uric acid metabolism.The combination of HAA-PSP and low-dose allopurinol can effectively alleviate the renal functional damage caused by hyperuricemia and the use of high-dose allopurinol,which is through the regulation of serum urea nitrogen and creatinine levels in mice,as well as the reduction of inflammatory cell infiltration observed by histopathological sections of kidneys,the restoration of morphological structure of glomeruli and proximal convoluted tubules,and the normalization of glomeruli.The proliferation of basement membrane and mesangial matrix was also inhibited.In addition,the combination of HAA-PSP and low-dose allopurinol can effectively enhance the oxidative defense ability of hyperuricemia mice,thereby alleviating the oxidative stress damage caused by HUA.Meanwhile,the combination of HAA-PSP and low-dose allopurinol can hinder the activation of NF-?B pathway,reduce and inhibit the inflammatory cytokines TNF-?,TGF-?1,IL-6 and IL-1? in the interleukin family,adhesion molecule ICAM-1 and the synthetase COX-2 in the kidney,thereby alleviating the renal inflammation in hyperuricemia mice,and effectively reducing the adverse reactions caused by high dose of allopurinol.It can be inferred that HAA-PSP,as a natural dietary functional component,can effectively reduce the dosage of allopurinol in the adjuvant synergistic treatment with allopurinol,while alleviating the additional renal damage caused by drugs,thus delaying the occurrence and development of chronic kidney disease caused by hyperuricemia patients in the course of treatment.The results of this study are expected to provide a scientific basis for the nutritional intervention of dietary anthocyanins and other natural components of flavonoids in the prevention of hyperuricemia and abnormal uric acid metabolism.