Competitive Substitution Effect Of Polyphenolic Compounds On The HSA-bound ?-zearalenol

?-Zearalenol(?-ZOL),the major metabolite of ZEN(one of the most widespread mycotoxins contaminated in corn,wheat,rice and other agricultural products)in human and animals.It has been reported that the toxic effect of ?-ZOL is greater than that of its parent ZEN.?-ZOL has immunotoxicity,genotoxicity,estrogenic effect,hematotoxicity,cytotoxicity,hepatotoxicity,teratogenicity and even carcinogenicity,this will pose a threat to the health of human and animals,thereby bringing huge economic losses.Consequently,it is very important to control the toxicity of ?-ZOL that exists in human and animal bodies.The metabolism of ?-ZOL is mainly performed via binding with human serum albumin(HSA)and then transported to target organs and cells in human body.It has been reported that certain polyphenolic compounds could be able to competitively replace mycotoxins in body.Therefore,the effects of different polyphenolic compounds such as flavonols,flavonoids,phenolic acids and dihydroflavonoids on the HSA-bound ?-ZOL were studied by the methods of fluorescence spectrum,ultraviolet-visible absorption spectrometry(UV-vis),infrared spectrum,ultrafiltration and high performance liquid chromatography(HPLC).It aims to explore which polyphenolic compounds have the obvious competitive substitution effect by simulating the pH of human blood in the basic metabolic system of HSA,reveal how polyphenolic compounds replace and free ?-ZOL through competition,and eventually achieve the purpose of promoting excretion and reducing the amount of accumulation of ?-ZOL in body.This is of great significance for the control of mycotoxins in the body through developing foodborne active substance and ultimately realizes the goal of a wide concept of health.The primary conclusions of this study are as follows:(1)Quercetin of flavonol,baicalin of flavone,rosmarinic acid of phenolic acid and naringin of dihydroflavone can affect the binding of ?-ZOL with HSA under the condition of simulated blood(pH=7.4).It was found that the binding sites on HSA of all the polyphenolic compounds studied in this study(including flavonoids,phenolic acids and stilbene polyphenols)are all located at site ?(?A),which was consistent with the binding site of ?-ZOL on HSA through competitive probe experiments.Consequently,it is possible for the above compounds to replace ?-ZOL by competition.After the polyphenolic compounds participating in the competitive system,the decrease in the binding constant of the polyphenol-protein system is low,indicating that the four typical structural polyphenolic compounds(quercetin,baicalin,rosmarinic acid and naringenin)have obvious competitive effects on the HSA-?-ZOL system.Competitive substitution experiments of four typical polyphenolic compounds on ?-ZOL found that quercetin,baicalin,rosmarinic acid and naringenin revealed the highest substitution rate of ?-ZOL at the ratio of 2:1,which was 54.21%,52.44%,49.93% and 40.41%,respectively.(2)The four typical polyphenolic compounds(quercetin,baicalin,rosmarinic acid and naringenin)can cause significant changes in the conformation of HSA-?-ZOL system.The study was performed using the methods of fluorescence quenching,synchronous fluorescence,ultraviolet-visible absorption spectroscopy,circular dichroism spectrum and infrared spectroscopy.It was found that quercetin and baicalin can bind to tryptophan residues of the HSA hydrophobic cavity,resulting in the enhancement of the hydrophilicity and the polarity of HSA amino acid residue microenvironment in the HSA-?-ZOL system,which indicated that HSA molecules have aggregated.However,when rosmarinic acid and naringenin were added to the HSA-?-ZOL binary system,the hydrophobicity of the microenvironment of tyrosine and tryptophan residues in HSA increased with the decrease of its polarity,illustrating that rosmarinic acid and naringenin can cause the unfold of the HSA peptide chain and result in the exposure of hydrophobic amino acids.The results of studies on the chemical forces of different systems indicated that quercetin,baicalin and naringenin can induce the hydrophobic interaction as the dominant role in the HSA-?-ZOL system,while rosmarinic acid induces the primary role of hydrogen bond in the HSA-?-ZOL system.(3)The research on the competitive substitution modes and its mechanisms of ?-ZOL interacted with polyphenolic compounds revealed that quercetin,baicalin and naringenin can directly completely replace with ?-ZOL on HSA and gradually take up the binding site of ?-ZOL on HSA to remove all the ?-ZOL from HSA.Rosmarinic acid should firstly bind to the site of HSA which is not occupied by ?-ZOL,and then compete with ?-ZOL to remove it from HSA.In addition,the dynamic process of quercetin competive binding to HSA with ?-ZOL showed that HSA could preferentially combine with polyphenolic compounds and forms a complex with a high stability when the two molecules coexist.As the concentration of quercetin or duration of competition increases,its competitive substitution rate for ?-ZOL gradually increases firstly and then gradually being stable after reaching the maximum competitive substitution rate,manifesting that the competitive substitution of polyphenolic compounds represented by quercetin to ?-ZOL may be a spontaneous process and then gradually tend to be dynamic equilibrium.In summary,quercetin,baicalin,rosmarinic acid,and naringenin have good competitive substitution effects on HSA-bound ?-ZOL,especially quercetin with the most significant competitive substitution effect.This research can lay a theoretical foundation,to some degree,for the further development of food-derived polyphenols,the study of their physiological activities and in vivo detoxification by metabolic competition,and the promotion of human health.