Study On The Interaction Of Ellagic Acid,urolithin B With Serum Albumin And The Cytotoxicity Of The Complex To HepG2 Cells

Serum albumin is the most abundant plasma protein in mammalian plasma.It can not only maintain blood p H value and osmotic pressure,but also transport a variety of endogenous and exogenous compounds.Small molecular compounds with pharmacological effects need to be transported by serum albumin to reach the site of action before they can exert their pharmacological effects.In clinical studies,the combination of drugs and serum albumin rate is one of the important parameters of drug safety evaluation,so the small molecular compounds and the interaction of serum albumin is considered to help clarify pharmacokinetic process of small molecular compound in the body.The small molecular compounds and the binding process between serum albumin and binding sites can provide theoretical basis for further study on the clinical research and drug,as well as small molecular compound drug molecular design and a preliminary basis.Bovine serum albumin and human serum albumin has a homology of 76%,and two proteins have two active Sites I andⅡ,so often bovine serum albumin is considered as the pattern protein to study the interaction between small molecules and protein.Ellagic acid,a natural polyphenolic small molecule,is widely found in chestnut,pomegranate,raspberry and other fruits,which has anti-inflammatory,anti-oxidative,anti-bacterial and anti-tumor effects.The ellagic acid is metabolized by intestinal microorganisms to produce a variety of Urolithin.Urolithin B is one of the end products of metabolism of ellagic acid to remove one lactone ring and remove three hydroxyl groups.In terms of pharmacological activity,it has anti-inflammatory,anti-tumor and therapeutic effects on cardiovascular diseases.They are similar in structure and pharmacological activity.The study of the interaction between ellagic acid and urolithin B and serum albumin is helpful to understand the binding mechanism of these two drugs in the transport of serum albumin fraction.On the basis of previous studies,this study systematically studied the interaction between ellagic acid,urolithin B and serum albumin,analyzed the effects of the two drugs on serum albumin absorption spectrum and kinetic simulation,and studied the effects of drug and BSA complex on Hep G2cytotoxicity.The effects of ellagic acid and urolithin B on the ultraviolet absorption of bovine serum albumin were studied by ultraviolet-visible spectroscopy.The results of ultraviolet spectroscopy showed that both ellagic acid and urolithin B enhanced bovine serum albumin at 280 nm.The absorption peak of the two small molecule compounds relaxes and unfolds the conformation of bovine serum albumin.Using fluorescence spectroscopy,combined with the Stern-Volmer equation and the double reciprocal equation,the effects of ellagic acid and urolithin B on the fluorescence quenching of bovine serum albumin were studied,and the quenching mechanism,binding constant and number of binding sites were determined.It shows that ellagic acid and urolithin B can quench the fluorescence of bovine serum albumin,and the fluorescence quenching process is static quenching.The binding constant Ka of ellagic acid and BSA is2.31×10⁴ L·mol^-1.The number of binding sites n is 0.92.When urolithin B interacts with BSA,the Ka value is 3.37×10⁵ L·mol^-1,and n is 1.08.The results indicate that one BSA molecule can interact with one ellagic acid or urolithin B.The comparison of binding constants shows that urolithin B has a higher quenching efficiency than ellagic acid.The effect of two small molecule compounds on the microenvironment of tyrosine and tryptophan residues in bovine serum albumin was studied by synchronous fluorescence spectroscopy.The results showed that both ellagic acid and urolithin B can make both the polarity of the residues around tyrosine and tryptophane changes,and the binding sites of the two small molecule drugs and bovine serum albumin are mainly concentrated on tryptophan residues.The molecular docking and molecular dynamics simulation results of ellagic acid,urolithin B,HSA’s SiteⅠand SiteⅡshowed that the combination of ellagic acid,urolithin B and HSA mainly uses hydrogen bonds and hydrophobic interactions.And the preferential binding site is Site I.The MTT method was used to study the effect of ellagic acid,urolithin B and the complex of these two small molecule compounds with BSA on the cytotoxicity of hepatoma cells Hep G2.The results showed that the IC₅₀ value of the complex of ellagic acid and BSA is reduced by 2.2 times compared with the IC₅₀ value of ellagic acid alone.The IC₅₀ value of the compound of urolithin B and BSA is reduced by 1.7 times compared with the IC₅₀ value of urolithin B alone,indicating that the compound has stronger cytotoxicity.Serum albumin has a enhancement effect on the inhibition of Hep G2 cell proliferation by ellagic acid and urolithin B.