Interaction Of Quercetin And Rutin With Buckwheat Trypsin Inhibitor

Protease inhibitor plays an important role in the study of enzyme function research and new drug design.It has been widely recognized as a potential cancer treatment agent in the medical field.PI can be widely found in plants,It also can be related to physiological activities including insect resistance,disease resistance and stress resistance.Most foods,such as legumes,cereals,nuts,fruits,vegetables,and some dairy products,contain protease inhibitors in varying amounts that can form complexes with enzymes in the gut,resulting lossing of enzyme activity,which significantly reduce the digestion and absorption of essential nutrients,at the same time,it also would have an adversely affecting for human or animal health.Therefore,these inhibitors are also known as anti-nutritional factors in the human diet.Recombinant buckwheat trypsin inhibitor(rBTI)is a 7.9 k D Potato type I inhibitor obtained by gene cloning and prokaryotic expression.It has a specific inhibitory effect on trypsin.Kunitz and Bowman-Birk inhibitors have been reported to combine with small molecules to form complexes that reduce their inhibitory activity and promote nutrient absorption in the body.However,it is not clear whether rBTI,as a type I inhibitor of potato,can reduce the content of its inhibitor by binding with small molecule ligands.The purpose of this study was to study the interaction between rutin,quercetin and rBTI inhibitor in buckwheat and its effect on rBTI inhibitor activity,so as to provide a theoretical basis for buckwheat as a nutritional additive.1.In this article,we make use of sequence alignments which can have an accurate analysis of the tryptophan residues and hydrophobic cavities of rBTI structure.WPELVG and DRVWV motifs are conserved in the PI-I family.The amino acid sequences of the PI-I family are highly similar,and the two conserved Cys residues form a disulfide bond that stabilizes the rBTI structure.There are two tryptophan residues in the rBTI sequence,one(W10)located near the hydrophobic cavity and the other(W53)located near the rBTI inhibition site.rBTI-W10 A and rBTI-W53 A were mutated into alanine A by site-directed mutagenesis to obtain rBTI-W10 A and rBTI-W53 A.SDS-PAGE and circular dichromatography showed that the structure of the mutant rBTI-W10 A had some changes,while the structure of rBTI-W53 A had no obvious changes.Fluorescence spectra showed that Trp 53 was the main source of rBTI fluorescence.These two mutants provide a convenient explanation for the subsequent analysis of the precise binding sites of small flavonoid molecules and rBTI.2.The interaction between aloe and quercetin and rBTI was analyzed by fluorescence spectroscopy and circular dichroism chromatography.The results show that both rutin and quercetin can interact with rBTI and form stable complexes through static quenching.Rutin or quercetin binds to rBTI by different forces,resulting in a conformational change.Rutin mainly uses van der Waals force and hydrogen bond,quercetin mainly uses hydrophobicity.3.The inhibitory effects of rutin and quercetin on rBTI inhibition was analyzed by trypsin activity inhibition test.The results showed that rutin and quercetin also had significant effects on rBTI inhibitory activity: both rutin and quercetin could reduce the trypsin inhibitory activity of rBTI,but the effect of quercetin was more obvious.Finally,the binding sites of rutin and quercetin with rBTI were revealed by molecular docking.These findings contribute to understanding the interaction of flavonoids with protease inhibitors and provide strategies for reducing the anti-nutritional effects of protease inhibitors.