Study On The Interaction Mechanism Between Shikonin And Four Chronic Metabolic Disease-related Enzymes

Chronic metabolic diseases mainly include type II diabetes mellitus,hyperuricemia and obesity,which have become an important threat to human health due to their high morbidity,high mortality and younger trend.Takingα-glucosidase,α-amylase,xanthine oxidase and pancreatic lipase as targets,seeking natural substances that are less toxic and side effects is a research hotspot in the field of prevention and treatment of these chronic metabolic diseases.Shikonin is a natural active substance derived from Lithospermum erythrorhizon Sieb.et Zucc.which has some medicinal values such as anti-inflammatory,antibacterial and anti-tumor.Exploring the interaction mechanism between shikonin and several chronic metabolic disease-related enzymes is of great significance for expanding the application scope of shikonin,the prevention and treatment of type II diabetes,hyperuricemia and obesity.In this study,the fluorescence quenching mechanism of shikonin withα-glucosidase,α-amylase,xanthine oxidase and pancreatic lipase were investigated by fluorescence spectroscopy experiments,and the binding constants and site numbers of shikonin and the four enzymes were calculated.The main forces of the interaction between shikonin with the four enzymes were analyzed.Synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy and circular dichroism techniques were used to study the effects of shikonin on the conformation of the four enzymes.The effects of shikonin on the microenvironment of the aromatic amino acid residues in the four enzymes were investigated through synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy experiments.The effects of shikonin on the secondary structure of the four enzymes were studied by circular dichroism.The interaction between shikonin with the four enzymes was predicted by molecular docking technology.The main experimental results of this paper were as follows:1.The binding was spontaneous between shikonin withα-glucosidase,and the main binding forces were hydrogen bonds and van der Waals forces.The quenching mechanism toα-glucosidase was static quenching,and there was only one binding site.At 298 K,the quenching rate constant,binding constant and number of binding site were（1.00±0.06）×10¹² L mol^-1 s^-1,（2.32±0.04）×10~4L mol^-1 and 1.08±0.01,respectively.The binding of shikonin toα-glucosidase could lead to changes in the microenvironment of the amino acid residues and the secondary structure of the enzyme.Molecular docking revealed that shikonin could interact with the amino acid residues surroundingα-glucosidase,and there was a hydrogen bond interaction with residue Arg1510.2.The binding of shikonin toα-amylase was a spontaneous reaction.The main driving force of binding was hydrogen bond and van der Waals force.The quenching mechanism toα-amylase was static quenching,and there was only one binding site.At 298 K,the quenching rate constant,binding constant and number of binding site were（2.04±0.04）×10¹² L mol^-1 s^-1,（2.72±0.25）×10~5 L mol^-1 and 1.26±0.01,respectively.The combination of shikonin andα-amylase changed the conformation ofα-amylase,amino acid residue microenvironment and secondary structure.Molecular docking revealed that shikonin could interact with the amino acid residues surroundingα-amylase,and there was a hydrogen bond interaction with residue Trp59.3.The binding was spontaneous between shikonin with xanthine oxidase,and the main binding force was hydrophobic interaction.The quenching mechanism to xanthine oxidase was static quenching,and there was only one binding site.At 298 K,the quenching rate constant,binding constant and number of binding site were（1.77±0.03）×10¹² L mol^-1 s^-1,（7.17±0.40）×10~3 L mol^-1 and 0.91±0.01,respectively.The binding of shikonin with xanthine oxidase could alter the microenvironment of amino acid residues and lead to changes in secondary structure.Shikonin could also interact with amino acid residues around xanthine oxidase and form hydrogen bonds with Gln767 and Ser1080 residues.4.The binding was spontaneous between shikonin and pancreatic lipase,and the main binding forces were hydrogen bonds and van der Waals forces.The quenching mechanism of pancreatic lipase was static quenching,and there was only one binding site.At 298 K,the quenching rate constant,binding constant and number of binding site were（2.14±0.07）×10¹¹ L mol^-1 s^-1,（6.79±0.33）×10~3 L mol^-1 and 1.14±0.01,respectively.The binding of shikonin with pancreatic lipase could alter the microenvironment of amino acid residues and lead to changes in secondary structure.Molecular docking revealed that shikonin could interact with the amino acid residues surrounding pancreatic lipase,and there was a hydrogen bond interaction with Ser152,His151,Phe77 and Thr78 residues.The above results showed that shikonin could spontaneously quench the endogenous fluorescence ofα-glucosidase,α-amylase,xanthine oxidase and pancreatic lipase by a static quenching mechanism.As a multi-target active substance,shikonin can be combined withα-glucosidase,α-amylase,xanthine oxidase and pancreatic lipase,respectively.It can change the conformation of the enzymes.This study will provide a certain theoretical basis and experimental basis for its research in the prevention and treatment of chronic metabolic diseases,such as type II diabetes,hyperuricemia and obesity.