The Binding Mechanism Of Hyperoside To Lipase And Preparation And Evaluation Of β-cyclodextrin Inclusion Complex

Obesity is not only strongly associated with the development of metabolic syndromes such as type II diabetes and non-alcoholic fatty liver disease but is also a major factor in the development of various cancers by disrupting the human immune system,posing a significant health risk to obese patients.Lipase,which is synthesized and secreted by the pancreas,has been identified as a key digestive enzyme for the digestion of dietary lipids in humans and a key target for the treatment of obesity.Inhibition of pancreatic lipase is effective in controlling lipid absorption and reducing calorie intake.Long-term administration of chemically synthesized pancreatic lipase inhibitors can cause many associated adverse effects.Therefore,dietary polyphenols(e.g.,flavonoids)with many biological activities but no side effects are promising alternatives for controlling calorie intake.In this paper,a flavonoid compound,Hyperoside(HYP),was selected for study and has received increasing attention from researchers due to its potential health benefits.However,the instability and low uptake rate of HYP limit their use in food and pharmaceutical applications.The use of cyclodextrins as a delivery system for bioactive substances due to their superior structural properties can effectively improve the bioavailability of hydrophobic bioactive substances.Based on this,this paper investigated the binding mechanism of HYP to lipase and the kinetics of lipase inhibition.The characterization of small molecule-lipase interactions not only helps to decipher the biological functions of lipase but also provides useful information for the development of new and promising therapeutic drugs.The water solubility and stability properties of the threeβ-cyclodextrin and HYP inclusion complexes were prepared and tested to broaden the scope of application of hydrophobic bioactive compounds.In addition,the investigation of the binding mechanism of the threeβ-cyclodextrin inclusion complexes containing HYP with lipase contributed to the understanding of the effect ofβ-cyclodextrin on the nature and function of the active small molecules with lipase.The main results were as follows.(1)The binding mechanism and potential inhibition of HYP with lipase were investigated by multi-spectroscopic techniques,isothermal titration calorimetry,enzyme inhibition kinetics,STD-NMR,and molecular modeling techniques(molecular docking,molecular dynamics).The results of isothermal titration calorimetry showed that the binding affinity was medium.Circular dichroism and Fourier-transform infrared spectroscopy showed that the binding of HYP slightly changed the conformation of lipase.The interaction disrupted the protective water layer on the lipase surface and reduced the protein particle size.Furthermore,enzyme inhibition kinetics experiments revealed that HYP inhibited lipase activity in a concentration-dependent manner.The results of computer simulation predictions and thermodynamic experiments confirmed that hydrogen bonding was the main driving force and that the addition of HYP rendered the lipase structure relatively unstable.(2)The threeβ-cyclodextrin/HYP inclusion complexes in a ratio of 1:1 were prepared by freeze-drying method and characterized by a series of analytical tools such as Fourier-transform infrared spectroscopy,X-ray diffraction,differential scanning calorimetry,scanning electron microscopy,fluorescence spectroscopy,and molecular docking,and the threeβ-cyclodextrin inclusion complexes were successfully encapsulated.(3)The DPPH and ABTS~+radical scavenging assays were performed to characterize the antioxidant capacity of the inclusion complexes,which increased with the concentration of HYP in the inclusion complexes.In addition,the results of both the solubility and stability experiments indicated that the inclusion complexes had greater stability than free HYP.The inclusion complexes improved the aqueous solubility and stability of HYP.(4)The threeβ-cyclodextrin/HYP inclusion complexes were investigated for binding mechanism with lipase,and the results showed that the threeβ-cyclodextrins encapsulated with HYP interacted with lipase,and the lipase underwent fluorescence quenching and formed the new complexes.In addition,UV-visible and circular dichroism spectroscopy confirmed that the binding of the threeβ-cyclodextrin inclusion complexes resulted in no significant change in protein conformation.