Study On ?-glucosidase Inhibitors Screening,Purification And Inhibition Mechanism In Artemisia Selengensis Turcz

Artemisia selengnesis Turcz?AST?is a special food resource of poyang lake in Jiangxi province,and the tender stems of it are often processed into delicious food and is popular in china.The leaves and root of AST are often discarded as directly and leading to the environment pollution and waste of natural resource.AST extracts have been reported to show many bioactive properties,such as antioxidation,anticancer,hypoglycemic,antimicrobial and so on.At present,the study on AST is mainly facus on the aerial parts?leaves and stems?of it,and there has few research about its roots.Diabetes is the third largest chronic disease in the world,?-glucosidase inhibitors was used to prevent and treat diabetes mellitus,and the inhibitors from natural product have become hotspot of research due to highly active,low side effect and toxicity.Our group has studied the antioxidation and hypoglycemic activity of the leaves of AST,and revealed it had a good antioxidation and?-glucosidase inhibitory activities.Therefore,in this paper,we studied the acetylcholinesterase inhibition,?-glucosidase,tyrosinase and xanthine oxidase inhibition of wild and cultivated AST leaves,stems and wild AST root,and then bioactivities-guided isolation and identification of?-glucosidase inhibitors from wild AST roots was used by liquid-liquid extraction,column separation and semi-HPLC.At last,the inhibitory mechanism of 3-caffeyl-5-feruloylquinic acid on?-glucosidase was studied.The conclusion was listed as follow:1.Wild and cultivated AST leaves had the higest phenolics?30.12 mg GAE/g DM?and flavonoids?0.63 mg QuE/g DM?,respectively.The strongest tyrosinase inhibition was observed in cultivated AST leaves extract.Wild AST leaves and roots extract have the best acetylcholinesterase inhibition and xanthine oxidase inhibition compared with other AST extract,respectively,but the IC₅₀ values of them is far lower than standards.Wild AST roots extract showed the highest?-glucosidase inhibition,the IC₅₀ values of it was 14.08 mg DM/mL.In the whole,the enzyme inhibition abilities of wild AST extracts was better than cultivated AST extracts.Wild AST roots can be used as the material for screening the high actively?-glucosidase inhibitors,and this research was expected to provide reference to the development and utilization of AST in functional food industry.2.Six compound was isolated and identified from wild AST roots by NMR and ESIMS,including 3,5-dicaffeoylquinic acid?1?,3-caffeoyl,5-fyruloylquinic acid?2?,1,3-dicaffeoylquinic acid?3?,neochlorogenic acid?4?,4-fyruloyl,5-caffeoylquinic acid?5?and 3,5-dicaffeoylquinic acid methyl ester?6?.Compounds 2,4,and 6 were identified in the AST for the first time.3-caffeoyl-5-fyruloylquinic acid had the best?-glucosidase inhibition in all compounds,and the IC₅₀ values of it was 289.96?M,which was two-fold higher than acarbose.What's more,in addition to1,3-dicaffeoylquinic acid,other compounds had good?-glucosidase abilities,the more caffeoyl groups in caffeoylquinic acid,the?-glucosidase inhibition of compounds is much stronger.As a result,3-caffeoyl-5-fyruloylquinic acid of AST roots has the potential of be development as an antidiabetic drug in the furture.3.3-caffeyl-5-fyruloylquinic acid was a mixed?-glucosidase inhibitor,and the inhibiting process by it was reverisable.The inactivation processe of?-glucosidase inhibitor was one-way and followed first-order kinetics in the presence of 3-caffeyl-5-fyruloylquinic acid.?-glucosidase was quenched by the interactions with 3-caffeyl-5-fyruloylquinic acid through a static quenching mode and formed 3-caffeyl--fyruloylquinic acid-?-glucosidase complex,there only had a single bingding site in?-glucosidase.The process of?-glucosidase combine with 3-caffeyl-5-fyruloylquinic acid was spontaneous and an exothermic reaction,and the main the driving forces of it was van der Waals force and hydrogen bond.The study of synchronous fluorescence spectroscopy and CD showed that the interactions between3-caffeyl-5-fyruloylquinic acid and?-glucosidase could change the micro-environments and conformation of the enzyme.3-caffeyl-5-fyruloylquinic acid could induce the increase in hydrophobicity around tyrosine residue of?-glucosidase.With the add of 3-caffeyl-5-fyruloylquinic acid,the content of?-turn and?-sheet were increased,but the content of?-helix and random coil were decreased.These result to close the active area of?-glucosidase,and then leading to lower the activities of?-glucosidase.