Research On The Hydrolysis Of Flavonoid Glycosides Of Glycosidase By Using Deep Eutectic Solvents

Flavonoids are a kind of compounds with anti-inflammatory,anti-cancer,anti-hypertensive,anti-diabetic and other pharmacological activities.The majority of flavonoids are the glycosides in plants,and only a small part of them is its corresponding aglycones.Studies have shown that most flavonoid aglycones are superior to their corresponding glycosides in various activities,due to their better solubility and oral bioavailability.In order to make use of flavonoid resources and improve the bioavailability of flavonoids,a lot of studies have been carried out to obtain highly active flavonoid aglycones.Among them,the structural modification of natural flavonoids by chemical and biological methods has become a research hotspot.Compared with chemical methods,the enzyme hydrolysis has the advantages of strong specificity,high catalytic efficiency,and no by-products.There are many factors that affect the wide application of the enzyme hydrolysis,among which the solvent is an important factor.Solvents can affect the rate of enzymatic reaction,and their physical and chemical properties such as polarity and pH can also significantly affect the activity and stability of the enzyme.At present,most of enzyme reaction occurs in buffer solution or organic solvent.However,these conventional solvents are not the best solvents for the catalytic activity of the enzyme,and organic solvent may even inactivate enzymes.Therefore,finding a suitable enzymatic reaction solvent is an urgent task in the practical production.Deep Eutectic Solvents?DESs?are a kind of eutectic solvents with a low melting point formed by hydrogen bond donors and hydrogen bond acceptors through hydrogen bonding.They can be simply prepared with cheap and non-toxic natural components.Similar to ILs,they have good physicochemical properties.Therefore,DESs can be used as novel solvents to solve the key problems of lower enzyme activity and poor substrate solubility in the field of biocatalysis.It is promising that using DESs can obtain more active aglycones in the hydrolysis of flavonoid glycosides.This study carried out a series of experiments on the activity and stability of?-glycosidase in DESs.Meanwhile,due to their unique solvents properties,DESs were used as green media in the hydrolysis of flavonoid glycosides.This article mainly includes the following three aspects:?1?In this paper,nine different types of DESs were synthesized by the heating method.The tested DESs consisted of mixtures of a choline salt and various hydrogen bond donors,such as amides,organic alcohols,sugars and organic acids.The low melting point of DESs was caused by hydrogen bonds in the two components.The formation processes of the new solvents were monitored by using ¹³C-NMR,¹H-NMR,FT-IR and DSC.The interactions between the components were discussed,and the mechanisms of low melting point of the solvents were further explained.?2?DESs were investigated as the enzymatic reaction media to explore the catalytic behavior of?-glucosidase.Studies have shown that DESs can significantly improve the enzyme activity and stability.Different types of DESs have different effects on the enzymatic hydrolysis,which is mainly determined by the type of hydrogen bond donors.At the same time,we studied that the water content in the solvent system affected the activity of the enzymes.A certain amount of water can reduce the viscosity of DESs and maintain the molecular flexibility of the enzyme in DESs.In addition,the kinetic analysis further described that an increase in the affinity of the enzyme towards the substrate in the DESs system was responsible for the enhancement of the enzymatic efficiency.?3?In this paper,we initially developed a green,efficient,and rapid DESs-based microwave-assisted enzymatic hydrolysis method of preparing the flavonoid agylcones.DESs can be used as green media in the biocatalysis due to their unique properties.They can siginificantly activate the activity of the enzymes and dissolve the insoluble substrates.Response surface method was used to optimize the hydrolysis conditions,as the following:ChCl-LA?46%,v/v?,pH 4.8,5 mg/mL enzyme,2.2 mM flavonoid glycosides,microwave heating?70 W,72°C?reaction for 20 min.Under the optimized conditions,the hydrolysis rate of baicalin may reach 96.63%.In this study,DESs was used as the enzymatic reaction media to improve the activity of?-glucosidase and the solubility of flavonoids.The microwave-assisted method with DESs as solvents and?-glucosidase as a catalyst was used for the first time,and 90%-97%of the flavonoids glycoside are converted to aglycones.Therefore,this study is based on the theory of green chemistry,and provides a theoretical guidance and practical significance for the non-aqueous enzyme catalysis.It also opens up new ideas and new methods for the practical production and application.