Enzymatic Synthesis Of Astragalin And Its Bioactivity Evaluation

Astragalin is a type of flavonoid compound that possesses various pharmacological activities,such as antioxidant,antibacterial,anti-inflammatory,anti-tumor,antiviral,lipid-lowering,and anti-angiogenic effects.Therefore,it has extensive applications in the fields of medicine,health products,and cosmetics.With the increasing awareness of health and the growing aging population,the demand for drugs in the fields of cancer prevention,immune regulation,and anti-inflammation is gradually increasing.As a natural medicine with good pharmacological activity and application prospects,astragalin has received more and more attention.Currently,the market size of astragalin is relatively small,but with further research and the expansion of application areas,its market demand is expected to gradually increase.In this study,kaempferol was used as a substrate and converted to astragalin through the catalysis of the glycosyltransferase FGT,along with UDP-glucose.However,the high cost of UDP-glucose resulted in a high reaction cost.To address this issue,the study used inexpensive sucrose and a small amount of UDP to replace UDP-glucose.By introducing the sucrose synthase Gm SUS and sucrose,a UDP recycling system was established,which reduced the reaction cost.Subsequently,the reaction product was identified as astragalin through product analysis,and its antioxidant activity was evaluated.The specific details are as follows:（1）Kaempferol was used as the substrate and converted into astragalin by the catalysis of the glycosyltransferase FGT with UDP-glucose.The FGT enzyme was expressed and purified using a GST tag column.The p H,temperature,and DMSO content were investigated for their effects on FGT enzyme activity,and the maximum enzyme activity was achieved at p H 9.5,40°C,and 10%DMSO.Additionally,to verify the feasibility of cascading sucrose synthase with FGT for the biosynthesis of astragalin,the effects of different concentrations of sucrose,fructose,and UDP on FGT enzyme activity were explored.（2）Construct the cascade reaction of glycosyltransferase FGT and sucrose synthase Gm SUS to synthesize ascarin.The sucrose synthase Gm SUS was expressed and purified using a GST-labeled column.The influence of different factors on the cascade reaction was explored.By changing the enzyme activity ratio of FGT and Gm SUS,the most suitable enzyme amount for the reaction was determined.The final enzyme activities of FGT and Gm SUS to be added were 36.8 m U/m L and 1660 m U/m L,respectively.When the p H is 8.0,the reaction temperature is 35°C,the volume content of DMSO is 10%,the concentration of sucrose is 200 m M,the concentration of UDP is 0.1 m M,and the concentration of kaempferol is 1.0 m M,the production of astragalin reaches the maximum.（3）The yield of ascarin was increased using fed-batch operation.In order to avoid the inhibition of sucrose synthase by high concentration of kaempferol,asifinin was synthesized by fed-batch operation.During the reaction,kaempferol was added three times and fresh enzyme solution was added once.Within 6 hours,the production of ascarinin reached 1550.1 mg/L,the conversion rate was 86.5%,and UDP cycled 34.8times.（4）Separation,purification and detection of astragalus glycosides.First,the reaction product was purified,and 16.2 mg of light yellow solid powder was obtained by using an LH-20 Sephadex column.Then,it was verified by using HPLC,LC-MS,H-NMR and C-NMR.High performance liquid chromatography showed that the peak position of the reaction product was around 4.8 min,which was consistent with the peak time of the standard product of ascarin.Liquid chromatography-mass chromatography showed that the molecular weight of the purified product was 448,which was the same as the theoretical value.The integration results of ¹H-NMR and ¹³C-NMR spectra are consistent with those recorded in the literature.Therefore,it can be confirmed that the reaction product is ascarin,and its purity is 94.91%.（5）In vitro antioxidant activity evaluation of astragalus glycosides.Four antioxidant activity experiments were used in this study,including ABTS free radical scavenging activity,DPPH free radical scavenging activity,superoxide anion free radical scavenging activity and hydroxyl free radical scavenging activity.The results showed that the antioxidant activity of kaempferol was significantly higher than that of ascarin in all experiments.Overall,these results provide an important reference for further research on the pharmacological effects of ascarin.