| Lycium barbarum leaves are rich in bioactive ingredients,including flavonoids.This study investigated the effect of growth ages and seasons on the flavonoids content of Lycium barbarum L.leaves(LL),and starch-utilizing enzyme inhibition effect of these flavonoids.The flavonoids with the highest bioactive activity was purified through resin and identified by LC-MS to elucidate the composition of flavonoids.And molecular modeling was used to clarify the mechanism underlying the effect of flavonoids on starch-utilization enzymes.The main results are as follows:(1)Crude flavonoids was first obtained from biennial LL harvested in August through ultrasonic extraction.The polysaccharide content in the crude extract was 5.57±0.34%,the protein 14.8310.49%,and the polyphenol 12.37±0.31%.The crude extract was then purified by AB-8 resin tandem polyamide resin columns,the purity of the resulting extract reached 73.1%,use this method to explore the next step of flavonoid content.(2)The flavonoid content in LL of different years and different seasons was determined(from 0.51±0.05 to 1.97±0.18 g/100g).Comparing and summarizing the accumulation patterns and changing characteristics of flavonoids:The content of phenolic compounds in leaves of flavonoids from leaves of Lycium barbarum was high for one to three years and the difference was not significant during the three years(P>0.05),the content decreased when it was grown to four years.The content of flavonoids in leaves is related to the growing season.In the early summer,the content was the highest(up to 1.97±0.18 g/100g),and gradually decreased in the late summer(up to 0.5 ±10.05g/100g).(3)Two starch enzymes were used to determine enzyme inhibitory activity.The IC50 value of a-amylase ranged from 0.52 to 4.23 mg/mL,and the IC50 value of a-glucoside ranged from 1.25 to 4.73 mg/mL.This shows that the LL flavonoids have strong inhibitory effects on a-amylase andα-glucosidase.BMLE had the best inhibitory effects(IC50 values of only 0.52 and 1.25 mg/mL),and the leaves of the BMLE were selected for the next study(4)By enzyme kinetics experiments,it was found that BMLE can combine with starch-utilization enzyme active sites.The inhibition type was determined as competitive inhibition and Ki was 9.19 mg/mL and 11.77 mg/mL.This shows that BMLE has high affinity with a-amylase.This is consistent with the inhibitory effect of BMLE on a-amylase stronger than that of a-glucosidase.(5)BMLE identified the flavonoid composition of LL by LC-MS.The identification results were 11 flavonoids,among which there were 3 flavonoid glycosides(rutin,naringin,quercitrin)and 8 flavonoid aglycones(catechins,isorhamnetin,kaempferol,luteolin,morin,myricetin,naringenin,quercetin).Eleven phenolic acids were also detected in the extract,including 4-hydroxycinnamic acid,caffeic acid,chlorogenic acid,gallic acid,gentisic acid,poly 4-hydroxybenzoic acid,rosmarinic acid,sinapic acid,syringic acid Vanillic acid,p-hydroxybenzoic acid.(6)Molecular simulations determine the binding energy of flavonoids on two starch-utilizing enzymes.The binding energies of 11 kinds of flavonoids and two kinds of starch inhibiting enzymes were found to be close to the binding energy of acarbose(-8.1 kcal/mol and-9.6 kcal/mol),indicating that the inhibitory effect was similar to that of acarbose.At the same time,it was observed in the molecular simulation effect diagram that most of the flavonoids can bind stably at the active site of the enzymeIn summary,the content of flavonoids in LL is related to the growth year and month.Based on the content of flavonoids and the inhibitory effect of enzyme activity,the best harvest time of loquat leaves was determined in early summer.This study also showed that wolfberry leaves may achieve hypoglycemic effects by inhibiting the mechanisms of alpha-amylase and alpha-glucosidase activity Therefore,the Lycium barbarum leaves can be used as a good flavonoid resource to develop and provide more effective ways to control blood sugar in diabetic patients. |
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