| The extract of Lycium barbarum bud tea was prepared by hot water extraction,their basic chemical contents were determined,and compositions of free amino acids as well as soluble sugars in Lycium barbarum bud tea infusion were analyzed by HPLC.The crude extract(CE,hot water extract)of Lycium barbarum bud tea and its four fractions of chloroform,ethyl acetate,butanol and water were prepared by fractional extraction.CE and its four fractions were detected by HPLC,and the main polyphenols of Lycium barbarum bud tea infusion were investigated by HPLC and HPLC-MS.The total polyphenols contents,antioxidant activities in vitro of CE and its four fractions were determined by Folin-Ciocalteu method and DPPH assay,TEAC assay,FRAP assay,assay for superoxide anion,total reducing power and PC 12 cell model,respectively.Furthermore,the inhibitory effects and types of CE and its four fractions on ?-glucosidase were investigated.The main findings are as following:1.Chemical compositions of Lycium barbarum bud tea infusionLycium barbarum bud tea infusion was prepared by hot water extraction(ratio of solid to liquid 1:40,temperature 96 ?,extraction time 30 min).Their chemical contents were determined,the main free amino acids and soluble sugars in Lycium barbarum bud and leaf teas were analyzed by HPLC.The chemical compositions and contents were determined as follows:6.32%polyphenol,1.88%flavonoids,4.59%carbohydrate,1.53%protein,1.89%free amino acids and catechins were not detected.The free amino acids and soluble sugars were analyzed with HPLC-DAD and ELSD-HPLC method,respectively.The results showed that Asp(3230.41 ?g/g),Asn(3754.77 ?g/g),His(5585.26 ?g/g)and Leu(4376.09?g/g)were relatively abundant in Lycium barbarum bud tea infusion.Meanwhile,the contents of fructose and sucrose were 31.44 and 10.99 mg/g,respectively,while none glucose was detected.2.Preparation and the antioxidant activities of CE and its four fractionsCE of Lycium barbarum bud tea was fractionated through successive partition to afford four fractions and the polyphenols contents were determined.Afterwards,the antioxidant activities of CE and its fractions were evaluated by assays for DPPH,TEAC,FRAP,superoxide and total reducing power.The polyphenols contents and antioxidant activities were all found decreasing in the order of n-butanol fraction(nBF,35.64%)>ethyl acetate fraction(EaF,16.24%)>crude extract(CE,14.72%)>chloroform fraction(CfF,13.99%)>water fraction(WtF,7.89%).All the results of the five antioxidant assays regarding to the antioxidant abilities exhibited the same sequence as polyphenol content,nBF exerted outstanding antioxidant activities according to the relatively lowest IC50 value.Furthermore,CE and all its fractions showed a certain degree of protection on PC 12 cell against the oxidative damage induced by H2O2,and nBF exerted better protective effect comparaed with CE and other three fractions.3.Polyphenol components in Lycium barbarum bud teaCE was purified with AB-8 macroporous resin to afford AB-8 sample,CE and its four samples together with several standards were then analyzed by HPLC.The results showed that all the chromatographic peaks obtained from CE,its four fractions and AB-8 sample were consistent,indicating that the polyphenol components in all the Lycium barbarum bud tea samples detected were the same.3-Caffeoylquinic acid,5-caffeoylquinic acid and lutin were identified as main polyphenol components of Lycium barbarum bud tea according the retention time of the standards and the corresponding molecular weight information obtained from analysis of AB-8 purified sample by HPLC-MS.4.Inhibitory effect of CE and its four fractions on ?-glucosidaseThe inhibitory effects of CE and its four fractions were evaluated by enzyme kinetics experiment,the results showed that all the samples detected exerted more powerful inhibitory effect than positive control acarbose.nBF exerted outstanding inhibitory effect according to the relatively lowest IC50 value of 48.9 ?g/mL while the values of EaF,CE and CfF were 344.2,381.3 and 478.2 ?g/mL respectively,WtF exerted the weakest inhibitory effect on ?-glucosidase with the largest IC50 value of 871.2 ?g/mL.Furthermore,the concrete inhibition type was studied with enzyme kinetics experiment by changing the concentration of enzyme and substrate,successively.The results exhibited that the inhibition type of all the samples detected were reversible noncompetitive inhibition. |
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