| Flavonoids are a class of secondary metabolites of plants,widely distributed in the various plants,have been receiving more and more attentions,recently,owing to their versatile bioactivities.Therefore,surface plasmon resonance?SPR?-based real-time interaction analysis methods were implemented to explore the binding between three Citrus flavonoids?hesperidin,neohesperidin,naringin?and?-amylase/starch in detail,respectively.The effects of pH,NaCl and CaCl2 on the binding affinity between flavonoids and?-amylase were also examined.The structure-activity relationship between flavonoids-?-amylase or flavonoids-starch and the effect of citrus flavonoids on the digestion of starch were discussed.The main study contents were summarized as followed:?The real-time interactions between three Citrus flavonoids?naringin,neohesperidin,hesperidin?and?-amylase were investigated with acacoside and atrazine as the contrast by SPR sensor,and the real-time kinetic curve and the kinetic constants was obtained.The effects of extrinsic factors,including pH and salt concentration,on the binding affinity of three Citrus flavonoids with?-amylase were also examined by SPR.These results indicate that acarbose,naringin,neohesperidin,hesperidin and atrazine can all bind with?-amylase to form a complex.The binding ability between three Citrus flavonoids was acarbose>naringin>neohesperidin>hesperidin>atrazine.The KD values was 0.78±0.12 mM,2.27±0.18 mM,3.09±0.20 mM,3.51±0.09 mM and 12.65±0.13 mM,respectively,which suggests that the binding of three Citrus flavonoids to?-amylase depends strongly on both the number and position of hydroxyl group.In addition,binding affinities of three Citrus flavonoids to?-amylase reached the highest at pH 6,and can be reinforced with 0.2 M NaCl and 0.1 M CaCl2.?Firstly,the inhibition of three Citrus flavonoids?naringin,neohesperidin,hesperidin?on?-amylase activity was studied by using the starch-iodine complex spectrum experience,and a reasonable inhibiting mode was proposed by using Lineweaver-Burk plots.Secondly,the real-time interaction kinetics of the three Citrus flavonoids with starch was investigated in detail using SPR sensor.Thirdly,the effect of three Citrus flavonoids on the starch retrogradation were examined during the interaction with starch based on scanning electron microscopy?SEM?.The results shows that three Citrus flavonoids can dose-inhibit?-amylase,when the concentrations of Citrus flavonoids were increased,the?-amylase activities were decreased,the ability of inhibition is naringin>neohesperidin>hesperidin.The inhibitory modes of naringin,neohesperidin and hesperidin toward?-amylase are non-competitive type.SPR assay showed three Citrus flavonoids can all bind with starch to form a complex,with the order of naringin>neohesperidin>hespeyeridin,and with KD values of respectively3.98±0.20 mM,4.92±0.15 mM,and 5.07±0.17 mM.These results suggest that the binding ability was mainly affected by the position of hydroxyl group.In,addition,SEM results show that three Citrus flavonoids can retard the retrogradation of starch.Therefore,we speculate that the effect of three Citrus flavonoids on the digestion of starch is not only due to the inhibition of?-amylase but also through binding with it.?The evolution of antioxidant activities of three Citrus flavonoids were also investigated during the interaction with?-amylase/starch,based on the 1,1-diphenyl-2-picryl hydrazyl?DPPH?and superoxide anion radical assay.The results showed that inhibition effects of?-amylase and starch on three Citrus flavonoids scavenging DPPH and superoxide anion is different,and the order of inhibition was naringin>neohesperidin>hespeyeridin.The inhibition rate of?-amylase and starch on three Citrus flavonoids scavenging DPPH was 31.10%?23.12%?22.07%?17.13%?9.7%?10.53%,respectively,while on scavening superoxide anion was 25.41%?18.57%?18.24%?13.08%?5.20%?5.67%.The order of the inhibition percentage of antioxidant activity is the same as that of the binding affinity,when the three Citrus flavonoids bind with?-amylase/starch.It was concluded that some of the hydroxyl groups related to antioxidant activities of three Citrus flavonoids based on the structure of them may have been masked on binding with?-amylase/starch,causing the decreased antioxidant activity of three Citrus flavonoids. |
PDF Full Text Request