| Broad Bean(Vicia faba L.)contains considerable amounts of polyphenols,which display superb antioxidant capacity and possess the potential to be exploited as function food ingredients.The aim of this work was to optimize the ultrasound-assisted extraction parameters of polyphenols from five typical broad beans cultivated by Jiangsu Academy of Agricultural Science,as well as their composition and physiological effects such as a-glucosidase inhibitory activity,bile salt binding capacity,xanthine oxidase inhibitory activity,etc.The main results of this word were addressed below.1.Polyphenols were extracted from broad bean with the assist of ultrasound technique.The effect of extraction parameters,including ethanol content,solvent/material ratio,extraction time and pH on the yield of polyphenols was investigated and further optimized by response surface methodology.The optimal extraction parameters were as follows:ethanol content 45%,solvent/material ratio 1:25(g/mL),extraction time 20 min and extraction pH 5.A polyphenol yield of 2.03 ±0.16 mg/g d.w.was obtained under the optimized condition.2.The content of polyphenol,flavonoids and procyanidins in broad beans were determined by Folin-Ciocalteu,aluminum chloride and vanillin-hydrochloric acid mehods,respectively.The free phenol content,bound phenol content and total phenol content(TPC)in five broad beans were in the range of 183.4-203.1,34.7-81.1 and 232.8-269.3 mg GAE/100 g DW,respectivley.The TPC of Sucan Miandouxuan was the highest,followed by Wanwuhe Daqingpi,Sucan Ganlinxuan,Sucan No.3 and Sucan 05-1,respectivley.The content of free flavonoids,bound flavonoids and total flavonoids(TFC)were in the range of 90.8-117.8,44.6-71.2 and 137.9-179.2 mg RE/100 g DW,respectively.The TFC in Sucan No.3 was the much higher than that in other four varieties.The content of free procyanidins,bound procyanidins and total procyanidins were in the range of 76.1-191.5,45.1-135.7 and 125.6-297.5 mg CE/100 g DW,respectively.The total procyanidins content,ranging from highest to lowest,was Sucan Miandouxuan,Sucan Ganlinxuan,Wanwuhe Daqingpi,Sucan 05-1 and Sucan No.3.There were significant differences(p<0.05)in the content of polyphenol,flavonoids and procyanidins among five broad beans.Besides,polyphenols,flavonoids and procyanidins in broad beans were predominately presented in free form rather than bound with biomacromolecules such as protein and polysaccharide.3.Five polyphenol monomers,gallic acid,catechin gallate,epicatechin glucoside,feruloyl-glucoside and caffeic acid 4-glucoside salidroside were identified in broad bean with HPLC-MS/MS technique.In addition,the antioxidant activities of free and bound polyphenols were evaluated by a combination of chemical methods(such as DPPH,ABTS,ORAC,FRAP,reducing force.etc)and HepG2 cell model.Results indicated that the free radical-scavenging activity of broad beans were positively correlated with the polyphenols content and the free polyphenols displayed significantly higher antioxidant activity than the bound ones,the results showed that the antioxidant activity(FRAP?reducing force?DPPH?ABTS)was highly correlated writh the content of polyphenol.There were significant differences in the antioxidant activity among polyphenols from five broad beans(P<0.05).On the other hand,both free and bound polyphenols could efficiently protect the HepG2 cell against the oxidative damage caused by H2O2 and the protective effect was dose-dependent.4.Polyphenols from five kinds of broad beans could significantly inhibit the activity of a-glucosidase.The IC50 values of free and bound polyphenols from 5 broad beans were in the range of 0.87-1.44 and 1.17-2.07 mg/mL,respectively,which were comparable to that of acarbose(1.04 mg/mL).This result suggested that broad bean polyphenols held the promise as blood glucose-decreasing ingredients.Besides,the inhibition of a-glucosidase by acarbose and free and bound polyphenols from Sucan 05-1 was a reversible reaction with remaining enzyme activity.From the results of Lineweaver-Burk plot,the a-glucosidase inhibition by acarbose was characterized to be a competitive process with an inhibition constant(Ki)of 1.64 mg/mL.By contrast,both the free and bound polyphenols from Sucan 05-1 displayed a noncompetitive inhibition,and the Ki were calculated to be 1.28 and 2.03 mg/mL,respectively.5.The binding capacity of free and bound polyphenols with bile salts were investigated by spectrophotometry.Results showed that the binding capacity of free polyphenols with sodium taurocholate,sodium glycolate and sodium cholate were in the range of 0.78-1.09,0.67-0.70 and 1.10-1.22 ?mol,respectively,while these of bound polyphenols were in the range of 0.98-1.20,0.67-0.92 and 0.89-1.06 ?mol,respectively.There were significant differences(p<0.05)in bile salt-binding ability among polyphenols from five broad beans and polyohenols from Sucan No.3 and Sucan Ganlinxuan exhibited much higher bile salt-binding capacity than any others.6.The xanthine oxidase-inhibitory activity of polyphenols from broad beans was investigated utilizing xanthine as a substrate.Results showed that more than 90%of the enzymatic activity of xanthine oxidase maintained in the presence of free polyphenols from broad beans.On the contrary,the inhibition rate of xanthine oxidase by bound ones on was generally higher than 30%.Polyphenols from Wanwuhe Daqingpi showed the highest xanthine oxidase-inhibitory activity,followe by Sucan Miandouxuan,Sucan 05-1,Sucan Ganlinxuan and Sucan No.3,respectively.In addition,there were significant differences(p<0.05)in xanthine oxidase-inhibitory activity among polyphenols from five broad beans. |
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