Studies On The Phaseolus Vulgaris Alpha-Amylase Inhibitor: Potential For Nutraceutical Application

The kidney bean(Phaseolus vulgaris)α-amylase inhibitor isoform 1(α-AI1) starch blockers is a potential widely used remedy against obesity and diabetes. Consumption of theα-amylase inhibitor causes marginal intraluminalα-amylase activity facilitated by the inhibitor's appropriate structural,physico-chemical and functional properties.As a result there is decreased postprandial plasma hyperglycaemia and insulin levels,increased resistance of starch to digestion and increased activity of colorectal bacteria.The efficacy and safety of the amylase inhibitor extracts,however,depend on the processing and extraction techniques used.The extracts are potential ingredients in foods for increased carbohydrate tolerance in diabetics,decreased energy intake for reducing obesity and for increased resistant starch.A crude extraction procedure involving water extraction,pH fractionation,and alcohol precipitation(75%) was used to select an appropriate variety for inhibitor extraction.Speckled kidney beans had the highestα-amylase inhibitory activity followed by red while the black kidney beans had the lowest inhibitory activity.The resultant SDS-PAGE pattern for the crude extract in the present research reduced the number of component proteins to two major band areas.It gave 1.4-2.6 grams of the crude speckled kidney bean P.vulgarisα-amylase inhibitor with an average specific hemagglutinating activity of 14222.2 U/mg protein.For chromatographic purification DEAE-Sepharose CL-6B and Sephacryl S-200 chromatography were used.The inhibitor was purified to electrophoretic purity without detectable hemagglutinating activity at 0.09%w/w of original seed flour and an increase in inhibitor specific activity from 0.32 to 80.78 units/mg protein.The inhibitor showed two fused broad bands in the region 14400-21000 Da on SDS-PAGE and a single band on Native-PAGE gels.The inhibitor had an IC50 value of 0.0098 mg/ml and reached maximum activity after 15-20 minutes of reaction. The inhibitor showed activity in the range 3.5-7.5 with a sharp increase in activity after pH 4.0 to a maximum at 5.8.The optimum conditions for stability were pH 5.5,time 16 minutes,and temperature 74.23℃.The starch blocking heat stability of speckled kidney beans(Phaseolus vulgaris)α-amylase inhibitor(α-AI1) for appropriate before-heat-treatment application as a nutraceutical additive against diabetes and obesity was assessed.The interactive effect of pH(A),Temperature(B) and Time(C) on residual inhibitory activity was modeled using Response surface methodology with the Box-Behnken design as; Re sidual Inhibitory Activity(%)=0.39278 A²C-1.115367 A²-0.070961 B²-0.27061 C² -0.064474 B+3.96313 AC-8.87121×10^-3BC+23.76840 A+8.21374 B-1.22720 C -251.48409Intrinsic fluorescence and ANS-assisted surface hydrophobicity indicated activity loss is accompanied tertiary structural unfolding.Chaotropic salts at high(1.0 M) and kosmotropic salts at low(0.1-0.01 M) concentration stabilized the inhibitor in the order CH₃COO^-＞Cl^-＞Br^-＞I^-＞SCN^- and vice-versa respectively.Differential scanning calorimetry showed the inhibitor denaturation temperature as 84.1℃,86.7℃and 87.6℃for onset(To),peak(Tp),and end(Tc) transition temperatures,respectively.