Screening for α-Amylase Inhibitors in Plants

Screening of plant material for α-amylase inhibitors was carried out in a microtitre plate according to Xiao et al (2006) based on the starch-iodine test [23 (link)]. The total assay mixture composed of 40 μl 0.02 M sodium phosphate buffer (pH 6.9 containing 6 mM sodium chloride), 0.02 units of PPA solution and plant extracts at concentration from 0.1-1.5 mgml^-1 (w/v) were incubated at 37°C for 10 min. Then soluble starch (1%, w/v) was added to each reaction well and incubated at 37°C for 15 min. 1 M HCl (20 μl) was added to stop the enzymatic reaction, followed by the addition of 100 μl of iodine reagent (5 mM I₂ and 5 mM KI). The colour change was noted and the absorbance was read at 620 nm on a microplate reader. The control reaction representing 100% enzyme activity did not contain any plant extract. To eliminate the absorbance produced by plant extract, appropriate extract controls without the enzyme were also included. The known PPA inhibitor, acarbose, was used a positive control at a concentration range of 6.5 - 32.8 μgml^-1. A dark-blue colour indicates the presence of starch; a yellow colour indicates the absence of starch while a brownish colour indicates partially degraded starch in the reaction mixture. In the presence of inhibitors from the extracts the starch added to the enzyme assay mixture is not degraded and gives a dark-blue colour complex whereas no colour complex is developed in the absence of the inhibitor, indicating that starch is completely hydrolysed by α-amylase.