Study Of New Methods For α-glucosidase Activity Assay And Its Inhibitors Screening

Type II diabetes mellitus, known as non insulin-dependent diabetes mellitus, is diagnosed on the basis of sustained hyperglycemia, and intensive control of blood glucose level is an effective way to halt diabetes development. It is well-known that α-glucosidase is a membrane-bound enzyme located at the epithelium of the small intestine, and the key enzyme in carbohydrate digestion. It specifically hydrolyzes the α-glucopyranoside bond, thereby releasing α-D-glucose from the non-reducing end of sugar. Hence, the inhibition of α-glucosidase may reduce intestinal glucose digestion and absorption, consequently controlling the post-prandial glycaemic response. Therefore, it is of great importance to assay α-glucosidase activity quickly and accurately and screen inhibitors from natural or synthetic compounds. Currently, the screening model at molecular level in vitro and the animal model using hyperglycemia rats are routinely employed for α-glucosidase inhibitor screening. However, for the current methods, the sensitivity and accuracy are limited. Additional, hyperglycemic animal model involves long-term animal experiments, which can’t meet the requirements of fast screening. In view of these drawbacks, two new methods have been established based on specific recognition between boronic acid derivatives and saccharides and can well detected α-glucosidase acitivity and the inhibitors at both molecular level and cellular level in vitro.1. A simple and visible colorimetric assay for α-glucosidase activity and its inhibitors screeningCurrent methods possibly give false positive results for screening of α-glucosidase inhibitors at molecular level in vitro, so a colorimetric method has been established for α-glucosidase activity assay and its inhibitors screening by using gold nanoparticles(Au NPs) owing excellent optical properties. The method is based on the specific recognition between 1,4-phenylenediboronic acid(PDBA) and 4-aminophenyl-α-D-glucopyranoside(p APG), which may induce aggregation of p APG-functionalized gold nanoparticles(Au NPs) to achieve color change of thetesting solution. The aggregation of Au NPs can be influenced by α-glucosidase since there is no coordination reactivity between PDBA and p-aminophenol, the hydrolyzed product of p APG, catalyzed by the enzyme. Therefore, a simple and easily-operated colorimetric method for the assay of α-glucosidase activity can be developed. Under the optimized experimental conditions, the ratios of absorbance at a wavelength of 650 nm to that at 520 nm vary linearly with the α-glucosidase activity within a range from 0.05 to 1.1 U/m L with a lowest detection limit of 0.004 U/m L. Moreover, using the proposed method, the inhibition effect of gallic acid and quercetin on α-glucosidase activity can be tested with IC50 values of 1.16 m M and 1.82 μM, respectively. The method overcomes the drawback of current enzyme-inhibitor model for the detection of α-glucosidase activity. Thus, with wide linear detection range, low detection limit, and high accuracy, the method not only has a great potential for the detection of α-glucosidase activity, but also for the screening of its inhibitors.2. Dual-electrochemical assay for α-glucosidase activity and the inhibitor at both molecular and cellular levels in vitroIn view of current methods with some defects, an electrochemical method is established based on competitive binding between magnetic nanoparticles(MNPs) / 4-aminophenyl-α-D-glucopyranoside(p APG) and sliver nanoparticles(Ag NPs) / dopamine(DA) with pyrene boric acid(PBA) immobilized on the surface of graphite electrode(GE) for the assay of α-glucosidase activity and the inhibitor screening at both cellular and molecular levels in vitro. Firstly, DA moiety at the outer layer of modified Ag NPs is designed to react with PBA, resulting in the formation of diphenol pyreneboronate ester on the electrode surface, so a very strong signal can be obtained. Then, substrate of the enzyme, p APG, is designed to be immobilized onto the surface of magnetic nanoparticles(MNPs). Since p APG can react with PBA as well, p APG pyreneboronate ester can be also formed. In the meantime, since p APG pyreneboronate ester has a lower dissociation constant than diphenol pyreneboronate ester, the MNPs/p APG complex can replace the Ag NPs/DA complex, i.e., the signal probe, thus the strong signal will be weakened. On the other hand, since α-glucosidasecan specifically catalyze the breakage of α-1,4 glycosidic bond, leading to the release of glucose moieties containing cis-diol group and the formation of MNPs/p AP, in the presence of the enzyme, displacement reaction induced by competitive coordination with PBA cannot be achieved. Meanwhile, the modified MNPs including MNPs/p APG and MNPs/p AP with unique magnetic separation property can eliminate interferences of other substrates derived from enzyme and cell reaction systems so as to increase the detection accuracy. More importantly, the activities of both isolated and membrane bound enzymes can be well evaluated by using this proposed method. Consequently, this method can be utilized for the assay of α-glucosidase activity and the inhibitor screening at the molecular and cellular levels in vitro.