Detection of α-amylase inhibitors using thin-layer chromatography

Abstract

There are many natural compounds in the treatment of type 2 diabetes mellitus, and the nature of most of them has not yet been studied. Because the diversity and sources of natural compounds are large. Also, their examination requires a lot of time and expense. We carried out experiments on endophytic fungi Penicillium brevicaule alba-CC200 and Aspergillus egypticus-HT166S, isolated from local plants Celosia cristata and Helianthus tuberosus. Secondary metabolites of both strains were separated into fractions using polar and non-polar solvents. Eluent systems were optimized to ensure complete separation of metabolites using thin-layer chromatography (TLC). The optimal separation of secondary metabolites was provided by an eluent system with a ratio of benzene:methanol (5:1). In particular, the methanol fraction of P. brevicaule alba-CC200 yielded 6 Rf values (0.83; 0.74; 0.59; 0.57; 0.49; 0.45) and A. egypticus-HT166S yielded 12 Rf values (0.97; 0.86; 0.74; 0.60; 0.54; 0.52; 0.48; 0.44; 0.37; 0.30; 0.22; 0.13). The spots on the chromatographic plates were then incubated with the enzyme and the plates were placed in a starch medium. The α-amylase inhibitory activity of the substances on the TLC plate was determined by washing with iodine. Qualitative reactions were performed to study the nature of the blue spots on the surface of the plates by isolating the Rf values. It was found that saponins were present in P. brevicaule alba-CC200 and flavonoids in A. egypticus-HT166S. This method is very simple, requires little time and expense. At the same time, it allows for rapid and visual screening of the inhibitory activity of many natural compounds.