Fast Equivalency Estimation of Unknown Enzyme Inhibitors in Situ the Effect-Directed Fingerprint, Shown for Bacillus Lipopeptide Extracts.

The hyphenation of high-performance thin-layer chromatography (HPTLC) with enzyme inhibition assays followed by high-resolution mass spectrometry (HRMS) represents a targeted profiling of complex natural samples required in the development of new natural pharmaceuticals, functional foods, and cosmetics. This direct combination of a chromatogram with an enzymatic assay substantially extents the understanding of inhibitor properties in vitro. For the first time, a straightforward workflow was established for estimating the equivalency of unknown inhibitors directly in the autogram. Exemplarily, lipopeptides produced as secondary metabolites by five different Bacillus strains were analyzed by HPTLC hyphenated with the tyrosinase and acetylcholinesterase (AChE) assays. Lipopeptides that showed an inhibition were characterized by HPTLC-HRMS. Among the many reports about the biological properties of lipopeptides, their enzyme inhibitory properties are new. The most intense inhibitors were identified as surfactin and iturin A according to reference substances and exact masses. Three further inhibitors were supposedly assigned as fengycin, iturin C, and surfactin methyl ester according to their exact masses. The inhibitory activities of surfactin and iturin A were quantitatively compared with kojic acid and piperine, as references for common natural inhibitors. Their equivalently calculated tyrosinase inhibition showed that 1 μg kojic acid was equal to 1.8 μg and 3.2 μg of iturin A and surfactin standards, respectively; regarding to AChE inhibition, 1 μg piperine was equal to 1.7 μg and 0.6 μg of iturin A and surfactin, respectively. Further unknown enzyme inhibitors found in the sample were exemplarily calculated as surfactin, iturin A, kojic acid, and piperine equivalents to estimate their importance.