Chair of Food Science, Institute of Nutritional Science, and Interdisciplinary Research Center , Justus Liebig University Giessen , Heinrich-Buff-Ring 26-32 , 35392 Giessen , Germany.
Anal Chem. 2018 Dec 18;90(24):14260-14268. doi: 10.1021/acs.analchem.8b03407. Epub 2018 Nov 27.
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.
高效薄层色谱（HPTLC）与酶抑制测定的联合应用，随后结合高分辨率质谱（HRMS），代表了对新天然药物、功能性食品和化妆品开发所需复杂天然样品的靶向分析。这种色谱图与酶测定的直接结合，大大扩展了对体外抑制剂特性的理解。本文首次建立了一种直接在自动图谱中估算未知抑制剂等效性的简单工作流程。例如，通过 HPTLC 与酪氨酸酶和乙酰胆碱酯酶（AChE）测定的联用，分析了来自五种不同芽孢杆菌菌株的脂肽作为次级代谢产物。显示出抑制作用的脂肽通过 HPTLC-HRMS 进行表征。在许多关于脂肽生物特性的报道中，其酶抑制特性是新的。根据参考物质和精确质量，鉴定出最强烈的抑制剂为表面活性素和伊枯草菌素 A。根据其精确质量，推测另外三个抑制剂为丰原素、伊枯草菌素 C 和表面活性素甲酯。通过与曲酸和胡椒碱（作为常见天然抑制剂的参考物质）定量比较，表面活性素和伊枯草菌素 A 的抑制活性。将计算出的等效酪氨酸酶抑制活性进行比较后发现，1μg 曲酸相当于 1.8μg 和 3.2μg 的伊枯草菌素 A 和表面活性素标准品；对于 AChE 抑制，1μg 胡椒碱相当于 1.7μg 和 0.6μg 的伊枯草菌素 A 和表面活性素标准品。进一步对样品中发现的未知酶抑制剂进行计算，作为表面活性素、伊枯草菌素 A、曲酸和胡椒碱的等效物，以评估它们的重要性。
