Innovation Centre of the Faculty of Chemistry Ltd., Studentski Trg 12-16, Belgrade 11158, Serbia.
University of Belgrade - Faculty of Chemistry, Studentski Trg 12-16, Belgrade 11158, Serbia.
J Chromatogr A. 2023 Aug 16;1703:464082. doi: 10.1016/j.chroma.2023.464082. Epub 2023 May 25.
High-Performance Thin-Layer Chromatography (HPTLC)-radical scavenging capacity (RSC) assays are standard techniques for the separation and identification of antioxidants from complex mixtures. HPTLC coupled with DPPH visualization of chromatograms allows for the detection of individual antioxidants. However, other HPTLC-RSC assays that recognize compounds exhibiting different mechanisms of radical-scavenging activity are rarely reported. In this study, we developed an integrated approach that combines five HPTLC-RSC assays, principal component analysis (PCA) and quantum chemical calculations to assess the antioxidant capacity of Sempervivum tectorum L. leaf extracts. Two HPTLC assays - potassium hexacyanoferrate(III) total reducing power assay (TRP) and total antioxidant capacity by phosphomolybdenum method (TAC) - were developed for the first time. The method supports a more in-depth study of the RSC of natural products, as it compares the radical scavenging fingerprints of S. tectorum leaf extracts and recognizes differences in their individual bioactive constituents. Kaempferol, kaempferol 3-O-glucoside, quercetin 3-O-glucoside, caffeic acid, and gallic acid were identified as the compounds that discriminate HPTLC-RSC assays according to their mechanism of action and capture the similarities between 20 S. tectorum samples. Additionally, DFT calculations on M06-2X/6-31+G(d,p) level were applied to map thermodynamic feasibility of hydrogen atom transfer (HAT) and single electron transfer (SET) mechanisms of the identified compounds. Based on experimental and theoretical results, a combination of HPTLC-ABTS and HPTLC-TAC assays were proposed as the optimal method for mapping the antioxidants from S. tectorum. This study represents a step forward in identifying and quantifying individual antioxidants from complex food and natural product matrices in a more rational manner.
高效薄层色谱法（HPTLC）-自由基清除能力（RSC）测定法是分离和鉴定复杂混合物中抗氧化剂的标准技术。HPTLC 与 DPPH 图谱可视化相结合，可检测单个抗氧化剂。然而，很少有报道其他识别具有不同自由基清除活性机制的化合物的 HPTLC-RSC 测定法。在这项研究中，我们开发了一种综合方法，该方法结合了五种 HPTLC-RSC 测定法、主成分分析（PCA）和量子化学计算，以评估景天属植物叶提取物的抗氧化能力。首次开发了两种 HPTLC 测定法 - 六氰合铁(III)酸钾总还原能力测定法（TRP）和磷钼酸盐法总抗氧化能力测定法（TAC）。该方法支持更深入地研究天然产物的 RSC，因为它比较了景天属植物叶提取物的自由基清除指纹，并识别出它们各自生物活性成分的差异。根据作用机制和对 20 个景天属植物样本的相似性，鉴定出山奈酚、山奈酚 3-O-葡萄糖苷、槲皮素 3-O-葡萄糖苷、咖啡酸和没食子酸是区分 HPTLC-RSC 测定法的化合物。此外，还应用 M06-2X/6-31+G(d,p)水平的 DFT 计算来映射所鉴定化合物的氢原子转移（HAT）和单电子转移（SET）机制的热力学可行性。基于实验和理论结果，提出了 HPTLC-ABTS 和 HPTLC-TAC 测定法的组合作为从景天属植物中映射抗氧化剂的最佳方法。本研究代表了在更合理的方式下从复杂的食品和天然产物基质中鉴定和定量单个抗氧化剂的一个进步。
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