Glavnik Vesna, Vovk Irena
Department of Food Chemistry, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia.
Plants (Basel). 2020 Dec 11;9(12):1753. doi: 10.3390/plants9121753.
Anthraquinones (yellow dyes) were extracted from Japanese knotweed rhizomes with twelve extraction solvents (water; ethanol (20%, 40%, 60%, 70% and 80%), ethanol, 70% methanol, methanol, 70% acetone, acetone and dichloromethane). The obtained sample test solutions (STSs) were analyzed using high-performance thin-layer chromatography (HPTLC) coupled to densitometry and mass spectrometry (HPTLC-MS/MS) on HPTLC silica gel plates. Identical qualitative densitometric profiles (with anthraquinone aglycones and glycosylated anthraquinones) were obtained for STSs in all the solvents except for the STS in dichloromethane, which enabled the most selective extractions of anthraquinone aglycones emodin and physcion. The highest extraction efficiency, evaluated by comparison of the total peak areas in the densitograms of all STSs scanned at 442 nm, was achieved for 70% acetone. In STS prepared with 70% acetone, the separation of non-glycosylated and glycosylated anthraquinones was achieved with developing solvents toluene-acetone-formic acid (6:6:1, 3:6:1 and 3:3:1 /) and dichloromethane-acetone-formic acid (1:1:0.1, /). Non-glycosylated anthraquinones were separated only with toluene-acetone-formic acid, among which the best resolution between emodin and physcion gave the ratio 6:6:1 (/). This solvent and dichloromethane-acetone-formic acid (1:1:0.1, /) enabled the best separation of glycosylated anthraquinones. Four HPTLC-MS/MS methods enabled the identification of emodin and tentative identification of its three glycosylated analogs (emodin-8--hexoside, emodin--acetyl-hexoside and emodin--malonyl-hexoside), while only the HPTLC-MS/MS method with toluene-acetone-formic acid (6:6:1, /) enabled the identification of physcion. Changes of the shapes and the absorption maxima (bathochromic shifts) in the absorption spectra after post-chromatographic derivatization provided additional proof for the detection of physcion and rejection of the presence of chrysophanol in STS.
采用十二种提取溶剂（水；乙醇（20%、40%、60%、70%和80%）、乙醇、70%甲醇、甲醇、70%丙酮、丙酮和二氯甲烷）从虎杖根茎中提取蒽醌（黄色染料）。使用高效薄层色谱（HPTLC）结合密度测定法和质谱法（HPTLC-MS/MS）在HPTLC硅胶板上对所得样品测试溶液（STSs）进行分析。除二氯甲烷中的STSs外，所有溶剂中的STSs均获得了相同的定性密度测定图谱（含有蒽醌苷元和糖基化蒽醌），二氯甲烷中的STSs能够最有选择性地提取蒽醌苷元大黄素和大黄素甲醚。通过比较在442 nm处扫描的所有STSs密度图中的总峰面积来评估，70%丙酮的提取效率最高。在用70%丙酮制备的STSs中，使用展开剂甲苯 - 丙酮 - 甲酸（6:6:1、3:6:1和3:3:1 /）和二氯甲烷 - 丙酮 - 甲酸（1:1:0.1、/）实现了非糖基化和糖基化蒽醌的分离。非糖基化蒽醌仅用甲苯 - 丙酮 - 甲酸分离，其中大黄素和大黄素甲醚之间最佳分离比例为6:6:1（/）。这种溶剂和二氯甲烷 - 丙酮 - 甲酸（1:1:0.1、/）能够实现糖基化蒽醌的最佳分离。四种HPTLC-MS/MS方法能够鉴定大黄素并初步鉴定其三种糖基化类似物（大黄素 - 8 - 己糖苷、大黄素 - 乙酰己糖苷和大黄素 - 丙二酰己糖苷），而只有采用甲苯 - 丙酮 - 甲酸（6:6:1、/）的HPTLC-MS/MS方法能够鉴定大黄素甲醚。色谱后衍生化后吸收光谱的形状和吸收最大值的变化（红移）为大黄素甲醚的检测以及STSs中大黄酚不存在的排除提供了额外证据。
