Radix isatidis has been widely used as a Chinese traditional medicine for its antivirus and anticancer activities where the minor components may contribute to these beneficial pharmaceutical effects. In order to enrich the target minor compounds effectively and rapidly, extraction, medium-pressure liquid chromatography (MPLC), high-speed countercurrent chromatography (HSCCC), and preparative high-performance liquid chromatography (pre-HPLC) were integratively used for separation and purification of two target minor compounds indole-3-acetonitrile-6-O-β-D-glucopyranoside (target 1) and clemastanin B (target 2) in the present study. Radix isatidis was dried, pulverized, and extracted with 50% methanol at room temperature, then concentrated, and subjected to pretreatment with D-101 macroporous resin chromatography and extraction by MPLC. The first target compound was separated by MPLC at the purity raised to 70–80%, but without the second minor compounds which were irreversibly adsorbed by C18 solid support. Therefore, the second target compound in the crude extract was directly separated by HSCCC at a purity of 80–90%. Finally, these refined samples were further separated by pre-HPLC to obtain a high purity at 98–99%. The chemical structure identification of each target compound was carried out by infrared radiation (IR), electrospray ionization mass spectrometry (ESI-MS), and 1H-NMR.

中文翻译：

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集成 MPLC 和 HSCCC 与制备型 HPLC 分离纯化板蓝根中的两种微量化合物

板蓝根因其抗病毒和抗癌活性而被广泛用作中药，其中微量成分可能有助于这些有益的药效。为了有效、快速地富集目标微量化合物，将萃取、中压液相色谱 (MPLC)、高速逆流色谱 (HSCCC) 和制备型高效液相色谱 (pre-HPLC) 相结合进行分离和分离。在本研究中纯化两种目标微量化合物 indole-3-acetonitrile-6-O-β-D-glucopyranoside（目标 1）和 clemastanin B（目标 2）。板蓝根干燥粉碎，室温下用50%甲醇提取，浓缩，D-101大孔树脂层析预处理，MPLC提取。第一个目标化合物通过 MPLC 分离，纯度提高到 70-80%，但没有被 C18 固体支持物不可逆吸附的第二个次要化合物。因此，粗提取物中的第二个目标化合物直接通过 HSCCC 分离，纯度为 80-90%。最后，这些精制样品通过前级 HPLC 进一步分离，以获得 98-99% 的高纯度。通过红外辐射(IR)、电喷雾电离质谱(ESI-MS)和1H-NMR对各目标化合物进行化学结构鉴定。这些精制样品通过预 HPLC 进一步分离，以获得 98-99% 的高纯度。通过红外辐射(IR)、电喷雾电离质谱(ESI-MS)和1H-NMR对各目标化合物进行化学结构鉴定。这些精制样品通过前级 HPLC 进一步分离，以获得 98-99% 的高纯度。通过红外辐射(IR)、电喷雾电离质谱(ESI-MS)和1H-NMR对各目标化合物进行化学结构鉴定。