Cell membrane chromatography (CMC) has been successfully applied to screen bioactive compounds from Chinese herbs for many years, and some offline and online two-dimensional (2D) CMC-high performance liquid chromatography (HPLC) hyphenated systems have been established to perform screening assays. However, the requirement of sample preparation steps for the second-dimensional analysis in offline systems and the need for an interface device and technical expertise in the online system limit their extensive use. In the present study, an offline 2D CMC-HPLC analysis combined with the XCMS (various forms of chromatography coupled to mass spectrometry) Online statistical tool for data processing was established. First, our previously reported online 2D screening system was used to analyze three Chinese herbs that were reported to have potential anti-inflammatory effects, and two binding components were identified. By contrast, the proposed offline 2D screening method with XCMS Online analysis was applied, and three more ingredients were discovered in addition to the two compounds revealed by the online system. Then, cross-validation of the three compounds was performed, and they were confirmed to be included in the online data as well, but were not identified there because of their low concentrations and lack of credible statistical approaches. Last, pharmacological experiments showed that these five ingredients could inhibit IL-6 release and IL-6 gene expression on LPS-induced RAW cells in a dose-dependent manner. Compared with previous 2D CMC screening systems, this newly developed offline 2D method needs no sample preparation steps for the second-dimensional analysis, and it is sensitive, efficient, and convenient. It will be applicable in identifying active components from Chinese herbs and practical in discovery of lead compounds derived from herbs.

细胞膜色谱（CMC）已成功应用于筛选中草药中的生物活性化合物，并且已经建立了一些离线和在线二维（2D）CMC高效液相色谱（HPLC）联用系统来进行筛选测定。但是，离线系统中用于二维分析的样品制备步骤的要求以及在线系统中对接口设备和技术专业知识的需求限制了它们的广泛使用。在本研究中，建立了离线XD CMC-HPLC分析与XCMS（各种形式的色谱法与质谱联用）相结合的在线统计工具，用于数据处理。第一的，我们先前报道的在线二维筛查系统用于分析三种据报道具有潜在抗炎作用的中草药，并鉴定出两种结合成分。相比之下，采用了建议的具有XCMS在线分析功能的离线2D筛选方法，除在线系统显示的两种化合物外，还发现了三种其他成分。然后，对这三种化合物进行了交叉验证，并确认它们也都包含在在线数据中，但是由于它们的浓度低且缺乏可靠的统计方法，因此未在其中进行鉴定。最后，药理实验表明这五种成分可以剂量依赖的方式抑制LPS诱导的RAW细胞上IL-6的释放和IL-6基因的表达。与以前的2D CMC筛选系统相比，这种新开发的离线2D方法不需要样品制备步骤即可进行二维分析，并且它灵敏，高效且方便。它将适用于从中草药中鉴定活性成分，并在发现中草药中的铅化合物方面具有实际意义。