Lippia origanoides (Verbenaceae) is an important Brazilian medicinal plant, also used for culinary purposes. Most chemical studies with this plant have been focused on its volatile composition. In this work, we combined High-Speed Counter-current Chromatography (HSCCC) and High Performance Liquid Chromatography coupled to Ultra Violet detection and High Resolution Mass Spectrometry (HPLC-UV-HRMSⁿ) methodologies to access the non-volatile chemical composition of L. origanoides. The crude ethanol extract of L. origanoides (LOEF) was first analyzed by HPLC-UV-HRMSⁿ and allowed the identification of 7 major compounds. Among them, eriodictyol, naringenin and pinocembrin, were determined and are phytochemical markers of this plant. However, owing to the complexity of this plant matrix, LOEF was fractionated by HSCCC (hexane-ethanol-water, 4:3:1) as a tool for preparative pre-purification, affording a flavonoid-rich fraction. A column screening with the chromatographic stationary phases ZIC-HILIC, monolithic and particulate RP18 was performed. The best column separation was achieved with a Purospher STAR RP18e, which was used for HPLC-DAD-HRMSⁿ studies. By this approach 12 compounds were further identified in addition to the major ones identified in the raw extract. Two of them, 6,8-di-C-hexosyl-luteolin and 6,8-di-C-glucosyl-apigenin, are being reported for the first time in the family Verbenaceae. This work shows the integration of HSCCC as a preparative tool for the fractionation and purification of natural products from a complex plant extract with other analytical techniques, with the purpose of showing each technique’s potential.

紫薇（马鞭草科）是巴西重要的药用植物，也用于烹饪。该植物的大多数化学研究都集中在其挥发性成分上。在这项工作中，我们结合了高速逆流色谱（HSCCC）和高效液相色谱，以及紫外检测和高分辨率质谱（HPLC-UV-HRMS ⁿ）方法，以访问L的非挥发性化学成分。origanoides。首先通过HPLC-UV-HRMS ⁿ分析牛至的粗乙醇提取物（LOEF）。并鉴定了7种主要化合物。其中，确定了雌雄醇，柚皮苷和松胚霉素，它们是该植物的植物化学标记。但是，由于这种植物基质的复杂性，LOEF被HSCCC（己烷-乙醇-水，4：3：1）分馏，作为制备型预纯化工具，提供了富含黄酮的馏分。用色谱固定相ZIC-HILIC，整体和颗粒RP18进行柱筛选。使用Purospher STAR RP18e可获得最佳的色谱柱分离效果，该色谱柱用于HPLC-DAD-HRMS ⁿ研究。通过这种方法，除了在原料提取物中鉴定出主要化合物外，还鉴定出12种化合物。其中两个是6,8- di- C-己糖木犀草素和6,8-di- C-葡糖基芹菜素是马鞭草科中的首次报道。这项工作显示了将HSCCC作为一种制备工具的集成，该分离工具可通过其他分析技术从复杂的植物提取物中分离和纯化天然产物，目的是展示每种技术的潜力。