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The high‐polar compounds from natural products are often used as medicines due to their good bioactivities. However, owing to the complexity and diversity of their structure, the separation of high‐polar compounds is still a challenging work. For this, an efficient method for enrichment and separation of the high‐polar compounds from Saussurea obvallata was developed. First, the target compounds were enriched from the total extract using a solid‐phase extraction method. An offline two‐dimensional liquid chromatography method was used for the separation of pure compounds from the enriched sample. After optimization of chromatographic conditions, high separation selectivity of target compounds was obtained on a polar‐modified C18 column and a HILIC XAmide column. Hence, a two‐dimensional reversed‐phase × hydrophilic interaction liquid chromatography system was constructed and enlarged from the analytical level to the preparative level. In the first dimension, four fractions were obtained on the XCharge C18 column with a recovery rate of 71.2%. In the second‐dimension preparation on the XAmide column, eight high‐polar compounds with more than 96% purity were isolated. All compounds were isolated from Saussurea obvallata for the first time. The results demonstrated that this developed strategy is effective for preparative‐scale isolation of high‐polar compounds from natural products.

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

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