One-step Separation and Purification of Four Phenolic Acids from Stenoloma chusanum (L.) Ching by Medium-pressure Liquid Chromatography and High-speed Counter-current Chromatography

Background: Stenoloma chusanum (L.) Ching is a Chinese traditional medicinal fern with high total flavonoid and total phenolic content. Traditionally, phenolic compounds were separated by using column chromatography, which is relatively inefficient. Objective: This study aims to use an efficient method to separate natural products from S. chusanum by Medium-Pressure Liquid Chromatography (MPLC) and High-Speed Counter-Current Chromatography (HSCCC). Methods: In the present research, firstly, a sample (2.5 g) from the dichloromethane extract of S. chusanum was separated by MPLC. Next, fraction P5 was purified by HSCCC with a two-phase solvent system composed of hexane-ethyl acetate-methanol-water (HEMWat) at a volume ratio of 2:4:1:4 (v/v/v/v). Result: Four phenolic acids were obtained and their structures were identified by means of NMR and ESI-mass analysis. They were identified as: 1) protocatechuic acid (34 mg, purity 90.1%), 2) syringic acid (66 mg, purity 99.0%), 3) p-hydroxybenzoic acid (5 mg, purity 91.2%) and 4) vanillic acid (6 mg, purity 99.3%). Conclusion: The combination of MPLC and HSCCC is a high-efficiency separation method for natural products. This is the first report with regard to the separation of four phenolic acids in one step by MPLC and HSCCC from S. chusanum (L.) Ching.