Preparative Isolation and Purification of Seven Isoflavones from Belamcanda chinensis

Abstract

Isoflavonoids from Belamcanda chinensis are known to have a number of physiological benefits including anti-inflammatory, anti-angiogenic and anti-mutagenic properties. However, there have been no reports on the effective isolation and purification of isoflavonoids from B. chinensis. To develop an efficient method for the preparative isolation and purification of isoflavones from B. chinensis by high-speed counter-current chromatography (HSCCC). A two-step HSCCC isolation method was developed using solvent system of n-hexane-ethyl acetate-2-propanol-methanol-water (5:6:2:3.5:6, v/v) and of ethyl acetate-methanol-water (10:2:9, v/v). FLASH purification system (45% methanol, isocratic) was also used for further purification. The purities and chemical structures of the isolated compounds were determined by high-performance liquid chromatography-photodiode array detection (HPLC-PDA), electrospray ionisation-mass spectrometry (ESI-MS), ¹H- and ¹³C-nuclear magnetic resonance spectrometry (NMR) and nuclear overhauser enhancement (NOE). HSCCC was successfully used for the preparative separation and purification of seven isoflavones, including tectoridin (145.4 mg, 97.5%), iridin (77.9 mg, 94.0%), irilin D (42.0 mg, 92.0%), tectorigenin (294.1 mg, 98.6%), iristectorigenin A (86.8 mg, 93.4%), irigenin (141.8 mg, 95.8%) and irisflorentin (73.4 mg, 94.7%) from the rhizomes of B. chinensis. Two isoflavone glycosides and five isoflavone derivatives were successfully isolated and purified from the crude methanol extract of dried rhizomes of the B. chinensis by HSCCC.