INTRODUCTION: 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. OBJECTIVE: To develop an efficient method for the preparative isolation and purification of isoflavones from B. chinensis by high-speed counter-current chromatography (HSCCC). METHODOLOGY: 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). RESULTS: 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. CONCLUSION: 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.
INTRODUCTION: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. OBJECTIVE: To develop an efficient method for the preparative isolation and purification of isoflavones from B. chinensis by high-speed counter-current chromatography (HSCCC). METHODOLOGY: 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). RESULTS: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. CONCLUSION: 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.