title: Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography.
abstract: Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.
authors.affiliation: Shanghai Key Laboratory for Pharmaceutical Metabolite Research, School of Pharmacy, Second Military Medical University, No. 325 Guohe Road, Shanghai 200433, China.
authors.forename: Jinyong
authors.initials: J
authors.lastname: Peng
authors.forename: Guorong
authors.initials: G
authors.lastname: Fan
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Chai
authors.forename: Yutian
authors.initials: Y
authors.lastname: Wu
mesh_terms: D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D003377:Countercurrent Distribution / Q000379:methods*; D005419:Flavonoids / Q000302:isolation & purification*; D032405:Patrinia / Q000737:chemistry*
chemical_list: D005419:Flavonoids