title: Orthogonal test design for optimization of supercritical fluid extraction of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone from Stellera chamaejasme L. and subsequent isolation by high-speed counter-current chromatography.
abstract: Supercritical fluid extraction (SFE) of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1- pentanone from Stellera chamaejasme L. was performed. An orthogonal L9 (3)4 test design was applied to select the optimum extraction parameters including pressure, temperature, modifier and sample particle size on yield 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 of pressure, 45 degrees C of temperature, 40-60 mesh of sample particle size and modified CO2 with 20% methanol. The yield of the crude extract from preparative SFE was 2.65%, which contained daphnoretin 25.2%, 7-methoxy-daphnoretin 22.8% and 1,5-diphenyl-1-pentanone 21.1%, respectively. Then the crude extract was successfully isolated and separated by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:13:13:10, v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 90 min. The target compounds isolated and purified by HSCCC were analyzed by high-performance liquid chromatography (HPLC). The separation produced total of 69.2mg of daphnoretin at 99.2% purity, 63.4 mg of 7-methoxy-daphnoretin at 98.7% purity and 58.3 mg of 1,5-diphenyl-1-pentanone at 98.1% purity from 300 mg of the crude extract in one-step separation. The recoveries of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone were 90.8, 91.5 and 90.4%, respectively, in HSCCC isolation step and the chemical structure identification was carried out by MS, 1H NMR and 13C NMR.
authors.affiliation: School of Pharmacy, Dalian Medical University, No. 465 Zhongshan Road, Dalian 116027, China. jinyongpeng2005@163.com
authors.forename: Jinyong
authors.initials: J
authors.lastname: Peng
authors.forename: Fuqiu
authors.initials: F
authors.lastname: Dong
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Xu
authors.forename: Youwei
authors.initials: Y
authors.lastname: Xu
authors.forename: Yan
authors.initials: Y
authors.lastname: Qi
authors.forename: Xu
authors.initials: X
authors.lastname: Han
authors.forename: Lina
authors.initials: L
authors.lastname: Xu
authors.forename: Guorong
authors.initials: G
authors.lastname: Fan
authors.forename: Kexin
authors.initials: K
authors.lastname: Liu
mesh_terms: D002851:Chromatography, High Pressure Liquid; D025924:Chromatography, Supercritical Fluid / Q000379:methods*; D003374:Coumarins / Q000302:isolation & purification*; D003377:Countercurrent Distribution / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D010316:Particle Size; D010422:Pentanones / Q000302:isolation & purification*; D021241:Spectrometry, Mass, Electrospray Ionization; D029645:Thymelaeaceae / Q000737:chemistry*
chemical_list: C433344:1,5-diphenyl-1-pentanone; C515752:7-methoxy-daphnoretin; D003374:Coumarins; D010422:Pentanones; C035316:daphnoretin