Study On Preparative Isolation And Purification Of Acetophenones From Three Asclepiadaceae Plant C. Bungei, C. Auriculatum And C. Paniculatum By High-Speed Counter-Current Chromatography

Cynanchum bungei Decne, Cynanchum auriculatum Royle ex Wight and Cynanchum paniculatum Bunge are famous traditional Chinese medicine of genus Cynanchum from family Asclepiadaceae. Because of their medical activities, more and more researchers have drawn great attention on them.High speed counter-current chromatography (HSCCC) is a unique liquid-liquid partition chromatography that uses no solid support matrix. Therefore, it eliminates irreversible adsorptive loss of samples onto the solid support matrix used in the conventional chromatographic column and the metamorphic change of samples. It is simple, economical, quick and with good repeatability. The method has been successfully applied to the analysis and separation of several natural products.The objectives of this study were to optimize the extraction conditions of components by a SFE system and combined with HSCCC to explore the separation and purification of three Asclepiadaceae plants in deep. Then the content of the components of C. bungei obtained by HSCCC were determined by HPLC. Five extraction fractions of C. bungei and C. auriculatum were compared and analysised by their chromatograms. It was investigated that similarities and differences of some main components in C. bungei and C. auriculatum. The investigations provided the theoretic foundation for exploring and applying the three plants.1 Isolation and purification of acetophenones from C. bungei by high-speed counter-current chromatographyA pair of two-phase stepwise solvent system composed of light petroleum–ethyl acetate–methanol–water (2:5:3:3, v/v) and (2:4:3:3, v/v), stationary phase: upper organic phase of 2:5:3:3 (v/v); mobile phase: lower phase of 2:5:3:3 (v/v) in 0–120 min, lower phase of 2:4:3:3 (v/v) after 120 min; flow rate: 1.7 mL/min; revolution speed: 850 rpm/min; detection wavelength: 254 nm; time: 270 min. A total of 62.3 mg (1), 18.9 mg (2), 15.0 mg (3) and 4.5 mg (4) were purified from the n-butanol extract (600 mg) of crude sample of C. bungei. in one-step separation, each at over 96% purity as determined by high performance liquid chromatography (HPLC).2 Determination of four acetophenones in Radix C. Bungei by HPLCThe HPLC-PDA was carried out with Symmetry-C18 (4.6 mm×250 mm, 5μm), as separation column; CH₃OH-H₂O (26:74, v/v) as mobile phase; 1.0 mL/min as flow rate; 30℃as temperature control. The compounds 1,3,4 were monitored at 280 nm, and the compound 2 was at 224 nm. The results indicated that the contents of 1, 2, 3 and 4 were 80.8, 8.1, 447.0, 67.0μg/g, respectively.3 Compared and analyzed the chromatogram of C. bungei and C. auriculatumIn order to found out the differences between them, we compared and analyzed the light petroleum, chloroform, ethyl acetate, methanol and water extract of C. bungei and C. auriculatum by UV-Vis, FT-IR and HPLC.The UV apectrum results showed that the main components exist in the C. bungei Decne and C. auriculatum are nearly the same at the same concentrations, but the UV absorption of the light petroleum and water fractions were obviously different.The IR results showed the different extraction fractions of C. bungei and C. auriculatum, we found that the chromatograms of light petroleum, chloroform and water extract were obviously different, especially in 2300 cm^-1-2400cm^-1. When light petroleum was used as extraction solvent, sample of C. bungei Decne has absorption, but C. auriculatum has no absorption. When chloroform was used as extraction solvent, sample of C. auriculatum has absorption, but C. bungei Decne has no absorption.280 nm and 254 nm were selected as the wavelenghs for HPLC analyze. At 280nm, a total of 22 components were detected, among them, 14 components existed in C. auriculatum. At 254 nm, a total of 26 were detected, 21 components existed in C. auriculatum and 25 components exist in C. bungei. It was found that the components existed in C. bungei Decne and C. auriculatum are nearly the same, but the contents of different components were differently.4 Isolation and purification of acetophenones from C. auriculatum by HSCCCA two-phase solvent system composed of light petroleum (b.p. 60-90℃)–ethyl acetate–methanol–water (4:9:6:6, v/v) was used, the lower phase was used as mobile phase and the upper phase was used as stationary phase. Flow rate: 1.8 mL/min; revolution speed: 900 rpm/min; detection wavelength: 280 nm; Time: 300 min. About 20.2 mg 4-hydroxyacetophenone, 35.0 mg baishouwubenzophenone and 8.3 mg 2,4-dihydroxyacetophenone, each at over 95% purity as determined by HPLC, were obtained from 400 mg of the ethanol extract.5 Isolation and purification of acetophenones from C. paniculatum by CO₂-SFE-HSCCCThe extraction conditions including pressure, temperature, time and sample particle size were optimized with an L9(3)4 orthogonal test. Under the optimized conditions of 15 MPa, 55℃, 1.5 h and a sample particle size of 40–60 mesh. The yield of the paeonol was 5.905%. HSCCC separation with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (2:6:3:4, v/v). The lower phase was used as mobile phase and the upper phase was used as stationary phase. Flow rate: 1.7 mL/min; revolution speed: 850 rpm/min; detection wavelength: 280 nm; Time: <4h. From 2.42 g of the crude extract, 1.07 g of paeonol was obtained at 99.4% purity as determined by HPLC-PDA.