Chapter 9 Separation of C u c u r b i t a c i n Β a n d C u c u r b i t a c i n Ε from F r u i t Base of Cucumis melo L. by H i g h - S p e e d Countercurrent Chromatography 1
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Downloaded by CORNELL UNIV on August 17, 2016 | http://pubs.acs.org Publication Date: May 15, 1995 | doi: 10.1021/bk-1995-0593.ch009
Q. Z. Du , X. P. Xiong , and Yoichiro Ito
1Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, Zhejiang 310008, China Laboratory of Biophysical Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 7N322, Bethesda, MD 20892
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Cucurbitacin Β (CuB) and cucurbitacin Ε (CuE) were separated from the fruit base of Cucumis melo L. by high -speed countercurrent chromatography (CCC) using a two -phase solvent system composed of hexane/ethyl acetate/ methanol/water (12:24:16:9 by volume). Three successive 300mg injections of sample, indicated by HPLC assay to contain 61.4% CuB and 35.1% CuE, were made at leap-frog intervals, prior to emergence of the second peak, thereby greatly reducing the overall separation time. Measurement of the stationary phase retention indicated that many more sample injections can be made without renewing the column contents. Guadi, the fruit base of Cucumis melo L., is a Chinese traditional herbal drug used for treating chronic hepatitis (I). Recent studies (2,3) have shown that cucurbitacin Β (CuB) and cucurbitacin Ε (CuE) (Fig. 1) are the main bioactive components in Guadi, and Cucurbitacin Tablet containing a mixture of CuB and CuE extracted from the fruit base of Cucumis melo L. is currently substituted for Guadi in China. However, it has been found that the curative effect of CuB is different from that of CuE (4), hence the separation of these two compounds is desired. In this paper, we report isolation of CuB and CuE from a crude fruit extract of Cucumis melo L. by high-speed CCC (5). Experimental Apparatus. High-speed CCC experiments were performed using a coil planet centrifuge equipped with a multilayer coil column that was designed and fabricated at the Beijing Institute of New Technology Application, Beijing, China. The multilayer coil was prepared by winding a 1.6mm ID PTFE (polytetrafluoroethylene) tube coaxially onto the column holder hub. The total column capacity measured 230 ml. The high-speed CCC centrifuge was rotated at 800 rpm with an 8-cm revolution radius. The system was equipped with an FMI pump (Zhejiang Instrument Factory, Hangzhou, China), a variable UV detector (UV-752, Shanghai Analytical Instrument Factory, Shanghai, China), a recorder, an injection valve and a fraction collector. 0097-6156/95/0593-0107$12.00/0 © 1995 American Chemical Society Conway and Petroski; Modern Countercurrent Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1995.
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MODERN COUNTERCURRENT
CHROMATOGRAPHY
Reagents. A l l organic solvents were of a n a l y t i c a l grade and purchased from Shanghai Chemical Factory, Shanghai, China. Standards f o r cucurbitacin Β (CuB) and cucurbitacin Ε (CuE) were purchased from T i a n j i n I n s t i t u t e of Pharmaceutical Research, State Pharmaceutical Administration of China, T i a n j i n , China.
Downloaded by CORNELL UNIV on August 17, 2016 | http://pubs.acs.org Publication Date: May 15, 1995 | doi: 10.1021/bk-1995-0593.ch009
Extraction of CuB and CuE from C. melo F r u i t . The extraction was i n i t i a t e d by refluxing 1 Kg of C. melo f r u i t powder i n 10 l i t e r s of 95% ethanol at 80°C f o r 2 hours. The ethanol extract was concentrated to a syrup i n a rotary evaporator. The syrup was extracted with ether. The ether extract was evaporated to dryness, defatted with petroleum ether, then redissolved i n ethanol and decolorized with active carbon. The ethanol solution was evaporated to dryness to obtain a crude CuB and CuE mixture. The crude mixture was r e c r y s t a l l i z e d from methanol to y i e l d 2.2 g of white powder containing CuB and CuE. High-Speed CCC Separation Procedure. The experiment was performed with a two-phase solvent system composed of hexane/ethyl acetate/methanol/ water (12:24:16:9, v/v). The solvent mixture was thoroughly e q u i l i b r a t e d i n a separatory funnel at room temperature and the two phases separated shortly before use. In each separation the multilayer c o i l was f i r s t e n t i r e l y f i l l e d with the lower aqueous stationary phase. Then the upper organic mobile phase was pumped into the i n l e t of the column at a flow rate of 1.5 ml/min, while the apparatus was rotated at 800 rpm. After the mobile phase front emerged and the two phases had established hydrodynamic equilibrium throughout the column, three successive injections of the sample solution (300mg of a CuB and CuE mixture i n 20ml of mobile phase) was made through the i n j e c t i o n valve i n 100 minute i n t e r v a l s . The effluent from the outlet of the column was continuously monitored with a UV detector at 234 nm and the f r a c t i o n s were collected with a f r a c t i o n c o l l e c t o r . HPLC Analysis. The o r i g i n a l sample solution and CCC fractions were analyzed by reversed-phase HPLC using Shimadzu HPLC equipment (Shimadzu Corporation, Kyoto, Japan) consisting of an LC-10AD pump, an SPD-10A UV-VIS detector, a manual injector and a C-R10A recording processor. HPLC separations were performed on a Shim-pack CLC-ODS-C18 column, 15 X 0.60 cm ID (Shimadzu Corporation). The mobile phase composed of methanol-ethyl acetate-water (3:1:1, v/v) was eluted i s o c r a t i c a l l y at a flow-rate of 1 ml/min and the effluent was monitored at 234 nm. Results and Discussion HPLC analysis of the o r i g i n a l sample shown i n F i g . 2 indicates that the sample contained 35.1% CuE (retention time: 4.56 min) and 61.4% CuB (retention time: 5.75 min). The high-speed CCC separation consisted of three successive injections of the sample at 100 minute i n t e r v a l s . This i n t e r v a l was chosen to leap-frog the second peak, so that the o v e r a l l separation time for the three injections was s i g n i f i c a n t l y shorter than three times the time required f o r separation of a single sample. After the separation was completed, the column contents were expelled into a graduated cylinder to estimate the retention of the stationary phase. I t was found that 161 ml (70% of the t o t a l column capacity) of the stationary phase s t i l l remained i n the column, suggesting that many more sample injections could be made without replenishing the stationary phase. F i g . 3 shows the chromatogram r e s u l t i n g from three successive sample i n j e c t i o n s . The fractions corresponding to peak 1 and peak 2 contained CuE and CuB, respectively, as confirmed by the HPLC analysis using the CuE and CuB standards. Peak fractions corresponding to each component were combined and evaporated to dryness i n vacuum to y i e l d 307 mg of CuE from peak 1 and 501 mg of CuB from peak 2, a recovery of 89.8% of t o t a l sample weight. The recovered amounts correspond to a sample composition of 37.9% CuE and 61.2% CuB, i n good agreement with the HPLC assay.
Conway and Petroski; Modern Countercurrent Chromatography ACS Symposium Series; American Chemical Society: Washington, DC, 1995.
9. DU ET AL.
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Separation of Cucurbitacin B and Cucurbitacin E
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Downloaded by CORNELL UNIV on August 17, 2016 | http://pubs.acs.org Publication Date: May 15, 1995 | doi: 10.1021/bk-1995-0593.ch009
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