Biotechnol. Prog. 1992, 8, 583-586
589
Isolation of Vindoline from Catharanthus roseus by Supercritical Fluid Extraction Kyu-Min Song, Sang-Woo Park, Won-Hi Hong, and Huen Lee* Department of Chemical Engineering, Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, Daejon, Korea
Sang-Soo Kwak and Jang-Ryol Liu Plant Cell Biology Laboratory, Genetic Engineering Research Institute, Taeduc Science Town, Daejon, Korea
Vindoline was extracted from the leaves of Catharanthus roseus over the ranges of 35-70 "C and 100-300 bar using supercritical carbon dioxide with and without the addition of 3 w t % ethanol as a cosolvent. The vindoline contents in the extracts were determined by HPLC and identified by LC/MS. The remarkable highest vindoline concentration, 58 w t % ,was obtained at the lowest temperature, 35 O C , and the highest pressure, 300 bar, of this study. The use of a cosolvent only slightly improved the extraction yields or selectivities at some experimental conditions.
Introduction The medicinal plant Catharanthus roseus, Madagascar periwinkle, contains various indole alkaloids, of which vindoline and catharanthine are the most important. Vindoline and catharanthine are well-known as precursors in the biosynthetic pathways of valuable dimeric indole alkaloids such as vinblastine and vincristine. These two dimeric alkaloids have been used for many years as chemotherapeutic agents in the treatment of acute leukemia and Hodgkin's disease (Svoboda et al., 1959). However, bulk production of vindoline has not been successful by plant cell and tissue cultures, although catharanthinehas been so produced (Drapeau et al., 1987; Smith et al., 1987; Tallevi and DiCosmo, 1988). In addition, as vindoline content in the intact plant is quite small, its isolation by column chromatography on a large scale is tedious and time consuming. Supercritical fluid extraction (SFE) has received much attention as a technique for separating relatively nonvolatile materials. The extraction of natural products using a supercritical fluid as the extraction agent is a nondestructive method for isolating valuable constituents from natural materials. In an isothermal process it is possible to separate sensitive ingredients under thermally mild conditions without decomposition. In the case of biochemical and pharmaceutical products, the use of supercritical carbon dioxide is most suitable since this solvent is toxicologically harmless. In this connection the objective of this study was to test the applicability of the SFE process for selectively extracting vindoline from the leaves of Catharanthus roseus by using carbon dioxide as a supercritical solvent. The effect of a cosolvent, ethanol, on the selectivity was also examined. Materials and Methods Sample Preparation. The leaves of Catharanthus roseus were dried in an oven with sufficient time and then ground to a fine powder to increase the mass-transfer efficiency between supercritical carbon dioxide (SC-CO2) and sample particles. SFE Procedure. A continuous-flow-through SFE system, shown schematically in Figure 1,was used for the 87567938/92/3008-0583$03.00/0
tests. Carbon dioxide was supplied from a gas cylinder and was directed to an electrically driven diaphragm-type compressor (Model 554-2121,Nova Werke Ag., Effretikon, Switzerland). ApLC-500 micro flow syringe pump (Model 1240-018, ISCO Inc., Lincoln, NE) was used to inject the cosolvent, ethanol, into the solvent gas, carbon dioxide, at a constant volumetric flow rate. The flow rates of ethanol and carbon dioxide used in this experiment were 20 std pL/min and 0.3 std L/min, respectively. The line filter (Model 60-51HF2, High Pressure Equipment Co., Erie, PA), incorporating 10-pm filter discs, was used to remove contaminants contained in the carbon dioxide. After compression, the carbon dioxide was introduced into a surge vessel to dampen the fluctuations generated by the compressor. In order to maintain a constant pressure within the system, a back pressure regulator (Model 261700, Tescom Co., Elk River, MN) with a stated accuracy of f l % of the relief pressure range was employed. The equilibrium cell is a 200 cm3high-pressure stainless steel vessel packed with 3-mm glass beads. To increase the extraction efficiency between carbon dioxide and the sample powder, a metal filter was installed at the end of the inlet tube. The temperature inside the air bath was controlled to within f O . l O C by using a proportional temperature controller (Model 4202PC2, Omega Engineering Inc., Stamford, CT). The carbon dioxide-solute mixture leaving the top of the extractor was expanded to atmospheric pressure through a micrometering valve into cold traps where the solute was condensed. The flow rate and volume of carbon dioxide were measured by a flow meter and a dry test meter (Model 63115, Precision Scientific, Inc., Chicago, IL). The dry test meter was equipped with gauges to measure flow temperature and pressure. The use of low solvent flow rates (between 0.1 and 0.5 std L/min) guarantees the attainment of equilibrium conditions at the extractor's outlet. QuantitativeAnalysis by HPLC. The crude alkaloid extract obtained from the SFE experiment was analyzed by HPLC (Model 8800, Spectra-Physics, San Jose, CA). The extract was dissolved in a small volume of methanol and filtered through a 0.5-pm FH-type Millipore filter. This sample was then loaded onto a reversed-phase
0 1992 American Chemical Society and American Institute of Chemical Englneers
Blotechnol. hog., 1992, Vol. 8, No. 6
sa4
Figure 1. Schematic diagram of the supercriticalfluid extraction system used in this study: (1)COz cylinder; (2) high-pressure valve; (3)filter; (4)compressor; (5) check valve; (6) rupture disk; (7) damper; (8)regulator; (9) preheater; (10)extractor; (11)sampling valve; (12) heater; (13) collector; (14) removal; (15) rotameter; (16) wet test meter; (17) constant temperature circulator; (18) cosolvent reservoir; (19)micro pump; (20) cosolvent injection valve. 2'5]
Vindoline-
2.0
II
Catharanthine
I
1.5 u
s
W
3
589
Isolation of Vindoline from Catharanthus roseus by Supercritical Fluid Extraction Kyu-Min Song, Sang-Woo Park, Won-Hi Hong, and Huen Lee* Department of Chemical Engineering, Bioprocess Engineering Research Center, Korea Advanced Institute of Science and Technology, Daejon, Korea
Sang-Soo Kwak and Jang-Ryol Liu Plant Cell Biology Laboratory, Genetic Engineering Research Institute, Taeduc Science Town, Daejon, Korea
Vindoline was extracted from the leaves of Catharanthus roseus over the ranges of 35-70 "C and 100-300 bar using supercritical carbon dioxide with and without the addition of 3 w t % ethanol as a cosolvent. The vindoline contents in the extracts were determined by HPLC and identified by LC/MS. The remarkable highest vindoline concentration, 58 w t % ,was obtained at the lowest temperature, 35 O C , and the highest pressure, 300 bar, of this study. The use of a cosolvent only slightly improved the extraction yields or selectivities at some experimental conditions.
Introduction The medicinal plant Catharanthus roseus, Madagascar periwinkle, contains various indole alkaloids, of which vindoline and catharanthine are the most important. Vindoline and catharanthine are well-known as precursors in the biosynthetic pathways of valuable dimeric indole alkaloids such as vinblastine and vincristine. These two dimeric alkaloids have been used for many years as chemotherapeutic agents in the treatment of acute leukemia and Hodgkin's disease (Svoboda et al., 1959). However, bulk production of vindoline has not been successful by plant cell and tissue cultures, although catharanthinehas been so produced (Drapeau et al., 1987; Smith et al., 1987; Tallevi and DiCosmo, 1988). In addition, as vindoline content in the intact plant is quite small, its isolation by column chromatography on a large scale is tedious and time consuming. Supercritical fluid extraction (SFE) has received much attention as a technique for separating relatively nonvolatile materials. The extraction of natural products using a supercritical fluid as the extraction agent is a nondestructive method for isolating valuable constituents from natural materials. In an isothermal process it is possible to separate sensitive ingredients under thermally mild conditions without decomposition. In the case of biochemical and pharmaceutical products, the use of supercritical carbon dioxide is most suitable since this solvent is toxicologically harmless. In this connection the objective of this study was to test the applicability of the SFE process for selectively extracting vindoline from the leaves of Catharanthus roseus by using carbon dioxide as a supercritical solvent. The effect of a cosolvent, ethanol, on the selectivity was also examined. Materials and Methods Sample Preparation. The leaves of Catharanthus roseus were dried in an oven with sufficient time and then ground to a fine powder to increase the mass-transfer efficiency between supercritical carbon dioxide (SC-CO2) and sample particles. SFE Procedure. A continuous-flow-through SFE system, shown schematically in Figure 1,was used for the 87567938/92/3008-0583$03.00/0
tests. Carbon dioxide was supplied from a gas cylinder and was directed to an electrically driven diaphragm-type compressor (Model 554-2121,Nova Werke Ag., Effretikon, Switzerland). ApLC-500 micro flow syringe pump (Model 1240-018, ISCO Inc., Lincoln, NE) was used to inject the cosolvent, ethanol, into the solvent gas, carbon dioxide, at a constant volumetric flow rate. The flow rates of ethanol and carbon dioxide used in this experiment were 20 std pL/min and 0.3 std L/min, respectively. The line filter (Model 60-51HF2, High Pressure Equipment Co., Erie, PA), incorporating 10-pm filter discs, was used to remove contaminants contained in the carbon dioxide. After compression, the carbon dioxide was introduced into a surge vessel to dampen the fluctuations generated by the compressor. In order to maintain a constant pressure within the system, a back pressure regulator (Model 261700, Tescom Co., Elk River, MN) with a stated accuracy of f l % of the relief pressure range was employed. The equilibrium cell is a 200 cm3high-pressure stainless steel vessel packed with 3-mm glass beads. To increase the extraction efficiency between carbon dioxide and the sample powder, a metal filter was installed at the end of the inlet tube. The temperature inside the air bath was controlled to within f O . l O C by using a proportional temperature controller (Model 4202PC2, Omega Engineering Inc., Stamford, CT). The carbon dioxide-solute mixture leaving the top of the extractor was expanded to atmospheric pressure through a micrometering valve into cold traps where the solute was condensed. The flow rate and volume of carbon dioxide were measured by a flow meter and a dry test meter (Model 63115, Precision Scientific, Inc., Chicago, IL). The dry test meter was equipped with gauges to measure flow temperature and pressure. The use of low solvent flow rates (between 0.1 and 0.5 std L/min) guarantees the attainment of equilibrium conditions at the extractor's outlet. QuantitativeAnalysis by HPLC. The crude alkaloid extract obtained from the SFE experiment was analyzed by HPLC (Model 8800, Spectra-Physics, San Jose, CA). The extract was dissolved in a small volume of methanol and filtered through a 0.5-pm FH-type Millipore filter. This sample was then loaded onto a reversed-phase
0 1992 American Chemical Society and American Institute of Chemical Englneers
Blotechnol. hog., 1992, Vol. 8, No. 6
sa4
Figure 1. Schematic diagram of the supercriticalfluid extraction system used in this study: (1)COz cylinder; (2) high-pressure valve; (3)filter; (4)compressor; (5) check valve; (6) rupture disk; (7) damper; (8)regulator; (9) preheater; (10)extractor; (11)sampling valve; (12) heater; (13) collector; (14) removal; (15) rotameter; (16) wet test meter; (17) constant temperature circulator; (18) cosolvent reservoir; (19)micro pump; (20) cosolvent injection valve. 2'5]
Vindoline-
2.0
II
Catharanthine
I
1.5 u
s
W
3
