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The Study of Supercritical Carbon Dioxide Extraction for Ganoderma Lucidum† Ruey Chi Hsu,* Bin Hwae Lin, and Chi Wei Chen Department of Chemical Engineering, University of Chang Gung, 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan, Taiwan
This study is about extraction of Ganoderma lucidum by using supercritical CO2 and the modifier ethanol. It demonstrates that modified supercritical extraction is suitable for the extraction of Ganoderma lucidum. The advantage of modified supercritical extraction over nonmodified supercritical extraction was in the polarity component extraction and enhancement of the fluidity of extracts. Modifier supercritical extraction also has higher extraction yield and lower extraction temperature than those of conventional solvent extractions. Although the extraction yield seems to have not increased significantly when the pressure increased, the extraction pressure controlled the extraction selectivity of triterpenoid components of Ganoderma lucidum. 1. Introduction The fruiting body of Ganoderma lucidum has been used as a folk medicine in the oriental country. Currently, the use of traditional Chinese herbs is a principle topic in our enterprise and country. Ganoderma lucidum is one of the focal points in the development of Chinese herbs. Ganoderma lucidum contains several triterpenoids and polysaccharides, which have been investigated in relation to their physiological effects.1,2 Those components can be used to cure various human diseases. A preliminary procedure for extraction of this component from Ganoderma lucidum involves solvent extraction and solvent adsorption.3 The solvents used in such methods was either methanol, which is hazardous, or hot water, which may degrade some of the energy-sensitive matrix. Here, we describe a new extraction method, which uses supercritical carbon dioxide as the solvent. Carbon dioxide is a clean and inexpensive solvent. As a supercritical fluid, the solvent strength of CO2 can be easily adjusted with small changes in pressure and without phase transition. Supercritical CO2 can be easily separated from the desired extraction product by reduction of pressure. In addition, the lower supercritical temperature of CO2 is good for energy-sensitive components such as triterpenoids. Although CO2 is an apolar molecular, the addition of polar organic solvents such as a modifier can enhance the extraction effect.4 Because of those properties, supercritical CO2 is a useful processing solvent for Ganoderma lucidum extraction. The following sections present a study of the extraction of Ganoderma lucidum by using supercritical CO2 and the modifier ethanol. 2. Experimental Apparatus and Procedure The experimental apparatus is schematically shown in Figure 1. CO2 from a cylinder with a siphon attachment is passed through a cooling coil and compressed to the operating pressure by a high-pressure diaphragm * To whom correspondence should be addressed. E-mail:
[email protected]. † Originally submitted for publication in the Supercritical Fluids special issue, published in December 2000.
pump (LEWA, EK-1). The flow rate of the fluid is regulated at the diaphragm pump. The compressed fluid is heated by passing through a jacked-type heat exchanger and then flowing to the extractor. The extractor column is 80-mm long and 40-mm i.d. and like a shelltube type heat exchanger in which compressed fluid flowed through the tube side and the hot water passed the shell side to maintain the extraction temperature. The fluid exiting from the extractor is expanded to ambient pressure through a back-pressure regulator and then the extracted material is collected. One heated jacket is used as the exit tube to avoid overcooling during the rapid expansion procedure. The CO2 flow rate was measured by a CO2 mass flowmeter. For runs with a modifier, pure ethanol was mixed with the CO2 by a HPLC pump. About 8 g of dried and crushed Ganoderma lucidum is placed into the extractor column for the extraction run. The extraction runs were carried out in a pressure range of 1500-3000 psi, and the CO2 flow rate was kept at approximately 4 × 10-6 m3/min, a modifier flow rate range of 0.15-0.5 mL/min. To avoid degradation of temperature-sensitive material, the temperatures of 40 and 50 °C were used for extraction. The extracted materials were weighted after the ethanol evaporated. 3. Results and Discussion 3.1. Conventional Solvent Extraction and Supercritical CO2 Extraction. A typical comparison of extraction yield from various extraction methods is tabulated in Table 1. Conventionally, Ganoderma lucidum extraction involved the methanol, ethanol, or hot water method. The conventional extraction methods used in this study have been mentioned5 and must extract at higher temperatures. The yield of methanol extraction (0.1083 g) is better than that of two traditional methods. Contemporarily, the yield of methanol extraction is also higher than that of nonmodified supercritical extraction (0.0956 g). It, due to methanol, has the higher polarity and solubility. However, methanol is a toxic solvent that avoids remaining in food or medicine. Nevertheless, the yield of modified supercritical extraction (0.1373 g) is higher than that of the methanol method. It should also be noted that supercritical extraction required a lower
10.1021/ie000203w CCC: $20.00 © 2001 American Chemical Society Published on Web 09/08/2001
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Figure 1. Experimental apparatus. Table 1. Comparison of Extraction Yields of Various Extraction Methodsa extraction method 90 °C hot water 60 °C ethanol 60 °C methanol 40 °C, 1500psib 40 °C, 1500psic
extraction amount of time (min) solvent (ml) 120 120 120 120 120
240 240 240 18 18
yield (g)
specific yield (g/mL solvent)
0.0748 0.0876 0.1083 0.0956 0.1373
3.117 × 10-4 3.650 × 10-4 4.513 × 10-4 5.311 × 10-3 7.628 × 10-3
a The yield is based on 8 g of Ganoderma lucidum. b Nonmodified supercritical fluid extraction; 18 mL of ethanol was used to elute. c Modified supercritical fluid extraction, EtOH flow rate ) 0.15 mL/min.
temperature than the typical extraction methods. This characteristic is good for heat-sensitive materials that exist in Ganoderma lucidum (like triterpenoid components). Furthermore, the amount of solvent used in conventional extraction methods (like hot water, methanol, and ethanol extraction) was 240 mL. The amount of solvent was based on the number of times (30) the Ganoderma lucidum was used; the ratio was used usually in conventional extraction. Overall extraction processes that were used in this study were carried out within 2 h. Thus, 18 mL of ethanol was used in supercritical fluid extraction under an ethanol flow rate equal to 0.15 mL/min. Consequently, in a comparison of the specific yield (ratio of yield and unit volume of organic solvent), it can be found that the supercritical fluid extraction, either nonmodified or modified, was approximately 1 order higher than that of conventional extraction methods. 3.2. Nonmodified and Modified Supercritical CO2 Extraction. Primarily, the supercritical extraction was carried out under nonmodifier conditions. It was found that a large amount of the extracts deposited at the exit line of the extractor and the extracts could not be collected from the exit under nonmodified supercritical extraction. Thus, we adjusted the entry line of the modifier from upstream to downstream of the extractor and then we were able to obtain the extracts. From
Figure 2. Chromatogram of triterpenoid components extracted by nonmodified supercritical fluid extraction.
previous results, we considered that the modifier effect not only increases the solubility of the extracts of Ganoderma lucidum but also enhances the fluidity of the extracts. To study the polarity effect of the modifier, a series of HPLC analyses were carried out to analyze the supercritical extracts of triterpenoid components. The HPLC analysis of the supercritical extracts involved a C18 Merck reversed phase column incorporating a gradient elution of the mobile phase that was described in Lin.5 A typical chromatogram for the results of the nonmodified and modified supercritical methods are shown in Figures 2 and 3, respectively. It can be found that the triterpenoid patterns of the modified supercritical extraction have higher absorbance than those of nonmodified supercritical extraction. It indicates that modified supercritical extraction can yield more of the triterpenoids. In addition, the characteristic of a reversed phase column is that the higher polar component can be eluted easier than the less polar component. Therefore, the higher polarity components have the shorter retention time in the chromatogram. The triterpenoids that abound in Ganoderma lucidum that
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Figure 3. Chromatogram of triterpenoid components extracted by modified supercritical fluid extraction.
Figure 5. Extraction yields under 40 °C and various modifier flow rates.
Figure 4. Extraction yields under various temperatures.
have polarity have been investigated.6 Consequently, it should be stressed that there are more peaks and higher absorbances for the shorter retention times in the chromatogram of modified supercritical extraction. This result means that ethanol can enhance the polarity of supercritical CO2 to extract more polar triterpenoid components from Ganoderma lucidum. Some triterpenoid components, as illustrated in Figure 3, can be identified from a comparison with similar analysis conditions of HPLC analysis in previous research,7,8 like peak 1 may be Ganoderic acid C2, peak 2 may be Ganoderic acid B, peak 3 may be Ganoderic acid B, and peak 4 may be Ganoderic acid C1. 3.3. Temperature and Pressure Effects in Modified Supercritical Extraction. To avoid the degradation of temperature-sensitive materials (like triterpenoid components), the modified supercritical extraction was carried out at lower temperature. A comparison of extraction under 40 and 50 °C is shown in Figure 4, which shows that the higher temperature had the higher extraction yield. This could be explained as the effect of increased diffusion and fluidity of extracts from the matrix to the supercritical medium. But it may be noted that the yield difference obtained from the different temperature is less than that of the modifier flow rate difference.
Figure 6. Triterpenoids chromatogram for extraction pressure at 1500 psi.
A comparison of the extraction yield under different pressures is shown in Figure 5. As expected, the higher pressure has the higher extraction yield. It is more reasonable that the density of the supercritical fluid is dependent on pressure and the solubility of the solvent is to first approximation related to density. Furthermore, it has to be noted that the extraction yield increases like the modifier increases are significant. In a comparison of the solvent effect, that of the cosolvent is higher than that of pressure. In previous results, it has been mentioned that a modifier can enhance the fluidity of viscous extracts of Ganoderma lucidum. Thus, we considered that the fluidity of the viscous extracts within Ganoderma lucidum can be enhanced by the dilute effect of modifier and consequently increase the extraction yield under supercritical extraction with a modifier. From this result, the higher pressure operating, which is the major problem encountered in supercritical fluid extraction, can be overcome by adding a modifier. Although the extraction yield seems to have not increased significantly when the pressure increased, the importance of pressure on extraction selectivity of triterpanoid components was shown in the chromatograms in Figures 6 and 7. In this study, a C18 reverse phase column was used in HPLC analysis; thus, the more polar material had less retention time. In a
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extraction was in the polarity component extraction and enhancement of the fluidity of extracts. The extraction yield was found to be increased with an increase in modifier flow rate from 0.15 to 0.5 mL/min. Although the extraction yield seems to have not increased significantly when the pressure increased, the extraction pressure controlled the extraction selectivity of triterpenoid components of Ganoderma lucidum. Literature Cited
Figure 7. Triterpenoids chromatogram for extraction pressure at 3000 psi.
comparison of the results of Figures 6 and 7, it can be found that the yields (expressed as absorbance) of triterpenoid components (3-5) increased with the increase in retention time under 3000 psi, whereas those decreased with the increase in retention time under 1500 psi. It can be seen that the yield of the less polar triterpenoid components increased with an increase in pressure. This also means that the extraction pressure controlled the extraction selectivity of trierpenoids. 4. Conclusion The extraction of Ganoderma lucidum in supercritical CO2 was measured with no modifier and with a modifier. This study demonstrates that the modified supercritical extraction is suitable for the extraction of Ganoderma lucidum. It also has higher extraction yield and lower extraction temperature than those of conventional solvent extractions. The advantage of modified supercritical extraction over nonmodified supercritical
(1) Shao, M. S. Triterpenoid Natural Products in Fungus Ganoderma lucidum. J. Chin. Chem. Soc. 1992, 39, 669. (2) Komoda, Y. M.; Shimizu, M.; Sonoda, Y.; Sato, Y. Ganoderic Acid and its Derivatives as Cholesterol Synthesis Inhibitors. Chem. Pharm. Bull. 1989, 37, 531. (3) Sye, W. F. Improvement Method of Extraction and HighPerformance Liquid Chromatographic Separation of Ganoderic Acids from Ganoderma Lucidum. J. Chin. Chem. Soc. 1991, 38, 179. (4) Kiran, E.; Brennecke, J. F. Supercritical Fluid Engineering Science Fundamentals and Applications; American Chemistry Society: Washington, DC, 1993. (5) Lin, B. H. Using Supercritical Fluid to Extract the Triterpenoids from Ganoderma lucidum. M.S. Thesis, Chang Gung University, Taiwan, 1998. (6) Nishitoba, T.; Sato, H.; Shirasu, S.; Sakamura, S. Evidence on the Strain-specific Triterpenoid Pattern of Ganoderma lucidum. Agric. Biol. Chem. 1986, 50, 2151. (7) Chen, M. T.; Leu, S. F.; Ho, Y. P.; Rai, S. G.; Yang, C. D.; Fu, W. K. The Establishment of Analytical Methods for Specific Compounds in Ganoderma tsuga and Database of Ganoderma Products; Food Industrial Research and Development Institute: Hsinchu, Taiwan, 1996. (8) Lin, L. J.; Shiao, M. S. J. Separation of Oxygenated Triterpenoid from Ganoderma lucidum by High Performance Liquid Chromatography. J. Chromatogr. 1987, 410, 195.
Received for review February 8, 2000 Revised manuscript received July 11, 2001 Accepted July 18, 2001 IE000203W
