Supercritical Carbon Dioxide Extraction Enhances Flavonoids in Water-Soluble Propolis

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1 J. Chin. Inst. Chem. Engrs., Vol. 33, No. 3, , 2002 Supercritical Carbon Dioxide Extraction Enhances Flavonoids in Water-Soluble Propolis Geng-Shian You [1], Sun-Che Lin [2], Chao-Ruey Chen [3], Wei-Chen Tsai [4], Chiehming J. Chang [5] Department of Chemical Engineering, National Chung Hsing University 250, Kuo-Kuang Road, Taichung, Taiwan 402, R.O.C. Wen-Wen Huang [6] Department of Biology, China Medical College 91, Shiue-Sh Road, Taichung, Taiwan 404, R.O.C. Abstract Two propolis, obtained from Brazil and China, were ground and individually treated using supercritical carbon dioxide to enhance the extraction and water solubility of nine flavonoids. Co-solvent effects of ethanol and water were examined. Extractions were carried out semi-batchly at 5 ml/min CO 2, at pressures ranging from 2000 to 5000 psig, at temperatures ranging from 35 to 65ºC, and with propolis to co-solvent ratios of 1 : 2 and 1 : 5 (w/v). After supercritical extraction of 500 liters of CO 2, the residual propolis in the extractor was further extracted by means of normal solvent extraction using 95% ethanol (ReE extract was obtained) and de-ionized water (ReW extract was obtained). Nine active propolis flavonoids in the extracts were analyzed to verify their inhibitory effects on leukemia cancer cells. Experimental results indicated that the dry SC-CO 2 extract mainly contained low-polar wax-like materials. The crude yield of Brazilian propolis in the dry SC-CO 2 extract reached around 10% at 45ºC and 4000 psig extraction. The amounts of the nine flavonoids in the Brazilian ReE and ReW extracts were 5% and 120%, higher than those obtained by means of conventional ethanol and water extraction, respectively. The Brazilian ReW extracts clearly contained a few water-soluble compounds and, especially, enhanced amounts of the nine flavonoids. All the extracts were tested for viability and cell proliferation of leukemia HL-60 and U937 cancer cells, and most exhibited a positive inhibitory effect. The results also suggested that something other than the nine flavonoids in dry SC-CO 2 extract was importantly involved in the inhibitory effect. Key Words : Carbon dioxide, Extraction, Propolis, Flavonoids, Anticancer INTRODUCTION Propolis is a complex resinous mixture collected and used by bees to seal holes in their honeycombs, and to protect the entrance against intruders. It has recently been reported that propolis possesses a variety of pharmaceutical activities, such as antibacterial, anti-fungi, anti-protozoan, antiviral, tumor cytotoxic, antioxidant, and other activities (Marcucci, 1995; Burdoc, 1998; Kujumgiev et al., 1999; Hayashi et al., 1999). Propolis has been used as a remedy or an additive in health foods worldwide. Most propolis is exported by Brazil and China. The properties of the propolis from these countries differ a little because the composition of the propolis depends on the time of harvesting, the surrounding vegetation and the area of collection. Nevertheless, propolis usually contains flavonoids, phenolics and their derivatives, and lignan (Bankova et al., 1998; Christov et al., 1999). Moreover, flavonoids, for example ganlangin, pinocembrin, quercetin, rhamnetin and pinostrobin, comprise the majority of pharmaceutically active compounds, with about existing 10% in propolis (Campos et al., 1990). Other aromatic acids and esters, such as caffeic acid phenethyl ester (CAPE), isopentyl ferulate and benzyl caffeate, also exhibit pharmaceutical activities (Marcucci, 1995). UV, TLC, HPLC and GC analyses are the methods most often used to quantify these compounds (Gwoisky and Salatino, 1998; Bankova et al., 1998; Markham et al., 1996). Propolis for edible and commercial purposes usually undergoes a series of biological [1], To whom all correspondence should be addressed [2] [3] [4] [5] [6]

2 234 J. Chin. Inst. Chem. Engrs., Vol. 33, No. 3, 2002 tests, such as those for in-vitro cytotoxicity, antioxidant activity, and hepatoprotective activity (Banskota et al., 2000). The typical in-vitro test for tumor cytotoxicity is the test of the viability of growth of human leukemia cells HL-60 and U937 with propolis (Chen et al., 1996; Park et al., 2001). Most of the commercially available propolis is an ethanolic extract of propolis (EEP). However, ethanolic extracts of propolis usually contain significant amounts of wax-like materials and other undesired compounds, which may cause allergic reactions. Examples are cinnamic acid and 1,1-dimethylallyl caffeic acid (Hausen et al., 1987; Miyataka et al., 1997). Thus, EEP is not suitable for children or sensitive people. The other type of propolis, called water-soluble propolis (WP), is miscible in water and thus has few side effects on the human body. However, wax-like propolis and flavonoid-type compounds are not very soluble in water. Therefore, direct water extraction of propolis is not as effective as ethanolic extraction, and the amounts of pharmaceutical compounds in WP extract are quite low. Supercritical carbon dioxide has attracted considerable attention because of its desirable properties, such as low viscosity, high diffusivity, non-toxicity and low critical points. It has become the most promising alternative medium in chemical, food, pharmaceutical, environmental and material engineering processes, for example, concentrating tocopherols from soybean oil scum (Chang et al., 2000), removing metal from water, and separating isomers (Tai et al., 2000a, 2000b). This study aimed to increase the amounts of flavonoids in water-soluble propolis by using supercritical carbon dioxide and adding a co-solvent. Nine active propolis flavonoids were analyzed to indicate the amounts of active compounds in propolis. The effects of the operating condition on the composition and on the inhibitory activity, which tested the viability and cell proliferation of leukemia cancer cells, were also investigated. This is the first published report describing the extraction of propolis with supercritical carbon dioxide. EXPERIMENTAL SECTION Materials Lumps of propolis, donated by a local supplier in Taiwan, were stored at 5 C in a refrigerator. Before each experiment, a propolis lump was ground down to 2 mm particles. Liquid CO 2 (purity 99.5%) was purchased from the LIEN-HWA Industrial Gases Corp. in Taiwan. Ethanol (95%), produced by the Taiwan Tobacco and Wine Monopoly Bureau, was used as the solvent/co-solvent for extraction. De-ionized water was prepared with a Milli-Q system. The following nine chemicals were used to make a standard mixture for HPLC analysis. Kaempferol, caffeic acid phenethyl ester (CAPE), acacetin, quercetin and naringenin were purchased from Sigma (Germany). Galangin and chrysin were obtained from Aldrich (U.S.A.). Isorhamnetin and pinocembrin were obtained from ROTH (Karlsruhe,Germany) and the INDOFINE Chemicals Corp. (U.S.A.), respectively. Mobile phases of HPLC grade (methanol, ethanol, phosphoric acid) were obtained from Baker (Paris). All the solutions were filtered through 0.45 µm membranes and degassed by an ultrasonic bath before HPLC analysis. Supercritical fluids extraction Figure 1 schematically depicts the highpressure apparatus used for SC-CO 2 extraction. The apparatus consists of a high-pressure pump (positivedisplacement type, CM-3200, LDC, U.S.A.), a 75 ml extractor (L/D=30) and a 750 ml absorber (L/D=10). At the beginning of a dry SC-CO 2 extraction experiment, 33 g of ground propolis was placed in the extractor (8), and 400 ml of ethanol (95%) was loaded into the absorber (11). 5 cm-length layers of glass wool were placed on both the top and the bottom of the extractor to prevent the entrainment of fine particles. Co-solvent was preloaded into the extractor to study the modified effect. Co-solvent ratios (w/v) of 1 : 2 and 1 : 5 were considered in this study. SC-CO 2 experiments were conducted in a semi-batch operation mode, and the high-pressure pump (4) delivered the liquid CO 2 into the extractor at a fixed flow rate of 5 ml/min. The extraction pressure, regulated by a back-pressure regulator (9-1), varied from 2000 psig to 5000 psig, and extraction temperatures ranging from 35 C to 65 C were used. The absorber pressure was regulated at 700 psig by a backpressure regulator (9-2), and propolis-laden supercritical fluid from the extractor was absorbed and collected at room temperature. A wet gas meter (13) measured the volume of CO 2. The variations in pressure were within ± 50 psig during each experiment, and those in temperature were within ± 1.0 C. Solid residues left after SC-CO 2 extraction were individually extracted by 1 : 20 (w/v) de-ionized water and 95% ethanol to facilitate comparison of the water and ethanol extracts of residues (ReW and ReE) with the water and ethanol extracts of raw propolis (WEP and EEP), in terms of the total yield, recovery, concentration factor and separation factor for nine flavonoids. After extraction, a certain amount of extract was placed in a vacuum oven for 24 hr, and the total weight of the dry materials was measured to calculate the crude yield, which was defined as the weight ratio of these dry materials to the propolis preloaded in the extractor. The number of experiments replicated at a fixed condition was listed in Table 2 and the errors in extractions were within ± 5%.

3 Geng-Shian You, Sun-Che Lin, Chao-Ruey Chen, Wei-Chen Tsai, Chiehming J. Chang and Wen-Wen Huang: 235 Supercritical Carbon Dioxide Extraction Enhances Flavonoids in Water-Soluble Propolis 1. CO 2 Cylinder 9-1~9-2. Backpressure regulator 2-1~2-4. Pressure gauge 10. Metering valve 3. Gas dryer 11. Absorber 4. High-pressure pump 12. Float flow meter 5-1~5-2. Temperature controller 13. Wet gas meter 6-1~6-3. Needle valve 14-1~14-5.Thermocouple 7. Heat exchanger 15. Sample vial 8. Extractor Fig. 1. Schematic diagram of high-pressure experimental apparatus. Table 1. The gradient solvent program of HPLC analysis used in this study. Time Flow Rate 0.1% H 3 PO 4 (aq) Methanol (min) (ml/min) % % Mode of Change to Next Flow Linear Step Linear Step Linear Linear Linear HPLC analysis of flavonoids A high precision liquid chromatograph system, consisting of a Waters 600E multi-solvent delivery pump, a Waters 486 UV/Vis detector, and a Nucleosil RP-C8 5U ( mm i.d.) column in a Waters temperature control module, was employed to analyze the flavonoids in the extracts, using a gradient solvent program technique. The mobile phase consisted of 0.1% phosphoric acid and methanol. The details of the gradient profile are listed in Table 1. The injection volume was 20 µl, and the UV wavelength was set at 211 nm. The column temperature was controlled at 55 C. The external-standard curves were constructed by the synthesized solutions consisting of the nine flavonoids at 100 ppm, 50 ppm and 10 ppm. Every sample was analyzed at least twice and the errors in HPLC analysis were within ± 5.0%. Cell culture and viability test Following Chen et al. (1996), U937 and HL-60 cells ( cell/ml) were grown in 100 mm diameter culture dishes containing RPMI-1640 medium (GIBCO, U.S.A.), supplemented with 10% fetal bovine serum (FBS), 100 µg/ml penicillin and

4 236 J. Chin. Inst. Chem. Engrs., Vol. 33, No. 3, 2002 Fig. 2. Temperature effect on the yield of dry SC-CO 2 extraction at 3000 psig (Brazilian propolis extracted with 500 liters CO 2 ). 100 µg/ml streptomycin sulfate, and incubated at 37 C in a 5% CO 2 humidified atmosphere. Cells were inoculated every three days to maintain normal growth. At the beginning of the two-day tests, various concentrations of the extracts, from 10 µg/ml to 250 µg/ml, were added to a series of culture dishes containing U937 or HL-60 cells in 15 ml RPMI medium; cell numbers were then counted every 24 hrs, using a hemocytometer (FACS, Becton Dickinson, U.S.A.). The viability test replicated three times at each fixed condition. The errors are listed in Table 4 individually. RESULTS AND DISCUSSION Brazilian propolis was first extracted using supercritical carbon dioxide at temperatures ranging from 35 C to 65 C and pressures ranging from 2000 psig to 5000 psig to find the optimal operating conditions. The Chinese propolis was then extracted under these optimal conditions. Temperature effect Figure 2 shows the temperature effect on the yield of SC-CO 2 extraction of Brazilian propolis at 3000 psig and 500 L CO 2. The maximal extraction yield of Brazilian propolis was only 6.6% for dried SC-CO 2 at 45 C. Increasing or decreasing the temperature from 45 C lowered the extraction yield. The reason for this might be that the dissolving capability of supercritical fluids is usually influenced by the competition between the increase in the solute volatility and the decrease in the SC-CO 2 density due to the rise in temperature. At temperatures below 45 C, the effect wherein the solute volatility increased comparatively rapidly with an isobaric increase in temperature dominated the change of solubility; thus, an increase in the extraction yield with temperature was observed. At temperatures higher than 45 C, the Fig. 3. Pressure effect on SC-CO 2 extraction at 45ºC ( and : Dry SC-CO 2 extraction; and : SFE with the addition of 1 : 5 (w/v) ethanol; Brazilian propolis extracted with 500 liters of CO 2 ). CO 2 density fell significantly, and the extraction yield fell as the temperature rose. Therefore, 45 C was regarded as the optimum temperature for removing wax from propolis using SC-CO 2 extraction, and was chosen for all the subsequent SC-CO 2 experiments. Pressure effect The solid lines in Fig. 3 indicate the pressure effect on the dry SC-CO 2 extraction of Brazilian propolis at 45ºC. Under isothermal conditions, the solubility of the solutes depended on the density of the SC-CO 2. Thus, the extraction yield increased as the pressure increased from 2000 psig to 5000 psig. However, the yield of the chosen nine flavonoids reached a maximum at around 4000 psig, and the extraction yield did not significantly change as the pressure increased from 4000 psig to 5000 psig; therefore, 4000 psig was chosed as the optimum pressure for SC-CO 2 extraction. Modifier effect Dry SC-CO 2 extractions revealed that most of the extracts were low-polar wax-like materials, which sparsely dissolved in water and ethanol, partially condensed a trace in the backpressure regulator, and melted between 59.6 C and 60.8 C. Figure 4 displays HPLC spectrums showing that few flavonoids were present in the dry SC-CO 2 extracts. Polar flavonoids were not effectively extracted by the dry SC-CO 2. Commercial propolis products are usually in ethanolic or water-soluble form. Hence, ethanol and water were chosen as modifiers to enhance the recovery of flavonoids. The dashed lines in Fig. 3 indicate the SC-CO 2 extraction of Brazilian propolis with 1 : 5 (w/v) ethanol at 45 C. Adding ethanol enhanced the efficiency of SC-CO 2 extraction; the op-

5 Geng-Shian You, Sun-Che Lin, Chao-Ruey Chen, Wei-Chen Tsai, Chiehming J. Chang and Wen-Wen Huang: 237 Supercritical Carbon Dioxide Extraction Enhances Flavonoids in Water-Soluble Propolis Fig. 4. Typical HPLC spectrums of Brazilian propolis extracts: (a) standard mixture of nine flavonoids; (b) SC-CO 2 extract; (c) water extract; (d) 95% ethanol extract; (1) naringenin; (2) quercetin; (3) kaempferol; (4) isorhamnetin; (5) pinocembrin; (6) caffeic acid phenethyl ester; (7) galangin; (8) chrysin; (9) acacetin. timal pressure was close 4000 psig. However, adding water to the propolis sample did not enhance the extraction yield above the best extraction yield of 9.6% obtained with dry SC-CO 2 as shown in Table 2. The extraction yield obtained with the addition of 1 : 5 (w/v) modifier was the highest in all the tests on the co-solvent effect. The extraction yield obtained with the addition of 1 : 5 (w/v) ethanol modifier reached only 77.5% of that obtained by means of normal ethanol extraction at one atmosphere and room temperature (42.7% vs. 55.1%). However, the amount of ethanol used was only 1/4 of that used in normal ethanol extraction. Concentration factor and separation factor of flavonoids The above findings reveal that dry SC-CO 2 extraction primarily removed low-polar wax-like materials from propolis. The propolis residue lift after dry SC-CO 2 extraction was further extracted by ethanol and water to recover flavonoids. ReE is the ethanol extract of residue propolis, and ReW is the water extract of residue propolis treated with SC-CO 2 extraction. EEP refers to the normal ethanol extract of propolis, and WEP is the normal water extract. The concentration and separation factors defined below describe the recovery efficiency of flavonoids from propolis: concentration factor: β = flavonoids recovery ; weight of extract (1) weight of propolis separation factor: α = flavonoids recovery weight of other compounds in extract, (2) weight of other compounds in propolis where flavonoids recovery = weight of nine flavonoidsin extract. (3) weight of nine flavonoidsin propolis Table 2 lists the experimental results of SC- CO 2 treatment and the co-solvent effect for both the Brazilian and Chinese propolis. The Brazilian propolis contained around 2-3% of the nine chosen flavonoids, and Chinese propolis contained 9-10%. Thus, the extracts of the Chinese propolis contained more flavonoids than did those of the Brazilian propolis. Furthermore, comparing the WEP and EEP extracts of both the Brazilian and Chinese propolis clearly reveals that the Chinese propolis contained a much larger amount of hydrophobic compounds that were soluble in ethanol (56.6% 5.03% = 51.57% vs. 55.1% 20.5% = 34.9%). Although SC-CO 2 extraction did not significantly affect the amounts of the nine flavonoids in the ReE extracts, SC-CO 2 pretreatment did increase the amounts of the nine flavonoids in the Brazilian ReW extracts. The amounts of the flavonoids in the Brazilian ReW extracts increased to 220% higher than those in the Brazilian WEP extracts (3323 µg/g vs µg/g). Figures 5 and 6 show the ratios of the concentration and separation factors ( β 2 β1 and α 2 α1, respectively) of extracts treated with SC-CO 2 to those of extracts

6 238 J. Chin. Inst. Chem. Engrs., Vol. 33, No. 3, 2002 Table 2. Experimental results for the SC-CO 2 treatment and co-solvent effect. Run no. Solvent / Cosolvent Yield (%) Flavonoids µg/g propolis β α Replicates B-EEP 1:20 EtOH (95%) B-WEP 1:20 Water a B25-CO 2 SC-CO B15-CO b 2 SC-CO 2 / 1 : 2 Water B16-CO b 2 SC-CO 2 / 1 : 5 Water B17-CO b 2 SC-CO 2 / 1 : 2 EtOH (95%) a B26-CO 2 SC-CO 2 / 1 : 5 EtOH (95%) B25-ReE a 1:20 EtOH (95%) B25-ReW a 1:20 Water M-EEP 1:20 EtOH (95%) M-WEP 1:20 Water a M2-CO 2 SC-CO a M3-CO 2 SC-CO 2 / 1 : 5 EtOH (95%) M2-ReE a 1:20 EtOH (95%) M2-ReW a 1:20 Water a SFE experiments were conducted at 45 C and 4000 psig. b SFE experiments were conducted at 45 C and 3000 psig. B: Brazilian propolis; M: Chinese propolis. sig- Fig. 5. Relative α and of β Brazilian ReE extract versus EEP extract at 45ºC ( ) β ReE / β EEP ; ( ) α ReE /α EEP. treated without SC-CO 2, at various pressures and at 45 C. The concentration and separation factors of the flavonoids in the Brazilian ReW extracts became at least twice those of the WEP extracts under SC-CO 2 extraction at 45 C and 4000 psig, perhaps because the SC-CO 2 extractions effectively removed nonpolar wax-like and volatile compounds, which hinder the extraction of water from flavonoids, thus increasing the amounts of the flavanoids in the Brazilian ReW extracts. However, SC-CO 2 pre-treatment negatively affected the ReW and ReE extracts from the Chinese propolis since the Chinese propolis contained about four times as many hydrophobic flavonoids as the Brazilian propolis. Ethanol extracted the flavonoids from the propolis so effectively that the enhancement in SC-CO 2 extraction was not Fig. 6. Relative α and β of Brazilian ReW extract versus WEP extract at 45 C ( ) β ReW / β WEP ; ( ) α ReW / α WEP. nificant. Inhibitory effect of extracts on human leukemia cancer cells The experimental results indicate that phenolic acid and flavonoids were major compounds in both the water and ethanol extracts. The proliferation rates of HL-60 and U937 at various dosages are listed in Tables 3 and 4, respectively. Table 5 gives the dosages of IC 50 determined by regression analysis. Most of the Chinese propolis extracts, except for the SC- CO 2 extract, were superior to the Brazilian propolis extracts, according to inhibitory tests in the U937 and HL-60 leukemia cancer cell viability experiments. This result was predictable because the ex-

7 Geng-Shian You, Sun-Che Lin, Chao-Ruey Chen, Wei-Chen Tsai, Chiehming J. Chang and Wen-Wen Huang: 239 Supercritical Carbon Dioxide Extraction Enhances Flavonoids in Water-Soluble Propolis tract of the Chinese propolis (i.e., M-EEP) contained Table 3. Inhibitory effects of various extracts on HL-60 cells. Extract Conc. (µg/ml) Proliferation After 24 hr (% of control ) Proliferation After 48 hr (% of control ) Control ± ± 3.79 B3-ReE ± ± ± ± ± ± 3.03 B3-CO ± ± ± ± ± ± 0.21 M1-ReE ± ± ± ± ± ± 1.46 M1-ReW ± ± ± ± ± ± 6.81 M1-CO ± ± ± ± 3.40 B: Brazilian propolis; M: Chinese propolis. Table 4. Inhibitory effects of various extracts on U937 cells. Extract Conc. (µg/ml) Proliferation After 24 hr (% of control ) Proliferation After 48 hr (% of control ) Control ± ± 6.12 B3-ReE ± ± ± ± ± ± 4.03 B3- CO ± ± ± ± ± ± 5.32 M1-ReE ± ± ± ± ± ± 2.36 M1-ReW ± ± ± ± ± ± 2.49 M1-CO ± ± ± ± 5.29 B: Brazilian propolis; M: Chinese propolis. Table 5. Inhibitory IC 50 dosages of various extracts on the growth of HL-60 and U937 cells. Cell B-CO 2 M-CO 2 B-ReE (µg/ml) M-ReE B-ReW M-ReW U HL B: Brazilian propolis; M: Chinese propolis. more flavonoids than did that of the Brazilian propolis (i.e., B-EEP). Whereas the result wherein SC-CO 2 extracted Brazilian propolis inhibited leukemia cell growth more effectively than did SC-CO 2 extracted Chinese propolis suggests that something other than the nine flavonoids dominated the inhibitory effect of the SC-CO 2 extract from the Brazilian propolis.

8 240 J. Chin. Inst. Chem. Engrs., Vol. 33, No. 3, 2002 CONCLUSION Dry supercritical carbon dioxide extraction removed primarily volatile and wax-like compounds from propolis. The optimal conditions for SC-CO 2 extraction of propolis were 45ºC and 4000 psig. The Brazilian propolis contained more water-soluble flavonoids than did the Chinese propolis; thus, SC-CO 2 pretreatment enhanced the concentration factor ( β ) and separation factor ( α ) of the nine flavonoids from the Brazilian propolis more than did extraction only with normal ethanol and water. The Brazilian ReW under SC-CO2 extraction at 45ºC and 4000 psig might be a potentially water-soluble propolis, because the Brazilian ReW extracts contained about 2.2 times as much of the nine flavonoids as were present in the Brazilian WEP. The experimental results show that adding 1 : 5 (w/v) ratio ethanol as a cosolvent in the propolis increased the efficiency of SC-CO 2 extraction, but that adding water did not. The extracts of Chinese propolis outperformed those of Brazilian propolis in inhibiting leukemia cancer cells. However, SC-CO 2 extracts from the Brazilian propolis inhibited leukemia cancer cells better than did extracts from Chinese propolis, suggesting that something other than the nine flavonoids in the SC- CO 2 extracts dominated the inhibitory effect. Further investigations are required to identify these active compounds in SC-CO 2 extracts. ACKNOWLEDGEMENT The authors gratefully acknowledge the financial support of the National Science Council of the Republic of China. α β Subscripts EEP WEP ReE ReW NOMENCLATURE separation factor, defined in Eq.(2). concentration factor, defined in Eq.(1). the normal ethanol extract of propolis the normal water extract of propolis the ethanol extract of residue propolis the water extract of residue propolis REFERENCES Bankova, V. S., R. S. Christov and A. D. Tejera, Lignans and Other Constituents of Propolis from the Canary Islands, Phytochemistry, 49, 1411 (1998). Banskota, A. H., Y. Tezuka, I. K. Adnyana, K. Midorikawa, K. Matsushige, D. Message, A. A. G. Huertas and S. Kadota, Cytotoxic, Hepatoprotective and Free Radical Scavenging Effects of Propolis from Brazil, Peru, the Netherlands and China, J. Ethnopharmacology, 72, 239 (2000). Burdoc, G. A., Review of the Biological Properties and Toxicity of Bee Propolis (Propolis), Food Chem. Toxicol., 36, 347 (1998). Campos, M. G. R., S. Sabatier, M. J. Amiot and S. Aubert, Characterization of Flavonoids in Three Hive Products: Bee Pollen, Propolis, and Honey. Planta Med., 56, 580 (1990). Chang, C. J., Y. F. Chang, H. Z. Lee, J. L. Lin and P. W. Yang, Supercritical Carbon Dioxide Extraction of High- Value Substances from Soybean Oil Deodorizer Distillate, Ind. Eng. Chem. Res., 39, 4521 (2000). Chen, J. H., Y. Shao, M. T. Huang, C. K. Chin and C. T. Ho, Inhibitory Effect of Caffeic Acid Phenethyl Ester on Human Leukemia HL-60 Cells, Cancer Lett., 108, 211 (1996). Christov, R. S, V. S. Bankova, I. Tsvetkova, A. Kujumgiev and A. D. Tejera, Antibacterial Furofuran Lignans from Canary Islands Propolis, Fitoterapia, 70, 89 (1999). Gwoisky, R. and A. Salatino, Analysis of Propolis: Some Parameters and Procedures for Chemical Quality Control, J. Apic. Res., 37, 99 (1998). Hausen, B. M., E. Wollenweber, H. Seniff and B. Post, Propolis Allergy. II. The Sensitizing Properties of 1,1- Dimethylallyl Caffeic Acid Ester, Contact Dermatitis, 17, 171 (1987). Hayashi, K., S. Komura, N. Isaji, N. Ohishi and K. Yagi, Isolation of Antioxidative Compounds from Brazilian Propolis: 3,4-Dihydroxy-5-Prenylcinnamic Acid, a Novel Potent Antioxidant, Chem. and Pharm. Bulletin, 47, 1521 (1999). Kujumgiev, A., I. Tsvetkova, Yu. Serkedjieva, V. S. Bankova, R. S. Christov and S. Popov, Antibacterial, Antifungal and Antiviral Activity of Propolis of Different Geographic Origin, J. Ethnopharmacology, 64, 235 (1999). Marcucci, M., Propolis: Chemical Composition, Biological Properties and Therapeutic Activity, Apidologie, 26, 83 (1995). Markham, K. R., K. A. Mitchell, A. L. Wilkins, J. A. Daldy and Y. Lu, HPLC and GC-MS Identification of the Major Organic Constituents in New Zealand Propolis, Phytochemistry, 42, 205 (1996). Miyataka, H., M. Nishiki, H. Matsumoto, T. Fujimoto, M. Matsuka and T. Satoh, Evaluation of Propolis. I. Evaluation of Brazilian and Chinese Propolis by Enzymatic and Physico-Chemical Methods, Biological and Pharmaceutical Bulletin, 20, 496 (1997). Park, J. W., Y. J. Choi, M. A. Jang, Y. S. Lee, D. Y. Jun, S. I. Suh, W. K. Baek, M. H. Suh, I. N. Jin and T. K. Kwon, Chemo-Preventive Agent Resveratrol, a Natural Product Derived from Grapes, Reversibly Inhibits Progression

9 Geng-Shian You, Sun-Che Lin, Chao-Ruey Chen, Wei-Chen Tsai, Chiehming J. Chang and Wen-Wen Huang: 241 Supercritical Carbon Dioxide Extraction Enhances Flavonoids in Water-Soluble Propolis through S and G2 Phases of the Cell Cycle in U937 Cells, Cancer Lett., 163, 43 (2001). Tai, C. Y., G. S. You and D. C. Wang, Modified Retrograde Crystallization Process for Separation of Binary Solid Mixtures Exploiting the Crossover Region of Supercritical Carbon Dioxide, Ind. Eng. Chem. Res., 39, 4357 (2000b). Tai, C. Y., G. S. You and S. L. Chen, Kinetics Study on Supercritical Fluid Extraction of Zinc(II) Ion form Aqueous Solutions, J. Supercrit. Fluids, 18, 201 (2000a). (Manuscript Received December 3, 2001) C psig 5 ml/min 500 L 1:2 1:5 ( ReE ReW ) (dry) 45 C 4000 psig 10% ReE ReW 5% ReW 120% HL-60 U937