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Effect of Ethanol Content on Carbon Dioxide Extraction of Polyphenols from Tea
JOURNAL OF FOOD COMPOSITION AND ANALYSIS (2001) 14, 75}82 doi:10.1006/jfca.2000.0949 Available online at http://www.idealibrary.com on
ORIGINAL ARTICLE Effect of Ethanol Content on Carbon Dioxide Extraction of Polyphenols from Tea C. J. Chang* , K. L. Chiu*, Y. L. Chen-, and P. W. Yang? Department of Chemical Engineering, National Chung-Hsing University, Taichung, Taiwan 40227, R.O.C. -Taiwan Tea Experiment Station, Pushin, Taiwan 32613, R.O.C. and ?Products Developing Department, Taiwan Sugar Company, Taipei, Taiwan 106, R.O.C. Received May 26, 1999, and in revised form June 23, 2000
This study presents a novel packed-column extractor coupled with an absorption system to improve the quality of oleoresin oils for Oolong and green teas, extracted by using carbon dioxide. The e!ect of various co-solvents on the extract is also examined. In addition, gravimetrical measurement and HPLC chromatographic analysis individually determine the amount of oleoresin oil and the concentration of four major catechins. According to those results, the mean contents in the extract are 7.0- and 10.0-fold higher in an addition of 95% ethanol than in no addition for green tea and Oolong tea, respectively. The ratio of four major catechins to ca!eine is the highest in Soxhlet ethanol extraction for both green and Oolong teas. 2001 Academic Press
Key Words: carbon dioxide; catechins; ca!eine; polyphenols; extraction.
INTRODUCTION Numerous epidemiological and pharmacological studies demonstrate that green tea extract possesses strong antioxidant e!ects (Roedig-Penman and Gordon, 1997; Vinson and Dabbagh, 1998; Lin et al., 1996, 1998; Tanaka et al., 1998) and antimutagenic activity (Paschka et al., 1998; Otsuka et al., 1998; Chen et al., 1998; Leanderson et al., 1997). Recently, great e!ort has been made to utilize polyphenolic compounds present in teas and a large number of polyphenolic monomers (catechins) have been characterized (Davis et al., 1997). According to those studies, four polyphenol compounds, epigallocatechin gallate (EGCg), epicatechin gallate (ECg), epigallocatechin (EGC), and epicatechin (EC) are signi"cant antioxidant constituents (Goto et al., 1996). Among those, EGCg is the most luxuriant component in tea extract (Bronner and Beecher, 1998; Zhu et al., 1998; Dalluge et al., 1998; Owuor and Obanda, 1998) and the most potent chemical tested for biological activity (Chen et al., 1998). High-performance liquid chromatography is the conventional means of analyzing catechins in tea and additional biological component constituents (Price and Spitzer, 1993; Bronner and Beecher, 1998; Goto et al., 1996; Ho et al., 1995; Carando et al., 1998; Tsuchiya et al., 1998; Zhu et al., 1998; Leanderson et al., 1997). Recent e!orts To whom correspondence and reprint requests should be addressed. Tel.: 886(04)-285-2592. Fax.: 886(04)-285-4734. E-mail: [email protected] 0889}1575/01/010075#08 $35.00/0
2001 Academic Press
76
CHANG E¹ A¸.
successfully analyzed tea catechins using capillary electrophoresis (Horie and Kohata, 1998), mass spectrometer (Poon, 1998), and micellar electrokinetic chromatography (Larger et al., 1998). However, to our knowledge, no study has separated catechins and ca!eine by using supercritical carbon dioxide extraction (King and Bott, 1993). In the light of the above discussion, this study presents a novel custom-designed supercritical #uid extraction technique coupled with an absorption system to extract polyphenols from green tea leaf. Controlling the extractive conditions allows us to evaluate the separation of ca!eine and polyphenols. Furthermore, the gravimetrical method can be applied to determine the amount in the extract. Moreover, HPLC chromatography is performed to quantify four major catechins (TC), along with a spectrophotometer used to measure the amount of total polyphenols (TPP). MATERIALS AND METHODS Green tea and Oolong tea were obtained from a commercial market. Phosphoric acid (85.1%), epicatechin gallate (ECg, 98%), epigallocatechin (EGC, 98%), epigallocatechin gallate (EGCg, 95%), epicatechin (EC, 98%), GR grade of gallic acid and ca!eine were purchased from Sigma Company (U.S.). All materials were used without further puri"cation. HPLC-grade acetonitrile (BDH, U.K.) was used as the mobilephase solvent. Absolute ethanol (99.8%) was obtained from RDH (Germany). 95% (v/v) ethanol was donated by Taiwan Tobacco and Wine Monopoly Bureau. Deionized water was obtained from a reverse-osmosis and ion exchange puri"ed water system (Barnstead, U.K.). Figure 1 schematically depicts a high-pressure apparatus used for CO extraction of tea polyphenols. The whole system consists mainly of a 100 mL-syringe ISCO pump, a refrigeration module, a 300 mL extraction vessel, one sight-gauge precipitator, two 1-L absorbent vessels, and a wet gas meter. A coil immersed in a water bath preheated liquid CO so that CO could attain the desired temperature prior to entering the extraction vessel. The electrically evenly heated extraction vessel was packed with 90 g of tea powder. The extractive temperature was measured by one K-type thermocouple and controlled by a proportional-integrator controller. The absorbing system (one precipitator and two absorbent vessels) was "lled with 1.4 L of 50% ethanol}water absorbent and maintained at 5.0 MPa and 297 K. The precipitator provides visual assurance that the saturated tea oils-laden supercritical CO , comes from the extraction vessel through a 1/16 in i.d. nozzle, sprayed into and well mixed with the absorbent. In this situation, the absorption pressure was maintained at 5.0 MPa and measured by a digital pressure transducer (Druck, PDCR910). Extraction and absorption pressures were controlled via two back-pressure regulators. Ten milliliter samples were taken at every 100 L CO volume, as measured by using a wet gas meter. Stock solutions of four catechins, gallic acid, and ca!eine were prepared by dissolving weighted quantities of commercial standards into water. Less concentrated solutions were prepared by diluting with deionized water. The soluble tea extracts from SFE-CO and Soxhlet extractions were centrifuged at 5000 rpm for 10 min. The supernatant was taken into a 10 mL syringe and "ltered through a 0.45-lm two-phase nylon membrane. A 10-lL volume of the "ltered supernatant was injected into the HPLC system. Calibration curves were constructed by linear regression of the peak} area ratio versus concentration. The measurement accuracy was within $2 mg/kg. The HPLC system consists of a Waters 600E multi-solvent delivery pump (Millipore, U.S.A.), a Waters 486 tunable wavelength detector (Millipore, U.S.A.), and a Waters U6K 100-lL sample injector (Millipore, U.S.A.). The analytical column was a reverse-phase Develosil ODS-HG column (150;4.5 mm, Nomura, Japan)
EXTRACTION OF POLYPHENOLS FROM TEA
77
FIGURE 1. Schematic of the diagram equipment used for CO extraction of tea polyphenols.
equipped with a guard column (10;4 mm, Nomura). The column was thermostatted at 403C and the #ow rate of the mobile phase was 1 mL/min. UV detection was achieved at a wavelength of 231 nm. Classic Millennium 2010 software was used for manipulation of the pump and data processing. The mobile phase used for analysis
78
CHANG E¹ A¸. TABLE 1 Gradient elution program for analysis of four catechins, ca!eine, and gallic acid
Time (min) Initial 3 7 12 17 20
A (%)
B (%)
95 90 90 70 60 60
5 10 10 30 40 40
FIGURE 2. HPLC chromatograms of green tea extracts. (a) Standard mixture; (b) extract at CO #95% EtOH; (c) extract at Soxhlet EtOH; (d) extract at Soxhlet H O.
was solvent A: 0.05% phosphoric acid solution; solvent B: acetonitrile. Table 1 lists the solvent gradient conditions. A spectrophotometer was used to measure total polyphenols and ca!eine, with the details outlined in Lin et al. (1996). All extracted samples were dried inside a vacuum oven, operated at 160 mm Hg, 333 K. Finally, the gravimetrical measurement was used to obtain the amount of the extract. Standard deviation (S.D.) was within $10%. RESULTS AND DISCUSSION Tea extracts were analyzed to understand the contents of four catechins, gallic acid, and ca!eine by HPLC. For carbon dioxide extraction a 10 mL sample was taken at every 100 L S.T.P. CO volume and the last sample of each run was analyzed. For Soxhlet extraction 250 mL solvent was used in each run and a 10 mL sample was taken for the analysis. Each datum represents the average of two replicates. Figures 2 and 3 display the chromatograms of these standard samples. Table 2 summarizes the extracted amounts and the concentrations of total polyphenols (TPP), four
EXTRACTION OF POLYPHENOLS FROM TEA
79
FIGURE 3. HPLC chromatograms of Oolong tea extracts. (a) Standard mixture; (b) extract at CO #95% EtOH; (c) extract at Soxhlet EtOH; (d) extract at Soxhlet H O.
major catechins (TC), gallic acid (GA), ca!eine (Caf ) in tea extracts. Soxhlet pure water and 95% (v/v) ethanol extractions acted as reference runs at their boiling points for 12 h, with the details outlined in Price and Spitzer (1993). The mean contents of total oleoresin oils were 7.0- and 10.0-fold higher with adding 95% ethanol than without addition for green tea and Oolong tea, respectively. According to our results, adding 95% ethanol co-solvent (9.0 wt%) allowed us to extract more oleoresin oils. Also, Soxhlet ethanol extraction produced the highest ratios of TC/Caf for both teas as shown in Table 2. Numerous investigations have con"rmed the feasibility of extracting tea catechins with conventional solvents like acetonitrile}water (Goto et al., 1996), hot water (Lin et al., 1996; Price and Spitzer, 1993; Dalluge et al. 1998). Roedig-Penman and Gordon (1997) showed that methanolic tea extract contained higher levels of EGCg and ECg. These studies indicated that the ratio of total catechins to ca!eine (i.e., TC/Caf ) ranges from 2.5 to 5.4 (Goto et al., 1996), 1.3 to 5.4 (Lin et al., 1996), 3.3 to 3.7 (Price and Spitzer, 1993). Our results indicated that the ratio of TC/Caf was around 1.0 and above 5.0, for CO #95% ethanol (9.0 wt%) extraction and Soxhlet ethanol extrac tion, respectively. At the same time, a few experiments have attempted in this study to extract the oleoresin oil by Soxhlet ethanol extraction "rst, and then, extract ca!eine from the Soxhlet's solution by using supercritical carbon dioxide extraction. However, the ratio of TC/Caf did not improve at all. CONCLUSION This study has extracted the oleoresin oils from green and Oolong teas by using high-pressure carbon dioxide with the co-solvent addition. According to our results, both the extract and the ratio of TC/Caf increase with the ethanol content. Our results further demonstrate that the ratio of four major catechins to ca!eine (i.e., TC/Caf ) is the highest in Soxhlet ethanol extraction for both green and Oolong teas.
80
TABLE 2 Concentrations of ca!eine (Caf ), four major catechins (TC), gallic acid (GA), and total polyphenols (TPP) in tea extracts. (CO extractions at 400 L CO , 31 MPa, and 333 K) Solvent (co-solvent: 90 mL)
0201 0203 0605 0259 0202 0204 0302 0223
Green tea CO CO #H O CO #18% EtOH CO #70% EtOH CO #95% EtOH CO #99.8% EtOH Soxhlet H O Soxhlet 95% EtOH
0129 0130 0623 0201 0301 0312
Oolong tea CO CO #H O CO #45% EtOH CO #99.8% EtOH Soxhlet H O Soxhlet 95% EtOH
ND: not detectable.
X !- (w/w)
Weight (g/100 mL)
Ca!eine (mg/kg)
EGC (mg/kg)
EGCg (mg/kg)
ECg (mg/kg)
EC (mg/kg)
GA (mg/kg)
TC/Caf (HPLC)
TPP/Caf (Spectrometer)
1.00 0.91 0.89 0.90 0.91 0.91
0.05 0.08 0.09 0.31 0.35 0.32 2.06 5.20
218 223 394 1369 755 880 1056 951
93 122 96 96 290 290 610 1312
18 83 34 30 510 492 1336 2992
ND 8 3 11 105 89 202 459
ND 8 2 16 90 80 183 273
ND 10 3 1 7 7 380 120
0.15 0.99 0.34 0.11 1.33 1.08 2.20 5.29
0.73 1.17
1.00 0.91 0.90 0.91
0.04 0.06 0.07 0.40 2.86 4.92
171 281 318 608 919 546
92 96 97 273 697 1266
17 20 45 167 813 1428
ND ND 9 44 165 235
ND ND 7 95 222 253
2 7 4 5 235 36
0.64 0.41 0.50 0.95 2.06 5.82
1.50 1.28 2.96 5.81 ND ND 0.95 6.26 8.42
CHANG E¹ A¸.
Run no.
EXTRACTION OF POLYPHENOLS FROM TEA
81
ACKNOWLEDGEMENT The authors would like to thank the National Science Council (NSC) of the Republic of China for "nancially supporting this research under Contract No. NSC88-2214-E005-003.
REFERENCES Bronner, W. E. and Beecher, G. R. (1998). Method for determining the content of catechins in tea infusions by high-performance liquid chromatography. J. Chromatogr. A 805, 137}142. Carando, S., Teissedre, P. L., and Cabanis, J. C. (1998). Comparison of (#)-catechin determination in human plasma by high-performance liquid chromatography with two types of detection: #uorescence and ultraviolet. J. Chromatogr. B 707, 195}201. Chen, Z. P., Schell, J. B., Ho, C. T., and Chen, K. Y. (1998). Green tea epigallocatechin gallate shows a pronounced growth inhibitory e!ect on cancerous cells but not on their normal counterparts. Cancer ¸ett. 129, 173}179. Dalluge, J. J., Nelson, B. C., Thomas, J. B., and Sander, L. C. (1998). Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography. J. Chromatogr. A 793, 265}274. Davis, A. L., Lewis, J. R., Cai, Y., Powell, C., Davis, A. P., Wilkins, J. P. G., Pudney, P., and Cli!ord, M. N. (1997). A polyphenolic pigment from black tea. Phytochemistry 46(8), 1397}1402. Goto, T., Yoshida,Y., Kiso, M., and Nagashima, H. (1996). Simultaneous analysis of individual catechins and ca!eine in green tea. J. Chromatogr. A 749, 295}299. Ho, Y., Lee, Y. L., and Hsu, K. Y. (1995). Determination of (#)-catechin in plasma by high-performance liquid chromatography using #uorescence detection. J. Chromatogr. B 665, 383}389. Horie, H. and Kohata, K. (1998). Application of capillary electrophoresis to tea quality estimation. J Chromatogr. A 802, 219}223. King, M. B. and Bott, T. R. (1993). Extraction of Natural Products ;sing Near-Critical Solvents. Blackie, Glasgow. Larger, P. J., Jones, A. D., and Dacombe, C. (1998). Separation of polyphenols using micellar electrokinetic chromatography with diode array detection. J. Chromatogr. A 799, 309}320. Leanderson P., Faresjo A. O., and Tagesson C. (1997). Green tea polyphenols inhibit oxidant-induced DNA strand breakage in cultured lung cells. Free Radical Biol. Med. 23, 235}242. Lin, Y. L., Juan, I. M., Chen, Y. L., Liang, Y. C., and Lin, J. K. (1996). Composition of polyphenols in fresh tea leaves and associations of their oxygen-radical-absorbing capacity with antiproliferative actions in "broblast cells. J. Agric. Food Chem. 44, 1387}1394. Lin, J. K., Lin, C. L., Liang, Y. C., Lin-Shiau, S. Y., and Juan, I. M. (1998). Survey of catechins, gallic acid and methylxanthines in green, oolong, puerh and black teas. J. Agric. Food Chem. 46, 3635}3642. Otsuka, T., Ogo, T., Eto, T., Asano, Y., Suganuma, M., and Niho, Y. (1998). Growth inhibition of leukemia cells by (-)-epigallocatechin gallate, the main constituent of green tea. ¸ife Sci. 63, 1397}1403. Owuor, P.O. and Obanda, M. (1998). The changes in black tea quality due to variations of plunking standard and fermentation time. Food Chem. 61, 435}441. Paschka, A. G., Butler, R., and Young, C. Y.-F. (1998). Induction of apoptosis in prostate cancer cell lines by the green tea component, (-)-epigallocatechin-3-gallate. Cancer ¸ett. 130, 1}7. Poon, G. K. (1998). Analysis of catechins in tea extracts by liquid chromatography-electrospray ionization mass spectrometry. J. Chromatogr. A 794, 63}74. Price, W. E. and Spitzer, J. C. (1993). Variations in the amounts of individual #avanols in a range of green teas. Food Chem. 47, 271}276. Roedig-Penman A. and Gordon, M. H. (1997). Antioxidant properties of catechins and green tea extracts in model food emulsions. J. Agric. Food Chem. 45, 4267}4270. Tanaka, T., Kusano, R., and Kouno, I. (1998). Synthesis and antioxidant activity of novel amphipathic derivatives of tea polyphenol. Bioorg. Med. Chem. ¸ett. 8, 1801}1806. Tsuchiya, H., Sato, M., Kato, H., Kureshiro, H., and Takagi, N. (1998). Nanoscale analysis of pharmacologically active catechins in body #uids by HPLC using borate complex extraction pretreatment. ¹alanta 46, 717}726.
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CHANG E¹ A¸.
Vinson, J. A. and Dabbagh, Y. A. (1998). Tea phenols: antioxidant e!ectiveness of teas, tea components, tea fractions and their binding with lipoproteins. Nutr. Res. 18, 1067}1075. Zhu, B. T., Taneja, N., and Loder, D. P. (1998). E!ects of tea polyphenols and #avonoids on liver microsomal glycuronidation of estradiol and estrone. J. Steroid Biochem. Mol. Biol. 64, 207}215.
ORIGINAL ARTICLE Effect of Ethanol Content on Carbon Dioxide Extraction of Polyphenols from Tea C. J. Chang* , K. L. Chiu*, Y. L. Chen-, and P. W. Yang? Department of Chemical Engineering, National Chung-Hsing University, Taichung, Taiwan 40227, R.O.C. -Taiwan Tea Experiment Station, Pushin, Taiwan 32613, R.O.C. and ?Products Developing Department, Taiwan Sugar Company, Taipei, Taiwan 106, R.O.C. Received May 26, 1999, and in revised form June 23, 2000
This study presents a novel packed-column extractor coupled with an absorption system to improve the quality of oleoresin oils for Oolong and green teas, extracted by using carbon dioxide. The e!ect of various co-solvents on the extract is also examined. In addition, gravimetrical measurement and HPLC chromatographic analysis individually determine the amount of oleoresin oil and the concentration of four major catechins. According to those results, the mean contents in the extract are 7.0- and 10.0-fold higher in an addition of 95% ethanol than in no addition for green tea and Oolong tea, respectively. The ratio of four major catechins to ca!eine is the highest in Soxhlet ethanol extraction for both green and Oolong teas. 2001 Academic Press
Key Words: carbon dioxide; catechins; ca!eine; polyphenols; extraction.
INTRODUCTION Numerous epidemiological and pharmacological studies demonstrate that green tea extract possesses strong antioxidant e!ects (Roedig-Penman and Gordon, 1997; Vinson and Dabbagh, 1998; Lin et al., 1996, 1998; Tanaka et al., 1998) and antimutagenic activity (Paschka et al., 1998; Otsuka et al., 1998; Chen et al., 1998; Leanderson et al., 1997). Recently, great e!ort has been made to utilize polyphenolic compounds present in teas and a large number of polyphenolic monomers (catechins) have been characterized (Davis et al., 1997). According to those studies, four polyphenol compounds, epigallocatechin gallate (EGCg), epicatechin gallate (ECg), epigallocatechin (EGC), and epicatechin (EC) are signi"cant antioxidant constituents (Goto et al., 1996). Among those, EGCg is the most luxuriant component in tea extract (Bronner and Beecher, 1998; Zhu et al., 1998; Dalluge et al., 1998; Owuor and Obanda, 1998) and the most potent chemical tested for biological activity (Chen et al., 1998). High-performance liquid chromatography is the conventional means of analyzing catechins in tea and additional biological component constituents (Price and Spitzer, 1993; Bronner and Beecher, 1998; Goto et al., 1996; Ho et al., 1995; Carando et al., 1998; Tsuchiya et al., 1998; Zhu et al., 1998; Leanderson et al., 1997). Recent e!orts To whom correspondence and reprint requests should be addressed. Tel.: 886(04)-285-2592. Fax.: 886(04)-285-4734. E-mail: [email protected] 0889}1575/01/010075#08 $35.00/0
2001 Academic Press
76
CHANG E¹ A¸.
successfully analyzed tea catechins using capillary electrophoresis (Horie and Kohata, 1998), mass spectrometer (Poon, 1998), and micellar electrokinetic chromatography (Larger et al., 1998). However, to our knowledge, no study has separated catechins and ca!eine by using supercritical carbon dioxide extraction (King and Bott, 1993). In the light of the above discussion, this study presents a novel custom-designed supercritical #uid extraction technique coupled with an absorption system to extract polyphenols from green tea leaf. Controlling the extractive conditions allows us to evaluate the separation of ca!eine and polyphenols. Furthermore, the gravimetrical method can be applied to determine the amount in the extract. Moreover, HPLC chromatography is performed to quantify four major catechins (TC), along with a spectrophotometer used to measure the amount of total polyphenols (TPP). MATERIALS AND METHODS Green tea and Oolong tea were obtained from a commercial market. Phosphoric acid (85.1%), epicatechin gallate (ECg, 98%), epigallocatechin (EGC, 98%), epigallocatechin gallate (EGCg, 95%), epicatechin (EC, 98%), GR grade of gallic acid and ca!eine were purchased from Sigma Company (U.S.). All materials were used without further puri"cation. HPLC-grade acetonitrile (BDH, U.K.) was used as the mobilephase solvent. Absolute ethanol (99.8%) was obtained from RDH (Germany). 95% (v/v) ethanol was donated by Taiwan Tobacco and Wine Monopoly Bureau. Deionized water was obtained from a reverse-osmosis and ion exchange puri"ed water system (Barnstead, U.K.). Figure 1 schematically depicts a high-pressure apparatus used for CO extraction of tea polyphenols. The whole system consists mainly of a 100 mL-syringe ISCO pump, a refrigeration module, a 300 mL extraction vessel, one sight-gauge precipitator, two 1-L absorbent vessels, and a wet gas meter. A coil immersed in a water bath preheated liquid CO so that CO could attain the desired temperature prior to entering the extraction vessel. The electrically evenly heated extraction vessel was packed with 90 g of tea powder. The extractive temperature was measured by one K-type thermocouple and controlled by a proportional-integrator controller. The absorbing system (one precipitator and two absorbent vessels) was "lled with 1.4 L of 50% ethanol}water absorbent and maintained at 5.0 MPa and 297 K. The precipitator provides visual assurance that the saturated tea oils-laden supercritical CO , comes from the extraction vessel through a 1/16 in i.d. nozzle, sprayed into and well mixed with the absorbent. In this situation, the absorption pressure was maintained at 5.0 MPa and measured by a digital pressure transducer (Druck, PDCR910). Extraction and absorption pressures were controlled via two back-pressure regulators. Ten milliliter samples were taken at every 100 L CO volume, as measured by using a wet gas meter. Stock solutions of four catechins, gallic acid, and ca!eine were prepared by dissolving weighted quantities of commercial standards into water. Less concentrated solutions were prepared by diluting with deionized water. The soluble tea extracts from SFE-CO and Soxhlet extractions were centrifuged at 5000 rpm for 10 min. The supernatant was taken into a 10 mL syringe and "ltered through a 0.45-lm two-phase nylon membrane. A 10-lL volume of the "ltered supernatant was injected into the HPLC system. Calibration curves were constructed by linear regression of the peak} area ratio versus concentration. The measurement accuracy was within $2 mg/kg. The HPLC system consists of a Waters 600E multi-solvent delivery pump (Millipore, U.S.A.), a Waters 486 tunable wavelength detector (Millipore, U.S.A.), and a Waters U6K 100-lL sample injector (Millipore, U.S.A.). The analytical column was a reverse-phase Develosil ODS-HG column (150;4.5 mm, Nomura, Japan)
EXTRACTION OF POLYPHENOLS FROM TEA
77
FIGURE 1. Schematic of the diagram equipment used for CO extraction of tea polyphenols.
equipped with a guard column (10;4 mm, Nomura). The column was thermostatted at 403C and the #ow rate of the mobile phase was 1 mL/min. UV detection was achieved at a wavelength of 231 nm. Classic Millennium 2010 software was used for manipulation of the pump and data processing. The mobile phase used for analysis
78
CHANG E¹ A¸. TABLE 1 Gradient elution program for analysis of four catechins, ca!eine, and gallic acid
Time (min) Initial 3 7 12 17 20
A (%)
B (%)
95 90 90 70 60 60
5 10 10 30 40 40
FIGURE 2. HPLC chromatograms of green tea extracts. (a) Standard mixture; (b) extract at CO #95% EtOH; (c) extract at Soxhlet EtOH; (d) extract at Soxhlet H O.
was solvent A: 0.05% phosphoric acid solution; solvent B: acetonitrile. Table 1 lists the solvent gradient conditions. A spectrophotometer was used to measure total polyphenols and ca!eine, with the details outlined in Lin et al. (1996). All extracted samples were dried inside a vacuum oven, operated at 160 mm Hg, 333 K. Finally, the gravimetrical measurement was used to obtain the amount of the extract. Standard deviation (S.D.) was within $10%. RESULTS AND DISCUSSION Tea extracts were analyzed to understand the contents of four catechins, gallic acid, and ca!eine by HPLC. For carbon dioxide extraction a 10 mL sample was taken at every 100 L S.T.P. CO volume and the last sample of each run was analyzed. For Soxhlet extraction 250 mL solvent was used in each run and a 10 mL sample was taken for the analysis. Each datum represents the average of two replicates. Figures 2 and 3 display the chromatograms of these standard samples. Table 2 summarizes the extracted amounts and the concentrations of total polyphenols (TPP), four
EXTRACTION OF POLYPHENOLS FROM TEA
79
FIGURE 3. HPLC chromatograms of Oolong tea extracts. (a) Standard mixture; (b) extract at CO #95% EtOH; (c) extract at Soxhlet EtOH; (d) extract at Soxhlet H O.
major catechins (TC), gallic acid (GA), ca!eine (Caf ) in tea extracts. Soxhlet pure water and 95% (v/v) ethanol extractions acted as reference runs at their boiling points for 12 h, with the details outlined in Price and Spitzer (1993). The mean contents of total oleoresin oils were 7.0- and 10.0-fold higher with adding 95% ethanol than without addition for green tea and Oolong tea, respectively. According to our results, adding 95% ethanol co-solvent (9.0 wt%) allowed us to extract more oleoresin oils. Also, Soxhlet ethanol extraction produced the highest ratios of TC/Caf for both teas as shown in Table 2. Numerous investigations have con"rmed the feasibility of extracting tea catechins with conventional solvents like acetonitrile}water (Goto et al., 1996), hot water (Lin et al., 1996; Price and Spitzer, 1993; Dalluge et al. 1998). Roedig-Penman and Gordon (1997) showed that methanolic tea extract contained higher levels of EGCg and ECg. These studies indicated that the ratio of total catechins to ca!eine (i.e., TC/Caf ) ranges from 2.5 to 5.4 (Goto et al., 1996), 1.3 to 5.4 (Lin et al., 1996), 3.3 to 3.7 (Price and Spitzer, 1993). Our results indicated that the ratio of TC/Caf was around 1.0 and above 5.0, for CO #95% ethanol (9.0 wt%) extraction and Soxhlet ethanol extrac tion, respectively. At the same time, a few experiments have attempted in this study to extract the oleoresin oil by Soxhlet ethanol extraction "rst, and then, extract ca!eine from the Soxhlet's solution by using supercritical carbon dioxide extraction. However, the ratio of TC/Caf did not improve at all. CONCLUSION This study has extracted the oleoresin oils from green and Oolong teas by using high-pressure carbon dioxide with the co-solvent addition. According to our results, both the extract and the ratio of TC/Caf increase with the ethanol content. Our results further demonstrate that the ratio of four major catechins to ca!eine (i.e., TC/Caf ) is the highest in Soxhlet ethanol extraction for both green and Oolong teas.
80
TABLE 2 Concentrations of ca!eine (Caf ), four major catechins (TC), gallic acid (GA), and total polyphenols (TPP) in tea extracts. (CO extractions at 400 L CO , 31 MPa, and 333 K) Solvent (co-solvent: 90 mL)
0201 0203 0605 0259 0202 0204 0302 0223
Green tea CO CO #H O CO #18% EtOH CO #70% EtOH CO #95% EtOH CO #99.8% EtOH Soxhlet H O Soxhlet 95% EtOH
0129 0130 0623 0201 0301 0312
Oolong tea CO CO #H O CO #45% EtOH CO #99.8% EtOH Soxhlet H O Soxhlet 95% EtOH
ND: not detectable.
X !- (w/w)
Weight (g/100 mL)
Ca!eine (mg/kg)
EGC (mg/kg)
EGCg (mg/kg)
ECg (mg/kg)
EC (mg/kg)
GA (mg/kg)
TC/Caf (HPLC)
TPP/Caf (Spectrometer)
1.00 0.91 0.89 0.90 0.91 0.91
0.05 0.08 0.09 0.31 0.35 0.32 2.06 5.20
218 223 394 1369 755 880 1056 951
93 122 96 96 290 290 610 1312
18 83 34 30 510 492 1336 2992
ND 8 3 11 105 89 202 459
ND 8 2 16 90 80 183 273
ND 10 3 1 7 7 380 120
0.15 0.99 0.34 0.11 1.33 1.08 2.20 5.29
0.73 1.17
1.00 0.91 0.90 0.91
0.04 0.06 0.07 0.40 2.86 4.92
171 281 318 608 919 546
92 96 97 273 697 1266
17 20 45 167 813 1428
ND ND 9 44 165 235
ND ND 7 95 222 253
2 7 4 5 235 36
0.64 0.41 0.50 0.95 2.06 5.82
1.50 1.28 2.96 5.81 ND ND 0.95 6.26 8.42
CHANG E¹ A¸.
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ACKNOWLEDGEMENT The authors would like to thank the National Science Council (NSC) of the Republic of China for "nancially supporting this research under Contract No. NSC88-2214-E005-003.
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