Article pubs.acs.org/JAFC
Optimization of Subcritical Water Extraction of Flavanols from Green Tea Leaves Min-Jung Ko,† Chan-Ick Cheigh,‡ and Myong-Soo Chung*,† †
Department of Food Science and Engineering, Ewha Womans University, Seoul 120-750, South Korea Department of Food and Food Service Industry, Kyungpook National University, Sangju 742-711, South Korea
‡
ABSTRACT: The subcritical-water extraction (SWE) of six kinds of flavanols from green tea leaves and the effect of extraction conditions were investigated by varying the temperature and time. The maximum yield of total flavanols, 71.36 ± 4.23 mg/g green tea leaves (mean ± SD), was obtained under extraction temperature/time conditions of 150 °C/5 min. The efficiency of SWE for total flavanols was slightly higher than that of the conventional extraction solvents such as methanol and ethanol. The extraction of flavanols via SWE was specifically adequate for epimer structures such as catechin, catechin gallate, and gallocatechin gallate due to the epimerization of epicatechins. The extraction efficiency of epimers was increased at temperatures up to 170 °C, whereas that of epicatechins was decreased. Thus, most epicatechins were converted to epimers during SWE, leading to some flavanol destruction at high temperatures, except when a short extraction time of 5 min was used. KEYWORDS: subcritical-water extraction, flavanols, epimerization, green tea leaves
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INTRODUCTION The health benefits of green tea, which are reportedly attributable to its high content of flavanols, have been studied extensively. The findings of many experiments support their strong physiological actions, including antioxidant, anticarcinogenic, antimutagenic, and anti-inflammatory activities.1,2 Flavanols have a nuclear structure of C6−C3−C6, comprising two benzene rings connected by a pyrene ring. However, flavanols have no oxygen molecule at position 4, unlike other oxygen-containing flavonoids (Table 1). 3 Flavanols are epicatechin (EC), epicatechin gallate (ECG), epigallocatechin gallate (EGCG), catechin (Ct), catechin gallate (CG), and gallocatechin gallate (GCG). Epicatechins (EC, ECG, and EGCG) and epicatechin epimers (Ct, CG, and GCG) are partially converted into their isomers under high temperature.4 These compounds are usually extracted using organic solvents (such as ethanol and methanol) and pH differences, in a process that generally takes >30 min.5−8 The use of organic solvents in food products is limited by strict legal statutes because they can be toxic and harmful. However, subcritical purified water (≈10 MPa) can be used to extract compounds by varying the temperature-dependent dielectric constant (ε). As the temperature of water is increased, its polarity decreases (from ε = 53 at 110 °C to ε = 36.5 at 190 °C) and becomes similar to that of organic solvents such as ethanol (ε = 25 at 20 °C).9 Our previous studies10,11 have determined the extraction yields for flavonoids and similar flavanols from plants when using the subcritical-water extraction (SWE) process. The extraction yields obtained using subcritical water were more than 8-, 6-, and 4-fold higher than those obtained using ethanol, methanol, and hot water, respectively, for an extraction time of 150 °C. These results are analogous to those of Yang and Hildebrand,14 who found that temperatures >190 °C resulted in degradation, oxidation, and odorous extracts of flavonoids such as flavanols. The efficiency of flavanol extraction depends upon factors such as the type of solvent, extraction temperature, extraction time, ratio of solvent to solute, and sample particle size. In the present study, the effects of SWE temperature and processing time on flavonol yields from GTL were investigated. The major flavanols extracted from GTL were EGCG and GCG. As shown in Figure 2, the concentration of EGCG extracted decreased dramatically for longer extraction times. The SWE yield of the other epicatechins, EC and ECG, was also time-dependent. However, the concentration of GCG first increased and then decreased with a 15 min extraction time. The SWE yields of the epimers Ct and CG were dependent upon both processing temperature and time. These results are similar to those of Vuong et al.,15 who found that the epicatechin structure was more unstable than the epimer structure at temperatures >80 °C. Effect of Epimerization of Flavanols on Subcritical Water. The epicatechins (EC, ECG, and EGCG) and epimers (Ct, CG, and GCG) can in part be converted back to their isomers via a process of epimerization. An epimerization change is likely to occur in a high-temperature environment, such as when using SWE, because heating results in the conversions of EGCG to GCG, ECG to CG, and EC to Ct.16−18 Table 2 lists the effects of extraction time and temperature on the epimerization of flavanols in subcritical water. The proportion of flavanol was calculated as follows:
Figure 3. Typical phase diagram of ECs (■) and epimers (●) during SWE (solid line). The flavanol yield during a 5 min extraction time at various temperatures is shown (dotted-dashed line).
a few ECs were converted to epimers; in particular, the extraction efficiency of ECs for a short extraction time of 5 min (dotteddashed line of phase I) was increased because the optimum extraction conditions had been reached and then subsequently decreased due to epimerization. In phase II there was an increase in the extraction efficiency of epimers continued to 170 °C, whereas that of ECs decreased. This means that most ECs were converted to epimers and the optimization of temperature for extracting epimers was attained, leading to some destruction at high temperature. In phase III, at extremely high temperatures the unstable flavanols, including both ECs and epimers, were destroyed. This is likely to have resulted in negligible epimerization of flavanols, with the exception of epimers at an extraction time of 5 min (dotted-dashed line of phase III), in which there was increased extraction due to epimerization of ECs. Therefore, extracting flavanols based on chemical structure is adequate for epimer structures such as Ct, CG, and GCG due to the epimerization of flavanols at high temperatures in subcritical water. Comparison of Extraction Yields of Flavanols among Extraction Solvents. The adequacy of SWE relative to conventional extraction methods (using ethanol and methanol as solvents with extraction conditions of 60 °C and 2 h) was evaluated. As indicated in Figure 4, the maximum total yield of the six flavanols studied was 71.36 ± 4.23 mg/g GTL (mean ± SD) under extraction temperature/time conditions of 150 °C/5 min. The efficiency of SWE was slightly higher than that obtained using conventional extraction with either methanol (yield = 70.87 ± 0.57 mg/g GTL) or ethanol (yield = 60.43 ± 1.14 mg/g GTL). The data presented in Figure 4 indicate that SWE is more effective at extracting epimers than epicatechins, which is due to the relatively high extraction temperature. The efficiency of SWE (yield = 21.69 ± 1.41 mg/g GTL) for epimers including Ct, CG, and GCG was higher than that of the conventional extraction solvents such as methanol (yield = 7.68 ± 0.09 mg/g GTL) and ethanol (yield = 6.98 ± 0.01 mg/g GTL). Therefore, the concentration of epimers greatly influences the efficiency of the total flavanols yield for optimize extraction conditions. GCG is an epimer of EGCG; an epimerization change is likely to occur in a high-temperature environment such as when using SWE, because heating results in the conversion of EGCG to GCG.4 In the case of using organic solvents, small reductions were
proportion of flavanol (%) = compound concentration/(compound concentration + their isomer concentration) × 100
The conversion of flavanols to their isomers is reversible, but flavanols are usually only epimerized from ECs (2R,3R) to epimers (2S,3R) under high-temperature and high-pressure conditions with a long extraction time. As indicated by the results in Table 2, epimerization was accelerated over a long extraction time, thus reducing the proportions of ECs but gradually increasing those of epimers. In addition, ECs were epimerized more rapidly at higher extraction temperatures, thus again reducing the proportions of ECs and increasing those of epimers. However, at temperatures >170 °C there was very little epimerization, so that any remaining flavanols were destroyed. Figure 3 shows a typical phase diagram for the SWE of flavanol at different temperatures (110−190 °C). The extraction of flavanols can be divided into three phases (I, II, and III) on the basis of the predominating effect of extraction efficiency, epimerization, and destruction. In phase I there was a significant increase in epimer extraction efficiency by 150 °C. Furthermore, 6831
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Figure 4. Comparison of yields of EC, ECG, EGCG, Ct, CG, and GCG obtained using conventional methanol and ethanol extraction methods (under temperature/time conditions of 60 °C/2 h) and SWE method (under temperature/time conditions of 150 °C/5 min). All experiments were conducted in triplicate. Data are the mean and SD values.
molecule at position 4 were not adequate solutes at temperatures >170 °C in SWE. This finding is similar to that obtained with anthrone, whereby the presence of oxygen increases the solubility by 10 times over that of anthracene at SWE temperatures between 150 and 160 °C.9 These results confirm that flavonols and flavanones that have an oxygen molecule at position 4 are more favorably extracted at higher temperatures than are flavanol catechins in SWE.12 The yield of total flavanols from GTL was maximal under extraction temperature/time conditions of 150 °C/5 min. Pineiro et al.24 also reported that the extraction of catechins and epicatechins from grape seeds was optimum under SWE conditions of 130 °C/10 min. In addition, the chemical compositions and flavanols concentration of the extract obtained by SWE were compared with the initial composition of the original material. The proximate analysis including carbohydrates, protein, lipids, ash, fiber, catechins, and caffeine contents is presented in Table 3. These results indicate that approximately 86.26% of flavanols was recovered from the original GTL material. Especially, epicatechin epimers (Ct, CG, and GCG) of approximately 95.43% were extracted by using the SWE method from the initial GTL.
observed in the yield of unstable EGCG with long extraction times. The epimers Ct, CG, and GCG were poorly extracted with both methanol and ethanol. Because gallocatechin (GC) and epigallocatechin (EGC), which are known secondary major flavonols in green tea leaves, were very unstable at high operating temperatures (110−190 °C), the extraction yields by the SWE were not reproducible and those two compounds were excluded from consideration in this study. Wang and Helliwell19 also reported that GC and EGC were taking place in other reactions such as oxidation or degradation for a given set of SWE conditions. Zhu et al.20 and Yoshida et al.5 demonstrated that flavanols in alkaline solutions were extremely unstable and degraded almost completely, whereas in acidic solutions they were stable. Although high extraction yields of other epicatechins could be obtained in SWE due to the relatively acidic character of subcritical water, GC and EGC were easily degraded at high temperature and the extraction yields of those two compounds did not influence the efficiency of the total flavanols yield. Moreover, SWE has some excellent advantages: it is ecofriendly and nontoxic (using only purified water) and requires a shorter extraction time than conventional solventbased methods, which make it an efficient recovery method. Adequacy of Flavanol Extraction Based on Chemical Structure. The extraction of flavanols was affected by differences in physicochemical characteristics of the compounds, including their melting points and molecular weights. Flavanols have a lower melting point (at around 200 °C) than flavonoids.16 Thus, EGCG, with the lowest melting point, has been considered too unstable to allow extraction.21 Flavanols including the gallates have large molecular weights and a stable structure. In addition, like gallate, gallic acid is soluble in alcohol, ether, acetone, and water (at 1.5 g/100 mL and 20 °C). Therefore, gallate catechins such as CG, GCG, ECG, and EGCG were extracted using subcritical water at higher optimum temperatures (i.e., >150 °C), indicating a relatively low dielectric constant. The solubility in subcritical water is quantified by the solvent conditions and is influenced by the solute.9 The polarity of the subcritical water solvent is determined by the dielectric constant (ε), which is lowered by increasing the temperature; less polar compounds can thus be extracted by subcritical water acting as an organic polar solvent.22 Flavanols dissolve well in organic solvents such as ethanol;23 however, flavanols with no oxygen
Table 3. Chemical Composition of the GTL Used in This Study and the Extracts Yielded by SWEa component
/g GTL
/g freeze-dried SWE extracts
total carbohydrate (%) crude protein (%) crude lipids (%) crude ash (%) crude fiber (%) EC (mg) ECG (mg) EGCG (mg) Ct (mg) CG (mg) GCG (mg) caffeine (mg)
36.67 ± 0.76 15.03 ± 0.82 1.54 ± 0.14 5.82 ± 0.07 35.93 ± 0.25 7.16 ± 0.21 9.93 ± 0.37 42.90 ± 3.67 9.02 ± 0.41 2.01 ± 0.02 11.71 ± 0.93 24.23 ± 0.55
33.72 ± 2.23 24.18 ± 0.52 1.54 ± 0.35 17.59 ± 0.15 17.97 ± 2.22 6.05 ± 0.02 8.89 ± 0.85 34.74 ± 3.52 8.52 ± 0.19 1.76 ± 0.29 11.42 ± 1.80 12.36 ± 0.56
a
SWE was operated under optimum extraction temperature/time conditions of 150 °C/5 min. Data are the mean ± SD values of at least three measurements. 6832
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The ecofriendly SWE of flavanols is affected by both the subcritical-water solvent and the structure of the compounds. Flavanols lacking an oxygen molecule at position 4 are not adequate solutes at temperatures above 170 °C in SWE. However, the SWE was found to be slightly more efficient than extraction with two conventional solvents. SWE of the epicatechins EC, EGCG, and ECG was time-dependent, and SWE of the epimer catechins Ct, GCG, and CG was both temperature- and time-dependent. In addition, SWE appears to be a more effective solvent for extracting epimers compared to the precursor epicatechins. Thus, SWE appears to be an environmentally friendly, nontoxic, efficient, and selective extraction method that requires shorter extraction times than its conventional organic-solvent counterpart. SWE could have practical applications in the effective extraction of antioxidant polyphenolic compounds from various types of plant. Furthermore, this method can be easily implemented on an industrial scale.
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AUTHOR INFORMATION
Corresponding Author
*(M.-S.C.) Phone: +82-2-32774508. Fax: +82-2-32774508. Email:
[email protected]. Funding
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2013R1A1A2A10060325). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED Ct, catechin; CG, catechin gallate; EC, epicatechin; ECG, epicatechin gallate; EGC, epigallocatechin; EGCG, epigallocatechin gallate; GC, gallocatechin; GCG, gallocatechin gallate; GTL, green tea leaves; HPLC, high-performance liquid chromatography; SWE, subcritical-water extraction
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