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DOI: 10.1039/C5RA15830C (Paper) RSC Adv., 2015, 5, 93937-93944

Efficient extraction of major catechins in Camellia sinensis leaves using green choline chloride-based deep eutectic solvents

Ji Li† ab, Zhigang Han†a, Yongpeng Zoua and Bo Yu*ab
aDepartment of Cardiology, 2nd Affiliated Hospital of Harbin Medical University, Harbin 150001, PR China. E-mail: dryu_hmu@163.com; Fax: +86 451 86297221; Tel: +86 451 86297221
bKey Laboratory of Myocardial Ischemia, Ministry of Education, Harbin Medical University, Harbin 150001, PR China

Received 7th August 2015 , Accepted 28th October 2015

First published on 28th October 2015

Abstract

In this study, an efficient microwave-assisted extraction (MAE) method using green deep eutectic solvents (DESs) was developed for the extraction of the major catechins in Camellia sinensis leaves. A series of choline chloride-based DESs were prepared, and DES-4, which was composed of choline chloride and lactic acid, was observed to possess the best extraction performance for the target catechins. Moreover, the main factors affecting the extraction efficiency were optimized using Box–Behnken design (BBD) combined with response surface methodology (RSM), and the optimal conditions were as follows: temperature 66 °C, duration 8 min and solvent/material ratio of 35 mL g−1. Under these conditions, a total extraction yield of 153.7 ± 5.2 mg g−1 was obtained. In addition, the separation of the target catechins from DES-based extraction solution was performed using AB-8 macroporous resin with recovery yields from 75.2% to 86.1%. The present study provided fundamental data for the development of DES-based extraction methods in food and pharmaceutical fields.

1. Introduction

Camellia sinensis, belongs to the Theaceae family, is mainly cultivated in China and Southeast Asia.1 It has been demonstrated that Camellia sinensis leaves, which can be processed into several varieties of tea, have various health benefits, such as preventing cardiovascular disease, chronic gastritis and several cancers, due to the presence of polyphenolic compounds, especially catechins.2–4 According to previous research, Camellia sinensis leaves are rich in catechins, among which (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) are the major components.5 Pharmacological studies have shown that these catechins can exert outstanding antioxidant activity as well as anti-bacterial, anti-viral, anti-inflammatory, anti-allergic, anti-hypertensive, anti-obesity and anti-diabetic properties.6–8 Nowadays, they have been widely used in food and pharmaceutical fields.9 Therefore, it is of practical significance to develop an efficient extraction method for obtaining the major catechins in Camellia sinensis leaves.

At present, conventional organic solvents, such as methanol, ethyl acetate and chloroform, remain the most frequently used solvents for extraction of bioactive compounds from plant materials. However, from the viewpoint of environmental friendliness, these organic solvents can no longer meet the trend of green chemistry. For this reason, it is required to develop green solvents to improve the existing extraction methods. In recent years, deep eutectic solvent (DES) has attracted increasing attentions as an excellent alternative to the conventional organic solvents.10 DES is generally composed of two safe components that are capable of associating with each other through hydrogen bonds and often contains water in certain ratio. It is mostly formed by mixing quaternary ammonium salts with a range of hydrogen bond donors, such as alcohols, organic acids, saccharides and amino acids.11,12 Compared with conventional organic solvents, DES possesses many preferable characteristics, including safety, non-toxicity, biodegradability, sustainability, low cost and easy preparation. Moreover, it shows good physicochemical properties as an ideal solvent: adjustable viscosity, negligible volatility, wide polarity range and high dissolving capacity for a variety of compounds, including the poorly water-soluble ones.13,14 Actually, DES has been applied for chemical synthesis, analytical preparation, electrochemistry and nanotechnology, aiming at increasing efficiency and reducing pollution.15–17

Besides solvent, extraction method significantly affects the extraction efficiency. As a high-performance extraction method, microwave-assisted extraction (MAE) has many advantages, such as short extraction duration, low solvent consumption and high extraction efficiency, and we have studied the rapid extraction of triterpene saponins and organic acids using MAE method.18–21 However, to the best of our knowledge, the combination of MAE and DES for the extraction of catechins has not been reported yet. Therefore, in this study, an efficient DES–MAE method for the extraction of the major catechins in Camellia sinensis leaves was developed. A series of choline chloride-based DESs were prepared, and their extraction performances for the target catechins were investigated. Furthermore, the main factors of DES–MAE were optimized, and their effects on the extraction efficiency were analyzed. In addition, the separation of the target catechins from extraction solution was attempted using AB-8 macroporous resin.

2. Materials and methods

2.1. Materials

Camellia sinensis leaves were purchased from local market in Laoshan (Shandong, China). The leaves were dried to constant weight using a vacuum oven, and then pulverized by a disintegrator. After that, the plant material was sieved between 10- and 20-mesh and stored in a sealed plastic bag at room temperature.

2.2. Chemicals and reagents

Choline chloride (>98.0%), ethylene glycol (>99.0%), glycerol (>99.0%), 1,4-butanediol (>98.0%), lactic acid (>98.0%), malic acid (>98.0%), citric acid (>98.0%), glucose (>99.0%), fructose (>99.0%) and sucrose (>99.0%) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

Methanol and formic acid of chromatographic grade were purchased from J&K Scientific Ltd. (Beijing, China). Ethanol of analytical grade was purchased from Beijing Chemical Reagents Co. (Beijing, China). Deionized water was produced by Milli-Q system (Bedford, MA, USA). AB-8 macroporous resin was purchased from the Chemical Plant of Nankai University (Tianjin, China).

The standards of (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) were purchased from Yuanye Biotechnology Ltd. (Shanghai, China) and their purities were ≥98%.

2.3. HPLC analysis

The quantification of EC, EGC, ECG and EGCG was performed using an Agilent 1200 series HPLC system (Agilent, San Jose, CA, USA) equipped with a quaternary solvent delivery system, a diode array detector and a column temperature controller. Chemical Station software (Rev B. 03. 02, Agilent Technologies) was used for system control and data acquisition. The chromatographic separation of the target catechins was performed on a Luna C18 column (250 mm × 4.6 mm i.d., 5 μm, Phenomenex, Torrance, CA, USA). The column temperature was kept at 30 °C and the injection volume was 5 μL. The mobile phase was composed of water containing 0.1% (v/v) formic acid (A) and methanol (B), which were applied in the following gradient elution program for separation: 0–5 min, A–B (80[thin space (1/6-em)]:[thin space (1/6-em)]20, v/v); 5–10 min, linear gradient to A–B (70[thin space (1/6-em)]:[thin space (1/6-em)]30, v/v); 10–20 min, linear gradient to A–B (60[thin space (1/6-em)]:[thin space (1/6-em)]40, v/v); 20–25 min, linear gradient to A–B (50[thin space (1/6-em)]:[thin space (1/6-em)]50, v/v). The flow rate was 1.0 mL min−1 and the ultraviolet spectrum was monitored at 280 nm.

2.4. DESs preparation

In this study, three kinds of hydrogen bond donors, including alcohols, organic acids and saccharides, were used for the preparation of choline chloride-based DESs. Each of the choline chloride-based DESs was prepared by heating choline chloride and one of the hydrogen bond donors to 80 °C with stirring until a homogeneous liquid was formed. The prepared DESs were listed in Table 1.
Table 1 List of the prepared choline chloride based-DESs
Abbreviation Component 1 Component 2 Molar ratio
DES-1 Choline chloride Ethylene glycol 1[thin space (1/6-em)]:[thin space (1/6-em)]2
DES-2 Glycerol
DES-3 1,4-Butanediol
DES-4 Lactic acid
DES-5 Malic acid
DES-6 Citric acid
DES-7 Glucose
DES-8 Fructose
DES-9 Sucrose

2.5. MAE procedure

The MAE process was performed using a digital microwave-assisted extractor (Sineo Microwave Chemistry Technology Co., Ltd., Shanghai, China) which allowed the variations of temperature and duration by a digital control panel. One gram of the plant material was placed into an extraction flask, and afterwards certain amount of extraction solvent was added. Besides the prepared DESs, as widely used food grade solvents, aqueous ethanol solution (80% ethanol) and water were used for the extraction of catechins according to previous research.22 Then, the extraction was automatically carried out by a presetting program. After extraction, the mixture was vacuum filtered, and the obtained extraction solution was analyzed by HPLC. The chromatogram of an extraction solution using DES–MAE was shown in Fig. 1. The preliminary MAE conditions were as follow: temperature 50 °C, duration 5 min and solvent/material ratio 30 mL g−1. Each of the extraction process was performed in triplicate and the experimental data were expressed as the mean ± SD of three parallel measurements.
image file: c5ra15830c-f1.tif
Fig. 1 HPLC chromatogram of an extraction solution using DES–MAE. Peaks: (A) EGC; (B) EGCG; (C) EC; (D) ECG.

2.6. Experimental design and statistical analysis

In order to obtain a high extraction yield of the target catechins, the main factors affecting the extraction efficiency of DES–MAE were optimized. As the factors affecting the physicochemical properties of extraction solvent, choline chloride/lactic acid molar ratio (1[thin space (1/6-em)]:[thin space (1/6-em)]1, 1[thin space (1/6-em)]:[thin space (1/6-em)]2, 1[thin space (1/6-em)]:[thin space (1/6-em)]3, and 1[thin space (1/6-em)]:[thin space (1/6-em)]4) and water content (v/v) in DES-4 (0%, 20%, 40% and 60%) were first studied by single factor experiments. Furthermore, based on the pre-experimental results (data not shown), Box–Behnken design (BBD) combined with response surface methodology (RSM) was applied to investigate the effects of three extraction parameters (independent variables), including temperature, duration and solvent/material ratio, at three levels (−1, 0 and +1), on the total extraction yield of the target catechins, which was taken as the response. In this test, 15 experimental runs included three replicates at the center point were conducted in random order. The coded and the actual levels of the variables set in the present experimental design were shown in Table 2, and the experimental data were employed to fit a second-order polynomial model.
Table 2 The coded and actual levels of the variables (temperature, duration and solvent/material ratio) set in BBD and the response values (the extraction yields of the target catechins)
Run Extraction parameters Extraction yield (mg g−1)
Temperature (°C) Duration (min) Solvent/material ratio (mL g−1) EGCG EGC ECG EC Total Predicted value
a Center point.
1 −1 (45) −1 (3) 0 (30) 32.2 15.3 6.5 5.3 59.3 59.1
2 1 (75) −1 (3) 0 (30) 41.6 20.8 8.2 6.9 77.5 76.3
3 −1 (45) 1 (9) 0 (30) 60.7 26.3 10.6 8.8 106.4 107.5
4 1 (75) 1 (9) 0 (30) 76.9 35.8 15.1 10.7 138.5 136.9
5 −1 (45) 0 (6) −1 (20) 29.8 12.5 5.3 5.0 52.6 55.0
6 1 (75) 0 (6) −1 (20) 35.1 15.0 7.4 6.8 64.3 66.7
7 −1 (45) 0 (6) 1 (40) 48.3 23.4 9.0 8.3 89.0 86.6
8 1 (75) 0 (6) 1 (40) 72.5 32.2 13.5 9.7 127.9 125.4
9 0 (60) −1 (3) −1 (20) 26.2 10.1 5.3 4.8 46.4 44.1
10 0 (60) 1 (9) −1 (20) 47.0 29.4 8.8 8.6 93.8 91.2
11 0 (60) −1 (3) 1 (40) 43.1 21.3 7.7 7.2 79.3 81.9
12 0 (60) 1 (9) 1 (40) 78.6 35.9 15.2 11.5 141.2 143.5
13a 0 (60) 0 (6) 0 (30) 77.2 35.0 13.3 12.6 138.1 138.5
14a 0 (60) 0 (6) 0 (30) 75.4 34.5 14.8 12.2 136.9 138.5
15a 0 (60) 0 (6) 0 (30) 76.3 36.7 15.6 11.4 140.0 138.5

Design-Expert 8.0.5 software (Trial version, State-Ease Inc., Minneapolis, MN, USA) was used for the experimental design and regression analysis. The statistical significance of the regression coefficient was checked by F-test. Analysis of variance (ANOVA) was applied to estimate the adequacy of the proposed model by evaluating lack of fit, coefficient of determination (R2) and F-value.

2.7. Heating reflux extraction procedure

Conventional heating reflux extraction (HRE) method using DES was performed for the comparison of extraction efficiency with DES–MAE. One gram of the plant material was placed into an extraction flask, and afterwards 35 mL of DES-4 (20% water content) was added. Then, the extraction was carried out at 66 °C (the optimal temperature for MAE) using a warmer for 8, 30 and 60 min, respectively. After each extraction, the mixture was filtered, and the obtained solution was analyzed by HPLC. Each of the extraction process was performed in triplicate and the experimental data were expressed as the mean ± SD of three parallel measurements.

2.8. Separation of the target catechins from extraction solution

The separation of the target catechins from DES-based extraction solution was carried out using a glass column (10 mm × 500 mm) wet-packed with 10 g of AB-8 macroporous resin (dry weight). The bed volume (BV) was 20 mL.

The extraction solution flowed through the column at the flow rate of 3 BV per h, and the concentrations of the target catechins in 0.5 mL effluents collected at 5 mL intervals were monitored by HPLC. While adsorptive equilibrium, the adsorbate-laden column was first washed with 3 BV of deionized water and afterwards eluted with 90% aqueous ethanol solution (v/v) at the flow rate of 6 BV per h. The concentrations of the target catechins in 0.5 mL eluents collected at 10 mL intervals were determined by HPLC. Then, the ethanolic eluents were concentrated to dryness through vacuum rotary evaporation, and the recoveries of the target catechins were calculated. The separation process was performed in triplicate and the results were expressed as the means of three parallel measurements.

3. Results and discussion

3.1. Selection of DESs

The composition of DES has significant effect on its extraction performance.23 In order to select a suitable DES for the extraction of the target catechins, nine choline chloride-based DESs were prepared, and their extraction performances for the target catechins were investigated. Moreover, it has been reported that the viscosity of DES is generally high, which hinders the mass transfer of compounds from plant matrix to extraction solvent, hence 20% (v/v) water was added into the prepared DESs for decreasing their viscosities.24 The extraction processes were performed as described in Section 2.5. As shown in Fig. 2, the differences in the total extraction yields using the prepared DESs and the reference solvents were obvious, and DES-4 represented the predominant extraction performance on account of its physicochemical properties, such as solubility, polarity and viscosity. The results indicated that the extraction performance of choline chloride-based DES was depended on its hydrogen bond donor, and the effect of lactic acid, which contributed to higher extraction yields of the target catechins, was superior to those of the other hydrogen bond donors. Based on the results, DES-4, composed of choline chloride and lactic acid, was selected as the suitable extraction solvent.
image file: c5ra15830c-f2.tif
Fig. 2 Extraction yields of the target catechins using different DESs and reference solvents.

3.2. Effect of choline chloride/lactic acid molar ratio

In order to investigate the effect of choline chloride/lactic acid molar ratio on the extraction performance of DES-4, the extraction processes were performed with DESs of different choline chloride/lactic acid molar ratios from 1[thin space (1/6-em)]:[thin space (1/6-em)]1 to 1[thin space (1/6-em)]:[thin space (1/6-em)]4. Fig. 3A showed that the extraction yields of the target catechins were found to increase with the change of choline chloride/lactic acid molar ratio from 1[thin space (1/6-em)]:[thin space (1/6-em)]1 to 1[thin space (1/6-em)]:[thin space (1/6-em)]2. It was mainly because of the decrease in the viscosity of DES-4, which facilitated the mass transfer of the target catechins from plant material to extraction solvent. However, the extraction yields gradually decreased when the choline chloride/lactic acid molar ratio changed from 1[thin space (1/6-em)]:[thin space (1/6-em)]2 to 1[thin space (1/6-em)]:[thin space (1/6-em)]4. This phenomenon was similar to the reported results by Wei et al.,24 and it was probably due to the decrease in the amount of choline chloride, which led to the reduction of proportion of hydrogen bond receptors in the DES system. Therefore, the choline chloride/lactic acid molar ratio of 1[thin space (1/6-em)]:[thin space (1/6-em)]2 was selected for the subsequent experiments.
image file: c5ra15830c-f3.tif
Fig. 3 Extraction yields of the target catechins using DESs of different factors: (A) choline chloride/lactic acid molar ratio; (B) water content.

3.3. Effect of water content in DES

In order to investigate the effect of water content on the extraction performance of DES, the extraction processes were performed with DESs of different water contents (0, 20%, 40% and 60%). It was observed in Fig. 3B that, as 20% water was added into DES-4, the extraction yields of the target catechins increased by about 20%. However, sequentially increasing the water content in DES-4 caused a significant decrease in the extraction yields. It has been reported that the addition of water will lead to a decrease in the viscosity of DES, and a decrease in the hydrogen-bonding interactions between the components of DES as well. Aiming at this problem, Dai et al. carried out detailed experiments and demonstrated that although the addition of water would cause the hydrogen-bonding interactions gradually weakened, DES could still possess the supermolecular characteristics with less than 50% water content.25 Consequently, proper addition of water not only can effectively reduce the viscosity of DES but also keep its structure, which may greatly facilitate the mass transfer process, thus 20% water content in DES-4 was selected for the subsequent experiments.

3.4. Optimization of DES–MAE

3.4.1. Statistical analysis and model fitting. To determine the optimal combination of the extraction parameters, the results obtained from 15 experimental runs of BBD were shown in Table 2. By applying multiple regression analysis on the experimental data, the proposed model was expressed as the following second-order polynomial equation for the response and variables in terms of coded levels:
Y = 138.50 + 12.61A + 27.18B + 22.54C + 3.48AB + 6.80AC + 3.62BC − 24.90A2 − 18.17B2 − 30.15C2
where Y was the total extraction yield of the target catechins, A was temperature, B was duration and C was solvent/material ratio. The results of the regression analysis indicated that a maximal total extraction yield of 157.3 mg g−1 target catechins (87.3 mg g−1 EGCG, 40.5 mg g−1 EGC, 16.8 mg g−1 ECG and 12.7 mg g−1 EC) could be obtained under the optimal conditions as follow: temperature 65.6 °C, duration 8.3 min and solvent/material ratio 34.7 mL g−1, and it was close to the predicted data which were respectively optimized for the target catechins (Table 3). For convenience, the actual extraction conditions were simplified as follow: temperature 66 °C, duration 8 min and solvent/material ratio 35 mL g−1.
Table 3 The optimal conditions for the extraction of the target catechins and the predicted extraction yields
Target catechins Temperature (°C) Duration (min) Solvent/material ratio (mL g−1) Extraction yield (mg g−1)
EGCG 66.3 8.5 35.3 87.6
EGC 65.1 8.6 34.5 40.7
ECG 67.8 8.7 34.8 16.9
EC 62.7 8.3 34.3 12.9

ANOVA for the proposed model was shown in Table 4. The significance of each coefficient was checked using F-test and by determining the p-value. A model term was considered significant when its p-value was less than 0.05. According to the ANOVA results, the high F-value (252.85) and low p-value (<0.0001) indicated that the model was suitable for the extraction process. Furthermore, the lack of fit was used to measure the failure of the model to represent the experimental data that were not included in the regression analysis, and the F-value of 0.78 implied the lack of fit was not significant relative to the pure error. The coefficient of determination (R2) was 0.9978, which suggested a high agreement between the observed and the predicted values by the model. Meanwhile, a low value 2.69 of the coefficient of variation (C.V.) showed a high degree of precision and a good deal of reliability of the experimental data. In summary, the results demonstrated that the model was adequate to represent the relationship between the response and the variables. Therefore, we concluded the proposed model was statistically sound.

Table 4 ANOVA of the proposed model for the extraction of the target catechins
Source Sum of squares Degree of freedom Mean square F-value p-value
Model 17[thin space (1/6-em)]537.39 9 1948.60 182.04 <0.0001
A 1272.60 1 1272.60 118.88 0.0001
B 5907.84 1 5907.84 551.90 <0.0001
C 4063.51 1 4063.51 379.61 <0.0001
AB 48.30 1 48.30 4.51 0.0870
AC 184.96 1 184.96 17.28 0.0089
BC 52.56 1 52.56 4.91 0.0775
A2 2289.27 1 2289.27 213.86 <0.0001
B2 1219.68 1 1219.68 113.94 0.0001
C2 3356.39 1 3356.39 313.55 <0.0001
Residual 53.52 5 10.70    
Lack of fit 48.70 3 16.23 6.74 0.1320
Pure error 4.82 2 2.41    
R-squared = 0.9970 C.V. = 3.29

In addition, it was observed in Table 4 that the linear coefficients (A, B and C), cross product coefficients (AC) and quadratic term coefficients (A2, B2 and C2) were considered to be significant (p < 0.05), but the cross product coefficients (AB and BC) were considered to be not significant (p > 0.05). The results indicated that temperature, duration and solvent/material ratio had important effects on the total extraction yield of the target catechins, and there was a great interaction between temperature and solvent during the extraction process.

3.4.2. Response surface analysis. As the graphical representations of the model equation, response surface plots were used to visualize the effects of variables on the response. The ANOVA results indicated that temperature, duration and solvent/material ratio were all significant variables, and their effects on the total extraction yield of the target catechins were shown in Fig. 4.
image file: c5ra15830c-f4.tif
Fig. 4 Response surface plots for the extraction of the target catechins: (A) varying temperature and duration; (B) varying temperature and solvent/material ratio; (C) varying duration and solvent/material ratio.

Temperature is always a key parameter of an extraction method, especially for the extraction of thermosensitive compounds. It was observed in Fig. 4A and B that the total extraction yield increased by raising temperature from the beginning of 45 °C and reached a maximum at about 65 °C. According to previous research, increasing temperature generally decreases the viscosity and increases the diffusivity of DES, which can facilitate the penetration of DES into the plant matrix, leading to more destruction of intermolecular interactions, and thus enhancing the dissolution of the target compounds.26 However, when temperature exceeded 65 °C, the total extraction yield tended to decrease, which was mainly due to the isomerization of the target catechins under exorbitant temperature. Prasad et al. also reported that a further raise in extraction temperature (over 58 °C) caused the chemical degradation of phenolic compounds.27

In the viewpoint of efficient extraction and saving energy, extraction duration was optimized in the present study. As shown in Fig. 4A and C, the extension of duration had a positive effect on the total extraction yield. Moreover, the plots indicated that the total extraction yield changed little when the duration was extended over 8 min. Thus, it was considered that 8 min was enough to extract most of the target catechins in Camellia sinensis leaves using DES–MAE method.

Solvent/material ratio is another important parameter affecting the extraction efficiency, because the target compounds in plant material could not be extracted completely using a low solvent/material ratio, while excessive amount of solvent will cause higher cost. Fig. 4B and C showed that the total extraction yield increased with the increase in solvent/material ratio from 20 to 35 mL g−1 and decreased with the further increase in solvent/material ratio. Tsiaka et al. also found such phenomenon in their study of the extraction of carotenoids using MAE method.28 Generally, higher solvent volumes increase the recovery rates in conventional extraction methods. However, it has been reported that a higher solvent volume may lead to a lower extraction yield using MAE method, because the distribution of microwave power is more uniform in a suitable level of solvent/material ratio than in a higher level.29

The response surface plots were also used to represent the interactions between the variables. As shown in Fig. 4B, the elliptical contour plot indicated that the interaction between temperature and solvent/material ratio was significant, which coincided with the ANOVA results. This result was probably related to the effect of temperature on the viscosity, diffusivity and solubility of DES. Meanwhile, the circular contour plots (Fig. 4A and C) showed that the interactions between duration and each other extraction factors were not significant.

3.5. Verification of the predictive model

The adequacy of the proposed model can be verified by comparing the predicted and the experimental values of the total extraction yield of the target catechins under the optimal conditions, so a verification experiment was carried out in three replicates. Under the simplified optimal conditions, a total extraction yield of 153.7 ± 5.2 mg g−1 target catechins (85.9 ± 2.3 mg g−1 EGCG, 39.7 ± 1.5 mg g−1 EGC, 15.6 ± 0.8 mg g−1 ECG and 12.5 ± 0.6 mg g−1 EC) was obtained, which was in good agreement with the predicted values (total 157.3 mg g−1: 87.3 mg g−1 EGCG, 40.5 mg g−1 EGC, 16.8 mg g−1 ECG and 12.7 mg g−1 EC). The results demonstrated that the proposed model was adequate for the extraction process.

3.6. Comparison of extraction methods

The comparison of MAE with HRE using DES for the extraction of the target catechins was carried out. It was observed in Table 5 that, in the case of the same extraction parameters, the total extraction yield of the target catechins using MAE was about 2-fold to that using HRE. Moreover, the results indicated that the target catechins were almost completely extracted using HRE for 60 min, and the total extraction yield was similar to that using MAE for 8 min. The better extraction efficiency of MAE was concerned with the mechanical effects of internal heating based on conduction and dielectric polarization caused by microwave irradiation, and the pressure built up within the cells leading to an efficient delivery to the plant material through the molecular interaction with the electromagnetic field.30 Therefore, MAE facilitated desorption and release of the target catechins from plant matrix, thus accelerating the extraction process as well as increasing the extraction yield.
Table 5 Comparison of the extraction yields of the target catechins by MAE and HRE using DES
Extraction method Temperature (°C) Solvent/material ratio (mL g−1) Duration (min) Extraction yield (mg g−1)
EGCG EGC ECG EC Total
MAE 66 35 8 85.9 ± 2.3 39.7 ± 1.5 15.6 ± 0.8 12.5 ± 0.6 153.7 ± 5.2
8 47.4 ± 2.0 20.2 ± 1.1 8.5 ± 0.3 7.3 ± 0.4 83.4 ± 3.8
HRE 66 35 30 78.2 ± 1.7 37.6 ± 1.6 13.0 ± 0.7 11.8 ± 0.3 140.6 ± 4.3
60 84.1 ± 1.4 40.5 ± 0.9 14.3 ± 0.5 12.9 ± 0.4 151.8 ± 3.2

3.7. Separation of target catechins from extraction solution

In this study, the separation of the target catechins from DES-based extraction solution was carried out using AB-8 macroporous resin. The target catechins completely flowed out of the column in about 9 BV, and after one run treatment with the resin, the contents of EGCG, EGC, ECG and EC in the product reached 42.8%, 12.4%, 7.9% and 4.5%, which were 4.9, 3.2, 5.1 and 3.6-fold to those in the extract under the optimal conditions, and the recovery yields of the four target catechins were 86.1%, 76.3%, 84.5% and 75.2%, respectively. It was indicated that the polar components of DES could be eluted with deionized water, and the target catechins could be easily obtained by eluting with 90% aqueous ethanol. The results demonstrated that the resin was effective for the adsorption and desorption of the target catechins. Therefore, it was efficient to separate the target catechins from the extraction solution of DES–MAE using AB-8 macroporous resin.

4. Conclusion

In this study, an efficient and green DES–MAE method was developed for the extraction of the major catechins in Camellia sinensis leaves. It was proved that the prepared DES, composed of choline chloride and lactic acid, possessed an excellent extraction performance for the catechins, which suggested that it had great potential to be used for the extraction of polyphenolic compounds. The results support that DES is an ideal extraction solvent, which meets the trend of green chemistry, due to its good physicochemical properties, green characteristics and outstanding extraction performances. Moreover, the separation of the target catechins from DES-based extraction solution was conveniently performed using AB-8 macroporous resin with high recovery yields. In conclusion, DES–MAE combined with macroporous resin separation has the potential to be widely extended for obtaining bioactive compounds from plant materials in food and pharmaceutical fields.

Acknowledgements

The authors gratefully acknowledge the financial supports by Special Fund of National Natural Science Foundation of China (81171430), Natural Science Foundation of Heilongjiang Province (H2015005) and Fund of Health and Family Planning Commission of Heilongjiang Province (2014-338).

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Footnote

† These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2015
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