1. Introduction
Currently, the tea plant (
Camellia sinensis) is cultivated in many countries, such as China and Japan, and tea has become a universally consumed beverage around the world [
1]. Green tea has the simplest manufacturing process but the most excellent benefits, and tea antioxidant polyphenols (TPs) constitute the majority of advantageous components in green tea [
2]. Catechins are the principal components of tea antioxidant polyphenols, which mainly include two non-ester catechins, (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), and two ester catechins, (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG) [
3,
4]. These catechins show potential health benefits, such as anticancer, anti-obesity, antibacterial, antioxidant, and antiviral effects [
5,
6,
7,
8]. Nowadays, catechins have been widely applied in the pharmaceutical and food industry. Therefore, it is of great importance to establish an eco-friendly and efficient extraction method for extracting antioxidant polyphenols in green tea.
To date, conventional organic solvents, such as ethanol, acetone, and chloroform, remain the most commonly used solvents to extract bioactive compounds from plant materials [
9,
10]. However, some organic solvents have several distinct disadvantages, such as high cost, high level of residue, and toxicity [
10,
11]. Therefore, it is of great necessity to develop new environmentally friendly “green” media to meet the requirements of sustainable development. Recently, the concepts of the eco-sustainability and green economy have led to increased concern being paid to deep eutectic solvents (DESs), new solvents that are emerging as a greener and promising substitute for conventional organic solvents [
12,
13,
14,
15,
16]. DESs fully meet the principles of green chemistry and are generally recognized as safe (GRAS) [
17]. DESs possess some distinct advantages compared to conventional solvents, such as favorable thermal stability, high biodegradability, easy preparation, low cost, and toxicity [
18,
19,
20]. Generally, DESs are prepared by the self-association of hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA). In the preparation of DESs, ChCl is the most widely used HBA, and ChCl-based DESs have been frequently used in various research fields, including the extraction of bioactive compounds from plant materials [
21,
22,
23,
24]. Another principle of green extraction is reduced energy consumption by innovative technologies, such as ultrasound [
25]. DES-based ultrasound-assisted extraction (UAE) can not only greatly reduce the consumption of solvents, energy and labor, but also can destroy the structure of plant cell walls by acoustic cavitation, and then enhance the yield of bio-active constituents [
26,
27].
In this study, we developed a rapid and efficient method to extract antioxidant polyphenols from green tea by combining the ultrasonic technique with DESs (UAE-DES), which was rarely reported previously. Among the 12 prepared choline chloride (ChCl)-based DESs, the ChCl-glycerol was finally screened as the most suitable candidate solvent for extracting antioxidant polyphenols from green tea, and the extraction conditions, including liquid to solid, ultrasonic power, and ultrasonic time, were further optimized by the response surface methodology (RSM). Under the optimal conditions, it was observed that the total phenolic content (TPC), the total amount of four major catechins, and antioxidant activity of green tea extract obtained by UAE-DES were all higher than those of the extracts obtained by conventional methods (UAE-ethanol extraction, ethanol extraction, and hot water extraction). In addition, scanning electron microscopy (SEM) analysis revealed that UAE-DES effectively disrupted the green tea leaf cells, thereby improving tea polyphenols yield, indicating a high efficiency of the new method.
2. Materials and Methods
2.1. Chemicals and Reagents
All the solvents or reagents used were of HPLC or analytical grade. Ethanol, FeCl3·6H2O, HCl, K2S2O8, and acetic acid were purchased from Titan Scientific Co., Ltd. (Shanghai, China). Gallic acid was purchased from Energy-Chemical Co., Ltd. (Shanghai, China). ChCl and xylitol were obtained from Adamas Co., Ltd. (Shanghai, China). Na2CO3 was purchased from Sinopharm Chemical Reagent (Shanghai, China). D-sorbitol, glucose, maleic acid, malonic acid, malic acid, sucrose, and Folin–Ciocalteu’s phenol reagent were all obtained from Macklin Biochemical Co., Ltd. (Shanghai, China). Citric acid, ethylene glycol (EG), and 1,2-propanediol (PD) were purchased from TCI Shanghai (Shanghai, China). Lactic acid and sodium acetate were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Glycerol was purchased from MP Biomedicals Co., Ltd. (Shanghai, China). 2, 4, 6-Tri(2-pyridyl)-s-triazine (TPTZ), [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were purchased from Sigma Aldrich (St. Louis, MO, USA). Commercial standards of catechins [(−)-EC, (−)-EGC, (−)-ECG, and (−)-EGCG, purity ≥ 98%] were purchased from Chengdu RefMedic Biotech Co., Ltd. (Chengdu, China).
2.2. Sample Preparation
The selenium-enriched green tea (Enshi Yulu, 1000 g) purchased from Enshi Selenium Impression Agricultural Development Co., Ltd. (Hubei, China), was ground into a fine powder using a tube mill (IKA Tube Mill 100 control) and stored at 4 °C and used within one month.
2.3. Preparation of DESs
As shown in
Table 1, the two components (ChCl and HBD) of DESs were first mixed with a certain molar ratio (ChCl/HBD). After the addition of 20% (
m/v) deionized water, the mixture was then heated at 60 °C under gentle stirring until a homogeneous solution was formed. This homogeneous solution was DES, which was finally stored at room temperature.
2.4. Extraction of Tea Polyphenols
The initial weight of green tea powder (0.50 g) was put into a 50 mL centrifuge tube, and was then mixed with 10 mL DES solution. The UAE process was conducted by using an ultrasonic processor (JY92-IIN, Ningbo Scientz Biotechnology Co., Ltd. China), and the initial ultrasonication conditions were as follows: an ultrasonic probe with a diameter of 6 mm; ultrasonic power of 325 W; ultrasonic time of 10 min. During the UAE process, the sample was always kept on ice in order to avoid the effect of high temperature on degrading tea polyphenols. All experiments were carried out in triplicate.
For UAE-DES or UAE with ethanol extraction, the green tea powder (0.50 g) and DES or ethanol (18 mL) were mixed in a 50 mL centrifuge tube with a certain liquid/solid ratio. After vortexing, the mixture was immediately treated using the ultrasonic processor.
For ethanol extraction, the green tea powder (0.50 g) and ethanol (18 mL) were mixed in a 50 mL centrifuge tube with a certain liquid/solid ratio, and the extraction of green tea polyphenols was carried out at room temperature (25 °C) shaking for 24 h.
For hot water extraction, the green tea powder (0.50 g) and 85 °C water (18 mL) were mixed in a 50 mL centrifuge tube with a certain liquid/solid ratio, and the extraction was carried out at room temperature (25 °C) for a certain time.
After extraction, the crude extract was centrifuged at 12,000× g for 10 min. The finally recovered supernatant was used as the sample for the determination of TPC and antioxidant activity, as well as HPLC analysis. The treated green tea leaf powder was freeze-dried for SEM analysis.
2.5. Determination of Total Phenolic Content (TPC)
The TPC was determined using the Folin–Ciocalteu method as reported with minor modifications [
28]. Briefly, the Folin–Ciocalteu solution agent (2.5 mL) was added to the properly diluted sample (500 µL) and incubated for 4 min, and then 2 mL Na
2CO
3 solution (75 g/L) was added to the mixture and the mixture was incubated for 2 h in the dark. The absorbance of the mixture was measured at 760 nm using a UV-visible spectrophotometer (UV1800, Jinghua Instrument Co., Ltd., Shanghai, China). Gallic acid was used as a standard and the results were expressed as milligrams of gallic acid equivalents (mg GAE)/g dry weight (DW) of samples. All experiments were carried out in triplicate.
2.6. Determination of Antioxidant Activity
The antioxidant activity of green tea extracts was determined using ferric-reducing antioxidant power (FRAP) assay, DPPH free radical scavenging assay, and ABTS free radical scavenging assay as described with some modifications by Tang et al. [
29] and Yang et al. [
30]. All experiments were carried out in triplicate.
For the FRAP assay, a 100 µL sample or FeSO4 standard solution was added into 3.0 mL fresh FRAP reagent (sodium acetate buffer: TPTZ solution: FeCl3 solution= 10:1:1, v/v/v). The reaction mixture was vortexed and incubated at room temperature (25 °C) in the dark for 4 min, and the absorbance was immediately recorded at 593 nm. The results were expressed as mmol Fe (II)/100 g DW.
For the DPPH assay, a 100 µL sample of Trolox standard solution was added into 3.9 mL of DPPH working solution. The reaction mixture was vortexed and incubated at room temperature (25 °C) in the dark for 2 h, and the absorbance was immediately recorded at 515 nm. The results were expressed as mmol Trolox/100 g DW.
For the ABTS assay, a 100 µL sample of Trolox standard solution was added into 3.9 mL of ABTS solution. The reaction mixture was vortexed and incubated at room temperature (25 °C) for 6 min, and the absorbance was immediately detected at 734 nm. The results were expressed as mmol Trolox/100 g DW.
2.7. High Performance Liquid Chromatography (HPLC) Analysis
The green tea extract obtained under the optimal condition of UAE-DES was analyzed by HPLC using an Agilent XDB C18 column (5 µm, 4.6 mm × 250 mm; Agilent Technologies, SantaClara, CA, USA). The mobile phase consists of solvent A (0.1% (v/v) formic acid in water) and solvent B (acetonitrile). The analysis was performed by a gradient elution program: 0 min, 5% B (v/v); 10 min, 16% B (v/v); 15 min, 16% B (v/v); 20 min, 30% B (v/v); 25 min, 100% B (v/v); 28 min, 100% B (v/v); 29–35 min, 5% B (v/v). The sample injection volume was 5 µL, and the flow rate was set at 1.0 mL/min. The retention time and spectra of phenolic compounds were compared with the standard compounds and were quantified basing on the peak areas. The results were expressed as mg/g DW.
2.8. Experimental Design
2.8.1. Single-Factor Experiments
The single-factor experiments were performed to analyze the effects of five factors, including liquid/solid ratio, ChCl/glycerol molar ratio, water content in ChCl-glycerol, ultrasonic power, and ultrasonic time, on TPC values, in order to obtain the major factors and their levels.
2.8.2. Response Surface Methodology
A three-factor-three-level Box–Behnken design (BBD) was applied to optimize the extraction conditions. Based on the single-factor experiments, liquid to solid ratio (X
1), ultrasonic power (X
2), and ultrasonic time (X
3) were chosen as independent variables in BBD and tested in a 17-run experiments (with 5 central point runs). As shown in
Table 2, each independent variable was coded at three levels, −1, 0, and +1, for low, medium, and high levels, respectively.
2.9. Morphology
The surface structures of treated samples were obtained and compared to that of unprocessed green tea leaves powder using a FEI Sirion 200 field-emission scanning electron microscope (FEI Co., Hillsboro, OR, USA).
2.10. Statistical Analysis
All values were expressed as the mean ± standard deviation (SD). Design-Expert 8.0.6 software (Trial version, State-Ease, Minneapolis, MN, USA) was used to analyze the multiple regression and estimate the coefficients of the regression model. The statistical importance of the regression coefficient was evaluated by F-test. One-way analysis of variance (ANOVA) was employed to evaluate the accuracy of the conducted model by estimate F-value, P-value, coefficient of determination (R2), and lack of fit. All statistical analyses were performed using the software SPSS (version 25.0, IBM SPSS Statistics, IBM Corp, Somers, NY, USA).
4. Conclusions
In this work, an environmentally friendly and powerful UAE-DES method was developed to extract antioxidant polyphenols from green tea. The DES ChCl-glycerol was finally selected as the most suitable DES. Subsequently, single-factor experiments were performed and the main factors influencing the extraction were optimized by RSM. The TPC value was 243 ± 7 mg GAE/g under the following optimized conditions: liquid to solid ratio of 36:1, ultrasonic power of 461.5 W, and ultrasonic time of 21 min. The antioxidant activity and content of four major catechins [(−)-EGC, (−)-EC, (−)-EGCG and (−)-ECG)] of the green tea extracts were also found to be higher than those of the extracts obtained by UAE with ethanol, ethanol extraction, and hot water extraction. In addition, SEM analysis indicated that UAE-DES led to a loose structure and surface erosion of the green tea leaves, which could result in greater penetration of the solvent into the plant material. The results presented in this work indicate that the combination of sustainable green solvents (DESs) and ultrasound-assisted extraction method (UAE) is a good method for the extraction of antioxidant polyphenols from green tea, which has potential applications in the food industry.