Ecofriendly Mechanochemical Extraction of Bioactive Compounds

Research Article pubs.acs.org/journal/ascecg

Ecofriendly Mechanochemical Extraction of Bioactive Compounds from Plants with Deep Eutectic Solvents Man Wang,† Jiaqin Wang,† Yanying Zhou,† Mingyue Zhang,† Qian Xia,† Wentao Bi,*,† and David Da Yong Chen*,†,‡ †

Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, Jiangsu Key Laboratory of Biomedical Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China ‡ Department of Chemistry, University of British Columbia, Vancouver, BC V6T 1Z1, Canada S Supporting Information *

ABSTRACT: A fast, highly efficient, and ecofriendly extraction method using deep eutectic solvents (DESs) for mechanochemical extraction (MCE) was developed to extract bioactive compounds from plants. Tea leaves containing bioactive compounds such as alkaloids, flavonoids, and catechins were used to evaluate this method. Dozens of DESs and DESs/water mixtures were systematically studied and optimized to select optimized extraction conditions. The results showed that the extractions can be completed within 20 s. Moreover, the developed extraction method is more ecofriendly, faster, gentler, and more efficient than conventional methods. For many compounds, we could simply use the described method without optimization. On the other hand, the target compounds were extracted with various interferences because of the wide ranging high extraction efficiency. Ultrahigh performance liquid chromatography coupled with highresolution mass spectrometry was therefore used for qualitative and quantitative analysis to characterize the efficiency for individual compounds. To avoid the negative effect of DESs on chromatographic separation, the analytical performances of this method, including reproducibility (RSD, n = 5), correlation of determination (r2), and the limit of detection, were determined. KEYWORDS: Deep eutectic solvents, Mechanochemical extraction, Bioactive compounds, UPLC-MS

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INTRODUCTION Bioactive natural products with pharmacological or biological activities can have a significant therapeutic effect and are often candidates in drug discovery.1,2 Many extraction methods have been developed to obtain these compounds for biological activity testing in vitro and in vivo. Standards of these compounds are also needed to perform quality control of medicinal plants or formulations.3 In many cases, a large amount of a variety of organic solvents need to be used in the extraction process. The large quantities of organic solvents are usually toxic and volatile and contribute to environmental pollution.4 Within the framework of green chemistry, solvent recovery is an important challenge. Thus, developing new green solvents is one of the key subjects in making chemical processes more ecofriendly. Metal-free ionic liquids (ILs) have emerged as a new class of promising solvents because they exhibit negligible volatility and nonflammability and have adjustable physicochemical properties and tailor-made selectivity for extraction of active ingredients. However, most ILs are hazardous/toxic and are poorly biodegradable.5,6 Additionally, the synthesis of ILs is harmful to the environment because it generally requires a large amount of salts and solvents to facilitate anion exchange.7 Deep eutectic solvents are emerging © 2017 American Chemical Society

as a new type of green solvents with similar properties to ILs and have the potential to replace traditional solvents and ILs.8 DESs are generally produced by mixing two or more naturally occurring, inexpensive, and biodegradable components that are capable of self-association, often through hydrogen bond interactions, to form a eutectic mixture with a melting point that is far below that of the individual compounds.9 As a result, most DESs are inexpensive and easy to prepare; they generally have a low vapor pressure, low toxicity, good thermal stability, wide liquid range, and are nonvolatile, nonflammable, and biodegradable.10 In addition to their applications in catalysis, electrochemistry, metal processing, and gas separations, DESs have been used as solvents in chemical and biological synthesis, electrochemistry, and preparation of inorganic materials.11−15 In particular, DESs have been used as solvents to extract bioactive compounds from plant materials in combination with other extraction methods, such as ultrasound-assisted extraction (UAE), heating extraction (HE), microwave-assisted extraction (MAE), negative pressure cavitation, and vortexing. UnfortuReceived: May 3, 2017 Revised: June 1, 2017 Published: June 5, 2017 6297

DOI: 10.1021/acssuschemeng.7b01378 ACS Sustainable Chem. Eng. 2017, 5, 6297−6303

Research Article

ACS Sustainable Chemistry & Engineering

98%), (−)-epigallotechin gallate (EGCg, ≥ 98%), choline chloride (ChCl, 98%), 1,3-butanediol (99%), 1,4-butanediol (99%), 1,6hexanediol (98%), malonic acid (98%), malic acid (>99%), acetamine (99%), methylurea (98%) and urea (≥99.5%) were purchased from Aladdin Industrial, Inc. (Shanghai, China). Ethyl glycol (EG, > 99%) and lactic acid (>98%) were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2,3-Butanediol (>98%) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Distilled water was vacuum filtered (HA-0.45, Division of Millipore, USA) prior to use. All other solvents were of HPLC or analytical grade. UPLC-MS Analysis for Qualitative Analysis of Bioactive Compounds. All UPLC experiments were carried out on a Thermo UltiMate 3000 Series:UPLC system which consisted of an HPG3400RS pump, WPS-3000TRS autosampler, diode array, and multiple wavelength detectors coupled to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Data processing was performed using Chromeleon 7 and Xcalibur software. The UPLC analysis was performed using a commercial C18 column (2.1 mm × 100 mm, 1.9 μm) purchased from Thermo Fisher Scientific, Inc. (Shanghai, China). Two solvent gradient elutions were performed at a flow rate of 0.3 mL/min. The solvent compositions used were (A) water containing 0.3% formic acid and (B) acetonitrile. The gradient programs were as follows: −5−0 min, 2% B; 0−2 min, 2% B; 2−3 min, 2%−3% B; 3−5 min, 3%−5% B; 5−7 min, 5%−10% B; 7−12 min, 10% B; 12−15 min, 10%−30% B; 15−17 min, 30%− 40% B; 17−20 min, 40% B. UV absorbance detection was performed at wavelengths of 231, 274, and 365 nm. The MS used is a high resolution tribrid system consisting of a quadrupole, an orbitrap, and ion trap mass analyzers. The MS operated with the full-scan mode; m/z range of 100−800; resolution of 120,000; automatic gain control (AGC) target set at 2e5, and maximum injection time of 100 ms. For limit of detection (LOD) determination, the system is set to single ion monitoring (SIM) mode, where the m/z value used for the seven target compounds are shown in Table S1 in the Supporting Information with an isolation width of 5 m/z. The ion transfer tube temperature was 300 °C. The mass spectrometer is controlled by Orbitrap Fusion Lumos 2.0 Tune (Thermo Fisher Scientific, USA). All of the MS data were processed using Xcalibur software, and the data were exported to Origin 8.5 (Originlab, USA) to produce the presented figures. Preparation of DESs. Different molar ratios of hydrogen bond donors (HBDs) and hydrogen bond acceptors (HBAs) were mixed at 80.0 °C with magnetic agitation until a clear and homogeneous DES liquid was formed, and a known amount of water was added when necessary.21 The prepared DESs are listed in Table 1. Preparation of Standard Solutions. Standard stock solutions were prepared by dissolving each compound in methanol at a concentration of 0.1 mg/mL for CAF, TBM, KAE, EC, ECg, EGC, and EGCg and stored at 4 °C. The standard working solutions were prepared by diluting the stock solutions with methanol. Extraction of Bioactive Compounds from Tea. Tea leaves were crushed, and individual 0.05 g samples were mixed with different

nately all of these generally require long extraction times.16−20 Some of these extraction methods, e.g., HE and MAE, may cause a loss or change in property for the bioactive molecules by thermal degradation, ionization, hydrolysis, or oxidation. UAE can be noisy and time-consuming. Accelerated solvent extraction (ASE) is an automated extraction technique that uses conventional liquid solvents at high temperatures and high pressures to extract compounds from solid samples. It is a rapid method that provides good recoveries, adequate precision, and reduced solvent use. However, the required cleaning procedures can be too tedious for practical applications. To overcome these limitations and prevent heat-induced alteration of the chemical structure and bioactivity, we introduce in this paper a DESs-based mechanochemical extraction (DESs-MCE) method as a better alternative for effective extraction of bioactive compounds. In plant materials, bioactive compounds usually exist on the cell wall and within the cytoplasm inside vacuoles containing cellular fluid. The mechanism of conventional extraction methods uses organic solvents combined with a pulsed electric field, microwave and ultrasonic irradiation, and extrusion treatment to dissolve bioactive compounds from the cell wall or permeate lipid membranes to separate individual cellular components. Alternatively, MCE, a room temperature mechanical treatment, can provide a unique motion to disrupt biomolecular lipid layers and cell walls using a multidirectional, simultaneous mechanical in-liquid smashing with specialized beads on the sample to achieve full release of bioactive compounds into the surrounding solvents for extraction. Meanwhile, some cellulose or lignin of the cell wall may dissolve in DESs because of hydrogen-bonding interactions, which accelerate the release of bioactive compounds to solvents. Commonly used DES-based extractions are operated at a high temperature to decrease the viscosity of the DESs. Instead, our DESs-MCE is a room-temperature extraction method that can prevent structural alteration. Compared to traditional extraction methods, DESs-MCE is an ecofriendly method that decreases the processing time while avoiding the consumption of organic solvents and providing high extraction yields. Tea leaves containing various bioactive compounds, such as alkaloids, flavonoids, and catechins, are used to demonstrate the feasibility of this new DESs-MCE method. These compounds are ideal for evaluating the performance of DES-MCE because flavonoids and catechins are especially sensitive to oxygen and temperature. The performance of the extraction is usually evaluated by the extracted amounts of target compounds, which are determined by high performance liquid chromatography (HPLC). However, the target compounds are extracted with many materials because of the high extraction efficiency of DESs-MCE. Thus, ultrahigh performance liquid chromatography (UPLC) coupled with high-resolution mass spectrometry (MS) was used for qualitative and quantitative analysis to evaluate the performance of the extraction process. The effects of the type of DESs, solid/liquid ratio, MCE vibration speed, and extraction time were systematically optimized. This work demonstrates a new green extraction strategy for obtaining bioactive compounds from plants.

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Table 1. Abbreviation of DESs Studied abbreviation DESs-1 DESs-2 DESs-3 DESs-4 DESs-5 DESs-6 DESs-7 DESs-8 DESs-9 DESs-10 DESs-11 DESs-12

EXPERIMENTAL SECTION

Materials. Caffenine (CAF), theobromine (TBM, ≥ 99.5%), kaempferol (KAE, ≥ 98%), (−)-epicatechin (EC, ≥ 98%), (−)-epicatechin gallate (ECg, ≥ 98%), (−)-epigallotechin (EGC, ≥ 6298

HBA

choline chloride

HBD ethyl glycol 1,2-butanediol 1,3-butanediol 1,4-butanediol 2,3-butanediol 1,6-hexanediol malonic acid lactic acid malic acid acetamine methylurea urea


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Figure 1. Extracted amounts of bioactive compounds by DESs-MCE. Types of DESs: DESs-4, DESs-7, and DESs-10; concentration of DES: 10−100 wt %; HBAs/HBDs = 1/3, mol/mol; vibration speed = 4 m/s; time = 60 s; left vertical represent values of KAE and TBM; right vertical represent values of EGC, CAF, EC, EGCg, and ECg. concentrations of aqueous DESs solutions in 2.0 mL Lysing Matrix D tubes with 1.4 mm ceramic spheres. The crushed tea leaves were mixed with certain amount of DESs and water. After a quick process in the MCE system (FastPrep-24, MP Biomedicals LLC, USA), the suspension was centrifuged, diluted, and collected for analysis. The extraction conditions were systematically optimized. To obtain the total amounts of target compounds in tea leaves, the feed was extracted several times with the developed and conventional extraction (HRE and UAE) method until no target compounds were detected by UPLC-MS. The total extractable amounts for the targets are TBM, 0.3573 ± 0.00486 mg/g; EGC, 32.6470 ± 0.007239 mg/g; CAF, 22.6461 ± 0.00578 mg/g; EC, 7.1831 ± 0.00578 mg/g; EGCg, 72.0777 ± 0.06176 mg/g; ECg, 12.3889 ± 0.03137 mg/g; KAE, 0.0035 ± 0.00099 mg/g.

higher than that of the other DESs because it has the lowest viscosity. Therefore, the addition of water to DESs can significantly benefit the extraction by decreasing the viscosity and adjusting the polarity. Formulations containing 10−100 wt % of DESs in the DESs/water mixture were used for extraction (Figure 1 and Table S2). The highest extracted amounts for these seven compounds were obtained when the DESs content in the DESs/water mixture was around 40−60 wt %. This is because high contents of DESs exhibit a high viscosity, whereas low contents of DESs show a high polarity. Thus, a suitable content of DESs could provide a DESs/water mixture with an excellent performance. Another phenomenon is that the extracted amount of KAE significantly decreased with decreasing DESs content, unlike the other target compounds. This may be due to the much stronger hydrophobicity of KAE than that of the other compounds. The increased amount of water significantly increased the polarity and hydrophilicity of the DESs/water mixture. Among the alcohol-based DESs and their aqueous mixtures, the DESs-4/water mixtures with better extraction performances were selected for further study. Among the carboxylic acid-based DESs, DESs-7 (HBAs/ HBDs = 1/1, mol/mol) and DESs-8 (HBAs/HBDs = 1/1−1/ 4, mol/mol) are clear and transparent liquids with relatively low viscosities at room temperature. The others are solids or liquids with high viscosities. Similar to the alcohol-based DESs, the viscosity of the carboxylic acid-based DESs is affected by the hydrogen bonding interaction, which increases with increasing numbers of carboxyl and hydroxyl groups. Therefore, the order of the viscosities is correlated with the number of carboxyl and hydroxyl groups in the HBDs (malic acid > malonic acid > lactic acid). These transparent liquid DESs were used to extract bioactive compounds with low extraction efficiencies. To increase the extraction efficiency, different amounts of water were added to the DESs (Figure 1 and Table S2). As shown in Table S2, the extracted amount of target compounds using the DESs/water mixture was markedly higher than that using the 100 wt % DESs. Similarly, the highest extraction efficiencies were obtained when the DESs contents were around 40−60 wt %. Another phenomenon is that the extracted amount of KAE significantly decreased with the addition of water. This may also be due to the change of polarity and hydrophilicity of the carboxylic acid-based DESs/water mixtures. For the subsequent investigation, DESs-7/water mixtures with slightly higher extraction efficiencies were selected. For the amine-based DESs, the different ratios of HBAs and HBDs did not all produce clear solutions. Similarly, the viscosity of the amine-based DESs increases with increasing

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RESULTS AND DISCUSSION Effect of Type of DESs. The type of DESs is vital for extracting the target compounds from a sample matrix based on the following properties: diffusion, solubility, viscosity, surface tension, polarity, and physicochemical interactions. Three groups of DESs, including alcohol-based DESs, carboxylic acid-based DESs, and amine-based DESs, were selected to extract alkaloids (CAF and TBM), flavonoid (KAE), and catechins (EGC, EC, EGCg, and ECg) from tea leaves (Table S2). For the alcohol-based DESs, DESs-1 (HBAs/HBDs = 1/2− 1/4, mol/mol), DESs-2 (HBAs/HBDs = 1/3−1/4, mol/mol), DESs-3 (HBAs/HBDs = 1/2−1/4, mol/mol), DESs-4 (HBAs/ HBDs = 1/2−1/4, mol/mol), and DESs-5 (HBAs/HBDs = 1/ 3−1/4, mol/mol) are clear and transparent liquids with relatively low viscosities at room temperature. The other alcohol-based DESs are either opaque or have higher viscosities. This phenomenon may be related to the position of hydroxyl groups and the carbon chain length of the HBDs. At HBAs/ HBDs molar ratios of 1/2, the viscosities of DESs-2 and DESs5 are higher than those of DESs-3 and DESs-4. The four DESs have the same carbon chain lengths and number of hydroxyl groups, but the positions of the hydroxyl groups are different. The position of two hydroxyl groups in DESs-2 and DESs-5 are closer to each other than the hydroxyl groups in the other DESs. Thus, the frameworks formed by hydrogen bonds in these two solvents are relatively rigid, and these DESs have higher viscosities. For DESs-1, DESs-4, and DESs-6, the carbon chain length increases from DESs-1 to DESs-6, which increases the viscosity. Thus, longer carbon chain lengths in the HBDs result in higher viscosities. The DESs with relative low viscosities were used to extract target bioactive compounds. As shown in Table S2, the extraction efficiency of DESs-1 is 6299


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Figure 2. Chromatograms of tea extracts. Types of DESs: DESs-4, DESs-7, and DESs-10.

numbers of amine groups and the molecular weight of the HBDs. To evaluate their extraction performances, it is necessary to add a suitable amount of water (0−90 wt % of water) to the amine-based DESs. The extraction results are shown in Figure 1 and Table S2. The DESs/water mixtures with DESs contents of 40−60 wt % exhibit better performances than the other mixtures. The extraction behavior of KAE is similar to that of alcohol-based and carboxylic acid-based DESs and their aqueous mixtures. On the basis of these results, the DESs-10 series was selected for further research. After investigating the extraction behaviors of these three groups of DESs, several other interesting phenomena were observed. The extraction efficiencies exhibit little difference among different groups of DESs for DESs contents of 10−20 wt %. Typically, when the DESs are diluted with water, some of the acid or amine HBDs may ionize H+ and OH− in the aqueous solution and thus affect the solubility of the target compounds. The similar results obtained might indicate that the solubility of the target compounds in the DESs-based solvents was dominated by solvent polarity. The other phenomenon is that the extracted amounts were rarely affected by the DESs type under the same conditions (Figure 1 and Table S2). Traditionally, the extraction mechanism is that organic solvents, such as methanol, acetonitrile, and acetone, infiltrate the cell to extract its internal bioactive compounds by distillation, radiation, ultrasound, etc. As for the MCE employed in this research, bioactive compounds were fully released into the extraction solvents from inside of the cell by breaking down the plant tissues and cell walls. Thus, compared to other extraction methods, MCE has many advantages, such as providing higher extraction yields, consuming less time, and decreasing the solvent consumption. This can also explain the resulting extraction efficiencies that are quite similar for different groups of DESs. In other words, the extraction performance of DESs-MCE is dominated by the dissolving capacity of the solvents. When the solubility of the target compounds reached the upper limit of the dissolving capacity of the solvents, different extraction performances were observed. Even by careful examination of the extraction efficiency of the DESs-based solvents, including DESs-4, DESs-7, and DESs10, the selection of best performing DESs was still difficult (Figure 1 and Table S2). However, another assessment

criterion was found when the chromatogram was evaluated. As shown in Figure 2, the acid-based DESs and the aminebased DESs may affect the separation of some bioactive compounds and could cause tailing on the C18 column. This may be due to the adsorption of amine and acid on the remaining silanol groups on C18, thus affecting the chromatographic separation. To obtain better extraction efficiencies, 10−100 wt % (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%) DESs in the DESs-4/water mixture were further investigated. As displayed in Table S3, a ChCl/HBD ratio of 1/3 (mol/mol) of DESs-4 was the most effective solvent, and the extracted amounts were highest at a concentration of 50 wt % water. Effect of Solid/Liquid Ratio. The solid/liquid ratio plays an important role in the completeness of the extraction. Thus, extractions were performed at different solid/liquid ratios (0.01, 0.03, 0.05, 0.07, 0.10, 0.12, 0.15, and 0.20 g/mL) to find the optimum condition (Figure 3). The extracted amount remained almost constant or slightly decreased from 0.01−0.20 g/mL.

Figure 3. Extracted amounts of bioactive compounds at different solid/liquid ratios. Concentration of DESs-4 (HBAs/HBDs = 1/3, mol/mol) = 50 wt %, vibration speed = 4 m/s, time = 60 s; left vertical represent values of KAE and TBM; right vertical represent values of EGC, CAF, EC, EGCg and ECg. 6300


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ACS Sustainable Chemistry & Engineering This may be because the target compounds were completely extracted in the range of 0.01−0.20 g/mL. When the solid/ liquid ratio exceeded 0.20 g/mL, the resulting extract was not sufficient for analysis. This phenomenon also demonstrates that the extraction efficiency is mainly dominated by the dissolving capacity of the solvents. In some cases, the dissolution of interference compounds, e.g., lignin and cellulose, may affect the extraction when too many plant powders are added to the DESs-MCE. Through this investigation, a solid/liquid ratio of 0.05 g/mL was selected for further investigation. Effect of Vibration Speed. The MCE speed is also a key factor affecting the grinding and cell disruption of tea samples. A higher speed may lead to a better extraction efficiency. Therefore, different MCE speeds (4, 4.5, 5, 5.5, and 6 m/s) were investigated. However, the vibration speed had no significant effect on the extracted amounts of seven bioactive compounds from tea leaves (Figure S1). The rest of the extraction experiments were performed at 4 m/s to minimize energy consumption and preserve the integrity of the ceramic balls. Effect of Extraction Duration. To optimize the extraction duration, extractions were performed at 4 m/s for 10, 20, 40, 60, and 80 s. As displayed in Figure S2, the extraction amounts increased as the extraction duration increasing from 10 to 20 s but became constant above 20 s. Therefore, 20 s was selected for the rest of the extraction experiments. Another advantage of this method was exhibited. Typically, long-term MCE generated heat, which altered the chemical structures of the target compounds. In this research, the extraction time was short, and the presence of DES stabilized the temperature. Overall, a ChCl/HBD ratio of 1/3 (mol/mol) for DESs-4, a concentration of 50 wt % water in the DES-water mixture, and a vibration speed of 4 m/s for 20 s at a solid/liquid ratio of 0.05 g/mL were selected as the conditions for further research. Comparison of Extraction Solvents and Methods. Ethanol, methanol, acetonitrile, acetone, and ethyl acetate were used to extract catechins, flavonoids, and alkaloid from tea leaves under optimal conditions to compare the efficiencies of the DESs and conventional solvent extractions (Figure 4). The

DESs aqueous solution demonstrated an excellent extraction efficiency compared to that using conventional solvents. This could be attributed to the suitable polarity of the target compounds, which can act as an HBD and interact with the DESs. Furthermore, the dried tea powder (0.10 g) was ultrasonicated in 2.0 mL of methanol and DESs-4 (a ChCl/ HBD ratio of 1/3 (mol/mol) at a concentration of 50 wt %) for 1 and 30 min. Then, the dried tea powder (0.50 g) was heat reflux extracted using 10.0 mL of methanol and DESs-4 (a ChCl/HBD ratio of 1/3 (mol/mol) at a concentration of 50 wt %) for 1 and 30 min at 80 °C. Conventional extraction methods provided similar or reduced extracted amounts of bioactive compounds than DESs-MCE (Table 2). The extraction time for DESs-MCE was much shorter than that used for the other methods. Table 2 shows that the DESs-MCE method is faster and more efficient than conventional methods. Stability of Bioactive Compounds in DESs Extract. Bioactive compounds are typically sensitive to oxygen and temperature. To evaluate the stability of this method, seven bioactive compounds were extracted by DESs-MCE under the selected experimental conditions. The obtained extract was placed in air at 20 °C and analyzed by UPLC-MS every 24 h. As displayed in Figure S3, the amounts of bioactive compounds in the extract show no evident changes after 96 h. Thus, DESs may maintain the structural stability of bioactive compounds because of the low solubility of oxygen in DES-based solvents and the surrounding ionic solution. Analytical Performance and Application. To evaluate the extraction performance, the extracts should be qualitatively and quantitatively analyzed. HPLC or UPLC with a UV or diode array detector were not sensitive enough for analysis, especially for qualitative analysis. For the extracts with complex components, the most effective solution is to use MS. Therefore, high-resolution UPLC-MS was employed for this research. The total ion chromatogram of tea (Tieguanyin) extract in positive and negative ion modes, and the selected ion monitor chromatograms of TBM, EGC, CAF, EC, EGCg, ECg, and KAE are shown in Figure S3. A series of experiments were designed to obtain the linearity, precision, detection limits, and other characteristics of the analytical method under the optimized conditions. Table S4 shows that all of the analytes had linear determination coefficients (r2) between 0.9940 and 0.9999. The precision was determined by extraction of a standard solution of a single concentration six times with the proposed method, and the RSD was 0.20−5.4%. On the basis of a signal-to-noise ratio of 3, the detection limits of the seven target compounds were 4.0−10.0 ng/mL (obtained by using LC-MS) and 4−200 ng/mL (obtained by using LC-DAD) with the equipment used as described in the Experimental Section. These results show that the proposed analytical method is robust. The extraction and analytical methods were combined and used to extract and determine alkaloids, flavonoids, and catechins from five tea samples (black tea, Hei-zhuan tea, white tea, Gua Pian Tea, and Tieguanyin). Figure 5) demonstrates that white tea, Gua Pian tea, and Tieguanyin contain higher levels of catechins than the other two types of teas, which is in line with the degree of fermentation during the manufacturing process. Green teas (white tea and Gua Pian tea) are derived directly from drying and steaming fresh tea leaves, and Tieguanyin is derived from a partial fermentation. However, black tea undergoes a full fermentation stage, which oxidizes or condenses the tea catechins to other large

Figure 4. Extracted amounts of bioactive compounds from Tieguanyin by different solvents. DESs-4 (HBAs/HBDs = 1/3, mol/mol) = 50 wt %; left vertical represent values of KAE and TBM; right vertical represent values of EGC, CAF, EC, EGCg and ECg. 6301


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0.0035 ± 0.00099 12.3889 ± 0.03137 72.0777 ± 0.06176 7.1831 ± 0.00578

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22.6461 ± 0.01157

CONCLUSION The DESs-MCE method was successfully applied to extract TBM, EGC, CAF, EC, EGCg, ECg, and KAE from plants. This method is greener, faster, gentler, and more efficient than conventional extraction methods. We show that this green method can be used without complicated operation or optimization because many factors do not significantly affect the extraction efficiency. The DESs-MCE method showed its potential to become a powerful tool for efficient extraction of oxygen-sensitive and thermal-sensitive bioactive compounds from different plant matrices.

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32.6470 ± 0.07239

0.0017 0.0021 0.0020 0.0031 8.6822 11.0018 6.5819 9.7813 3.57381 0.47497 1.53594 0.03571 ± ± ± ± 44.4529 57.9258 30.1312 47.0208 0.31267 0.03370 0.18242 0.01178 ± ± ± ± 4.7492 6.1116 3.8053 5.7973 1.32031 0.12697 0.58245 0.14394 ± ± ± ± 16.4721 19.9374 13.6801 19.3262 0.86081 0.10435 0.90769 0.20558 ± ± ± ± 23.5767 29.1845 18.6819 27.2791 0.01949 0.00416 0.00686 0.00350 ± ± ± ± 0.2549 0.3149 0.1922 0.2874

polyphenols. There is more caffeine in green teas than in the other teas, and there are few flavonoids in each of the tea studied. Black tea and Hei-zhuan tea contain slightly higher levels of KAE as determined in this study.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b01378. Some LC and MS parameters, DESs compositions and extraction efficiencies, extraction mechanical vibration velocity and extraction duration, and selected MS spectra data. (PDF)

0.3573 ± 0.00486

0.57044 0.06831 0.32534 0.12526 ± ± ± ±

0.00027 0.00018 0.00000 0.00001

± ± ± ±

± ± ± ±

KAE

0.75527 0.12326 0.22255 0.12439 ± ± ± ± 10.4937 12.0084 7.9970 10.9341 4.24335 0.85137 0.81985 0.37800

EGCg

± ± ± ± 0.41461 0.05169 0.08594 0.06554 ± ± ± ±

EC

5.8965 6.6858 4.6995 6.3454 1.36036 0.22994 0.42585 0.22224 ± ± ± ±

CAF

19.5684 21.8727 16.6508 21.0044 1.78352 0.22911 0.57052 0.48997 ± ± ± ±

EGC

28.5660 31.2823 22.6274 29.5131 0.02337 0.00352 0.00536 0.00344 ± ± ± ±

TBM

0.3059 0.3423 0.2432 0.3217

Figure 5. Analysis of five types of tea samples by using DESs-MCE method. DESs-4 (HBAs/HBDs = 1/3, mol/mol) = 50 wt %; vibration speed = 4 m/s, time = 20 s, solid/liquid ratio = 0.05 g/mL.

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AUTHOR INFORMATION

20 s DESs-4 (50%)

*Tel.: +86 25 85891705. Fax: +86 25 85891707. E-mail: [email protected] (W. Bi). *Tel.: +86 25 85891705. Fax: +86 25 85891707. E-mail: chen@ chem.ubc.ca (D.D.Y. Chen). ORCID

David Da Yong Chen: 0000-0002-3669-6041 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Foundation of China (Grants No. 21475061 and No.

BM

1 min 30 min 1 min 30 min DESs-4 (50%) DESs-4 (50%) methanol methanol UAE

time

1 min 30 min 1 min 30 min

solvent method

DESs-4 (50%) DESs-4 (50%) methanol methanol

Corresponding Authors

HRE

extracted amount (mg/g)

Table 2. Comparison of DESs-MCE Method with UAE and HRE Methods (n = 3)

55.8270 63.1488 37.5397 52.7313

0.0019 0.0024 0.0027 0.0039 ECg

0.00030 0.00030 0.00017 0.00011


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21507062), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry ([2015] 1098), Program of Natural Science Research of Jiangsu Higher Education Institutions of China (Grant No. 16KJB150022), Priority Academic Program Development of Jiangsu Higher Education Institutions, Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, and a Chinese National Scholarship for Graduate Students (Man Wang).

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