Research Article pubs.acs.org/journal/ascecg
Well-Designed Hydrophobic Deep Eutectic Solvents As Green and Efficient Media for the Extraction of Artemisinin from Artemisia annua Leaves Jun Cao,† Meng Yang,† Fuliang Cao,*,‡ Jiahong Wang,† and Erzheng Su*,†,§ College of Light Industry Science and Engineering and ‡Co-innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China § State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, China Downloaded via UNIV OF CALIFORNIA SANTA BARBARA on July 4, 2018 at 20:12:48 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
S Supporting Information *
ABSTRACT: The green and efficient extraction of bioactive compounds from plant biomass is an important area of interest in the pharmaceutical industries. Hydrophilic deep eutectic solvents (DESs) have been considered as green alternatives to conventional solvents for bioactive compound extraction. In this study, we aimed to provide a practical example demonstrating the tunability of hydrophobic DESs as designer solvents to efficiently extract bioactive compounds from plant biomass. Artemisinin, known as the only drug effective in the treatment of malaria, was chosen for extraction from Artemisia annua leaves. A hydrophobic DES named N81Cl-NBA that was tailor-made from methyl trioctyl ammonium chloride and 1-butanol at a molar ratio of 1:4 showed the highest extraction yield. With N81Cl-NBA-based ultrasound-assisted extraction (UAE), the main factors affecting the extraction yield were statistically optimized using a central composite design combined with a response surface methodology. The optimal conditions were obtained as follows: solvent/solid ratio 17.5:1, ultrasonic power 180 W, temperature 45 °C, particle size 80 mesh, and extraction time 70 min. Under these conditions, an extraction yield of 7.9936 ± 0.0364 mg/g was obtained, which was distinctly higher than that obtained using the conventional organic solvent petroleum ether. Moreover, the recovery of the target artemisinin from the N81Cl-NBA extraction solution was achieved by AB-8 macroporous resin with a recovery yield of 85.65%. N81Cl-NBA could be reused at least two times without a significant decrease in extraction yield. This study suggests that not only hydrophilic DESs but also hydrophobic DESs are truly designer solvents that can be used as green and safe extraction solvents for pharmaceutical applications. KEYWORDS: Artemisinin, Artemisia annua leaves, Extraction, Hydrophobic deep eutectic solvents, Designer solvent
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INTRODUCTION
acetone, and ethyl acetate; and soluble in ethanol. Moreover, despite the high solubility of artemisinin in ethanol, the amount of impurities extracted by ethanol have been shown to be much higher than with petroleum ether.5 The more impurities are present within crude extracts, the more expensive purification methods are needed. Extraction from A. annua is presently the sole realistic source of artemisinin.6 There is a great need to improve the efficiency of artemisinin extraction. The need is heightened by the fact that the artemisinin level in A. annua is very low, with a concentration of 0.01−1.4%, and the amount of artemisinin currently extracted through conventional methods is only 60− 80%.7 One potential method to improve the efficiency of
The World Health Organization (WHO) has recommended artemisinin-based combination therapies (ACT) for the treatment of malaria.1 Artemisinin derivatives used in ACT are synthesized from artemisinin in one or two synthetic steps. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, extracted from the herb Artemisia annua.2 With the recent development of ACT, there is a real opportunity to provide cures for malarial infections and potentially create significant decreases in the prevalence and morbidity of this disease. A barrier to the development of ACT is the high cost due to protracted traditional extraction methods.3 A well-established procedure for extracting artemisinin from its plant source involves extraction with nonpolar organic solvents such as petroleum ether and hexane.2,4 However, artemisinin is slightly soluble in cool petroleum ether; easily soluble in chloroform, © 2017 American Chemical Society
Received: December 18, 2016 Revised: February 6, 2017 Published: February 14, 2017 3270
DOI: 10.1021/acssuschemeng.6b03092 ACS Sustainable Chem. Eng. 2017, 5, 3270−3278
Research Article
ACS Sustainable Chemistry & Engineering
and calculated by means of a calibration curve established with a regression equation y = 0.3612x − 6.1299 (R2 = 0.9984) (Figure S1 in the Supporting Information). Optimization of Precolumn Reaction for HPLC Analysis. A 2.0 g portion of A. annua leaves powder was mixed with 20 mL of petroleum ether in a 50 mL centrifuge tube, and the mixture was executed in an ultrasonic facilities at 200 W and 50 °C for 2 h. The extracted solution was centrifuged at 10 000 rpm for 30 min. 0.5 mL of supernatant was mixed with 3.0 mL of 0.2% (w/v) sodium hydroxide (NaOH) methanol solution in a water bath at 45 °C for 25 min. After cooling to room temperature, the mixture was acidified with 2.0 mL of acetic acid methanol solution. The acetic acid concentration range from 0.06 to 0.16 mol/L at an interval of 0.02 mol/L was investigated. Next, under the optimum acetic acid concentration, the acidification time was investigated from 0.5 to 2.5 h at an interval of 0.5 h. At last, the concentration of NaOH was investigated from 0.1 to 0.4% (w/v). Preparation of Hydrophobic DESs. Traditionally, artemisinin is extracted from its plant source using hydrophobic organic solvents such as petroleum ether and hexane.2,4 Thus, methyl trioctyl ammonium chloride (N81Cl) was chosen as the HBA to prepare hydrophobic DESs with 13 different HBDs, which were alcohols with different side chains in this work.17 Hydrophobic DESs were prepared by heating the mixture of HBA and HBD to 80 °C with constant stirring until a homogeneous liquid was formed. The prepared DESs are listed in Table 1.
artemisinin extraction is utilizing new green solvents. If an extraction solvent has a relatively high extraction efficiency of artemisinin and minimizes the coextraction of impurities, then the extent of purification can be reduced. There are alternative extraction solvents involving the use of supercritical fluids, ionic liquids and hydrofluorocarbons.2,8,9 Thus far, none of the alternatives have provided a clear incentive to change. Deep eutectic solvents (DESs) have attracted broad attention as a new generation of green solvents, which can substitute for organic solvents.10 Most DESs are commonly made up of two or more nontoxic, biodegradable, inexpensive, and nonflammable components that are able to connect with each other through hydrogen bonding.11 DESs are regarded as designer solvents with a wide selection of hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). DESs hold great promise as solvents for use in many fields because of their biodegradability, low toxicity, easy preparation, and novel properties.12,13 There are ranges of reports using hydrophilic DESs as extraction media to extract bioactive compounds from plants.14−16 Consequently, this work looks at the possibility that DESs could increase the concentration of extracted artemisinin. In such a framework, DESs were tailored to examine their extraction ability. After an initial screening, DESs with high extraction efficiency were selected. Following that, response surface methodology (RSM) was used to optimize the extraction condition parameters relevant to the extraction efficiency. Finally, the recovery of artemisinin from the DESbased extraction solution and the reuse of the DES were investigated.
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Table 1. List of the Prepared N81Cl-Based Hydrophobic DESs abbreviation
HBA
HBD
DES-1
N81Cl
ethylene glycol 1-propanol 1,3propanediol glycerol 1-butanol 1,2-butanediol hexyl alcohol capryl alcohol decyl alcohol dodecyl alcohol 1-tetradecanol cyclohexanol DL-menthol
DES-2 DES-3
EXPERIMENTAL SECTION
Materials. The A. annua leaves were purchased from Nanyang (Henan, China). The leaves were dried at 40 °C to constant weight using a vacuum oven, pulverized by a disintegrator, sieved (30−40 mesh), and stored in a desiccator. Methyl trioctyl ammonium chloride (≥99.0%) was obtained from Adams-Beta Co., Ltd. (Shanghai, China). Artemisinin (≥99.0%), decyl alcohol (≥98.0%), hexyl alcohol (≥98.0%), and cyclohexanol (≥97.0%) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). 1-Hexadecanol (≥99.0%), capryl alcohol (≥99.0%), 1-tetradecanol (≥99.0%), dodecyl alcohol (≥99.0%), and tetrapropylammonium bromide (≥98.0%) were purchased from Chinese Medicine Group Chemical Reagent Co., Ltd. (Shanghai, China). Methanol (chromatographic grade) was purchased from Tedia Company, Inc. (Shanghai, China). Deionized water was obtained by a Milli-Q water purification system (Millipore, Billerica, MA). All other reagents and chemicals used in the experiment were of analytical reagent grade. The macroporous resins (HPD-17, D101, DM130, HPD-450, ADS17, and AB-8) used to recover the target artemisinin from the DES extraction solution were donated by the Cangzhou Bon Adsorber Technology Co., Ltd. (Cangzhou, China) and pretreated before use according to the instructions provided by the manufacturer. HPLC Analysis. HPLC analysis was performed using an Elite HPLC system (Dalian, China) equipped with a high-pressure gradient (P1201), a UV/vis detector (UV1201), an autosampler (AS1201), and a column oven. Data processing was carried out using an EC2006 chromatography data processing workstation. HPLC analysis was conducted on a SinoChrom ODS-BP column (4.6 nm × 200 mm, 5.0 μm) connected with a Jiajie protection column purchased from Elite (Dalian, China). The mobile phase was CH3OH-0.01 mol/L NaACHAC buffer (pH = 5.8, 60:40, v/v) at a flow rate of 1 mL/min. The column temperature was controlled at 27 °C. Detection was performed at a wavelength of 260 nm. The content of artemisinin was determined by HPLC using the method of precolumn reaction
DES-4 DES-5 DES-6 DES-7 DES-8 DES-9 DES-10 DES-11 DES-12 DES-13
molar ratio
appearance at room temperature
1:2
transparent liquid
1:2 1:2
transparent liquid transparent liquid
1:2 1:2 1:2 1:2 1:2 1:2 1:2
transparent transparent transparent transparent transparent transparent transparent
1:2 1:2 1:2
transparent liquid transparent liquid transparent liquid
liquid liquid liquid liquid liquid liquid liquid
Extraction of Artemisinin from A. annua Leaves Employing Hydrophobic DESs. For the initial DES screening and tailoring, an accurately weighed 80 mg sample of A. annua leaves powder was added into 0.80 mL of a DES in a 2.0 mL centrifuge tube. After brief vortexing, the mixture was extracted in an air-bath shaker at 250 rpm and 30 °C for 15 min, followed by centrifugation at 10 000 rpm for 10 min. Then, 0.5 mL of supernatant was sampled and mixed with 3.0 mL of 0.3% (w/v) sodium hydroxide methanol solution and then heated in a water bath at 45 °C for 25 min.18,19 The reactant was cooled to room temperature and acidified with 2.0 mL of a 0.1 mol/L acetic acid methanol solution. The resulting solution was filtered through a 0.45 μm membrane filter and analyzed by HPLC. Parallel experiments were performed three times, and the artemisinin was quantified and the deviation evaluated. DES-Based Extraction of Artemisinin Employing Different Methods. After initial DES screening and tailoring, the most efficient DES was chosen as the extraction solvent to compare the extraction efficiency of different extraction methods. Air-bath shaking, water-bath shaking, magnetic stirring, heating, and ultrasonic methods were tested for comparison. An 80 mg sample of A. annua leaves powder was mixed with 0.80 mL of the selected DES. The mixture was extracted by 3271
DOI: 10.1021/acssuschemeng.6b03092 ACS Sustainable Chem. Eng. 2017, 5, 3270−3278
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Table 2. Experimental Data and the Observed Responses with Different Combinations of Solvent to Solid Ratio (A, mL/g), Ultrasonic Power (B, W), Extraction Temperature (C, deg C), Particle Size of A. annua Powder (D, mesh), and Extraction Time (E, min) Used in CCD factor
extraction yield (mg/g)
run
A
B
C
D
E
actual value
predicted value
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
17.5(1) 12.5(−1) 17.5(1) 17.5(1) 15(0) 12.5(−1) 15(0) 15(0) 15(0) 12.5(−1) 17.5(1) 17.5(1) 10(−2) 17.5(1) 12.5(−1) 15(0) 17.5(1) 17.5(1) 12.5(−1) 12.5(−1) 12.5(−1) 15(0) 15(0) 17.5(1) 12.5(−1) 15(0) 15(0) 17.5(1) 15(0) 15(0) 12.5(−1) 17.5(1) 17.5(1) 12.5(−1) 15(0) 17.5(1) 15(0) 12.5(−1) 20(2) 15(0) 15(0) 17.5(1) 12.5(−1) 12.5(−1) 12.5(−1) 17.5(1) 17.5(1) 12.5(−1) 12.5(−1) 15(0)
140(−1) 180(1) 180(1) 180(1) 160(0) 140(−1) 160(0) 160(0) 160(0) 180(1) 140(−1) 180(1) 160(0) 180(1) 140(−1) 160(0) 180(1) 180(1) 140(−1) 180(1) 180(1) 160(0) 160(0) 180(1) 180(1) 160(0) 160(0) 140(−1) 160(0) 160(0) 140(−1) 140(−1) 140(−1) 180(1) 160(0) 140(−1) 200(2) 140(−1) 160(0) 160(0) 120(−2) 140(−1) 140(−1) 180(1) 140(−1) 140(−1) 180(1) 140(−1) 180(1) 160(0)
45(−1) 55(1) 45(−1) 55(1) 50(0) 55(1) 50(0) 50(0) 50(0) 45(−1) 45(−1) 55(1) 50(0) 45(−1) 45(−1) 50(0) 45(−1) 45(−1) 45(−1) 45(−1) 45(−1) 50(0) 50(0) 55(1) 55(1) 50(0) 60(2) 45(−1) 50(0) 50(0) 55(1) 55(1) 45(−1) 55(1) 40(−2) 55(1) 50(0) 55(1) 50(0) 50(0) 50(0) 55(1) 55(1) 55(1) 45(−1) 55(1) 55(1) 45(−1) 45(−1) 50(0)
60(1) 30(−1) 60(1) 30(−1) 40(−0.33) 60(1) 40(−0.33) 40(−0.33) 40(−0.33) 60(1) 60(1) 60(1) 40(−0.33) 30(−1) 60(1) 40(−0.33) 30(−1) 60(1) 60(1) 60(1) 30(−1) 20(−1.67) 80(2.33) 30(−1) 60(1) 40(−0.33) 40(−0.33) 30(−1) 40(−0.33) 40(−0.33) 30(−1) 30(−1) 30(−1) 60(1) 40(−0.33) 60(1) 40(−0.33) 30(−1) 40(−0.33) 40(−0.33) 40(−0.33) 30(−1) 60(1) 30(−1) 30(−1) 60(1) 60(1) 30(−1) 30(−1) 40(−0.33)
70(1) 50(−1) 70(1) 50(−1) 60(0) 50(−1) 80(2) 60(0) 60(0) 70(1) 50(−1) 50(−1) 60(0) 50(−1) 50(−1) 60(0) 70(1) 50(−1) 70(1) 50(−1) 50(−1) 60(0) 60(0) 70(1) 70(1) 40(−2) 60(0) 70(1) 60(0) 60(0) 70(1) 70(1) 50(−1) 50(−1) 60(0) 70(1) 60(0) 50(−1) 60(0) 60(0) 60(0) 50(−1) 70(1) 70(1) 50(−1) 50(−1) 70(1) 70(1) 70(1) 60(0)
7.3727 7.0319 7.9334 7.0930 6.8398 7.6314 8.1607 6.2643 6.2314 7.6388 7.1201 7.7269 6.4326 6.4928 6.8630 6.4153 7.8927 7.3428 7.3843 7.2685 6.0854 5.9246 7.4781 7.8800 7.7817 6.3634 8.1914 6.6761 6.6481 6.3660 7.1510 7.2025 5.9216 7.6025 6.8877 7.4608 7.8979 6.7368 6.7974 6.1885 6.3649 7.0526 7.5861 7.7735 5.9513 7.7574 7.8011 6.7115 7.6780 6.0026
7.3353 6.9823 8.0031 7.2021 6.3641 7.7036 7.9148 6.3641 6.3641 7.8739 7.0811 7.7670 6.4526 6.4971 6.9434 6.3641 7.8102 7.3457 7.3004 7.1138 6.2158 6.0819 7.3638 7.9855 7.8269 6.5558 8.0278 6.7766 6.3641 6.3641 7.2065 7.2293 5.8666 7.5965 6.9978 7.5043 7.6289 6.7234 6.7238 6.3641 6.5804 6.8490 7.5309 7.8684 5.6795 7.7797 7.8946 6.6922 7.6316 6.3641
air-bath shaking at 250 rpm and 30 or 60 °C, water-bath shaking at 150 rpm and 30 or 60 °C, magnetic stirring at 150 rpm and 30 or 60 °C, heating at 60 °C and 0 rpm, or ultrasonication at 200 W and 30 or 60 °C. Experimental Design and Statistical Analysis. To select an appropriate variable range for optimization by an experimental design based on the response surface methodology (RSM) approach, single-
factor experiments were performed (presented in the Supporting Information). After that, the main factors affecting the extraction efficiency were optimized to obtain the highest extraction yield of the target artemisinin. Central composite design (CCD) combined with RSM was applied to research the effects of the five parameters including solvent/solid ratio (A), ultrasonic power (B), extraction temperature (C), particle size of A. annua leaves powder (D), and 3272
DOI: 10.1021/acssuschemeng.6b03092 ACS Sustainable Chem. Eng. 2017, 5, 3270−3278
Research Article
ACS Sustainable Chemistry & Engineering extraction time (E) at five levels on the extraction yield of the target artemisinin, which was taken as the response. Fifty experimental runs were conducted in random order. The coded and the actual levels of the variables set in the present experimental design are shown in Table 2. Furthermore, a second-order polynomial model was applied to analyze the experimental data. The experimental design and regression analysis were accomplished by Design-Expert 8.0.5 software (Trial version, Stat-Ease Inc., Minneapolis, MN, USA). An F-test was applied to check the statistical significance of the regression coefficient. The adequacy of the proposed model was estimated through analysis of variance (ANOVA) by evaluating lack of fit, coefficient of determination (R2) and F-value Comparison between the Optimal DES and a Conventional Organic Solvent. In order to evaluate the extraction efficiency of the optimal DES, an organic solvent that has conventionally been used as the solvent for extracting artemisinin from A. annua leaves was chosen for comparison after RSM optimization. The comparison was carried out using the ultrasonic method under the optimized extraction conditions. Recovery of the Target Artemisinin from DES Extraction Solution and the Reuse of DES. Macroporous resins including HPD-17, D101, DM130, HPD-450, ADS-17, and AB-8 were used to recover the target artemisinin from the DES extraction solution. Macroporous resin (0.1 g) was added into 1.0 mL of the DES extraction solution. The mixture was shaken at 150 rpm and 25 °C for 12 h, and the concentration of the artemisinin in the supernatant was monitored by HPLC. After adsorption equilibrium was attained, the artemisinin-laden resin was filtered out and then desorbed with 1.0 mL of desorption solvent at 150 rpm and 25 °C for 6 h. The artemisinin contents in the DES extraction solution, the solution after adsorption, and the solution after desorption were determined separately. Accordingly, the adsorption yield of the macroporous resin and the desorption yield of the desorption solvent were calculated. The DES was collected and reused for the next extraction.
The heating method was chosen to prepare the DESs in this work. The prepared DESs are listed in Table 1. N81Cl could form stable DESs with the 13 HBDs, which were all transparent liquids at room temperature (Table 1). After preparation, the DESs were stored in sealed vials and dehydrated by incubating with 3 Å molecular sieves for several days before use. The 13 hydrophobic DESs produced were initially screened for extraction of artemisinin from A. annua leaves. The screening results are displayed in Figure 1. The type of HBD
Figure 1. Extraction yields (milligrams of artemisinin per gram of A. annua leaves powder) of the 13 initially prepared hydrophobic DESs. Error bars indicate the SEM (n = 3).
showed a clear effect on the extraction yield of artemisinin. In this study, N81Cl was designated the HBA, and thus, the properties of the DES were mainly affected by the HBD. Comparison among DES-2, DES-3, and DES-4 showed that extraction yield of DES formed with trihydroxy-HBD (DES-4) was lower than that of DES formed with dihydroxy-HBD (DES-3), and that of DES formed with monohydroxy-HBD (DES-2) was the highest. Comparison among DESs formed with dihydroxy-HBDs (DES-1, DES-3, and DES-6) indicated that extraction yield increased a few along with the increase of the length of fatty alcohol chain. Substituted cyclohexanol (DLmenthol) as HBD (DES-13) produced a lower extraction yield than that of cyclohexanol as HBD (DES-12). As for DESs formed with monohydroxy-HBDs (DES-2, DES-5, and DES7−11), the extraction yield increased with the increase of the length of fatty alcohol chain, and attained the maximum at C4 of the length of fatty alcohol chain, then decreased with the increase of the length of fatty alcohol chain. The DESs formed with C3−C6 of the length of fatty alcohol chain might hold similar polarity to artemisinin, leading to higher extraction yield. In addition, when the HBD was solid, the viscosity of the produced DES was high. On the contrary, the viscosity of the produced DES was relatively low in the case that the HBD used was liquid. The viscosities of DESs formed with 1-propanol, 1butanol, and hexyl alcohol were lower, and thus, facilitated the mass transfer of the artemisinin from plant material to DESs and enhanced the extraction yield. Changes in the type and length of the alkyl chain of the HBDs would change the polarity and viscosity of DESs and thus influence the extraction efficiency of different hydrophobic DESs. Based on careful examination of the extraction efficiency of various DESs, DES2, DES-5, and DES-7 were chosen for the next DES design. Tailoring the DES for Higher Extraction Efficiency. Artemisinin is easily soluble in chloroform, acetone, and ethyl acetate; soluble in ethanol; slightly soluble in petroleum ether; and hardly soluble in water. It can be seen that artemisinin is a
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RESULTS AND DISCUSSION Optimization of Precolumn Reaction for HPLC Analysis. According to previous reports,20,21 artemisinin has weaker ultraviolet absorption at 203 nm due to the lack of a conjugated radical in the molecular structure. After reacting with alkali, the endoperoxide bridge in the artemisinin will be destroyed and rearranged into a new chemical compound Q292 with conjugated structure (Figure S2 in the Supporting Information). The maximum absorption wavelength of Q292 is 292 nm. Q292 can be quantitatively converted to another chemical compound Q260 at pH 5.58−6.04. Q260 has a maximum ultraviolet absorption at 260 nm. Therefore, the amount of artemisinin can be quantified by the sum of the two chromatographic peaks of Q260 and Q292.20 To obtain a stable precolumn process for HPLC analysis, the concentration of sodium hydroxide, concentration of acetic acid, and acidification time for the precolumn reaction were optimized based on the previous method.20 The results are shown in Figure S3 (Supporting Information). Finally, the precolumn process was established as follows: 0.5 mL of sample was derived with 3.0 mL of 0.3% (w/v) sodium hydroxide methanol solution in a water bath at 45 °C for 25 min. After cooling to room temperature, the reaction mixture was acidified with 2.0 mL of 0.1 mol/L acetic acid methanol solution. Hydrophobic DESs Preparation and Initial Screening. According to previous reports,2,4 DESs with hydrophobicity like petroleum ether or hexane may be used as alternatives for artemisinin extraction. Based on the literature,17 N81Cl was chosen as the HBA to prepare hydrophobic DESs with 13 alcohol HBDs with different side chains at selected molar ratios. 3273
DOI: 10.1021/acssuschemeng.6b03092 ACS Sustainable Chem. Eng. 2017, 5, 3270−3278
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enhance their extraction efficiency.23 When N81Cl to HBD molar ratios decreased from 1:4 to 1:5, too many HBDs would lead to the reduction of N81Cl concentration, which reduced the interactions between HBA and HBD and between the artemisinin and chloride anion, thus decreasing the extraction efficiency.14 Comparatively, the N81Cl to HBD molar ratio of 1:4 gave the highest extraction yield for all three DESs, and DES N81Cl-NBA (1:4) showed the highest extraction yield of the DESs. The HBA type is also an important factor influencing the physicochemical properties of a DES.23,24 Therefore, tetrapropylammonium bromide (TPAB) and tetrabutylammonium bromide (TBAB), bearing similar structures to N81Cl but having shorter alkyl chains, were chosen to replace N81Cl as the HBA to prepare DESs at varied HBA to HBD molar ratios. When TPAB was used as the HBA, HBA to HBD molar ratios of 1:1 to 1:8 were tested, and 18 DESs were successfully prepared. The extraction yields of the 18 DESs for artemisinin are shown in Figure 2B. It was found that TPAB could substitute for the N81Cl as the HBA to prepare DESs for the extraction of artemisinin. The TPAB to HBD molar ratio influenced the extraction yield to some degree. The DES TPAB-NBA (1:3) showed the highest extraction yield. When TBAB was used as the HBA, HBA to HBD molar ratios of 1:1 to 1:6 were tried, and 18 DESs were successfully prepared. The extraction yields of the 18 DESs for artemisinin are shown in Figure 2C. It was found that TBAB could also substitute for the N81Cl as the HBA to prepare DESs for the extraction of artemisinin. The TBAB to HBD molar ratio clearly influenced the extraction yield. The DES TBAB-NBA (1:2) showed the highest extraction yield. Through the above tailoring, 1-butanol (NBA) was found to be the most effective HBD. DESs N81ClNBA (1:4), TPAB-NBA (1:3), and TBAB-NBA (1:2) were chosen for further tailoring. DESs can be prepared from two or more components, and their properties can be tailored by changing the HBD numbers.25 Therefore, ternary DESs were prepared by addition of a second HBD to N81Cl-NBA (1:4), TPAB-NBA (1:3), and TBAB-NBA (1:2), hoping to tailor the DESs for higher extraction efficiency. According to the initial screening results shown in Figure 1, 1-propanol, 1,2-propanediol, 1,3-butanediol, hexyl alcohol, and capryl alcohol were chosen as the second HBDs to prepare ternary DESs. As a result, 60 ternary DESs with different molar ratios of HBA:HBD1:HBD2 were prepared and used to extract artemisinin from A. annua leaves (Figure 3). Compared to the binary DES N81Cl-NBA (1:4), the second HBD addition could indeed change the extraction efficiency but could not improve the extraction efficiency for most of the ternary DESs (Figure 3A). Only the addition of hexyl alcohol and capryl alcohol, producing the ternary DESs N81Cl-NBA-HA (1:3:1) and N81Cl-NBA-CA (1:3:1), gave higher extraction yields (Figure 3A). Similar results were obtained for TPAB-NBA (1:3), and only capryl alcohol addition, producing a ternary DES TPAB-NBA-CA (1:1:2), showed a higher extraction yield (Figure 3B). For TBAB-NBA (1:2), the second HBD addition could not improve the extraction efficiency for any of the ternary DESs (Figure 3C). Therefore, N81Cl-NBA-CA (1:3:1) and TPAB-NBA-CA (1:1:2) and TBAB-NBA (1:2), with the highest extraction yield in each group, were chosen, and their stabilities were investigated before using them for the next optimization. The results showed that the ternary DES TPAB-NBA-CA (1:1:2) would salt out after storing at room temperature for more than
compound with moderate hydrophobicity. Therefore, tailoring the DES may provide a DES that is more suitable for artemisinin extraction from A. annua leaves. As we know, the HBA to HBD molar ratio is one of the important factors influencing the physicochemical properties of a DES.22 Therefore, the effects of HBA to HBD molar ratios of DES2, DES-5, and DES-7 on the extraction yields of artemisinin were first investigated. DES-2, DES-5, and DES-7 with N81Cl to HBD molar ratios ranging from 1:2 to 1:5 were prepared and tested for artemisinin extraction. The results are shown in Figure 2A. The N81Cl to HBD molar ratios of the three DESs
Figure 2. Extraction yields (milligrams of artemisinin per gram of A. annua leaves powder) of the hydrophobic DESs with different N81Cl/ HBD molar ratios (A), TPAB/HBD molar ratios (B), and TBAB/ HBD molar ratios (C). Error bars indicate the SEM (n = 3). Abbreviations: N81Cl methyl trioctyl ammonium chloride, TPAB tetrapropylammonium bromide, TBAB tetrabutylammonium bromide, NPA 1-propanol, NBA 1-butanol, HA hexyl alcohol, CA capryl alcohol, and DA decyl alcohol.
showed some influence on the extraction yields. The extraction yields increased with decrease of N81Cl to HBD molar ratios from 1:2 to 1:4 and, then, decreased with decrease of N81Cl to HBD molar ratios from 1:4 to 1:5. The HBDs used to form DES-2, DES-5, and DES-7 are liquid. When N81Cl to HBD molar ratios decreased from 1:2 to 1:4, the amounts of HBDs increased, and thus resulted in the decrease in viscosity and surface tension of DES-2, DES-5, and DES-7, which would 3274
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TBAB-NBA (1:2), and N81Cl-NBA-CA (1:3:1), including magnetic stirring, heating, water-bath shaking, and UAE were compared with air-bath shaking under the same conditions. As displayed in Figure 4, different extraction methods exhibited
Figure 4. Comparison of the extraction efficiencies of different extraction methods using N81Cl-NBA (1:4), TBAB-NBA (1:2), and N81Cl-NBA-CA (1:3:1) as the extraction solvents at 30 (A) and 60 °C (B). Experimental conditions are described in the section Extraction of Artemisinin from A. annua Leaves Employing Hydrophobic DESs. Error bars indicate the SEM (n = 3).
discernible differences in extraction yields regardless of the DESs and temperature used. At 30 °C, the UAE method was the most efficient extraction method (Figure 4A). At 60 °C, the amount of artemisinin extracted by the UAE method was close to that extracted by the magnetic stirring method and higher than those of the other four extraction methods (Figure 4B). The extraction temperature was also an important factor contributing to the increase in extraction yield. When the temperature increased from 30 to 60 °C, the extraction yield increased notably for all DESs and extraction methods (Figure 4). The magnetic stirring and UAE methods were selected as the standby methods for the next optimization. In addition, N81Cl-NBA-CA (1:3:1) was excluded from the next optimization because it showed lower extraction efficiency at 60 °C when the magnetic stirring and UAE methods were used. Optimization of the Extraction Conditions Using RSM. As reported in previous DES-based extractions,13,15,26 several independent variables such as extraction temperature, solvent to solid ratio, and extraction time have usually been investigated for optimization. RSM-based optimization of parameters is often adopted because it allows the evaluation of the interaction effects between variables and variable optimized in their full scope through fewer experiments.27 To choose appropriate parameter ranges for RSM optimization, preliminary singlefactor experiments were carried out, and the results are shown in Figures S4−S9 in the Supporting Information. As a result, solvent/solid ratios (A) ranging from 10:1 to 20:1 (mL/g),
Figure 3. Extraction yields (milligrams of artemisinin per gram of A. annua leaves powder) of the ternary DESs prepared based on N81ClNBA (1:4) (A), TPAB-NBA (1:3) (B), and TBAB-NBA (1:2) (C) by addition of the second HBD at different molar ratios. Error bars indicate the SEM (n = 3). Abbreviations NPA 1-propanol, HA hexyl alcohol, CA capryl alcohol, 1,2-PDO 1,2-propanediol, and 1,3-BDO 1,3-butanediol.
10 days (data not shown) and was thus abandoned. At the end, two binary DESs N81Cl-NBA (1:4) and TBAB-NBA (1:2) and one ternary DES N81Cl-NBA-CA (1:3:1) were chosen for the following optimization. N81Cl-NBA-HA (1:3:1) was not chosen, because it only gave a slightly higher extraction yield than N81Cl-NBA (1:4) but had a more complex composition. The above tailoring process shows that DESs can be tuned to achieve the desired extractability against a certain bioactive compound. Comparison of Different Extraction Methods. Magnetic stirring, heating, water-bath shaking, air-bath shaking, and ultrasound-assisted extraction (UAE) can be employed for extraction using DESs as extraction solvents. In this study, airbath shaking was employed as the extraction method in the initial screening and the solvent tailoring procedure because of its simplicity. Prior to optimization of the final extraction conditions, other types of extraction methods that are compatible with the tailored DESs, N81Cl-NBA (1:4), 3275
DOI: 10.1021/acssuschemeng.6b03092 ACS Sustainable Chem. Eng. 2017, 5, 3270−3278
Research Article
ACS Sustainable Chemistry & Engineering
experimental data were in high agreement with the predicted extraction. Additionally, it is shown in Table 3 that the linear coefficients (A, B, C, D, and E), cross product coefficients (BD, BE, CD, CE, and DE), and quadratic term coefficients (B2, C2, and E2) were considered to be significant (p < 0.05), whereas the other cross product coefficients (AB, AC, AD, AE, and BC) had no significant influence (p > 0.05). These results indicated that solvent/solid ratio, ultrasonic power, temperature, particle size of A. annua leaves power, and extraction time had important effects on the total extraction yield of the target artemisinin and that there were interactions between ultrasonic power and particle size, ultrasonic power and extraction time, temperature and particle size, temperature and extraction time, and particle size and extraction time. The effects of variables on the extraction yield and the interaction effects between the variables were visualized by graphical representations of the model equation, which were 3D response surface plots, shown in Figure S10 (Supporting Information). Analysis of Figure S10 gave the same results as ANOVA. The results of the regression analysis indicated that a maximal extraction yield of 8.0031 mg/g of artemisinin could be acquired under the following optimal conditions: solvent/ solid ratio 17.5:1, ultrasonic power 180 W, temperature 45 °C, particle size 80 mesh, and extraction time 70 min. To verify the fitness of the predicted response values, verification experiments were performed under the above optimal conditions in five replicates. The observed extraction yields were 7.9453, 7.9724, 8.0267, 7.9925, and 8.0311 mg/g, which were largely consistent with the predicted value. This indicated that the established quadratic model was statistically reliable and reasonable. Evaluation of Extraction Efficiency of the N81Cl-NBA (1:4) in Comparison to Petroleum Ether. Conventionally, petroleum ether (60−90 °C) has been used as the extraction solvent for extracting artemisinin from A. annua leaves.29,30 After obtaining the optimal extraction conditions, the extraction efficiency of hydrophobic DES N81Cl-NBA (1:4) was compared with that of petroleum ether (60−90 °C). The comparison was carried out using the ultrasonic method under the optimized extraction conditions obtained in the above section. The extraction yield of N81Cl-NBA (1:4) was 7.9936 ± 0.0364 mg/g, which was notably higher than that (6.1830 ± 0.1956 mg/g) of petroleum ether (60−90 °C). Petroleum ether is volatile, flammable, and toxic. In consideration of its sustainability, biodegradability and pharmaceutically acceptable toxicity, the newly optimized method using the tailor-made N81Cl-NBA (1:4) developed in this study is clearly efficient, ecofriendly and nontoxic and thus can be used as a green substitute for extracting artemisinin from A. annua leaves. Recovery of Artemisinin from the DES Extraction Solution and Reuse of the DES. Distillation is not suitable for the recovery of artemisinin from DES N81Cl-NBA due to the negligible vapor pressure of the DES. In recent years, several methods have been reported to recover the extracted compounds from DESs, including the applications of supercritical carbon dioxide, antisolvents, solid-phase extraction (SPE), and adsorption chromatography.13,15,31 The resin adsorption method has been found to be a very simple and efficient method for the recovery of several bioactive compounds from hydrophilic DES extraction solutions.14,26,32 In the present study, six macroporous resins were assessed to
ultrasonic powers (B) ranging from 120 to 200 W, temperature ranging from 40 to 60 °C, particle sizes of A. annua leaves powder (D) ranging from 20 to 80 mesh, and extraction times (E) ranging from 40 to 80 min were chosen for RSM optimization using N81Cl-NBA (1:4) and UAE as the extraction solvent and method. Magnetic stirring and TBABNBA (1:2) were excluded during the single-factor experiments. Extraction yields of artemisinin were used as the response function of the CCD method, which is one of the most common methods to define optimum values in multilevel design.27,28 The experimental orders, levels of coded and uncoded variables and extraction yields are listed in Table 2. Through employing multiple regression analysis to analyze the experimental data, the following second-order polynomial equation was used to express the relationship between the extraction yield (Y) and variables in terms of coded levels: Y = 6.46 + 0.064A + 0.23B + 0.23C + 0.29D + 0.29E + 0.024AB − 0.015AC − 0.012AD − 0.026AE − 0.069BC − 0.091BD + 0.1BE − 0.071CD − 0.013CE − 0.016DE + 0.056A2 + 0.19B2 + 0.29C2 + 0.041D2 + 0.22E2. ANOVA for the proposed model is shown in Table 3. F-test and p-value were applied to check Table 3. ANOVA of the Proposed Model for the Extraction of Artemisinin source
sum of squares
degree of freedom
mean square
Model A B C D E AB AC AD AE BC BD BE CD CE DE A2 B2 C2 D2 E2 residual lack of fit pure error total
20.30 0.16 2.13 2.18 3.23 3.23 0.02 0.01 0.00 0.02 0.15 0.27 0.32 0.16 0.56 0.88 0.10 1.09 2.63 0.05 1.51 1.11 0.62 0.50 21.41
20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 29 22 7 49
1.02 0.16 2.13 2.18 3.23 3.23 0.02 0.01 0.00 0.02 0.15 0.27 0.32 0.16 0.56 0.88 0.10 1.09 2.63 0.05 1.51 0.04 0.03 0.07
F-value
p-value
26.42 4.20 55.56 56.63 84.06 84.15 0.46 0.20 0.13 0.55 4.01 7.12 8.46 4.28 14.61 22.88 2.61 28.49 68.55 1.34 39.43
