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Design and Performance Evaluation of Ionic-Liquids-Based Microwave-Assisted Environmentally Friendly Extraction Technique for Camptothecin and 10-Hydroxycamptothecin from Samara of Camptotheca acuminata Shu-ya Wang,† Lei Yang,*,† Yuan-gang Zu,* Chun-jian Zhao, Xiao-wei Sun, Lin Zhang, and Zhong-hua Zhang Key Laboratory of Forest Plant Ecology, Northeast Forestry University, 150040 Harbin, People’s Republic of China
bS Supporting Information ABSTRACT: Ionic-liquids-based, microwave-assisted extraction (ILMAE) was successfully applied to the extraction of two alkaloids: camptothecin (CPT) and 10-hydroxycamptothecin (HCPT) from samara of Camptotheca acuminata. Ten different types of 1-alkyl-3-methylimidazolium, with different cations and anions, were investigated in this work, and 1-hexyl-3-methylimidazolium bromide ([C8MIM]Br) solution was selected as the optimal solvent. In addition, the extraction parameters, including ionic liquid concentration, soak time, microwave irradiation power, irradiation time, solid liquid ratio, and number of extraction cycles, were optimized. The optimal conditions were as follows: 0.8 M [C8MIM]Br; soak time, 2 h; microwave irradiation power, 385 W; irradiation time, 8 min; solid liquid ratio, 1:12 (g/mL); and two of extraction cycles. No degradation of the target analytes have been observed at the optimum conditions, as evidenced from the stability studies performed with standard CPT and HCPT. The proposed method also shows high reproducibility. Compared with the traditional methods, the proposed approach demonstrates higher efficiency in a shorter extraction time (from 4 h to 8 min), which demonstrates that ILMAE is a rapid, efficient, and simple sample extraction technique.
1. INTRODUCTION Over the past several years, ionic liquids have received everincreasing interest as environmental benign green solvents. Ionic liquids are known as salts with low melting temperatures. They are composed of organic cations and inorganic or organic anions. Because of their unique chemical and physical properties, such as low vapor pressure, high thermal stability, no flammability, and no ignition point,1 4 ionic liquids are becoming increasingly attractive as alternative extraction media.5 7 The extraction of effective components from botanical materials is usually achieved by using traditional methods such as maceration extraction at room temperature (ME), stirring extraction (SRE), Soxhlet extraction (SE), and heat-reflux extraction (HRE).8 However, these extraction processes have some drawbacks, including being time-consuming, having low efficiency, being discommodious, and requiring large volumes of toxic organic solvents. Therefore, an increasing amount of attention is being paid to the development of more-efficient extraction methods for the rapid extraction of effective components from botanical materials. Microwave-assisted extraction (MAE), with the main advantages of shorter extraction time, high efficiency, simple operation, and low cost,9 12 has the potential to be an alternative to traditional extraction methods. Microwave energy acts as nonionizing radiation that causes molecular motion by causing migration of ions and rotation of dipoles.13 It has been found that ionic liquids as solvents and co-solvents can efficiently absorb microwave energy.14 Camptotheca acuminata is a species indigenous to China, among the known biologically active components of the samara; r 2011 American Chemical Society
the two main quinoline alkaloids are named camptothecin (CPT) and 10-hydroxycamptothecin (HCPT).15 Because of the promising antitumor characteristics of CPT, it has attracted considerable attention worldwide. HCPT has been demonstrated to be more potent and less toxic than CPT. It exhibits strong antitumor characteristics when used against hepatoma, gastric carcinoma, colon cancer, and leukemia, and it is widely used in experimental and clinical studies.16 18 In addition, other various anticancer drugs (9-nitrocamptothecin, irinotecan,19 9-aminocamptothecin, topotecan,20,21 and so on) are semisynthesized by CPT and HCPT to overcome the drawbacks of intolerable toxicity and insolubility in water and increase antitumor activity. CPT and its analogues have demonstrated effectiveness in killing various cancer cells such as small and nonsmall cells of lung cancer, ovarian cancer, pancreatic cancer, myelomonocytic leukemia, and related disorders. Ionic liquids have shown potential as solvents in the extraction of various useful substances from plant samples, such as phenolic compounds,22 25 lignans,26 and terpene lactones.22,27 Ionic liquids have also been used to extract several types of alkaloids, such as aporphine alkaloid,12 piperidine alkaloid,28 terpenoid indole alkaloid,29 and phenolic alkaloid.30 Therefore, to the best of our knowledge, the extraction of quinoline alkaloids with ionic liquid as the solvent has not yet been reported in the literature. Received: June 26, 2011 Accepted: November 10, 2011 Revised: October 30, 2011 Published: November 10, 2011 13620
dx.doi.org/10.1021/ie201370m | Ind. Eng. Chem. Res. 2011, 50, 13620–13627
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Figure 1. HPLC chromatogram of two alkaloids in the [C8MIM]Br extract. Inset shows an HPLC chromatogram of a mixture of two standard alkaloids.
Hence, it is of interest to investigate the microwave extraction of quinoline alkaloids using ionic liquids. The objective of this study was to develop an effective and environmental friendly ionic liquid based microwave-assisted approach for the extraction of CPT and HCPT from samara of C. acuminata. Herein, we describe our investigations on the performance of various ionic liquids with different cations and anions in an ionic-liquids-based microwave-assisted extraction (ILMAE) method. It was found that parameters—including the concentration of ionic liquid, the soak time, the microwave irradiation power, the irradiation time, the solid liquid ratio, and the number of extraction cycles—were influential on the final yield, and these parameters were optimized systematically. At the same time, the ILMAE was compared with traditional solvents and traditional extraction methods for extracting CPT and HCPT. This approach may provide a set of optimum conditions for ILMAE of CPT and HCPT from samara of C. acuminata.
2. EXPERIMENTAL SECTION 2.1. Reagents and Samples. CPT and HCPT standards (98%), purchased from Wako Chemical Co. (Osaka, Japan), were used as received. Acetonitrile was high-performance liquid chromatography (HPLC) grade (J&K Chemical, Ltd., China). All of the ionic liquids that were purchased from Chengjie (Shanghai, PRC) were used as received. All of the other solvents and chemicals used in this study were of analytical grade, purchased from Beijing Chemical Reagents Co. (Beijing, PRC). The pure water was purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA). Samara of C. acuminata was purchased from a local Chinese medicinal materials market in Guanghan (Sichuan, PRC) and authenticated by Prof. Shao-quan Nie of the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, China. Voucher specimens were deposited in the herbarium of this facility. Dried samaras were powdered to a homogeneous size and then sieved (60 80 mesh).
2.2. HPLC Analysis and Quantification. The HPLC system consists of a Waters 717 automatic sample handling system, 1525 Bin pump, 2487 UV-detector and Analytical-2-Prep column temperature control box (Waters, USA). Chromatographic separation was performed on a HiQ sil-C18 reversed-phase column (4.6 mm 250 mm, 5 μm, KYA TECH). A stock solution of CPT and HCPT was prepared in ethanol, at concentrations of 95 μg/mL for CPT and 106 μg/mL for HCPT. Working standard solutions were prepared by serial dilution of stock solutions in ethanol and then stored at 1 4 °C in darkness. The extracts were directly injected into the liquid chromatograph. The injection volume was 10 μL, and the column temperature was maintained at 25 °C. The mobile phase consisted of acetonitrile and pure water (3:7, v/v), with a flow rate of 1 mL/min. The detection wavelengths were 254 nm for CPT and 266 nm for HCPT. Under these conditions, the two alkaloids were separated at the baseline. CPT and HCPT were identified by comparing their retention times with corresponding peaks in the standard solution. A comparison of chromatograms of the two alkaloids obtained from [C8MIM]Br extract with those contained in standard solutions is shown in Figure 1. No influence attributable to the ionic liquids used was observed on the peak resolution, elution order, or elution time. Corresponding calibration curves for each compound are YCPT = 38743x + 97809 (r = 0.9994) and YHCPT = 35572x + 15747 (r = 0.9999). Good linearities were found for CPT and HCPT in the ranges of 2.97 95.00 and 3.31 106.00 μg/mL, respectively. 2.3. Ionic Liquids-Based Microwave-Assisted Extraction (ILMAE). The experimental setup is shown schematically in Figure S1 in the Supporting Information. A household microwave oven (Glanz, Shunde, PRC) was used for ILMAE with a microwave irradiation frequency of 2450 MHz. The maximum output power of the oven was 700 W. In order to examine the effect of microwave power, five power levels were studied: 100% (700 W), 77% (540 W), 55% (385 W), 33% (230 W), and 17% (120 W). The entire system was operated at atmospheric pressure. 13621
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Industrial & Engineering Chemistry Research The dimensions of the interior cavity of the oven are 215 mm 350 mm 330 mm. The microwave oven was modified by drilling a hole at the top. A round-bottom flask with a capacity of 50 mL is placed in the oven and connected to a reflux condenser through the hole. After the reflux condenser is placed into the oven, the hole around the neck of the flask is covered with polytetrafluoroethylene, to prevent the leakage of microwaves. A quantity of 1.0 g of dried sample was mixed with ionic liquid solutions, which we prepared in pure water, soaking for a while, and then the suspension was extracted by MAE. The optimum anion, cation, concentration of selected ionic liquid, soak time, microwave irradiation power, irradiation time, solid liquid ratio, and number of extraction cycles were systematically studied in this work. After MAE, the extracts obtained were cooled to room temperature rapidly by a cold bath and filtrated through a 0.45-μm microporous membrane (Guangfu Chemical Reagents Co. Tianjin, PRC) for subsequent HPLC analysis. 2.4. Optimum Microwave-Assisted Extraction Methods by Orthogonal Design. To further study the interaction between the factors, we optimize the operating condition by orthogonal design with four factors applied using Design-Expert 7.0 software (Stat-Ease, Minneapolis, MN, USA). The bounds of the factors were as follows: [C8MIM]Br concentration, 0.8 1.2 M; microwave power, 230 540 W; extraction time, 8 12 min; and solid liquid ratio, 1:8 1:12. 2.5. Stability, Recovery, and Repeatability of ILMAE. Stability tests were performed by determing CPT and HCPT standards, which were dissolved in 0.8 M [C8MIM]Br and extracted by MAE under the optimum conditions (microwave power = 385 W, extraction time = 8 min, solution volume = 12 mL). The recoveries of CPT and HCPT were taken as the indicative markers for the stability of CPT and HCPT under the derived operating extraction conditions. To determine the repeatability of the novel extraction method, five samples of the same weight (0.5 g) were processed under the same optimum extraction conditions as those obtained from the systematic study of different extraction parameters. 2.6. Reference Extraction Methods. All the conventional extraction methods were conducted under their optimal conditions. 2.6.1. Ethanol-Based Microwave-Assisted Extraction (EMAE). Ethanol (85%) was taken as the solvent in the microwaveassisted extraction of CPT and HCPT from samara of C. acuminata. The extraction experiment was operated under the optimum conditions, except for solvent type. The subsequent steps were as same as those in ILMAE. 2.6.2. Ethanol-Based Heat Reflux Extraction (EHRE). Dried sample (1.0 g) was mixed with 12 mL of ethanol (85%) in a round-bottom flask; then, the flask was placed into a water bath, connected to cooling water, and then allowed to reflux for 4 h under a temperature of 90 °C. The subsequent steps were the same as those in ILMAE. 2.6.3. Ethanol-Based Soxhlet Extraction (ESE). Dried sample (1.0 g) was mixed with 120 mL of ethanol (85%); then, the mixture was placed in a Soxhlet apparatus and extracted for 12 h under a temperature of 90 °C. The ethanol extraction was concentrated to dryness, and then redissolved in 12 mL of ethanol (85%). The subsequent steps were the same as those in ILMAE. 2.6.4. Ethanol-Based Stirring Extraction at 50 °C (ESRE). Stirring extraction was carried out on an electric stirrer. Dried sample (1.0 g) was placed in a flask and mixed with 12 mL of ethanol (85%). The extraction time was limited to 8 h, and the stirring speed was determined to be 120 rpm and the extraction
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Table 1. Some Physicochemical Properties of the Studied Ionic Liquidsa
a
TSO = p-toluenesulfonate.
temperature was set as 50 °C. The subsequent steps were the same as those in ILMAE. 2.6.5. Ethanol-Based Maceration Extraction (EME). Dried sample (1.0 g) and 12 mL of ethanol (85%) was placed in a 50-mL flask. The extraction time was set as 24 h. The subsequent steps were the same as those in ILMAE. 2.7. Statistical Analysis. One way of ANOVA testing was used to calculate the significance of the differences of the extraction yield of alkaloids. The results of HPLC analysis were expressed as means of extraction yield ( the standard deviation (SD).
3. RESULTS AND DISCUSSION 3.1. Screening of Ionic Liquids. The structure of ionic liquids has appreciable impact on its physicochemical properties, which might greatly affect the yields of target analytes.22 To evaluate the performance of 1-alkyl-3-methylimidazolium-type ionic liquids (see Table 1) in the MAE process, the impact of the identity of the anion and the alkyl chain length of the cation on the yields of CPT and HCPT are researched and recorded in this paper. 3.1.1. Effect of the Anion. The anion identity is considered to be a great influence on the properties of the ionic liquid, particularly influencing the water miscibility of the ionic liquid.31 Therefore, 1-butyl-3-methylimidazolium ionic liquids with seven h, and HSOh) different anions (Clh, Brh, BFh, 4 NOh, 3 ClOh, 4 TSO 4 were tested. The results obtained are given in Figure 2a. Figure 2a indicates that TSOh was more efficient than others for CPT and Brh was more efficient for HCPT. This result indicates that the extraction yields of alkaloids are mainly anion-dependent. The reasons are that [C4MIM]Br and [C4MIM]TSO have stronger multi-interactions including hydrogen bonding and hydrophobic interactions with alkaloids.27 Besides, some ionic liquid solutions, such as [C4MIM]Br and[C4MIM]TSO, have slightly higher acidity, which might facilitate the extraction of weakly basic alkaloids from C. acuminata.32 34 In comparison, Brh was more efficient than TSOh for CPT and HCPT, so Brh was taken for the subsequent evaluation. 13622
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Figure 2. Effect of (a) the ionic liquid anion and (b) the alkyl chain length of the cation on the extraction yield of target analytes with 1.0 M ionic liquids. Sample amount, 1.0 g; extractant volume, 10 mL; soak time, 2 h; irradiation power, 230 W; and microwave irradiation time, 10 min.
3.1.2. Effect of the Alkyl Chain Length of the Cation. With the same anion of Brh, a series of 1-alkyl-3-methylimidazolium cations, increasing the alkyl chain length of cation, from ethyl ([C2MIM]Br) to octyl ([C8MIM]Br), were evaluated. As shown in Figure 2b, increasing the alkyl chain length of the cation from ethyl to hexyl dramatically raises the yields of CPT and HCPT. When the alkyl chain length of the cation was increased from hexyl to octyl, the yields increase slowly. The length of the alkyl chain of the cation is related to the water miscibility of the Brh ionic liquid,31 and the water miscibility of Brh ionic liquid may be related to the yields. Considering the above results, [C8MIM]Br was chosen for the subsequent evaluation. 3.2. Optimization of Extraction Conditions. The univariate method was used in all instances for optimization of the six parameters, which might extensively affect the extraction step: ionic liquid concentration, soak time, microwave irradiation power, irradiation time, solid liquid ratio, and number of extraction cycles. There are many factors affecting the extraction of CPT and HCPT from the samara of C. acuminata. We worked out the optimal level of each factor by the single factor test. However, because of the interaction among the factors, the combination impacts of the optimal level of each factor may not be the optimum extraction conditions. Orthogonal test is a scientific method which can provide arrangement of experimentation with multifactors. Based on single factor tests, the optimum extraction conditions can be worked out via orthogonal testing.
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3.2.1. Single-Factor Experiments. 3.2.1.1. Ionic Liquid Concentration. It has been demonstrated that polar molecules can strongly absorb microwave energy;35,36 for this reason, water is always chosen as the co-solvent in MAE processes. In the experiment, 1.0 g of sample was soaked in 10 mL [C8MIM]Br for 2 h and then extracted for one cycle under a microwave irradiation power of 230 W and 10 min of irradiation. The effect of the concentration of [C8MIM]Br ionic liquid is obvious. As the results show in Figure 3a, the yields of CPT and HCPT increase gradually when the concentration of ionic liquid is over the range of 0.25 1.0 M. Upon further increases, however, a slight decrease in extraction yield is observed. This is most probably due to the longer length of the alkyl chain, which makes its water miscibility and viscosity more sensitive to the concentration. Finally, a [C8MIM]Br concentration range of 0.8 1.2 M was adopted in further optimization studies. 3.2.1.2. Soak Time. Because the sample is dry powder, enough soak time to absorb sufficient microwave energy when performing the extraction is indispensable. In the experiment, 1.0 g of sample was soaked in 10 mL of solvent (1.0 M [C8MIM]Br) for 1, 2, 3, 4, and 8 h before being microwave-irradiated (see Figure 3b), extracted for one cycle under a microwave irradiation power of 230 W and 10 min of irradiation. When the soak time is
