Desalination 286 (2012) Contents lists available at SciVerse ScienceDirect. Desalination. journal homepage:

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1 Desalination 286 (12) Contents lists available at SciVerse ScienceDirect Desalination journal homepage: Simultaneous extraction and purification of aloe polysaccharides and using ionic liquid based aqueous two-phase system coupled with dialysis membrane Zhi-jian Tan, Fen-fang Li, Xue-lei Xu, Jian-min Xing College of Chemistry and Chemical Engineering, Central South University, Changsha, 483, China article info abstract Article history: Received 25 September 11 Received in revised form 23 November 11 Accepted 24 November 11 Available online 22 December 11 Keywords: Aloe polysaccharides Purification Ionic liquid Aqueous two-phase system Dialysis membrane In this paper, an ionic liquid based aqueous two-phase system (ILATPS) was employed in simultaneous extraction and purification of aloe polysaccharides () and by a single-step procedure. Parameters affecting the extraction efficiency were investigated in detail, such as type and concentration of phaseforming salt, temperature, ph, inorganic electrolyte, etc. Under the optimal extraction conditions, can be extracted into salt-rich phase with high extraction efficiency, while majority and other impurities were extracted into IL-rich phase. were further purified using a dialysis membrane to remove the salt and IL. In addition, the monosaccharide constitutes of were analyzed by a HPLC method. The purity of the final product was demonstrated by a thermogravimetric analysis (TGA). IL in IL-rich phase was recycled by a simple solvent extraction method using dichloromethane. Compared with other liquid liquid extraction, ILATPS is much simpler and greener, it will open up new possibilities in the separation of other active ingredients in natural plants. 11 Elsevier B.V. All rights reserved. 1. Introduction Aloe vera L. is a plant belonging to the Liliaceae family. About a total of 36 aloe species are growing in the dry regions of North American, Europe and Asia [1]. It has long been used as nutraceutical, medicine and cosmetic [2]. The aloe vera plant contains 75 potentially active substances, including polysaccharides,, minerals, phenolic compounds, vitamins, sugars, saponins, and amino acids et al. [3,4]. are the major active constitutes in the aloe gel. They are responsible for the pharmacological activities of wound healing, antiinflammation, and immunomodulating properties [5]. Various methods were used for separation and purification of, such as ethanol precipitation, ion-exchange chromatography [6], gel permeation chromatography [7], and membrane separation [8]. Ethanol precipitation seems to be a simple method for obtaining crude, but in order to obtain the product with high purity, a further processing must be performed. Ion-exchange chromatography method consumes great time and plenty of organic solvent. Gel permeation chromatography is an efficient further purification method, but this procedure requires high cost and complex operations, which restricts its large scale application. Membrane separation is an effective method for purification of, but the viscosity of aloe gel is very high and the membrane is easy to be fouled by the aloe gel without further Corresponding author. Tel.: ; fax: address: lfflqq@mail.csu.edu.cn (F. Li). treatment. Therefore, it is of great interest to develop a simple and efficient pretreatment method. In recent years, as a novel liquid liquid extraction technique, aqueous two-phase system (ATPS) constitutes a greener and potentially more efficient pretreatment solution, which is applied to separate and purify lots of compounds in a single-step procedure. ATPS based on IL and salt was first reported by Rogers [9]. The ILATPS has advantages with combination of IL and ATPS, such as negligible viscosity, little emulsion formation, free of volatile organic solvent, quick phase separation, high extraction efficiency, and gentle biocompatible environment [1 13]. It had been applied in extraction and purification of [11], antibiotics [12,14], alkaloids [15], drugs [16], and small organic molecules [17]. But there were few reports about the extraction of active ingredients in natural plants. In this paper, we developed a simple, efficient and green technique in simultaneous extraction and isolation of and. had migrated into the salt-rich phase, while majority impurities of, minerals and phenolic compounds were extracted into the IL-rich phase. Through investigating the partitioning behavior of and in ILATPS, the optimal extraction conditions were obtained, which demonstrated that this method was indeed capable of obtaining with high purity. Furthermore, existed in saltrich phase can be purified using a dialysis membrane, salt and IL were filtrated, leaving the in the dialysis tubing. After lyophilization, were analyzed by TG. [Bmim]BF 4 in the IL-rich phase could be extracted into the CH 2 Cl 2, after removing CH 2 Cl 2, [Bmim]BF 4 was recycled. The whole flow chart of extraction, separation and purification of and recovery of IL is shown in Fig /$ see front matter 11 Elsevier B.V. All rights reserved. doi:1.116/j.desal

2 39 Z. Tan et al. / Desalination 286 (12) Experimental 2.1. Materials and reagents Fresh aloe leaves were obtained from Hainan province in China. [Bmim]BF 4 (1-butyl-3-methylimidazoliumtetrafluoroborate) was purchased from ChengJie Chemical Co., Ltd. (Shanghai, China) with a purity>99% and was used without further purification. Mannose, glucose, xylose, galactose and bovine serum albumin (BSA) were all biological reagent; Coomassie Brilliant Blue G25; 1-phenyl-3-methyl-5- pyrazolone (PMP); PEG were all purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Triton X-114 was purchased from Union Carbide Company (Germany). NaH 2 PO 4,(NH 4 ) 2 SO 4,andphenol were of analytical grade and purchased from TianJin Kermel Chemical Reagent Co., Ltd. (Tianjin, China). HPLC acetonitrile was purchased from TianJin HengXing Chemical Preparation Co., Ltd. (Tianjin, China). Other reagents were all analytical grade and used without further treatment. De-ionized water was used to prepare the sample solutions Preparation of crude The procedures were reported in our previous work [18]. Fresh aloe leaves were washed out, after the peel was removed, pulp with high viscosity was obtained. The pulp was homogenized using an agitator, then was centrifuged at rpm for 15 min. The aloe gel juice was filtrate by gauze and then condensed under vacuum condition. Five times the volume of 95% (v/v) ethanol was added and left overnight at 4 C. The precipitate was collected by centrifugation and repeatedly washed sequentially with lesser amounts of ethanol. After drying, the crude were obtained and stored at 4 C Simultaneous extraction of and To a 15 ml centrifuge tube, 1. g [Bmim]BF 4, 3. ml water, a given amount of salt and 1. ml solution were added. Another mixture with the same phase components but without crude solution was prepared as a blank to avoid interference. The mixture was stirred well to make the salt dissolve completely. The phase-separation was speeded up by centrifugation at rpm for 5 min, two clear phases formed. The partitioning experiments were done at room temperature, the top phase was mainly composed of IL,, minerals and other impurities with a small volume, and the bottom phase was the salt-rich solution containing with a large volume. The volume of each phase was noted down Analysis of and The bottom phase was withdrawn for analysis of by the phenol H 2 SO 4 method [19]. The whole procedure was as follows:.2 ml salt-rich phase was withdrawn to a 1 ml colorimeter tube, 1. ml 5% phenol was added, then 5. ml of concentrated sulfuric acid was added. The tube was well shaken and allowed to stand for min before analysis. The absorbance was measured at the wavelength of 49 nm by a 721-Vis spectrophotometer (HengPing, Shanghai). The calibration curve for analysis of was Y= X.658 with r=.9994 (RSD=2.%, n=6) using the mannose as the standard solution where Y is the absorbance, and X is the concentration of mannose in the range of.2.12 mg ml 1. The protein concentration was determined by the Bradford method [] with Coomassie Brilliant Blue G25. Total protein concentration was determined using BSA as standard. The whole procedure was as follows:.1 ml of IL-rich phase was withdrawn into the a 1 ml colorimeter tube and diluted to 1. ml, then 5. ml of Coomassie Brilliant Blue G25 solution was added, and the tube was well shaken and allowed to stand for 5 min before analysis. Samples were measured at 595 nm by a 721-visible spectrophotometer. The calibration curve for analysis of was Y=4.76X+.322 with r=.9991 (RSD=1.87%, n=5) where Y is the absorbance, and X is the concentration of BSA in the range of.2.32 mg ml 1. The phase ratio (R) was defined in Eq. (1): R ¼ V t V b where V t and V b are the phase volume in top and bottom phases, respectively. The extraction efficiency of (E a ) and (E p ) was calculated according to Eqs. (2) and (3), respectively. E a ¼ C bv b m a % ð2þ E p ¼ C tv t m p % ð3þ where m a and m p represent the amount of and in the crude solution added to the ATPS. C b and V b represent the concentration and volume in the bottom phase. C t and V t represent the concentration and volume in the top phase. ð1þ Fig. 1. Flow chart of extraction, separation and purification of and recovery of IL.

3 Z. Tan et al. / Desalination 286 (12) Results and discussion 3.1. Selection of the optimal ATPS NaH 2 PO 4 concentration (%, w/w) (NH 4 ) 2 SO 4 concentration (%, w/w) Fig. 2. Effect of the salts concentration to extraction efficiency. Conditions: ILATPS contained 1. g [Bmim]BF 4, 1.4 g NaH 2 PO 4, 3. ml water, 1. ml solution, all experiments were done at room temperature, the ph was not adjusted. To find out the optimal ATPS, PEG-based, surfactant-based, alcohol-based and IL-based ATPSs were tested in preliminary studies. As shown in Table 1, the extraction efficiency of ILATPS was higher than other ATPSs. Furthermore, the ILATPS has smaller viscosity than the PEG-based and surfactant-based ATPSs. [Bmim]BF 4 is a widely used IL, which can form ATPS with various salts. In this paper, (NH 4 ) 2 SO 4 and NaH 2 PO 4 were considered as the phase-forming salts, the reasons are as follow: firstly, they possess strong phase-formation ability with IL; secondly, they have good solubility in aqueous solution; thirdly, the two salts can form weakly acidic aqueous solutions, which is more stable for the existence of ; and fourthly, they can't react with H 2 SO 4 when determination of uses the phenol H 2 SO 4 method. The effect of salt concentration to the extraction efficiency is shown in Fig. 2, it was observed that NaH 2 PO 4 showed higher extraction efficiency and was more beneficial as the phase-forming salt. In addition, it can result in an appropriate ph range for determination of the samples. To optimize the NaH 2 PO 4 concentration, the amount of NaH 2 PO 4 in the range of g was investigated. When 1.4 g NaH 2 PO 4 (concentration is 25.93%, w/w) was added, the maximal extraction efficiency was obtained. With increase of salts, the extraction efficiency decreased, this is because of the strong salting-out effect, less free water is available to dissolve the resulting in the decrease of extraction efficiency. Phase ratio Effect of temperature The partitioning of and in [Bmim]BF 4 /NaH 2 PO 4 system at the temperature range of 25 5 C was investigated. As shown in Fig. 3, the phase ratio decreased with the increase of temperature, higher extraction efficiencies of and were obtained at lower temperature. As other literatures reported [21 23], an increase in temperature resulted in a transfer of water from the top to the bottom phase in ATPS. Therefore, when the volume of IL-rich phase decreases and salt-rich phase increases, the phase ratio decreases accordingly. IL concentration increases in the IL-rich phase, and the salt concentration decreases in the salt-rich phase. The salting-out effect decreases in salt-rich phase, the extraction efficiency of decreases. However, increase of temperature will make more IL redissolve in the aqueous solution [24], the result is the net IL concentration in IL-rich phase decreases, so the extraction efficiency of decreases accordingly. Proteins have a satisfactory extraction efficiency closing to the mild room temperature. Under this condition, IL/salt ATPS have good extraction ability for, in addition, have good solubility in IL-rich phase. also suffer from the disadvantage of thermal instability at high temperatures. In the light of these results, all the experiments were performed at room temperature Effect of ph Temperature ( o C) phase ratio Fig. 3. Effect of the temperature to extraction efficiency. Conditions: ILATPS contained 1. g [Bmim]BF 4, 1.4 g NaH 2 PO 4, 3. ml water, 1. ml solution, the ph was not adjusted. Na 2 HPO 4 H 3 PO 4 buffer solution was used to adjust the ph value of the ATPS. The effect of ph to extraction efficiency in the range of was investigated. As shown in Fig. 4, the extraction efficiency of was slightly influenced by ph, but the weakly acidic circumstance was more suitable for the existence of, that is because there are some weakly acidic existing in aloe. Proteins are easily affected by the change of ph, more can be extracted at lower Table 1 Simultaneous extraction of and in different ATPSs. Component 1 Component 2 E a (%) E p (%) PEG (13.33%, w/w) NaH 2 PO 4 (%, w/w) (salt-rich phase) 7.53 (PEG-rich phase) PEG (13.33%, w/w) NaH 2 PO 4 (%, w/w) (salt-rich phase) (PEG-rich phase) PEG6 (13.33%, w/w) NaH 2 PO 4 (%, w/w) (salt-rich phase) (PEG-rich phase) Triton X-114 (9.9%, w/w) Nil (aqueous phase) (surfactant-rich phase) Ethanol (18.75%, w/w) NaH 2 PO 4 (31.25%, w/w) (salt-rich phase) (ethanol-rich phase) [Bmim]BF 4 (18.52%, w/w) NaH 2 PO 4 (25.93%, w/w) 9.69 (salt-rich phase) (IL-rich phase)

4 392 Z. Tan et al. / Desalination 286 (12) ph values, maybe that is most charged have stronger electrostatic interaction with the anion and cation of ionic liquid under that condition. The [Bmim]BF 4 /NaH 2 PO 4 system is at an appropriate ph about 5. 6., so the ph of this system was not adjusted Effect of addition of electrolyte Neutral inorganic salts are usually used in ATPS to direct partitioning of target molecules, they can change the charge balance between the phases. Typically, salts added to the phase systems of.1.2 mol L 1 can give rise to electrostatic potential differences of the order of 5 1 mv [25]. The effect of salt to extraction efficiency was studied by adding NaCl in the range of mol L 1 into ATPS. As shown in Fig. 5, the extraction efficiency of varied little with value of % by addition of NaCl. Probably this is because are uncharged, the main acting force between and ATPS is hydrophilicity other than the electrostatic interactions. Whereas, are easily influenced by electrostatic potential difference in ILATPS, they are electric and easily affected by the inorganic electrolyte, so the extraction efficiency of increased sharply with ph Fig. 4. Effect of the ph to extraction efficiency. Conditions: ILATPS contains 1. g [Bmim]BF 4, 1.4 g NaH 2 PO 4, 3. ml water, 1. ml solution, all experiments were done at room temperature Concentration of NaCl (mol L -1 ) Fig. 5. Effect of NaCl to extraction efficiency. Conditions: ILATPS contains 1. g [Bmim]BF 4, 1.4 g NaH 2 PO 4, 3. ml water, 1. ml solution, all experiments were done at room temperature, the ph was not adjusted. addition of NaCl. In this paper, when 6.25 mmol NaCl (the concentration was 1.33 mol L 1 ) was added, both the two compounds can obtain higher extraction efficiencies, so this condition was chosen for further studies Analysis of monosaccharide The monosaccharide constitutes were analyzed after the hydrolysis of by a HPLC method. Chromatographic analyses were performed by a LC-1A HPLC (Shimadzu, Japan) with a C 18 column ( mm 4.6 mm I.D.; 5 μm) from HanBon Sci. & Tech. The mobile phase consisted of.1 mol L 1 ammonium acetate buffer solution (ph 5.5) and acetonitrile (78:22, v/v). The flow-rate was.8 ml min 1 and the UV detection wavelength was set at 245 nm. The column temperature was maintained at 3 C and the injection volume was 1 μl. Four standard monosaccharides with the same concentration and crude were pretreated by PMP solution. As shown in Fig. 6 the main constitutes of were mannose and few other kinds of monosaccharide. a mau b mau c mau PMP Man PMP Time (min) Man PMP Time (min) Time (min) Glc Gal Xyl Glc Gal Fig. 6. Analysis of monosaccharide constituents in : (a) PMP solution as a blank sample; (b) standard monosaccharide solution; (c) after hydrolysis.

5 Z. Tan et al. / Desalination 286 (12) Weight (%) Temperature ( o C) after dialysis crude a b aloe polysaccharides HPLC high performance liquid chromatography [Bmim]BF 4 1-butyl-3-methylimidazoliumtetrafluoroborate PEG polyethylene glycol BSA bovine serum albumin TGA thermogravimetric analysis Rpm revolutions per minute PMP 1-phenyl-3-methyl-5-pyrazolone MWCO molecular weight cutoff R correlation coefficient RSD relative standard deviation R volume ratio E a extraction efficiency of extraction efficiency of E p Fig. 7. TGA curve of (a) crude and (b) after ATPS extraction and dialysis Desalination and recovery of IL When were extracted into the salt-rich phase, a dialysis membrane (D45 mm, MWCO ) was used to filter the salt and IL. This membrane can be recycled. Under the optimal extraction conditions, with high purity can be obtained on a large scale. Analysis of by TG is shown in Fig. 7, it can be seen that after ATPS extraction and dialysis had a larger rate of weight loss at the weight loss scope of. This demonstrates that the with higher purity was obtained after the processing of ATPS extraction and desalination. Majority of [Bmim]BF 4 stay in the IL-rich phase after ATPS extraction. We recovered [Bmim]BF 4 using the method as Deng reported [26]. [Bmim]BF 4 was extracted into CH 2 Cl 2 solution, IL can be recycled after removing CH 2 Cl Conclusions A [Bmim]BF 4 /NaH 2 PO 4 ILATPS coupled with dialysis membrane was used to extract and purify. The crude were obtained by ethanol precipitation then was added into the ILATPS. The affecting factors of salts type and concentration, temperature, ph, addition of NaCl were studied. Under the following condition: ILATPS contained 1. g [Bmim]BF 4, 1.4 g NaH 2 PO 4, 3. ml water, 1. ml crude solution and 6.25 mmol NaCl, the temperature and ph were not adjusted, 93.12% and 95.85% were extracted in salt-rich phase and IL-rich phase, respectively. in salt-rich phase were separated from salt by a dialysis membrane. The purity of was demonstrated by TGA. Furthermore, monosaccharide constitutes of were analyzed by HPLC. The results demonstrated that almost completely consisted of mannose. This proposed method opens up new possibilities in the large-scale separation and purification of other active ingredients in natural plants. Nomenclature IL ionic liquid ATPS aqueous two-phase system ILATPS ionic liquid based aqueous two-phase system Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Project No. 9561), Hunan Provincial Innovation Foundation for Postgraduate (Project No. CX11B83) and Plan Project of Changsha Science and Technology Bureau (Project No: K ). References [1] Z.H. Yu, C. Jin, M. Xin, J.M. He, Carbohydr. Polym. 75 (9) [2] X.L. Chang, C.H. Wang, Y.M. Feng, Z.P. Liu, J. Food Eng. 75 (6) [3] B.S. Khatkar, K.S. Ahlawat, J. Food Sci. Technol. Mys. 48 (11) [4] E. Aysan, H. Bektas, F. Ersoz, Eur. J. Obstet. Gynecol. Reprod. Biol. 149 (1) [5] N. Pugh, S.A. Ross, M.A. ElSohly, D.S. Pasco, J. Agric. Food Chem. 49 (1) [6] X.L. Chang, Y.M. Feng, W.H. Wang, J. Taiwan Inst. Chem. Eng. 42 (11) [7] A. Femenia, P. Garcia-Pascual, S. Simal, C. Rossello, Carbohydr. Polym. 51 (3) [8] F.F. Li, J.M. Xing, Appl. Biochem. Biotechnol. 158 (9) [9] K.E. Gutowski, G.A. Broker, H.D. Willauer, J.G. Huddleston, R.P. Swatloski, J.D. Holbrey, R.D. Rogers, J. Am. Chem. Soc. 125 (3) [1] M. Soylak, E. Yilmaz, Desalination 275 (11) [11] Y.C. Pei, J.J. Wang, K. Wu, X.P. Xuan, X.J. Lu, Sep. Purif. Technol. 64 (9) [12] Y. Wang, X.H. Xu, J.A. Han, Y.S. Yan, Desalination 266 (11) [13] J. Han, Y. Wang, C.L. Yu, Y.S. Yan, X.Q. Xie, Anal. Bioanal. Chem. 399 (11) [14] M.K. Khoshkbarchi, A. Soto, A. Arce, Sep. Purif. Technol. 44 (5) [15] M.G. Freire, C.M.S.S. Neves, I.M. Marrucho, J.N.C. Lopes, L.P.N. Rebelo, J.A.P. Coutinho, Green Chem. 12 (1) [16] S.H. Li, C.Y. He, H.W. Liu, K.A. Li, F. Liu, Chin. Chem. Lett. 16 (5) [17] J.A.P. Coutinho, A.F.M. Claudio, M.G. Freire, C.S.R. Freire, A.J.D. Silvestre, Sep. Purif. Technol. 75 (1) [18] F.F. Li, J.M. Xing, Nat. Prod. Res. 23 (9) [19] M. DuBois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Anal. Chem. 28 (1956) [] M.M. Bradford, Anal. Biochem. 72 (1972) [21] Y.S. Yan, X.Q. Xie, J.A. Han, Y. Wang, G.W. Yin, W.S. Guan, J. Chem. Eng. Data 55 (1) [22] Y.S. Yan, X.G. Xie, Y. Wang, J. Han, Anal. Chim. Acta 687 (11) [23] M.T. Zafarani-Moattar, D. Nikjoo, J. Chem. Eng. Data 53 (8) [24] Y. Wang, J. Han, X.Q. Xie, C.X. Li, Cent. Eur. J. Chem. 8 (1) [25] K. Berggren, M.R. Egmond, F. Tjerneld, Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol () [26] F.Z. Deng, D.F. Guo, Chin. J. Anal. Chem. 34 (6)

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