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An efficient method for extraction, separation and purification of eugenol from Eugenia caryophyllata by supercritical fluid extraction and high-speed counter-current chromatography
Separation and Purification Technology 57 (2007) 237–241
An efficient method for extraction, separation and purification of eugenol from Eugenia caryophyllata by supercritical fluid extraction and high-speed counter-current chromatography Yanling Geng, Jianhua Liu, Ruimin Lv, Jinpeng Yuan, Yunliang Lin, Xiao Wang ∗ Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China Received 8 December 2006; received in revised form 10 April 2007; accepted 10 April 2007
Abstract Supercritical fluid extraction (SFE) of eugenol from Eugenia caryophyllata was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction system. Then the extraction was scaled up by 60× using a preparative SFE system under the optimized conditions of 50 ◦ C, 30 MPa and a sample particle size of 40–60 mesh. The yield of the preparative SFE was 17.1% and the yield of eugenol was 94 mg/g of dry buds. Eugenol in the extract was separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:0.5:1:0.5, v/v). From 1.5 g of crude extract, 804 mg of eugenol was obtained at 98.5% purity as determined by HPLC. © 2007 Elsevier B.V. All rights reserved. Keywords: Supercritical fluid extraction (SFE); Counter-current chromatography; Eugenia caryophyllata; Eugenol; Preparative chromatography
1. Introduction Eugenol (Fig. 1) is a major component of essential oil isolated from the Eugenia caryophyllata (Syzigium aromaticum, Myrtaceae), which has been widely used as herbal drug to treat dyspepsia, acute/chronic gastritis and diarrhea. It was the first component of an essential oil proved to be a significant germicide and sedative used in dentistry, and today is still in use [1]. Eugenol is also an important flavoring agent in cosmetic and food products [2]. Its scavenging properties against different radicals such as DPPH [3], ABTS [4], superoxide [5] and azide, hydroxyl, and haloperoxyl radicals [6] have been reported. It has been demonstrated to inhibit prostaglandin biosynthesis [7] and to block COX-2 activity with an IC50 value of 129 mol [8]. In long-term carcinogenicity experiments by various groups in CD-1 mice and F344 rats, eugenol was not associated with tumor formation [9]. In a skin painting study by Van Duuren and Goldschmidt [10], eugenol was reported as being partially effective in inhibiting benzo(a)pyrene-induced skin carcinomas. Eugenol was shown to inhibit DMBA-croton oil-induced papillomas by
∗
Corresponding author. Tel.: +86 531 8260 5319; fax: +86 531 8296 4889. E-mail address: [email protected] (X. Wang).
1383-5866/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2007.04.015
about 84% [11]. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point [12]. The preparative separation and purification of eugenol from plant materials by classical methods are tedious and usually require multiple chromatographic steps on silica gel. Because of the important biological properties and broad applications, it is urgent to develop an efficient method to extract, separate and purify eugenol. Eugenol can be chemically synthesized; however, in view of the increasing environmental and health concerns about the use of organic solvents and the production of toxic waste in organic synthesis, there has been growing interest in using supercritical fluids for extraction and isolation of products from natural sources since it requires less solvent, has a short extraction time and is capable of extracting thermally labile compounds under mild conditions [13]. In the present study, the extraction condition was optimized first with an analytical-scale SFE system with an orthogonal test. Then, the extraction was scaled up by 60 times with a preparative-scale SFE system. The crude extract obtained was then purified by high-speed counter current chromatography (HSCCC). HSCCC is a unique liquid–liquid partition chromatography technique that uses no solid support matrix. HSCCC
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Y. Geng et al. / Separation and Purification Technology 57 (2007) 237–241 Table 1 L9 (3)3 orthogonal test design
Fig. 1. Chemical structure of eugenol.
eliminates the irreversible adsorptive loss of samples onto the solid support matrix as used in the conventional chromatographic column [14]. This method has been successfully applied to the analysis and separation of various natural products [15–18]. However, no report has been published on the use of SFE to extract and HSCCC to isolate eugenol from natural plants. We herein report an efficient new method for extraction, separation and purification of eugenol from the Chinese medicinal plant E. caryophyllata. 2. Experimental 2.1. Reagents Carbon dioxide (99.9%) was obtained from Tianhai Gas Company, Jinan, China. Organic solvents including ethanol, hexane, ethyl acetate, and methanol were all of analytical grade and were purchased from Juye Chemical Factory, Jinan, China. HPLC-grade methanol was from Siyou Tianjin Chemical Factory, Tianjin, China. Standard eugenol was purchased from the National Institute of the Control of Pharmaceutical and Biological Products, Ministry of Health, Beijing, China. The dry buds of E. caryophyllata Thunb were obtained from a local drug store and identified by Prof. F. Zhou (College of Pharmacy, Shandong University of Traditional Chinese Medicine, Shandong, China). 2.2. Optimization of SFE conditions A Spe-edTM SFE system (Applied Separations, Inc., USA) in the SFE mode was used for optimization the extraction conditions. A 50-ml extraction cell was used to optimize extraction conditions. In order to determine a suitable extraction condition in a wide range with a minimum number of trials, an orthogonal test design L9 (3)3 was employed where temperature, pressure and the sample’s particle size were considered to be three major factors for effective extraction. Combinations of the three different levels of each factor were listed in Table 1. In each test, 5 g of the milled and sieved E. caryophyllata buds was placed into an extraction cell. Carbon dioxide with a purity of 99.9% was used as a solvent. After 10 min of static extraction (no liquid flow), the sample was then subjected to dynamic extraction by flowing gaseous carbon dioxide at a rate of 1.5 l/min for 20 min. The extract was trapped into a collection vessel containing about 100 ml of ethanol, and the sample was then analyzed by HPLC.
Test no.
A, Pressure (MPa)
B, Temperature (◦ C)
1 2 3 4 5 6 7 8 9
A1 A1 A1 A2 A2 A2 A3 A3 A3
B1 B2 B3 B1 B2 B3 B1 B2 B3
20 20 20 30 30 30 40 40 40
C, Particle size (mesh) 30 40 50 30 40 50 30 40 50
C1 C2 C3 C2 C3 C1 C3 C1 C2
10–20 20–40 40–60 20–40 40–60 10–20 40–60 10–20 20–40
2.3. SFE scaling up After the extraction conditions were optimized, the extraction was then scaled up by approximate 60 times with a preparativescale SFE system. The buds (300 g, 40–60 mesh) were placed into a 1-l extraction vessel, and were extracted statically for 20 min followed by another 6 h dynamically under the optimized conditions at 50 ◦ C and 30 MPa. The flow-rate of carbon dioxide supercritical fluid was set at 2.5 l/min, and the extract in the supercritical fluid was depressed directly into a separate vessel. The extract was then subjected to purification by HSCCC. 2.4. Selection of two-phase solvent system A number of two-phase solvent systems were tested by changing the volume ratio of the solvent to obtain the optimum composition that gave suitable partition coefficient (K) values. The partition coefficient values were determined according to the literature [16]. In brief, 2 ml of each phase of the equilibrated two-phase solvent system was added to approximately 1 mg of crude extract placed in a 10-ml test tube. The test tube was caped, and was shaken vigorously for 1 min to equilibrate the sample thoroughly. An equal volume of each phase was then analyzed by HPLC to obtain the partition coefficients. The partition coefficient value was expressed as the peak area of the compound in the upper phase divided by the peak area of the compound in the lower phase. 2.5. Preparation of two-phase solvent system and sample solution The selected solvent system was thoroughly equilibrated in a separation funnel by repeatedly vigorously shaking at room temperature. The two phases were separated shortly prior to use. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase. The sample solution was prepared by dissolving the crude extract in the mixture solution of lower phase and upper phase (1:1, v/v) of the solvent system. 2.6. HSCCC separation procedure Preparative HSCCC was carried out using a Model GS10A-2, with a multilayer coil of 1.6 mm i.d. and 110 m in length with a
Y. Geng et al. / Separation and Purification Technology 57 (2007) 237–241
total capacity of 230 ml. The β values of this preparative column range from 0.5 at internal to 0.8 at the external (β = r/R, where r is the rotation radius or the distance from the coil to the holder shaft and R is the revolution radius or the distances between the holder axis and central axis of the centrifuge) (Beijing Institute of New Technology Application, Beijing, China). The solvent was pumped into the column with a Model NS-1007 constantflow pump (Beijing Institute of New Technology Application, Beijing, China). Continuous monitoring of the effluent was achieved with a Model 8823A-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at 254 nm. A manual sample injection valve with a 10-ml loop (for the preparative HSCCC) (Tianjin High New Science Technology Company, Tianjin, China) was used to introduce the sample into the column. A portable recorder (Yokogawa Model 3057, Sichuan Instrument Factory, Chongqing, China) was used to draw the chromatogram. In each separation, the multiplayer coiled column was first filled entirely with the upper organic phase as the stationary phase. Then, the lower aqueous phase was pumped into the head end of the column at a suitable flow-rate of 2 ml/min for Model GS10A-2, while the apparatus was rotated at a speed of 800 rpm. After hydrodynamic equilibrium was reached, as indicated by a clear mobile phase eluting from the tail outlet, the sample solution was injected through the injection valve. The effluent from the tail end of the column was continuously monitored with a UV detector at 254 nm and the chromatogram was recorded. Each peak fraction was collected according to the elution profile and determined by HPLC. After the separation was completed, retention of the stationary phase was measured by collecting the column contents by forcing them out of the column with pressurized nitrogen gas. 2.7. HPLC analyses of HSCCC fractions The HPLC system used throughout this study consisted of a Waters 600 pump, a Waters 600 controller (Waters, USA), a sample injector (Rheodyne, USA) with a 10-l loop, and a Waters 996 photodiode array detector. Evaluation and quantification were made on a Millenium 32 workstation (Waters). The crude extract and each purified fraction from the preparative HSCCC separation were analyzed by HPLC with a
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Shim-pack VP-ODS column (250 mm × 4.6 mm, i.d.) at 280 nm and at a column temperature of 25 ◦ C. The mobile phase, a solution of methanol and water (65:35, v/v), was set at a flow-rate of 1 ml/min. The effluent was monitored by a photodiode array detector. Routine sample calculations were made by comparison of the peak area with that of the standard. The identification of HSCCC peak fractions was carried out by EI-MS on an Agilent 5973N mass spectrograph and by 1 H NMR and 13 C NMR spectra on a Varian NMR spectrometer. 3. Results and discussion 3.1. Optimization of temperature, pressure and sample particle size for maximum SFE efficiency The first step in the SFE of eugenol is to optimize the operating conditions to obtain an efficient extraction of target compound. Since various parameters potentially affect the extraction process, the optimization of the experimental conditions is a critical step in the development of a SFE method. In fact, the fluid pressure, temperature and sample particle size are generally considered as the most important factors. The optimization of the method can be carried out step-by-step or by using an experimental design. In the present study, all selected factors were examined using an orthogonal L9 (3)3 test design. The products obtained from each L9 (3)3 test of the analytical SFE were quantitatively analyzed, and the results were shown in Table 2. The maximum extraction yield of crude extract was 19.6% and the maximum concentration of eugenol in the crude extract was 593 mg/g. Extraction efficiencies at different sets of temperature, sample particle size and pressure were examined under L9 (3)3 test design. The results shown in Table 2 indicate that there are great yield differences among each set of SFE conditions. If the yield of eugenol was expressed as a control index, the results in Table 2 are transformed to Table 3 after orthogonal analysis. The sample particle size was found to be the most important determinant of the yield. The yield of eugenol significantly increased as the particle size decreased (Fig. 2). Pressure and temperature have no significant influence on the yield of eugenol, the 30 MPa of pressure and 50 ◦ C of temperature, however, seem favorable for the extraction of eugenol
Table 2 L9 (3)3 test results Test no.
A (1)
B (2)
C (3)
Yield (%)a
Yield (mg/g)b
Yield (mg/g)c
1 2 3 4 5 6 7 8 9
A1 A1 A1 A2 A2 A2 A3 A3 A3
B1 B2 B3 B1 B2 B3 B1 B2 B3
C1 C2 C3 C2 C3 C1 C3 C1 C2
9.1 12.7 19.6 12.0 19.4 9.3 18.8 9.7 14.2
583.50 438.6 578.6 532.5 593.3 515.1 541.5 535.1 454.2
53.1 55.7 113.4 63.9 115.1 47.9 101.8 51.9 64.5
a b c
Extraction yield (%) = (the amount of extract/sample mass) × 100. Extraction yield (mg/g) = the amount of eugenol in extract/crude extract mass. Extraction yield (mg/g) = the amount of eugenol in extract/sample mass.
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Table 3 Analysis of L9 (3)3 test results Eugenol yield (mg/g)
K1 K2 K3 k1 k2 k3 R Optimal level a b c
A
B
C
222.2a
218.8 222.7 225.8 72.9 74.2 75.3 2.4 B3
152.9 184.1 330.3 50.9 61.4 110.1 59.2 C3
226.9 218.2 74.1b 75.6 72.7 2.9c A2
KiA = extraction yield at Ai . kiA = KiA /3. A A RA i = max{ki } − min{ki }.
(Fig. 2 and Table 3). These results indicate that the optimal conditions for extraction of eugenol by SFE was 30 MPa of pressure, 50 ◦ C of temperature and 40–60 mesh of sample particle size.
Fig. 3. (A) HPLC chromatogram of the extract from preparative SFE; (B) HPLC analyses and UV spectrum of the eugenol purified with HSCCC. Experimental conditions: a Shim-pack VP-ODS column (250 mm × 4.6 mm, i.d.); column temperature: 25 ◦ C; mobile phase: methanol and water (65:35, v/v); flow rate: 1.0 ml/min; detection: 280 nm; injection volume: 10 l.
3.2. Preparative-scale SFE Under the above-optimized SFE extract conditions, 51.3 g crude extract was obtained from 300 g sample. HPLC analysis in Fig. 3A shows the SFE extract contained 54.8% of eugenol. 3.3. Selecting the suitable solvent system The selection of the two-phase solvent system is the most important step in performing HSCCC method. Successful separation necessitates the careful search for a suitable two-phase solvent system, which provides an ideal range of the partition coefficient (K) for the applied sample. Based on the physical properties of eugenol, we selected a two-phase solvent system composed of n-hexane, ethyl acetate, methanol and water because it provides a broad range of hydrophobicity by modifying the volume ratio of the four solvents. The ideal K values of the target compound should be close to 1. A smaller K value may result in a loss of peak resolution, while a larger one produces excessive sample band broadening. Table 4 shows that n-hexane–ethyl acetate–methanol–water ratios (1:0.5:1:0.5, 1:0.8:1.5:1 and 1:1:2:1) could be used to separate the sample. After trying all the above solvent systems, the ratio 1:0.5:1:0.5 Table 4 Partition coefficients (K) of eugenol
Fig. 2. Effects of pressure, temperature, and sample particle size on yield of eugenol.
Solvent systems
K
n-Hexane–ethyl acetate–methanol–water 1:1:1:1 1:0.8:1.5:1 1:1:2:1 1:0.8:1:0.8 1:0.5:1:0.5
3.96 1.26 1.21 1.17 0.92
Y. Geng et al. / Separation and Purification Technology 57 (2007) 237–241
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fluid extraction technique. Under optimal conditions, i.e. a pressure of 30 MPa, a temperature of 50 ◦ C, and a sample particle size of 40–60 mesh, the yield of eugenol was 94 mg/g of dry buds. From a crude SFE extract, eugenol was obtained with 98.5% purity by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:0.5:1:0.5, v/v) in one step. The results of the present study demonstrate that SFE and HSCCC are very useful techniques for the extraction, separation and purification of eugenol from E. caryophyllata. Acknowledgements
Fig. 4. Chromatogram of the crude extract by preparative HSCCC. Experimental conditions: multilayer coil of 1.6 mm i.d. PTFE tube with a total capacity of 230 ml; revolution speed: 800 rpm; solvent system: n-hexane–ethyl acetate–methanol–water (1:0.5:1:0.5, v/v); stationary phase: upper phase; mobile phase: lower phase; flow-rate: 2.0 ml/min; detection: 254 nm; sample size: 1.5 g; retention of stationary phase: 55%. Peak A: Eugenol.
(v/v) was best to effect the separation of the extract. Fig. 4 shows the separation of HSCCC using this solvent system. 3.4. HSCCC separation of eugenol Using the selected solvent system, the crude extract (1.5 g) was separated and purified in one-step by preparative HSCCC, which was shown in Fig. 4. The retention of the stationary phase was 55%, and the separation time was about 150 min in each separation run. Based on the HPLC analysis and the elution curve of the preparative HSCCC, all collected fractions were combined into different pooled fractions. Fig. 3B shows the HPLC analysis of the combined fractions. Peak A (Fig. 4) was identified as eugenol by EI-MS and NMR spectra as follows: EI-MS m/z: 164 (100%), 149, 137, 131, 121, 103, 91, 77, 55. 1 H NMR (600 MHz, CDCl3 ) δ ppm: 3.31 (2H, d, H1 ), 3.88 (OCH3 ), 5.05 (2H, t, H3 ), 5.53 (1H, s, OH), 5.95 (1H, m, H2 ), 6.67 (2H, d, H5, H6 ), 6.84 (1H, d, H4 ). 13 C NMR (600 MHz, CDCl3 ) δ ppm: 137.8 (C-1), 115.5 (C-2), 143.9 (C-3), 146.4 (C-4), 111.1 (C-5), 121.2(C-6), 39.9 (C-1 ), 131.9 (C-2 ), 114.3 (C-3 ), 55.8 (OCH3 ). The crude extract from E. caryophyllata was analyzed by HPLC (Fig. 3A). The result indicated that the crude sample contained several compounds among which eugenol represented 54.8% of the total. After only one-step of operation by HSCCC, 804 mg of eugenol (98.5% purity) was obtained from 1.5 g of SFE extract. These results demonstrate the high resolving power of HSCCC. 4. Conclusion Eugenol from the traditional Chinese medicine E. caryophyllata was extracted, separated and purified by the supercritical
Financial supports from Key Science and Technology Program of Shandong Province (2006GG1109078, 2006GG1108097-05) and Shandong Academy of Sciences are gratefully acknowledged. References [1] K. Markowitz, M. Moynihan, M. Liu, S. Kim, Oral Surg. Oral Med. Oral Pathol. 73 (1992) 729–737. [2] K. Krishnaswamy, N. Raghuramulu, Indian J. Med. Res. 108 (1998) 167–181. [3] V. Bondet, W. Brand-Williams, C. Berset, Lebensm. Wiss. Technol. 30 (1997) 609–615. [4] D.O. Kim, C.Y. Lee, Crit. Rev. Food Sci. Nutr. 44 (2004) 253–273. [5] S. Fujisawa, T. Atsumi, K. Satoh, Y. Kadoma, M. Ishihara, N. Okada, M. Nagasaki, I. Yokoe, H. Sakagami, In Vitro Mol. Toxicol. 13 (2000) 269–280. [6] K.I. Priyadarsini, S.N. Guha, M.N. Rao, Free Radic. Biol. Med. 24 (1998) 933–941. [7] A. Rasheed, G. Laekeman, J. Totte, A. Vietinch, A.G. Herman, N. Engl. J. Med. 310 (1984) 50–51. [8] U. Huss, T. Ringbom, P. Perera, L. Bohlin, M.J. Vasange, J. Nat. Prod. 65 (2002) 1517–1521. [9] J.A. Miller, A.B. Swanson, E.C. Miller, Tokyo Japan Science Society Press/Baltimore University Park Press, Tokyo/Baltimore, MD, 1979, pp. 111–125. [10] B.L. Van Duuren, B.M. Goldschmidt, J. Natl. Cancer Inst. 56 (1976) 1237–1242. [11] K. Sukumaran, M.C. Unnikrishnan, R. Kuttan, Indian J. Physiol. Pharmacol. 38 (1994) 306–308. [12] R. Ghosh, N. Nadiminty, J.E. Fitzpatrick, W.L. Alworth, J. Thomas Slaga, A.P. Kumar, J. Biol. Chem. 280 (2005) 5812–5819. [13] E. Martinelli, K. Schulz, G.A. Mansoori, in: T.J. Bruno, J.F. Ely (Eds.), Supercritical Fluid Technology, CRC, Boca Raton, FL, 1991, p. 451. [14] Y. Ito, J. Chromatogr. A 1065 (2005) 145–168. [15] T.Y. Zhang, in: A. Berthod (Ed.), Countercurrent Chromatography—The Support-Free Liquid Stationary Phase, Elsevier, Amsterdam, 2002 (Chapter 8). [16] X. Wang, Y. Wang, J. Yuan, Q. Sun, C. Zheng, J. Chromatogr. A 1055 (2004) 135–140. [17] J.Y. Peng, Y.Y. Jiang, G.R. Fan, B. Chen, Q.Y. Zhang, Y.F. Chai, Y.T. Wu, Sep. Purif. Technol. 52 (2006) 22–28. [18] Z.L. Liu, J. Wang, P.N. Shen, C.Y. Wang, Y.J. Shen, Sep. Purif. Technol. 52 (2006) 18–21.
An efficient method for extraction, separation and purification of eugenol from Eugenia caryophyllata by supercritical fluid extraction and high-speed counter-current chromatography Yanling Geng, Jianhua Liu, Ruimin Lv, Jinpeng Yuan, Yunliang Lin, Xiao Wang ∗ Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China Received 8 December 2006; received in revised form 10 April 2007; accepted 10 April 2007
Abstract Supercritical fluid extraction (SFE) of eugenol from Eugenia caryophyllata was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction system. Then the extraction was scaled up by 60× using a preparative SFE system under the optimized conditions of 50 ◦ C, 30 MPa and a sample particle size of 40–60 mesh. The yield of the preparative SFE was 17.1% and the yield of eugenol was 94 mg/g of dry buds. Eugenol in the extract was separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:0.5:1:0.5, v/v). From 1.5 g of crude extract, 804 mg of eugenol was obtained at 98.5% purity as determined by HPLC. © 2007 Elsevier B.V. All rights reserved. Keywords: Supercritical fluid extraction (SFE); Counter-current chromatography; Eugenia caryophyllata; Eugenol; Preparative chromatography
1. Introduction Eugenol (Fig. 1) is a major component of essential oil isolated from the Eugenia caryophyllata (Syzigium aromaticum, Myrtaceae), which has been widely used as herbal drug to treat dyspepsia, acute/chronic gastritis and diarrhea. It was the first component of an essential oil proved to be a significant germicide and sedative used in dentistry, and today is still in use [1]. Eugenol is also an important flavoring agent in cosmetic and food products [2]. Its scavenging properties against different radicals such as DPPH [3], ABTS [4], superoxide [5] and azide, hydroxyl, and haloperoxyl radicals [6] have been reported. It has been demonstrated to inhibit prostaglandin biosynthesis [7] and to block COX-2 activity with an IC50 value of 129 mol [8]. In long-term carcinogenicity experiments by various groups in CD-1 mice and F344 rats, eugenol was not associated with tumor formation [9]. In a skin painting study by Van Duuren and Goldschmidt [10], eugenol was reported as being partially effective in inhibiting benzo(a)pyrene-induced skin carcinomas. Eugenol was shown to inhibit DMBA-croton oil-induced papillomas by
∗
Corresponding author. Tel.: +86 531 8260 5319; fax: +86 531 8296 4889. E-mail address: [email protected] (X. Wang).
1383-5866/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2007.04.015
about 84% [11]. In a B16 xenograft study, eugenol treatment produced a significant tumor growth delay (p = 0.0057), an almost 40% decrease in tumor size, and a 19% increase in the median time to end point [12]. The preparative separation and purification of eugenol from plant materials by classical methods are tedious and usually require multiple chromatographic steps on silica gel. Because of the important biological properties and broad applications, it is urgent to develop an efficient method to extract, separate and purify eugenol. Eugenol can be chemically synthesized; however, in view of the increasing environmental and health concerns about the use of organic solvents and the production of toxic waste in organic synthesis, there has been growing interest in using supercritical fluids for extraction and isolation of products from natural sources since it requires less solvent, has a short extraction time and is capable of extracting thermally labile compounds under mild conditions [13]. In the present study, the extraction condition was optimized first with an analytical-scale SFE system with an orthogonal test. Then, the extraction was scaled up by 60 times with a preparative-scale SFE system. The crude extract obtained was then purified by high-speed counter current chromatography (HSCCC). HSCCC is a unique liquid–liquid partition chromatography technique that uses no solid support matrix. HSCCC
238
Y. Geng et al. / Separation and Purification Technology 57 (2007) 237–241 Table 1 L9 (3)3 orthogonal test design
Fig. 1. Chemical structure of eugenol.
eliminates the irreversible adsorptive loss of samples onto the solid support matrix as used in the conventional chromatographic column [14]. This method has been successfully applied to the analysis and separation of various natural products [15–18]. However, no report has been published on the use of SFE to extract and HSCCC to isolate eugenol from natural plants. We herein report an efficient new method for extraction, separation and purification of eugenol from the Chinese medicinal plant E. caryophyllata. 2. Experimental 2.1. Reagents Carbon dioxide (99.9%) was obtained from Tianhai Gas Company, Jinan, China. Organic solvents including ethanol, hexane, ethyl acetate, and methanol were all of analytical grade and were purchased from Juye Chemical Factory, Jinan, China. HPLC-grade methanol was from Siyou Tianjin Chemical Factory, Tianjin, China. Standard eugenol was purchased from the National Institute of the Control of Pharmaceutical and Biological Products, Ministry of Health, Beijing, China. The dry buds of E. caryophyllata Thunb were obtained from a local drug store and identified by Prof. F. Zhou (College of Pharmacy, Shandong University of Traditional Chinese Medicine, Shandong, China). 2.2. Optimization of SFE conditions A Spe-edTM SFE system (Applied Separations, Inc., USA) in the SFE mode was used for optimization the extraction conditions. A 50-ml extraction cell was used to optimize extraction conditions. In order to determine a suitable extraction condition in a wide range with a minimum number of trials, an orthogonal test design L9 (3)3 was employed where temperature, pressure and the sample’s particle size were considered to be three major factors for effective extraction. Combinations of the three different levels of each factor were listed in Table 1. In each test, 5 g of the milled and sieved E. caryophyllata buds was placed into an extraction cell. Carbon dioxide with a purity of 99.9% was used as a solvent. After 10 min of static extraction (no liquid flow), the sample was then subjected to dynamic extraction by flowing gaseous carbon dioxide at a rate of 1.5 l/min for 20 min. The extract was trapped into a collection vessel containing about 100 ml of ethanol, and the sample was then analyzed by HPLC.
Test no.
A, Pressure (MPa)
B, Temperature (◦ C)
1 2 3 4 5 6 7 8 9
A1 A1 A1 A2 A2 A2 A3 A3 A3
B1 B2 B3 B1 B2 B3 B1 B2 B3
20 20 20 30 30 30 40 40 40
C, Particle size (mesh) 30 40 50 30 40 50 30 40 50
C1 C2 C3 C2 C3 C1 C3 C1 C2
10–20 20–40 40–60 20–40 40–60 10–20 40–60 10–20 20–40
2.3. SFE scaling up After the extraction conditions were optimized, the extraction was then scaled up by approximate 60 times with a preparativescale SFE system. The buds (300 g, 40–60 mesh) were placed into a 1-l extraction vessel, and were extracted statically for 20 min followed by another 6 h dynamically under the optimized conditions at 50 ◦ C and 30 MPa. The flow-rate of carbon dioxide supercritical fluid was set at 2.5 l/min, and the extract in the supercritical fluid was depressed directly into a separate vessel. The extract was then subjected to purification by HSCCC. 2.4. Selection of two-phase solvent system A number of two-phase solvent systems were tested by changing the volume ratio of the solvent to obtain the optimum composition that gave suitable partition coefficient (K) values. The partition coefficient values were determined according to the literature [16]. In brief, 2 ml of each phase of the equilibrated two-phase solvent system was added to approximately 1 mg of crude extract placed in a 10-ml test tube. The test tube was caped, and was shaken vigorously for 1 min to equilibrate the sample thoroughly. An equal volume of each phase was then analyzed by HPLC to obtain the partition coefficients. The partition coefficient value was expressed as the peak area of the compound in the upper phase divided by the peak area of the compound in the lower phase. 2.5. Preparation of two-phase solvent system and sample solution The selected solvent system was thoroughly equilibrated in a separation funnel by repeatedly vigorously shaking at room temperature. The two phases were separated shortly prior to use. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase. The sample solution was prepared by dissolving the crude extract in the mixture solution of lower phase and upper phase (1:1, v/v) of the solvent system. 2.6. HSCCC separation procedure Preparative HSCCC was carried out using a Model GS10A-2, with a multilayer coil of 1.6 mm i.d. and 110 m in length with a
Y. Geng et al. / Separation and Purification Technology 57 (2007) 237–241
total capacity of 230 ml. The β values of this preparative column range from 0.5 at internal to 0.8 at the external (β = r/R, where r is the rotation radius or the distance from the coil to the holder shaft and R is the revolution radius or the distances between the holder axis and central axis of the centrifuge) (Beijing Institute of New Technology Application, Beijing, China). The solvent was pumped into the column with a Model NS-1007 constantflow pump (Beijing Institute of New Technology Application, Beijing, China). Continuous monitoring of the effluent was achieved with a Model 8823A-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at 254 nm. A manual sample injection valve with a 10-ml loop (for the preparative HSCCC) (Tianjin High New Science Technology Company, Tianjin, China) was used to introduce the sample into the column. A portable recorder (Yokogawa Model 3057, Sichuan Instrument Factory, Chongqing, China) was used to draw the chromatogram. In each separation, the multiplayer coiled column was first filled entirely with the upper organic phase as the stationary phase. Then, the lower aqueous phase was pumped into the head end of the column at a suitable flow-rate of 2 ml/min for Model GS10A-2, while the apparatus was rotated at a speed of 800 rpm. After hydrodynamic equilibrium was reached, as indicated by a clear mobile phase eluting from the tail outlet, the sample solution was injected through the injection valve. The effluent from the tail end of the column was continuously monitored with a UV detector at 254 nm and the chromatogram was recorded. Each peak fraction was collected according to the elution profile and determined by HPLC. After the separation was completed, retention of the stationary phase was measured by collecting the column contents by forcing them out of the column with pressurized nitrogen gas. 2.7. HPLC analyses of HSCCC fractions The HPLC system used throughout this study consisted of a Waters 600 pump, a Waters 600 controller (Waters, USA), a sample injector (Rheodyne, USA) with a 10-l loop, and a Waters 996 photodiode array detector. Evaluation and quantification were made on a Millenium 32 workstation (Waters). The crude extract and each purified fraction from the preparative HSCCC separation were analyzed by HPLC with a
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Shim-pack VP-ODS column (250 mm × 4.6 mm, i.d.) at 280 nm and at a column temperature of 25 ◦ C. The mobile phase, a solution of methanol and water (65:35, v/v), was set at a flow-rate of 1 ml/min. The effluent was monitored by a photodiode array detector. Routine sample calculations were made by comparison of the peak area with that of the standard. The identification of HSCCC peak fractions was carried out by EI-MS on an Agilent 5973N mass spectrograph and by 1 H NMR and 13 C NMR spectra on a Varian NMR spectrometer. 3. Results and discussion 3.1. Optimization of temperature, pressure and sample particle size for maximum SFE efficiency The first step in the SFE of eugenol is to optimize the operating conditions to obtain an efficient extraction of target compound. Since various parameters potentially affect the extraction process, the optimization of the experimental conditions is a critical step in the development of a SFE method. In fact, the fluid pressure, temperature and sample particle size are generally considered as the most important factors. The optimization of the method can be carried out step-by-step or by using an experimental design. In the present study, all selected factors were examined using an orthogonal L9 (3)3 test design. The products obtained from each L9 (3)3 test of the analytical SFE were quantitatively analyzed, and the results were shown in Table 2. The maximum extraction yield of crude extract was 19.6% and the maximum concentration of eugenol in the crude extract was 593 mg/g. Extraction efficiencies at different sets of temperature, sample particle size and pressure were examined under L9 (3)3 test design. The results shown in Table 2 indicate that there are great yield differences among each set of SFE conditions. If the yield of eugenol was expressed as a control index, the results in Table 2 are transformed to Table 3 after orthogonal analysis. The sample particle size was found to be the most important determinant of the yield. The yield of eugenol significantly increased as the particle size decreased (Fig. 2). Pressure and temperature have no significant influence on the yield of eugenol, the 30 MPa of pressure and 50 ◦ C of temperature, however, seem favorable for the extraction of eugenol
Table 2 L9 (3)3 test results Test no.
A (1)
B (2)
C (3)
Yield (%)a
Yield (mg/g)b
Yield (mg/g)c
1 2 3 4 5 6 7 8 9
A1 A1 A1 A2 A2 A2 A3 A3 A3
B1 B2 B3 B1 B2 B3 B1 B2 B3
C1 C2 C3 C2 C3 C1 C3 C1 C2
9.1 12.7 19.6 12.0 19.4 9.3 18.8 9.7 14.2
583.50 438.6 578.6 532.5 593.3 515.1 541.5 535.1 454.2
53.1 55.7 113.4 63.9 115.1 47.9 101.8 51.9 64.5
a b c
Extraction yield (%) = (the amount of extract/sample mass) × 100. Extraction yield (mg/g) = the amount of eugenol in extract/crude extract mass. Extraction yield (mg/g) = the amount of eugenol in extract/sample mass.
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Table 3 Analysis of L9 (3)3 test results Eugenol yield (mg/g)
K1 K2 K3 k1 k2 k3 R Optimal level a b c
A
B
C
222.2a
218.8 222.7 225.8 72.9 74.2 75.3 2.4 B3
152.9 184.1 330.3 50.9 61.4 110.1 59.2 C3
226.9 218.2 74.1b 75.6 72.7 2.9c A2
KiA = extraction yield at Ai . kiA = KiA /3. A A RA i = max{ki } − min{ki }.
(Fig. 2 and Table 3). These results indicate that the optimal conditions for extraction of eugenol by SFE was 30 MPa of pressure, 50 ◦ C of temperature and 40–60 mesh of sample particle size.
Fig. 3. (A) HPLC chromatogram of the extract from preparative SFE; (B) HPLC analyses and UV spectrum of the eugenol purified with HSCCC. Experimental conditions: a Shim-pack VP-ODS column (250 mm × 4.6 mm, i.d.); column temperature: 25 ◦ C; mobile phase: methanol and water (65:35, v/v); flow rate: 1.0 ml/min; detection: 280 nm; injection volume: 10 l.
3.2. Preparative-scale SFE Under the above-optimized SFE extract conditions, 51.3 g crude extract was obtained from 300 g sample. HPLC analysis in Fig. 3A shows the SFE extract contained 54.8% of eugenol. 3.3. Selecting the suitable solvent system The selection of the two-phase solvent system is the most important step in performing HSCCC method. Successful separation necessitates the careful search for a suitable two-phase solvent system, which provides an ideal range of the partition coefficient (K) for the applied sample. Based on the physical properties of eugenol, we selected a two-phase solvent system composed of n-hexane, ethyl acetate, methanol and water because it provides a broad range of hydrophobicity by modifying the volume ratio of the four solvents. The ideal K values of the target compound should be close to 1. A smaller K value may result in a loss of peak resolution, while a larger one produces excessive sample band broadening. Table 4 shows that n-hexane–ethyl acetate–methanol–water ratios (1:0.5:1:0.5, 1:0.8:1.5:1 and 1:1:2:1) could be used to separate the sample. After trying all the above solvent systems, the ratio 1:0.5:1:0.5 Table 4 Partition coefficients (K) of eugenol
Fig. 2. Effects of pressure, temperature, and sample particle size on yield of eugenol.
Solvent systems
K
n-Hexane–ethyl acetate–methanol–water 1:1:1:1 1:0.8:1.5:1 1:1:2:1 1:0.8:1:0.8 1:0.5:1:0.5
3.96 1.26 1.21 1.17 0.92
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fluid extraction technique. Under optimal conditions, i.e. a pressure of 30 MPa, a temperature of 50 ◦ C, and a sample particle size of 40–60 mesh, the yield of eugenol was 94 mg/g of dry buds. From a crude SFE extract, eugenol was obtained with 98.5% purity by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:0.5:1:0.5, v/v) in one step. The results of the present study demonstrate that SFE and HSCCC are very useful techniques for the extraction, separation and purification of eugenol from E. caryophyllata. Acknowledgements
Fig. 4. Chromatogram of the crude extract by preparative HSCCC. Experimental conditions: multilayer coil of 1.6 mm i.d. PTFE tube with a total capacity of 230 ml; revolution speed: 800 rpm; solvent system: n-hexane–ethyl acetate–methanol–water (1:0.5:1:0.5, v/v); stationary phase: upper phase; mobile phase: lower phase; flow-rate: 2.0 ml/min; detection: 254 nm; sample size: 1.5 g; retention of stationary phase: 55%. Peak A: Eugenol.
(v/v) was best to effect the separation of the extract. Fig. 4 shows the separation of HSCCC using this solvent system. 3.4. HSCCC separation of eugenol Using the selected solvent system, the crude extract (1.5 g) was separated and purified in one-step by preparative HSCCC, which was shown in Fig. 4. The retention of the stationary phase was 55%, and the separation time was about 150 min in each separation run. Based on the HPLC analysis and the elution curve of the preparative HSCCC, all collected fractions were combined into different pooled fractions. Fig. 3B shows the HPLC analysis of the combined fractions. Peak A (Fig. 4) was identified as eugenol by EI-MS and NMR spectra as follows: EI-MS m/z: 164 (100%), 149, 137, 131, 121, 103, 91, 77, 55. 1 H NMR (600 MHz, CDCl3 ) δ ppm: 3.31 (2H, d, H1 ), 3.88 (OCH3 ), 5.05 (2H, t, H3 ), 5.53 (1H, s, OH), 5.95 (1H, m, H2 ), 6.67 (2H, d, H5, H6 ), 6.84 (1H, d, H4 ). 13 C NMR (600 MHz, CDCl3 ) δ ppm: 137.8 (C-1), 115.5 (C-2), 143.9 (C-3), 146.4 (C-4), 111.1 (C-5), 121.2(C-6), 39.9 (C-1 ), 131.9 (C-2 ), 114.3 (C-3 ), 55.8 (OCH3 ). The crude extract from E. caryophyllata was analyzed by HPLC (Fig. 3A). The result indicated that the crude sample contained several compounds among which eugenol represented 54.8% of the total. After only one-step of operation by HSCCC, 804 mg of eugenol (98.5% purity) was obtained from 1.5 g of SFE extract. These results demonstrate the high resolving power of HSCCC. 4. Conclusion Eugenol from the traditional Chinese medicine E. caryophyllata was extracted, separated and purified by the supercritical
Financial supports from Key Science and Technology Program of Shandong Province (2006GG1109078, 2006GG1108097-05) and Shandong Academy of Sciences are gratefully acknowledged. References [1] K. Markowitz, M. Moynihan, M. Liu, S. Kim, Oral Surg. Oral Med. Oral Pathol. 73 (1992) 729–737. [2] K. Krishnaswamy, N. Raghuramulu, Indian J. Med. Res. 108 (1998) 167–181. [3] V. Bondet, W. Brand-Williams, C. Berset, Lebensm. Wiss. Technol. 30 (1997) 609–615. [4] D.O. Kim, C.Y. Lee, Crit. Rev. Food Sci. Nutr. 44 (2004) 253–273. [5] S. Fujisawa, T. Atsumi, K. Satoh, Y. Kadoma, M. Ishihara, N. Okada, M. Nagasaki, I. Yokoe, H. Sakagami, In Vitro Mol. Toxicol. 13 (2000) 269–280. [6] K.I. Priyadarsini, S.N. Guha, M.N. Rao, Free Radic. Biol. Med. 24 (1998) 933–941. [7] A. Rasheed, G. Laekeman, J. Totte, A. Vietinch, A.G. Herman, N. Engl. J. Med. 310 (1984) 50–51. [8] U. Huss, T. Ringbom, P. Perera, L. Bohlin, M.J. Vasange, J. Nat. Prod. 65 (2002) 1517–1521. [9] J.A. Miller, A.B. Swanson, E.C. Miller, Tokyo Japan Science Society Press/Baltimore University Park Press, Tokyo/Baltimore, MD, 1979, pp. 111–125. [10] B.L. Van Duuren, B.M. Goldschmidt, J. Natl. Cancer Inst. 56 (1976) 1237–1242. [11] K. Sukumaran, M.C. Unnikrishnan, R. Kuttan, Indian J. Physiol. Pharmacol. 38 (1994) 306–308. [12] R. Ghosh, N. Nadiminty, J.E. Fitzpatrick, W.L. Alworth, J. Thomas Slaga, A.P. Kumar, J. Biol. Chem. 280 (2005) 5812–5819. [13] E. Martinelli, K. Schulz, G.A. Mansoori, in: T.J. Bruno, J.F. Ely (Eds.), Supercritical Fluid Technology, CRC, Boca Raton, FL, 1991, p. 451. [14] Y. Ito, J. Chromatogr. A 1065 (2005) 145–168. [15] T.Y. Zhang, in: A. Berthod (Ed.), Countercurrent Chromatography—The Support-Free Liquid Stationary Phase, Elsevier, Amsterdam, 2002 (Chapter 8). [16] X. Wang, Y. Wang, J. Yuan, Q. Sun, C. Zheng, J. Chromatogr. A 1055 (2004) 135–140. [17] J.Y. Peng, Y.Y. Jiang, G.R. Fan, B. Chen, Q.Y. Zhang, Y.F. Chai, Y.T. Wu, Sep. Purif. Technol. 52 (2006) 22–28. [18] Z.L. Liu, J. Wang, P.N. Shen, C.Y. Wang, Y.J. Shen, Sep. Purif. Technol. 52 (2006) 18–21.