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Purification of caffeic acid, chlorogenic acid and luteolin from Caulis Lonicerae by high-speed counter-current chromatography
Separation and Purification Technology 63 (2008) 721–724
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Short communication
Purification of caffeic acid, chlorogenic acid and luteolin from Caulis Lonicerae by high-speed counter-current chromatography Zhilong Wang a,c , Jianhua Wang b,c,∗ , Yinshi Sun b,c , Shengbo Li d , Hanzhong Wang a,c a
College of Life Science, Shandong Agricultural University (SDAU), Taian, Shandong 271018, China College of Agronomy, SDAU, Taian, Shandong 271018, China c State Key Laboratory of Crop Biology, SDAU, Taian, Shandong 271018, China d Shandong Yate Eco-tech Co., Ltd., Linyi, Shandong 266071, China b
a r t i c l e
i n f o
Article history: Received 10 March 2008 Received in revised form 1 August 2008 Accepted 5 August 2008 Keywords: Caulis Lonicerae Counter-current chromatography Chlorogenic acid Caffeic acid Luteolin
a b s t r a c t Chlorogenic acid, caffeic acid and luteolin are the major phenolic compounds found in the traditional Chinese herbal medicine Caulis Lonicerae. The separation and purification of the three compounds from the crude extract of Caulis Lonicerae was achieved by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate–ethanol–water (4:1:5, v/v). Caffeic acid (7.2 mg), chlorogenic acid (15.7 mg) and luteolin (18.8 mg) from 110 mg crude extract of Caulis Lonicerae were obtained and their purities were 95.55%, 97.24% and 98.11%, respectively. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Caulis Lonicerae, rendongteng in Chinese, is the dry stem and branch of Caprifoliaceae plant Lonicera japonica Thunb. It plays major roles in clearing body heat and detoxification, dispelling wind and removing obstruction in channels. Caulis Lonicerae is usually used to treat epidemic febrile diseases and fever, thermotoxin and bloody flux, abscess and sore, rheumatism, joint red and swollen and arthralgia [1]. However, for a long time, people have paid more attention to the research and exploitation of Flos Lonicerae, the dry flower of L. japonica Thunb. In recent years, people have found phenolic compounds and flavonoids in Caulis Lonicerae, especially that the contents of caffeic acid and luteolin in Caulis Lonicerae were also higher than those in Flos Lonicerae [2]. Therefore, an efficient method for the preparative separation and purification of chlorogenic acid, caffeic acid and luteolin (Fig. 1) from Caulis Lonicerae is much needed. High-speed counter-current chromatography (HSCCC) is a useful method for rapid chromatographic purification employing highly efficient fraction by a hybrid technique of liquid–liquid counter-current distribution and liquid chromatography that was
first invented by Ito [3]. In conjunction with the use of centrifugal force, the centrifugal force field generated from both rotational and synchronous planetary motion of coiled columns containing two immiscible liquid phases provides vigorous mixing between stationary and mobile phases, as well as retention of a very large fraction of the stationary phase. HSCCC uses no solid support matrix and eliminates the irreversible absorptive loss of samples onto the solid support matrix used in the conventional chromatographic column [4]. This method has been successfully used for the preparative separation of a number of natural products, such as Forsythia suspensa [5], Pueraria lobata [6] and Tangerine peel [7]. It was reported that chlorogenic acid could be obtained from Flos Lonicerae by two-step HSCCC separation [4], however, no report has been published on the simultaneous isolation and purification of chlorogenic acid, caffeic acid and luteolin from natural products. The present paper described the successful preparative separation and purification of chlorogenic acid, caffeic acid and luteolin from Caulis Lonicerae by one-step HSCCC. 2. Experimental 2.1. Reagents and materials
∗ Corresponding author at: College of Agronomy, Shandong Agricultural University, Taian, Shandong 271018, China. Tel.: +86 538 8242226; fax: +86 538 8242226. E-mail address: [email protected] (J. Wang). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.08.006
Chlorogenic acid, caffeic acid and luteolin standard samples were obtained from the National Institute of the Control of Pharmaceutical and Biological Products, Ministry of Health, Beijing, China.
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Z. Wang et al. / Separation and Purification Technology 63 (2008) 721–724
Fig. 2. HSCCC chromatogram of the crude extract from Caulis Lonicerae. Conditions: revolution speed: 800 rpm; solvent system: ethyl acetate–ethanol–water (4:1:5, v/v); stationary phase: upper organic phase; mobile phase: upper organic phase; mobile phase: lower aqueous phase; sample size: 110 mg; injection volume: 5 ml; detection wavelength: 254 nm; flow rate: 2.0 ml/min; retention of stationary phase: 48.9%.
Fig. 1. Chemical structures of chlorogenic acid, luteolin and caffeic acid.
Organic solvents including ethanol, ethyl acetate, phosphoric acid and methanol were all of analytical grade and were purchased from Tianjin YongDa Chemical Reagent Development Center, Tianjin, China. HPLC-grade acetonitrile was from Tianjin Kermel Chemical Reagents Development Center, Tianjin, China. Caulis Lonicerae was obtained from the Garden of Medcinal Plant, Shandong Agricultural University, Taian, China and identified by Prof. Jianhua Wang, Shandong Agricultural University. 2.2. Apparatus The preparative HSCCC instrument employed in the present study is the Model GS10A, with a multilayer coil planet centrifuge (Beijing Institute of New Technology Application, Beijing, China) equipped with a polytetrafluoroethylene multilayer coil of 110 m × 1.6 mm, i.d., with a total capacity of 234 ml. The ˇ value
of the preparative column varied from 0.5 at internal to 0.7 at the external (ˇ = 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). The rotation speed is adjustable from 0 to 1000 rpm, and 800 rpm was used in the present studies. The system was also equipped with one NS-1007 constant flow pump, a Model 8823B-UV monitor operating at 254 nm, a manual injection valve with a 10 ml sample loop. The HPLC system used throughout this study consisted of a Waters 600 pump, a Waters 600 controller (Waters, USA), a sample injector (made in Australia) with a 25 l loop, a Waters In-line Degasser AF, and a Waters 2996 photodiode array detector system. Evaluation and quantification were made on an EmpowerTM 2 Chromatography Data Software system (Waters, USA). 2.3. Preparation of crude extract The dried powder of Caulis Lonicerae (5 kg) were extracted with 2000 ml of boiling ethanol four times. The extraction time was 3, 3, 2 and 1 h, respectively. Then the ethanol solutions were combined and evaporated to about 200 ml by rotary vaporization at 40 ◦ C under reduced pressure. The deposit was separated and dried crude sample was obtained. It was stored in a refrigerator for subsequent HSCCC separation. 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 solvent
Table 1 Partition coefficients (K) and separation factors (˛) of chlorogenic acid, caffeic acid and luteolin in different two-phase solvent systems Solvent systems (v/v)
Ethyl acetate–n-butanol–water (2:1:3) Petroleum ather–ethyl acetate–methanol–water (1:0.8:1:0.8) Chloroform–n-butanol–methanol–water (4:0.5:3: 2) Ethyl acetate–methanol–water (4:1:5) Ethyl acetate–ethanol–water (2:1:3) Ethyl acetate–ethanol–water (4:1:5) Ethyl acetate–ethanol–acetate acid–water (4:1:0.25:5)
K-value Chlorogenic acid
˛
Caffeic acid
˛
Luteolin
1.86 0.17 2.91 0.76 0.67 0.59 1.14
1.43 1.09 0.34 3.91 2.37 1.80 4.09
2.67 0.19 1.00 2.97 1.58 1.06 4.64
5.84 0.62 2.04 6.27 3.97 2.29 1.88
15.60 0.11 2.03 18.62 6.26 2.43 8.74
Z. Wang et al. / Separation and Purification Technology 63 (2008) 721–724
723
system that gave suitable partition coefficient (K) values. The partition coefficient (K) values were determined by HPLC according to the literature [8]. Two milliliters of each phase of the equilibrated two-phase solvent system was added to approximately 1 mg of a test sample 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 (K). The K 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 aqueous phase was used as the mobile phase, while the organic phase was used as the stationary phase. The sample solution was prepared by dissolving the crude sample in the mixture solution of organic phase and aqueous phase (1:1, v/v) of the solvent system used for HSCCC separation. 2.6. HSCCC separation In the crude sample isolation and separation, the coil column was first entirely filled with the upper phase of the solvent system. Then the apparatus was rotated at 800 rpm, while the lower phase was pumped into the column at a flow rate of 2.0 ml/min. After the mobile phase front emerged and hydrodynamic equilibrium was established in the column, about 5 ml sample solution containing 110 mg of the crude extract was injected through the injection value. The effluent of the column was continuously monitored with a UV detector at 254 nm. Peak fractions were collected according to the elution profile. The retention of the stationary phase relative to the total column capacity was computed from the volume of the stationary phase collected from the column after the separation was completed. 2.7. HPLC analysis The crude extract and each purified fraction from the preparative HSCCC separation were analyzed by HPLC with a Symmetry C18 column (5 m, 250 mm × 4.6 mm, i.d.) at 350 nm and at a column temperature of 25 ◦ C. The mobile phase, a solution of acetonitrile and 0.1% phosphoric acid (20:80, v/v), was eluted at a flow rate of 1.0 ml/min. The effluent was monitored by a photodiode array detector, respectively. 3. Results and discussion 3.1. HSCCC separation of caffeic acid, chlorogenic acid and luteolin from the crude extract A successful separation of the target compounds using HSCCC requires a careful search for a suitable two-phase solvent system
Fig. 3. HPLC chromatogram of the crude extract from Caulis Lonicerae (E), HPLC of standard samples of chlorogenic acid, caffeic acid and luteolin (A), HPLC chromatogram and UV spectrum of peak 1 from preparative HSCCC (B), HPLC chromatogram and UV spectrum of peak 2 from preparative HSCCC (C), HPLC chromatogram and UV spectrum of peak 3 from preparative HSCCC (D). Experimental conditions: symmetry C18 column (5 m, 4.6 mm × 250 mm); column temperature: 25 ◦ C; mobile phase: acetonitrile–0.1% phosphoric acid (20:80, v/v); flow rate: 1.0 ml/min; detection wavelength: 350 nm; injection volume: 10 l.
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to provide an ideal range of partition coefficients for the applied material. Generally, the two-phase solvent system needs to satisfy the following requirements: (1) for ensuring a satisfactory retention of the stationary phase, the settling time of the solvent system should be considerably shorter than 30 s; (2) for an efficient separation, the partition coefficient (K) value of the target compounds should be close to 1, and the separation factor between two components (˛ = K2 /K1 , K2 > K1 ) should be greater than 1.5 [7,8]. In general, both low or high K values usually result in a poor peak resolution. Since chlorogenic acid is not soluble in non-polar solvent, but has some solubility in methanol and ethanol, and is freely soluble in water. In order to select a suitable two-phase solvent system, several two-phase solvent systems were tested and their K values were summarized in Table 1. Our results indicated: (1) Ethyl acetate–n-butanol–water, ethyl acetate–ethanol–acetate acid–water and chloroform–nbutanol–methanol–water had high K values, indicating that the components of peaks 2 and 3 were mostly partitioned in the upper phase (Table 1). When they were used for the HSCCC separation, chlorogenic acid and luteolin would not be separated. Thus, these two-phase solvent systems were not suitable for the separation of chlorogenic acid and luteolin from the crude sample. (2) Petroleum aether–ethyl acetate–methanol–water had a low K-value, also indicating that the components of peaks 1–3 were mostly partitioned in the aqueous phase (Table 1). When they were used for the HSCCC separation, chlorogenic acid and luteolin would be eluted together with other compounds near the solvent front resulting in a poor resolution. (3) Ethyl acetate–methanol–water or ethyl acetate–ethanol–water had suitable K values and all target components showed ˛ values of over 1.5. However, when they were used in the HSCCC separation, the two-phase solvent system composed of ethyl acetate–ethanol–water had a better retention of stationary phase than that of ethyl acetate–methanol–water. Furthermore, the two-phase solvent system of ethyl acetate–ethanol–water at a volume ratio of 4:1:5 was found to be satisfactory for the separation of chlorogenic acid, caffeic acid and luteolin from the crude extract. Fig. 2 shows the preparative HSCCC separation of 110 mg of the crude sample using ethyl acetate–ethanol–water (4:1:5, v/v/v) solvent system. The retention of stationary phase was 48.9%. Based on the HPLC analysis and the elution curve of the preparative HSCCC, peak 1 corresponded to caffeic acid, peak 2 corresponded to chlorogenic acid and peak 3 corresponded to luteolin. 7.2 mg caffeic acid, 15.7 mg chlorogenic acid and 18.8 mg luteolin were obtained.
3.2. HPLC analysis Peak fractions from HSCCC together with the crude extracts were analyzed by HPLC. The chromatograms of HPLC and UV spectra of these compounds were shown in Fig. 3. The purified samples of caffeic acid, chlorogenic acid and luteolin calculations were made by comparison of the peak area with the standard. The results of HPLC showed that peak 1 corresponded to caffeic acid, peak 2 corresponded to chlorogenic acid and peak 3 corresponded to luteolin, and their purity were 95.55%, 97.24% and 98.11%, respectively. 4. Conclusion In conclusion, HSCCC was successfully used for the separation and purification of caffeic acid, chlorogenic acid and luteolin from Caulis Lonicerae. Caffeic acid (7.2 mg), chlorogenic acid (15.7 mg) and luteolin (18.8 mg) were obtained from 110 mg of the crude extract in a one-step separation with a two-phase solvent system comprised of ethyl acetate–ethanol–water (4:1:5, v/v). Acknowledgements Financial supports from the National Development and Reform Commission (No. Guofa 018, 2006) and the Department of Science and Technology of Shandong Province (No. SDGP2004-54) are gratefully acknowledged. References [1] R.S. Chen (Ed.), National Pharmacopoeia Chinese Traditional Medicine Practicality Handbook, Jiangsu Technology Press, Jiangsu, 2004, p. 129. [2] Z.M. Qian, H.J. Li, P. Li, D. Tang, S.J. Qin, Determination of eight bioactive compounds in Caulis Lonicerae Japonicae and Folium Lonicerae Japonicae by high performance liquid chromatography, Chin. J. Anal. Chem. 35 (2007) 1159–1163. [3] Y. Ito, Efficient preparative counter-current chromatography with a coil planet centrifuge, J. Chromatogr. 214 (1981) 122–125. [4] H.T. Lu, Y. Jiang, F. Chen, Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae, J. Chromatogr. A 1026 (2004) 185–190. [5] H.B. Li, F. Chen, Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography, J. Chromatogr. A 1083 (2005) 102–105. [6] X.L. Cao, Y. Tian, T.Y. Zhang, X. Li, Y. Ito, Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography, J. Chromatogr. A 855 (1999) 709–713. [7] X. Wang, F.W. Li, H.X. Zhang, Y.L. Geng, J.P. Yuan, T. Jiang, Preparative isolation and purification of polymethoxylated flavones from Tangerine peel using high-speed counter-current chromatography, J. Chromatogr. A 1090 (2005) 188–192. [8] H. Oka, K.-i. Harada, Y. Ito, Y. Ito, Separation of antibiotics by counter-current chromatography, J. Chromatogr. A 812 (1998) 35–52.
Contents lists available at ScienceDirect
Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Short communication
Purification of caffeic acid, chlorogenic acid and luteolin from Caulis Lonicerae by high-speed counter-current chromatography Zhilong Wang a,c , Jianhua Wang b,c,∗ , Yinshi Sun b,c , Shengbo Li d , Hanzhong Wang a,c a
College of Life Science, Shandong Agricultural University (SDAU), Taian, Shandong 271018, China College of Agronomy, SDAU, Taian, Shandong 271018, China c State Key Laboratory of Crop Biology, SDAU, Taian, Shandong 271018, China d Shandong Yate Eco-tech Co., Ltd., Linyi, Shandong 266071, China b
a r t i c l e
i n f o
Article history: Received 10 March 2008 Received in revised form 1 August 2008 Accepted 5 August 2008 Keywords: Caulis Lonicerae Counter-current chromatography Chlorogenic acid Caffeic acid Luteolin
a b s t r a c t Chlorogenic acid, caffeic acid and luteolin are the major phenolic compounds found in the traditional Chinese herbal medicine Caulis Lonicerae. The separation and purification of the three compounds from the crude extract of Caulis Lonicerae was achieved by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of ethyl acetate–ethanol–water (4:1:5, v/v). Caffeic acid (7.2 mg), chlorogenic acid (15.7 mg) and luteolin (18.8 mg) from 110 mg crude extract of Caulis Lonicerae were obtained and their purities were 95.55%, 97.24% and 98.11%, respectively. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Caulis Lonicerae, rendongteng in Chinese, is the dry stem and branch of Caprifoliaceae plant Lonicera japonica Thunb. It plays major roles in clearing body heat and detoxification, dispelling wind and removing obstruction in channels. Caulis Lonicerae is usually used to treat epidemic febrile diseases and fever, thermotoxin and bloody flux, abscess and sore, rheumatism, joint red and swollen and arthralgia [1]. However, for a long time, people have paid more attention to the research and exploitation of Flos Lonicerae, the dry flower of L. japonica Thunb. In recent years, people have found phenolic compounds and flavonoids in Caulis Lonicerae, especially that the contents of caffeic acid and luteolin in Caulis Lonicerae were also higher than those in Flos Lonicerae [2]. Therefore, an efficient method for the preparative separation and purification of chlorogenic acid, caffeic acid and luteolin (Fig. 1) from Caulis Lonicerae is much needed. High-speed counter-current chromatography (HSCCC) is a useful method for rapid chromatographic purification employing highly efficient fraction by a hybrid technique of liquid–liquid counter-current distribution and liquid chromatography that was
first invented by Ito [3]. In conjunction with the use of centrifugal force, the centrifugal force field generated from both rotational and synchronous planetary motion of coiled columns containing two immiscible liquid phases provides vigorous mixing between stationary and mobile phases, as well as retention of a very large fraction of the stationary phase. HSCCC uses no solid support matrix and eliminates the irreversible absorptive loss of samples onto the solid support matrix used in the conventional chromatographic column [4]. This method has been successfully used for the preparative separation of a number of natural products, such as Forsythia suspensa [5], Pueraria lobata [6] and Tangerine peel [7]. It was reported that chlorogenic acid could be obtained from Flos Lonicerae by two-step HSCCC separation [4], however, no report has been published on the simultaneous isolation and purification of chlorogenic acid, caffeic acid and luteolin from natural products. The present paper described the successful preparative separation and purification of chlorogenic acid, caffeic acid and luteolin from Caulis Lonicerae by one-step HSCCC. 2. Experimental 2.1. Reagents and materials
∗ Corresponding author at: College of Agronomy, Shandong Agricultural University, Taian, Shandong 271018, China. Tel.: +86 538 8242226; fax: +86 538 8242226. E-mail address: [email protected] (J. Wang). 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.08.006
Chlorogenic acid, caffeic acid and luteolin standard samples were obtained from the National Institute of the Control of Pharmaceutical and Biological Products, Ministry of Health, Beijing, China.
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Z. Wang et al. / Separation and Purification Technology 63 (2008) 721–724
Fig. 2. HSCCC chromatogram of the crude extract from Caulis Lonicerae. Conditions: revolution speed: 800 rpm; solvent system: ethyl acetate–ethanol–water (4:1:5, v/v); stationary phase: upper organic phase; mobile phase: upper organic phase; mobile phase: lower aqueous phase; sample size: 110 mg; injection volume: 5 ml; detection wavelength: 254 nm; flow rate: 2.0 ml/min; retention of stationary phase: 48.9%.
Fig. 1. Chemical structures of chlorogenic acid, luteolin and caffeic acid.
Organic solvents including ethanol, ethyl acetate, phosphoric acid and methanol were all of analytical grade and were purchased from Tianjin YongDa Chemical Reagent Development Center, Tianjin, China. HPLC-grade acetonitrile was from Tianjin Kermel Chemical Reagents Development Center, Tianjin, China. Caulis Lonicerae was obtained from the Garden of Medcinal Plant, Shandong Agricultural University, Taian, China and identified by Prof. Jianhua Wang, Shandong Agricultural University. 2.2. Apparatus The preparative HSCCC instrument employed in the present study is the Model GS10A, with a multilayer coil planet centrifuge (Beijing Institute of New Technology Application, Beijing, China) equipped with a polytetrafluoroethylene multilayer coil of 110 m × 1.6 mm, i.d., with a total capacity of 234 ml. The ˇ value
of the preparative column varied from 0.5 at internal to 0.7 at the external (ˇ = 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). The rotation speed is adjustable from 0 to 1000 rpm, and 800 rpm was used in the present studies. The system was also equipped with one NS-1007 constant flow pump, a Model 8823B-UV monitor operating at 254 nm, a manual injection valve with a 10 ml sample loop. The HPLC system used throughout this study consisted of a Waters 600 pump, a Waters 600 controller (Waters, USA), a sample injector (made in Australia) with a 25 l loop, a Waters In-line Degasser AF, and a Waters 2996 photodiode array detector system. Evaluation and quantification were made on an EmpowerTM 2 Chromatography Data Software system (Waters, USA). 2.3. Preparation of crude extract The dried powder of Caulis Lonicerae (5 kg) were extracted with 2000 ml of boiling ethanol four times. The extraction time was 3, 3, 2 and 1 h, respectively. Then the ethanol solutions were combined and evaporated to about 200 ml by rotary vaporization at 40 ◦ C under reduced pressure. The deposit was separated and dried crude sample was obtained. It was stored in a refrigerator for subsequent HSCCC separation. 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 solvent
Table 1 Partition coefficients (K) and separation factors (˛) of chlorogenic acid, caffeic acid and luteolin in different two-phase solvent systems Solvent systems (v/v)
Ethyl acetate–n-butanol–water (2:1:3) Petroleum ather–ethyl acetate–methanol–water (1:0.8:1:0.8) Chloroform–n-butanol–methanol–water (4:0.5:3: 2) Ethyl acetate–methanol–water (4:1:5) Ethyl acetate–ethanol–water (2:1:3) Ethyl acetate–ethanol–water (4:1:5) Ethyl acetate–ethanol–acetate acid–water (4:1:0.25:5)
K-value Chlorogenic acid
˛
Caffeic acid
˛
Luteolin
1.86 0.17 2.91 0.76 0.67 0.59 1.14
1.43 1.09 0.34 3.91 2.37 1.80 4.09
2.67 0.19 1.00 2.97 1.58 1.06 4.64
5.84 0.62 2.04 6.27 3.97 2.29 1.88
15.60 0.11 2.03 18.62 6.26 2.43 8.74
Z. Wang et al. / Separation and Purification Technology 63 (2008) 721–724
723
system that gave suitable partition coefficient (K) values. The partition coefficient (K) values were determined by HPLC according to the literature [8]. Two milliliters of each phase of the equilibrated two-phase solvent system was added to approximately 1 mg of a test sample 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 (K). The K 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 aqueous phase was used as the mobile phase, while the organic phase was used as the stationary phase. The sample solution was prepared by dissolving the crude sample in the mixture solution of organic phase and aqueous phase (1:1, v/v) of the solvent system used for HSCCC separation. 2.6. HSCCC separation In the crude sample isolation and separation, the coil column was first entirely filled with the upper phase of the solvent system. Then the apparatus was rotated at 800 rpm, while the lower phase was pumped into the column at a flow rate of 2.0 ml/min. After the mobile phase front emerged and hydrodynamic equilibrium was established in the column, about 5 ml sample solution containing 110 mg of the crude extract was injected through the injection value. The effluent of the column was continuously monitored with a UV detector at 254 nm. Peak fractions were collected according to the elution profile. The retention of the stationary phase relative to the total column capacity was computed from the volume of the stationary phase collected from the column after the separation was completed. 2.7. HPLC analysis The crude extract and each purified fraction from the preparative HSCCC separation were analyzed by HPLC with a Symmetry C18 column (5 m, 250 mm × 4.6 mm, i.d.) at 350 nm and at a column temperature of 25 ◦ C. The mobile phase, a solution of acetonitrile and 0.1% phosphoric acid (20:80, v/v), was eluted at a flow rate of 1.0 ml/min. The effluent was monitored by a photodiode array detector, respectively. 3. Results and discussion 3.1. HSCCC separation of caffeic acid, chlorogenic acid and luteolin from the crude extract A successful separation of the target compounds using HSCCC requires a careful search for a suitable two-phase solvent system
Fig. 3. HPLC chromatogram of the crude extract from Caulis Lonicerae (E), HPLC of standard samples of chlorogenic acid, caffeic acid and luteolin (A), HPLC chromatogram and UV spectrum of peak 1 from preparative HSCCC (B), HPLC chromatogram and UV spectrum of peak 2 from preparative HSCCC (C), HPLC chromatogram and UV spectrum of peak 3 from preparative HSCCC (D). Experimental conditions: symmetry C18 column (5 m, 4.6 mm × 250 mm); column temperature: 25 ◦ C; mobile phase: acetonitrile–0.1% phosphoric acid (20:80, v/v); flow rate: 1.0 ml/min; detection wavelength: 350 nm; injection volume: 10 l.
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to provide an ideal range of partition coefficients for the applied material. Generally, the two-phase solvent system needs to satisfy the following requirements: (1) for ensuring a satisfactory retention of the stationary phase, the settling time of the solvent system should be considerably shorter than 30 s; (2) for an efficient separation, the partition coefficient (K) value of the target compounds should be close to 1, and the separation factor between two components (˛ = K2 /K1 , K2 > K1 ) should be greater than 1.5 [7,8]. In general, both low or high K values usually result in a poor peak resolution. Since chlorogenic acid is not soluble in non-polar solvent, but has some solubility in methanol and ethanol, and is freely soluble in water. In order to select a suitable two-phase solvent system, several two-phase solvent systems were tested and their K values were summarized in Table 1. Our results indicated: (1) Ethyl acetate–n-butanol–water, ethyl acetate–ethanol–acetate acid–water and chloroform–nbutanol–methanol–water had high K values, indicating that the components of peaks 2 and 3 were mostly partitioned in the upper phase (Table 1). When they were used for the HSCCC separation, chlorogenic acid and luteolin would not be separated. Thus, these two-phase solvent systems were not suitable for the separation of chlorogenic acid and luteolin from the crude sample. (2) Petroleum aether–ethyl acetate–methanol–water had a low K-value, also indicating that the components of peaks 1–3 were mostly partitioned in the aqueous phase (Table 1). When they were used for the HSCCC separation, chlorogenic acid and luteolin would be eluted together with other compounds near the solvent front resulting in a poor resolution. (3) Ethyl acetate–methanol–water or ethyl acetate–ethanol–water had suitable K values and all target components showed ˛ values of over 1.5. However, when they were used in the HSCCC separation, the two-phase solvent system composed of ethyl acetate–ethanol–water had a better retention of stationary phase than that of ethyl acetate–methanol–water. Furthermore, the two-phase solvent system of ethyl acetate–ethanol–water at a volume ratio of 4:1:5 was found to be satisfactory for the separation of chlorogenic acid, caffeic acid and luteolin from the crude extract. Fig. 2 shows the preparative HSCCC separation of 110 mg of the crude sample using ethyl acetate–ethanol–water (4:1:5, v/v/v) solvent system. The retention of stationary phase was 48.9%. Based on the HPLC analysis and the elution curve of the preparative HSCCC, peak 1 corresponded to caffeic acid, peak 2 corresponded to chlorogenic acid and peak 3 corresponded to luteolin. 7.2 mg caffeic acid, 15.7 mg chlorogenic acid and 18.8 mg luteolin were obtained.
3.2. HPLC analysis Peak fractions from HSCCC together with the crude extracts were analyzed by HPLC. The chromatograms of HPLC and UV spectra of these compounds were shown in Fig. 3. The purified samples of caffeic acid, chlorogenic acid and luteolin calculations were made by comparison of the peak area with the standard. The results of HPLC showed that peak 1 corresponded to caffeic acid, peak 2 corresponded to chlorogenic acid and peak 3 corresponded to luteolin, and their purity were 95.55%, 97.24% and 98.11%, respectively. 4. Conclusion In conclusion, HSCCC was successfully used for the separation and purification of caffeic acid, chlorogenic acid and luteolin from Caulis Lonicerae. Caffeic acid (7.2 mg), chlorogenic acid (15.7 mg) and luteolin (18.8 mg) were obtained from 110 mg of the crude extract in a one-step separation with a two-phase solvent system comprised of ethyl acetate–ethanol–water (4:1:5, v/v). Acknowledgements Financial supports from the National Development and Reform Commission (No. Guofa 018, 2006) and the Department of Science and Technology of Shandong Province (No. SDGP2004-54) are gratefully acknowledged. References [1] R.S. Chen (Ed.), National Pharmacopoeia Chinese Traditional Medicine Practicality Handbook, Jiangsu Technology Press, Jiangsu, 2004, p. 129. [2] Z.M. Qian, H.J. Li, P. Li, D. Tang, S.J. Qin, Determination of eight bioactive compounds in Caulis Lonicerae Japonicae and Folium Lonicerae Japonicae by high performance liquid chromatography, Chin. J. Anal. Chem. 35 (2007) 1159–1163. [3] Y. Ito, Efficient preparative counter-current chromatography with a coil planet centrifuge, J. Chromatogr. 214 (1981) 122–125. [4] H.T. Lu, Y. Jiang, F. Chen, Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae, J. Chromatogr. A 1026 (2004) 185–190. [5] H.B. Li, F. Chen, Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography, J. Chromatogr. A 1083 (2005) 102–105. [6] X.L. Cao, Y. Tian, T.Y. Zhang, X. Li, Y. Ito, Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography, J. Chromatogr. A 855 (1999) 709–713. [7] X. Wang, F.W. Li, H.X. Zhang, Y.L. Geng, J.P. Yuan, T. Jiang, Preparative isolation and purification of polymethoxylated flavones from Tangerine peel using high-speed counter-current chromatography, J. Chromatogr. A 1090 (2005) 188–192. [8] H. Oka, K.-i. Harada, Y. Ito, Y. Ito, Separation of antibiotics by counter-current chromatography, J. Chromatogr. A 812 (1998) 35–52.