2320 Yarong Wang1 Shining Cai1 Yang Chen1 Liang Deng2 ∗ Xumei Zhou3 Jia Liu4 Xin Xu1 Qiang Xia1 Mao Lin1 Jili Zhang1 Weili Huang1 Wenjun Wang1 Canhui Xiang1 Guozhen Cui1 Lianfeng Du1 Huan He1 Baohui Qi1 1 Department
of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai, Guangdong, China 2 School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University Chenggong New City, Kunming, Yunnan, China 3 Department of Pharmaceutical Analysis, Zunyi Medical University, Zunyi, Guizhou, China 4 Department of English, Zunyi Medical University, Zhuhai, Guangdong, China
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Short Communication
Separation and purification of five alkaloids from Aconitum duclouxii by counter-current chromatography C19 -diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter-current chromatography. In the process of separation, the critical conditions of counter-current chromatography were optimized. The two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1, 24.1 mg of 2, 250.6 mg of 3, 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra-high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation. Keywords: Aconitum duclouxii L / C19 -diterpenoid alkaloids / Counter-current chromatography DOI 10.1002/jssc.201500059
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Received January 16, 2015 Revised March 27, 2015 Accepted April 12, 2015
1 Introduction Aconitum L. (Ranunculacea) is a large genus comprising 400 species and widely distributed in the temperate region of the northern hemisphere. This genus is well known for toxicity and is usually regarded as medicinal plants. In China, there are about 76 Aconitum species, and some of them have been used for the treatment of rheumatoid arthritis, arrhythmia, pains, and various cancers [1, 2]. Characteristic compounds of these plants are the diterpenoid alkaloids [3]. Diterpenoid alkaloids, classified as the C18 -, C19 -, and C20 -subtypes, have been reported to be the main bioactive constituents of plants from aconitum genus. The important pharmacological activities and structural complexity of these diterpenoid alkaloids have long aroused scientists’ strong interest in the
Correspondence: Prof. Yang Chen, Department of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai Campus, Zhuhai, Guangdong 519041, China E-mail: [email protected] Fax: +86-756-7637616 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
investigation of phytochemistry, synthesis, and medicinal chemistry [4]. Aconitum duclouxii Levl (Baicaowu in Chinese), a perennial herb distributed in Dali Bai Autonomous Prefecture, Yunnan Province of China, has long been used as a folk medicine to treat rheumatism and pains [5]. Previous investigations on this herb led to the isolation of six new C19 -diterpenoid alkaloids and various known alkaloids [6–9]. Because of their complex structures, pharmacological activities [10, 11], interesting chemical reactions [12], and economic importance [13], the C19 -diterpenoid alkaloids have aroused scientists’ considerable interest. However, these studies, which adopted classical methods including silica gel, SiO2 and Sephadex LH-20 in the preparative separation and purification of alkaloids from this herb, are proved to be tedious and time consuming. They required multiple chromatographic steps and thus resulted in lower recovery and higher cost. Consequently, it is important to develop an efficient method to separate and purify main C19 -diterpenoid alkaloids from the roots of this herb. CCC (Counter-current chromatography) was first introduced by Ito in the late 1960s [14]. Since then, it has ∗ Additional corresponding author: Lecturer Liang Deng. E-mail: [email protected]
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Sample Preparation
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Figure 1. Chemical structures of target alkaloids obtained from Aconitum duclouxii.
been widely used in the field of natural product chemistry to effectively separate a large variety of compounds. The technique, an all-liquid method without solid phases, relies on the partition of a sample between two immiscible solvents to achieve separation. The relative proportions of the solute that dissolves in two phases are determined by the respective distribution ratios (K). It has great advantages over the traditional liquid–solid separation methods. It can eliminate the complications resulting from the solid support matrix, such as irreversible adsorptive sample loss and deactivation, tailing of solute peaks, and contamination [15]. CCC has the unique features of high recovery, high efficiency, and ease of scale-up. Thus it has been widely used in the separation and purification of various natural and synthetic products [16–19]. To the best of our knowledge, many studies have been published, reporting the successful use of CCC for the separation and purification of C20 - and C18 -diterpenoid alkaloids from genus aconitum [20–26]. More recently, three C19 -diterpenoid alkaloids were isolated from the lateral roots of Aconitum carmichaeli Debx by CCC [27]. However, there is no article gthus far reporting the separation of C19 -diterpenoid alkaloids from A. duclouxii by CCC. This paper, for the first time, describes the successful preparative separation and purification of five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), from A. duclouxii by CCC. Among these components, ducloudine A is a novel C19 -diterpenoid alkaloid recently obtained from the roots of A. duclouxii [6]. The chemical structures of the five alkaloids are shown in Fig. 1.
2 Materials and methods 2.1 Materials and chemicals Acetonitrile (Sigma, USA) was of chromatographic purity and pure water (Watson, China) was used for UHPLC. All reagents and solvents, used for preparation of crude extract, UHPLC, and CCC separation, were of analytical grade (Fuyu Fine Chemical Reagent, Tianjin, China). The roots C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of A. duclouxii were collected from Dali Bai Autonomous Prefecture, Yunnan Provinces of China, in December 2012. The samples were identified by Professor Shu-Gang Lu from the School of Life Sciences, Yunnan University, Kunming, China. The voucher specimens are deposited at the Department of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai Campus, China. 2.2 Preparation of total alkaloids The air-dried and powdered roots (0.5 kg) of A. duclouxii Levl were percolated with 0.5% HCl (2 L).The aqueous acidic solution was basified with 10% aqueous NH3 ·H2 O to pH 9– 10 and then extracted with ethyl acetate (3 × 2.5 L). Removal of the solvent under reduced pressure afforded a total of 5.5 g of crude alkaloids as a yellowish amorphous powder, which was then stored in a refrigerator (4⬚C) for CCC separation. 2.3 Selection of the solvent system The suitable solvent systems were evaluated by UHPLC according to the partition coefficients (0.5 < K < 2.0) in a series of solvent systems as follows: about 1.0 mg sample was added to the test tubes, and then 2 mL of each phase of a preequilibrated two-phase solvent system was added and thoroughly mixed. Each test tube was rigorously shaken for several minutes and left to stand at room temperature until equilibrium was attained. Then 3 L of the upper and lower phases were analyzed by UHPLC at 233 nm. The partition coefficient (K) is defined as Aupper /Alower , where Aupper and Alower were the UHPLC peak areas of target compounds in the upper and lower phases, respectively.
2.4 CCC separation 2.4.1 CCC apparatus The preparative CCC was performed using a model TBE1000A CCC (Shanghai Tauto Biotechnique, Shanghai, www.jss-journal.com
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Figure 2. UHPLC chromatograms of total alkaloids extract from Aconitum duclouxii sample. Column: Symmetry 1 C18 (50 × 2.1 mm id, 1.8 m); A (acetonitrile/tetrahydrofuran, 25:15) and B (0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid) in a gradient mode 16–19% A (0–5.0 min), 19–25% A (5.0–15.0 min), 25– 35% A (15.0–20.0 min), 35– 16% A (20.0–24.0 min), 16% A (24.0–25.0 min); column temperature: 30⬚C; flow rate: 0.3 mL/min; detection wavelength: 233 nm. Table 1. The K (partition coefficients) values of the compounds 1–5 in different solvent systems
Solvent systems
Ethyl acetate/n-butanol/water
n-Hexane/ethyl acetate/methanol/water
n-Hexane/ethyl acetate/methanol/water/ NH3 ·H2 O (25%)
Volume ratio
K values
(v/v)
1
2
3
4
5
4:1:5 4:1.5:5 4:2:5 1:1:1:1 1.5:0.5:1:0.5 1.5:0.2:1:0.2 1:1:1:1:0.05 1:1:1:1:0.1 1:1:1:1:0.2
2.15 2.02 1.76 1.08 0.56 0.37
2.36 2.34 2.01 1.54 0.97 0.55
2.78 2.49 1.89 1.97 1.36 0.76
3.18 2.59 1.78 2.89 1.71 0.99
3.05 2.78 1.98 3.54 2.16 1.13
China). The apparatus consisted of an upright coil type-J planet centrifuge with three multilayered coils (Each coil in this configuration had a beta range of 0.6–0.78) connected in series (diameter of tube, 3.0 mm, total capacity 1000 mL) and a 80 mL manual sample loop. The rotation speed was adjustable, ranging from 0 to 600 rpm. The CCC system was equipped with a TBP-5002 pump, a TBD-2000 D-UV detector, a DC-0506 constant temperature regulator (Shanghai Tauto Biotechnique, Shanghai, China), which was used to control the separation temperature. The outflow of CCC was detected by a Model 2000D-UV monitor (Sanotac Scientific Instruments, Shanghai, China). The data were collected with a Sanotac chromatography workstation. In the separation process, the temperature of separation columns was maintained at 25⬚C, and the effluents were monitored at 233 nm for C19 -diterpenoid alkaloids of A. duclouxii. 2.4.2 Preparation of solvent system and sample solution The solvent system composed of n-hexane/ethyl acetate/ methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was used for CCC separation in one run. The preparation of each C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
two-phase solvent system was performed in a separation funnel according to the volume ratios and thoroughly equilibrated after shaking at room temperature. The upper phase and lower phase were then separated and degassed by ultrasonication for 30 min shortly before use. The sample solution of for CCC separation was prepared by dissolving 1000 mg of total alkaloids extract in 20 mL lower phase of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v). 2.4.3 CCC separation procedure The CCC was performed as follows in the whole separation: the column was first entirely filled with the upper phase as the stationary phase; subsequently, the lower mobile phase was then pumped into the inlet of the column as the mobile phase at the flow rate of 8.0 mL/min, while the apparatus was run at 600 rpm; after a clear mobile phase eluted at the tail outlet and the hydrodynamic equilibrium was reached, samples were then injected into the injection valve; the effluent from the outlet of the column was continuously monitored with a UV detector at 233 nm for C19 -diterpenoid alkaloid from the roots of A. duclouxii. The peak fractions were collected manually according to the chromatographic profile; after www.jss-journal.com
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Figure 3. Chromatogram of total alkaloids extract from Aconitum duclouxii by preparative CCC. Solvent system: n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v). The detection wavelength of chromatograms was 233 nm. The sample weight was 1000 mg. Retention of stationary phase was 42% and the rotation speed was 600 rpm. The temperature of separation columns was maintained at 25⬚C and the flow rate of mobile phase was 8.0 mL/min.
target compounds were eluted, the centrifuge was stopped and stationary phase was pumped out from the column with pressured air and collected in a graduated cylinder to measure the retention volume.
2.5 UHPLC Analysis The UHPLC equipment was an ACQUITY UPLC H Class system, equipped with a QSM quaternary solvent manager, a UPL PDA detector, a CHA column thermostat, a SDI sample manager, a RP Symmetry1 C18 (2.1 mm × 100 mm T3 1.8 m, Milford, MA, USA) column from Phenomenex (Torrance, California, USA), and an Empower 3 UPLC workstation (Waters, USA). The mobile phase was consisted of A (acetonitrile/tetrahydrofuran, 25:15) and B (0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid), which was programmed as follows: from 0 to 5 min, 16–19% A, 5–15.0 min, linear increase from 19–25% A. 15.0–20.0 min, linear increase from 25–35% A. 20.0–24.0 min, linear decrease from 35–16% A. 24.0–25.0 min, isocratic for 16% A. The flow rate was 0.3 mL/min while spectra were recorded from 200 to 500 nm. The pooled fraction was concentrated by a rotary evaporator and each fraction was analyzed by analytical UHPLC to check the purity before characterization. The purities of the collected fractions were determined by UHPLC based on the peak area of the target species normalized to the sum of all observed peaks.
2.6 Structure elucidation The target peak fractions obtained by CCC were identified by their UV, high-resolution ESI-MS and NMR spectra. The MS analysis of purified alkaloids were performed by ESI-Q-TOF mass spectrometer (Agilent 6530 Series, Agilent C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Technologies, Santa Clara, CA, USA) in a positive ionization mode. Parameters for the Agilent Jet Stream Technology are the superheated nitrogen sheath gas temperature (350⬚C) and flow rate (12 L/min). Electrospray conditions were the following: capillary: 4000 V; nebulizer: 20 psi; drying gas: 10 L/min; gas temperature: 350⬚C; skimmer voltage: 60 V; octapole RFPeak: 750 V; fragmentor: 135 V. The data recorded was processed with the Mass profiler (Agilent, Santa Clara, CA, USA) with accurate mass application specific additions from Agilent MSD TOF software. A second orthogonal sprayer with a reference solution was used as a continuous calibration using the following reference masses: 121.0509 and 922.0098 m/z. Spectra were acquired over the m/z 50–1000 range at a scan rate of 1 s per spectrum. NMR spectra were recorded on Bruker AM-400 spectrometers (Bruker, Karlsruhe, Germany) using TMS as the internal reference.
3 Results and discussion 3.1 Optimization of UHPLC analysis The whole separation of all target compounds is a crucial and challenging task for LC analysis. In present work, a UHPLC– DAD mode for the analysis of Aconitum L was established. In the course of optimizing the conditions of separation, the system conditions were investigated, including the mobile phase system (acetonitrile/water and methanol/water with different concentrations of tetrahydrofuran, ammonium acetate, and acetic acid), gradient program (gradient time, gradient shape, and initial composition of the mobile phase), and column temperature. Acetonitrile/tetrahydrofuran, 25:15 (A) and 0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid (B) was used as the mobile phase in gradient mode as mentioned in Section 2.5. The flow rate was 0.3 mL/min while www.jss-journal.com
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3.2 Selection of two-phase solvent system and other conditions of CCC
Figure 4. UHPLC chromatograms of peak fractions obtained by CCC preparation. Column: Symmetry 1 C18 (50 × 2.1 mm id, 1.8 m); A (acetonitrile/tetrahydrofuran, 25:15) and B (0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid) in the gradient mode (see Fig. 2 caption); column temperature: 30⬚C; flow rate: 0.3 mL/min; detection wavelength: 233 nm.
spectra were recorded from 200 to 500 nm. Under this condition, all alkaloids could reach almost baseline separation, and a chromatogram is shown in Fig. 2.
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Successful separation by CCC depends upon the selection of a suitable two-phase solvent system, which provides an ideal range of the partition coefficient (K) for the target compounds [28]. If the K value is much smaller than 1, the solutes will be eluted close to each other near the solvent front, which may result in loss of peak resolution; on the contrary if the K value is much greater than 1, the solutes will be eluted in excessively broad peaks, and may lead to extended elution time [29]. When the K value of the target compound is between 0.4 and 2.5, the solvent system is suitable for CCC separation [30]. In the present investigation, the K values of alkaloids 1–5 partitioned between the upper and lower phases of a series of solvent system were evaluated with UHPLC (Table 1). At first, the two-phase solvent system, ethyl acetate/n-butanol/water with different volume ratios, was carefully screened to optimize the solvent system for CCC separation and the K values of the target alkaloids were measured. The results indicated that the K values of the five target compounds, which were mostly distributed in the upper phase, are too large in the two-phase solvent system (Table 1). When the search changed to n-hexane/ethyl acetate/methanol/water, K values of five chemicals decreased significantly but remained too large (Table 1). Therefore, NH3 ·H2 O (25%) was added to modify the K values of five components. When the NH3 ·H2 O fraction increased, the K values of all target alkaloids decreased. Among the solvent systems with different concentration of the modifier, n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1) was proved to achieve suitable K values (1, K = 0.56; 2, K = 0.97; 3, K = 1.36; 4, K = 1.71; 3, K = 2.16), which meets the K value requirements and means the five components could be well separated. Adding NH3· H2 O as a modifier to the solvent system makes target alkaloids much better soluble in the lower phase if compared with those without NH3 ·H2 O. This condition was further evidenced by analytical CCC to be satisfactory for separation of all target compounds from the total alkaloids of Aconitum L (data not shown). Other factors, such as the flow rate of the mobile phase, the temperature and the revolution speed of the separation column, were also investigated. As expected, the flow rate affected the stationary phase retention as well as separation time and the peak resolution. To shorten the separation time while still maintaining an adequate resolution, the flow rate of mobile phase of preparative CCC was selected at 8.0 mL/min for separation (retention of stationary phase was 42%). The results of CCC separations also indicated that increasing the temperature could improve the CCC separation, but a high temperature may run the risk of stripping away the stationary phase due to the emulsification of the solvent system, so 25⬚C was selected for separation. In this work, separations of preparative CCC were performed at 600 rpm.
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Figure 5. Positive ESI-Q-TOF-MS analyses of purified alkaloids from Aconitum duclouxii.
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3.3 Preparative CCC separation As shown in Fig. 3, under the optimum CCC condition, satisfactory effect of separation was achieved and five pure alkaloids were obtained by CCC separation of 1000 mg total alkaloids extract of Aconitum L., namely, alkaloids 1 (148.2 mg, collected during 81–96 min), 2 (24.1 mg, collected during 103–110 min), 3 (250.6 mg, collected during 114–144 min), 4 (73.9 mg, collected during 158–177 min), and 5 (31.4 mg, collected during184–200 min). In addition, the purities of all target compounds 1–5 were 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by UHPLC peak area percentage (Fig. 4). Moreover, the chemical structures of the prepared compounds are shown in Fig. 1 and details are listed in the Supporting Information. The results of current study clearly demonstrated that CCC could provide highly efficient preparative separation of alkaloids from Aconitum L.
4 Conclusions A novel method for systematic separation and purification of C19 -diterpenoid alkaloids from the roots of A. duclouxii has been described in this research. The results of our study clearly demonstrated that CCC could provide highly efficient preparative separation of main alkaloids from A. duclouxii. Generally, five alkaloids were obtained from the crude extract of A. duclouxii. Their purities were 96.6–98.4%. The presented separation and purification method was simple, fast, and efficient, and the obtained high pure alkaloids are suitable for biochemical and toxicological investigation. The authors acknowledge the financial support from the Project Natural Science Foundation of China (No. 21162046), the Project of High Level Talents of Guizhou Province (TZJF2010-062) and the Project of Guizhou Provincial Administration of Traditional Chinese Medicine (2012-GZYY20). We would like to show our sincere gratitude to Le Cai for his help in structure identification. In addition, our cordial thanks go to all of our colleagues for their excellent assistance.
The authors have declared no conflict of interest.
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[5] People’s Government of Dali Bai Autonomous Prefecture, The Natural Resources of Chinese Medicinal Materials of Dali, Yunnan Ethnic Publishing House, Kunming 1991, p. 77. [6] Yin, T. P., Cai, L., Lei, G., Dong, J. W., Liu, Y. X., Ding, Z. T., Chin. J. Org. Chem. 2013, 33, 2528–2532. [7] Yin, T. P., Cai, L., Lei, G., He, J. M., Dong, J. W., Fang, H. X., Zhou, H., Ding, Z. T., J. Asian. Nat. Prod. Res. 2014, 16, 345–350. [8] Yin, T. P., Cai, L., Zhou, H., Zhu, X. F., Chen, Y., Ding, Z. T., Nat. Prod. Res. 2014, 28, 1649–1654. [9] Wang, C. Y., Chen, J. B., Zhu, R. H., Acta. Pharm. Sin. 1984, 19, 445. [10] Dzhakhagirov, F. N., Sultankhodzhaev, M. N., Tashkhodzhaev, B., Silmov, B. T., Khim. Prir. Soedin. 1997, 33, 254–270. [11] Pelletier, S. W., The Alkaloids: Chemical and Biological Perspectives, Springer Verlag Press, New York, 1983, pp. 120–153. [12] Wang, F. P., Liang, X. T., The Alkaloids: Chemistry and Pharmacology, Academic Press, New York, 1992, pp. 151–247. [13] Panter, K. E., Manners, G. D., Stegelmeier, B. L., Gardner, D. R., Ralphs, M. H., Pfister, J. A., James, L. F., Biochem. Syst. Ecol. 2002, 30, 113–128. [14] Ito, Y., Weinstein, M., Aoki, I., Harada, R., Kimura, E., Nunogaki, K., Nature 1966, 212, 985–987. [15] Jubert, C., Baiiley, G., J. Chromatogr. A 2007, 1140, 95– 100. [16] Cao, X. L., Ito, Y., J. Chromatogr. A 2003, 1021, 117–124. [17] Du, Q. Z., Li, L., Jerz, G., J. Chromatogr. A 2005, 1077, 98–101. [18] Sun, A. L., Sun, Q. H., Liu, R. M., J. Sep. Sci. 2007, 30, 1013–1018. [19] Xu, H. N., He, C. H., Sep. Purif. Technol. 2007, 56, 85–89. [20] Tang, Q., Yang, C., Ye, W., Liu, J., Zhao, S., Phytochem. Anal. 2008, 19, 155–159. [21] Wang, X., Shu, X., Wang, X., Yu, J., Jing, F., Molecules 2014, 19, 12619–12629. [22] Yang, F., Ito, Y., J. Chromatogr. A 2002, 943, 219–225. [23] Tang, Q., Yang, C., Ye, W., Liu, J., Zhao, S., J. Chromatogr. A 2007, 1144, 203–207. [24] Yang, F., Ito, Y., J. Chromatogr. A 2002, 923, 281–285. [25] Liu, Y., Chen, T., Chen, C., Zou, D., Li, Y., Chin. J. Chromatogr. 2014, 32, 543–546.
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of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai, Guangdong, China 2 School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products, Kunming Medical University Chenggong New City, Kunming, Yunnan, China 3 Department of Pharmaceutical Analysis, Zunyi Medical University, Zunyi, Guizhou, China 4 Department of English, Zunyi Medical University, Zhuhai, Guangdong, China
J. Sep. Sci. 2015, 38, 2320–2326
Short Communication
Separation and purification of five alkaloids from Aconitum duclouxii by counter-current chromatography C19 -diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter-current chromatography. In the process of separation, the critical conditions of counter-current chromatography were optimized. The two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1, 24.1 mg of 2, 250.6 mg of 3, 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra-high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation. Keywords: Aconitum duclouxii L / C19 -diterpenoid alkaloids / Counter-current chromatography DOI 10.1002/jssc.201500059
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Received January 16, 2015 Revised March 27, 2015 Accepted April 12, 2015
1 Introduction Aconitum L. (Ranunculacea) is a large genus comprising 400 species and widely distributed in the temperate region of the northern hemisphere. This genus is well known for toxicity and is usually regarded as medicinal plants. In China, there are about 76 Aconitum species, and some of them have been used for the treatment of rheumatoid arthritis, arrhythmia, pains, and various cancers [1, 2]. Characteristic compounds of these plants are the diterpenoid alkaloids [3]. Diterpenoid alkaloids, classified as the C18 -, C19 -, and C20 -subtypes, have been reported to be the main bioactive constituents of plants from aconitum genus. The important pharmacological activities and structural complexity of these diterpenoid alkaloids have long aroused scientists’ strong interest in the
Correspondence: Prof. Yang Chen, Department of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai Campus, Zhuhai, Guangdong 519041, China E-mail: [email protected] Fax: +86-756-7637616 C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
investigation of phytochemistry, synthesis, and medicinal chemistry [4]. Aconitum duclouxii Levl (Baicaowu in Chinese), a perennial herb distributed in Dali Bai Autonomous Prefecture, Yunnan Province of China, has long been used as a folk medicine to treat rheumatism and pains [5]. Previous investigations on this herb led to the isolation of six new C19 -diterpenoid alkaloids and various known alkaloids [6–9]. Because of their complex structures, pharmacological activities [10, 11], interesting chemical reactions [12], and economic importance [13], the C19 -diterpenoid alkaloids have aroused scientists’ considerable interest. However, these studies, which adopted classical methods including silica gel, SiO2 and Sephadex LH-20 in the preparative separation and purification of alkaloids from this herb, are proved to be tedious and time consuming. They required multiple chromatographic steps and thus resulted in lower recovery and higher cost. Consequently, it is important to develop an efficient method to separate and purify main C19 -diterpenoid alkaloids from the roots of this herb. CCC (Counter-current chromatography) was first introduced by Ito in the late 1960s [14]. Since then, it has ∗ Additional corresponding author: Lecturer Liang Deng. E-mail: [email protected]
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Figure 1. Chemical structures of target alkaloids obtained from Aconitum duclouxii.
been widely used in the field of natural product chemistry to effectively separate a large variety of compounds. The technique, an all-liquid method without solid phases, relies on the partition of a sample between two immiscible solvents to achieve separation. The relative proportions of the solute that dissolves in two phases are determined by the respective distribution ratios (K). It has great advantages over the traditional liquid–solid separation methods. It can eliminate the complications resulting from the solid support matrix, such as irreversible adsorptive sample loss and deactivation, tailing of solute peaks, and contamination [15]. CCC has the unique features of high recovery, high efficiency, and ease of scale-up. Thus it has been widely used in the separation and purification of various natural and synthetic products [16–19]. To the best of our knowledge, many studies have been published, reporting the successful use of CCC for the separation and purification of C20 - and C18 -diterpenoid alkaloids from genus aconitum [20–26]. More recently, three C19 -diterpenoid alkaloids were isolated from the lateral roots of Aconitum carmichaeli Debx by CCC [27]. However, there is no article gthus far reporting the separation of C19 -diterpenoid alkaloids from A. duclouxii by CCC. This paper, for the first time, describes the successful preparative separation and purification of five C19 -diterpenoid alkaloids, namely, benzoylaconine (1), N-deethylaconitine (2), aconitine (3), deoxyaconitine (4), and ducloudine A (5), from A. duclouxii by CCC. Among these components, ducloudine A is a novel C19 -diterpenoid alkaloid recently obtained from the roots of A. duclouxii [6]. The chemical structures of the five alkaloids are shown in Fig. 1.
2 Materials and methods 2.1 Materials and chemicals Acetonitrile (Sigma, USA) was of chromatographic purity and pure water (Watson, China) was used for UHPLC. All reagents and solvents, used for preparation of crude extract, UHPLC, and CCC separation, were of analytical grade (Fuyu Fine Chemical Reagent, Tianjin, China). The roots C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
of A. duclouxii were collected from Dali Bai Autonomous Prefecture, Yunnan Provinces of China, in December 2012. The samples were identified by Professor Shu-Gang Lu from the School of Life Sciences, Yunnan University, Kunming, China. The voucher specimens are deposited at the Department of Pharmaceutical Sciences, Zunyi Medical University, Zhuhai Campus, China. 2.2 Preparation of total alkaloids The air-dried and powdered roots (0.5 kg) of A. duclouxii Levl were percolated with 0.5% HCl (2 L).The aqueous acidic solution was basified with 10% aqueous NH3 ·H2 O to pH 9– 10 and then extracted with ethyl acetate (3 × 2.5 L). Removal of the solvent under reduced pressure afforded a total of 5.5 g of crude alkaloids as a yellowish amorphous powder, which was then stored in a refrigerator (4⬚C) for CCC separation. 2.3 Selection of the solvent system The suitable solvent systems were evaluated by UHPLC according to the partition coefficients (0.5 < K < 2.0) in a series of solvent systems as follows: about 1.0 mg sample was added to the test tubes, and then 2 mL of each phase of a preequilibrated two-phase solvent system was added and thoroughly mixed. Each test tube was rigorously shaken for several minutes and left to stand at room temperature until equilibrium was attained. Then 3 L of the upper and lower phases were analyzed by UHPLC at 233 nm. The partition coefficient (K) is defined as Aupper /Alower , where Aupper and Alower were the UHPLC peak areas of target compounds in the upper and lower phases, respectively.
2.4 CCC separation 2.4.1 CCC apparatus The preparative CCC was performed using a model TBE1000A CCC (Shanghai Tauto Biotechnique, Shanghai, www.jss-journal.com
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Figure 2. UHPLC chromatograms of total alkaloids extract from Aconitum duclouxii sample. Column: Symmetry 1 C18 (50 × 2.1 mm id, 1.8 m); A (acetonitrile/tetrahydrofuran, 25:15) and B (0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid) in a gradient mode 16–19% A (0–5.0 min), 19–25% A (5.0–15.0 min), 25– 35% A (15.0–20.0 min), 35– 16% A (20.0–24.0 min), 16% A (24.0–25.0 min); column temperature: 30⬚C; flow rate: 0.3 mL/min; detection wavelength: 233 nm. Table 1. The K (partition coefficients) values of the compounds 1–5 in different solvent systems
Solvent systems
Ethyl acetate/n-butanol/water
n-Hexane/ethyl acetate/methanol/water
n-Hexane/ethyl acetate/methanol/water/ NH3 ·H2 O (25%)
Volume ratio
K values
(v/v)
1
2
3
4
5
4:1:5 4:1.5:5 4:2:5 1:1:1:1 1.5:0.5:1:0.5 1.5:0.2:1:0.2 1:1:1:1:0.05 1:1:1:1:0.1 1:1:1:1:0.2
2.15 2.02 1.76 1.08 0.56 0.37
2.36 2.34 2.01 1.54 0.97 0.55
2.78 2.49 1.89 1.97 1.36 0.76
3.18 2.59 1.78 2.89 1.71 0.99
3.05 2.78 1.98 3.54 2.16 1.13
China). The apparatus consisted of an upright coil type-J planet centrifuge with three multilayered coils (Each coil in this configuration had a beta range of 0.6–0.78) connected in series (diameter of tube, 3.0 mm, total capacity 1000 mL) and a 80 mL manual sample loop. The rotation speed was adjustable, ranging from 0 to 600 rpm. The CCC system was equipped with a TBP-5002 pump, a TBD-2000 D-UV detector, a DC-0506 constant temperature regulator (Shanghai Tauto Biotechnique, Shanghai, China), which was used to control the separation temperature. The outflow of CCC was detected by a Model 2000D-UV monitor (Sanotac Scientific Instruments, Shanghai, China). The data were collected with a Sanotac chromatography workstation. In the separation process, the temperature of separation columns was maintained at 25⬚C, and the effluents were monitored at 233 nm for C19 -diterpenoid alkaloids of A. duclouxii. 2.4.2 Preparation of solvent system and sample solution The solvent system composed of n-hexane/ethyl acetate/ methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v) was used for CCC separation in one run. The preparation of each C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
two-phase solvent system was performed in a separation funnel according to the volume ratios and thoroughly equilibrated after shaking at room temperature. The upper phase and lower phase were then separated and degassed by ultrasonication for 30 min shortly before use. The sample solution of for CCC separation was prepared by dissolving 1000 mg of total alkaloids extract in 20 mL lower phase of n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v). 2.4.3 CCC separation procedure The CCC was performed as follows in the whole separation: the column was first entirely filled with the upper phase as the stationary phase; subsequently, the lower mobile phase was then pumped into the inlet of the column as the mobile phase at the flow rate of 8.0 mL/min, while the apparatus was run at 600 rpm; after a clear mobile phase eluted at the tail outlet and the hydrodynamic equilibrium was reached, samples were then injected into the injection valve; the effluent from the outlet of the column was continuously monitored with a UV detector at 233 nm for C19 -diterpenoid alkaloid from the roots of A. duclouxii. The peak fractions were collected manually according to the chromatographic profile; after www.jss-journal.com
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Figure 3. Chromatogram of total alkaloids extract from Aconitum duclouxii by preparative CCC. Solvent system: n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1, v/v). The detection wavelength of chromatograms was 233 nm. The sample weight was 1000 mg. Retention of stationary phase was 42% and the rotation speed was 600 rpm. The temperature of separation columns was maintained at 25⬚C and the flow rate of mobile phase was 8.0 mL/min.
target compounds were eluted, the centrifuge was stopped and stationary phase was pumped out from the column with pressured air and collected in a graduated cylinder to measure the retention volume.
2.5 UHPLC Analysis The UHPLC equipment was an ACQUITY UPLC H Class system, equipped with a QSM quaternary solvent manager, a UPL PDA detector, a CHA column thermostat, a SDI sample manager, a RP Symmetry1 C18 (2.1 mm × 100 mm T3 1.8 m, Milford, MA, USA) column from Phenomenex (Torrance, California, USA), and an Empower 3 UPLC workstation (Waters, USA). The mobile phase was consisted of A (acetonitrile/tetrahydrofuran, 25:15) and B (0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid), which was programmed as follows: from 0 to 5 min, 16–19% A, 5–15.0 min, linear increase from 19–25% A. 15.0–20.0 min, linear increase from 25–35% A. 20.0–24.0 min, linear decrease from 35–16% A. 24.0–25.0 min, isocratic for 16% A. The flow rate was 0.3 mL/min while spectra were recorded from 200 to 500 nm. The pooled fraction was concentrated by a rotary evaporator and each fraction was analyzed by analytical UHPLC to check the purity before characterization. The purities of the collected fractions were determined by UHPLC based on the peak area of the target species normalized to the sum of all observed peaks.
2.6 Structure elucidation The target peak fractions obtained by CCC were identified by their UV, high-resolution ESI-MS and NMR spectra. The MS analysis of purified alkaloids were performed by ESI-Q-TOF mass spectrometer (Agilent 6530 Series, Agilent C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Technologies, Santa Clara, CA, USA) in a positive ionization mode. Parameters for the Agilent Jet Stream Technology are the superheated nitrogen sheath gas temperature (350⬚C) and flow rate (12 L/min). Electrospray conditions were the following: capillary: 4000 V; nebulizer: 20 psi; drying gas: 10 L/min; gas temperature: 350⬚C; skimmer voltage: 60 V; octapole RFPeak: 750 V; fragmentor: 135 V. The data recorded was processed with the Mass profiler (Agilent, Santa Clara, CA, USA) with accurate mass application specific additions from Agilent MSD TOF software. A second orthogonal sprayer with a reference solution was used as a continuous calibration using the following reference masses: 121.0509 and 922.0098 m/z. Spectra were acquired over the m/z 50–1000 range at a scan rate of 1 s per spectrum. NMR spectra were recorded on Bruker AM-400 spectrometers (Bruker, Karlsruhe, Germany) using TMS as the internal reference.
3 Results and discussion 3.1 Optimization of UHPLC analysis The whole separation of all target compounds is a crucial and challenging task for LC analysis. In present work, a UHPLC– DAD mode for the analysis of Aconitum L was established. In the course of optimizing the conditions of separation, the system conditions were investigated, including the mobile phase system (acetonitrile/water and methanol/water with different concentrations of tetrahydrofuran, ammonium acetate, and acetic acid), gradient program (gradient time, gradient shape, and initial composition of the mobile phase), and column temperature. Acetonitrile/tetrahydrofuran, 25:15 (A) and 0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid (B) was used as the mobile phase in gradient mode as mentioned in Section 2.5. The flow rate was 0.3 mL/min while www.jss-journal.com
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3.2 Selection of two-phase solvent system and other conditions of CCC
Figure 4. UHPLC chromatograms of peak fractions obtained by CCC preparation. Column: Symmetry 1 C18 (50 × 2.1 mm id, 1.8 m); A (acetonitrile/tetrahydrofuran, 25:15) and B (0.1 mol/L ammonium acetate with 0.07 mol/L acetic acid) in the gradient mode (see Fig. 2 caption); column temperature: 30⬚C; flow rate: 0.3 mL/min; detection wavelength: 233 nm.
spectra were recorded from 200 to 500 nm. Under this condition, all alkaloids could reach almost baseline separation, and a chromatogram is shown in Fig. 2.
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Successful separation by CCC depends upon the selection of a suitable two-phase solvent system, which provides an ideal range of the partition coefficient (K) for the target compounds [28]. If the K value is much smaller than 1, the solutes will be eluted close to each other near the solvent front, which may result in loss of peak resolution; on the contrary if the K value is much greater than 1, the solutes will be eluted in excessively broad peaks, and may lead to extended elution time [29]. When the K value of the target compound is between 0.4 and 2.5, the solvent system is suitable for CCC separation [30]. In the present investigation, the K values of alkaloids 1–5 partitioned between the upper and lower phases of a series of solvent system were evaluated with UHPLC (Table 1). At first, the two-phase solvent system, ethyl acetate/n-butanol/water with different volume ratios, was carefully screened to optimize the solvent system for CCC separation and the K values of the target alkaloids were measured. The results indicated that the K values of the five target compounds, which were mostly distributed in the upper phase, are too large in the two-phase solvent system (Table 1). When the search changed to n-hexane/ethyl acetate/methanol/water, K values of five chemicals decreased significantly but remained too large (Table 1). Therefore, NH3 ·H2 O (25%) was added to modify the K values of five components. When the NH3 ·H2 O fraction increased, the K values of all target alkaloids decreased. Among the solvent systems with different concentration of the modifier, n-hexane/ethyl acetate/methanol/water/NH3 ·H2 O (25%) (1:1:1:1:0.1) was proved to achieve suitable K values (1, K = 0.56; 2, K = 0.97; 3, K = 1.36; 4, K = 1.71; 3, K = 2.16), which meets the K value requirements and means the five components could be well separated. Adding NH3· H2 O as a modifier to the solvent system makes target alkaloids much better soluble in the lower phase if compared with those without NH3 ·H2 O. This condition was further evidenced by analytical CCC to be satisfactory for separation of all target compounds from the total alkaloids of Aconitum L (data not shown). Other factors, such as the flow rate of the mobile phase, the temperature and the revolution speed of the separation column, were also investigated. As expected, the flow rate affected the stationary phase retention as well as separation time and the peak resolution. To shorten the separation time while still maintaining an adequate resolution, the flow rate of mobile phase of preparative CCC was selected at 8.0 mL/min for separation (retention of stationary phase was 42%). The results of CCC separations also indicated that increasing the temperature could improve the CCC separation, but a high temperature may run the risk of stripping away the stationary phase due to the emulsification of the solvent system, so 25⬚C was selected for separation. In this work, separations of preparative CCC were performed at 600 rpm.
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Figure 5. Positive ESI-Q-TOF-MS analyses of purified alkaloids from Aconitum duclouxii.
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3.3 Preparative CCC separation As shown in Fig. 3, under the optimum CCC condition, satisfactory effect of separation was achieved and five pure alkaloids were obtained by CCC separation of 1000 mg total alkaloids extract of Aconitum L., namely, alkaloids 1 (148.2 mg, collected during 81–96 min), 2 (24.1 mg, collected during 103–110 min), 3 (250.6 mg, collected during 114–144 min), 4 (73.9 mg, collected during 158–177 min), and 5 (31.4 mg, collected during184–200 min). In addition, the purities of all target compounds 1–5 were 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by UHPLC peak area percentage (Fig. 4). Moreover, the chemical structures of the prepared compounds are shown in Fig. 1 and details are listed in the Supporting Information. The results of current study clearly demonstrated that CCC could provide highly efficient preparative separation of alkaloids from Aconitum L.
4 Conclusions A novel method for systematic separation and purification of C19 -diterpenoid alkaloids from the roots of A. duclouxii has been described in this research. The results of our study clearly demonstrated that CCC could provide highly efficient preparative separation of main alkaloids from A. duclouxii. Generally, five alkaloids were obtained from the crude extract of A. duclouxii. Their purities were 96.6–98.4%. The presented separation and purification method was simple, fast, and efficient, and the obtained high pure alkaloids are suitable for biochemical and toxicological investigation. The authors acknowledge the financial support from the Project Natural Science Foundation of China (No. 21162046), the Project of High Level Talents of Guizhou Province (TZJF2010-062) and the Project of Guizhou Provincial Administration of Traditional Chinese Medicine (2012-GZYY20). We would like to show our sincere gratitude to Le Cai for his help in structure identification. In addition, our cordial thanks go to all of our colleagues for their excellent assistance.
The authors have declared no conflict of interest.
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[5] People’s Government of Dali Bai Autonomous Prefecture, The Natural Resources of Chinese Medicinal Materials of Dali, Yunnan Ethnic Publishing House, Kunming 1991, p. 77. [6] Yin, T. P., Cai, L., Lei, G., Dong, J. W., Liu, Y. X., Ding, Z. T., Chin. J. Org. Chem. 2013, 33, 2528–2532. [7] Yin, T. P., Cai, L., Lei, G., He, J. M., Dong, J. W., Fang, H. X., Zhou, H., Ding, Z. T., J. Asian. Nat. Prod. Res. 2014, 16, 345–350. [8] Yin, T. P., Cai, L., Zhou, H., Zhu, X. F., Chen, Y., Ding, Z. T., Nat. Prod. Res. 2014, 28, 1649–1654. [9] Wang, C. Y., Chen, J. B., Zhu, R. H., Acta. Pharm. Sin. 1984, 19, 445. [10] Dzhakhagirov, F. N., Sultankhodzhaev, M. N., Tashkhodzhaev, B., Silmov, B. T., Khim. Prir. Soedin. 1997, 33, 254–270. [11] Pelletier, S. W., The Alkaloids: Chemical and Biological Perspectives, Springer Verlag Press, New York, 1983, pp. 120–153. [12] Wang, F. P., Liang, X. T., The Alkaloids: Chemistry and Pharmacology, Academic Press, New York, 1992, pp. 151–247. [13] Panter, K. E., Manners, G. D., Stegelmeier, B. L., Gardner, D. R., Ralphs, M. H., Pfister, J. A., James, L. F., Biochem. Syst. Ecol. 2002, 30, 113–128. [14] Ito, Y., Weinstein, M., Aoki, I., Harada, R., Kimura, E., Nunogaki, K., Nature 1966, 212, 985–987. [15] Jubert, C., Baiiley, G., J. Chromatogr. A 2007, 1140, 95– 100. [16] Cao, X. L., Ito, Y., J. Chromatogr. A 2003, 1021, 117–124. [17] Du, Q. Z., Li, L., Jerz, G., J. Chromatogr. A 2005, 1077, 98–101. [18] Sun, A. L., Sun, Q. H., Liu, R. M., J. Sep. Sci. 2007, 30, 1013–1018. [19] Xu, H. N., He, C. H., Sep. Purif. Technol. 2007, 56, 85–89. [20] Tang, Q., Yang, C., Ye, W., Liu, J., Zhao, S., Phytochem. Anal. 2008, 19, 155–159. [21] Wang, X., Shu, X., Wang, X., Yu, J., Jing, F., Molecules 2014, 19, 12619–12629. [22] Yang, F., Ito, Y., J. Chromatogr. A 2002, 943, 219–225. [23] Tang, Q., Yang, C., Ye, W., Liu, J., Zhao, S., J. Chromatogr. A 2007, 1144, 203–207. [24] Yang, F., Ito, Y., J. Chromatogr. A 2002, 923, 281–285. [25] Liu, Y., Chen, T., Chen, C., Zou, D., Li, Y., Chin. J. Chromatogr. 2014, 32, 543–546.
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