G Model

ARTICLE IN PRESS

CHROMA-356039; No. of Pages 8

Journal of Chromatography A, xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn夽 Jing-Lan Liu, Xin-Yuan Wang, Ling-Ling Zhang, Mei-Juan Fang, Yun-Long Wu, Zhen Wu, Ying-Kun Qiu ∗ School of Pharmaceutical Sciences, Xiamen University, South Xiang-An Road, Xiamen 361102, China

a r t i c l e

i n f o

Article history: Received 21 September 2014 Received in revised form 19 November 2014 Accepted 19 November 2014 Available online xxx Keywords: 2D CCC × LC Stop-and-go Heart-cutting Peucedanum praeruptorum Coumarins

a b s t r a c t Pure compounds isolated from complex natural plants are important for drug discovery. This study describes a novel two-dimensional hyphenation of counter-current chromatography and highperformance liquid chromatography (2D CCC × HPLC) with heart-cutting and stop-and-go techniques for preparative isolation of multiple targets components from Peucedanum praeruptorum Dunn (Umbelliferae) crude extracts in a single step. The CCC and HPLC were hyphenated via a 4-port valve equipped at the post-end of the CCC column, to heart cut the impure fractions to the 2nd dimensional HPLC for further separation. Furthermore, the stop-and-go flow scheme was applied in the 1st dimensional CCC to fit with the time constraints of the 2nd dimensional preparative HPLC. Last but not least, an optimal biphasic solvent system composed of n-heptane/acetone/water (31:50:19, v/v/v) with suitable Kd values and a higher retention of the stationary phase was chosen to separate target compounds, resulting in the improvement of the CCC column efficiency. By taking the advantages of this rationally designed system, sixteen coumarins were isolated from 1.0 g of P. praeruptorum crude extract, with HPLC purity from 90.1% to 99.5%, in a single 2D separation run. More interestingly, two minor linear coumarins and one angular coumarin were isolated from P. praeruptorum Dunn for the first time. As far as we known, this is the first report on the combination of heart-cutting technique and stop-and-go protocol in 2D CCC × HPLC system, by which good separations on comprehensive matrix were achieved. We expect that this approach may have broad applications for simultaneous isolation and purification of multiple components from other complex plant-derived natural products. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Pure compounds from medicinal plants have served as an important source of drugs for combating diseases since ancient times and are playing an increasing crucial roles in clinical therapy because of their high pharmacological activity and low toxicity [1,2]. Due to its complex matrix, it remains a challenge to isolate and purify target compounds in rapid, efficient and economical manner for accelerating the processes of drug discovery.

夽 Presented at the 8th International Conference on Countercurrent Chromatography – CCC 2014, 23–25 July 2014, Uxbridge, United Kingdom. ∗ Corresponding author. Tel.: +86 592 2189868; fax: +86 592 2189868. E-mail address: [email protected] (Y.-K. Qiu).

As a typical example, Baihuaqianhu, the dried roots of Peucedanum praeruptorum Dunn (Umbelliferae), is recognized as one of the most important traditional medicines and has been compiled in the Chinese Pharmacopeia [3]. Its major constituents are coumarins, such as praeruptorin A, praeruptorin B, Pd-Ib, qianhucoumarin J, and d-laserpitin, some of which have been reported to show certain effects on reducing blood pressure, anti-leukemia, anti-cancer, reducing platelet aggregation and thrombus [4,5]. The traditional method for separation of coumarins from P. praeruptorum Dunn is silica gel column and reversed-phase ODS column chromatography, which is time-consuming and results in loss of many potentially interesting compounds, due to the adsorptive effect of the solid matrix [6]. In order to eliminate irreversible absorptive loss of samples onto the solid support matrix in conventional chromatography, countercurrent chromatography (CCC), a unique liquid–liquid partition

http://dx.doi.org/10.1016/j.chroma.2014.11.053 0021-9673/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model CHROMA-356039; No. of Pages 8 2

ARTICLE IN PRESS J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

chromatographic method with a support-free liquid stationary phase, has been developed. It relies simply on the partition of a sample between the two phases of an immiscible solvent system. In addition, crude plant extracts can be chromatographed with sample loads ranging from milligrams to grams, with relatively high purity in a single CCC separation [7,8]. Successful applications of CCC have also been reported to separate and purify plant-derived natural products [9]. Recent reports have also revealed that many natural products could be isolated by flow-rate gradient elution CCC or stepwise solvent gradient CCC [10–12]. On the other hand, the recently developed high performance countercurrent chromatography (HPCCC), whose separation times are minutes rather than hours, has shown powerful separation ability [13]. The preparative HPLC, although being an effective means for separation of complicated constituents with a broad range of polarity from medicinal plants, is difficult to isolate highly lipophilic samples dissolved in organic solvents, because the solvent effects exhibit negative impact on HPLC resolution and eventually result in the failure of preparative isolation. The CCC techniques, in which samples are dissolved in two-phase solvent for injection, have been successfully and widely applied in separations of all sorts of components including lipophilic compounds [14]. Two-dimensional liquid chromatography (2D-LC), which is one of the most common multi-dimensional liquid chromatography (MDLC) and provides for higher resolution of complex samples and larger peak capacity than a single LC process, has been recently developed to largely improve separation capacity and sample recovery rate. The advantage of this 2D-LC system locates on the orthogonal selectivity furnished by two different columns. Depending upon whether all parts of a sample are transferred to the second dimension, 2D-LC methods can be classified as comprehensive (all components are transferred) or heart-cutting (specific parts of the sample are transferred) [15]. In addition, three available schemes may be implemented when coupling separations in LC × LC, namely on-line, off-line and stop-flow (also known as stop-and-go) modes and all of them have been successfully utilized to separate the target constituents of plant-derived natural products [16–18]. Recently, several different kinds of coumarins have been successfully separated from P. praeruptorumc by using CCC [6,19]. Similar report by coupling CCC and prep-HPLC in off-line mode has also successfully seperated d-laserpitin from P. praeruptorum [20]. In addition, combination of CCC with ESI-MS resulted in the isolation of seven coumarins from P. praeruptorum [21]. The light petroleum–ethyl acetate–methanol–water solvent system was applied in all of these CCC separations. However, even the gradient elution CCC technique was applied, only a few compounds could be isolated in a single separation process, with a relative long separation time ranging from 450 to 600 min. Hence, a rapid and on-line method to achieve one-step separation of target compounds from crude plant extract was highly desirable. In order to address this issue, we have recently developed a novel comprehensive 2D CCC × HPLC system in on-line mode to preparative isolate toad venom (Bufo bufo gargarizans) [14], where first dimensional CCC column was interfaced with the second dimensional preparative HPLC column by using a makeup pump and a 2-position 10-port switching valve in conjunction with two reversed phase holding columns. This streamlined design of preparative 2D CCC × HPLC has great potentials for the fast and highly efficient preparation and purification of organic compounds from complex nature products. In the current study, a novel on-line 2D CCC × LC system with heart-cutting and stop-and-go techniques, which is different from our previous 2D CCC × LC working in the comprehensive and flowprogramming mode, has been developed for ultra-fast and effective separation of multiple target components from a crude herbal extract in a single step. The reason of applying heart-cutting mode is because it is less time-consuming than comprehensive mode,

especially in the case of preparative scale purification. Furthermore, the stop-and-go flow scheme [17] was employed in the CCC separation to increase residence time, which was designed to fit the preparative time of the second dimension and provided good chances for separating more comprehensive components. To be highlighted here, the stop-and-go CCC × HPLC provides an attractive alternative to off-line separation as it offers automation, minimal sample handling and avoids sample exposure between the two dimensions. To the best of our knowledge, it is the first report for the application of heart-cutting and stop-and-go techniques in the 2D CCC × HPLC system for the successful online isolation and purification of target coumarins from P. praeruptorum crude extracts.

2. Experimental 2.1. Chemicals and materials All solvents used for the preparation of crude extracts and CCC separations were of analytical grade (Jinan Reagent Factory, Jinan, China). HPLC grade solvents for HPLC were purchased from Merck, Darmstadt, Germany and all water was redistilled. The dried root of P. praeruptorum Dunn were purchased from Hangzhou Traditional Chinese Medicine Decoction Pieces Factory (Hangzhou, China) and identified by Professor Quan-Cheng, Chen (Xiamen University, Xiamen, China). A voucher specimen (2013102401-QH) has been deposited at School of Pharmaceutical Sciences, Xiamen University. 2.2. Instrumentation All instruments used in this study are commercially available. The CCC instrument employed in the present study was a TBE-300 high-speed counter-current chromatograph (Tauto Biotechnique, Shanghai, China) with three multilayer coil separation columns connected in series (i.d. of the tubing = 1.5 mm, total volume = 300 ml) and a 20 ml sample loop. The revolution radius was 5 cm, and the ˇ values (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) of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal. The revolution speed of the apparatus can be regulated with a speed controller in the range between 0 and 1000 rpm. The system was also equipped with a Büchi C-601 pump, a C-615 pump controller (Büchi Labortechnik AG, Flawil, Switzerland), and a ProStar 218 photodiode array detector (Varian Inc. Corporate, Santa Clara, USA). The data were collected with a Varian Star Workstation 6.41 (Varian Inc. Corporate, Santa Clara, USA). The LC system using a Varian binary gradient LC system (Varian Inc. Corporate, Santa Clara, USA) containing two solvent deliver modules (PrepStar 218), a photodiode array detector (ProStar 335) and a fraction collector (ProStar 701). Preparative-HPLC control and data acquisition were also performed by Varian Star Workstation. An Agilent 1100 system HPLC has been used for the analysis of the crude extract and the isolated fractions. It was equipped with a G1379A degasser, a G1311A QuatPump, a G1367A Wpals, a G1315B diode assay detector (DAD), and an Agilent ChemStation for LC. 2.3. Preparation of P. praeruptorum extract The 1.0 kg dried roots of P. praeruptorum were extracted with 5 L of ethanol twice and concentrated under reduced pressure at 40 ◦ C to rendering 148.2 g residue. The residue was stored in a refrigerator (5 ◦ C) for further use.

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model

ARTICLE IN PRESS

CHROMA-356039; No. of Pages 8

J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

3

Fig. 1. Scheme for a CCC × preparative HPLC system with heart-cutting and stop-and-go techniques.

2.4. Preparation of two-phase solvent system and sample solutions In this study, three wildely used biphasic solvent systems were selected and evaluated according to the Kd values for CCC separation, and they were: Arizona system composed of n-heptane/ethyl acetate/methanol/water [22], the expanded Arizona system composed of n-heptane/acetonitrile/methyl tert-butyl ether (MTBE)/water [23] and the acetone scale composed of nheptane/methyl isobutyl ketone (MIBK)/acetone/water [22]. They were prepared by adding the solvents to a separation funnel according to the volume ratios and mixed thoroughly. After equilibrium was established, the upper phase and the lower phase were separated and degassed by sonication for 30 min shortly prior to use. The sample solution for CCC separation was prepared by dissolving 0.5 or 1.0 g of P. praeruptorum crude extract into 2 ml of the biphasic solvent systems (1:1, v/v) before use.

2.5. Distribution ratios of target components in different two-phase solvent systems The two-phase solvent system was selected according to the distribution ratio (Kd) of each target component, whose value was determined by HPLC analysis. A suitable amount of crude extract was dissolved in the lower phase of a two-phase solvent system. The solution was analyzed by HPLC. The peak area was recorded as A1 . Then equal volume of the upper phase was added to the solution and the two phases were mixed thoroughly. After partition equilibration, the lower phase solution was determined by HPLC again, and the peak area was recorded as A2 . The Kd value was calculated according to the following equation: Kd = (A1 − A2 )/A2 .

2.6. Classical CCC separation procedure CCC separation was performed as follows: the multilayer coiled column was first entirely filled with the upper phase as stationary phase. Afterward, the apparatus was rotated at 900 rpm and the mobile lower phase was pumped into the head end of the column at a flow-rate of 2 ml min−1 . When the solvent front of mobile phase appeared in outlet pipe, which means a hydrodynamic equilibrium was established in the column. The sample extract solution was injected into the column through the sample valve. The effluent was continuously monitored by the Varian DAD detector at 350 nm.

2.7. Interface between CCC and preparative HPLC A 2-position 4-port switching valve (model EDU4UW, VICI, Schenkon, Switzerland) was equipped at the post-end of the CCC to discard the effluent without targeted components until the first CCC peak emerged. On the other hand, fractions with high purity were also collected through the 4-port valve based on its previous analytical data. By switching the 4-port valve, other impure fractions was shunted and transported to a dynamic mixer (ChuangXinTongHeng Science and Technology, Beijing, China), to which a medium pressure pump, used for the addition of water as makeup fluid to dilute the elution from 1st dimensional CCC, was connected. Then the tandem CCC dilution and HPLC columns were interfaced by two equivalent holding columns (15 mm × 30 mm i.d.) and an electronically controlled Valco Cheminert 2-position 10-port switching valve (model EDU10UW, VICI, Schenkon, Switzerland) that make up an integrated online column switching CCC × preparative HPLC system. In order that the dilution actually induced a good accumulation (peak concentration) on the solid-phase cartridge entrance, the trapped analytes were washed out of the holding column in the back-flush mode (Fig. 1). 2.8. 2D CCC × LC separation procedures The present 2D CCC × LC system configuration was shown as Fig. 1. Several time dependent system configurations had been designed according to the elution sequence of targeted components. A representative CCC × LC had been performed as following: CCC pumps delivered the desired upper or lower phase of selected biphasic solvent system for the first dimensional CCC separation. When the first interested CCC peak containing the first targeted component emerged, the 4-port valve was switched and the effluent was firstly diluted at a certain ratio with water and subjected to the first holding column (15 mm × 30 mm i.d.). After all effluents of the first impure fraction had been eluted and adsorbed on the first holding column, the 2-position 10-port switching valve was switched and HPLC pumps began to deliver mobile phase solution to desorb and isolate the first targeted peak into its constituent components as the start of a preparation HPLC process. Meanwhile, when all the effluent of the second mixture fraction was trapped on the second holding column, the CCC was kept on the same rotation but the CCC pump was stop according to the program in Fig. 2, in order that the second interested CCC peak width could fit with the demand of the first interested CCC peak’s HPLC separation. Once the first isolation had finished, the 2-position 10-port switching valve

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model CHROMA-356039; No. of Pages 8

ARTICLE IN PRESS J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

4

Fig. 2. CCC for the separation of water extract from Peucedanum praeruptorum Dunn. (a) The classical CCC purification and (b) the stop-and-go CCC profile. (i) CCC conditions: (a, b) CCC conditions: solvent system: n-heptane/acetone/water (31:50:19, v/v/v); stationary phase: upper phase; mobile phase: lower phase; column temperature: 30 ◦ C; rotational speed: 900 rpm; initial stationary phase retention: 78%; detection wavelength: 350 nm; injection mode: injection after equilibrium; injection volume: 2 ml; flowrate: 2.0 ml min−1 ; elution mode: head to tail. (ii) Sample weight: (a) 0.5 and 1.0 g; (b) 1.0 g. (iii) Valve switching and CCC pump ON/OFF program for the stop-and-go CCC profile: The 2-position 4-port switching valve (Valve A) position A: where the waste go to discard and the pure CCC fractions go to collect (Fr.5, Fr.7 and Fr.8); Valve A position B: where the entire CCC mixture fractions go to the two equivalent holding columns conjunction with preparative HPLC; The 2 position 10-port switching valve (Valve B) alternates the enrichment or HPLC isolation status of the two holding columns. After the Fr. 2, 3 and 4 were eluted completely from the 1st dimensional column, the CCC pump was kept stopping until the 2nd dimensional HPLC isolation of previous fraction completed.

was switched again and the CCC pumps were turned on again for eluting the other components successively, as the second mixture fraction trapped on the second holding column was isolating on the HPLC column. As for the other pure fractions (Fr.5, Fr.7, and Fr.8), whose purities were confirmed in preliminary CCC separation and HPLC analysis data, they were collected to test tubes by switching the 4-port valve at the post-end of CCC. A reversed-phase preparative column (COSMOSIL MSII C18 column, 250 mm × 20.0 mm i.d.) was used as the 2nd dimensional stationary phase. The mobile phase was methanol (A) and water (B) in a linear gradient mode and programmed as shown in Fig. 3, which was optimized according to each fraction. The flowrate of the mobile phase was 8.0 ml min−1 . All effluents through the HPLC column were monitored by a DAD detector at 321 nm and automatically collected into test tubes. The preparative HPLC chromatograms were recorded and cascaded by Varian Star Workstation v6.41 as shown in Fig. 3.

mode as follows: A from 40% to 100% and B from 60% to 0% during 0–30 min. The flow-rate of the mobile phase was 1.0 ml min−1 the effluents were monitored at 321 nm by a DAD detector (Fig. 4).

2.9. Qualitative and quantitative HPLC analysis

Strategy of solvent system selection is a key issue for CCC separation, which is exclusively based on the partition of a target compound in two immiscible liquid phases. The solvent system composed of light petroleum/ethyl acetate/methanol/water used for the CCC separation of P. praeruptorum was previously reported [6,19–21]. However, the selectivity and resolution of CCC

The total extract and purified fragments isolated by the 2D CCC × HPLC were analyzed with a COSMOSIL MSII C18 column (250 mm × 4.6 mm i.d., 5 ␮m) at room temperature. The mobile phase used was methanol (A) and water (B) in a linear gradient

2.10. Structural identification Identification of the compounds was carried out by electrospray ionization mass spectrometry (ESI-MS), one dimensional (1D) and two dimensional (2D) nuclear magnetic resonance spectra. Positive ESI-MS were measured on a Thermo Q Exactive LC–MS/MS spectrometer. NMR experiments were carried out using a Bruker Avance III 600 FT-NMR spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard. 3. Results and discussion 3.1. Selection of solvent system

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model CHROMA-356039; No. of Pages 8

ARTICLE IN PRESS J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

5

Fig. 3. Integrated CCC × reverse phase preparative HPLC separation of P. praeruptorum crude extracts with heart-cutting and stop-and-go technique. (i). The same CCC protocol as that of Fig. 2(b) was applied in the 1st dimensional CCC isolation. (ii). The makeup pump was kept turned on at flow rate of ml min−1 after the first CCC peak emerged, in order that the CCC eluent was diluted by water at ratio of 1:3. (iii). Condition of 2nd dimensional HPLC: Mobile phase system was methanol (A) and water (B) in a linear gradient mode. Fr. 1: A:B from 40:60 to 60:40 for 35 min and 60:40 to 55:45 kept until 40 min; Fr.2: A:B from 55:45 to 75:25 for 35 min and run back to 65:35 at 40 min; Fr.3: A:B from 65:35 to 100:0 for 30 min and run to 70:30 at 35 min; Fr.4: A:B from 70:30 to 80:20 for 30 min and run to 75:25 at 35 min. Fr.6: A:B from 75:25 to 80:20 for 30 min and 75:25 to 100:0 kept until 40 min. The flow-rate of the mobile phase was 8.0 ml min−1 (iv). Sample weight of isolated compounds: 3.0 mg (1), 2.7 mg (2), 3.3 mg (3), 2.4 mg (4), 2.6 mg (5), 2.8 mg (6), 2.6 mg (7), 3.5 mg (8), 2.7 mg (9), 158.6 mg (10), 56.8 mg (11), 3.3 mg (12), 4.0 mg (13), 5.2 mg (14), 3.6 mg (15) and 26.7 mg (16).

separation may be improved, if the solvent system was further comprehensively evaluated by considering the Kd values, stationary phase fraction (Sf), as well as the intermolecular properties of the solvents. Thus, optimization of the biphasic solvent systems was the first step before coupling the CCC column with HPLC. In the present study, three practical scales of biphasic solvent systems were selected for evaluation, and they were: Arizona system [22], expanded Arizona system [23] and the acetone solvent system [22]. It is important to note that each solvent system has a different influence on the solute–solvent interaction due to the difference in dipole moment, polarizability, hydrogen bond and proton force. For example, methanol used in the Arizona solvent system is a protic solvent, which works as a proton donor or a hydrogen bond donor (HBD). The introduction of acetonitrile in the expanded Arizona system is due to its weak proton acceptor character and the ability to serve a dipolar aprotic solvent with a dipole moment of 3.92D [23], which is much bigger than acetone (2.91D) and methanol (1.69D). With respect to the acetone solvent system, acetone is taken into account because it serves as a medium polar, aprotic solvent which acts only as a hydrogen bond acceptor. In accordance with the classification of the Fuzzy group analysis method [24], acetone also exerts electron acceptor capacity, while acetonitrile is an electron donor solvent. These three different solvent systems were prepared by adding the corresponding solvents into different volume ratios for further investigation. 3.2. Evaluation of solvent system based on distribution ratio (Kd) A satisfying CCC separation is determined by the distribution ratio (Kd) of target components, that is a key index used to evaluate whether a solvent system is suitable for separation. Generally,

the Kd value of the target compounds should lie in the range from 0.5 to 2, which is the optimal range for significantly enhanced CCC separation efficiency. The Kd values of the target compounds in previously mentioned three biphasic solvent systems were carefully tested by using the simple test tube method (Table 1). The results showed that neither the Arizona system nor the expanded Arizona system could achieve satisfactory Kd values for all of the target compounds (6–16), while the acetone solvent system works well. For example, in solvent systems S1 and S3, the target compounds exhibited much higher Kd values, which were deemed unsuitable for CCC separation. Whereas the Kd values for compounds 6 and 7 in S2 and S4 systems were not higher than 0.2, which would lead to separation failure for these two compounds. As for the acetone solvent systems S5 and S6, most compounds showed moderate Kd values. Especially for the solvent system S5, the Kd values of most target compounds were close to 1, which implied good separation efficiency with high resolution. Therefore, we chose n-heptane/acetone/water (31:50:19, v/v/v) as solvent system for CCC separation, which provided optimum Kd values. Furthermore, this solvent system was simple and the two phases would reach equilibrium very quickly, which produced a good preliminary separation of P. praeruptorum. It should be pointed out that the acetone solvent system was excellent in terms of the orthogonality between the 2D CCC and HPLC system. As shown in Table 1, compound 10 and 13 showed similar Kd values that were difficult to separate by the 1st dimensional CCC, while their retention time (tR ) were different in the HPLC chromatogram. On the contrary, the retention time of compound 10 and 11 were close but exhibited difference in Kd values, indicating that good separation could be achieved in the CCC rather than HPLC. Similar observations of a few compound

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model

ARTICLE IN PRESS

CHROMA-356039; No. of Pages 8

J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

6

Fig. 4. HPLC analysis of the crude extracts and isolated fractions by CCC × HPLC system with heart-cutting and stop-and-go techniques. The analysis was performed on a COSMOSIL MSII C18 column (250 mm × 4.6 mm i.d., 5 ␮m) with a guard column (10 mm × 4.6 mm i.d., 5 ␮m). The mobile phase was methanol (A) and water (B) in a linear gradient mode as follows: A from 40% to 100% and B from 60% to 0% during 0–30 min. The flow rate of the mobile phase was 1.0 ml min−1 and the effluents were monitored at 321 nm by a DAD detector. The column temperature was kept at 30 ◦ C. Compound purity: 94.5% (1), 97.1% (2), 96.1% (3), 96.5% (4), 97.7% (5), 98.3% (6), 96.4% (7), 95.6% (8), 93.8% (9), 90.1% (10), 97.5% (11), 99.4% (12), 98.1%(13), 99.2 (14), 95.7% (15) and 98.1% (16). The relative contents in percentage were calculated with area normalization method.

pairs (i.e. compounds 8 and 9, as well as compounds 11 and 12) with identical HPLC retention time but different Kd values were found, indicating that these pairs could be easily separated by 1st dimensional CCC, but difficult to be distinguished by single HPLC. To be mentioned, their actual Kd values could only be obtained after separation by this 2D CCC × HPLC system. In short, after careful investigation, a biphasic CCC solvent system composed of acetone/n-heptane/water (50:30:19, v/v/v),

which showed moderate Kd values for most of the target compounds, was selected for further 2D CCC × HPLC separation. 3.3. Classical CCC separation In the CCC separation techniques, the volume of the stationary phase retained in the column (stationary phase fraction, Sf) is an essential parameter that directly influences the separation quality,

Table 1 Kd values and HPLC retention time (tR ) of the main components in various solvent systems. Solvent systemsa

No.

Ratio

Kd valueb 6

H:E:M:W H:N:T:W H:A:K:W HPLC tR (min) a b c d

S1 S2 S3 S4 S5d S6

6:5:6:5 2:1:2:1 2:5:2:5 1:2:1:2 31:50:0:19 28:50:1:21

7

8c

9c

10

11c

12c

13

14

15

16

0.41 0.10 0.30 0.12 0.25 0.33

0.76 0.18 0.57 0.20 0.38 0.56

1.21 0.29 1.12 0.42 0.50 0.76

1.69 0.40 1.48 0.55 0.66 0.97

2.40 0.53 2.16 0.83 0.90 1.32

2.06 0.49 1.92 0.72 0.79 1.12

2.49 0.56 2.22 0.88 0.93 1.38

2.80 0.62 2.66 1.26 1.10 1.75

3.80 0.75 3.10 1.51 1.23 2.08

5.62 1.06 4.58 2.39 1.67 2.70

8.30 1.56 7.06 3.20 2.78 4.00

13.52

16.30

21.09

21.09

23.69

23.94

23.94

24.90

25.56

27.27

28.38

H: n-heptane; E: ethyl acetate; M: methanol; W: water; N: acetonitrile; T: MTBE; A: acetone; K: MIBK. The Kd values of compounds 1–5 were too small to be listed here, owing to their high hydrophilicity. The Kd values of compounds 8, 9, 11 and 12 were measured by their pure products, instead of crude extract. The selected CCC solvent system with optimum Kd values.

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model CHROMA-356039; No. of Pages 8

ARTICLE IN PRESS J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

which plays a key role on the number of theoretical plates and resolution of CCC column. In addition, the peak resolution was influenced by mass load, since the hydrodynamic equilibrium between the immiscible phases could be strongly affected by overloaded sample, which would result in a drastic loss of stationary phase and a significant decrease in process capacity. Using the optimized solvent system composed of nheptane/acetone/water (31:50:19, v/v/v), with the upper phase as stationary phase and lower one as mobile phase, the classical CCC separations had been performed as Fig. 2(a). Experimental result showed that chromatographic performance was almost stable with little stationary phase loss. In detail, with the initial stationary phase retention of about 76–78%, the final stationary phase retention was 72–75% in the case of 0.5 g sample loading and 65–70% in the case of 1.0 g sample loading on the CCC column. In the operating condition of 2.0 ml min−1 flow rate at 900 rpm, all fractions could be well resolved even when 1.0 g of sample was loaded. However, there was still a restriction of 1.0 g on the CCC sample loading amount, in order to obtain good 2nd dimensional HPLC resolution. In this sample loading range, the CCC chromatogram showed only slight peak broadening and acceptable full elution time (about 250 min) by using the selected acetone solvent system. 3.4. On-line stop-and-go and heart-cutting CCC scheme Experimental results showed that the second-dimensional chromatography required a certain period of time for preparative isolation in the 2D CCC × HPLC system. Therefore, the stop-and-go technique was employed into this system to solve this operation time limitation especially in the cases of multi-target components. When the stop-and-go scheme was applied according to the program in Fig. 2(b), the elution time from the 1st dimensional CCC chromatography could be easily controlled during the transferring and separation process in the 2nd dimensional HPLC. The preparative time of each fraction in the 2nd dimension was tuned to 30 or 40 min, which shortened the CCC pump parking time to less than 30 min and avoided 1st dimensional CCC peak’s band broadening. Moreover, we employed heart-cutting mode instead of comprehensive mode in this 2D CCC × HPLC system, thanks to the high purity of selected CCC fractions (≥95%) according to HPLC analysis. In order to work at heart-cutting mode, the pure fractions were collected through the 2-position 4-port valve (Valve A), equipped at the post-end of CCC column. In this case, the impure fractions from 1st dimensional CCC column could also be transferred to 2nd dimensional reversed-phases C18 column for subsequent separation and purification. It is worth mentioning that the overall separation time was greatly shortened by using heart-cutting technique. In summary, with combination of stop-and-go and heart-cutting scheme, the coupling of CCC and HPLC is convenient and efficient, with a full experimental period of less than 330 min in a single run. 3.5. Integrated CCC × reverse phase HPLC separation of P. praeruptorum The integrated CCC × HPLC separation of the P. praeruptorum crude extracts is illustrated as Fig. 3. In the 1st dimensional CCC, with biphasic solvent system composed of n-heptane/acetone/water (31:50:19, v/v/v), three fractions (Fr. 5, Fr. 7 and Fr. 8) were well-separated with purity higher than 95% and they were directly collected to test tube through the 2-position 4-port valve (Valve A) at position B. By switching Valve A to position A, the other five impure fractions were online heartcut, concentrated, and introduced to two identical solid-phases holding columns, controlled by the 2-position 10-port valve (Valve

7

B). Higher performance purification was then conducted in the second dimensional preparative HPLC. The two valves (Valve A, B), as well as the CCC pump, were controlled by the program shown in Fig. 2(b), according to the HPLC preparative time and purity of each CCC fraction. As a result, more interested effluents could be isolated in a single step, with greatly improved separation efficiency. After less than 330 min separation, 8 fractions were directly produced according to the heart-cutting and stop-and-go CCC × HPLC elution profile, from 1.0 g of crude extract using the proposed column-switching CCC × HPLC protocol. More interestingly, further HPLC analysis showed that 16 compounds, with high purity of 90.1–99.5%, were purified in a single-step separation (Fig. 4). To the best of our knowledge, this is the first report on the combination of heart-cutting technique and stop-flow protocol in the 2D CCC × HPLC system to achieve one-step separation and purification of multiple target compounds from natural products. 3.6. Identification of isolated compounds The chemical structures of those isolated compounds with purity higher than 90% were identified as praeroside III (1) [25], umbelliferone (2) [26], rutaretin (3) [27], (−)-cis-khellactone (4) [27], nodakenetin (5) [19,28], bergapten (6) [28,29], xanthotoxin (7) [29], Pd-Ib (8) [21], d-laserpitin (9) [20], nodakenetin tiglate (10) [30], praeruptorin A (11) [21], decursinol angelate (12) [28], corymbocoumarin (13) [31], qianhucoumarin J (14) [20], praeruptorin B (15) [21] and praeruptorin E (16) [21], by ESI-MS and 1 H, 13 C NMR data (Supporting Information), which were consistent with the authentic samples or literature data. Among the isolated compounds, two minor linear coumarins, nodakenetin tiglate (10) and decursinol angelate (12); one angle coumarin named as corymbocoumarin (13), were firstly isolated from P. praeruptorum. 4. Conclusions In this work, a novel on-line CCC × HPLC system with heartcutting and stop-and-go techniques has been designed and implemented to enhance capability and resolving power for the separation and purification of P. praeruptorum. The system integrates CCC and HPLC into a single automated run to perform the purification of target compounds from the complex mixture, without intermediate steps of sample preparation. By using the stop-and-go scheme, the CCC elution program fitted the demand of the 2nd dimensional HPLC isolation well. Application of the heart-cutting technique, which only subjected to the impure CCC fractions for further separation, greatly reduced the total operation time to less than 330 min. The present 2D CCC × HPLC system combination of the heart-cutting technique and stop-and-go protocol has successfully accomplished the goal of one-step separation of 16 compounds from P. praeruptorum ethanol extract with high purity and recovery rate in an economic and time efficient manner. It is worth mentioning that more than 4 compounds were successfully further isolated in the 2nd dimensional chromatography from two CCC fractions (Fr. 1 and Fr. 6) respectively, by taking advantage of our rationally built CCC × LC scheme with an optimal solvent system. While two pairs of compounds in identical HPLC retention time, compounds 8 and 9, as well as compounds 11 and 12, were separated by 1st dimensional CCC into different fractions. The result indicates the peak capacity improvement of this 2D CCC × HPLC system, when compared with the single CCC or HPLC column. The above results clearly show that this novel 2D CCC × HPLC system with heart-cutting and stop-and-go protocols is an effective and high-yield technique, which has potential applications

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053

G Model CHROMA-356039; No. of Pages 8

ARTICLE IN PRESS J.-L. Liu et al. / J. Chromatogr. A xxx (2014) xxx–xxx

8

in the preparative extraction and purification of multiple target components from complex natural products. We envisaged that this approach could be useful for not only P. praeruptorum, but also many other multi-component plant-derived natural products, which might contribute a lot to drug discovery. Acknowledgments The project was supported by the National Natural Science Foundation of China (Nos. 81102333 and 81273400), as well as Fujian Natural Science Foundation for Distinguished Young Scholars (No. 2012J06020). The authors would also like to acknowledge the financial supports from the Science and Technology Program of Xiamen, Fujian Province, China (No. 3502Z20143001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chroma.2014. 11.053. References [1] F.E. Koehn, G.T. Carter, The evolving role of natural products in drug discovery, Nat. Rev. Drug. Discov. 4 (2005) 206–220. [2] I. Paterson, E.A. Anderson, The renaissance of natural products as drug candidates, Science 310 (2005) 451–453. [3] Chinese Pharmacopoeia Committee, in: Pharmacopoeia of the People’s Republic of China, The Medicine Science and Technology Press of China, Beijing, 2010. [4] S.G. Zhou, H.Q. Huang, M.R. Rao, Effect of Praeruptorin C on cardiac remodelling, cardiac function in two-kidney two-clip renovascular hypertensive rats, Acta Pharm. Sin. 22 (2006) 543–547. [5] M.R. Rao, L. Sun, X.W. Zhang, Effect of praeruptorum coumarin on heart hemodynamics, myocardial compliance and collagen content in heart hypertrophy rats, Chin. J. Pharmacol. Toxicol. 16 (2002) 265–269. [6] R.M. Liu, L. Feng, A.L. Sun, L.Y. Kong, Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed countercurrent chromatography, J. Chromatogr. A 1057 (2004) 89–94. [7] O. Sticher, Natural product isolation, Nat. Prod. Rep. 25 (2008) 517–554. [8] Y.J. Pan, Y.B. Lu, Recent progress in countercurrent chromatography, J. Liq. Chromatogr. Relat. Technol. 30 (2007) 649–679. [9] I.A. Sutherland, D. Fisher, Role of counter-current chromatography in the modernisation of Chinese herbal medicines, J. Chromatogr. A 1216 (2009) 740–753. [10] S.C. He, S.C. Li, J.H. Yang, H.Y. Ye, S.J. Zhong, H. Song, Y.K. Zhang, C. Peng, A.H. Peng, L.J. Chen, Application of step-wise gradient high-performance countercurrent chromatography for rapid preparative separation and purification of diterpene components from Pseudolarix kaempferi Gordon, J. Chromatogr. A 1235 (2012) 34–38. [11] O. Shehzad, S. Khan, I.J. Ha, Y. Park, A. Tosun, Y.S. Kim, Application of stepwise gradients in counter-current chromatography: a rapid and economical strategy for the one-step separation of eight coumarins from Seseli resinosum, J. Chromatogr. A 1310 (2013) 66–73.

[12] S. Ignatova, N. Sumner, N. Colclough, I. Sutherland, Gradient elution in countercurrent chromatography: a new layout for an old path, J. Chromatogr. A 1218 (2011) 6053–6060. [13] C. DeAmicis, N.A. Edwards, M.B. Giles, G.H. Harris, P. Hewitson, L. Janaway, S. Ignatova, Comparison of preparative reversed phase liquid chromatography and countercurrent chromatography for the kilogram scale purification of crude spinetoram insecticide, J. Chromatogr. A 1218 (2011) 6122–6127. [14] Y.K. Qiu, X. Yan, M.J. Fang, L. Chen, Z. Wu, Y.F. Zhao, Two-dimensional countercurrent chromatography × high performance liquid chromatography for preparative isolation of toad venom, J. Chromatogr. A 1331 (2014) 80–89. [15] Q. Ren, C.S. Wu, J.L. Zhang, Use of on-line stop-flow heart-cutting twodimensional high performance liquid chromatography for simultaneous determination of 12 major constituents in tartary buckwheat (Fagopyrum tataricum Gaertn), J. Chromatogr. A 1304 (2013) 257–262. [16] G. Guiochon, N. Marchetti, K. Mriziq, R.A. Shalliker, Implementations of twodimensional liquid chromatography, J. Chromatogr. A 1189 (2008) 109–168. [17] J.N. Fairchild, K. Horvath, G. Guiochon, Approaches to comprehensive multidimensional liquid chromatography systems, J. Chromatogr. A 1216 (2009) 1363–1371. [18] Y. Wei, W.W. Huang, Y.X. Gu, Online isolation and purification of four phthalide compounds from Chuanxiong rhizoma using high-speed counter-current chromatography coupled with semi-preparative liquid chromatography, J. Chromatogr. A 1284 (2013) 53–58. [19] R.M. Liu, Q.H. Sun, Y.R. Shi, L.Y. Kong, Isolation and purification of cournarin compounds from the root of Peucedanum decursivum (Miq.) Maxim by high-speed counter-current chromatography, J. Chromatogr. A 1076 (2005) 127–132. [20] Z.G. Hou, J.G. Luo, J.S. Wang, L.Y. Kong, Separation of minor coumarins from Peucedanum praeruptorum using HSCCC and preparative HPLC guided by HPLC/MS, Sep. Purif. Technol. 75 (2010) 132–137. [21] Z.G. Hou, D.R. Xu, S. Yao, J.G. Luo, L.Y. Kong, An application of high-speed counter-current chromatography coupled with electrospray ionization mass spectrometry for separation and online identification of coumarins from Peucedanum praeruptorum Dunn, J. Chromatogr. B 877 (2009) 2571–2578. [22] J.H. Renault, J.M. Nuzillard, O. Intes, A. Maciuk, Comprehensive Analytical Chemistry: Countercurrent Chromatography, Elsevier, Amsterdam, 2002. [23] A.P. Foucault, L. Chevolot, Counter-current chromatography: instrumentation, solvent selection and some recent applications to natural product purification, J. Chromatogr. A 808 (1998) 3–22. [24] M. Vitha, P.W. Carr, The chemical interpretation and practice of linear solvation energy relationships in chromatography, J. Chromatogr. A 1126 (2006) 143–194. [25] M. Hisamoto, H. Kikuzaki, H. Ohigashi, N. Nakatani, Antioxidant compounds from the leaves of Peucedanum japonicum Thunb, J. Agric. Food Chem. 51 (2003) 5255–5261. [26] S.S. Sankar, R.D. Gilbert, R.E. Fornes, C-13 Nmr-studies of some hydroxycoumarins and related-compounds, Org. Magn. Reson. 19 (1982) 222–224. [27] J. Reisch, A.A.W. Voerste, Natural product chemistry. 181. Investigations on the synthesis of dihydropyrano-coumarins and dihydrofurano-coumarins by application of catalytic enantioselective cis-dihydroxylation, J. Chem. Soc. Perkin Trans. 1 (1994) 3251–3256. [28] S.Y. Lee, D.S. Shin, J.S. Kim, K.B. Oh, S.S. Kang, Antibacterial coumarins from Angelica gigas roots, Arch. Pharm. Res. 26 (2003) 449–452. [29] S.W. Yoo, J.S. Kim, S.S. Kang, K.H. Son, H.W. Chang, H.P. Kim, K. Bae, C.O. Lee, Constituents of the fruits and leaves of Euodia daniellii, Arch. Pharm. Res. 25 (2002) 824–830. [30] S. Chacko, O.V. Singh, M.G. Sethuraman, V. George, Coumarins from Heracleum candolleanum, Indian Drugs 38 (2001) 594–596. [31] A. Tosun, N. Ozkal, M. Baba, T. Okuyama, Pyranocoumarins from Seseli gummiferum subsp corymbosum growing in Turkey, Turk. J. Chem. 29 (2005) 327–334.

Please cite this article in press as: J.-L. Liu, et al., Two-dimensional countercurrent chromatography × high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.11.053