Isolation and Purification of Sesquiterpene Lactones from Ixeris sonchifolia (Bunge) Hance by High-Speed Counter- Current Chromatography and Semi-Preparative High Performance Liquid Chromatography

Purpose: To isolate and purify sesquiterpene lactones from Ixeris sonchifolia (Bunge) Hance by highspeed counter-current chromatography (HSCCC). Methods: I. sonchifolia was extracted with water and then loaded on a glass column (10 × 1500 cm containing 3000g D101 macroporous resin) where various concentrations of aqueous ethanol (0, 10, 30, 50, and 95 %) were used to elute the column successively. The 50 % ethanol fraction was purified by HSCCC using a solvent system comprised of ethyl acetate: n-butanol: methanol: water (4: 6: 1: 20, v/v), and semi-preparative high performance liquid chromatography (HPLC). The chemical structures of the components obtained were further confirmed by high-resolution mass spectroscopy (MS) and nuclear magnetic resonance spectroscopy (NMR). Results: Three compounds, including ixerin Z1 (0.7 mg), ixerin Z (11.4 mg), and 11, 13α-dihydroixerin Z (8.2 mg), with purity of 96.2, 98.2, and 98.4 %, respectively, were obtained from 200 mg each of the 50 % ethanol fraction. Conclusion: HSCCC is a rapid and effective method for isolating and purifying sesquiterpene lactones from I. sonchifolia.


INTRODUCTION
Ixeris sonchifolia (Bunge) Hance, commonly called kudiezi in China, which belongs to the family Compositae, is mainly distributed in Northeastern areas of China [1]. It is widely used as a folk medicine in China for its remarkable medical effects, such as dissipating blood stasis, improving microcirculation, and antineoplastic activity [2][3][4][5].
The chemical compounds of this plant are sesquiterpene lactones, organic acids, flavonoids, and triterpenes [6][7][8][9]. Ixerin Z and 11,13α-dihydroixerin Z are the major sesquiterpene lactones [10], which have been reported to have various activities for instance, anti-inflammatory, anti-microbial, and anti-tumor activities [11,12]. In order to gain enough reference substance to perform further investigation on its metabolism, it is important to use an efficient method to isolate and purify ixerin Z and 11,13 α-dihydroixerin Z from their natural sources.
Some conventional techniques including silica gel column chromatography have been successfully used to separate these chemicals from this plant [10]. However, these classic chromatography methods are complicated, inefficient, and time-consuming. Thus, an efficient and low-cost technique to obtain these compounds is required.
In the past few years, high-speed counter-current chromatography (HSCCC), a support-free liquidliquid partition chromatographic technique, has been reported to eliminate irreversible adsorption of sample onto solid support matrix, stationaryphase deactivation, and contamination, and has made great progress in preparative isolation and purification of active components from natural products [13][14][15]. Therefore, our aim is to establish a protocol for enriching and isolating sesquiterpene lactones effectively from I. sonchifolia by HSCCC and to provide enough reference substance for in vitro and in vivo studies.

EXPERIMENTAL Apparatus
The instrument used was a TBE-300B HSCCC (Tauto Biotech, Shanghai, China) equipped with three multi-layer coil separation columns connected in series (total volume of 300 mL) and a 20 mL sample loop. The β value of the preparative column varied from 0.5 at the internal layer to 0.8 at the external layer. The revolution speed of the apparatus was regulated with a speed controller in the range from 0 to 1000 rpm. The HSCCC system was equipped with a TBP-1002 pump (Aglient, Santa Clara, USA), an HX-1050 constant temperature regulator (Beijing Boyikang Lab Implement, Beijing, China), a TBD-2000 UV detector (Aglient, Santa Clara, USA) operating at 254 nm.
The analytical TBE-20A HSCCC instrument (Tauto Biotech, Shanghai, China) used was equipped with three polytetrafluoroethylene preparative coils (diameter of tube, 0.8 mm; total volume, 16 mL). The β value varied from 0.6 at the internal terminal to 0.78 at the external terminal.
HPLC analysis was performed on an Agilent system equipped with ultraviolet detector (Agilent, Santa Clara, USA). The MS analyses were performed with a LTQ-Orbitrap coupled with an ESI source (Thermo Electron, Bremen, Germany). The structures of the compounds were identified by Bruker Avance 500 NMR (Bruker Biospin, Rheinstetten, Germany).

Reagents and materials
I. sonchifolia was collected from Haicheng city, Liaoning province of China in September 2013. It was authenticated by Professor Jian-Qiu Lu, Center of Scientific Experiment, Beijing University of Chinese Medicine. A voucher specimen (no. 20130901) was deposited in the same department. All organic solvents used for HSCCC separation were of analytical grade (Beijing chemicals works, Beijing, China). Acetonitrile used for HPLC was of HPLC grade (Fisher, NJ, USA), and water was prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA) in our laboratory. D101 macroporous resin was purchased from the Chemical Plant of Nankai University (Tianjin, China).

Preparation of crude extract
The whole herb of I. sonchifolia (2 kg) was chopped and extracted with 12 L pure water by refluxing for 1 h, which was repeated three times. The extract was loaded on a glass column (10 × 1500 cm) containing 3000 g D101 macroporous resin. Various concentrations of aqueous ethanol (0, 10, 30, 50, and 95 %) were used to elute the column successively. The 50 % ethanol fraction was concentrated under reduced pressure to achieve a brown residue.

HSCCC separation and semi-preparative HPLC purification procedures
During HSCCC separation, a two-phase solvent system of ethyl acetate n-butanol methanol water (4 : 6 : 1 : 20, v/v) was chosen to separate the compounds. The multilayer coil column was first completely filled with the upper phase as the stationary phase, and the lower phase was then pumped into the head end of the inlet column at a flow rate of 2.0 mL/min, while the apparatus was rotated at 900 rpm. After hydrodynamic equilibrium was established throughout the coil, as indicated by a clear mobile phase eluting at the tail outlet, the sample solution (200 mg of the 50 % ethanol fraction in solvent composed of 10 mL the lower and 10 mL upper phases) was injected into the column through the sample loop. During the separation process, the column temperature was set at 30 o C. The detection wavelength was 254 nm. The effluent was continuously monitored and the fractions were collected according to the chromatograms obtained.

HPLC analysis and identification of compounds
The 50 % ethanol fraction and each peak fraction from HSCCC were all analyzed by HPLC using a Zorbax SB-C18 column (

Under the HSCCC conditions, fractions A and B
were separated from sample in a single HSCCC step (shown in Figure 1) within 240 min. The purities of compound 1 in fractions A was 79.5 % based on HPLC peak area percentage. After crystallization with dichloromethane, compound 1 with higher purity was obtained from fractions A. Fraction B was a mixture of two compounds, which was further purified on semi-preparative HPLC using acetonitrile water (17:83, v/v) at 2.0 mL/min to yield compounds 2 and 3.
The chemical structures of compounds separated by HSCCC were identified according to the HRMS, H-NMR, and C-NMR data. Their chemical structures are displayed in Figure 2. The quantity and purity of the three compounds are shown in Table 1.

DISCUSSION
An HPLC method for the determination of the purities of sesquiterpene lactones separated by HSCCC was established at first. In order to obtain an appropriate elution system, mobile systems such as methanol water and acetonitrile water were tested. The result demonstrated that acetonitrile water with a gradient elution mode could afford better resolution of chromatographic peaks among the targets and the other compounds.
The important step in the successful separation of HSCCC is the selection of suitable two-phase solvent system [19]. In our experiments, nbutanol methanol water at different volume ratios were first selected as solvent system [20], and tested by analytical HSCCC. The analytes were eluted close to the solvent front with poor separation, whereas an addition of ethyl acetate could yield a suitable separation time and a much better resolution.
Due to the similar structures and largely polarities of ixerin Z and 11,13α-dihydroixerin Z, it is difficult to separate them from complex system in a single HSCCC procedure with these two-phase solvent systems [21]. Thus semipreparative HPLC was chosen to isolate and purify the mixture of ixerin Z and 11, 13αdihydroixerin Z.

CONCLUSION
A rapid and effective method for the isolation and purification of the major sesquiterpene lactones from I. sonchifolia, which could provide reference substances for further metabolism studies, is reported in this work. To the best of our knowledge, this is the first report on separation of sesquiterpene lactones in medical plants using HSCCC method.