Isolation of chemical compounds from Sorbus tianschanica Rupr by high-speed counter-current chromatography

Sorbus tianschanica Rupr has been used as a pharmacologically valuable plant in Xinjiang to treat tuberculosis, asthma, cough and gastritis for a long time. Four compounds including three flavonoids and chlorogenic acid were isolated and purified by high-speed counter-current chromatography (HSCCC) from S. tianschanica Rupr. The two-phase solvent system composed of n-hexane ethyl acetate n-butanol water (2:6:1:8, v/v). Consequently, chlorogenic acid (1), Quercetin-3-O-(6''-Omalonyl)-β-D-glucoside (2), Kaempferol-3-O-(6''-O-malonyl)-β-D-glucopyranoside (3) and Kaempferol-3O-β-D-glucopyranoside (4) were obtained from 250 mg crude extracts with purities of 93, 91, 89 and 96%, respectively in a single run. The chemical structures were confirmed by mass spectrometer (MS), 1 H nuclear magnetic resonance (NMR) and 13 C NMR. To our knowledge, compounds (2) and (3) were obtained from S. tianschanica Rupr for the first time.


INTRODUCTION
Sorbus tianschanica Rupr, a member of family Rosaceae, is distributed in mountainous regions of Central Asia and west of China.It has been widely used as a pharmacologically valuable plant in Xinjiang to treat tuberculosis, asthma, cough and gastritis (Zhang et al., 1973).Pharmacological research indicates that crude extracts of flavonoids from S. tianschanica exhibits a wide range of biological activities (Yu et al., 2004), such as against myocardial ischemia reperfusion injury in rats (Fu et al., 2010), anti-myocardial ischemia (Zhang et al., 2009) and antitussive.It is reported that major constituents in S. tianschanica are phenolic acids, aglycones, flavonoids, which include quercetin, tutin, hyperin and flavonols (Yu et al., 2004;Li et al., 2010).However, few new constituents in this plant have been *Corresponding author.E-mail: yu5406@hotmail.com.described in recent years.Considering such bioactive compounds of flavonoids from S. tianschanica, it is prerequisite to develop an efficient method of isolating each compound with high purity for further pharmacological research and quality control.
Although column chromatography and preparative liquid chromatography (LC) are widely used in isolation and purification of flavonoids (Rijke et al., 2006;Salib et al., 2006;Gil-Izquierdo et al., 2001), the methods are time-consuming and sample-losing during the separation and purification processes.Thus, a more efficient strategy is required in order to obtain compounds of low content in original plant with high sample recovery.Highspeed counter-current chromatography (HSCCC), a type of liquid-liquid partition chromatography, has great merits of higher yield and recovery in extracting target compounds than those of conventional column chromatography (Ito, 2005).Separation process of HSCCC is based on the composition of two solvent system, providing flexibility in choosing separation solvents for different target compounds.HSCCC has great advantages of 100% sample recovery, simplicity to scale up and high yield of sample.Therefore, this technique has been extensively applied for separation and purification of natural products (Ito et al., 2011;Lee et al., 2011;Shi et al., 2011;Hu et al., 2008), especially for flavonoids (Liu et al., 2005;Yang et al., 2009;Wu et al., 2009;Wei et al., 2011).
The study has succeeded in developing an efficient approach for isolation and purification of four bioactive compounds from S. tianschanica by HSCCC in one single run.Their chemical structures have been confirmed by high performance liquid chromatographymass spectrometer (HPLC-MS),

MATERIALS AND METHODS
The HSCCC instrument was TBE-300B (Tauto Biotechnique Company, Shanghai, China) equipped with a 280 ml coil column (i.d. of the tubing 1.9 mm) and a 20 ml sample loop.The revolution speed could be adjusted from 0 to 1000 rpm.TBP 5002 constant flow pump (Tauto Biotechnique Company, Shanghai, China), a Model ultraviolet (UV) 20 detector was used to record absorption (Tianzhao, Hangzhou, China).
Acetonitrile applied for HPLC were chromatographic grade (Fisher Scientific, USA), and water was distilled water.All organic solvents were analytical grade (GuangFu, Tianjin, China).AB-8 macroporous resin (Nankai University, Tianjin, China) were employed for crude extracts preparation.

Preparation of crude extracts
ST leaves (1 kg) were extracted twice with 60% ethanol by ultrasonic means.The extracts were mixed together and concentrated to remove ethanol.After filtrated with cotton, the filtrate was loaded onto AB-8 macroporous resin, and then washed successively with water and 60% ethanol, respectively.The eluant of 60% ethanol was concentrated by using a rotary evaporator and then a freeze-dryer to give dried crude powder (74 g).

Selection of two-phase solvent system
The two-phase solvent system was selected according to the partition coefficient (K) of each target component.Acceptable K of the target compounds should be in the range 0.2 to 5.0 (Ito, 2005).Yu et al. 5143 The K-values were determined by HPLC as follows: Two-phase solvent system was prepared by mixing various solvent thoroughly in a separatory funnel and left for a period at room temperature.
Then equal volume of the upper and the lower phase were taken out and mixed again, afterwards, 120 mg crude extracts were added into the two-phase system.Then both the upper and the lower phase solution were detected by HPLC.The obtained peak area of the upper phase (A1) and the lower phase (A2).K-values according to the equation: K=A1/A2.

Preparation of two-phase solvent system and sample solution
The two-phase solvent system composed of n-hexane -ethyl acetate -n-butanol -water (2:6:1:8, v/v) was used for HSCCC separation.When all the solvent mixed thoroughly, the two-phases were separated and degassed for 30 min before using.
The sample solution for HSCCC separation was prepared by dissolving 250 mg crude extracts in 20 ml lower phase solvent.

HSCCC separation procedure
The upper phase was used as the stationary phase; the lower phase was used as the mobile phase.Firstly, the column was fully filled with the upper phase, and then the HSCCC rotated at 800 rpm, while the lower phase was pumped into the column at a flow rate of 2.0 ml min -1 .When the hydrodynamic equilibrium, which can be directly recognized by observing the record from the workstation, was established in the column, 20 ml of the sample solution containing 250 mg of the crude extract was injected into the column.The temperature was controlled at 25°C.The effluent was continuously monitored with a UV detector at 254 nm.Peak fractions were collected according to the UV chromatogram.

HPLC analysis and identification of HSCCC peak fractions
The crude extracts and each peak fraction were obtained by HSCCC were analyzed by HPLC with a Waters C18 column (150 mm  4.6 mm i.d. 5 μm).The mobile phase was acetonitrile (A) and 0.4% phosphoric acid solution (B) in gradient mode as follows: 0 to 10 min, 15 to 22% (A), 10 to 20 min, 22 to 30% (A), flow rate 1.0 ml min -1 .The effluent was monitored at 254 nm, 25°C.Purities of each peak fraction were given by HPLC.The chromatograms of crude extracts and peak fractions separated by HSCCC are shown in Figure 1.

Optimization of two-phase solvent system and other conditions of HSCCC
The two-phase solvent system was selected by the Kvalues of each target compound.Table 1 shows the Kvalues, which were also determined by HPLC or tested in HSCCC.The results indicated that when chloroformmethanol -water (4:3:2, v/v) was used as the two-phase solvent, K-values of each target compound were too low to suit each compound to be separated.The system consisting of ethyl acetate -n-butanol -water (3:1:3, v/v) (Zhou et al., 2005) was then tested and the results displayed K-values were too high for each compound.Thus, it took a great of time for separation, and the retention percentage of the stationary phase was low.
Then optimization was done at the volume ratio of (4:1:6, v/v), and got better results.However, since the fact that the volume ratio of n-butanol was still large resulted in high viscosity of solvent system, the obtained retention percentage of the stationary phase was too low.K-values was acceptable with ethyl acetate -n-butanol -water (6:1:8, v/v) as solvent system, but two compounds of the extract cannot be fully separated, giving purities both lower than 65%.When n-hexane -ethyl acetate -nbutanol -water (1:6:1:8, v/v) and (1.5:6:1:8, v/v) was used, separation of four compounds can be realized but required quite a long time.When n-hexane -ethyl acetate -n-butanol -water (2:6:1:8, v/v) was applied, the four compounds were well separated, and the time was acceptable.The purities of each target compound were higher than 89%.Thereby, n-hexane -ethyl acetate -nbutanol -water (2:6:1:8, v/v) was used as the two-phase solvent system of HSCCC in the following study.Other factors, for instance, the flow rate of mobile phase, the temperature and the revolution speed of separation process, were also considered.Increasing flow rate of mobile phase will reduce the time of separation, while it will sacrifice retention percentage of the stationary phase and resolution of each compound.In the study, we used flow rate 2.0 ml min -1 , a good resolution can be obtained with retention percentage of the stationary phase 63.5%.For the Peak V, since it is the last one in the chromatogram, the flow rate was increased from 2.0 to 3.5 ml min -1 after 300 min.This compound can be collected with high purity in less time as well.Theoretically, rise in temperature could improve the HSCCC separation, but in practice we found it displayed poor effect, so 25°C was selected for separation.The experiment was performed at the revolution speed of 800 rpm.
By using this method, we can not only get at least four compounds in one time, but obtaining sufficient amounts ready for further scientific study can be also expected, which is hardly to realize with column chromatography and preparative HPLC, wherein repeating work is needed to get the comparable quantity.These compounds will be employed for further pharmacological studies or as reference substances.
Up to now, there are no previous scientific literature reports of using HSCCC for isolation and purification flavonoids from S. tianschanica.As far as we know, two compounds obtained in this research, quercetin-3-O-(6''-O-malonyl)-β-D-glucoside and kaempferol-3-O-(6''-O-malonyl)-β-D-glucopyranoside are for the first time extracted from the plant-S.tianschanica.

Figure 1 .
Figure 1.HPLC chromatogram of crude extract and HSCCC peak fractions.(A), Crude extract from S. tianschanica Rupr; (B-E), HPLC analyses of the purified HSCCC peak fractions I-III and V.

Table 1 .
The K-values of target components measured in several solvent systems.