A Strategy for Preparative Separation of 10 Lignans from Justicia procumbens L. by High-Speed Counter-Current Chromatography

Ten compounds, including three lignan glycosides and seven lignans, were purified from Justicia procumbens L. in 8 h using an efficient strategy based on high-speed counter-current chromatography (HSCCC). The two-phase solvent system composed of petroleum–ethyl acetate–methanol–H2O (1:0.7:1:0.7, v/v) was firstly employed to separate the crude extract (320 mg), from which 19.3 mg of justicidin B (f), 10.8 mg of justicidin A (g), 13.9 mg of 6′-hydroxyjusticidin C (h), 7.7 mg of justicidin E (i), 6.3 mg of lignan J1 (j) were obtained with 91.3 mg of enriched mixture of compounds a–e. The enriched mixture (91.3 mg) was further separated using the solvent system consisting of petroleum–ethyl acetate–methanol–H2O (3:3.8:3:3.8, v/v), yielding 12.1 mg of procumbenoside E (a); 7.6 mg of diphyllin-1-O-β-d-apiofuranoside (b); 7.4 mg of diphyllin (c); 8.3 mg of 6′-hydroxy justicidin B (d); and 7.9 mg of diphyllin acetyl apioside (e). The purities of the 10 components were all above 94%, and their structures were identified by NMR and ESI-MS spectra. The results demonstrated that the strategy based on HSCCC for the separation of lignans and their glycosides was efficient and rapid.


Introduction
Justicia procumbens L. (Chinese name Juechuang) is one of the famous traditional Chinese herbal medicine, which stemmed from the dried whole plant of the genus Acanthaceae [1]. It has detoxification and diuretic swelling effect for the treatment of fever, cough, sore throat, cirrhosis of the ascites [2,3]. Previous phytochemical studies indicated that J. procumbens contains various ingredients, including lignans, lignan glycosides, alkaloids, flavonoids, triterpenes, steroids, etc. [4][5][6]. Modern pharmacological investigations indicated that lignans and their glycosides are the main chemical components in J. procumbens, which exhibit various pharmacological activities, including antitumor, antivirus, antihepatitis, inhibition of platelet aggregation, and cytotoxicity [7][8][9][10]. In order to better understand the biochemical properties of lignans and their glycosides from J. procumbens and to ascertain their clinical applications, it is essential to develop an efficient method for the preparative separation of lignans and their glycosides.

Selection of the Two-Phase Solvent System
Selection of solvent system is the key step for the HSCCC separation, usually by measuring the KD value of target compounds to determine the suitable two-phase solvent system. For the isolation of HSCCC, the optimum range of the KD value of target compounds in two-phase solvent system is 0.5-2 [25][26][27]. According to the chemical properties of lignans, in this paper, the KD values of 10 components in J. procumbens were measured by HPLC in a series of different solvent systems compose of petroleum (Pet)-ethyl acetate (EtOAc)-methanol (MeOH)-H2O at various volume ratios.
The KD value of the 10 target compounds were summarized in Table 1. As shown in Table 1, there are no satisfactory KD values for the 10 components in a single two-phase solvent system, and it is impossible that the 10 components were isolated by a single-step CCC separation. However, Pet-EtOAc-MeOH-H2O (1:0.7:1:0.7, v/v) provided suitable KD values for compounds f-j (Figure 2), when this solvent system was applied in CCC apparatus at the conventional flow-rate of 2.0 mL·min −1 , compounds f-j could be purified with good resolution and compounds a-e ( Figure 2) were eluted together as an enriched mixture near the solvent front. The whole separation process took more than 6 h, but it would be shortened by increasing the flow rate. However, the flow rate reached up to 4 mL·min −1 , loss of the stationary phase caused the low resolution between compounds f and g. However, when the flow rate was 3.0 mL·min −1 , satisfactory isolation with high efficiency and resolutions was acquired in 5 h ( Figure 3A).
Based on the KD values of compounds a-e in Pet-EtOAc-MeOH-H2O (1:0.7:1:0.7, v/v), the solvent system composed of Pet-EtOAc-MeOH-H2O may be valid for the separation of the enriched mixture. In further research, the ratio of EtOAc and H2O were increased to reduce the elution ability of the mobile phase. The KD values of compounds a-e in a series of solvent system composed of Pet-EtOAc-MeOH-H2O at different ratios were evaluated (Table 1), and Pet-EtOAc-MeOH-H2O

Selection of the Two-Phase Solvent System
Selection of solvent system is the key step for the HSCCC separation, usually by measuring the K D value of target compounds to determine the suitable two-phase solvent system. For the isolation of HSCCC, the optimum range of the K D value of target compounds in two-phase solvent system is 0.5-2 [25][26][27]. According to the chemical properties of lignans, in this paper, the K D values of 10 components in J. procumbens were measured by HPLC in a series of different solvent systems compose of petroleum (Pet)-ethyl acetate (EtOAc)-methanol (MeOH)-H 2 O at various volume ratios.
The K D value of the 10 target compounds were summarized in Table 1. As shown in Table 1, there are no satisfactory K D values for the 10 components in a single two-phase solvent system, and it is impossible that the 10 components were isolated by a single-step CCC separation. However, Pet-EtOAc-MeOH-H 2 O (1:0.7:1:0.7, v/v) provided suitable K D values for compounds f-j (Figure 2), when this solvent system was applied in CCC apparatus at the conventional flow-rate of 2.0 mL·min −1 , compounds f-j could be purified with good resolution and compounds a-e ( Figure 2) were eluted together as an enriched mixture near the solvent front. The whole separation process took more than 6 h, but it would be shortened by increasing the flow rate. However, the flow rate reached up to 4 mL·min −1 , loss of the stationary phase caused the low resolution between compounds f and g. However, when the flow rate was 3.0 mL·min −1 , satisfactory isolation with high efficiency and resolutions was acquired in 5 h ( Figure 3A).  (Table 1), and Pet-EtOAc-MeOH-H 2 O (3:3.8:3:3.8, v/v) provide suitable K D values for compounds a-e, and when this solvent system was used for separation of the enriched mixture at the flow rate of 3.0 mL·min −1 , five irregular peaks were presented in 3 h, which indicating the successful separation of compounds a-e ( Figure 3B). Therefore, the solvent system composed of Pet-EtOAc-MeOH-H 2 O (1:0.7:1:0.7, v/v) was applied to purify the crude sample, and the HSCCC absorption curve was presented in Figure 3A. Subsequent HPLC analysis indicated that a mixture, justicidin B (peak f), justicidin A (peak g), 6 -hydroxyjusticidin C (peak h), justicidin E (peak i), and lignan J 1 (peak j) were obtained. For the further separation, the enriched mixture was purified by HSCCC using Pet-EtOAc-MeOH-H 2 O (3:3.8:3:3.8, v/v) as the solvent system. Figure 3B shows that five major peaks were gained. Five HSCCC peaks were analyzed by HPLC showed that the five peaks were procumbenoside E (peak a), diphyllin-1-O-β-D-apiofuranoside (peak b), diphyllin (peak c), 6 -hydroxy justicidin B (peak d), and diphyllin acetyl apioside (peak e), respectively.  Therefore, the solvent system composed of Pet-EtOAc-MeOH-H2O (1:0.7:1:0.7, v/v) was applied to purify the crude sample, and the HSCCC absorption curve was presented in Figure 3A. Subsequent HPLC analysis indicated that a mixture, justicidin B (peak f), justicidin A (peak g), 6′-hydroxyjusticidin C (peak h), justicidin E (peak i), and lignan J1 (peak j) were obtained. For the further separation, the enriched mixture was purified by HSCCC using Pet-EtOAc-MeOH-H2O (3:3.8:3:3.8, v/v) as the solvent system. Figure 3B shows that five major peaks were gained. Five HSCCC peaks were analyzed by HPLC showed that the five peaks were procumbenoside E (peak a), diphyllin-1-O-β-D-apiofuranoside (peak b), diphyllin (peak c), 6′-hydroxy justicidin B (peak d), and diphyllin acetyl apioside (peak e), respectively.

Material and Reagents
Dried whole plant of J. procumbens was purchased from Bozhou drug market (Bozhou, Anhui province, China), and identified by Professor Jia Li (Shandong University of Traditional Chinese Medicine, Jinan, China).

Apparatus
HSCCC separation was performed by TBE-300C (Tauto Biotech, Shanghai, China), which was armed with three multilayer coils connected in series (total volume: 320 mL, the diameter: 2.6 mm) with a sample loop (20 mL). Rotation speed of the apparatus can be controlled up to 1000 rpm with a speed controller. During the experiment, the temperature of the HSCCC equipment is controlled at

Apparatus
HSCCC separation was performed by TBE-300C (Tauto Biotech, Shanghai, China), which was armed with three multilayer coils connected in series (total volume: 320 mL, the diameter: 2.6 mm) with a sample loop (20 mL). Rotation speed of the apparatus can be controlled up to 1000 rpm with a speed controller. During the experiment, the temperature of the HSCCC equipment is controlled at 25 • C by the DCW-0506 low constant temp monitor (Tauto Biotech, Shanghai, China). The pumping of the two-phase system was performed by the BT100-2J constant flow pump (Longer Pump Co., Ltd., Baoding, China). Spectra were produced from an 8823B-UV detector (Asahi Technology Co., Ltd., Hangzhou, China) at 254 nm. The description of the CCC diagram was implemented by a model MTK 1000 recorder (Hangzhou Mico Technology Co., Ltd., Hangzhou, China).
The sample was analyzed by HPLC Agilent 1120 (Agilent Technologies, Santa Clara, CA, USA) equipped with a G1315C diode array detector (DAD) system, an Agilent 1120 binary-solvent delivery system, an automatic sample injection, and an Empower 3 work-station.

Pre-Processing of Crude Sample
The dried whole plant (5 kg) of J. procumbens was fully crushed into powder and extracted three times (3 h, 2 h, 2 h) with 95% ethanol under reflux. The extract was filtered and concentrated by rotary evaporation at 55 • C to remove ethanol completely Then, equal amount of ethyl acetate (1 L) was applied to extract the concentrate three times, yielding 87.5 g of crude sample which was stored in refrigerator at 4 • C for further HSCCC purification.

Measurement of the Partition Coefficients (K D )
Pet-EtAc-MeOH-H 2 O as the optimized solvent system was used to measure the partition coefficients (K D ) value of the target compounds by HPLC as follows: Approximately 2 mg of crude sample was placed in a test tube, to which 1.5 mL equilibrated two-phase solvent system were added. The sample was fully dissolved in the solvent system by violently shaking the tube. Then, equal amounts of the organic and aqueous phases were separately analyzed by HPLC. The K D values of target compounds are defined as The HPLC peak area of the target components in the stationary phase; A m : The HPLC peak area of target components in the mobile phase.

Preparation of Solvent Systems and Sample Solutions
Two-phase solvent systems composed of Pet-EtOAc-MeOH-H 2 O (1:0.7:1:0.7, v/v) and Pet-EtOAc-MeOH-H 2 O (3:3.8:3:3.8, v/v) were applied in the HSCCC separation. Each solvent mixture was thoroughly equilibrated in a separatory funnel for 2 h at room temperature. Shortly before use, each solvent system was separated into two phases and each phase was degassed separately.
The sample solutions were prepared by dissolving 320 mg of the crude sample in 5 mL of each phase (total of 10 mL) used for separation.

Separation Procedure
In each separation process, the upper phase was firstly pumped into the three multilayer-coil combined in series at 40 mL·min −1 . Then, when the CCC instrument rotational speed reaches 810 rpm in the positive direction, the mobile phase was introduced into the column with 3.0 mL·min −1 . When the hydrodynamic equilibrium between the two phases was reached, the sample solution was put into the sample loop, while the UV detector (254 nm) monitored the effluent continuously. The peaks were gathered manually in accordance with the UV absorption curve and then subjected to HPLC analysis. Finally, measure the ratio of the organic phase to the whole volume in the multilayer-coiled columns to obtain the retention of the organic phase.

HPLC Analysis of CCC Separation Products
The peak fraction from CCC separation, crude sample, and the enriched mixture were analyzed by HPLC equipped with a G1315C diode array detector (DAD) system. A RP-C 18 column (5 µm, 4.6 mm × 250 mm; Waters Technologies, Milford, MA, USA) at 25 • C was applied for all the analyses. The flow-rate was get to 1 mL·min −1 , and the monitor wavelength was 254 nm. Using the mobile phase composed of A (acetonitrile) and B (water including of 0.1% TFA), and the gradient elution was carried out as follows: 0 min, 30% A; 0-10 min, 30-45% A; 10-25 min, 45-60% A; 25-40 min, 60-100% A.

Identification of CCC Fractions
The pure compounds obtained by the CCC separation were analyzed by 1 H-NMR and 13 C-NMR with a Varian-600 spectrometer (Varian, Palo Alto, CA, USA)and detected the molecular weight by an Agilent 1100/MS-G1946 (Agilent, Santa Clara, CA, USA) mass selective detector. The detail data of each component was as follows: Compound a (peak a in Figure 3B

Conclusions
The satisfactory isolation strategy of 10 lignans from a crude extract of J. procumbens was successfully optimized and established by HSCCC. From 320 mg of the crude sample, 10 lignans were obtained, including procumbenoside E (12.1 mg), diphyllin-1-O-β-D-apiofuranoside (7.6 mg), diphyllin (7.4 mg), 6 -hydroxy justicidin B (8.3 mg), diphyllin acetyl apioside (7.9 mg), justicidin B (19.3 mg), justicidin A (10.8 mg), 6 -hydroxyjusticidin C (13.9 mg), justicidin E (7.7 mg), and lignan J 1 (6.3 mg) with purity all over 94.0%. The study is of great reference value for obtaining high purity lignans and their glycosides from J. procumbens, which also indicated that HSCCC is a powerful technique for the separation and purification of active compounds with a wide range of polarity from natural products.