J S S

ISSN 1615-9306 · JSSCCJ 38 (12) 2007–2192 (2015) · Vol. 38 · No. 12 · June 2015 · D 10609

JOURNAL OF

SEPARATION SCIENCE

Methods Chromatography · Electroseparation Applications Biomedicine · Foods · Environment

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2038 Yanjuan Liu1,2 Xiaofen Chen3 JunXi Liu1 Duolong Di1 1 Key

Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Chinese Academy of Sciences, Lanzhou Institute of Chemical Physics, Lanzhou, China 2 Graduate University of the Chinese Academy of Sciences, Beijing, China 3 Lanzhou University, Analysis and Testing Center, Lanzhou, China Received December 22, 2014 Revised March 8, 2015 Accepted March 29, 2015

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Research Article

Three-phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high-speed counter-current chromatography A three-phase solvent system was efficiently applied for high-speed counter-current chromatography to separate secondary metabolites with a wide range of hydrophobicity in Dicranostigma leptopodum. The three-phase solvent system of n-hexane/methyl tert-butyl ether/acetonitrile/0.5% triethylamine (2:2:3:2, v/v/v/v) was selected for high-speed countercurrent chromatography separation. The separation was initiated by filling the column with a mixture of intermediate phase and lower phase as a stationary phase followed by elution with upper phase to separate the hydrophobic compounds. Then the mobile phase was switched to the intermediate phase to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with the lower phase. In this research, 12 peaks were eluted out in one-step operation within 110 min, among them, eight compounds with acceptable purity were obtained and identified. The purities of ␤-sitosterol, protopine, allocryptopine, isocorydione, isocorydine, coptisine, berberrubine, and berberine were 94.7, 96.5, 97.9, 86.6, 98.9, 97.6, 95.7, and 92.8%, respectively. Keywords: Dicranostigma leptopodum / High-speed counter-current chromatography / Solvent systems DOI 10.1002/jssc.201401466



Additional supporting information may be found in the online version of this article at the publisher’s web-site

1 Introduction The plant of Dicranostigma leptopodum (fam. papaveraceae) has been used as a traditional medicine to treat tonsillitis, hepatitis, inflammation, and so on in China for a long time [1–4]. D. leptopodum (Maxim) Fedde, as an ornamental plant, is mainly distributed in Qinling mountain area, northwest of China. With development of natural product chemistry, recent research shows that D. leptopodum has excellent biological activities. Extracts of D. leptopodum have been reported to exhibit antimicrobial activity [4], antiviral [5], antitumor [6], Correspondence: Dr. Duolong Di, Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Chinese Academy of Sciences, Lanzhou Institute of Chemical Physics, No. 18, Tianshui Middle Road, Lanzhou 730000, China Fax: +86-931-8277088 E-mail: [email protected]

Abbreviations: DAD, diode array detector; HSCCC, highspeed counter-current chromatography; IP, intermediate phase; LP, lower phase; PTFE, polytetrafluroethylene; TEA, triethylamine; UP, upper phase  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

anti-liver fibrosis activity [7], and anti-inflammatory activity [8]. Moreover, recent research demonstrated that isocorydine obtained from D. leptopodum not only inhibited cell proliferation in hepatocellular carcinoma cell lines by inducing G2/M cell-cycle arrest and apoptosis, but also targeted the drug-resistant cellular side population (or cancer stem cells) through PDCD4-related apoptosis [6, 9]. In the phytochemical investigations on this plant, some isoquinoline alkaloids have been isolated and identified, such as dihydrosanguinaline, 6-acetonyl-5,6-dihydrosanguinaline, sinoacutine, isocorydine, corydine, isocorydione N-methylhernovine, protopinium, protopine, allocryptopine, and berberrubine [10]. Moreover, several terpene compounds have also been obtained from D. leptopodum [3, 4]. But a further investigation is still necessary to find the chemical basis of bioactivities of this plant. The existing methods for separating and purifying components of D. leptopodum are time-consuming, labor-intensive, and material is lost. This work aimed to find an effective method to isolate more compounds from whole plant of D. leptopodum by high-speed counter-current chromatography (HSCCC). Since the 1980s, HSCCC has been widely used for the separation of natural and synthetic products [11]. Typically, www.jss-journal.com

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this separation technique is based on the partition of analytes between two immiscible organic–aqueous two-phase solvent systems without solid support and irreversible adsorption. HSCCC is a essentially form of liquid–liquid partition chromatography in which one of liquid phase of the immiscible two-phase system is retained in the column as stationary phase with the aid of gravity or a centrifugal force field while the other liquid phase is used as mobile phase [12–15]. However, most two-phase solvent systems are limited to separate constituents with a narrow range of polarity, and in fact, plant extracts usually contain very complicated constituents with a broad range of polarity. So, it is necessary to find a method to separate natural products with a wide range of polarity. Recently, several applications of three-phase solvent systems in HSCCC separation have been reported. This kind of solvent system has potential in separation of constituents with a wide range of polarity. Shibusawa et al. found that the aqueous-organic solvent mixtures, such as nhexane/methyl acetate/acetonitrile/water, n-hexane/ethyl acetate/acetonitrile/water, and n-hexane/methyl t-butyl ether/ acetonitrile/water can form three-phase solvent systems at specific volume ratios [16]. Ito and Shinomiya showed that a mixture of hydrophilic thiamine, nicotinamide, and hydrophobic vitamin K1 and K3 could be separated by threephase HSCCC using intermediate phase (IP) as stationary phase and both lower phase (LP) and upper phase (UP) as mobile phases [17]. A three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water at a volume ratio of 4:4:3:4 was applied successfully to the HSCCC separation of 15 standards with a broad range of hydrophobicity in one-step operation on one hand, on the other hand was applied to comprehensive separation of a wide variety of secondary metabolites in tea extract. In this method, all three phases of the three-phase solvent system were used for HSCCC separation [16, 18]. In the HSCCC separation of Catechin oligomers with different degrees of polymerization from apple condensed tannins, the best separation was achieved using a three-phase solvent system composed of n-hexane/methyl acetate/acetonitrile/water at a volume ratio of 1:1:1:1. After the elution of monomers, dimers, trimers, and a part of tetramers using the middle phase as mobile phase, methyl acetate was used as mobile phase to facilitate elution the oligomers from tetramers to decamers [19]. In the research of Yin et al., the LP and IP of three-phase solvent system, n-hexane/acetonitrile/dichloromethane/water, at a volume ratio of 5:5:1:5, were used as stationary phases, while UP was used as mobile phase. Because two phases of a three-phase solvent system were retained in a rotating column and act as the stationary phases during a HSCCC separation, so, they thought this HSCCC column can be considered as a combination column, and they successfully prepared three flavonoids from P. pinnata using the combination column [20]. In the present study, we demonstrate a new effective HSCCC method by utilizing all three phases of the threephase solvent system composed of n-hexane/methyl t-butyl  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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ether/acetonitrile/water to achieve a comprehensive isolation and purification of multiple compounds with a broad range of hydrophobicity from D. leptopodum in a one-step operation.

2 Materials and methods 2.1 Apparatus In this study, a Spectrum HSCCC instrument (DE Spectrum Centrifuge; Dynamic Extractions, Slough, UK) equipped with two bobbins was employed. Each bobbin of the HSCCC fits one analytical column and one preparative column made of polytetrafluroethylene (PTFE). The column volume of each analytical column is 14.0 mL of 0.8 mm id. The column volume of two preparative columns are 72.0 and 71.0 mL, respectively (1.6 mm id). The β value is defined as β = r/R, where r is the coiled tubing radius and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge. In this case, β values of the multilayer coil ranged from 0.64 (internal terminal) to 0.81 (external terminal). The maximum revolution speed of the Spectrum HSCCC instrument is limited to 1600 rpm. The HSCCC system was equipped with two preparative pumps NP7000 (Hanbon Sci. & Tec., Jiangsu, China), a NU3000 UV–Vis detector (Hanbon Sci. & Tec., Jiangsu, China), a DLSB-10/40⬚C constant temperature circulating instrument (Yarong Instruments, Zhengzhou, China) and a CBS-A automatic fraction collector (Shanghai Huxi Analysis Instrument Factory, Shanghai, China) to collect the fractions. For HPLC analysis, we used an Agilent 1200 HPLC system (Agilent Technologies, Palo Alto, CA, USA) equipped with a G1322A vacuum degasser, a G1311A quaternary pump, a G1315D diode array detector (DAD) and a G1328B manual injection valve with a 20.0 ␮L loop. The system was controlled by Agilent Chemstation software (version A.10.02; Agilent Technologies, Palo Alto, CA, USA). In addition, a SinoChrom ODS-BP analytical column (250 mm × 4.6 mm id, 5 ␮m, Elite, Dalian, China) was used.

2.2 Materials and reagents n-Hexane (HEX) used for HSCCC separation was of analytical grade and purchased from Zhongqin Chemical Reagent (Shanghai, China). Methyl tert-butyl ether (MBE) and methyl acetate (MeOAc) used in HSCCC were of analytical grade and purchased from Tianjin Damao Chemical (Tianjin, China) and Sinopharm Chemical Reagent (Ningbo, China), respectively. Acetonitrile (ACN) used for HSCCC separation and HPLC analysis was of chromatographic grade and purchased from Jiangsu Hanbon Sci. & Tech. (Jiangsu, China). Triethylamine was of analytical grade and purchased from Rionlon Bohua Pharmaceutical Chemical (Tianjin, China). Methanol used for HPLC analysis was of chromatographic grade and purchased from Yuwang Group (Shandong, China). www.jss-journal.com

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Table 1. Solvent systems composed of HEX/MBE/ACN/water and HEX/MeOAc/ACN/water

Three-phase solvent systems N

HEX

MBE

MeOAc

ACN

Water

Phase formation

a) UP/IP/LP (v/v/v)

1 2 3 4 5 6 7 8 9 10 11 12

2 2.5 2.5 1 2 2 2 5 5 5 5 4

3 3.5 3.5 1 1 1 2 0 0 0 0 0

0 0 0 0 0 0 0 5 5 5 4 4

3 3 3.5 1 1 2 3 1 2 5 5 3

2 2 2 1 1 1 2 5 5 5 5 4

Two Two Two Three Three Three Three Two Two Three Three Three

b) n.d b) n.d b) n.d

7:2:7 15.5:1:8.5 11:9.5:4.5 1.2:1.2:1 b) n.d b) n.d 1.3:2.3:1.4 1.8:2.4:3 1.4:1.7:1.9

HEX, n-hexane; MeOAc, methyl acetate; ACN, acetonitrile; MBE, methyl tert-butyl ether. a) Upper phase; intermediate phase; lower phase. b) Not detected.

Table 2. The effect of initial filling condition of binary stationary phases on final retention volume of each phase and Sf in rotating HSCCC column

Before rotation pumped volume ratio (LP/IP)

At steady-state during rotating HSCCC

into HSCCC column Volumes in rotating HSCCC column (mL)a)

5:5 4:6 3:7 2:8 1:9

VUP

VIP

VLP

45.0 58.0 50.0 44.0 41.0

37.5 38.2 50.1 70.4 87.7

60.5 47.8 42.9 28.6 14.3

Stationary phase

Fractional combination of

fraction (Sf )b)

the stationary phases (m)c)

0.69 0.60 0.65 0.69 0.71

0.34 0.56 0.59 0.71 0.86

a) The capacity of the column was 143 mL. b) Sf = (VI P + VL P)/143 c) m = VI P/(VI P + VL P)

Ultrapure water, used for preparation of all the samples and solutions, was obtained from a Spring-R10 water purification system (Research Scientific Instrument, Xiamen, China).

2.3 Plant collection and extraction The whole plant of D. leptopodum was collected from Chunxing, Gansu, northwest of China. It was authenticated by Prof. Zhigang Ma of Lanzhou University, China. A voucher sample (ZYC20120525) is kept in Key Laboratory of Chemistry of Northwestern Plant Resources, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences. Before performing

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comprehensive HSCCC separation, the extract of D. leptopodum was prepared according to the following method: D. leptopodum was powdered by a homogenizer, and 30.0 g of D. leptopodum powder was soaked in the three-phase solvent mixture (UP + IP + LP; 180.0 mL of each, total 540.0 mL) at 40⬚C for 30 min in a round-bottomed flask, and then extracted by ultrasonication at 25⬚C and 60 Hz for 40 min. The extract was filtered through nylon membranes with a pore size of 0.45 ␮m, and the filtrate forming three phases (UP/IP/LP) was delivered into another test tube. An aliquot of UP, IP and LP in the filtered extract (2.0 mL of each phase of crude drug extract) was mixed in a small vial (a total of 6.0 mL of crude drug extract) which was applied to the following HSCCC separation.

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2.4 Three-phase solvent system

2.7 HPLC analysis

The three-phase solvent system was prepared in a separatory funnel according to the volume ratio. It was thoroughly mixed and then completely equilibrated at room temperature. Then the three phases, consisting of the upper phase, the intermediate phase, and the lower phase, were divided and degassed by ultrasonication for 30 min shortly before use.

Crude sample and fractions separated by three-phase HSCCC were analyzed by HPLC. The HPLC chromatograms are shown in Supporting Information Figs. S1 and S2, respectively. The mobile phase was composed of water (500.0 mL) + phosphoric acid (1.0 mL) + triethylamine (2.4 mL) (A) and acetonitrile (B), the gradient used for elution: 0–20 min, 20– 26% (B); 20–45 min, 26–45% (B). The injection volume was 20.0 ␮L. The flow rate was kept at 1.0 mL/min for the over run and the effluent was monitored by a DAD detector at 280 nm. All of the sample solution and HPLC mobile phase was filtered through a 0.45 ␮m Millipore filter before use.

2.5 Determination of the effect of initial filling condition of the binary stationary phases on the final retention volume of each phase in HSCCC column In each experiment, both the LP and IP were simultaneously pumped into the coiled PTFE column at a constant volume ratio (VIP /VLP : 5:5, 6:4, 7:3, 8:2, and 9:1). Then, the coiled column was rotated at 1600 rpm, while the UP mobile phase as an initial mobile phase was pumped into the column in a tail to head direction at a flow rate of 5.0 mL/min. When the hydrodynamic mixing between the three phases reached a steady state of equilibrium in the rotating column, the rotation was stopped and the column contents were immediately collected into a graduated cylinder. The volume ratio and retention of the stationary phases (Sf ) in the rotated column were then calculated from the collected IP and LP volumes (VIP and VLP ) and the column capacity (143 mL) according to the following equation: Sf = (VIP + VLP )/143 [21].

2.6 HSCCC procedure In the case of HSCCC separation of the constituents in the extract, as described in the previous section, both the IP and LP were simultaneously pumped into the column at a total flow rate of 12.0 mL/min maintaining a constant volume ratio of 7:3 (IP/LP). After the column was entirely filled with both phases, the column was rotated at 1600 rpm while UP as an initial mobile phase was pumped into the column in a tail-tohead direction at a constant flow rate of 5.0 mL/min. When the hydrodynamic equilibrium between the three phases was established in the rotating column, 6.0 mL sample solution (mixture of 2.0 mL of each phase) was injected into the column through the sample port. The compounds in D. leptopodum extract were separated by HSCCC with stepwise elution using all three phases as mobile phase as follows: firstly, the hydrophobic compounds in the sample were separated and eluted out by the initial UP mobile phase for 25 min; then, the mobile phase was switched to IP, and moderately hydrophobic compounds were separated and eluted out with the IP mobile phase for 50 min; at last, LP was used as the mobile phase to elute the polar compounds that were already separated but still remained in the column for 40 min. During the whole elution process, the effluent from the head end of the column was monitored using a UV detector at 280 nm and was collected in 2 min intervals from the time when sample was injected.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2.8 Compound identification by NMR spectroscopy The 1 H NMR and 13 C NMR spectral of the isolated compounds were recorded on a Varian Inova-400 FT-NMR spectrometer (USA) in CDCl3 or DMSO using TMS as an internal reference. The chemical shifts (␦) are reported in ppm and the coupling constants (J) are reported in Hertz (Hz).

3 Results and discussion 3.1 Optimization of HSCCC conditions before sample injection 3.1.1 Selection of three-phase solvent system for HSCCC It is same as two-phase HSCCC, solvent system also plays a significant role in three-phase HSCCC. So, to select an appropriate solvent system is vital before HSCCC separation. Table 1 shows 12 kinds of three-phase solvent systems comprising HEX/MBE/ACN/water and HEX/MeOAc/ACN/ water with different volume ratios together with volume percentage of each phase. As shown in Table 1, there are only two phases in numbers 1, 2, 3, 8, and 9 solvent systems; as far as numbers 4, 5, 6, 10, and 11 solvent systems, their phasevolume percentage distinctions are too large, so these solvent systems cannot be used for three-phase HSCCC separation. The solvent system of number 12 is not appropriate to separate D. leptopodum either, because its polarity is high and the lipophilic compounds eluted out at the first stage cannot be separated with this solvent. At last, the solvent system of number 7 was selected for three-phase HSCCC separation. 3.1.2 Selection of initial filling condition of the binary stationary phases It is different from the conventional HSCCC method with a two-phase system, this method uses two phases (IP and LP) as the stationary phases where the volume ratio of these two phases retained in the rotating column has direct relationship with the HCCC separation effect. It is mainly determined by www.jss-journal.com

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Table 3. The effect of flow rate of UP mobile phase on final retention volume of each phase and Sf in the rotating HSCCC column

Flow rate of UP mobile phase (mL/min)

3.0 4.0 5.0 6.0

Volume in rotating HSCCC column (mL) VUP

VIP

VLP

32.0 44.0 50.0 60.0

66.0 55.8 50.1 49.1

45.0 43.2 42.9 33.9

Stationary phase fraction (Sf )

0.78 0.69 0.65 0.58

the volume ratio of the two stationary phases (IP/LP) present in the column before rotation, and a flow rate of UP mobile phase after rotation at a certain revolution speed. Table 2 displays the effect of the initial filling conditions of the binary stationary phases (IP and LP) on the final retention volume of each phase in the HSCCC column. As shown in Table 2, the initial pumped volume ratio between IP and LP into the HSCCC column had only a minor effect on the final Sf values (approximately 0.60–0.71). However, it obviously affected each phase volume retained in the column. When the pumped volume ratio of IP and LP was 5:5, the retained volume of LP (60.5 mL) in the column after the rotation was much more than that of IP (37.5 mL), because LP is more viscous than IP [18]. With the increase of the initial volume ratio of IP, the retained volume of IP increased. In view of Sf and each phase volume retained in the column, the initial volume ratio (IP/LP) of 7:3 was selected for the separation of target compounds from the complex crude extract.

3.1.3 Optimization of an appropriate flow rate of UP mobile phase Table 3 displays the effect of flow rate of UP mobile phase on Sf in the rotating column. All data were obtained under the identical conditions for the starting volume ratio of both stationary phases (IP/LP = 7:3), the revolution speed at 1600 rpm and the experiment temperature was 30⬚C. Under these conditions, an increase of the flow rate of UP mobile phase obviously caused final Sf value decrease, however, it scarcely affected the final volume ratio of IP/LP. As shown in Table 3, the lower the flow rate was, the higher the Sf was. However, a lower flow rate of mobile phase made longer separation time, and the separation effect was low. A higher flow rate of mobile phase gave shorter separation time of the analyte, but when the flow rate was increased to 7.0 mL/min, hydrodynamic mixing between the three phases failed to reach steady-state hydrodynamic equilibrium in the column. In consideration of Sf , separation time and final retention volume of each phase, 5.0 mL/min was selected as the flow rate of the UP mobile phase.  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Separation profile of D. leptopodum by HSCCC using three phase solvent system. UP, upper phase; IP, intermediate phase; LP, lower phase.

3.2 HSCCC separation of several compounds with a broad range of hydrophobicity from D. leptopodum using three-phase solvent system The three-phase solvent system composed of HEX/MBE/ ACN/water (2:2:3:2) was selected for HSCCC separation. From our previous studies and the published literatures [22, 23] we learned that the main constituents of D. Leptopodum are alkaloids, so 0.5% triethylamine (TEA) v/v instead of water was used in solvent preparation. Therefore, solvent system composed of HEX/MBE/ACN/0.5% TEA (2:2:3:2) was used in D. Leptopodum separation. The column was first entirely filled with a mixture of IP and LP (at a volume ratio of 7:3). Then the UP was pumped into the column at a flow rate of 5.0 mL/min while apparatus was run at a revolution speed of 1600 rpm. After hydrodynamic equilibrium of the three-phase was reached as indicated by a clear mobile phase eluting at the outlet in the rotating column (the retention of the stationary phase at 65.0%), the sample solution composed of 2.0 mL of each phase was injected into the column using a sample injector. Figure 1 shows the HSCCC chromatogram of D. leptopodum obtained by using the three-phase system under the optimized conditions described in the previous section. The starting mobile UP was eluted for 25 min, and its flow rate was 5.0 mL/min. At this stage, peak a containing several compounds was first eluted out from the column, because these compounds contained in peak a were too hydrophobic and scarcely distributed to the IP stationary phase. At this time, other hydrophobic compounds 1–3 distributed between UP mobile phase and IP stationary phase were well separated in the column and then eluted out by UP mobile phase, and the elution of these compounds proceeds from the tail toward the head of the column. After UP mobile phase was switched to IP at 25 min and the flow rate of the mobile phase was adjusted to 4.0 mL/min at the same www.jss-journal.com

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Figure 2. Structures of the isolated compounds from D. leptopodum. 1, ␤Sitosterol; 2, Protopine; 3, Allocryptopine; 7, Isocorydione; 8, Isocorydine; 10, Coptisine; 11, Berberrubine; 12, Berberine.

time, compounds 4–10 with medium polarity retained at the IP and/or LP stationary phase were distributed between the new IP mobile phase and the LP stationary phase in the column and were eluted out from tail to head direction. Finally, the mobile phase was switched to LP at 75 min after the sample injection, at the same time the flow rate of mobile phase was adjusted to 5.0 mL/min. Compounds 11 and 12 and compounds contained in peak b that are hydrophilic and dissolved only in LP were eluted out by this phase toward the head of the column. The last peak b was a mixed peak.

3.3 Structure identification The structure identification of the isolated compounds was carried out by 1 H NMR and 13 C NMR spectroscopy as well  C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

as comparison with the published literatures. Data of each compound is as follows: ␤-Sitosterol (1): white crystals, 1 H NMR (400 MHz, DMSOd6) ␦: 5.43 (d, J = 5.0 Hz, 6-H), 3.67 (m, H-3), 1.08 (s, 19-H), 0.99 (d, J = 6.3 Hz, 21-H), 0.92 (d, J = 6.5 Hz, 27H), 0.87 (d, J = 6.5 Hz, 26-H), 0.81 (t, J = 6.0 Hz, 29-H), 0.75 ppm (s, 18-H); 13 C NMR (100 MHz, DMSO-d6) ␦: 143.0 (C-5), 127.2 (C-6), 75.1 (C-3), 57.2 (C-14), 56.2 (C-17), 51.3 (C-9), 47.2 (C-24), 42.9 (C-13), 41.3 (C-12), 40.2 (C-4), 38.0 (C-1), 37.6 (C-10), 36.7 (C-20), 35.0 (C-22), 33.2 (C-7), 32.8 (C-8), 30.0 (C-2), 29.7 (C-25), 28.8 (C-16), 27.0 (C-23), 25.0 (C-15), 24.2 (C-28), 22.5 (C-11), 20.9 (C-26), 20.1 (C27), 19.7 (C-19), 19.1 (C-21), 13.2 (C-29), 12.4 ppm (C-18). These data are in agreement with earlier published data for ␤-sitosterol [24]. www.jss-journal.com

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Protopine (2): colorless crystals, 1 H NMR (400 MHz, CDCl3 ) ␦: 6.91 (1H, S, H-4), 6.68 (1H, d, J = 8.5 Hz, H-11), 6.67 (1H, d, J = 8.5 Hz, H-12), 6.62 (1H, S, H-1), 5.95 (2H, S, – OCH2 O–), 1.95 ppm (3H, S, N–CH3 ); 13 C NMR (100 MHz, CDCl3 ) ␦: 194.1 (C-14), 148.0 (C-2), 146.3 (C-3), 146.0 (C9), 145.7 (C-10), 136.9 (C-4a), 132.5 (C-14a), 129.8 (C-12a), 125.0 (C-12), 117.7 (C-8a), 110.4 (C-1), 108.1 (C-4), 106.7 (C-11), 101.2 (2,3-OCH2 O–), 100.8 (9,10-OCH2 O–), 57.7 (C-6), 50.9 (C-8), 46.3 (C-13), 41.5 (N–CH3 ), 31.5 ppm (C5). These data are in agreement with earlier published data for protopine [25]. Allocryptopine (3): white crystals, 1 H NMR (400 MHz, CDCl3 ) ␦: 6.98 (1H, s, H-4), 6.90 (1H, d, J = 8.5Hz, H-11), 6.80 (1H, d, J = 8.5Hz, H-12), 6.63 (1H, s, H-1), 5.95 (2H, s, -OCH2 O-), 3.83 (3H, s, 10-OCH3 ), 3.76 (3H, s, 9-OCH3 ), 3.60 (2H, s, H-8), 3.363.20 (2H, m, H-6), 3.002.69 (2H, m, H-5), 2.05 (2H, s, H-13), 1.93 ppm (3H, s, N-CH3 ); 13 C NMR (100 MHz, CDCl3 ) ␦: 151.5 (C-9), 148.1 (C-2), 147.1 (C10), 146.2 (C-3), 134.8 (C-4a), 132.6 (C-14a), 131.4 (C-12a), 128.2 (C-8a), 127.6 (C-12), 111.0 (C-11), 110.1 (C-1), 108.8 (C-4), 101.3 (–OCH2 O–), 60.7 (–OCH3 ), 57.1 (C-6), 55.7 (–OCH3 ), 51.1 (C-8), 45.0 (C-13), 41.5 (N–CH3 ), 31.2 ppm (C-5). These data are in agreement with earlier published data for allocryptopine [22]. Isocorydione (7): purple acicular crystals, 1 H NMR (400 MHz, CDCl3 ) ␦: 6.98 (1H, s, H-7), 6.94 (1H, s, H-3), 5.92 (1H, s, H-9), 3.99 (3H, s, 1-OCH3 ), 3.93 (3H, s, 10-OCH3 ), 3.87 (3H, s, 2-OCH3 ), 3.49 (2H, t, J = 6.5 Hz, H-5), 3.20 (3H, s, N-CH3 ), 3.15 ppm (2H, t, J = 6.5 Hz, H-4); 13 C NMR (100 MHz, CDCl3 ) ␦: 186.5 (C-8), 178.4 (C-11), 163.9 (C-10), 152.2 (C-2), 150.3 (C-6a), 143.9 (C-1), 136.5 (C-7a), 128.4 (C-3a), 127.0 (C-1a), 126.8 (C-11b), 119.3 (C-1b), 118.1 (C11a), 112.9 (C-3), 105.2 (C-9), 98.3 (C-7), 60.7 (1-OCH3 ), 56.5 (2-OCH3 ), 56.3 (10-OCH3 ), 50.3 (C-5), 40.2 (N–CH3 ), 29.3 ppm (C-4). These data are in agreement with earlier published data for isocorydione [26]. Isocorydine (8): white crystals, 1 H NMR (400 MHz, CDCl3 ) ␦: 6.87 (1H, d, J = 8.0 Hz, H-9), 6.84 (1H, d, J = 8.0 Hz, H-8), 6.73 (1H, s, H-3), 3.92 (3H, s, 2-OCH3 ), 3.89 (3H, s, 10-OCH3 ), 3.73 (3H, s, 1-OCH3 ), 3.14 (1H, dd, J = 16.4, 3.6 Hz, H-7), 2.96 (2H, m, H-4), 2,92 (2H, m, H-5), 2.67 (1H, dd, J = 16.4, 3.6 Hz, H-6a), 2.49 ppm (3H, s, N–CH3 ); 13 C NMR (100 MHz, CDCl3 ) ␦: 153.1 (C-2), 150.2 (C-10), 144.3 (C-11), 142.2 (C-1), 129.9 (C-1b), 129.2 (C-3a), 126.9 (C-7a), 126.1 (C-1a), 119.7 (C-11a), 119.2 (C-8), 111.7 (C9), 111.0 (C-3), 62.4 (1-OCH3 ), 62.0 (C-6a), 56.2 (2-OCH3 ), 55.8 (11-OCH3 ), 52.8 (C-5), 33.2 (C-7), 26.1 ppm (C-4). These data are in agreement with earlier published data for isocorydine [27]. Coptisine (10): yellow powder, 1 H NMR (400 MHz, CDCl3 ) ␦: 9.83 (1H, s, H-8), 8.77 (1H, s, H-13), 8.04 (1H, d, J = 9.0 Hz, H-11), 7.81 (1H, d, J = 9.0 Hz, H-12), 7.77 (1H, s, H-1), 7.09 (1H, s, H-4), 6.54 (2H, s, 9, 10-OCH2 O), 6.18 (2H, s, 2, 3-OCH2 O), 4.71 (2H, t, J = 6.0 Hz, H-6), 3.12 ppm (2H, t, J = 6.0 Hz, H-5); 13 C NMR (100 MHz, CDCl3 ) ␦: 152.3 (C-2), 149.0 (C-3), 148.3 (C-8), 146.3 (C-9), 145.5 (C-10), 138.1 (C-14), 134.7 (C-4a), 132.0 (C-12a), 124.3 (C C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

12), 122.9 (C-13), 122.2 (C-11), 121.7 (C-14a), 115.1 (C-8a), 109.6 (C-1), 106.8 (C-4), 105.7, 103.8 (2×OCH2 O), 56.2 (C-6), 27.8 ppm (C-5). These data are in agreement with earlier published data for coptisine [28]. Berberrubine (11): red crystals, 1 H NMR (400 MHz, CDCl3 ) ␦: 9.23 (1H, s, H-8), 8.01 (1H, s, H-13), 7.63 (1H, d, J = 9.0 Hz, H-12), 7.42 (1H, s, H-1), 6.90 (1H, d, J = 9.0 Hz, H-11), 6.81 (1H, s, H-4), 6.05 (1H, s, H-15), 4.72 (1H, t, J = 6.0 Hz, H6), 3.87 (1H, s, H-16), 3.14 ppm (1H, t, J = 6.0 Hz, H-5); 13 C NMR (100 MHz, CDCl3 ) ␦: 152.1 (C-2), 150.0 (C-3), 147.8 (C-9), 147.8 (C-10), 143.8 (C-8), 138.2 (C-14), 135.3 (C-8a), 132.6 (C-4a), 131.2 (C-12a), 125.3 (C-13), 123.7 (C-12), 121,9 (C-1a), 120.5 (C-11), 110.2 (C-4), 106.9 (C-1), 104.4 (C-15), 59.3 (C-6), 57.4 ppm (C-16). These data are in agreement with earlier published data for berberrubine [29]. Berberine (12): yellow powder, 1 H NMR (400 MHz, DMSOd6) ␦: 9.92 (1H, s, H-8), 8.96 (1H, s, H-13), 8.33 (1H, d, J = 9.0 Hz, H-11), 8.10 (1H, d, J = 9.0 Hz, H-12), 7.92 (1H, s, H-1), 7.11 (1H, s, H-4), 6.20 (2H, s, 2, 3-OCH2 O), 5.03 (2H, t, J = 6.0 Hz, H-6), 4.32 (3H, s, 10-OCH3 ), 4.13 (3H, s, 9-OCH3 ), 3.21 ppm (2H, t, J = 6.0 Hz, H-5); 13 C NMR (100 MHz, DMSO-d6) ␦: 151.3 (C-2), 150.7 (C-3), 148.9 (C9), 146.5 (C-10), 144.9(C-8), 138.4 (C-14), 133.7 (C-4a), 131.9 (C-12), 128.3 (C-12a), 125.1 (C-14a), 122.2 (C-13), 121.3 (C1), 120.7 (C-11), 108.9 (C-8a), 106.4 (C-4), 103.2 (OCH2 O), 63.2 (C-6), 58.2 (9-OCH3 ), 56.0 (10-OCH3 ), 27.2 ppm (C-5). These data are in agreement with earlier published data for berberine [28]. The structures of the isolated compounds are shown in Fig. 2.

4 Conclusion The present study indicated that three-phase HSCCC can be used to separate multiple compounds with a broad range of polarity. Eight compounds including highly hydrophobic ␤-sitosterol to polar berberine were separated in a one-step operation in about 110 min. Although the resolution is not good enough, this method is efficient to separate sample without any pretreatment. The structures of eight isolated compounds were identified. Because the purity of the other four compounds is low, their structures were not identified. We are undertaking further studies on three-phase HSCCC separation of D. leptopodum and intend to report on the outcome of the studies in due course. This research was financially supported by The National Natural Sciences Foundation of China (NSFC No. 20775083 and No. 21175142) and Open Fund of Key Laboratory of Chemistry of Northwestern Plant Resources of The Chinese Academy of Science (No. CNPR-2011kfkt-02). The authors have declared no conflict of interest. www.jss-journal.com

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Three-phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high-speed counter-current chromatography.

A three-phase solvent system was efficiently applied for high-speed counter-current chromatography to separate secondary metabolites with a wide range...
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