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DOI: 10.1039/C6RA14056D (Paper) RSC Adv., 2016, 6, 83343-83349

Preparative separation of quaternary ammonium alkaloids from Caulis Mahoniae by conventional and pH-zone-refining counter-current chromatography

Heng Zhu† a, Daijie Wang†b, Lei Wena, Jinqian Yub, Yanling Gengb, Hengqiang Zhaob, Ruixuan Zhaob and Xiao Wang*ab
aCollege of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan 250355, China. E-mail: wangx@sdas.org; Fax: +86 531 82964889; Tel: +86 531 82605319
bShandong Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan 250014, China

Received 30th May 2016 , Accepted 14th August 2016

First published on 17th August 2016

Abstract

In this work, the preparative separation of quaternary ammonium alkaloids from Caulis Mahoniae by pH-zone-refining counter-current chromatography (PZRCCC) was compared to a conventional high-speed counter-current chromatography (HSCCC). A flow-rate changing strategy was performed in conventional CCC separation with two-phase solvent systems with a solution composed of chloroform–methanol–0.5 mM HCl and water (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v). Compared to conventional CCC separation, the separation of 3.0 g crude alkaloid extracts was carried out by PZRCCC with solvent system of chloroform–methanol–water (4[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]3, v/v). The retainer acid and eluter base were optimized by changing the concentration ratios to 60 mM HCl in upper aqueous phase and 7.5 mM TEA in lower phase. From 3.0 g of the crude alkaloid extracts, stepharanine (53.7 mg, 97.4%), columbamine (28.1 mg, 96.5%), jatrorrhizine (150.6 mg, 99.0%), palmatine (169.8 mg, 99.0%) and berberine (157.2 mg, 99.5%) were obtained. The compounds were identified by ESI-MS and 1H-NMR data. The results indicated that PZRCCC is an excellent separation mode for separating quaternary ammonium alkaloids compared with conventional CCC.

1. Introduction

Caulis Mahoniae, the dried stem of Mahoniabealei (Fort) Carr. or Mahoniafortune (Lindl.) Fedde, is widely distributed in southeast of China. As a famous folk medicine, Caulis Mahoniae is widely used for treating jaundice hepatitis, jaundice, skin ulcer, etc.1,2 Caulis Mahoniae is also known for its effects of anti-cancer, anti-viral, anti-inflammatory, anti-arrhythmia, anti-bacterial, and hypoglycemic. Major effective compounds in Caulis Mahoniae are quaternary ammonium3–6 alkaloids.

A number of traditional chromatography techniques are used for the separation of quaternary ammonium alkaloids (e.g. pre-HPLC, alkaline silica gel and sephadex LH-20). Although traditional methods are widely used, their disadvantages cannot be neglected, such as large solvent consumption, irreversible sample adsorption and the requirement of complex multiple steps. It is necessary to establish an efficient method to separate quaternary ammonium alkaloids from Caulis Mahoniae.

High-speed counter-current chromatography (HSCCC) is a liquid–liquid partition chromatography which can eliminate the irreversible adsorption without solid packing support. Due to the advantages of HSCCC, such as large sample injection, high recovery rate, simple sample preparation, and excellent repeatability,7 HSCCC has been developed to separate and isolate various samples.8–16 pH-zone-refining counter-current chromatography (PZRCCC), derived from HSCCC and initially invented by Ito, can separate ionic compounds including alkaloids and organic acids.17–26 The chromatography peaks are rectangular with highly concentrated sample, depending on their hydrophobicities and pKa values. Compared to conventional CCC, PZRCCC provides approximately 10-fold increase in sample loading, enrichment of minor impurities, and high concentration of peaks.

In this paper, two CCC methods including conventional CCC and PZRCCC are compared and employed for preparative separation of quaternary ammonium alkaloids from Caulis Mahoniae. Fig. 1 shows the chemical structures of quaternary ammonium alkaloids.


image file: c6ra14056d-f1.tif
Fig. 1 Chemical structures of compounds separated from Caulis Mahoniae.

image file: c6ra14056d-f2.tif
Fig. 2 The HPLC chromatogram of crude alkaloids from Caulis Mahoniae. Experimental conditions: column, Waters Symmetry® C18 column (4.6 mm × 250 mm, i.d., 5 μm); mobile phase, acetonitrile–1% TEA (adjust the pH value to 3 with phosphoric acid) solution (25[thin space (1/6-em)]:[thin space (1/6-em)]75, v/v); detection, 265 nm; flow-rate, 1.0 mL min−1.

2. Experimental

2.1 Reagents and materials

Petroleum ether, chloroform, methanol, alcohol, triethylamine (TEA), and hydrochloric acid (HCl) were analytical grade and purchased from Siyou Chemical Reagent Factory. Acetonitrile, phosphoric acid and TEA used for HPLC were of chromatographic grade (Tedia Company, Inc., Fairfield, USA). Ultrapure water (18.2 MΩ) was purified by osmosis Milli-Q water system (Millipore, Bedford, MA, USA). Caulis Mahoniae was purchased from JinanJian-lian TCM store and the specie was identified by Professor Jia Li (Shandong University of Traditional Chinese Medicine, Jinan, China). The voucher specimen (no. MD201509) was stored in Shandong Analysis and Test Center, Jinan, Shandong, China.

2.2 Apparatus

HSCCC separation was performed by TBE-300A (Shanghai Tauto Biotechnique, Shanghai, China), which was equipped with three multilayer coil separation PTFE columns connected in series (total volume 300 mL, i.d. of the tube diameter 2.6 mm) with a 20 mL sample loop. The rotation speed was adjusted from 0 to 1000 rpm by speed controller. During the separation process, the temperature of separation columns was maintained at 25 °C using a model HX series constant temperature circulator instrument (Changcheng Company, Zhengzhou, China). TBP-5002 constant-flow pump (Shanghai Tauto Biotechnique, Shanghai China) was used to pump the two solvent systems. The chromatogram data of the alkaloid extracts was obtained using Model 3057 portable recorder (Yokogawa, Sichuan Instrument Factory, Sichuan, China). Spectra were generated from 8823A-UV detector (Beijing Emilion Technology, Beijing, China) at a wavelength of 254 nm.

HPLC was performed on Waters e2695 equipment (Waters, Milford, MA, USA) including a Waters 2998 diode assay detector (DAD) system, an automatic sample injection, a Waters 2695 quaternary-solvent delivery system, and a millennium 32 workstation.

2.3 Preparation of crude extracts from Caulis Mahoniae

The dried rhizomes of Caulis Mahoniae (4 kg, 40–60 mesh) were extracted under reflux (70 °C) with 2 L of 95% ethanol twice for two hours. After the extraction, the extracts were filtered under decompression, and combined together to further concentrate them via rotary evaporation at 50 °C.

The pH value of the liquid concentration was adjusted to 3 with 2% HCl. After that, the acid solution was extracted with petroleum ether for five times. The extracted solution was slowly alkalized with 10% ammonia until the pH value was 10. After rotary evaporation, 10.2 g of yellow precipitate was obtained and stored in refrigerator at 5 °C.

2.4 Preparation of solvent systems and sample solutions

In conventional CCC, flow-rate changing strategy was used to separate total alkaloids in Caulis Mahoniae. The two-phase solvent system used for conventional CCC was chloroform–methanol–0.5 mM HCl and water (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, volume ratio, the same as follows). After equilibration in a separating funnel, the solvent system that was used for the experiment was separated into two phases. 250 mg of crude alkaloid extracts were dissolved in 5 mL stationary phase and 5 mL mobile phase.

In PZRCCC separation, the two phase solvent system consisted of chloroform-methanol–water (4[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]3, v/v). After equilibration in a separating funnel, the solution was divided into two phases for the experiment. The upper phase was acidified with 60 mM HCl (stationary phase) and lower organic phase was alkalified with 7.5 mM TEA (mobile phase). 3.0 g of crude alkaloid extracts were dissolved in the lower phase without alkalization and acidified upper phase.

2.5 Separation procedure

2.5.1 Conventional CCC separation. The multilayer-coiled columns were first filled with upper phase at 20.0 mL min−1. Then the HSCCC equipment was rotated at 800 rpm in a clockwise mode, whereas, the lower mobile phase was pumped into the apparatus at 2.0 mL min−1. After hydrodynamic equilibrium was established, the sample solution with 250 mg total alkaloids from Caulis Mahoniae was injected via the sample loop. The effluent was monitored continuously with the UV detector at 254 nm. When elution time reached 200 minutes, the mobile phase was changed into 10.0 mL min−1 until 310 min. The stationary phase retention was defined as the ratio of stationary phase to the total volume in the column after separation.
2.5.2 PZRCCC separation. The PZRCCC was first filled the with upper phase, and sample solution with 3.0 g of total alkaloids from Caulis Mahoniae was injected. The lower mobile phase was pumped into the CCC column at 2.0 mL min−1 and rotated at 800 rpm in a clockwise mode. The effluent was monitored at 254 nm and recorded in a portable recorder. pH values of all fractions were obtained at the room temperature. The stationary phase retention in PZRCCC was defined the same as conventional CCC.

2.6 Analysis and identification of separation products

Analysis used for HPLC was Waters Symmetry® C18 column (4.6 mm × 250 mm, i.d., 5 μm) at 25 °C. The mobile phase was acetonitrile–1% TEA (pH value was adjusted to 3 using phosphoric acid) solution (25[thin space (1/6-em)]:[thin space (1/6-em)]75, v/v) and the flow-rate was 1.0 mL min−1. The detector wavelength was set at 265 nm.

Agilent 5973N mass selective detector was used to detect the molecular weight of pure compounds with ESI interface. The NMR spectrum was recorded by Varian-600 spectrometer with TMS as an internal standard and d6-DMSO as the solvent (Varian, Palo Alto, CA, USA).

3. Results and discussion

3.1 Conventional CCC separation

According to the alkaloid properties and previous test on HSCCC separation, the solvent systems composed of petroleum ether–ethyl acetate–methanol–water (1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1.1, v/v), ethyl acetate–n-butanol–0.5 mM HCl, water solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]5, v/v), and chloroform–methanol–0.5 mM HCl, water solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v) were designed to get an efficient separation of the alkaloid compounds. The KD values of solvent systems of HSCCC are presented in Table 1. Table 1 shows that the KD values were too small and unsuitable to separate alkaloids from Caulis Mahoniae using petroleum ether–ethyl acetate–methanol–water (1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1, v/v) and ethyl acetate–n-butanol–0.5 mM HCl solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]5, v/v) as the solvent system. In the view of alkaloids being soluble in chloroform, the solvent system composed of chloroform–methanol–0.5 mM HCl water solution for the separation. When chloroform–methanol–0.5 mM HCl and water solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v) was used, compounds A, D and E afforded a suitable KD value for separation but the elution time of compounds B and C were too long. Since increasing the flow-rate had little effect on the stationary phase retention in chloroform series solvent systems, a flow-rate changing strategy was used in compounds B and C separation.
Table 1 The KD-values of target compounds in different solvent systems
Solvent system KD-Values of compounds A–E
A B C D E
Petroleum ether–ethyl acetate–methanol–water 1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]1.1 0.24 0.32 0.22 0.19 0.33
Ethyl acetate–n-butanol–0.5 mM HCl water solution 4[thin space (1/6-em)]:[thin space (1/6-em)]1[thin space (1/6-em)]:[thin space (1/6-em)]5 0.07 0.12 0.16 0.11 0.29
Chloroform–methanol–0.5 mM HCl water solution 4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2 1.27 4.75 5.50 0.40 0.57

It was then assayed on HSCCC, where the separation was carried out with chloroform–methanol–0.5 mM HCl and water solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v). The lower phase comprising of chloroform–methanol–0.5 mM HCl and water solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v) was used as the mobile phase in flow-rate changing strategy (0–200 min, 2.0 mL min−1; 200–240 min, 10.0 mL min−1), and a good separation result was expected. After that, five compounds were successfully isolated from 250 mg crude alkaloid extracts of Caulis Mahoniae (shown in Fig. 3), including stepharanine (compound A, 13.2 mg), columbamine (compound B, 6.6 mg), jatrorrhizine (compound C, 17.3 mg), palmatine (compound D, 13.4 mg), and berberine (compound E, 14.7 mg) with the purities of 97.5%, 96.0%, 98.2%, 99.0%, and 99.5% as determined by HPLC. The compound recoveries were 98%, 97%, 98%, 93%, and 97%, respectively. The productivities of the organic solvent were also calculated. In conventional CCC, the productivities per hour of the five compounds (compound A, B, C, D, E) were 2.6, 1.3, 3.4, 2.6, and 2.9 mg h−1, respectively, and 9.4, 4.7, 12.4, 9.6, and 10.5 mg L−1 per liter of the solvent, respectively.


image file: c6ra14056d-f3.tif
Fig. 3 Conventional CCC separation and HPLC analysis to the HSCCC peak fractions. Conditions: stationary phase, the upper phase of chloroform–methanol–0.5 mM HCl water solution (4[thin space (1/6-em)]:[thin space (1/6-em)]1.5[thin space (1/6-em)]:[thin space (1/6-em)]2, v/v); 0–200 min, flow-rate 2.0 mL min−1, 200–240 min, flow-rate 10.0 mL min−1; sample size, 250 mg; detection, 254 nm; stationary phase retention, 66.7% (flow-rate 2.0 mL min−1), 52.8% (flow-rate 10.0 mL min−1); revolution speed, 800 rpm. HPLC conditions are as in Fig. 2.

3.2 PZRCCC separation

Through preliminary experiment, the solvent system composed of chloroform–methanol–water (4[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]3, v/v) was selected with different concentration of retainer acid and eluter base in PZRCCC separation. When 1.0 g crude alkaloid extracts were separated with 30 mM HCl as retainer acid (upper phase) and 10 mM TEA as eluter base (lower phase), compound A was successfully separated with a purity over 98% (Fig. 4I), whereas, compounds B, C and D, E were not fully separated. When 60 mM HCl and 10 mM TEA was used, the peak resolution improved with a longer elution time. As shown in (Fig. 4II), 1.0 g crude alkaloid extracts were completely separated. Subsequently, the sample amount increased to 3.0 g. Compound A was separated successfully while compounds B, C and D, E were partly separated. When the stationary phase was acidified with 60 mM HCl and the mobile phase was alkalified with 7.5 mM TEA, 3.0 g crude alkaloid extracts were successfully separated, as shown in (Fig. 4III). Ultimately, five compounds were isolated and were identified as stepharanine (compound A, 53.7 mg), columbamine (compound B, 28.1 mg), jatrorrhizine (compound C, 150.6 mg), palmatine (compound D, 169.8 mg), and berberine (compound E, 157.2 mg) with the purities of 97.4%, 96.5%, 99.0%, 98.7%, and 98.5%, respectively, and were determined by HPLC. The recoveries of those five compounds were 33%, 34%, 71%, 98%, and 86%, respectively. In PZRCCC, the productivities per hour of the five compounds (compound A, B, C, D, E) were 7.4, 3.9, 20.6, 23.3, and 21.5 mg h−1, respectively. The productivities of the organic solvent of the five compounds were 44.8, 23.4, 125.5, 141.5, and 131.0 mg L−1, respectively.
image file: c6ra14056d-f4.tif
Fig. 4 PZRCCC separation and HPLC analysis to the HSCCC peak fractions. Conditions: stationary phase, the upper phase of chloroform–methanol–water (4[thin space (1/6-em)]:[thin space (1/6-em)]3[thin space (1/6-em)]:[thin space (1/6-em)]3, v/v); detection, 254 nm; flow-rate, 2.0 mL min−1; revolution speed, 800 rpm. I: 30 mM HCl in upper stationary phase and 10 mM TEA in lower phase, sample loading 1.0 g, stationary phase retention 40%; II: 60 mM HCl in upper stationary phase and 10 mM TEA in lower phase, sample loading 1.0 g, stationary phase retention 41.7%; III: 60 mM HCl in upper stationary phase and 10 mM TEA in lower phase, sample loading 3.0 g, stationary phase retention 36.9%; IV: 60 mM HCl in upper phase and 7.5 mM TEA in lower phase, sample loading 3.0 g, retention of stationary phase 36.4%.

3.3 Identification of the isolated compounds

Compound A (peak A in Fig. 3, peak A in Fig. 4III): the compound appeared as a light yellow colored crystal in chloroform methanol, and showed a positive result to the bismuth potassium iodide reaction. UV (λMeOHmax): 227, 282, 348 nm. Positive ESI-MS (m/z): 324.1 [M + H]+. 1H-NMR (125 MHz, DMSO-d6) δppm: 9.70 (1H, s, H-8), 8.73 (1H, s, H-13), 7.89 (1H, d, J = 9.0 Hz, H-12), 7.85 (1H, d, J = 9.0 Hz, H-11), 7.54 (1H, s, H-1), 7.03 (1H, s, H-4), 4.89 (2H, t, J = 6.0 Hz, H-6), 3.17 (2H, t, J = 6.0 Hz, H-5), 4.04 (3H, s, 9-OCH3), 3.88 (3H, s, 3-OCH3). When these results were compared to literature,27 the compound was identified as stepharanine.

Compound B (peak B in Fig. 3, peak B in Fig. 4III): the compound appeared as light yellow spiculas in chloroform methanol, and gave a positive result with bismuth potassium iodide reaction. UV (λMeOHmax): 263, 345 nm. Positive ESI-MS (m/z): 338.4 [M + H]+. 1H-NMR (125 MHz, DMSO-d6) δppm: 9.87 (1H, s, H-8), 8.80 (1H, s, H-13), 8.21 (1H, d, J = 8.4 Hz, H-11), 8.03 (1H, d, J = 8.4 Hz, H-12), 7.58 (1H, s, H-1), 7.10 (1H, s, H-4), 4.96 (2H, t, J = 5.4 Hz, H-6), 4.10 (3H, s, 10-OCH3), 4.08 (3H, s, 9-OCH3), 3.91 (3H, s, 3-OCH3), 3.20 (2H, t, J = 5.4 Hz, H-5). When these results were compared to literature,28 the compound was identified as columbamine.

Compound C (peak C in Fig. 3, peak C in Fig. 4III): the compound appeared as light red spiculas in chloroform methanol, and gave a positive result with bismuth potassium iodide reaction. UV (λMeOHmax): 265, 345 nm. Positive ESI-MS (m/z): 338.4 [M + H]+. 1H-NMR (125 MHz, DMSO-d6) δppm: 9.85 (1H, s, H-8), 9.01 (1H, s, H-13), 8.11 (1H, d, J = 8.4 Hz, H-12), 8.00 (1H, d, J = 8.4 Hz, H-11), 7.29 (1H, s, H-1), 6.96 (1H, s, H-4), 4.91 (2H, t, J = 5.4 Hz, H-6), 4.08 (3H, s, 10-OCH3), 4.06 (3H, s, 9-OCH3), 3.95 (9H, s, 2-OCH3), 3.15 (2H, t, J = 5.4 Hz, H-5). When these results were compared to literature,29 the compound was identified as jatrorrhizine.

Compound D (peak D in Fig. 3, peak D in Fig. 4III): the compound appeared as a yellow needle crystal in chloroform methanol, and gave a positive result with bismuth potassium iodide reaction. UV (λMeOHmax): 273, 346 nm. Positive ESI-MS (m/z): 352.4 [M + H]+. 1H-NMR (125 MHz, DMSO-d6) δppm: 7.72 (1H, s, H-1), 7.09 (1H, s, H-4), 9.89 (1H, s, H-8), 8.22 (1H, d, J = 8.4 Hz, H-11), 8.02 (1H, d, J = 8.4 Hz, H-12), 9.04 (1H, s, H-13), 4.95 (2H, t, J = 5.4 Hz, H-6), 4.12 (3H, s, 10-OCH3), 4.07 (3H, s, 9-OCH3), 3.96 (3H, s, 2-OCH3), 3.87 (3H, s, 3-OCH3), 3.24 (2H, t, J = 5.4 Hz, H-5). These results were compared to literature,30 and the compound was identified as palmatine.

Compound E (peak E in Fig. 3, peak E in Fig. 4III): the compound appeared as a yellow needle crystal in chloroform methanol, and gave a positive result with bismuth potassium iodide reaction. UV (λMeOHmax): 263, 346 nm. Positive ESI-MS (m/z): 336.4 [M + H]+. 1H-NMR (125 MHz, DMSO-d6) δppm: 9.91 (1H, s, H-8), 8.95 (1H, s, H-13), 8.20 (1H, d, J = 8.4 Hz, H-11), 8.01 (1H, d, J = 8.4 Hz, H-12), 7.78 (1H, s, H-1), 7.07 (1H, s, H-4), 6.16 (2H, s, 2,3-OCH2O), 4.93 (2H, t, J = 5.4 Hz, H-6), 4.08 (3H, s, 10-OCH3), 4.06 (3H, s, 9-OCH3), 3.22 (2H, t, J = 5.4 Hz, H-5). These results were compared to literature,31 and the compound was identified as berberine.

4. Conclusions

In this paper, two separation models conventional HSCCC and PZRCCC were successfully used for the preparative separation of quaternary ammonium alkaloids from Caulis Mahoniae. Five compounds were obtained in one-step separation with two CCC separation models and were identified as stepharanine, columbamine, jatrorrhizine, palmatine and berberine with purities over 96%. The results demonstrated that in order to save elution time, flow-rate changing strategy may be employed in conventional HSCCC separation. Due to the high polarity of quaternary ammonium alkaloids, reverse elution PZRCCC can be used for the low organic phase as mobile phase. And the concentration of the eluate (mobile phase) and retainer (stationary phase) are optimized by alkali and acid. Compared with the conventional CCC, PZRCCC is an efficient and rapid method for separating quaternary ammonium alkaloids because of high concentration of fractions and large sample loading capacity.

Acknowledgements

The article was financially supported by National Natural Science Foundation of China (21506119) and Science and Technology Development Program of Shandong Province (2014GSF118156).

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Footnote

† These authors have equal contribution to this work. Both persons are the first authors.
This journal is © The Royal Society of Chemistry 2016
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