ABSTRACT
Alkaloids, widespread in plants, have a series of pharmacological activities and have been widely used to treat various diseases. Because alkaloids are usually presented in multicomponent mixtures and are deeply low in content, they are very difficult to extract and separate by traditional methods. High-speed counter current chromatography(HSCCC) is a kind of liquid-liquid chromatography without solid support phase, which has the advantages of large injection volume, low cost, and no irreversible adsorption. Compared with the traditional methods of extraction and separation of alkaloids, HSCCC can ensure the separation of many different alkaloids at one time, with a high recovery and large amount. In this paper, the advantages and disadvantages of HSCCC compared with traditional separation methods were discussed and the solvent system and elution mode of HSCCC used to separate alkaloids in recent years were summarized by referring to the relevant literature to provide some references for the separation of alkaloids by HSCCC.
Subject(s)
Biological Products , Countercurrent Distribution/methods , Chromatography, High Pressure Liquid/methods , Alkaloids/analysis , Solvents/chemistryABSTRACT
Objective: To develop a new method for the determination of calcipotriol ointment by high-speed counter-current chro-matography(HSCCC). Methods: An HSCCC method was used coupled with two-phase solvent system consisting of petroleum benzine-ethanol -water(2: 1: 0. 5, v/v/v). The injection volume was 1. 0 ml and the detection wavelength was set at 265 nm. Results: The proposed method showed good linearity within the range of 4.02-40.20 μg·ml-1(r=0.999 4). The average recovery was 99.2%, and the RSD was 0. 4% (n=9) . Conclusion: The HSCCC method is simple and rapid, and suitable for the determination of calcipo-triol ointment.
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Objective: To develop an effective and rapid method for the preparation of emodin-8-O-β-D-glucopyranoside (EG) reference substance from Cuspidati Rhizoma et Radix (PCRR). Methods: The target fraction was isolated by macroporous resin column chromatography and ODS column chromatography, and then the target compound was purified by high-speed counter-current chromatography (HSCCC). The structure was identified by ESI-MS and NMR spectral techniques, and its purity was determined by HPLC analysis using the main component self-comparison with no correction factor method. Results: A solvent system that consisted of dichloromethane-ethyl acetate-methanol-water (8:1:6:5) was applied to the separation based on assessment using HPLC partition coefficient method. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase. The reference substance of EG was prepared, and its purity was up to 98% in line with the standard of quantitative analysis. Conclusion: The method is suitable for the preparation of EG from PCRR, which provides a basis for the quality control of natural product and their preparations.
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Objective: To develop a suitable method for the large-scale separation of quinolone alkaloids from the fruits of Evodia rutaecarpa by high-speed counter-current chromatography (HSCCC). Methods: Two solvent systems were developed for the separation method. The upper phase was used as the stationary phase, and the lower phase was used as the mobile phase at 35 °C with the flow rate of 2 mL/min and rotation speed of 855 r/min. Results Using the described method, 1-methyl-2-undecyl-4(1H)-quinolone (1), evocarpine (2), 1-methy-2-[(6Z,9Z)]-6,9-pentade-cadienyl-4-(1H)-quinolone (3), dihydroevocarpine (4), and the mixture (5) of 1-methyl-2-[(Z)-10-pentadecenyl]-4(1H)-quinolone (Va) and 1-methyl-2-[(Z)-6-pentadecenyl]-4(1H)-quinolone (Vb) could be isolated from a petroleum ether extract. They were identified by 1H-NMR, 13 C-NMR, and MS/MS, and the purities were 94.3%, 95.2%, 96.8%, 98.3%, and 96.8%, respectively. Conclusion: Five quinolone alkaloids from the fruits of E. rutaecarpa could be systematically isolated and purified using HSCCC. The presented method is simple and efficient with good potentials on the preparation of reference substances, especially on the quality control of Chinese materia medica. © 2014 Tianjin Press of Chinese Herbal Medicines.
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Objective: To develop an efficient preparative method for the separation of Gelsemium alkaloids from Gelsemium elegans. Methods: High-speed counter-current chromatography (HSCCC) with several two-phase solvent systems was investigated for the separation of Gelsemium alkaloids. The purity and structure identification of the purified compounds were performed with HPLC and NMR spectra, respectively. Results: In a single operation, 206.6 mg of crude alkaloid sample was separated to yield 28.7 mg of koumine, 24.9 mg of gelsemine, 26.9 mg of humantenine, and 7.2 mg of gelsevirine, with the purities of 97.8%, 95.4%, 97.4%, and 93.5%, respectively. Conclusion: A preparative HSCCC method is successfully established for the separation of four Gelsemium alkaloids from G. elegans with a modified two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-0.5% triethylamine-H2O (3:5:3:4).
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To optimize the solvent system of high-speed counter-current chromatography (HSCCC) for diterpenoidtanshinone separation and define it's antitumor activity in vitro. Total diterpenoidtanshinone was made by CO2 supercritical fluid extraction (SFE), UPLC was applied to determining the peak area of tanshinone IIA and cryptotanshinone in different solvents up or under phase, and the partition coefficient K values of them were calculated. CKK-8 was used to observe the inhibitory effects of diterpenoidtanshinone on human liver cancer (QGY-7703), lung cancer (PC9, A549), gastric cancer (MKN-45, HGC-27), colon cancer (HCT116), myeloma (U266, RPMI8226), and breast cancer (MCF-7). The best solvent system for HSCCC was petroleum ether-ethyl acetate-methanol-aqua (12∶8∶ 13∶7). The yield of diterpenoidtanshinone was 8.65%. Diterpenoidtanshinone has good effect of antitumor in vitro especially on human PC9 cell. The selected solvent system is suitable for diterpenoidtanshinone separation by HSCCC, and the established HSCCC method is reliable and easy for operating. Diterpenoidtanshinone has good antitumor effect in vitro.
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Objective: Echinacoside and verbascoside are known for their excellent neuroprotective, anti-inflammatory, and anti-oxidative activities, therefore large amount of pure compounds are urgently needed as authentic standards for various in vivo and in vitro studies. Nowadays, they are abundenty extracted from endangered Cistanche spp. Phytochemical studies revealed that Penstemon spp., comprising about 280 species, were rich natural sources for the exploitation and large scale preparation of phenylethanoid glycosides. Thus, rapid isolation and purification of various phenylethanoid glycosides from Penstemon spp. were necessary. Methods: The crude extract of P. digitalis was first enriched by AB-8, and then the high-speed counter-current chromatography (HSCCC) combined with semi-preparative high performance liquid chromatography (HPLC) method was adopted to isolate and purify echinacoside and verbascoside. Results: Eventually, verbascoside (67.2 mg) with the purity of 92.6% and echinacoside (3.96 mg) with the purity of 98.9% were obtained from 5 g powdered leaves of P. digitalis. Conclusion: The present mode of HSCCC coupled with semi-preparative HPLC could be a powerful method for the rapid isolation and purification of other phenylethanoid glycosides. © 2013 Tianjin Press of Chinese Herbal Medicines.
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Objective: To develop a new method for the isolation and preparation of cordycepin by high speed counter-current chromatography (HSCCC) with Cordyceps militaris fermentation broth as raw material. Methods: The solvent systems for cordycepin separation were assessed and selected by HPLC partition coefficient method and analytical HSCCC. A solvent system that consisted of ethyl acetate-n-butanol-0.5% ammonia water (2:3:5) was applied to the separation. The upper phase was used as the stationary phase, while the lower phase was used as the mobile phase for the cordycepin separation by HSCCC. Results: A high efficiency of HSCCC separation was achieved. Finally, cordycepin (43.8 mg) was obtained from 400 mg crude extract of C. militaris fermentation broth in one-step separation with a purity of 98.7%. Conclusion: HSCCC is an efficient and simple method for the large- scale preparation of cordycepin from C. militaris fermentation broth.
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Objective: To develop a method for separation and purification of tanshinone from Salvia miltiorrhiza by combination of silica gel and high-speed counter-current chromatography (HSCCC). Methods: The crude extract of S. miltiorrhiza was separated by silica gel chromatography and F1 and F2 were obtained. Then, F1 and F2 were separated by HSCCC with a two-phase solvent system composed of petroleum ether-methanol-water (4:3:4:2 and 8:5:8:3), respectively. The lower phase was used as the mobile phase with a flow rate of 2.0 mL/min, while the apparatus rotated at 850 r/min and the eluates were detected at 254 nm. The structures of the target compounds were identified by ESI-MS and NMR. Results: From 80 mg of F1, three compounds with tanshinone I (14 mg), dihydrotanshinone I (22 mg), and tanshinone IIA (26 mg) were obtained. And from 80 mg of F2, dihydrotanshinone (11 mg), trijuganone B (15 mg), and cryptotanshinone (30 mg) were obtained. The purities of these six compounds determined by HPLC were all over 96%, respectively. Conclusion: Combination of silica gel and HSCCC is an efficient method for separation of tanshinone from S. miltiorrhiza.
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Objective: The aim of the study was to establish a high speed counter-current chromatography (HSCCC) method for the isolation and purification of alpinetin and cardamomin from Alpinia katsumadai. Methods: Two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5 : 5 : 7 : 3) was used. The flow rate of the mobile phase was 2.0 mL/min, the revolution speed was 800 r/min, the separation temperature was controlled at 25 °C, the reservation ratio of the stationary phase was 50%, and the detection wavelength was 300 nm. Results: Alpinetin (17.2 mg) and cardamomin (25.1 mg) could be obtained from 100 mg of the crude extract in one-step separation by the method. The purities of them were 98.1% and 99.2%, respectively, as determined by HPLC and their chemical structures were identified by 1H-NMR and 13C-NMR. Conclusion: The traditional method, column elution, could not eliminate irreversible adsorption, while the HSCCC method used for the isolation and purification of alpinetin and cardamomin from A. katsumadai has many advantages, such as facility, high efficiency, and high recovery as well.
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Objective To develop an efficient method to isolate and purify the main components isoaloeresin D and aloin from Aloe vera for its industrial production. Methods High-speed counter-current chromatography was used to isolate isoaloeresin D and aloin in a one-step separation from dried crude extract ofA. vera. The biphasic solvent system the lipophilic phase was selected as the mobile phase and the apparatus was rotated at 840 r/min. The effluent was detected at 254 nm. Results Isoaloeresin D (53.1 mg) and aloin (106.9 mg) were separated from the crude extract (384.7 mg) with the purities of 98.6% and 99.5%, respectively. Conclusion HSCCC is a powerful technique for isolation and separation of chemical composition from aloe.
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AIM: To research how to separate the active component in Ligusticum chuanxiong Hort by high speed counter current chromatography. METHODS: Senkyunolide A and Z-ligustilide,the main components of volatile oil in Ligusticum chuanxiong Hort were one step separated by high speed counter current chromatography. n-hexane-ethyl acetate-ethanol-water,1 ∶ 1 ∶ 1 ∶ 1(v/v),was used as the solvent system for HSCCC. Top phase and bottom phase were respectively used as static phase and mobile phase. Optimum speed and flow rate were 900 r/min and 1. 2 mL/min respectively. RESULTS: Collected fractions were analyzed by HPLC and identified by EI-MS and 1HNMR. Purity could reach more than 95% . CONCLUSION: Lactone is fit to be separated and prepared by high speed counter current chromatography with good resolution and high purity. We find a fast and efficient way to separate these.