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.
Biological Products ; Countercurrent Distribution/methods* ; Chromatography, High Pressure Liquid/methods* ; Alkaloids/analysis* ; Solvents/chemistry*

High-performance countercurrent chromatography (HPCCC) coupled with reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to isolate dihydrophaseic acid 3′-O-β-D-glucopyranoside (DHPAG) from the extract of Nelumbo nucifera seeds. Enriched DHPAG sample (2.3 g) was separated by HPCCC using ethyl acetate/n-butanol/water system (6:4:10, v/v/v, normal-phase mode, flow rate: 4.0 mL/min) to give 23.1 mg of DHPAG with purity of 88.7%. Further preparative RP-HPLC experiment gave pure DHPAG (16.3 mg, purity > 98%). The current study demonstrates that utilization of CCC method maximizes the isolation efficiency compared with that of solid-based conventional column chromatography.
Chromatography ; Chromatography, Liquid ; Countercurrent Distribution ; Methods ; Nelumbo

Anthocyanins are water soluble plant pigments which are responsible for the blue, red, pink, violet colors in several plant organs such as flowers, fruits, leaves and roots. In recent years, anthocyanin-rich foods have been favored as dietary supplements and health care products due to diverse biological activities of anthocyanins including antioxidant, anti-allergic, anti-diabetic, anti-microbial, anti-cancer and preventing cardiovascular disease. High-performance countercurrent chromatography (HPCCC) coupled with reversed-phase medium pressure liquid chromatography (RP MPLC) method was applied for the rapid and efficient isolation of cyanidin 3-glucoside (C3G) and peonidin 3-glucoside (P3G) from black rice (Oryza sativa L., Poaceae). The crude black rice extract (500 mg) was subjected to HPCCC using two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid (TBME/B/A/0.01% TFA, 1 : 3 : 1 : 5, v/v, flow rate - 4.5 mL/min, reversed phase mode) to give enriched anthocyanin extract (37.4 mg), and enriched anthocyanin extract was sequentially chromatographed on RP-MPLC to yield C3G (16.5 mg) and P3G (8.7 mg). The recovery rate and purity of isolated C3G were 76.0% and 98.2%, respectively, and those of P3G were 58.3% and 96.3%, respectively. The present study indicates that HPCCC coupled with RP-MPLC method is more rapid and efficient than multi-step conventional column chromatography for the separation of anthocyanins.
Anthocyanins ; Cardiovascular Diseases ; Chromatography* ; Chromatography, Liquid ; Countercurrent Distribution* ; Delivery of Health Care ; Dietary Supplements ; Flowers ; Fruit ; Plants ; Trifluoroacetic Acid ; Viola

Cyanidin-3-glucoside (C3G) and cyanidin-3-rutinoside (C3R) were isolated by high-performance countercurrent chromatography (HPCCC) using a two-phase solvent system composed of tert-butyl methyl ether/n-butanol/acetonitrile/water/trifluoroacetic acid (1 : 3 : 1 : 5 : 0.01, v/v) to give pure C3G (34.1 mg) and C3R (14.3 mg) from 1.5 g crude mulberry fruit extract. Using the pure C3G and C3R, a reliable high-performance liquid chromatography (HPLC) method was developed and validated to determine the C3G and C3R contents in mulberry fruit. C3G and C3R were separated simultaneously using an Eclipse XDB-C18 column (4.6 x 250 mm I.D., 5 microm) coupled with a photodiode array detector (PDA). The gradient elution of the mobile phase consisting of acetonitrile (0.5% formic acid) and water (0.5% formic acid) was applied (1.0 mL/min), and the detection wavelength was 520 nm. The calibration curves of C3G and C3R showed good linearity (both with r2 = 0.9996) in the concentration range 15.625 - 500 microg/mL, and the relative standard deviations (RSD%) of intra- and interday variability were in the ranges 2.1 - 8.2% and 4.1 - 17.1%, respectively. The accuracies were ranged 96.5 - 102.6% for C3G and C3R, respectively. The developed HPLC method was used to determine the contents of C3G and C3R in newly harvested mulberry from eight different provinces of Korea.
Calibration ; Chromatography, High Pressure Liquid ; Chromatography, Liquid* ; Countercurrent Distribution* ; Fruit* ; Korea ; Morus* ; Water

High-speed counter-current chromatography (HSCCC) was used to high performance separate and prepare lignans from Schisandrae chinensis fructus. The solvent system is composed of n-hexane-ethyl acetate-methanol-water (9 : 1 : 5 : 5) and n-hexane-ethyl acetate-methanol-water (9 : 1 : 9 : 5), speed is at 900 r.min-1, and flow rate is at 2.0 mL.min-1. Five fractions from Schisandrae chinensis fructus extract were separated and prepared with one HSCCC process. They were identified as schisandrin, gomisin J, schisandrol B, schisantherin A and deoxyschizandrin by electrospray ionization-multiple tandem mass spectrometry (ESI-MSn), respectively. Their contents were obtained in 98.74%, 94.32%, 99.53%, 94.23% and 98.68% by ultra high performance liquid chromatography (UPLC), separately. The rapid and simple method can be applied for the preparation of lignans from Schisandrae chinensis fructus.
Countercurrent Distribution ; Cyclooctanes ; chemistry ; isolation & purification ; Dioxoles ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Fruit ; chemistry ; Lignans ; chemistry ; isolation & purification ; Molecular Structure ; Plants, Medicinal ; chemistry ; Polycyclic Compounds ; chemistry ; isolation & purification ; Schisandra ; chemistry ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry

OBJECTIVETo isolate and purify gallic acid and brevifolincarboxylic acid simultaneously by high-speed counter-current chromatography (HSCCC) from a crude extract of Polygonum capitatum.

METHODThe biphasic solvent system composed of ethyl acetate-n-butanol-0.44% acetic acid (3:1:5) was used at a flow rate of 2.0 mL x min(-1), while the aqueous phase was selected as the mobile phase and the apparatus was rotated at 860 r x min(-1). The effluent was detected at 272 nm.

RESULT51.5 mg of gallic acid and 5.9 mg of brevifolincarboxylic acid were separated from 1.07 g of the crude extract with the purities of 99.7% and 97.5%, respectively, while brevifolincarboxylic acid was obtained firstly from the genus Polygonum. The structures of the compounds were identified by ultraviolet spectrometry (UV), infra-red spectrometry (IR), liquid chromatography/mass spectrometry (LC/MS), time-of-flight mass spectrometry( TOF-MS), 1H-nuclear magnetic resonance (NMR) and 13C-NMR.

CONCLUSIONThis method is feasible and rapid for isolation and purification of gallice acid and brevifolincarboxylil acid.

Carboxylic Acids ; analysis ; isolation & purification ; Countercurrent Distribution ; methods ; Gallic Acid ; analysis ; isolation & purification ; Plant Extracts ; analysis ; isolation & purification ; Polygonum ; chemistry

In order to improve the stationary phase retention of polar solvent systems and aqueous two-phase systems (ATPSs), we designed a multiple spiral disk assembly for type-J high-speed counter-current chromatography (HSCCC). The stationary phase retention was studied under different elution modes by using two solvent systems that contained 1-butanol-acetic acid-water (4:1:5, V/V/V) and polyethylene glycol (PEG) 1000-K2HPO4-water (12.5:12.5:75, W/W/W). The best retention was obtained in L-I-T, U-O-H, L-I-H three modes by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a relatively high flow rate. Meanwhile, the relationship between retention percentage of the stationary phase (Sf) and various parameters such as flow-rate (F), rotation speed (w) and column temperature (T) was also studied. Sf increased with the increase of w and decreased with the increase of F. Regression analysis showed a linear relationship between Sf and F1/2/w. The influence of T on Sf was not obvious between 20 degrees C and 40 degrees C, lower temperature than 20 degrees C was not suitable for viscous ATPSs. Acceptable resolutions were achieved when it was applied for the separation of dipeptides including Leu-Tyr and Val-Tyr by using 1-butanol-acetic acid-water (4:1:5, V/V/V) solvent system. The proteins including cytochrome C and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins were well separated by 12.5% PEG1000-12.5% K2HPO4-75% water (pH 9.0) and 16% PEG 1000-12.5% K2HPO4-71.5% water (pH 8.0) system.
Countercurrent Distribution ; instrumentation ; methods ; Peptides ; isolation & purification ; Proteins ; isolation & purification

OBJECTIVETo study the optimum technical conditions of extracting Hydroxysafflor yellow A (HSYA) from Carthmus tinctorius by multi-stage countercurrent extraction technology.

METHODThe effects of extraction time of each stage, extraction temperature, ethanol concentration and solid-liquid ratio (g x mL(-1)) on extraction yield of HSYA were studied by orthogonal test design and the comparison of other extraction methods were presented.

RESULTExtraction time and solid-liquid ratio had significant influence on the extraction yield, and the optimum parameters were as follows: Extraction time of each stage was 120 min, solid-liquid ratio was 1 : 10 (g x mL(-1)), ethanol concentration was 30%, and extracted at room temperature. Under the optimum conditions, the extraction yield of HSYA was 1.56% and the purity of the extract was 6.06%. Compared with the traditional extraction method and the ultrasonic extraction method of the pharmacopoeia, the extraction yield was increased by 6.12% and 9.09%, the purity of extract was increased by 42.9% and 27.0%, respectively.

CONCLUSIONThe multi-stage countercurrent extraction technology has many advantages such as simple operation, less solvent consumption, higher extraction yield and purity of extract and it has wide industrial application prospect.

Carthamus ; chemistry ; Chalcone ; analogs & derivatives ; analysis ; isolation & purification ; Countercurrent Distribution ; methods ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Quinones ; analysis ; isolation & purification

OBJECTIVETo develop a method for the preparative separation of gentiopicrin from Radix Gentianae by high-speed counter-current chromatography (HSCCC).

METHODThe crude alcohol extracts were eluted on a macroporous resin column and then purified by high speed counter-current chromatography (HSCCC). A two-phase solvent system composed of ethyl acetate: n-butanol: water (2 : 1 : 3) was used, and the lower phase was used as the mobile phase at a flow rate of 1.5 mL x min(-1), while the apparatus rotated at 800 r x min(-1) and the eluate was detected at 254 nm.

RESULT136 mg gentiopicrin with purity of 99.6% determined by HPLC were obtained from 300 mg crude extraction only in one-step separation and less than 200 minutes.

CONCLUSIONThe established method is simple, high efficiency and suitable for large-scale separation of gentiopicrin.

Chromatography, High Pressure Liquid ; Countercurrent Distribution ; Gentiana ; chemistry ; Glucosides ; isolation & purification ; Iridoid Glucosides ; Iridoids ; isolation & purification ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Resins, Synthetic ; Rhizome ; chemistry

Multi-stage countercurrent extraction technology, integrating solvent extraction, repercolation with dynamic and countercurrent extraction, is a novel extraction technology for the traditional Chinese medicine. This solvent-saving, energy-saving and high-extraction-efficiency technology can at the most drive active compounds to diffuse from the herbal materials into the solvent stage by stage by creating concentration differences between the herbal materials and the solvents. This paper reviewed the basic principle, the influence factors and the research progress and trends of the equipments and the application of the multi-stage countercurrent extraction.
Countercurrent Distribution ; methods ; Drugs, Chinese Herbal ; isolation & purification ; Particle Size ; Plants, Medicinal ; chemistry ; Solvents ; chemistry ; Technology, Pharmaceutical ; instrumentation ; methods ; Temperature ; Time Factors