Application of high-speed counter current chromatography in extraction and separation of alkaloids in natural products / 中国中药杂志

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.

Efficient Isolation of Dihydrophaseic acid 3′-O-β-D-Glucopyranoside from Nelumbo nucifera Seeds Using High-performance Countercurrent Chromatography and Reverse-phased High-performance Liquid Chromatography

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.

Isolation And Development Of Quantification Method For Cyanidin-3-glucoside And Cyanidin-3-rutinoside In Mulberry Fruit By High-performance Countercurrent Chromatography And High-performance Liquid Chromatography

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.

Rapid Isolation of Cyanidin 3-Glucoside and Peonidin 3-Glucoside from Black Rice (Oryza sativa) Using High-Performance Countercurrent Chromatography and Reversed-Phase Column Chromatography

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.

High performance preparation and structural confirmation of lignans from Schisandrae chinensis fructus by using HSCCC combined with ESI-MSn method / 药学学报

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.

Simultaneous isolation and purification of gallic acid and brevifolincarboxylic acid from Polygonum capitatum by high-speed counter-current chromatography / 中国中药杂志

<p><b>OBJECTIVE</b>To isolate and purify gallic acid and brevifolincarboxylic acid simultaneously by high-speed counter-current chromatography (HSCCC) from a crude extract of Polygonum capitatum.</p><p><b>METHOD</b>The 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.</p><p><b>RESULT</b>51.5 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.</p><p><b>CONCLUSION</b>This method is feasible and rapid for isolation and purification of gallice acid and brevifolincarboxylil acid.</p>

Research on technological process of two-pot countercurrent extraction of hydroxysafflor yellow A / 中国中药杂志

<p><b>OBJECTIVE</b>To study the optimum technical conditions of extracting Hydroxysafflor yellow A (HSYA) from Carthmus tinctorius by multi-stage countercurrent extraction technology.</p><p><b>METHOD</b>The 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.</p><p><b>RESULT</b>Extraction 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.</p><p><b>CONCLUSION</b>The 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.</p>

Application of spiral disk column in high-speed counter-current chromatography for peptide and protein separation / 生物工程学报

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.

Preparative separation of gentiopicrin from Radix Gentianae by high-speed counter-current chromatography with macroporous resin / 中国中药杂志

<p><b>OBJECTIVE</b>To develop a method for the preparative separation of gentiopicrin from Radix Gentianae by high-speed counter-current chromatography (HSCCC).</p><p><b>METHOD</b>The 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.</p><p><b>RESULT</b>136 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.</p><p><b>CONCLUSION</b>The established method is simple, high efficiency and suitable for large-scale separation of gentiopicrin.</p>

Advances in studies on multi-stage countercurrent extraction technology in traditional Chinese medicine / 中国中药杂志

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.

Isolation and identification of hetisine-type alkaloids from Aconitum coreanum by high speed countercurrent chromatography / 药学学报

<p><b>AIM</b>To search for more bioactive compounds from the roots of Aconitum coreanum (Lèvl.) Rapaics.</p><p><b>METHODS</b>High speed countercurrent chromatography was successfully applied to the separation of alkaloids from Aconitum coreanum. The structures were elucidated by their physicochemical properties and spectroscopic analysis.</p><p><b>RESULTS</b>Two-phase solvent system composed of CHCl3-CH3OH-0.2 mol x L(-1) HCl (10:3:3, volume ratio) was used in this experiment, eight alkaloids were obtained from the roots of Aconitum coreanum, which were identified as: 2alpha-propionyl-11alpha,13beta-diacetyl-14-hydroxyhetisine (I), Guanfu base P (II), Guanfu base G (III), Guanfu base F (IV), Guanfu base Z (V), Guanfu base O (VI), Guanfu base A (VII), Guanfu base B (VIII).</p><p><b>CONCLUSION</b>Compound I is a new alkaloid, named Guanfu base R.</p>

Purification of ovalbumin from hen egg white by high-speed counter-current aqueous two-phase chromatography / 生物工程学报

High-speed counte-recurrent chromatography (HSCCC) is a continuous liquid-liquid partition chromatography without solid matrix, which has the significant features of high resolution and high recovery. The separation of bio-macromolecule in aqueous two-phase systems (ATPs) with HSCCC is still under research, and the establishment of high-speed counter-current aqueous two-phase chromatography (HSCCC-ATP) relies on the improvement of equipment structure and optimization of operation parameters. By using a multi-column high-speed counter-current chromatograph, the separation of protein mixture and the purification of ovalbumin from hen egg white were studied. The effects of pH and PEG concentration on the partition coefficients of proteins were tested in PEG1000-phosphate ATPs, and distinct differences among partition coefficients of proteins were found at pH 9.2 and 15.0% (W/W) PEG concentration in said system. The separation of protein mixture, consisting of cytochrome C, lysozyme and myoglobin was successfully performed in 15.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 0.8mL/min, using upper phase as stationary phase. pH and PEG concentration also had distinct effects on the partition coefficients of the major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme. The optimal pH value and PEG concentration for the purification of ovalbumin by HSCCC-ATP were found to be 9.2 and 16.0% (W/W) respectively. Ovalbumin was successfully purified to homogeneity from the hen egg white sample in 16.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 1.8mL/min, using upper phase as stationary phase. The purification recovery of ovalbumin was around 95%.

Fast preparative separation of flavones from capitula of Eriocaulon ligulatum (Vell.) L. B. Smith (Eriocaulaceae) by High-speed countercurrent chromatography (HSCCC)

High-speed countercurrent chromatography (HSCCC) is a leading method for the fast separation of natural products from plants. It was used for the preparative isolation of two flavone monoglucosides present in the capitula of Eriocaulon ligulatum (Vell.) L.B.Smith (Eriocaulaceae). This species, know locally as botão-dourado, is exported to Europe, Japan and North America as an ornamental species, constituting an important source of income for the local population of Minas Gerais State, Brazil. The solvent system, optimized in tests prior to the HSCCC run, consisted of the two phases of the mixture ethyl acetate: n-propanol: water (140:8:80, v/v/v), which led to the successful separation of 6-methoxyluteolin-7-O-Beta-D-allopyranoside and 6-methoxyapigenin-7-O-Beta-D-allopyranoside in only 3 hours. The two flavonoids were identified by NMR (1-D and 2-D) and ESI-MS, comparing their spectra with published data

Purification and fingerprinting development of Salvia miltiorrhiza Bunge by high-speed counter-current chromatography / 生物工程学报

In an attempt to apply high-speed counter-current chromatography HSCCC for TCM fingerprints, the separation and purification of the Chinese medicinal plant Salvia miltiorrhiza Bunge of different localities was realized using the technique. The equipments used include a HSCCC (TBE-300) of Shenzhen Tauto Biotech containing three connected preparative coils (diameter of tube = 2.6mm, total volume = 300mL) and a 20mL sample loop and a HPLC from Shimadzu of Japan with a Ultrasphere C18 column (150 x 4.6mm ID, 5microm) and a 20microL sample loop. Salvia miltiorrhiza Bunge samples from 3 locations were separated by HSCCC in a Step-wise elution program with solvent systems A (hexane:ethanol: water = 10:5.5:4.5) and B (hexane:ethanol: water = 10:7:3) at a speed of 900 r/min and a flow-rate of 2mL/min. All the 12 peak fractions were eluted within 13 hours. The contents of each component varied greatly in different samples, which confirmed previous observation that the locations and climates have a great impact on the TCM quality and also indicated a quality control system is necessary to safeguard the quality of the herb. The retention times of the 12 peak fractions from crude extracts of the samples were collected by HPLC and the absorption spectrums of the corresponding peaks were identified. The 12 components of the three crude samples were readily distinguishable and can be used as fingerprints of S. miltiorrhiza Bunge. The relative standard deviation of the HSCCC retention times was less than 3%, which satisfies the requirement of the national standard reference index. The components 7, 8 and 11 from the standards were identified to be crypototanshinone, tanshinone I and tanshinone II A respectively. This study demonstrates that if it is possible to apply HSCCC for TCM fingerprinting, especially with samples of high viscosity and highly absorptive components. The precision and the run time of fingerprinting can be further improved if larger volume and a temperature control system is used. With these and other improvements, HSCCC is expected to play an important role in TCM development.

Dermostatin A and B: chromatography, structural and configurational studies using HPLC, CCD, 13C (125 MHz) and 1H (500 MHz) NMR spectroscopy.

HPLC of crude Dermostatin indicated presence of three pairs of components. Hence, attempts were made to purify Dermostatin. Purification of crude Dermostatin has been carried out using column chromatography and counter current distribution methods. Each of these fractions were tested for activity. The major fraction which showed greater activity was taken for the preparation of Dermostatin nona-acetate. Structural characterisation of Dermostatin nona-acetate has been carried out using UV-visible spectroscopy in different solvents to obtain the characteristic spectrum of a carbonyl conjugated hexaene at room temperature. Structural and configurational studies of Dermostatin nona-acetate using 500 MHz 1H NMR and 125 MHz 13C NMR has been used in the assignment of various functional groups in Dermostatin A and B as well as to provide corroboration to the earlier structural elucidation.

Aortografía a contracorriente por inyección de contraste en arteria periférica / Countercurrent aortography with contrast injection in peripheral artery

Se practicó una aortografía a contracorriente, por inyección de contraste en la arteria radial izquierda, en 8 niños con edades entre 27 días y 42 meses de edad. Con ello fué posible evaluar anastomosis sistémico-pulmonares en 3 e 4 casos, incluyendo uno de 42 meses de edad. En otros 3 casos se pudo visualizar una coartación de la aorta. En el último caso con sospecha clínica de anillo vascular, la aortografía evidenció una arteria subclavia derecha anómala. Concluimos, que el procedimiento es seguro y muy útil para estudiar algunas patologías que comprometen la aorta torácica incluso en niños mayores

Counter-current flow in artificial kidneys

The he parameters controlling the design and function of artificial 1 kidney systems, namely surface area of the transfer medium, [membrane], its permeability as well as operating conditions such as the rate of flow of both the blood and dialysate were correlated, The obtained correlation provides a means For calculating the artificial kidney performance [dialysance] in terms of blood flow rat blood to dialysate flow rate ratio, membrane surface area and membrane permeability : D= Q B[1- [1-k] / [e[1-k] PA / Q[B-k]] < 1 Considering the equation commonly used for calculating membrane permeability i.e. Renkin formula it is worth noting that it can only be applied for specific cases where blood to dialysate flow rates are extremely low and it may be considered as a specific case of the predicted equation