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.

Determination of Calcipotriol Ointment by High-speed Counter-current Chromatography / 中国药师

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.

Application of Multi-stage Countercurrent Extraction Technology in the Extraction Process of Aesculi / 世界科学技术-中医药现代化

This study was aimed to optimize the extraction process of Aesculi. The extraction process of Aesculi wasoptimized through orthogonal experiment while the transfer rate of escin Ia and escin Ib. And the yield of dry extract. wasadopted as marks. And ratio of solid to liquid, the ethanol concentration, extraction time and extraction temperature werestudies. The results showed that optimized extraction conditions of Aesculi were determined as follows. The ratio of solidto liquid was 1: 10, the ethanol concentration was 70％, extracted for 20 minutes ever stage and the extractiontemperature was 60℃. It was concluded that the extraction process is efficacious for general flavone and icariinextraction. The method is suitable for the standardized production technology of Aesculi.

Isolation of quinoline alkaloids from three Choisya species by high-speed countercurrent chromatography and the determination of their antioxidant capacity

Abstract Choisya ternata Kunth, C. ternata var. sundance Kunth and the hybrid Choisya 'Aztec-Pearl' are three related species belonging to the Rutaceae family. Ethanol extracts were prepared from the leaves of these three species and evaluated in relation to their antioxidant activity using in vitro and ex vivo models. The ethanol extracts belonging to the three species produced a very high antioxidant profile as evidenced by the DPPH radical scavenging activity, the determination of total phenolics and flavonoid equivalent. The generation of reactive species of oxygen in leukocytes stimulated with LPS was dramatically reduced when the three ethanol extracts were used. The alkaloids anhydroevoxine and choisyine were isolated from the ethanol extract of C. ternata using HEMWat (4:6:5:5) as the solvent system by means of high-speed countercurrent chromatography. This was the first time quinoline alkaloids were isolated from this species using HSCCC. These compounds were also assayed for their capacity to inhibit the generation of ROS in leukocytes stimulated by LPS and the results also suggested that they are reactive oxygenase inhibitors.

Diterpenes and a new benzaldehyde from the mangrove plant Rhizophora mangle

ABSTRACT This work describes the isolation, by high-speed counter-current chromatography, of the diterpenes manool, jhanol and steviol and the benzaldehyde p-oxy-2-ethylhexyl benzaldehyde from the stilt roots hexane extract of the mangrove plant Rhizophora mangle L., Rhizophoraceae. For this, a non-aqueous biphasic solvent system composed of hexane–acetonitrile–methanol 1:1:0.5 (v/v/v) was applied. As far as we know, only steviol was previously isolated in Rhizophoraceae and this is the first time that p-oxy-2-ethylhexyl benzaldehyde is reported.

Preparative Isolation and Purification of Garcinol by High-Speed Countercurrent Chromatography / 世界科学技术-中医药现代化

This study aimed at preparing the chemical reference substance of garcinol.A preparative high-speed countercurrent chromatography (HSCCC) was adopted for the isolation of garcinol from the twigs of Garcinia multifiora Champ,G.esculenta Y.H.Li and the fruits of G.xanthochymus Hook.f.ex T.Anders.The crude extracts were separated by HSCCC with two phase solvent systems composed of n-hexane-ethyl acetate-95％ ethanol-water (8∶8∶12∶4,volume ratio) using the upper stationary phase and the lower mobile phase.Under the conditions of a flow rate of 2.0 and 4.0 mL· min-1,and the apparatus rotation at 850 rpm,the column temperature at 25℃ and the detection wavelength at 254 nm,the fraction containing garcinol was purified by the Sephadex LH20 chromatography.The purity test in the extracts was determined by high-performance liquid chromatography.As a result,the contents of garcinol were 45,281 and 4080mg respectively separated from 104.765 g twigs of G.multiflora Champ,105.270 g G.esculenta Y.H.Li and 102.318 g the fruits of G.xanthochymus Hook.f.ex T.with the purity of 98.72％,98.36％ and 98.42％.Compared with the sample pretreatments and separation efficiency of the three plants,it was found that the fat-soluble extracts rapidly and efficiently extracted using n-hexane-ethyl acetate-95％ ethanol-water (5∶5∶5∶5,volume ratio) contained abundant garcinol taking fruits of G.xanthochymus Hook.f.ex T as the raw material with the combination of HSCCC and Sephadex LH-20 chromatography.In conclusion,it was indicated that the combination method is efficient with high operability and large preparation quantity.

Isolation and preparation of leonurine from Leonurus japonicus by high speed countercurrent chromatography / 中草药

Objective: To establish a simple and effective method for the isolation of leonurine from Leonurus japonicus by high speed countercurrent chromatography (HSCCC). Methods: The extraction conditions of leonurine were optimized by one-factor experimental design. After comparing several different solvent systems, the two phase system of ethyl acetate-n-butanol-water (3∶2∶5) was finally chosen as operating solvent of HSCCC for the separation of leonurine, in which the lower phase was determined as the mobile phase and the upper phase as stationary. The detection of eluates was performed with an ultraviolet detector at 277 nm. The rotation speed was adjusted at 850 r/min, and the flow rate was 2.2 mL/min. Results: Leonurine was successively isolated from n-butanol fraction by HSCCC, and the above established method was also successively applied to the crude extact of L. japonicus. Finally, 68 mg of leonurine with purity about 96.2% could be obtained from 2.48 g crude extract of L. japonicus in a single injection. Conclusion: The described approaches actively promote efficient preparation strategy to obtain high purity of leonurine.

Separation and purification of emodin-8-O-β-D-glucopyranoside from Polygoni Cuspidati Rhizoma et Radix by column and high-speed counter-current chromatography / 中草药

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.

Relevant factors analysis of hysterosalpingography countercurrent / 实用放射学杂志

Objective To study on the factors of countercurrent occured in hysterosalpingography to improve the understanding of countercurrent.Methods 180 patients who underwent hysterosalpingography due to infertility were recruited,and 63 of them who were involved in countercurrent in the process of hysterosalpingography were analyzed statistically.Results The single factor analy-sis demonstrated that such four factors of primary/secondary infertility,menstrual clean days,tubal obstruction or not,and depth of cannula were associated with countercurrent,while Logistic regression analysis indicated that the factors of menstrual clean days, tubal obstruction or not,and depth of cannula during the hysterosalpingography operation were closely related.Conclusion Counter-current are caused by the comprehensive impact of several comprehensive factors like menstrual clean day,tubal obstruction or not, and cannula operation.Therefore adequate preparation should be made before and during the operation,to reduce the occurrences of countercurrent.

Advances in Technology of Countercurrent Chromatography for Separation of Protein and Peptide / 分析化学

Countercurrent chromatography us a kund of contunuous and effectuve luquud-luquud partutuon chromatography wuth many unuque characterustucs such as large load capacuty, no urreversuble adsorptuon, hugh recovery rate, low rusk of sample denaturatuon and so on, whuch has urreplaceable advantages un separatuon of proteun and peptude. Thus revuew presents the advances of several kunds of new technology of countercurrent chromatography un the separatuon of proteun and peptude. The developung prospect of thus fueld us also duscussed.

Purification of Puerarin from Pueraria lobata by FCPC versus HSCCC Using Small-volume Columns / 中草药·英文版

Objective: To develop an efficient method for separating and purifying puerarin from the roots of Pueraria lobata. Methods: Separation by fast centrifugal partition chromatography (FCPC) was processed with a biphasic solvent system composed of ethyl acetate- n-butanol-water (2:1:3). The separation conditions were determined as follows: sample loading of 10 mg, flow rate of 2 mL/min, rotation speed of 2200 r/min, ascending mode, and detection wavelength of 254 nm. High speed countercurrent chromatography (HSCCC) was used as a comparative method with the rotation speed of 800 r/min, flow rate of 2.0 mL/min, and sample loading of 10 mg in tail-to-head mode. Results: Puerarin was obtained by FCPC with a resolution of 0.90 and a purity above 99%, while a resolution below 0.50 and a purity below 90% by HSCCC. Compared with HSCCC, FCPC has the advantages with higher purity and better resolution. Conclusion: FCPC is a powerful method to separate and purify puerarin. © 2014 Tianjin Press of Chinese Herbal Medicines.

Separation of Five Quinolone Alkaloids from Fruits of Evodia rutaecarpa by High-speed Counter-current Chromatography / 中草药·英文版

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.

Preparative Separation of Four Alkaloids from Gelsemium elegans by High-speed Counter-current Chromatography / 中草药·英文版

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).

Study on Extraction Process of Ferulic Acid from Angelicae Sinensis Radix with Technology of Three Can Group Dynamic Countercurrent / 中国中医药信息杂志

Objective To optimize technology of three can group dynamic countercurrent extraction process of ferulic acid from Angelicae Sinensis Radix.Methods The content of ferulic acid was determined by HPLC. With content of ferulic acid as index, comprehensive test was used to investigate effect of extraction solvent and extraction time on extraction efficiency.Results The optimum process parameters were as follows:extraction solvent with 10 times of water;20 minutes for each extraction time.Conclusion The process which uses method of three can group of dynamic countercurrent extraction of ferulic acid from Angelicae Sinensis Radix is reliable, highly efficient and energy saving.

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.

Research progress in new technologies for extraction and separation of Chinese materia medica / 中草药

Extraction and separation technologies appeared in recent years are particularly reviewed in the principles, features, latest research and application results, and existing problems as well. These new techniques include ultrahigh pressure extraction (UPE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), pressurized solvent extraction (PSE), smashing tissue extraction (STE), heating-free extraction (HFE), high-speed countercurrent chromatography (HSCCC), and molecular imprinting technique (MIT). Further research topics and application prospects of these extraction and separation technologies are also suggested, so as to provide the reference for the research and production of modern Chinese materia medica.

Studies on HSCCC preparation technology of diterpenoidtanshinone and its antitumor activity in vitro / 中草药

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.

Separation and purification of coumarins from Heracleum millefolium by high-speed counter-current chromatography / 中草药

Objective: To separate and purify coumarins from Heracleum millefolium by high-speed counter-current chromatography (HSCCC). Methods: HPLC was used to analyze and optimize the solvent system and hexane-ethyl acetate-water-methanol (1.5:2.5:2:1.5) was chosen as the two-phase solvent system of HSCCC, in which the upper phase was used as the stationary phase, while the lower phase was used as the mobile phase with flow rate of 3 mL/min, the revolution speed was set at 850 r/min, and detected at 323 nm. Three compounds were obtained as crystals by vacuum concentration at 50℃, and determined through HPLC, then their structures were identified by IR, ESI-MS, 1H-NMR, and 13C-NMR. Results: Columbianetin acetate (6.3 mg), osthole (10.6 mg), and columbianadin (6.4 mg) were obtained from crude sample (300 mg) extracts and their purity were above 96.0%. Conclusion: HSCCC is a powerful technique for the rapid separation and purification of coumarins from crude extract of H. millefolium.

Application of Dynamic Ultrasound Countercurrent Extraction Technology in Industrialization of Lianhua Qingwen Capsule / 中国中医药信息杂志

Objective To study the application of dynamic ultrasound countercurrent extraction technology in industrialization of Lianhua Qingwen Capsule by multi-index optimization. Methods Ultrasonic temperature and ultrasonic time were investigated by using single factor design. The three factors of dry extract yield, the active ingredient content, and fingerprints were selected as the optimization indexes to investigate the solvent consumption and extraction time between the dynamic ultrasound countercurrent extraction process and the original reflux extraction process under the fixed conditions of process parameters. Results Compared with traditional reflux extraction technology, dynamic ultrasound countercurrent extraction technology effectively reduced the alcohol consumption and saved extraction time on the basis of guarantee of the active ingredient content. Conclusion Dynamic ultrasound countercurrent extraction process, with simple and fast advantages, can be used to Lianhua Qingwen Capsule production, which can significantly reduce production costs and increase productivity.