Isolation and purification of macrocyclic components from Penicillium fermentation broth by high‐speed counter‐current chromatography

In this paper, high‐speed counter‐current chromatography (HSCCC), assisted with ESI‐MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7′‐O‐formylbrefeldin A (6.5 mg, 95.0%) and 7′‐O‐acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK‐15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two‐step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two‐step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n‐hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X‐ray crystallography, ESI‐MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.     

Enrichment and isolation of barbigerone from Millettia pachycarpa Benth. using high‐speed counter‐current chromatography and preparative HPLC

Enrichment of the anti‐tumor compound barbigerone along with a rotenoid derivative from Millettia pachycarpa Benth. was performed by a two‐step high‐speed counter‐current chromatography (HSCCC) separation process. In the first step, 155.8 mg of target fraction (Fra6) was obtained from 400 mg ethyl acetate extract of M. pachycarpa Benth. with an increase in barbigerone from 5.1 to 13% via HSCCC using a solvent system of n‐hexane–ethyl acetate–methanol–water (5:4:5:3, v/v) under normal phase head to tail elution. HSCCC was repeated to eliminate the major contaminant in this initial fraction 6. After a separation time of 65 min, 22.1 mg barbigerone of 87.7% purity was obtained from Fra6 with the ternary solvent system of n‐hexane–methanol–water (2:2:1, v/v) under normal phase elution. Finally, preparative HPLC was employed for the further isolation of barbigerone and the rotenoid derivative. The structures were confirmed by ESI‐MS, ¹H NMR and ¹³C NMR.     

Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high‐speed counter‐current chromatography in stepwise elution mode

Preparative high‐speed counter‐current chromatography (HSCCC) was successfully applied to the isolation and purification of three stilbene oligomers from Vitis chunganeniss using stepwise elution with a pair of two‐phase solvent systems composed of n‐hexane–ethyl acetate–methanol–water at (2:5:2:5, v/v) and (1:2:1:2, v/v). The preparative HSCCC separation was performed on 800 mg of crude sample yielding hopeaphenol (21.1 mg), amurensin G (37.2 mg) and vitisin A (95.6 mg) in a one‐step separation, with purities over 95% as determined by HPLC. The structures of these three compounds were identified by MS, ¹H NMR and ¹³C NMR. In addition, their antioxidant activities were screened by DPPH assay, where vitisin A showed strong antioxidant activity. Further EPR experiments with spin‐trapping technique demonstrated that vitisin A is a potent and selective singlet oxygen quencher, which may be used in singlet oxygen‐mediated diseases as a pharmacological agent.     

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high‐speed counter‐current chromatography

An efficient strategy for extracting and separating five lignans from Schisandra chinensis (Turcz.) Baill has been developed using supercritical fluid extraction (SFE) and high‐speed counter‐current chromatography (HSCCC) in the present study. First, the extraction was performed by a preparative SFE system under 15 MPa of pressure at 36°C for 4 h. Then, the SFE extract was successfully separated and purified by HSCCC with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (6:4:5:5, 6:4:6:4, 6:4:8:2, v/v) in a stepwise elution mode. The fractions were analyzed by HPLC, and the chemical structures of the products were identified by ESI‐MS and ¹H NMR spectroscopy. As a result, a total of 12.5 mg of schisandrin at 98.0% purity, 7.1 mg of gomisin A at 98.1% purity, 1.8 mg of schisantherin B at 93.3% purity, 4.4 mg of deoxyschisandrin at 92.9% purity, and 6.8 mg of γ‐schisandrin at 89.1% purity were obtained from 300 mg crude extract in a one‐step purification.     

Rapid preparative separation of six bioactive compounds from Gentiana crassicaulis Duthie ex Burk. using microwave‐assisted extraction coupled with high‐speed counter‐current chromatography

A rapid method combining microwave‐assisted extraction (MAE) and high‐speed counter‐current chromatography (HSCCC) was applied for preparative separation of six bioactive compounds including loganic acid ( I ), isoorientin‐4′‐O‐glucoside ( II ), 6′‐O‐β‐d ‐glucopyranosyl gentiopicroside ( III ), swertiamarin ( IV ), gentiopicroside ( V ), sweroside ( VI ) from traditional Tibetan medicine Gentiana crassicaulis Duthie ex Burk. MAE parameters were predicted by central composite design response surface methodology. That is, 5.0 g dried roots of G. crassicaulis were extracted with 50 mL 57.5% aqueous ethanol under 630 W for 3.39 min. The extract (gentian total glycosides) was separated by HSCCC with n‐butanol/ethyl acetate/methanol/1% acetic acid water (7.5:0.5:0.5:3.5, v/v/v/v) using upper phase mobile in tail‐to‐head elution mode. 16.3, 8.8, 12., 25.1, 40.7, and 21.8 mg of compounds I–VI were obtained with high purities in one run from 500 mg of original sample. The purities and identities of separated components were confirmed using HPLC with photo diode array detection and quadrupole TOF‐MS and NMR spectroscopy. The study reveals that response surface methodology is convenient and highly predictive for optimizing extraction process, MAE coupled with HSCCC could be an expeditious method for extraction and separation of phytochemicals from ethnomedicine.     

Preparative separation of capsaicin and dihydrocapsaicin from Capsicum frutescens by high‐speed counter‐current chromatography

Capsaicin and dihydrocapsaicin are two main bioactive components of Capsicum frutescens and are widely used as food additives and drugs in China and India. Due to their similarity in structures, isolation of capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography, normal‐phase thin‐layer chromatography (TLC) becomes difficult. This study involves separating capsaicin and dihydrocapsaicin with sufficient purity and recovery using high‐speed counter‐current chromatography (HSCCC) with a solvent system composed of n‐hexane–ethyl acetate–methanol–water–acetic acid (20:20:20:20:2, v/v/v/v/v). Separation parameters such as sample volume, and sample concentration were first optimized on analytical HSCCC, and then scaled up to preparative HSCCC. 0.65 g capsaicin and 0.28 g dihydrocapsaicin were obtained from 1.2 g crude extract and their purities were 98.5 and 97.8%, respectively. The recoveries of the two compounds were 86.3 and 85.4%, respectively. The purity of the isolated compounds was analyzed by high‐performance liquid chromatography (HPLC) and their structures were identified by ¹H nuclear magnetic resonance (NMR) and ¹³C NMR analysis.     

A consecutive preparation method based upon accelerated solvent extraction and high‐speed counter‐current chromatography for isolation of aesculin from Cortex fraxinus

A consecutive preparation method based upon accelerated solvent extraction (ASE) coupled with high‐speed counter‐current chromatography (HSCCC) was presented and aesculin was obtained from Cortex fraxinus. The extraction condition of ASE was optimized with response surface methodology; some significant parameters such as the solvent system and its stability, the amount of loading sample in HSCCC were also investigated. The original sample was first extracted with methanol at 105°C and 104 bar for 7 min using ASE, then the extracts were consecutively introduced into the HSCCC system and separated and purified with the same ethyl acetate/n‐butanol/water (7:3:10, v/v/v) solvent system for five times without further exchange and equilibrium. About 3.1 ± 0.2 mg/g in each time and total of 15.4 mg/g aesculin with purity over 95% was isolated from Cortex fraxinus. The results demonstrated that the consecutive preparation method was time and solvent saving and high throughput, it was suitable for isolation of aesculin from Cortex fraxinus, and also has good potential on the separation and purification of effective compounds from natural product.     

Separation and purification of five phenylpropanoid glycosides from Lamiophlomis rotata (Benth.) Kudo by a macroporous resin column combined with high‐speed counter‐current chromatography

Five phenylethanoid glycosides (PhGs), forsythoside B, verbascoside, alyssonoside, isoverbascoside, and leucosceptoside B, were isolated and purified from Lamiophlomis rotata (Benth.) Kudo by high‐speed counter‐current chromatography (HSCCC) combined with macroporous resin (MR) column separation. In the present study, the two‐phase solvent system composed of ethyl acetate/n‐butanol/water (13:3:10, v/v/v) was used for HSCCC separation. A total of 27 mg of forsythoside B, 41 mg of verbascoside, 29 mg of alyssonoside, 23 mg of isoverbascoside, and 13 mg of leucosceptoside B with purities of 97.7, 99.2, 99.5, 99.3, and 97.3%, respectively, were obtained in a one‐step separation within 4 h from 150 mg of crude extract. The recoveries of the five PhGs after MR‐HSCCC separation were 74.5, 76.5, 72.5, 76.4, and 77.0%, respectively. The chemical structures of all five compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Separation of phenylsuccinic acid enantiomers using biphasic chiral recognition high‐speed countercurrent chromatography

High‐speed countercurrent chromatography (HSCCC) combined with biphasic chiral recognition was successfully applied to the resolution of phenylsuccinic acid enantiomers. d ‐Isobutyl tartrate and hydroxypropyl‐β‐cyclodextrin were employed as lipophilic and hydrophilic selectors dissolved in the organic stationary phase and aqueous mobile phase, respectively. The two‐phase solvent system was made up of n‐hexane/methyl tert‐butyl ether/water (0.5:1.5:2, v/v/v). Impacts of the type and concentration of chiral selectors, the pH value of the aqueous phase solution as well as the temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomer was obtained by separating 810 mg of racemate with a purity >99.5% and a recovery rate between 82 and 85%. The experimental results indicate that biphasic recognition HSCCC provide a promising means for efficient separation of racemates.     

Preparative isolation of ganoderic acid S,ganoderic acid T and ganoderol B from Ganoderma lucidum mycelia by high‐speed counter‐current chromatography

Ganoderic acid S, ganoderic acid T and ganoderal B are the main bioactive triterpenes of Ganoderma lucidum. In this study, mycelia of G. lucidum were obtained by two‐stage fermentation and then extracted by ethanol and petroleum ether sequentially to obtain crude triterpenes. The crude sample was further purified by recycling high‐speed counter‐current chromatography with n‐hexane–ethyl acetate–methanol–water (7:12:11:5, v/v/v/v) as the optimized two‐phase solvent system. A 16.4 mg aliquot of ganoderol B with a purity of 90.4% was separated from 300 mg of the crude sample in a single run. After employing the recycling elution mode of HSCCC with n‐hexane–ethyl acetate–methanol–water (6:10:8:4.5, v/v/v/v) for five cycles, 25.7 mg ganoderic acid T and 3.7 mg ganoderic acid S with purities of 97.8 and 83.0%, respectively, were obtained. The purities of three compounds were determined by high‐performance liquid chromatography and their chemical structures were identified by NMR and MS data.     

Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.     

An efficient combination of supercritical fluid extraction and high‐speed counter‐current chromatography to extract and purify homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker‐Gawler

Supercritical fluid extraction (SFE) was used to extract homoisoflavonoids from Ophiopogon japonicus (Thunb.) Ker‐Gawler. The optimization of parameters was carried out using an orthogonal test L₉ (3)⁴ including pressure, temperature, dynamic extraction time and the amount of modifier. The process was then scaled up by 100 times with a preparative SFE system under the optimized conditions of 25 MPa, 55°C, 4.0 h and 25% methanol as a modifier. Then crude extracts were separated and purified by high‐speed counter‐current chromatography (HSCCC) with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/ACN/water (1.8:1.0:1.0:1.2:1.0 v/v). There three homoisoflavonoidal compounds including methylophiopogonanone A 6‐aldehydo‐isoophiopogonone A, and 6‐formyl‐isoophiopogonanone A, were successfully isolated and purified in one step. The collected fractions were analyzed by HPLC. In each operation, 140 mg crude extracts was separated and yielded 15.3 mg of methylophiopogonanone A (96.9% purity), 4.1 mg of 6‐aldehydo‐isoophiopogonone A (98.3% purity) and 13.5 mg of 6‐formyl‐isoophiopogonanone A (97.3% purity) respectively. The chemical structure of the three homoisoflavonoids are identified by means of ESI‐MS and NMR analysis.     

Efficient method for screening and identification of radical scavengers in the leaves of Olea europaea L

In this article, an efficient method was developed to screen, isolate and identify the major radical scavengers in the leaves of Olea europaea L. by DPPH‐HPLC‐DAD, HSCCC and NMR. The method of DPPH‐HPLC‐DAD was used to screen the major radical scavengers. It was found that three major constituents (A, B, C) in the extract of the leaves of O. europaea L. possessed potential antioxidant activities. In order to identify the chemical structures of those compounds, the HSCCC method with a two‐phase solvent system composed of petroleum ether–ethyl acetate–water at an optimized volume ratio of 6:600:700 (v/v/v) together with column chromatography was developed to isolate and purify the active compounds. Pure compounds A (225 mg), B (10 mg) and C (12 mg) with purities 92.6, 95.1 and 96.4%, respectively, were obtained from the crude sample (500 mg). Their structures were identified as oleuropein (A), luteolin‐7‐O‐glucoside (B) and verbascoside (C) by ¹H‐NMR and ¹³C‐NMR. Copyright © 2010 John Wiley & Sons, Ltd.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

Separation of five oligostilbenes from Vitis amurensis by flow‐rate gradient high‐performance counter‐current chromatography

A rapid and efficient high‐performance counter‐current chromatography (HPCCC) method was developed to separate five oligostilbenes from the roots of Vitis amurensis. An n‐hexane/ethyl acetate/methanol/water system (4:8:4:10, v/v/v/v) was selected as an optimal two‐phase solvent system of which the upper phase was used as the stationary phase and the lower phase was used as the mobile one. Partition coefficient values for the target compounds under these optimized conditions were 0.28 ( 1 , ampleosin A), 7.12 ( 2 , (+)‐g‐viniferin), 2.26 ( 3 , vitisin A), 5.38 ( 4 , wilsonol C), and 11.23 ( 5 , vitisin B). Flow‐rate gradient HPCCC (4 mL/min in 0–70 min, 8 mL/min in 70–250 min) was applied to isolate the target compounds in as high purity as possible within the shortest possible run time. Under these conditions, ampelopsin A (12.1 mg), (+)‐g‐viniferin (10.4 mg), vitisin A (2.8 mg), wilsonol C (3.2 mg), and vitisin B (37 mg) were isolated with >95% purity from 150 mg of enriched oligostilbene extract. Although the K_D of the last eluted compound, vitisin B (K_D = 11.23), was relatively large, it was eluted in 115–145 min using the two‐phase solvent system. This study shows that HPCCC is an efficient tool for the isolation and purification of natural products.     

Separation of phenolic acids from sugarcane rind by online solid‐phase extraction with high‐speed counter‐current chromatography

Sugarcane rind contains some functional phenolic acids. The separation of these compounds from sugarcane rind is able to realize the integrated utilization of the crop and reduce environment pollution. In this paper, a novel protocol based on interfacing online solid‐phase extraction with high‐speed counter‐current chromatography (HSCCC) was established, aiming at improving and simplifying the process of phenolic acids separation from sugarcane rind. The conditions of online solid‐phase extraction with HSCCC involving solvent system, flow rate of mobile phase as well as saturated extent of absorption of solid‐phase extraction were optimized to improve extraction efficiency and reduce separation time. The separation of phenolic acids was performed with a two‐phase solvent system composed of butanol/acetic acid/water at a volume ratio of 4:1:5, and the developed online solid‐phase extraction with HSCCC method was validated and successfully applied for sugarcane rind, and three phenolic acids including 6.73 mg of gallic acid, 10.85 mg of p‐coumaric acid, and 2.78 mg of ferulic acid with purities of 60.2, 95.4, and 84%, respectively, were obtained from 150 mg sugarcane rind crude extracts. In addition, the three different elution methods of phenolic acids purification including HSCCC, elution–extrusion counter‐current chromatography and back‐extrusion counter‐current chromatography were compared.     

Separation of three phenolic high‐molecular‐weight compounds from the crude extract of Terminalia Chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography

This study presents an efficient strategy for separation of three phenolic compounds with high molecular weight from the crude extract of Terminalia chebula Retz. by ultrasound‐assisted extraction and high‐speed counter‐current chromatography. The ultrasound‐assisted extraction conditions were optimized by response surface methodology and the results showed the target compounds could be well enriched under the optimized extraction conditions. Then the crude extract was directly separated by high‐speed counter‐current chromatography without any pretreatment using n‐hexane/ethyl acetate/methanol/water (1:7:0.5:3, v/v/v/v) as the solvent system. In 180 min, 13 mg of A, 18 mg of B, and 9 mg of C were obtained from 200 mg of crude sample. Their structures were identified as Chebulagic acid (A, 954 Da), Chebulinic acid (B, 956 Da), and Ellagic acid (C) by ¹H NMR spectroscopy.     

Application of supercritical fluid extraction coupled with counter–current chromatography for extraction and online isolation of unstable chemical components from Rosa damascena

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and online isolation of the unstable compounds from Rosa damascene in a single extraction and separation operation in two stages. The solvent systems of SFE/HSCCC were optimized with the help of multiexponential function model. At the first stage, the upper phase of the solvent system of n‐butanol–tert‐butyl methyl ether–acetonitrile–0.1% aqueous TFA (1.7:1.0:0.8:4.0, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase to separate the hydrophobic compounds. At the second stage, the upper phase of the solvent system of n‐hexane–ethyl acetate–methanol–water (3.2:1.0:2.8:2.6, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the moderate hydrophobic compounds. Six compounds including formononetin, delphinidin, cyaniding, 5,6,4′‐trihydroxy‐7,8‐dimethoxy flavone, 5,3′‐dihydroxy‐7,8‐dimethoxy flavone, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxy flavone were successfully separated in one extraction–separation operation within 300 min. The targeted compounds were identified by MS and NMR spectroscopy. This research has opened up great prospects for industrial application of SFE/HSCCC to the extraction and separation of unstable compounds.     

Preparative isolation of pseudolaric acids A and B,and their glucosides from the root bark of Pseudolarix kaempferi using high‐speed counter‐current chromatography

In order to provide the chemical markers for the quality control of herbal medicines, four diterpenoids, pseudolaric acids A and B (PAA and PAB), and their glucosides were isolated from the methanol extract of the Chinese herb Pseudolarix kaempferi using high‐speed counter‐current chromatography (HSCCC). The diphase solvent system was n‐hexane/EtOAc/MeOH/H₂O which was used at two ratios (5:5:5:5 and 1:9:4:6 by volume) in the separation of pseudolaric acids and their glycosides, respectively. As a result, PAA (14 mg), PAB (129 mg), PAA‐O‐β‐D ‐glucopyranoside (8 mg, PAAG), and PAB‐O‐β‐D ‐glucopyranoside (42 mg, PABG) were obtained from 0.5 g of the crude extract. Their purities were determined to be above 97% by HPLC analysis. Their chemical structures were confirmed by ¹H and ¹³C NMR analysis or HPLC comparison with the reference compounds.     

Preparative separation and purification of bufadienolides from Chinese traditional medicine of ChanSu using high‐speed counter‐current chromatography

A preparative high‐speed counter‐current chromatography method for isolation and purification of bufadienolides from ChanSu was developed by using a stepwise elution with two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at the ratios of 4:6:2:4 v/v, 4:6:2.5:4 v/v and 4:6:3.2:4 v/v. A total of 3.8 mg of gamabufotalin (1), 7.2 mg of arenobufagin (2), 3.4 mg of telocinobufagin (3), 5.3 mg of bufotalin (4), 8.5 mg of cinobufotalin (5) and 8 mg of bufalin (6) were obtained in one‐step separation from 80 mg of the crude extract with purity of 92.7, 96.7, 87.2, 97.3, 94.9 and 99.4%, respectively. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology.