Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

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.     

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.     

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.     

Enantioseparation of chiral aromatic acids by multiple dual mode counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin as chiral selector

This work concentrates on extending the utilization of multiple dual mode (MDM) counter‐current chromatography in chiral separations. Two aromatic acids, 2‐(6‐methoxy‐2‐naphthyl)propionic acid (NAP) and 2‐phenylpropionic acid (2‐PPA), were enantioseparated by MDM counter‐current chromatography using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as chiral selector. The two‐phase solvent systems consisting of n‐hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP‐β‐CD (7.5:2.5:10 for NAP and 7:3:10 for 2‐PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first‐phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2‐PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter‐current chromatography, MDM elution greatly improved peak resolution in chiral separations.     

Effective on‐line high‐speed shear dispersing emulsifier technique coupled with high‐performance countercurrent chromatography method for simultaneous extraction and isolation of carotenoids from Lycium barbarum L. fruits

An efficient combination strategy based on high‐speed shear dispersing emulsifier technique and high‐performance countercurrent chromatography was developed for on‐line extraction and isolation of carotenoids from the fruits of Lycium barbarum. In this work, the high‐speed shear dispersing emulsifier technique has been employed to extract crude extracts using the upper phase of high‐performance countercurrent chromatography solvent system composed of n‐hexane?dichloromethane?acetonitrile (10:4:6.5, v/v) as the extraction solvent. At the separation stage, the high‐performance counter‐current chromatography process adopts elution–extrusion mode and the upper phase of the solvent system as stationary phase (reverse‐phase mode). As a result, three compounds including zeaxanthin, zeaxanthin monopalmitate, and zeaxanthin dipalmitate with purities of 89, 90, and 93% were successfully obtained in one extraction‐separation operation within 120 min. The targeted compounds were analyzed and identified by high‐performance liquid chromatography, mass spectrometry, and NMR spectroscopy. The results indicated that the present on‐line combination method could serve as a simple, rapid, and effective way to achieve weak polar and unstable compounds from natural products.     

Enrichment and separation of antitumor triterpene acids from the epidermis of Poria cocos by pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography

Triterpene acids were extracted from the epidermis of Poria cocos (Schw.) Wolf. These acids were found to inhibit the growth of lung cancer cells in vitro and in vivo. An efficient method for the preparative separation of antitumor triterpene acids was established that involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography. We used pH‐zone‐refining counter‐current chromatography to concentrate the triterpene acids using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (3:7:5:5, v/v/v/v), trifluoroacetic acid (10 mM) was added to the upper phase as a retainer, and ammonia (10 mM) was added to the lower phase as an eluter. As a result, 200 mg concentrate of triterpene acids was obtained from 1.0 g of crude extract. The concentrate was further separated by conventional high‐speed counter‐current chromatography using a solvent system composed of petroleum ether/ethyl acetate/methanol/water (0.8:1.2:1.2:0.9, v/v), yielding 50 mg of poricoic acid A and 5 mg of poricoic acid B from 120 mg concentrate, respectively. The inhibitory activity of the major compound on lung A549 cells was examined and poricoic acid A was found to significantly inhibit the growth of A 549 cells.     

Elution–extrusion counter‐current chromatography for the separation of two pairs of isomeric monoterpenes from Paeoniae Alba Radix

In this work, a simple and efficient protocol for the rapid separation of two pairs of isomeric monoterpenes from Paeoniae Alba Radix was developed by combining macroporous resin and elution–extrusion counter‐current chromatography. The crude extract was firstly subjected to a D101 macroporous resin column eluted with water and a series of different concentrations of ethanol. Then, effluents of 30 and 95% ethanol were collected as sample 1 and sample 2 for further counter‐current chromatography purification. Finally, a pair of isomers, 96 mg of compound 1 and 48 mg of compound 2 with purities of 91.1 and 96.2%, respectively, was isolated from 200 mg of sample 1. The other pair of isomers, 14 mg of compound 3 and 8 mg of compound 4 with purities of 93.6 and 88.9%, respectively, was isolated from 48 mg of sample 2. Their purities were analyzed by high‐performance liquid chromatography, and their chemical structures were identified by mass spectrometry and ¹H NMR spectroscopy. Compared to a normal counter‐current chromatography separation, the separation time and solvent consumption of elution–extrusion counter‐current chromatography were reduced while the resolutions were still good. The established protocol is promising for the separation of natural products with great disparity of content in herbal medicines.     

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.     

An efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment,separation, and purification of alkaloids and organic Acids from natural products

This study presents an efficient strategy based on liquid‐liquid extraction and pH‐zone‐refining counter‐current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two‐phase solvent system with maximum or minimum partition coefficient was developed for the liquid‐liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH‐zone‐refining counter‐current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin‐3‐adipoyl‐l ‐arginine, 42 mg of telocinobufagin‐3‐pimeloyl‐l ‐arginine, 51 mg of telocinobufagin‐3‐suberoyl‐l ‐arginine, 132 mg of marinobufagin‐3‐suberoyl‐l ‐arginine, and 57 mg of bufalin‐3‐suberoyl‐l ‐arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.     

Direct from solid natural products to pure compounds in a single step: Coupling online extraction with high‐speed counter‐current chromatography

Extraction is the most important step in the purification of bioactive compounds from natural products. This study introduces a simple online extraction strategy coupled with high‐speed counter‐current chromatography for efficient extraction and purification of bioactive components from solid natural products. For online extraction strategy, 1.0 g of ground Mangnolia officinalis or Piper nigrum was loaded into a guard column, which was then positioned on the manual injection valve instead of the sample loop. Bioactive components were directly extracted by the mobile phase of high‐speed counter‐current chromatography, and then transferred into high‐speed counter‐current chromatography for purification. In addition, the compatibility of the developed methodology for direct purification of bioactive components from fresh M. officinalis was successfully demonstrated. Obviously, in comparison with traditional offline heat‐reflux extraction, online extraction avoided the instrument, time, solvent, and energy consumption, and purified two phenolic compounds (honokiol and magnolol) from M. officinalis and three alkaloids (piperyline, piperine, and piperanine) from P. nigrum with high extraction efficiency. The superiority of the developed methodology is to establish an easy, rapid, and efficient technique for the purification of a wide variety of bioactive components from solid natural products.     

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.     

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.     

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 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.     

Full use of the liquid nature of the stationary phase: The development of elution‐extrusion counter current chromatography

Elution‐extrusion counter current chromatography extrudes the most solute retained in the column with the highest possible peak resolution. It can greatly improve the hydrophobic window. In recent years, elution‐extrusion counter current chromatography has received extensive attention in the separation of complex samples. This article first reviews the development and application of elution‐extrusion counter current chromatography, including its origin, mechanism, advantages and disadvantages, and some representative applications. At the same time, this review also shared our visions and ideas on how to improve the elution‐extrusion mode. This article aims to provide certain reference for the research of this technology.     

Accelerated solvent extraction and pH‐zone‐refining counter‐current chromatographic purification of yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom

This study aimed to seek an efficient method to extract and purify yunaconitine and 8‐deacetylyunaconitine from Aconitum vilmorinianum Kom. by accelerated solvent extraction combined with pH‐zone‐refining counter‐current chromatography. The major extraction parameters for accelerated solvent extraction were optimized by an orthogonal test design L₉ (3)⁴. Then a separation and purification method was established using pH‐zone‐refining counter‐current chromatography with a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water (5:5:2:8, v/v) with 10 mM triethylamine in the upper phase and 10 mM HCl in the lower phase. From 2 g crude extract, 224 mg of 8‐deacetylyunaconitine (I) and 841 mg of yunaconitine (II) were obtained with a purity of over 98.0%. The chemical structures were identified by ESI‐MS and ¹H and ¹³C NMR spectroscopy.     

Combinative application of pH‐zone‐refining and conventional high‐speed counter‐current chromatography for preparative separation of caged polyprenylated xanthones from gamboge

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography for the first time. pH‐zone‐refining counter‐current chromatography was performed with the solvent system composed of n‐hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high‐speed counter‐current chromatography with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n‐hexane/methyl tert‐butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β‐morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high‐performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH‐zone‐refining counter‐current chromatography and conventional high‐speed counter‐current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.     

Enantiomeric separation of oxybutynin by recycling high‐speed counter‐current chromatography with hydroxypropyl‐β‐cyclodextrin as chiral selector

Recycling high‐speed counter‐current chromatography was successfully applied to the preparative separation of oxybutynin enantiomers. The two‐phase solvent system consisted of n‐hexane, methyl tert‐butyl ether, and 0.1 mol/L phosphate buffer solution (pH = 5.0) with the volume ratio of 6:4:10. Hydroxypropyl‐β‐cyclodextrin was employed as the chiral selector. The influence of factors on the chiral separation process, including the concentration of chiral selector, the equilibrium temperature, the pH value of the aqueous phase were investigated. Under optimum separation conditions, 15 mg of oxybutynin racemate was separated with the purities of both the enantiomers over 96.5% determined by high‐performance liquid chromatography. Recovery for the target compounds reached 80–82% yielding 6.00 mg of (R)‐oxybutynin and 6.15 mg of (S)‐oxybutynin. Technical details for recycling elution mode were discussed.     

Screening of antioxidant phenolic compounds in Chinese Rhubarb combining fast counter‐current chromatography fractionation and liquid chromatography/mass spectrometry analysis

In this paper, an effective method combing fast elution‐extrusion counter‐current chromatography (CCC) and LC/MS for rapid screening of antioxidative phenolic compounds in Chinese Rhubarb is presented. An integrated three‐coil CCC column (40 mL each coil) was used to accomplish the optimization of biphasic liquid system. In a single run (approximately 40 min), the solvent system composed of n‐hexane/ethyl acetate/methanol/water (1:1:1:1, v/v) was selected as optimum CCC liquid system for fast fractionation of the crude ethanol extract. With a 140 mL‐capacity CCC instrument, 100 mg Chinese Rhubarb extract was separated under the optimized conditions, producing six fractions in only 100 min. The quantities of each fraction were ～15 mg. In addition, each fraction was subjected to antioxidant activity assay and characterized by LC/MS analysis. Fifty compounds, including phenolic acids, phenolic glucosides and hydroxyanthraquinones, were detected by LC/MS/MS analysis. As a result, gallic acid together with Fr I showed excellent antioxidant activity, which was well consistent with previous studies and exhibited great potential for natural drug discovery program of the present method.