Efficient methods for isolating five phytochemicals from Gentiana macrophylla using high‐performance countercurrent chromatography

Efficient high‐performance countercurrent chromatography methods were developed to isolate five typical compounds from the extracts of Gentiana macrophylla. n‐Butanol‐soluble extract of G. macrophylla contained three hydrophilic iridoids, loganic acid ( 1 ), swertiamarin ( 2 ) and gentiopicroside ( 3 ), and a chromene derivative, macrophylloside D ( 4 ) which were successfully isolated by flow rate gradient (1.5 mL/min in 0–60 min, 5.0 mL/min in 60–120 min), and consecutive flow rate gradient HPCCC using n‐butanol/0.1% aqueous trifluoroacetic acid (1:1, v/v, normal phase mode) system. The yields of 1 – 4 were 22, 16, 122, and 6 mg, respectively, with purities over 97% in a flow rate gradient high‐performance countercurrent chromatography, and consecutive flow rate gradient high‐performance countercurrent chromatography gave 1 , 2 , 3 (54, 41, 348 mg, respectively, purities over 97%) and 4 (13 mg, purity at 95%) from 750 mg of sample. The main compound in methylene chloride soluble extract, 2‐methoxyanofinic acid, was successfully separated by n‐hexane/ethyl acetate/methanol/water (4:6:4:6, v/v/v/v, flow‐rate: 4 mL/min, reversed phase mode) condition. The structures of five isolates were elucidated by ¹H, ¹³C NMR and ESI‐Q‐TOF‐MS spectroscopic data which were compared with previously reported values.     

Developments in high‐speed countercurrent chromatography and its applications in the separation of terpenoids and saponins

High‐speed countercurrent chromatography is a liquid–liquid separation chromatographic technique, which has the unique feature of eliminating irreversible adsorption using liquid support medium, and is widely used in research and development of traditional Chinese medicine, biochemistry, food, environment analysis, and so on. In this review, some new developments of countercurrent chromatography, for instance cross‐axis countercurrent chromatography, dual countercurrent chromatography, foam countercurrent chromatography, and pH‐zone‐refining countercurrent chromatography are presented. Furthermore, the research and progress in high‐speed countercurrent chromatography techniques and its application in the separation and purification of terpenoids and saponins are reviewed.     

Separation and purification of steroidal saponins from Paris polyphylla by microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection

A method of microwave‐assisted extraction coupled with countercurrent chromatography using evaporative light scattering detection was successfully developed for the separation and purification of steroidal saponins from Paris polyphylla. The main extraction conditions including microwave power, liquid/solid ratio, irradiation time, and extraction temperature were optimized using an orthogonal array design method. A suitable two‐phase solvent system consisting of n‐heptane/n‐butanol/acetonitrile/water (10:19:6:20, v/v/v/v) was employed in the separation and purification of the extracts of P. polyphylla. A total of 7.1 mg polyphyllin VII, 4.3 mg gracillin, 9.2 mg dioscin, and 10.2 mg polyphyllin I were obtained from 1.5 g P. polyphylla in less than 300 min, the purities of which determined by HPLC were 96.7, 97.3, 98.7, and 98.6%, respectively. The identification and characterization of these compounds were performed by LC–ESI‐MS and ¹H NMR spectroscopy. The results demonstrated that the proposed method is feasible, economical and efficient for the extraction, separation and purification of effective compounds from natural products.     

Quick method for separating target compounds from the bark of Maqian (Zanthoxylum myriacanthum var. pubescens) by high‐performance countercurrent chromatography

Choosing a suitable solvent system for a countercurrent chromatography separation presents a challenge for many researchers. In this study, we introduce a quick method of separating a target compound from the bark of Zanthoxylum myriacanthum var. pubescens by countercurrent chromatography. This method relies on the thin‐layer chromatography based generally useful estimation of solvent systems. This paper will present how to quickly choose a suitable solvent system with a thin‐layer chromatography based generally useful estimation of solvent systems working chart. O‐Methyltembamide ( 1 ) was enriched by countercurrent chromatography using n‐hexane/ethyl acetate/methanol/water (6:4:6:4) as the solvent system. Further purification was achieved by high‐performance liquid chromatography with purities of 98.2% from Z. myriacanthum var. pubescens bark.     

Evaluation of gardenia yellow using crocetin from alkaline hydrolysis based on ultra high performance liquid chromatography and high‐speed countercurrent chromatography

Gardenia yellow is globally the most valuable spice and food color. It is generally a mixture of water‐soluble carotenoid glycosyl esters which consist of crocetin bis(gentiobiosyl) ester as the main component. Crocetin is a natural carotenoid dicarboxylic acid that may be a candidate drug for pharmaceutical development, however, it is either present in trace amounts or is absent in natural gardenia yellow products. We here propose that crocetin produced by alkaline hydrolysis can be used to qualitatively evaluate gardenia yellow products using an ultra high performance liquid chromatographic assay. A useful and efficient isolation technique for isolating high‐purity crocetin from gardenia yellow using high‐speed countercurrent chromatography is described. High‐speed countercurrent chromatographic fractionation followed by an ultra high performance liquid chromatographic assay showed that trans‐crocetin is easily converted to about 15% cis‐crocetin (85% trans‐crocetin). Crocetin in gardenia yellow was quantitatively evaluated. Our approach is based on the hydrolysis process for converting crocetin glycosyl esters to crocetin before evaluation and isolation using the ultra high performance liquid chromatographic and high‐speed countercurrent chromatographic methods. The combination of hydrolysis and chromatographic methods allows evaluation of the purity and quantity of crocetin in gardenia yellow.     

Separation of three anthraquinone glycosides including two isomers by preparative high‐performance liquid chromatography and high‐speed countercurrent chromatography from Rheum tanguticum Maxim. ex Balf

Anthraquinone glycosides, such as chrysophanol 1‐O‐β‐d‐ glucoside, chrysophanol 8‐O‐β‐d‐ glucoside, and physion 8‐O‐β‐d‐ glucoside, are the accepted important active components of Rheum tanguticum Maxim. ex Balf. due to their pharmacological properties: antifungal, antimicrobial, cytotoxic, and antioxidant activities. However, an effective method for the separation of the above‐mentioned anthraquinone glycosides from this herb is not currently available. Especially, greater difficulty existed in the separation of the two isomers chrysophanol 1‐O‐β‐d‐ glucoside and chrysophanol 8‐O‐β‐d‐ glucoside. This study demonstrated an efficient strategy based on preparative high‐performance liquid chromatography and high‐speed countercurrent chromatography for the separation of the above‐mentioned anthraquinone glycosides from Rheum tanguticum Maxim.ex Balf.     

Single‐step isolation of embelin using high‐performance countercurrent chromatography and determination of the fatty acid composition of seeds of Embelia schimperi

Embelin (2,5‐dihydroxy‐3‐undecyl‐p ‐benzoquinone) is known for its potent anthelmintic activity, but also for wound‐healing, antidiabetic, anticonvulsant, antitumour, anti‐inflammatory, analgesic, hepatoprotective, antioxidant, antibacterial and antispermatogenic activities. A high‐performance countercurrent chromatography method was developed for the purification of embelin from an extract of the seeds of Embelia schimperi fruit. The optimized solvent system (n ‐hexane–ethylacetate–ethanol–water, 7:3:7:3) resulted in the isolation of 13.9 mg of embelin, directly from 100 mg of crude extract, in a single step within a short time (40 min). Although the compound appeared to be completely pure when analysed by ultra‐performance liquid chromatography (UPLC) with photo diode array detection, the purity was established as ~90% by UPLC–mass spectrometry. Furthermore, we report the fatty acid composition of the seeds of E. schimperi fruit. Nine fatty acids were quantified from the fruit seed extract by gas chromatography–mass spectrometry, with linoleic (46.4%), palmitic (21.5%) and oleic (19.6%) acids making up the largest proportions.     

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.     

Comprehensive separation of iridoid glycosides and triterpenoid saponins from Dipsacus asper with salt‐containing solvent by high‐speed countercurrent chromatography coupled with recycling mode

The roots of Dipsacus asper Wall as a commonly used traditional Chinese medicine are used for tonifying liver and kidney and strengthening bones and muscles. However, an effective separation strategy for comprehensive and rapid separation of the main active compounds from the roots of D. asper is nonexistent. This investigation provided an effective separation method based on AB‐8 macroporous resin column chromatography using different ratios of ethanol in water and two different modes of high‐speed countercurrent chromatography with salt‐containing solvent system for rapid enrichment and separation from the roots of D. asper. The macroporous resin column chromatography was performed on AB‐8 resin using ethanol in water ratios of 10, 30, 40, 50, and 80% as the optimized enrichment conditions for iridoid glycosides and triterpenoid saponins with different polarities. For high‐speed countercurrent chromatography separation, the conventional and recycling modes were combined together to develop a strategy for 12 compounds ( 1 – 12 ) from the enriched parts of 30, 40, and 80% ethanol, including six high‐polarity iridoid glycosides ( 1 – 6 ) using inorganic salt‐containing solvent system and six triterpenoid saponins ( 7 – 12 ). Recycling high‐speed countercurrent chromatography separation was successfully applied to separate two isomers ( 9 and 10 ) after 11 cycles.     

Preparative separation of five flavones from flowers of Polygonum cuspidatum by high‐speed countercurrent chromatography

A preparative high‐speed countercurrent chromatography method was successfully used for the isolation of five minor flavones from Polygonum cuspidatum flowers. Among them, three compounds were obtained from P. cuspidatum for the first time. A twin two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (1:6:3:6, v/v/v/v) and petroleum ether/ethyl acetate/methanol/water (2:4:3:3, v/v/v/v) was developed. Compounds were obtained from the fraction B and fraction C prepurified by silica gel column chromatography. Five minor compositions, 6.8 mg of hesperidin, 11.2 mg of phloridzin, 4.9 mg of luteolin, 5.3 mg of hyperin, and 3.7 mg of luteoloside were obtained from 140 mg of the fraction B and 110 mg of fraction C with a purity of 95.3, 96.4, 98.0, 96.8, and 95.3%, respectively, as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Preparative isolation of neomangiferin and mangiferin from Rhizoma anemarrhenae by high‐speed countercurrent chromatography using ionic liquids as a two‐phase solvent system modifier

A preparative high‐speed countercurrent chromatography method for isolation and purification of neomangiferin and mangiferin from Rhizoma anemarrhenae was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Neomangiferin and mangiferin were purified from the crude extract of R. anemarrhenae by using ethyl acetate‐water‐[C₄mim][PF₆] (5:5:0.2 v/v) as two‐phase solvent system. In total, 22.5 mg of neomangiferin and 70.6 mg of mangiferin were obtained from 150 mg of the crude extract. The purities of neomangiferin and mangiferin were 97.2 and 98.1%, respectively, as determined by HPLC. The chemical structures of the isolated compounds were identified by ¹H‐NMR and ¹³C‐NMR.     

High‐speed countercurrent chromatography isolation of flavans from Ixeris chinensis and their identification by NMR spectroscopy

High‐speed countercurrent chromatography, combined with macroporous resin chromatography were applied to the separation and purification of flavans from Ixeris chinensis. Four flavans, namely, 5‐methoxy‐7,4′‐dihydroxyflavan‐3‐ol ( 1 ), 5,7‐dimethoxy‐4′‐hydroxyflavan‐3‐ol ( 2 ), 5,7‐dimethoxy‐4′‐hydroxyflavan ( 3 ), and 5,7‐dimethoxy‐8‐methyl‐4′‐hydroxyflavan ( 4 ), were obtained from I. chinensis for the first time. Their chemical structural identification was carried out by spectroscopic methods, including 1D and 2D NMR spectroscopy. Amounts of 13.2 mg of compound 1 , 6.4 mg of compound 2 , 5.8 mg of compound 3 , and 14.5 mg of compound 4 were separated from 120 mg 75% ethanol fraction. The purities of 1 – 4 were 99.1, 99.2, 97.3, and 98.6 %, respectively.     

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.     

Enzymatic transformation of platycosides and one‐step separation of platycodin D by high‐speed countercurrent chromatography

Platycosides, the saponins found in the roots of Platycodon grandiflorum (Platycodi Radix), are typically composed of oleanane triterpenes with two side chains. In platycosides, platycodin D, a glucose unit at C‐3, is a major component, which has several pharmacological activities. Because of the high demand for this compound, we attempted to enzymatically convert platycodin D₃ and platycoside E, having two and three glucose units at C‐3, respectively, into platycodin D. In this study, we tested the ability of several glycosidases to transform platycosides, or more specifically, the ability to transform platycoside E and platycodin D₃ into platycodin D. To obtain pure platycodin D on a preparative scale, high‐speed countercurrent chromatography with a solvent system of ethyl acetate/n‐butanol/water (1.2:1:2, v/v/v) was used for the separation of the enzymatically transformed product. Approximately 39.4 mg of platycodin D (99.8% purity) was obtained from 200 mg of the product in a one‐step separation. The results strongly support the advantage of enzymatic transformation of the platycosides for the efficient enrichment of platycodin D in the complicated extract of the medicinal plant.     

Rapid separation of cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside from crude mulberry extract using high‐performance countercurrent chromatography and establishment of a volumetric scale‐up process

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside, using high‐performance countercurrent chromatography, and the establishment of a volumetric scale‐up process from semi‐preparative to preparative‐scale. To optimize the separation parameters, biphasic solvent systems composed of tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi‐preparative‐scale high‐performance countercurrent chromatography. The optimized semi‐preparative‐scale high‐performance countercurrent chromatography parameters (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200–1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative‐scale (tert‐butyl methyl ether/n‐butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0–10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin‐3‐glucoside and cyanidin‐3‐rutinoside. The separation of mulberry anthocyanins using semi‐preparative high‐performance countercurrent chromatography and its volumetric scale‐up to preparative‐scale was addressed for the first time in this report.     

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two‐dimensional contour plots for countercurrent chromatography with high‐performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two‐dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high‐performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off‐line comprehensive two‐dimensional countercurrent chromatography with high‐performance liquid chromatography provided a promising and powerful method for separation of complex natural products.     

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high‐speed countercurrent chromatography

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi‐preparative high‐speed countercurrent chromatography with a two‐phase solvent system composed of cyclohexane‐ethyl acetate‐methanol‐water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance (¹H‐NMR), HMBC, ESI‐MS and HR‐MS.     

Uncertainty in the determination of glucose in aqueous solutions by high‐performance liquid chromatography with evaporative light scattering detection

The determination of glucose and other carbohydrates is the most widespread chemical analysis that is performed within the industries of food, beverage, forage, biomass, pulp and paper, pharmaceuticals among others. Besides that, sugar refineries need to control their products, by‐products and effluents, and furthermore, glucose in the sucrose refining process, is considered an impurity, which shall be controlled. Being HPLC the most currently instrumental technique used for glucose analysis, the evaporative light scattering detector (ELSD) offers advantages (sensitivity, possibility for operating in gradient mode) over the also used refractive index detector. In this work, an HPLC‐ELSD methodology was optimised and validated, aiming the estimate of the uncertainty associated with the results at low levels of concentration of glucose to be measured. Linearity of the response was obtained in the range of glucose concentrations from 20 to 300 mg/L, with an analysis time of 10 min. The global uncertainty was estimated accordingly to the bottom‐up approach used by Eurachem. It was 13% on average for concentrations from 100 to 300 mg/L. For lower concentrations, uncertainty increased significantly up to 30% in the vicinity of the LOD of the method.     

Isolation of terpenoids from Pimpinella anisum essential oil by high‐performance counter‐current chromatography

High‐performance counter‐current chromatography was successfully used for the isolation and purification of terpenoid compounds from the essential oil of Pimpinella anisum L. A two‐phase solvent system composed of n‐heptane/methanol/ethyl acetate/water (5:2:5:2, v/v/v/v) was suitable for the purification of linalool, terpinen‐4‐ol, α‐terpineol, p‐anisaldehyde, while n‐heptane/methanol (1:1, v/v) was used for the isolation of anethole and foeniculin. A scale‐up process from analytical to preparative was developed. Additionally, a stepwise gradient elution was applied and instead of two different runs, 40 min each, one 80 min separation was performed; although the time of separation remains the same, it was possible to repeat the efficiency even if the water‐containing mobile phase was changed to a nonaqueous system. The obtained essential oil, as well as purified compounds, was analyzed by GC. A total of 0.64 mg of linalool, 0.52 mg of terpinen‐4‐ol, 0.10 mg of α‐terpineol, 0.62 mg of p‐anisaldehyde, 15 mg of anethole, and 2.12 mg of foeniculin were obtained from 210 mg of the essential oil of P. anisum L. in a short time with purities of 99, 98, 94, 93.54, 93, and 93.6%, respectively.     

Separation of six xanthones from Swertia franchetiana by high‐speed countercurrent chromatography

In our present study, two groups of xanthones isomers (1‐hydroxy‐3,5,8‐trimethoxyxanthone and 1‐hydroxy‐3,7,8‐trimethoxyxanthone; 1,8‐dihydroxy‐3,7‐dimethoxyxanthone and 1,8‐dihydroxy‐3,5‐dimethanolxanthone) and other two xanthones (3‐methoxy‐1,5,8‐trihydroxyxanthone and 3,5‐dimethoxy‐1‐hydroxyxanthone) were separated from Swertia franchetiana . First, a solvent system composed of petroleum ether/methanol/water (2:1:0.6, v/v) was developed for the liquid–liquid extraction of these xanthones from the crude extract. Then, an efficient method was established for the one‐step separation of these six xanthones by high‐speed countercurrent chromatography using n‐hexane/ethyl acetate/methanol/ethanol/water (HEMEW; 6:4:4:2:4, v/v) as the solvent system. The results showed that liquid–liquid extraction could be well developed for efficient enrichment of target compounds. Additionally, high‐speed countercurrent chromatography could be a powerful technology for separation xanthones isomers. It was found ethanol could be a good methanol substitute when the HEMEW system could not provide good separation factors.