Novel linear and step‐gradient counter‐current chromatography for bio‐guided isolation and purification of cytotoxic podophyllotoxins from Dysosma versipellis (Hance)

Dysosma versipellis (Hance) is a famous traditional Chinese medicine for the treatment of snakebite, weakness, condyloma accuminata, lymphadenopathy, and tumors for thousands of years. In this work, four podophyllotoxin‐like lignans including 4′‐demethylpodophyllotoxin (1), α‐peltatin (2), podophyllotoxin (3), β‐peltatin (4) as major cytotoxic principles of D. versipellis were successfully isolated and purified by several novel linear and step gradient counter‐current chromatography methods using the systems of hexane/ethyl acetate/methanol/water (4:6:3:7 and 4:6:4:6, v/v/v/v). Compared with isocratic elution, linear and step‐gradient elution can provide better resolution and save more time for the separation of photophyllotoxin and its congeners. Their cytotoxicities were further evaluated and their structures were validated by high‐resolution electrospray TOF MS and nuclear magnetic resonance spectra. All components showed potent anticancer activity against human hepatoma cells HepG2.     

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.     

Preparative separation of phytosterol analogues from green alga Chlorella vulgaris using recycling counter‐current chromatography

The unicellular alga Chlorella vulgaris is a well‐known health food. It has been proven that the minor phytosterols, ergosterol and its analogue, are an important class of bioactive substances in C. vulgaris . In this work, a recycling counter‐current chromatographic approach was proposed for preparative separation of two analogue sterols from crude extract of C. vulgaris . The separation unit was set up with a type‐J instrument coupled with a column switching valve. A two‐phase solvent system composed of n‐hexane/dichloromethane/acetonitrile (10:3:7, v/v/v) was selected and optimized. After five cycles of separation, two analogue sterols were baseline separated, producing 11.7 mg 26‐nor‐25‐isopropyl‐5,7,22‐trien‐3β‐ol and 20.3 mg ergosterol from 300 mg of C. vulgaris extract. Their purities were both above 95%. The structures of two sterols were identified by using NMR spectroscopy.     

Preparative isolation of flavonoid compounds from Oroxylum indicum by high‐speed counter‐current chromatography by using ionic liquids as the modifier of two‐phase solvent system

A preparative high‐speed counter‐current chromatography method for isolation and purification of flavonoid compounds from Oroxylum indicum was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Two flavonoid compounds including baicalein‐7‐O‐diglucoside and baicalein‐7‐O‐glucoside were purified from the crude extract of O. indicum by using ethyl acetate–water–[C₄mim][PF₆] (5:5:0.2, v/v) as two‐phase solvent system. 36.4 mg of baicalein‐7‐O‐diglucoside and 60.5 mg of baicalein‐7‐O‐glucoside were obtained from 120 mg of the crude extract. Their purities were 98.7 and 99.1%, respectively, as determined by HPLC area normalization method. The chemical structures of the isolated compounds were identified by ¹H‐NMR and ¹³C‐NMR.     

Effect of inorganic salt on partition of high‐polarity parishins in two‐phase solvent systems and separation by high‐speed counter‐current chromatography from Gastrodia elata Blume

Parishins are high‐polarity and major bioactive constituents in Gastrodia elata Blume. In this study, the effect of several inorganic salts on the partition of parishins in two‐phase solvent systems was investigated. Adding ammonium sulfate, which has a higher solubility in water, was found to significantly promote the partition of parishins in the upper organic polar solvents. Based on the results, a two‐phase solvent system composed of butyl alcohol/acetonitrile/near‐saturated ammonium sulfate solution/water (1.5:0.5:1.2:1, v/v/v/v) was used for the purification of parishins by high‐speed counter‐current chromatography. Fractions obtained from high‐speed counter‐current chromatography were subjected to semi‐preparative high‐performance liquid chromatography to remove salt and impurities. As a result, parishin E (6.0 mg), parishin B (7.8 mg), parishin C (3.2 mg), gastrodin (15.3 mg), and parishin A (7.3 mg) were isolated from water extract of Gastrodia elata Blume (400 mg). These results demonstrated that adding inorganic salt that has high solubility in water to the two‐phase solvent system in high‐speed counter‐current chromatography was a suitable approach for the purification of high‐polarity compounds.     

Separation of caffeoylquinic acids and flavonoids from Asteris souliei by high‐performance counter‐current chromatography and their anti‐inflammatory activity in vitro

Eleven compounds were successfully separated from Asteris souliei by using a two‐step high‐performance counter‐current chromatography method. The first step involved a reversed phase isocratic counter‐current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step‐gradient reversed‐phase counter‐current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI‐MS and NMR spectroscopy (¹H and ¹³C). Baicalin ( 5 ), eriodictyol ( 7 ), apigenin‐7‐glycoside ( 8 ), quercetin ( 9 ), luteolin ( 10 ), and apigenin ( 11 ) showed obvious inhibitory effects on lipopolysaccharide‐induced nitric oxide production in RAW264.7 cells at a concentration of 10 μg/mL.     

Recent progress in separation prediction of counter‐current chromatography

As a liquid‐liquid partition chromatography, counter‐current chromatography has advantages in large sample loading capacity without irreversible adsorption, which has been widely applied in separation and purification fields. The main factors, including partition coefficient, two‐phase solvent systems, apparatus, and operating parameters greatly affect the separation process of counter‐current chromatography. To promote the applications of counter‐current chromatography, it is essential to develop theoretical research to master the principles of counter‐current chromatographic separations so as to achieve predictions before laborious trials. In this article, recent progress about separation prediction methods are reviewed from a point of the steady and unsteady state of the mass transfer process of counter‐current chromatography and its mass transfer characteristics, and then it is divided into three aspects: prediction of partition coefficient, modeling the thermodynamic process of counter‐current chromatography, and modeling the dynamic process of counter‐current chromatography.     

Application of high‐speed counter‐current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high‐speed counter‐current chromatography using a two‐phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high‐performance liquid chromatography with an Agilent ZORBAX® SB‐C18 column (4.6 mm × 150 mm, 5 μm). After separation by high‐speed counter‐current chromatography and high‐performance liquid chromatography, we obtained 3.5 mg of platetyolin A, 4.1 mg of platetyolin B, and 18.1 mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high‐performance liquid chromatography and the chemical structures were evaluated by high‐resolution electrospray ionization time‐of‐flight mass spectrometry and one‐ and two‐dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.     

Screening and separation of α‐amylase inhibitors from Solanum nigrum with amylase‐functionalized magnetic graphene oxide combined with high‐speed counter‐current chromatography

A screening method using α‐amylase‐functionalized magnetic graphene oxide combined with high‐speed counter‐current chromatography was proposed and utilized to screen and separate α‐amylase inhibitors from extract of Solanum nigrum . The α‐amylase‐functionalized magnetic graphene oxide was characterized and found to demonstrate satisfactory structure, magnetic response (24.5 emu/g), and reusability (retained 90% of initial activity after five cycles). The conditions for the screening with α‐amylase functionalized magnetic graphene oxide were optimized and set at pH 7.0 and 25°C. As a result, two potent flavonoid compounds, apigenin‐7‐O‐glucuronide ( 1 ) and astragalin ( 2 ), were separated and collected through high‐speed counter‐current chromatography and subjected to high‐performance liquid chromatography analysis with purity higher than 90% (according to HPLC data), which were identified as α‐amylase inhibitors. These results suggested that utilization of α‐amylase functionalized magnetic graphene oxide in the rapid screening and isolation bioactive compounds from complex natural products is a feasible and environmentally friendly method.     

Three‐phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high‐speed counter‐current chromatography

A three‐phase solvent system was efficiently applied for high‐speed counter‐current chromatography to separate secondary metabolites with a wide range of hydrophobicity in Dicranostigma leptopodum. The three‐phase solvent system of n‐hexane/methyl tert‐butyl ether/acetonitrile/0.5% triethylamine (2:2:3:2, v/v/v/v) was selected for high‐speed counter‐current chromatography separation. The separation was initiated by filling the column with a mixture of intermediate phase and lower phase as a stationary phase followed by elution with upper phase to separate the hydrophobic compounds. Then the mobile phase was switched to the intermediate phase to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with the lower phase. In this research, 12 peaks were eluted out in one‐step operation within 110 min, among them, eight compounds with acceptable purity were obtained and identified. The purities of β‐sitosterol, protopine, allocryptopine, isocorydione, isocorydine, coptisine, berberrubine, and berberine were 94.7, 96.5, 97.9, 86.6, 98.9, 97.6, 95.7, and 92.8%, respectively.     

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.     

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.     

Preparative separation of bioactive constitutes from Zanthoxylum planispinum using linear gradient counter‐current chromatography

Counter‐current chromatography is a chromatographic technique with a support‐free liquid stationary phase. In the present study, a successful application of linear gradient counter‐current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n‐hexane/ethyl acetate/methanol/water quaternary solvents, in terms of “HEMWat” or “Arizona” solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter‐current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n‐hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter‐current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.     

Preparative separation of anti‐oxidative constituents from Rubia cordifolia by column‐switching counter‐current chromatography

An effective column‐switching counter‐current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti‐oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column‐switching CCC system was interfaced by a commercial low‐pressure six‐port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1^st‐D) to the second dimension (2^nd‐D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1^st‐D. By valve switching technique the whole interested region of 1^st‐D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column‐switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram‐amounts of four anti‐oxidative components over 90% pure. Structures of purified compounds were identified by ¹H and ¹³C NMR.     

Preparative separation of flavonoids in plant extract of Smilacis Glabrae Roxb. by high performance counter‐current chromatography

Four flavonoids, isoastilbin, astilbin, isoengelitin, and engelitin were isolated and purified simultaneously from Smilacis Glabrae Roxb. for the first time by high performance counter‐current chromatography using a system consisting of n‐hexane–n‐butanol–water (1:2:3, v/v/v). A total of 392.6 mg of astilbin, 71.4 mg of isoastilbin, 47.4 mg of engelitin, and 10.3 mg of isoengelitin were purified from 1.89 g of the ethyl acetate extract of Smilacis Glabrae Roxb. in six runs, each at over 94.51% purity as determined by HPLC. The structures of the four compounds were identified by their retention time, the LC‐ESI‐MSⁿ in the negative ion mode, and confirmed by ¹H‐NMR experiments. The characteristic LC‐ESI‐MS fragmentation patterns of the four compounds were discussed.     

Separation of betacyanins from flowers of Amaranthus cruentus L. in a polar solvent system by high‐speed counter‐current chromatography

Betacyanin extract of Amaranthus cruentus L. flowers was fractionated by semi‐preparative high‐speed counter‐current chromatography in a highly polar solvent system: propan‐1‐ol/acetonitrile/(NH₄)₂SO_{4satd. soln}/H₂O (1.0:0.5:1.2:1.0, v/v/v/v) in tail‐to‐head mode with 76% retention of the stationary phase. The crude extract as well as the fractions containing betacyanins were analyzed by liquid chromatography with tandem mass spectrometry as well as by high‐resolution ion‐trap time‐of‐flight mass spectrometry detection technique for the molecular formulae and multi‐step fragmentation pattern elucidation. Four betacyanins; namely, amaranthin, betanin, 6′‐O‐formyl‐amaranthin, and 6′‐O‐malonyl‐amaranthin as well as their diastereomeric forms differing in the configuration of the C‐15 carbon atom were identified in the fractions. Amaranthin was the dominant pigment in the extract and was additionally analyzed by nuclear magnetic resonance correlation techniques after the counter‐current chromatographic and high‐performance liquid chromatographic isolation. Betacyanins were highly enriched during a single high‐speed counter‐current chromatographic step; therefore, the tentative identification of new compounds for the whole Amaranthaceae family, 6′‐O‐formyl‐amaranthin and 6′‐O‐malonyl‐amaranthin was possible. Different elution profiles of the pigments observed in the counter‐current chromatographic system in comparison to high‐performance liquid chromatography system confirm a complementarity of both the techniques especially in the separation of diastereomeric pairs of betacyanins.     

Preparative separation and purification of liensinine,isoliensinine and neferine from seed embryo of Nelumbo nucifera GAERTN using high‐speed counter‐current chromatography

A preparative high‐speed counter‐current chromatography method for separation and purification of liensinine, isoliensinine and neferine from seed embryo of Nelumbo nucifera GAERTN was successfully established by using n‐hexane‐ethyl acetate‐methanol‐water (5:8:4:5, v/v, containing 0.5% NH₄OH) as the two‐phase solvent system. From 200 mg of crude extract, 18.4 mg of liensinine, 19.6 mg of isoliensinine and 58.4 mg of neferine were obtained with the purity of 96.8, 95.9, and 98.6%, respectively. The identification of the three alkaloids was performed with ¹H NMR and ¹³C NMR.     

Separation and purification of four compounds from Desmodium styracifolium using off‐line two‐dimensional high‐speed counter‐current chromatography

An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and ¹H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples.     

Development of a method to screen and isolate bioactive constituents from Stellera chamaejasme by ultrafiltration and liquid chromatography combined with semi‐preparative high‐performance liquid chromatography and high‐speed counter current chromatography

A simple and efficient method based on ultrafiltration with liquid chromatography and mass spectrometry was used for the rapid screening and identification of ligands in the extracts of Stellera chamaejasme. The bound ligands, i.e. daphnoretin, isopimpinellin, chamaechromone, neochamaejasmin A, and chamaejasmine (purity of 96.8, 90.75, 91.41, 93.98, and 98.91%, respectively), were separated by semi‐preparative high‐performance liquid chromatography combined with high‐speed counter‐current chromatography. To the best of our knowledge, this is the first study to report the detection of potent lipoxidase and lactate dehydrogenase inhibitors in Stellera chamaejasme extracts. The results demonstrate that our method of ultrafiltration with liquid chromatography and mass spectrometry combined with mixed chromatography can be used to screen and confirm the bioactivity of all isolated compounds. This method also eliminates the need for separation of inactive compounds, thereby improving efficiency when studying bioactive substances. For some complex mixtures, neither semi‐preparative high‐performance liquid chromatography nor high‐speed counter‐current chromatography can purify all the target active compounds with high purity in a one‐step separation. The combination of the two methods allow for efficient purification of target bioactive compounds with different polarities and physicochemical properties based on their complementary properties.     

Systematic characterization of flavonoids from Siraitia grosvenorii leaf extract using an integrated strategy of high‐speed counter‐current chromatography combined with ultra high performance liquid chromatography and electrospray ionization quadrupole time‐of‐flight mass spectrometry

The chemical constituents of the Siraitia grosvenorii leaf extract were studied. Firstly, high‐speed counter‐current chromatography was applied to the one‐step separation of four compounds from S. grosvenorii leaf extract with the solvent system composed of 0.01% acetic acid water/n‐butanol/n‐hexane/methanol (5:3:1:1, v/v/v/v). In this work, 270 mg of crude sample yielded four compounds, a new kaempferol O‐glycoside derivative, kaempferol 3‐O‐α‐L‐[4‐O‐(4‐carboxy‐3‐hydroxy‐3‐methylbutanoyl)]‐rhamnopyranoside‐7‐O‐α‐L‐rhamnopyranoside, named kaempferitrin A (2.1 mg, 90%), and three known compounds, grosvenorine (3.4 mg, 93%), kaempferitrin (14.4 mg, 99%) and afzelin (4 mg, 98%), and the structures of these compounds were identified by NMR spectroscopy and mass spectrometry. Then, ultra high performance liquid chromatography with electrospray ionization quadrupole time‐of‐flight mass spectrometry was used to illustrate the dominant flavonoids in S. grosvenorii leaf extract. 34 flavonoids including 19 kaempferol O‐glycosides, 4 quercetin O‐glycosides, 6 flavanone derivatives, and 5 polymethoxyflavones, were accurately or tentatively identified by carefully comparing their retention times, UV data, precise masses, the typical fragments of the standards and literature data. Most of these compounds were reported for the first time. This study establishes a foundation for the further development and utilization of S. grosvenorii leaves in future.