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 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.     

Changes in the mobile phase composition on a stepwise counter‐current chromatography elution for the isolation of flavonoids from Siparuna glycycarpa

This paper describes the isolation of flavonoids and other aromatic compounds from an ethyl acetate extract of leaves of Siparuna glycycarpa using stepwise elution counter‐current chromatography (CCC). The elution profile yielded the following compounds: diglycosylated flavonoids, quercetin 3‐O‐rutinoside and quercetin 7‐O‐rutinoside, followed by monoglycosylated flavonoids, kaempferol‐3‐O‐β‐glucopyranoside, kaempferol‐3‐O‐β‐rhamnopiranoside, kaempferol‐3‐O‐β‐6′′(p‐coumaroyl) glucopyranoside, and quercetin‐3‐O‐β‐glucopyranoside, and then free phenolics, protocatechuic acid, and 2′,6′‐dihydroxy‐4, 4′‐dimethoxydihydrochalcone, which shows that this type of elution covers a broader range of polarity than the traditional isocratic mode. This makes it more suitable to perform separations of mixtures containing large differences in hydrophobicity. A GC analysis of a blank CCC run was performed to determine if changes in the mobile phase composition affect the chromatographic process. Results showed a gradual variation of the composition of the mobile phase emerging after the step gradient, favoring the selectivity of the solvent system.     

Separation and purification of antioxidants from Ampelopsis heterophylla by counter‐current chromatography

In this study, bioactive components from Ampelopsis heterophylla were separated by counter‐current chromatography (CCC). The antioxidant activity of the crude extract was initially evaluated by an online HPLC method. Five compounds in the crude extract exhibited good antioxidant activities, namely, hyperoside ( 1 ), isoquercitrin ( 2 ), rutin ( 3 ), kaempferol‐3‐rutinoside ( 4 ), and quercetin ( 5 ). These compounds were further separated by CCC with biphasic solvent systems and their structures were identified by MS and NMR spectroscopy. All the compounds exhibited significant 1,1‐diphenyl‐2‐picryl‐hydrazyl radical scavenging activities with IC₅₀ values at 18.2 ± 1.3, 17.0 ± 1.4, 24.2 ± 1.2, 38.1 ± 1.7, and 9.0 ± 1.2 μM, respectively. The scavenging ratios of the compounds against hydroxyl radicals were 65 ± 5, 68 ± 4, 96 ± 2, 70 ± 4, and 98 ± 2%, respectively.     

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.     

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.     

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.     

Separation and purification of five alkaloids from Aconitum duclouxii by counter‐current chromatography

C₁₉‐diterpenoid alkaloids are the main components of Aconitum duclouxii Levl. The process of separation and purification of these compounds in previous studies was tedious and time consuming, requiring multiple chromatographic steps, thus resulted in low recovery and high cost. In the present work, five C₁₉‐diterpenoid alkaloids, namely, benzoylaconine ( 1 ), N‐deethylaconitine ( 2 ), aconitine ( 3 ), deoxyaconitine ( 4 ), and ducloudine A ( 5 ), were efficiently prepared from A. duclouxii Levl (Aconitum L.) by ethyl acetate extraction followed with counter‐current chromatography. In the process of separation, the critical conditions of counter‐current chromatography were optimized. The two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water/NH₃·H₂O (25%) (1:1:1:1:0.1, v/v) was selected and 148.2 mg of 1 , 24.1 mg of 2 , 250.6 mg of 3 , 73.9 mg of 4, and 31.4 mg of 5 were obtained from 1 g total Aconitum alkaloids extract, respectively, in a single run within 4 h. Their purities were found to be 98.4, 97.2, 98.2, 96.8, and 96.6%, respectively, by ultra‐high performance liquid chromatography analysis. The presented separation and purification method was simple, fast, and efficient, and the obtained highly pure alkaloids are suitable for biochemical and toxicological investigation.     

A new tropane alkaloid from the leaves of Erythroxylum subsessile isolated by pH‐zone‐refining counter‐current chromatography

Tropane alkaloids are bioactive metabolites with great importance in the pharmaceutical industry and the most important class of natural products found in the Erythroxylum genus. However, these compounds are usually separated by traditional chromatographic techniques, in which the sample is progressively purified in multiple chromatographic steps, resulting in a time‐ and solvent‐consuming procedure. In this work we present the isolation of a novel alkaloid, 6β,7β‐dibenzoyloxytropan‐3α‐ol, together with the two known 3α‐benzoyloxynortropan‐6β‐ol and 3α,6β‐dibenzoyloxytropane alkaloids, directly from the crude alkaloid fraction from the leaves of Erythroxylum subsessile, by using a single run pH‐zone‐refining counter‐current chromatography method. The ethyl acetate/water (1:1, v/v) biphasic solvent system with triethylamine and HCl as retention and eluter agents, respectively, was used to isolate tropane alkaloids for the first time. The structures of the isolated alkaloids were elucidated by spectroscopic methods.     

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.     

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.     

Simultaneous separation of three isomeric sennosides from senna leaf (Cassia acutifolia) using counter‐current chromatography

Senna leaf is widely consumed as tea to treat constipation or to aid in weight loss. Sennoside A, A₁, and B are dirheinanthrone glucosides that are abundant and the bioactive constituents in the plant. They are isomers that refer to the (R*R*), (S*S*), and (R*S*) forms of protons on C‐10 and C‐10′ centers and it is difficult to refine them individually due to their structural similarities. The new separation method using counter‐current chromatography successfully purified sennoside A, A₁, and B from senna leaf (Cassia acutifolia) while reversed‐phase medium‐pressure liquid chromatography yielded sennoside A only. n‐Butanol/isopropanol/water (5:1:6, v/v/v) was selected as the solvent system for counter‐current chromatography operation, and the partition coefficients were carefully determined by adding different concentrations of formic acid. High‐resolution mass spectrometry and NMR spectroscopy were performed to verify the chemical properties of the compounds.     

Semi‐preparative high‐speed counter‐current chromatography separation of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn by pH‐gradient elution

A new high‐speed counter‐current chromatography method for semi‐preparative separation and purification of alkaloids from embryo of the seed of Nelumbo nucifera Gaertn was developed by using pH‐gradient elution mode. Diethyl ether was used as the stationary phase of the two‐phase solvent system and Na₂HPO₄/NaH₂PO₄ buffer solution with pH values of 7.5 and 7.2 in gradient mode as the mobile phase. Consequently, 33 mg of liensinine, 42 mg of isoliensinine, and 67 mg of neferine were obtained from 200 mg of crude extracts. The purities of them were all over 98% as determined by HPLC area normalization method, and the structures were identified by ¹H‐NMR and ¹³C‐NMR.     

Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter‐current chromatography

Resveratrol dimers belong to a group of compounds called stilbenes, which along with proanthocyanidins, anthocyanins, catechins, and flavonols are natural phenolic compounds found in grapes and red wine. Stilbenes have a variety of structural isomers, all of which exhibit various biological properties. Counter‐current chromatography with a two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water (2:5:4:5, v/v/v/v) was applied to isolate and purify stilbene from the stems of wine grape. Two isomers of resveratrol dimers trans‐ε‐viniferin and trans‐δ‐viniferin were obtained from the crude sample in a one‐step separation, with purities of 93.2 and 97.5%, respectively, as determined by high‐performance liquid chromatography. The structures of these two compounds were identified by ¹H and ¹³C NMR spectroscopy. In addition, their antioxidant activities were assessed by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) assay. The antioxidant activities of trans‐δ‐viniferin were higher than that of trans‐ε‐viniferin in this model. This work demonstrated that counter‐current chromatography is a powerful and effective method for the isolation and purification of polyphenols from wine grape. Additionally, the DPPH radical assay showed that the isolated component trans‐δ‐viniferin exhibited stronger antioxidant activities than trans‐ε‐viniferin and a little bit weaker than vitamin E at the same concentration.     

Preparative enantioseparation of propafenone by counter‐current chromatography using di‐n‐butyl l‐tartrate combined with boric acid as the chiral selector

This paper extends the research of the utilization of borate coordination complexes in chiral separation by counter‐current chromatography (CCC). Racemic propafenone was successfully enantioseparated by CCC with di‐n‐butyl l ‐tartrate combined with boric acid as the chiral selector. The two‐phase solvent system was composed of chloroform/ 0.05 mol/L acetate buffer pH 3.4 containing 0.10 mol/L boric acid (1:1, v/v), in which 0.10 mol/L di‐n‐butyl l ‐tartrate was added in the organic phase. The influence of factors in the enantioseparation of propafenone were investigated and optimized. A total of 92 mg of racemic propafenone was completely enantioseparated using high‐speed CCC in a single run, yielding 40–42 mg of (R)‐ and (S)‐propafenone enantiomers with an HPLC purity over 90–95%. The recovery for propafenone enantiomers from fractions of CCC was in the range of 85–90%.     

Target‐guided isolation of polar antioxidants from Abelmoschus esculentus (L). Moench by high‐speed counter‐current chromatography method coupled with wavelength switching and extrusion elution mode

An off‐line two‐dimensional high‐speed counter‐current chromatography strategy combined with the wavelength switching technique and extrusion elution mode was successfully developed and applied to the isolation of polar antioxidants from Abelmoschus esculentus (L).Moench. Target‐guided by the result of 2,2‐diphenyl‐1‐picrylhydrazyl screening assay, four antioxidants were obtained with purities over 90% through orthogonal high‐speed counter‐current chromatography separation. UV spectroscopy, mass spectrometry and ¹H NMR spectroscopy were employed to identify their structures, which were assigned as l ‐tryptophan, quercetin‐3‐O‐sophoroside, 5,7,3′,4′‐tetrahydroxyflavonol‐3‐O‐[β‐d ‐rhamnopyranosil‐(1→2)]‐β‐d ‐glucopyranoside, and quercetin‐3‐O‐glucoside. Each monomer exhibited significant antioxidant activity. The results demonstrated that proposed method could be an effective approach to isolate bioactive compounds from complex natural products.     

An interesting two‐phase solvent system and its use in preparative isolation of aconitines from aconite roots by counter‐current chromatography

Two‐phase solvent system plays crucial role in successful separation of organic compounds using counter‐current chromatography (CCC). An interesting two‐phase solvent system, composed of chloroform/ethyl acetate/methanol/water, is reported here, in which both phases contain sufficient organic solvents to balance their dissolving capacities. Adjusting the solvent system to get satisfactory partition coefficients (K values) for target compounds becomes relatively simple. This solvent system succeeded in sample preparation of aconitine (8.07 mg, 93.69%), hypaconitine (7.74 mg, 93.17%), mesaconitine (1.95 mg, 94.52%) from raw aconite roots (102.24 mg, crude extract), benzoylmesaconine (34.79 mg, 98.67%) from processed aconite roots (400.01 mg, crude extract), and yunaconitine (253.59 mg, 98.65%) from a crude extract of Aconitum forrestii (326.69 mg, crude extract).     

Simultaneous separation of triterpenoid saponins and flavonoid glycosides from the roots of Glycyrrhiza uralensis Fisch by pH‐zone‐refining counter‐current chromatography

Glycosides including triterpenoid saponins and flavonoid glycosides are the main constituents of Glycyrrhiza uralensis Fisch (licorice) and exhibit prominent pharmacological activities. However, conventional methods for the separation of glycosides always cause irreversible adsorption and unavoidable loss of sample due to their high hydrophilicities. The present paper describes a convenient method for the simultaneous separation of triterpenoid saponins and flavonoid glycosides from licorice by pH‐zone‐refining counter‐current chromatography. Ethyl acetate/n‐butanol/water (2:3:5, v/v) with 10 mM TFA in the upper organic stationary phase and 10 mM ammonia in the lower aqueous mobile phase was used as the biphasic solvent system. Three triterpenoid saponins and two flavonoid glycosides including licorice‐saponin A3 (63.3 mg), glycyrrhizic acid (342.2 mg), 3‐O‐[β‐d ‐glucuronopyranosyl‐(1 → 2)‐β‐d ‐galactopyranosyl]glycyrrhetic acid (56.0 mg), liquiritin apioside (232.6 mg), and liquiritin (386.5 mg) were successfully obtained from licorice ethanol extract (2 g) in one step. This method subtly takes advantage of the common acidic properties of triterpenoid saponins and flavonoid glycosides, and obviously is much more efficient and convenient than the previous methods. It is also the first time that the separation of acidic triterpenoid saponins by using pH‐zone‐refining counter‐current chromatography has been reported.     

A rapid and convenient method for preparative separation of three indissolvable polyphenols from Euphorbia pekinensis by the flexible application of solvent extraction combined with counter‐current chromatography

A rapid and convenient method was established to preparatively isolate the three ellagic acid types of compounds, which were the main polyphenols in Euphorbia pekinensis, by flexibly applying solvent extraction combined with counter‐current chromatography (CCC). The total extract (extracted using 95% ethanol) of E. pekinensis was pretreated by two simple steps before CCC isolation, following the procedure: the total extract was extracted by classical solvent extraction using petroleum ether and ethyl acetate, respectively, and then the ethyl acetate extract was suspended using 95% ethanol, after being allowed to stand overnight, the sediment was obtained. Partial sediment (100 mg) was then directly separated by CCC with a two‐phase solvent system composed of chloroform‐95% ethanol‐water‐85% formic acid (50:50:50:5, v/v/v/v). About 22 mg of 3,3′‐dimethoxy ellagic acid (1), 12 mg of 3,3′‐di‐O‐methyl‐4‐O‐(β‐d ‐xylopyranosyl)ellagic acid (2), and 35 mg of ellagic acid (3) with purities of 96.0, 95.2, and 95.4% were obtained respectively in one step within 4 h. After being purified by washing with methanol, the purities of the three compounds obtained were all above 98%. The purities were determined by HPLC and their chemical structures were further identified by ¹H and ¹³C NMR spectroscopy. The recoveries were calculated as 84.6, 85.7, and 89.5%, respectively. The result demonstrated that the present isolation method was rapid, economical and efficient for the preparative separation of polyphenols from E. pekinensis.     

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.