One-step High-speed Counter-current Chromatography Separation Research Articles

Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.

Purification of Four Caffeoylquinic Acid Derivatives from the Flowers of Gynura Procumbens by HSCCC.

Four caffeoylquinic acid derivatives from the Gunura procumbens flowers (GPF) were successfully isolated and purified by high-speed counter-current chromatography (HSCCC). Ethyl acetate-methanol-water (3:1:3, v/v/v) was the optimum biphasic solvent system, which was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Chlorogenic acid (3.83 mg), Isochlorogenic acid A (6.51 mg), Isochlorogenic acid B (4.38 mg) and Isochlorogenic acid C (4.47 mg) were obtained for the first time in an one-step HSCCC separation from 800 mg of the crude extracts. The purities of four compounds were determined to be >95% by HPLC. Chemical structures of each isolated compounds were identified by nuclear magnetic resonance and electrospray ionization mass spectrometry methods. It is worth noting that all the four compounds were isolated here for the first time from GPF and this work confirms the effectiveness of HSCCC for the separation of compounds contained in complex samples, and provides a foundation for further exploitation of G. procumbens.

Separation of acteoside and linarin from Buddlejae Flos by high‐speed countercurrent chromatography and their anti‐inflammatory activities

Buddleja officinalis Maxim., a deciduous, flowering shrub, is used as a traditional Chinese medicine; the bioactivity of B. officinalis is primarily due to flavonoids and phenylethanoid glycosides. In the study, acteoside and linarin were successfully isolated from B. officinalis by high-speed countercurrent chromatography with a two-phase solvent system composed of ethyl acetate: n-butanol: water (5:0.8:5, v/v/v). The purities of acteoside and linarin were determined to be 97.3 and 98.2%, respectively, using one-step high-speed countercurrent chromatography separation. The chemical structures of the two compounds were identified by electrospray ionization-mass spectrometry and nuclear magnetic resonance. After separation, the anti-inflammatory effects of the two compounds were evaluated using lipopolysaccharide-induced human umbilical vein endothelial cells. Acteoside and linarin inhibited the expression of nitric oxide, tumor necrosis factor α and interleukin 1β, which demonstrated that acteoside and linarin possessed anti-inflammatory activity.

Purification of Four Flavonoid Glycosides from Lotus (Nelumbo nucifera Gaertn) plumule by Macroporous Resin Combined with HSCCC.

High-speed counter-current chromatography (HSCCC) combined with macroporous resin (MR) column was successfully applied to the isolation and purification of four flavonoid glycosides from the medicinal herb Lotus plumule (LP). A polar two-phase solvent system composed of ethyl acetate-n-butanol-water (1:2:3, v/v/v) was selected by high-performance liquid chromatography (HPLC) and run on a preparative scale where the lower aqueous phase was used as the mobile phase with a head-to-tail elution mode. Quercetin-3-O-β-D-glucopyranoside (15 mg), isorhamnetin-3-O-β-D-glucopyranoside (13 mg), apigenin 6-C-β-D-glucopyranosyl-8-C-α-L-arabinopyranoside (18 mg) and apigenin 6,8-di-C-β-D-glucopyranoside (48 mg) were obtained in a one-step HSCCC separation from 240 mg of the sample. The purity of each compound was over 95% as determined by HPLC. Chemical structures of the isolated compounds were identified by electrospray ionization mass spectrometry (ESI-MS-MS) and nuclear magnetic resonance (NMR) methods. Moreover, the four compounds were isolated from LP for the first time.

Identifying Natural syNergist from Pongamia pinnata Using High-Speed Counter-Current Chromatography Combined with Isobolographic Analysis.

For identifying the synergistic compounds from Pongamia pinnata, an approach based on high-speed counter-current chromatography (HSCCC) combined with isobolographic analysis was designed to detect the synergistic effects in the complex mixture. In the approach, the complex mixture was considered as the combination of two individual samples for isobolographic analysis: the target compound and the mixture with complete removal of the target compound (subtracted residue). The two samples were prepared by HSCCC, and were used for the calculation of the expected effect of their combination. Using this approach, three compounds representing the major peaks in the HPLC of the brine shrimp toxic extract from P. pinnata (brine shrimp lethality test (BST) LC50 36.5 μg/mL), pinnatin (1), 3,7-Dimethoxy-3′,4′-methylenedioxy flavone (2), and karanjin (3), were prepared from the extract, and were tested for their synergistic potency by BST. The two-phase solvent system containing n-hexane-ethyl acetate–MeOH–water (14:7:10:10, v/v/v/v) was selected for the one-step HSCCC separation according to the partition coefficient values (K). The extract was separated into seven fractions (Fr1–7) by HSCCC with a total mass recovery of 96.3%. Fr2, 4, and 6 were the peak fractions corresponding to compounds 3, 2, and 1, respectively. The purities and recoveries of the target compounds after the chromatographic analysis were 95.9%–97.5% and 92.2%–96.1%, respectively. The subtracted residue of each target compound was performed by recombining all HSCCC fractions except the fraction containing the target compound. Isobolographic analysis disclosed a significant synergistic effect between karanjin and its subtracted residue (potency ratio of 0.47), which gave clear evidence that the toxicity of the extract results from synergistic interactions, and karanjin was one of the synergists participating in the interaction. The other two compounds were excluded from the synergism because these two compounds showed additive effects with their subtracted residues. Karanjin was the first synergistic compound identified from P. pinnata.

Separation and Purification of Four Stilbenes from Vitis vinifera L. cv. Cabernet Sauvignon Roots Through High-speed Counter-current Chromatography

A method for preparative separation and purification of trans-resveratrol, δ-viniferin, e-viniferin and trans-vitisin B from the roots of Vitis vinifera L. cv. Cabernet Sauvignon was successfully established and is reported on in this paper. The four important stilbenes were purified by high-speed counter-current chromatography (HSCCC) with a suitable quaternary solvent system composed of chloroform–methanol–n-butanol–water (4:3:0.05:2, v/v). A total of 7.1 mg ± 0.2 mg of trans-resveratrol, 1.1 mg ± 0.1 mg of δ-viniferin, 18.7 mg ± 0.5 mg of e-viniferin, and 12.2 mg ± 0.2 mg of trans-vitisin B, with purities of 97.89%, 90.61%, 94.37% and 78.38% respectively, were obtained from 241 mg of crude sample in a one-step HSCCC separation. The chemical structures of trans-resveratrol and δ-viniferin were further confirmed with the retention time using the method of standard addition, while the structural identification of e-viniferin and trans-vitisin B was performed with LC-ESI/MS, 1H-NMR, and 13C-NMR.

Isolation and Purification of Isochlorogenic Acid A fromLonicera japonicaThunb Using High-Speed Counter-Current Chromatography

A high-speed counter-current chromatography (HSCCC) method was established for the isolation and purification of isochlorogenic acid A from Lonicera japonica Thunb. The two-phase solvent system was composed of n-hexane:ethyl acetate: isopropanol:water (2:3:2:5, v/v/v/v). From 150 mg of the ethyl acetate fraction of L. japonica Thunb, 19.65 mg of isochlorogenic acid A was obtained in a one-step HSCCC separation, with a purity of 99.1%, as determined by high-performance liquid chromatography (HPLC). The structure was further identified by ultraviolet (UV), mass spectrometry (MS) and nuclear magnetic resonance (NMR).

One-step separation of antioxidant compounds from Erythrina variegata by high speed counter-current chromatography.

High speed counter-current chromatography (HSCCC) was used for separation and purification of antioxidant compounds from ethyl acetate fraction of the stem bark of Erythrina variegata. The optimal two-phase solvent system was composed of n-hexane-ethyl acetate-methanol-water (1:4:1:4, v/v/v/v). After one-step HSCCC separation, 75 mg of protocatechuic acid ( 1: ), 32 mg of chlorogenic acid ( 2: ) and 44 mg of caffeic acid ( 3: ) were purified from 420 mg of the ethyl acetate fraction. The purity of isolated compounds was determined up to 99.7% as determined by high-performance liquid chromatography (HPLC). The chemical structures of the three compounds were confirmed by UV, HPLC-MS/MS and (1)H NMR. The IC50 values of scavenging DPPH free radical for the three compounds were 22.5, 41.9 and 20.9 μg/mL, respectively. Protocatechuic acid and chlorogenic acid were obtained from the stem bark of E. variegata for the first time.

One-step Separation and Purification of Two Chromones and One Pyrone from Aloe barbadensis Miller: a Comparison Between Reversed-phase Flash Chromatography and High-speed Counter Current Chromatography

Chromones and pyrones are the major secondary metabolites of Aloe barbadensis Miller. As they are minor components of the plant, an efficient purification procedure for them is of great importance for promoting their pharmacological studies. To develop efficient methods for one-step separation and purification of two chromones (5-((S)-2'-oxo-4'-hydroxypentyl)-2-hydroxymethylchromone (1) and 5-((4E)-2'-oxo-pentenyl)-2-hydroxymethylchromone (3)) and one pyrone (aloenin aglycone (2)) from A. barbadensis via reversed-phase flash chromatography (RP-FC) and high-speed counter current chromatography (HSCCC). The RP-FC separation was performed using methanol:water (26:74, v/v) as the mobile phase at a flow rate of 20 mL/min. A solvent system composed of dichloromethane:methanol:water (3:1.5:1, v/v/v) was used for the HSCCC separation, at a flow rate of 2.0 mL/min. A one-step RP-FC operation within 110 min was successfully used for the purification of compounds 1 (27.9 mg, 96.5%), 2 (32.4 mg, 98.2%) and 3 (4.1 mg, 99.0%) from 129 mg of crude sample, and a one-step HSCCC separation within 95 min was successfully implemented for the purification of compounds 1 (31.1 mg, 97.6%), 2 (35.8 mg, 96.7%) and 3 (2.7 mg, 98.1%) from 134 mg of crude sample. The developed procedures were efficient, with low cost and high yield, which would afford sufficient amounts of high-purity compounds for chromatographic purposes and pharmacological activity screening.

Purification of Two Triterpenoids from Schisandra chinensis by Macroporous Resin Combined with High-Speed Counter-Current Chromatography

A method for preparative purification of corosolic acid and nigranoic acid from Schisandra chinensis (SC) was established using a combination of macroporous absorption resin column separation and high-speed counter-current chromatography (HSCCC). The crude extracts obtained from SC using 70% ethanol were separated on a macroporous resin column and then eluted with a graded ethanol series. The 70% ethanol fraction was used as the sample for separation of the two triterpenoids by HSCCC. The two-phase solvent system used for HSCCC separation was chloroform-n-butanol-methanol-water (10:0.5:7:4, v/v/v/v). The upper phase was used as the stationary phase of HSCCC. Corosolic acid (16.4 mg) of 96.3% purity and nigranoic acid (9.5 mg) of 98.9% purity were obtained in a one-step HSCCC separation from 100 mg of the sample. The structures of corosolic acid and nigranoic acid were identified by (1)H-nuclear magnetic resonance (NMR) and (13)C-NMR.

CONNECTION OF HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY WITH EVAPORATIVE LIGHT SCATTERING DETECTOR BY FLOW INJECTION AND ITS APPLICATION FOR PREPARATIVE ISOLATION AND PURIFICATION OF GINKGOLIDE COMPOUNDS FROM GINKGO BILOBA L.

High-speed counter-current chromatography (HSCCC) and evaporative light scattering detector (ELSD) were connected together by flow injection for the first time. A new method for isolation and purification of ginkgolide compounds from Ginkgo biloba L. was developed. Two pairs of two-phase solvent system, n-hexane-ethyl acetate-methanol-water (4:5:1:5, v/v) and n-hexane-ethyl acetate-methanol-water (4:5:2:5, v/v), were used in HSCCC separation. The stationary phase was the upper phase of n-hexane-ethyl acetate-methanol-water (4:5:1:5, v/v). The mobile phase was the lower phase of n-hexane-ethyl acetate-methanol-water (4:5:1:5, v/v) and n-hexane-ethyl acetate-methanol-water (4:5:2:5, v/v) in gradient elution mode. Ginkgo biloba L. powders were degreased by light petroleum and extracted with 70% ethanol. The extracts were purified with ethyl acetate extraction and acidic alumina oxide column separation, and 2.4 g of purified extracts were obtained from 500 g of Ginkgo biloba L. 107 mg of ginkgolides A, 64 mg of ginkgolides B, 52 mg of ginkgolides C, and 83 mg of bilobalide were obtained in one-step HSCCC separation from 500 mg of the purified extracts. The purities of the obtained compounds were all above 98% as determined by HPLC-ELSD area normalization method and their chemical structures were identified by 1H-NMR and 13C-NMR.

PREPARATIVE ISOLATION AND PURIFICATION OF PHENOLIC ACIDS FROM THE DRIED BUDS OF LONICERA JAPONICA THUNB BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY IN GRADIENT ELUTION MODE

A new high speed counter-current chromatography (HSCCC) method for preparative separation and purification of three phenolic acids from the dried buds of Lonicera japonica Thunb was developed by using gradient elution mode. Two pairs of two-phase solvent system composed of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid at volume ratios of 1:5:1:5 and 1:5:2:5 were used in HSCCC separation. The upper phase of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid (1:5:1:5, v/v) was used as the stationary phase. The lower phase of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid (1:5:1:5, v/v) and (1:5:2:5, v/v) were used as the mobile phase in gradient elution mode. From 200 mg of the crude extract 51.6 mg of 3-caffeoylquinic acid, 7.5 mg of caffeic acid, and 42.4 mg of 3,5-O-dicaffeoylquinic acid were obtained in one-step HSCCC separation. The purities of the obtained compounds were all above 95% as determined by HPLC area normalization method, and their chemical structures were identified by 1H-NMR and 13C-NMR.

Preparative Isolation and Purification of Three Sesquiterpenoid Lactones from Eupatorium lindleyanum DC. by High-Speed Counter-Current Chromatography

A high-speed counter-current chromatography (HSCCC) method was established for the preparative separation of three sesquiterpenoid lactones from Eupatorium lindleyanum DC. The two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (1:4:2:3, v/v/v/v) was selected. From 540 mg of the n-butanol fraction of Eupatorium lindleyanum DC., 10.8 mg of 3β-hydroxy-8β-[4'-hydroxy-tigloyloxy]-costunolide, 17.9 mg of eupalinolide A and 19.3 mg of eupalinolide B were obtained in a one-step HSCCC separation, with purities of 91.8%, 97.9% and 97.1%, respectively, as determined by HPLC. Their structures were further identified by ESI-MS and 1H-NMR.

Preparative isolation and purification of 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis by high-speed counter-current chromatography

An efficient method for the isolation and purification of 12,13-dihydroxyeuparin from Radix Eupatorii Chinensis by high speed counter-current chromatography (HSCCC) was established in this paper. The ether extracts of Radix Eupatorii Chinensis were purified by HSCCC with a solvent system of hexyl hydride–ethyl acetate–methanol–water (1:2:1:2, v/v/v/v). The upper phase was used as the stationary phase and the lower phase as the mobile phase. About 8.4mg of 12,13-dihydroxyeuparin was obtained from 200mg of ether extracts from Radix Eupatorii Chinensis in one-step HSCCC separation, with the purity of 96.71%, as determined by HPLC. After methanol–water recrystallization, the purity of 12,13-dihydroxyeuparin reached 99.83%. Such a simple and effective method was fairly useful to prepare pure compound as reference substances for related study on Radix Eupatorii Chinensis.

Extraction and preparative purification of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography

A method for extraction and preparative separation of tanshinones from Salvia miltiorrhiza Bunge was successfully established in this paper. Tanshinones from Salvia miltiorrhiza Bunge were extracted using ethyl acetate as the extractant under reflux. The extracts were then purified by high speed counter-current chromatography (HSCCC) with light petroleum–ethyl acetate–methanol–water (6:4:6.5:3.5, v/v) as the two phase solvent system. The upper phase was used as the stationary phase and the lower phase as the mobile phase. 8.2 mg of dihydrotanshinone I, 5.8 mg of 1,2,15,16-tetrahydrotanshiquinone, 26.3 mg of cryptotanshinone, 16.2 mg of tanshinone I, 25.6 mg of neo-przewaquinone A, 68.8 mg of tanshinone IIA and 9.3 mg of miltirone were obtained from 400 mg of extracts from Salvia miltiorrhiza Bunge in one-step HSCCC separation, with the purity of 97. 6%, 95.1%, 99.0%, 99.1%, 93.2%, 99.3% and 98.7%, respectively, as determined by HPLC area normalization method. Their chemical structures were identified by 1H NMR.

Preparative Purification of Morroniside and Loganin from Fructus corni by Combination of Macroporous Absorption Resin and HSCCC

A method for preparative purification of loganin and morroniside from Fructus corni was established by combination of macroporous absorption resin column separation and high-speed counter-current chromatography (HSCCC). The crude extracts obtained from Fructus corni by ultrasonic extraction with 50% methanol were subjected to separation on a macroporous resin column and then eluted with 15% and 40% ethanol, respectively. A fraction of 40% ethanol was used as the sample for separation of morroniside and loganin by HSCCC. The two-phase solvent system used for HSCCC separation was n-butanol-methanol-1% acetic acid water (4:1:6, v/v). The upper phase was used as the stationary phase of HSCCC. Morroniside (28.7 mg) of 97.8% purity and loganin (11.5 mg) of 98.6% purity were obtained in a one-step HSCCC separation from 50 mg of sample. The structures of morroniside and loganin were identified by 1H-NMR and 13C-NMR.

Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography

The first preparative separation of two benzoxazinoids, (2 R)-2- O-β- d-glucopyranosyl-2 H-1,4-benzoxazin-3(4 H)-one (HBOA-Glc) and (2 R)-2- O-β- d-glucopyranosyl-4-hydroxy-2 H-1,4-benzoxazin-3(4 H)-one (DIBOA-Glc), by means of high-speed counter-current chromatography (HSCCC) from the n-butanol extract of Acanthus ilicifolius L. is presented. The two-phase solvent system containing ethyl acetate– n-butanol–0.5%NH 4OH (2:3:5, v/v/v, system B) was selected for the one-step HSCCC separation of HBOA-Glc and DIBOA-Glc according to the partition coefficient values ( K) for target compounds and the separation factor ( α) between the two target compounds. In the one-step HSCCC separation using solvent B, from 100 mg n-butanol extract of A. ilicifolius, 6.3 mg HBOA-Glc and 6.8 mg DIBOA-Glc were isolated with purities of 90.3% and 80.2%, respectively. In order to obtain the two target compounds with higher purity, a second separation process was developed comprising two steps. In the two-step separation, the sample was first pre-purified by HSCCC using ethyl acetate– n-butanol–water (2:3:5, v/v/v, system A) solvent system and then purified using solvent system B. A 100-mg amount of the n-butanol extracts of A. ilicifolius was separated to yield 5.8 mg of HBOA-Glc and 4.8 mg of DIBOA-Glc with purities of 97.1% and 94.8%, respectively, which were directly used for NMR analyses.