Application of High-Speed Counter-Current Chromatography Preparative Separation of Flavone C-Glycosides From Lophatherum gracile

Preparative high-speed counter-current chromatography (HSCCC) was used to separate and purify bioactive constituents from the stems and leaves of Lophatherum gracile Brongn. Six flavone C-glycosides each at over 95% purity including two new compounds were obtained in one-step separation by HSCCC with an optimized two-phase solvent system composed of ethyl acetate-n-butanol-ethanol-water at volume ratio of 4:2:1.5:8.5 (v/v/v/v). The experiment yielded 19.9 mg of luteolin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (1), 28.5 mg of luteolin 6-C-α-L-arabinopyranosyl-7-O-β-D-glucopyranoside (2), 31.5 mg of isoorientin (3), 44.8 mg of orientin (4), 25.3 mg of swertiajaponin (5) and 12.1 mg of apigenin 6-C-β-D-galactopyranosiduronic acid (1→2)-β-D-glucopyranoside (6) from 500 mg of crude extracts. The purity of these compounds was determined by high-performance liquid chromatography (HPLC). Their chemical structures were identified by electron spray ionization mass spectroscopy (ESI-MS), ¹H and ¹³C nuclear magnetic resonance spectroscopy (NMR).     

Application and Comparison of High-Speed Countercurrent Chromatography and High Performance Liquid Chromatography in Preparative Enantioseparation of α-Substitution Mandelic Acids

This paper is mainly about extending research on application and comparison of preparative high-speed countercurrent chromatography (HSCCC) and preparative high performance liquid chromatography (HPLC) in chiral separations. Preparative enantioseparations of α-cyclopentylmandelic acid and α-methylmandelic acid by HSCCC and HPLC were compared using hydroxypropy-β-cyclodextrin (HP-β-CD) and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as the chiral mobile phase additives. In preparative HPLC the enantioseparation was achieved on the ODS C₁₈ reverse phase column with the mobile phase composed of a mixture of acetonitrile and 0.10 mol L^?1 phosphate buffer at pH 2.68 containing 20 mmol L^?1 HP-β-CD for α-cyclopentylmandelic acid and 20 mmol L^?1 SBE-β-CD for α-methylmandelic acid. The maximum sample size for α-cyclopentylmandelic acid and α-methylmandelic acid was only about 10 mg and 5 mg, respectively. In preparative HSCCC the enantioseparations of these two racemates were performed with the two-phase solvent system composed of n-hexane-methyl tert.-butyl ether-0.1 molL^?1 phosphate buffer solution at pH 2.67 containing 0.1 mol L^?1 HP-β-CD for α-cyclopentylmandelic acid (8.5:1.5:10, v/v/v) and 0.1 mol L^?1 SBE-β-CD for α-methylmandelic acid (3:7:10, v/v/v). Under the optimum separation conditions, totally 250 mg of racemic α-cyclopentylmandelic acid could be completely enantioseparated by HSCCC with HP-β-CD as a chiral mobile phase additive in a single run, yielding 114-116 mg of enantiomers with 98-99% purity and 89-92% recovery. But, no complete enantioseparation of α-methylmandelic acid was achieved by preparative HSCCC with either of the chiral selectors due to their limited enantioselectivity. In this paper, preparative enantioseparation by HSCCC and HPLC was compared from various aspects.     

Isolation and Purification of Kaempferol-3,7-O-α-L-Dirhamnopyranoside from Siraitia grosvenori Leaves by High-Speed Counter-Current Chromatograph and Its Free Radical Scavenging Activity

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of flavonoid glycoside from the leaves of Siraitia grosvenori by using a two-phase-solvent system composed of ethyl acetate–n-butanol–water (4:1:5, v/v/v). kaempferol-3,7-O-α-L-dirhamnopyranoside was obtained in one-step separation and less than 5.5 h from 90 mg of crude extract from the S. grosvenori leaves. The chemical structure of this compound was identified by MS, ¹H NMR, and ¹³C NMR. Free radical scavenging activity of kaempferol-3,7-O-α-L-dirhamnopyranoside was also evaluated and the results showed that it had good free radical scavenging activity with its IC₅₀ value being 3.97 mg/ml.     

Separation and Purification of Z Ligustilide and Senkyunolide A from Ligusticum chuanxiong Hort. with Supercritical Fluid Extraction and High‐Speed Counter‐Current Chromatography

Abstract

The essential oil was obtained by supercritical fluid extraction from dried roots of Ligusticum chuanxiong. Different solvent systems for high‐speed counter‐current chromatography (HSCCC) were compared. A system composed of n hexane–ethyl acetate–methanol–water–acetonitrile in the ratio of 8:2:5:5:3 (v/v) was found to be optimum for HSCCC of the essential oil. Z ligustilide and senkyunolide A were separated by HSCCC with purity of 98% determined by GC. The chemical structures of these two components were identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS).     

Separation and Purification of Bioactive Flavonol Glycosides from Hedyotis diffusa Willd by High-Speed Counter-Current Chromatography

Three flavonoid glycosides including quercetin-3-O-[2″-O-(6″′-O-E-sinapoyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(I), quercetin-3-O-[2″-O-(6″′-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(II) and quercetin-3-O-[2″-O-(6″′-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-galactopyranoside(III) were isolated and purified from Hedyotis diffusa Willd by high-speed counter-current chromatography (HSCCC). This run was carried out with a two-phase solvent system composed of n-hexane–ethyl acetate–n-butanol–methanol–1.0% acetic acid (1:1:3.5:1:4.5, v/v) by eluting the lower phase as the mobile phase with a flow-rate at 2.0 ml/min. Consequently, 29.6 mg of I, 35.1 mg of II, 41.3 mg of III with purities of over 95% were obtained from 200 mg of the crude extracts in a single run in less than 130 min. The structure of the isolated compounds was confirmed by MS, ¹H NMR, and ¹³C NMR analysis.     

Separation and Purification of Gardecin from Gardenia jasminoides Ellis by High-Speed Countercurrent Chromatography

In the present study, a high-speed counter-current chromatography (HSCCC) was investigated for separation and purification of gardecin on a preparative scale. Hexane–ethyl acetate–methanol–water (0.3:1:0.3:1, v/v) was selected as the optimum solvent system to purify gardecin from a fraction obtained from HPD100 column chromatography fractionation of gardenia. After HSCCC isolation, 20.1 mg of gardecin with purity of 97.4% was obtained from 322 g of dry gardenia fruits. Chemical structure identification of this pigment was carried out by MS, ¹H NMR, and ¹³C NMR. Additionally, antioxidant activity of gardecin in comparison with crocin-1 was investigated. The present results demonstrated that gardecin could be efficiently obtained using HSCCC from this herb and this compound features strong antioxidant activity.     

Separation and Preparation of Five Cyclopamine Analogs from Rhizomes of Veratrum oxysepalum Turcz. by Two-Step High-Speed Counter-Current Chromatography

Five cyclopamine analogs including jervine, veratramine, pseudojervine, veratrosine, and verdine were isolated from the rhizomes of Veratrum oxysepalum Turcz. by an efficient two-step high-speed counter-current chromatography (HSCCC) method. In the filrst step, HSCCC, dichloromethane-methanol-water (4:3.5:2, v/v/v) was employed to obtain 24.5 mg of jervine, 18.2 mg of veratramine, 9.4 mg of pseudojervine, 20.8 mg of veratrosine, and 5.2 mg of verdine from 200.0 mg of crude alkaloid extracts, with the purities of 65.7, 97.5, 95.2, 98.3, and 98.1%, respectively. After the filrst HSCCC run, the jervine-fraction was subjected to the second step HSCCC using n-hexane-ethyl acetate-methanol-water (4:5:4:5, v/v/v/v) system for further purification and 12.7 mg of jervine with 95.8% purity was obtained. The structures of isolates were identified by liquid chromatography-quadruple time-of-flight tandem mass spectrometry (LC-QTOF MS/MS), ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy.     

Activity-Guided Isolation and Purification of Three Flavonoid Glycosides from Neo-Taraxacum siphonanthum by High-Speed Counter-Current Chromatography

DPPH (1,1-diphenyl-2-picryhydrazyl) radical scavenging assay was used to screen different fractions of Neo-Taraxacum siphonanthum ethanol extracts. The potent active fraction was isolated and purified by preparative high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-n-butanol-water (3:4:7, v/v/v). The flow rate was 1.5 mL/min and resolution speed was 800 rpm. Three flavonoid glycosides with the purity over 99% were obtained and identified as luteolin- 3′-O-β-D-glucopyranoside (I), luteolin-7-O-β-D-glucopyranoside (II), and luteolin-4′-O-β-D-glucopyranoside (III) by ESI-MS, ¹H NMR and ¹³C NMR analysis. Antioxidant activity of three flavonoid glycosides was assessed by DPPH assay, all of which showed potent activity.     

Isolation and Purification of Two Constitutes from Dendrobium fimbriatum Hook by High-Speed Counter-current Chromatography Using Stepwise Elution

Abstract

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of 2-hydroxyethyl caffeate and denhydroshizukanolide from Dendrobium fimbriatum Hook using stepwise elution with a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:1:1:1, v/v) and (3:1:3:1, v/v). Using a preparative unit of the HSCCC centrifuge, about a 100 mg amount of the sample was separated, yielding 13.3 mg of 2-hydroxyethyl caffeate and 18.0 mg of denhydroshizukanolide at a high purity of over 95%. The peak fraction of HSCCC was identified by ¹H NMR and ¹³C NMR.     

Separation and Purification of Two Isomeric Saponins from Albiziae Cortex by High-Speed Counter-Current Chromatography and Preparative High-Performance Liquid Chromatography

Following constituents’ enrichment steps with the AB-8 macroporous resin, silica gel, and ODS columns, high-speed counter-current chromatography (HSCCC) and preparative HPLC were successfully used for the isolation and purification of two complex isomeric saponins including a new one from albiziae cortex. The two-phase solvent system used for separation was composed of n -hexane/ n -butanol/water (1:10:5, v/v/v ). A total of 8.2 mg julibroside J _{5 a} and 11.6 mg julibroside J ₅ with purity of higher than 98%, respectively, as determined by HPLC-ELSD were obtained from the constituents enriched fraction (475.4 mg) of albiziae cortex. Their structures were identified by HR-MS, ¹ H NMR ¹³ C NMR, and 2D NMR. This is the first ever report on the separation of complex isomeric saponins from albiziae cortex by HSCCC.     

Separation and Purification of Arctiin,Arctigenin, Matairesinol,and Lappaol F from Fructus Arctii by High-Speed Counter-Current Chromatography

Arctiin (I), arctigenin (II), matairesinol (III), and lappaol F (IV) were isolated and purified from the traditional Chinese medicine Fructus Arctii by high-speed counter-current chromatography (HSCCC). The crude extracts from Fructus Arctii were treated with D101 macroporous resin first and divided into two parts: fraction 1 and fraction 2. Fraction 1 was separated by ethyl acetate-n-butanol-water (4:0.5:5, v/v/v) and yielded 164 mg of I from 250 mg of fraction 1. Fraction 2 was separated by n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v/v/v) and yielded 27 mg of II, 5 mg of III, and 3 mg of IV from 150 mg of fraction 2. The purities of the four compounds were 99.64%, 98.48%, 96.16%, and 91.41%, respectively, as determined by HPLC-DAD. The chemical structures of the isolated compounds were identified by MS, UV, ¹H NMR, and ¹³C NMR analysis.     

Separation and purification of four tannins from Potentilla parvifolia Fisch. (Rosaceae) using high-speed counter-current chromatography

Four tannins were isolated and identified from Potentilla parvifolia using high-speed counter-current chromatography (HSCCC) in this study. A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:11:1.2:11, v/v/v/v) was chosen and yielded 18.8 mg of procyanidin B3, 9.8 mg of procyanidin B6, 9.1 mg of procyanidin B7, and 6.9 mg of 2,3,4,6-tetra-O-galloyl-D-glucose, all at over 95% purity, from 120 mg of the crude sample powder. The identifications were performed with ¹H NMR and ¹³C NMR. This is the first report showing the presence of procyanidin B7 and 2,3,4,6-tetra-O-galloyl-D-glucose in the genus Potentilla, and procyanidin B3 and procyanidin B6 were isolated from P. parvifolia for the first time.     

One-Step Isolation and Purification of Four Xanthone Glycosides from Tibetan Medicinal Plant Halenia elliptica by High-Speed Counter-Current Chromatography

High-speed counter-current chromatography (HSCCC) was successfully applied to the isolation and purification of four xanthone glycosides from Halenia elliptica, a plant widely used in traditional Tibetan medicine. The introduction of HSCCC greatly improved the efficiency of compounds preparation from Halenia elliptica. The following were obtained from 100 mg of crude sample in one-step separation: 2.5 mg of 1-O-primeverosyl-2,3,4,5,7-pentamethoxyxanthone, 7.0 mg of 1-O-primeverosyl-2,3,4,7- tetramethoxyxanthone, 10.0 mg of 1-O-primeverosyl-2,3,5-trimethoxyxanthone (demethyoxyhaleniaside), and 8.5 mg of 1-O-primeverosyl-2,3,4,5-tetramethoxyxanthone. HPLC analysis showed that each target compound had a purity of over 98%, and UV, ¹H NMR, and ¹³C NMR data confirmed the component chemical structures.     

Preparative Separation and Purification of Three Flavonoids from the Anti-Inflammatory Effective Fraction of Smilax china L. by High-Speed Counter-Current Chromatography

Flavonoids are the main chemical constituents of Smilax china L. and thought to be responsible for the anti-inflammatory activity of Smilax china L. In this study, a suitable HSCCC method was developed to separate the three main flavonoids in a one-separation from the anti-chronic pelvic inflammation disease (CPID) effective fraction of Smilax china L. with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v/v/v). 42 milligrams of astilbin, 10 mg neoisoastilbin, and 6 mg quercetin-3-O-α-L-rhamnoside were separated from 200 mg of the anti-CPID effective fraction of Smilax china L. with purity of 98.3%, 98.7%, 98.5%, respectively, by HPLC analysis. Orthogonal test L₉ (3⁴) was also applied to investigate the optimum extracting conditions of the three flavonoids. HSCCC was successfully used for the isolation and purification of the three flavonoids from the anti-CPID effective fraction of Smilax china L. and the neoisoastilbin was first separated from Smilax china L.     

Separation of Four Compounds from Forsythia suspensa by Counter-Current Chromatography with Stepwise Elution

High speed counter-current chromatography technique in preparative scale has been successfully applied to separate and purify main compounds from the ethyl acetate extract of Forsythia suspense using stepwise elution with two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:4:1:4,v/v) and (1:4:2:3,v/v). Under the optimized conditions, the preparative high-speed counter-current chromatography was performed on 350 mg of the ethyl acetate yielding phillyrin (12.8 mg), isolariciresinol-9’-O-β-D-glucopyranoside (5.3 mg), pinoresinol (21.2 mg), and phillygenin (8.3 mg) in a one-step separation, with purities over 90% as determined by HPLC. The structures of the separated compounds were identified by HPLC-MS and ¹H NMR.     

Isolation and Purification of Geniposide,Crocin-1, and Geniposidic Acid from the Fruit of Gardenia jasminoides Ellis by High-Speed Counter-Current Chromatography

Geniposide, crocin-1, and geniposidic acid were simultaneously separated from the fruit of Gardenia jasminoides Ellis by one step of high-speed counter-current chromatography with the solvent system composed of ethyl acetate-n-butanol-water(1:4:5, v/v/v) within 130 min. The purities of the three compounds were 98.7%, 97.1%, and 90.4%, respectively, as determined by HPLC. Their structures were confirmed by MS, UV, ¹H NMR, and¹³C NMR analysis.     

Combination of supercritical fluid extraction with high-speed countercurrent chromatography for extraction and isolation of ethyl p-methoxycinnamate and ethyl cinnamate from Kaempferia galanga L.

Supercritical fluid extraction (SFE) with high-speed countercurrent chromatography (HSCCC) was successfully used for the extraction and isolation of ethyl p-methoxycinnamate (EPMC) and ethyl cinnamate (EC) from Kaempferia galanga L. The SFE parameters including extraction temperature, extraction pressure and entrainer volume were optimized by central composite design (CCD). Then the crude extract was separated by HSCCC with a two-phase solvent system composed of n-hexane:ethyl acetate:methanol:water (7:3:8:2, v/v/v/v) in one-step within 60 min. As a result, 13 mg of EPMC and 2 mg of EC were isolated from 100 mg of crude extract with purities of 98.4% and 98.1%, as determined by HPLC. The structural identification was carried out by UV, MS and NMR spectra.     

Separation and Purification of Three High-Purity Isoflavonoids from Belamcanda chinensis (L.) DC. by Supercritical Fluid Extraction and High-Speed Counter-Current Chromatography

Supercritical fluid extraction (SFE) was used to extract three isoflavonoids including irigenin, irisfloretin and dichtomitin from Belamcanda chinensis (L.) DC. The parameters including pressure, temperature, sample particle size, and flow rate of CO₂ were optimized with an orthogonal test. Under the optimized conditions of 15 MPa, 55°C, a sample particle size of 20–40 mesh and CO₂ flow rate of 40 L h^?1. The process was then scaled up by 10 times using a preparative SFE system. The yield of the crude extract from SFE was 4.1%, which contained irigenin, irisfloretin, and dichtomitin 0.71%, 0.49%, and 0.05%, respectively. To compare the extraction methods, Soxhlet Extraction (SE) was performed. The results indicated that SFE was better than SE. Irigenin, irisfloretin, and dichtomitin in the SFE extract were then separated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (2:4:3:3, v/v). From 5.0 g of dry crude extract, 27.8 mg irigenin, 16.4 mg irisfloretin, and 2.1 mg dichtomitin were obtained at purities of 97.1%, 96.4%, and 98.0%, respectively, as determined by HPLC-PDA. These results well indicate that SFE and HSCCC are very powerful techniques for the extraction and purification of irigenin, irisfloretin, and dichtomitin from B. chinensis.     

Separation of 4’-demethyldeoxypodophyllotoxin from Sinopodophyllum emodi by medium-pressure LC and high-speed counter-current chromatography guided by HPLC-MS

A minor lignan, 4ˊ-demethyldeoxypodophyllotoxin, was isolated from Sinopodophyllum emodi. First, a crude extract of S. emodi was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to confirm the presence of the target compound. Then, the target lignan was successfully enriched in the crude extract precipitate by medium-pressure LC (MPLC) with high recovery (85%). Finally, an efficient separation was achieved by high-speed counter-current chromatography (HSCCC) using a two-phase solvent system of n-hexane-ethyl acetate–methanol–water (1:1:1:1, v/v/v/v). These results clearly demonstrate that the combination of HPLC-MS, MPLC, and HSCCC could be a powerful technique for the rapid screening, enrichment, and separation of minor compounds from complex natural products.     

Biological stereoselective reduction of 2,6‐ and 3,5‐dimethylcyclohexanones by Glomerella cingulata

The microbial transformations of 2,6‐ and 3,5‐dimethylcyclohexanone were investigated using the plant pathogenic fungus, Glomerella cingulata. With this organism 2,6‐ and 3,5‐dimethylcyclohexanone gave the corresponding 2,6‐ and 3,5‐dimethylcyclohexanol. The metabolites from 2,6‐dimethylcyclohexanone indicated enantioselective reduction by specific optical rotation of the products. The enantiomeric excesses of the microbiological reduction products were determined by ¹H‐NMR spectra of (+)‐MTPA‐esters of the alcohols produced. The reduction of 2,6‐dimethylcyclohexanone was stereospecific, with the (2R,6R)‐ketone being converted to the corresponding (2R,6R)‐(−)‐2,6‐dimethylcyclohexanol; absolute configuration, 70% ee. On the other hand, 3,5‐dimethylcyclohexanone gave the (1α,3α,5α)‐3,5‐dimethylcyclohexanol (74%) and (1α,3β,5β)‐3,5‐dimethylcyclohexanol (26%). © 1999 Society of Chemical Industry