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.     

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.     

Application of high-speed counter-current chromatography coupled with high performance liquid chromatography for the separation and purification of Quercetin-3-O-sambubioside from the leaves of Nelumbo nucifera

Quercetin-3-O-sambubioside [Quercetin-3-O-β-D-xylopyranosyl (1→2)-β-D-glucopyranoside] was separated and purified by semi-preparative high-speed counter-current chromatography (HSCCC) with a two-phase-solvent system composed of ethyl acetate-n-butanol-water (4:1:5, v/v) from the leaves of Nelumbo nucifera (Lotus). A total of 5.0 mg of the targeted compound with a purity of 98.6% as determined by high performance liquid chromatography (HPLC) was obtained from 100 mg of the crude extract cleaned up by AB-8 macroporous resin in a one-step separation. Quercetin-3-O-sambubioside was a novel flavonoid glycoside from the leaves of Nelumbo nucifera, and its chemical structure was identified by means of ESI-MS, 1D NMR and 2D 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.     

High-Purity Calycosin-7-O-β-D-glucopyranoside Recovered from Radix Astragali by Extraction,Fractionation and Recrystallization

Abstract

Calycosin-7-O-β-D-glucopyranoside (CG), one major isoflavonoid in Radix Astragali with promising pharmacological effects, was separated with a low-cost process in the present study. The sequential separation and purification procedures established involved extraction with 90% (v/v) aqueous ethanol at 75°C for 2 h twice followed by partition with ethyl acetate, elution with water, and 40% (v/v) aqueous ethanol on a styrene-based resin column and recrystallization at 4°C for 12 h with methanol. These conditions resulted in recovery of 81.6% of total CG with over 97% purity.     

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.     

One Step Purification of Piperine Directly from Piper nigrum L. by High Performance Centrifugal Partition Chromatography

Abstract

In this study, a preparative high performance centrifugal partition chromatography (HPCPC) method for isolation and purification of the bioactive component piperine directly from the ethanol extract of Piper nigrum L. was successfully established by using n-hexane-ethyl acetate-methanol-water as the two-phase solvent system. The upper phase of n-hexane-ethyl acetate-methanol-water (6:5:6:5, v/v) was used as the stationary phase of CPC. Under the optimum conditions, 40 mg of piperine at 98.5% purity, as determined by HPLC, was yielded from 300 mg of the crude extract in a single CPC separation. The peak fraction of CPC was identified by ¹H NMR and ¹³C NMR.     

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.     

An Efficient Strategy Based on Macroporous Resins and Semi-Preparative High Performance Liquid Chromatography for Rapid Separation of Five Flavonoids Components From Flaveria Bidentis(L.) Kuntze

In this study, a simple, rapid, and efficient purification method of five major flavonoids from crude Flaveria bidentis extracts was established. Five flavonoids components were initially obtained by ethanol-water extraction of Flaveria bidentis (L.) Kuntze, followed by using D4020 resin-based column chromatography and semi-preparative high performance liquid chromatography. 9.3 mg hyperoside, 6.5 mg patuletin-3-O-glucoside, 1.7 mg isorhamnetin 3-sulphate, 8.4 mg astragalin, and 4.9 mg 6-methoxykaempferol-3-O-galactoside at high purity of over 94% were obtained from 580 mg Flaveria bidentis extracts. This method is more efficient than HSCCC comparing with the results of these two methods regarding analysts’ purity, recovery, and running time.     

HPLC and 13C-NMR study of carboxymethyl-β-(1 → 6)-D-gluco-β-(1 → 3)-D-glucan derived from saccharomyces cerevisiae

Sodium salt of carboxymethyl-β-(1 → 6)-D-gluco-β-(1 → 3)-D-glucan (CMG-Na) was prepared from β-D-glucan isolated from baker's yeast (Saccharomyces cerevisiae). Three samples, Fractions I, II, and III, were further separated from the crude CMG-Na derivative. For the physicochemical characterization of the separated fractions, the methods of high-performance liquid chromatography (HPLC) in the size-exclusion mode and carbon?13 nuclear magnetic resonance (¹³C-NMR) spectroscopy were applied. The HPLC method revealed that the molecular weights, M_n, M_w, and M_z averages, of Fraction II were 9.71 × 10⁴, 2.27 × 10⁵, and 3.59 × 10⁵ Da, respectively, whereas those of Fraction III were 1.52 × 10⁴, 2.13 × 10⁴, and 3.57 × 10⁴ Da, respectively. The ¹³C-NMR spectra of Fraction II showed a ratio of 3 : 1 for β?(1 → 3) / β?(1 → 6), whereas for Fraction III, the content of β-(1 → 3) units was smaller. © 1993 John Wiley & Sons, Inc.     

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 of four sesquiterpenoids from Curcuma longa by high-speed counter-current chromatography

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation of four major sesquiterpenoids with similar structures, namely ar-turmerone, β-turmerone, α-turmerone, and E-α-atlantone, from the essential oil of Curcuma longa Linn. using a two-phase solvent system composed of n-heptane-ethyl acetate–acetonitrile–water (9.5/0.5/9/1, v/v). From a single HSCCC run, 1.7861 g of ar-turmerone, 0.4708 g of β-turmerone, 1.3427 g of α-turmerone, and 0.1650 g of E-α-atlantone were obtained. The purity of each compound was over 98% as determined by HPLC. The chemical structures of these compounds were determined by ¹H NMR and ¹³C NMR.     

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.     

Solvent extraction of Eu3+ with 4-oxaheptanediamide into ionic liquid system

ABSTRACT

Liquid–liquid extraction of Eu³⁺ from aqueous solution with 4-oxaheptanediamides (OHAs) as extractant into room temperature ionic liquids (RTILs) of 1-alkyl-3-methylimidazolium hexafluorophosphate (C_nmim⁺PF₆^–, n = 4, 6 and 8) was investigated. The strong affinity of OHAs to Eu³⁺ was observed in the present C_nmim⁺PF₆^– system. The extraction was assumed to proceed by cation-exchange mechanism and formed a 4:1 complex of the OHA extractants and Eu³⁺ in C₄mim⁺PF₆^– system. The preferable composition of extracted species was presumed to be Eu(OHA)₄(H₂O)₄(PF₆)₃ by ESI-MS.     

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.     

Structural Characterization of a Water-Soluble Polysaccharide from the Fruiting Bodies of Agaricus bisporus

An edible fungal polysaccharide termed as ABP was obtained by extraction with hot water, and followed successive chromatographic purification using DEAE-Sepharose Fast Flow column and Sephacryl S-300 High-Resolution column. A symmetrical peak was obtained on high-performance size-exclusion chromatography with an average molecular weight of 5.17 × 10⁴ Da, which was named ABP, and its main components were d-glucose and d-mannose. Based on the study of methylation analysis, along with FT-IR, GC, GC-MS, 1D ¹H and ¹³C NMR and 2D NMR (H-HCOSY, TOCSY, HMQC, and NOESY), its chemical structure was featured with a repeating unit (1→6) linking β-d-Glcp as the main backbone with (1→4)-linked α-d-Manp units. The structure of the mainly repeating units of ABP was established as:  → 6) - β - D - Glucp - (1 → 4) - α - D - Manp(1 → 6) - β - D - Glucp - (1 → 6) - β - D - Glucp - (1 →