Effective and Preparative Separation of Bioactive Flavonoids From Gynostemma Pentaphyllum Tea Using Elution-Extrusion Counter-Current Chromatography

Elution-extrusion counter-current chromatography (EECCC) was successfully applied for screen and separation of four flavonoids from Gynostemma pentaphyllum tea, a popular herbal tea extract in China. With the hexane/ethyl acetate/methanol/water 5/6/5/6 (v/v) system, 300 mg of G. pentaphyllum tea extract were fractionated on a 180 mL-capacity preparative hydrodynamic CCC column. Satisfactory separation efficiency was achieved, producing milligram-amounts of quercetin, isorhamnetin, and cirsiliol over 90% pure in one EECCC process. Due to the hydrophilic property, the major flavonoid glycoside, rutin, was co-eluted with the solvent front as a mixture. Therefore, another carefully selected biphasic liquid system composed of ethyl acetate/n-butanol/water (4/1/5, v/v) was employed, yielding 35 mg of rutin with 97.1% purity. Structures of all separated compounds were identified by ESI-MS, ¹H NMR, and ¹³C NMR.     

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.     

Isolation and Purification of Six Bioactive Compounds from the Seeds of Trigonella foenum-graecum L. using High-Speed Counter-Current Chromatography

It is difficult to simultaneously isolate and purify flavonoids and polyhydroxystilbenes due to their similar polarities. In this study, we isolated and purified these compounds from Trigonella foenum-graecum L. seed extracts using high-speed counter-current chromatography (HSCCC) in two steps with two different kinds of solvent systems. In the first step, n-hexane:ethyl acetate:methanol:water (1:5:1:5, v/v) was used as the two-phase solvent system, desoxy-rhaponticin and rhapontigenin were purified, and orientin, vitexin, isovitexin, and rhaponticin were eluted together. In the second step, ethyl acetate:water (1:1, v/v) was used as the two-phase solvent system, and orientin, vitexin, rhaponticin, and isovitexin were isolated and purified. Purities of the isolated compounds were determined using HPLC, while UV spectra, ¹ H-NMR, and ¹³ C-NMR aided in structure identification.     

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).     

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.     

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.     

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.     

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 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.     

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.     

Enrichment and Separation of Sinomenine and Acutumine from Sinomenium acutum by pH-Zone-Refining Counter-Current Chromatography

Enrichment and separation of alkaloids from a chloroform extract of Sinomenium acutum has been successfully performed for the first time using pH-zone-refining counter-current chromatography. The two-phase solvent system used for enrichment was composed of Methyl tert-butyl ether (MtBE)–acetonitrile (CH₃CN)–water (4:1:5, v/v), where 10 mM triethylamine (TEA) was added to the upper organic stationary phase as a retainer and 10 mM hydrochloric acid (HCl) to the aqueous mobile phase as an eluter, which could enrich the alkaloids from the crude extract well. For the preparative separation, the solvent system consisted of MtBE–CH₃CN–water (4:0.5:5, v/v) with 10 mM TEA in organic stationary phase and 5 mM HCl in the aqueous mobile phase, which could separate and purify the enriched crude alkaloids successfully. 0.82 g of crude alkaloids was enriched from 1.60 g of chloroform extract in the first step separation. From the enriched crude alkaloids, 376 mg of sinomenine and 85 mg of acutumine were obtained in the second step separation with the purity of 98.1% and 98.7%, respectively. The chemical structures of the isolated compounds were identified by UV, ESI-MS and ¹H NMR.     

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 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.     

Separation and Purification of Quinolone Alkaloids from the Chinese Herbal Medicine Evodia rutaecarpa (Juss.) Benth by High Performance Counter-Current Chromatography

Preparative separation of quinolone alkaloids in Evodia rutaecarpa (Juss.) Benth was conducted by high performance counter-current chromatography (HPCCC) with a pair of two solvent systems consisting of n-hexane-methanol-water-acetic acid (2:1:1:0.2, v/v) and (5:4:2:0.1, v/v). Consequently, 31.78 mg 1-methyl-2-nonyl-4 (1H)-quinolone (I), 59.25 mg 1-methyl-2-(6-undecenyl)-4 (1H)-quinolone (II), 333.27 mg evocarpine (III), 101.13 mg 1-methyl-2-(6,9-pentadecadienyl)-4(1H)-quinolone (IV), 132.17 mg dihydroevocarpine (V), and 86.99 mg 1-methyl-2-(10-pentadecenyl)-4(1H)-quinolone (VI) were obtained from 1.3 g of the crude extract. The structures of these compounds were identified by mass spectrometer (MS), nuclear magnetic resonance (¹H NMR and ¹³C NMR).     

Rapid Detection of Antioxidant Flavonoids in Azalea (Rhododendron mucronulatum) Flowers using On-line HPLC-ABTS+ System and Preparative Isolation of Three Flavonoids by Centrifugal Partition Chromatography

High-performance liquid chromatography coupled to an on-line ABTS⁺-based assay (on-line HPLC-ABTS⁺) system was used to determine the principle antioxidants in azalea flowers. Three flavonoids, myricetin, quercetin, and kaempferol, recovered in ethyl acetate extracts of azalea flowers were determined to have antioxidant activities. These three flavonoids were isolated and purified by successive centrifugal partition chromatography (CPC) using two different biphasic solvent systems, consisting of n-hexane-ethyl acetate-methanol-water (3:5:3:5 or 4:5:4:5, v/v). A total of 46.2 mg of myricetin, 28.9 mg of quercetin, and 10.6 mg of kaempferol with purities of 97.0%, 95.4%, and 93.9%, respectively, were purified from 500 mg of ethyl acetate soluble material from azalea flowers. The structures were identified by their retention time, UV spectra, and ESI-MS in the positive ion mode and were confirmed by NMR experiments.     

Preparative Isolation of Bioactive Nitrile Glycoside “Niazirin” from the Fruits of Moringa oleifera Using Fast Centrifugal Partition Chromatography

Fast centrifugal partition chromatography was successfully applied in the separation of bioactive constituent niazirin directly from the chloroform extract of Moringa oleifera. The experiment was performed with a two-phase solvent system composed of ethyl acetate/BuOH/water (6:0.5:4 v/v/v) in which the upper organic layer was used as stationary phase and lower aqueous phase was used as mobile phase. From 1 g of crude extract, 70 mg of niazirin was obtained in 94.8% purity as determined by HPLC. The total yield recovery was >94%. The isolated nitrile glycoside (niazirin) was characterized on the basis of its ¹H, ¹³C-NMR and ESI-MS data.     

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.     

Preparative-Scale Separation of Anticancer Triterpenes from Eucalyptus hybrid by Centrifugal Partition Chromatography

Centrifugal partition chromatography was successfully applied in the separation of close R _f complex anticancer triterpenes directly from a fraction of Eucalyptus hybrid (Myrtaceae). The experiment was performed with a two-phase solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1.5:1 v/v/v) where 2% ammonia solution was added in lower aqueous mobile phase to achieve pH 9.5. From 1.5 g of a fraction, 145 mg of ursolic acid and 72 mg of ursolic acid lactone were obtained in 95.4% and 94.8% purities. The total yield recovery was >94% and the isolated triterpenes were characterized on the basis of their ¹H, ¹³C-NMR, and ESI-MS data.     

One Step Large-Scale Separation and Purification of Rutin from Boenninghausenia sessilicarpa by Countercurrent Chromatography

In this study, a preparative countercurrent chromatography (CCC) method for isolation and purification of the bioactive component rutin directly from the ethanol extract of Boenninghausenia sessilicarpa was successfully established by using n-butanol-ethyl acetate-water as the two-phase solvent system. The upper phase of n-butanol-ethyl acetate-water (4:1:5, v/v) was used as the stationary phase of CCC. Under the optimum conditions, 112 mg of rutin at 98.6% purity was obtained from 2.0 g of the crude extract in a single CCC separation. The peak fraction of CCC was identified by negative ESI, ¹H NMR, and ¹³C NMR.     

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.