Preparative isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis by high-speed counter-current chromatography.

High-speed counter-current chromatography was applied to the isolation and purification of salidroside from the Chinese medicinal plant Rhodiola sachalinensis A. Bor. The crude salidroside was obtained by extraction with methanol from Rhodiola sachalinensis A. Bor. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v) was successfully performed yielding salidroside (32 mg) at 98% purity from 250 mg of the crude extract in a one-step separation.     

Bioassay-guided isolation of antioxidants from Astragalus altaicus by combination of chromatographic techniques

An efficient method for bioassay-guided preparative isolation of antioxidants from the n-butanol extract of Astragalus altaicus Bunge was ingeniously developed by combination of silica gel column chromatography and high-speed counter-current chromatography. Under the bioassay-guidance of antioxidant activities, the antioxidants were gradually separated from the crude sample of Astragalus altaicus Bunge by silica gel column chromatography and high-speed counter-current chromatography. Silica gel column chromatography separation was performed with chloroform, chloroform-methanol (100:1~5:1, v/v) and chloroform-methanol-water (5:1:0.1~2:1:0.1, v/v/v). High-speed counter-current chromatography separation was performed with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:6, v/v/v), which was successfully selected by thin layer chromatography analysis, at a flow rate of 1.5 mL/min. As a result, isorhamnetin-3-gentiobioside (20.8 mg), rutin (82.0 mg), and narcissin (12.8 mg) were obtained for the first time from 200 mg of the crude sample, ABS-5 of Astragalus altaicus Bunge. The purities were all at over 95% by high-performance liquid chromatography analysis, and their structures were unambiguously identified by mass spectroscopy, (1) H, and (13) C nuclear magnetic resonance spectroscopy. Antioxidant activities of the three compounds were also assayed by in vitro ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diamonium salt] radical cation scavenging activity. Among them, rutin possessed the highest antioxidant capacity with SC(50) value of 22.15 μg/mL.     

Simultaneous separation and purification of five bioactive coumarins from the Chinese medicinal plant Cnidium monnieri by high-speed counter-current chromatography

Cnidium monnieri (L.) Cusson is a well-known Chinese medicinal plant, which has been used for the treatment of impotence, frigidity, and skin-related diseases, and exhibits strong antipruritic, antiallergic, antidermatophytic, antibacterial, antifungal, and antiosteoporotic activities. A high-speed counter-current chromatography method was developed for the separation and purification of five bioactive coumarins from this plant. The crude coumarins were obtained by ethanol extraction from the dried fruits of Cnidium monnieri (L.) Cusson under sonication. High-speed counter-current chromatography with the two-phase solvent systems n-hexane-ethyl acetate-ethanol-water (5:5:4:6, v/v) and n-hexane-ethyl acetate-ethanol-water (5:5:6:4, v/v) was successfully performed with stepwise elution. The five relatively pure coumarins were obtained from 500 mg of the crude extract in a single run. Their purities were 90.6-98.9%, and the recoveries were 85.7-94.2%.     

Preparative isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza by high-speed counter-current chromatography

High-speed counter-current chromatography was applied to the isolation and purification of salvianolic acid B from the Chinese medicinal plant Salvia miltiorrhiza Bunge. The crude salvianolic acid B was obtained by extraction with ethanol-water from S. miltiorrhiza Bunge. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (3:7:1:9, v/v) was successfully performed yielding 342 mg salvianolic acid B at 98% purity from 500 mg of the crude extract in a one-step separation.     

Isolation and purification of the bioactive carotenoid zeaxanthin from the microalga Microcystis aeruginosa by high-speed counter-current chromatography

High-speed counter-current chromatography was successfully applied for the first time to the isolation and purification of the bioactive carotenoid zeaxanthin from the cyanobacterium Microcystis aeruginosa. The crude zeaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v/v/v) was successfully performed yielding zeaxanthin at 96.2% purity from 150 mg of the crude extract in a one-step separation. The recovery of zeaxanthin was 91.4%. This was also the first report that zeaxanthin was successfully separated and purified from microalgae.     

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave-assisted extraction coupled with high-speed counter-current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.     

Isolation and purification of oridonin from the whole plant of Isodon rubescens by high-speed counter-current chromatography

Semi-preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of oridonin from Isodon rubescens by using a two-phase-solvent system composed of n-hexane-ethyl acetate-methanol-water (2.8:5:2.8:5, v/v/v/v). The targeted compound isolated, collected and purified by HSCCC was analyzed by high performance liquid chromatography (HPLC). A total of 40.6 mg of oridonin with the purity of 73.5% was obtained in less than 100 min from 100 mg of crude Isodon rubescens extract. The chemical structure of the compound was identified by IR, 1H-NMR and 13C-NMR.     

Preparative isolation and purification of psoralen and isopsoralen from Psoralea corylifolia by high-speed counter-current chromatography

Psoralen and isopsoralen were separated from Psoralea corylifolia by high-speed counter-current chromatography (HSCCC). A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v) was used for HSCCC separation, and yielded, from 100 mg of crude extract, 39.6 mg of psoralen and 50.8 mg of isopsoralen each at over 99% purity as determined by high performance liquid chromatography (HPLC). The identification of psoralen and isopsoralen were performed with 1H NMR and 13C NMR.     

Preparative isolation and purification of bergapten and imperatorin from the medicinal plant Cnidium monnieri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of bergapten and imperatorin from the Chinese medicinal plant Cnidium monnieri (L.) Cusson. The crude extract was obtained by extraction with ethanol from the dried fruits of Cnidium monnieri (L.) Cusson under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (5:5:5:5, v/v/v/v) was successfully performed by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml min(-1) after 180 min. The components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 45.8 mg of bergapten at 96.5% purity and 118.3 mg of imperatorin at 98.2% purity from 500 mg of the crude extract in a single run. The recoveries of bergapten and imperatorin were 92.1 and 93.7%, respectively.     

Preparative isolation and purification of gastrodin from the Chinese medicinal plant Gastrodia elata by high-speed counter-current chromatography

Gastrodia elata Blume is a famous Chinese medicinal plant, which has been widely used for the treatment of rheumatism, epilepsy, paralysis, hemiplegia, lumbago, headache and vertigo. High-speed counter-current chromatography was successfully used for the first time for the preparative isolation and purification of the bioactive component gastrodin from G. elata Blume. The crude gastrodin was obtained by extraction with ethanol from the dried roots of G. elata Blume under sonication. Preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v/v) was successfully performed yielding 48 mg gastrodin at 96% purity from 500 mg of the crude extract (10.3% gastrodin) with the recovery of approximately 90% in a one-step separation.     

Preparative separation of rhein from Chinese traditional herb by repeated high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was repeatedly used for isolation and purification of rhein from Rheum officinale Baill (Dahuang) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:5:5, v/v), which had been selected by analytical (HSCCC). Using two preparative units of the HSCCC centrifuge, about a 500 mg amount of the crude extract was separated, yielding 6.7 mg of rhein at a high purity of over 97%.     

Separation of salidroside from Rhodiola crenulata by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was used to purify salidroside from an extract of Rhodiola crenulata with two steps using a two-phase solvent system composed of ethyl acetate-n-butanol-water (1:4:5, v/v) in the first run and chloroform-methanol-isopropanol-water (5:6:1:4) in the second run. The method yielded 21.9 mg of salidroside from 1.216 g of the crude sample at 98% purity determined by HPLC analyses. Identification was performed by 1H NMR, 13C NMR, and MS.     

Separations of flavonoids and alkaloids in medicinal herbs by high-speed counter-current chromatography

Counter-current chromatography is a new liquid-liquid partition chromatography without using solid support. Recently, the technique has been remarkably improved in both partition efficiency and separation time. In this paper the capability of this high-speed counter-current chromatography was demonstrated on separation of two sets of samples obtained from medicinal herbs: a synthetic mixture of 3'-hydroxygenkwanin, luteolin and apigenin was separated on a two-phase solvent system composed of chloroform-methanol-water (4:3:2, v/v/v) and a crude ethanol extract from Anisodus tangulicus (Maxin) Pasch on chloroform-0.07 M sodium phosphate (pH 6.4) (1:1, v/v). In the light of chromatograms obtained from these samples, advantages of high-speed counter-current chromatography over other chromatographic methods were discussed in terms of partition efficiency, peak resolution, separation time, sample loading capacity, etc.     

Preparative isolation and purification of celastrol from Celastrus orbiculatus Thunb. by a new counter-current chromatography method with an upright coil planet centrifuge

A new counter-current chromatography (CCC) method with an upright coil planet centrifuge, which holds four identical multilayer coil columns in the symmetrical positions around the centrifuge axis, was applied to the isolation and purification of celastrol from the roots of Celastrus orbiculatus Thunb. The crude celastrol was obtained by elution with light petroleum from ethanol extracts using 15 cm x 5 cm i.d. silica gel flash chromatography. Preparative CCC with a two-phase system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v) was successfully performed, yielding 798 mg celastrol at 99.5% purity from 1020 mg of the crude sample in one step separation.     

Separation and purification of alkaloids and phenolic acids from Phellodendron chinense by pH-zone refining and online-storage inner-recycling counter-current chromatography

In this work, eight compounds from Phellodendron chinense were separated and purified by pH-zone refining counter-current chromatography and traditional counter-current chromatography coupled with online-storage inner-recycling counter-current chromatography (IRCCC). The pH-zone-refining mode was adopted for separating 2.0 g of crude extract with the solvent system of chloroform–methanol–water (4:3:3, v/v), in which 10 mM hydrochloric acid and 10 mM triethylamine were added in the stationary and mobile phases, respectively. Meanwhile, traditional counter-current chromatography coupled with online-storage IRCCC separation was performed by the solvent system of n-hexane-ethyl acetate-methanol-water (5:5:2:8, v/v). Finally, eight compounds, including six alkaloids as 6-methylpiperidin-2-one( 1 ), isoplatydesmine( 4 ), berlambine( 5 ), epiberberine( 6 ), palmatine( 7 ), berberine( 8 ) and two phenolic acids as ferulic acid( 2 ), isoferulic acid( 3 ), were successfully obtained using these three different CCC modes with the purities over 95.0%.     

Preparative high-speed counter-current chromatography for purification of shikonin from the Chinese medicinal plant Lithospermum erythrorhizon

The bioactive compound shikonin was successfully isolated and purified from the crude extract of the traditional Chinese medicinal plant Lithospermum erythrorhizon Sieb. et Zucc. by preparative high-speed counter-current chromatography (HSCCC). The preparative HSCCC was performed using a two-phase solvent system composed of n-hexane-ethylacetate-ethanol-water (16:14:14:5 (v/v)). A total amount of 19.6 mg of shikonin at 98.9% purity was obtained from 52 mg of the crude extract (containing 38.9% shikonin) with 96.9% recovery. The preparative isolation and purification of shikonin by HSCCC was completed in 200 min in a one-step separation.     

Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the semipreparative separation and purification of puerarin and related isoflavones from a crude extract of Pueraria lobata. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). Using the above solvent system the preparative HSCCC was successfully performed yielding six relatively pure isoflavones including puerarin from 80 mg of the crude extract in one-step separation.     

Isolation of quercetin-3-O-L-rhamnoside from Acer truncatum Bunge by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of quercetin-3-O-L-rhamnoside from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase-system composed of ethyl acetate-ethanol-water at a volume ratio of 5:1:5 (v/v/v). In a single operation, 41.9mg of quercetin-3-O-L-rhamnoside was obtained from 366mg of the crude extract. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of quercetin-3-O-L-rhamnoside was over 96%. Its structure was identified by MS, 1H NMR and 13C NMR. Quercetin-3-O-L-rhamnoside was obtained from this plant for the first time.     

Purification of paeoniflorin from Paeonia lactiflora Pall. by high-speed counter-current chromatography

High-speed counter-current chromatography was successfully used for the first time for the preparative separation and purification of paeoniflorin from the Chinese medicinal plant Paeonia lactiflora Pall. using a two-phase solvent system composed of n-butanol-ethyl acetate-water (1:4:5, v/v) in a single run. From 160 mg of the crude sample containing 22.0% paeoniflorin, 33.2 mg of paeoniflorin was yielded at 98.2% purity as determined by HPLC analysis. The recovery of paeoniflorin was 94.3%.     

Large-scale separation of alkaloids from Corydalis decumbens by pH-zone-refining counter-current chromatography

pH-zone-refining counter-current chromatography was successfully applied to the separation of alkaloids from a crude extract of Corydalis decumbens (Thunb.) Pers. using a multilayer coil planet centrifuge (CPC). The experiment was performed with a two-phase solvent system composed of methyl tert-butyl ether (MtBE)-acetonitrile-water (2:2:3, v/v) where triethylamine (5-10 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5-10 mM) to the aqueous mobile phase as an eluter. From 3.1 g of the crude extract, 495 mg protopine, 626 mg tetrahydropalmatine and 423 mg bicuculline were obtained each with a purity of over 93% as determined by high performance liquid chromatography. The structures of the isolated compounds were identified by electron ionization mass spectrometry (EI-MS), high-performance liquid chromatography (HPLC)-electrospray ionisation-mass spectrometry (ESI-MS) and 1H NMR.