Isolation of dammarane saponins from Panax notoginseng by high-speed counter-current chromatography

The Chinese phytomedicinal formulation Sanqi Zongdai Pian, traditionally prepared from crude extracts from roots of Panax notoginseng (Araliaceae), contains highly polar dammarane saponins which were separated at a preparative scale using high-speed counter-current chromatography (HSCCC). In each operation, 283 mg methanolic extract of five tablets was separated and yielded pure 157, 17, 13 and 56 mg of ginsenoside-Rb1, notoginsenoside-R1, ginsenoside-Re and ginsenoside-Rg1, respectively, n-hexane-n-butanol-water (3:4:7, v/v/v) was used for the two-phase solvent system of the HSCCC separation. The chemical structures of three ginsenosides and one notoginsenoside were elaborated by means of electrospray ionization MS-MS and NMR analysis.     

Isolation and purification of honokiol and magnolol from cortex Magnoliae officinalis by high-speed counter-current chromatography

High-speed counter-current chromatography was used to isolate and purify honokiol and magnolol from cortex Magnoliae Officinalis (Magnolia officinalis Rehd. et Wils.), a plant used in the traditional Chinese medicine. A crude sample, 150 mg, was successfully separated with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.4:1:0.4, v/v), and the fractions were analyzed by high-performance liquid chromatography. The separation produced 80 and 45 mg of honokiol and magnolol with purities of 99.2 and 98.2%, respectively, in 2.5 h.     

Preparative purification of glycyrrhizin extracted from the root of liquorice using high-speed counter-current chromatography

Glycyrrhizin is one of the main bioactive components in liquorice (Glycyrrhiza uralensis Fisch) which has recently been found to be highly active in inhibiting replication of the severe acute respiratory syndrome (SARS)-associated virus. The separation and purification of glycyrrhizin from a methanol-water (70:30 (v/v)) extract of liquorice roots was achieved using high-speed counter-current chromatography. The separation was performed at a preparative scale in a one-step separation with a two-phase solvent system composed of ethyl acetate-methanol-water (5:2:5 (v/v)). The lower phase was used as the mobile phase in the head-to-tail elution mode. The present method yielded 42.2 mg glycyrrhizin at 96.8% purity from 130 mg of the crude exact with 95.2% recovery as determined by HPLC analysis.     

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.     

Preparative isolation of osthol and xanthotoxol from Common Cnidium Fruit (Chinese traditional herb) using stepwise elution by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of osthol and xanthotoxol from Cnidium monnieri (L.) Cusson (Common Cnidium Fruit) 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 (5:5:6:4, v/v), which had been selected by analytical high-speed counter-current chromatography. Using a preparative unit of the HSCCC centrifuge, about a 308 mg amount of the crude extract was separated, yielding 88.3 mg of osthol and 19.4 mg of xanthotoxol at a high purity of over 98%.     

Preparative isolation and purification of syringin and edgeworoside C from Edgeworthia chrysantha Lindl by high-speed counter-current chromatography

The bioactive compound syringin along with edgeworoside C were separated from the n-butanol extract of the stems and barks of Edgeworthia chrysantha Lindl (E. papyrifera) by high-speed counter-current chromatography (HSCCC) while it was difficult to purify each compound by silica gel column chromatography. Syringin was isolated from this plant for the first time. The two-phase solvent system used was composed of ethyl acetate-ethanol-water at an optimized volume ratio of 15:1:15 (v/v/v). Preparative HSCCC yielded, from 110mg of the partially purified extract, 28mg of syringin and 45 mg edgeworoside C each at over 96% purity by high-performance liquid chromatography analysis. Their structures were identified by electron impact ionization MS, 1H NMR and 13C NMR.     

Purification of astilbin and isoastilbin in the extract of smilax glabra rhizome by high-speed counter-current chromatography

Purification of astilbin from the rhizome extract of Smilax glabra was conducted using a high-speed counter-current chromatograph equipped with a 700 mL column. In a single operation, 1.5 g of crude sample was separated to yield 105 mg of component astilbin and 48 mg of isoastilbin while the upper phase of the two-phase solvent system composed of n-hexane-n-butanol-water (1:1:2, v/v/v) was used for stationary phase. The chemical structures of the two flavonoid glycosides were confirmed by electrospray ionization ion trap multiple mass spectrometry and nuclear magnetic resonance experiments.     

Large-scale isolation and purification of geniposide from the fruit of Gardenia jasminoides Ellis by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of geniposide from Gardenia jasminoides Ellis. 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). According to the above solvent system, preparative HSCCC was successfully performed with the optimal solvent system composed of ethyl acetate-n-butanol-water (2:1.5:3, v/v/v) yielding 389 mg of geniposide at over 98% purity from 1g of the partially purified extract with 38.9% recovery in a one-step separation.     

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.     

Isolation and purification of coumarin compounds from Cortex fraxinus by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully used for the isolation and purification of coumarin compounds from Cortex fraxinus, the Chinese herbal drug. n-Butanol-methanol-0.5% acetic acid (5:1.5:5, v/v) was used as the two-phase solvent system. 14.3 mg of fraxin, 26.5 mg of aesculin, 5.8 mg of fraxetin and 32.4 mg of aesculetin with the purity of 97.6, 99.5, 97.2 and 98.7%, respectively were obtained from 150 mg of crude extracts of C. fraxinus in a single run. The structures of the isolated compounds were identified by 1H NMR and 13C NMR.     

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.     

Preparative isolation and purification of isoflavan and pterocarpan glycosides from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography

(3R)-(-)-7,2'-Dihydroxy-3',4'-dimethyl isoflavan-7-O-beta-D-glucopyranoside and (6aR, 11aR) 9,10-di-methoxypterocarpan-3-O-beta-D-glucopyranoside were separated from the ethyl acetate extract of the root of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography (HSCCC). A two-phase system composed of ethyl acetate-ethanol-acetic acid-water (4:1:0.25:5, v/v) was selected by analytical HSCCC. Preparative HSCCC yielded, from 100 mg of the partially purified extract, 50 mg of isoflavan glycoside and 10 mg of pterocarpan glycoside each at over 95% purity by high-performance liquid chromatography (HPLC) analysis. Their structures were identified by MS, 1H NMR and 13C NMR.     

New 18-l process-scale counter-current chromatography centrifuge

A new Dynamic Extractions Maxi-counter-current chromatography (CCC) centrifuge with a column volume of 18-l has been installed in the Advanced Bioprocessing Centre at Brunel. This instrument has four times the capacity of the 4.6-l Maxi-CCC centrifuge which has been operating robustly for 3 years. Tests using the model sample system benzyl alcohol and p-cresol with a heptane:ethyl acetate:methanol:water (HEMWat) phase system (1.4:0.1:0.5:1.0) show that resolution is almost double with this new high capacity device. Commissioning tests with a mixture of caffeine, K_D = 0.21; ferulic acid, K_D = 0.82; umbelliferone, K_D = 1.2 and vanillin, K_D = 1.49 using a HEMWat phase system of 1:1.5:1:1.5 on the 9-l column show that resolutions equivalent to analytical instruments will be possible using the full 18-l capacity. They also show that predictable scale-up from simple test tube tests is feasible with knowledge of the stationary phase retention for the planned process scale run.     

Solid phase extraction conditions for the selective isolation of drugs from biological fluids predicted using liquid chromatography

Summary A new method to predict the most suitable conditions for the solid phase extraction of 1,4-benzodiazepines and related compounds using C18 Sep-Pak carridges is proposed. The composition of the washing and elution solvents for the solid phase extraction of a test compound can be obtained from its capacity factor on a C18 HPLC column and an equation which relates capacity factors and solid phase extraction data of other similar compounds. The solid phase extraction data given in this paper can be used by others, there by saving considerable time and effort in the development of sample preparation methods. The suitability of the method was checked with two test compounds, showing good results.     

Preparative isolation and purification of three flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of flavonoids from the Chinese medicinal plant Epimedium koreamum Nakai was successfully established by using chloroform-methanol-water (4:3.5:2, v/v) as the two-phase solvent system. The method yielded 11.4 mg of epimedokoreanoside I, 46.5 mg of icariin and 17.7 mg of icariside II from 200 mg of the crude sample in one-step separation with the purity of 98.2%, 99.7% and 98.5%, respectively, as determined by high-performance liquid chromatography (HPLC). The structures of the flavonoids were identified by 1H NMR and 13C NMR.     

Preparative isolation of novel antioxidant flavonoids of alfalfa by stop-and-go counter-current chromatography and following on-line liquid chromatography desalination

In this work, we have established a new stop-and-go two-dimensional chromatography coupling of counter-current chromatography and liquid chromatography (2D CCC × LC) for the preparative separation of two novel antioxidant flavonoids from the extract of alfalfa (Medicago sativa L.). The CCC column has been used as the first dimension to purify the target flavonoids using a solvent system of isopropanol and 20% sodium chloride aqueous solution (1:1, v/v) with the stop-and-go flow technique, and the LC column packed with macroporous resin has been employed as the second dimension for on-line absorption, desalination and desorption of the targeting effluents purified from the first CCC dimension. As a result, two novel flavonoids, 6,8-dihydroxy-flavone-7-O-β-D-glucuronide (15.3 mg) and 6-methoxy-8-hydroxy-flavone-7-O-β-D-glucuronide (13.7 mg), have been isolated from 126.8 mg of crude sample pre-enriched by macroporous resin column. Their structures have been identified by electrospray ionization mass spectrometry (ESI-MS), electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) and one- and two-dimensional nuclear magnetic resonance spectra (1D and 2D NMR). Further antioxidant assays showed that the first component possess a strong antioxidant activity. All the results demonstrated that the stop-and-go 2D CCC × LC method is very efficient for the separation of flavonoids of alfalfa and it can also be applied to isolate other comprehensive multi-component natural products.     

Advantages of a small-volume counter-current chromatography column

Counter-current chromatography (CCC) works with a support-free liquid stationary phase. This allows for preparative separations and purifications. However, there are serious technical constraints because of the need to keep a liquid stationary phase in a column. Centrifugal fields are used. A new commercial hydrodynamic 18 mL column made with a narrow-bore 0.8 mm Teflon tubing was evaluated by comparing it with older hydrodynamic CCC columns and a similar 19 mL column but made with 1.6 mm Teflon tubing. A small-volume CCC column allows for reliable and fast solute partition coefficient determination. When resolution is required, both high efficiency and liquid stationary phase retention are needed. Unfortunately, these two requirements bear technical contradictions. A column coiled with a narrow tubing bore will provide a high chromatographic efficiency while a column containing wider tubing bore will achieve higher stationary phase retention. In all cases, increasing the magnitude of the centrifugal field also increases the stationary phase retention. The solution is to build centrifuges able to produce high fields that will provide acceptable liquid phase retention with narrow-bore tubes. The new 18 mL 0.8 mm tubing bore column is able to rotate as fast as 2100 rpm generating a 240 × g field. The two older CCC columns cannot compete with the new one. However, the small 19 mL column with 1.6 mm bore tubing can be useful when fast results are desired without top resolution.     

Isolation and purification of flavonoid glycosides from Trollius ledebouri using high-speed counter-current chromatography by stepwise increasing the flow-rate of the mobile phase

Three flavonoid glycosides including orientin, vitexin, quercetin-3-O-neohesperidoside and one unknown compound were isolated and purified by high-speed counter-current chromatography (HSCCC) and semi-preparative HPLC from Trollius ledebouri Reichb., a traditional Chinese medicine. Preparative HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v) was successfully performed by increasing the flow-rate of the mobile phase from 1.5 to 2.5 ml/min after 190 min. Consequently, 95.8 mg orientin, 11.6 mg vitexin, 9.3 mg unknown compound with purities of over 97% and one partially purified peak fraction (contained quercetin-3-O-neohesperidoside at 85.1% purity) were obtained from 500 mg of the crude extract. Then the partially purified fraction was further purified by reversed-phase semi-preparative high-performance liquid chromatography. The structure identification of all pure fractions was carried out by UV, MS, 1H NMR and 13C NMR.     

Simulated counter-current moving column chromatography used in the continuous separation of carbohydrate mixtures

Summary A moving column-bundle counter-current chromatographic system consisting of twelve 0.9 cm id×72.5 cm long glass columns has been used for the separation of glucose-starch and glucose-maltose mixtures. The system was packed with Spherosil XOBO75 for the glucose-starch separation and with Dowex-5OW-X4K⁺ for the glucose-maltose separation.Purities of up to 100% were obtained and the operating throughputs were up to 4.4 kg sugar solids/m³ resin/hr.     

Purification of honokiol derivatives from one-pot synthesis by high-performance counter-current chromatography

This paper describes the application of high-performance counter-current chromatography (HPCCC) as a fast, useful and economic alternative for the separation and purification of seven honokiol derivatives (two of them are isomers), which were synthesized by a one-pot procedure. Five honokiol derivatives were successfully separated by n-hexane–ethyl acetate–methanol–water solvent system at three different volume ratios in a step-gradient elution. Two derivatives were obtained through a cycle elution mode. The whole separation process produced 366.3 mg, 323.6 mg, 242.8 mg, 216.2 mg, 203.5 mg, 185.8 mg and 279.3 mg of 3′-formylhonokiol (1), 2′-methoxy-3′-formylhonokiol (2), 2′-methoxyhonokiol (3), 4-methoxyhonokiol (4), 3′,5-diformylhonokiol (5), 2′,4-dimethoxy-3′-formylhonokiol (6) and 2′,4-dimethoxyhonokiol (7) from crude sample of 3 g with purities of 98.7%, 99.3%, 98.6%, 98.2%, 99.0%, 98.4% and 99.2%, respectively. The purities and structural identification were determined by HPLC, ¹H NMR, ¹³C NMR and mass spectroscopy.