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 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%.     

Preparative isolation and purification of lutein from the microalga chlorella vulgaris by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the isolation and purification of lutein from microalgae. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of n-hexane-ethanol-water (4:3:1, v/v). Using the above solvent system, preparative HSCCC was successfully performed yielding lutein at 98% purity from 200 mg of the crude extract in a one-step separation.     

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 of epigallocatechin and flavonoids from Hypericum perforatum L. by high-speed counter-current chromatography and preparative high-performance liquid chromatography

High-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of epigallocatechin and flavonoids from Hypericum perforatum L. The two-phase solvent system composed of ethyl acetate–methanol–water (10:1:10, v/v) was used for HSCCC. About 900 mg of the crude extract was separated by HSCCC, yielding 7.8 mg of quercitrin at a purity of over 97%, 12.6 mg of quercetin at a purity of over 93%, and 38.9 mg of a mixture of hyperoside, isoquercitrin and miquelianin constituting over 97% of the fraction. A mixture of epigallocatechin and avicularin pooled from three HSCCC runs, a total amount of 54.3 mg, was further separated by prep-HPLC yielding 23.4 mg of epigallocatechin and 15.3 mg of avicularin each at a purity of over 97%.     

高速逆流色谱法分离纯化续随子种子中的七叶内酯

建立了高速逆流色谱(HSCCC)技术分离纯化续随子种子中七叶内酯的方法。将续随子种子的乙酸乙酯萃取物直接进行高速逆流色谱分离,考察了不同溶剂系统的分离效果。结果表明,最佳的溶剂系统为氯仿-甲醇-水(体积比为4:3:2),以其上相为固定相,下相为流动相。从200 mg续随子种子乙酸乙酯萃取物中分离得到80 mg七叶内酯,纯度为99.04%。HSCCC技术可高效分离纯化续随子种子中的七叶内酯,为得到高纯度的七叶内酯提供了制备技术。     

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%.     

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 three sesquiterpene lactones from the roots of Cichorium glandulosum Boiss. et Huet. by high-speed counter-current chromatography

Because of the skeletal complexity and similarity of the polarity, little research was reported on the isolation of sesquiterpene lactones by high-speed counter-current chromatography (HSCCC). Herein, three sesquiterpene lactones were successfully purified from the ethyl acetate extract of the roots of the traditional Uyghur medicinal plant Cichorium glandulosum Boiss. et Huet. by HSCCC. The separation was performed in two steps with two solvent systems: n-hexane-ethyl acetate-methanol-water (1.5:5:2.75:5, v/v/v/v) and ethyl acetate-methanol-water (20:1:20, v/v/v). From 166 mg of the ethyl acetate extract, 19 mg of lactucopicrin was isolated with the first solvent system and 10 mg of 11beta,13-dihydrolactucin and 16 mg of lactucin were obtained with the second solvent system. All purified compounds were over 94% purity as determined by HPLC analysis, and these chemical structures were confirmed by (1)H NMR and (13)C 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.     

Isolation of high-purity casticin from Artemisia annua L. by high-speed counter-current chromatography

Following an initial clean-up step on silica, high-speed counter-current chromatography (HSCCC) was used to purify a flavone, casticin (5,3'-dihydroxy-3,6,7, 4'-tetramethoxyflavone), from an extract of the dried leaves of Artemisia annua L. The two-phase solvent system used was composed of n-hexane-ethyl acetate-methanol-water at an optimized volume ratio of 7:10:7:10 (v/v). HSCCC separation of 226.4 mg of crude sample (containing casticin at 16.5% purity after silica gel clean-up) yielded 36.3 mg of casticin with a purity of over 99% and 96.2% recovery. Identification of the target compound was performed by (1)H NMR, (13)C NMR, two-dimensional NMR, electrospray ionization MS, IR and UV.     

Preparative separation and purification of deoxyschisandrin and gamma-schisandrin from Schisandra chinensis (Turcz.) Baill by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully applied to the preparative separation and purification of deoxyschisandrin and gamma-schisandrin from the crude petroleum ether extracts of Schisandra chinensis (Turcz.) Baill. The optimum solvent system composed of n-hexane-methanol-water (35:30:3, v/v) led to the successful preparation of deoxyschisandrin and gamma-schisandrin. The analysis of HPLC for each peak fraction of preparative HSCCC showed that the purity of deoxyschisandrin (8 mg) was over 98% and gamma-schisandrin (12 mg) was over 96% from 100 mg of the crude petroleum ether extracts in one-step separation.     

Isolation and purification of canthaxanthin from the microalga Chlorella zofingiensis by high-speed counter-current chromatography

Certain microalgae are considered to be a potential source of canthaxanthin, which possesses strong antioxidant and anticancer activities. A high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of canthaxanthin from the microalga Chlorella zofingiensis. The crude canthaxanthin was obtained by extraction with organic solvents after the microalgal sample had been saponified. Preparative HSCCC, with a two-phase solvent system composed of n-hexane-ethanol-water (10:9:1 v/v), was successfully performed yielding canthaxanthin at 98.7% purity from 150 mg of the crude extract (2.1% canthaxanthin) in a one-step separation. The recovery of canthaxanthin was 92.3%. This was the first report that canthaxanthin was successfully separated and purified from microalgae.     

Application of preparative high-speed counter-current chromatography for separation of methyl gallate from Acer truncatum Bunge

Preparative separation of methyl gallate in leaves extract of Acer truncatum Bunge was conducted using high-speed counter-current chromatography (HSCCC) with a solvent system composed of ethyl acetate-ethanol-water at volume ratios of 5:1:5 (v/v/v). In a single operation, 57.5 mg of methyl gallate was obtained from 120 mg of the extract. HPLC analyses of the counter-current chromatography (CCC) fraction revealed that the methyl gallate was having over 97% purity. Its structure was identified by 1H NMR and 13C NMR.     

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.     

Preparative separation of high-purity cordycepin from Cordyceps militaris(L.) Link by high-speed countercurrent chromatography

A high-speed counter-current chromatography (HSCCC) technique in a preparative scale has been applied to separate and purify cordycepin from the extract of Cordyceps militaris(L.) Link by a one-step separation. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-n-butanol-methanol-water (23:80:30:155, v/v/v/v) by eluting the lower mobile phase at a flow rate of 2 ml/min under a revolution speed of 850 rpm. HSCCC separation of 216.2 mg crude sample (contained cordycepin at 44.7% purity after 732 cation-exchange resin clean-up) yielded 64.8 mg cordycepin with purity of 98.9% and 91.7% recovery. Identification of the target compound was performed by UV, IR, MS, (1)H NMR and (13)C NMR.     

Preparative isolation and purification of phillyrin from the medicinal plant Forsythia suspensa by high-speed counter-current chromatography

Forsythia suspensa (Thunb.) Vahl. has been used widely in traditional medicines to treat gonorrhea, erysipelas, inflammation, pyrexia and ulcer. It has also shown antioxidant activity, as well as antibacterial, antiviral, choleretic and antiemetic effects. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of the bioactive molecule phillyrin from F. suspensa (Thunb.) Vahl. The crude phillyrin was obtained by extraction with 50% ethanol from the dried fruits of F. suspensa (Thunb.) Vahl. under sonication. Preparative HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (1:9:1:9, v/v/v/v) was successfully performed, and the components purified and collected were analyzed by high-performance liquid chromatography. The method yielded 5.6 mg phillyrin at 98.6% purity from 500 mg of the crude extract (1.2% phillyrin) with the recovery of 92% in a one-step separation.     

Application of high-speed counter-current chromatography coupled with high-performance liquid chromatography-diode array detection for the preparative isolation and purification of hyperoside from Hypericum perforatum with online purity monitoring

Following preparative isolation and purification by high-speed counter-current chromatography (HSCCC), the collected fractions were generally analyzed by high-performance liquid chromatography (HPLC) to determine the relative purities of each fraction. Our paper reports for the first time a preparative isolation-purity detection hyphenated system: online coupling of HSCCC with high-performance liquid chromatography-diode array detection (HSCCC-HPLC-DAD). The introduction of online purity analysis in HSCCC has dramatically improved the efficiency of this technique by overcoming the drawbacks of post analysis in HSCCC isolation. The effluent from the outlet of HSCCC was splitted into two parts: one was collected, while the other was introduced directly into an HPLC-DAD system for purity analysis through a switch valve. Therefore, the purities of the obtained fractions from HSCCC were monitored, and fractions with high purities were collected. This strategy has been successfully demonstrated with the preparative isolation and purification of hyperoside from Hypericum perforatum (St. Jone's Wort); a model of TBE-300A HSCCC was used to isolate and separate hyperoside from H. perforatum with a two-phase solvent system composed of ethyl acetate-ethanol-water at the volume ratio of 5:1:5 (v/v) using online detection technique. The isolation was done in less than 3.5 h, and a total of 83.0-mg hyperoside at over 99.0% purity was yielded from 300 mg of the partially purified extract. This new strategy possesses general utility in the preparation of bioactive compounds from traditional Chinese medicine (TCM).     

Preparative isolation and purification of two benzoxazinoid glucosides from Acanthus ilicifolius L. by high-speed counter-current chromatography

The first preparative separation of two benzoxazinoids, (2R)-2-O-beta-d-glucopyranosyl-2H-1,4-benzoxazin-3(4H)-one (HBOA-Glc) and (2R)-2-O-beta-d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA-Glc), by means of high-speed counter-current chromatography (HSCCC) from the n-butanol extract of Acanthus ilicifolius L. is presented. The two-phase solvent system containing ethyl acetate-n-butanol-0.5%NH(4)OH (2:3:5, v/v/v, system B) was selected for the one-step HSCCC separation of HBOA-Glc and DIBOA-Glc according to the partition coefficient values (K) for target compounds and the separation factor (alpha) between the two target compounds. In the one-step HSCCC separation using solvent B, from 100mg n-butanol extract of A. ilicifolius, 6.3 mg HBOA-Glc and 6.8 mg DIBOA-Glc were isolated with purities of 90.3% and 80.2%, respectively. In order to obtain the two target compounds with higher purity, a second separation process was developed comprising two steps. In the two-step separation, the sample was first pre-purified by HSCCC using ethyl acetate-n-butanol-water (2:3:5, v/v/v, system A) solvent system and then purified using solvent system B. A 100-mg amount of the n-butanol extracts of A. ilicifolius was separated to yield 5.8 mg of HBOA-Glc and 4.8 mg of DIBOA-Glc with purities of 97.1% and 94.8%, respectively, which were directly used for NMR analyses.