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.     

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.     

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 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 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.     

Comparison of high-speed counter-current chromatography instruments for the separation of the extracts of the seeds of Oroxylum indicum

Analytical Milli high-speed counter-current chromatography (HSCCC) was used for the selection and optimization of the two-phase solvent system to separate flavonoids from the extracts of the seeds of Oroxylum indicum. The optimum solvent system obtained from Milli-CCC was also the best solvent system for preparative HSCCC and led to the successful separation of two crude flavonoids from the seeds of O. indicum by Lab/Prep (laboratory preparative) HSCCC using different sized coils. Four flavonoids were isolated by preparative HSCCC: baicalein-7-O-diglucoside (25.0 mg, 92% purity), baicalein-7-o-glucoside (50.4 mg; 95% purity), baicalein (75 mg; purity 98%) and chrysin (100 mg; purity 98%).     

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.     

Preparative separation and purification of squalene from the microalga Thraustochytrium ATCC 26185 by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was successfully applied to the preparative separation and purification of squalene from microalgae. Crude squalene was obtained from the microalga Thraustochytrium ATCC 26185 by extraction with organic solvents. The crude squalene was further separated using a waterless two-phase solvent system composed of n-hexane-methanol (2:1, v/v). The upper phase as the mobile phase was pumped into the column at a flow-rate of 2.0 ml min(-1) in the tail-to-head elution mode. The fractions purified and collected were analyzed by high-performance liquid chromatography. The method yielded 0.2 mg squalene at 96% purity from 150 mg of the crude squalene (0.14% squalene) with 95% recovery. The separation of squalene by HSCCC was completed in 90 min.     

Preparative isolation and purification of two amides from Mallotus lianus Croiz by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the preparative isolation and purification of two amides from Mallotus lianus Croiz. In a single HSCCC separation, using the two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (5:1:5:1 v/v), 247.5 mg of the enriched crude sample was separated to afford 10.3 mg of N-isobutyl-2E,4E,12Z-octadecatrienamide and 15.7 mg of (7Z,10Z,18Z)tricosa-7,10,18-trienamide, a novel compound, with the purities of 98.0 and 94.6%, respectively. The HSCCC fractions were analyzed by HPLC and chemical structures of the compounds were identified by 1D- and 2D-NMR, ESI-, and GC-MS.     

Flow rate gradient high-speed counter-current chromatography separation of five diterpenoids from Triperygium wilfordii and scale-up

In this paper, high-speed counter-current chromatography (HSCCC) instruments with different gravitational forces were applied for the separation of bioactive compounds from Triperygium wilfordii Hook.f. The critical parameters including sample concentration, sample volume and flow rate were first optimized on an analytical Mini-DE HSCCC system, and then scaled up to a preparative TBE 300A HSCCC system. Although this scale-up process was performed using different CCC instruments with different centrifuges and gravitational forces, the same resolutions were obtained and the elution time could be predictable. Five diterpenoid compounds and one unknown compound were separated from Triperygium wilfordii Hook.f. by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (HEMW) (3:2:3:2, v/v/v/v). This one-step flow gradient separation produced triptonide (25 mg), isoneotriptophenolide (77 mg), hypolide (83 mg), unknown compound (1 mg), triptophenolide (42 mg), triptonoterpene methyl ether VI (37 mg) from 320 mg crude extract with purities of 98.2%, 96.6%, 98.1%, 95.3%, 95.1%, and 96.5%, respectively. Their purities and structures were identified by high-performance liquid chromatography, mass spectrometry and NMR. This paper demonstrates that analytical CCC plays an important role in optimizing parameters and scale-up process when analytical CCC and preparative CCC are supplied by different manufacturers with different gravitational forces, and the scale-up process from analytical CCC to preparative CCC is still predictable.     

SEPARATION OF THE MINOR FLAVONOLS FROM FLOS GOSSYPII BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

An effective high-speed countercurrent chromatography (HSCCC) method was established for further separation and purification of four minor flavonols in addition to five major flavonols which were reported by our previous study from extracts of Flos Gossypii. HSCCC was performed with three two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (7.5:15:6:7, v/v), (2.5:15:2:7, v/v) and (0:1:0:1, v/v). The separation was repeated 3 times, and 3.8 mg of 8-methoxyl-kaempferol-7-O-β-D-rhamnoside (HPLC purity 98.27%), 6.7 mg of astragalin (HPLC purity 94.18%), 3.3 mg of 4'-methoxyl-quercetin-7-O-β-D-glucoside (HPLC purity 94.30%) and 8.2 mg of hyperoside (HPLC purity 93.48%) were separated from 150 mg of the crude sample. The chemical structures of the flavonols were confirmed by MS, (1)H NMR and (13)C NMR. Meanwhile, the results indicated that the target compound with smaller K value (<0.5) can be separated by increasing column length of HSCCC. And four separation rules of flavonols according to the present study and references were summarized, which can be used as a useful guide for separation of flavonols by HSCCC.     

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.     

Preparative isolation and purification of costunolide and dehydrocostuslactone from Aucklandia lappa Decne by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of costunolide and dehydrocostuslactone from the Chinese medicinal plant Aucklandia lappa Decne (Muxiang in Chinese) was successfully established by using light petroleum-methanol-water (5:6.5:3.5, v/v/v) as the two-phase solvent system. The upper phase of light petroleum-methanol-water (5:6.5:3.5, v/v/v) was used as the stationary phase of HSCCC. 35.7 mg of costunolide and 43.6 mg of dehydrocostuslactone with the purity of 100% and 99.6%, respectively, were separated successfully in one-step separation from 110 mg of crude sample from Aucklandia lappa Decne. The structures of costunolide and dehydrocostuslactone were identified by 1H NMR and 13C NMR.     

Preparative separation of lappaconitine, ranaconitine, N-deacetyllappaconitine and N-deacetylranaconitine from crude alkaloids of sample Aconitum sinomontanum Nakai by high-speed counter-current chromatography

Analytical high-speed counter-current chromatography (HSCCC) was used for the systematic selection and optimization of the two-phase solvent system to separate alkaloids from Aconitum sinomontanum Nakai. The optimum solvent systems CHCl3-MeOH-0.3 M/0.2 M HCl (4:1.5:2, v/v) thus obtained led to the successful separation of lappaconitine, ranaconitine, N-deacetyllappaconitine and N-deacetylranaconitine from 60 to 500 mg of crude alkaloid sample by preparative HSCCC separation.     

Separation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of baicalin and wogonoside from the Chinese medicinal plant Scutellaria baicalensis Georgi (Huang-qin in Chinese) was successfully established by using ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) as the two-phase solvent system. The upper phase of ethyl acetate-methanol-1% acetic acid water (5:0.5:5, v/v) was used as the stationary phase of HSCCC. Baicalin (58.1 mg) and wogonoside (17.0mg) with the purity of 99.2 and 99.0%, respectively, were separated successfully in one-step separation from 120 mg of crude sample from S. baicalensi, Georgi. The structures of baicalin and wogonoside were identified by 1H NMR and 13C NMR.     

Efficient new method for extraction and isolation of three flavonoids from Patrinia villosa Juss. by supercritical fluid extraction and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of orotinin, orotinin-5-methyl ether and licoagrochalcone B from Patrinia villosa was performed. The optimization of parameters including pressure, temperature, modifier and sample particle size on yield was carried out using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa, 45 degrees C, a sample particle size 40-60 mesh and modified CO2 with 20% methanol. The yield of the preparative SFE was 2.82% (crude extract I) and the combined yield of orotinin, orotinin-5-methyl ether and licoagrochalcone B was 0.82 mg/g of dry sample mass. Then the crude extract I was re-dissolved in methanol and methanol soluble fraction (crude extract II, 0.17%) was obtained, which was successfully isolated and separated by a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:6:6:6, v/v/v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 3 h. The target compounds isolated and purified by HSCCC were analyzed by high performance liquid chromatography. The separation produced total of 38.2 mg of orotinin at 99.2% purity, 19.8 mg of orotinin-5-methyl ether at 98.5% purity and 21.5 mg of licoagrochalcone B at 97.6% purity from 400 mg of the crude extract in a one-step separation. The recoveries of orotinin, orotinin-5-methyl ether and licoagrochalcone B were 91.1, 91.6 and 90.3%, respectively, and the chemical structure identification was carried out by UV, IR, MS, 1H NMR and 13C NMR.     

Preparative isolation of alkaloids from Corydalis bungeana Turcz. by high-speed counter-current chromatography using stepwise elution

High‐speed counter‐current chromatography (HSCCC) was successfully applied for the preparative separation and purification of alkaloids from Corydalis bungeana Turcz. (Kudiding in Chinese) for the first time. After the measurement of partition coefficient of seven target alkaloids in the nine two‐phase solvent systems composed of CHCl₃–MeOH–(0.1 M; 0.2 M; 0.3 M) HCl (4:1.5:2; 4:2:2; 4:3:2, v/v), CHCl₃–MeOH–0.2 M HCl (4:2:2, v/v) and CHCl₃–MeOH–0.3 M HCl (4:3:2, v/v) were finally selected for the HSCCC separation using the first upper phase as the stationary phase and the stepwise elution of the two lower mobile phases. Consequently, sanguinarine (10 mg), corynoline (25 mg), protopine (20 mg), corynoloxine (18 mg), and 12‐hydroxycorynoline (8 mg) were obtained from 200 mg of crude alkaloid extracts with purities of 94–99% as determined by HPLC. Their chemical structures were characterized on the basis of ¹H‐NMR, ¹³C‐NMR, and LC‐ESI‐Q‐TOF‐MS/MS analyses.     

Orthogonal test design for optimization of supercritical fluid extraction of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone from Stellera chamaejasme L. and subsequent isolation by high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1- pentanone from Stellera chamaejasme L. was performed. An orthogonal L9 (3)4 test design was applied to select the optimum extraction parameters including pressure, temperature, modifier and sample particle size on yield using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa of pressure, 45 degrees C of temperature, 40-60 mesh of sample particle size and modified CO2 with 20% methanol. The yield of the crude extract from preparative SFE was 2.65%, which contained daphnoretin 25.2%, 7-methoxy-daphnoretin 22.8% and 1,5-diphenyl-1-pentanone 21.1%, respectively. Then the crude extract was successfully isolated and separated by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:13:13:10, v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 90 min. The target compounds isolated and purified by HSCCC were analyzed by high-performance liquid chromatography (HPLC). The separation produced total of 69.2mg of daphnoretin at 99.2% purity, 63.4 mg of 7-methoxy-daphnoretin at 98.7% purity and 58.3 mg of 1,5-diphenyl-1-pentanone at 98.1% purity from 300 mg of the crude extract in one-step separation. The recoveries of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone were 90.8, 91.5 and 90.4%, respectively, in HSCCC isolation step and the chemical structure identification was carried out by MS, 1H NMR and 13C NMR.     

Preparative isolation and purification of three rotenoids and one isoflavone from the seeds of Millettia pachycarpa Benth by high-speed counter-current chromatography

Both analytical and preparative high-speed counter-current chromatography (HSCCC) were used to isolate and separate chemical bioactive constituents from the seeds of Millettia pachycarpa Benth, a famous traditional Chinese medicine. Three rotenoids and one isoflavone were successfully purified for the first time by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (HEMWat) (1:0.8:1:0.6, v/v/v/v). The separation parameters were first performed on the analytical HSCCC and optimized conditions were then scaled up to preparative HSCCC. The separation produced 160.2 mg tephrosin, 14.6 mg 4',5'-dimethoxy-6,6-dimethylpyranoisoflavone, 109.4 mg deguelin, 6.7 mg 6a,12a-dehydrodeguelin with respective purities of 95, 93, 95, 95%, in one single run from 400 mg crude extract of the seeds of M. pachycarpa Benth. The purity of the isolated compounds was analyzed by high-performance liquid chromatography (HPLC) and their structures were identified by electrospray ionization mass spectrometry (ESI-MS); (1)H nuclear magnetic resonance ((1)H NMR) and (13)C nuclear magnetic resonance ((13)C NMR) analysis. This paper is an excellent example of the role that CCC is playing in isolating active compounds for pre-clinical trials of new chemical entities, even when scaling up between centrifuges from different manufacturers.     

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.