Isolation of high purity 1-[2',4'-dihydroxy-3',5'-di-(3"-methylbut-2"-enyl)-6'-methoxy] phenylethanone from Acronychia pedunculata (L.) Miq. by high-speed counter-current chromatography

Following an initial clean-up step on silica, high-speed counter-current chromatography (HSCCC) was used to purify an aryl ketone, 1-[2',4'-dihydroxy-3',5'-di-(3"-methylbut-2"-enyl)-6'-methoxy] phenylethanone from an extract of the stem bark of the shrub Acronychia pedunculata. The two-phase solvent system used was composed of n-heptane-ethyl acetate-methanol-water at an optimized volume ratio of 4:1:4:1 (v/v/v/v). Target compound (58.1 mg) with a purity of 98.9% was obtained after HSCCC of 183.5 mg sample with a purity of 35.7% recovered after the silica clean-up step. Identification of the target compound was performed by 1H NMR, 13C NMR, two-dimensional NMR and LC-electrospray ionization MS.     

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.     

Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography

High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.     

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 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 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.     

High performance liquid chromatography equipped with a cathodic detector and column-switching device as a high-throughput method for a phosphatase assay with p-nitrophenyl phosphate

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarin compounds from the Chinese medicinal plant Peucedanum decursivum (Miq.) Maxim (Zihuaqianhu in Chinese) was successfully established by using light petroleum-ethyl acetate-methanol-water (5:5:7:4, v/v) as the two-phase solvent system. The upper phase of light petroleum-ethyl acetate-methanol-water (5:5:7:4, v/v) was used as the stationary phase of HSCCC. Nodakenetin (2.8 mg), 6.1 mg of Pd-C-IV, 7.3 mg of Pd-D-V, 4.7 mg of ostruthin, 7.8 mg of decursidin and 11.2 mg of decursitin C with the purity of 88.3%, 98.0%, 94.2%, 97.1%, 97.8% and 98.4%, respectively, were separated successfully in one-step separation from 150 mg of crude sample from P. decursivum (Miq.) Maxim. After purified by HSCCC again with light petroleum-ethyl acetate-methanol-water (5:5:4:5, v/v) as the two-phase solvent system, the purity of (I) can reach 99.4%. The structures of all the compounds were identified by 1H NMR and 13C NMR.     

PREPARATIVE ISOLATION AND PURIFICATION OF FIVE FLAVONOIDS FROM POGOSTEMON CABLIN BENTH BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY AND PREPARATIVE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

High-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were successively used for the separation of pogostone and four flavonoids from Pogostemon cablin (Blanco) Benth. An efficient HSCCC separation was achieved on a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (11:5:11:5, v/v/v/v). Three well-separated peaks were obtained in the HSCCC chromatogram. The first and the second fractions each contained two flavonoids which were further separated by preparative HPLC. Consequently, the separation yielded 11.5 mg of 4', 5-Dihydroxy-3', 7-dimethoxyflavanone at a purity of 99%, 20.3 mg of 5- Hydroxy-7, 3', 4'-trimethoxyflavanone at a purity of 98%, 18 mg of 5, 4'-Dihydroxy-3, 7, 3'-trimethoxyflavone at a purity of 96%, and 8 mg of 5-Hydroxy-3, 7, 4'-tetramethoxyflvone at a purity of 98%. The third HSCCC fraction yielded 18.5 mg of pogostone at a purity of 95%. The chemical structures of these compounds were identified by ESI-MS(n), (1)H-NMR, and (13)C-NMR.     

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 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.     

Application of preparative high-speed counter-current chromatography for separation and purification of arctiin from Fructus Arctii

Following an initial clean-up step on the AB-8 resin (polystyrene resin, 0.3-1.25 mm: NanKai Chemical Factory, Tianjin, China), high-speed counter-current chromatography (HSCCC) was used to purify an arctiin from an extract of the fruits of the Arctium lappa L. Arctiin is a major lignan compound in the traditional Chinese medicinal herb A. lappa L. The two-phase solvent system used was composed of ethyl acetate-n-butanol-ethanol-water at an optimized volume ratio of 5:0.5:1:5 (v/v/v/v). The upper phase was used as the mobile phase in the head to tail elution mode. A total amount of 159 mg of arctiin at 98% purity was obtained from 350 mg of the crude extract (containing 49% arctiin) with 91% recovery. The preparative isolation and purification of arctiin by HSCCC was completed in 5 h in a separation. Identification of the target compound was performed by LC-electrospray ionization MS and 13C-NMR. The structure of the product was further confirmed by comparison with authentic sample (National Institute of the Control of Pharmaceutical and Biological Products, Beijing, China).     

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 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%.     

Preparative isolation and purification of coumarins from Peucedanum praeruptorum Dunn by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of coumarins from Peucedanum praeruptorum Dunn (Baihuaqianhu in Chinese) was successfully established by using light petroleum-ethyl acetate-methanol-water as the two-phase solvent system in gradient elution mode. The upper phase of light petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v) was used as the stationary phase of HSCCC. The mobile phase used in HSCCC was the lower phase of light petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v) and light petroleum-ethyl acetate-methanol-water (5:5:6.5:3.5, v/v) that was changed in gradient. Four kinds of coumarins and another unknown compound were obtained and yielded 5.3 mg of qianhucoumarin D, 7.7 mg of Pd-Ib, 35.8 mg of (+)-praeruptorin A, 31.9 mg of (+)-praeruptorin B and 6.4 mg of unknown compound with the purity of 98.6%, 92.8%, 99.5%, 99.4% and 99.8% in one-step separation, respectively. The structures of the coumarins were identified by 1H NMR and 13C NMR.     

Isolation of antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography guided by thin layer chromatography-antioxidant autographic assay

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane–ethyl acetate–methanol–water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91 mg psoralen, 6.26 mg isopsoralen, 3.19 mg psoralidin, 0.92 mg corylifol A, and 2.43 mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250 mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4 mg 8-prenyldaidzein (purity 91.7%), 4.18 mg neobavaisoflavone (purity 97.4%) and 4.36 mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by ¹H NMR and ¹³C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC₅₀ value of 44.7 μM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.     

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.     

An effective high-speed countercurrent chromatographic method for preparative isolation and purification of mollugin directly from the ethanol extract of the Chinese medicinal plant Rubia cordifolia

The medicinal plant Rubia cordifolia has been used widely in traditional Chinese medicine (TCM) for its antibacterial, antioxidant and anti-inflammatory activities. In this study, a preparative high-speed countercurrent chromatography (HSCCC) method for isolation and purification of the bioactive component mollugin directly from the ethanol extract of R. cordifolia was successfully established by using light petroleum (bp 60-90 degrees C)/ethanol/diethyl ether/water as the two-phase solvent system. The upper phase of light petroleum/ethanol/diethyl ether/water (5:4:3:1 v/v) was used as the stationary phase of HSCCC. Under the optimum conditions, 46 mg of mollugin at 98.5% purity, as determined by HPLC, could be yielded from 500 mg of the crude extract in a single HSCCC separation. The peak fraction of HSCCC was identified by 1H NMR and 13C NMR.     

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.     

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.     

Supercritical fluid extraction of aurentiamide acetate from Patrinia villosa Juss and subsequent isolation by silica gel and high-speed counter-current chromatography

Supercritical fluid extraction (SFE) of aurentiamide acetate from Patrinia villosa Juss was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system. Then the extraction was scaled up by 100 times using a preparative SFE system under the optimized conditions of 55 degrees C, 35 MPa and modified CO2 with 10% methanol. Then, the crude extract I obtained by SFE was chromatographed on silica gel and the solvent system composed of petroleum ether-ethyl acetate (5:1, v/v) was used to produce the crude extract II, which was further isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:1.2:1.2:1, v/v/v/v). One hundred fifty-five milligrams of aurentiamide acetate was obtained from 400 mg crude extract II (contained 42% target) with a purity of 99.3% determined by HPLC and 92.3% recovery in one-step elution, and identification was performed by UV, MS, 1H NMR and 13C NMR. As far as we know, this is the first report of discovering aurentiamide acetate from the plant of Patrinia genius.