Isolation of a novel flavanone 6-glucoside from the flowers of Carthamus tinctorium (Honghua) by high-speed counter-current chromatography

A novel flavanone glycoside, (2S)-4',5,6,7-tetrahydroxyflavavone 6-O-beta-D-glucopyranoside was isolated from the ethyl acetate extract of the flowers of Carthamus tinctorium by high-speed counter-current chromatography (HSCCC). Using an optimized two-phase solvent system composed of ethyl acetate-methanol-water (5:1:5, v/v), target compound (52 mg) with purity of 98.0% was obtained from 2.0 g of sample by HSCCC in seven times run. The structure of the target compound was elucidated by means of spectroscopic methods including IR, MS, 1D and 2D NMR techniques.     

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.     

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.     

Preparative isolation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of five compounds from the Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. The crude extracts from P. cuspidatum Sieb. et Zucc were treated with light petroleum-ethyl acetate-methanol-water (2:5:4:6, v/v). Sample 1 was obtained from the lower phase and sample 2 from the upper phase. The sample 1 was separated with light petroleum-ethyl acetate-water (1:5:5, v/v) and yielded 19.3mg of piceid, 17.6 mg of anthraglycoside B from 200mg of sample 1. The sample 2 was separated with light petroleum-ethyl acetate-methanol-water (3:5:4:6, v/v) and light petroleum-ethyl acetate-methanol-water (3:5:7:3, v/v) in a gradient elution and yielded 18.5mg of resveratrol, 35.3mg of emodin and 8.2mg of physcion from 220 mg of sample 2. The purity of each compound is over 95% as determined by HPLC. The chemical structures of these components were identified by (1)H NMR and (13)C 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.     

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.     

新型取代苯乙醇葡萄糖氧苷的合成及其抗缺氧活性

以取代苯甲醛为原料,经Wittig反应、水解反应和NaBH₄还原反应制得5个取代苯乙醇衍生物（4a~4e）;以4a~4e和其他芳乙醇（4f~4r）为原料,依次与全乙酰化溴代葡萄糖经Koenigs-Knorr偶联和MeONa/MeOH脱除乙酰保护基,合成了18个取代苯乙醇葡萄糖氧苷类似物（5a~5r,其中5b~5r为新化合物）,其结构经¹H NMR和HR-MS（ESI）表征。采用MTT法研究了5a~5r对缺氧损伤的内皮细胞（EA.hy926）代谢活力的影响。结果表明：5e, 5g, 5m, 5p, 5q和5r对EA.hy926的保护作用优于红景天苷。     

Development of an HPLC method for the determination of salidroside in beagle dog plasma after administration of salidroside injection: application to a pharmacokinetics study

A simple RP-HPLC method was established for the determination of salidroside in dog plasma. Salidroside is one of the most active ingredients of Rhodiola L. The method had within-run precision values in the range of +/- 2.3 to +/- 9.1% (n = 5) and between-run precision in the range of +/- 3.2 to +/- 9.8%. A simple protein precipitation for salidroside extraction was processed using ACN at precipitant-to-plasma volume ratio (P-P ratio) of 3:2. The extraction recoveries of salidroside at seven concentrations were higher than 63.2%. There was a linear relationship between chromatographic area and concentration over the range of 0.83-520 microg/mL for salidroside in plasma (R = 0.9926). The LOQ (S/N = 10) of the method was 0.83 microg/mL. The method was applied in a study of the pharmacokinetics of salidroside injection in six beagle dogs. The major pharmacokinetic parameters of C(max), AUC(0-24), AUC(0-infinity), and t(1/2) of salidroside in beagle dogs after i.v. administration of a single 75 mg/kg (5 mL/kg) dose were 96.16 +/- 8.59 microg/mL, 180.3 +/- 30.6 microg h/mL, 189.3 +/- 32.1 microg h/mL, and 2.006 +/- 0.615 h, respectively.     

Isolation and purification of inflacoumarin A and licochalcone A from licorice by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to isolate and purify bioactive flavone compounds from the ethanol extract of Glycyrrhiza inflata Bat., a particular plant species of licorice. HSCCC separation was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (5:6:3:2, v/v) by eluting the lower mobile phase at a flow rate of 1.8 ml/min and a revolution speed of 800 rpm. Purification was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1.5:6:3:2, v/v) by eluting the lower mobile phase at a flow-rate of 1.5 ml/min and a revolution speed of 800 rpm. Two major flavone peaks: inflacoumarin A and licochalcone A were collected and the respective yields of the peaks amount to 6 mg (8.6%, w/w) and 8 mg (11.4%, w/w) from 70 mg of the crude extract sample. The purities of inflacoumarin A and licochalcone A reached 99.6% and 99.1%, respectively, after a sequential purification run. The structures of inflacoumarin A and licochalcone A were positively confirmed by 1H NMR and 13C NMR, 1H-13C-COSY, UV, FT-IR and electron ionization MS analyses.     

Preparative isolation and purification of trans-3,5,4'-trihydroxystilbene-4'-O-beta-D-glucopyranoside and (+)catechin from Rheum tanguticum Maxim. ex Balf. using high-speed counter-current chromatography by stepwise elution and stepwise increasing the flow-rate of the mobile phase

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of trans-3,5,4'-trihydroxystilbene-4'-O-beta-D-glucopyranoside (compound 1) and (+)catechin (compound 2) from Rheum tanguticum Maxim. ex Balf. by stepwise elution with a pair of two-phase solvent system composed of ethyl acetate-ethanol-water (25:1:25, v/v) and (5:1:5, v/v), and stepwise increasing the flow-rate of the mobile phase from 0.8 to 2.0 mlmin(-1) after 5 h. The preparative HSCCC separation was performed on 250 mg of crude extract yielding pure compound 1 (10.2 mg) and compound 2 (26.7 mg) all at purities of over 96% in a single run. The structures of the two compounds have been elucidated by means of spectroscopic methods including MS and 1H, 13C nuclear magnetic resonance spectroscopy.     

Isolation of hydroxycinnamoyltartaric acids from grape pomace by high-speed counter-current chromatography

A method for the isolation of caftaric, coutaric and fertaric acids from grape pomace by high-speed counter-current chromatography (HSCCC) was developed. Using a system of hexane/ethyl acetate/methanol/water 3:7:3:7 (v/v/v/v) and 0.5% trifluoroacetic acid (TFA) in the head-to-tail elution mode, the target compounds were separated from co-extracted polyphenolics and subsequently isolated in a second run (tert-butyl-methyl ether/acetonitrile/n-butanol/water, 2:2:1:5 (v/v/v/v) and 0.5% TFA; tail-to-head elution mode). The concomitant flavonoid quercetin 3-glucuronide was also isolated with the present method. The compounds were characterized by 1H NMR spectroscopy, by LC/electrospray ionization (ESI)-MS/MS in the negative ionization mode, and by UV spectroscopy. A purity of 97.0% (2.0% Z-isomer) for caftaric acid, 97.2% (4.8% Z-isomer) for coutaric acid, and 90.4% (13% Z-isomer) for fertaric acid was obtained from 10 g of grape pomace with yields of 62, 48 and 23%, respectively. Caftaric and coutaric acids may be used for in vitro and in vivo studies and as reference substances for analytical purposes.     

Preparative separation of six coumarins from the pummelo (Citrus maxima (Burm.) Merr. Cv. Shatian Yu) peel by high-speed countercurrent chromatography

In this work, six coumarins, including two new ones, 8-(3-hydroxy-2,2-dimethylpropyl)-7-methoxy-2H-chromen-2-one (2) and 5-[(7′,8′-dihydroxy-3′,8′-dimethyl-2-nonadienyl)oxy] psoralen (4), as well as four known ones, 5-[(6′,7′-dihydroxy-3′,7′-dimethyl-2-octenyl) oxy] psoralen (1), marmin (3), epoxybergamottin (5), and aurapten (6) were successfully separated from the crude extract of pummelo (Citrus maxima (Burm.) Merr. Cv. Shatian Yu) peel by high-speed countercurrent chromatography in a single run with petroleum-ether–ethyl acetate–methanol–water (4:6:6:4, v/v). The structures of these six coumarins were elucidated by ESI-MS, extensive 1D and 2D NMR spectroscopy.     

Preparative separation of gambogic acid and its C-2 epimer using recycling high-speed counter-current chromatography

A recycling counter-current chromatographic system was first set up with a high-speed counter-current chromatography instrument coupled with a column switching valve. This system was first successfully applied to the preparative separation of epimers, gambogic acid and epigambogic acid from Garcinia hanburyi using n-hexane-methanol-water (5:4:1, v/v/v) as the two-phase solvent system. As a result, 28.2 mg gambogic acid and 18.4 mg epigambogic acid were separated from 50 mg of mixture. Their purities were both above 97% as determined by HPLC. The chemical structures were then identified by their (1)H NMR and (13)C NMR spectra.     

Preparative separation and purification of bufadienolides from Chinese traditional medicine of ChanSu using high‐speed counter‐current chromatography

A preparative high‐speed counter‐current chromatography method for isolation and purification of bufadienolides from ChanSu was developed by using a stepwise elution with two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at the ratios of 4:6:2:4 v/v, 4:6:2.5:4 v/v and 4:6:3.2:4 v/v. A total of 3.8 mg of gamabufotalin (1), 7.2 mg of arenobufagin (2), 3.4 mg of telocinobufagin (3), 5.3 mg of bufotalin (4), 8.5 mg of cinobufotalin (5) and 8 mg of bufalin (6) were obtained in one‐step separation from 80 mg of the crude extract with purity of 92.7, 96.7, 87.2, 97.3, 94.9 and 99.4%, respectively. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology.     

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.     

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 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.     

离子液体对β-糖苷酶催化合成红景天苷的影响(英文)

研究了混合溶剂体系中阴离子分别为BF4-,PF6-,Cl-,Br-和I的14种离子液体对来源于黑布林糖苷酶催化合成红景天苷反应的影响.结果表明,在最佳反应条件下,β-糖苷酶的反应初速度和红景天苷的产率分别为3.3mmol/(L·h)和24.5%.离子液体咪唑阳离子的烷基链长(C2~C10)对β-糖苷酶的活性影响较大,当烷基链长为C6时,糖苷酶表现出较高的催化活性.     

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.     

Separation and Identification of Ergosta-4,6,8(14),22-tetraen-3-one from Ganoderma atrum by High-Speed Counter-Current Chromatography and Spectroscopic Methods

High speed counter-current chromatography in semi-preparative scale was used to separate and purify ergosta-4,6,8(14),22-tetraen-3-one from Ganoderma atrum, a famous traditional Chinese medicine. A two-phase solvent system composed of a mixture of n-hexane–ethanol–water (6: 5: 1, v/v/v) was used and the separation conditions were optimized. In a typical run in less than 400 min, 100 mg of samples can be separated to yield 14 mg of ergosta-4,6,8(14),22-tetraen-3-one with 99.1% purity. The structure of this compound was elucidated by UV, EI-MS, ¹H NMR and ¹³C NMR.