Preparative purification of five bioactive components from Agrimonia pilosa Ledeb by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) coupled with ultraviolet (UV) detection or evaporative light-scattering detection was successfully applied for preparative separation of five bioactive compounds from Agrimonia pilosa Ledeb. In preliminary process, D101 macroporous resin was used to separate the crude extract of the plant and four fractions (20, 40, 50, and 60% aqueous ethanol elutions) were produced. Then, these fractions were directly subjected to HSCCC purification. Five chemicals including taxifolin-3-glucoside (6.4 mg), quercetin-3-rhamnoside (13.0 mg), tiliroside (14.7 mg), agrimonolide (21.4 mg), and tormentic acid (29.8 mg) with the purities of 94.24, 95.37, 97.42, 95.29, and 96.34% were separated from each 200 mg prepared fraction. The purities were analyzed by high-performance liquid chromatography, and the chemical structures of the products were identified by UV detection, mass spectrometry, nuclear magnetic resonance, and the standards. This paper used a simple method to separate five bioactive compounds from A. pilosa Ledeb, and it could provide a new idea for the purification of bioactive compounds from other medicinal plants.     

Preparative isolation and purification of atractylon and atractylenolide III from the Chinese medicinal plant atractylodes macrocephala by high-speed counter-current chromatography

The medicinal plant Atractylodes macrocephala (Baizhu in Chinese) has been widely used in traditional Chinese medicine for energy and stomach complaints, treatment of dyspepsia and anorexia, anti-inflammation, anticancer and for increasing assimilation. A high-speed counter-current chromatography (HSCCC) method was developed for the preparative separation and purification of two main bioactive components, namely, atractylon and atractylenolide III from A. macrocephala by using light petroleum (60-90 degrees C)-ethyl acetate-ethanol-water (4:1:4:1 v/v) as the two-phase solvent system in dual-mode elution. Compared with the separation using the normal-mode elution, the dual-mode HSCCC can be achieved with shorter elution time. Atractylenolide III (32.1 mg) at 99.0% purity and 319.6 mg atractylon at 97.8% purity could be obtained from 1000 mg crude sample in a single run. The recoveries of atractylenolide III and atractylon were 95.4 and 92.6%, respectively.     

Preparative isolation and purification of antioxidative diarylheptanoid derivatives from Alnus japonica by high-speed counter-current chromatography

This study employed the online HPLC-2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS)(+) bioassay to rapidly determine the antioxidant compounds occurring in the crude extract of Alnus japonica. The negative peaks of the ABTS(+) radical scavenging detection system, which indicated the presence of antioxidant activity, were monitored by measuring the decrease in absorbance at 734 nm. The ABTS(+)-based antioxidant activity profile showed that three negative peaks exhibited antioxidant activity. High-speed counter-current chromatography (HSCCC) was used for preparative scale separation of the three active peaks from the extract. The purity of the isolated compounds was analyzed by HPLC and their structures were identified by (1)H- and (13)C-nuclear magnetic resonance spectrometry (NMR), heteronuclear multiple bond correlation (HMBC), and heteronuclear single quantum correlation (HSQC). Two solvent systems composed of n-hexane/ethylacetate/methanol/water (4:6:4:6, v/v) and of ethyl acetate/methanol/water (1:0.1:1, v/v) were performed in high-speed counter-current chromatography. Consequently, a total of 527 mg of hirsutanonol 5-O-β-D-glucopyranoside, 80.04 mg of 3-deoxohirsutenonol 5-O-β-D-glucopyranoside, and 91.0 mg of hirsutenone were obtained with purity of 94.7, 90.5, and 98.6%, respectively.     

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.     

Preparative purification of gentamicin components using high-speed counter-current chromatography coupled with electrospray mass spectrometry

We developed a useful and preparative method based on high-speed counter-current chromatography with mass spectrometry (HSCCC/MS) to purify gentamicin C1a, C2/2a and C1 from standard powder. The analytes were purified on the HSCCC model CCC-1000 (multi-layer coil planet centrifuge) with a volatile two-phase solvent system composed of n-butanol/10% aqueous ammonia solution (50:50, v/v) and detected on an LCMS-2020EV quadrupole mass spectrometer fitted with an electrospray ionization (ESI) source system in positive ionization following scan mode (m/z 100-500). The HSCCC/ESI-MS peaks indicated that gentamicin C1a (m/z 450: [M+H](+)), C2/2a (m/z 464: [M+H](+)) and C1 (m/z 478: [M+H](+)) have the peak resolution values of 1.3 and 1.7 from 30 mg of loaded gentamicin powder. The HSCCC yielded 3.9 mg of gentamicin C1a, 12.6 mg of gentamicin C2/2a and 12.0 mg of gentamicin C1. These purified substances were analyzed by LC/MS with scan positive-mode. Based on the LC/MS chromatograms and spectra of the fractions, analytes were estimated to be over 95% pure. These gentamicin isomers of C1a, C2/2a and C1 were evaluated for their antibacterial activities. The overall results indicate that this approach of HSCCC/MS is a powerful technique for the purification of gentamicin components.     

Preparative isolation and purification of flavone compounds from sophora japonica L. by high-speed counter-current chromatography combined with macroporous resin column separation

High-speed counter-current chromatography combined with macroporous resin column separation was applied to the isolation and purification of genistein-7,4'-di-O-beta-D-glucoside (I), genistein-7-O-beta-D-glucopyranoside-4'-O-[(alpha-L-rhamnopyransoyl)-(1-2)-beta-D-glucopyranoside] (II), kaempferol-3-O-beta-D-sophoroside(III), quercetin-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (IV), genistein-4'-beta-L-rhamnopyransoyl-(1 - 2)-alpha-D-glucopyranoside (V), and kaempferol-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (VI) from the Chinese medicinal herb Sophora japonica L. The crude extracts from the pericarps of Sophora japonica L. were pre-separated on a D-101 macroporous resin column and divided into two parts as sample 1 and sample 2. An 80-mg portion of sample 1 was separated by using n-butanol-acetic acid (1%) (5:5, v/v) as the two-phase solvent system and yielded 30.1 mg of compound I, 23.3 mg of compound II. A 120 mg portion of sample 2 was separated by using ethyl acetate-n-butanol-acetic acid (1%) (5:0.8:5, v/v) as the two-phase solvent system and yielded 5.5 mg of compound III, 31.7 mg of compound IV, 37.4 mg of compound V, and 6.2 mg of compound VI. The purities of compounds I, II, III, IV, V, and VI were 98.7, 98.2, 97.8, 98.5, 99.3, and 98.9%, respectively, as determined by HPLC. The chemical structures of these components were identified by 1H-NMR and 13C-NMR.     

Preparative separation of minor saponins from Platycodi Radix by high-speed counter-current chromatography

Platycosides (PSs), the saponins found in the root of Platycodon grandiflorum (Jacq.) A. DC. (Platycodi Radix), are typically composed of oleanene backbones with two side chains; one is a 3-O-glucose linked by a glycosidic bond, and the other is a 28-O-arabinose-rhamnose-xylose-apiose linked by an ester bond. Minor saponins, acetylated isomers of the major saponin on either the 2' or 3' position of rhamnose, were isolated from Platycodi Radix using a multi-step process including high-speed counter-current chromatography (HSCCC) and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). After the separation of the major components, the enriched minor saponin fraction was used for this study. A two-phase solvent system consisting of chloroform-methanol-isopropanol-water (3:2:2:3, v/v) was used for HSCCC. HSCCC separation of the enriched minor saponin fraction yielded 2'-O-acetylplatycodin D, 3'-O-acetylpolygalacin D, 2'-O-acetylpolygalacin and a mixture of 3'-O-acetylplatycodin D and polygalacin D. The mixture fraction from HSCCC separation was further purified by preparative RP-HPLC, giving 3'-O-acetylplatycodin D and polygalacin D at a purity of over 98.9%. The developed method provides the preparative and rapid separation of minor saponins in the crude extract of Platycodi Radix. To the best of our knowledge, this is the first on the separation of acetylated PSs by HSCCC.     

Isolation and purification of ginkgo flavonol glycosides from Ginkgo biloba leaves by high-speed counter-current chromatography

A high-speed counter-current chromatography method was developed for the separation and purification of bioactive flavonol glycosides from a crude ethanol extract of Ginkgo biloba leaves. The separation was performed with a two-phase solvent system composed of n-hexane-butanol-ethyl acetate-methanol-0.5% acetic acid (1:0.5:3.5:1:4, v/v) and three pure compounds were eluted in high purities in a one-step separation. Their purities were determined by HPLC and identified by MS,(1)H-NMR, and(13)C-NMR.     

Separation of alkaloids from herbs using high-speed counter-current chromatography

Alkaloids represent a most widespread group of bioactive natural products. Because of their alkalinity and structural diversity, the fractionation and purification of the alkaloids from herbs can often present a number of practical difficulties using the conventional chromatographic techniques. High-speed counter-current chromatography (HSCCC) is a liquid-liquid partition chromatography with a support-free liquid stationary phase, and is gaining more and more popularity as a viable separation technique for bioactive compounds from natural resources. In the present review, focus is placed on the separation of alkaloids by both conventional HSCCC and pH-zone-refining counter-current chromatography (CCC) techniques from herbs. The review presents the separation of over 120 different alkaloid compounds from more than 30 plant species by the conventional HSCCC and pH-zone-refining CCC. Based on the data from the literature, the proper solvent systems for the separation of alkaloids by the conventional HSCCC and pH-zone-refining CCC are also summarized.     

Preparative isolation and purification of three flavonoid glycosides from Taraxacum mongolicum by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) was successively applied to purify three flavonoid glycosides from the aerial part of Taraxacum mongolicum, a traditional Chinese medicine. Subsequent UV, MS, and NMR analyses have led to the characterization of three flavonoid glycosides including two new compounds isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-L-arabinopyranoside and isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside, and a known compound, isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-xyloypyranoside, which were first isolated from T. mongolicum. The two-phase solvent system composed of ethyl acetate/n-butanol/water (2:1:3, v/v/v) was performed in HSCCC. Consequently, a total of 25.7 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-L-arabinopyranoside, 19.1 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-glucopyranoside, and 10.6 mg isoetin-7-O-beta-D-glucopyranosyl-2'-O-alpha-D-xyloypyranoside were obtained with purity of 98.7, 98.3, and 99.1%, respectively, as determined by HPLC from 500 mg enriched extract after cleaning-up by polyamide resin.     

Preparative isolation of cyclolanostane-type saponins from Astragalus membranaceus bge. var. mongholicus (Bge.) Hsiao by TLC-MS/MS guided high-speed counter-current chromatography

Two cyclolanostane-type saponins, astragalosides I and II, were first identified by TLC-MS/MS in the ethyl acetate extract of the roots of Astragalus membranaceus Bge var. mongholicus (Bge.) Hsiao without chemical reference substances. They were then isolated by high-speed counter-current chromatography with a two-step two-phase solvent system of ethyl acetate-2-propanol-water (5:1:5, 50:1:50, v/v/v). The quantities of astragalosides I and II isolated from 1 g of the crude extract were 30.2 mg and 16.5 mg, respectively. Their purities were found to be over 95% by HPLC-ELSD analysis. Their chemical structures were confirmed by 1H and 13C nuclear magnetic resonance analysis.     

Rapid preparative isolation of a new phenylpropanoid glycoside and four minor compounds from Sparganium stoloniferum using high-speed counter-current chromatography as a fractionation tool

A rapid high-speed counter-current chromatography (HSCCC) method was used to isolate five minor compounds from rhizome of Sparganium stoloniferum namely San Leng in Chinese, including two phenylpropanoid glycosides, sparganiaside A (1) and 1-O-feruloyl-3-p-coumaroylglycerol (2), and three aromatic acids, vanillic acid (3), p-hydroxylcinnamic acid (4), and p-hydroxybenzoic acid (5), of which, compound 1 was a new one. Five compounds were preparatively enriched at top efficiency by one-step HSCCC operation in the isolation procedure. A suitable solvent system composed of chloroform-methanol-water (4:3.5:1.8, v/v/v) was used. And the operation time was less than 4 h. The purities of compounds (1-5) in the enriched fractions were determined to be 75.8%, 66.3%, 90.6%, 79.9%, and 98.2%, respectively. The mean recoveries of the five compounds were 84.8%, 87.3%, 81.8%, 90.3%, and 92.7%, respectively. Compounds 1-4 were further purified by semi-preparative high-performance liquid chromatography (HPLC). This is the first report on the use of HSCCC as a fractionation tool for preparative isolation of minor compounds from S. stoloniferum. The method was proved to be rapid, convenient, high yield, and low cost. HSCCC was shown to be a quick and effective tool in isolation of natural products even though the compounds were not abundant.     

Application of high-speed counter-current chromatography for the isolation of antiviral eremophilenolides from Ligularia atroviolacea

Ligularia is mainly distributed in the western regions of China. Most of the species have been traditionally used in folk medicine for the treatment of hepatitis B, asthma, hemoptysis and pulmonary tuberculosis. In our continuation of research on antiviral components from traditional Chinese medicine, Ligularia atroviolacea was tested for inhibitory effects on hepatitis B virus (HBV). A bioassay-guided phytochemical examination of L. atroviolacea disclosed that its ethyl acetate extract, which was made up of two eremophilenolides, showed suppressive activity on the expression of HBV surface antigen (HBsAg) in the HepG2.2.15 cell line. Then a simple and effective preparative high-speed counter-current chromatography method was successfully developed for the isolation and purification of two main active metabolites, 8beta-hydroxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide and 8beta-methoxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide from the ethyl acetate extract of L. atroviolacea by a one-step separation using a two-phase solvent system composed of light petroleum (60-90 degrees C)-ethyl acetate-methanol-water (9:1:8:2, v/v/v). The chemical structures of the two eremophilenolides were identified by ESI-MS, (1)H-NMR and (13)C-NMR analysis. The anti-HBV activity of the two purified compounds was measured; both of them showed suppressive activity on the expression of HBsAg in the HepG2.2.15 cell line. The results support the continued and expanded exploitation and utilization of L. atroviolacea. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave-assisted extraction coupled with high-speed counter-current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.     

高速逆流色谱分离制备陈皮中的黄酮类化合物

应用高速逆流色谱法分离制备了陈皮中3种黄酮类化合物。以石油醚-乙酸乙酯-甲醇-水(体积比为2∶4∶3∶3)为两相溶剂系统,在主机转速850 r/min、流动相流速1.7 mL/min、检测波长280 nm条件下进行分离制备,6 h内从4.0 g陈皮粗提物中一步分离制备得到橙皮苷10.1 mg、桔皮素49.8 mg和5-羟基-6,7,8,3′,4′-五甲氧基黄酮50.6 mg,纯度均达97.0%以上,各化合物结构经质谱和核磁共振氢谱、碳谱鉴定。利用该方法可以对陈皮中的黄酮类化合物进行快速的分离和纯化。     

Preparative isolation and purification of antioxidative stilbene oligomers from Vitis chunganeniss using high‐speed counter‐current chromatography in stepwise elution mode

Preparative high‐speed counter‐current chromatography (HSCCC) was successfully applied to the isolation and purification of three stilbene oligomers from Vitis chunganeniss using stepwise elution with a pair of two‐phase solvent systems composed of n‐hexane–ethyl acetate–methanol–water at (2:5:2:5, v/v) and (1:2:1:2, v/v). The preparative HSCCC separation was performed on 800 mg of crude sample yielding hopeaphenol (21.1 mg), amurensin G (37.2 mg) and vitisin A (95.6 mg) in a one‐step separation, with purities over 95% as determined by HPLC. The structures of these three compounds were identified by MS, ¹H NMR and ¹³C NMR. In addition, their antioxidant activities were screened by DPPH assay, where vitisin A showed strong antioxidant activity. Further EPR experiments with spin‐trapping technique demonstrated that vitisin A is a potent and selective singlet oxygen quencher, which may be used in singlet oxygen‐mediated diseases as a pharmacological agent.     

高速逆流色谱分离纯化蔓荆子中的活性成分

应用高速逆流色谱法(HSCCC)分离纯化蔓荆子中的活性成分。以石油醚-乙酸乙酯-甲醇-水(体积比为3:6:3.6:3)为两相溶剂体系,在转速为800 r/min、流速为1.5 mL/min、检测波长为254 nm的条件下进行分离,所得馏分经高效液相色谱法(HPLC)检测,并经电喷雾电离(ESI)质谱和核磁共振谱(NMR)鉴定化合物的结构。从250 mg蔓荆子粗提物中一次性分离得到4个化合物,分别为23 mg对羟基苯甲酸、15 mg 3,6,7-三甲基槲皮万寿菊素、24 mg蔓荆子黄素和5 mg蒿黄素,其纯度约为93.1%、 97.3%、 98.7%和98.5%。该法具有简便、快速、重复性好的优点,为分离蔓荆子中的活性成分提供了新的方法。     

Development of a rapid and convenient method to separate eight ginsenosides from Panax ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detection

Ginsenosides exhibit diverse biological activities and are major well-known components isolated from the radix of Panax ginseng C.A. Meyer. In the present work, a rapid and facile method for the separation and purification of eight ginsenosides from P. ginseng by high-speed counter-current chromatography coupled with evaporative light scattering detector (HSCCC-ELSD) was successfully developed. The crude samples for HSCCC separation were first purified from ginseng extract using a macroporous resin; the extract was loaded onto a Diaion-HP20 column and fractionated by methanol and water gradient elution. The ginsenosides-protopanaxadiol (PPD) and protopanaxatriol (PPT) fractions were subsequently eluted with 65 and 80% methanol and water gradient elution, respectively. Furthermore, these two fractions were separated by HSCCC-ELSD. The two-phase solvent system used for separation was composed of chloroform/methanol/water/isopropanol at a volume ratio of 4:3:2:1. Each fraction obtained was collected and dried, yielding the following eight ginsenosides: Rg(1), Re, Rf, Rh(1), Rb(1), Rc Rb(2) and Rd. The purity of these ginsenosides was greater than 97% as assessed by HPLC-ELSD, and their structures were characterized by electrospray-ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectroscopy. This is the first report regarding the separation of the ginsenosides Rh(1), Rb(2) and Rc from P. ginseng by HSCCC.     

Preparative isolation of hydrolysable tannins chebulagic acid and chebulinic acid from Terminalia chebula by high-speed counter-current chromatography

As a chromatographic column, the high-speed counter-current chromatography system was equipped with a preparative HPLC series, enabling the successful isolation of hydrolysable tannins from the fruits of Terminalia chebula, a traditional Chinese medicine. The two-phase solvent system was composed of n-hexane-ethyl acetate-methanol-water (1:20:1:20 v/v). As a result, 33.2 mg chebulagic and 15.8 mg chebulinic acids were obtained in one step from 300 mg of crude extract. Their purities were determined by HPLC to be 95.3 and 96.1%, respectively. The chemical structures were identified by their MS and 1H NMR spectra.     

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.