Counter-current Chromatography Method Research Articles

Establishing carbon dots assisted high speed countercurrent chromatography and its application for efficient separation of rare earth element ions

Rare earth elements with unique magnetic properties and optical properties, known as the ‘industrial vitamin’, has a very high commercial value. As a secondary resource of rare earth elements, low-concentration solution includes mixed rare earth ions, which need to realize efficient separation and recovery urgently. High speed countercurrent chromatography is suitable for the separation and purification of rare earth element ions due to its advantages of large loading, good tolerance to samples, and simple pretreatment. In this study, a carbon dots assisted high speed countercurrent chromatography method was designed and established, the carbon dots were applied to the mobile phase of high speed countercurrent chromatography for the first time. The low concentration of REEs solution was enriched, and the effective separation of La (III), Ce (III), Gd (III) and Er (III) was successfully achieved. The complete separation of La (III), Ce (III), Gd (III) and Er (III) was achieved with a solvent system of 0.05 mol L−1 P507 (PE), 0.05 mol L−1 HNO3, and 0.1 mol L−1 CDs2 carbon dots (1:1:0.01, v/v/v), the upper phase as stationary phase, the lower phase as mobile phase. Density functional theory result showed that the binding energy of REEs (III)-CDs2 was larger than that of REEs (III)-P507, so the affinity of CDs2 to REEs (III) was stronger than that of P507. Therefore, with the addition of CDs2, the ability of mobile phase to elute REEs from the stationary phase was enhanced, and the separation effect was improved.

The enrichment and separation of lanthanides ions from wastewater using solvent sublation coupled with high speed countercurrent chromatography

Rare earth elements are non-renewable resources. During their processing, a lot of wastewater are produced, which poses a danger to human beings and the environment. Herein, the enrichment method of solvent sublation for La (III), Ce (III), Gd (III) and Er (III) was designed and established. Enrichment parameters pH, flotation time, extractant concentration and air flow rate were optimized. The result showed that the highest recovery of La (III) reached up to 92.33 % under the optimized conditions of pH 8, 10 min of flotation time, 0.15 mol L-1 of the extractant 2-ethylhexyl phosphate acid-2-ethylhexyl ester and 30 mL min−1 of air flow rate. The light rare earth element Ce (III) and heavy rare earth elements Gd (III) and Er (III) were also examined and obtained with maximum recovery. The stability of the extractant complexation with rare earth ions and the mechanism of competitive extraction were studied by density functional theory calculations. The extraction ability was following sequence of La (III) < Ce (III) < Gd (III) < Er (III). The enrichment of rare earth elements from real water samples was investigated and obtained result was consistent with the experiment result of synthetic solutions. The established high speed countercurrent chromatography method was used to successfully separate La (III), Ce (III), Gd (III) and Er (III) from the solution of rare earth ions enriched by solvent sublation.

Effective separation of maslinic acid and oleanolic acid from olive pomace using high-speed shear off-line coupled with high-speed countercurrent chromatography and their antibacterial activity test

In this work, a high-speed shear extraction off-line coupling high-speed countercurrent chromatography method was developed to separate maslinic acid and oleanolic acid from olive pomace. To improve extraction efficiency, the polar disparity between maslinic acid and oleanolic acid necessitated the concurrent utilization of both polar and non-polar solvents during high-speed shear extraction. Then, the high-speed shear extraction was directly feed to high-speed countercurrent chromatography for subsequently separation. A total of 250 min were needed to complete the extraction and separation process. This yielded two molecules from 3.3 g of defatted olive pomace: 7.2 mg of 93.8 % pure maslinic acid and 2.3 mg of 90.1 % pure oleanolic acid, both determined by HPLC at 210 nm. Furthermore, the compounds exhibited inhibitory activity against Escherichia coli and Staphylococcus aureus. At a concentration of 100 μg/mL, its efficacy in inhibiting hyaluronidase was comparable to that of the standard drug indomethacin. Compared with the conventional separation method, this coupled technique reduced the whole time due to the direct injection of sample extraction solution. This technique provides a useful approach for the separation of natural products with significant polarity differences.

Capparis cartilaginea decne (capparaceae): isolation of flavonoids by high-speed countercurrent chromatography and their anti-inflammatory evaluation

Introduction:Capparis cartilaginea Decne. (CC) originates from the dry regions of Asia and the Mediterranean basin. In traditional medicine, tea of CC leaves is commonly used to treat inflammatory conditions such as rheumatism, arthritis, and gout. Due to the limited studies on the phytochemistry and biological activity of CC compared to other members of the Capparaceae family, this work aims to: 1) Identify the chemical composition of CC extract and 2) Investigate the potential anti-inflammatory effect of CC extract, tea and the isolated compounds.Methods: To guarantee aim 1, high-speed countercurrent chromatography (HSCC) method; Nuclear Magnetic Resonance (NMR) and High-Performance Liquid Chromatography coupled to Electrospray Ionisation and Quadrupole Time-of-Flight Mass Spectrometry (HPLC-ESIQTOF-MS/MS) were employed for this purpose. To guarantee aim 2, we studied the effect of the isolated flavonoids on matrix metalloproteinases (MMPs) −9 and −2 in murine macrophages. Molecular docking was initially performed to assess the binding affinity of the isolated flavonoids to the active site of MMP-9.Results and discussion:In silico model was a powerful tool to predict the compounds that could strongly bind and inhibit MMPs. CC extract and tea have shown to possess a significant antioxidant and anti-inflammatory effect, which can partially explain their traditional medicinal use.

Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P.umbellatus. First, the extraction of P.umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P.umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P.umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

A countercurrent chromatography solvent system based on deep eutectic solvents for separation of cis- and trans-crocetin from Gardenia jasminoides Ellis.

Due to the structural similarity and large difference in concentration, the separation of trans- and cis-crocetin has been challenging, and the cis-crocetin is usually neglected. In this work, a countercurrent chromatography method was developed for the quick separation of trans-crocetin and cis-crocetin from the hydrolytic extract of Gardenia jasminoides Ellis. High purity of trans-crocetin (>95%) and cis-crocetin (>91%) were prepared simultaneously for the first time through a novel biphasic system based on deep eutectic solvents, n-heptane/n-butyl alcohol/13mmol/L Na2 CO3 in water/acetamide-benzyltrimethylammonium chloride (4:1, mol/mol) (4:7:9:1, v/v). The addition of deep eutectic solvent significantly improved the separation efficiency. The two targets can be easily recovered from the separation system through simple acidification and precipitation. It has potential for preparative separations on a large scale.

Anti-Inflammatory Effect of Ebractenoid F, a Major Active Compound of Euphorbia ebracteolata Hayata, through Inhibition of Nuclear Factor-κB Activation.

Euphorbia ebracteolata Hayata (Euphorbiaceae family) is a perennial plant that is widely distributed in Korea, Japan, and China. Its roots contain bioactive diterpenes that have anti-inflammatory properties. However, the anti-inflammatory mechanisms are not yet fully understood. This study aimed to identify the most active anti-inflammatory compound from the roots of E. ebracteolata Hayata, using bioassay-guided fractionation and a combinative method of high-speed countercurrent chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). Then, we investigated its anti-inflammatory mechanism in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Ebractenoid F was identified as the most potent bioactive compound of E. ebracteolata Hayata. Ebractenoid F significantly decreased nitric oxide (NO) production and nuclear factor-κB (NF-κB) activation induced by LPS in RAW 264.7 macrophages. Moreover, ebractenoid F decreased the degradation of inhibitory κB-α, the nuclear translocation of the p65 and p50 subunits of NF-κB, and the expression of NF-κB downstream genes. Furthermore, ebractenoid F inhibited the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK) and c-Jun NH2 terminal kinase (JNK), in LPS-stimulated RAW 264.7 cells. In conclusion, ebractenoid F exerts the most potent anti-inflammatory effect by suppressing NF-κB-mediated NO production in LPS-stimulated RAW 264.7 cells. Ebractenoid F may be a useful therapeutic compound for the prevention or treatment of inflammation-associated diseases.

Ellagic acid from pomegranate peel: Consecutive countercurrent chromatographic separation and antioxidant effect.

Ellagic acid is one of the most representative natural antioxidants, and is rich in pomegranate peel. In this study, a consecutive countercurrent chromatographic (CCC) separation method was established to improve the preparative efficiency of ellagic acid from pomegranate peel. By optimizing the solvent system, sample size and flow rate, 280 mg of ellagic acid was obtained from 5g of crude sample from pomegranate peel by CCC after six consecutive injections. Moreover, the values of EC50 for ellagic acid in scavenging ABTS·+ and DPPH· were 4.59 ± 0.07 and 10.54 ± 0.07 μg/ml, respectively, indicating a strong antioxidant activity. This study not only established a high-throughput method for the preparation of ellagic acid, but also provided a successful example for the development of and research on other natural antioxidants.

Biphasic alcoholysis coupled with high-speed countercurrent chromatography for high performance on separating phorbol from Croton tiglium Linn extracts.

Phorbol is a tetracyclic diterpenoid found inEuphorbia tirucalli,Croton tiglium, andRehmannia glutinosa, and is nuclear of various phorbol esters. The rapid obtaining of phorbol with high purity highly contributes to its application, such as synthesizing phorbol esters with designable side chains and particular therapeutic efficacy. This study introduced a biphasic alcoholysis method for obtaining phorbol from croton oil by using polarity imparity organic solvents in both phases and established a high-speed countercurrent chromatography method for simultaneous separation and purification of phorbol. The optimized operation conditions of biphasic alcoholysis were a reaction time of 91min, a temperature of 14°C, and a croton oil-methanol ratio of 1:30 (g:ml). The phorbol during the biphasic alcoholysis was 3.2-fold higher in content than that obtained in conventional monophasic alcoholysis. The optimized high-speed countercurrent chromatography method was using the ethyl acetate/n-butyl alcohol/water at 4.7:0.3:5 (v:v:v) with Na2 SO4 at 0.36g/10ml as the solvent system, using the mobile phase flow rate of 2ml/min, the revolution of 800r/min, under which the retention of the stationary phase was achieved at 72.83%. The crystallized phorbol following high-speed countercurrent chromatography was obtained as high purity of 94%.

The Generally Useful Estimate of Solvent Systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots.

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional high-performance countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v) and chloroform/methanol/water (10:9:6, v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography. This article is protected by copyright. All rights reserved.

Preparation of Isoquercetin From Toona sinensis by Liquid-Liquid-Refining Extraction And Consecutive Counter-Current Chromatography.

A high-speed counter-current chromatographic (HSCCC) method using ethyl acetate-water as solvent system was established to separate isoquercetin from Toona sinensis. In an HSCCC single separation, the sample sizes of ethanol extract were optimized from 203to 1200mg. The results showed that the yield of the target compound increased from 4to 26mg, and the corresponding purity decreased from 93.30 to 81.82%. To further improve the yield and purity, liquid-liquid-refining extraction was introduced to pretreat the ethanol extract and enrich the target compound. The ethanol extract was extracted with n-hexane-ethyl acetate-method-water (1:5:1:5, v/v) and ethyl acetate-water in turn to remove the low-polarity and high-polarity impurities and obtain the crude sample. Under the similar conditions, 85.25mg of the target compound with the purity of 95.12% was separated from 240mg of the crude sample. Subsequently, a consecutive HSCCC was developed to obtain 257mg of the target compound from 720mg of crude sample, which was equivalent to 14.4g of ethanol extract. This method improved the purity of the target compound, but more importantly, the sample size can reach 12 times of the maximum sample size of the ethanol extract in a single run.

Separation of eight phenolic compounds from the over-ground parts of Aconitum pendulum Busch by repeated injection high-speed counter-current chromatography

ABSTRACT In this study, eight phenolic compounds were obtained from the over-ground parts of the Aconitum pendulum (A. pendulum) Busch through an efficient high-speed counter-current chromatography （HSCCC）method. First, the column chromatography for macroporous resin was used for the treatment of crude extract of over-ground parts of A. pendulum Busch. And then, 30% ethanol elution fraction was transferred to HSCCC for separation using the classic ethyl acetate, n-butanol and water system with the solvent ratio of 7:3:10. To improve parting efficiency, a mode of repeated injection was developed. Two groups of compounds existing in one HPLC peak were effectively separated by HSCCC. The results revealed that HSCCC could effectively isolate compounds with the same or similar polarity. Preparative HPLC was used to perform further purification, and then eight phenolic compounds were separated from the over-ground parts of the A. pendulum Busch for the first time. The current investigation successfully provides a reference for phytochemical research and, comprehensive development and utilization of A. pendulum Busch in the future. Furthermore, HSCCC efficiently separated compounds with similar polarity and its combination with preparative HPLC could play a great role in the separation of natural materials.

Isolation and purification of flavonoids from Euonymus alatus by high-speed countercurrent chromatography and neuroprotective effect of rhamnazin-3-O-rutinoside in vitro.

The flavonoids from Euonymus alatus exhibit many biological activities including significant antioxidant, anti-inflammatory, anti-cancer. In this work, a high-speed countercurrent chromatography method for the isolation and purification of flavonoids from crude extracts of Euonymus alatus was established. The effects of several solvent systems on the separation efficiency of target compounds in the extract of Euonymus alatus were studied. The solvent system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of (3:5:3:5, v/v) was chosen, in which the lower phase was used as the mobile phase at the rotation speed of 800 rpm and flow rate of 2.0 mL/min. The three flavonoids were obtained and identified as patuletin-3-O-rutinoside, rhamnazin-3-O-rutinoside, and dehydrodicatechin A by mass spectroscopy and nuclear magnetic resonance, and the quantities of patuletin-3-O-rutinoside, rhamnazin-3-O-rutinoside, and dehydrodicatechin A were 2.2, 9.7, and 1.8 mg, respectively. The results indicated that high-speed countercurrent chromatography was a simple and efficient method for the isolation and purification of flavonoids from the crude extracts of Euonymus alatus. The cellular antioxidant activity experimental result indicated that rhamnazin-3-O-rutinoside could alleviate H2 O2 -induced oxidative stress.

Isolation of Four Major Compounds of γ-Oryzanol from Rice Bran Oil by Ionic Liquids Modified High-Speed Countercurrent Chromatography and Antimicrobial Activity and Neuroprotective Effect of Cycloartenyl Ferulate In Vitro

This study presents an effective high-speed countercurrent chromatography method of two-phase solvent system combined with ionic liquids for the isolation and purification of four major compounds of γ-oryzanol from rice bran oil. The conditions of high-speed countercurrent chromatography, including solvent systems and the content of ionic liquids, were optimized to improve separation efficiency. The optimal solvent system was n-hexane–dichloromethane–acetonitrile–[C4mim][PF6] at a volume ratio of (10:1:10:0.15, v/v). Adding a small amount of ionic liquids significantly improved the separation efficiency. Four compounds of γ-oryzanol, cycloartenyl ferulate, 24-methylene cycloartanyl ferulate, campesteryl ferulate, and sitosteryl ferulate were successfully purified in one run from rice bran oil with the purities of 95.25%, 92.48%, 92.47%, and 93.43%, respectively. The result of bioassay showed that rice bran oil and separated individual cycloartenyl ferulate could significantly inhibit mycelium growth of crop diseases Rhizoctonia solani and Fusarium graminerum, the bioactivity of cycloartenyl was higher than that of the rice bran oil. The cellular antioxidant activity experimental result indicted that cycloartenyl ferulate could alleviate H2O2-induced oxidative stress.

An efficient high-speed countercurrent chromatography method for preparative isolation of highly potent anti-cancer compound antroquinonol from Antrodia camphorata after experimental design optimized extraction.

To avoid irreversible stationary phase adsorption and tedious and time-consuming separation steps, high-speed countercurrent chromatography was employed for the preparative separation of anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata for the first time. A Box-Behnken experimental design, based on three parameters including liquid-to-solid ratio, extraction time, and extraction temperature, was applied to optimize the ultrasonic extraction procedure. The optimal extraction condition was set as follows: liquid-to-solid ratio: 49.57:1; extraction time: 55.76 min; extraction temperature was arranged as 44.21°C. Meanwhile, an optimized solvent system containing petroleum ether, ethyl acetate, methanol, and water (4:1:4:1, v/v/v/v) was selected for the preparative separation of antroquinonol at a flow rate of 2.0mL/min. The yield of isolated antroquinonol was determined to be 6.0mg from 0.67g of ethyl acetate extracts. The isolated antroquinonol was elucidated by ultra-high-performance liquid chromatography-tandem mass spectrometry, and NMR spectroscopy, and by comparison with literature data. The purity of isolated antroquinonol was determined to be 97.12%. This study confirmed that high-speed countercurrent chromatography was powerful and cost-effective for the preparative separation of the high-potently anti-tumor compound antroquinonol from solid fermentation culture of A. camphorata.

A simple and highly efficient counter-current chromatography method for the isolation of concentrated fractions of compounds based on the sequential sample loading technique: Comparative theoretical study of conventional multiple and intermittent sample loading counter-current chromatography separations

A new modification of the conventional multiple sample loading (MSL) mode – sequential sample loading (SSL) – is suggested to enhance further the performance of the counter-current chromatography (CCC) separation processes. The sequential sample loading technique is simple and easy to implement: the continuous sample solution supply to a CCC column is alternated (interrupted) with short periods of the "pure" mobile phase supply. Periodic (batch) and continuous SSL CCC separations can be designed and implemented. In continuous processes, the sample solution loading is carried out in the form of separate series, consisting of a number of sequential sample solution loads. In this work, the modeling of the conventional multiple sample loading and the sequential sample loading counter-current chromatography is used to compare the two operating modes considered. Equations for the calculation of band profiles, the recovery yield and the purity are given. Equations are also derived permitting the calculation of the optimum operating parameters of the separation processes. It is shown that the use of sequential sample loading makes it possible to produce fractions of purified compounds with a much higher concentration than in the original sample solution. The simulations of the conventional multiple sample loading and the sequential sample loading counter-current chromatography separations are presented in “Mathcad” software.

Efficient separation of N-Alkylamides from Piper longum L. using off-line two-dimensional coupled with gradient high-speed counter-current chromatography

Piper longum L. (Piperaceae) is famous for flavoring food and treatment purposes. N-Alkylamides (NAAs) are the most common secondary metabolites found in Piper species with broad spectrum of biological properties. Because of the similar structures and lower polarities, it is difficult to separate NAAs by traditional chromatographic methods. Herein, an efficient off-line two-dimensional coupled with gradient high-speed counter-current chromatography (HSCCC) method is reported for preparative separation of N-alkylamides from P. longum. The HSCCC solvent system with n-hexane/ethyl acetate/methanol/water (5:4:5:4, v/v) was used for the first dimensional separation. Two pure compounds i.e. piperlonguminine (1) and piperine (2) were successfully separated, coupled with an enriched trace ingredient. Furthermore, the enriched ingredient was used for the second dimensional separation with a gradient HSCCC mode by adjustment of the mobile phase with more enhanced elution ability. Eight minor compounds were successfully separated including retrofractamide A (3), retrofractamide C (4), dehydropipernonaline (5), pipernonaline (6), piperundecalidine (7), guineesine (8), brachystamide B (9), and (2E,4E,12Z)-N-isobutyl-2,4,12- octadecatrienamide (10). The structures of the isolated pure compounds were characterized by ESI-MS, 1H, and 13C NMR spectral analysis.

Chromatographic behavior of six lanthanides on a centrifugal mixer-settler extractor cascade

This work furthers the development of counter-current chromatography as an industrial separation process method. It was demonstrated that the industrial counter-current chromatography methods, in particular, for the separation groups of rare earth metals, can be implemented in a modified cascade of centrifugal mixer-settler extractors. The retention behavior of rare earth elements (samarium, europium, gadolinium, terbium, dysprosium and yttrium) on the pilot chromatographic unit consisting of 70 serially connected centrifugal mixer-settler extractors was experimentally studied under isocratic elution conditions using the mixture of 30 vol.% CyanexⓇ572 + 10 vol.% tributylphosphate in a hydrocarbon diluent as the stationary phase and aqueous nitric acid as the mobile phase. Theoretical analysis of experimental studies showed an acceptable agreement between the assumptions of the theory and experimental results.

Theoretical study of industrial scale closed-loop recycling counter-current chromatography separations

Industrial separation technologies can be improved and greatly simplified by using the methods of counter-current chromatography (CCC). We have previously proposed the use of currently available solvent extraction equipment (a series of multistage columns, a cascade of centrifugal mixer-settler extractors) as large-scale CCC devices. For industrial separations, the application of closed-loop recycling counter-current chromatography (CLR CCC) methods seems to be the most promising. To improve the performance of the CLR CCC separations, semi-continuous three-stage processes (1 – continuous loading of the mixture solution over a definite time; 2 – separation of solutes in recycling closed-loop; 3 – elution of the fractions of the separated solutes with the mobile phase) can be used. The purpose of this study is to present a simple and easy to use mathematical model allowing the simulation and design of various options for implementing such separation processes and analyze the influence of its main parameters on separation efficiency.

Isolation of six anthraquinone diglucosides from cascara sagrada bark by high-performance countercurrent chromatography.

In this study, high-performance countercurrent chromatography was employed to isolate six anthraquinone diglucosides, namely, cascarosides A-F, from cascara sagrada (Rhamnus purshiana DC [Rhamnaceae]) bark. The n-butanol-soluble extract of cascara sagrada was separated by off-line two-dimensional high-performance countercurrent chromatography. The first-dimensional high-performance countercurrent chromatography resolved the n-butanol-soluble extract (510mg) of cascara sagrada using the flow-rate gradient method with a chloroform-methanol-isopropanol-water (6:6:1:4, v/v/v/v, normal-phase mode) system to afford four anthraquinone diglucoside fractions (groups I [cascarosides C-D, 71mg], II [cascarosides E-F, 56mg], III [cascaroside A, 53mg], and IV [cascaroside B, 31mg]). Groups I and II were separated by the second-dimensional high-performance countercurrent chromatography using an ethyl acetate-n-butanol-water (7:3:10, v/v/v, normal-phase mode) system to yield cascarosides C (34mg), D (26mg), E (19mg), and F (15mg). Additionally, one-step preparative-scale high-performance countercurrent chromatography method was developed to isolate large amounts of cascarosides A (389mg) and B (187mg) from the water-soluble extract (2.1g) of cascara sagrada using an ethyl acetate-n-butanol-water (2:8:10, v/v/v, normal-phase mode) system. The current study demonstrated that high-performance countercurrent chromatography is a powerful technique for the isolation of marker compounds from herbal materials.