Counter-current Chromatography Technique Research Articles

Ionic-liquid-based ultrasound-assisted extraction combined with countercurrent chromatography and semipreparative LC for the preparation of monoamine oxidase B inhibitors from Pueraria thomsonii.

A simple and efficient method was developed for the rapid screening and identification of ligands for monoamine oxidase B. A new ionic-liquid-based ultrasound-assisted extraction method for medicinal herbs was also developed and validated. In addition, the hyphenated technique of countercurrent chromatography and semipreparative-LC was developed and applied to the isolation of the chemical constituents for Pueraria thomsonii Benth. Three potent monoamine oxidase B inhibitors, namely, daidzein-4',7-diglucoside (42.2mg), puerarin 6''-O-xyloside (88.3mg), and 3'-hydroxypuerarin (48.5mg) with purities of 98.2, 96.3, and 97.1%, respectively, were obtained from 500g of P. thomsonii raw material using semi-preparative high-performance liquid chromatography, whereas 3'-methoxypuerarin (76.2mg), daidzein-8-C-apiosyl (1→6) glucoside (84.2mg), and tectorigenin (75.1mg) with purities of 98.5, 96.4, and 96.8%, respectively, were obtained from 500g raw material via countercurrent chromatography using a two-phase solvent system comprising n-hexane-ethyl acetate-methanol-water at a volume ratio of 1.85:1.00:0.86:3.69 (v/v/v/v). Then, the anti-Alzheimer activity of the phytochemicals was assessed using a PC12 cell model. Treatment with tectorigenin, daidzein-4',7-diglucoside, puerarin 6''-O-xyloside, 3'-hydroxypuerarin, 3'-methoxypuerarin, and daidzein-8-C-apiosyl (1→6) glucoside (100 μg/mL), resulted in cell viabilities of 69.00, 65.81, 59.69, 57.90, 55.61, and 54.59%, respectively (p<0.001). The protocol was proved to be very accurate and efficient.

Characterization, Large-Scale HSCCC Separation and Neuroprotective Effects of Polyphenols from Moringa oleifera Leaves

Moringa oleifera leaves have been widely used for the treatment of inflammation, diabetes, high blood pressure, and other diseases, due to being rich in polyphenols. The main objective of this work was to largely separate the main polyphenols from Moringa oleifera leaves using the technique of high-speed counter-current chromatography (HSCCC). The phenolic composition in Moringa oleifera leaves was first analyzed qualitatively and quantitatively by UPLC-Q-Exactive Orbitrap/MS and UPLC-QqQ/MS, respectively, indicating that quercetin and kaempferol derivatives, phenolic acid and apigenin are the main polyphenols in Moringa oleifera leaves, with quercetin and kaempferol derivatives predominating. Furthermore, the conditions of HSCCC for large-scale separation of polyphenols from Moringa oleifera leaves were optimized, which included the selection of the solvent system, flow rate and the sample load. Only by one-step HSCCC separation (within 120 min) under the optimized conditions, six quercetin and kaempferol derivatives, a phenolic acid and an apigenin could be individually isolated at a large scale (yield from 10% to 98%), each of which possessed high purity. Finally, the isolated polyphenols and phenolic extract from Moringa oleifera leaves (MLPE) were verified to have strong neuroprotective activities against H2O2-induced oxidative stress in PC-12 cells, suggesting that these compounds would contribute to the main beneficial effects of Moringa oleifera leaves.

Antimicrobial and antioxidant activities of flavonoids isolated from wood of sweet cherry tree (Prunus avium L.)

Sweet cherry (Prunus avium L.) is a tree widely cultivated in temperate regions for its tasty and healing fruits. Pruning works on the tree give each year considerable amounts of woody wastes that hardly have any utility. The aims of this work were to detect the most active antioxidants present in a cherry pruning wood sample, to isolate and characterize them, and study the antimicrobial and antibiofilm activities of components found in the wood ethyl acetate extract against a selection of foodborne microorganisms. The online HPLC–DPPH technique allowed the detection of two active antioxidant peaks that, after being isolated by a combination of countercurrent chromatography (FCPC) and conventional preparative techniques, and subsequent structural characterization by NMR, MS and polarimetry, resulted to be (‒)-catechin (1) and (‒)-taxifolin (4). Other components of the cherry wood extract were also isolated, among which compounds 1, 4, and (+)-dihydrowogonin (12) have never been reported in P. avium. A selection of the isolated flavonoids was submitted to antimicrobial and antibiofilm activity evaluations against strains from type culture collections, as well as on multi-resistant strains previously isolated in our laboratory. Those compounds with antimicrobial activity detected in preliminary screenings by standard agar diffusion tests ‒the flavan-3-ol 1, the flavanonols 4 and (+)-aromadendrin (5), the flavanone (+)-pinocembrin (15), and the flavone tectochrysin (17)‒ were subjected to the minimal inhibitory concentration (MIC) test, showing all of them MIC values of 100 μg/mL. Compound 4 also induced a significative inhibition on the formation of biofilms by Enterobacter sp. UJA37p at a concentration of 1 μg/mL and a significative disruption of preformed biofilm by this strain at 0.1 µg/mL. Similar results on biofilm disruption were observed with compound 17.

Target separation and antitumor metastasis activity of sesquiterpene-based lysine-specific demethylase 1 inhibitors from zedoary turmeric oil

Lysine-specific histone demethylase 1 (LSD1) was the first histone demethylase identified in epigenetics and has recently emerged as an attractive therapeutic target for treating tumors. To date, almost all reported LSD1 inhibitors have been chemosynthesized; however, natural products possess pharmacological and biological activity and can be sources for drug development. Here, we established a target separation countercurrent chromatography technique to isolate LSD1 inhibitors from zedoary turmeric oil. Four sesquiterpene-based LSD1 inhibitors were efficiently obtained with an inhibition ratio equal to or less than that of the positive control drug. Compound 2 showed the most potent inhibitory activity, with a half-maximal inhibitory concentration of 3.97 μM, and was further tested to determine its ability to inhibit LSD1 and its antitumor metastatic effects in MDA-MB-231 cells. These four compounds are the first sesquiterpene-based natural LSD1 inhibitors to be characterized. Our findings provide a new molecular framework for studying LSD1 inhibitors and offer a template for designing more sesquiterpene-based LSD1 inhibitors with potential antitumor activity.

Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography

Brazilein is among the main chemical constituents of Caesalpinia sappan. It has diverse pharmacological activities. Modern pharmacological studies have shown that the compound has antitumor, anti-inflammatory, antibacterial, antioxidant, immunomodulatory, and other pharmacological activities. Brazilein is often used as a stain in various industries. The separation of brazilein by traditional column chromatography will not only result in contamination of the chromatographic column materials, but also lead to loss of the active ingredient. Countercurrent chromatography is an advanced liquid-liquid chromatographic separation technique. It has been widely used for natural product separation and isolation as it offers several advantages, such as low solvent consumption, a highly selective solvent system, and high recoveries. Typical countercurrent chromatography techniques include centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and high performance countercurrent chromatography (HPCCC). It is well known that choosing a suitable solvent system is vital in countercurrent separation. Therefore, two methods were introduced for choosing a suitable solvent system. One is the generally useful estimation of solvent systems (GUESS) method, which employs thin-layer chromatography (TLC) to identify a suitable solvent system with minimal labor for the rapid purification of target compounds, and another is the Shake-Flash method. The solvent system could be determined by observing the distribution of the sample in the upper and lower phases. Two kinds of solvent systems were screened using the TLC-GUESS and Shake-Flash methods, and tested through the analysis mode of the HPCCC instrument. The results showed that chloroform-methanol-water (4:3:2, v/v/v) was the optimal solvent system for HPCCC separation. A total of 15.2 mg of brazilein and 5.7 mg of caesappanin C were obtained from an ethyl acetate extract with high purities (95.6% and 89.0%, analyzed by HPLC) in one step using the preparation mode of HPCCC, the reversed-phase liquid chromatography mode with the apparatus rotated at 1600 r/min, a flow rate of 10 mL/min, separation temperature of 25℃, and detection wavelength of 285 nm. Their structures were determined by spectroscopic and spectrometric analyses. Brazilein stained the solid packing material in the column and was difficult to elute. The results showed that the use of HPCCC for the separation of brazilein can not only prevent the loss of target active ingredients in Caesalpinia sappan, but also shorten the separation and purification times and improve the operating efficiency. Therefore, HPCCC can be used for the separation and preparation of other pigment compounds in Caesalpinia sappan and other dye plants.

Preparative Separation and Purification of Liquiritin and Glycyrrhizic Acid from Glycyrrhiza uralensis Fisch by High-Speed Countercurrent Chromatography.

The technique of high-speed countercurrent chromatography (HSCCC) was applied to the preparative isolation and purification of liquiritin and glycyrrhizic acid from a crude extract of Glycyrrhiza uralensis Fisch for the first time. Using single factor and orthogonal design experiments, the best extraction conditions were 70% ethanol, 1:25 ratio of solid-to-liquid (w/v) and extracted 1.5h at 80°C. The contents of liquiritin and glycyrrhizic acid in the crude extract were 1.3 and 5.3%, respectively. Using the two-phase solvent system of ethyl acetate-methanol-water (5:2:5, v/v), 6.0mg liquiritin (the purity was 96.7%, and the recovery was 89.3%), and 20.5mg glycyrrhizic acid (the purity was 98.9%, and the recovery was 77.1%) were obtained from 500mg crude extraction by HSCCC, respectively. The retention rate of stationary phase was 51.0%. Their structures were identified by high-performance liquid chromatography, melting points, ultraviolet radiation, Fourier transform infrared (FTIR), Electrospray ionization mass spectrometry (ESI-MS), 1H Nuclear Magnetic Resonance (NMR) and 13C NMR spectra. The scavenging abilities of glycyrrhizic acid to 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals were stronger than those of liquiritin.

Chiral Ionic Liquids: Structural Diversity, Properties and Applications in Selected Separation Techniques.

Ionic liquids (ILs) are chemical compounds composed of ions with melting points below 100 °C exhibiting a design feature. ILs are commonly used as the so-called green solvents, reagents or highly efficient catalysts in varied chemical processes. The huge application potential of ionic liquids (IL) justifies the growing interest in these compounds. In the last decade, increasing attention has been devoted to the development of new methods in the synthesis of stable chiral ionic liquids (CILs) and their application in various separation techniques. The beginnings of the successful use of CILs to separate enantiomers date back to the 1990 s. Most chiral ILs are based on chiral cations or chiral anions. There is also a limited number of CILs possessing both a chiral cation and a chiral anion. Due to the high molecular diversity of both ions, of which at least one has a chiral center, we have the possibility to design a large variety of optically active structures, thus expanding the range of CIL applications. Research utilizing chiral ionic liquids only recently has become more popular. However, it is the area that still has great potential for future development. This review aimed to describe the diversity of structures, properties and examples of applications of chiral ionic liquids as new chiral solid materials and chiral components of the anisotropic environment, providing chiral recognition of enantiomeric analytes, which is useful in liquid chromatography, countercurrent chromatography and other various CIL-based extraction techniques including aqueous biphasic (ABS) extraction systems, solid–liquid two-phase systems, liquid–liquid extraction systems with hydrophilic CILs, liquid–liquid extraction systems with hydrophobic CILs, solid-phase extraction and induced-precipitation techniques developed in the recent years. The growing demand for pure enantiomers in the pharmaceutical and food industries sparks further development in the field of extraction and separation systems modified with CILs highlighting them as affordable and environmentally friendly both chiral selectors and solvents.

Nigella damascena L. essential oil and its main constituents, damascenine and β-elemene modulate inflammatory response of human neutrophils ex vivo

Nigella damascena L belongs to Ranunculaceae family and is mentioned in Eastern traditional medicine for the treatment of high temperatures, regulation of menstruation or catarrhal affections. The anti-inflammatory activity of compounds present in the essential oil obtained from seeds of this plant can be found in literature, however no studies on immunomodulatory activity are provided. Hence, in this work anti-inflammatory activity of N. damascena seed essential oil as well as damascenine and main compound β-elemene was evaluated on ex-vivo lipopolysaccharide (LPS)–stimulated human neutrophils. For isolation of damascenine fast and efficient protocol was elaborated using high performance countercurrent chromatography technique for the first time. Also detailed spectroscopic characteristic of damascenine was provided for the first time. Damascenine was separated from the essential oil in a mixture of petroleum ether/acetonitrile/acetone (2:1.5:0.5 v/v/v) in reversed phase mode in 12 min with 99.47% purity. Essential oil, damascenine and β-elemene presented immunomodulatory activity evaluated in LPS–stimulated neutrophils ex vivo. All studied samples significantly inhibited release of interleukin 1 beta (IL-1β) and interleukin 8 (IL-8). What is more, damascenine and β-elemene decreased matrix metallopeptidase 9 (MMP-9) production similar to dexamethasone. The release of tumor necrosis factor (TNF-α) was also inhibited in all range of concentrations, however the activity was weaker then activity of dexametasone. The previously reported anti-inflammatory activity of damascenine and β-elemene investigated in murine models was confirmed in our study on human neuthrophils suggesting their possible strong inhibitory effect on inflammatory response progression.

Integrating Strategies of Herbal Metabolomics, Network Pharmacology, and Experiment Validation to Investigate Frankincense Processing Effects.

In-depth research on processing can promote the globalization of processed herbs. The purpose of this study is to propose an improved strategy for processing effect investigation. Frankincense and processed frankincense were used as research subjects. First, high-speed countercurrent chromatography (HSCCC) and preparation high-performance liquid chromatography (PHPLC) techniques were used for major compounds isolation and minor compounds concentration. Processed frankincense was subjected to two stepwise solvent systems, namely, n-hexane:ethanol:water (6:5:1) and n-hexane:methyl-acetate:acetonitrile:water (4:4:3:4), to yield 12 fractions, and 18 compounds were further separated. Second, a comprehensive metabolomic analysis conducted by ultrahigh-performance liquid-chromatography/electrospray-ionization mass spectrometry (UHPLC-Qtof-MS) coupled with multivariate statistics was performed to fully characterize the chemical components and discover the potential biomarkers between frankincense and processed frankincense. In total, 81 metabolites, including the 18 separated compounds, were selected as potential biomarkers between frankincense and processed frankincense among 153 detected compounds for their VIP values of greater than one. The tirucallane-type compounds and components with 9,11-dehydro structures clearly occurred at high levels in the processed frankincense, while lupine-type compounds and those with 11-keto structures were significantly higher in frankincense. Then, a network pharmacology model was constructed to decipher the potential mechanisms of processing. Intestinal absorption properties prediction indicated the possibility of processing-related absorption enhancement. A systematic analysis of the constructed networks showed that the C-T network was constructed with 18 potential biomarkers and 69 targets. TNF and IL-1β were among the top-ranked and were linked by 8 and 7 pathways, which were mainly involved in inflammation. The arachidonic acid metabolism pathway exhibited the highest number of target connections. Finally, the prediction was validated experimentally by an intestinal permeability and efficacy assay. The experiments provided convincing evidence that processed frankincense harbored stronger inhibition effects toward TNF-α-, IL-1β- and arachidonic acid-induced platelet aggregation. The processing procedure leads to changes of the chemical metabolites, which triggers the enhancement of absorption and cure efficiency. The global change of the metabolites, absorption and pharmacological effects of processing were depicted in a systematic manner.

Purification of Human Monoclonal Antibodies and Their Fragments.

This chapter summarizes the most common chromatographic mAb and mAb fragment purification methods, starting by elucidating the relevant properties of the compounds and introducing the various chromatography modes that are available and useful for this application. A focus is put on the capture step affinity and ion-exchange chromatography. Aspects of scalability play an important role in judging the suitability of the methods. The chapter introduces also analytical chromatographic methods that can be utilized for quantification and purity control of the product. In the case of mAbs, for most purposes the purity obtained using an affinity capture step is sufficient. Polishing steps are required if material of particularly high purity needs to be generated. For mAb fragments, affinity chromatography is not yet fully established, and the capture step potentially may not provide material of high purity. Therefore, the available polishing techniques are touched upon briefly. In the case of mAb isoform and bispecific antibody purification, countercurrent chromatography techniques have proven to be very useful and a part of this chapter has been dedicated to them, paying tribute to the rising interest in these antibody formats in research and industry.

Isolation of two major sesquiterpenes from the leaf essential oil of Eugenia uniflora by preparative‐scale high‐speed countercurrent chromatography

AbstractThe high‐speed countercurrent chromatography technique was employed to isolate two major compounds from the leaf essential oil of Eugenia uniflora L. (Myrtaceae). The volatile extract of Eugenia uniflora obtained by hydrodistillation was analyzed by gas chromatography coupled with flame ionization detection and gas chromatography coupled with mass spectrometry and shown to be almost exclusively constituted by sesquiterpenes, in which (+/‐)‐selina‐1,3,7(11)‐trien‐8‐one (A) and (+/‐)‐selina‐1,3,7(11)‐trien‐8‐one epoxide (B) were largely predominant. These two major compounds were separated by submitting the crude essential oil to countercurrent chromatography using hexane/acetonitrile 1:1 v/v as the solvent system, running in both the tail‐to‐head (mode I) and head‐to‐tail (mode II) modes. Upon running in mode I, a single application of the crude oil (800 mg) in the separation equipment coil afforded 78.2 mg of B (&gt; 97.5% purity). Running in mode II produced 24.7 mg of A (92.5% purity) and 46.3 mg (93.1% purity) of B. The purity of each compound was determined by GC. For the first time, this technique was shown to be efficient to separate sesquiterpenes that are structurally very similar, affording high‐purity products in a short time with significant solvent savings.

Separation of Volatile Metabolites from the Leaf-Derived Essential Oil of Piper mollicomum Kunth (Piperaceae) by High-Speed Countercurrent Chromatography.

The technique of high-speed countercurrent chromatography was applied to the isolation of compounds in essential oil derived from the leaves of Piper mollicomum species. Plant leaves (200.0 g) were submitted to hydrodistillation in a modified Clevenger apparatus. The resulting crude leaf essential oil was analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography-mass spectrometry (GC-MS) to determine the profile of the components. The purified fractions were composed of monoterpenes and sesquiterpenes such as camphor (85.0 mg at 98.5% purity), (E)-nerolidol (100.0 mg at 92.8% purity), and camphene (150.0 mg at 82.0% purity). A minor component of the essential oil, bornyl acetate (16.2 mg at 91.2% purity) was also isolated in the one-step separation protocol in 2 h. The countercurrent chromatography technique proved to be a fast and efficient method for the separation of volatile metabolites that conserved the solvent while delivering various fractions of high purity.

Elution-extrusion countercurrent chromatography separation of six pairs of isomeric iridoids from Cornus officinalis Sieb. et Zucc. guided by ion current extraction in mass spectrometry.

A mass spectrometry-guided elution-extrusion countercurrent chromatography protocol was developed to separate chemical components from Cornus officinalis Sieb. et Zucc. In this study, ion current extraction, a mass spectrometry-based data postacquisition method, was utilized to boost the separation power and scope of countercurrent chromatography technique. As a peak repicking and denoising tool, ion current extraction was carried out to process the liquid chromatography with mass spectrometry and the countercurrent chromatography with mass spectrometry data. So the target components were reacquired in the created extracted ion current patterns with enhanced responses and diminished background noise, which facilitated the distribution constant determination (by liquid chromatography with extracted ion current) and the targets fractionation (by countercurrent chromatography with extracted ion current). Under the guidance of the extracted ion currents of the target components and with the support of elution-extrusion mode in the countercurrent chromatography separation, six pairs of minor iridoid isomers were obtained in shortened experimental duration. Besides, a reciprocal shifted symmetry plot was established to represent the elution-extrusion countercurrent chromatography chromatogram. The results demonstrated the capability of the ion current extraction-guided elution-extrusion countercurrent chromatography protocol in discovery, analysis, and fractionation of low-concentration and structurally similar chemicals from a complicated sample.

Separation and Purification with a Liquid Stationary Phase

Yoichiro Ito introduced countercurrent chromatography (CCC) in 1966, reporting the separation of blood plasma cells with a sealed helical tube. Since then, CCC has been a fertile ground for instrumental and technical innovation. The key innovation of CCC was to use centrifugal forces to retain the stationary liquid phase in the column in such a way that it is able to interact dynamically with the mobile phase without any solid support. The broad diversity of countercurrent separation terminology reflects the innovative spirit of the field, as well as the global appeal of this technique. The selection of the appropriate biphasic liquid system is the core of the CCC technique. The CCC columns must generate the centrifugal field needed to maintain the liquid stationary phase; therefore, they cannot be a simple tube with frits at both ends. Rotors, motor, gears, spools, and rotating seals are very specific things that are not needed in a classical liquid chromatography column with a solid stationary phase. The differences between the two main types of CCC columns are described. The bases of the CCC theory are also given.

HSCCC separation and enantiomeric distribution of key volatile constituents of Piper claussenianum (Miq.) C. DC. (Piperaceae)

High Speed Countercurrent Chromatography (HSCCC) technique was used for the preparative isolation of the major leishmanicidal compounds from the essential oils of Piper claussenianum species in Brazil. The essential oils from inflorescences of P. claussenianum were analyzed by GC-FID and GC-MS. The enantiomeric ratio of the major constituents of the P. claussenianum essential oils were determined using a Rt-DEXsm chiral capillary column by GC-FID analysis. It was found an enantiomeric excess of (+)-(E)-nerolidol in the leaves, and (+)-linalool and (+)-(E)-nerolidol in the inflorescences essential oil. The major volatile terpenes alcohols were isolated in preparative scale from inflorescences: linalool (320.0 mg) and nerolidol (95.0 mg) in high purity level. The HSCCC, a support-free liquid–liquid partition chromatographic technique, proved to be an effective and useful method for fast isolation and purification of hydrophobic and similarly structured bioactive components from essential oils of Piper species.

Centrifugal partition chromatography - A review of recent applications and some classic references.

Countercurrent chromatography, based on liquid-liquid partitioning, has many technological variants. One of them is centrifugal partition chromatography, introduced by Wataru Murayama and Kanichi Nunogaki in 1982. This technique, like other countercurrent chromatography techniques, is based on the phenomenon of liquid-liquid partitioning between two immiscible liquid phases that stay at equilibrium. But the significant difference between this technique and others is the retention mechanism of stationary phase. In the case of centrifugal partition chromatography, this mechanism is based on hydrostatic force, formed by the centrifugal field in the rotor in one-axis centrifuge. Sometimes that allows more control of stationary phase, for example, when aqueous two-phase and other difficult solvent systems are used. However, the efficiency of the separation in centrifugal partition chromatography is also affected by a variety of parameters dependent on the sample properties in the solvent system, physical properties of the solvent system, parameters of the instrument, and the method. This article includes also recent ideas for improvements to the technique and broadening its application (e.g., (multiple) dual-mode or elution-extrusion procedure, pH-zone-refining centrifugal partition chromatography, ion-exchange centrifugal partition chromatography, online and offline coupling of centrifugal partition chromatography).

Isolation and Purification of Phenolic Acids from Sugarcane (Saccharum officinarum L.) Rinds by pH-Zone-Refining Counter-Current Chromatography and Their Antioxidant Activity Evaluation

The phenolic acids exhibit significant antioxidant, antiinflammatory, antimutation, and tyrosinase inhibitory effects. A high efficient and specific method for separation of phenolic acids from the sugarcane rinds based on pH-zone-refining counter-current chromatography technique was established. The separation was performed with a solvent system of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4:1:5, where 5 mM trifluoroacetic acid was added to the upper phase as a retainer and 3 mM NH4OH was added to the lower phase as an eluter. As a result, three phenolic acids including 4.8 mg of caffeic acid, 12.9 mg of ferulic acid, and 65.2 mg of p-coumaric acid were successfully purified in one run from 1.00 g crude extract with the purities of 93.4, 94.6, and 98.8%, respectively. 2,2-Diphenyl-1-picrylhydrazyl radical and total antioxidant capacity assay were used to evaluate antioxidant activities. The separated individual phenolic acid had higher antioxidant activity than their mixture. Caffeic acid showed the greatest antioxidant activity, followed by ferulic acid and p-coumaric acid.

HSCCC-based strategy for preparative separation of in vivo metabolites after administration of an herbal medicine: Saussurea laniceps, a case study.

This paper reports a novel strategy based on high-speed counter-current chromatography (HSCCC) technique to separate in vivo metabolites from refined extract of urine after administration of an herbal medicine. Saussurea laniceps (SL) was chosen as a model herbal medicine to be used to test the feasibility of our proposed strategy. This strategy succeeded in the case of separating four in vivo metabolites of SL from the urine of rats. Briefly, after oral administration of SL extract to three rats for ten days (2.0 g/kg/d), 269.1 mg of umbelliferone glucuronide (M1, purity, 92.5%), 432.5 mg of scopoletin glucuronide (M2, purity, 93.2%), 221.4 mg of scopoletin glucuronide (M3, purity, 92.9%) and 319.0 mg of scopoletin glucuronide (M4, purity, 90.4%) were separated from 420 mL of the rat urine by HSCCC using a two-phase solvent system composed of methyl tert-butyl ether–n-butanol–acetonitrile–water (MTBE–n-BuOH–ACN–H2O) at a volume ratio of 10:30:11:49. The chemical structures of the four metabolites, M1 to M4, were confirmed by MS and 1H, 13C NMR. As far as we know, this is the first report of the successful separation of in vivo metabolites by HSCCC after administration of an herbal medicine.

Developments in high-speed countercurrent chromatography and its applications in the separation of terpenoids and saponins.

High-speed countercurrent chromatography is a liquid-liquid separation chromatographic technique, which has the unique feature of eliminating irreversible adsorption using liquid support medium, and is widely used in research and development of traditional Chinese medicine, biochemistry, food, environment analysis, and so on. In this review, some new developments of countercurrent chromatography, for instance cross-axis countercurrent chromatography, dual countercurrent chromatography, foam countercurrent chromatography, and pH-zone-refining countercurrent chromatography are presented. Furthermore, the research and progress in high-speed countercurrent chromatography techniques and its application in the separation and purification of terpenoids and saponins are reviewed.

Separation and preparation of 6-gingerol from molecular distillation residue of Yunnan ginger rhizomes by high-speed counter-current chromatography and the antioxidant activity of ginger oils in vitro.

Molecular distillation residue (MD-R) from ginger had the most total phenol content of 247.6mg gallic acid equivalents per gram (GAE/g) among the ginger oils. High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale was successfully performed in separation and purification of 6-gingerol from MD-R by using a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:2:5:7, v/v/v/v). The target compound was isolated, collected, purified by HSCCC in the head-tail mode, and then analyzed by HPLC. A total of 90.38±0.53mg 6-gingerol was obtained from 600mg MD-R, with purity of 99.6%. In addition, the structural identification of 6-gingerol was performed by EI/MS, (1)H NMR and (13)C NMR. Moreover, the orders of antioxidant activity were vitamin E (VE)>supercritical fluid extraction oleoresin (SFE-O)=MD-R=6-gingerol>molecular distillation essential oil (MD-EO) and butylated hydroxytoluene (BHT)=VE>6-gingerol>MD-R=SFE-O>MD-EO, respectively in 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging and β-Carotene bleaching.