Preparative separation of bioactive constitutes from Zanthoxylum planispinum using linear gradient counter-current chromatography.

Counter-current chromatography is a chromatographic technique with a support-free liquid stationary phase. In the present study, a successful application of linear gradient counter-current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n-hexane/ethyl acetate/methanol/water quaternary solvents, in terms of "HEMWat" or "Arizona" solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter-current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n-hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter-current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.

Chemical composition and flavour characteristics of a liquid extract occurring as waste in crab (Ovalipes punctatus) processing.

BACKGROUND: The crab processing industry has generated a considerable quantity of by-products, and these untapped residues resulted in environmental problem and waste of natural resources. Therefore, the purpose of this research was to evaluate the further usage potential of Ovalipes punctatus extract. The proximate composition, minerals, fatty acids, amino acids, tasty components (free amino acid, flavour 5'-nucleotides, glycine betaine and inorganic ions) and volatile flavour components were studied. RESULTS: O. punctatus extract was found to have a high protein (31.2 g kg⁻¹), but a low fat content (0.13 g kg⁻¹). The protein contained high amounts of arginine (110.2 g kg⁻¹ protein) and glutamic acid (108.9 g kg⁻¹). The fatty acid profiles were dominated by saturated fatty acids, while C20 n-3 and n-6 fatty acids accounted for 85% of its polyunsaturated fatty acids. Arginine, alanine, glycine, glycine betaine, glutamic acid and chloridion (taste active value greater than 1) were primary taste-active components. A total of 77 volatiles were identified, and benzaldehyde and pyrazines were the major flavour contributors to the aroma of O. punctatus extract. Furthermore, sensory evaluation with a five-point hedonic scale showed that the overall flavour of O. punctatus extract had high acceptance. CONCLUSION: Results presented in this study indicated that O. punctatus extract could be utilised to produce nutritious food or value-added products.

Counter-current chromatographic method for preparative scale isolation of picrosides from traditional Chinese medicine Picrorhiza scrophulariiflora.

Apocynin, androsin, together with picroside I, II and III from crude extracts of Picrorhiza scrophulariiflora were isolated by means of high-speed counter-current chromatography (CCC) combining elution-extrusion (EE) and cycling-elution (CE) approach. The EECCC took full advantages of the liquid nature of the stationary phase for a complete sample recovery and extended the solute hydrophobicity window, while CECCC showed its unique advantage in achieving effective separation of special compounds through preventing stationary phase loss. In the present work, the biphasic liquid system composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, v/v/v/v) was used for separation of apocynin and androsin, ethyl acetate/n-butanol/water/formic acid (4:1:5:0.005, v/v/v/v) for picroside I, II and III. However, due to the extremely similar K values (K1 /K2 ≈1.2), picroside I and III were always eluted together by several biphasic solvent systems. In this case, the CECCC exhibited great superiority and baseline separated in the sixth cycle using ethyl acetate/water (1:1, v/v) as biphasic liquid system. Each fraction was analyzed by UPLC-UV and ESI-MS analysis, and identified by comparing with the data of reference substances. Compared with classical elution, the combination of EE and CE approach exhibits strong separation efficiency and great potential to be a high-throughput separation technique in the case of complex samples.

Multiwalled carbon nanotubes as sorbent for online solid-phase extraction of resveratrol in red wines prior to fused-core C18-based ultrahigh-performance liquid chromatography-tandem mass spectrometry quantification.

An ultrafast analytical protocol based on online solid-phase extraction (SPE)/high-performance liquid chromatography-tandem mass spectrometry for the determination of resveratrol in red wines has been developed. In the present work, multiwalled carbon nanotubes (MWCNTs) were used as SPE sorbents for the analytes' online extraction and cleanup. The target analytes were separated on a fused-core C18-silica column (Halo, 50 mm × 2.1 mm i.d., 2.7 µm) and quantified by triple-quadrupole linear ion trap mass spectrometry in negative ion multiple-reaction monitoring (MRM) mode. The proposed analytical procedures were carefully optimized and validated. The calibration function is linear from 0.37 to 370 ng mL(-1) and from 0.13 to 130 ng mL(-1) for trans- and cis-resveratrol, respectively. The limits of quantification (LOQs) of trans- and cis-resveratrol obtained were 0.05 and 0.06 ng mL(-1), which means that the proposed method is suitable for trace analysis of resveratrol at low-level concentration. At the three fortified levels (low, medium, and high), recoveries of resveratrol ranging from 76.9 to 108.3% were obtained. Eight red wine samples from different regions of China were analyzed. The results indicated that the present online SPE-LC-MS/MS system significantly increased sample throughput and decreased solvent consumption, exhibiting great potential to be applied for analyzing resveratrol in red wines.

Preparative separation of anti-oxidative constituents from Rubia cordifolia by column-switching counter-current chromatography.

An effective column-switching counter-current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti-oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column-switching CCC system was interfaced by a commercial low-pressure six-port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1st-D) to the second dimension (2nd-D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1st-D. By valve switching technique the whole interested region of 1st-D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column-switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram-amounts of four anti-oxidative components over 90% pure. Structures of purified compounds were identified by (1)H and (13)C NMR.

Preparative separation of isoquinoline alkaloids from Stephania yunnanensis by pH-zone-refining counter-current chromatography.

In this paper, five isoquinoline alkaloids were successfully separated from a crude extract of Stephania yunnanensis using pH-zone-refining counter-current chromatography in single-step. With a two-phase solvent system composed of methyl-tert-butyl ether (MtBE)-acetonitrile-water (2:2:3, v/v) where triethylamine (10 mM) was added to the upper organic phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. From 1.4 g crude extract, 68.7 mg isocorydine, 78.2 mg corydine, 583.4 mg tetrahydropalmatine, 36.3 mg N-methylasimilobine, and 47.3 mg anonaine were separated with purities over 90%. Their structures were identified by (1)H NMR, (13)C NMR, ESI-MS data.

Screening of antioxidant phenolic compounds in Chinese rhubarb combining fast counter-current chromatography fractionation and liquid chromatography/mass spectrometry analysis.

In this paper, an effective method combing fast elution-extrusion counter-current chromatography (CCC) and LC/MS for rapid screening of antioxidative phenolic compounds in Chinese Rhubarb is presented. An integrated three-coil CCC column (40 mL each coil) was used to accomplish the optimization of biphasic liquid system. In a single run (approximately 40 min), the solvent system composed of n-hexane/ethyl acetate/methanol/water (1:1:1:1, v/v) was selected as optimum CCC liquid system for fast fractionation of the crude ethanol extract. With a 140 mL-capacity CCC instrument, 100 mg Chinese Rhubarb extract was separated under the optimized conditions, producing six fractions in only 100 min. The quantities of each fraction were approximately 15 mg. In addition, each fraction was subjected to antioxidant activity assay and characterized by LC/MS analysis. Fifty compounds, including phenolic acids, phenolic glucosides and hydroxyanthraquinones, were detected by LC/MS/MS analysis. As a result, gallic acid together with Fr I showed excellent antioxidant activity, which was well consistent with previous studies and exhibited great potential for natural drug discovery program of the present method.

Integrated countercurrent extraction of natural products: a combination of liquid and solid supports.

An integrated online column-switching countercurrent chromatography (CCC) with a solid-phase trapping/preconcentration interface is presented. The interface is systematically evaluated in terms of sorbent type, column size, and kinetic factor from the view of the unique CCC process. Results indicate that satisfactory trapping efficiency can be achieved using a 25 mm x 10 mm i.d. column packed with Oasis HLB materials. In addition to the analyte focusing effect, large volume injection is avoided, thereby allowing the use of totally different biphasic liquid systems to enhance the system orthogonality. The present integrated system simply combines the liquid and solid supports and is successfully applied in a one-step preparative separation of four antioxidative compounds from the ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia L. (Rubiaceae), exhibiting great advantages in peak resolution, peak capacity, and instrument integration compared with conventional CCC separations.

Single-step purification of dracorhodin from dragon's blood resin of Daemonorops draco using high-speed counter-current chromatography combined with pH modulation.

Dracorhodin is a major constituent found in "Dragon's blood" resin of Daemonorops draco Willd. Blume. This natural flavylium compound is a potent pharmaceutical substance due to its biological and pharmacological activities such as antimicrobial, antiviral, antitumor and cytotoxic activity. An effective high-speed counter-current chromatography method was successfully established for the isolation and purification of dracorhodin directly from extract of D. draco by using a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (2:3:2:3 v/v). Under the optimal conditions, 6.6 mg dracorhodin was obtained from 100 mg crude resin. The isolated fraction of counter-current chromatography was determined by HPLC, NMR, UV/visible and ESI/MS combined with pH modulation, since dracorhodin is unstable in solution which exists in different forms depending on pH values. The data were compared with those of the reference substance, and the literatures as well. The purity of dracorhodin was over 98% based on the HPLC result.

Differentiation of configurations of the phenylbutenoid dimer derivatives from Zingiber cassumunar by tandem mass spectrometry.

High-performance liquid chromatography/electrospray tandem mass spectrometry was developed to distinguish isomers and compounds of similar structures with different configurations in the rhizomes of Z. cassumunar. Energy-resolved breakdown curves were utilized to differentiate four compounds. Compounds 2 (3R,4S) and 4 (3R,4R) were a pair of stereoisomers which could be distinguished easily by breakdown curves. The breakdown curve of compound 1 was identical to that of compound 2, which suggested that the configuration of compound 1 was (3R,4S) or (3S,4R). The breakdown curve of compound 3 was completely different from those of compounds 1, 2 and 4, and it might be that the configuration of the double bond of compound 3 was different from the other three compounds. Hence, the described method using breakdown curves has great potential in the distinguishing of isomers and compounds of similar structure with different configurations.

Screening of complex natural extracts by countercurrent chromatography using a parallel protocol.

In countercurrent chromatography (CCC) the choice of the liquid system is the heart of any separation. It corresponds to the selection of the mobile phase and the stationary phase at the same time. Any change in one phase composition induces a change in the other phase composition which renders the choice of the appropriate liquid system difficult and lengthy. A scale of compositions of the heptane-ethyl acetate-methanol-water quaternary liquid system was referred to by letters from A to Z and called the Arizona (AZ) liquid system. Each composition of the AZ system has the same heptane/ethyl acetate and methanol/water volume ratios. It is shown that there is a continuous polarity change from the hydrophilic A composition (ethyl acetate-water) to the hydrophobic Z (heptane-methanol) mixture by measuring the distribution constant K(D) of a known test mixture. For all compounds, the log K(D) is linearly increasing with the water content of the lower aqueous phase of the composition used. The slopes of the log K(D) versus percent H(2)O have very different values which means that the chromatographic selectivity changes with liquid system compositions. The AZ system was associated to the elution-extrusion method to design a procedure to identify rapidly the appropriate solvent composition able to fractionate correctly a complex natural extract. With the use of an integrated three-coil CCC column (40 mL each coil) able to test three AZ compositions in parallel, it is shown that the optimum AZ composition is found in half a day using less than a liter total volume of solvents. Two natural extracts are rapidly screened using the proposed protocol. An extract of Piper longum L. of intermediate polarity was fractionated in five usable portions using the 3/2/3/2 (Q) composition of the AZ system. A polar extract of Polygonum cuspidatum was also separated in five fractions using the 1/6/1/6 (D) composition. In both cases, a 140 mL CCC column was used for a direct scale-up transfer with the same liquid system. Purified fractions were subjected to an antioxidant activity assay and liquid chromatography with UV and mass spectrometry detection (LC-UV/MS) analysis to determine the molecular weight, number, and quantity of compounds in the active fractions. Four fractions of P. cuspidatum showed excellent antioxidant activity. They were rapidly produced at the milligram level by the 140 mL CCC column and fractionated by semipreparative high-performance liquid chromatography (HPLC) in individual compounds that were each identified by NMR and MS and reevaluated for confirmation of bioactivity. The rapid screening CCC protocol associated to the preparative capability of CCC allows for a fast identification and characterization of active compounds in natural products.

Rapid and preparative separation of traditional Chinese medicine Evodia rutaecarpa employing elution-extrusion and back-extrusion counter-current chromatography: comparative study.

Traditional Chinese medicines (TCMs) have attracted much attention in recent years. Elution-extrusion and/or back-extrusion counter-current chromatography (EECCC/BECCC) both take full advantage of the liquid nature of the stationary phase. They effectively extend the solute hydrophobicity window that can be studied and rendered the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, a popular traditional Chinese medicine, Evodia rutaecarpa, was used as the target complex mixture for extrusion CCC separations. With a carefully selected biphasic liquid system (n-hexane/ethyl acetate/methanol/water, 3/2/3/2, v/v) and optimized conditions (V(CM)=V(C), mobile phase flow rate: 3mL/min in descending mode, sample loading: 100mg), five fractions could be obtained in only 100min on a 140-mL capacity CCC instrument using both elution- and back-extrusion methods. Each fraction was analyzed and identified compared with the data of major standards using LC/MS. Moreover, the performance of both extrusion protocols was systematically compared and summarized. EECCC could be operated continuously and was found extremely suitable for high-throughput separation; however, post-column addition of a clarifying reagent is recommended to smooth the UV-signal during the extrusion process. Considering BECCC, the practical operation is very simple by just switching a 4-port valve to change the flow direction. The change of flowing direction should be done after a sufficient amount of mobile phase has flushed the column in the classical mode so that solutes with small and medium distribution constants have been eluted. Otherwise, a significant portion of the solutes will stay in the mobile phase inside the column, mix together and produce a broad peak showing in the mobile phase eluting after the stationary phase extrusion. Compared with classical CCC or other preparative separation tools, extrusion CCC approaches exhibit distinguished superiority in the modernization process of traditional Chinese medicines.

Preparative isolation and purification of two amides from Mallotus lianus Croiz by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was applied to the preparative isolation and purification of two amides from Mallotus lianus Croiz. In a single HSCCC separation, using the two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (5:1:5:1 v/v), 247.5 mg of the enriched crude sample was separated to afford 10.3 mg of N-isobutyl-2E,4E,12Z-octadecatrienamide and 15.7 mg of (7Z,10Z,18Z)tricosa-7,10,18-trienamide, a novel compound, with the purities of 98.0 and 94.6%, respectively. The HSCCC fractions were analyzed by HPLC and chemical structures of the compounds were identified by 1D- and 2D-NMR, ESI-, and GC-MS.

Ionic liquid-based microwave-assisted extraction of phenolic alkaloids from the medicinal plant Nelumbo nucifera Gaertn.

An ionic liquid-based microwave-assisted extraction (ILMAE) approach has been successfully applied to the effective extraction of the phenolic alkaloids present in samples of the medicinal plant Nelumbo nucifera Gaertn. The ionic liquids investigated comprised a range of four anions, four 1-alkyl-3-methylimidazolium derivatives differing in hydrophobic chain length. The results indicate that varying the anion has apparent effects on the overall extraction efficiency. In addition, the influence of some microwave parameters, such as irradiation power, extraction time and solid-liquid ratio, are also investigated. Under the optimized conditions, the proposed approach has been evaluated in comparison with the conventional heat-reflux extraction (HRE) and regular MAE. The reduction of the extraction times (from 2h to 90s) and remarkable higher efficiency (20-50% improved) supports the suitability of the proposed approach. In addition, the proposed method is validated by the recovery, correlation coefficient (R(2)), and reproducibility (RSD, n=5), which are in the range of 98-105%, 0.9994-0.9998, and 1.2-5.4%, respectively.

Rapid screening of bioactive components from Zingiber cassumunar using elution-extrusion counter-current chromatography.

Elution-extrusion counter-current chromatography (EECCC) takes full advantages of the liquid nature of the stationary phase. It effectively extends the solute hydrophobicity window that can be studied and renders the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, EECCC was used to screen the crude ethanol extract of Zingiber cassumunar and to isolate milligram-amounts of bioactive components. The two column volume (2V(C)) EECCC method was applied to rapidly optimize the composition of the biphasic liquid system in both reversed- and normal-phase separation mode. With the n-hexane/ethyl acetate/methanol/water 1/1/1/1 (v/v) system, 100mg of crude Z. cassumunar extract were fractionated on a 140 mL-capacity semi-preparative hydrodynamic CCC column and 0.5 g on a 1600 mL column for large-scale preparation. Satisfactory separation efficiency was achieved in both cases, producing milligram-amounts of four phenylbutenoids over 90% pure and of a mixture of diastereoisomers (phenylbutenoid dimers). However, the global throughputs of the two columns were 8 and 11 mg/h, not very different. This is due to the fact that the 1600 mL column could not retain the liquid stationary phase as well as the smaller 140 mL column. It was necessary to work at much lower flow rate than calculated. Methanol was added as a post-column clarifying reagent for stable continuous UV detection. A lipophilic biphasic liquid system composed of n-hexane/acetonitrile/water (5/3/2, v/v) allowed to resolve the pair of diastereoisomers with the larger preparative instrument producing 35 mg of the (+/-)-trans form 99.1% pure and 28 mg of the (+/-)-cis isomer 98.1% pure. Compared with classical elution, the EECCC approach exhibits strong separation efficiency and great potential to be a high-throughput separation technique in the case of complex samples.

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

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

Effective two-dimensional counter-current chromatographic method for simultaneous isolation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens.

A preparative two-dimensional counter-current chromatographic system (2D-CCC) for simultaneous separation and purification of oridonin and ponicidin from the crude extract of Rabdosia rubescens was presented. It was based on the use of a high-speed CCC (HSCCC, total column volume V(c)=146ml) instrument in the first dimension (1st-D) and a preparative upright CCC (UCCC, V(c)=1500ml) column in the second dimension (2nd-D). The interface was a homemade column-switching system with a 50-ml sample loop. In this way, almost the whole interested region from the first dimension was transferred on-line to the second dimension for further separation. The use of a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (1:5:1:5 and 3:5:3:5, v/v) in the two dimensions permitted the simultaneous separation of oridonin and ponicidin, making this 2D-CCC system with satisfactory resolution and peak capacity. During about 9h separation, two injections with about a 250mg amount of the crude extract for each, was accomplished using this 2D-CCC system, yielding 60mg of oridonin (1) and 10mg of ponicidin (2) all at a purity of over 95% as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization (ESI) MS, (1)H NMR and (13)C NMR.

A comparative study of upright counter-current chromatography and high-performance liquid chromatograpohy for preparative isolation and purification ofphenolic compounds from Magnoliae officinalis.

A comparative study of preparative isolation and purification of the phenolic compounds magnolol and honokiol from the Chinese medicinal plant Magnoliae officinalis by upright counter-current chromatography (CCC) and semi-preparative HPLC is presented. The comparison reveals that with a two-phase solvent system composed of light petroleum (bp 60-90 degrees C)-ethyl acetate-tetrachloromethane-methanol-water (1:1:8:6:1, v/v), 1250 mg of honokiol and 520 mg of magnolol, with a purity of 98.7 and 99.5%, respectively, were obtained from 2.0 g of a crude sample of Magnoliae officinalis in a single CCC separation. In contrast, semi-preparative HPLC allowed isolation and purification of these two phenolic compounds with significantly lower productivity and higher solvent consumption. Structures of the purified compounds were identified by 1H and 13C NMR.

Isolation and purification of oridonin from Rabdosia rubescens using upright counter-current chromatography.

A preparative counter-current chromatography (CCC) method for isolation and purification of oridonin, a new cancer chemoprevention agent, from the Chinese medicinal plant Rabdosia rubescens was successfully established. The crude oridonin was obtained by elution with a light petroleum/acetone solvent mixture from ethanol extracts of R. rubescens using column chromatography on silica gel. With a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (1:2:1:2, v/v), 120 mg of oridonin at the purity of 97.8% was obtained from 200 mg of the crude sample in a single-step CCC separation. The structure of oridonin was identified by ESI-MS, 1H NMR, and 13C NMR.

[Contrastive analysis of volatile oil from Serissa serissoides in different seasons].

OBJECTIVE: To provide the foundation for reasonable utilization by analysing the essential oils from Serissa serissoides in different seasons. METHOD: Essential oils were obtained by steam distillation. The chemical components were separated and identified by gas chromatography-mass spectrometer (GC-MS). The relative content of each component was determined by area normalization. RESULT: Forty-three peaks were identified from autumn material, representing 78.91% of the total oil. Main constituents of the essential oil from the autumn material were found to be 1b,5,5,6a-tetramethyl-octahydro-1-oxa-cyclopropa [a] inden-6-one (7.32%); methyl linolenate (4.14%); cubenol (5.97%); 2-methoxy-4-vinylphenol (10.87%); delta-9(10)-tetrahydrocostunolide-1-keto (35.51%). Seventy-two peaks were identified from spring material, representing 79.88% of the total oil. Main constituents of the essential oil from the spring material were found to be caryophyllene (3.315%); ethylbenzene (3.523%); 3-hexen-1-ol (4.537%); 2-methoxy-4-vinylphenol (6.513%); 5-propionyl-2-chlorobenzeneacetic acid, methyl ester (8.541%), germacrene D (12.311%). CONCLUSION: The same compounds in both materials are as follows: 2,2-dimethyl-6-methylene-cyclohexanepropanol; 2-methoxy-4-vinylphenol; 3,7-dimethyl-1,6-octadien-3-ol; cubenol; docosane and eicosane. It seems that they are the diagnostic components in these medicinal materials. Essential substances are different in quantity and quality in different seasons.