Monolithic column functionalized with quinine derivative for anion-exchange capillary electrochromatography.

A novel anion-exchange organic polymer monolithic column based on monomers N-benzylquininium chloride and acrylamide were firstly prepared by in situ copolymerization for capillary electrochromatography. Moreover, N-benzylquininium was firstly introduced as a strong anion-exchange functional group. A relatively strong anodic EOF was obtained in the pH values from 4.0 to 9.0, which was in the same direction with the electrophoretic mobility of acid compounds. Hence, the anion-exchange monolithic column was very suitable for the rapid separation of acid compounds. Eight acid compounds (2-chlorobenzoic acid, mandelic acid, 4-hydroxybenzoic acid, indole-3-acetic acid, 2-aminoterephthalic acid, 3,5-pyridinedicarboxylic acid, benzoic acid, and 4-aminobenzoic acid) were successfully separated on the monolithic column. The highest column efficiency was 4.60 × 105 plates/m (theoretical plates, N) for 3,5-pyridinedicarboxylic acid. The proposed monolithic column was characterized by SEM and FT-IR. The RSDs of the acid compounds migration time for run-to-run, day-to-day, and column-to-column were all less than 5.0%.

A general separation method of phenolic acids using pH-zone-refining counter-current chromatography and its application to oat bran.

pH-zone-refining counter-current chromatography technique for the separation of natural and synthetic mixtures has been widely used, especially for organic acids and alkaloids. Phenolic acids are very important compounds due to the potential treatment for a wide variety of diseases. However, there is not a general method for their separation. In this work, the conditions of pH-zone-refining counter-current chromatography, involving solvent systems, concentration of retainer and eluter, flow rate of mobile phase as well as sample pretreatment, were optimized to improve extraction efficiency and reduce separation time. Finally a general separation method for seven common phenolic acids has been established using pH-zone-refining counter-current chromatography. The separation of these phenolic acids was performed with a two-phase solvent system composed of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4.75: 0.25: 5, where 3mM trifluoroacetic acid was added to the organic stationary phase as a retainer and 3mM NH4OH was added to the aqueous mobile phase as an eluter. As a result, seven phenolic acids, including syringic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were successfully separated with the purities of 95.9%, 67.3%, 96.9%, 82.4%, 97.0%, 91.0%, and 97.2%, respectively. The established general method has been applied to the crude sample of oat bran pretreated with AB-8 resin. A total of 49.5mg of syringic acid, 109.2mg of p-coumaric acid and 184.5mg of ferulic acid were successfully purified in one run from 1.22g crude extract with the purities of 95.2%, 93.0%, and 91.8%, respectively.

Conservation strategy for Pelargonium sidoides DC: phenolic profile and pharmacological activity of acclimatized plants derived from tissue culture.

ETHNOPHARMACOLOGICAL RELEVANCE: Pelargonium sidoides DC (Geraniaceae), a popular medicinal plant used in folk medicine in the treatment of respiratory-related infections has gained international prominence due to its usage in several herbal formulations. This has led to high demand and the subsequent decimation of wild populations. AIM OF THE STUDY: Using plant tissue culture techniques, Pelargonium sidoides plants were cloned in vitro, acclimatized under greenhouse conditions and evaluated for their phytochemical content and pharmacological activity. METHODS: Phenolic content in extracts of in vitro-derived, greenhouse-acclimatized and wild Pelargonium sidoides plants were analyzed using UPLC-MS/MS. The oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging activity and minimum inhibitory concentration (MIC) of the extracts against bacterial and fungal strains were evaluated. RESULTS: Similarities in phenolic profiles were identified confirming the chemical signatures that characterize Pelargonium sidoides plants. Extracts of greenhouse-acclimatized and wild plants exhibited comparable antimicrobial and antioxidant properties. CONCLUSIONS: Overall, the study highlights the potential of integrating plant tissue culture technologies in conservation strategies of medicinal plants. In particular, the results strongly suggest the feasibility of both large-scale cultivation and plant part substitution as alternative solutions to the current destructive overharvesting practices of wild Pelargonium sidoides populations.

Enantioseparation of chiral aromatic acids by multiple dual mode counter-current chromatography using hydroxypropyl-ß-cyclodextrin as chiral selector.

This work concentrates on extending the utilization of multiple dual mode (MDM) counter-current chromatography in chiral separations. Two aromatic acids, 2-(6-methoxy-2-naphthyl)propionic acid (NAP) and 2-phenylpropionic acid (2-PPA), were enantioseparated by MDM counter-current chromatography using hydroxypropyl-ß-cyclodextrin (HP-ß-CD) as chiral selector. The two-phase solvent systems consisting of n-hexane/ethyl acetate 0.1 mol/L phosphate buffer pH 2.67 containing 0.1 mol/L HP-ß-CD (7.5:2.5:10 for NAP and 7:3:10 for 2-PPA, v/v/v) were used. Conventional MDM and modified MDM were compared according to peak resolution under current separation mechanism. The influence of elution time after the first-phase inversion and number of cycles for MDM were investigated. Peak resolution of NAP and 2-PPA increased from 0.62 to 1.05 and 0.72 to 0.84, respectively, using optimized MDM conditions. Being an alternative elution method for counter-current chromatography, MDM elution greatly improved peak resolution in chiral separations.

Theoretical and experimental investigations on migration behavior of weak monoprotic acids in continuous two-dimensional ion exchange/reversed phase CEC.

A continuous 2D ion exchange/RP CEC system was constructed in this report and retention characteristics of weak monoprotic acids in the column were investigated theoretically. The transport equations were deduced for predicting migration behavior of weak monoportic acids based on mixing model combining ion exchange, RP, and electrophoretic separation mechanism. The influences of separation voltage, length of capillary, pH value, and ionic strength of buffer and concentration of organic modifier in mobile phase on the separation were well described by the equations. It was verified that the migration rate of solute was linearly related to voltage in the case of lower voltage, decreased with the increase of the first-dimensional length, and [H+]. The retention time of solute increased with enhancing ionic strength and concentration of organic modifier. These theoretical results were all demonstrated by the relevant experiments. The relevant application was performed for the aqueous extract of Rhizoma gastrodiae, showing the obvious potential in method development for the analysis of complex samples.

In-vial liquid-liquid microextraction-capillary electrophoresis method for the determination of phenolic acids in vegetable oils.

An in-vial liquid-liquid microextraction method was developed for the selective extraction of the phenolic acids (caffeic, gallic, cinnamic, ferulic, chlorogenic, syringic, vanillic, benzoic, p-hydroxybenzoic, 2,4-dihydroxybenzoic, o-coumaric, m-coumaric and p-coumaric) in vegetable oil samples. The optimised extraction conditions for 20 g sample were: volume of diluent (n-hexane), 2 mL; extractant, methanol: 5 mM sodium hydroxide (60:40; v/v); volume of extractant, 300 µL (twice); vortex, 1 min; centrifugation, 5 min. Recoveries for the studied phenolic acids were 80.1-119.5%. The simultaneous determination of the phenolic acid extracts was investigated by capillary electrophoresis (CE). Separations were carried out on a bare fused-silica capillary (50 µm i.d.× 40 cm length) involving 25 mM sodium tetraborate (pH 9.15) and 5% methanol as CE background electrolyte in the normal polarity mode, voltage of 30 kV, temperature of 25°C, injection time of 4s (50 mbar) and electropherograms were recorded at 200 nm. The phenolic acids were successfully separated in less than 10 min. The validated in-vial LLME-CE method was applied to the determination of phenolic acids in vegetable oil samples (extra virgin olive oil, virgin olive oil, pure olive oil, walnut oil and grapeseed oil). The developed method shows significant advantages over the current methods as lengthy evaporation step is not required.

Cationic cyclodextrin as versatile chiral selector for enantiomeric separation in capillary electrophoresis.

A novel cationic cyclodextrin, mono-6(A)-(3-methoxypropan-1-ammonium)-6(A)-ß-cyclodextrin chloride, has been developed and used as chiral selector in capillary electrophoresis (CE). This chiral selector has three recognition sites: ß-CD, ammonium cation and methoxy group in the sidearm to contribute three corresponding driving forces including inclusion complexation, electrostatic interaction and hydrogen bonding. This elegantly designed CD exhibits outstanding enantioselectivities in CE for a wide range of acidic and ampholytic racemates due to the extra hydrogen bonding for the chiral recognition. Under optimum pH 6.0, chiral resolutions over 10 can be readily achieved for acidic racemates with CD concentration below 10 mM. This cationic CD exhibits great potential for versatile chiral separation in CE. The finding in this study may be applied to construct new host-guest systems for practical applications.

Cytotoxic phenylpropanoid glycosides from Fagopyrum tataricum (L.) Gaertn.

Fagopyrum tataricum (L.) Gaertn (tartary buckwheat) is an ancient dicotyledonous crop belonging to Polygonaceae family. Besides its benefits for human consumption, tartary buckwheat is also an important folk medicine in China for its antioxidant, antitumour, hypotensive, hypoglycaemic and hypolipidaemic activities. Phytochemical investigation of the ethyl acetate fraction of tartary buckwheat roots led to the isolation of seven new phenylpropanoid glycosides, tatarisides A-G (1-7), together with a known phenylpropanoid glycoside, diboside A (8). Their structures were elucidated by means of spectroscopic methods. All compounds (1-8) were evaluated for their cytotoxic activity against four human cancer cell lines (A-549, HCT116, ZR-75-30 and HL-60). Tatariside C (3) was the most active compound with IC50 values of 6.44-7.49µg/ml against the four tested cell lines.

Plant growth inhibitors isolated from sugarcane (Saccharum officinarum) straw.

Several compounds related with plant defense and pharmacological activities have been isolated from sugarcane. Straw phytotoxins and their possible mechanisms of growth inhibition are largely unknown. A bioassay-guided fractionation of the phytotoxic constituents leachated from a sugarcane straw led to the isolation of trans-ferulic (trans-FA), cis-ferulic (cis-FA), vanillic (VA) and syringic (SA) acids. The straw leachates and their identified constituents significantly inhibited root growth of lettuce and four weeds. VA was more phytotoxic to root elongation than FA and SA. The identified phenolic compounds significantly increased leakage of root cell constituents, inhibited dehydrogenase activity and reduced chlorophyll content in lettuce. VA and FA inhibited mitotic index while SA increased cell division. Additive (VA-FA and FA-SA) and synergistic (VA-SA) interactions on root growth were observed at the response level of EC(25). Although the isolated compounds differed in their relative phytotoxic activities, the observed physiological responses suggest that they have a common mode of action. HPLC analysis indicated that sugarcane straw can potentially release 1.43 (ratio 2:1, trans:cis), 1.14 and 0.14mmolkg(-1) (straw dry weight) of FA, VA and SA, respectively. As phenolic acids are often found spatially concentrated in the top soil layers under plant straws, further studies are needed to establish the impact of these compounds in natural settings.

Capillary zone electrophoretic chiral discrimination using 6-O-(2-hydroxy-3-trimethylammoniopropyl)-beta-cyclodextrin as a chiral selector.

A charged highly water-soluble CD derivative, 6-O-(2-hydroxy-3-trimethylammoniopropyl)-beta-CD (herein noted as 6-HPTMA-beta-CD) was synthesized and successfully used as a chiral selector for enantiomeric separation of some acidic compounds by CZE in an uncoated capillary. Substitution with 2-hydroxy-3-trimethylammoniopropyl groups at the primary hydroxyl group of the CD was aimed at influencing the magnitude and selectivity of analyte-CD interactions. The behavior of 6-HPTMA-beta-CD was compared with that of the commercially available quaternary ammonium-beta-CD (QA-beta-CD) under the same separating conditions. The experiments were carried out using a BGE consisting of 50 mM phosphate in the pH range of 4-6 by adding a relatively low concentration of chiral selector (less than 10 mM). The effects of the concentration of CD and the pH of the electrolyte on the resolution of these compounds were studied.

A sulfonated capillary that gives reproducible migration times for capillary zone electrophoresis and micellar electrokinetic chromatography.

To obtain reproducible migration times and rapid analyses of analytes, sulfonate groups were chemically introduced to the inner wall of untreated fused-silica capillary with 2-(4-chlorosulfonylphenyl)ethyltrichlorosilane. The sulfonated capillary showed relatively constant electroosmotic mobility which was greater than that obtained by an untreated fused-silica capillary over the pH range studied (pH 2-9). In both CZE and MEKC, the RSDs of the migration times of analytes with the sulfonated capillary were less than 0.2% which were significantly lower than those obtained with an untreated fused-silica capillary (0.5-3.5%). When BGE were set at pH 7.0 for CZE and MEKC, the analysis times with the sulfonated capillary were about half those obtained with an untreated fused-silica capillary. These results indicate that the sulfonated capillary can provide highly reproducible and rapid analyses in CE.

Separation, characterization, and quantitation of benzoic and phenolic antioxidants in American cranberry fruit by GC-MS.

A GC-MS method is reported for separation and characterization of widely different amounts of benzoic and phenolic acids as their trimethylsilyl derivatives simultaneously in cranberry. Fifteen benzoic and phenolic acids (benzoic, o-hydroxybenzoic, cinnamic, m-hydroxybenzoic, p-hydroxybenzoic, p-hydroxyphenyl acetic, phthalic, 2,3-dihydroxybenzoic, vanillic, o-hydroxycinnamic, 2,4-dihydroxybenzoic, p-coumaric, ferulic, caffeic, and sinapic acid) were identified in cranberry fruit in their free and bound forms on the basis of GC retention times and simultaneously recorded mass spectra. Except for benzoic, p-coumaric, caffeic, ferulic, and sinapic acids, 10 other phenolic acids identified have not been reported in cranberry before. The quantitation of the identified components was based on total ion current (TIC). The experimental results indicated cranberry fruit contains a high content of benzoic and phenolic acids (5.7 g/kg fresh weight) with benzoic acid being the most abundant (4.7 g/kg fresh weight). The next most abundant are p-coumaric (0.25 g/kg fresh weight) and sinapic (0.21 g/kg fresh weight) acid. Benzoic and phenolic acids occur mainly in bound forms and only about 10% occurs as free acid.

Isolation and characterization of novel benzoates, cinnamates, flavonoids, and lignans from Riesling wine and screening for antioxidant activity.

A German Riesling wine has been fractionated with the aid of countercurrent chromatography. After purification by HPLC, the structures of 101 compounds were established by mass spectrometry and NMR spectroscopy. Seventy-three of the isolated compounds exhibited a phenolic or benzylic structure. Fifty-four compounds were reported for the first time as Riesling wine constituents. New compounds identified in this work included twelve benzoic and cinnamic acid derivatives. In addition to two isomeric (E)-caffeoyl ethyl tartrates, the glucose esters of (E)-cinnamic, (E)-p-coumaric, and (E)-ferulic acid, as well as the 4-O-glucosides of (E)- and (Z)-ferulic acid, have been identified for the first time in Riesling wine. The structures of two additional phenylpropanoids were elucidated as 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one and 2,3-dihydroxy-1-(4-hydroxy-3-methoxyphenyl)-propan-1-one. Moreover, two ethyl esters, i.e., ethyl protocatechuate and ethyl gallate, as well as the glucose ester of vanillic acid, were newly detected in Riesling wine. Novel representatives in the flavonoid group were dihydrokaempferol, dihydroquercetin, and four dihydroflavonol glycoconjugates, i.e., the 3-O-glucosides of dihydrokaempferol and dihydroquercetin, as well as the 3-O-xyloside and the 3'-O-glucoside of dihydroquercetin. Additionally, six novel lignans, i.e., lariciresinol 4-O-glucoside, three isolariciresinol derivatives, and two secoisolariciresinols, as well as three neolignans were isolated. Structural elucidation of the newly isolated wine constituents is reported together with the determination of their antioxidant activity.