An on-line high performance liquid chromatography-crocin bleaching assay for detection of antioxidants
Bountagkidou, O. ; Klift, E.J.C. van der; Tsimidou, M.Z. ; Ordoudi, S.A. ; Beek, T.A. van - \ 2012
Journal of Chromatography. A, Including electrophoresis and other separation methods 1237 (2012). - ISSN 0021-9673 - p. 80 - 85.
radical scavenging compounds - chemiluminescence detection - natural antioxidants - complex-mixtures - identification - capacity - extracts - inhibition - plant
An on-line HPLC (high performance liquid chromatography) method for the rapid screening of individual antioxidants in mixtures was developed using crocin as a substrate (i.e. oxidation probe) and 2,2'-azobis(2-amidinopropane dihydrochloride (AAPH)) in phosphate buffer (pH 7.5) as a radical generator. The polyene structure of crocin and AAPH-derived peroxyl radicals resemble the lipidic substrates and radicals found in true food more closely than the popular, albeit artificial, DPPH (1,1-diphenyl-2-picrylhydrazyl) and ABTS+ (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)) do. After separation by a C18 (octadecyl silica) column and UV (ultraviolet) detection, antioxidative analytes react with peroxyl radicals at 90 °C and the inhibition of crocin oxidation (i.e. bleaching) is detected as a positive peak by an absorbance detector at 440 nm. The method is simple, uses standard instruments and inexpensive reagents. It can be applied for isocratic HPLC runs using mobile phases containing 10–90% organic solvent in water, weak acids or buffers (pH 3.5–8.5). With baseline correction, gradient runs are also feasible. The radical scavenging activity of several natural antioxidants and a green tea extract was studied. After optimisation of conditions such as reagent concentrations and flows, the limit of detection varied from 0.79 to 7.4 ng, depending on the antioxidant. --------------------------------------------------------------------------------
Isolation, identification and activity of natural antioxidants from costmary (Chrysanthemum balsamita) cultivated in Lithuania
Pukalskas, A. ; Venskutonis, P.R. ; Dijkgraaf, I. ; Beek, T.A. van - \ 2010
Food Chemistry 122 (2010)3. - ISSN 0308-8146 - p. 804 - 811.
radical scavenging compounds - chlorogenic acid isomers - liquid-chromatography - caffeic acid - online - plants - l. - derivatives - extracts - phenols
The sweet, minty-lemony leaves of costmary (Chrysanthemum balsamita) are used for salads and tea, and as flavourings in meats, sausages, cakes and ale. In this study, the extracts isolated from costmary aerial parts were investigated as antioxidants in rapeseed oil and as free radical-scavengers in DPPH and ABTS(+) assays. It was found that costmary extracts and their fractions were weak antioxidants in rapeseed oil; however, some fractions were active in scavenging synthetic free radicals. Crude methanol-water extract, its tertbutyl methyl ether and butanol fractions were the most effective in DPPH assay by scavenging 87.0%, 86.9% and 86.4% of radicals present in the reaction, respectively. Several active compounds were detected in these fractions, using HPLC with on-line radical-scavenging detection. After multi-step fractionation of these fractions, four radical-scavenging constituents were isolated and their properties were assessed by DPPH (antiradical power, ARP, calculated as an inverse value of the effective concentration, 1/EC50) and ABTS(+) (Trolox equivalent antioxidant capacity, TEAC(6min)) free radical-scavenging assays. The following structures were elucidated by NMR and MS: 5-O-caffeoylquinic acid (ARP = 3.85: TEAC(6min) = 0.60), 3,5-O-dicaffeoylquinic acid (ARP = 6.25: TEAC(6min) = 1.16), 5,7,4'-trihydroxy-3',8-dimethoxyflavone (ARP = 0.03) and 5,7,3',4'-tetrahydroxy-3,8-dimethoxyflavonol (ARP = 3.79; TEAC(6min) = 1.50).
Recent developments in the rapid analysis of plants and tracking their bioactive constituents
Beek, T.A. van; Tetala, K.K.R. ; Koleva, I. ; Dapkevicius, A. ; Exarchou, V. ; Jeurissen, S.M.F. ; Claassen, F.W. ; Klift, E.J.C. van der - \ 2009
Phytochemistry Reviews 8 (2009)2. - ISSN 1568-7767 - p. 387 - 399.
desorption electrospray-ionization - performance liquid-chromatography - 2-dimensional gas-chromatography - mass-spectrometric detection - radical scavenging compounds - nuclear-magnetic-resonance - thin-layer-chromatography - natural-products - biochemical detec
Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC, LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification. Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH¿ or ABTS¿+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced, non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore, new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 ¿m width with the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input.
Antioxidant activity assays on-line with liquid chromatography
Niederländer, H.A.G. ; Beek, T.A. van; Barsatute, A. ; Koleva, I. - \ 2008
Journal of Chromatography. A, Including electrophoresis and other separation methods 1210 (2008)2. - ISSN 0021-9673 - p. 121 - 134.
radical scavenging compounds - coulometric array detection - flow-injection system - dpph screening method - dad-spe-nmr - phenolic-compounds - electrochemical detection - natural-products - plant-extracts - chemiluminescence detection
Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPH and ABTS+ have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.
Hyphenated chromatographic techniques for the rapid screening and identification of antioxidants in methanolic extracts of pharmaceutically used plants .
Exarchou, V. ; Fiamegos, Y.C. ; Beek, T.A. van; Nanos, C.G. ; Vervoort, J.J.M. - \ 2006
Journal of Chromatography. A, Including electrophoresis and other separation methods 1112 (2006)1-2. - ISSN 0021-9673 - p. 293 - 302.
performance liquid-chromatography - nuclear-magnetic-resonance - solid-phase extraction - dad-spe-nmr - radical scavenging compounds - mass-spectrometry - hypericum-perforatum - online detection - lc-nmr - phenolic-compounds
Phytochemical analysis is an important scientific research area, which normally relies on a number of rather laborious and time-consuming techniques for compound identification. Isolation of the ingredients of plant extracts in adequate quantities for spectral and biological analysis was the basis of this research. In this paper the possibility of on-line rapid screening of antioxidant components in methanolic plant extracts and their subsequent identification is reported. Based exclusively on hyphenated chromatographic techniques the methanolic extracts of Tilia europea, Urtica dioica, Lonicera periclymenum and Hypericum perforatum are initially screened for their antioxidant components via an on-line DPPH and ABTS radical scavenging technique. Structural elucidation of the active analytes is achieved by means of LC-MS and LC-UV-SPE-NMR. After the determination of the appropriate LC gradient, a minimal number of chromatographic runs with these hyphenated techniques are adequate for the acquisition of the necessary data, leading to the identification of the targeted compounds. Based on their UV, NMR and MS spectra, the antioxidant compounds identified in the extracts under study were found to be either flavonoid glycosides or mono- and dicaffeoylquinic acids. Although the aim of the study was to show the great potential of the LC-UV-NMR-DPPH/ABTS approach for the rapid screening and identification of plant constituents, the results produced in the course of this study also have some merit by themselves. Some of the compounds detected are reported for the first time in the specific plant extracts