M. Helena Florêncio

Iron and copper chelation by flavonoids: an electrospray mass spectrometry study

Flavonoids are well known as effective free radical scavengers exhibiting therefore an antioxidant behaviour. Another antioxidant mechanism however may result from the ability they have to chelate metal ions, rendering them inactive to participate in free radical generating reactions. Electrospray mass spectrometry has been used to study metal ion interactions with a set of flavonoids from different classes. Complexes with a range of stoichiometries, of metal: flavonoid, 1:1, 1:2, 2:2, 2:3 have been observed. The stoichiometry 1:2 is in general the preferred one. It is established for flavones and for the flavanone naringenin that the binding metal sites are preferentially at the 5-hydroxyl and 4-oxo groups. Redox reactions are also observed through the change of the oxidation state of the metal, jointly with the oxidation of the flavonoid by loss of hydrogen. Structures of the oxidized species of some flavonoids are proposed.

Plant extracts with anti-inflammatory properties-A new approach for characterization of their bioactive compounds and establishment of structure-antioxidant activity relationships

Geranium robertianum L. (Geraniacea) and Uncaria tomentosa (Willd.) DC. (Rubiaceae) plant extracts, frequently used in traditional medicine for treatment of inflammatory and cancer diseases, were studied to identify potential bioactive compounds that may justify their therapeutic use and their underlying mechanisms of action. Since some of the pharmacological properties of these plant extracts may be linked to their antioxidant potential, the antioxidant activity, in relation to free radical scavenging, was measured by the ABTS/HRP and DPPH() assays, presenting U. tomentosa the higher activity. The antioxidant activity was also evaluated by scavenging of HOCl, the major strong oxidant produced by neutrophils and a potent pro-inflammatory agent. U. tomentosa was found to be a better protector against HOCl, which may justify its effectiveness against inflammatory diseases. SPE/LC-DAD was used for separation/purification purposes and ESI-MS/MS for identification/characterization of the major non-volatile components, mainly flavonoids and phenolic acids. The ESI-MS/MS methodology proposed can be used as a model procedure for identification/characterization of unknowns without the prerequisite for standard compounds analysis. The ESI-MS/MS data obtained were consistent with the antioxidant activity results and structure-activity relationships for the compounds identified were discussed.

Electrospray ionization tandem mass spectrometry fragmentation of protonated flavone and flavonol aglycones: a re‐examination

Flavonoids are important phytochemicals which have been intensively studied in the last decades in view of their antioxidant activity, which is of particular importance in the case of flavones and flavonols, that differ in a single 3-OH group. Mass spectrometry has been used to elucidate the structures of many types of flavonoids and their metabolites. The work we present here is focused on the electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of flavone and flavonols aglycones. Their fragmentation mechanisms in the positive ion mode are described and compared with previously reported mechanisms. We analyzed flavonoid derivatives produced by reaction of the flavonoids with chemically synthesized hypohalous acids (HOCl, HOBr and HOI) and peroxynitrite, reactive species involved in the inflammatory response. All the proposed pathways have been analyzed using computational chemistry methods in order to seek for possible variations and establish the most plausible ones. We observed that the losses of one and two CO molecules can be useful in terms of antioxidant activity prediction. Losses of one and two C(2)H(2)O groups are also informative in terms of structure and activity predictions. The retro-Diels-Alder fragmentations, and subsequent neutral losses, were reviewed and, according to our calculations, the most plausible structures for the product ions were established. These fingerprints will be of great value for differentiating flavonoids from other compounds in complex biological mixtures and for a thorough structural identification of flavonoid aglycones and their in vivo metabolites.

Titanium dioxide anatase as matrix for matrix‐assisted laser desorption/ionization analysis of small molecules

The use of inorganic species as assisting materials in matrix-assisted laser desorption/ionization (MALDI) analysis is an alternative approach to avoid interfering matrix ions in the low-mass region of the mass spectra. Reports of the application of inorganic species as matrices in MALDI analysis of small molecules are, however, scarce. Nevertheless, titanium dioxide (TiO(2)) powder has been reported to be a promising matrix medium. In this study we further explore the use of TiO(2) as a matrix for the MALDI analysis of low molecular weight compounds. We present results showing that nanosized TiO(2) anatase and TiO(2) rutile perform better as MALDI matrices than a commercial TiO(2) anatase/rutile mixture. Moreover, when using nanosized TiO(2) anatase as a matrix, high-quality mass spectra can be obtained with strong analyte signals and weak or non-existing matrix interference ions. Furthermore, our results show that the phase type plays an important role in the application of TiO(2) as a MALDI matrix.

Antioxidants in medicinal plant extracts. A research study of the antioxidant capacity of Crataegus, Hamamelis and Hydrastis.

The antioxidant capacity of extracts of Crataegus oxyacantha, Hamamelis virginiana, Hydrastis canadensis, plants native to Europe and North America which have long been used in herbal medicine for the treatment of cardiac and circulatory functions, has been investigated. The total antioxidant potential conferred by all hydrogen donating antioxidants present in these extracts has been assessed by the ABTS assay and the relative order of antioxidant potential has been established. Gas chromatography coupled to mass spectrometry (GC‐MS) has been used for the chemical identification of the antioxidant volatile compounds present in the extracts. The GC‐MS data were related to the results obtained using the ABTS assay. Copyright

Structure and antioxidant activity of brominated flavonols and flavanones

Hypobromous acid (HOBr) produced by both eosinophil peroxidase (EPO) and myeloperoxidase (MPO) is a stronger oxidant than HOCl, and is also essential for optimal and efficient microbial killing. Considering the potential cytotoxic effect of HOBr, if it is formed outside the phagosome, it should be useful to scavenge it in order to protect the nearby tissues. In this study the ability of selected flavonoids to protect against HOBr mediated oxidation reactions was performed through a competitive reaction, and the resulting products identified by high performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry(ESI-MS/MS). Several structural features were found to be important to confer high antioxidant activity to flavonoids towards HOBr: the C2=C3 double bond and the 3-OH group in the C-ring, and the presence of both 5-OH and 7-OH groups in the A-ring. The MS results showed that flavonoids are dibrominated in the A-ring, suggesting that (except for fisetin) bromination occurs at C6 and C8 positions, through an electrophilic aromatic substitution reaction. The chemical modifications achieved by bromination of flavonoids have changed their biological properties, presenting their brominated derivatives higher antioxidant activity, as radical scavengers, and higher lipophilicity, than the parent flavonoids. Brominated flavonoids may then diffuse easily through membranes increasing the intracellular concentration of the compounds. These locally formed metabolites may also interact with signaling cascades involving cytokines and regulatory transcription factors, thus playing a role in inflammation and in the regulation of immune response.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric and semi‐empirical calculations study of five isoflavone aglycones

Five isoflavones, daidzein, genistein, formononetin, prunetin and biochanin A, known for their biological properties, are investigated by electrospray ionization mass spectrometry in the positive ion mode. The most probable protonation sites are determined taking into account semi-empirical calculations using the PM6 Hamiltonian. Fragmentation mechanisms are proposed based on accurate mass measurements, MS(3) experiments and supported by the semi-empirical calculations. Some of the fragmentation pathways were found to be dependent on the substitution pattern of the B-ring and the ions afforded by these fragmentations can be considered as diagnostic. It was possible to distinguish between prunetin and biochanin A, two isobaric isoflavone aglycones included in this study. Furthermore, a comparison of the fragmentation patterns of genistein and biochanin A, two isoflavones, with those of their flavone counterparts, apigenin and acacetin, enabled us to identify some key ions mainly due to structural features, allowing distinction to be made between these two classes of compounds.

Flavonoid–matrix cluster ions in MALDI mass spectrometry

The behaviour of 2,5-dihydroxybenzoic acid (2,5-DHB) matrix under matrix-assisted laser desorption/ionisation (MALDI) conditions was investigated, and the formation of 2,5-DHB cluster ions, mainly dehydrated 2,5-DHB ions, is reported. Interestingly, in the mass spectra of this compound, besides dimers and trimers, protonated tetramers, pentamers, hexamers and heptamers were also found with significant abundance.The MALDI behaviour of four flavonoids, quercetin, myricetin, luteolin and kaempferol, using 2,5-DHB as matrix, was also investigated. The mass spectra of the flavonoids studied revealed a number of flavonoid-2,5-DHB cluster ions (mainly with the dehydrated 2,5-DHB). The number of clusters formed is dependent on the structure of the analyte. For luteolin and kaempferol, in particular, evidence was found for the formation of cluster ions involving retro Diels Alder fragments and intact flavonoids molecules, as well as the corresponding protonated retro Diels Alder fragments with dehydrated DHB molecules. All ion compositions were attributed taking into account high accuracy mass measurements and tandem mass spectrometry experiments.

ELECTROSPRAY MASS SPECTROMETRY OF ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

Electrospray ionisation (ESI) mass spectrometry combined with collision-induced decomposition of ions given by captopril, lisinopril and enalaprilat, angiotensin-converting enzyme (ACE) inhibitors, has been used to characterise these compounds. As expected, the ESI mass spectra are characterised by the presence of abundant [M + H]+ ions with very little fragmentation at low cone voltages. Fragment ions are produced by collision-induced decomposition of these ions at higher cone voltages, and in MS/MS experiments. Fragmentation pathways and structures of fragment ions observed have been proposed. Copyright

Electrospray ionization mass spectrometric analysis of newly synthesized α,β-unsaturated γ-lactones fused to sugars

Knowledge of the fragmentation mechanisms of lactones and their behaviour under electrospray ionization (ESI) conditions can be extended to larger and more complex natural products that contain an alpha,beta-unsaturated gamma-lactone moiety in their structure. Moreover, little is known about the gas-phase behaviour of alpha,beta-unsaturated gamma-lactones linked or fused to sugars. Therefore, five alpha,beta-unsaturated gamma-lactones (butenolides) fused to a pyranose ring, recently synthesized compounds with potential relevance regarding their biological properties, were investigated using ESI-MS and ESI-MS/MS in both positive and negative ion modes. Their fragmentation mechanisms and product ion structures were compared. It was observed that two isomers could be unambiguously distinguished in the negative ion mode by the fragmentation pathways of their deprotonated molecules as well as in the positive ion mode by the fragmentation pathways of either the protonated or the sodiated molecule. Fragmentation mechanisms are proposed taking into account the MS/MS data and semi-empirical calculations using the PM6 Hamiltonean. The semi-empirical calculations were also very useful in determining the most probable protonation and cationization sites.