Giangiacomo Beretta

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (beta-alanyl-L-histidine) acts as a quencher of cytotoxic alpha,beta-unsaturated aldehydes, using 4-hydroxy-trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than L-histidine and N-acetyl-carnosine while beta-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that the beta-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible alpha,beta-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of alpha,beta-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.

Non-covalent inclusion of ferulic acid with α-cyclodextrin improves photo-stability and delivery: NMR and modeling studies

Ferulic acid (FA) is a highly effective antioxidant and photo-protective agent, already approved in Japan as a sunscreen, but it is poorly suited for cosmetic application because of its low physicochemical stability. We prepared the inclusion complex of FA with alpha-cyclodextrin by co-precipitation from an aqueous solution, and used (1)H NMR and molecular dynamics to investigate the most probable structure of the inclusion complex. In rotating frame nuclear Overhouser effect spectroscopy (ROESY) experiments FA penetrated the alpha-CD hydrophobic cavity with the alpha,beta-unsaturated part of the molecule and some of its aromatic skeleton. In proton chemical shift measurements of FA and alpha-cyclodextrins we determined the stoichiometry of the association complex (1:1) by Jobs method, and its stability constant (K(1:1) 1162+/-140 M(-1)) and described the molecular dynamics of the complex on the basis of theoretical studies. Encapsulation with alpha-cyclodextrin improves (i) the chemical stability of FA against UVB stress (10 MED [Minimal Erythemal Dose: 1 MED=25 mJ/cm(2) for skin phototype II: 30]), since no degradation products are formed after irradiation, and (ii) the bioavailability of FA on the skin, slowing its delivery (Strainer cell model).

A solid-phase extraction procedure coupled to 1H NMR, with chemometric analysis, to seek reliable markers of the botanical origin of honey

The aim of this work was to establish an analytical method for identifying the botanical origin of honey, as an alternative to conventional melissopalynological, organoleptic and instrumental methods (gas-chromatography coupled to mass spectrometry (GC-MS), high-performance liquid chromatography HPLC). The procedure is based on the (1)H nuclear magnetic resonance (NMR) profile coupled, when necessary, with electrospray ionisation-mass spectrometry (ESI-MS) and two-dimensional NMR analyses of solid-phase extraction (SPE)-purified honey samples, followed by chemometric analyses. Extracts of 44 commercial Italian honeys from 20 different botanical sources were analyzed. Honeydew, chestnut and linden honeys showed constant, specific, well-resolved resonances, suitable for use as markers of origin. Honeydew honey contained the typical resonances of an aliphatic component, very likely deriving from the plant phloem sap or excreted into it by sap-sucking aphids. Chestnut honey contained the typical signals of kynurenic acid and some structurally related metabolite. In linden honey the (1)H NMR profile gave strong signals attributable to the mono-terpene derivative cyclohexa-1,3-diene-1-carboxylic acid (CDCA) and to its 1-O-beta-gentiobiosyl ester (CDCA-GBE). These markers were not detectable in the other honeys, except for the less common nectar honey from rosa mosqueta. We compared and analyzed the data by multivariate techniques. Principal component analysis found different clusters of honeys based on the presence of these specific markers. The results, although obviously only preliminary, suggest that the (1)H NMR profile (with HPLC-MS analysis when necessary) can be used as a reference framework for identifying the botanical origin of honey.

Parthenolide Inhibits the LPS‐induced Secretion of IL‐6 and TNF‐α and NF‐κB Nuclear Translocation in BV‐2 Microglia

Feverfew (Tanacetum parthenium [L.] Sch. Bip. [Asteraceae]) is a popular herbal treatment used to prevent and treat headache and migraine. Parthenolide (PTN), the sesquiterpene lactonic derivative that is the plants major component, might be one of the ingredients that act on mediators of inflammation. In the present study, in cultured lipopolysaccharide (LPS)‐stimulated BV‐2 microglia pretreatment with PTN caused a dose‐dependent reduction of interleukin‐6 (IL‐6) secretion (29% by 200 nm, p < 0.001; 45% by 1 µm, p < 0.001; 98% by 5 µm, p < 0.001); at 5 µm, the highest concentration tested, it also reduced the secretion of TNF‐α (54%, p < 0.001). Western blotting analysis on separate cytoplasmic and nuclear extracts showed that PTN strongly reduced the translocation of nuclear factor (NF)‐κB to the cell nucleus. The reduction of microglial activation by inhibition of proinflammatory agents may help attenuate the onset and intensity of acute migraine attacks. These in vitro results provide an additional explanation for the efficacy of orally administered T. parthenium as an antimigraine agent. Copyright

Quinoline alkaloids in honey: Further analytical (HPLC-DAD-ESI-MS, multidimensional diffusion-ordered NMR spectroscopy), theoretical and chemometric studies

The wound-healing properties of honey are well established and it has been suggested that, among its pharmaco-active constituents, kynurenic acid (KA) exerts antinociceptive action on injured tissue by antagonizing NMDA at peripheral GABA receptors. The aim of this study was to investigate the quantitative profile of KA and of two recently identified, structurally related derivatives, 3-pyrrolidinyl-kynurenic acid (3-PKA) and its gamma-lactamic derivative (gamma-LACT-3-PKA), by examining their mass spectrometric behavior, in honeys from different botanical sources. We used a combination of HPLC-DAD-ESI-MS and NMR techniques (one-dimensional (1)H NMR and diffusion-ordered spectroscopy NMR). Chestnut honey constantly contained KA (2114.9-23 g/kg), 3-PKA (482.8-80 mg/kg) and gamma-LACT-3-PKA (845.8-32 mg/kg), confirming their reliability as markers of origin. A new metabolite, 4-quinolone (4-QUIN), was identified for the first time in one chestnut honey sample (743.4 mg/kg). Small amounts of KA were found in honeydew, sunflower, multifloral, almond and eucalyptus honeys, in the range of 23.1-143 mg/kg, suggesting contamination with chestnut honey. Total phenol content (TPC) was in the range from 194.9 to 1636.3 mg(GAE)/kg and total antiradical activity (TAA) from 61 to 940 mg/(GAE)/kg), depending on the botanical origin. Principal component analysis (PCA) was then done on these data. The three different clusters depicted: (i) antinociceptive activity from KA and/or its derivatives, typical of chestnut honey; (ii) antioxidant/radical scavenging activity by antioxidants responsible for the antiinflammatory action (dark honeys); (iii) peroxide-dependent antibacterial activity due to H(2)O(2) production by glucose oxidase in honey. The PCA findings provide useful indications for the dermatologist for the treatment of topical diseases, and the profiling of KA and its derivatives may shed light on new aspects of the kynurenine pathway involved in tryptophan metabolism.

GC–MS profiling of the phytochemical constituents of the oleoresin from Copaifera langsdorffii Desf. and a preliminary in vivo evaluation of its antipsoriatic effect

Copaiba is the oleoresin (OR) obtained from Copaifera (Fabaceae), a neotropical tree which grows in Amazon regions. The balsam, constituted by an essential oil and a resinous fraction is used as folkloristic remedy in the treatment of several inflammatory diseases and for its antioxidant and antibacterial properties. Aim of this work was (a) to carry out a characterization by GC-MS of the volatile and nonvolatile constituents of Copaifera langsdorffii Desf. oleoresin (OR); (b) to investigate the mechanism of its anti-inflammatory activity; (c) to evaluate its antipsoriatic effect after oral intake/topical application. The volatile fraction (yield: 22.51%, w/w) shows: α-bergamotene (48.38%), α-himachalene (11.17%), β-selinene (5.00%) and β-caryophyllene (5.47%). The OR residue (77.49%, w/w), after derivatization, showed as main constituents the following compounds: copalic, abietic, daniellic, lambertinic, labd-7-en-15-oic, pimaric, isopimaric acids and kaur16-en18-oic acid. Preincubation of LPS-stimulated human THP-1 monocytes with increasing concentrations of the OR purified fraction (OR-PF), containing diterpene acids, diterpenes and sesquiterpenes, reduced the release of pro-inflammatory cytokines (IL-1β, IL-6, TNFα) in a dose-range of 0.1-10 μM. In addition, in cell culture system of human THP-1 monocytes, 1 μM OR-PF counteracts LPS-driven NF-κB nuclear translocation. In a preliminary clinical trial three patients affected by chronic psoriasis, treated with oral intake or topical application of the OR, exhibited a significant improvement of the typical signs of this disease, i.e. erythema, skin thickness, and scaliness. In conclusion, the results of this work, beside an extensive analytical characterization of the OR chemical composition, provide strong evidences that its anti-inflammatory activity is related to the inhibition of the NF-κB nuclear translocation, and consequently of proinflammatory cytokines secretion.

Anti-Ischemic Activity and Endothelium-Dependent Vasorelaxant Effect of Hydrolysable Tannins from the Leaves of Rhus coriaria (Sumac) in Isolated Rabbit Heart and Thoracic Aorta

The aim of this work was to investigate the cardioprotective activity of hydrolysable gallotannins from Rhus coriaria L. leaves extract (RCLE) in isolated rabbit heart preparations, submitted to low-flow ischemia/reperfusion damage. RCLE induces a dose-dependent normalization of coronary perfusion pressure (CPP), reducing left ventricular contracture during ischemia, and improving left ventricular developed pressure and the maximum rate of rise and fall of left ventricular pressure at reperfusion. Creatinine kinase (CK) and lactate dehydrogenase (LDH) outflow were significantly reduced during reperfusion. In parallel there was a rise in the release of the cytoprotective 6-ketoprostaglandin F (1alpha) (6-keto-PGF (1alpha)) and a decrease of tumor necrosis factor-alpha (TNF-alpha), both significant only at the highest RCLE concentrations (150-500 microg/mL). The vasorelaxant activity of RCLE was studied in isolated rabbit aorta rings precontracted with norepinephrine (NE) with and without endothelium. The vasorelaxation induced by RCLE was predominantly endothelium-dependent as demonstrated by the loss of RCLE vasorelaxant ability in i) de-endothelized rings and ii) in intact aortic rings after pretreatment with NG-monomethyl- L-arginine (L-NMMA) and 1 H-[1.2.4]oxadiazolo[4.3- A]quinoxalin-1-one (ODQ). The inhibition of vasorelaxation in intact rings by indomethacin (INDO) demonstrates the ability of RCLE to modulate the coronary endothelium cyclooxygenase (COX) pathway. The K-ATP channel antagonist glibenclamide (GLIB) was ineffective. The antioxidant activity of RCLE, investigated in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) model and in living cell systems (rat erythrocytes), was stronger than that of gallic acid, ascorbic acid and trolox. The structure of its main bioactive constituents, profiled by HPLC-ESI-HR-S, comprised a mixture of polygalloylated D-glucopyranose with different degrees of galloylation and 3- O-methylgallic acid. The cardiovascular protective effect of RCLE seems to be due to an interplay of different factors: COX pathway activation, TNF-alpha inhibition, endothelial nitric oxide synthase (eNOS) activation, and free radical and ROS scavenging.

Quenching of α,β-unsaturated aldehydes by green tea polyphenols: HPLC–ESI–MS/MS studies

The aim of this work was to investigate in vitro the quenching activity of green tea polyphenols against alpha,beta-unsaturated aldehyde, using 4-hydroxy-nonenal (HNE) as prototype and HPLC-ESI-MS/MS techniques. HNE is the most abundant and genotoxic product of oxidation of dietary polyunsaturated fatty acids, and is believed to be involved in the early stage of colorectal carcinogenesis on account of its genotoxic potential. Both epigallocatechin gallate (EGCG, 1.0-3.5mM), the main constituent of green tea polyphenols, and a green tea aqueous extract are able to quench HNE (50 microM) in colorectal physiomimetic conditions (10mM phosphate buffer, pH 8.0, 37 degrees C), giving rise to the formation of six diastereomeric covalent adducts at the ring A of EGCG, as indicated by their ESI-MS/MS fragmentation pathways. The specificity of the adduction positions was explained by (1)H NMR experiments. HNE quenching is pH-dependent and maximum at pH 8.0. ESI-MS analysis showed no formation of 4-hydroxy-2,3-epoxy-nonanal, or adduction of the epoxide to EGCG. This implies that too little hydrogen peroxide (1mM, 24h incubation, FOX-2 method) develops from auto-oxidation of EGCG in our aerobic experimental conditions to oxidize HNE to its corresponding epoxide, so this mechanism is not responsible for the compounds disappearance. EGCG and green tea extract also quenched acrolein, another genotoxic alpha,beta-unsaturated aldehyde, giving one predominant adduct and minor isobaric species, probably due the adduction of acrolein at different positions of the EGCG ring A. These results suggest that EGCG and green tea extract, beside the proposed mechanisms of chemoprevention that target multiple cell-signaling pathways that control cell proliferation and apoptosis in cancer cells, can also prevent protein carbonylation in the tumor tissue environment, depending on the pH of the medium surrounding the tissue, the type of tumor, the stage of dysregulation of lipid peroxidation and, finally, the stage of carcinoma development.

An analytical and theoretical approach for the profiling of the antioxidant activity of essential oils: the case of Rosmarinus officinalis L.

The antioxidant constituents of essential oils (EOs) of Rosmarinus officinalis L. (α-pinene chemotype) were isolated at the flowering (A), post-flowering (B), and vegetative stages (C). GC-MS was used to analyze total chemical composition, Folin-Ciocalteau and Prussian blue methods for reducing substances. Radical scavenging capacity (DPPH test, IC(50) 36.78±0.38, 79.69±1.54, 111.94±2.56μL) and anti-lipoperoxidant activity (TBARS, IC(50) 0.42±0.04, 1.20±0.06μL, 4.07±0.05μL) differed widely in the three stages. Antioxidant activity, identified after silica gel fractionation chromatography, was closely related (R(2)=0.9959) to each EOs content of hydroxilated derivatives, (containing alcohols, phenols and 1,8 cineole): 15.26±0.12%, 7.22±0.06%, and 5.09±0.10% in EOs from A, B, and C. Modeling the C, H, O terpenes in a simulated phospholipid bilayer indicated that anti-lipoperoxidant activity depended on the stability and rapidity of their interactions with the membrane bilayer components, and their positioning over its surface.

Total Antioxidant Performance Is Associated with Diet and Serum Antioxidants in Participants of the Diet and Physical Activity Substudy of the Jackson Heart Study

Total antioxidant performance (TAP) measures antioxidant capacities in both hydrophilic and lipophilic compartments of serum and interactions known to exist between them. Our objective was to assess TAP levels in a subset of Jackson Heart Study (JHS) participants and to examine associations with dietary and total (diet + supplement) intakes of alpha-tocopherol, gamma-tocopherol (diet only), beta-carotene, vitamin C, fruit, vegetables, and nuts, and serum concentrations of alpha-tocopherol, gamma-tocopherol, and beta-carotene. We conducted a cross-sectional analysis of 420 (mean age 61 y; 254 women) African American men and women participating in the Diet and Physical Activity Sub-Study of the JHS in Jackson, Mississippi. In multivariate-adjusted models, we observed positive associations between total alpha-tocopherol, total and dietary beta-carotene, and total vitamin C intakes and TAP levels (P-trend < 0.05). Positive associations were also observed for vegetable, fruit, and total fruit and vegetable intakes (P-trend < 0.05). For serum antioxidant nutrients, alpha-tocopherol but not beta-carotene was associated with serum TAP levels. There were inverse associations for serum gamma-tocopherol and TAP levels. Associations for alpha-tocopherol were seen at intake levels much higher than the current Recommended Dietary Allowance. It may, therefore, be prudent to focus on increasing consumption of fruit, vegetables, nuts, and seeds to increase total antioxidant capacity.