New anti-RNS and -RCS products for cosmetic treatment.

Oxidants and free radicals are known to be a very important factor in skin aging, taking an active part in lipidic peroxidation, breakage of proteins and DNA, etc. The most well-known are reactive oxygen species (ROS), for example, superoxide radical anion, or more commonly called, superoxide (O), hydroxyl radical (OH(*)) or hydrogen peroxide (H(2)O(2)). Both free radicals and other oxidants can be generated by metabolic activity within the cell and by other environmental challenges,. In addition, other dangerous species are known such as reactive nitrogen species (RNS) and reactive carbonyl species (RCS). Some of the most important RNS are peroxynitrite (ONOO(-)), nitrogen dioxide radical ((*)NO(2)) and the nitronium ion (NO). For RCS, some of the most important are 4-hydroxynonenal (HNE), acrolein (ACR), malondialdehyde (MDA) or glyoxal (GXL). Both compounds (RNS and RCS) are thought to play an important role in many diseases and in skin aging, for example, collagen cross-linking, DNA damage, protein tyrosine nitration, etc. This work investigates two new specific chemicals: Lipochroman-6((R))- an anti-RNS which shows good results in inhibiting the nitration of tyrosine by peroxynitrite, and Aldenine((R))- a tripeptide anti-RCS which protects cells from reactive carbonyl compounds such as HNE or ACR; it also shows the ability to prevent glycation of proteins, specifically by superoxide dismutase (SOD).

Procyanidins from Vitis vinifera seeds inhibit the respiratory burst of activated human neutrophils and lysosomal enzyme release.

The inhibitory properties of procyanidins (a standardized oligomeric catechin fraction) from Vitis vinifera L. seeds on the respiratory burst and on the release of granule components myeloperoxidase, beta-glucuronidase and elastase were studied in activated human neutrophils. Procyanidins strongly inhibit superoxide generation with an IC(50) of 7.2 microM, through a direct scavenging of superoxide and prevent the release from calcium ionophore activated neutrophils of beta-glucuronidase (IC(50) = 13.9 microM), myeloperoxidase (IC(50) = 7.2 microM) and elastase (IC(50) = 5.4 microM). In addition they dose-dependently inhibit the activity of myeloperoxidase released from calcium ionophore-stimulated cells with an IC(50) value of 2 microM. The monomeric constitutive unit (+)-catechin was far less active than procyanidins in all the models tested. These results evidence that procyanidins efficiently restrain the inflammatory response of activated neutrophils in vitro and whenever absorbed in vivo can prevent their oxidative discharge at the site(s) of their adhesion.

Nitrosylhemoglobin, an unequivocal index of nitric oxide release from nitroaspirin: in vitro and in vivo studies in the rat by ESR spectroscopy.

Electron spin resonance (ESR) spectroscopy was applied for the unequivocal detection/quantitation of nitric oxide (NO) as nitrosylhemoglobin (HbFe(II)NO) released from nitroaspirin, benzoic acid,2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX-4016; NO-ASA), the lead of a new class of nonsteroidal anti-inflammatory drugs. In both in vitro and in vivo experiments, the paramagnetic complex was detected at 100 K in the venous blood of the rat (microwave power, 20 mW) and characterized by a three-line hyperfine structure with coupling constants (A(x) and A(z)) of 17 G at g(x)=2.066 and g(z)=2.009. The kinetics of NO release from the drug were first determined in vitro by incubating rat blood with 1 mM NO-ASA and confirmed by the two-line hyperfine structure obtained with the labeled compound ((15)N-NO-ASA). In in vivo studies, the hematic levels of HbFe(II)NO were determined after oral (p.o.) and intraperitoneal (i.p.) administration of the drug (100 and 200 mg kg(-1)). In p.o. treated animals, the complex was detectable at 1 h post-dosing and its formation was maximal at 4-6 h, where the antithrombotic activity peaks. In i.p. treated animals, HbFe(II)NO complex peaks at the second hour to decline thereafter: in these animals, the ESR technique was applied to also detect nitrosylmyoglobin as an index of NO diffusion/compartmentalization in myocardial tissue. The results of this study emphasize the great potentiality of ESR spectroscopy for the study of the release, the metabolic fate and distribution of NO from nitrovasodilators.

Complexation of Ginkgo biloba extract with phosphatidylcholine improves cardioprotective activity and increases the plasma antioxidant capacity in the rat.

The aim of this work was to compare in the rat the cardioprotective efficacy and the total plasma antioxidant activity of a standardised Ginkgo biloba L. extract (GB) as such (300 mg/kg/day) or complexed with phosphatidylcholine (GB-PC; 1:2 w/w), after a 5 days oral administration. At the end of the treatment, the total plasma antioxidant defence was determined by the TRAP and FRAP assays, and the hearts from all groups of animals subjected to moderate ischemia (flow reduction to 1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min). The recovery of left ventricular developed pressure (LVDP) at the end of reperfusion was 35-40% of the preischemic values in both control and vehicle rats, 50.2% in the GB group and 72.5% in the GB-PC pre-treated animals. Creatine kinase (CK) outflow in the perfusate from the hearts of GB and GB-PC treated animals were restrained to a different extent vs. controls (by 71% GB-PC; by 22% GB); the rate of prostacyclin (6-keto-PGF1 alpha) release was far greater in GB-PC than in GB hearts. In parallel, the GB extract significantly increased the total antioxidant plasma capacity (by 24.5% TRAP; 27.9% FRAP) only when complexed with phospholipids. This indicates an increased bioavailability of phenolic antioxidants when suitably embedded within a lipophilic carrier. The results of this study demonstrate that complexation of Ginkgo biloba with phospholipids induces in the rat, even after a short treatment a greater resistance of the heart to ischemia/reperfusion damage in respect to the native extract, due to an increased plasma antioxidant activity.

LC coupled to ion-trap MS for the rapid screening and detection of polyphenol antioxidants from Helichrysum stoechas.

Liquid Chromatography-Ion Trap Mass Spectrometry with an atmospheric pressure chemical ionization (APCI) interface in the negative and positive-ion modes in parallel to UV-diode-array detection (DAD), was applied for the rapid detection/characterization in crude extracts of the water-soluble antioxidant phenolics from Helichrysum stoechas. APCI-MS provides unequivocal molecular weight data of these compounds and useful information about their structures (diagnostic fragments ions), which were confirmed by the UV-DAD fingerprints. This combined approach allows the identification of ten constituents, including the three naturally occurring isomers of caffeoylquinic acid (CGAs), namely neo-chlorogenic acid, chlorogenic acid and crypto-chlorogenic acid, 2 isomeric dicaffeoyl quinic acids, 2 isomeric naringenin glucosides, quercetin, kaempferol and apigenin glucosides and a tetrahydroxychalcone-glucoside. The water-soluble extract from H. stoechas, standardized in both total polyphenol and kaempferol-3-glucoside content, exhibits strong antioxidant activity in vitro when tested in both artificial membrane systems (phosphatidylcholine liposomes) and in a cell model (rat erythrocytes).

Fluorescent probes as markers of oxidative stress in keratinocyte cell lines following UVB exposure.

In this work we describe the development of specific markers for determination of both the membrane and intracellular damage induced by free radicals generated by UVB radiation (5-150 mJ/cm2) in cultured keratinocytes. This using simple, specific and sensitive fluorescent probes: cis-parinaric acid (PNA) to monitor membrane lipid peroxidation and 2',7'-dichloro-dihydrofluorescein diacetate (DCFH-DA) to evaluate the intracellular redox status, in parallel to the fluorimetric determination of the main intracellular antioxidant glutathione. To validate the methodologies, the changes in the intracellular oxidative status following exposure to low doses UVB were measured in both control and N-acetylcysteine-protected cells, in parallel with morphological analyses. UVB induces an early reduction of GSH inside the cell correlated with an increase in the intracellular peroxide content. The effects were time- and dose-dependent. In addition, using a sensitive fluorescent method, we quantitated the release of proteases, a family of proteolytic enzymes greatly involved in the onset/perpetuation of the free radical-induced skin damage from keratinocytes exposed to suberythemal UVB doses (5-15 mJ/cm2). The use of these fluorescent probes provides a reliable tool to detect the early signs of damage in keratinocyte cultures (when the apoptotic phenomenon has not yet been triggered) useful for future screening of protective molecules.

Diet enriched with procyanidins enhances antioxidant activity and reduces myocardial post-ischaemic damage in rats.

Aim of this work was to study the efficacy of procyanidins from Vitis vinifera seeds, a standardized mixture of polyphenol antioxidants, on cardiac mechanics following ischemia/reperfusion stunning in the rat, after 3 weeks supplementation. Young and aged male rats were fed a diet enriched with procyanidins complexed (1:3 w/w) with soybean lecithin (2.4%); control animals (CTR-young and CTR-aged) received an equal amount of lecithin and 2 additional groups of animals the standard diet. At the end of the treatment, the total plasma antioxidant defense (TRAP), vitamin E, ascorbic acid and uric acid were determined in plasma and the hearts from all groups of animals subjected to moderate ischemia (flow reduction to 1 ml/min for 20 min) and reperfusion (15 ml/min for 30 min). In both young and aged rats supplemented with procyanidins the recovery of left ventricular developed pressure (LVDP) at the end of reperfusion was 93% (p < 0.01) and 74% (p < 0.01) of the preischemic values and the values of coronary perfusion pressure (CPP) were maintained close to those of the preischemic period. Also creatine kinase (CK) outflow was restrained to baseline levels, while a 2-fold increase in prostacyclin (6-keto-PGF1alpha) in the perfusate from hearts of young and aged rats was elicited during both ischemia and reperfusion. In parallel, procyanidins significantly increased the total antioxidant plasma capacity (by 40% in young and by 30% in aged rats) and the plasma levels of ascorbic acid, while tend to reduce vitamin E levels; no significant differences were observed in uric acid levels. The results of this study demonstrate that procyanidins supplementation in the rat (young and aged) makes the heart less susceptible to ischemia/reperfusion damage and that this is positively associated to an increase in plasma antioxidant activity.

Mass spectrometric characterization and HPLC determination of the main urinary metabolites of nimesulide in man.

A study was undertaken for the characterization and quantitative determination of the main urinary metabolites of the non-steroidal anti-inflammatory drug (NSAID) nimesulide (4-nitro-2-phenoxy-methanesulfonanilide) in man following single oral administration (200 mg). Urines were collected from six healthy volunteers at 12, 24, 48, 72 and 96 h post-administration and submitted to liquid liquid extraction before (free metabolites) and after enzymatic hydrolysis (conjugated metabolites). The structure of the metabolites, isolated by TLC separation, was elucidated by mass spectrometry (electron impact ionization) and confirmed by synthesis. Five metabolites were identified: they arise from hydroxylation to the phenoxy nucleus (M1 = hydroxynimesulide); reduction of the nitro group to an amino derivative (M2); concomitant hydroxylation and reduction (M3); N-acetylation of the M2 (M4) and of the M3 (M5) metabolites. Quantitation was by reverse phase high performance liquid chromatography (Supelcosil LC-18 DB column; mobile phase: sodium phosphate buffer (pH 3.0, 50 mM)-acetonitrile (gradient elution); flow rate: 1 ml min(-1); UV detection, 230 nm), procedure which allows in a single chromatographic run the simultaneous determination of the unchanged drug and of its metabolites. The urinary excretion of the drug and metabolites (free + conjugated) in the overall 96 h-interval accounts for approximately 40% of the administered dose: 17.55 +/- 3.6% M1; 0.72 +/- 0.43% M2; 2.45 +/- 1.22% M3; 19.07 +/- 4.3% M5. The bulk of the metabolites was in conjugated form. Percentages excretion of the unchanged drug and of M4 metabolite were below 0.5%. The described method is suited to specifically and quantitatively measure nimesulide and metabolites in human urine with acceptable precision and accuracy.

Scavenging of free radicals by tenoxicam: a participating mechanism in the antirheumatic/antiinflammatory efficacy of the drug.

The radical scavenging activity of tenoxicam against hydroxyl (HO.), superoxide (O2.-), and peroxyl (LOO.) radicals, all of them involved in the inflammatory reactions, has been tested in different cell-free systems and by different techniques. Tenoxicam is a good scavenger of both HO. radicals (IC50 = 56.7 microM), as determined by Electron Spin Resonance (ESR) spectroscopy with the spin trapping (5,5-dimethyl-1-pyrroline N-oxide, DMPO) technique, and O2.- radicals generated by the phenazine methosulfate/reduced beta-nicotinamide adenine dinucleotide (PMS/NADH) system. The high reactivity of the drug towards HO. was confirmed by the rate constant of reaction with HO. (k approximately 10(10) M-1s-1), determined by competition kinetic studies with N,N-dimethyl-4-nitrosoaniline. In addition at a microM level (1-5 microM) it dose-dependently prevents the phycoerythrin peroxidation induced by the water-soluble azoinitiator 2,2-azobis (2-amidinopropane) dihydrochloride (ABAP), indicating a quenching effect on aqueous peroxyl radicals. The HO.-entrapping capacity was confirmed in models more close to the in vivo situation: tenoxicam inhibits the HO.-induced depolymerization of hyaluronic acid already at 15 microM and the HO.-driven lipid peroxidation in phosphatidylcholine liposomes (PCL) with an IC50 of 10 microM. In this membrane model it delays at 1-10 microM level the decomposition of phosphatidylcholine hydroperoxides to short-chain alkenals (markers: total carbonyl functions as 2,4-dinitrophenylhydrazones and conjugated dienes). The high susceptibility of the drug to HO. attack is also demonstrated by its extensive degradation (HPLC studies) when irradiated with HO. radicals. The antioxidant component of tenoxicam evidenced in this study sheds some light on the hitherto undefined mechanism of the antiinflammatory action of the drug.

Echinacoside and caffeoyl conjugates protect collagen from free radical-induced degradation: a potential use of Echinacea extracts in the prevention of skin photodamage.

The protective effect of caffeoyl derivatives (echinacoside, chlorogenic acid, chicoric acid, cynarine, and caffeic acid, typical constituents of Echinacea species) on the free radical-induced degradation of Type III collagen has been investigated. The macromolecule was exposed to a flux of oxygen radicals (superoxide anion and hydroxyl radical) generated by the xanthine/xanthine oxidase/Fe2+/EDTA system and its degradation assessed qualitatively by SDS-PAGE and quantitatively as the amount of soluble peptides (according to the 4-hydroxyproline method) released from native collagen after oxidative stress. The SDS-PAGE pattern of native collagen is markedly modified by free radical attack, with formation of a great number of peptide fragments with molecular masses below 97 kDa: in the presence of microM concentrations of echinacoside, there is a complete recovery of the native profile. Collagen degradation was, in fact, dose-dependently inhibited by all the compounds, with the following order of potency: echinacoside approximately chicoric acid > cynarine approximately caffeic acid > chlorogenic acid, with IC50 ranging from 15 to 90 microM. These results indicate that this representative class of polyphenols of Echinacea species protects collagen from free radical damage through a scavenging effect on reactive oxygen species and/or C-, N-, S-centered secondary radicals, and provide an indication for the topical use of extracts from Echinacea species for the prevention/treatment of photodamage of the skin by UVA/UVB radiation, in which oxidative stress plays a crucial role.

Anti-elastase and anti-hyaluronidase activities of saponins and sapogenins from Hedera helix, Aesculus hippocastanum, and Ruscus aculeatus: factors contributing to their efficacy in the treatment of venous insufficiency.

Triterpene and steroid saponins and sapogenins of medicinal plants (Aesculus hippocastanum L., Hedera helix L., Ruscus aculeatus L.) are claimed to be effective for the treatment/prevention of venous insufficiency. In this work we evaluated the inhibitory effects of these plant constituents on the activity of elastase and hyaluronidase, the enzyme systems involved in the turnover of the main components of the perivascular amorphous substance. The results evidence that for Hedera helix L., the sapogenins only non-competitively inhibit hyaluronidase activity in a dose-dependent fashion, showing comparable IC50 values (hederagenin IC50 = 280.4 microM; oleanolic acid IC50 = 300.2 microM); both the saponins hederacoside C and alpha-hederin are very weak inhibitors. The same behaviour is observed for serine protease porcine pancreatic elastase: the glycosides are devoid of inhibitory action, while genins are potent competitive inhibitors (oleanolic acid IC50 = 5.1 microM; hederagenin IC50 = 40.6 microM). Constituents from Aesculus hippocastanum L. show inhibitory effects only on hyaluronidase, and this activity is mainly linked to the saponin escin (IC50 = 149.9 microM), less to its genin escinol (IC50 = 1.65 mM). By contrast, ruscogenins from Ruscus aculeatus L., ineffective on hyaluronidase activity, exhibit remarkable anti-elastase activity (IC50 = 119.9 microM; competitive inhibition). The mechanism of elastase inhibition by triterpene and steroid aglycones, with a nitroanilide derivative as substrate, is discussed.

Characterization of cisplatin-glutathione adducts by liquid chromatography-mass spectrometry. Evidence for their formation in vitro but not in vivo after concomitant administration of cisplatin and glutathione to rats anc cancer patients.

After incubation of equimolar amounts of cisplatin (CDDP) and glutathione (GSH) in phosphate buffer pH 7.4 at 37 degrees C, we detected two CDDP-GSH adducts whose structures, characterized by LC-MS, corresponded to cis-[Pt(NH3)2Cl(SG)] and cis-([Pt(NH3)2Cl]2(mu-SG))+. The latter is a new CDDP-GSH adduct, which was postulated but never structurally characterized so far. Rats and patients were given a 15-min intravenous infusion of CDDP (10 mg/kg to rats and 25 mg/m2 to patients) preceded by a GSH intravenous administration (200 mg/kg to rats as a bolus and 1.5 g/m2 to patients as a 15-min infusion). After the administrations, CDDP-GSH adducts were absent in rat and human plasma ultrafiltrates. The discrepancy between in vitro and in vivo findings can be explained based on pharmacokinetic considerations.

Direct characterization of caffeoyl esters with antihyaluronidase activity in crude extracts from Echinacea angustifolia roots by fast atom bombardment tandem mass spectrometry.

Fast atom bombardment (FAB-MS) and fast atom bombardment tandem mass spectrometry (FAB-MS/MS) techniques (negative ions) have been successfully applied for identification of the constituents responsible for the antihyaluronidase activity of Echinacea angustifolia roots, whose extracts are widely employed for the adjuvant therapy of chronic inflammatory diseases. Crude extracts from different solvents were tested for antihyaluronidase activity, and those with the greatest inhibitory action (the ethylacetate, butylacetate and chloroform fractions, IC50 0.44, 0.50 e 0.62 mg/ml) were directly analyzed by MS. Full scan mass spectra produced intense molecular anions: collisional activation of these resulted in tandem mass spectra rich in significant product ions. Four main caffeoyl conjugates were detected and identified by tandem mass spectrometry (daughter and parent ion mode): 2,3-O-dicaffeoyltartaric acid (chicoric acid) and 5-O-dicaffeoylquinic acid (cynarine) and 2-O-caffeoyltartaric acid (caffaric acid) in the ethylacetate fraction. Among these caffeoyl conjugates, chicoric and caftaric acids had the greatest antihyaluronidase activity: IC50 = 0.42 and 0.61 mM, while the IC50 of cynarine and chlorogenic acid were 1.85 and 2.25 mM.

Antioxidant activity of nimesulide and its main metabolites.

The antioxidant activity of nimesulide and its main metabolites, 4'-hydroxynimesulide (M1) and 2-(4'-hydroxyphenoxy)-4-N-acetylamino-methansulfonanilide (M2), was investigated using 2 in vitro models: NADPH-supported lipid peroxidation in rat liver microsomes (marker MDA formation) and xanthine/xanthine oxidase, iron-promoted depolymerisation of hyaluronic acid, determined by gel permeation chromatographic analysis (marker molecular weight distribution). In the lipid peroxidation model, all the compounds inhibited MDA formation in a concentration-dependent manner, although with different potencies; the maximum scavenging effect was observed for M1 [50% inhibitory concentration (IC50) = 30 mumol/L; M2 IC50 = 0.5 mmol/L; nimesulide = 0.8 mmol/L]. Nimesulide was more active than its metabolites in preventing OH-induced depolymerisation of hyaluronic acid, with a 50% effective concentration of approximately 230 mumol/L, which was fairly comparable to that of tenoxicam. This protective effect was due to the OH.-entrapping capacity of the drug, which, in the Fenton-driven model, is easily converted, via OH. attack, to M1 and putatively to 2-hydroxy-4-nitro-methansulfonanilide.

Application of collisionally activated decomposition mass-analysed ion kinetic energy spectrometry to a survey of sheep milk for benzimidazole residues in relation to withdrawal periods.

A collisional spectroscopy procedure for simultaneous detection of five widely employed benzimidazole anthelmintics (laevamisole, thiabendazole, mebendazole, fenbendazole, febantel) in sheep milk was developed. The method which involves injection of crude milk extracts and selection and collision of the most abundant ionic species (M+. or fragments) obtained under electron impact ionization, is highly suitable for multi-residue analysis because of its sensitivity (limits of detection in the range of 0.6-2.8 p.p.b) and its rapidity (more than five samples per hour can be processed). The collisional approach was applied successfully for monitoring of anthelmintic residues in sheep milk.

Phytochemical characterization and radical scavenger activity of flavonoids from Helichrysum italicum G. Don (Compositae).

The glycosidic fraction of the flavonoids extracted from the flowering tops of the Helichrysum italicum G. Don was isolated, purified and characterized. This fraction was constituted by three compounds, which were assigned the structure of 4,2',4',6'-tetrahydroxychalcone-2'-glucoside, kaempferol-3-glucoside and naringenin-glycoside. Radical scavenger properties of the single glycosyl-flavonoids and of the in toto glycosidic fraction were tested with in vitro systems where different reactive oxygen species are generated (superoxide ions, hydroxyl radicals) and on lipid peroxidation induced by ADP/Fe2+ and NADPH or CCl4 in rat liver microsomes. The formation of reactive oxygen species was detected by cytochrome c reduction, salicylic acid hydroxylation and hyaluronic acid depolymerization. The action of the glycosidic fraction on the release of TXB2 and 12-HETE in human platelets, after collagen stimulation, was also evaluated. The glycosidic fraction inhibited in a dose dependent fashion lipid peroxidation in rat liver microsomes treated with ADP/Fe2+ or CCl4. This effect is due to the ability of flavonoids to scavenge free radicals at different stages of the process (superoxide ions, hydroxyl and lipid peroxide radicals). The single glycosyl-flavonoids exhibited a different scavenger activity, depending on the oxygen species and the chemical structure of the compounds. No effect of the fraction was observed on TXB2 and 12-HETE formation at 100 microns concentration.

Analysis of tiadenol in human plasma by capillary gas chromatography with electron capture detection.

A sensitive and specific method for the quantitative determination of tiadenol in human plasma is described. After addition of the internal standard, both compounds were quantitatively extracted into chloroform and then derivatized with heptafluorobutyric anhydride (the structures of both derivatives were confirmed by electron impact mass spectrometry). Quantitation was achieved by capillary gas chromatography, using a (63) Ni-electron capture detector. Linearity was observed in the concentration range 5-100 ng ml(-1) and the minimum concentration of tiadenol detectable in plasma was 2.0 ng ml(-1). The method was successfully applied to plasma specimens collected from healthy human volunteers following a single oral administration of 800 mg of tiadenol.

Perinatal development of styrene monooxygenase and epoxide hydrolase in rat liver microsomes and nuclei.

Nuclear enzymes were found to develop earlier than the corresponding microsomal activities. In fact styrene monooxygenase enzymatic activity at 18 days gestational age reached about half the values of adult animals, whereas fetal microsomal activity was only about 1/20 the adult level at the same age. In microsomes and nuclei the ontogenic development of epoxide hydrolase is slightly slower than styrene monooxygenase. This suggests that fetuses and newborn animals are exposed to higher risk of accumulation of styrene-7,8-oxide, a toxic and possibly teratogenic product of styrene monooxygenase.