Rita Petrucci

Recovery of natural antioxidants from spent coffee grounds.

Spent coffee grounds (SCG) were extracted with an environmentally friendly procedure and analyzed to evaluate the recovery of relevant natural antioxidants for use as nutritional supplements, foods, or cosmetic additives. SCG were characterized in terms of their total phenolic content by the Folin-Ciocalteu procedure and antioxidant activity by the DPPH scavenging assay. Flavonoid content was also determined by a colorimetric assay. The total phenolic content was strongly correlated with the DPPH scavenging activity, suggesting that phenolic compounds are mainly responsible for the antioxidant activity of SCG. An UHPLC-PDA-TOF-MS system was used to separate, identify, and quantify phenolic and nonphenolic compounds in the SCG extracts. Important amounts of chlorogenic acids (CGA) and related compounds as well as caffeine (CAF) evidenced the high potential of SCG, a waste material that is widely available in the world, as a source of natural phenolic antioxidants.

Hypochlorite scavenging activity of hydroxycinnamic acids evaluated by a rapid microplate method based on the measurement of chloramines

Scavengers of hypochlorite (XOCI) could have beneficial effects in diseases in which this oxidant plays a pathogenic role. It has been reported that ferulic acid and chlorogenic acid, the quinic ester of caffeic acid, are good hypochlorite scavengers, but a systematic evaluation of the naturally occurring hydroxycinnamic acids (HCAs), which these substances belong to, has not been performed yet. Thus, in this work we studied, by two different in‐vitro methods, the antioxidant activity of five HCAs: p‐coumaric acid, ferulic acid, sinapinic acid, caffeic acid and chlorogenic acid. The methods applied in this study were based on the oxidation of human serum albumin (HSA) by XOCI, a new microplate method based on the measurement of chloramines and a previously described carbonyl assay. Firstly, lysine‐derived chloramines, in the presence or absence of the HCAs, were detected using 5‐thio‐2‐nitrobenzoic acid (TNB), measuring the absorbance at 415nm by a microplate reader. To remove excess XOCI, Trolox, a known XOCI scavenger, was added before TNB. Secondly, lysine‐derived carbonyls, in the presence or absence of the HCAs, were detected by using 2,4‐dinitro‐phenylhydrazine. Hydroxycinnamic acids appeared active (caffeic≥sinapinic>chlorogenic≅ferulic>p‐coumaric acid) by both methods, suggesting possible pharmacological applications for these compounds, which are present at high concentrations in the plant kingdom.

Oxidative stress parameters in different systemic rheumatic diseases

The involvement of oxidative stress in the pathogenesis of rheumatic disorders, such as systemic sclerosis (SSc) and chronic polyarthritides, has been suggested yet not thoroughly verified experimentally. We analysed 4 plasmatic parameters of oxidative stress in patients with SSc (n = 17), psoriatic arthritis (PsA) (n = 10) and rheumatoid arthritis (RA) (n = 9) compared with healthy subjects (n = 22). The biomarkers were: total antioxidant capacity (TAC) measured by ferric reducing antioxidant power (FRAP) method, hydroperoxides determined by ferrous ion oxidation in presence of xylenol orange (FOX) method and sulfhydryl and carbonyl groups assessed by spectrophotometric assays. The results showed significantly increased hydroperoxides in SSc, PsA and RA (3.97 ± 2.25, 4.87 ± 2.18 and 5.13 ± 2.36 μmol L−1, respectively) compared with the control group, (2.31 ± 1.40 μmol L−1; P < 0.05). Sulfhydryls were significantly lower in SSc (0.466 ± 0.081 mmol L −1), PsA (0.477 ± 0.059 mmol L−1) and RA (0.439 ± 0.065 mmol L−1) compared with the control group) (0.547 ± 0.066 mmolL−1; P < 0.05). TAC in all three diseases showed no difference in comparison with controls. Carbonyls were significantly higher in RA than in the control group (32.1 ± 42 vs 2.21 ± 1.0 nmol (mg protein)−1; P < 0.05). The obtained data indicate augmented free radical‐mediated injury in these rheumatic diseases and suggest a role for the use of antioxidants in mediated prevention and treatment of these pathologies.

Hypochlorite scavenging activity of flavonoids.

Scavengers of hypochlorite, a highly reactive oxidant produced by activated phagocytes, could have potential therapeutic effects in diseases in which this oxidant plays a pathogenic role. Flavonoids are polyphenolic substances present in food plants and have been extensively studied for their antioxidant properties against various free radicals. Less is known about their reactivity with hypochlorite. In this study, the hypochlorite scavenging activity of flavonoids was investigated using a microplate assay recently developed in our laboratory. This method evaluates the ability of a substance to inhibit the formation of chloramines in human serum albumin upon oxidation by hypochlorite. Thirteen flavonoids were tested. Most of them inhibited human serum albumin oxidation at micro‐molar concentrations and appeared more active than Trolox, a water‐soluble equivalent of vitamin E. It was observed that the greater the number of hydroxyl substitutions, the greater the scavenging activity. The 3‐hydroxy substitution seemed to be particularly important for scavenging activity, whereas the presence of a 2,3‐double bond in the C ring did not. Flavonoids were found to be good hypochlorite scavengers in‐vitro and further information is provided about the chemical aspects important for scavenging activity. Thus, flavonoids could have beneficial effects in diseases such as atherosclerosis in which hypochlorite plays a pathogenic role.

Chemical and Electrochemical Study on the Interactions of Aminoxyls with Superoxide Anion

The electrochemical behaviour of tetramethyl-pyrrolinic, -piperidinic, indolinonic and quinolinic aminoxyls and of oxygen was studied in DMFAI20 in order to evaluate the feasibility of an electron transfer process between aminoxyls and superoxide anion. The rate constants of these reactions were calculated by applying the Marcus theory. A new mechanism for the reaction, which operates in competition with those already reported in the literature involving an electron transfer process, is proposed. Copyright

A study on the interactions between coenzyme Q 0 and superoxide anion. Could ubiquinones mimic superoxide dismutase (SOD)

An electrochemical study was carried out on 1,4-benzoquinone, duroquinone, coenzymes Q0 and Q10 in the absence and in the presence of molecular oxygen in aprotic (DMF) and protic (DMF/H2O 95:5 (v/v)) media. Water was added because the investigated reactions are deeply influenced by the presence of protons. Q0 and Q10 exhibited a similar electrochemical behaviour. Since Q0 is more soluble in protic medium than the biologically more important analogue Q10, it was chosen as a model for a more detailed investigation. Voltammetric studies of Q0 carried out in aprotic and protic media in the presence of oxygen showed that, besides simple O2·− dismutation, the Q0 promoted dismutation of O2·− should also be considered. Spectroelectrochemical experiments with the same experimental conditions support the electrochemical results, showing that in the presence of superoxide and in aprotic medium semiquinone Q0·− gives rise to a disproportionation equilibrium, while in the presence of water it tends to be reoxidized to the starting Q0 by OOH·. EPR measurements are also in agreement with these results.

Protein oxidation markers in the serum and synovial fluid of psoriatic arthritis patients

The role of oxidative stress has been studied in rheumatoid arthritis (RA) and other inflammatory joint diseases to some extent, but its importance in psoriatic arthritis (PsA) has rarely been investigated. The aim of this study was to analyze the levels of protein oxidation markers, sulfhydryl (SH) and carbonyl (CO) groups, in the synovial fluid (SF) and serum of PsA patients and compare them with the findings in RA and osteoarthritis (OA) patients. A total of 49 subjects with a knee‐joint effusion including 16  PsA, 18  RA, and 15 OA patients were studied. In all patients, the levels of SH groups measured in the serum and SF inversely correlated with the number of white blood cells (WBC) (P<0.05) and the percentage of polymorphonuclear leukocytes (PMN) (P<0.01) in SF. Serum SH levels inversely correlated with serum erythrocyte sedimentation rate (ESR) (P<0.02) and C‐reactive protein (CRP) (P<0.05) values. The SH levels in SF were significantly lower in patients affected by PsA and RA compared to OA cases (P<0.02). The serum SH levels in PsA were lower than OA (P<0.001) and higher than RA patients (P<0.05). The serum and synovial levels of CO groups in PsA, RA, and OA patients were similar. Our study provides novel evidence on the involvement of protein oxidation in PsA and confirms the important role of oxidative stress in the pathogenesis of RA. These data suggest that antioxidant agents can potentially be a useful addition to the conventional therapy in the management of these diseases. J. Clin. Lab. Anal. 22:210–215, 2008.

In vitro evaluation of antioxidant activity by electrophoresis and high performance liquid chromatography

Two methods for the analysis of antioxidants, based on polyacrylamide gel electrophoresis (PAGE) and gel permeation high performance liquid chromatography (HPLC) were developed. Both of them exploit the variations of the signal (band or peak) given by human serum albumin (0.2% w/v in 100 mM sodium phosphate pH 7) upon oxidation with hypochlorite (1% of a solution containing 4% active Cl), quantitatively determined by densitometric analysis or peak integration. Based on such changes, two formulas were defined which allowed the determination of the antioxidant activity of ascorbic acid (EC(50,PAGE)=4.8x10(-4) M, EC(50,HPLC)=3.6x10(-4) M), glutathione (EC(50,PAGE)=1.5x10(-4) M, EC(50,HPLC)=2.0x10(-4) M) and melatonin (EC(50,PAGE)=5.2x10(-4) M, EC(50,HPLC)=3.2x10(-4) M), chosen as reference compounds. A good correlation was found between the activities of these substances in the two assays, which are also in good agreement with literature data, indicating that the two methods are essentially equivalent. These assays could be useful for the screening of new antioxidant drugs for pathological conditions such as cataract, rheumatic diseases, atherosclerosis and Alzheimers disease.

A spectroelectrochemical and chemical study on oxidation of 7,8-dihydroxy-4-methylcoumarin (DHMC) and some related compounds in aprotic medium

Electrochemical and chemical oxidation of 7,8-hydroxy-4-methylcoumarin (DHMC 1) and 7,8-diacetoxy-4-methylcoumarin (DAMC 4) were studied to investigate the mechanisms occurring in their antioxidant activities in acetonitrile, under electron transfer and H-atom transfer conditions. Electrolysis and chemical reactions were followed on-line by monitoring the UV spectral changes with time. The anodic oxidation of DHMC, studied by cyclic voltammetry and controlled potential electrolysis, occurs via a reversible one-step two-electrons process, yielding the corresponding stable phenoxonium cation. Moreover, the chemical oxidation with an H-atom acceptor also follows a similar path, yielding the stable neutral quinonic product. Intermediates were never evidenced in both cases. Only in the presence of a strong base, an anodic oxidation product mono-electronic was evidenced, likely the DHMC radical anion. However, the anodic oxidation of the acetoxy derivative DAMC occurs at very high potential values, ruling out the possibility that the antioxidant activity observed in vivo might occur via an electron transfer mechanism; no reactions were evidenced with an H-atom acceptor.

Reactions of indolic nitrones and N-heteroaromatic bases under irradiation and chemical oxidation

The spin adducts formed under UVA irradiation or in the presence of mild oxidants in the reaction of a number of heteroaromatic bases and two indolic nitrones have been characterized by means of ESR spectroscopy. The formation of the spin adducts is explained via the Forrester–Hepburn mechanism, while the occurrence of inverted spin trapping is excluded. Photolysis of nitrones 1 and 2 or of the corresponding cyclic hydroxamic acids resulted in the formation of the related acyl nitroxides.