Rinaldo Cervellati

The Briggs‐Rauscher Reaction as a Test to Measure the Activity of Antioxidants

A new method for monitoring the relative activity of antioxidants is presented, and its advantages and limits are discussed. The method is based on the previously reported inhibitory effects of free-radical scavengers on the oscillations of the Briggs-Rauscher reaction. The effect consists of an immediate cessation of oscillations, an inhibition time that linearly depends on the concentration of the antioxidant added, and subsequent regeneration of oscillations. Here the effects of ten antioxidants (pyrocatechol (=benzene-1,2-diol), ferulic acid (=3-(4-hydroxy-3-methoxyphenyl)prop-2-enoic acid), caffeic acid (=3-(3,4-dihydroxyphenyl)prop-2-enoic acid), 2,6-, 3,4-, 2,4-, 3,5-, and 2,5-dihydroxybenzoic acids, homovanillic acid (=4-hydroxy-3-methoxybenzeneacetic acid), and resorcinol (=benzene-1,3-diol)) were studied in detail. Relative antioxidant activities of these substances with respect to resorcinol were determined in different ways on the basis of inhibition times. The limits of the calculated values of relative activity based on the Briggs-Rauscher reaction are the same as those obtained with other analytical procedures and are discussed here. The new method is inexpensive: reagents and apparatus are commonly used in all chemical laboratories. The thermochemical behavior of the Briggs-Rauscher reaction and the dependence of inhibition time on the temperature were also carefully investigated and taken into account. A semiquantitative mechanistic interpretation of the inhibitory effects based on a suitable kinetic model is given.

Cyanidin-3- O -β-glucopyranoside, a natural free-radical scavenger against aflatoxin B1- and ochratoxin A-induced cell damage in a human hepatoma cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2)

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B1 (AFB1). Induction of oxidative stress also plays an important role in the toxicity of another mycotoxin, ochratoxin A (OTA). In the present study, the protective effect of cyanidin-3-O-beta-glucopyranoside (C-3-G; an anthocyanin contained in oranges, blackberries, strawberries and cranberries) against AFB1- and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2). The ability of C-3-G to reduce the production of reactive oxygen species (ROS), the inhibition of protein and DNA synthesis and the apoptosis caused by the two mycotoxins was also investigated in both cell lines. Our experiments proved the significant cytoprotective effect of C-3-G in vitro against OTA- and AFB1-induced cell damage. In particular, 24 h of pretreatment with 50 microm-C-3-G inhibited the cytotoxicity of 10 microm-AFB1 (by 35 %) and of 10 microm-OTA (by 25 %) in Hep G2 cells (P < 0.001) and of 10 microm-AFB1 (by 10 %, P < 0.01) and of 10 microm-OTA (by 14 %, P < 0.05) in CaCo-2 cells. Moreover, 50 microm-C-3-G attenuated ROS production induced by the two toxins in both cell lines (P < 0.05). Inhibition of DNA and protein synthesis induced by the mycotoxins was counteracted by pretreatment with the antioxidant at 50 microm. Similarly, apoptotic cell death was prevented as demonstrated by a reduction of DNA fragmentation and inhibition of caspase-3 activation. The in vitro free-radical scavenging capacity of the anthocyanin was tested with the Briggs-Rauscher oscillating reaction. This system works at pH approximately 2. The results showed good scavenging power, in accordance with the observed inhibition of ROS production.

New isatin derivatives with antioxidant activity

The reaction between isatin and 2,5-dimethoxyaniline is described. The main product was identified as 3,3-bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one. The antioxidant activity of the compounds isolated was evaluated with two methods. Three published antitumor E-3-(2-chloro-3-indolylmethylene)1,3-dihydroindol-2-ones entered the same tests to search whether they are endowed with antioxidant activity too. 3,3-Bis(4-amino-2,5-dimethoxyphenyl)-1,3-dihydroindol-2-one and the three antitumor agents showed a good chemical antioxidant activity.

Phytochemical composition and antioxidant activity of Laurus nobilis L. leaf infusion.

Laurus nobilis L. (laurel) leaves are frequently used as a spice for cooking purposes. Folk medicine in many countries uses the infusion of the plant in stomachic and carminative remedies, as well as for the treatment of gastric diseases. Little information is available about the phytochemical composition of the infusion of dried leaves, which is a way to consume this aromatic and medicinal plant. Phytochemical investigations on the infusion were performed by high-performance liquid chromatography (HPLC) with a diode array detector (DAD) and direct electrospray ionization-tandem mass spectrometry. Several flavonoid derivatives were detected. Semipreparative HPLC from the infusion of laurel leaves isolated 10 flavonoid O-glycosides, one flavonoid C-glycoside, catechin, and cinnamtannin B1. Structures of the isolated compounds were computed on the basis of spectral measurements including high-resolution mass spectrometry spectroscopy and one- and two-dimensional nuclear magnetic resonance techniques. The amount of the flavonoids was also determined by HPLC-DAD. The antioxidant activity of the tea and the isolated compounds was also measured using two different in vitro methods: the Briggs-Rauscher oscillating reaction test, at a pH similar to that of the gastric juice, and the Trolox equivalent antioxidant capacity assay, at the pH of blood. For the infusion and the methanol extract the total phenolic content was also measured using the Folin-Ciocalteu reagent.

In vitro evaluation of the chemoprotective action mechanisms of leontopodic acid against aflatoxin B1 and deoxynivalenol-induced cell damage.

Several in vitro studies showed that free radical scavengers possess chemopreventive properties against mycotoxin‐induced cell damage which are at least partially associated with the induction of phase II detoxifying enzymes and antioxidant enzymes like glutathione S‐transferase (GST) and glutathione peroxidase (GPx). The aim of this project was to study the chemopreventive effects of leontopodic acid (LA), a potent natural occurring free radical scavenger isolated from the aerial parts of Leontopodium alpinum. Different mycotoxins were evaluated in two different cell lines on the basis of their specific toxicity: aflatoxin B1 (AFB1) on HepG2 cells and deoxynivalenol (DON) on U937 cells. Cell viability and reactive oxygen species concentration were determined, and the effects of pre‐treatment with LA on these parameters were investigated together with the GST and GPx activity as well as the concentration of reduced glutathione. The results show that LA protects U937 cells from DON‐induced cell damage but not HepG2 cells from AFB1. Moreover LA is able to enhance GPx activity in U937, but not GST activity in HepG2. We hypothesize that the increase in detoxifying enzymes is probably the main mechanism of antioxidant mediated chemoprevention. Copyright

Experimental and mechanistic investigation of an iodomalonic acid-based Briggs-Rauscher oscillator and its perturbations by resorcinol.

Classic Briggs-Rauscher oscillators use malonic acid (MA) as a substrate. The first organic product is iodomalonic acid. Iodomalonic acid (IMA) can serve as a substrate also; thus, the first product in that case is diiodomalonic acid (I(2)MA). Nonoscillating iodination kinetics can be followed by absorbance at 462 nm in acidic KIO(3) so long as IMA is in substantial excess over [I(2)]. At 25 °C, simulations lead to the two most important rate laws, and related rate constant estimates are reported. I(2)MA eventually decomposes by unknown processes, but I(2), O(2), H(2)O(2), and Mn(2+) speed up that decomposition, liberating most of the iodine back to the solution. Resorcinol is an effective inhibitor of oscillations both in MA oscillators and in IMA oscillators. Response of an IMA oscillator to varying amounts of resorcinol is shown herein and is similar to that for MA-based oscillators. The inhibitory effect of resorcinol is diminished by addition of IMA to a MA-based oscillator. The iodination reaction between IMA and resorcinol is too slow (0.043 M(-1) s(-1)) to account for the decreased inhibitory effectiveness of resorcinol. Rather, the decomposition of I(2)MA is responsible for the inhibition decrease.

Chemopreventive and antioxidant activity of 6-substituted imidazo[2,1-b]thiazoles

The synthesis of new imidazo[2,1-b]thiazoles bearing phenolic groups is reported. These compounds and some previously described analogs were evaluated as antioxidant agents with three chemical model systems, and cancer chemopreventive potential was examined by inhibition of NO production, TNF-α activated NFκB activity, and aromatase activity, as well as induction of QR1 and RXRE binding. Two of the test compounds, 9 and 12, displayed promising activity by inhibiting iNOS, NFκB and aromatase in dose-dependent manner, with IC50 values in low micromolar range. The same compounds activated QR1 in a bifunctional manner. When incubated with human liver microsomes, the active compounds were further hydroxylated on the parent ring system, suggesting the next logical step in the development of these promising leads will entail synthetic production of metabolites followed by additional assessment of biological activity.

Inhibitory Effects by Soy Antioxidants on the Oscillations of the Briggs‐Rauscher Reaction

Inhibitory effects by addition of aqueous extracts of soy flour to an active Briggs-Rauscher mixture are reported. The effect consists of an immediate cessation of oscillations, but, after some time, the oscillatory behavior is regenerated with amplitude and frequency different from those observed in a reference mixture. The inhibition time depends linearly on the concentration of substances contained in the extract in a wide range of concentration. The inhibitory effects are due to the high free-radical scavenging activity of substances contained in the soy flour. Two preponderant products contained in the soy flour extract were identified and characterized as malonyldaidzin and malonylgenistin. The antioxidant activity of these isoflavones contained in the extracts was determined on the basis of the inhibition time. A qualitative mechanistic explanation of the inhibitory effects is given. Our findings are decisive indirect evidence of involvement and important role played by HOO. radicals in establishing oscillations in the Briggs-Rauscher (BR) system. The linear relationship between the inhibition time and the whole-mass concentration of antioxidant contained in soy extracts added to a BR mixture is an indication of the possibility to develop and implement an analytical procedure for monitoring the activity of antioxidant scavengers of free radicals based on the oscillating Briggs-Rauscher reaction.

Monocyclic β-lactams as antibacterial agents: Facing antioxidant activity of N-methylthio-azetidinones

A series of N-methylthio-β-lactams with antibacterial activity were thoroughly evaluated as antioxidants. We found that only the presence of a polyphenolic moiety anchored to the β-lactam ring ensured an adequate antioxidant potency. New compounds, efficiently combining in one structure antioxidant and antibacterial activity, may provide a promising basis for the development of new leads useful in adverse clinical conditions such as in cystic fibrosis patients, in whom colonization by MRSA and epithelial damage by chronic pulmonary oxidative stress take place.

Gastroprotective Effect and Antioxidant Properties of Different Laurus nobilis L. Leaf Extracts

Laurus nobilis L. (Family Lauraceae) is an evergreen tree widely distributed in the Mediterranean area and Europe. It is used in folk medicine of different countries as a stomachic and carminative as well as in treatment of gastric diseases. Extracts obtained with different methods (methanol and chloroform) from laurel leaves were evaluated for their gastroprotective activities in the rat. The antioxidant capacity of the different extracts has been also measured in vitro. In order to confirm the activities investigated, histological observations were performed. The gastric damage was significantly reduced by all extracts administered. The more effective protection was produced by chloroformic and methanolic crude extracts. The results obtained after oral administration of L. nobilis leaf extracts are in good agreement with their antioxidant capacity, confirming the relationship between pharmacological efficacy and antiradical activity. Histological evidences confirm the results evaluated with the animal procedures.