Neurochem Res (2014) 39:758–769 DOI 10.1007/s11064-014-1267-5

ORIGINAL PAPER

Is There a Correlation Between In Vitro Antioxidant Potential and In Vivo Effect of Carvacryl Acetate Against Oxidative Stress in Mice Hippocampus? Lu´cio Fernandes Pires • Luciana Muratori Costa • Antonia Amanda Cardoso de Almeida Oskar Almeida Silva • Gilberto Santos Cerqueira • Damia˜o Pergentino de Sousa • Rivelilson Mendes de Freitas

•

Received: 29 September 2013 / Revised: 23 February 2014 / Accepted: 25 February 2014 / Published online: 12 March 2014 Ó Springer Science+Business Media New York 2014

Abstract This study investigated in vitro and in vivo antioxidant potential of carvacryl acetate (CA), a derivative of carvacrol, monoterpenic component of oregano. The correlation between in vitro and in vivo CA effects was also determined. In vitro tests measured thiobarbituric acid reactive species content, nitrite formation and hydroxyl radical levels. In vivo tests measured thiobarbituric acid reactive species content, nitrite concentration and reduced glutathione (GSH) levels, as well as glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase activities were measured, using mice hippocampus. The CA administrations for in vivo tests were intraperitoneally and acutely improved. CA reduced lipid peroxidation, nitrite and hydroxyl radical contents in vitro as well as lipid peroxidation and nitrite content in vivo. It also increased reduced GSH levels and GPx as well as catalase activities. Moreover, CA required a lower concentration to inhibit

50 % of free radicals measured in vitro than trolox. There was significant negative correlation between in vitro nitrite levels and in vivo reduced GSH levels; in vitro nitrite content and in vivo GPx activity as well as in vitro hydroxyl radical levels and in vivo SOD activity. To date, this is the first study which suggests vitro and in vivo antioxidant potential to this monoterpene and the correlation between these parameters.

L. F. Pires  R. M. de Freitas Postgraduate Program of Pharmacology, Federal University of Piauı´, Teresina, PI, Brazil

G. S. Cerqueira Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceara´, Rua Coronel Nunes de Melo, Fortaleza, CE 1127, Brazil

L. F. Pires (&)  R. M. de Freitas (&) Laborato´rio de Pesquisa em Neuroquı´mica Experimental, Curso de Farma´cia, Universidade Federal do Piauı´-UFPI, Campus Universita´rio Ministro Petroˆnio Portella, Bairro Ininga, Teresina, PI CEP 64049-550, Brazil e-mail: [email protected] R. M. de Freitas e-mail: [email protected]; [email protected]; [email protected] L. M. Costa  A. A. C. de Almeida  O. A. Silva  R. M. de Freitas Laboratory of Experimental Neurochemistry Research, Department of Biochemistry and Pharmacology, Center of Pharmaceutical Technology, Federal University of Piauı´, Teresina, PI, Brazil

123

Keywords Catalase  Free radicals  Glutathione peroxidase  Hippocampus  Mouse  Reduced glutathione

Introduction High oxygen consumption, low antioxidant defense and lipid-rich constitution make the brain highly vulnerable to

D. P. de Sousa Department of Pharmaceutical Science, Federal University of Paraı´ba, Joa˜o Pessoa, PI, Brazil

Neurochem Res (2014) 39:758–769

O

O

Fig. 1 Chemical structure of carvacryl acetate (ethyl 5-isopropyl-2methyl-phenyl)

oxidative damage, implicated in several diseases. Human brain uses 20 % of the oxygen consumed by the body, although this organ constitutes only 2 % of the body weight. In presence of oxidative stress, lipid-rich constitution of the brain is subjected to peroxidation, which results in a decrease on membrane fluidity and damage, compromising receptors, enzymes and ion channels in brain cells. As a result, oxidative stress can alter neurotransmission, neuronal function and all brain activity [1]. In animal models of epilepsy, there have been observed significant increases on oxidative stress in hippocampus, cerebral cortex and striatum as well as decreased antioxidant enzymatic activity [2, 3]. Furthermore, oxidative stress has also been associated with neuropsychiatric and neurodegenerative diseases. Therefore, intrinsic oxidative vulnerability of the brain has led some authors to suggest that oxidative damage may be a plausible pathogenic factor for those diseases [1, 4]. Additionally, several studies have demonstrated antioxidant potential of natural products such as essential oil of Citrus limon L. [5], Bellis perennis L. extract [6] and monoterpenes like (-)-a-terpineol [7], a-thujene [8], 1,8cineole, a-pinene [9], c-terpinene [10], sesquiterpene nerolodiol like [11], carvone, cyano-carvone [12, 13], carvacrol [14], isopulegol [15], isopulegone [16], limonene, geraniol and nerol [17], and polyphenols, as a derivative of green tea (-)-epigallocatechin gallate [18]. This study aimed to suggest the antioxidant potential of carvacryl acetate (CA), a monoterpene ester (Fig. 1)

759

derived from carvacrol [14]. In its chemical structure, CA has three benzylic hydrogens and the three a-hydrogens, which may bind to free radicals (Fig. 1). Despite being currently understudied, CA has already been reported on its potential for anthelmintic activity against Schistosoma mansoni [19]. In this study, CA was tested for in vitro and in vivo antioxidant properties. In vitro tests involved measuring lipid peroxidation and scavenging activity against hydroxyl radical (OH-) and nitric oxide (NO). For in vivo tests, mice hippocampi were used to measure lipid peroxidation, nitrite content, reduced glutathione (GSH) and glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase activities. In vitro results were compared with Trolox 140 lg/ml and in vivo results were compared with ascorbic acid (AA) 250 mg/kg intraperitoneal (ip). For in vivo tests, hippocampus was used due to its high sensibility to oxidative stress as well as the high affinity of lipophilic compounds to this neuroanatomical region. Therefore, an aromatic ring and an ester group probably give CA a property to penetrate the blood–brain barrier and to exert antioxidant effects in the hippocampus [14]. After results, correlation between CA in vitro antioxidant potential and in vivo antioxidant effect was established. To date, this is the first study to test and correlate these parameters of CA in mice hippocampus.

Materials and Methods Drugs and Reagents Reagents were polyoxyethylene sorbitate monooleate (Tween 80), ascorbic acid, 2-deoxyribose, 2,20 -azobis (2amidinopropane), Griess reagent, hydrogen peroxide, phosphate buffer, sodium nitroprusside, xanthine, xanthine oxidase, Triton X-100, TBA (thiobarbituric acid) and trichloroacetic acid. All of them were purchased by Sigma Aldrich (USA). Preparation of Carvacryl Acetate Chemically defined as ethyl 5-isopropyl-2-methyl-phenyl (Fig. 1), with a purity of 98 %, CA has a molecular weight of 192.26 g/mol and a refractive index of 1.497, boiling point of 94.56 °C at 760 mmHg, 48.414 kJ/mol of vaporization enthalpy and density of 0.994 g/cm3. Its color is yellow-green, and it has a pungent and astringent flavor, with a characteristic odor like oregano (Origanum vulgare L.). CA is found in liquid state at ambient temperature, with a density of 0.994 ± 0.06 g/cm3. It was obtained by carvacrol acetylation in which acetic anhydride was used as acylating agent and pyridine as catalyst. Carvacrol (5 g;

123

760

0.033 mol)—pyridine (7.5 ml) and acetic anhydride (12.5 ml)—purchased by Sigma Aldrich (USA)—were added in a flask (50 ml) equipped with magnetic stirrer, coupled to a Friedrich condenser. Then, it was subjected to magnetic stirring and under constant reflux for 24 h. Reaction mixture was poured into ice water (60 ml). The reaction product was extracted in a dropping funnel and chloroform was used as solvent extractor (3 times of 60 ml). Chloroform phases were combined and washed with saturated copper sulfate (3 times of 60 ml) and was also washed with water (3 times of 60 ml) and dried with Na2SO4 anhydrous. Subsequently, solvent was evaporated in a rotary evaporator. Reaction product was subjected to column chromatography using silica gel as stationary phase and a mixture of hexane, ethyl acetate (95:5), as mobile phase. There were obtained 4.779 g (0.025 mol) of CA with 76 % yield [20]. Confirmation of the chemical structure of CA was performed by infrared (IR) spectroscopic data, 1H NMR and 13C NMR DEPT: IR (4,000–400 cm-1): 3050; 2950; 2850; 1750; 1500; 850. 1H NMR (200 MHz, CDCl3): 7.20 (d, J = 7.80 Hz, 1H); 7.00 (d, J = 7.80 Hz, 1H); 6.90 (s, 1H); 2.95–2.75 (m, 1H); 2.30 (s, 3H); 2.15 (s, 3H); 1.26 (d, J = 6.80 Hz, 6H); 13C NMR DEPT (50 MHz-CDCl3): 169.1; 149.1; 147.9; 130.7; 127.0; 124.0; 119.6; 67.3; 33.4; 23.7; 20.6; 15.6. Referring to its yellow-green color, we tested a control group with CA, without positive controls, and it has not affected any of the spectrophotometric assays used in this study. Animals For in vivo tests, male Swiss albino mice (Mus musculus), weighing between 25 and 30 g, approximately 2 months old from the animal bioterium of the Center of Agricultural Sciences—CCA, Federal University of Piauı´ (UFPI), were used. Animals were kept under monitored conditions of temperature equivalent to 25 ± 1 °C, in acrylic cages (maximum six animals per cage) with free access to food pellets and water type, maintained in light–dark cycle of 12 h with light phase of 6 h–18 h, and were observed for 24 h on similar environmental conditions. Experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals of the Department of Health and Human Services of the United States of America (USA). All the experiments were previously submitted to approval by the Ethics Committee on Animal Experimentation, UFPI (EAEC/UFPI in 013/2011). Procedures relating euthanasia of animals were in accordance with the Sole Paragraph of 2th Article of Resolution number 714, of June

123

Neurochem Res (2014) 39:758–769

20, 2002 of the Federal Council of Veterinary Medicine – CFMV, avoiding animal suffering at each stage of the experiments. Treatment of Animal’s Experimental Groups for In Vivo Tests For in vivo tests, the animals were divided into six experimental groups. In order to obtain the first group, mice were treated with vehicle 0.01 ml/g intraperitoneally (negative control group, n = 10). The second group received AA at the dose of 250 mg/kg (positive control, ip, 250 AA group, n = 10). The other four groups were treated with CA (25, 50, 75 and 100 mg/kg, ip) (n = 10). CA was emulsified with Tween 80 0.05 % and dissolved in 0.9 % saline (vehicle). All the administrations were acutely, by a single intraperitoneal injection, in just one day. In order to perform the neurochemical studies, after evaluation of parameters related to acute toxicity during observation period, the animals were sacrificed by administering sodium pentobarbital. Neurochemical Studies in Mice Hippocampi for In Vivo Tests For neurochemical studies, we adopted the following methods: after 1 h of each single ip administration (vehicle, positive control or CA), the mice were subjected to euthanasia by decapitation with a guillotine (Harvard, USA). Then, the brains were quickly removed and placed on aluminum foil in a petri dish on ice. After dissection, each hippocampus was placed in foil, identified, weighed and stored at -80 °C for subsequent preparation of homogenates. Homogenates (10 % w/v) to the hippocampi were prepared with potassium phosphate buffer 50 mM, pH 7,8 and then centrifuged at 15,000 rpm for 15 min at 4 °C. Supernatants were removed to determine TBARS, nitrite and GSH levels as well as GPx, SOD and catalase activities. Evaluation of Antioxidant Activity Determining Antioxidant Potential Against the Formation of Thiobarbituric acid Reactive Substances In Vitro and In Vivo Thiobarbituric acid reactive substances (TBARS) assay was used to quantify lipid peroxidation and adapted according to a previously described method by Esterbauer and Cheeseman [21]. For in vitro tests, lipid peroxidation was induced by adding 0,1 ml of 2,20 -azobis-2-methylpropanimidamide dihydrochloride (AAPH, 0.12 M). The rich in lipids substrate was composed by an homogenate of

Neurochem Res (2014) 39:758–769

egg yolk (1 % w/v) in 50 mM phosphate buffer (pH 7.4). An aliquot of 0.5 ml of the substrate was sonicated, and then homogenized with 0.1 ml of CA at different concentrations (0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml). In the control group, there was tested only the vehicle (0.05 % Tween 80 dissolved in 0.9 % of saline solution). Reactions were performed for 30 min at 37 °C. The samples (0.5 ml) were centrifuged with 0.5 ml of trichloroacetic acid (15 %) at an acceleration of 1.2009g for 10 min. An aliquot of 0.5 ml of supernatant was mixed with 0.5 ml of thiobarbituric acid (0.67 %) and heated at 95 °C for 30 min. Absorbance of samples was measured using a UV–Vis spectrophotometer at 532 nm. Results were expressed as percentage of TBARS formed from AAPH only (induced control). Trolox (6-hydroxy-2,5,7,8tetramethylchromane-2-carboxylic acid) was used as a standard drug. In exception of vehicle group, all the other groups contain 0.1 ml of AAPH 0.12 M. For in vivo tests, samples were mixed to 1 ml of 10 % trichloroacetic acid and 1 ml of 0.67 % thiobarbituric acid. The samples were then heated in a water bath for 15 min. Butanol (2:1 v/v) solution was added. After centrifugation (8009g, 5 min), TBARS were determined. Malonaldehyde with TBA reaction produces a chromophore which can be measured photometrically at 532 nm. Results were expressed as nmol of malondialdehyde/g of tissue [22]. Determining In Vitro and In Vivo Antioxidant Potential of Carvacryl Acetate on Removing Nitrite Content Nitric oxide was generated from sodium nitroprusside (SNP), Na2[Fe(CN)5NO]2H2O decomposition in 20 mM phosphate buffer (pH 7.4). Once formed, NO interacts with oxygen to produce nitrite ions (NO2-), which were detected by Griess test [23]. For in vitro tests, the reaction mixture (1 ml) containing SNP 10 mM in standard phosphate and CA at different concentrations (0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml) was incubated at 37 °C for 1 h. An aliquot of 0.5 mL was taken and homogenized with 0.5 ml of Griess reagent. The absorbance of the chromophore was measured at 540 nm. Results were expressed as percentage of nitrite formed by the reaction medium. In exception of vehicle group, all the other groups contain SNP in decomposition in 20 mM phosphate buffer (pH 7.4) by Griess reaction. For in vivo tests, measurement of in vivo nitrite content in vehicle, AA 250 and CA 25, 50, 75, 100 (n = 10) hippocampi was also determined by the Griess reaction [24]. Nitrite was detected and analyzed through formation of a pink reddish color treating a sample containing NO2- with Griess reagent. Reagent contained 0.2 % of naftilethylenodiamine dihydrocloride and 2 % of sulfanilamide in 5 % of phosphoric acid.

761

In a blank tube, 500 ll of reagent was added to 500 ll of distilled water and, in another tube, 500 ll of reagent was added to 500 ll of tissue homogenate in a percentage of 10 %. Readings were taken at 560 nm in a spectrophotometer and results were expressed as lM. Determination of In Vitro Antioxidant Potential of Carvacryl Acetate Against the Formation of Hydroxyl Radical Formation of hydroxyl radical from Fenton’s reaction was quantified by oxidative degradation of 2-deoxyribose [25]. It was determined malonaldehyde level generated by its condensation with 2-thiobarbituric acid (TBA). Briefly, reactions were initiated by addition of Fe2? 6 mM final solutions containing 5 mM 2-deoxyribose, 100 mM of H2O2 and 20 mM phosphate buffer (pH 7.2) to determine antioxidant activity of CA at concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml against the formation of hydroxyl radical. CA was added before addition of Fe2?. Reactions were performed for 15 min at room temperature and was stationed by addition of H3PO4 at 4 % (v/v) followed by 1 % TBA (w/v) in 50 mM NAOH. The solutions were heated for 15 min at 95 °C, and then cooled to room temperature. Absorbance was measured at 532 nm using a SP 220 Biospectro spectrophotometer and results were expressed as percentage of levels of 2-deoxyribose degradation. In exception of vehicle group, all the other groups were composed with oxidative degradation of 2-deoxyribose, which allowed to quantify the formation of hydroxyl radical by the malonaldehyde generated. Determination of In Vivo Reduced Glutathione Levels and Glutathione Peroxidase Activity GSH was measured in CA 25, 50, 75, 100 groups, vehicle and AA 250 (n = 10) using the method described by Sedlak and Lindsay [26]. Hippocampi were homogenized in 0.02 M EDTA. Results were expressed in ng per gram of tissue. GSH was measured by enzymatic recycling process wherein reduced GSH is sequentially oxidized by 5,50 dithiobis-2-nitrobenzoic acid for oxidized GSH, which is then reduced in presence of NADPH by glutathione reductase (GR) [27]. Glutathione peroxidase was also measured in CA 25, 50, 75, 100 groups, vehicle and AA 250 (n = 10) using a method described by Sinet et al. [28], and protein concentration was measured according to a method described by Lowry et al. [29]. Determination of In Vivo Superoxide Dismutase Activity This method was prepared through reaction buffer containing 50 mM of potassium phosphate (pH 7.8), 500 mM

123

762

of xanthine, 200 lM of potassium cyanide and 1 mM of EDTA. Homogenates supernatants were removed for determination of SOD activity. Xanthine oxidase (XO - 5 U/ml) was used in reaction solution prepared from standard XO (1 lL for 80 lL of potassium phosphate buffer 50 mM, pH 7.8). Then, an assay containing 975 lL of reaction medium, 20 lL of sample and 5 lL of XO was performed. Mixture was stirred and the reading was performed for 6 min. Kinetic blank reading was done at 550 nm. SOD activity amount of samples was calculated using means of linear absorptions obtained by curve for 6 min. Protein concentration was measured by a method described by Lowry and collaborators [29]. One unit (U) of the SOD activity corresponds to 50 % inhibition of reaction of O2 with cytochrome C. Superoxide dismutase activity in the CA 25, 50, 75 and 100 as well as vehicle and AA 250 groups (n = 10) was tested using xanthine and xanthine oxidase to generate superoxide radicals, and was determined from a schematic curve. Results were expressed as U/mg of protein.

Neurochem Res (2014) 39:758–769

Results Antioxidant Potential of Carvacryl Acetate to Reduce Lipid Peroxidation Level In Vitro CA effects for in vitro lipid peroxidation are shown in Fig. 2a. For CA 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml groups, decreases of TBARS levels were respectively by 61, 66, 69, 70 and 76 % when compared with 2,2 ‘-azobis-2aminopropane (AAPH) group (p \ 0.001). For comparative purposes, Trolox 140 lg/ml reduced TBARS levels by 61 % (p \ 0.001). Comparing CA 1.8; 3.6; 5.4 and 7.2 lg/ ml with Trolox 140 lg/ml, there was a better effect than Trolox respectively by 13 % (p \ 0.05); 20 % (p \ 0.001); 26 % (p \ 0.0001) and 38 % (p \ 0.0001). The 50 % inhibitory effective concentration (EC50) of CA was 0.1785 lg/ml (r2 = 0.9654), with a variation between 0.0864 and 0.3691 lg/ml (confidence interval of 95 %). Trolox showed an EC50 of 10.45 lg/ml (r2 = 0.8660), with a margin of variation between 4.953 and 22.6 lg/ml (confidence interval of 95 %).

Determination of In Vivo Catalase Activity Catalase activity was measured in CA 25, 50, 75, 100 groups, vehicle and AA 250 (n = 10) by a method using H2O2 to generate H2O and O2 [30]. Protein concentration was measured by the method of Lowry and collaborators [29]. Results were expressed in mmol/min/mg of protein [31]. Statistical Analyses All results were presented as mean ± standard error of mean (SEM). Data were evaluated by analysis of variance (ANOVA) followed by Student’s t test-Neuman–Keuls as post hoc test. Data were analyzed using the GraphPad Prism 5.0 (San Diego, CA, USA). Experimental groups were compared with vehicle group and positive control. For in vitro tests, TBARS levels, production of nitrite and percentage of 2-deoxyribose degradation were measured. Trolox (6-hydroxy-2,5,7,8-tetrametilchrome-2-carboxylic acid), an analogue from vitamin E, which has antioxidant effects on the nervous cells [32] was considered positive control [33]. For in vivo tests, levels of TBARS, nitrite content, reduced GSH levels and GPx, SOD and catalase concentrations were measured. AA was considered positive control. Differences were considered statistically significant when p \ 0.05. In order to establish linear statistical relationship between in vitro antioxidant potential and in vivo antioxidant effect of CA, Pearson correlation coefficient was used. Confidence interval was established and statistical significance was considered when p \ 0.05.

123

Antioxidant Potential of Carvacryl Acetate to Remove Nitrite Content In Vitro CA effects on nitrite production in vitro are shown in Fig. 2b. For CA 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml groups, there was a reduction of 43, 45, 52, 57 and 60 %, respectively, when compared with SNP (p \ 0.001). Trolox reduced nitrite production by 60 % when compared with SNP (p \ 0.001). The EC50 of CA against NO formation was 0.1940 lg/ml (r2 = 0.9684), with a margin of variation in concentration of 0.09312–0.4043 lg/ml, with confidence interval of 95 %. Trolox showed an EC50 of 5.802 lg/ml (r2 = 0.8200) against nitrite ion formation with confidence interval of 95 % (2.807–11.99 lg/ml). Antioxidant Potential of Carvacryl Acetate in Reducing In Vitro Hydroxyl Radical Formation CA hydroxyl radical scavenging activity is shown in Fig. 2c. At concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ ml, CA reduced the 2-deoxyribose degradation respectively by 44, 50, 55, 59 and 74 % when compared with System (p \ 0.001) while Trolox reduced it by 75 % when compared with System (p \ 0.001). In addition to these results, a concentration-dependent effect related to CA groups was observed. When CA 0.9 lg/ ml was compared with CA 1.8 lg/ml, a 11 % superior effect was observed in the latter concentration (p \ 0.0001). When CA 1.8 was compared with 3.6 lg/ml, a 10 % greater effect was observed in the latter concentration (p \ 0.001). The comparison between CA 3.6 lg/ml with CA 5.4 lg/ml

Neurochem Res (2014) 39:758–769

763

showed a 9 % greater effect with the latter concentration (p \ 0.001). And finally, when compared CA 5.4 lg/ml with CA 7.2 lg/ml, a 37 % greater effect in the latter concentration was observed (p \ 0.0001). EC50 of CA against of hydroxyl radical formation was 0.1397 lg/ml (r2 = 0.9796), with a variation of 0.07363–0.2651 lg/ml and a confidence interval of 95 %. Trolox showed an EC50 of 5.288 lg/ml (r2 = 0.8742) against the formation of the hydroxyl radical with a confidence interval of 95 % (2.988–9.356 lg/ml). Antioxidant Potential of Carvacryl Acetate to Reduce In Vivo Thiobarbituric acid Reactive Substances Content CA at all doses (25, 50, 75 and 100 mg/kg ip – acute administration) suggested antioxidant effect, which was superior to AA 250 (Fig. 3a). AA 250 reduced the levels of TBARS by 47 % when compared with vehicle group (p \ 0.0001). CA 25, 50, 75 and 100 reduced TBARS levels respectively by 65, 70, 72 and 85 % when compared with vehicle group (p \ 0.0001). All CA groups had also a greater effect in reducing levels of TBARS when compared with AA 250 group. CA 25, 50, 75 and 100 group reduced TBARS levels by 33, 43, 48 and 71 % when compared with AA 250, respectively (p \ 0.0001). Antioxidant Potential of Carvacryl Acetate to Reduce In Vivo Nitrite Content CA (acute administration) reduced in vivo nitrite content when compared with vehicle group and had a superior effect to AA 250 (Fig. 3b). AA 250 reduced nitrite content by 49 % when compared with vehicle (p \ 0.0001) while CA at doses of 25, 50, 75 and 100 reduced it respectively by 78, 82, 85 and 92 % when compared with vehicle (p \ 0.0001). When CA 25, 50, 75 and 100 mg/kg were compared with positive control group (AA 250), decreases of nitrite content were respectively by 56, 65, 71 and 85 % (p \ 0.0001). Antioxidant Potential of Carvacryl Acetate for In Vivo Reduced Glutathione Levels

Fig. 2 Antioxidant in vitro potential of carvacryl acetate against thiobarbituric acid reactive substances (TBARS) production (a), nitrite ion formation (b) and hydroxyl radical (c). Values represent mean ± standard error of the mean, n = 5, experiments in duplicate. a p \ 0.001 versus vehicle; bp \ 0.001 versus generating system oxidative stress (AAPH, or SNP System), respectively (ANOVA and t-Student–Neuman–Keuls as post hoc test)

Both CA and AA 250 (acute administration) increased in vivo GSH levels when compared with vehicle group (p \ 0.05) (Fig. 3c). CA25 increased GSH by 26 % when compared with vehicle (p \ 0.05). CA50 increased this parameter by 33 % when compared with vehicle (p \ 0.05). CA75 increased this parameter by 40 % when compared with vehicle (p \ 0.05) and CA100 increased this parameter by 50 % when compared with vehicle (p \ 0.001).

123

764

Neurochem Res (2014) 39:758–769

Fig. 3 Carvacryl acetate (CA) effect on thiobarbituric acid reactive species (TBARS) (a), nitrite (b), reduced glutathione (GSH) levels (c) and on glutathione peroxidase activity (d) in mice hippocampus. Results represent mean ± standard error of the mean of number of

animals used in the experiments (n = 10/group). ap \ 0.0001 when compared with vehicle, bp \ 0.0001 when compared with ascorbic acid group (AA 250) (ANOVA and t-Student–Neuman–Keuls as post hoc test)

Antioxidant Potential of Carvacryl Acetate for In Vivo Glutathione Peroxidase Activity

Antioxidant Potential of Carvacryl Acetate for In Vivo Catalase Activity

CA 25, 50, 75, 100 groups (acute administration) increased GPx activity respectively by 49, 52, 71 and 102 % when compared with vehicle group (p \ 0.0001) (Fig. 3d). With a 37 % increase, when compared with vehicle group (p \ 0.0001), AA 250 augmented GPx activity by an inferior percentual when compared with all CA doses. When CA 75 mg/kg was compared with 50 mg/kg, there was a superior effect for the higher dose (p \ 0.001). When CA 100 mg/kg was compared with CA 75 mg/kg, there was a superior effect for the higher dose (p \ 0.0001) (Fig. 3d).

Both CA and AA 250 (acute administration) increased catalase activity (Fig. 4b). CA 25, 50, 75, 100 groups increased catalase activity respectively by 57, 63, 57 and 88 % when compared with vehicle (p \ 0.0001). AA 250 increased it by 62 % when compared with vehicle (p \ 0.0001).

Antioxidant Potential of Carvacryl Acetate for In Vivo Superoxide Dismutase Activity In this parameter, neither CA nor AA 250 significantly changed SOD activity when compared with vehicle group (Fig. 4a).

123

Discussion The brain has a lipid-rich constitution and is more vulnerable to lipid peroxidation products than any other tissue [12]. Thus, lipid peroxidation corresponds to an index of neuronal damage on phospholipid cell membrane of neurons. Therefore, a substance which decreases the formation of peroxyl radicals may have potential to prevent neuronal damage related to lipid peroxidation [34, 35]. CA at all concentrations protected lipids from oxidation when compared with AAPH.

Neurochem Res (2014) 39:758–769

Fig. 4 Carvacryl acetate (CA) effect on superoxide dismutase (SOD) (a) and catalase (b) activities in mice hippocampus. Results represent mean ± standard error of the mean of number of animals used in experiments (n = 10) (ANOVA and t-Student–Neuman–Keuls as post hoc test). ap \ 0.0001 when compared with vehicle

Cyano-carvone (CC), a derivative of monoterpene carvone, also showed inhibitory activity for in vitro lipid peroxidation. At concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml, CC reduced TBARS respectively by 56; 62; 66; 69.6 and 72.1 % when compared with AAPH (p \ 0.001) [12]. In this study, CA had better improves in comparison with CC at same concentrations (61, 66, 69, 70 and 76 %, respectively). Additionally, comparing CA with isopulegone at concentrations of 0.9; 1.8; 3.6; 5.4; 7.2 lg/ml, the last one reduced TBARS respectively by 59.35; 62.81; 63.97; 65.04 and 65.96 % when compared with AAPH (p \ 0.001) [16]. And, comparing CA with nerolidol, a sesquiterpene found in many essential oils of medicinal plants, the last one at concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml decreased TBARS respectively by 63.41; 66; 67.14; 68.72 and 73.22 % when compared with AAPH (p \ 0.001) [11]. In this parameter, only CA 0.9 lg/ml showed a lower percentage than nerolidol in reducing

765

TBARS. However, when TBARS in vitro and in vivo levels were correlated in this study, there was no statistical significance, since r2 = 0.7280, p C 0.1467 and confidence interval was between -0.5996 and 0.9969 (Fig. 5a). These findings indicate that, despite CA suggests in vitro and in vivo potential to reduce TBARS levels, there is no correlation between those two parameters. The investigation for CA in vitro antioxidant potential to nitrite scavenging demonstrated that CA at all five concentrations reduced nitrite production when compared with SNP group (43, 45, 52, 57 and 60 %, respectively). For comparative purpose, a study which measured scavenging activity of CC in concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml for in vitro NO- production demonstrated an inhibition respectively by 19.6; 22.6; 26.9; 32.3 and 32.1 % when compared with SNP (p \ 0.001) [12]. Isopulegone reduced nitrite radical contents by 24.76; 27.81; 34.35; 40.98 and 49.18 %, at respective concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml, when compared with SNP (p \ 0.001) [16]. Thus, the percentages of reduction on nitrite content with CC and isopulegone treatment were lower than those of CA, which means that CA could be more effective at the same concentrations. Under normal conditions, there is a regulation on the balance between the rate of production of NO and its metabolites with antioxidant systems. However, in neurodegenerative conditions, this balance can be impaired, with increased production of NO [36, 37]. Thus, a substance which rebalances this rate and combat reactive oxygen and reactive nitrogen species may have a preventive potential against diseases like diabetes, cancer, multiple sclerosis, ischemic damage, Alzheimer’s and Parkinson0 s disease [38–43]. Regarding the ability to scavenging hydroxyl radical, CA at the five concentrations reduced the levels of 2-deoxyribose when compared with System, similarly with it was observed in Trolox group. For comparative purpose, CC 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml displays antioxidant activity against formation of in vitro hydroxyl radicals respectively by 47.2; 51.1; 52.2; 57.6 and 64.6 when compared with System (p \ 0.001) [12]. CA had higher percentage values of hydroxyl radical reduction than CC at the three highest concentrations (44, 50, 55, 59 and 74 %, respectively). Isopulegone 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ml removed hydroxyl radicals respectively by 45.28; 48.62; 53.67; 60.19 and 63.87 % when compared with system [p \ 0.001]. It was inferior to CA when the four highest concentrations were compared [16]. In another study, nerolildol at concentrations of 0.9; 1.8; 3.6; 5.4 and 7.2 lg/ ml reduced hydroxyl radical formation by 40.39; 47.44; 48.41; 49.5 and 52.9 %, respectively, when compared with System (p \ 0.001) [11], while CA had better effect when the four highest concentrations were compared.

123

766

Neurochem Res (2014) 39:758–769

A

B

C

D

E

Fig. 5 Carvacryl acetate (CA) effect on the correlation between TBARS content in vitro and in vivo (r2 = 0,7280; p C 0,1467) (a); Nitrite content in vitro and reduced glutathione (GSH) levels in vivo (r2 = 0.9671, p B 0.0166) (b); Nitrite content in vitro and glutathione peroxidase (GPx) activity in vivo (r2 = 0.9520, p B 0.0243) (c); hydroxyl radical (OH) levels in vitro and in vivo superoxide

dismutase (SOD) activity (r2 = 0.9479, p B 0.0264) (d); and Nitrite content in vitro and Catalase activity in vivo (r2 = 0,5741; p C 0,2423) (e). To be established linear statistical relationship, the Pearson correlation coefficient was used, with statistical significance when p \ 0.05

Hydroxyl radical reacts readily with various essential biomolecules to cell survival, particularly DNA, proteins and lipids polysaccharide chain [38–40, 43]. Such damage can cause degenerative diseases. Therefore, a substance which will increase scavenging of these radicals and/or increase activity of the enzymatic system which scavenge these radicals implicates a potential to combat degenerative diseases [42]. In addition, CA demonstrated a lower EC50 than trolox for all measurements of TBARS, nitrite and hydroxyl radical. This is an important result, since the EC50 is concentration required to inhibit 50 % of formation of these radicals within 1 h—the lower the value of this parameter, the more potency has the substance to scavenge these radicals.

Referring to in vivo lipid peroxidation and reactive nitrogen species scavenging, CA 25, 50, 75 and 100 mg/kg suggested more effectiveness and more potency than AA 250 mg/kg. For in vivo GSH levels, AA 250 increased this parameter better than all CA doses, but it should be noted that AA dose (250 mg/kg) was proportionally superior to CA highest dose (100 mg/kg) (Fig. 3c). Pearson correlation between reduction on in vitro nitrite content and in vivo GSH by CA demonstrated a strong negative correlation, since r2 = 0.9671, p B 0.0166 with a negative confidence interval (between -0.9997 and -0.4067). Pearson correlation coefficient was greater than 0.8, which is a strong linear correlation between the parameters in question (Fig. 5b). It suggests that CA may have its antioxidant effect not only on reduction of in vitro nitrite and

123

Neurochem Res (2014) 39:758–769

GSH levels improvement activity per se, but a potentiating effect of interaction with these two parameters. Due to the fact that endogenous GSH is an antioxidant, reacting with free radicals and protecting nerve cells [44, 45], we consider that the higher the concentration of reduced GSH, lower should be nitrite levels in mice hippocampi. Referring to in vivo GPx activity, a greater effect of CA was observed at the four doses when compared with AA 250. CA also displays a dose-dependent effect for this parameter. The Pearson correlation between reduction for in vitro nitrite content and in vivo activity of GPx by CA demonstrated a strong negative correlation, since r2 = 0.9520, p B 0.0243 with a negative confidence interval (between -0.9995 and -0.2339). Thus, this coefficient was greater than 0.8, a strong linear correlation between the parameters in question (Fig. 5c). This correlation was expected as GPx is part of enzymatic defense system, which protects the brain against oxidative stress [46] and, therefore, it has the property to reduce free radical contents. It suggests that CA may have its antioxidant effect not only on reduction of in vitro nitrite levels and increasing GPx activity per se, but a potentiating effect of interaction with these two parameters in mice hippocampi. Regarding SOD activity, it is known that, although superoxide anion is a weak oxidant, it produces potentially damaging hydroxyl radicals and singlet oxygen. Both OHand singlet oxygen radicals contribute to oxidative stress [47]. SOD enzyme catalyzes dismutation of superoxide into oxygen and hydrogen peroxide, being an important antioxidant defense on the CNS [42]. In this study, this parameter was not altered by any group treated with CA. Statistical significance was neither observed for AA 250 (Fig. 4a). However, there was a strong negative Pearson correlation between reduction on in vitro OH- content and in vivo SOD activity, since r2 = 0.9479, p B 0.0264 and with a negative confidence interval (between -0.9995 to -0.1947), with a coefficient greater than 0.8 (Fig. 5d). It corroborates that, despite CA did not alter SOD activity, the higher this parameter, lowest must be content of free radicals like hydroxyl radical. Finally, catalase activity in mice hippocampus was increased in all four doses of CA when compared with vehicle. Catalase is responsible for H2O2 decomposition, direct product of dismutation of superoxide radical in H2O and O2 [42, 48–50]. Along with GSH, SOD, GPx and glutathione reductase (GSH-Rd), catalase is part of enzymatic defense system which protects the brain against oxidative stress [46]. Thus, a substance that will increase catalase activity may also potentially increase free radicals scavenging and, therefore, prevent cellular damage. However, when in vivo Catalase was correlated with in vitro Nitrite, there was no statistical significance, since r2 = 0.5741, p C 0.2423 and confidence interval was

767

between -0.9945 and 0.7486 (Fig. 5e). These findings indicate that, despite CA increased Catalase in vivo, and reduced in vitro Nitrite levels, there is no correlation between those two parameters. Probably, all these action mechanism of CA may be, at least in part, due to its chemical structure, considering its four benzylic hydrogens and its three a-hydrogens, which bind to free radicals. Furthermore, the addition of an ester group to carvacrol gives CA an additional lipophilic property, which allows an easier penetration on the blood– brain barrier and exerts its antioxidant potential more safely and effectively when compared with other antioxidants already available [51, 52].

Conclusion This study allows us to conclude that CA demonstrated an in vitro antioxidant activity by reducing lipid peroxidation, nitrite production and hydroxyl radical formation, as well as in vivo effects. In many procedures, CA demonstrated better antioxidant effects than standard substances and monoterpenes. It reduced lipid peroxidation, nitrite content as well as increased reduced GSH levels. Additionally, CA improved GPx and catalase activities in mice hippocampus. There was also a negative correlation between in vitro nitrite levels and in vivo GSH levels as well as between in vitro nitrite content and in vivo GPx activity and a significant correlation between in vitro hydroxyl radical content and in vivo SOD activity. All these findings suggest that CA could have a potential to prevent and treat neurodegenerative diseases, which have in their pathophysiologic hypotheses an overproduction of free radicals and deficiencies in enzymatic defense system against oxidative stress. Acknowledgments We would like to thank the National Council of Technological and Scientific Development (CNPq/Brazil) and the Research Supporting Foundation of State of Piaui (FAPEPI/Brazil) for the financial support and Steˆnio Gardel Maia for technical assistance. Conflict of interest

The authors state no conflict of interest.

References 1. Bouayed J, Rammal H, Soulimani R (2009) Oxidative stress and anxiety. Oxid Med Cell Longev 2:63–67 2. Santos IMS, Tome´ AR, Saldanha GB, Ferreira PMP, Milita˜o GCG, Freitas RM (2009) Oxidative stress in the hippocampus during experimental seizures can be ameliorated with the antioxidant ascorbic acid. Oxid Med Cell Longev 2:214–221 3. Nobre Ju´nior HV, Fonteles MMF, Freitas RM (2009) Acute seizure activity promotes lipid peroxidation, increased nitrite levels and adaptive pathways against oxidative stress in the frontal cortex and striatum. Oxid Med Cell Longev 2:130–137

123

768 4. Freitas RLM, Santos IMS, Souza GF, Saldanha GB, Tome´ AR, Freitas RM (2010) Oxidative stress in rat hippocampus caused by pilocarpine-induced seizures is reversed by buspirone. Brain Res Bull 81:505–509 5. Campeˆlo LM, Gonc¸alves FC, Feitosa CM, Freitas RM (2011) Antioxidant activity of Citrus limon essential oil in mouse hippocampus. Pharm Biol 49:709–715 6. Marques THC, Melo CHS, Freitas RM (2012) In vitro evaluation of antioxidant, anxiolytic and antidepressant-like effect of the Bellis perennis extract. Rev Bras Farmacogn 22:1044– 1052 7. Silva OA, Almeida AAC, Carvalho RBF, Nogueira Neto JD, Sousa DP, Freitas RM (2012) Potencial antioxidante in vitro do (-)-a-terpineol. Biofar Rev Biol Farm 8:140–152 8. Mothana RAA, Hasson SS, Schultze W, Mowitz A, Lindequist U (2012) Phytochemical composition and in vitro antimicrobial and antioxidant activities of essential oils of three endemic Soqotraen Boswellia species. Food Chem 126:1149–1154 9. Wannes WA, Mhamdi B, Sriti J, Jemia MB, Ouchikh O, Hamdaoui G, Kchouk ME, Marzouk B (2010) Antioxidant activities of the essential oils and methanol extracts from myrtle (Myrtus communis var. italica L.) leaf, stem and flower. Food Chem Toxicol 48:1362–1370 10. Ruberto G, Baratta MT (2000) Antioxidant activity of selected essential oil components in two lipid model systems. Food Chem 69:167–174 11. Nogueira Neto JD, Sousa DP, Freitas RM (2013) Avaliac¸a˜o do potencial antioxidante in vitro do nerolidol. Rev Cieˆnc Farm Ba´sica Apl 34:125–130 12. Costa DM, Oliveira GAL, Sousa DP, Freitas RM (2012) Avaliac¸a˜o do potencial antioxidante in vitro do composto ciano-carvona. J Basic Appl Pharm Sci 33:567–575 13. Costa DA, Oliveira GAL, Lima TC, Santos PS, Sousa DP, Freitas RM (2012) Anticonvulsant and antioxidant effects of cyanocarvone and its action on acetylcholinesterase activity in mice hippocampus. Cell Mol Neurobiol 32:633–640 14. Baser KH (2008) Biological and pharmacological activities of carvacrol and carvacrol bearing essential oils. Curr Pharm Des 14:3106–3119 15. Silva MIG, Silva MAG, Neto MRA, Moura BA, Sousa HL, Lavor EPH, Vasconcelos PF, Maceˆdo DS, Sousa DP, Vasconcelos SMM, Sousa FCF (2009) Effects of isopulegol on pentylenetrazol-induced convulsions in mice: possible involvement of GABAergic system and antioxidant activity. Fitoterapia 80:506–513 16. Silva AO, Oliveira FRAM, Lima TC, Sousa DP, Souza AA, Freitas RM (2012) Evaluation of the antioxidant effects in vitro of the isopulegone. Free Rad Antiox 2:50–55 17. Conforti F, Statti GA, Tundis R, Loizzo MR, Menichini F (2007) In vitro activities of Citrus medica L. vc. Diamante (Diamante citron) relevant to treatment of diabetes and Alzheimer’s disease. Phytother Res 21:427–433 18. Vignes M, Maurice T, Lante F, Nedjar M, Thethi K, Guiramand J, Recasens M (2006) Anxiolytic properties of green tea polyphenol (x)-epigallocatechin gallate (EGCG). Brain Res 1110:102–115 19. Moraes J, Carvalho AA, Nakano E, Almeida AA, Marques TH, Andrade LN, Freitas RM, Sousa DP (2013) Anthelmintic activity of carvacryl acetate against Schistosoma mansoni. Parasitol Res 112:603–610 20. Vogel AI, Tatchell AR, Furnis BS, Hannaford AJ, Smith PWG (1996) Vogel’s textbook of practical organic chemistry, 5th edn. Prentice Hall, Englewood Cliffs, New Jersey 21. Esterbauer H, Cheeseman KH (1990) Determination of aldehydic lipid peroxidation products: malonaldehyde and 4-hydroxynonenal. Methods Enzymol 186:407–421

123

Neurochem Res (2014) 39:758–769 22. Draper HH, Hadley M (1990) Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 186:421–431 23. Basu S, Hazra B (2006) Evaluation of nitric oxide scavenging activity, in vitro and ex vivo, of selected medicinal plants traditionally used in inflammatory diseases. Phytother Res 20:896–900 24. Green LC, Tannenbaum SR, Goldman P (1981) Nitrate synthesis in the germfree and conventional rat. Science 212:56–58 25. Lopes GKB, Schulman H, Hermes-Lima M (1999) Polyphenol tannic acid inhibits hydroxyl radical formation from Fenton reaction by complexing ferrous ions. Biochim Biophys Acta 1472:142–152 26. Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal Biochem 25:192–205 27. Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem 106:207–212 28. Sinet PM, Michelson AM, Bazin A, Lejeune J, Jerome H (1975) Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 subjects. Biochem Biophys Res Commun 67:910–915 29. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275 30. Chance B, Maehly AC (1955) Assay of catalases and peroxidases. Methods Enzymol 2:764–775 31. Maehly AC, Chance B (1954) The assay of catalases and peroxidases. Methods Biochem Anal 1:357–359 32. Spanevello R, Mazzanti CM, Schmatz R, Bagatini M, Stefanello N, Correa M, Kaizer R, Maldonado P, Mazzanti A, Grac¸a DL, Martins TB, Danesi C, Morsch VM, Schetinger MRC (2009) Effect of vitamin E on ectonucleotidase activities in synaptosomes and platelets and parameters of oxidative stress in rats experimentally demyelinated. Brain Res Bull 80:45–51 33. Marı´n-Prida J, Pento´n-Rol G, Rodrigues FP, Alberici LC, Stringhetta K, Leopoldino AM, Naal Z, Polizello ACM, Llo´pzArzuaga A, Rosa MN, Liberato JL, dos Santos WF, Uyemura SA, Pento´n-Arias E, Curti C, Pardo-Andreu GL (2012) C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2?/phosphate-induced impairment. Brain Res Bull 89:159L–167L 34. Cruz VP, Gonza´lez-Corte´s C, Pedraza-Chaverrı´ J, Maldonado PD, Andre´s-Martı´nez L, Santamarı´a A (2006) Protective effect of S-allylcysteine on 3-nitropropionic acid-induced lipid peroxidation and mitochondrial dysfunction in rat brain synaptosomes. Brain Res Bull 68:379–383 35. Freitas RM, Vasconcelos SMM, Souza FCCF, Viana GSB, Fonteles MMF (2005) Oxidative stress in the hippocampus after pilocarpine-induced status epilepticus in Wistar rats. FEBS J 272:1307–1312 36. Mello FAC, Hoffamn ME, Meneghini R (1983) Cell killing and DNA damage by hydrogen peroxide are mediated by intracellular iron. Biochem J 218:273–275 37. Pazdernik TL, Emerson MR, Cross R, Nelson SR, Samson FE (2001) Soman-induced seizures: limbic activity, oxidative stress and neuroprotective proteins. J Appl Toxicol 21:87–94 38. Balausubramanian B, Pogozelski WK, Tullius TD (1998) DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. Proc Natl Acad Science USA 95:9738–9743 39. Begusova M, Giliberto S, Gras J, Sy D, Charlier M, SpotheimMaurizot M (2003) DNA radiolysis in DNA-protein complexes: a stochastic simulation of attack bu hydroxyl radicals. Int J Radiat Biol 79:385–391 40. Ogino T, Okada S (1995) Oxidative damage of bovine serum albumin and other enzyme proteins by iron–chelate complexes. Biochim Biophys Acta 1245:359–365

Neurochem Res (2014) 39:758–769 41. Oyagi A, Oida Y, Hara H, Izuta H, Shimazawa M, Matsunaga N, Adachi T, Hara H (2008) Protective effects of SUN N8075, a novel agent with antioxidant propertires, in in vitro and in vivo models of Parkinson0 s disease. Brain Res 1214:169–176 42. Schereibelt G, Van Horssen J, Van Rossum S, Dijkstra CD, Drukarch B, De Vries HE (2007) Therapeutic potential and biological role of endogenous antioxidant enzymes in multiple sclerosis pathology. Brain Res Rev 56:322–330 43. Sutton HC, Winterbourn CC (1989) On the participation of higher oxidation states of iron and copper in Fenton reactions. Freed Rad Biol Med 6:53–58 44. Oliveira A, Almeida JPC, Freitas RM, Nascimento VS, Aguiar LMV, Ju´nior HVN, Fonseca FN, Viana GSB, Sousa FCF, Fonteles MMF (2007) Effects of levetiracetam in lipid peroxidation level, nitrite–nitrate formation and antioxidant enzymatic activity in mice brain after pilocarpine-induced seizures. Cell Mol Neurobiol 27:395–406 45. Sapakal VD, Shikalgar TS, Ghadge RV, Adnaik RS, Naikwade NS, Magdum CS (2008) In vivo screening of antioxidant profile: a review. J Herbal Med Toxicol 2:1–8 46. Dallaqua B, Damasceno DC (2011) Comprovac¸a˜o do efeito antioxidante de plantas medicinais utilizadas no tratamento do

769

47. 48.

49.

50. 51.

52.

Diabetes mellitus em animais: artigo de atualizac¸a˜o. Rev Bras Plantas Med 13:367–373 Meyer AS, Isaksen A (1995) Application of enzymes as food antioxidants. Trends Food Sci Technol 6:300–304 Lanfocazal M, Culcasi M, Gaven F, Pietri S, Bockaert J (1993) Nitric-oxide, superoxide and peroxynitrite—putative mediators od NMDA-induced cell-death in cerebellar granule cells. Neuropharmacol 32:1259–1266 Miles AM, Gibson MF, Kirshna M, Pacelli R, Wink D, Cook JC, Grisham MB (1995) Effect of superoxide on nitric oxidedependent N-nitrosation reactions. Free Radic Res 23:379–390 Olanow CW (1993) A radical hypothesis for neurodegeneration. Trends Neurosci 16:439–444 Okada Y, Tanaka K, Sato E, Okajima H (2008) Antioxidant activity of the new thiosulfinate derivative, S-benzylphenylmethanethiosulfinate, from Petiveria alliacea. L Org Biomol Chem 6:1097–1102 Be´nardais K, Pul R, Singh V, Skripuletz T, Lee DH, Linker RA, Gudi V, Stangel M (2013) Effects of fumaric acid esters on blood–brain barrier tight junction proteins. Neurosci Lett 555:165–170

123