(−)-α-Bisabolol-induced gastroprotection is associated with reduction in lipid peroxidation, superoxide dismutase activity and neutrophil migration

European Journal of Pharmaceutical Sciences 44 (2011) 455–461

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( )-a-Bisabolol-induced gastroprotection is associated with reduction in lipid peroxidation, superoxide dismutase activity and neutrophil migration Nayrton Flávio Moura Rocha a,⇑, Gersilene Valente de Oliveira a, Fernanda Yvelize Ramos de Araújo a, Emiliano Ricardo Vasconcelos Rios a, Alyne Mara Rodrigues Carvalho a, Leonardo Freire Vasconcelos a, Danielle Silveira Macêdo a, Pedro Marcos Gomes Soares a, Damião Pergentino De Sousa b, Francisca Cléa Florenço de Sousa a a b

Universidade Federal do Ceará, Dep. Fisiologia e Farmacologia, Cel. Nunes de Melo 1127, Rodolfo Teofilo, 60416010 Fortaleza, Ceara, Brazil Universidade Federal de Sergipe, Centro de Ciências Biológicas e da Saúde, Departamento de Fisiologia. Universidade Federal de Sergipe, Campus I 49100-000 - Sao Cristovao, Brzsil

a r t i c l e

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Article history: Received 5 May 2011 Received in revised form 18 August 2011 Accepted 22 August 2011 Available online 7 September 2011 Keywords: ( )-a-Bisabolol Ulcer Super oxide dismutase Gastroprotection Natural compound

a b s t r a c t This work examined the gastroprotection of ( )-a-bisabolol, an unsaturated optically active sesquiterpene alcohol obtained by the direct distillation essential oil from plants. ( )-a-Bisabolol has been described as a compound capable of reducing the gastric ulcer area in response to absolute ethanol. We evaluated the gastroprotection of ( )-a-bisabolol in ethanol-induced gastric lesions model through histopathological assessment, measurement of the membrane lipids peroxidation (MDA), myeloperoxidase (MPO) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity and the nitrite amount. Our results showed that ( )-a-bisabolol was able to reduce injuries associated with the administration of ethanol and the formation of thiobarbituric acid reactive substances (MDA) was also able to increase SOD activity and reduce the influx of cells inflammatory (neutrophils) in the gastric mucosa. The effect of ( )a-bisabolol seems to be unrelated to the nitric oxide. ( )-a-Bisabolol caused a reduction of catalase activity. These findings show that ( )-a-bisabolol is able to decrease oxidative stress and inflammatory event associated with the lesions induced by ethanol. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction ( )-a-Bisabolol is an unsaturated, optically active sesquiterpene alcohol obtained by the direct distillation essential oil from plants. The most common source utilized is the chamomile, Matricaria chamomilla (Reynolds, 1996). However, there are others sources from which ( )-a -bisabolol can be extracted, for example, the Brazilian plant species Vanillosmopsis erythropappa, is a rich supply of this substance (Vichnewski et al., 1989). In recent work our demonstrated that ( )-a-bisabolol has a gastroprotective effect against ethanol and indomethacin-induced ulcer in mice related to capacity to reduce the decrease GSH amount in gastric mucosa, showing an antioxidant activity (Rocha et al., 2010). The Reactive Oxygen Species (ROS) has been described as one of the probable pathogenic factors of gastric mucosal lesions associated with water immersion stress, anti-inflammatory drugs and ulcers induced by ethanol (Bilici et al., 2002). Under these conditions, there is an imbalance between formation and degradation of these ⇑ Corresponding author. Address: Universidade Federal do Ceará, Dep. Fisiologia e Farmacologia, Cel. Nunes de Melo 1127, Rodolfo Teófilo, 60416010 Fortaleza, Ceará, Brazil. Tel.: +55 85 33668337. E-mail address: [email protected] (N.F.M. Rocha). 0928-0987/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2011.08.020

species: the enzymatic antioxidant defenses and non-enzymatic can not restrain the ROS increase, thus, may exert deleterious actions on the gastric mucosal epithelium. The formation of (ROS) is a constant event in the cells and occurs as a result of normal metabolism, as in cellular respiration or in pathological conditions such as inflammation. The ROS include the superoxide anion (O2 ), hydrogen peroxide (H2O2) and hydroxyl radical (O2 ) (Cnubben et al., 2001). Under physiological conditions, approximately 95% of molecular oxygen undergoes controlled reduction in mitochondrial cytochrome oxidase system to form water. The rest of the molecular oxygen undergoes incomplete reduction leading to formation of ROS (Fesharaki et al., 2006). These species are highly damaging to cells, because they interact indiscriminately with DNA, lipids and proteins and can change their functions. They can react and lead to damage to membrane proteins changing cell permeability and consequently the whole cell physiology may be compromised, as well as interactions with DNA can lead to mutations and cancer development (Cnubben et al., 2001; Koc et al., 2008). Superoxide anions and hydroxyl radicals are involved in a number of degenerative changes, frequently associated with an increase in peroxidation processes linked to low concentration of antioxidants (Tamagno et al., 1998).

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The cells of the gastrointestinal tract have an antioxidant defense system capable of preventing the cytotoxicity of ROS through mechanisms that involve the action of enzymes and compounds with potential to scavenge free radicals. In the list of enzymes involved in this action are superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (Cat). The mucosa is also protected by a system of ‘‘hijackers’’ of ROS as thiols, reduced glutathione (GSH), alpha-tocopherol (vitamin E), vitamin C, carotenoids, methionine and taurine, which bind to oxygen radicals and prevent their harmful actions (Fesharaki et al., 2006). The enzyme SOD has the function of removing the superoxide anion of the cell environment by catalyzing of dismutation reaction of these anions, generating oxygen and hydrogen peroxide (H2O2) (Fattman et al., 2003). The peroxide hydrogen is mainly detoxified by the action of catalase which then generates oxygen and water. GSH-px selenium dependent is also capable of catalyze the reaction of breaking of hydrogen peroxide into oxygen and water (Matés, 2000). GSH also occupies an important place in the antioxidant mechanisms of cells. This tripeptide binds quickly and directly with hydroxyl radical, peroxynitrite and nitrosating species preventing the cells from interacting with these citotoxic products (Griffith, 1999). Moreover, GSH is the cofactor of the enzyme GSH-px and, enzymatically, through the action of glutathione S-transferase binds to electrophilic compounds potentially harmful to the cells (Cnubben et al., 2001). Ethanol-induced lesions model has been used to evaluate possible anti-ulcer activity of some substances, this agent produces characteristic necrotic lesions in the gastric mucosa of rodents, and the quantitative analysis of lesions’ area is the index utilized to assess a potential gastroprotective action. The ( )-a-bisabolol has been described as a naturally occurring compound capable of reducing the gastric ulcer area in response to absolute ethanol (Bezerra et al., 2009; Rocha et al., 2010). Acute administration of absolute ethanol generates an inflammatory response that, as such, is the result of a complicated chain of events involving the immune response, which release great content of free radicals, enzymes caustic and inflammatory cytokines. In this context, the antioxidant defense system is not fully effective in protecting these tissues. It is common observation that the administration of additional antioxidants substances that exert their action directly or indirectly, have the potential to slow or even prevent the damage caused by increased oxidative stress (Silva et al., 2009; Bilici et al., 2002) so the investigation of a possible antioxidant associated with a compound effective in reducing gastric damage induced by ethanol becomes essential for better characterization and mechanistic study of drug action. 2. Methods 2.1. Animals Male Swiss mice (24–32 g) were used in this study. Animals were kept in a temperature-controlled room at 25 ± 2 °C with a 12-h light/dark cycle, with food and water ad libitum. The study was approved by the Ethics Committee for Animal Research at the Federal University of Ceará in Brazil, and it was conducted in accordance with the National Institute of Health in Bethesda, USA. 2.2. Histopathological assessment Histological evaluation was performed on the glandular face of the stomach. Tissue samples were preserved in 10% buffered formalin and processed for routine paraffin block preparation. Sections about 4 mm thick were cut and stained with hematoxylin

and eosin. The mucosal injury evaluation was performed under light microscopy by an experienced histologist blinded to the treatment regimen. The histopathological changes were assessed according to the following criteria that were previously described by Laine and Weinstein (1988): (1) edema (score 0–4), (2) hemorrhagic damage (score 0–4), and (3) epithelial cell loss (score 0–3). 2.3. Measurement of the membrane lipids peroxidation The rate of lipoperoxidation in the gastric mucous membrane was estimated by determination of malondialdehyde (MDA) using the Thiobarbituric Acid Reactive Substances (TBARS) test. The stomachs were washed with saline to minimize the interference of hemoglobin with free radicals and to remove blood adhered to the mucous membrane. The stomachs were homogenized to 10% of tissue with potassium phosphate buffer. Then, 250 lL was removed and stored at 37 °C for 1 h, after which 400 lL of 35% perchloric acid was added, and the mixture was centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was removed, mixed with 400 lL of 0.6% thiobarbituric acid and incubated at 95–100 °C for 1 h. After cooling, the absorbance at 532 nm was measured. A standard curve was generated using 1,1,3,3-tetrametoxypropane. The results were expressed as nmol of MDA/mg of protein. The concentration of proteins was measured using the method described by Bradford, 1976. Measurement of total protein in the stomach sample after ethanol-induced lesions. 2.4. Protein quantification The method is based on the interaction of the Coomassie Blue G250 dye with proteins. At the pH of the reaction, the interaction between proteins of high molecular weight and the dye causes a shift in the dye to the anionic form, which absorbs strongly at 595 nm. Solutions of albumin standard, distilled water, buffer and samples were added to the wells. For sample preparation, 2 lL of sample and 38 lL of buffer were added to each well. Then, 200 lL Bradford’s solution (diluted 5) was added to each well. After 5 min, a reading was taken at the wavelength of 595 nm Bradford, 1976. 2.5. Measurement of the myeloperoxidase (MPO) activity The animals were divided into groups of eight and were sacrificed 30 min after administration of the ethanol. Stomach samples were removed and immediately frozen in liquid nitrogen. Samples were then homogenized in a solution of 0.5% hexadecyltrimethylammonium bromide (HTAB) in 50 mM phosphate buffer pH 6.0 (1 mL per 50 mg of the tissue) and centrifuged at 4000 rpm for 15 min at 4 °C. The amount of the enzyme in the supernatant (30 lL) was analyzed by spectrophotometry after addition of 200 lL of phosphate buffer (50 mM, pH 6), containing 0.167 mg/ ml of h-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The absorbance at 470 nm was measured at time 0 and 5 min (Bradley et al., 1982). 2.6. Measurement of the superoxide dismutase (SOD) activity SOD activity was measured according to Sun et al. (1988). The activity of the enzyme was evaluated by measuring its capacity to inhibit the photochemical reduction of nitro-blue tetrazolium (NBT). In this assay, the photochemical reduction of riboflavin generates O2 that reduces the NBT to produce formazan salt, which absorbs at a wavelength of 560 nm. In the presence of SOD, the reduction of the NBT is inhibited because the enzyme converts the superoxide radical to peroxide. The results are expressed as the quantity of SOD necessary to inhibit the rate of reduction of

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the NBT by 50% in units of the enzyme per gram of protein. Homogenates (10% of tissue in buffer phosphate) were centrifuged (10 min, 3600 rpm, 4 °C), and the supernatant was removed and centrifuged a second time (20 min, 12,000 rpm, 4 °C). The resulting supernatant was assayed. In a dark chamber, 1 mL of the reactant (50 mM phosphate buffer, 100 nM EDTA and 13 mM l-methionine, pH 7.8) was mixed with 30 lL of the sample, 150 lL of 75 lM NBT and 300 lL of 2 lM riboflavin. The tubes containing the resulting solution were exposed to fluorescent light bulbs (15 W) for 15 min and then read using a spectrophotometer at 560 nm.

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The activity of the enzyme is measured at 230 nm using spectrophotometry (Beckman DU) by reading the variation of the absorbance between the first and sixth minutes and then expressing the results in lM/min/lg of protein. The reaction environment was prepared with H2O2 (18 mL), 1 M Tris HCl buffer 5 mM EDTA pH 8 (1 mL) and Milli-Q H2O (0.8 mL). Immediately, 980 lL of the reaction environment was added to 20 lL of the homogenate (10%) in a quartz cuvette.

2.8. Measurement of the nitrite amount 2.7. Measurement of catalase (CAT) activity Catalase activity is proportional to the rate of output of O2 and H2O. Hydrogen peroxide (H2O2) is used as the substrate and is hydrolyzed according to the Maehly and Chance method (1954).

The amount of stable nitrite, the end product of NO metabolism, in the gastric mucosa was determined by a colorimetric assay as described by Green et al. (2000). Briefly, 100 lL of gastric mucosa homogenate was mixed with an equal volume of Griess reagent that consists of equal parts of 1% sulfanilamide and 0.1% naphthyl

Fig. 1. Macroscopic (A-Non lesioned; B-BIS 100 and C-Vehicle) and microscopic aspect of gastric mucosa of mice pre-treated with vehicle (Tween 80 3% in distilled water, p.o.) (Ulcer), ( )-a-bisabolol 100 and 200 mg/kg (BIS 100, BIS 200) or N-acetyl cisteine(NAC). Animals treated with ethanol, showing intense inflammatory infiltration, edema, hemorrhage and epithelial cell loss. (Ulcer). Pretreatment with ( )-a-bisabolol (100 and 200 mg/kg, p.o., respectively) before the administration of ethanol, showing preservation of gastric mucosa. Quantitative results from these assessments are shown in Fig. 2.

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ethylenediamine dihydrochloride (NEED), 5% H3PO4 and distilled water and incubated at room temperature for 10 min. The absorbance was read at 540 nm on a microplate reader (UVM-340, Asys Hitech, Netherlands). The amount of nitrite was calculated from a NaNO2 standard curve. 3. Results 3.1. Histopathological assessment after ethanol-induced lesions Histopathological analyses of the gastric mucosa are shown in Figs. 1 and 2. Animals pretreated with ( )-a-bisabolol (100 and 200 mg/kg, p.o.) showed less macro- and microscopic mucosal damage when compared with the control group treated only with ethanol (Fig. 1), in which severe lesions were characterized by hemorrhagic injury, edema and loss of epithelial cells. Pretreatment with NAC also inhibited lesions promoted by ethanol. ( )-a-Bisabolol significantly decreased the ethanol-induced lesions to a similar extent as NAC Fig. 2. 3.2. Measurement of the myeloperoxidase (MPO) activity The analyses of MPO activity in the animals ethanol-injured pre-treated only with vehicle showed an increase (25.85 ± 4.25 uMPO/mg of tissue, p < 0.05) compared with animals non-injured by ethanol (9.84 ± 0.63). The treatment with ( )-abisabolol diminished the MPO activity in gastric mucosa of animals injured by absolute ethanol (0.2 mL) compared with animals ethanol-injured pre-treated only with vehicle, in both doses utilized in this study, the reduction was significant (⁄p < 0.05) as can be seen in the Fig. 3. In the dose of 100 mg/kg the reduction was in mean 37.67% (16.11 ± 2.45) and in the dose of 200 mg/kg was 39.84% (15.55 ± 0.485) NAC 300 mg/kg reduced the MPO activity with significance (11.50 ± 1.10) compared with animals ethanol-injured pre-treated only with vehicle. 3.3. Measurement of membrane lipid peroxidation (MDA) The quantification of MDA in the animals ethanol-injured pretreated only with vehicle showed an increase

Total scores

10 8 6

uMPO/mg of wet tissue

458

30 25 20

*

15 10

#

5 0

Vehicle Vehicle

NAC

Bis 100 Bis 200

Ethanol Fig. 3. Effect of ( )-a-bisabolol(Bis) 100 and 200 mg/kg on MPO activity in the gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to ethanol. The results are presented as mean ± SEM. ⁄p < 0.05 and # p < 0.05, significant difference compared with vehicle (ethanol-lesioned) group. ANOVA followed by Newman–Keuls as the post hoc test was used.

(12.17 ± 0.80 nMols/g of tissue, p < 0,05) compared with animals non-injured by ethanol (4.24 ± 1.18). The treatment with ( )a-bisabolol was capable to decrease the MDA amount in gastric mucosa of animals injured by absolute ethanol (0.2 mL) in comparison with animals ethanol-injured pre-treated only with vehicle, in both doses utilized in this study, the reduction was significant (⁄p < 0.05) as can be seen in the Fig. 4. In the dose of 100 mg/kg the reduction was in mean 67.33% (3.975 ± 0.78) and in the dose of 200 mg/kg was 58.60% (5.038 ± 0.87) NAC 300 mg/kg reduced the MDA amount with significance (7.89 ± 1.58) compared with animals ethanol-injured pre-treated only with vehicle. 3.4. Nitrite content in the injured stomach after ethanol-induced lesions The quantification of nitrite in the animals ethanol-injured pretreated only with vehicle showed a decrease (1.64 ± 0.12 nM, ⁄ p < 0.05) compared with animals non-injured by ethanol (2.88 ± 0.3). The treatment with ( )-a-bisabolol did not show difference in the nitrite amount in gastric mucosa of animals injured by absolute ethanol (0.2 mL) in comparison with animals ethanolinjured pre-treated only with vehicle, in both doses utilized in this study, as can be seen in the Fig. 5.

*

4 2 0

Vehicle

Bis 200

Bis 100

NAC

Ethanol Fig. 2. Scores of histopathological changes in the gastric mucosa of mice subjected to gastric lesions induced by ethanol pretreated or not with ( )-a-bisabolol. Animals were treated orally with vehicle (3% Tween 80), ( )-a-bisabolol (BIS 100 and 200 mg/kg), or N-acetylcysteine (NAC 300 mg/kg), 60 min before administration of ethanol (0.2 mL, p.o.). Histopathological changes of glandular portion of stomach were assessed as described by Laine and Weinstein (1988). The results are presented as median with minimum and maximum values of the total scores [edema (score 0–4), hemorrhagic damage (score 0–4), and epithelial cell loss (score 0–3)] shown in parentheses (n = 4). ⁄p < 0.05, #p < 0.05 vs. vehicle + ethanol. We used the nonparametric Kruskal–Wallis followed by Dunn’s test as post hoc.

Fig. 4. Effect of ( )-a-bisabolol(Bis) 100 and 200 mg/kg on lipoperoxidation level (content of MDA) in the gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to ethanol. The results are presented as mean ± SEM. ⁄p < 0.05 and #p < 0.05, significant difference compared with vehicle (ethanol-lesioned) group. ANOVA followed by Newman–Keuls as the post hoc test was used.

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µMols/min/µg of protein

5.0×10 4

*

4.0×10 4 3.0×10 4

#

2.0×10 4 1.0×10 4 0 Vehicle Vehicle

NAC

Bis 100 Bis 200

Ethanol

Fig. 5. Effect of ( )-a-bisabolol(Bis) 100 and 200 mg/kg on nitrite content of gastric tissue in mice (n = 8/group). One additional group received vehicle and was not exposed to ethanol. The results are presented as mean ± SEM. #p < 0.05, significant difference compared with vehicle (ethanol-lesioned). ANOVA followed by Newman–Keuls as the post hoc test was used.

*

uSOD/mg of protein

12 # 8

Fig. 7. Effect of ( )-a-bisabolol(Bis) 100 and 200 mg/kg on catalase activity in mice (n = 8/group). One additional group received vehicle and was not exposed to ethanol. The results are presented as mean ± SEM. ⁄p < 0.05 and and #p < 0.05, significant difference compared with vehicle (ethanol-lesioned). ANOVA followed by Newman–Keuls as the post hoc test was used.

comparison with animals ethanol-injured pre-treated only with vehicle, in both doses utilized in this study, the reduction was significant (⁄p < 0.05) as can be seen in the Fig. 7. In the dose of 100 mg/kg the reduction was in mean 35.24% (27493 ± 2485) and in the dose of 200 mg/kg was 28.23% (30943 ± 2852) NAC 300 mg/kg reduced the catalase activity with significance (32654 ± 2214) compared with animals ethanol-injured pretreated only with vehicle. 4. Discussion

4

0

Vehicle Vehicle

NAC

Bis100 Bis 200

Ethanol Fig. 6. Effect of ( )-a-bisabolol(Bis) 100 and 200 mg/kg on superoxide dismutase activity in mice (n = 10/group). One additional group received vehicle and was not exposed to ethanol. The results are presented as mean ± SEM. ⁄p < 0.05 and and # p < 0.05, significant difference compared with vehicle (ethanol-lesioned). ANOVA followed by Newman–Keuls as the post hoc test was used.

3.5. Measurement of the superoxide dismutase (SOD) activity The quantification of SOD activity in the animals ethanol-injured pre-treated only with vehicle showed a decrease (6.59 ± 0.38 uSOD/mg of protein, p < 0.05) compared with animals non-injured by ethanol (8.05 ± 0.212). The treatment with ( )-abisabolol was capable to increase SOD activity in gastric mucosa of animals injured by absolute ethanol (0.2 mL) in comparison with animals ethanol-injured pre-treated only with vehicle, in both doses utilized in this study, the increase was significant (⁄p < 0.05) as can be seen in the Fig. 6. In the dose of 100 mg/kg the increase was in average 31.03% (8.63 ± 0.56) and in the dose of 200 mg/kg was 36.42% (8.99 ± 0.71). 3.6. Measurement of the catalase (CAT) activity The measurement of catalase activity in the animals ethanol-injured pre-treated only with vehicle showed an increase (43120 ± 5791 lmols/min/lg of protein, p < 0.05) compared with animals non-injured by ethanol (26232 ± 1952), The treatment with ( )-a-bisabolol was able to decrease the catalase activity in gastric mucosa of animals injured by absolute ethanol (0.2 mL) in

The mechanism of injury induced by ethanol is very varied, including the reduction of bicarbonate secretion and mucus production (Marhuenda et al., 1993), damage to blood flow and gastric mucosal cell lesion (Birdane et al., 2007). The ethanol-induced damage is also associated with excessive production of free radicals that attack cellular essentials constituents such as nucleic acids, proteins and lipids (La casa et al., 2000). The increased contents of lipid peroxides and free radicals derived from oxygen results in significant changes in cellular levels, membranes damage, cell death and epithelial erosion (Birdane et al., 2007). Ethanol-induced lesions were microscopically characterized by hemorrhage, edema, inflammatory infiltrate, and loss of epithelial cells; this result is consistent with other studies (Silva et al., 2009; Medeiros et al., 2008). Our findings showed that ( )-abisabolol was able to maintain the integrity of the gastric mucosa against the damaging effects of ethanol evidenced by analyzes of total scores of histophatological marker of injury and by MPO measurement (inflammatory infiltrate indicator). The degree of lipid peroxidation in tissues was measured by determining the amount of MDA, this analysis may relate directly to the level of injury to a tissue (Kanter et al., 2005). It is well described in literature the ability of absolute ethanol increase the amount of MDA, which is closely related with gastric damage associated. (Li et al., 2011). The increase in levels of MDA was also significantly associated with increased apoptosis in gastric tissue of animals subjected to orogastric treatment with absolute ethanol (Li et al., 2011). Our findings show that the absolute ethanol (0.2 mL/animal, po) significantly increased the production of malonidialdeído (MDA), however, the ( )-a-bisabolol was able to prevent increase in the amount of MDA induced by ethanol which shows an antioxidant activity and is consistent with the findings of the histopathological as well as with previous studies that showed gastroprotective ability of this substance. The superoxide anions (O2 ), products of the interaction between molecules of oxygen and electrons from the transport chain

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in mitochondria, is converted by the action of superoxide dismutase (SOD) to hydrogen peroxide, which in turn is detoxified by glutathione peroxidase (GPx) and catalase (CAT). These enzymes constitute an endogenous antioxidant system, which acts by preventing cell damage induced by free radicals and ROS (Remmen et al., 2004). Ethanol has been linked to increased production of superoxide anions and decreased activity of SOD, both factors contribute to the generation of oxidative damage, because the ethanol increases the production of a harmful factor and simultaneously reduces its detoxification capacity (Mutoh et al., 1990; Potrich 2009). In our study, the ( )-a-bisabolol showed a beneficial role increasing SOD activity (compared with animals ethanol-lesioned treated only with vehicle) and thereby enhance the dismutation of superoxide anion. In animals without associated gastric damage the SOD activity is higher than in animals treated with ethanol, which is consistent with previous reports (Rios et al., 2010). The SOD enzyme may be induced by oxidative stress, which constitutes a mechanistic of defense, in case of stress induced by ethanol this ability of induction and, therefore, the defense strategy is diminished (Nordberg and Arner, 2001). The ability to increase SOD activity makes the ( )-a-bisabolol, a compound with gastroprotective activity acting under antioxidant defense system and this fact may constitutes one of the mechanisms of action of this substance. Hydrogen peroxide (H2O2) is a degradation product of superoxide anion under action of SOD and is the substrate of the enzyme catalase (CAT). This peroxide is an oxidizing agent that under the action of CAT is detoxified in H2O and O2. Hydrogen peroxide is a molecular species, unlike other reactive oxygen species (ROS), such as hydroxyl (OH) and superoxide anion (O2 ) being both radicals. Hydrogen peroxide and superoxide anions do not exert their greatest toxic effects by direct action; but they participate in the generation of more harmful specie – hydroxyl radical. (OH) (Campos and Yoshida, 2004). The Superoxide and H2O2 give rise to hydroxyl radical (OH), by entering the Haber–Weiss and Fenton reaction, respectively, both being dependent on iron ions. (Halliwell, 1989). Our results showed that administration of absolute ethanol at a dose (0.2 mL/animal, po) increased the enzymatic activity of catalase in comparison to healthy animals. The understanding of the change in catalase activity in gastric mucosa by the acute administration of ethanol is not peaceful in the literature, Mitchell et al. 2010 found a decrease in this enzyme activity in the presence of ethanol, Rios et al. 2010 showed an increase in activity in the presence of ethanol and Ineu et al. 2008 reported no change in the activity of Cat in the presence of ethanol. In our study, ethanol-lesioned animals that were pretreated with ( )-a-bisabolol or NAC had a reduction in Cat activity compared to ethanol-lesioned animals treated with vehicle, treatment with ( )-a-bisabolol showed catalase activity similar to healthy animals (non lesioned). Despite the recognized deleterious role of H2O2 in the living system and particularly when applied directly on the gastric mucosa (Mohamadin et al., 2009) the background on the role of the enzyme Cat under ulcers induced by ethanol makes it difficult to interpret these results, because, in this model, gastroprotective substances do not follow a pattern of activity. Considering the fact that real role of Cat in the model of gastric damage induced by ethanol is somewhat obscure, the increased degradation of superoxide anions is shown as a parameter more closely associated with the antioxidants able to reduce gastric injury associated with absolute ethanol. The enzyme myeloperoxidase (MPO) is the main marker of neutrophil infiltration in ulcerogenic lesions, this enzyme is found within the neutrophils and catalyzes the oxidation of chloride ion by H2O2 to form hypochlorous acid (HOCl) potent reactive oxygen species (ROS) (Nian-sheng et al., 2010). Neutrophils represent the first line of defense of innate immune response phagocyting, killing

and digesting bacteria and fungi; however there is free radical generation and other forms of ROS such as superoxide anion (Segal, 2005; Klebanoff, 2005). Some studies have shown that ethanol induces the infiltration of neutrophils in gastric mucosa correlating directly the genesis of ulcerative lesions with the amount of migrant neutrophils and the induction of an acute inflammation process. This study showed that in ethanol-induced gastric lesions, administration of ( )-a-bisabolol at doses of 100 and 200 mg/kg orally reduced migration of neutrophils to the gastric mucosa compared to animals not receiving this treatment. This finding supports the proposition that the ethanol-induced lesions have an inflammatory characteristic by inducing neutrophil migration in these lesions and allow us to relate the anti-ulcer action of ( )a-bisabolol to the reduction of number of neutrophils in the gastric mucosa. This data come together harmoniously to others obtained by our group (sent to publication), which describe an anti-inflammatory action this substance. Thus, ( )-a-bisabolol prevent the activation of these cells and induction of oxidative stress and inflammatory damage, as occurs with fucoidin, a substance capable of preventing the migration of neutrophils and that displays an outstanding ability to reduce gastric injuries associated with the anti-inflammatory drugs (Souza et al., 2008). Nitric oxide is a key mediator in the defense of the gastrointestinal mucosa (Muscará and Wallace, 1999). Kato et al. (1998) demonstrated the inhibitory effect of NO on acid production and Brown et al. (1993) associated the NO with increased secretion of bicarbonate by gastric epithelium. Nitric oxide is also involved in maintaining basal blood flow of gastric microcirculation (Guth, 1992). Khattab et al. (2001) reported that local NO levels are reduced in models of gastric lesions induced by ethanol and indomethacin, suggesting that the decrease of NO content can be a decisive factor to facilitate gastric mucosal injury. In our study, absolute ethanol decreased the amount of nitrite, a product of NO metabolism, reinforcing the idea that the decrement of NO is an important factor in gastric lesions induced by ethanol. Treatment with ( )-a-bisabolol not caused significant changes in the amount of nitrite compared to injured animals that received only vehicle. This finding is in agreement with previous data, where the pre-treatment with LNAME, NO synthesis antagonist, did not affect the ability of ( )a-bisabolol protect the gastric mucosa. Thus, it seems that the metabolic pathway of nitric oxide is not involved in the gastroprotective effect of ( )-a-bisabolol. In conclusion, the ( )-a-bisabolol was able to reduce injuries associated with the administration of ethanol and the formation of thiobarbituric acid reactive substances (MDA) was also able to increase SOD activity and reduce the influx of cells inflammatory (neutrophils) in the gastric mucosa, these findings show that ( )a-bisabolol is able to decrease oxidative stress and inflammatory event associated with the lesions induced by ethanol. The effect of ( )-a-bisabolol seems to be unrelated to the nitric oxide and the capacity of reduction of catalase activity seems to be less important for explaining the activity antiulcerogenic molecule.

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