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«Universidade Federal de Goiás, Goiânia, GO, Brasil Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brasil Corresponding ...»

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Antigenotoxic, and anticytotoxic activities

of an ethanolic extract of Lafoensia pacari

(Lythraceae) stem bark in bacteria and mice

D.C.S. Lima1, C.R. Silva1, B.L. Sampaio2, J.R. de Paula2 and

L. Chen-Chen1

Departamento de Biologia Geral, Instituto de Ciências Biológicas,

Universidade Federal de Goiás, Goiânia, GO, Brasil

Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO, Brasil

Corresponding author: L. Chen-Chen

E-mail: chenleego@yahoo.com.br

Genet. Mol. Res. 12 (3): 3887-3896 (2013) Received January 26, 2013 Accepted July 29, 2013 Published September 23, 2013 DOI http://dx.doi.org/10.4238/2013.September.23.7 ABSTRACT. Lafoensia pacari (Lythraceae), popularly known in Brazil as “pacari”, is a small tree native to the Cerrado that is used in folk medicine to treat cancer and as an anti-inflammatory and cicatrizing agent.

We evaluated the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activities of an ethanol extract of L. pacari stem bark (EESB) using the Ames test and the mouse bone marrow micronucleus test. In the Ames test, EESB did not significantly increase the number of His+ revertants in Salmonella typhimurium tester strains TA98 and TA100 at all doses, demonstrating lack of mutagenicity. Only the highest dose of EESB significantly increased the micronucleated polychromatic erythrocyte frequency in the micronucleus test, indicating mild genotoxicity.

EESB produced a mutagenic index lower than the negative control in the Ames test. In the micronucleus test, at all doses, EESB caused a significant decrease in the polychromatic/normochromatic erythrocyte ratio (PCE/NCE) at 24 h compared with the negative control. EESB co-administered together with the respective positive control caused ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (3): 3887-3896 (2013) D.C.S. Lima et al.

a significant decrease in the number of His+ revertant colonies in the Ames test and in the frequency of micronucleated polychromatic erythrocytes in the micronucleus test, demonstrating a D

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INTRODUCTION

Brazil is a country that has the greatest biodiversity on the planet. Recent studies indicate that between 170,000 and 210,000 Brazilian species are known to science, approximately 13% of the world’s biota (Nascimento et al., 2011). A large portion of this biodiversity is found in the Brazilian Cerrado, a region that covers approximately 25% of the total land area of the country and is characterized by a gradient of grassland to savanna and forest formations, as well as high richness of animal and plant species (Ribeiro et al., 2011).

The Cerrado is home to more than 7000 native species of vascular plants and many of them have been widely used in folk medicine by local people to treat a variety of diseases (Hiruma-Lima et al., 2006). In fact, several plants provide health benefits by exhibiting antihemorrhagic (Mazzolin et al., 2010), antioxidant (Souza et al., 2012), and antimutagenic activities (Vilar et al., 2009b). Moreover, several studies have demonstrated that numerous active compounds from Brazilian Cerrado plants show promising biological properties for cancer therapy (de Mesquita et al., 2009).

However, many plants can also pose health risks since they exhibit cytotoxic (Silva et al., 2012), genotoxic (Vilar et al., 2009a), and mutagenic activities (Ferreira et al., 2009).

Long-term use of genotoxic compounds can cause DNA damage and promote the development of degenerative diseases, such as cancer (Santos et al., 2011).

Lafoensia pacari A. St.-Hil., popularly known in Brazil as “pacari”, “dedaleiro”, “didal” and “mangabeira-brava”, is a medicinal plant of the family Lythraceae that is widely used as an antipyretic, wound healing, anti-inflammatory, and antidiarrheal agent, as well as in the treatment of gastritis and cancer (Sampaio et al., 2011). Infusion or maceration in white wine or in water for oral administration are the most common methods for preparing a remedy with this plant (Solon et al., 2000; Galdino et al., 2009).

The phytochemical analysis of L. pacari has revealed that this species contains phenolic compounds, mainly tannins and flavonoids (Solon et al., 2000; Galdino et al., 2009;

Sampaio et al., 2011). It also possesses many biological properties, such as free radical scavenging (Solon et al., 2000), antidyspeptic (da Mota et al., 2006), anti-inflammatory (Rogerio et al., 2008), antidepressant-like (Galdino et al., 2009), and antifungal (Silva Junior et al., 2010) activities. Nevertheless, so far and to the best of our knowledge, no studies of the genotoxic and antigenotoxic effects of L. pacari have been published.

Due to the broad biological activity of L. pacari and aiming to contribute to the safe and efficient use of this plant in folk medicine, the objective of the present study was to evaluate the genotoxic, cytotoxic, and protective effects of the ethanolic extract from L. pacari stem ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (3): 3887-3896 (2013) Lafoensia pacari antigenotoxic and anticytotoxic activities bark (EESB) using the Ames test and the mouse bone marrow micronucleus test.

The Ames mutagenicity test uses several different tester strains of Salmonella typhimurium to measure two classes of gene mutation, namely base pair substitution and small frameshifts (Mortelmans and Zeiger, 2000). This test serves as a model for predicting and understanding the toxicological properties of the test substance (Claxton et al., 2010).





The micronucleus test is widely used to detect clastogenicity (chromosome breakage) and aneugenicity (chromosome lagging resulting from damage to the mitotic apparatus), and therefore, it has been the primary in vivo test accepted and recommended by the regulatory agencies around the globe for product safety assessment (Krishna and Hayashi, 2000).

MATERIAL AND METHODS

Plant material Samples of L. pacari stem bark were collected in Urutaí (17°22ꞌ04.4ꞌꞌS; 48°12ꞌ19.6ꞌ ꞌW), in the state of Goiás, Brazil. The air-dried and powdered stem bark was exhaustively extracted with 95% aqueous ethanol at room temperature for three days, and the resultant alcohol solution was filtered and concentrated to dryness. The EESB powder obtained was transferred to glass flasks filled to the top and kept at 5°C until the moment of use. For the Ames test, the EESB powder was dissolved in sterile distilled water and dimethyl sulfoxide (DMSO) (3:1), whereas for the micronucleus test it was dissolved in DMSO alone.

Ames test - Salmonella mutagenicity assay Strains Salmonella typhimurium tester strains TA98 and TA100 were kindly supplied by the Laboratório de Radiobiologia Molecular of the Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Experimental procedure The S. typhimurium histidine point mutation assay proposed by Maron and Ames (1983) was followed. A 0.1-mL aliquot of bacterial suspension (1-2 x 109 cells/mL) of each strain (TA98 and TA100) was incubated with 0.5, 1.0, 2.0, and 4.0 mg/plate EESB at 37°C for 25 min. A 2.0-mL aliquot of top agar (0.6% Difco agar, 0.5% NaCl, 50 μM L-histidine and 50 μΜ biotin, at 45°C) was added to the test tubes and poured onto Petri dishes containing minimal agar medium (1.5% agar, 2% glucose, and Vogel-Bonner medium E). Each assay was performed three times in triplicate and included a negative [100 µL sterile distilled water and DMSO (3:1)] and a positive control [0.5 μg 4-nitroquinoline 1-oxide (4-NQO) per plate for TA98 and 1.5 μg sodium azide for TA100]. For the evaluation of antimutagenicity, doses of 0.5, 1.0, 2.0 and 4.0 mg/plate EESB were incubated and combined with their respective positive controls. After incubation at 37°C for 48 h, the His+ revertant colonies were counted.

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Statistical analysis The softwares Excel and/or Sigma Stat 3.5 were used in all analyses. All the results were tabulated and the experimental values were expressed as mean ± standard deviation (SD). The data obtained from the experiments of mutagenicity were evaluated by ANOVA and Tukey’s test for difference of means. The magnitude of the mutagenicity induction was measured by the mutagenic index (MI), calculated as the ratio between the number of colonies in the test treatment and the number of colonies in the negative control treatment. The inhibition percentage of mutagenicity (IP) induced by each mutagen was calculated in relation to the number of revertant colonies obtained in the control group treated with the mutagen alone,

using the following formula (Sghaier et al., 2011):

test plates: plates incubated with mutagen and extract control plates: plates incubated with the mutagen alone SR: spontaneous revertants (test strains incubated in the absence of extract and mutagen) Mouse bone marrow micronucleus test Animals This study was approved by the Human and Animal Research Ethics Committee of the Universidade Federal de Goiás (CoEP/UFG No. 224/2011). Healthy, young male adult (8-12 weeks) outbred mice (Mus musculus, Swiss Webster), weighing 25-30 g, obtained from the Central Animal House of the Universidade Federal de Goiás were used in the study. All animals were brought to the laboratory five days before the experiments and housed in plastic cages (40 x 30 x 16 cm) at 24 ± 2°C and 55 ± 10% humidity, with a light-dark natural cycle of 12 h. Food (appropriate commercial rodent diet Labina, Ecibra Ltda., Santo Amaro, SP, Brazil) and water were given ad libitum.

Experimental procedure The experiments were performed according to von Ledebur and Schmid (1973). For the evaluation of genotoxicity, three doses of EESB (100, 200, and 300 mg/kg body weight) were orally administered to groups of five animals for each treatment. The same doses were administered in combination with mitomycin C (MMC, 4 mg/kg i.p., Lot No. 237AEL, Bristol, Meyers Squibb, São Paulo, SP, Brazil) to groups of five animals for each treatment for the evaluation of antigenotoxicity.

A positive control group (MMC) and a negative control group (DMSO) were also included.

The animals were euthanized by cervical dislocation 24 or 48 h after the administration of EESB, and their bone marrow cells were flushed from both femurs in fetal bovine serum (FBS, Lot No. 30721063, Laborclin, Campinas, SP, Brazil). After centrifugation (300 x g, 5 min), the bone marrow cells were smeared on glass slides, coded for blind analysis, air-dried, and fixed with absolute methanol (CH4O, Lot No. 55026, Synth, Diadema, SP, Brazil) for 5 min at room temperature. The smears were stained with Giemsa (Lot No. 1081, Doles, Goiânia, GO, Brazil), dibasic sodium phosphate (Na2HPO4·12H2O, Lot No. 982162, ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (3): 3887-3896 (2013) Lafoensia pacari antigenotoxic and anticytotoxic activities Vetec, Duque de Caxias, RJ, Brazil) and monobasic sodium phosphate (NaH2PO4·H2O, Lot No. 983831, Vetec) to detect micronucleated polychromatic erythrocytes (MNPCE).

For each animal, four slides were prepared and 2000 polychromatic erythrocytes (PCE) were counted to determine the frequency of MNPCE using light microscope (Olympus BH-2 10 x 100, Tokyo, Japan). Genotoxicity and antigenotoxicity were assessed by the frequency of MNPCE, whereas cytotoxicity and anticytotoxicity were evaluated by the PCE and normochromatic erythrocytes (NCE) ratio (PCE/NCE).

Statistical analysis

To analyze the genotoxic activity of EESB using the mouse bone marrow micronucleus test, the frequencies of MNPCE of the treated groups were compared with the results obtained for the negative control groups using one-way ANOVA. P 0.05 was considered indicative of significance. To assess its cytotoxicity, the PCE/NCE ratio obtained at different concentrations of EESB was compared with the negative control by the chi-square test (χ2). P

0.05 was considered indicative of significance.

RESULTS

Ames test - Salmonella mutagenicity assay The results of the mutagenic and antimutagenic evaluation are presented in Table 1.

All results were from three independent experiments carried out in triplicate. The data obtained for the positive and negative control groups indicate that the strains were in agreement with the guidance established both by Maron and Ames (1983) and Mortelmans and Zeiger (2000).

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µg sodium azide for TA100. Mutagenicity: aSignificant difference compared to the negative control (P 0.05). bNo significant difference compared to the negative control (P 0.05). Antimutagenicity: cNo significant difference compared to the positive control (P 0.05). dSignificant difference compared to the positive control (P 0.05).

In the evaluation of mutagenicity, the doses of EESB tested (0.5, 1.0, 2.0 and 4.0 mg/ plate) did not cause an increase in the number of His+ revertant colonies in either tester strain, TA98 or TA100, since no statistically significant difference was observed between the negative

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control group and any dose of EESB (P 0.05). Furthermore, none of the strains tested reached MI ≥ 2 or dose-response effect with EESB treatment, with the highest induction occurring for strain TA100, which reached MI = 1.12 at the dose of 1.0 mg/plate. The results of the Ames test demonstrated that EESB did not exhibit a mutagenic effect. Tester strains TA98 and TA100 exhibited an MI lower than with the negative control group (MI = 1.00), at almost all doses, indicating that this extract possibly had a slight toxic effect against S. typhimurium cells.

The antimutagenic analyses demonstrated that at all doses tested (0.5, 1.0, 2.0 and 4.0 mg/plate) EESB showed a significant decrease in the number of His+ revertant colonies in tester strains TA98 and TA100 compared with the respective positive controls (P 0.05). At the doses of 0.5, 1.0, 2.0, and 4.0 mg/plate, EESB showed an IP of 28, 44, 80 and 92% in strain TA98, and 42, 44, 54 and 67% in strain TA100, respectively. These results demonstrated that EESB exhibited relevant antimutagenic action at all doses, but mainly at the dose of 4.0 mg/plate, which produced an IP of 92 and 67%, in TA98 and TA100, respectively. Therefore EESB was able to significantly protect DNA from the mutagens 4-NQO and sodium azide at all doses tested.



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