EFFECTS CYTOTOXIC AND GENOTOXIC OF AQUEOUS EXTRACT OF FENNEL (Foeniculum vulgare var. vulgare Mill.)

Fennel (Foeniculum vulgare Mill), originating in the basin of the Eastern Mediterranean and Caucasian, is now cultivated in many varieties selected by the sweetness and low concentrations of anethole, which allows consumption in salad dressings and condiments. Other varieties were selected to obtain high concentrations of essential oils and are used in folk medicine pharmaceutical industry. Despite the long history of application ethnomedicina l F. vulgare, no reports of serious side effects, several studies have shown that they can present cytotoxic and genotoxic activity. Therefore, this study aimed to analyze the cytotoxic and genotoxic effects of aqueous extract of fennel (Foeniculum vulgarevar. vulgare Mill.), Prepared in 2%, 4% and 8%, compared to bioindicators (Allium cepa L. and Musmusculus L.). The aqueous extract of F. vulgarea concentration of 2% cytotoxic effects caused significantly inhibiting mitotic division and genotoxic effects, providing chromosomal damage induced micronucleus formation in meristematic cells of Allium cepa L. In experimental animals in vivo genotoxic potential was found in cell bone marrow of mice. In this way, fennel tea should be consumed with moderations and medical care, especially for infants who have not yet developed the digestive tract, it may be harmful to health.

A long history in etnomédica application without any reports of serious side effects, suggests that F. vulgare can be considered safe (6). However, no work described above on the phenotypic plasticity, which F. vulgare, can be provided for different concentrations of metabolic compounds according to the environment in which it is planted.
Severalstudies performed with F. vulgare from different geographical origins and different varieties indicate that the contents of estragole in fruits can vary from 2% to 86% and the trans-anethole from 0% to 89% (7).According to Simõeset al. (1999), the environment in which the plant grows exertsgreat influence on production and the compositionofthe chemistry included in the essential oils (8). Temperature, relative humidity, duration of sun exposure and wind regime can have a direct influence, especially on species that have histological structures of oil storage on the leaf surface, such asF. vulgare.
According to Gross et al. (2009)  Phytochemical phenotypic plasticity and failure to identify varieties is likely to be a major cause of disputes found in the scientific literature regarding the toxic effects of F. vulgare.
In acute toxicity tests F. vulgare, determined the median lethal dose (LD50) of 1,326 mg/kg, with the occurrence of prostration, sedation, respiratory discomfort, movement disorder, apathy to external stimulation, weakness, tremors and fasciculations in the dorsal muscles of the guinea pigs during the first 24 hours of treatment(11).
The Unified Health System (SUS), said in its medicinal plant program publications Central medicines (CEME) that F. vulgare is toxic in preclinical toxicology studies Detection of potentially cytotoxic and genotoxic substances and their likely effects on organisms, it is important in the sense of the impact that they can bring to people, animals, plants and humans. v.6, n.1, p.80-94, Jan-Mar, 2019 Considering the wide use of F. vulgare in Brazilian folk medicine and in cooking; and little information on their potential citogenotóxics and genotoxic effects, the use of bioassays are needed to provide reliable information to the public.
Thus, this study aimed to assess and verify the cytotoxic effects and genotóxic effects of aqueous leaf extract Foeniculum vulgare var. vulgare Mill. on bioindicators.
2.0 Results and Discussion.

2.1System Test plant
In Table 1, it appears that aqueous leaf extract of fennel to 2%, relative to the

TABLE 1
With respect to IMT, IP, IM, IT IA and the control group (1% glyphosate), it was found that the aqueous extract of fennel 2% showed similar results (p> 0.05) in reference to inhibition of cell cycle.
According, glyphosate mechanism of action is rather unique because it is the only herbicide capable of specifically inhibiting the enzyme 5-enolpyruvylshikimate-3phosphate synthase (EPSPS), which catalyzes the condensation of shikimic acid and pyruvate phosphate, thus preventing synthesis of three essential amino acidstryptophan, phenylalanine and tyrosine, which are precursors of phenolic compounds (14).
The cytotoxic activity of fennel was verified studies , in which the authors found a significant decrease in significant IMT in animal testing system of mice, indicating a significant antimitotic activity, furthermore, the ethanolic extract of the fruits of the plant at doses of 0.5, 1.0 and 3.0 g.kg -1 administered orally, did not cause any deaths in mice (20).
Apart from causing potential cytotoxic effects of the substances extracted in the aqueous extract of fennel 2% promoted damage on the chromosomes, inducing the formation of micronucleous (0.25%) in total analyzed blades, the roots of A. cepa (FIGURE 1). Other chromosomal abnormalities were observed, probably the small number of cells in the mitotic division in this concentration (TABLE 1).
With regard to the percentage changes found, the CP group (1% glyphosate), who has caused the largest percentage of damage to chromosomes (0.87%) significantly different from the other treatments studied, as expected (p <0.05).
Similar results were also observed by (21).

FIGURE 1
The micronuclei usually result in production or acentric chromosome fragments entire chromosomes in which lag anaphases. When the cell enters telophase, these are included in the daughter cells.
the especialist demonstrated the relevance of tonea system strain for the assessment of cytotoxicity and genotoxicity of organic substances, claiming the cells of roots strain have an important system of enzymes necessary for the activation of mutagens (22) Currently several studies have proven the test efficiency A. strain for monitoring the cytotoxic and genotoxic effects of various organic substances (23) (24-26), as observed in the present study with aqueous extract of Foeniculum vulgare in 2%.

Animal Testing System
The results within and between groups were analyzed by ANOVA and Tukey test (p<0.05) for comparison of means. After analysis it was found that there was a significant difference in the mean micronuclei (MN's) of the treatments tested for the groups CN and CP. Among the averages MN's concentrations of 4% and 8%, there was no significant difference, but the same were significantly higher than the average of the CN group to 2%; and smaller, as expected, that the CP, showing, in this way, the average number NM increased as the concentration increased the aqueous leaf extract Foeniculum vulgare Mill (TABLE 2).

TABLE 2
These results indicate that the aqueous extract of fennel, at the concentrations tested, have potential lclastogenic to promote chromosomal damage or damage to the mitotic apparatus in the formation of erythroblasts in bone marrow of mice.
The NRBC mammalian Extrude its core in the terminal stage of maturation, which are subsequently phagocytized by macrophages. However, when the nuclei of erythroblasts DNA is damaged by clastogenic substances are formed and remain micronuclei in polychromatic erythrocytes and are readily identified after staining (FIGURE 2).

FIGURE 2
Regarding the presence of many active biological constituents in DNA fragmentation was also verified in the present study through the micronucleus test in mice erythrocytes (FIGURE 2 AND TABLE 2). Tang and Edenharder (1997) showed that the fennel leaves extracts show moderate activity mutagenic in strains of S. typhimurium TA 98 (28). Several studies suggest beneficial effects and adverse F. vulgare in relation to this work, such as effects antioxidants and anti-clastogenic(31-34) however, all these studies were performed withessential oil, whose substances are not extracted in aqueous and alcoholic extracts and / or seeds. Despite the long history of application etnomedicinal F. vulgare, no reports of serious side effects, the present study and other above, one can notconsider it a safe species, particularly for infants who do not have the digestive tract developed.

However, the diverse and proven pharmacological activities Foeniculum vulgare
show there is still a huge scope to chemical exploration.
In many homes in Brazil it is possible to find fennel, this in turn, is widely used in traditional Brazilian medicine indiscriminately, in several places is also consumed as food, it is expected that this document will awaken several researchers to deepen their knowledge regarding this plant.

3.1Collection of botanical material
Parties flights Foeniculum vulgarevar. vulgare Mill.(Stems and leaves) were collected at the site of medicinal plants of the Center for Biological Studies -UNEC (19 ° 47 '23 "S, 42 ° 08' 21" W), in May, the morning in shaded conditions. Immediately after collection, the upper parts were packed in sterile plastic bags and brought to the lab pharmacobotanics UNEC to prepare the aqueous extract, the sample specimen is deposited in the UNEC-0305042016 * and was identified by Juscelio Clemente Abreu .

Preparation of the aqueous extract of fennel
The aqueous extract was prepared according to popular Brazilian ethnobotany 20g of fresh leaves in 1L of boiled water, making up the extract at 2%, after reaching room temperature. Later, using the same methodology, the extracts a 4% and 8%, aiming the determination of the dose response relationship.

System test plant (Allium cepa L.)
Experimental It took only the concentration of the extract to 2%, this test system, because it did not occur cytotoxic and genotoxic changes, the test animal's system would not be realized, avoiding in this way the use of guinea pigs.

3.4System Test Animals (Musmusculus L.)
mice were used in theSwiss strain, Adults with an average weight of 28g.The animals spent 6 days adjustment period, with water and food ad libitum commercial environment with a photoperiod of 12h light and 12h dark, average temperature of 23 °C mice Groups of 4 rats were treated with aqueous extracts of fennel, orally, in acute treatment, the three experimental concentrations (2%, 4% and 8%). For the negative control (NC) was used distilled water and the positive control (PC) was used cyclophosphamide, 24hours before euthanasia. The genotoxicity was evaluated by counting all erythrocytes micronuclei (MN) in the bone marrow smears from each femur in each treatment, comprising two blades repeats (38).
All solutions (extracts, CN and CP) were administeredby gavage a single dose of 50mg/kg, introducing special needle through the mouth into the stomach of the animal, and the like via the accidental or intentional ingestion of the test substance.
This research is duly authorized by the CEP/UNEC.

Statistical analysis
The experimental design for each evaluation test system was randomized (DIC) using variance analysis method (ANOVA) and subsequent Tukey test for the comparison of averages, at 5% probability.