Antiproliferative action of aqueous extracts of Hymenaea stigonocarpa Mart . ( Fabaceae ) on the cell cycle of Allium cepa L .

In this study we evaluated the action of crude aqueous extracts obtained from rhytidome of Hymenaea stigonocarpa (jatobá-do-cerrado) on Allium cepa meristematic root cells in three concentrations: 0.082, 0.164, 0.328g/mL, at exposure times of 24 and 48 h. The slides were prepared by the crushing technique, and cells analyzed throughout the cell cycle, totaling 5000 for each control group and concentration. It was found that all three concentrations, including the lowest which is considered ideal for use, in all exposure times, had significant antiproliferative action on the cell cycle of this test system. For cells under division, we observed a high number of cells in prophase. Therefore, under the conditions studied H. stigonocarpa indicated to be cytotoxic.


INTRODUCTION
Leguminosae is a plant family of great economic and medical importance to temperate and tropical regions worldwide.Among these plant species of this genus Hymenaea, used in Brazil as medicinal plants.
Worldwide, many plant species are used for the treatment and prevention of diseases, but most of them have not yet been evaluated for their cytotoxic, genotoxic and mutagenic potential, which is essential for an effective use of herbal medicines by the population (Bagatini et al. 2007).
The outer surface of the stem or rhytidome of Hymenaea stigonocarpa Mart.(jatobá-do-cerrado) is widely used in the form of tea in folk medicine in the states of northern and northeastern Brazil to reduce cholesterol and glucose levels, ease pain and stomach cramps, bronchitis and asthma, and to cure urinary tract infections (Ramos et al. 2007).The chemical composition of the rhytidome of this plant contains diterpene acids, anthraquinones, high concentration of mineral salts, tannins, flavonoids, oligosaccharides and xyloglucans (Cartaxo et al. 2010).No data was found in scientific literature on the cytotoxic potential of this medicinal plant.
The Allium cepa test plant system is an excellent biomarker for the first screening of medicinal plant cytotoxicity due to its kinetic properties of proliferation for having large chromosomes which are few in number (2n = 16) (Fachinetto et al. 2007) and its reliability and agreement with other toxicity tests, greatly aiding human health damage prevention studies (Bagatini et al. 2007, Leme andMarin-Morales 2009).For this cytotoxicity evaluation, 03 concentrations were established, 0.082; 0.164 and 0.328g/ mL, of which of 0.082 g/mL, is considered normal, according to Sousa et al. (2006).For the preparation of concentrations, rhytidome pieces, 80g each, were placed in boiling water for infusion where they remained for 20 minutes.After this time, the crude aqueous extracts were filtered out and placed to cool at room temperature.The teas were prepared in the same way as the population would prepare them.
The A. cepa bulbs were placed for rooting in flasks with distilled water at 25°C and aerated constantly, until obtaining roots of about 1.0cm long.For the analysis of each concentration we stipulated an experimental group with five bulbs.Before placing the roots in contact with their respective concentrations, some roots were collected and fixed to serve as control (CO) of the bulb itself.Then the remaining roots were placed on their respective concentrations, for 24 hours, this procedure being called the exposure time of 24 hours (ET 24 h).
After this time some roots were removed and fixed.Subsequently, the remaining roots of each bulb were once again placed in their respective concentrations where they remained for 24 more hours, procedure being called the exposure time of 48 hours (ET 48 h).Fixing occurred in Carnoy 3:1 (ethanol: acetic acid) at room temperature for about 24 hours.For each collection root retired, on average, three roots per bulb onion.
Slides, average of 03 per bulb, were prepared following the protocol proposed by Guerra and Souza (2002) each slide was stained with drops of 2% acetic orcein, and analyzed by light microscopy, in a 40X objective lens.For each bulb we analyzed 1,000 cells, totaling 5,000 for each concentration.Values of the average number of cells in each A. cepa cell cycle phase were calculated and the mitotic index (MI) determined.The statistical analysis was performed by the χ 2 test, with a probability level <0.05, using the BioEstat 3.0 statistical software (Ayres 2007).
Table I shows the number of cells in interphase and at different stages of cell division, and mitotic index values obtained for the root cells of A. cepa treated with water (CO) and with the concentrations of H. stigonocarpa teas for 24 and 48 hours.Significant χ 2 values are also shown.
From the results obtained (Table I), it can be observed that the concentrations tested, including normal, greatly drecreased the mitotic index of A. cepa meristematic root cells compared to the MI obtained for the respective controls.One could also observe that most of the dividing cells were in prophase.These results suggest a cytotoxic action of the H. stigonocarpa aqueous extracts studied at the two exposure times evaluated, and in the test system used.MI values obtained for ET of each concentration were not significant among themselves.
To date, few studies have been conducted to evaluate Hymenaea genus cytotoxicity, however the results of these studies corroborate those obtained here for H. stigonocarpa.Among the studies is that by Pettit et al. (2003) who found that the flavonoid palstatin, in joint action with diterpenes extracted from the leaves of Hymenaea palustri, dramatically inhibited cell division in human stomach cancer cell lines.Likewise, Closa et al. (1997) found that the flavonoid astilbin and diterpenes extracted from leaves of the Hymenaea martiana species have cytotoxic action on rodent liver cells treated with 1149 CYTOTOXICITY OF Hymenaea stigonocarpa MART. a clastogenic drug, significantly inhibiting their cell division rate and indicated as hepatoprotective.Abdel-Kader et al. (2002) found that diterpenes indicated in the rhytidome of the Hymenaea courbaril trunk had the potential to reduce the mitotic index of human ovarian cancer cells.These authors suggest that the rhytidome of this species has chemopreventive potential.
Thus, from the results obtained in A. cepa for H. stigonocarpa as well as those reported in the literature for other species of the Hymenaea genus, it becomes relevant to conduct other studies to evaluate toxicity of this plant with other test systems, different exposure times and different treatments to thereby establish what the optimal, efficient and safe concentrations are for the utilization of this plant, and verify, with proprietary, its antiproliferative capacity.
The plant identification and collection of rhytidomes were performed by Prof. Maria do Socorro Meireles de Deus, Master in botany and Professor at the Federal University of Piauí.

A. cepa treated with water (control) and three concentrations rhytidome tea of H. stigonocarpa, 0.082, 0.164, 0.328g/mL under ET of 24 and 48 h. 5,000 cells were analyzed for each control group and concentration.
Exposure time, P -Prophase, M -Metaphase A -Anaphase, T -Telophase, MI -Mitotic Index.Means followed by the same letter do not differ significantly at 5% by the χ 2 test.