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

An Acad Bras Cienc Anais da Academia Brasileira de Ciências An. Acad. Bras. Ciênc. 0001-3765 1678-2690 Academia Brasileira de Ciências Neste estudo avaliou-se a ação de extratos aquosos brutos obtidos do ritidoma de Hymenaea stigonocarpa (jatobá-do-cerrado) sobre as células meristemáticas de raízes de Allium cepa, em três concentrações: 0,082; 0,164; 0,328g/mL, nos tempos de exposição de 24 e 48 h. As lâminas foram feitas pela técnica de esmagamento, e analisaram-se células em todo ciclo celular, totalizando 5.000 para cada grupo controle e concentração. Verificouse que as três concentrações, inclusive a menor e considerada ideal para consumo, em todos os tempos de exposição tiveram ação antiproliferativa significativa sobre o ciclo celular deste sistema teste. Para as células em divisão observou-se um grande número de células em prófase. Portanto, nas condições analisadas a H. stigonocarpa mostrou-se citotóxica. 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 and Marin-Morales 2009). The present study aimed to evaluate the toxic effects of crude aqueous extracts of the jatobá-do-cerrado rhytidome, on the cell cycle of Allium cepa roots at different concentrations and exposure times. For this work, samples of the H. stigonocarpa stem rhytidomes were collected in a medicinal garden in the city of Teresina, in the state of Piauí, in May 2012. 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. 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 χ2 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 χ2 values are also shown. TABLE I Number of cells in interphase and at different stages of cell division, total cell analyzed in cell cycle root tips of analyzed in cell cycle root tips of 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. Concentration (g/mL) ET Intephase Cells P M A T Cells under Division MI (%) 0.082 CO 3566 452 394 378 270 1494 28.7a 24h 4270 474 71 91 94 730 14.6b 48h 4320 413 91 90 82 676 13.5b 0.164 CO 3131 500 493 487 389 1869 37.4a 24h 4201 535 91 80 93 799 16.0b 48h 4140 587 88 93 92 860 17.2b 0.328 CO 3279 462 455 394 410 1721 34.4a 24h 4277 469 80 93 81 723 14.5b 48h 4427 438 48 55 32 573 11.5b CO – Control; ET – 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 χ2 test. 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 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. REFERENCES Abdel-Kader M, Berger JM, Slebodnick C, Malone S, Wisse JH, Werkhoven MC, Mamber S and Kingston DG. 2002. Isolation and absolute configuration of ent-Halimane diterpenoids from Hymenaea courbaril from the Suriname rain forest. J Nat Prod 65: 11-15. Abdel-Kader M Berger JM Slebodnick C Malone S Wisse JH Werkhoven MC Mamber S Kingston DG 2002 Isolation and absolute configuration of ent-Halimane diterpenoids from Hymenaea courbaril from the Suriname rain forest J Nat Prod 65 11 15 Ayres M. 2007. BioEstat 5.0: Aplicações estatísticas nas áreas das ciências biológicas e médicas. 5a ed., Belém: Sociedade Civil Mamirauá: Brasília CNPq. Ayres M 2007 BioEstat 5.0: Aplicações estatísticas nas áreas das ciências biológicas e médicas 5a ed Belém Sociedade Civil Mamirauá Brasília CNPq Bagatini MD, Silva ACF and Tedesco SB. 2007. Uso do sistema-teste Allium cepa como bioindicador de genotoxicidade de infusões de plantas medicinais. Rev Bras Farmacogn 18: 509-516. Bagatini MD Silva ACF Tedesco SB 2007 Uso do sistema-teste Allium cepa como bioindicador de genotoxicidade de infusões de plantas medicinais Rev Bras Farmacogn 18 509 516 Cartaxo SL, De Almeida Souza MM and De Albuquerque UP. 2010. Medicinal plants with bioprospecting potential used in semi-arid northeastern Brazil. J Ethnopharmacol 131: 326-342. Cartaxo SL De Almeida Souza MM De Albuquerque UP 2010 Medicinal plants with bioprospecting potential used in semi-arid northeastern Brazil J Ethnopharmacol 131 326 342 Closa D, Torres M, Hotter G, Bioque G, Léon OS, Gelpí E and Rósello-Catafau J. 1997. Prostanoids and free radicals in Cl4C- induced hepatotoxicity in rats: effect astilbin. Prostaglandins Leukot Essent Fatty Acids 56: 331-334. Closa D Torres M Hotter G Bioque G Léon OS Gelpí E Rósello-Catafau J 1997 Prostanoids and free radicals in Cl4C- induced hepatotoxicity in rats: effect astilbin Prostaglandins Leukot Essent Fatty Acids 56 331 334 Fachinetto JM, Bagatini MD, Durigon ACFS and Tedesco SB. 2007. Efeito anti-proliferativo das infusões de Achyrocline satureioides DC (Asteraceae) sobre o ciclo de celular de Allium cepa. Rev Bras Farmacogn 17: 49-54. Fachinetto JM Bagatini MD Durigon ACFS Tedesco SB 2007 Efeito anti-proliferativo das infusões de Achyrocline satureioides DC (Asteraceae) sobre o ciclo de celular de Allium cepa Rev Bras Farmacogn 17 49 54 Guerra M and Souza M. 2002. Como observar os cromossomos: um guia de técnicas em citogenética vegetal, animal e humana. Ribeirão Preto: FUNPEC, 2002, 191 p. Guerra M Souza M 2002 Como observar os cromossomos: um guia de técnicas em citogenética vegetal, animal e humana Ribeirão Preto FUNPEC 2002 191 Leme DM and Marin-Morales MA. 2009. Allium cepa test in environmental monitoring: A review on its application. Mut Res 682: 71-81. Leme DM Marin-Morales MA 2009 Allium cepa test in environmental monitoring: A review on its application Mut Res 682 71 81 Pettit GR, Meng Y, Stevenson CA, Doubek DL, Knight JC, Cichacz Z, Pettit RK, Chapuis JC and Schmidt JM. 2003. Isolation and structure of palstatin from the Amazon tree Hymenaea palustri. J Nat Prod 66: 259-162. Pettit GR Meng Y Stevenson CA Doubek DL Knight JC Cichacz Z Pettit RK Chapuis JC Schmidt JM 2003 Isolation and structure of palstatin from the Amazon tree Hymenaea palustri J Nat Prod 66 259 162 Ramos ACS, Lemos-Filho JP, Ribeiro RA, Santos FR and Lovato MB. 2007. Phylogeography of the tree Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) and the influence of Quaternary climate changes in the Brazilian Cerrado. Ann Bot 100: 1219-1228. Ramos ACS Lemos JP Filho Ribeiro RA Santos FR Lovato MB 2007 Phylogeography of the tree Hymenaea stigonocarpa (Fabaceae: Caesalpinioideae) and the influence of Quaternary climate changes in the Brazilian Cerrado Ann Bot 100 1219 1228