Abstract: Aloysia gratissima is used in Brazilian folk medicine for the treatment of digestive and respiratory diseases. The infusions (6 and 24 g L-1) and essential oils (0.25%, on ethanol) were prepared and we used groups of five Allium cepa bulbs for each treatment. A total of 2500 cells per treatment were analyzed and the mitotic indexes were calculated. The antiproliferative effect of infusions and essential oils of Aloysia gratissima on the Allium cepa (onion) cell cycle was evaluated using the leaves of studied specimens. The infusions presented a significant decrease in the mitotic index (4.55% at 6 g L-1 and 2.04% at 24 g L-1) compared to the control-water (6.83%), as well as for the essential oil (2.58%), in comparison to the control-ethanol (3.65%). This investigation showed that the infusions and essential oil of Aloysia gratissima present important antiproliferative effects on the Allium cepa cell cycle.
INTRODUCTION
The knowledge about medicinal plants represents, in some cases, the only therapeutic treatment for many communities. However, the indiscriminate use of some species can be prejudicial to the health of the population and the effect of the action of extracts of plants on cell division in other organisms is important. The possible adverse and mutagenic effects can be detected by inhibiting cell division, like chromosomal breakages in metaphases, anaphasics bridges and micronuclei in telophase (Vieira and Vicentini, 1997).
The World Health Organization estimates that up to 80% of the world’s population relies on traditional medicinal system for some aspect of primary health care (Farnsworth et al., 1985). In developing or non-industrialized countries, like Brazil, the use of infusions of plants for treatment of diseases is common (Teixeira et al., 2003).
Essential oils are obtained from many aromatic plant species and are used in industries for the production of soaps, cosmetics, perfumes and foods. Investigations concerning the evaluation of the biological activities of essential oils of some medicinal plants have revealed that some exhibited antibacterial, antifungal, anti-inflammatory, analgesic and insecticidal properties (Burt, 2004; Bakkali et al., 2008). They are usually used topically, in massages, therapeutic baths, compresses, or inhalation (Steflitsch and Steflitsch, 2008).
Aloysia gratissima Gilles and Hook Tronc. (Verbenaceae), commonly known as erva-santa, alfazema-do-brasil, erva-da-graça and erva-de-nossa-senhora, is an herb used in Brazilian popular medicine, especially in the Southern state of Rio Grande do Sul. Traditionally, it is used as gastrointestinal and respiratory medicine (Pascual et al., 2001). The tea made from its leaves is popularly used as an analgesic, antiflu, anti-rheumatic, balsamic and stomaquic, to decrease the arterial pressure and cholesterol values, as well as for pneumatic diseases.
Investigations with this plant demonstrated high content of essential oil and differences in principal chemical composition (Agostini et al., 2003; Ricciardi et al., 2005; Franco et al., 2007).
The use of medicinal plants for the treatment of diseases is an exploratory practice and widespread in Brazil. Considering the extensive medicinal use of Aloysia gratissima, studies using bioindicators of toxicity and of mutagenicity, with Allium cepa test in vivo are necessary.
Based on this context, the purpose of this study was to evaluate the antiproliferative effect of infusions and essential oil of A. gratissima using the A. cepa cell cycle.
MATERIALS AND METHODS
Plant material: The leaves of A. gratissima were collected in Santa Maria (Rio Grande do Sul), Brazil, in July 2007. A voucher specimen was deposited in the Herbarium SMDB (Santa Maria Department of Biology), at UFSM, in Santa Maria, Rio Grande do Sul, Brazil.
Preparation of the aqueous extracts and essential oil: Fresh leaves of A. gratissima were infused in water for 5 min and the aqueous extracts were strained and placed at room temperature for cooling. These infusions were prepared in two concentrations: 6 and 24 g L-1. The 6 g L-1 is a normal concentration used during the preparation of the medicinal tea. The essential oil was obtained from fresh leaves of A. gratissima by hydro-distillation with Clevenger’s apparatus. The oil concentration used in the test was 0.25% (w/v) in ethanol.
Evaluation of antiproliferative effect on Allium cepa assay: For the meristematic onion root-tip cell test, we used 25 A. cepa bulbs divided into five groups of five onion bulbs for each treatment (distilled water, ethanol, aqueous extracts at 6 and 24 g L-1 and essential oil at 0.25%). For each treatment, all bulbs were rooted in distilled water for three days and then they were placed in their respective treatment for 24 h. The two control groups, which had not received treatment, remained in distilled water and ethanol, respectively. After 24 h, controls and experimental bulbs were collected and fixed in 3:1 (v/v) ethanol-acetic acid for 24 h. Subsequently, they were placed in 70% (v/v) aqueous ethanol under refrigeration (4±2°C) until analyzed (Fiskesjö, 1994; Knoll et al., 2006). For each bulb, five slides were made using five root-tips hydrolyzed in 1 N HCl for 5 min and washed in distilled water. The fragmented meristematic regions were stained with 2% (w/v) acetic orcein (Guerra and Souza, 2002). Each slide was assessed by bright-field optical microscopy at 500x magnification and the number of interphase, prophase, metaphase, anaphase and telophase cells was recorded. At least 2500 cells for each treatment and the controls were scored. Mean values for the different cell cycle phases and the mitotic index (%) were calculated.
| Table 1: | No. of cells in different phases of the cell cycle and the mitotic index of onion root-tip cells. The total number of cells analyzed for each treatment was 2500 |
| *Means, in column, with the same letter are not significantly different (χ2 test, p≤0.05) | |
Statistical analysis: The statistical data analysis was performed using the Chi-squared (χ2) test at p≤0.05, with the statistical program BioEstat (Version 3.0) (Ayres et al., 2003).
RESULTS
Table 1 shows the total number of analyzed cells and the number of cells observed in interphase and in the different phases of cell division during the A. cepa cell cycle. In this same table, the mitotic index (number of cells in division/number of cells in interphase) is also demonstrated.
The mitotic index of the A. cepa cell cycle decreased significantly after 24 h in both concentrations of aqueous extracts and in the essential oil of A. gratissima when compared to the control group-distilled water. The values obtained were: 4.55% for aqueous extract at 6 g L-1 (χ2 = 10.22); 2.04% for aqueous extract at 24 g L-1 (χ2 = 60.14) and 2.58% for essential oil (χ2 = 44.16). Between the control group-ethanol and treatments, significant differences were also observed (aqueous extract at 6 g L-1, χ2 = 2.33; aqueous extract at 24 g L-1, χ2 = 10.76; essential oil, χ2 = 4.27). Likewise, significant difference was also obtained between the control groups (distilled water and ethanol) (χ2 = 21.99). After analyzing the three treatments, significant differences in the mitotic index were obtained: aqueous extract at 6 g L-1 x aqueous extract at 24 g L-1 x essential oil (χ2 = 26.76); aqueous extract at 6 g L-1 x aqueous extract at 24 g L-1, (χ2 = 22.61); aqueous extract at 6 g L-1 x essential oil (χ2 = 12.74); aqueous extract at 24 g L-1 x essential oil (χ2 = 1.53).
DISCUSSION
The cytotoxic and genotoxic effects of aqueous extracts and essential oils of A. gratissima on A. cepa were evaluated and we observed that the teas and essential oil significantly inhibited cell division, in both analyzed concentrations (Table 1). This result indicates the existence of antiproliferative activity in the tea made from this species. Comparatively, similar results were obtained from the essential oil and aqueous extracts at 24 g L-1, though the tea presented higher potential of inhibiting cell division. Considering that this difference is significant, but the values are numerically adjacent, we accept the hypothesis that substances present in the leaves act in inhibiting cell division.
According to the results observed in Table 1, it is important to consider essential oil composition and to indicate the existence of a relationship with the increase of the content of leaves used in tea preparation. However, it is difficult to compare the qualitative and quantitative chemical composition present in the samples, due to the extraction processes used in this study (aqueous extract and essential oil). Da Silva et al. (2006) evaluated the chemical composition of the aerial parts of A. gratissima and the results demonstrated the presence of sesquiterpenoids compounds (guaiol, bisabolol and spathulenol), triterpenoids (α-amirin, betulinic acid, oleanolic acid and ursolic acid), flavonoids and phenylethanoids (verbascoside and arenarioside).
The literature points the chemical composition of the essential oil of A. gratissima and that differences were observed, due to the site of collection, environmental conditions and genetic variable. Duschatzky et al. (2004) evaluated the essential oil composition of the species collected from Argentina and the main components were: cadinol (17.4%), caryophyllene oxide (15.8%), limonene oxide (5.3%), chrysanthenyl acetate (5.6%) and β-caryophyllene (4.8%); in another research, the chemical composition of aerial parts of A. gratissima in three localities of the province of Corrientes (Argentina), during different seasonal stages was studied and the principal constituents were: β-elemene (tr to 35.7%), viridiflorol (0.9-33.6%), β-caryophyllene (1.8-28%), α-thujone (6.8-17.5%), 10-epi-cubebol (0.1-13.4%), bicyclogermacrene (3.8-12.8%), (E)-nerolidol (tr to 11.6%) and germacrene D (1.9-10.1%). The variation was associated with the ontogeny of the plant population and the geographical origin of the material (Ricciardi et al., 2005). The analysis of the constituents present in essential oils of plant collected from Goiânia State (Brazil) were evaluated by Franco et al. (2007) and found the principal components to be β-pinene (14.06%), trans-pinocamphone (18.42%), trans-pinocarvila acetate (13.55%), pinocamphone (6.59%), guaiol (6.27%), β-caryophyllene (4.99%) and caryophyllene oxide (4.15%). Through these observations it is difficult to state which compound present in the oil is responsible for the decrease in the mitotic index values obtained from meristematic cells of A. cepa analysis, but the presence of antioxidative substances can be related to that activity. The antiproliferative capacity of other medicinal species that present antioxidative substances in their composition has been studied, as shown for Pterocaulon polystachyum DC, Achyrocline satureoides (Lam.) DC, Psychotria myranthia Mull. Arg. and P. leiocarpa Cham. et Schlecht (Knoll et al., 2006; Fachinetto et al., 2007; Lubini et al., 2008).
The cytotoxicity tests, employing a plant test in vivo, such as the A. cepa test, are validated by several researchers with a good correlation to the in vitro test using animal cells (Vicentini et al., 2001; Teixeira et al., 2003).
The mitotic index is used as an indicator of adequate cell proliferation (Gadano et al., 2002), which can be measured using the A. cepa plant system. The chromosomal aberration method in A. cepa roots is validated by the International Programme on Chemical Safety (IPCS, WHO) and the United Nations Environmental Programme (UNEP), as a test efficient for the analysis and monitoring in situ of genotoxicity of environmental substances (Silva et al., 2004).
In this study, our results demonstrated that infusions and the essential oil of leaves of A. gratissima showed high antiproliferative activity using the in vivo A. cepa cell test, indicating their potential use for inhibiting the cell cycle in tumors and anti-aging. It should be noted that there are no works that demonstrate the antiproliferative activity of the plant. However, this study is bioindicative and more research will be necessary to establish the use of extracts of A. gratissima as antimutagenic in humans.
ACKNOWLEDGMENTS
We are grateful to Professor Melânia Palermo from the Department of Industrial Pharmacy (UFSM) and Mrs. Vera Pereira Pagliarin for collaboration in the essential oil extraction. The authors thank H.D. Laughinghouse for their contribution in the interpretation of results and review of botanical terms.