Cytotoxic effects of infusions (tea) of Solidago microglossa DC. (Asteraceae) on the cell cycle of Allium cepa

Efeitos citotóxicos das infusões (chá) de Solidago microglossa DC. (Asteraceae) no ciclo celular de Allium cepa

ARTIGO

Cytotoxic effects of infusions (tea) of Solidago microglossa DC. (Asteraceae) on the cell cycle of Allium cepa

Efeitos citotóxicos das infusões (chá) de Solidago microglossa DC. (Asteraceae) no ciclo celular de Allium cepa

Margarete Dulce Bagatini^I; Juliana Maria Fachinetto^II; Antonio Carlos Ferreira da Silva^II; Solange Bosio Tedesco^{II, *}

^IDepartamento de Química, Laboratório de Enzimologia, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio18, Sala 2208, 971105-900 Santa Maria-RS, Brasil

^IIDepartamento de Biologia, Laboratório de Citogenética Vegetal e Genotoxicidade, Universidade Federal de Santa Maria, Av. Roraima, 1000, Prédio16, Sala 3118, 97105-900 Santa Maria-RS, Brasil

ABSTRACT

The medicinal specie Solidago microglossa DC., which is native from South America, is used as infusion (tea) for stomach disorders in popular medicine. This study aimed to evaluate the potential cytotoxic effect of these infusions on the cell cycle of Allium cepa. In natura leaves of three populations of this species were used in preparing infusions at two concentrations: 1.75 mg/mL and 14 mg/mL. Onion (Allium cepa) root-tip cells were used as test system in in vivo assays. Slides were done through smashing technique. Cells were analyzed in all cell cycles of A. cepa, totalizing 6000 cells for each group of bulbs. The mitotic index (MI) was calculated, and the statistical analysis was carried out through the Chi-square test (χ² = 0.05). The results show that the infusions of S. microglossa at a highest concentration (14 mg/mL) caused a significant reduction in the MI compared with control in the studied populations. In concentrations commonly used by people there was a significant increase of MI in relation to controlled populations 1 and 3 and a decrease in population 2. In conclusion, infusions of S. microglossa presented antiproliferative effect at a highest concentration.

Keywords: Test system vegetal, medicinal plant, cytotoxic, Solidago microglossa, Asteraceae.

RESUMO

A espécie medicinal Solidago micrglossa DC., a qual é nativa da América do Sul, é usada como infusão (chá) para desordens estomacais na medicina popular. O objetivo desse estudo foi avaliar o potencial efeito citotóxico dessas infusões no ciclo celular de Allium cepa. Folhas in natura de três populações dessa espécie foram utilizadas no preparo das infusões em duas concentrações: 1,75 mg/mL e 14 mg/mL. Células de raízes de cebola (Allium cepa) foram usadas como sistema teste nos ensaios in vivo. As lâminas foram feitas através da técnica de esmagamento. As células foram analisadas em todos os ciclos celulares de A. cepa, totalizando 6000 células para cada grupo de bulbos. O índice mitótico (IM) foi calculado e a análise estatística foi realizada através do teste Qui-quadrado (χ² = 0,05). Os resultados mostraram que as infusões de S. microglossa na maior concentração (14 mg/mL) causou uma redução no IM comparado com os controles nas populações estudadas. Nas concentrações comumente usadas pelas pessoas houve um aumento significativo do IM em relação ao controle nas populações 1 e 3 e foi reduzido na população 2. Em conclusão, infusões de S. microglossa apresentam efeito antiproliferativo na maior concentração testada.

Unitermos: Sistema teste vegetal, planta medicinal, citotóxico, Solidago microglossa, Asteraceae.

INTRODUCTION

In Brazil, the use of crude plant extracts, infusions or plasters is a widespread practice in the treatment of pathologies (Bighetti et al., 2005; Marliére et al., 2008; Veiga-Junior, 2008). However, their inappropriate and not controlled use may cause more damages than benefits to human health (Amorim et al., 2007; Lanini et al., 2009). So, having a deep knowledge of these plants is important even from cell levels to the effects upon live organisms.

According to the World Health Organization (WHO), about 65-80% of the developing countries depend on plants to heal from diseases, due to poverty and lack of modern medicine access. However, few plants have been studied scientifically in order to evaluate their safe and efficacy (Calixto, 2005; Silveira et al., 2008; Tagliati et al., 2008; Nascimento et al., 2009).

Solidago microglossa (Asteraceae) is among the species of medicinal plants used as tea in alternative medicine in South America. In Brazil, it is known by popular names such as "arnica-silvestre", "erva-lanceta", "arnica" and "brazilian-arnica". S. microglossa leaves are used for stomach disorders, adstringent, cicatrizing, acnes and bruises (Lorenzi, 2000). The phytochemical study of this species demonstrated the presence of quercitrin, a glicosidic flavonoid, and also tanines, saponines, resins and essential oils in the tips, as well as inulin and rutine diterpines, quinine acid, rhamnoides and caffeic acid, chlorogenic and hydrocynamic and their derivates in the roots (Torres et al., 1989). Some studies have reported the cicatrizing and antimicrobial activity of the S. microglossa (Neto et al., 2004; Morel et al., 2006). However, there are no data in the literature, at least of our knowledge, with regard to the cytotoxicity of this medicinal plant popularly used.

Studies concerning the toxicity and mutagenicity of medicinal plants are needed to verify the efficacy and safety of their use in the treatment of some diseases (Macêdo et al., 2008; Ferreira et al., 2009). In this context, meristematic cells of Allium cepa are often used as a vegetal test system to indicate the genotoxic potential of the medicinal plant extracts. Moreover, this system can be useful to evaluate the mutagenic activity of specific drugs due to its sensitivity and its relationship with the mammal test system (Grant, 1978; Fiskesjo, 1993; Grisolia & Takahashi, 1990; Grover et al., 1990; Rank & Nilsen, 1997; Chauhan et al., 1999; Bagatini et al., 2007).

Thus, the present study aimed to evaluate the cytotoxic potential of infusion (tea) of S. microglossa on the A. cepa cell cycle.

MATERIAL AND METHODS

Plant collection

Leaves of S. microglossa were collected from three sites in Rio Grande do Sul State, Brazil. The sites were: Boca do Monte/Santa Maria (population 1), São Sepé Road - BR 392 (population 2), São Pedro (population 3). From each collected population, there was a voucher sample at the university herbarium, under register number 9928, 9686 and 9689, respectively. The plants were identified by Professor Dr. do Canto-Dorow.

Infusions preparation

In natura leaves of S. microglossa were put in boiling water, remaining in infusion for 10 min. The aqueous extracts were filtered and cooled at room temperature. The infusions were prepared at two concentrations, each one corresponding to one treatment: 1.75 mg/mL (treatment 1) and 14 mg/mL (treatment 2). The concentration popularly used to prepare the medicinal tea is 1.75 mg/mL.

In this study, three groups of six onion bulbs were used for each population of S. microglossa, each group represents one of the treatments. All bulbs were put in distilled water to take root, after that they were transferred to infusions that were at room temperature, remaining for 24 h (control bulbs remained in distilled water). After, root-tips from 5 to 10 mm were collected and fixed in ethanol:acetic acid (3:1) for 24 h. Next, they were withdrawn from the fixer and were kept in ethanol 70% and maintained in the cooler to their use (modified from Camparoto et al., 2002).

The effect of infusion on the cell division of the anion root-tips

An average of five slides were made for each bulb of each treatment and control of the three population studied. In preparing the slides, root-tips were hydrolyzed in HCI 1M for 5 min. The meristematic region of the root-tips was fragmented and stained with acetic orcein 2% by using squashing technique (Guerra, 1999).

Analyses of cells from each bulb/population were carried out. The total number of analyzed cells per bulb group was 6000. Slides were evaluated by observing cells in interphase, prophase, metaphase, anaphase and telophase with the use of an optical microscope with a 40x objective. The average number of cells in each phase of the division cycle of A. cepa and the mitotic index (MI) was calculated.

Statistical analysis

Statistical analysis of the data was performed through the use of χ² test at probability level of <0.05 BioEstat 4.0 (Ayres et al., 2005) has found the results as statistically significant.

RESULTS AND DISCUSSION

The total number of analyzed cells, the number of cells in division during the cell cycle of A. cepa and the mitotic index (MI) values are displayed in Table 1. The results presented in Table 1 show that the infusions of S. microglossa at a highest concentration (14 mg/mL) caused a significant reduction in the MI compared with control in the studied populations (population 1 (χ²= 17.96), population 2 (χ²= 374.6) and population 3 (χ²= 7.36)). In population 1 and 3, the values of MI increased in the concentrations of 1.75 mg/mL (χ²= 12.58 and χ²= 7.66), compared with control group. In population 2, it was observed a significant reduction in the MI as the concentrations of infusion of S. microglossa were increased (χ²= 32.63).

Taken together, these results indicate that infusions of S. microglossa present cytotoxic but not mutagenic effect, based in an in vivo vegetal test system. The cytotoxic and antiproliferative effects of S. microglossa were observed through the diminishing of the mitotic index that occurred in all populations tested, when the highest concentration of S. microglossa infusion was used. According with our data, it has been reported that the seasonal variation, the climate, soil and collection period may be responsible for the differences in the chemical composition of plants of this species (Dias, 2001).

Besides the great number of cells analyzed, it was not observed any kind of chromosomal alteration in the cells from onion root-tips neither in control nor in treatment with infusion of S. microglossa, which is visible with the use of an optical microscope. These results suggest the absence of mutagenic effects of S. microglossa infusion in the concentrations used in this study.

It is worth highlighting that the test system of A. cepa is well known and recognized for cytotoxic studies. This system permits the direct contact of root-tips with the tested substance, becoming possible to run the evaluation of different concentrations in different periods of treatment. The studies carried out with the A. cepa test system are considered satisfactory as indicators of the genotoxic potential of the plant extract. In addition, this system has been used as an indicator of the chromosomal alterations in order to warn human people about some product consumption (Vicentini et al., 2001). Being particularly considered important, several researchers have performed the in vitro animal test together with the test system of A. cepa, obtaining similar results (Chauhan et al., 1999; Vicentini et al., 2001; Teixeira et al., 2003), providing valuable information for the human health.

Solidago microglossa is a vegetal species which contains tanines and flavonoids among other substances in its chemical composition. Tanines acts in the protein precipitation and enzymatic inhibition. The enzymatic activity assigned to these compounds can be responsible for the inhibition of cell division in meristematic cells of A. cepa (Teixeira et al., 2003). This species also contains flavonoids, which have been reported to possess cytotoxic activities (Ya-Qin et al., 2001). The flavonoids from Humulus lupulus L. and Lonchocarpus spp. presented cytotoxic and antiproliferative activity in cell cycle on cancer prostate cells (Delmulle et al., 2006; Borges-Argáez et al., 2007). However, the infusions from Maytenus ilicifolia and Bauhinia candicans, despite contain tanines and flavonoids, did not present inhibition of cell division. In addition, according to Bu-Abbas et al. (1996), the high concentration of these substances could be responsible for the antimutagenic activity and probably anticancerigen of the green tea.

Knoll et al. (2006) studied the genotoxic potential of different populations of Pterocaulon polystachyum, displaying their antiproliferative capacity, by using the A. cepa test. During the test, the inhibition of A. cepa cell division increased in a concentration dependent manner. Similar results were obtained by Fachinetto et al. (2007) using the A. cepa test system to analyze infusions of Achyrocline satureidoides. Moreover, it was observed an increase in the antiproliferative action of Achyrocline satureidoides infusions depending on the plant storage time.

The results of this study with S. microglossa infusions at several concentrations and in different populations in the system test A. cepa show the existence of antiproliferative and cytotoxic activities of this species, suggesting a possible therapeutic potential for the inhibition of the cell cycle in eukaryotic organisms. Further studies should be carried out to establish the main anti-genotoxic components of the S. microglossa.

ACKNOWLEDGEMENT

The authors are thankful to Professor Dr. Thais Sctotti do Canto-Dorow for the botanic identification of the species used in the research.

Received 2 June 2008; Accepted 29 November 2008

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Publication Dates

• Publication in this collection
  24 Nov 2009
• Date of issue
  June 2009

History

• Accepted
  29 Nov 2008
• Received
  02 June 2008