Action of Aqueous Extracts of Phyllanthus niruri L . ( Euphorbiaceae ) leaves on Meristematic Root Cells of Allium cepa L .

This study aimed to evaluate the effects of aqueous extracts of dried Phyllanthus niruri L. (stonebreaker) leaves on Allium cepa L. root meristem cells at four concentrations, 0.02 (usual concentration), 0.04, 0.06 and 0.08mg/mL and exposure times of 24 and 48 hours. For each concentration we used a group of five onion bulbs that were first embedded in distilled water and then transferred to their respective concentrations. The radicles were collected and fixed in acetic acid (3:1) for 24 hours. The slides were prepared by the crushing technique and stained with 2% acetic orcein. Cells were analyzed throughout the cell cycle, totaling 5000 for each control and exposure time. The calculated mitotic indices were subjected to the Chi-squared statistical analysis (p<0.05). From the results obtained it was observed that all four concentrations tested had significant antiproliferative effect on the cell cycle of this test system. We also found the presence of cellular aberrations such as colchicined metaphases, anaphasic and telophasic bridges, and micronuclei in the two exposure times for all concentrations evaluated. Therefore, under the conditions studied the concentrations of aqueous extracts of leaves of P. niruri showed to be cytotoxic and genotoxic.


INTRODUCTION
Currently, about 70% of the world population uses medicinal plants in primary health care (Frescura et al. 2012).However, many of them have not been sufficiently studied for their potential toxic effects at the cellular level (Bagatini et al. 2007, Meyer et al. 2011).These studies are of great importance for contributing towards standardizing quantities for safe and effective use of these plants by the population (Asare et al. 2012).
Phyllanthus niruri, a species belonging to the family Euphorbiaceae, subfamily Phyllanthoideae, originated in India and is widely distributed in the Americas.This medicinal plant, popularly known in Brazil as stonebreaker, is classified as herbaceous, measuring on average 60 cm tall, with horizontally ramified stem.Its flowers, yellowgreen, are minutel, dioecious and inserted in the http://dx.doi.org/10.1590/0001-37652014201301701131-1136 ERASMOVLANE S.B. NEVES et al. leaf axils (Moreira et al. 2013).Its phytochemical constituents have been well established, particularly lignans, tannins and flavonoids, the latter two being present in high concentrations in the leaves (Devi et al. 2005, Asare et al. 2012).
Aqueous extracts obtained by infusion of stonebreaker dried leaves are widely used in popular medicine for the removal of calcium oxalate kidney stones (Álvarez et al. 2009, Nascimento-Barros andAlbuquerque 2012), as an eupeptic (Asare et al. 2012), for liver disorders (Murugaiyah and Chan 2009), in alleviating jaundice and in fighting bladder and bowel infections (Abdulla et al. 2010).However, there is a lack of studies in the literature on its toxic effects at the cellular level.
Bioassays with plants are considered appropriate for monitoring of toxic effects of chemical compounds (USEPA) (Grant 1999, Iganci et al. 2006, Herrero et al. 2012).Allium cepa (onion) is an efficient bioindicator of the cytotoxicity of aqueous extracts of medicinal plants (Fachinetto et al. 2007, Sabini et al. 2011).This is due to its kinetic properties of proliferation and because it processes large chromosomes in reduced number (2n=16), which facilitates their analysis (Matsumoto et al. 2006, Caritá and Marin-Morales 2008, Herrero et al. 2012).This test system is also effective in the evaluation of mutagenicity of aqueous extracts of plants with medicinal properties since it enables the observation of abnormalities of the mitotic cycle, such as colchicined metaphases, anaphasic and telophasic bridges, and interphase anomalies, such as micronuclei and binucleate cells (Leme andMarin-Morales 2008, Sabini et al. 2011).
Thus, due to the widespread use of P. niruri by the population, and the need for further studies on the action of this plant at a cellular level, and also considering that the A. cepa system is suitable for assessment of cytotoxicity and mutagenicity of aqueous extracts of medicinal plants, this study aimed to evaluate the effect of different concentrations of aqueous extracts of dried stonebreaker leaves, obtained by popular use, on A. cepa meristematic root cells in two exposure times.

MATERIALS AND METHODS
This work was developed on the Senador Helvídio Nunes de Barros Campus of the Universidade Federal do Piauí (UFPI), municipality of Picos, state of Piauí.
PLANT COLLECTION Samples of P. niruri were collected in a medicinal nursery local in the city of Teresina, state of Piauí, in May of 2012 and identified by Prof Ms Maria do Socorro Meireles de Deus, who holds a master in botany and is a professor at UFPI.The leaves of these samples were then stored under environmental conditions for 6 months.Soon after identification, a control sample of this species was taken to the Graziela Barroso Herbarium of UFPI.
PREPARATION OF INFUSIONS Dry stonebreaker leaves were placed in boiling water where they remained in infusion for 10 minutes.Subsequently, the aqueous extracts were filtered and cooled to room temperature.Four concentrations were established, 0.02; 0.04; 0.06 and 0.08mg/mL, of which 0.02mg/mL is considered the most common and recommended by the Center for Drug Information Base of Medicinal and Toxic Plants (CIMPLAMT 2012).

ANALYSIS
The bulbs of Allium cepa, acquired in the produce market in the city of Picos -Piauí, were placed to root in flasks with distilled water, at room temperature (± 25°C), constantly aerated and with a period of twelve hours of light and twelve hours of darkness, until roots with about 1.0cm of length were obtained.For analysis of each concentration, an experimental group was stipulated with five bulbs, according to the protocol described by Fiskesjo (1994).

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ACTION OF P. niruri PLANT SYSTEM-TEST Before putting the roots in contact with their respective concentrations, some roots were collected and fixed to serve as control (CO) of the bulb itself.Soon afterwards, the remaining roots were placed in their respective concentrations, for 24 hours, this procedure denominated as 24-hour exposure time (ET 24h).
After this time some roots were removed and fixed.Afterwards, the remaining roots from each bulb were returned to their respective concentrations where they remained for 24 hours, which we called 48-hour exposure time (48h ET).After this period, roots were again collected and fixed.Exposure times of 24 and 48 h were chosen in order to evaluate the effect of the concen trations studied in more than one cell cycle.
The fixing of the roots occurred in Carnoy 3:1 (ethanol: acetic acid) at room temperature for 24 hours.For each root collection, an average of three roots onion bulb were removed.
PREPARATION AND READING OF THE SLIDES, AND DATA ANALYSIS The slides, three per bulb on average, were made following the protocol proposed by Guerra and Souza (2002).Each slide was stained with two drops of 2% acetic orcein (Fachinetto and Tedesco 2009) and examined under an optical microscope at 40X.For each bulb 1,000 cells were analyzed, totaling 5000 cells for each control and concentration.During the analysis we observed cells in interphase, prophase, metaphase, anaphase and telophase.We calculated the number of cells in interphase and under division for each control and exposure time and determined and mitotic index.
We also evaluated the presence of cellular aberrations such as mitotic cycle anomalies (colchicined metaphases, anaphasic and telophasic bridges) interphasic anomalies (micronucleated and binucleated cells).For this evaluation 1,000 cells were analyzed for each control and exposure time.
The statistical analysis of all the data was conducted through the Chi-square (χ 2 ), with a level of probability < 0.05, through the statistical software BioEstat 3.0 (Ayres 2007).The results obtained showed that all four tested concentrations of P. niruri significantly inhibited MI of meristematic root cells of A. cepa under the two ET when compared to the MI of their respective controls.As can be seen, at all concentrations, inhibition of cell division is maintained with the increase of ET.However, when comparing the MI between the ET of a same concentration, we found that they were not statistically significant.

RESULTS AND DISCUSSION
Table II presents the number of colchicined metaphases, bridges in anaphase and telophase, micronucleated cells, and total cellular aberrations present in the root meristem cells of A. cepa with water and treated with different concentrations of P. niruri in exposure times of 24 and 48 hours.
In both ET evaluated for the four concentrations, the presence of colchicined metaphases, anaphase bridges, telophase bridges and micronuclei were verified.All concentrations induced a number of cellular aberrations that differed significantly from their respective CO, but did not differ among themselves.The cytotoxicity results obtained here corroborate those observed by Barros et al. (2006), who evaluated the bone marrow of rats provided with alcoholic extracts of stonebreaker leaves at a dose of 300mg/ml via gavagem and chronic treatment, and found that this plant decreased the cell division rate of marrow cells of these animals, thus being cytotoxic.However, Asare et al. ( 2012) evaluated the action of alcoholic extracts of P. niruri at doses of 30 and 300mg/ml on peripheral blood of white mice for ninety days, and found that the extracts were not cytotoxic nor mutagenic in these animals.There are no other studies in the scientific literature that evaluated cytotoxicity and mutagenicity of extracts of P. niruri in the test systems with normal cells, i.e those without and previous treatment with clastogenic drugs or with a cell disorder.
Thus, taking into account the results obtained in this present work where the aqueous extracts of P. niruri had antiproliferative and mutagenic action, the few studies done to date on the toxic effects of this plant at the cellular level, recognizing that common sense often considers medicinal plants free from adverse reactions to the body often leading to their indiscriminate use, and that the P. niruri plant is easily found in medicinal plant nurseries, herbalists, natural food stores and farmers markets, it is highly relevant to carry out work similar to this one with A. cepa, with other test-systems, exposure times and different treatments to thereby establish, with propriety, what the ideal and safe concentrations are for the use of this plant.
Table I presents the number of cells in interphase and at different stages of cell division, and the mitotic

TABLE I Cell cycle analyzes of Allium cepa root tips treated with the infusion of Phyllanthus niruri leaves at concentrations of 0.02, 0.04, 0.06 and 0.08 mg/mL after 24 h and 48 h. Analysis of 5,000 cells per group.
Means followed by the same letter do not differ significantly at the level of 0.05 by the χ 2 test.ERASMOVLANE S.B. NEVES et al. index values obtained from root meristem cells of A. cepa treated with water and with different concentrations of P. niruri under exposure times of 24 and 48 hours.

TABLE II Number of cells with colchined metaphases, bridges in anaphase and telophase and micronucleated cells, and total of aberrant cells treated with the infusion of Phyllanthus niruri leaves at concentrations of 0.02, 0.04, 0.06 and 0.08 mg/mL after 24 h, 48 h and 7 days of exposure.
Means followed by the same letter do not differ significantly at the level of 5% by the χ 2 test.