Post-treatment with plant extracts used in Brazilian folk medicine caused a partial reversal of the antiproliferative effect of glyphosate in the Allium cepa test

Species of the genus Psychotria are used for multiple purposes in Brazilian folk medicine, either as water infusions, baths or poultices. This study was aimed to evaluate the genotoxic and antiproliferative effects of infusions of Psychotria brachypoda and P. birotula on the Allium cepa test. Exposure to distilled water was used as a negative control, while exposure to glyphosate was used as a positive control. The interaction of extracts (as a post-treatment) with the effects of glyphosate was also studied. Results showed that glyphosate and the extracts of both P. brachypoda and P. birotula reduced the mitotic index as compared with the negative control (distilled water). Surprisingly, however, both extracts from P. brachypoda and P. birotula caused a partial reversal of the antiproliferative effect of glyphosate when used as a post-treatment. Glyphosate also induced the highest number of cells with chromosomal alterations, which was followed by that of P. birotula extracts. However, the extracts from P. brachypoda did not show any signifi cant genotoxic effect. Post-treatment of glyphosate-treated samples with distilled water allowed a partial recovery of the genotoxic effect of glyphosate, and some of the Psychotria extracts also did so. Notably, post-treatment of glyphosatetreated samples with P. brachypoda extracts induced a statistically signifi cant apoptotic effect. It is concluded that P. brachypoda extracts show antiproliferative effects and are not genotoxic, while extracts of P. birotula show a less potent antiproliferative effect and may induce chromosomal abnormalities. The fi nding of a partial reversion of the effects of glyphosate by a post-treatment with extracts from both plants should be followed up. BIOCELL 2013, 37(2): 23-28 ISSN 0327 -9545 PRINTED IN ARGENTINA


Introduction
The economic potential of medicinal species that are native to Brazil is huge and these species are considered a natural resource worthy of preservation and optimization of their use (Pereira et al., 2006).
The most popular internal uses of infusions of Psychotria species include bronchial, gastrointestinal and female reproductive disorders, as well as being considered an aid in the pre-and postpartum periods.External uses as poultices and baths are also popular for the treatment of fever, headaches and earaches and for skin and eye disorders (Adjibadé, 1989apud Paranhos, 2003;Lajis et al., 1993;Perry, 1980).
P. brachypoda produces the alkaloid psycholatine, with high pharmacological potential, because it has analgesic activity of the opioid, anxiolytic and antipsychotic types, interacting with receptors of several neurotransmitter systems in the central nervous system (Fragoso, 2007).P. birotula contains pyrrolidinoindole alkaloids, along with meso-chimonanthine and chimonanthine, also used pharmaceutically (Brand et al., 2009).
According to Silva et al. (2004) tea consumption would suppress the effects of mutagenic agents, but Vicentini et al. (2001) reported that teas and herbal infusions may contain toxic mutagenic substances.So, studies of toxicity and mutagenic activity are needed to contribute to the safe use of these folk medicines.
Thus, this study was aimed to assess the antiproliferative, genotoxic, and antimutagenic effects of the extracts of P. brachypoda and P. birotula, using the Allium cepa test.This test, fi rst introduced by Levan (1938), was used since it has been recognized by the  (Teixeira et al., 2003;Vicentini et al., 2001).

Materials and Methods
Leaves of P. brachypoda and P. birotula were collected in the municipality of Dom Pedro de Alcântara (Rio Grande do Sul, Brazil) in September 2010, i.e., during the period of vegetative development.The species were identifi ed according to Mori et al. (1989).The leaves were dried at room temperature for 90 days, and then aqueous extracts were prepared by infusion for 10 minutes (tea) in two concentrations for each species (5 and 20 g of dried leaves per liter).
At the end of the different treatments the roots (2 cm samples, meristematic region) were collected and fi xed in ethanol: acetic acid (3:1) during 24 hours and then stored in 70% alcohol in the refrigerator.Afterwards the samples were hydrolyzed in HCl 1N for 5 minutes and were stained with acetic orcein 2% after squashing the meristematic region with a glass rod (Guerra and Souza, 2002).The slides were studied with a light microscope LEICA 400X.One thousand cells were counted per bulb and the MI and the percent oc-currence of chromosomal alterations were calculated.
We counted 5000 cells for each group of bulbs, analyzing cells in mitosis (prophase, metaphase, anaphase, and telophase) and interphase, and also recording the number of apoptotic cells observed during the count of 5000 cells.
Statistical analysis of the effect of treatments was performed by the Chi-square test, using BioEstat 5.O (Ayres, 2007).

Results
Table 1 shows the effect of the different treatments on the number of cells in interphase and the different phases of cell division, as well as on the MI values.
Glyphosate, as wells as extracts from both P. brachypoda and P. birotula caused signifi cant decreases in the MI as compared with the distilled water control, but both doses of P. brachypodaand the lower dose of P. birotula were more effective than glyphosate.
The antiproliferative effect of glyphosate treatment was not modifi ed by the post-treatment with distilled water for 24 h, but post-treatment with both doses of P. brachypoda and P. birotula extracts caused a partial reversal of the effect of glyphosate.
Glyphosate treatment induced the highest number of cells with chromosomal alterations, while treatment with P. brachypoda extracts did not differ from the distilled water controls.The extracts of P. birotula, however, induced a signifi cantly higher number of alterations than either distilled water or P. brachypoda extracts.Post-treatment with distilled water, as well as with two extracts (P.brachypoda, 20 mg/L, and P. birotula, 5 mg/L) caused a partial reversal of the genotoxic effect of glyphosate.
Also, glyphosate treated samples showed a signifi cantly higher number of cells in apoptosis when post-treated with both P. brachypoda extracts, and this apoptotic effect was dose dependent.

Discussion
Glyphosate is known for its antiproliferative action and for inducing chromosomal alterations in meristematic cells of A. cepa (Souza et al., 2010).These effects were confi rmed in the current study.
We are reporting here that water extracts from both P. brachypoda and P. birotula signifi cantly reduced the mitotic index as compared with the negative control (distilled water).Surprisingly, however, extracts from both P. brachypoda and P. birotula caused a partial reversal of the antiproliferative effect of glyphosate when used as a post-treatment.Glyphosate also induced the highest number of cells with chromosomal alterations, which was followed by that of P. birotula extracts.However, the extracts from P. brachypoda did not show any genotoxic effect.Post-treatment of glyphosate-treated samples with distilled water allowed a partial recovery of the genotoxic effect of glyphosate, and some of the Psychotria extracts also did so.It is concluded that P. brachypoda extracts show antiproliferative effects and are not genotoxic, while extracts of P. birotula show a less potent antiproliferative effect and may induce chromosomal abnormalities.
It should also be mentioned that genotoxic activity was shown by extracts from P. birotula in the current study, although the effect was signifi cantly lower than that of glyphosate.On the contrary, extracts from P. brachypoda did not produce any signifi cant genotoxicity.Lubini et al. (2008) also found genotoxicity in extracts from Psychotria myriantha, but not in those of P. leiocarpa.
Notably, post-treatment with extracts from P. brachypoda and P. birotula (at some but not all doses) caused a partial reversal of the antiproliferative and genotoxic effects of glyphosate.Post-treatment with distilled water was ineffective.These results are interesting but diffi cult to interpret at the present stage of knowledge: indeed, a reversal of an antiproliferative action of glyphosate by an extract which is in itself antiproliferative is intriguing and may indicate that the antiproliferative action of glyphosate and Psychotria extracts are using different mechanisms.
Also notably, post-treatment of glyphosate treated bulbs with both concentrations of P. brachypoda was followed by induction of apoptosis in a statistically signifi cant number of cells.
Finally, it should be mentioned that a partial but signifi cant reversal of the genotoxic effect of glyphosate was produced by post-treatment with extracts from P. brachypoda (20 g/L) or P. birotula (5 g/L).However, post-treatment with distilled water was similarly effective.

TABLE 1 . Number of cells in either interphase or the mitotic phases, and on the mitotic index (Allium cepa test; 5000 cells were analyzed per treatment).
UNEP) as an effi cient test for the analysis and monitoring in situ of the genotoxicity of environmental substances.It has also been validated by comparison with animal tests

TABLE 2 . Number of cells with chromosomal alterations or apoptosis (Allium cepa test; 5000 cells were ana- lyzed per treatment).
cMeans followed by the same letter do not differ signifi cantly at the 5% level (Chi-square test).* Negative control; ** Positive control