Growth Inhibition and Pro-apoptotic Action of Eleusine indica (L) Gaertn Extracts in Allium test

Background: E. indica is a gramineae largely used in Brazilian traditional medicine for treatment of respiratory diseases. Previous research work has shown this plant to have glicoflavonoids constituents. Place and Duration of Study: The study was carried out during four months in 2014 in the Laboratory of Molecular Biology, Department of Pharmacy, Lutheran University of Brazil (CEULJI/ULBRA), Ji-Paraná, Rondônia, Brazil. Aims: This study aimed to determine possible mutagenicity and/or cytotoxicity activity of E. indica, using the Allium test to investigate root growth, mitotic index and micronuclei formation. Methodology: We applied the Allium test to analyze the effects of E. indica compounds on genetic material. The plant aerial parts were dried, pulverized and prepared as aqueous extract. The experiment consisted in 5 assays: E. indica extracts inconcentrations of 25%, 50%, and100%; positive control (CuSO4); and negative control (distilled water). Each assay was performed with 10 repetitions. Results: Initial results showed inhibition of meristems growth and stable mitotic index. Differential analysis indicated influence in cell division process with a significant number of cells in metaphase Original Research Article Oliveira and Romão; EJMP, 8(3): 121-127, 2015; Article no.EJMP.2015.097 122 that did not progress to the next mitotic cycle stage, and no significant presence of micronuclei. Conclusion: Our results strongly suggest the plant having cytotoxic compounds with microtubule affinity interaction without mutagenicity activity.


INTRODUCTION
Medicinal plants have been used in diseases' treatment in the form of teas and infusions since the beginning of human history [1][2][3]. The Amazon region has a legacy of great genetic variability in plants and many of them are not yet cataloged species. A large array of plant species that have been used for a long time in traditional medicine have not yet been studied. One species that occurs in this region is the grass E. indica, pertaining to the Poaceae family, popularly known as Indian goosegrass; it is characterized as an upright plant, consisting of stem, hairless and striated, with heights ranging from 30 to 70cm, annual, herbal, with reproduction by seed, and with the ability to grow in any type of soil, preferably in environments with high temperatures and humidity [4].
In studies on medicinal purposes, this plant is cited for treatment of diseases related to the respiratory tract [5]. This fact was already confirmed in earlier work through the isolation of two flavones: the vitexin and the schaftoside, both with significant positive results in asthma treatment [6,7]. Studies have reported E. indica use in inflammatory processes, as nasal decongestant, as therapeutic for osteoporosis and plantar fasciitis, and in the form of infusion or decoction against pneumonia, influenza, and fever [8][9][10][11].
The compounds isolated from E. indica belong to the group of flavonoids which comprises more than 6000 substances known to have close relation with physiological processes in plants. Flavonoids regulate growth and development playing a role as a protective mechanism against pathogens and solar radiation. This substances' group has extensive ubiquity and diverse biological activities, which can be beneficial or harmful, and only a limited number of them have been studied with this regard [12,13,9].
The Allium test is an excellent biomarker for first screening of genotoxicity and mutagenicity of medicinal plants due to its low cost, reliability, and compliance with other tests. It is also appropriated to study cytotoxic effects of medicinal plants, whereas the Allium cepa roots are in direct contact with the tested substance, allowing the evaluation in different concentrations. Chromosomal changes and division of meristematic cells in onion root are often used as an alert signal to advert people about the consumption of certain products [14,1]. Therefore, this study aimed to determine possible mutagenicity and/or citotoxity activity of E. indica, using the Allium test to investigate root growth, mitotic index and micronuclei formation.

Phytochemical Compounds Extraction
The extracts were prepared following the folk medicine usage of E. indica. The fresh aerial parts were oven dried with air circulation for 24 hours at 44°C, and subsequently pulverized. The phytochemical compounds extraction was made by decoction. Distilled water (pH=7.2) was heated up to 90°C, then E. indica pulverized parts were added in a proportion of 100g per 1L during 15 minutes [15]. Thereafter, the solution was cooled to room temperature and filtered in filter paper (PRO Lab. Quantitative Filter Paper). The resultant solution consisted in the 100% concentration assay, here and after denominated Eleusine 1 . For the 50% concentration assay, distilled water was added to the Eleusine 1 solution in 1:1 ratio, here and after denominated Eleusine 2 . Finally for the 25% concentration assay, distilled water were used in 3:1 ratio, here and after denominated Eleusine 3 .

Allium Test
The A. cepa (2n = 16) specimens were acquired in the local popular market, all of small size, uniform, same origin, not sprouted and healthy. The bulbs were germinated with the bottom dipped in a 30mL solution which were the 3 E. indica assays, the negative and positive control for a period of 72 hours at 24°C. The experiment had as negative control (NC), distilled water, and as positive control (PC), copper sulfate (CAS Nº. 7758998; Sigma Chemical Co., St Louis, MO, USA) at a concentration of 0.006g/L -1 [16,17].
The experiment was conducted using 10 replicates for each assay. After the exposure period, the meristems were collected and placed in a solution of 3:1 (v/v) methanol/acetic acid at a temperature of 4°C during 12 hours for chemical fixation. Afterwards, the hydrolysis was performed in a 1 mol/L -1 Hydrochloric acid (HCl) solution for 5 minutes in water bath at 60°C followed by distilled water wash.

Slides were made in duplicate and stained with Panotipo
Rapido LB Kit (Record Nº. 80002670065; Reny Lab Chemical and Pharmaceutical Co., Barbacena, MG, Brazil). The squash was performed after the coverslip addition [18]. Slide analysis was done by optical microscopy with 1000x magnification. The variables studied were: number of micronuclei formation per 1000 cells per slide, root number and root length measured after 5 days of exposure, and mitotic index (MI). The MI was calculated as shown in Equation 1, where CM is a number of cells in mitosis and TC is a total number of analyzed cells.

Statistical Analysis
For statistical analysis, the collected data was treated in the statistical software R Core Team, version 3.0.2. The computed tests were ANOVA followed by Tukey's test and the nonparametric Pearson correlation test, with an adopted significance level of P≤0.05.

Physicochemical Characterization
The root number and root length levels are presented in Table 1. These results show that all E. indica assays caused significant root growth inhibition in comparison with negative and positive control assays. The inhibitions in root number and root length were greater in increasing concentrations of E. indica extracts. Average root length was 3.99 with a Standard Deviation (SD) of 1.71 (3.99±1.71 cm) in negative control and 1.4±0.55 cm in positive control. However, average root length in Eleusine 3 assay decreased significantly in comparison to negative control (Table 1). Average root length in treatment assays also decreased significantly according to concentration. Statistical analysis shows that the values observed in Eleusine 3 and Eleusine 2 assays are significant (P≤0.05) and the values of Eleusine 1 are highly significant (P<0.001) 1 . When comparing data from treatment assays among themselves, no significant correlation was found (r=0.27 and P>0.05) 2 .
Inhibition in germination process is not contingent upon extract concentration and dose escalation may not interfere in the inhibitory process [19]. Studies with Cymbopogon citratus popularly known as lemon grass and also belonging to the Poaceae family, found the plant aqueous extract having compounds with allelochemical effects on Lactuca sativa root growth [20]. The influence of some plants on root growth occurs by the production of secondary metabolic compounds that act inhibiting or promoting the germination process and the cell division process [21]. The allelochemicals are released into the atmosphere by plants in various ways: by volatilization, root exudation, leaching and decomposition of waste vegetable structures [21,22].

Mitotic Index
The number of cells in mitosis and respective mitotic index are presented in Table 2, the results are not considered statistically significant (P>0.05). Considering that root growth was inhibited, we analyzed the mitotic cell phase by cell differential analysis and we found that E. indica assays had a large number of cells in metaphase, as shown in Fig. 1, with severe depression values for anaphase and telophase which was proven by comparison with negative and positive controls (P≤0.001). 1 ANOVA and Tukey's test. 2 Pearson Correlation coefficient.  MI measures the proportion of cells in the cell cycle M-phase and its inhibition can be interpreted as cellular death or a delay in the cell proliferation kinetics [23]. Reduction in the mitotic activity could be due to DNA synthesis inhibition or a blocking in the cell cycle G2 phase, preventing the cell from entering in mitosis [24]. Mitodepressive effects of some herbal extracts, including the ability to block DNA synthesis and nuclear proteins, were reported earlier [25,26]. Several other herbal extracts have been reported to inhibit mitosis [27][28][29].
The same effect was observed in earlier studies with chamomile inflorescence, with significant increase in the MI, and cell death concomitant with increased testing doses [30]. In a study conducted by [20], different results were reported for C. citratus extracts, in which root growth inhibition decreased MI. Flavonoids may interfere in the mitotic process, therefore induce apoptosis depending on its nature and concentration [13].
Cell differential analysis determined the absence of membrane cell boundaries in E. indica assays as shown in Fig. 1a. In cells with membrane absence the chromatin was in apoptotic process as in Fig. 1b. initially sparse and in transcription, beginning a rapid process of condensation and inactivation. Coarse lumps of heterochromatin were formed into the nucleus, with some changes in nuclear boundary. The inactivation of genetic material led to dismantling the cytoskeleton and to its disorganization, as in Fig.  1c. causing cell deformation on its contours and undoing its joints [31].
Apoptosis is an essential process to maintain the development of living beings, as it is important to eliminate unnecessary or defective cells. During apoptosis, the cell undergoes morphological changes including cell shrinkage, loss of adhesion to the extracellular matrix and to neighboring cells, chromatin condensation, internucleosomal DNA fragmentation and formation of apoptotic bodies. This biological phenomenon, in addition to playing an important role in controlling many vital processes, is associated with numerous diseases such as cancer [31].

Micronuclei
The negative control assay showed 5

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable. brizanthiae, also a reported scarce micronucleus' formation, that occurs due to the meiotic behavior with slight formation of diploid accessions in the first division which is corrected in meiosis II leading to a normal cell division guided studies with C. citratus in Allium test did not lead to micronuclei formation Aloe vera extract, an average of 5.25 micronuclei was reported, which , although not belonging to , also leads to low e values for micronucleus formation . The low number of micronucleus formation extract may be linked to its flavonol compounds: vitexin and schaftoside, which are antioxidants agents that act by [1,12].
Ultimately, our results indicate that when applied E. indica extract has cytotoxic compounds with microtubule affinity interaction without mutagenicity activity. We used aerial parts. Experiments with crude extracts are appropriate because traditional medicinal herbs are generally used in the same way. Our findings in this study E. indica requires additional investigation with isolated compounds regarding microtubules interaction in the cell