Genotoxic and Chemopreventive Effects of Vochysia divergens Leaves (Pantanal, Brazil)

The medicinal plant Vochysia divergens is a colonizing tree species of the Pantanal, a unique and little explored wetland region in Brazil. This species is used in folk medicine as syrups and teas to treat respiratory infections, digestive disorders, asthma, scarring, and skin diseases. The objectives of this study were to evaluate the antioxidant, cytotoxic, and genotoxic potential of the ethanolic extract of Vochysia divergens leaves (VdE), as well as the influence of VdE and its major component (the flavone 3′,5-dimethoxy luteolin-7-O-β-glucopyranoside; 3′5 DL) on MMS-induced genotoxicity. The extract significantly reduced the viability of V79 cells in the colorimetric XTT assay at concentrations ≥ 39 μg/mL. A significant increase in micronucleus frequencies was observed in V79 cell cultures treated with VdE concentrations of 160 and 320 μg/mL. However, animals treated with the tested doses of VdE (500, 1000, and 2000 mg/kg b.w.) exhibited frequencies that did not differ significantly from those of the negative control group, indicating the absence of genotoxicity. The results also showed that VdE was effective in reducing MMS-induced genotoxicity at concentrations of 20, 40, and 80 μg/mL in the in vitro test system and at a dose of 15 mg/kg b.w. in the in vivo test system. Its major component 3′5 DL exerted no protective effect, suggesting that it is not responsible for the effect of the extract. The results of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay showed that VdE was able to scavenge 92.6% of free radicals. In conclusion, the results suggest that the protective effect of VdE may be related, at least in part, to the antioxidant activity of its chemical constituents.


Introduction
The use of medicinal products derived from plants has increased considerably over the last three decades, with about 80% of people using these products for primary health care [1]. The Brazilian Cerrado, located in the central region of the country, is one of the most extensive biomes (204 million hectares) and is considered the richest tropical grassland in the world in terms of biodiversity and the second largest biome in South America [2]. It is estimated that only 30% of this biodiversity is reasonably known [3]. Plants endemic to the Cerrado have been receiving increased attention as a source of bioactive compounds, especially phenolic compounds [4], substances with known antioxidant, chemopreventive, cytoprotective, antimutagenic, antiestrogenic, and antiangiogenic activities [5].
Vochysia divergens Pohl (Vochysiaceae), commonly known as Cambará, is native to the Amazon Basin. As settlers of the wetlands of the Brazilian Pantanal, this species is considered an invasive plant that is widely tolerant to seasonal variations in hydrological conditions and is therefore resistant to the dry season as well as to the seasonal floods that occur during the rainy season in the Pantanal [6]. The plant is used in folk medicine to treat respiratory infections, digestive disorders, and asthma. There are also reports of its use for wound healing and treatment of skin diseases. The main nutritional reserves of its seeds are proteins, oils, and few carbohydrates [7]. The extracts of the stem bark of V. divergens and some compounds isolated from this plant, beta-sitosterol, betulinic acid, and sericic acid, have been evaluated for antibacterial activity. Sericic acid was the most active compound, a finding that may explain in part the popular use of this plant for the treatment of infectious diseases [8]. Another study identified the schistosomicidal potential of the ethanolic extract of V. divergens leaves and isolated flavones [9]. However, few studies have explored V. divergens. Considering the importance of this species in folk medicine, we performed a toxicogenetic evaluation of the ethanolic extract of V. divergens leaves (VdE) and of its major component, the flavone 3 5-dimethoxy luteolin-7-O--glucopyranoside (3 5 DL), and evaluated their effects on genomic stability and oxidative stress.

Extract Preparation and Flavone Isolation and Quantification.
The leaves of V. divergens (1.27 kg) were air-dried, powdered, and exhaustively extracted by maceration in EtOH. These procedures were performed at room temperature. The solution was then filtered and the solvent was removed under reduced pressure, yielding the crude extract (82.83 g VdE). Part of the extract obtained (3 g) was purified over a Sephadex LH-205 column and eluted with methanol to afford seven fractions. Reverse-phase ODS chromatography was performed with Fraction 3 (80 mg), which was purified by preparative RPHPLC [CH3OH-H2O-CH3COOH (50:49.9:0.1, v/v/v)] to yield 3 5 DL (10 mg) [9]. The flavone was quantified in VdE by HPLC-DAD according to Pimenta et al. [10].

Experimental Design.
Concentrations of VdE of 20, 40, 80, 160, and 320 g/mL were used for genotoxicity assessment. This choice was based on the XTT assay using the criterion of cytotoxicity. Concentrations of 20, 40, and 80 g/mL were used to study the influence of VdE on the genotoxicity induced by the mutagenic agent methyl methanesulfonate (MMS, 44 g/mL; Sigma-Aldrich, St. Louis, MO, USA). The concentrations of 3 5 DL were selected based on the proportion of this compound in VdE, corresponding to 0.77%. Thus, the cell cultures were treated with 0.616 g/mL 3 5 DL for genotoxicity evaluation. The different concentrations of the compound (0.154, 0.308, and 0.616 g/mL) were combined with MMS for the assessment of its influence on genomic stability. Negative (no treatment), solvent (DMSO 1%), and positive (MMS, 44 g/mL) controls were included.

Micronucleus Test.
A total of 500,000 V79 cells were seeded in a culture flask (25 cm 2 ) with 5 mL HAM-F10 + DMEM medium and incubated for 25 h. The cells were then washed with PBS and treated for 3 h with different concentrations of V. divergens and the controls in culture medium without fetal bovine serum. The cells were washed twice with PBS and medium containing cytochalasin-B (3 g/mL; Sigma-Aldrich, St. Louis, MO, USA) and fetal bovine serum was added. The cultures were incubated for 17 h. For micronucleus analysis as described by Fenech [11], 3,000 binucleated cells were analyzed per treatment (1,000 cells/treatment/repetition). The cytotoxicity of the treatments was measured by the nuclear division index (NDI) after the analysis of 1,500 cells (500 cells/repetition). Cells with wellpreserved cytoplasm containing 1 to 4 nuclei were scored. The NDI was calculated according to Eastmond and Tucker [12] using the following formula: where M1 to M4 are the number of cells with 1, 2, 3, and 4 nuclei, respectively, and is the total number of viable cells. In addition, the cytotoxicity index (CI) was calculated as proposed by Kirsch-Volders et al. [13]:  percent mean values obtained in triplicate using the following formula: where is the absorbance without sample (only solvent and free radical) and sample is the absorbance obtained with the crude extract or gallic acid [15].

Statistical
Analysis. The data were analyzed by analysis of variance for completely randomized experiments, with calculation of P values. In cases in which P < 0.05, treatment means were compared by the Tukey test and the minimum significant difference was calculated for =0.05. All statistical analyses were performed using the GraphPad Prism 5.0 program (GraphPad Software, San Diego, CA, USA). 3.2. Colorimetric XTT Assay. Figure 3 shows the cytotoxicity of VdE evaluated by the colorimetric XTT assay in V79 cells at concentrations ranging from 2.4 to 5,000 g/mL. Significant differences were found for concentrations equal to or higher than 39 g/mL when compared to the negative control, demonstrating a cytotoxic effect (Figure 3).   with a CI of 35.61%. No significant differences in NDI were observed between the other cultures treated with VdE, alone or combined with MMS, and untreated cultures, indicating the absence of cytotoxicity ( Table 1). Evaluation of the major component of the extract, 3 5 DL, revealed the absence of genotoxicity and cytotoxicity at the highest concentration tested (0.606 g/mL). No differences in micronucleus frequencies were observed between cultures treated with 3 5 DL plus MMS and those treated with MMS alone (Table 1).

In Vivo Micronucleus
Test. The frequencies of MNPCEs in Swiss mouse bone marrow treated with different doses of VdE alone or combined with MMS are shown in Table 2. There was no significant difference in the frequencies between animals treated with the tested doses of VdE (500, 1,000, and 2,000 mg/kg b.w.) and the negative and solvent control groups, indicating the absence of genotoxicity. In addition, the oral administration of 15 mg/kg b.w. of VdE concomitantly with the injection of MMS led to a significant reduction in the frequency of MNPCEs when compared to the group treated with MMS alone. Evaluation of cytotoxicity revealed no significant differences in the ratios of PCE/total erythrocytes between the different treatments and the negative control, indicating the absence of cytotoxicity. Figure 4 shows the mean sequestration frequency of the DPPH radical obtained for the different concentrations of VdE used to evaluate its possible antioxidant activity. The results demonstrate antioxidant capacity of the extract, with a dose-dependent response and maximum inhibition of 92.6% at the highest concentration.

Discussion
The present results showed that VdE exerted genotoxic activity at the highest concentrations tested in the in vitro test system. The VdE presents flavones in its chemical composition, being the flavone 3 5 DL its major component [9,10]. Polyphenolic compounds, as flavones, can act as prooxidants in some in vitro systems [16]. Some polyphenols may have carcinogenic or genotoxic effects at high doses or concentrations. It is possible that the genotoxic effects observed in vitro may be attributable to the high concentration used, at which polyphenols may become prooxidants [17,18].
Evidence-Based Complementary and Alternative Medicine 5   However, when the extract was evaluated in the in vivo test, no genotoxicity was observed. These divergences in the results between the two test systems may be explained by factors such as metabolism, pharmacokinetics, and DNA repair processes, which are active in the in vivo test system and contribute to the responses observed.
Analysis of the influence of VdE on the genotoxicity induced by MMS revealed a chemopreventive effect of the 6 Evidence-Based Complementary and Alternative Medicine extract. The major component present in VdE, 3 5 DL, was also evaluated in vitro to correlate the data obtained with the possible activity of its chemical constituents. However, no protective effect of the flavone on MMS-induced genotoxicity was found. This finding suggests that the chemopreventive activity of VdE may be due to the synergistic effect of its chemical constituents.
Our study demonstrated that VdE exhibits the characteristics of Janus compounds, i.e., substances that behave as genotoxic or antigenotoxic agent at different concentrations depending on the conditions used. The extract was genotoxic at the highest concentrations tested, whereas it exerted a chemopreventive effect in the in vitro test system at the lower concentrations. Several molecular mechanisms underlying the Janus effect have been postulated; however, the specific induction and consequent saturation of certain enzymes of an antimutagenic system such as DNA repair seem to be more likely [16].
Chemical studies of the genus Vochysia (Vochysiaceae) reported the presence of ellagic acid, physcion, 2,6dimethoxy,4-benzoquinone [17], a pyrrolidinoflavone [18], 3-O-B-D-glucopyranosyl--sitosterol, and two dicarboxylated triterpenes (bartogenic acid and vismiaefolic acid) [19]. Pimenta et al. [9,10] demonstrated flavones as constituents of Vochysia extracts. Flavones are a class of flavonoids that are a subject of increasing interest because of their biological activities in vitro and in vivo, especially their antioxidant activity. Flavones from plants are typically bound to sugar units such as glycosides, especially 7-O-glycosides [20], and may also contain acetyl or malonyl moieties. Flavone O-glycosides are composed of the aglycone moiety and one or more sugars attached through a -linkage [21].
MMS is an SN2 class type mutagenic agent that causes N-alkylation of purines [22]. Additionally, MMS is known to facilitate the formation of adducts such as N7methylguanine (N7MeG), N3-methylguanine (N3MeG), and N3-methyladenine (N3MeA), as well as crosslinks expressed as base substitution mutations. Although DNA adducts do not directly block replication, they produce apurinic sites, with consequent breaks in the double strands [23] that are repaired by base excision, the main defense mechanism against SN2 agents [24]. Regarding the mechanism of action underlying MMS-induced genotoxicity, VdE may act as a chemoprotector by competing with DNA as a target for alkylation, reducing MMS-induced genotoxic damage.
Alkylating agents have been shown to deplete the enzyme glutathione S-transferase in mammalian cells, leading to oxidative stress as a byproduct of normal cellular function which can compromise cellular antioxidant defenses [25]. Considering that alkylating agents may play a role in the generation of reactive oxygen species, the antioxidant compounds present in VdE may be responsible for the reduction in the alkylation damage induced by MMS. The antioxidant activity of VdE was demonstrated by the DPPH reduction assay in the present study. This assay is widely used as a model system of free radical scavenging activity in plants.
Research on natural sources of antioxidant compounds is being conducted because of their importance for preventing the onset of oxidative reactions. Antioxidants act by delaying or preventing the oxidation of substrates involved in oxidative processes, inhibiting the formation of free radicals. As observed for VdE, phytochemical studies have demonstrated the presence of triterpenoids, steroids, and polyphenols in the genus Vochysia and in the family Vochysiaceae. In addition, polyphenolic compounds such as ellagic acid derivatives are considered chemical markers of the family Vochysiaceae [7].
Erratic absorption by the cell membrane and the consequent inconstant bioavailability of the compounds in the cell explain the absence of a significant dose-dependent effect of VdE. In addition, the assessment of dose effects is complicated by the fact that many chemoprotective compounds act simultaneously at different levels of protection [26]. Thus, the lack of observation of a dose-response relationship might be attributed to the activation of different mechanisms depending on the dose of the extract used.

Conclusions
The present results showed a genotoxic effect of VdE only at the higher concentrations tested (160 and 320 g/mL), and no genotoxicity was observed in the in vivo test system. On the other hand, VdE was effective in reducing the genotoxicity induced by MMS. The major component 3 5 DL exerted no protective effect, indicating that it is not responsible for the effect of the extract. The results suggest that the protective effect of VdE may be related to the antioxidant activity of its chemical constituents.

Data Availability
The readers can access the data that support the conclusions of the present study through the tables and figures presented.