The essential oils from M. piperita, C. citratus, R. officinalis, P. boldus and F. vulgare were extracted in the Laboratory of Essential Oils of the Department of Chemistry of the Federal University of Lavras by hydrodistillation over a 2-h period using a modified Clevenger apparatus. They were identified by gas chromatography coupled to a mass spectrometer (CG-MS) and quantified by gas chromatography coupled to a flame ionization detector (FID) according to the procedure previously described by Rezende et al. (2017) [7] .

The analyses involving human blood were approved by the Human Research Ethics Committee (COEP) of the Federal University of Lavras, with registration number 48793115.0.0000.5148. The erythrocyte suspension was prepared using 10 mL of blood collected in tubes containing sodium citrate, which were centrifuged for 10 min at 4˚C and 2500 g. After centrifugation, the plasma was removed, and the erythrocytes were suspended in phosphate-buffered saline (PBS) (pH = 7.2 - 7.4) and centrifuged again under the same conditions. This procedure was repeated three times to obtain a packed red blood cell pellet.

The analysis of the hemolytic activity of the essential oils was accomplished using the methodology of Price, Wilkinson and Gentry (1982) [8] , with modifications. The medium was prepared with 1% agar in PBS and 0.01 M calcium chloride, 0.1 g sodium azide, and 1% blood erythrocytes. Cavities 3 mm in diameter were made in the medium after solidification in Petri dishes for the application of 0.6, 1.2 and 1.8 μL aliquots of the essential oils, and the plates remained in a cell culture chamber at 37˚C for 16 hours.

The essential oils (0.6, 1.2 and 1.8 μL aliquots) were incubated with the Bothrops jararacussu venom in a water bath at 37˚C for 30 minutes to evaluate a possible inhibitory action of the oils on the hemolysis induced by the venom. The formation of a translucent halo around the cavity in the gel is indicative of activity, and this halo was measured in millimeters for the quantification of hemolytic activity.

The phospolipase activity was determined in a solid medium in accordance with the method described by Gutiérrez et al. (1988) [9] . A gel similar to that described for the determination of hemolytic activity was prepared except that 1% lecithin from egg yolk was substituted for the erythrocytes. After solidification of the medium, cavities 3 mm in diameter were prepared for appliction of the samples.

The essential oils (0.6, 1.2 and 1.8 μL) were incubated with B. jararacusu venom in a water bath at 37˚C for 30 minutes and then applied to the plates, which were maintained for 16 hours at 37˚C in a cell culture chamber. The formation of a translucent halo around the hole in the gel was indicative of activity, and this halo was measured in millimeters for the quantification of phospholipase activity.

For the cytotoxic and phospholipase activities, the test was performed by comparing averages one at a time (each volume was separately compared to the control). The data were submitted to analysis of variance, and the means were compared by the Scott-Knott test at the 5% probability level. The statistical program used was SISVAR [10] .

With the exception of the essential oil from R. officinalis, all the oils evaluated

induced hemolysis (Figure 6); halos between 9 and 15 mm in diameter were observed for the oils from C. citratus and P. boldus. The 15 mm halo, referring to the 1.8 μL volume of oil from P. boldus, does not differ significantly from the control containing only venom.

Pre-incubation of the essential oil from P. boldus with B. jararacussu venom resulted in potentiation of hemolytic activity by approximately 30% for all the volumes of oil evaluated (Figure 7). An inhibition of approximately 45% occurred with the 0.6-μL aliquot of the essential oil from M. piperita, whereas the 1.2- and 1.8-μL volumes potentiated the action of the hemolytic enzymes present in the venom, represented mainly by proteases and phospholipases A₂.

Contrary to the oil from R. officinalis, the lowest volume (0.6 μL) of M. piperita oil evaluated potentiated the lithotripsy induced by venom (20%) and inhibited this activity (35%) when the highest volumes (1.2- and 1.8-μL) were tested. For the essential oil from F. vulgare, 100% inhibition was observed with the 0.6- and 1.2-μL volumes. However, the essential oil from C. citratus did not significantly alter the hemolytic activity induced by the venom (Table 1). These differentiated activities for each essential oil can be explained by the difference in their compositions.

The different performances of the essential oils evaluated for the hemolytic activity induced by B. jararacussu venom are presented in Table 1. The data suggest the presence of specific interactions between the constituents of some oils and the hemolytic toxins present in the venom because potentiation was not observed for C. citratus oil (hemolytic in volumes of 0.6-, 1.2- and 1.8-μL) and F. vulgare (hemolytic with the 1.8 μL volume). Potentiation would be expected if there was a sum of the effect of the oils with that of the venom. In addition, significant potentiation was observed with the 0.6-μL volume of R. officinalis oil and significant inhibition with the 1.2- and 1.8-μL aliquots. These results differ from those observed with the oil from M. piperita, which caused inhibition at the lowest volume evaluated and potentiation at higher volumes.

*0% value represents the absence of an effect in tests in which the oils did not inhibit or potentiate the action of the venom. **Differ from the positive control (activity of the snake venom considered as 100%) at 5% of significance.

The total or partial inhibition of hemolytic activity may be related mainly to the action of phospholipases A₂, which may represent the presence or absence of interactions between the molecules present in the toxins and essential oil constituents, as well as the possibility that the inhibitory action might also result from antioxidant mechanisms [11] . These mechanisms are closely related to the number of molecules (enzymes and active plant compounds) present in the reaction environment and justify the different actions (inhibitory, potentiating or no effect) observed for the various oil volumes analyzed.

According to Figure 8, the action of the phospholipases A₂ present in the venom was potentiated only by the 1.8-μL aliquots of the essential oils from M. piperita and F. vulgare and the 0.6-μL volume of the C. citratus oil. The smallest volume evaluated (0.6-μL) of the essential oil from R. officinalis caused a 10% inhibition of the phospholipase activity, whereas the essential oil from P. boldus did not alter the activity induced by the venom.

Some studies have described the action of plant compounds on the different classes of enzymes present in snake venoms. Silva et al. (2017) [12] evaluated the inhibitory potential of essential oils from Mentha viridis (L). L. and Mentha pulegium L. on phospholipase A₂ present in snake venoms and observed that both essential oils were able to inhibit the degradation of phospholipids induced by Bothrops venoms. The essential oils also presented hemolytic activity, the activity of the oil from Mentha viridis (L). L. being observed only at the highest concentrations (14.6 and 29 μL∙mL⁻¹).

Miranda et al. (2016) [13] investigated the inhibitory properties of the essential oils from Baccharis dracunculifolia, Conyza bonariensis, Tithonia diversifolia and Ambrosia polystachya by means of the coagulation and fibrinogenolytic activities induced by Bothrops and Lachesis snake venoms. They observed that the essential oils exhibited some therapeutic properties because they inhibited the coagulation and fibrinogenolysis induced by the poisons. The authors suggested that the topical use of the oils, in general, does not require specific pharmaceutical preparations and can be applied directly after the extraction. Many oils with antimicrobial, anti-inflammatory and curative properties are described in the literature, and these actions are of great value in the treatment of snake poisonings.

Miranda et al. (2014) [14] studied the effect of the essential oil from Hedychium coronarium on the fibrinogenolytic and coagulant activities induced by Bothrops and Lachesis venoms. They observed significant inhibition of the coagulation induced by both venoms, suggesting their possible use as a complementary alternative to serum therapy because the essential oils do not require specific formulations and their topical use can be performed immediately upon extraction.

Yamaguchi and Veiga-Junior (2013) [15] evaluated the hemolytic capacity of essential oils obtained from Endlicheria citriodora branches and leaves and observed no damage to the membrane. They also reported that both oils were composed basically of methyl geranate (monoterpenoid ester), which corresponds to 95.15% and 93.75% of the oils from the branches and leaves, respectively.

Phospholipases A₂ in Bothrops snake venom induce the hydrolysis of membrane phospholipids and can generate arachidonic acid, which is a precursor of prostaglandins, thromboxanes, leukotrienes and other bioactive lipids that act mainly in inflammatory processes and in the blood coagulation cascade, thereby altering hemostasis [16] . The different venom toxins can be inhibited by several molecules, including chelating agents such as heparin, plasma factors of animal origin and plant extracts [17] .

Studies by Silva et al. (2017) [12] using Bothrops venom showed that both the essential oil from Mentha pulegium and that from Mentha viridis had an inhibitory effect on phospholipases A₂ on the order of 4.1% at the concentration of 14.6 μL∙mL⁻¹.

In the year 2016, Oliveira et al. [11] evaluated the possible interactions between vitamins and enzymes present in Bothrops atrox and Crotalus durissus terrificus venoms in vitro. Inhibition assays for proteolysis, hemolysis, coagulation, and hemagglutination were performed using different proportions of vitamins to inhibit the minimum effective dose of each venom. The authors observed that the vitamins were responsible for the 100% reduction in the cleavage of azocasein by C.d.t. poison, induced thrombolysis by the B. atrox venom, and also observed the induction of fibrinogenolysis by both poisons.

Oliveira et al. (2016) [11] observed possible interactions between the vitamins and the active site of the enzymes. These interactions may occur in the hydrophobic regions present in the enzymes and vitamins, as well as in the inhibitions exerted by the antioxidant mechanism.

According to reports by Borges et al. (2000) [18] , the aqueous extract of Casearia sylvestris inhibited the hemorrhagic activity caused by the venom of several snakes of the Bothrops genus. An aqueous extract of Mandevilla velutina was an effective inhibitor of phospholipase A₂ and inhibited some of its toxic effects, such as hemorrhage [19] .

Carvalho et al. (2013) [3] reported the importance of plant species in treating snakebites, especially in places that do not have access to serotherapic treatment. Phospholipases A₂, being among the main constituents of Bothrops snake venoms, can be inhibited by components of these plants, such as phenolic compounds, flavonoids, alkaloids, steroids, terpenoids (mono-, di- and triterpenes), and polyphenols (vegetable tannins).

In the present work, approximately 10% inhibition of phospholipases occurred in the presence of the oil from R. officinalis (rosemary). This oil is composed of terpenes, alcohols and ethers. This result agrees with the work reported by Mors, Nascimento and Pereira (2000) [20] that demonstrated the inhibitory action of several pentacyclic triterpenes, such as oleanolic acid, lupeol, ursolic acid, taraxerol, taraxasterol, α, β-amirina and friedeline, on snake venoms. Considering that phospholipase activity is only exerted by PLA₂s and that hemolytic activity is exerted by both PLA₂s and proteases, the observed results point to the presence of specific protease inhibitors in the evaluated oils, especially in the oils from M. piperita, R. officinalis and F. vulgare, which induced significant inhibition of the hemolytic activity exerted by the B. jararacussu venom.