Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom

Background Snakebite is a significant public health issue in tropical countries. In Brazil, some of the most common snake envenomations are from Bothrops. Bothrops bites trigger local and systemic effects including edema, pain, erythema, cyanosis, infections, and necrosis. Vellozia flavicans is a plant from the Brazilian “cerrado” (savanna) that is popularly used as an anti-inflammatory medicine. Since inflammation develops quickly after Bothrops bites, which can lead to infection, the aim of the present study was to observe possible anti-snake venom and antimicrobial activities of V. flavicans (Vf). Methods The chromatographic profile of the main constituents from the Vf leaf hydroalcoholic extract was obtained by thin-layer chromatography (TLC). The anti-snake venom activity was measured by Vf’s ability to neutralize the in vitro neuromuscular blockade caused by Bothrops jararacussu venom (Bjssu) in a mouse phrenic nerve-diaphragm model (PND). After a 20 min incubation, preparations of PND were added to Tyrode’s solution (control); Vf (0.2, 0.5, 1, and 2 mg/mL); 40 μg/mL Bjssu; pre-incubation for 30 min with Bjssu and 1 mg/mL Vf; and a Bjssu pretreated preparation (for 10 min) followed by 1 mg/mL Vf. Myographic recording was performed, and the contractile responses were recorded. The antimicrobial activity (minimum inhibitory concentration [MIC] and minimum bactericidal concentration [MBC]) was obtained for Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis, using gentamicin and vancomycin as positive controls. Results TLC analysis yielded several compounds from Vf, such as flavonoids (quercetin) and phenolic acids (chlorogenic acid). Bjssu completely blocked the contractile responses of PND preparations, while Vf preserved 97% (±10%) of the contractile responses when incubated with Bjssu. In the PND pretreated with Bjssu, Vf was able to inhibit the neuromuscular blockade progress. MIC and MBC of Vf ranged from 2.5 to 5.0 mg/mL for P. aeruginosa and S. aureus strains, while no antimicrobial activity was observed for E. coli and E. faecalis. Conclusions The hydroalcoholic extract from Vf leaves was able to neutralize and decrease the in vitro neuromuscular blockade caused by Bjssu. However, it did not show significant antimicrobial activity against the tested bacteria.


Background
Envenomation from snake attacks is a significant public health problem in rural areas of tropical and subtropical countries. According to the World Health Organization, there are at least 421,000 accidents involving snakes and 20,000 deaths worldwide from snakebite each year [1].
In Brazil, the genus Bothrops is a major group of snakes that commonly cause envenomation. Snakebites from this genus, which includes those of Bothrops jararacussu, are characterized by local and systemic effects, such as inflammation at the bite site, ecchymosis, bleeding, and skin infection with abscess. Snakebites are usually associated with inoculation of bacteria present in the snake's mouth, which can lead to necrosis, gangrene, and amputation [2].
There are few reports on the in vivo neurotoxicity induced by B. jararacussu venom. Some reports detail unspecified signs [3][4][5] or describe systemic effects including blindness, blurred vision, difficulty in swallowing, and paralysis, which are reminiscent of the actions of Crotalus venom [6]. However, the in vitro irreversible neuromuscular blockade induced by B. jararacussu venom, which was first demonstrated by Rodrigues-Simioni et al. [7], has inspired other studies with other species of Bothrops.
Vellozia flavicans Mart. ex Schult. (Velloziaceae) is a native plant from the Brazilian "cerrado" (savanna) vegetation, popularly known as "canela-de-ema". It is an herbaceousshrub used as an anti-inflammatory and anti-rheumatic in folk medicine [8]. The Velloziaceae family contains about 270 species of tropical monocotyledonous plants, many of which are found in the Brazilian tropical scrub growing on rock outcrops [9]. Branco et al. [10] characterized the chemical constituents of V. graminifolia, which shares phytochemical groups already found in other Vellozia plants, such as flavonoids [11,12], diterpenoids [13][14][15], and triterpenoids [16,17]. Although the phytochemical elucidation is relatively advanced for this family, little is known about the pharmacological properties of specific species.
Plants used as anti-inflammatory medicines could be potentially effective for treating snakebites. This pharmacological relation has been confirmed by studies that found both anti-inflammatory and anti-venom properties from some plant extracts and their compounds [18,19]. Therefore, as V. flavicans is popularly used as anti-inflammatory medicine, it could have also some anti-venom activity. To test this hypothesis, a model using B. jararacussu venom is better than other venoms, such as Crotalus durissus terrificus, because the latter does not induce significant inflammation in the tissues around the snake bite [20]. B. jararacussu venom causes inflammation at the bite site and induces an irreversible neuromuscular blockade in vitro, but not in vivo [7].
The aim of the present study was to observe the ability of a hydroalcoholic extract obtained from V. flavicans leaves to neutralize the in vitro neuromuscular blockade caused by B. jararacussu venom in a mouse phrenic nerve-diaphragm model. In addition, the antimicrobial activity of the same extract was verified against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Enterococcus faecalis strains, using gentamicin and vancomycin as positive controls. Thin-layer chromatography (TLC) was used to observe the main chemical constituents of the extract.

Plant material
The leaves of V. flavicans were collected at Porto Nacional, Tocantins State, Brazil, in September 2011, at S10°44' 08.3" and W048°21' 46.7". The plant was identified by the Botany Section from Biology Department of Tocantins Federal University, where a voucher specimen was deposited (HTO 8533). The V. flavicans leaves were dried at 37°C over 7 days and then powdered, ground in a mill, and macerated over 5 days in 70% ethanol. Then, the suspension was percolated (under protection against light) at 20 drops/min, resulting in a 10% (w/v) hydroalcoholic extract [21]. The extract was concentrated under reduced pressure, lyophilized, and stored at room temperature without light and humidity until the assays were performed.

Antiophidian activity V. flavicans hydroalcoholic extract solubilization
The lyophilized extract was solubilized in 30 μL dimethyl sulfoxide (DMSO; Sigma) and added to Tyrode's solution (5 mL) before addition to the bath containing the biological preparation (see below). This DMSO concentration did not cause changes in the basal response of the neuromuscular preparation [23].

Venom
Crude venoms were collected from male and female adult B. jararacussu specimens (Bjssu, DL50 mouse, e.v. = 5.18 mg/kg), in a glass receptacle covered with plastic paraffin film to avoid saliva contamination. The snake specimens are kept in the Serpentário do Centro de Estudos da Natureza -CEN at the University of Vale do Paraiba (UNIVAP, São José dos Campos, SP, Brazil) [24]. After collection, the venom was immediately placed in an ice bath, lyophilized, and kept in a refrigerator at 4°C. This process was certified by Professor Dr José Carlos Cogo from UNIVAP.

Animals
Male Swiss white mice (26-32 g) were provided by Animais de Laboratorio (Anilab, Paulinia, Brazil) and housed at 25 ± 3°C on a 12 h light/dark cycle, with food and water ad libitum. This study was approved by the institutional Commission for Ethics in the Use of Animals (CEUA) at the Federal University of São Carlos (UFSCAR) under protocol number 063/2012. All the experiments were performed following the guidelines of the Brazilian Society of Laboratory Animal Science (SBCAL).

Mouse phrenic nerve-diaphragm muscle (PND) preparation
The phrenic nerve-diaphragm [25] was obtained from mice anesthetized with halothane and killed by exsanguination. The diaphragm was removed and mounted under a tension of 5 g/cm in a 5 mL organ bath containing aerated Tyrode's solution (control). This solution was composed of the following constituents (in mM): NaCl 137, KCl 2.7, CaCl 2 1.8, MgCl 2 0.49, NaH 2 PO 4 0.42, NaHCO 3 11.9, and glucose 11.1.

Antimicrobial activity
Bacterial strains and culture conditions

Determination of minimum inhibitory (MIC) and bactericidal (MBC) concentrations
MIC and MBC values were determined by using the broth microdilution method according to the guidelines from the Clinical and Laboratory Standards Institute (CLSI) protocol M07-A9. Briefly, four to five colonies were harvested from pure cultures growing on TSA and were used to prepare a bacterial inoculum. Colonies were transferred into tubes containing 5 mL of TSB and cultured at 37°C until reaching turbidity equivalent to a 0.5 McFarland standard (approximately 1.5 × 10 8 CFU/mL).
The bacterial inoculum (100 μL) was then added into each well, allowing a final concentration of 5 × 10 5 CFU/ mL. Plates were incubated under aerobic conditions at 37°C for 18 h and visually inspected for turbidity after incubation. MIC was recorded as the lowest extract concentration that caused no turbidity. Solvent controls were assayed with the treatments to verify their possible antibacterial activity. Results were also compared with vancomycin, gentamicin (positive controls), and ethanol (negative control).
For MBC determination, 10 μL from each well with no visible growth were plated on TSA and incubated at 37°C for 24 h. MBC was considered as the lowest extract concentration that completely inhibited (100%) the bacterial growth. Both MIC and MBC values (mg/mL) were averaged from two different experiments performed in replicate.

Statistical analysis
Results are shown as the mean ± SEM. Number of experiments (n) is indicated in the legend of the figure. Student's t-test was used to compare all data and the significance level was set at 5%.

Results and discussion
One of the criteria for selecting plants for pharmacological studies is their traditional use in folk medicine (ethnopharmacology). Selection is also based on chemical composition, especially considering certain compounds with well-known pharmacological activity [28,29]. In the present study both criteria were considered, since V. flavicans is traditionally used as an anti-inflammatory and antirheumatic. In addition, its chemical composition has been studied. Velloziaceae mainly contain flavonoids (phenolic compounds), diterpenes, and triterpenoids as the main secondary metabolites. Terpenoid is a term used to indicate that the substances have a common biosynthetic origin, the isoprene molecule. Flavonols are related to natural resistance factors and have some biological effects including antimicrobial and cardiovascular activities [30]. Terpenoids have other functions including growth-regulating properties, communication, and defense against insects [22].
Chlorogenic and caffeic acids may act as antidotes against snake venoms by binding to proteins through hydrophobic interactions and hydrogen bonds [32]. Quercetin is as a potent inhibitor of lipoxygenase, which explains its anti-inflammatory properties. Quercetin has a phenolic hydroxyl group on carbon 5, a pyronic carbonyl in close proximity, and coplanarity [33].
Even using other solvent systems, terpenoids were not found when compared with their representative βsitosterol, the most abundant of the phytosteroids. Thus, the TLC profile, acting as a plant quality control, allowed the characterization of V. flavicans extract and comparison with literature data. However, the pharmacological activity is mainly attributed to flavonols. Mors et al. [33] proposed a correlation between the ability of plants and their chemical components to neutralize snake venoms, and their anti-inflammatory and antihepatotoxic properties. In the present study, B. jararacussu venom was chosen to test if a plant with anti-inflammatory properties also has antiophidian potential, because B. jararacussu venom causes inflammation at the bite site [34] and causes an irreversible neuromuscular blockade in vitro [7,35,36].
The clinical effects of B. jararacussu venom are well described by Milani-Júnior et al. [37] and are similar to those of other Bothrops species. Although the mechanism of cytokine production induced by Bothrops venoms is not completely understood [38], the compounds leading to inflammation are well established. Therefore, since the anti-inflammatory properties are already attributed to V. flavicans [8], we decided to test the ability of this plant extract in neutralize the envenomation caused by B. jararacussu venom in a well-controlled and previously validated experimental design.
The World Health Organization has included snakebite envenoming as a neglected disease [39]. Few efforts have been carried out to change this, even considering the existing serum therapy. As observed in the present  study and some other studies, plants could be an alternative or a complementary therapy to serum treatment. Figure 2 shows the dose-response curve (in mg/mL) of V. flavicans hydroalcoholic extract. Concentrations of 0.2 mg/mL (p < 0.05); 0.5 mg/mL (p < 0.05) and 2 mg/mL (p < 0.05), but not 1 mg/mL (p > 0.05), showed significantly less twitch tension than Tyrode's control solution, indicating that these concentrations were able to interfere with the normal contraction of PND. A better result was obtained with 1 mg/mL V. flavicans extract, since this concentration did not change the basal response of neuromuscular preparation. Curiously, the low concentrations (0.2 and 0.5 mg/mL) caused a decrease in the baseline (Tyrode's solution). This result suggests a parallel regulation on skeletal muscle excitability by an unclear mechanism, since other endogenous signaling pathways are capable to compensate the cholinergic control of movement. Glutamate, for example, was recently suggested as a co-transmitter of acetylcholine in motoneurons of mammalian neuromuscular junction [40,41]. Figure 3 shows the pharmacological profile of preincubated (30 min) venom (40 μg/mL) plus V. flavicans extract (1 mg/mL) compared with the venom alone and Tyrode's solution. The extract was able to completely abolish the decreased twitch tension caused by B. jararacussu venom at all times. At the end of experiment (120 min), the preparation with V. flavicans extract was 97% (±10%) responsive, indicating extract efficacy.
Effectiveness of mixing a plant extract or an isolated phytochemical with a venom to abolish the venom toxic effects is not guaranteed. Success depends on the venom and plant constituents. The main constituents of V. flavicans very closely reproduce the results obtained with the hydroalcoholic extract of Casearia sylvestris Sw. [42] or Casearia gossypiosperma Briq. [43], which are rich in flavonoids and polyphenols. However, the results were not similar with those observed for Dipteryx alata, which is rich in triterpenoids [26,44]; Mikania laevigata, which is rich in coumarin [45]; Plathymenia reticulata Benth., which is rich in tannins [46]; or Camellia sinensis, which is rich in catechins [36]. Nevertheless, except for tannins  and tannic acid, which act by a clearly visible precipitation [45], most of the mechanisms of action of other phytochemicals remain unclear. Figure 4 shows the representative myographic register of B. jararacussu venom (A, 40 μg/mL) exhibiting the characteristic irreversible muscle paralysis. When venom (40 μg/mL) + V. flavicans extract (1 mg/mL) are preincubated together for 30 min before addition into the bath, the toxic effect of the venom was not expressed (B). This result shows the total protection given by the plant extract against the venom, probably by a mechanism that excludes protein precipitation, as seen in tannins and tannic acid [45]. Even in preparations pretreated with B. jararacussu (40 μg/mL) for 10 min, which showed contracture and 40% of visible paralysis, the addition of 1 mg/mLV. flavicans extract into the bath was able to delay the paralysis progression, in a non-preincubated model (C). The mechanism of the in vitro, but not the in vivo, neuromuscular paralysis caused by B. jararacussu venom was recently demonstrated [36]. There is a direct relationship between the cell damage level and the percentage of twitch tension response [43].
The antimicrobial activities (MIC and MBC) of V. flavicans extract against E. coli, E. faecalis, P. aeruginosa, and S. aureus strains values are shown in Table 1. All susceptible bacterial species were inhibited or killed by vancomycin and/or gentamicin, in accordance to the recommendations of CLSI. Ethanol (extract solvent) did not interfere with bacterial growth in the concentrations tested.
To assess the antimicrobial activity of a plant extract, strict endpoint criteria for MIC, IC 50 and/or IC 90 must be considered. Generally an extract can be considered effective if it shows IC 50 lower than 100 μg/mL [47]. The MIC values found in the present study were up to 50 times higher than the concentration considered as the maximum concentration acceptable for IC 50 . Therefore, V. flavicans extract does not possess relevant antibacterial activity, considering the high values of MIC and MBC for P. aeruginosa and S. aureus strains (2.5-5.0 mg/ml) and the absence of inhibition of E. coli and E. faecalis strains.
Some specific plants extracts and metabolites can be effective against microorganisms and snake envenomation. Andrographis paniculata demonstrated activity against different pathogenic microorganisms [48] and it has anti-venom activity against Naja naja venom [49]. Vitis vinifera can effectively inhibit toxic effects, such as edema, hemorrhage, myonecrosis, and coagulation of human plasma induced by Echis carinatus venom [50]. V. vinifera can also inhibit the growth of S. aureus, E. coli, and Bacillus cereus [51]. A plant extract showing both anti-venom and antimicrobial activities would be very useful since it could be used to prevent or to treat local/systemic infection caused by snakebite [2]. Although V. flavicans extract did not show good antimicrobial activity and a more appropriate experimental inflammation model was not used [34], the extract showed potential for treating Bothrops snake envenomation.

Conclusions
In conclusion, the V. flavicans hydroalcoholic extract showed promising results against the in vitro neuromuscular blockade-induced by B. jararacussu venom at the nerve-muscle apparatus. The extract reduced the characteristic cell damage induced by the venom and avoided cell damage progression.