Inhibitory Effect of Plant Manilkara subsericea against Biological Activities of Lachesis muta Snake Venom

Snake venom is composed of a mixture of substances that caused in victims a variety of pathophysiological effects. Besides antivenom, literature has described plants able to inhibit injuries and lethal activities induced by snake venoms. This work describes the inhibitory potential of ethanol, hexane, ethyl acetate, or dichloromethane extracts and fractions from stem and leaves of Manilkara subsericea against in vivo (hemorrhagic and edema) and in vitro (clotting, hemolysis, and proteolysis) activities caused by Lachesis muta venom. All the tested activities were totally or at least partially reduced by M. subsericea. However, when L. muta venom was injected into mice 15 min first or after the materials, hemorrhage and edema were not inhibited. Thus, M. subsericea could be used as antivenom in snakebites of L. muta. And, this work also highlights Brazilian flora as a rich source of molecules with antivenom properties.


Introduction
Snakebites are a public health problem that affects mainly poor people who live in rural areas of Africa, Asia, Latin America, and Oceania [1,2]. According to World Health Organization, it is estimated that up to five million people are bitten by snakes every year, and of these, 100,000 deaths occur annually, with 400,000 amputations and other severe health consequences (such as infection, tetanus, and scarring). Poor access to health care and scarcity of antivenoms increase the severity of the injuries and their outcomes. Snake venoms are a complex mixture of proteins, including metalloproteinase, serine protease, phospholipase A 2 , disintegrins, Ctype lectins, myotoxins, and others [3,4]. Lachesis muta (Bushmaster) is the longest venomous snake in the Americas and is distributed in the equatorial forests east of the Andes, ranging from eastern Ecuador, Colombia, Peru, northern Bolivia, and eastern and northern Venezuela, to Guyana, French Guyana, Surinam, and northern Brazil. Within their range, they are often abundant and are an important cause of snakebites [5]. L. muta snakebites are mainly characterized by systemic (generalized bleeding, coagulopathy, renal failure, and shock) and local effects (pain, hemorrhage, edema, and necrosis) [6,7]. In South America, B. jararaca has a higher incidence of accidents (95%) than L. muta (2%), but, on the other hand, L. muta bites led to more severe symptoms (listed above) and have lethality indexes three times higher than B. jararaca [8]. Thus, reducing these indexes is necessary and must be fast.
Although antivenoms are effective against systemic effects, they do not inhibit the local effects. And, a few countries produce antivenom of adequate quality as well. Moreover, antivenoms may produce side effects and their cost of production may be expensive. In fact, worldwide the production of antivenoms is a global challenge. Due to all these limitations, the search for alternative or complementary treatment for snakebites deserves attention.
The present work investigates the ability of different ethanolic extracts of leaves and stem of M. subsericea and solvent-partitioned fractions to neutralize some in vitro and in vivo biological activities induced by L. muta snake venom. The leaves (1.930 kg) and stems (0.960 kg) of M. subsericea were dried at 40 ∘ C for two days, ground, and macerated with ethanol 96% (v/v) at room temperature until exhaustion. Then, both materials were filtered and solvent evaporated using a rotary evaporator, yielding 530 g of ethanolic extract of leaves (ETL) and 169.3 g ethanolic extract of stems (ETS). These extracts were sequentially washed with solvents of increasing polarities (hexane and ethyl acetate). After complete removal of solvent under reduced pressure, using a rotary evaporator, 21.6 g hexane-soluble (HEL) and 11.5 g ethyl acetate-soluble (EAL) fractions from leaves and 12.1 g hexane (HES) and 13.7 g ethyl acetate (EAS) fractions from stems were obtained. All materials were dissolved in DMSO (30% v/v) to perform the biological assays.

Venom and
Animals. Lachesis muta lyophilized venom was kindly supplied from Fundação Ezequiel Dias (FUNED), Brazil. Venom was diluted in physiological saline (1 mg/mL) and stored at −20 ∘ C until experiments. Male Swiss mice (18-20 g) were obtained from the Center of Laboratory Animals (NAL) of the Federal Fluminense University (UFF). All experiments were approved by the UFF Institutional Committee for Ethics in Animal Experimentation (protocol number 212) that were in accordance with the guidelines of the Brazilian Committee for Animal Experimentation (COBEA).

Proteolytic Activity.
Proteolytic activity of L. muta venom was determined according to the method of Garcia et al. [24], using azocasein as substrate (0.2%, w/v, in 20 mM Tris-HCl, 8 mM CaCl 2 , and pH 8.8). Aliquots of L. muta venom (5-50 g/mL) were incubated with 0.4 mL azocasein at 37 ∘ C for 90 min in a total volume of 1.2 mL. The enzymatic reaction was stopped by adding trichloroacetic acid (5%, v/v, final concentration). The tubes were centrifuged at 15,000 ×g for 3 min. Then, the supernatant was removed and mixed with 2 N NaOH, and the tubes were read at A 420 nm (spectrophotometer Hitachi U-5100) to measure the release of azo dye and recorded from the samples. An effective concentration (EC) was defined as the amount of venom ( g/mL) able to produce a variation of 0.2 units at A 420 nm. For the inhibitory experiments, one EC of L. muta venom (8.75 g/mL) was incubated at room temperature with the extracts or fractions of M. subsericea at 1 : 10 venom : plant (w/w). Then, proteolytic activity was determined accordingly. Positive control experiments were performed by incubating L. muta venom with saline or DMSO (1% v/v).

Clotting Activity.
A pool of citrated normal human plasma (diluted with equal volume of saline) was obtained from the local blood bank (University Hospital Antônio Pedro of UFF) and was mixed with L. muta snake venom (2.5-70 g/mL) and the clotting time was monitored using an Amelung coagulometer, model KC4A (Labcon, Germany). The concentration of venom ( g/mL) that clotted plasma in 60 seconds was considered as the minimum coagulant dose (MCD). To evaluate the inhibitory effect, the extracts or fractions of M. subsericea were incubated at room temperature for 30 minutes with one MCD of venom (25 g/mL) at 1 : 10 venom : plant (w/w), and then the mixture was added to plasma and clotting time was recorded. Negative control experiments were performed in parallel by mixing extracts or DMSO (0.5% v/v) with plasma in the absence of venom.
2.6. Hemolytic Indirect Activity. Hemolytic activity of L. muta venom was determined by the indirect hemolytic test using human erythrocytes and hen's egg yolk emulsion as substrate [25]. One minimum indirect hemolytic dose (MIHD) was defined as the lowest amount of L. muta venom ( g/mL) able to produce 100% hemolysis. To verify the inhibitory action of M. subsericea, one MIDH (17.6 g/mL) was incubated at room temperature for 30 minutes with M. subsericea extracts or fractions at 1 : 10 venom : plant (w/w) ratio. Then, the hemolytic assay was performed. One hundred percent of hemolysis was obtained after lysing erythrocytes with distilled water. Negative control experiments were performed in parallel by mixing extracts or DMSO (1% v/v) with erythrocytes in the absence of venom.

Hemorrhagic Activity.
Hemorrhagic lesions produced by L. muta crude venom were quantified using a procedure described by Kondo et al. [26], with minor modifications. Briefly, samples of venom (100 L) were injected intradermally (i.d.) into abdominal skin of mice. Two hours later, animals were euthanized and the skin was removed, stretched, and inspected for visual changes in the inner surface or subcutaneous layers to localize hemorrhagic spots. Hemorrhage was quantified as the minimum hemorrhagic dose (MHD), defined as the amount of venom ( g/g) able to produce a hemorrhagic halo of 10 millimeters (mm) and that was the positive control. To evaluate the inhibitory effect, the extracts or fractions were incubated with two MHD (1.56 g/g) of venom at room temperature for 30 minutes at 1 : 10 venom : plant ratio (w/w). This mixture was injected i.d. into animals and hemorrhagic activity evaluated, as described above. In another set of experiments, L. muta venom was injected i.d. 15 min first or after injection of M. subsericea extract or fractions. Hemorrhage was expressed as the mean diameter (in millimeters) of the hemorrhagic halo induced by venom in the absence and presence of the extract. Negative control experiments were performed in parallel by injecting i.d. extracts or DMSO (1% v/v) in the absence of venom.

Edematogenic Activity.
Edema-inducing activity of L. muta venom was determined according to Yamakawa et al. [27], with modifications. Groups of mice received subcutaneously (s.c.) 50 L of L. muta venom (0.74 g/g) in the right paw, while the left paw received 50 L of saline, DMSO (1% v/v), or extracts. One hour after injection, edema was evaluated as the percentage increase in weight of the right paw compared to the left one. Antiedematogenic activity was performed by incubating the extracts or fractions of M. subsericea with L. muta venom for 30 min at room temperature at 1 : 10 venom : plant ratio (w/w), and then mixture was injected s.c. into mice. Also, venom was also injected s.c. prior to or after the extract. Positive control experiments were performed by incubating L. muta venom with saline or DMSO (1% v/v) prior to injection.

The L. muta Venom-Induced
Clotting. ETL (250 g/mL) fully prevented L-muta-induced clotting and ETS delayed clotting so that it took around eight times longer, when compared to control value (L. muta incubated with DMSO or saline) that clotted plasma in around 60 seconds ( Figure 2). When venom was incubated with EAS or EAL, plasma clotted around 300 and 150 seconds, respectively ( Figure 2). On the other hand, HES or HEL did not inhibit clotting induced by venom. DMSO (0.5% v/v, final concentration) did not interfere on clotting time induced by L. muta venom (Figure 2).

The L. muta Venom-Induced Hemolysis.
The extracts ETL or EAS (176 g/mL) inhibited 100% hemolysis induced by L. muta venom, while ETS or HES inhibited circa of 95% and HEL and ETL inhibited 75% and 60% hemolysis, respectively ( Figure 3). Significance level ( < 0.05) when compared to C1 or C2 columns. # means that plasma did not clot until 600 seconds of observation. the mice were fully protected from hemorrhage induced by the venom (Figure 4). HEL inhibited the hemorrhage halo by 46% such activity and HES did not protect mice from L. muta-induced hemorrhage ( Figure 4). However, when L. muta venom was injected into mice 15 minutes prior to or after ETS or ETL, protection was not observed anymore (results not shown).
3.5. The L. muta Venom-Induced Edema. ETL (7.3 g/g) fully inhibited the formation of edema induced by L. muta venom (0.73 g/g), and ETS inhibited around 70% ( Figure 5). HES and EAS inhibited edema below 5% and for EAS or EAL, 40 and 60% inhibition was observed. DMSO (1% v/v) neither interfered on hemorrhagic or edema of venom nor induced hemorrhage.

Discussion
Snakebites represent an important public health problem, mainly in rural and poor areas of developing countries, and because of that, they are considered neglected diseases according to World Health Organization. L. muta snakebites cause pain, edema, necrosis, hemorrhage, vascular disturbances, and death. Since the antiserum treatment is not so effective against local effects, it is necessary to search for alternative or complementary methods in order to efficiently neutralize such effects. Native people have used plants and their metabolites to treat several diseases, including snake bite [9], and the literature has proven their efficacy as well as their metabolites against some harmful activities of snake venoms [28]. Thus, in this work, we tested the ability of ethanolic crude extracts from stem and leaves of M. subsericea and also their soluble fractions (hexane-soluble and ethyl acetate-soluble) to inhibit some toxic and harmful activities induced by L. muta venom. Overall, extracts of the stem and leaves of M. subsericea as well as its fractions inhibited such activities. Their inhibition occurred around 90% the proteolytic activity of L. muta venom that has been associated with some symptoms that follow snakebites, as hemorrhage and/or clotting. As seen, extracts of stem or leaves inhibited clotting and hemorrhage induced by L. muta venom and thus would prevent bleeding or vascular disturbances caused by L. muta bites. ETL inhibited clotting more efficiently than ETS, but EAS was the most powerful to prevent clotting. In addition, ETS, ETL, EAS, and EAL inhibited 100% hemorrhage induced by L. muta. Hemorrhage induced by snake venoms may occur through activating blood clotting and/or degrading specific proteins (as fibrinogen) of the blood cascade system. Metalloproteases and serine proteases are responsible for such harmful effects. Data from the literature state that plants or their products, mainly those enriched in phenolic compounds [29], inhibit hemorrhage by chelating metal ions that are essential for the action of metalloproteases and serine proteases [9,30,31]. Other plants constituents, as flavonoids, xanthenes, and terpenoids, may also bind to the catalytic site of enzymes and, in turn, inhibit their enzymatic activity [32,33]. Previous reports have identified components isolated from the root of Hemidesmus indicus [34] or from Vitex negundo and Emblica officinalis [35] able to neutralize lethal, hemorrhagic, clotting, and inflammatory activities of Viper venom, probably through an antioxidant action [36]. Phospholipases A 2 promote several harmful activities, including hemolysis, platelet aggregation, edema, and myotoxicity [25,37,38], and as seen, M. subsericea interfered on some of these effects. ETS, ETL, HES, and HEL of M. subsericea inhibited 100% hemolysis, but only ETL fully protected mice from edema. ETS or EAL inhibited edema above 60% and EAS inhibited it by 40%. Polar and nonpolar secondary metabolites have been described as responsible for the inhibition of PLA 2 in snake venom [39]. Besides plants, other natural products are inhibitors of PLA 2 enzymes, such as diterpenes from the marine brown algae Spatoglossum schröederi [31] or Canistrocarpus cervicornis [40]. In addition, these algae also inhibited clotting and hemorrhagic activities induced by L. muta venom [31,40]. Proteolysis, edema, and hemorrhage contribute to and/or are responsible for the local effects observed in victims after snakebite, which lead to amputation or morbidity in victims. Previous reports have indicated the presence of beta-and alpha-amyrin esters, such as their acetates, caprylates, and caproates in the hexanic extracts of leaves and stems [13,22] that had antivenom properties. Moreover, spectroscopic analysis indicates the presence of some flavonoids, as quercetin, myricetin, and kaempferol-like in the ethyl acetate-soluble fraction of stems or leaves of M. subsericea (unpublished data). It should be mentioned that these substances displayed antivenom activity [9] and may be partially responsible for the inhibitory properties observed at the present study.

Conclusion
Since M. subsericea extracts inhibited in vivo (hemorrhagic and edematogenic) and in vitro (hemolytic, proteolytic, and clotting) harmful activities induced by L. muta venom, they may have a potential to the development of an alternative or complementary treatment for snakebites caused by L. muta.