Antiophidian properties of plant extracts against Lachesis muta venom

ORIGINAL PAPER

Antiophidian properties of plant extracts against Lachesis muta venom

De Paula RC^I; Sanchez EF^II; Costa TR^III; Martins CHG^IV; Pereira PS^V; Lourenço MV^V; Soares AM^III; Fuly AL^I

^IDepartment of Cell Biology, Institute of Biology, Fluminense Federal University, Niterói, Rio de Janeiro State, Brazil

^IICenter for Research and Development, Ezequiel Dias Foundation, Belo Horizonte, Minas Gerais State, Brazil

^IIIDepartment of Clinical, Toxicological and Bromatological Analysis, School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil

^IVResearch Group on Natural Products, Laboratory of Applied Microbiology, University of Franca, Franca, São Paulo State, Brazil

^VBiotechnology Unit, University of Ribeirão Preto, Ribeirão Preto, São Paulo State, Brazil

^{Correspondence to}

ABSTRACT

Snakebites comprise a serious health problem in several countries due to their global incidence, which exceeds 2.5 million per year, and the elevated number of victim fatalities. To counteract envenomations, antivenoms have been used regularly for more than a century. Apart from side effects including anaphylactic reactions, antivenoms are not able to efficiently neutralize local tissue damage, which contributes to increasing the severity and morbidity observed in patients. This fact, in turn, may be responsible for economic hardship, particularly in rural populations of developing countries. In the present work, we evaluated the antiophidian properties of 12 Brazilian plant extracts against the hemolytic, coagulant, hemorrhagic and proteolytic effects of Lachesis muta venom. Taken together, our data revealed that most of these aqueous products were capable of inhibiting those activities at different levels, except for Sapindus saponaria extract. In contrast, Stryphnodendron barbatiman extract completely neutralized all the analyzed biological activities. Thus, we may conclude that Brazilian flora may also be useful against L. muta accidents.

Key words: snake venom, Lachesis muta, plant extract, antivenom, biological activities.

INTRODUCTION

Snake venoms are a complex mixture of components that affect a wide range of tissues and organs. We may consider the main active ones to be phospholipases A₂ enzymes, metallo- and serine proteases, phosphodiesterases, myotoxins, lectins, L-amino acid oxidases, disintegrins and neurotoxins, which are responsible for hemorrhage, necrosis, edema, blood clotting, hypotension and neurotoxicity effects in victims (1). In Brazil, Bothrops, Crotalus, Lachesis and Micrurus genus are responsible for, respectively, 90, 7, 2 and 0.5% of the accidents. But, although the accidents caused by Lachesis are rare, the incidence of death (also denominated lethality or fatality index) should be considered (2).

Envenomation by snakes is often treated by parenteral administration of antiophidian serum (antivenoms) obtained from hyperimmunized equine serum. This serum therapy efficiently neutralizes the systemic toxic effects, thus preventing victim fatalities. However, antivenoms present some disadvantages. They can induce adverse reactions ranging from mild symptoms to serious ones and they do not neutralize the local tissue damage (3). Thus, it is important to search for new venom inhibitors, whether synthetic or from a natural source, to complement the traditional serum therapy. Hence, the aim of this work was to evaluate the antiophidian properties of twelve aqueous plant extracts from Brazilian flora against biological effects caused by L. muta snake venom.

MATERIALS AND METHODS

Venom and Animals

Lachesis muta snake venom was kindly supplied by the Ezequiel Dias Foundation, Belo Horizonte, Minas Gerais state, Brazil, vacuum dried and stored at -20ºC until experiments. Male Swiss Wistar mice (18-20 g) were obtained from the Center of Laboratory Animals (NAL) of the Fluminense Federal University (UFF).

Plant Extracts

The number of columns, which appears in parenthesis, represents the plant extracts in the graphs displayed in the Results section. Leaves, stems and roots of Eclipta alba (column 1), Mandevilla velutina (column 2), Mikania glomerata (column 3), Jatropha ellyptica (leaves, column 4), Jatropha ellyptica (stems, column 5), Miconia fallax (column 6), Miconia albicans (column 7), Miconia sellowiana (column 8), Stryphnodendron barbatiman (column 9), Tibouchina stenocarpa (column 10), Casearia sylvestris (column 11) and Sapindus saponaria (column 12) were collected from the Biotechnology Unit of the University of Ribeirão Preto (UNAERP), São Paulo state, Brazil. A voucher specimen of each plant has been deposited at UNAERP and authenticated by Professor Hermógenes de Freitas Leitão Filho of the Institute of Biology, State University of Campinas (UNICAMP). Plant extracts were prepared according to Maiorano et al. (4) and further dissolved in physiological saline solution yielding aqueous extracts.

Inhibition of the Venom

For inhibition experiments, a fixed amount of L. muta snake venom was mixed with varying amounts of plant extracts to obtain two venom:plant ratios (w/w), 1:10 and 1:20. Then, the mixture was incubated for 30 minutes at room temperature (r.t.), after which the biological assays were performed.

Phospholipase A₂ (PLA₂) Activity

PLA₂ activity was determined by the indirect hemolytic test using human erythrocytes and hen's egg yolk emulsion as substrate (5). One minimum indirect hemolytic dose (MIHD) was defined as the amount of L. muta venom (μg/mL) able to produce 70-80% hemolysis. To verify the inhibitory action of plant extracts, they were preincubated with one MIHD and the hemolytic assay was performed. The maximal effect (100% hemolysis) was obtained after lysing erythrocytes with distilled water to constitute the control.

Clotting Activity

A normal pool of human plasma from healthy volunteers was mixed with L. muta crude venom and clotting experiments were performed in an Organon Teknika BE Coagulator 1® (Germany) (6). The amount of venom (μg/mL) that clots plasma in 60 seconds was denoted as the minimum coagulant dose (MCD). The anticlotting effect of plant extracts was determined against one MCD. Plant extracts were used as control instead of venom.

Hemorrhagic Activity

Hemorrhagic lesions produced by L. muta venom were quantified by intradermally injecting samples into abdominal skin of mice two hours before the animals were euthanized according to the UFF Institutional Committee for Ethics in Animal Experimentation (7). Then, abdominal skin was removed, stretched and the site of injection analyzed. One minimum hemorrhagic dose (MHD) was defined as the amount of venom (μg/g) that produced a hemorrhagic halo of 10 mm. Hemorrhagic activity was expressed by the means (in mm) of the hemorrhagic halos induced by L. muta venom in the absence or presence of plant extracts. Negative control experiments were performed by injecting saline, in parallel.

Proteolytic Activity

Proteolytic activity was determined according to the method of Garcia et al. (8) using azocasein as substrate (0.2% w/v, in 20 M Tris-HCl, 8 mM CaCl₂, pH 8.8) in the presence orabsence of plant extracts. One minimum proteolytic dose (MPD) was defined as the amount of venom (μg/mL) able to produce a variation of 0.2 at A405 nm.

Ethics Committee

The experiments were approved by the UFF Institutional Committee for Ethics in Animal Experimentation and are in accordance with the guidelines of the Brazilian College for Animal Experimentation (COBEA).

Statistical Analysis

Results are expressed as means ± SD obtained with the indicated number of animals or experiments performed. The statistical significance of differences among experimental groups was evaluated using the ANOVA test and p values less than 0.05 were considered statistically significant.

RESULTS

A previous study found that L. muta venom presented phospholipase A₂ (indirect hemolytic activity), hemorrhagic and proteolytic activities and also induced blood clotting (9). Herein, we evaluated the ability of different plant extracts to neutralize those related biological activities of L. muta crude venom.

Antihemolytic and Anticlotting Effects

As shown in Figure 1, plant extracts were able to inhibit the indirect hemolytic (Panel A) and clotting (Panel B) activities caused by L. muta venom (12 μg/mL and 15 μg/mL), but with different potencies. The venom concentrations used above represent the MIHD and MCD, respectively. However, the extracts of E. alba (column 1), M. velutina (column 2), M. fallax (column 6), M. albicans (column 7), S. barbatiman (column 9) and T. stenocarpa (column10) inhibited the indirect hemolytic activity (100%), in contrast to S. saponaria (column 12) which did not neutralize this effect. Meanwhile, E. alba (column 1), M. velutina (column 2), S. barbatiman (column 9) and T. stenocarpa (column 10) fully inhibited coagulation of L. muta (100%), since no clotting was observed throughout ten minutes of observation. However, M. glomerata (column 3), J. ellyptica (column 4), J. ellyptica (column 5), M. albicans (column 7), M. sellowiana (column 8), S. barbatiman (column 9), T. stenocarpa (column 10), C. sylvestris (column 11) and again S. saponaria (column 12) did not counteract the clotting effect of L. muta venom. The M. fallax extract (column 6) was able to delay clotting fourfold (at 1:20 venom:plant ratio) when compared with venom. None of the plant extracts alone produced hemolysis or induced blood clotting (data not shown).

Antihemorrhagic and Antiproteolytic Effects

The effects of aqueous extracts on hemorrhagic (Figure 2A) and proteolytic (Figure 2B) activities induced by L. muta venom were also analyzed. As observed in Figure 2A, the extracts of M. glomerata (column 3) and S. saponaria (column 12) did not counteract L. muta venom-induced hemorrhage (12.5 μg/g). In contrast, all of the other plant extracts (J. ellyptica, column 4; J. ellyptica, column 5; M. fallax, column 6; M. sellowiana, column 8; S. barbatiman, column 9 and T. stenocarpa, column 10) did neutralize such lesions induced by L. muta venom and produced inhibition of around 100 % (Figure 2A).

It is worth noting that S. barbatiman extract (column 9) produced 100% inhibition of a three-fold MHD (30 μg/g) of L. muta (data not shown). In parallel, plant extracts also inhibited one MPD of L. muta venom (10 μg/mL) with different potencies (Figure 2B). As shown, the extracts of M. velutina (column 2) and M. glomerata (column 3) presented the lowest inhibitory effect (60 and 65 % for 1:10 venom:plant ratio; 85% and 65% for 1:20) while S. sapindus (column 12) was not capable of inhibiting this phenomenon. The other plant extracts - J. ellyptica (column 4), J. ellyptica (column 5), M. fallax (column 6), M. albicans (column 7), M. sellowiana (column 8), S. barbatiman (column 9) and T. stenocarpa (column 10) - inhibited 100% of L. muta proteolytic activity (10 μg/mL) according to both venom:plant ratios evaluated (Figure 2B). Again, plant extracts or saline alone did not produce hemorrhagic lesions or interfere in the proteolytic assay (data not shown).

Inhibitory Profile of Plant Extracts

Furthermore, we compared the inhibitory profile of plant extracts according to their indirect hemolytic (hemolysis), clotting, hemorrhagic and proteolytic biological activities (Table 1). As shown in Table 1, none of these activities was affected by S. saponaria. However, M. glomerata and J. ellyptica (stems) extracts were not able to completely inhibit any activity; but M. velutina, J.ellyptica (leaves) or C. sylvestris extracts fully inhibited at least one activity (clotting, proteolytic and proteolytic, respectively). On the other hand, the extracts of M. fallax (hemolysis and hemorrhagic), M. albicans (hemorrhagic and proteolytic), M. sellowiana (hemolysis and proteolytic), S. barbatiman (hemolysis, hemorrhagic and proteolytic) and T. stenocarpa (hemolysis, hemorrhagic and proteolytic), inhibited 100% of at least two of those activities. Also, it is worth highlighting that only the S. barbatiman extract fully abolished all of the activities assessed in this work (Table 1).

DISCUSSION

Given the high incidence of accidents caused by Bothrops and Crotalus species, data from the literature have focused much attention on them (10). But, although the accidents caused by L. muta snake venom are rare, they also represent a medical problem in Brazil, especially because of the high lethality index (1.0%) when compared to approximately 0.3% for the Bothrops genus (2).

Envenomation by L. muta promotes a wide range of symptoms including nociception, edema and muscle necrosis, as well as neurotoxic, hemorrhagic, hemolytic and blood clotting disorders. These effects are related to the presence of phospholipase A₂ enzymes, serino- and metalloproteases, L-amino acid oxidases, nucleotidases and others. The pathophysiological alterations may cause victims to die. To counteract those systemic and local effects of venoms, an antiserum is used as treatment.

According to the Brazilian Health Ministry and World Health Organization (11) guidelines, the antiophidic serum is produced by the following three public institutes in Brazil: the Butantan Institute in São Paulo city, São Paulo state; Ezequiel Dias Foundation in Belo Horizonte city, Minas Gerais state; and the Vital Brasil Institute, in Niterói city, Rio de Janeiro state; and then distributed free of charge to hospitals. As far as we know, this treatment is not efficient enough to neutralize local effects, thus contributing to high morbidity indexes observed in Brazil (2, 12-15).

Indeed, antisera possess some disadvantages such as side effects in victims, poor availability in distant areas, delays in administration, need for refrigeration, high cost and low efficacy (3, 10). Furthermore, snake venoms have both inter- and intraspecific variation in composition that, in turn, may affect antivenom neutralization ability (16). Because of these factors, alternative treatments for snakebite are under investigation and plant extracts constitute a rich source of molecules with several pharmacological properties (4, 17, 18).

In several countries, plants have been used by indigenous groups and in folk medicine against snake bite envenomation. However, in most of the cases, scientific validation of their antiophidian properties is needed. As far as we know, there are many examples of medicinal plants in folk medicine and it is believed that 80% of the world population uses plants as the first-line treatment in cases of envenomation (18). Although the action mechanism remains unknown, several reports have shown that plant-derived compounds including rosmarinic acid, quercetin, aristolochic acid pterocarpans and terpenoids and crude extracts are able to neutralize toxic snake venom effects (17, 19-23).

In the present experiment we demonstrated for the first time the ability of different aqueous plant extracts to neutralize biological effects of L. muta snake venom, due to the scarcity of data on the use of plants to neutralize its venom. All of the plant extracts tested in this work inhibited at least one biological activity, except for S. saponaria that failed to neutralize any activity. The biological activities evaluated herein are relevant to envenomation symptoms such as necrosis, hemolysis, hemorrhage, inflammation, edema and pain, which obviously may contribute to victim fatalities. Therefore, vegetal extracts appears as an alternative treatment against local damage to complement the serum therapy or may be useful as prototypes for designing new antiophidian molecules to improve the current treatment used for L. muta bites.

ACKNOWLEDGMENTS

This work was supported by the State of Rio de Janeiro Research Foundation (FAPERJ), the State of São Paulo Research Foundation (FAPESP), the National Council for Scientific and Technological Development (CNPq), the Coordination for the Improvement of Higher Education Personnel (CAPES) and the International Foundation for Science (IFS grant F/4571-1).

Received: September 14, 2009

Accepted: February 10, 2010

Abstract published online: February 12, 2010

Full paper published online: May 30, 2010

Conflicts of interest: There is no conflict.

Financial Source: FAPERJ, FAPESP, CNPq, CAPES and IFS (grant F/4571-1).

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Publication Dates

• Publication in this collection
  07 June 2010
• Date of issue
  2010

History

• Received
  14 Sept 2009
• Accepted
  10 Feb 2010