Effect of Diterpenes Isolated of the Marine Alga Canistrocarpus cervicornis against Some Toxic Effects of the Venom of the Bothrops jararaca Snake

Snake venoms are composed of a complex mixture of active proteins and peptides which induce a wide range of toxic effects. Envenomation by Bothrops jararaca venom results in hemorrhage, edema, pain, tissue necrosis and hemolysis. In this work, the effect of a mixture of two secodolastane diterpenes (linearol/isolinearol), previously isolated from the Brazilian marine brown alga, Canistrocarpus cervicornis, was evaluated against some of the toxic effects induced by B. jararaca venom. The mixture of diterpenes was dissolved in dimethylsulfoxide and incubated with venom for 30 min at room temperature, and then several in vivo (hemorrhage, edema and lethality) and in vitro (hemolysis, plasma clotting and proteolysis) assays were performed. The diterpenes inhibited hemolysis, proteolysis and hemorrhage, but failed to inhibit clotting and edema induced by B. jararaca venom. Moreover, diterpenes partially protected mice from lethality caused by B. jararaca venom. The search for natural inhibitors of B. jararaca venom in C. cervicornis algae is a relevant subject, since seaweeds are a rich and powerful source of active molecules which are as yet but poorly explored. Our results suggest that these diterpenes have the potential to be used against Bothropic envenomation accidents or to improve traditional treatments for snake bites.


Results and Discussion
Marine organisms are among the richest sources of natural products, with a variety of properties that have led to their use by human beings as sources of medicines and food, as well as for biotechnological applications [22][23][24]. In Brazil, the brown marine algae are the most widely studied, with more than 300 diterpenes isolated from at least 35 species all over the world [19]. Although some biological activities have been studied [21,[25][26][27], the potential of brown seaweeds to neutralize the toxic effects of snake venoms has not been widely investigated.

Antihemolytic and Antiproteolytic Effect
The mixture of diterpenes linearol/isolinearol inhibited hemolysis ( Figure 1A) and proteolysis ( Figure 1B) caused by B. jararaca venom in a concentration-dependent manner. Neither the single diterpenes nor dimethylsulfoxide (DMSO, the vehicle) induced or inhibited hemolysis or proteolysis. PLA 2 enzymes are one of the most active components in snake venoms [28]. These enzymes induce a wide range of pharmacological and toxic effects, including hemolysis, neurotoxicity, cardiotoxicity, effects on platelet aggregation, myotoxicity and edema, that contribute to the envenomation symptoms [29,30]. Domingos et al. [19] have reported the ability of the mixture of diterpenes (linearol/isolinearol) to inhibit the crude venom as well as a PLA 2 isolated from L. muta venom; but, its mechanism of action has not been investigated yet. Moreover, other reports have cited PLA 2 inhibitors from marine organisms, and some of them are terpenes [31].  . jararaca venom (18 µg/mL) was incubated for 30 min at room temperature with a mixture of diterpenes at 45 µg/mL, 90 µg/mL or 180 µg/mL; and (B) B. jararaca venom (2 µg/mL) was incubated with diterpenes at 16 µg/mL, 40 µg/mL or 80 µg/mL, and then hemolytic (A) and proteolytic (B) activities were measured, as described in the Experimental Section. Data are expressed as mean ± SEM of three individual experiments (n = 3). * p < 0.05 when compared to first column.

Anticoagulation
B. jararaca venom induces plasma clotting that it is associated with the action of a specific group of enzymes, the metallo-and serine enzymes. The snake venom serine proteinases (SVSPs) are among the best-characterized enzymes to affect the hemostatic system [32][33][34]. Some may affect platelet function or disrupt specific factors of the coagulation cascade, causing an imbalance in the hemostatic system of the victim [35]. As seen in Figure 2, the mixture of linearol/isolinearol (360 and 720 µg/mL) did not inhibit plasma clotting or fibrinogen triggered by B. jararaca venom, regardless of the concentration of diterpenes tested.  Figure 2. B. jararaca venom (12 µg/mL) was incubated for 30 min at room temperature with saline (1), DMSO (1% v/v) (2) or diterpenes at 360 µg/mL (gray columns) (3) or at 720 µg/mL (white columns, 3). Then, the mixture was added to plasma (A) or to fibrinogen (B), and coagulation time was recorded. Data are expressed as mean ± SEM of three individual experiments (n = 3).

Antihemorrhagic and Antiedematogenic Activities
The mixture of linearol/isolinearol (87 µg/g) inhibited 100% of the hemorrhagic activity of B. jararaca venom (at 3 µg/g, Figure 3). Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes that cleave, in a highly selective fashion, key peptide bonds of some plasma and extracellular matrix proteins, leading to hemorrhage [35]. Besides hemorrhage, SVMPs activate blood clotting, have apoptotic activity, inhibit platelet aggregation and are pro-inflammatory [34]. According to proteomic approaches, SVMPs represent the major components of snake venoms [34] and contribute to the toxicity of venoms. The inhibition of hemorrhagic activity may suggest an interaction between diterpenes with Zn 2+ which is located at the active site of such enzymes [36], thus blocking the hemorrhagic effect.
Moreover, another experimental protocol was employed in order to mimic a real envenomation situation, whereby. B. jararaca venom (3 µg/g) was injected subcutaneously (s.c.), and 20 min later the diterpenes (87 µg/g) were injected at the same site where the venom was injected. Again, B. jararaca-induced hemorrhage was inhibited by around 80% (data not shown). This result is very interesting, since hemorrhage is one of the main effects responsible for amputations or deformity in envenomed victims. On the other hand, at the highest antibothropic serum dose (4 mL/mg), hemorrhagic activity of B. jararaca was only partially inhibited (30%, Figure 3). This low antivenom efficacy to neutralize hemorrhage caused by Bothrops venom has been described elsewhere [9,30].  Envenomation by snakes usually promotes an intense inflammatory reaction and edema, and PLA 2 enzymes and other enzymes are responsible for such effects. The diterpenes (87 µg/g) did not inhibit edematogenic activity of B. jararaca venom (at 2 µg/g, data not shown); and thus, would not prevent inflammation induced by snake bites. On the other hand, in our previous work, this mixture of diterpenes inhibited hemolysis and edema induced by a PLA 2 isolated from L. muta [21]. Despite the similarity in composition between the B. jararaca and L. muta venoms, the presence of specific components in each venom may explain the different inhibitory profiles of the diterpenes.

Antilethality Activity
Injection of B. jararaca venom (30 µg/g) incubated either with saline or DMSO killed all mice after around 330 min (Table 1). When diterpenes (60 µg/g) were incubated with B. jararaca venom, all animals died around the same time period as well. However, the animals that received antibothropic serum, at any concentration (0.4, 1.2 or 4 mL/mg), did not die (Table 1). Therefore, the diterpenes did not protect mice from the toxic effects of B. jararaca venom, unlike the antibothropic serum used. Such results do not discourage the use of diterpenes as antivenom agents, since the mixture of diterpenes, unlike the antiserum, fully inhibited local effects (hemorrhage and proteolysis); that are responsible for amputation and morbidity.

Algal Material
Specimens of C. cervicornis (Dictyotaceae, Phaeophyta) were collected during May, 2006, at Praia do Forno, in the city of Armação de Búzios, located in the north of Rio de Janeiro State (22°45′42″S and 41°52′27″W), Brazil, at depths ranging from 0.3 to 2 m. The seaweeds were washed with local sea water, separated from sediments, epiphytes and other associated organisms. Voucher specimens were deposited at the herbarium of the Universidade do Estado do Rio de Janeiro (HRJ 10754).

Antihemolytic Activity
The degree of hemolysis of B. jararaca venom was determined by the indirect hemolytic test using human erythrocytes and hen's egg yolk emulsion as substrate [38]. The amount of B. jararaca venom (µg/mL) that produced 100% hemolysis was designed as the Minimum Indirect Hemolytic Dose (MIHD). Inhibitory experiments were determined by incubating the mixture of diterpenes (linearol/isolinearol) with one MIHD for 30 min at room temperature, and then hemolytic activity was assayed.

Anticoagulant Activity
The clotting of human plasma or fibrinogen induced by B. jararaca venom was determined on a digital Amelung Model KC4A coagulometer (Labcon, Bavaria, Germany). Different concentrations of B. jararaca venom were mixed with fibrinogen solution (2 mg/mL, final concentration) or with human plasma (donated from healthy volunteers of the local blood bank of the Hospital of the Federal Fluminense University), and the amount of venom (µg/mL) that clotted either fibrinogen or plasma in 60 s was designed as the Minimum Coagulant Dose (MCD). To evaluate the inhibitory effect, the diterpenes were incubated for 30 min at room temperature with one MCD of venom, and then, the mixture was added to fibrinogen or plasma and clotting time recorded. Control experiments were performed in parallel by mixing DMSO (0.5% v/v, final concentration) or saline with venom, instead of diterpenes.

Antiproteolytic Activity
Proteolytic activity of B. jararaca venom was determined using azocasein as substrate (0.2% w/v, in 20 mM Tris-HCl, 8 mM CaCl 2 , pH 8.8), with minor modification [39]. An Effective Concentration (EC) was defined as the amount of venom (µg/mL) able to produce a variation of about 0.2 OD units at A 420 nm. The diterpenes were incubated with one EC of B. jararaca venom for 30 min at room temperature and then proteolytic activity was measured.

Antihemorrhagic Activity
Hemorrhagic lesions produced by B. jararaca venom were quantified using a procedure described elsewhere [40], with minor modifications. Briefly, samples were injected subcutaneously (s.c) into the abdominal skin of mice. Two hours later, the animals were euthanized, the abdominal skin removed, stretched and inspected for visual changes in the inner layer in order to localize hemorrhagic spots. Hemorrhage was expressed as Minimum Hemorrhagic Dose (MHD), defined as the amount of venom (µg/g) able to produce a hemorrhagic halo of 10 mm [41]. The effect of diterpenes was investigated by incubating them with one MHD of B. jararaca venom for 30 min at room temperature. Then, the mixture of diterpenes was injected s.c. into mice and hemorrhage measured. Hemorrhagic activity was expressed as the mean diameter (in millimeters) of the hemorrhage halo induced by B. jararaca venom in the absence and presence of the diterpenes. Antibothropic serum 0.4, 1.2, 4.0 mL/mg was also incubated with venom, and the mixture was injected s.c. into mice. In another set of experiment, B. jararaca venom was injected s.c., and 20 min later, the diterpenes were injected at the same site of venom injection. Negative controls were performed by injecting s.c. DMSO (0.9% v/v, final concentration) or saline. The total number of mice for each experimental group was 4-5, and experiments were repeated twice.

Antiedematogenic Activity
The edema-inducing activity of B. jararaca venom was determined according to Yamakawa et al. [42], with modifications. Groups of mice received 50 µL of venom subcutaneously (s.c) in the right foot pad, whereas the left food pad received 50 µL of saline or DMSO. One hour after injection, edema was evaluated and expressed as the percentage of increase in the weight of the right foot pad compared to the left one. The inhibitory effect was investigated by incubating diterpenes with B. jararaca venom for 30 min at room temperature, and then the mixtures were injected s.c. into mice and edema was measured. Negative controls were performed by mixing venom with DMSO (0.9% v/v, final concentration) or with saline. The total number of mice for each experimental group was 4-5, and experiments were repeated twice.

Antilethal Activity
Different doses B. jararaca venom were injected intraperitoneally (i.p) into groups of mice and the number of deaths for each dose of venom was observed over a period of 24 to 48 h. For neutralization assays, the diterpenes were incubated for 30 min at room temperature with B. jararaca venom, and then, the mixture was injected i.p. and lethal activity was assessed. The lethality was determined and compared with the control group that received B. jararaca venom. Antibothropic serum 0.4, 1.2, 4.0 mL/mg was also incubated with venom, and the mixture was injected i.p. into mice. In control experiments, DMSO (0.9% v/v, final concentration) or saline was incubated with venom instead of diterpenes, and injected i.p. into mice. The total number of mice for each experimental group was 5-6, and experiments were repeated twice.

Statistical Analysis
Results are expressed as mean ± SEM obtained with the indicated number of animals or experiments performed. The statistical significance of differences among experimental groups was evaluated using the Student's t test and p values of ≤0.05 were considered statistically significant.

Conclusions
Our results suggest that diterpenes from the marine alga, C. cervicornis, are a promising source of molecules to improve the regular treatment for envenomation by B. jararaca snake bites, and/or useful as prototypes for designing alternative/new antiophidian molecules. However, an in-depth scientific investigation is imperative to evaluate the antivenom potential of natural products in order to derive therapeutically effective natural products for snake bites.