Hypericum brasiliense plant extract neutralizes some biological effects of Bothrops jararaca snake venom.

Alternative treatments for snake bite are currently being extensively studied, and plant metabolites are considered good candidates for such purpose. Here, the ability of a crude ethanolic extract of Hypericum brasiliense plant in neutralizing Bothrops jararaca snake venom was investigated by in vitro (coagulation, hemolysis or proteolysis) and in vivo (hemorrhage, lethality and edema) biological assays. We describe for the first time the ability of H. brasiliense extracts to inhibit some pharmacological effects of a Brazilian snake venom. Inhibitory assays were performed by incubating B. jararaca venom with H. brasiliense extracts for 30min at room temperature before the assays were performed. The results showed that H. brasiliense extracts impaired lethality, edema, hemorrhage, hemolysis, proteolysis as well as fibrinogen or plasma clotting induced by B. jararaca venom. This indicates that H. brasiliense extracts can provide promising agents to treat B. jararaca envenomation.

INTRODUCTION

Snake bites pose a major health risk in many countries, with the global incidence of snake bites exceeding 5,000,000 per year (Kasturiratne et al, 2010). This problem is more profound in the developing countries, in particular in areas where the access to medical service and to the antiophidic treatment is challenging (Pierini et al, 1996).

In Brazil, the Bothrops genus is responsible for 90% of the registered accidents, with Bothrops jararaca species representing nearly 95% of these accidents. The victims present local (pain, edema, tissue damage, myonecrosis, and ecchymosis) and systemic symptoms (hemorrhage, blood incoagulability, nefrotoxicity). The snake venoms interfere with the hemostatic system, interacting with blood coagulation factors and platelet aggregation leading to persistent bleeding in victims (Clemetson et al, 2007).

In many parts of the world, regular treatment for snake venom accidents is serum therapy, which involves the parentheral administration of antiophidian serum (antivenoms). This therapy efficiently neutralizes the systemic toxic effects, preventing death of the victims. However, antivenoms have some disadvantages, thus limiting their efficient use (Chippaux and Goyffon, 1998; Heard et al, 1999; da Silva et al, 2007): For example, they can induce adverse reactions ranging from mild symptoms to serious (anaphylaxis), and in addition, they do not neutralize the local tissue damage (Gutiérrez et al, 2009). Thus, complementary therapeutics need to be investigated, with plants being considered as a major source (Soares et al, 2007). In many countries, plant extracts have long been in use traditionally to treat envenomation (Mors et al, 2000). The exact mechanisms of action of the plant extracts remain largely illusive, however, a number of previous reports indicate that plant-derived compounds, such as rosmarinic acid (Ticli et al, 2005; Aung et al, 2010), quercetin (Nishijima et al, 2009) and glycyrrhizin (Assafim et al, 2006) can inhibit biological activities of some snake venoms in vivo and in vitro, including B. jararaca venom.

H. brasiliense (also called H. laxiusculum; Brandão et al, 2009) is an annual bush known in the Brazilian folk medicine as “milfacadas” or “alecrim bravo”. Its extracts have been shown to display anti-inflammatory (Perazzo et al, 2008a) and antibacterial properties (Rocha et al, 1995; 1996; França et al, 2009), antidepressant activity (Perazzo et al, 2008b), effects on central nervous system (Mendes et al, 2002), and protection of mice against lethality of B. jararaca venom (Rocha et al, 1991). The aim of this work was to evaluate the ability of extract of H. brasiliense to inhibit edema, lethality, hemorrhage, clotting, hemolysis and proteolysis induced by B. jararaca snake venom.

MATERIAL AND METHODS

Materials

Bovine fibrinogen and azocasein were purchased from Sigma Chemical Co (St Louis, Missouri, USA). All other reagents were of the best grade available.

Plant materials and preparation of H. brasiliense standardized extract

H. brasiliense was collected from the city of Nova Friburgo, RJ, Brazil in 2001. A voucher specimen (nº19980) has been deposited at the herbarium of the Museu Nacional, Universidade Federal do Rio de Janeiro (Brazil). H. brasiliense standardized extract (HBSE) of the whole plant was prepared by selective extraction of flavonoid compounds and xanthone derivates (Rocha et al, 1995) in order to contain, after hydrolysis, not less than 6.5% of total flavonoids, expressed as quercetin. The ethanolic crude extract was further dissolved in a 30% dimethylsulfoxide (DMSO) solution to perform the biological assays.

Venom and animals

Lyophilized B. jararaca snake venom was kindly supplied by Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil. The venom was prepared in physiological saline at 1mg/ml concentration and was stored at -20°C until used. Balb/c mice (18-20gm) were obtained from the Center of Laboratory Animals (NAL) of the Fluminense Federal University (UFF). They were housed under controlled conditions of temperature (24 ±1°C) and light. All animal experiments were approved by the UFF Institutional Committee for Ethics in Animal Experimentation (protocol n° 00029) that were in accordance with the guidelines of the Brazilian Committee for Animal Experimentation (COBEA).

Biological assays

Antiproteolytic activity

Proteolytic activity of B. jararaca venom was determined according to the method of Garcia et al (1978), using azocasein as substrate (0.2%, w/v, in 20mM Tris-HCl, 8mM CaCl2, pH 8.8). Aliquots of B. jararaca venom (5-50µg/ml) were incubated with 0.4ml azocasein at 37°C for 90min in a total volume of 1.2ml. The enzymatic reaction was stopped by adding trichloracetic acid (5%, v/v, final concentration). The tubes were centrifuged at 15,000xg for 2min. The supernatant was removed and mixed with 2N NaOH. After 10min, tubes were read at A 420nm. An Effective Concentration (EC) was defined as the amount of venom (μg/ml) able to produce a variation of ~0.2OD units at A 420nm. For inhibition experiments, two EC of B. jararaca venom (24μg/ml) were mixed with varying amounts of HBSE (120-480μg/ml) to obtain four different ratios (w/w) venom:plant, 1:5; 1:10; 1:15 and 1:20. Then, the proteolytic activity was determined.

Anticoagulant activity

A pool of citrated normal human plasma (diluted with an equal volume of saline) from healthy volunteers or fibrinogen (2mg/ml, final concentration) was mixed with B. jararaca snake venom (2.5-70μg/ml) and the clotting time was assessed on a Amelung coagulometer, model KC4A (Labcon, Germany). The concentration of venom (μg/ml) that clotted plasma or fibrinogen in 60sec was designated the Minimun Coagulant Dose (MCD). To evaluate the inhibitory effect, HBSE was preincubated for 30min at room temperature with one MCD of venom. The mixture was then added to plasma or fibrinogen and the clotting time was recorded. Negative control experiments were performed in parallel by mixing HBSE or saline with plasma alone.

Antihemolytic activity

Hemolysis of B. jararaca venom was determined by the indirect hemolytic test using human erythrocytes and hen egg yolk emulsion as substrate (Fuly et al, 1997). One Minimum Indirect Hemolytic Dose (MIHD) was defined as the amount of B. jararaca venom (μg/ml) able to produce 100% hemolysis. To verify the inhibitory action of HBSE, the extract (150-1500μg/ml) was preincubated with one MIHD (30μg/ml) and the hemolytic assay was performed. The maximum effect (100% hemolysis) was obtained after lysing erythrocytes with distilled water to constitute the control. Control experiments were performed by incubating B. jararaca venom with either saline or DMSO.

Antihemorrhagic activity

Hemorrhagic lesions produced by B. jararaca venom were quantified as described by Kondo et al, 1960), with minor modifications. Briefly, the samples were injected intradermally (i.d.) into the abdominal skin of mice. Two hours later, the animals were euthanized, abdominal skin removed, stretched and inspected for visual changes in the internal aspect in order to localize hemorrhagic spots. Hemorrhage was quantified as the Minimum Hemorrhagic Dose (MHD), defined as the amount of venom (mg/kg) able to produce a hemorrhagic halo of 10mm (Nikai et al, 1984). The inhibitory effect of HBSE was investigated by incubating plant (800mg/kg) with two MHD of B. jararaca venom (100mg/kg) for 30min at room temperature and then, the mixture was injected into mice and hemorrhage was measured. Hemorrhagic activity was expressed as the mean diameter (in millimeter) of the hemorrhagic halo induced by B. jararaca venom in the absence and presence of the plant. Negative control experiments were performed by injecting DMSO (1%, v/v, final concentration) or saline.

Antilethality

Groups of five mice were injected intraperitoneally (i.p.) with B. jararaca venom (70mg/kg), and observation was followed during 24hr. The antilethality experiment was performed by mixing B. jararaca venom with HBSE (1140mg/kg) or with quercetin (1140mg/kg) for 30min. at 37°C, and then mixtures were injected i.p. into mice. Control experiments were performed by incubating venom with DMSO (1%, v/v, final concentration) or with saline. The volume of the injection was 0.5ml.

Antiedema

Edema-inducing activity of B. jararaca venom was determined according to Yamakawa et al (1976). Groups of five mice received subcutaneously (s.c.) 50µL of B. jararaca venom (7mg/kg) in the right paw, while the left paw received 50µl of saline or DMSO. 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 HBSE (140mg/kg) or quercetin (140mg/kg) with B. jararaca venom for 30min at room temperature, and then mixture was injected into mice. Control experiments were performed by incubating B. jararaca venom with DMSO or saline.

Statistical analysis

Results are presented as means ±S.E.M. The statistical significance of differences between tests was evaluated by using Student's unpaired t-test. P values of <0.05 were considered statistically significant.

RESULTS

Chemical composition of H. brasiliense extract

The chemical composition of the ethanolic extract of H. brasiliense was tested by RP-HPLC chromatography. As shown in Figure 1, seven major peaks of phytochemicals were obtained and in relation to their elution times were recorded as follows when compared with a mixture of molecular weight standards (Rocha et al, 1995): chlorogenic acid (1), hyperoside (2), isoquercitrin (3), guaijaverin (4); quercitrin (5), quercetin (6), kaempferol (7).

Figure 1.

Chromatography profile of HBSE. Ethanolic extract of H. brasiliense (HBSE) was injected into a Nucleosil MN 120-5 C18 silica column mounted on an HPLC apparatus. The elution was carried out at room temperature using a linear gradient from 10-60% of acetonitrile/water mixture in trifluoracetic acid (0.05%, v/v) at a flow rate of 1ml/min in 30min. Peaks were monitored at 254nm and were denoted as follows: peak 1, chlorogenic acid; peak 2, hyperoside; peak 3, isoquercitrin; peak 4, guaijaverin; peak 5, quercitrin; peak 6, quercetin; peak 7, kaempferol.

Antiproteolytic activity of H. brasiliense

B. jararaca crude venom was able to hydrolyze azocasein in a concentration-dependent manner with an EC of 12μg/ml (data not shown). Two EC of B. jararaca venom (24μg/ml) were mixed with HBSE to give final venom:plant ratio (w/w) of 1:5, 1:10, 1:15 and 1:20, respectively. As shown in Figure 2, HBSE inhibited proteolytic activity of B. jararaca venom with different potencies; the ratio 1:20 being the most effective, where a complete inhibition was observed. In contrast, the 1:5 ratio inhibited around 60% of proteolysis (Figure 2). HBSE alone (500μg/ml) did not hydrolyze azocasein or DMSO (1%, v/v, final concentration) did not interfere in proteolytic activity of B. jararaca.

Figure 2.

Inhibitory effect of HBSE on proteolysis induced by B. jararaca venom. B. jararaca venom (24μg/ml) was mixed with different concentrations of HBSE (120-480μg/ml) to give venom:plant ratio (w/w) of 1:5 (column 1); 1:10 (column 2); 1:15 (column 3); and 1:20 (column 4), respectively. Data are expressed as means ±S.E.M of three individual experiments (n=3). *, significance level (p <0.05) when compared to column 1.

Anticoagulant effect of H. brasiliense

HBSE was able to inhibit B.jararaca-induced clotting upon human plasma (Figure 3A) or upon purified bovine fibrinogen (Figure 3B) in a concentration-dependent manner. The MCD was 25μg/ml or 40μg/ml for plasma or fibrinogen assay, respectively. As observed, at the ratio 1:50 (venom:plant) (Figure 3A, column 4) and 1:0.25 (Figure 3B, column 4), plasma or fibrinogen did not clot. However, at 1:10 or 1:0.05, HBSE did not have any effect, regardless of the clotting method employed (Figure 3, column 2). The HBSE or DMSO alone did not interfere in clotting times (data not shown).

Figure 3.

Inhibitory effect of HBSE on clotting induced by B. jararaca venom. A. B. jararaca venom (25μg/ml) was mixed with HBSE (250-1250μg/ml) to give different venom:plant ratio (w/w): column 2 (1:10); column 3 (1:20); column 4 (1:50), respectively. B. B. jararaca crude venom (40μg/ml) was mixed with HBSE at venom:plant ratio, column 2 (1:0.05); column 3 (1:0.15); column 4 (1:0.25). Column 1, on both panels represents one MCD of B. jararaca venom mixed with DMSO, instead of HBSE. Data are expressed as means ±S.E.M of two individual experiments (n=4). ##, means that plasma did not clot for the observed 800sec. *, significance level (p <0.05) when compared to column 1.

Antihemolytic effect of H. brasiliense

HBSE completely inhibited the hemolysis caused by one MIHD of B.jararaca venom (30μg/ml) at the 1:50 venom:plant ratio (Figure 4, column 4), while a 30% to 80% inhibition was achieved with lower venom:plant ratios (Figure 4, columns 1, 2 and 3). Neither HBSE alone induce hemolysis, nor the hemolytic activity induced by B. jararaca venom mixed with DMSO was affected (data not shown).

Figure 4.

Inhibitory effect of HBSE on hemolysis induced by B. jararaca venom. B. jararaca venom (30μg/ml) was mixed with HBSE (150-600μg/ml) to give different venom:plant ratio (w/w), as follow: column 1 (1:5); column 2 (1:10); column 3 (1:20) and column 4 (1:50), respectively. The hemolytic assay was performed as described in methods. Data are expressed as means ±S.E.M of two individual experiments (n=3).

Antihemorrhagic effect of H. brasiliense

The effect of HBSE on hemorrhagic activity of B. jararaca venom was evaluated (Figure 5). Intradermal injection of B. jararaca venom (100mg/kg) induced a hemorrhage halo of 20mm in mice that corresponds to two MHD. As shown in Figure 5, when HBSE (800mg/kg) was preincubated with B. jararaca venom (ratio 1:8, venom:HBSE, w/w), no hemorrhage was detected (Figure 5, column 3). DMSO did not interfere in B. jararaca-induced hemorrhage (Figure 5, column 2) and HBSE did not induce hemorrhage in mice (data not shown).

Figure 5.

Inhibitory effect of HBSE on hemorrhage induced by B. jararaca venom. B. jararaca venom (100mg/kg) was preincubated for 30min at room temperature with saline (column 1), with DMSO (column 2) or with 800µg/ml HBSE (column 3). The mixtures were injected into mice and hemorrhage assay performed. Data are expressed as means SEM of two individual experiments (n=3).

Antilethality effect of H. brasiliense

B. jararaca venom (70mg/kg) was injected i.p. into mice, and one hundred percent of lethality was observed within 3hr (data not shown). When such dose of B. jararaca venom was mixed with H. brasiliense (1140mg/kg) giving a 1:16 venom:plant ratio (w/w), no death was observed (data not shown). On the other hand, quercetin (1140mg/kg) did not protect mice from death caused by B. jararaca venom. HBSE or quercetin also was not lethal to mice.

Antiedematogenic effect of H. brasiliense

B. jararaca venom (7mg/kg) produced a rapid onset in paw edema of mice within 60min after injection. HBSE (140mg/kg) mixed with venom (ration 1:2, venom:plant, w/w) abolished its edematogenic activity (data not shown).

DISCUSSION

The H. brasiliense Choisy plant belongs to the Guttiferae family and the genus Hypericum comprises about of 380 species worldwide (Santos et al, 2004). In Brazil, 17 species are found, mainly in the regions Southeastern and South (Slusarski et al, 2007). A wide range of pharmacological properties and medicinal applications have been indicated for this plant, such as astringent, prevention of the alimentary tract ailments, antimicrobial, wound-healing, anticancer, anti-inflammatory, antispasmodic, antidepressant and also antiophidian (Rocha et al, 1994,1995; Apaydin et al, 1999; Perazzo et al, 2008a; 2008b, França et al, 2009). These biological activities are due to several bioactive compounds, such as flavonoids, xanthones, phloroglucinols and terpenes that are produced by their metabolism (Abreu et al, 2004). Previous reports showed that H. brasiliense extract (HBSE) had a potent anti-inflammatory and peripheral analgesic action probably due to inhibition of production of arachdonic acid derivatives (Perazzo et al, 2008a). Pre-clinical studies have demonstrated that the extract of H. brasiliense exhibit low toxicity at doses under 1000mg/kg (Rieli et al, 2002), and no genotoxic effects in in vivo systems (Espósito et al, 2005). It is to be noted that doses used in this work were lower than those used by Rieli et al (2002).

The search for bioactive molecules in plants used in folk medicine has been growing in the past few years. Here, we have reported that HBSE neutralized some biological effects of B. jararaca venom, named hemolysis, coagulation, proteolysis, lethality, edema and hemorrhage. These reactions really contribute significantly to symptoms that follow a bite by this snake. Several workers have studied the ability of plants as well as their purified fractions to inhibit biological activities of snake venoms (Melo et al, 1994; Maiorano et al, 2005; Oliveira, et al, 2005; Cavalcante et al, 2007; Lomonte et al, 2009; de Paula et al, 2010). However, only a few have investigated the neutralizing mechanism of their action. In some cases a direct interaction with catalytic sites of enzymes or with metal ions which are essential for enzymes enzyme activities may be involved (Borges et al, 2005; Núñez et al, 2005). Regardless of the precise mechanisms, HBSE appears to be a promising clinical agent for use as first aid treatment, or in combination with antiserum. Intriguingly, quercetin – one of the most abundant compounds in this plant (Abreu and Mazzafera, 2005; Abreu et al, 2004), which has been shown to display some pharmacological properties, such as anti-inflammatory, reduction in heart failure and anticancer properties (Lee et al, 2011; Xavier et al, 2011) – did not inhibit hemorrhage, lethality and edema induced by B. jararaca venom. This suggests that the antivenom property of HBSE cannot be attributed to quercetin alone; others flavonoids, dianthrones, and hyperforin may either be acting alone or in combination with quercetin. However, this demands further investigation.

CONCLUSIONS

Since H. brasiliense extract inhibited several biological activities of B. jararaca venom, it has the potential of an alternative or complementary treatment strategy of envenomation by B. jararaca snake. However, further specific studies need to be conducted to discover the exact compounds responsible for these observations, their efficacy, safety and the antiophidian mechanisms of action.