SciELO - Scientific Electronic Library Online

 
vol.52 issue1Glycoprotein nonmetastatic melanoma protein B accelerates tumorigenesis of cervical cancer in vitro by regulating the Wnt/β-catenin pathwayEffects of nanoparticles of hydroxy-aluminum phthalocyanine on markers of liver injury and glucose metabolism in diabetic mice author indexsubject indexarticles search
Home Pagealphabetic serial listing  

Services on Demand

Journal

Article

Indicators

Related links

Share


Brazilian Journal of Medical and Biological Research

Print version ISSN 0100-879XOn-line version ISSN 1414-431X

Braz J Med Biol Res vol.52 no.1 Ribeirão Preto  2019  Epub Dec 03, 2018

http://dx.doi.org/10.1590/1414-431x20187581 

Research Article

Anti-ophidian activity of Bredemeyera floribunda Willd. (Polygalaceae) root extract on the local effects induced by Bothrops jararacussu venom

N.T.Q. Alves1 

R.M. Ximenes2 

R.J.B. Jorge1  3 

J.A.M. Silveira1 

J.V.A. Santos1 

F.A.P. Rodrigues1 

P.H.S. Costa1 

F.A.F. Xavier Jr.4 

J.S.A.M. Evangelista4 

A. Havt1 

V.C.G. Soares5 

M.H. Toyama6 

A.N.A. Oliveira7 

R.M. Araújo7 

R.S. Alves8 

H.S.A. Monteiro1 

1Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceará, Fortaleza, CE, Brasil

2Departamento de Antibióticos, Universidade Federal de Pernambuco, Recife, PE, Brasil

3Centro Acadêmico de Vitória, Universidade Federal de Pernambuco, Vitória de Santo Antão, PE, Brasil

4Faculdade de Veterinária, Universidade Estadual do Ceará, Fortaleza, CE, Brasil

5Universidade Paulista, Campus Jundiaí, Jundiaí, SP, Brasil

6Instituto de Biociências, Universidade Estadual Paulista, São Vicente, SP, Brasil

7Instituto de Química, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil

8Departamento de Análises Clínicas e Toxicológicas, Universidade Federal do Ceará, Fortaleza, CE, Brasil

ABSTRACT

Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom’s toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3′,4′-OH flavonoids are known inhibitors of phospholipases A2.

Key words: Bredemeyera floribunda; Snakeroot; Snake venom; Rutin; Antihemorrhagic; Anti-edematogenic

Introduction

Snakebites are a neglected health problem in tropical regions of South America, Africa, Oceania, and Asia (1,2). In Brazil, the main genus of snakes responsible for envenomation is Bothrops. Among Bothrops species, B. jararacussu is responsible for up to 10% of the cases in its area of occurrence, being one of the most feared snakes of Brazil. The effects of envenomation from this genus are characterized by systemic and local effects including, edema, blisters, necrosis, bleeding, and local inflammation (3,4).

The use of plants for treating envenomation caused by snakebites is an age-old practice found in many cultures, long before commercial anti-venoms were developed (5). Plant extracts, which have multiple biochemical constituents, are an extremely rich source of pharmacologically active compounds. Interactions of certain active compounds with toxins and lethal enzymes found in snake venom can lead to neutralization of their toxic activities (6).

The only effective snakebite treatment is the use of mono- or polyvalent antiserum, but it is ineffective for mitigating local damage induced by the venom (7). This has motivated the search for alternative neutralizing agents from a variety of natural sources and development of synthetic compounds aimed at obtaining novel therapeutic tools to complement and improve conventional serum therapy (8,9).

In Brazil, several plant species have been classified as anti-ophidian in ethnobotanical surveys, but only a few have been scientifically evaluated (10,11). Among these species, Bredemeyera floribunda Willd. (Polygalaceae), known as "raiz-de-cobra" (Portuguese for snakeroot), is widely used to treat snakebites in Brazilian folk medicine. Its anti-ophidian activity is in part attributed to the triterpenoid saponins found in its roots (5,10,12). The aim of this study was to characterize the anti-ophidian activity of an ethanol extract of B. floribunda roots on the local effects induced by B. jararacussu venom both in vitro and in vivo.

Material and Methods

Plant material, venom, and reagents

Roots of B. floribunda Willd. (Polygalaceae) were collected in March 2013 in Serra do Araripe, Ceará, Brazil. Plant identification was performed by taxonomists from Herbarium Prisco Bezerra of Universidade Federal do Ceará, and a voucher was deposited (No. 58399). B. jararacussu venom (BjuV) was donated by the Butantan Institute (Brazil). All substrates and reagents used in this work were obtained from Sigma (USA).

Preparation and phytochemical characterization of B. floribunda roots extract (BFRE)

Dried and crushed roots (200 g) were extracted for 7 days with ethanol 70%, three consecutive times. The ethanol was evaporated at 40°C at low pressure and then the extract was lyophilized yielding 14 g of yellow powder. Secondary metabolites in the BFRE were assessed by thin-layer chromatography (TLC) using chemical developers as described in Table S1 (13). Gel chromatography using Sephadex LH-20 (Pharmacia Fine Chemicals, Sweden) eluted with methanol was performed four times yielding five fractions after TLC analysis: A (6.1 g), B (0.5 g), C (0.9 g), D (2.6 g), and E (3.2 g). These fractions were further analyzed by 1H and 13C nuclear magnetic resonance (NMR).

The 1D and 2D NMR spectra were collected on a Bruker Avance DRX-500 (500 MHz for 1H and 125 MHz for 13C) spectrometer (USA) equipped with a 5 mm inverse detection z-gradient probe. Chemical shifts (δ) are reported in ppm relative to the residual MeOD (δ 3.31), and to the central peak arising from the MeOD carbon (δ 49.15).

In vitro enzymatic inhibition

Anti-phospholipase A2 activity. Phospholipase A2 (PLA2) activity was assessed using 4-nitro-3-(octanoyloxy)benzoic acid (4N3OBA, Biomol, USA) as a chromogenic substrate in 96-well plates. First, BjuV or mixtures of BjuV and BFRE (1:1, 1:2, 1:3, w:w) were diluted in buffer (10 mM Tris-HCl, 10 mM CaCl2, 100 mM NaCl, pH 8.0) to a final concentration of 2 mg/mL. Reaction mixtures were prepared using 200 µL of buffer, 20 µL of substrate, 20 µL of extract, and 20 µL of BjuV (9). The absorbance was measured at 425 nm and read every 10 min using a Spectramax 340 plate reader (Molecular Devices®, USA).

Anti-proteolytic activity. Proteolytic activity was determined using the synthetic chromogenic substrate Nα-benzoyl-DL-arginine p-nitroanilide (DL-BApNA), in 96-well plates, according to Ponce-Soto et al. (14). BjuV or mixtures of BjuV and BFRE (1:1, 1:2, 1:3, w:w) were diluted with buffer (Tris-HCl, 10 mM, 10 mM CaCl2, 100 mM NaCl, pH 7.8) at 2 mg/mL. Reaction mixtures were prepared using 200 µL of buffer, 20 µL of substrate, 20 µL of extract, and 20 µL of BjuV. The microplate was subsequently incubated at 37°C and after 30 min, the reaction was stopped by adding 200 µL of 5% trichloroacetic acid and was centrifuged at 2205 g for 5 min at room temperature. The microplate was read at 410 nm using a microplate reader VERSA Max plate reader (Molecular Devices®).

Animals

Swiss male mice (30-35 g, n=6 for each group) were used. Animals were housed and kept in a temperature-controlled room (23±2°C) under a 12/12 h light/dark cycle with food and water ad libitum. This study was approved by the Ethics Committee on Animal Research of Universidade Federal do Ceará under protocol number 52/13.

Acute toxicity

A limit test of 2000 mg/kg was performed according to the OECD 425 guideline. First, one animal was administered a dose of 2000 mg/kg followed by 24 h of observation. Since this animal did not die, two more animals received the BFRE (2000 mg/kg, ip) under the same conditions. A total of five animals were used, and after a long-term observation of 14 days, the animals were euthanized, and necropsy was performed with histological analysis of their organs (15).

Experimental groups

Mice were anesthetized with sodium pentobarbital (50 mg/kg, ip) and randomized into 6 groups: I: Saline, 50 μL/site; II: BjuV, 50 μg/site; III: BFRE, 150 mg/kg, ip, 30 min before BjuV; IV: BFRE, 150 mg/kg, ip, 30 min after BjuV; V: BjuV (50 μg) + BFRE (150 μg) (1:3, w/w) pre-incubated for 15 min in total volume of 50 μL; and VI: antibothropic serum (ABS, ip) in a sufficient quantity to neutralize 500 mg of venom 30 min before BjuV administration.

For the investigation of carrageenan-induced edema, four additional groups were created: VII: carrageenan group, 1% carrageenan dissolved in 0.9% NaCl solution, 50 μL/paw; VIII: BFRE, 150 mg/kg, ip, 30 min before carrageenan; IX: BFRE, 150 mg/kg, ip, 30 min after carrageenan; X: indomethacin, 10 mg/kg, ip, 30 min before carrageenan.

Inhibition of paw edema induced by B. jararacussu venom or carrageenan

After a sub-plantar injection of 50 μL of BjuV or carrageenan and treatment according to each previously described group (n=6), paw volume was measured at 10, 30, 60, 120, 240, and 480 min using a plethysmometer (Ugo Basile®, Italy). The contralateral hind paw was injected with sterile saline (50 μL/paw) in all animals as a volume control. Paw edema was estimated by calculating volume difference compared to basal paw volume (16). For each time-point, values were compared among experimental groups. The total edema was calculated as area under the curve (AUC) of the increase in paw volume.

Hemorrhage and necrosis inhibition

Mice were anesthetized with sodium pentobarbital (50 mg/kg, ip) and dorsal skin was shaved. The venom solution (50 μL) was administered intradermally and treatments were performed according to the previously described experimental groups I to VI (n=12). After 2 h, half of the animals in each group were euthanized, and their dorsal skin was removed. The inner surface of the skin was examined, photographed, and the hemorrhagic area size (mm2) was measured using ImageTool 3.00 software (University of Texas Health Science Center, USA) (17). To determine the necrotic area (mm2), the remaining animals were euthanized 72 h after their respective treatments, and the same procedure was performed.

Histopathological analysis

Tissues samples from paw edema experiments were collected immediately after euthanizing the animals and stored in 10% buffered formalin for 24 h, followed by 70% ethanol. Fragments were subjected to dehydration, diaphanization, and cut to a thickness of 4 μm. Slides were stained with hematoxylin-eosin (HE) and analyzed using a trinocular optical microscope Motic® BA310 (China) with an attached camera (2.0 MP Live Motic® 2000 resolution), using the Motic® Image Plus 2.0 software.

Statistical analysis

All results are reported as means±SE. The normal distribution was analyzed by D’Agostino-Pearson test and the data were analyzed by ANOVA followed by Bonferroni post-test when several experimental groups were compared to the control group. Results were considered significant when P<0.05.

Results

Phytochemical characterization of B. floribunda roots extract

TLC analysis of BFRE revealed the presence of flavonoids, saponins, and sugars. Sephadex LH-20 gel chromatography provided five fractions (A to E) containing chemically distinct groups of compounds. Subsequent 1H and 13C NMR analysis identified fraction A as a mixture of triterpene saponins and saccharose, fraction B as saccharose, and fraction C as a mixture of rutin and saccharose. After rechromatography, fraction D and E yielded 5.3 g of a pure orange powder soluble in methanol (B1, 37.8% of the extract).

The 1H NMR spectrum (MeOD, 500 MHz, Figure S1) of compound B1 showed characteristic glycoside hydrogen signals between δ 3.26 and 3.81 ppm. The chemical shifts characteristic of aromatic hydrogens between δ 6.19 and 7.67 ppm were also observed. Upon analyzing the region between δ 6.19 and 7.67 (Figure S2), it was possible to identify the peaks at δ 6.19 (H6, d, J=1, 8 Hz) and δ 6.38, but it was not possible to calculate J due to lack of multiplicity. However, the J values for H6 and 13C NMR signals indicated meta coupling between them. Signals for H5′ and H6′ located in ring B, at δ 6.87 (H5′, d, J=6.7 Hz) and δ 7.62 (H6′, dd, J=1.4 Hz e 6.7 Hz) ppm, with ortho and meta coupling were observed. In ring B, a signal at δ 7.67 (H2′, d, J=1.36 Hz) arising from meta coupling with H6' was also found.

The 13C NMR spectrum (100 MHz, MeOD, Figure S3) exhibited a signal at δ 18.0 ppm, characteristic of methyl carbon. Between δ 68.6 and 104.8 ppm, signals from oxygenated carbons with sp3 hybridization were observed, which are characteristic of glycosides, along with signals originated from anomeric carbons at δ 102.5 (C-1-′) and 104.8 (C-1-). Between δ 105.7 and 166.0 ppm, signals arising from sp2 carbons belonging to aromatic rings were observed. An α,β-unsaturated ketone carbonyl signal at δ 179.4 ppm was also seen in NMR spectrum.

Heteronuclear single quantum correlation (HSQC) analysis (C5D5N, 500 MHz, Figure S4) corroborated the proposed hydrogenation profile for each carbon (Table 1). The NMR spectra, in the context of literature data, supported the identification of compound B1 as the flavonoid rutin (18).

Table 1 1H nuclear magnetic resonance (NMR) (MeOD, 400 MHz) and 13C NMR (MeOD, 100 MHz) data of compound B1. 

C NMR 13C NMR 13C (Moura et al., 2011) NMR 1H (int., mult., JH,H) NMR 1H (Moura et al., 2011) (int., mult., JH,H)
2 159.4 156.5
3 145.9 133.2
4 179.4 177.3
5 163.0 161.2
6 100.0 98.7 6.19 (1 H, d, J = 1.4 Hz) 6.20 (1 H, d, J = 2.1 Hz)
7 166.0 164.5
8 95.0 93.6 6.38 (1 H, d, J = 1.4 Hz) 6.40 (1 H, d, J = 2.1 Hz)
9 158.5 156.4
10 105.7 103.7
1′ 135.7 121.5
2′ 123.4 116.2 7.67 (1 H, d, 1.4 Hz) 7.53 (1 H, d, J = 2.0 Hz)
3′ 117.8 115.2
4′ 149.8 148.5
5′ 116.1 115.2 6.87 (1 H, d, J = 6.7 Hz) 6.83 (1 H, d, J = 9.0 Hz)
6′ 123.2 121.0 7.62 (1 H, dd, J = 1.4 Hz and J = 6.7 Hz) 7.75 (1 H, dd, J = 2.4 Hz and J = 7.8 Hz)
1″ 104.8 101.2
2″ 77.2 75.8
3″ 75.8 74.0
4″ 71.4 69.9
5″ 78.2 76.4
6″ 68.6 66.9
1‴ 102.5 100.7
2‴ 72.3 70.5
3‴ 72.1 70.3
4‴ 74.0 71.8
5‴ 68.9 68.2
6‴ 18.0 17.6

Acute toxicity test

No deaths occurred up to a 2000 mg/kg (ip) BFRE dosage, but the animals showed mild drowsiness in the first 6 h after administration. Neither necropsy nor histopathological analysis indicated any changes in the organs. The LD50 of BFRE is greater than 2000 mg/kg, indicating that BFRE has low acute toxicity (data not shown).

In vitro enzymatic inhibition

PLA2 and proteolytic activities of BjuV were inhibited in vitro by BFRE at different ratios (1:1, 1:2, and 1:3, w/w) as shown in Figures 1 and 2, respectively. Proteolytic activity was inhibited in a concentration-dependent manner, while PLA2 activity did not show a concentration-dependent inhibitory relationship.

Figure 1 Effect of Bredemeyera floribunda root extract (BFRE) on the phospholipase A2 activity of Bothrops jararacussu venom (BjuV). BFRE was preincubated with BjuV at ratios of 1:1, 1:2, and 1:3 (BjuV:BFRE). 4N3OBA was used as a substrate and increasing absorbance was measured at 425 nm. Data are reported as means±SE. *P<0.05 compared to BjuV (ANOVA with Bonferroni post-test). 

Figure 2 Effect of Bredemeyera floribunda root extract (BFRE) on the proteolytic activity of Bothrops jararacussu venom (BjuV). BFRE was preincubated with BjuV at ratios of 1:1, 1:2, and 1:3 (BjuV:BFRE). DL-BApNA was used as a substrate and increasing absorbance was measured at 410 nm. Data are reported as means±SE. *P<0.05, ***P<0.001 compared to BjuV; #P<0.05 compared among BFRE 1:1 ratio (ANOVA with Bonferroni post-test). 

Anti-edematogenic activity

Intraperitoneal treatment with BRFE (150 mg/kg) either before or after BjuV administration reduced venom-induced increases in paw volume (Figure 3A). When BjuV was mixed with BFRE (1:3) before injection into the mouse paw, edema was also inhibited. Edema inhibition by BFRE administered before BjuV injection was long lasting compared to the other experimental conditions.

Figure 3 Effect of Bredemeyera floribunda root extract (BFRE) on Bothrops jararacussu venom (BjuV)-induced (A), and carrageenan-induced (B) paw edema. Paw volume was measured at 10, 30, 60, 120, 240, and 480 min after BjuV inoculation and total edema was calculated as AUC (area under curve). Data are reported as means±SE. *P<0.05, **P<0.01, and ***P<0.001 compared to BjuV/carrageenan (control) (ANOVA with a Bonferroni post-test). BT: group treated with BFRE (150 mg/kg, ip) 30 min before BjuV/carrageenan inoculation; AT: group treated with BFRE (150 mg/kg, ip) 30 min after BjuV/carrageenan inoculation; PI: BjuV and BFRE (1:3) preincubated for 15 min at 37°C before inoculation; ABS: antibothropic serum; Indo: indomethacin (10 mg/kg, ip) 30 min before carrageenan inoculation. 

To characterize a possible anti-inflammatory effect of BFRE, another experimental group of animals was treated with 1% carrageenan. BFRE administered 30 min before carrageenan inhibited the increase in paw volume seen at 240 min, as also observed in the indomethacin group. However, total edema as determined by AUC was only inhibited in the indomethacin group (Figure 3B).

Histopathological analysis of paw tissue indicated edema, inflammatory infiltrate, necrosis, and degeneration in muscle fibers in the BjuV group. BFRE treatment before or after BjuV inoculation decreased the number of inflammatory cells and mitigated muscle degeneration. When BjuV and BFRE were mixed in a 1:3 ratio before inoculation, histological analysis indicated the presence of inflammatory cells and muscle degeneration. In the ABS group, edema, inflammatory cells infiltrating the dermis and subcutaneous tissue, and muscle degeneration were observed (Figure 4).

Figure 4 Histological analysis of edema induced by Bredemeyera jararacussu venom and neutralization by Bothrops floribunda extract. Epidermis/dermis (1) and Hypoderm/muscle tissue (2). A.1/A.2: Sub-plantar injection of BjuV 50 µg/50 uL; B.1/B.2: Pre-treatment ip with BFRE 150 mg/kg; C.1/C.2: Post-treatment ip with BFRE 150 mg/kg; D.1/D.2: Pre-incubated BFRE:BjuV, 1:3 w/w; E1/E.2: antivenom serum, sufficient quantity to neutralize 500 g of the venom; F.1/F.2: saline control. A: Edema (arrow head). A to E: Edema, inflammatory cells, and destruction of muscle fibers at different levels (asterisks). A, B, and E: mild hemorrhagic areas (arrows). F: no alterations. Light microscope at ×400, HE staining. Scale bar: 10 µm. 

Hemorrhagic and necrotic activity

Figure 5A shows a decrease in the hemorrhagic area induced by BjuV due to treatment with BFRE in all experimental groups (III, IV and V). ABS treatment (VI) was not able to inhibit hemorrhaging induced by BjuV. In terms of necrotizing activity, BFRE blocked the venom action in the treated groups, even after 72 h of administration. At this time, a positive control performed with the ABS decreased necrotic areas (Figure 5B), indicating that ABS required some time to reach an effective concentration in tissues.

Figure 5 Effect of Bredemeyera floribunda root extract (BFRE) on hemorrhagic (A) and necrotizing (B) effects of Bothrops jararacussu venom (BjuV). Lesion areas were calculated in mm2. Data are reported as means±SE. *P<0.05, ***P<0.001 compared to BjuV; #P<0.05 compared to ABS (ANOVA with Bonferroni post-test). BT: group treated with BFRE (150 mg/kg, ip) 30 min before BjuV inoculation; AT: group treated with BFRE (150 mg/kg, ip) 30 min after BjuV inoculation; PI: BjuV and BFRE (1:3) preincubated for 15 min at 37°C before inoculation; ABS: antibothropic serum. 

Discussion

The anti-ophidian properties of natural products have been exhaustively studied. Ubiquitous secondary metabolites possess anti-ophidian activity and include flavonoids, steroids, and triterpenes (10). Bredemeyerosides B and D, triterpene saponins isolated from the roots of B. floribunda Willd., exhibited significant anti-ophidian activity in mice, although the therapeutic activity of the associated extract remains poorly characterized (12,19). Flavonoids and other phenolics, such as rutin, quercetin, and chlorogenic acid, have been suggested to be responsible for the anti-ophidian activity of several plants used in traditional medicines to treat snakebites, such as Casearia sylvestris Sw. (Flacourtiaceae), Vellozia flavicans Mart. ex Schult. (Velloziaceae), and Vochysia haenkeana Mart. (Vochysiaceae) (4,20,21).

The local toxic effects caused by bites of snakes from the Viperidae family, especially those from the Crotalinae subfamily, are mainly due to the actions of PLA2, metalloproteases, and serine proteases. Snake venom metalloproteases and PLA2 are poor immunogenic and fast-acting toxins, making complete inhibition of local effects in a clinical setting very difficult (22). Snake venom PLA2 can be catalytically active (Asp49) or inactive (Lys49), but both are myotoxic and can trigger inflammatory responses causing edema, which is mediated to different degrees by amines (histamine and serotonin) released by degranulating mast cells (23). The in vitro PLA2 activity of BjuV on 4N3OBA was significantly inhibited by BFRE. Flavonoids with 3′- and 4′-OH groups (such as quercetin and rutin) are well-known inhibitors of snake venom PLA2 enzymatic and toxic activities (24,25,26).

BFRE also inhibited the paw edema induced by BjuV. Wanderley et al. (27) characterized the local inflammation caused by B. jararacussu and inferred that the venom produces an early edema onset dependent on prostanoid production and neutrophil migration. These effects were suggested to be mediated by increased local production of IL-1β, COX-2 expression, and neutrophil chemotaxis induced by the venom. To determine if the effect of BFRE on paw edema was due to a general anti-inflammatory action or direct inhibition of BjuV toxins, the effect of BFRE on carrageenan-induced paw edema was evaluated. Paw edema induced by carrageenan is a widely used model to investigate the pathophysiology of acute inflammation and to assess anti-inflammatory effects of anti-inflammatory drug candidates (28). Intraplantar injection of carrageenan produced acute inflammation characterized by increased water in the tissue, exudation of plasma proteins, infiltration of neutrophils, and arachidonic acid metabolism through the cyclooxygenase and lipoxygenase pathways. The maximum effects of carrageenan administration can be observed after approximately 3 h and involve the synthesis and release of mediators such as prostaglandins, histamine, bradykinin, serotonin, and leukotrienes (29).

BFRE treatment decreased the edematogenic peak effects at 240 min but did not inhibit the total edema reported as the AUC. Rutin has been shown to exert anti-inflammatory effects in rat paw edema induced by carrageenan through the inhibition of neutrophil chemotaxis (30). In addition to enzymatic inhibition of venom toxins, BFRE may decrease neutrophil migration to the site as well as the production of inflammatory mediators, decreasing the edema severity in carrageenan- and BjuV-induced paw edema.

The other major group of toxins responsible for the local effects in bothropic envenomation are snake venom zinc-dependent metalloproteases (SVMPs). To evaluate the inhibitory effects of BFRE on SVMP, in vitro inhibition using DL-BApNA as a substrate was monitored. BFRE inhibited BjuV proteolytic activity in a concentration-dependent manner. SVMPs are the main toxins involved in hemorrhaging and necrosis caused by bothropic envenomation. The pathogenesis of venom-induced bleeding involves direct damage to microvessels, which is related to the proteolytic activity of SVMPs (31). SVMPs are mainly responsible for the hemorrhaging through the degradation of the endothelial layer of basement membrane proteins such as laminin, fibronectin, type IV collagen, and proteoglycans. Furthermore, SVMPs may also inhibit platelet aggregation and trigger cytokine release (31,32).

In this study, BFRE was shown to inhibit both the hemorrhagic and necrotizing effects of BjuV. It is likely that BFRE chelated the zinc of the metalloproteinases, or inhibit degradation of the components of the basement membrane, preventing the action of these enzymes and mitigating the hemorrhagic, proteolytic, and necrotizing effects of BjuV. Flavonoids isolated from Schizolobium parahyba have been shown to inhibit the proteolytic, hemorrhagic, and necrotizing activities of B. jararacussu and B. neuwiedi venoms (33).

Previous studies indicated that the efficacy of extracts is greater than that of single constituents, likely due to the synergism among molecules present in the extract. Extracts rich in proanthocyanidins, triterpenoid saponins, polysaccharides, and coumarins are potent inhibitors of SVMPs found in Bothrops venom (22). On the other hand, the inhibition of snake venom PLA2 by natural compounds is often due to the association of PLA2 and flavonoids present in these plants. BFRE contains both flavonoids and saponins, which may contribute in a synergistic manner to the observed effects (34).

In conclusion, BFRE inhibited B. jararacussu whole venom PLA2 and protease enzymatic activities in vitro, and their related toxic effects in vivo (edema and hemorrhage/necrosis, respectively). Previous reports indicated the anti-ophidian action of triterpenoid saponins found in B. floribunda roots (19). The large amounts of rutin found in B. floribunda roots could potentiate the effect of saponins, since 3′,4′-OH flavonoids are well-known inhibitors of PLA2. These results provide some evidence for the use of B. floribunda root extract in Brazilian traditional medicine. More studies are required to elucidate the mode of action of B. floribunda and to consider its use as a possible adjuvant in treatment of local effects of bothropic envenomation.

Supplementary material

Click here to view [pdf].

References

1. Feitosa ES, Sampaio V, Sachett J, Castro DB, Noronha MD, Lozano JL, et al. Snakebites as a largely neglected problem in the Brazilian Amazon: highlights of the epidemiological trends in the State of Amazonas. Rev Soc Bras Med Trop 2015; 48: 34–41, doi: 10.1590/0037-8682-0105-2013. [ Links ]

2. Laustsen AH, Johansen KH, Engmark M, Andersen MR. Recombinant snakebite antivenoms: a cost-competitive solution to a neglected tropical disease. Plos Negl Trop Dis 2017; 11: e0005361, doi: 10.1371/journal.pntd.0005361. [ Links ]

3. Santoro ML, Sano-martins IS, Fan HW, Cardoso JLC, Theakston RDG, Warrel DA, et al. Haematological evaluation of patients bitten by the jararaca, Bothrops jararaca, in Brazil. Toxicon 2008; 51: 1440–1448, doi: 10.1016/j.toxicon.2008.03.018. [ Links ]

4. Tribuiani N, Silva AM, Ferraz MC, Silva MG, Bentes APG, Graziano TS, et al. Vellozia flavicans Mart. ex Schult. hydroalcoholic extract inhibits the neuromuscular blockade induced by Bothrops jararacussu venom. BMC Complement Altern Med 2014; 14: 48, doi: 10.1186/1472-6882-14-48. [ Links ]

5. Pereira NA, Pereira BM, Nascimento MC, Parente JP, Mors WB. Pharmacological screening of plants recommended by folk medicine as anti-snake venom. IV. Protection against jararaca venom by isolated constituents. Planta Med 1994; 60: 99–100, doi: 10.1055/s-2006-959425. [ Links ]

6. Ambikabothy J, Ibrahim H, Ambu S, Chakravarthi S, Awang K, Vejayan J. Efficacy evaluations of Mimosa pudica tannin isolate (MPT) for its anti-ophidian properties. J Ethnopharmacol 2011; 137: 257–262, doi: 10.1016/j.jep.2011.05.013. [ Links ]

7. Molander M, Staerk D, Mørck Nielsen H, Brandner JM, Diallo D, Kusamba Zacharie C, et al. Investigation of skin permeation, ex vivo inhibition of venom-induced tissue destruction, and wound healing of African plants used against snakebites. J Ethnopharmacol 2015; 165: 1–8, doi: 10.1016/j.jep.2015.02.014. [ Links ]

8. Lomonte B, Angulo Y, Moreno E. Synthetic peptides derived from the C-terminal region of Lys49 phospholipase A2 homologues from Viperidae snake venoms: biomimetic activities and potential applications. Curr Pharm Des 2010; 16: 3224–3230, doi: 10.2174/138161210793292456. [ Links ]

9. Cotrim CA, de Oliveira SC, Diz Filho EB, Fonseca FV, Baldissera L Jr, Antunes E, et al. Quercetin as an inhibitor of snake venom secretory phospholipase A2. Chem Biol Interac 2011; 189: 9–16, doi: 10.1016/j.cbi.2010.10.016. [ Links ]

10. Mors WB, Nascimento MC, Pereira BM, Pereira NA. Plant natural products active against snake bite – the molecular approach. Phytochemistry 2000; 55: 627–642, doi: 10.1016/S0031-9422(00)00229-6. [ Links ]

11. Sajon SR, Sana S, Rana S. Anti-venoms for snake bite: A synthetic and traditional drugs review. J Pharmacogn Phytochem 2017; 6: 190–197. [ Links ]

12. Pereira BMR, Daros MR, Parente JP, Matos FJA. Bredemeyeroside D, a novel triterpenoid saponin from Bredemeyera floribunda: a potent snake venom antidote activity on mice. Phytother Res 1996; 10: 666–669, doi: 10.1002/(SICI)1099-1573(199612)10:8<666::AID-PTR937>3.0.CO;2-H. [ Links ]

13. Wagner H, Bladt S. Plant Drug Analysis: a Thin Layer Chromatography Atlas. 2 ed. Munich: Springer; 1996. [ Links ]

14. Ponce-Soto LA, Bonfim VL, Novello JC, Navarro Oviedo R, Yarlequé CA, Marangoni S. Isolation and characterization of a serine protease, Ba III-4, from Peruvian Bothrops atrox venom. Protein J 2007; 26: 387–394, doi: 10.1007/s10930-007-9078-z. [ Links ]

15. OECD Guidelines for the Testing of Chemicals/Section 4: health effects. Test No. 425: acute oral toxicity: up-and-down procedure. OECD Publishing: Paris; 2008. p. 1–27. [ Links ]

16. Guimarães AQ, Cruz-Hofling MA, Araújo PMF, Bom C, Araújo AL. Pharmacological and histopathological characterization of Bothrops lanceolatus (Fer de lance) venom-induced edema. Inflamm Res 2004; 53: 284–291. [ Links ]

17. Esmeraldino LE, Souza AM, Sampaio SV. Evaluation of the effect of aqueous extract of Croton urucurana Baillon (Euphorbiaceae) on the hemorrhagic activity induced by the venom of Bothrops jararaca, using new techniques to quantify hemorrhagic activity in rat skin. Phytomedicine 2005; 12: 570–576, doi: 10.1016/j.phymed.2004.01.012. [ Links ]

18. Moura ACS, Vilegas W, Santos LC. Identificação de alguns constituintes químicos de Indigofera hirsute Linn. (Fabaceae) por CLAE-IES-EM (TOF) e avaliação da Atividade antirradicalar. Química Nova 2011; 34: 1136–1140. [ Links ]

19. Daros MR, Matos FJA, Parente JP. A new triterpenoid saponin, Bredemeyeroside B, from the roots of Bredemeyera floribunda. Planta Med 1996; 62: 523–527, doi: 10.1055/s-2006-957962. [ Links ]

20. Cintra-Francischinelli M, Silva MG, Andréo-Filho N, Gerenutti M, Cintra ACO, Giglio JR, et al. Antibothropic action of Casearia sylvestris Sw. (Flacourtiaceae) extracts. Phytother Res 2008; 22: 784–790, doi: 10.1002/ptr.2365. [ Links ]

21. Harder C, de Oliveira AL, Scriboni AB, Cintra ACO, Schezaro-Ramos R, dos Santos MG, et al. Pharmacological properties of Vochysia Haenkeana (Vochysiaceae) extract to neutralize the neuromuscular blockade induced by Bothropstoxin-I (Lys49 Phospholipase A2) myotoxin. Adv Pharm Bull 2017; 7: 433–439, doi: 10.15171/apb.2017.052. [ Links ]

22. Nishijima CM, Rodrigues CM, Silva MA, Ferreira ML, Vilegas W, Hiruma-Lima C. Anti-hemorrhagic activity of four Brazilian vegetable species against Bothrops jararaca venom. Molecules 2009; 14: 1072–1080, doi: 10.3390/molecules14031072. [ Links ]

23. Barbosa AM, do Amaral RO, Teixeira CF, Hyslop S, Cogo JC. Pharmacological characterization of mouse hind paw oedema induced by Bothrops insularis (jararaca ilhoa) snake venom. Toxicon 2003; 42: 515–523, doi: 10.1016/S0041-0101(03)00230-7. [ Links ]

24. Lindahl M, Tagesson C. Flavonoids as phospholipase A2 inhibitors: importance of their structure for selective inhibition of group II Phospholipase A2. Inflammation 1997; 21: 347–356, doi: 10.1023/A:1027306118026. [ Links ]

25. Cotrim CA, Oliveira SCB, Diz-Filho EBS, Fonseca FV, Baldissera Jr L, Antunes E, et al. Quercetin as an inhibitor of snake venom secretory phospholipase A2. Chem Biol Interact 2011; 189: 9–16, doi: 10.1016/j.cbi.2010.10.016. [ Links ]

26. Toyama Dde O, Gaeta HH, de Pinho MV, Ferreira MJ, Romoff P, Matioli FF, et al. An evaluation of 3-Rhamnosylquercetin, a glycosylated form of quercetin, against the myotoxic and edematogenic effects of sPLA2 from Crotalus durissus terrificus. Biomed Res Int 2014; 2014: 341270. [ Links ]

27. Wanderley CW, Silva CM, Wong DV, Ximenes RM, Morelo DF, Cosker F, et al. Bothrops jararacussu snake venom-induces a local inflammatory response in a prostanoid-and-neutrophil-dependent manner. Toxicon 2014; 90: 134–147, doi: 10.1016/j.toxicon.2014.08.001. [ Links ]

28. Nunes FP, Sampaio SC, Santoro ML, Sousa-e-Silva MCC. Long-lasting anti-inflammatory properties of Crotalus durissus terrificus snake venom in mice. Toxicon 2007; 49: 1090–1098, doi: 10.1016/j.toxicon.2007.01.017. [ Links ]

29. Zhang LT, Zhang YW, Takaishi Y, Duan HQ. Antitumor triterpene saponins from Anemone flaccida. Chin Chem Lett 2008; 19: 190–192, doi: 10.1016/j.cclet.2007.11.029. [ Links ]

30. Selloum L, Bouriche H, Tigrine C, Boudoukha C. Anti-inflammatory effect of rutin on rat paw oedema, and on neutrophils chemotaxis and degranulation. Exp Toxicol Pathol 2003; 54: 313–318, doi: 10.1078/0940-2993-00260. [ Links ]

31. Gutiérrez JM, Escalante T, Rucavado A, Herrera C. Hemorrhage caused by snake venom metalloproteinases: a journey of discovery and understanding. Toxins (Basel) 2016; 8: 93. [ Links ]

32. Gómez-Betancur I, Benjumea D. Traditional use of the genus Renealmia and Renealmia alpinia (Rottb.) Maas (Zingiberaceae) – a review in the treatment of snakebites. Asian Pac J Trop Med 2014; S1: S574-S578. [ Links ]

33. F Vale LH, Mendes MM, Fernandes RS, Costa TR, S Hage-Melim LI, A Sousa M, et al. Protective effect of schizolobium parahyba flavonoids against snake venoms and isolated toxins. Curr Top Med Chem 2011; 11: 2566–2577, doi: 10.2174/156802611797633438. [ Links ]

34. Carvalho BM, Santos JD, Xavier BM, Almeida JR, Resende LM, Martins W, et al. Snake venom PLA2s inhibitors isolated from Brazilian plants: synthetic and natural molecules. Biomed Res Int 2013; 2013: 53045, doi: 10.1155/2013/153045. [ Links ]

Received: April 7, 2018; Accepted: October 8, 2018

Correspondence: R.M. Ximenes: <rafael.ximenes@ufpe.br>

Creative Commons License This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.