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

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.

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).

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.

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.

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.