Mice were anesthetized with sodium pentobarbital (50 mg/kg, ip) and randomized into 6 groups: I: Saline, 50 [micro]L/site; II: BjuV, 50 mg/site; III: BFRE, 150 mg/kg, ip, 30 min before BjuV; IV: BFRE, 150 mg/kg, ip, 30 min after BjuV; V: BjuV (50 [micro]g) + BFRE (150 [micro]g) (1:3, w/w) preincubated for 15 min in total volume of 50 [micro]L; 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 [micro]L/ 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.
TLC analysis of BFRE revealed the presence of flavonoids, saponins, and sugars.
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.
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.
When BjuV was mixed with BFRE (1:3) before injection into the mouse paw, edema was also inhibited.
To characterize a possible anti-inflammatory effect of BFRE, another experimental group of animals was treated with 1% carrageenan.
BFRE treatment before or after BjuV inoculation decreased the number of inflammatory cells and mitigated muscle degeneration.
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).
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 treatment decreased the edematogenic peak effects at 240 min but did not inhibit the total edema reported as the AUC.
To evaluate the inhibitory effects of BFRE on SVMP, in vitro inhibition using DL-BApNA as a substrate was monitored.