Figure 1.
Schematic representation for the synthesis of apigenin structural analogues.
Figure 2.
Stereo view of 3D structure of the SVMP (Bothropasin) showing molecular interaction with the compound 5d.
The interaction between the compound and active site residues are shown using a line representation colored by parent atoms except for carbon (green color). The ligand is presented in the ball and stick form with their parent color except for carbon (white color). Hydrogen bonds are presented as white dotted lines.
Table 1.
Computational analysis of binding of compounds towards SVMP.
Figure 3.
Effect of compound 5d on caseinolytic and gelatinolytic activities of EC venom.
EC venom (3 µg) was pre-incubated separately with varied doses of compound 5d (1∶0; 1∶1; 1∶2; 1∶5; 1∶10; 1∶25 and 1∶50; venom: compound; w/w; Lane 1–7 respectively, Lane-8 represents 100 µg of compound 5d alone) for 10 min at 37°C. Samples were electrophoresed on gels impregnated separately with (A) casein (0.2%) and (B) gelatin (1%) as substrate. The gels were then processed as described in materials and methods section.
Figure 4.
Neutralization of hemorrhagic activity of EC venom by compound 5d.
EC venom (2 µg) was pre-incubated separately with various doses of compound 5d (1∶0 to 1∶10; venom: compound; w/w) in a total volume of 50 µL PBS for 10 min at 37°C. The samples were then injected intradermally into experimental animals (n = 5) and the assay was performed as described in materials and methods section. Results are expressed as repetitive pictures of three independent experiments.
Table 2.
Effect of compound 5d on EC venom-induced hemorrhage in an independent injection experiment.
Figure 5.
Light microphotograph of mice skin sections and blood vessels.
EC venom (2 µg) was pre-incubated separately with compound 5d for 10 min at 37°C. The samples were injected intradermally into a groups of mice (n = 5) in a total volume of 50 µL saline. Mice were anaesthetized and sacrificed after 3 h, the dorsal patch of skin tissue was removed and the injected spot was processed for histopathological studies as described in materials and methods section. (A) Saline injected control section- note the intact ECM and the basement membrane surrounding the blood vessels. (B) EC venom-injected section shows the extensive disorganized dermis and epidermis layers. (C) EC venom pre-incubated with compound 5d (1∶5; venom: compound; w/w) injected section shows restoration of normal basement membrane. Original magnification 40x. The inset shows high power view of damaged and intact blood vessels.
Figure 6.
Light microphotograph of mice skeletal muscle sections.
EC venom (5 µg) was pre-incubated separately with various doses of compound 5d (1∶0; 1∶5; 1∶10 and 1∶25; venom: compound; w/w) for 10 min at 37°C. The samples were injected intramuscularly into groups of mice (n = 5) in a total volume of 50 µL saline. Mice were anaesthetized and sacrificed after 3 h, the muscle tissue from the injected site was removed and processed for histopathological studies as described in materials and methods section. (A) Saline injected control section- note the intact basement membrane with striated myofibrils (B) EC venom-injected section shows the extensive disorganized myofibroblasts with infiltration of inflammatory leucocytes. (C)–(E) EC venom pre-incubated with the compound 5d injected section shows the inhibition and restoration of normal histology of muscle tissue (with respective doses of 1∶5; 1∶10 and 1∶25; venom: compound; w/w). (F) 150 µg Compound 5d alone injected section similar to saline control. Original magnification 40x.
Figure 7.
Effect of compound 5d on EC venom induced myotoxicity.
EC venom (5 µg) was pre-incubated separately with varied doses of compound 5d (1∶0; 1∶5; 1∶10 and 1∶25; venom: compound; w/w) for 10 min at 37°C. The samples were injected intramuscularly into group of mice (n = 5) in a total volume of 50 µL saline. Mice were anaesthetized and sacrificed after 3 h and the assay was performed as described in materials and methods section. Results are expressed as mean ± SEM of three independent experiments. *** p<0.001, ** p<0.01, a - significant compared to saline control group and b - significant compared to venom alone group.
Table 3.
Effect of compound 5d on EC venom-induced serum LDH and CPK levels in experimental animals in an independent injection experiment.
Table 4.
Effect of compound 5d on EC venom-induced edema activity.
Table 5.
Effect of compound 5d on procoagulant activity of EC venom.
Figure 8.
Effect of compound 5d on EC venom-induced fibrino(geno)lytic activity.
EC venom (0.2 µg) was pre-incubated separately with different doses of compound 5d (1∶0; 1∶1; 1∶5; 1∶10; 1∶25 and 1∶50; venom: compound; w/w; Lane 2–7 respectively, Lane-1 represents substrate alone and Lane-8 represents substrate with 10 µg of compound 5d alone) for 10 min at 37°C. The reaction was initiated by adding respective substrates (A) fibrin and (B) fibrinogen and incubated for 30 min. Samples were electrophoresed on 10% SDS-PAGE and the gels were processed as described in materials and methods section.
Figure 9.
UV-VIS spectral studies of compound 5d in presence of CaCl2 and ZnCl2.
The mixture of compound 5d (1 mM) and different concentrations of (A) CaCl2 and (B) ZnCl2 (0–2.0 mM) in a final volume of 1 mL PBS. The samples were monitored by spectroscopic scanning with the wavelength range of 200 to 300 nm.