Inhibitory potential of important phytochemicals from Pergularia daemia (Forsk.) chiov., on snake venom (Naja naja)

Abstract

Pergularia daemia (Forsk.) chiov., is a milk weed of Asclepiadaceae family. In the present study β-sitosterol, β-amyrin, α-amyrin and lupeol were identified in the leaf by GC-MS. Molecular docking studies were performed to evaluate their activities on phospholipase A2 (PLA2) and l-amino acid oxidase enzymes which constituted a rich source in snake venoms (Naja naja). Snake venom Phospholipase A2 with PDB code 1A3D devoid of co-crystallized ligand was extracted from Protein Data Bank. Using Molegro Virtual Docker two cavities are formed by cocrystallization. l-Amino acid oxidase (PDB code 4E0V) was a receptor model with a co-crystallized ligand FAD. Among the phytochemicals analysed, β-sitosterol displayed high affinity of binding to the active site regions of phospholipase A2 and l-amino acid oxidase, respectively. The affinity of binding was -125.939 and -157.521 kcal/mole identified by gold scores. α-Amyrin and β-amyrin had two hydrogen bond interactions with PLA2. Hence this study suggests that β-sitosterol identified in P. daemia can antagonize PLA2 and LAAO activities and forms a theoretical basis for the folk use of the plant against snake venom.

Keywords: Molecular docking; Pergularia daemia; Phospholipase A2; Snake venom; l-Amino acid oxidase.

Figure 1

Twig of Pergularia daemia bearing flowers and fruit. The leaves are almost glabrous above and velvety below. The flower corolla forms a greenish-yellow or dull white tube. The fruits release ovate seeds covered with velvety hairs.

Figure 2

By using the Pergularia daemia trnL-trnF intergenic spacer, partial sequence; plastid a phylogenetic tree was constructed. Maximum likelihood phylogram was discovered in 8 independent ratchet analyses. Note the broken branch to the outgroup, Cynanchum, with the exception of a few weakly supported nodes, this topology is identical to the strict consensus of most parsimonious trees discovered.

Figure 4

GC–MS chromatogram of P. daemia leaf extract showing β-sitosterol, α-amyrin, β-amyrin and lupeol Chromatogram b, c, d, e shows compounds identified compared to inbuilt NIST library.

Figure 3

In-vitro PLA2 activity of the extract in agarose gel impregnated with 1.2% sheep erythrocytes and 1.2% egg yolk and 10 mM CaCl₂. 1–8 Cavities show inhibition of haemolytic activity of naja venom by increasing dose of plant extract (5, 10, 15, 20, 25, 30, 35, 40 μl of plant extract) in series.

Figure 5

Tannic acid standard graph for the estimation of phenols from Pergularia daemia extract using the Folins method.

Figure 6

Standard graph of quercetin for the estimation of flavonoids from Pergularia daemia extract.

Figure 7

Graph shows percentage inhibition of PLA2 activity corresponding to phenolic content of the extract.

Figure 8

Venom protease inhibition activity of Pergularia daemia plotted against the corresponding concentration of the phenolic component in the extract.

Figure 9

Percentage inhibition of hyaluronidase activity of Pergularia daemia plotted against corresponding concentration of phenolic component in the extract.

Figure 10

Energy contributions of active site amino acids of 1A3D with β-sitosterol. Atoms contributing energy are represented as spheres (size corresponds to the energy associated).

Figure 11

Energy contributions of active site amino acids of 4E0V with β-sitosterol. Atoms contributing energy are represented as spheres (size corresponds to the energy associated).