Materials and methods
Venom from Naja naja was purchased from Irula Co-operative Society Ltd., Chennai, India. All other reagents and chemicals used were of analytical grades purchased from Sisco Research Laboratories (SRL), Bangalore, India.
Results and discussion
Snake venom PLA2s belonging to elapidae family are known to be multi-toxic and lethal (Doley et al., 2010; Kini, 2003). Due to the prominent role of PLA2s play in snake envenomation, there is enormous pharmacological interest in search of PLA2 inhibitors (Narendra Sharath Chandra et al., 2007; Nanda et al., 2007). Further, considering the limitations of antiserum therapy (Dhananjaya et al., 2011; Girish and Kemparaju, 2011), it is justified that research has to focus on developing alternatives and in this regard finding inhibitors of the multi-toxic svPLA2s from medicinal plants have gained much interest in the recent past (Carvalho et al., 2013; Gomes et al., 2012). Although many sPLA2 inhibitors have been isolated from various medicinal plants (Springer, 2001; Nanda et al., 2007; Narendra Sharath Chandra et al., 2007), however, still effective and specific inhibitors of sPLA2 are not available. In these line of studies, the aqueous steam thapsigargin Supplier extract of M. indica is evaluated for its potential to inhibit phospholipase A2 (PLA2) belonging to group IA i.e. NN-XIb-PLA2, which was isolated from R. viper venom as per the previously described method (Rudrammaji and Gowda, 1998).
The sPLA2 belonging to group IA i.e. NN-XIb-PLA2 gave a specific activity of around 172.4±3.1, when measured using PC as substrate (Table 1). When pre-incubated with different concentration of extract it was observed that the aqueous extract of M. indica, inhibited the enzymatic activity in a concentration dependent manner as shown in Fig. 1. The results show that the extent of inhibition was >95% at the 40μg/ml of extract used. The IC50 values calculated by linear XY scattered plot were 7.6μg/ml (Table 1). Most of the sPLA2 inhibitors are known to inhibit the activity either by binding to substrate or by chelating calcium, which is required for activity. Also, it is observed that the sPLA2 inhibitors affect the “Quality of interface” by modifying the phospholipid bilayer properties which render the phospholipids inaccessible to the enzyme. The steroid inducible inhibitors of PLA2 like lipocortin I and II are shown to inhibit PLA2s by nonspecific binding to the membrane phospholipids. It is observed that their inhibition is relieved by increasing the substrate concentration (Davidson et al., 1987). In this study, it was observed that, when examined as a function of substrate concentration, there was no relieve of inhibition of the extract pre-incubated, as the substrate concentration was increased from 20 to 120nM (Fig. 2). This suggests that the inhibition is independent of substrate concentration. Further, in the calcium dependent activity test, it was observed that an increase in calcium concentration from 2.5 to 15mM, increased NN-XIb-PLA2 enzymatic activity in a dose dependent manner. However, while when IC50 concentration of M. indica extract was used along with varying concentration of calcium, there was no relieve of inhibition (Fig. 3), suggesting that the inhibition by M. indica extract is independent on calcium concentration. These all studies show that inhibition by aqueous extract of M. indica is independent on substrate and calcium concentration. Further, it is reported that some of the PLA2 inhibitors are shown to mediate displacement of catalytically essential calcium from the enzyme and thus inhibition of enzymatic activity (Pruzanski et al., 1992). In the calcium binding studies experiments, it was found that the PLA2 enzyme activity both before and after the dialysis of the enzyme inhibitor mixture was unaltered i.e., the % of inhibitory activity of M. indica extract was not decreased upon extensive dialysis, suggesting that the inhibition is irreversible, supporting the observation that inhibition by M. indica extract is independent on substrate and calcium concentration. These studies indicate that the inhibition could be due to direct interaction of components/molecules present in M. indica extract at active site residues of the sPLA2 enzyme. NN-XIb-PLA2 enzyme exhibited indirect hemolytic activity, which is an indirect way of measuring PLA2 activity using egg yolk and washed erythrocytes are used as substrates. When the effect of aqueous extract of M. indica at different concentrations (0–40μg/ml) was tested it was found that the extract in general effectively inhibited indirect hemolytic activity up to 98% at ∼40μg/ml concentration (Fig. 4). This in situ inhibition activity is well correlated with the inhibitory activity of the in vitro PLA2 enzyme. Therefore, the inhibition of NN-XIb-PLA2 activity by molecules in M. indica extract could be attributed to the modulation of the catalytic activity of PLA2 at the interface itself, i.e., beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization of the membrane components. It is well known that secretory PLA2s cause cell membrane asymmetry by degradation of glycerol phospholipids of the membranes.
Materials and methods