1.1.Statistical Fig 1).AOAgNPs displayed significant antiproteinase activity at different

1.1.Statistical analysisResults of anti-inflammatory activity were
expressed as Mean ± SEM. Results were analyzed using one way ANOVA. Differences
were considered as statistically significant at p<0.05 level, compared to control.2.     Results and DiscussionAnti-inflammatory potential of AgNPs synthesized from Anacardium occidentale leaf was assessed in vitro assays mainly comprising of inhibition of protein denaturation, RBC membrane stabilization and inhibition of proteinase activity. Previous studies Sangita et al., 2012 proved that cell membrane destabilization specially lysosomes, denaturation of proteins, in particular blood proteins and activation of proteinases from dying cells are the major causes of arthritis and inflammation. The result of the human red blood cell membrane stabilizing activity was displayed in Table 1.AOAgNPs were used to study HRBC test at 50,100,150,200 and 500?g/ml. A concentration dependent anti-inflammatory activity of AOAgNPs was found and the protection percentage was increased with the increase in drug concentration. AOAgNPs showed 81.63% HRBC cell membrane stabilizing activity at 500?g/ml dose which is comparable to that of standard drug diclofenac sodium (87.69%)(Table1). Membrane stabilization effect of AOAgNPs is through inhibition of hypotonicity induced lysis of erythrocyte membrane. As the erythrocytic membrane is analogous to the lysosomal membrane and so stabilization of erythrocyte membrane implies that AOAgNPs also stabilizes lysosomal membranes Kumar et al., 2011. Stabilization of lysosomal membrane is important in limiting the inflammatory process by prevention of the release of lysosomal constituents of activated neutrophil containing bactericidal enzymes and proteases, which cause further inflammation and tissue damage upon extra cellular release Yurugasan et al., 1981. Yet the exact mechanism of the membrane stabilization is not known. Hypotonicity induced haemolysis may be happened from the shrinkage of the cell due to osmotic loss of intracellular electrolyte and fluid components. AOAgNPs may inhibit the processes by decreasing the efflux of these intracellular components Yang et al., 2010; Vadivu and Lakhsmi, 2008. In this study it was found that in a dose dependent manner AOAgNPs inhibited protein denaturation and showed 82.1% inhibition at 500?g ml-1 whereas standard drug diclofenac sodium exhibited 83.53% protein denaturation ((Table 2, Fig 1).AOAgNPs displayed significant antiproteinase activity at different concentrations as shown in Table 3. It showed maximum inhibition of 76% at 500?g ml-1 and indomethacin showed the maximum inhibition (82%) at 500?g ml-1.Inhibition of lipoxygenase by plants extracts are reported Khasawneh et al., 2011; Ben-Nasr, 2015. Synthesis of silver nanoparticles using extract of endophytic fungi exert maximum lipoxygenase activity (68%) compared with standard ascorbic acid (38%).Reactive oxygen species is acknowledged to spread inflammation by stimulating the release of cytokines and also by secretion of enzymes such as lipoxygenase from inflammatory cells. In the present study, AOAgNPs exhibited potent lipoxygenase inhibitory activity comparable to the standard indomethacin used. These results suggest that AOAgNPs have a potentially high anti-inflammatory effect. Therefore, plants rich in antioxidant constituents with potential antioxidant activity are found to be beneficial to counter inflammatory reactions.On the basis of the findings in the study of in-vitro anti-inflammatory activity of AOAgNPs, we examined its in vivo anti-inflammatory activity. Carrageenan-induced paw edema in rodent is a suitable model to evaluate the acute anti-inflammatory effect of test compounds Amdekar et al., 2015 . The decrease in paw volume of the treated groups with respect to time is presented in AOAgNPs significantly inhibited paw edema in the carrageenan induced mice in time-dependent manner (Table 3).Significant in vivo anti-inflammatory was exhibited by AOAgNPs compared to carrageenan-control after 30 min which was comparable to standard drug indomethacin. At the end of 4 h, there was significant decrease in paw odema in AOAgNPs treated groups at 50,100 and 200 µg/kg concentration (Table 3) compared to the control group. It was noted that anti-inflammatory effect of silver nanoparticles started at 1 hour and peaked at 4 hrs and the maximum inhibitory effect was found between 2 and 4 hours. Carrageenan induced paw edema is characterized by biphasic events with involvement of different inflammatory mediators. In the first phase (during the first 2 h after carrageenan injection), chemical mediators such as histamine and serotonin play a great role, and in second phase (3-4 h after carrageenan injection), Kinin and prostaglandins are involved Yang et al.,2010;Emamuzo et al.,2010;Shenoy et al.,2010;Georgewill et al.,2010. In a previous study, preadministration of silver nanoparticles synthesized using European black elderberry reduced paw edema and cytokines levels in mice/ rat, early after the induction of inflammation (David et al., 2014).Our results also showed that inhibition of oedema started from the first hour and continued during all phases of inflammation after AOAgNPs administration, which is probably due to inhibition of various chemical mediators of inflammation. Carrageenan-induced paw edema is also sensitive to cycloxygenase inhibitors and is used to evaluate the effect of non steroidal anti-inflammatory agents (like AOAgNPs) those primarily inhibit the cyclooxygenase, enzyme for prostaglandin synthesis (Sarkhel 2016). Both from in-vitro and in-vivo studies, it is revealed that the AOAgNPs possess potent anti-inflammatory activity and this may be attributed to both silver as well as the phytochemicals that cap and stabilize the biogenic silver nanoparticles, AOAgNPs.