Nitric oxide induced by Indian ginseng root extract inhibits Infectious Bursal Disease virus in chicken embryo fibroblasts in vitro

Ganguly, Bhaskar; Umapathi, Vijaypillai; Rastogi, Sunil Kumar

Nitric oxide levels were determined on the basis of a sodium nitrite standard curve (y = 0.0102x + 0.0139, R[2] = 0.9877; Fig. 1 ) using Griess reagent. Nitric oxide levels, expressed as micromolar equivalents of sodium nitrite, at different time intervals are shown in Fig. 2 . MCW extract, alone or with the virus, caused a rapid and significant (p < 0.01) increase in NO levels within 2 h of extract supplementation. The increased levels of NO declined significantly faster in the uninfected CEF than in the virus-infected CEF. CEF infected with the virus and left untreated showed a more gradual but persisting increase in NO levels. These findings suggest that MCW extract causes a rapid rise in NO levels in CEF that is inhibitory to the virus. Our findings are supported by previous findings of Jena [17] , where it has been shown that NO is inhibitory to IBDV in vitro. It has been stated that IBDV infection alone is insufficient to induce NO production in CEF in vitro; cytokines or chemical donors are essentially required to achieve NO production in CEF [17] . Our results establish that IBDV infection induces production of NO by CEF. Moreover, WS can induce NO in CEF irrespective of IBDV infection. Control CEF also showed a late rise in NO levels; this may possibly have been due to senescence-associated changes. Studies involving aminoguanidine (AMG), a known inhibitor of NO induction, were undertaken to further elucidate the interplay of NO, MCW extract, and IBDV. The non-cytotoxic dose of AMG was determined at 118 μg/mL whereas the LD 50 was determined at about 11.88 mg/mL (Fig. 3) . For the ease of dilution and dispensing, AMG was supplemented at a concentration of 100 μg/mL that was well within the range of concentrations known to inhibit inducible NO [21] . IBDVinduced cytopathy increased significantly with the delay in MCW supplementation (Fig. 4) . The presence of AMG significantly attenuated the virus-inhibitory activity of MCW. These findings of AMG supplementation studies suggest that anti-IBDV activity of MCW extract involves NO production, which gets diminished in the presence of AMG. Nonetheless, MCW extract must be capable of inhibiting IBDV in vitro through additional mechanisms, not necessarily involving induction of NO, as some inhibition of the virus by MCW extract occurred even in the presence of AMG.