2.5. Anti-HSV activity of oxyresveratrol in cream preparation by plaque reduction assay
To evaluate the anti-HSV activity of oxyresveratrol in cream preparation, 20% oxyresveratrol in cream was diluted in completed media and its anti-HSV activity was assessed in Vero cells against HSV-1 (KOS) and HSV-2 (Baylor186) by the plaque reduction assay. In post treatment assay, triplicate cultures of Vero cells in 24 well tissue culture plates were infected with 50 plaque forming units (PFU)/0.1 ml of HSV-1 or HSV-2 strains for 1 h at room temperature.
Cells were added with overlay medium containing various concentrations of oxyresveratrol cream and then incubated at 37 C for 2 days. The infected cells were fixed with 5% formalin solution and stained with 0.03% methylene blue solution. The number of plaques was counted under a microscope and the 50% inhibitory concentration for plaque formation (IC50) was determined
(Shiraki et al., 1991; Lipipun et al., 2003 and Suzuki et al.,2006). In the inactivation plaque reduction assay, the virus was incubated with each dilution of oxyresveratrol in cream vehicle
for 1 h and the mixture was added into the cells for 1 h. Then the cells were overlaid and further performed as post treatment assay.Cream base without oxyresveratrol was diluted in the same manner as oxyresveratrol in cream vehicle and was subjected to the plaque reduction assay as above.