<i>In</i><i>vivo</i> efficacy of

<p>HM. BALB/c mice were fed with HM (0.25 or 0.5 mg/kg) or ACV (5 mg/kg) and after 8 h of drug treatment the animals were infected with HSV-2G (9 X 10⁵ pfu per animal) intravaginally. The challenged animals of test groups were fed with HM twice daily for 7 days. Development of lesions and death were observed three times daily, while brain and vaginal tissue were collected after sacrification on days 2, 4, 6 or 8 after infection, homogenized and centrifuged. The supernatant was used for the determination of virus yield by plaque assay. Mean lesion score [A], Mean±S.D. of virus yield at log₁₀ (PFU/organ) in vaginal tissue [B] and brain [C]. </p

Anti-HSV efficacy of HM.

<p>[A] <b>Plaque </b><b>Reduction </b><b>Assay</b>. Infected cells were treated with HM or ACV at 0.5-50 μg/ml, overlaid with methylcellulose and plaques developed after 2-3 days were stained. The % of plaque number reduction was calculated, and the effective concentration of drug that inhibited the number of viral plaques was interpolated from the dose-response curve. [B] <b>Time </b><b>course </b><b>analysis</b>. Inhibitory effects of HM and ACV at various time points prior to infection (-3 to -1 h), at the time of infection (0 h) and post-infection (2-24 h) with HSV-2 were evaluated by plaque reduction assay. Each bar represents the mean ± S.E.M of three independent experiments.</p

Histopathology of Genital tissue of Balb/C mice

<p>: uninfected [A], infected with HSV-2G [B], HSV-2G infected animals treated with 0.5 µg/ml of HM [C], and ACV at 5 µg/ml [D]. </p

Effect of HM on viral IE transcriptional events.

<p>[A] <b>EMSA</b>: HSV-2G infected Vero cells were treated with HM for 2 and 4 h and assayed for EMSA. Biotin-labelled oligo was present in Lanes 1-6; P, biotin labelled oligo, M, mock control. [B] <b>Supershift </b><b>assay</b>: nuclear extracts from HSV-2G infected HM treated cells for 4 h p.i. was pre-incubated with HCF-1 polyclonal antibodies, added with reaction mixture, applied to non-denaturing 4% polyacrylamide gels and visualized by autoradiography. P, free biotin labelled probe; ns, nonspecific binding. [C] HCF-1 or LSD1 were immunoprecipitated in HM treated virus infected Vero cell lysate and the association was confirmed by immunoblotting with anti-HCF-1 and anti-LSD1 antibodies. </p