Therefore, each drug by itself or in combination was tested at an equipotency ratio based on its corresponding IC50 value. The interaction degree between glucoevatromonoside and acyclovir was calculated through combination index (CI) equation, based on the median-effect principle of the mass-action law, using Calcusyn software (version 2.1,
Biosoft). According to the CI theorem, CI values <1, =1, and >1 indicate synergism, additive effect, and antagonism, respectively. This assay followed the procedures described earlier (Hu et al., 2009). Adenosine 5′-triphosphatase (NKATPase) activity assay was determined using a Quantichrom ATPase/GTPase assay kit (Bioassay, Hayward, CA, USA), according to the manufacture’s instructions. The initial screening of 65 cardenolide derivatives for anti-HSV activity was performed only with HSV-1(KOS strain) using a plaque reduction assay. Following the same strategy DNA Damage inhibitor proposed by Su et al. (2008), we decided that compounds showing IC50 values ⩽1 μM would be chosen to proceed another screening for anti-HSV-1 (29R strain) and anti-HSV-2 (333 strain) activity. Among the 65 tested compounds, 16 were found to possess antiherpes activity
with IC50 values ⩽1 μM, and a natural compound, glucoevatromonoside, isolated from a Brazilian cultivar of Digitalis lanata ( Braga et al., 1996) was chosen for further evaluation of its mode of action ( Table 1) due to its high SI and lower IC50, when compared to acyclovir, and also because there was enough quantity selleck chemicals to perform all designed assays necessary Pregnenolone for this purpose. Digoxigenin showed an IC50 ⩾1 μM, but it was tested in the second screening because it is the aglycone of some tested cardenolide derivatives and showed antiherpes effects in a previous work ( Su et al., 2008). As shown in Table 1, HSV-1 (29R strain, resistant to acyclovir) was highly sensitive to the treatment with the tested cardenolides, which implicates that the targets of these compounds are probably different
from those of acyclovir. Hence, they might represent a novel group of drugs with distinct antiviral mechanism from those of conventional drugs. Firstly, in order to compare the potency of glucoevatromonoside antiherpes activity, at different MOI, a yield reduction assay was conducted. As shown in Fig. 2, both concentrations of glucoevatromonoside were able to inhibit HSV-1 replication at all tested conditions, even at the MOI 0.4 which is thousand times higher than that used in the initial screening. At the higher tested MOI, the glucoevatromonoside at 0.26 μM, which is two times higher than its IC50 value, showed a reduction of 5.1 Log, in comparison with viral controls. Remarkably, this reduction was higher than that obtained with acyclovir (2.5 Log) at the same conditions Since this antiviral potency is not commonly observed for other antiviral agents, this compound holds a clear advantage over them (Talarico and Damonte, 2007).