Nepeta nuda ssp. nuda L. water extract: Inhibition of replication of some strains of human alpha herpes virus (genus simplex virus) in vitro, mode of action and NMR-based metabolomics

Abstract

Nepeta nuda L. has been used in traditional folk medicine for its diuretic, anti-asthmatic, antioxidant, spasmolytic, sedative and analgesic properties (attributed to the nepetalactones). In the present study, water extract from Nepeta nuda ssp. nuda L. was tested in order to study its’ effect on the replication of Human Alpha herpes virus (HHV) type 1, strain F (ACV-sensitive) and type 2, strain DD (ACV-resistant) in vitro. Toxicity was measured at 48 h (CC50 = 7.35 mg/ml ±0.06) and 72 h (CC50 = 4.488 mg/ml ±0.308) after infection. The extract showed potent anti-herpesvirus activity in both antiviral tests performed (MTT-based colorimetric assay and yield reduction assay). By the time the extract was added, two experimental arrangements were applied. The antiviral activity increased when water extract was added simultaneously with the inoculation of the cell monolayer (EC50 of 0.66 mg/ml ±0.04 and 0.788 mg/ml ±0.009 for the F and the DD strains, respectively, measured via colorimetric assay). The IC50 value was 0.181 mg/ml ±0.073 and 0.0888 mg/ml ±0.014 for the F and the DD strains, respectively, measured via yield reduction assay. Unfortunately, selectivity for viral versus cellular molecular targets (SI) was low except for the SI values (40.60 and 82.77 for the F and the DD strains, respectively) obtained via the yield reduction assay when water extract was added simultaneously with the inoculation of the cell monolayer. In both types of antiviral assays water extract retained activity against the ACV-resistant DD strain. The virucidal assay showed that water extract did not reduce the infectivity of either of the strains used at a concentration equal to the maximum non-toxic concentration. Therefore, the above-mentioned rise in the antiviral activity detected in experimental settings when the extract was added immediately after inoculation is not due to direct inactivation of the extracellular virions. Rather, it is due to interference with the adsorption but not the penetration (according to the results of the conducted experiment). The time of addition test demonstrated that the water extract continued to exhibit antiviral activity even when added 10 h after infection. All these observations suggest that water extract exibits its anti-herpesvirus activity by influencing both early (adsorption) and late events of HHV replication. Metabolomic studies of the extract showed that the major phenolic acids present in the extract include rosmarinic, chlorogenic, gallic, vanillic, caffeic, protocatechuic, ferulic and cinnamic acids; while the presence of flavonoids was marked by cirsimaritin, chrysoeriol, vanillin, rutin and quercetin.