The present study for the first time, demonstrated the anti-HSV activity of crude methanolic extract of M. peltatus leaf, an ethnomedicine of Onge tribes of Andaman and Nicobar Islands, India. Phytochemical study revealed that the crude methanolic extract contain two major fractions, fraction A and B, of which fraction A had significant anti-HSV activity. Chromatographic separation and spectral analysis revealed that fraction A contains a known triterpene ursolic acid, which possesses strong antiviral activity against HSV-1 and HSV-2 in vitro. The antiviral activity of the crude methanolic extract was weak compared to fraction A, probably due to its low concentrations of bioactive compound(s). While the higher antiviral activity of fraction A, than the crude methanolic extract, is due to the higher concentration of bioactive compounds within the fraction. Earlier study with the crude methanolic extract of M. peltatus showed moderate antibacterial and antifungal , analgesic and antiinflammatory [20, 21, 38] activity. On the otherhand fraction B do not showed any anti-HSV activity, hence not included in further study.
The cytotoxicity study revealed that the crude methanolic extract, fraction A and isolated ursolic acid had different CC50 due to the variable concentration of bioactive compound(s), and the antiviral activity was found far below the CC50 dose. Further, fraction A and isolated ursolic acid on both HSV-1 F and HSV-2 G revealed dose-dependent antiviral activity. Infection of Vero cell by HSV-1 and HSV-2 was significantly prevented by the fraction A, or isolated ursolic acid with higher SI values. However, the CC50 of fraction B was just double the EC50, giving an insignificant SI for both HSV-1 and HSV-2.
The dose-dependent activity and time course analysis was done to understand how the fraction A and isolated ursolic acid inhibit the viral infection. Interestingly, fraction A and isolated ursolic acid was found to inhibit both HSV-1 and HSV-2 infection(s) in dose-dependent manner, with an EC50 of 7.8 μg/ml and 5.5 μg/ml for HSV-1 F and 8.2 and 5.8 μg/ml for HSV-2 G, respectively. However, 100% inhibition of HSV-1 was recorded at 14.5 μg/ml of fraction A and 9.0 μg/ml for isolated ursolic acid; while for HSV-2 it was 15 μg/ml of fraction A and 12.5 μg/ml of isolated ursolic acid. Results on the time-course effect between 0 and 5 h post-infection revealed that the fraction A and isolated ursolic acid possess a similar inhibitory effect. This suggests that the mode of action is not due to inhibition of vial adsorption, but inhibition of viral replication. The time response study, also demonstrated that both fraction A and isolated ursolic acid probably interfere early stage of HSV replication, as the maximum inhibition was noted at 2-5 h post-infection. However, the real antiviral mechanism of fraction A and isolated ursolic acid remains to be further elucidated.
The indirect immunofluorescence assay was carried out to determine the kinetics of fraction A on antigen expression of HSV-1. Maximum reduction in number of infected fluorescent cells was observed at 4 h, along with a characteristic pattern of small foci of positive cells and even single fluorescent cells. This suggests that fraction A can inhibit viral dissemination. When fraction A was subsequently added at 2 h and 4 h time period, a significant reduction of positive fluorescent cells was observed (Figure 6). The non-amplification of fraction A treated HSV-1 infected cells by PCR further complemented and strengthened the antiviral activity of this plant. Detection and amplification of pol gene (control gene) in fraction A or isolated ursolic acid treated and virus infected cells (Figure 7) indicated that there was no cytotoxicity after treatment of cells with this plant product.
The widely used anti-herpes virus drug acyclovir is a nucleoside analogue, specifically targets the thymidine kinase of HSV . However, its extensive and long term use yielded drug-resistant strains [9, 11], due to mutations in viral thymidine kinase and/or DNA polymerase, that alter substrate sensitivity , and thus, become chromosome mutagen. Moreover, efficacy of therapeutic vaccines against primary and recurrent HSV infection has failed  and thus, search for natural alternative is the top priority to control and prevent HSV infections and its transmission. The earlier reports indicated that different species of Mallotus (M. philippinensis
M. repandus) contain several secondary metabolites like terpenoids (mallotucin and malloripine), diterpenic lactones (mallotucin B,C,D), triterpene alcohol (moretenol), saponins (crotoxigenin, coroglusagenine), cardinolipids, resins (rottlerine, isorottlerine), flavonoids, and β-sitosterol [41, 42]. However, there were no reports on the bioactivity and phytochemistry of M. peltatus, except the isolation of ursolic acid and β-sitosterol alongwith some fatty acids by this group . The ursolic acid is a pentacyclic amphiphilic triterpene with planner hydroxylated polycyclic [(3b)-3-hydroxyurs-12-en-28-oic acid] structure, ubiquitous in medicinal plants as free acid or aglycones for triterpenoid saponins, and have been used since antiquity due to multiple bioactivities . Contemporary research revealed that the ursolic acid, isolated from plants, is cytotoxic to some tumor and cancer cells [43–45], including skin tumor  and recommended for skin cancer therapy . Other studies reported its antiviral , antibacterial , and potent anti-inflammatory [50–52] activities. It is a highly selective inhibitor of cyclic AMP-dependent protein kinase , human DNA polymerases and DNA topoisomerases  and has antioxidative  and apoptotic [56, 57] activities. Ursolic acid isolated from Rosmarinus officinalis L. leaves is reported to inhibit the motility of Trypanosoma cruzi epimastigotes ; while ursolic acid isolated from Ocimum sanctum
O. basilicum and O. americanum showed anti-HSV activity with ED50 of 35–47 μg/ml by interfering at various steps of viral multiplication .
Thus, our results showed that fraction A, which contain ursolic acid as one of the compound, might be a potential therapeutic candidate against HSV infections, as indicated by its SI value (7.86 – 22.3). Ursolic acid is known to be less toxic, can restore skin’s collagen bundle and elasticity, and is dermatologically innocuous , while the antivirals presently used for herpes virus treatment have high toxicity, several side effects and problem of frequent drug resistance development. Therefore, our study demonstrated that the fraction A and its component(s) can serve as an alternative agent in herpes virus infection and thus, merit a greater attention.