## 2 Results and Discussion
### Antiviral Properties  
The three fractions of _I. aquifolium_ Silver Queen (polyphenols, saponins, and higher terpenids) were tested for antiviral properties against HSV-1 and HAdV-5 (Table 4). It was observed that the degree of reduction of replication of both viruses by polyphenols and saponins was 4 log (99.99% reduction). For the terpenoid fraction, a similar efficacy (99.99%) was observed against HSV-1, while the reduction level was 2 log (99% virucidal efficacy) against HAdV-5 [34; 35]. A parallel evaluation of the cytotoxicity of the formulations at the concentrations used in the virucidal tests showed no changes in the cultured cells. The exact mechanism of the observed action of the tested extracts and fractions is not yet known. Presumably, it is based on the action of a nonpolar (terpenoid) and a saponin fraction. Terpenoids are a diverse class of phytochemicals derived from isoprene units, which numerous authors have reported to show significant potential as antiviral agents against a broad spectrum of enveloped and nonenveloped viruses [36]. According to [PERSON] et al., they interfere with virus entry into the host cell in the early stages of infection, which hinders its replication [37] and modulates the host's immune responses. For example, \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Virus** & **PLHA** & **SAP** & **TERP** \\ \hline
**HSV-1** & 4 log (99.99\%) & 4 log (99.99\%) & 4 log (99.99\%) \\ \hline
**HAdV-5** & 4 log (99.99\%) & 4 log (99.99\%) & 2 log (99.00\%) \\ \hline \hline \end{tabular} PLHA—polyphenol and phenolic acid extract, SAP—saponin extract, TERP—higher terpenids.  
\end{table} 
Table 4: Comparison of antiviral properties of fractions of _I. aquifolium_ Silver Queen leaves (expressed in logarithmic scale and % reduction). Figure 7: Principal component analysis of _Ilex_ taxons (PC1 vs. PC2) based on saponin content; AM—_I. aquifolium_ Aurea Marginata, BG—_I. meserveae_ Blue Girl, BP—_I. meserveae_ Blue Prince, BPR—_I. meserveae_ Blue Princess, HNS—_I. aquifolium_ Handsworth New Silver, PA—_I. paruguariensis, SQ—_I. aquifolium_ Silver Queen. anti-HIV effects are believed to include activation of protein kinase C isoenzymes [38]. Based on the tests carried out in this project, the substances in the fractions have virucidal potential, probably linked to blocking or destroying the virus proteins, which prevents them from attaching to the cell, although we cannot exclude a parallel antiviral effect. This type of multidirectional action of _Garcinia parvifolia_ leaf extract was demonstrated in a study by [PERSON] et al. [39]. Their multifaceted mechanisms of action make them attractive agents for combating viral infections and offer opportunities for synergy with existing antiviral drugs. So far, they have been confirmed to be effective against influenza viruses, SARS-CoV-2, HCV (hepatitis C), and HSV, among others [36]. Furthermore, according to a study analysing HSV-1 therapy using simultaneous administration of acyclovir and betulin, these substances were found to have a strong synergistic effect against HSV [40]. However, further research is needed to clarify the mechanisms of action of _Ilex_ terpenoids against different viruses. According to [PERSON] and [PERSON], terpenoids showed potent antiviral activity against a wide range of human and animal viruses. Their diverse structural and chemical properties offer promising opportunities for the development of novel antiviral agents [36]. The second group of compounds in _Ilex_ extracts is saponins, which have a variety of biological activities, including antiviral activity. In a study by [PERSON] et al., two substances isolated from natural sources due to activities associated with their amphiphilic constituents were tested for their effects on the replication of herpes simplex virus type 1. A triterpen saponin representing the oleanane group inhibited viral DNA synthesis, while a triterpen saponin representing the ursane group inhibited viral HSV-1 capsid protein synthesis [41].