*2.2. Antioxidant Activity of the Formulations Against LPS-Induced ROS Formation in Vero Cells*

To determine whether Ech alone or combined with other antioxidants is able to decrease intracellular ROS level in Vero cells, we used the model of *E. coli* lipopolysaccharide (LPS)-induced ROS formation. The ROS levels in Vero cells treated with LPS increased by 20% in comparison to control–untreated cells (Figure 1). Ech, Ech + Asc + Toc, and Asc + Toc decreased the ROS formation by 61%, 68%, and 50% in Vero cells, correspondingly, in comparison to LPS-treated cells.

**Figure 1.** Influence of Ech and studied formulations on the lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formations in Vero cells. The formulations were tested at a concentration of five μg/mL. \* *p* < 0.05; \*\* statistically significant differences between Asc + Toc and Ech (*p* ≤ 0.05).

Ech and its composition with Asc and Toc showed significant antioxidant effects on both experimental models, which makes them promising agents for further investigations on TBEV and HSV-1 replications accompanied by oxidative stress.

#### *2.3. Cytotoxicity and Antiviral Activity of Formulations.*

Cytotoxicity assay was carried out to determine the concentration range of formulations for the subsequent study of its antiviral activity in the non-toxic range for pig embryo kidney (PK) and Vero cells. Acyclovir and ribavirin were used as standard antivirals for HSV-1 and TBEV, respectively. Based on the obtained methylthiazolyltetrazolium bromide (MTT) assay results, 50% cytotoxic concentrations (CC50) against PK and Vero cells were determined for all of the studied formulations (Table 2). Further antiviral activity assay was performed at the concentrations of the formulations below 400 μg/mL.

**Table 2.** Cytotoxic and antiviral activities of formulations against tick-borne encephalitis virus (TBEV) and herpes simplex virus type 1 (HSV-1).


CC50-50% cytotoxic concentration of a formulation, IC50-50% virus-inhibiting concentration of a formulation, SI: selective index of the formulation. \* Statistically significant differences between Asc + Toc and other formulation (*p* ≤ 0.05), \*\* statistically significant differences between antioxidant composition and Ech (*p* ≤ 0.05).

The anti-TBEV and anti-HSV-1 activity of tested formulations were assessed using cytopathic effect (CPE) inhibition assay. PK and Vero cells infected with the 10-fold dilutions of corresponding virus were simultaneously treated with different concentrations of the formulations. It was found that the formulations inhibited virus-induced CPE in a dose-dependent manner, and values of the 50% inhibitory concentrations (IC50) and selective indices (SI) of the tested formulations for both viruses are presented in the Table 2. Ech and the Ech + Asc + Toc composition revealed moderate antiviral activities against TBEV and HSV-1 compared with Asc + Toc. Furthermore, based on IC50 and SI values, the Ech + Asc + Toc composition was more active toward TBEV and HSV-1 than Ech and Asc + Toc (*p* ≤ 0.05) (Table 2). The obtained data revealed that the presence of Asc and Toc in composition with Ech enhances antiviral activity of this formulation up to two times compared with Ech alone.

#### *2.4. Time-of-Formulation-Addition Assay*

The inhibitory effects of tested formulations on different stages of TBEV and HSV-1 replication cycles were studied by time-of-addition experiments via MTT assay (Figure 2). Cells were pretreated with formulations before viral infection (pretreatment of cells), viruses were incubated with formulations before cell infection (pretreatment of virus), or infected cells were incubated with formulations after penetration of the virus into host cells (treatment of infected cells).

**Figure 2.** Antiviral action of the formulations on different stages of virus replication cycles. \* Statistically significant differences between Asc + Toc and other formulation (*p* ≤ 0.05), \*\* statistically significant differences between antioxidant composition and Ech (*p* ≤ 0.05).

In the case of the pretreatment of viruses with the formulations (direct virucidal effect), Ech and the Ech + Asc + Toc composition considerably suppressed TBEV infection: inhibition rates (IR) were of 75 ± 4% and 89 ± 5%, respectively (*p* < 0.05). The corresponding pretreatment of HSV-1 by Ech and the Ech + Asc + Toc composition completely protected cells against this infection. However, only a minor effect on HSV-1 infection was detected when the virus was pretreated with acyclovir (Figure 2).

The treatment of PK and Vero cells with the tested formulations before infection (preventive effect) was much less effective. Ech, the Ech + Asc + Toc composition, and the Asc + Toc composition showed almost no preventive action against both virus infections. The same results were found when Vero cells were pretreated with acyclovir prior to infection.

When the formulations were added at an early stage of virus replication (one hour after infection), Ech and the Ech + Asc + Toc composition possessed moderate virus-inhibiting effects against TBEV infection with an IR of 21 ± 2% and 36 ± 3%, respectively, and against HSV-1 with an IR of 28 ± 3% and 43 ± 4%, respectively, compared to an inactive Asc + Toc formulation (~10%, *p* < 0.05). Meanwhile, acyclovir showed the highest antiviral activity, with an inhibition of the HSV-1 replication of 79 ± 4%.

#### **3. Discussion**

It was of interest that the Ech + Asc + Toc composition, which included three different antioxidants, demonstrated the most potent antioxidant action. Therefore, this composition showed a pronounced synergistic effect, which means that its antioxidant activity was much higher compared to each of the components added in the same amount to stabilize the lipid substrate due to the continuous regeneration of Toc from both the Ech and Asc. Moreover, the synergistic action of Ech in combination with other antioxidants was not so far reported. Ech and the Ech + Asc + Toc composition demonstrated high antioxidant activity on the model of LPS-induced ROS formation in Vero cells.

Since Ech and its composition with Asc and Toc showed significant antioxidant effects on both experimental models, and because of the ability of Ech to overcome the blood–brain barrier [23], further investigations of their effects on the replication cycles of neurotropic viruses such as TBEV and HSV-1 accompanied by oxidative stress were performed. Earlier combinations of antiviral agents with antioxidants have been used for the treatment of some other viral infections, for example, at influenza-associated complications [24]. In this study, we have shown the possibility of enhancing the antioxidant and antiviral effects of Ech due to combination with other antioxidants. We have found that the most effective method of application of Ech and the studied composition is the pretreatment of viruses with the formulations (virucidal action). The antioxidant composition Ech + Asc + Toc also demonstrated stronger antiviral activity against TBEV and HSV-1 compared to Ech. The inhibitory concentrations (IC50) of the composition were half that of Ech, while the selective indices (SI) were twice as large as those of Ech (Table 2). It should be noted that regardless of the method of exposure of viruses and cells to the formulations, the virus inhibition rates of the composition of antioxidants was higher than that by Ech itself (*p* < 0.05, Figure 1).

It was shown that the main mechanism of the in vitro action of Ech and the composition of antioxidants at the stages of the life cycles of TBEV and HSV-1 is a direct inactivation of virus particles (Figure 2). Many authors have suggested that the virucidal activity of polyphenols (Ech is considered a polyphenol as well) might be caused by direct action on the viral particles inhibiting the adsorption of the virus to the host cell receptors [25–27]. At the same time, Li et al. reported that polyphenols can cause irreversible damage or the reversible blocking of certain regions of the viral capsid protein [28]. We suppose that Ech and its composition with antioxidants can bind with some envelope virus proteins that are necessary for the adsorption of the virus to cells. Since many polyphenols exhibit antioxidant and antiviral properties, we can assume that the activity of Ech and its composition in relation to TBEV and HSV-1 can also be caused by interfering with the redox imbalance caused by these viruses [16]. Thus, Ech, either alone or in varying compositions, can both directly affect virus particles and indirectly enhance the antioxidant defense mechanisms in the hosting cell.

Thus, the ability of Ech and its compositions to inactivate HSV-1 and TBEV virus particles makes it useful as an antiviral agent in preventing de novo viral infection, and thereby could help control viral spread and limit recurrent infections.

Our data on the antioxidant and antiviral activities of Ech—which earlier have been applied to the treatment of cardiovascular and eye diseases—and antioxidant composition based on Ech indicate the necessity of further studies of these formulations for the development of promising antiviral drugs.

#### **4. Materials and Methods**
