**1. Introduction**

Oxidative stress, arising through production of free radicals including reactive oxygen species (ROS), is usually defined as a disturbance in the balance between the level of ROS and antioxidant defenses [1]. Viral infections, along with other numerous human diseases, are accompanied by oxidative stress, which plays an important role in their pathogenesis [2,3]. Oxidative processes promote virus replication in infected cells, decrease cell proliferation, and induce cell apoptosis [4]. Intensification of the processes of free radical lipid oxidation and the sharp suppression of the antioxidant and antiradical protection system of the body are observed in patients with neurotropic virus infections such as tick-borne encephalitis [5] and herpes simplex [6,7]. Central nervous system tissues are especially sensitive to lipid peroxidation due to their high lipid content [8]. The lipid peroxides resulting from the ROS-induced peroxidation of membrane phospholipids, such as malondialdehyde, can transverse the circulation and cell membranes, with the resultant dysfunction of vital cellular processes such as membrane transport and mitochondrial respiration [9].

Antioxidants with different mechanisms of action are used to prevent or treat various diseases that are associated with oxidative stress and possess therapeutic effects in many cases [10–12]. Since the most important aspect of the treatment of viral diseases is the suppression of viral replication followed by cell survival, the search for drugs that have antiviral properties among antioxidants is promising. There are many examples showing that natural antioxidants such as vitamins C and E (ascorbic acid and α-tocopherol, respectively), curcumin, various polyphenols, and others are promising agents for antiviral therapy, since they decrease ROS levels in infected cells, the expression of pro-apoptotic

signaling molecules, and modulate the cellular levels of stress-related proteins such as c-Jun N-terminal kinases (JNK), phospho-p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinases (ERK-1/2), and transcription factor NF-kB [13–18].

A well-known natural antioxidant echinochrome A (naphthoquinonoid pigment of sea urchins) is the active substance of the Russian drug Histochrome®, which is used in cardiology for the treatment of ischemic heart disease and myocardial infarction, and in ophthalmology for the treatment of degenerative diseases of the retina and cornea, macular degeneration, primary open-angle glaucoma, and others [19,20].

The aim of this research was to study the in vitro antioxidant and antiviral activities of echinochrome A (Ech) and the compositions based on Ech, including also other antioxidants, against RNA-containing tick-borne encephalitis virus (TBEV) and DNA-containing herpes simplex virus type 1 (HSV-1).

This paper was prepared for printing on the basis of materials presented as a lecture on the Third International Symposium on Life Science, Vladivostok, Russia, September 2018.

## **2. Results**

#### *2.1. Antioxidant Activity of Ech Formulations Alone or Combined with Other Antioxidants*

We have compared antioxidant properties of Ech, α-tocopherol (Toc), and ascorbic acid (Asc), as well as their combinations, using the model of linetol peroxidation. The procedure that we applied relates to simple gravimetric methods via the measurement of weight increases following oxygen fixation on fatty acids [21]. Action of the studied substances on linetol was characterized as the induction time of the lipid auto-oxidation reaction (Δτ, h-difference between times necessary for linetol oxidation in the presence and absence of an antioxidant). The determination of antioxidant activities made it possible not only to compare the antioxidant activities of the studied substances with each other, but also to find the optimal ratio of antioxidants in the most active compositions. It was established that Toc was the most effective antioxidant in this experiment (Δτ 125 h) (Table 1). Ech was some less effective (Δτ 100 h), while Asc showed no antioxidant effect on this model. The low efficiency of Asc may be explained by its high susceptibility to auto-oxidation in linetol solution. It is known that in experiments in vitro, Asc lacks antioxidant activity in the absence of Toc. This observation was confirmed by our experiments (Δτ of the mixture Asc + Toc (2:1) was 195 h, which is more than effect of Toc itself). A mixture of all three antioxidants (Ech + Asc + Toc) demonstrated a stronger effect on a model of linetol auto-oxidation as a result of the synergy of these compounds (Δτ 223 h) (Table 1). We calculated the effect of synergism (in %) according to Kancheva et al. by the formulas for binary and ternary mixtures of antioxidants [22].


**Table 1.** Antioxidant activity of the formulations on a model of linetol auto-oxidation. <sup>1</sup>

<sup>1</sup> The concentration of Ech, Asc, Toc and their compositions in test medium was of 0.05 mg/mL. \* Statistically significant differences between Ech and antioxidant compositions (*p* ≤ 0.05); \*\* statistically significant differences between three-component and two-component mixtures of antioxidants (*p* ≤ 0.05). Asc: ascorbic acid, Ech: echinochrome A, Toc: α-tocopherol.
