Effect of castalagin against HSV-1 infection in newborn mice

Abstract We tested in vivo anti-herpetic effect of castalagin, an ellagitannin compound, extracted from pedunculate oak (Quercus robur). Previous investigations found that castalagin possesses a strong inhibitory effect in vitro against HSV-1/2 equal to acyclovir (ACV). It is also effective against ACV-resistant mutants and shows a synergistic effect with ACV. We study castalagin’s activity towards HSV-1 infection in newborn mice. Acute toxicity determination in mice showed LD50 value of 295 mg/kg. Prolonged toxicity was also constructed. Castalagin manifested a marked activity against HSV-1 (LD90/0.02 ml) administered in 7-day course at 0.02 ml s.c. doses of 7.5 or 10 mg/kg (PI 57-58%). ACV course demonstrated a marked activity at 20 mg/kg. The selectivity ratio LD50/ED50 (295/7.5) could be accepted as ≥ 33. Graphical Abstract


Introduction
Herpes simplex viruses (HSVs) are ubiquitous and have been known since ancient times.They often infect humans, causing a number of diseases from mild, uncomplicated mucosal infections to life-threatening conditions.
ß 2023 Informa UK Limited, trading as Taylor & Francis Group NATURAL PRODUCT RESEARCH 2023, VOL. 37, NO. 24, 4156-4161 https://doi.org/10.1080/14786419.2023.2173191Therapeutic possibilities for the treatment of infections caused by HSVs include a huge assortment of antiviral pharmaceuticals.The most largely distributed antiherpetic drugs belong to the nucleoside analogues group, whose therapeutic potential is based on inhibition of the virus genome.Four other classes of antiherpetic drugs are the pyrophosphate analogue foscarnet, the helicase-primase inhibitors amenamevir and pritelivir, the saturated alcohol doconasol, and the HIV-1 protease inhibitor nelfinavir mesylate.
Recently, tannins have drawn special interest as antiherpetic agents.These polyphenolic biomolecules are divided into two broad groups of condensed and hydrolysable compounds.Ellagitannins belong to the group of hydrolysable tannins.There is a lot of evidence in the literature that different types of ellagitannins show antiherpesvirus activity (Kurokawa et al. 2001;Chattopadhyay et al. 2010).Our previous studies have also shown that three ellagitanninscastalagin, vescalagin and grandininpossess remarkable activity against HSV-1 and HSV-2 strains that are sensitive and resistant to ACV (Vilhelmova et al. 2011).Moreover, we found that the most effective ellagitannins, castalagin, demonstrates a marked synergistic combination effect with ACV (Vilhelmova et al. 2011).In stark contrast to the wide range of HSV-1/2-sensitive cell cultures that make a wide variety of studies of these viruses possible, the scope of in vivo experimental models is quite limited.Such models include encephalitis in mice (Barr on and Vela 1995), dermatitis in adult mice (Galabov et al. 1982;Mastikova et al. 2019), intrauterine infection in guinea pigs (Stanberry et al. 1982) and keratitis in rabbit eye (Webre et al. 2012).
The present work aimed to study the effect of castalagin against HSV-1 in experimental neuroinfection in newborn mice.To do so, we first had to determine castalagin's acute single dose toxicity.
According to the available literature, castalagin has not been assessed in vivo in isolated form for acute or prolonged toxicity.It is reasonable to use available data from chemically similar ellagitannins as a starting point for planning experiments to assess castalagin's toxicity and bio-tolerance.We used the available pharmacodynamics data of the chemically similar compound 7, which differs from castalagin in just one moiety (Figure 1).

Acute toxicity of castalagin in mice
Acute toxicity of castalagin in mice was evaluated at 1 and 24 h after the intraperitoneal (i.p.) injection.No mortality was found until 24 hr or later.Mortality rate was evaluated at 24 hr after the injection.The LD 50 value of 295 mg/kg i.p. was calculated using Prozorovskii et al. (1978) express method.

Toxicological picture of the intoxication
After the first minute, we observed strong abdominal spasms that continued for over 40 min.These were accompanied by more frequent breathing, tremors, shaky walking, adynamia, hiding in corners, cataleptic posture (resembling a frog) and relaxed rear limbs while the front limbs were "folded in prayerful gesture".We also observed the Schtraub phenomenon (vertically erected tail, probably due to spasms in the sphincters), shuffling of the rear limbs while moving the body and bristled up hair.There was no mortality at 1 hr after i.p. application of castalagin.Animals died at 24 hr or later with convulsions and asphyxia.We speculate that the toxic effect of castalagin may be partly due to its active metabolite formed in vivo in the liver.
After one hour, we observed partial recovery and muscle tonus improvement of the rear limbs, but the mice still had difficulty breathing.Animals died with convulsions and asphyxia.Macroscopic examination of the internal organs after death found bloated stomach, congealed blood in the peritoneum due to the strong local irritating action of the compound, white spots in the liver as sing of hepatoxic effect.
Castalagin produced strong local irritating effect observed by strong abdominal convulsions for a period of time over 40 min post i.p. injection.

Prolonged toxicity
These results are also supported by data from additional mortality on Day 5 and changes in the serum transaminases on Day 5 after treatment with toxic doses of castalagin, ASAT was more than 10 times higher and ALAT was 5 times higher vs. controls, indicating moderate liver toxicity.

Histological examinations.
No visible changes in animals' internal organs were found after dissection on Day 5.
2.1.2.3.Biochemical studies.Specific parameters of the serum transaminases were studied as indicators of potential liver toxicity.ASAT was more than 10 times higher than normal values.ALAT was 100 U per L, a 5 times increase indicating moderate liver toxicity.GGTP was 5 U per L, a borderline value also indicating moderate liver toxicity.Alkaline phosphatase was 389 U per L. Serum cholinesterase was 620 U per L, indicating light chronic toxicity.

Effect of castalagin in HSV-1 infection in newborn mice
Newborn mice were inoculated by HSV-1, Victoria strain, at an inoculation dose of LD 90 in 0.02 ml (Hristova et al. 2022).The treatment courses, with castalagin or ACV, were initiated 1 hr after inoculation and consisted of subcutaneous injection of one dose per day for 7 days.Mortality, protection index (PI), mean survival time (MST), animal body weight and virus content in the mouse brain were measured.
Within 7 days post-inoculation, 91% of mice in the placebo group were dead (Table 1).The MST was 3.8 days.Castalagin manifested a marked activity in HSV-1 infected mice (PI 57-58%) at the administered daily doses of 7.5 and 10 mg/kg.Some protective effect was recorded at the 20 mg/kg dose.The dose of 5 mg/kg was ineffective.The MST in all castalagin treated groups was prolonged compared to placebo controls.The reference course of ACV demonstrated an activity at 20 mg/kg.ACV at doses of 40 mg/kg and 20 mg/kg per day yielded survival rates of 32% and 50%, respectively.The MST was increased with the 20 mg/kg daily dose of ACV compared to the placebo group.The higher daily dose had a weaker protective effect against HSV-1 infection, which may be due to drug toxicity (King 1988).
On the basis of the established acute LD 50 dose, the selectivity ratio LD 50 /ED 50 could be accepted as being !33.However, this value is probably markedly higher given that the antiviral testing was carried out in HSV-1 infected newborn mice and the acute LD 50 measurement involved mice weighing 15-18 g.
Treatment with castalagin or ACV reduced the infectious virus content in the pooled brains (Table 1), in agreement with the established protective effect.This reduction was exceptionally expressed with the daily dose of 10 mg/kg castalagin.A decrease by 1.5 Dlogs was established with 7.5 mg/kg.ACV also reduced the virus titres by 1.2-1.5 Dlogs.
Castalagin and ACV treatments for virus-inoculated mice resulted in no significant differences or dose-dependent deviations in weight between these groups and the placebo group on Day 7 of the study (Figure S1).
To date, very little is known about the pharmacology and pharmacokinetics of the type of ellagitannins that include castalagin, vescalagin, etc. (Vilhelmova et al. 2011;Landete 2011).Many of these compounds have been evaluated metabolically as part of complex mixtures derived from the extracts of various plants such as oaks, walnuts, some Myrtaceae, and others.However, most of them were applied via oral routes, making comparisons between the data from those studies and the present study rather problematic, as most of those ellagitannins would have been decomposed into smaller components in the gastrointestinal tract by the available microflora (Garcia-Munos and Vaillant 2014;Burlacu et al. 2020).
From the in vivo data presented in this study, we can deduce that at concentrations close to LD 50, castalagin causes blood congealing near the injection area (in this case, the peritoneum).This is not very surprising because one of the common characteristics of tannins is their ability to bind to the soluble globular proteins found in the serum.We can also speculate that castalagin or its metabolites act detrimentally on the liver at the concentrations used here.Our histopathological data suggests that they probably caused congealing of blood in the liver or some enzyme arrest, or both.Overall, our results established a relatively high LD 50 (295 mg/kg i.p.), which means castalagin has a moderate acute toxicity.
Our study contributes by offering data regarding prolonged toxicity on Day 5 after acute castalagin treatment.
In previous studies using cell cultures, this ellagitannin demonstrated a very high antiherpetic activity (Vilhelmova et al. 2011;Vilhelmova-Ilieva et al. 2020).Castalagin possesses a strong effect on the replication of HSV-1 (Victoria strain), greater than that of acyclovir (Vilhelmova et al. 2011).Of interest is the convincing data showing the synergistic combination effect of castalagin and ACV against HSV-1 (Vilhelmova et al.

2011).
All this evidence strongly supports the characterization of castalagin as a prospective anti-HSV antiviral.Moreover, the very beneficial result obtained with the combination of castalagin and ACV could increase the role of ACV as an antiherpetic agent.

Experimental
All details are provided in the supplementary material.

Conclusions
Evidently, the results of the present study on the effect of castalagin in experimental infection of HSV-1 in newborn mice, are of exceptional importance.The marked protection index and especially selectivity (at 33) show demonstrable promise.We therefore conclude that there is a coinciding of in vitro and in vivo effectivity of the compounds, what is a very hopeful measure of its anti-HSV effectivity.So, this initial in vivo testing of castalagin emphasises its value and quality as antiherpes antiviral and is a decisive step in its preparation for use in the clinical praxis.

Figure 1 .
Figure 1.Common chemical structure of castalagin and compound 7 (a).R moiety corresponds to -OH in castalagin and to lyxose (connected at the first carbon atom) (b) in compound 7 (shown with dashed arrow).

Table 1 .
Effect of treatment with castalagin or acyclovir in infection by HSV-1 in newborn mice.Data were obtained from two repeats at minimum.Placebodaily s.c.administration of saline; SDstandard deviation; PIprotection index; i.cer.-intracerebral;ID 50infectious dose 50.One-way ANOVA (Bonferroni's multiple comparison post-test) ns (not significant) p > .05 vs. Placebo c Mean value of two assays of mouse brain samples a Two-tailed Fisher's exact test.ÃÃÃ p < .0001 vs. Placebo; ÃÃ p < .005 vs. Placebo; Ã p < .05 vs. Placebo.b