2.2.2. Determination of Anti-Inflammatory Activity
The results of the studies (
Figure 1) indicate that the DECV showed anti-inflammatory activity. Compared to the control, an anti-exudative effect of DECV was observed within the first hour after the start of treatment and reached its maximum values at the third and fifth hour, depending on the solvent of the extract. The hydroethanolic DECV showed the highest anti-inflammatory activity among all tested extracts.
During the first hour of the experiment, the most pronounced anti-inflammatory activity was shown by the reference drug (Hypericum tincture), which reduced the oedema by 37.30% compared to the control. However, at the third and fifth hour of the experiment, the anti-exudative activity of the reference drug decreased slightly compared to the hydroethanolic DECV. In the group of animals treated with this extract, the volume of the rat paw decreased compared to the control group by 36.00% within the first hour of the experiment, by 46.4% at 3 h and by 65.6% at 5 h after the phlogogenic agent injection.
After the experiment, all animals had peripheral blood taken from their tail vein and the content of hemoglobin and the number of red blood cells and leukocytes were determined (
Table 3). It is a well-known fact that one of the indicators of an inflammatory process is leukocytosis. According to the data given in
Table 3, the number of leukocytes in the control group of animals increased by 1.47 times. The use of all investigated substances in various ways contributed to the reliable normalization of the number of leukocytes, and at the end of the experiment, the indicators in the peripheral blood of the tested animals were brought to the level of the untreated controls.
Thus, the results of the study indicate that both DECVs have anti-inflammatory activity. The hydroethanolic DECV had the most pronounced anti-exudative activity; it reduced the inflammatory reaction by 65.60% and had approximately the same effect as the reference drug (
Hypericum tincture). This is consistent with Villanueva-Bermejo et al. (2019) [
19], who showed a carbon dioxide extract using ethanol as a co-solvent showed a certain anti-inflammatory effect.
2.2.3. Determination of Antimicrobial Activity
As a result of the conducted studies, it was established that the DECV inhibited the growth of microorganisms and had a bacteriostatic effect against Pseudomonas aeruginosa (the zone of inhibition of microorganism was 8–9 mm), Escherichia coli (8–10 mm), and Proteus vulgaris (6–7 mm), and partially against Staphylococcus epidermidis (0 mm for water and 7 mm for hydroethanolic DECV). The hydroethanolic DECV had the most pronounced antimicrobial activity against all tested microorganisms and had approximately the same effect as the reference substances ampicillin (0–14 mm) and oleandomycin (0–5 mm), if we exclude their activity against S. aureus and S. epidermidis.
Kakouri et al. (2017) [
20] reported that hydroethanolic extracts rich in flavonoids and other phenolic compounds did not showed remarkable activity against the microorganisms mentioned above. On the other hand, the hydroquinone glycoside arbutin displayed relatively strong antibacterial activity against
S. aureus but was absent in both DECVs containing arbutin [
10]. According to Jurica et al. (2017) [
9], the antimicrobial activity of leaf extracts of
Arbutus unedo, other than arbutin itself, could be related to the presence of phenolic acids such as cinnamic, ferulic, and caffeic, but their content in both DECVs studied by us is not very high (
Table 1). Arbutin also showed antibacterial activity against
P. aeruginosa and
E. coli (MIC > 25.6 mg/mL) in other study [
9].
2.2.4. Determination of Neurotropic Activity
The results of the study of the behavioral responses of mice in the “Open Field” test indicated a significant effect of DECV on the motor activity of animals (
Figure 2). The novelty stress showed the greatest impact on the orienting-research behavior of mice in the “Open Field” arena within the first minute of the experiment.
C. vulgaris extracts activated the locomotor behavior of mice due to their increased psycho-emotional excitation. The total motor activity increased mainly due to the large number of horizontal movements. Grooming increased significantly compared to the control in the group of mice treated with hydroethanolic DECV. Some authors have described grooming as an “indicator of comfort” for animals [
21,
22]. The latent period of exit from the center of the arena was not significantly different for all studied groups. The fading indicator in the “Open Field” test was of great interest. The hydroethanolic DECV did not cause fading of the animals at all, and the water DECV had approximately the same effect as the
Valeriana officinalis extract.
Thus, both the water and hydroethanolic DECV activated the exploratory behavior of mice under the conditions of novelty stress without inhibition of their orienting-research activity. Valeriana officinalis extract reduced vertical motor activity and grooming, which means inhibition of orienting-research behavior.
2.2.5. Determination of Anti-Anxiety and Antidepressant Activities
The “Elevated Plus Maze” test. The investigated DECVs influenced the level of anxiety, fear and perception caused by the novelty of its environment, its reaction to the height and to the illuminated open space (
Table 4). In the analysis of the results, special attention was paid to the group of animals that were administrated hydroethanolic DECV. The total number of appearances of animals of this group in the dark chamber was 1.30 times higher compared to the control. The total time spent in the dark branches of the maze was determined in seconds. The mean value for animals treated with hydroethanolic DECV was the lowest (103.29 s) among all tested substances. The total number of appearances in the central location increased by 1.25 times compared to the control. The total number of appearances of animals in the illuminated branches of the maze increased by 1.33 times compared to the control.
The total time of staying in the illuminated branches was determined in seconds and was the highest for the animals of this group (138.43 s). The total time of staying in the central location was calculated in seconds and was the highest for the animals that were administered extract 2 (56.86 s). The total number of cases when the animals looked down from the maze was calculated to evaluate the risk of the animals. This indicator was the highest in this group of animals (19.86).
Thus, the administration of the hydroethanolic DECV activated the orienting-research behavior of the animals and showed an anti-anxiety effect, which was determined by the large number of movements of animals in the illuminated branches of the maze and the number of cases of looking down. Animals in this group preferred to stay in the illuminated branches and in the center of the maze, which could be interpreted as a stress resistance. The large number of cases of looking down meant that the animals were not afraid to explore all ways of leaving the maze. The large number of appearances in the dark branches and the shortest time of staying there may indicate the high motor activity of the animals and favoring the illuminated branches of the maze. Considerable attention should be drawn to the number of appearances of the animals in the illuminated branches. This indicator was the highest during all 5 min of the experiment in the group of animals that were administrated hydroethanolic DECV.
The “Porsolt Forced Swim Test”. The antidepressant effect of the investigated DECV was evaluated by their ability to reduce the time of immobility and time of passive swimming of animals, and to increase the latent period of the first “fading” and the time of active swimming. In the conditions of the “Porsolt Forced Swim Test”, the administration of the tested extracts showed an antidepressant effect on the tested animals (
Table 5). Thus, on the first day of the experiment, the latent period of the first “fading” of the animals treated with extract 2 was 1.18 times shorter compared to the control. The latent period of the first “fading” was extended on the third day of the experiment and lasted 1.10 times longer compared to the control. On the fifth day of the experiment, the latent period of the first “fading” of animals was reduced and lasted 1.22 times less compared to the control. The total time of active swimming of the animals of this group on the first and third days of the experiment was the longest (190.29 s and 99.57 s). On the fifth day of the experiment, the total active swimming time was 1.79 times shorter compared to the control. On the third day of the experiment, the time of total active swimming of animals treated with extract 1 was 1.17 times longer compared to the control.
Therefore, the hydroethanolic DECV exhibited antidepressant activity on the first and third days of the experiment, as evidenced by the longest period of active swimming and the shortest time of immobility of the animals. The time of passive swimming of the animals that were administered hydroethanolic DECV was the shortest on the first day of the experiment.
The “Cube” test. The anxiolytic effect of the investigated extracts was evaluated by their ability to increase the number of approaches of animals to the cube and the total time of the cube examination, as well as to reduce the latent period of the first approach of the animal to the cube. In the conditions of the “Cube” test, the administration of both DECVs to the animals affected the level of their anxiety and fear of a new object in their home cage (
Table 6). The latent period of the first approach to the cube was the shortest in the group of animals receiving extract 2 (0.46 s). In this group of animals, both the number of approaches to the cube (5.00) and the total time of the cube examination (14.57 s) were the highest.
It is interesting to mention that
C. vulgaris is known in Estonian ethnomedicine for its sedative and anti-diabetes activities, but it does not have the anti-cancer activity found in the other 44 species [
23,
24,
25].
Hypericum perforatum, Leonurus cardiaca and
Piper methysticum are well known natural antidepressants and/or anxiolytics, and also the species
Epimedium L. genus (Berberidaceae) belonging to the Chinese pharmacopoeia possesses antidepressant activity [
26,
27,
28]. Their BASs are totally different, and also different than the
Valeriana genus. Dhiman et al. (2020) [
29] found that
Valeriana jatamansi possesses sedative and antidepressant properties and is an alternative choice among existing drugs like benzodiazepines to treat insomnia.
Additionally, C. vulgaris seems to be promising species to treat these health problems. The water and hydroethanolic DECVs increased the number of approaches of the animals to the cube, the total time of the cube examination, and reduced the latent period of the first approach to the cube, which indicates the activation of orienting-research behavior of animals and the presence of an anti-anxiety effect.
2.2.6. Discussion of the Results
Considering the chemical composition of the obtained extracts, we can conclude that the content of hydrocinnamic acids and flavonoids in the hydroethanolic DECV are, respectively, in 1.34 and 2.61 times more than the aqueous one, and the sum of phenolic compounds, in general, is 1.87 times more. The extracts contain a high content of flavonoids—rutin, hyperoside and quercetin-3-D-glucoside—which also prevail in motherwort grass
Leonurus cardiaca and preparations based on it [
26,
30,
31], which extent determines the effect of extracts on the nervous system. There are more hydroquinone (1.33 times) derivatives in the aqueous extract which determines the activity of the extracts to different groups of microorganisms [
8,
10,
11]. Phenolic compounds have anti-inflammatory activity [
19,
32,
33], and as there are more of them in the hydroethanolic DECV, it is more active. In general, a greater amount of phenolic compounds in the hydroethanolic extract and it determines its advantages. The experiments showed that the hydroethanolic DECV possess anti-inflammatory, antimicrobial, anxiolytic, stress-protective, anti-anxiety and anti-depressant effects due the optimal concentration of ethanol (70%) used by us.