**3. Results**

First, the antioxidant activity in essential oils was compared. The two samples of each category were analyzed: the essential oil without excipient, or pure essential oil (EO1), and essential oil with 1% of magnesium aluminometasilicate (EO2). The results are presented in Table 1. The range of essential oil concentration in this examination was from 0.2 to 20%.


**Table 1.** Antioxidant activity of nutmeg essential oils applied at different concentrations.

\*—significant difference compared to EO1, *p* < 0.05, *n* = 3.

Both essential oils demonstrated similar antioxidant activity increasing in a concentrationdependent manner, except for some small fluctuations at 2% (EO2 had slightly higher antioxidant activity compared to EO1) and 20% (EO2 antioxidant activity was slightly lower than EO1). Both essential oil preparations at 10% concentration had higher than 50% antioxidant activity (more than half of DPPH radicals were bound).

Next in the study, the antioxidant activity of essential oil hydrolats was tested by using DPPH radical scavenging method. Hydrolat from EO1 was named EOH1, and from hydrolat from EO2 was named EOH2. The data are provided in Table 2.


**Table 2.** Antioxidant activity of nutmeg essential oil hydrolats.

\*—significant difference compared to EOH1, *p* < 0.05, *n* =

At small quantities of up to 0.3 mL, the antioxidant activities of both hydrolat preparations were similar, but at 0.5 mL and 1 mL, the EOH1 antioxidant activity was significantly higher compared to that of EOH2. EOH1 at 1 mL had an antioxidant activity greater than 50%, and this activity level was similar to 5%–10% of EO1 activity. The results show that free radical scavenging activity of 0.2 mL of hydrolat is higher than that of 1% or less concentrated essential oil.

Summarizing the antioxidant activity results, magnesium aluminometasilicate did not improve, and even slightly decreased the antioxidant activity of nutmeg essential oil and its hydrolat.

Next in the study, antibacterial properties of nutmeg essential oil and hydrolats were investigated on 15 pathogenic clinical isolate strains by using a dilution range assay. The results are presented in Table 3.


**Table 3.** Antimicrobial study results of nutmeg essential oil and its hydrolats.

+ means the pathogens growth. 1. Klebsiella pneumoniae, 2. Salmonella enterica 24 SPn06, 3. Pseudomonas aeruginosa 17-331, 4. Acinetobacter baumanni 17-380, 5. Proteus mirabilis, 6. 6MRSA M87fox, 7. Enterococcus faecalis 86, 8. Enterococcus faecium 103, 9. Bacillus cereus 18 01, 10. Streptococcus mutans (referent), 11. Enterobacter cloacae, 12. Citrobacter freundii, 13. Staphylococcus epidermidis, 14. Staphylococcus haemolyticus, 15. Pasteurella multocida. Where the growth of bacteria were inhibited, minimal inhibitory concentrations were provided in %.

EO1 only suppressed *Pasteurella multocida* growth, with the minimal concentration to achieve this effect being 0.2%. However, nutmeg essential oil with 1% of magnesium aluminometasilicate (EO2) had a broader effect. Next to *P. multocida*, it inhibited *E. faecalis* and *S. mutans*, and the efficient concentrations were rather low. A mere 0.5% was enough to completely suppress *E. faecalis*, and for the *S. mutans* strain even less than 0.1% was effective. In the case of EOH1 and EOH2, only the hydrolat with aluminometasilicate suppressed the growth of *S. mutans.* Thus, the results indicate that the excipient magnesium aluminometasilicate broadens the spectrum of antimicrobial activity of nutmeg essential oil.

One of the most important pharmacological activities of plant essential oils is related to anti-inflammatory properties. Nutmeg essential oil is also known for inflammation reducing activity [24]. Therefore, next in the study, we have assessed nutmeg seed essential oil and hydrolat preparations in a virus mimetic Poly I:C-induced inflammation in vitro model by using human fibroblast cell culture. Before starting the treatments, the general toxicity test of the oils and hydrolats was performed and LD50 doses as well as safe concentrations were established.

As indicated in Figure 1a, there were no significant di fference in cell viability detected after treatment with both EO1 and EO2 essential oil solutions up to the dilution ratio 1:100. Further increases in concentration up to the dilution ratio 1:40 significantly decreased viability of the fibroblasts. After treatment with essential oil solutions at 1:40, the viability dropped from 97 ± 2% in control to 70 ± 12% in the case of EO1, and to 42 ± 10% in the case of EO2. At the dilution ratio of 1:5, the percentage of viable cells in the cultures was lower than 10% in the case of both essential oil preparations. The dilution ratios corresponding to LD50 calculated for EO1, EO2, and 96% ethanol were 0.047, 0.022, and 0.055, respectively. Thus, EO2 was significantly more toxic for the cells compared to EO1, and also to ethanol. In contrast, the toxicity pattern of EO1 was very close to that of ethanol, indicating there were no or very little toxic compounds in this essential oil preparation.

**Figure 1.** The e ffect of nutmeg essential oil ethanol solutions (**a**) and nutmeg essential oil hydrolats (**b**) on viability of cultured human fibroblasts. EO1—essential oil without excipient solution, EO2—essential oil with 1% magnesium aluminometasilicate solution, EOH1—hydrolat from EO1, and EOH2—hydrolat from EO2. In addition, 96% ethanol was assessed as solvent control for the essential oil. Punctured lines indicate the dilution ratios corresponding to LD50. \*—statistically significant di fference compared to untreated control, #—compared to EO1 in (**a**) or EOH1 in (**b**), respectively, when *p* < 0.05.

Evaluation of cell viability after 24 h treatment with nutmeg seed essential oil hydrolats revealed that both EOH1 and EOH2 were not toxic up to a dilution of 1:20 (Figure 1b). After cell incubation with 1:10 EOH2, the percentage of viable cells in the cultures decreased to 57 ± 19%, making a significant di fference compared with the untreated control. A significant viability drop in EOH1 treatment series was achieved when the dilution ratio 1:5 was applied. The level of viable cells in this treated cultures was 44 ± 14%. After treatment with EOH1 and EOH2 at the ratio 1:2, nearly all cells in the cultures were found to be necrotic. The dilution ratios corresponding to LD50 calculated for EOH1 and EOH2 were 0.160 and 0.105, respectively. Toxicity evaluation of the hydrolats indicated that EOH2 is slightly more toxic compared to EOH1.

The next task in this work was to evaluate the e fficiency of nutmeg seed essential oil and hydrolat preparations to reduce toxicity and signaling in viral inflammation in vitro model. To stimulate inflammatory response, human fibroblast cell culture was treated with 1 μg/mL virus double stranded RNR mimetic polyinosinic: polycytidylic acid (Poly I:C) for 24 h, with or without nutmeg seed essential oil solutions or hydrolats. After toxicity assessment, the dilution ratios selected for anti-inflammatory property testing were 1:200 and 1:100 for the essential oil solutions, and 1:100 and 1:40 for the hydrolats. Anti-inflammatory assessment results are presented in Figure 2.

**Figure 2.** The e ffect of nutmeg seed essential oil ethanol solutions and the essential oil hydrolats on Poly I:C-treated human fibroblast cell viability (**a**) and cytokine IL-6 release from the cells (**b**). EO1—essential oil without the excipient solution, EO2—essential oil with 1% magnesium aluminometasilicate solution, EOH1—hydrolat from EO1, and EOH2—hydrolat from EO2. \*—statistically significant di fference compared to untreated control, #—compared to Poly I:C-only treatment, when *p* < 0.05; *n* = 5–7.

After fibroblast cell culture treatment with 1 mg/mL Poly I:C, the amount of viable cells decreased by 59% (Figure 1a). Addition of nutmeg essential oil preparations to the cell culture medium increased cell viability in the Poly I:C-a ffected cultures. Statistically significant di fferences compared with Poly I:C samples were found after treatment with 1:200 EO1, 1:200 and 1:100 EO2, as well as 1:40 EOH1 and 1:40 EOH2. The percentages of viable nuclei in these samples were 81 ± 9%, 82 ± 16%, 76 ± 10%, 72 ± 14%, and 79 ± 12%, respectively. Thus, EO2 has demonstrated the highest cytoprotective capacity in a virus mimetic inflammation model.

Evaluation of the release of IL-6 to the incubation medium revealed that after 24 h of Poly I:C treatment, the level of this pro-inflammatory cytokine jumped from nearly a "zero" value to 883 ± 273 pg/mL (Figure 2b). Nutmeg essential oil preparations applied together with Poly I:C significantly reduced the concentration of IL-6 in the medium. The significant drop in the IL-6 level was in the samples incubated with 1:100 EO1, 1:200 and 1:100 EO2, 1:40 EOH1, and 1:100 and 1:40 EOH2. IL-6 concentration in these samples was found in the range between 162 ± 123 pg/mL (with 1:40 EOH2) and 206 ± 83 pg/mL (with 1:100 EO1). The assessment of IL-6 release indicates that both the solution of nutmeg seed essential oil with magnesium aluminometasilicate and the hydrolat from this essential oil are most e fficient against Poly I:C-induced release of this inflammatory cytokine.
