**4. Discussion**

The purpose of our study was to compare the biological activity of nutmeg seed essential oil and hydrolat without excipient and using magnesium aluminometasilicate as the excipient. To our knowledge, it is the first application of aluminometasilicate as an excipient in essential oil studies. Essential oils have strong antioxidant activity, and some of them are used as preservation agents protecting food or cosmetics from oxidation-induced spoilage [25,26]. Antioxidant activity studies help to elucidate essential oil capacity to protect food from free radical damage [27]. The DPPH radical scavenging method is widely used for this purpose because it is simple and cost-e fficient, and gives reliable results. Therefore, it was selected to evaluate the essential oil and hydrolat preparations in our study. Our previous study [22] showed that magnesium aluminometasilicate had influence not only on the yield of essential oil, but also on its chemical composition. Magnesium aluminometasilicate significantly increased the quantity of sabinene, α-pinene, and limonene. Dai et al. (2013) study with *Wedella Prostrata* essential oil (containing 11.38% limonene and 10.74% α-pinene) had a lower antioxidant activity than 100 μg/mL limonene but a higher antioxidant activity than the pure α-pinene [28]. Such a result suggests that limonene is a more prominent antioxidant compared to α-pinene. Our nutmeg seed essential oil (EO2) had 11.66 ± 3.39% α-pinene and 4.91 ± 0.71% limonene [22]. Based on the results of the study where the presence of limonenen together with α-pinene resulted in higher antioxidant activity [28], we can predict that our EO2 has higher antioxidant activity than the pure α-pinene sample. The *Juniperus scopulorum* 10% essential oil had a 54.7% antioxidant activity (composition: sabinene 50.7%, α-pinene 3.23%, limonene 2.22%, cis sabinene hydrate 0.58%) [29]. Our study shows that 10% essential oils EO1 and EO2 have 61.01 ± 0.26% and 62.11 ± 0.43% antioxidant activity, respectively. Predominant compounds in EO2 were sabinene 61.42%, cis sabinene hydrate 0.3%, limonene 5.62%, and α-pinene 15.05%. The EO1 essential oil had more β-pinene [22], which could increase its antioxidant activity. Other authors have demonstrated nutmeg essential oil with higher antioxidant activity besides α-pinene had also β-pinene [27]. Misharina et al. (2009) have also studied antioxidant properties of nutmeg essential oil and found that 16.5% concentrated solution had approximately 50% antioxidant activity [30]. Such di fferences may be due to the distinct technique of the research and the variation in nutmeg seed material.

Hydrolats analyzed in our study had lower antioxidant activity compared to essential oils, most likely because of the lower concentrations of volatile compounds. We have searched the literature data, but could not find any studies about the antioxidant activity of nutmeg seed hydrolats so far. Hydrolats prepared from other plant sources had di fferent antioxidant properties. For example, *Salvia o*ffi*cinalis* 0.1 g/mL had about 30% radical scavenging activity. The same concentration of *Rosmarinus o*ffi*cinalis* hydrolat had about 50% activity [31]. Our hydrolats (0.1 g/mL) had 31.43 ± 1.55% (EO1) and 27.24 ± 1.63% (EO2) antioxidant activity—the same as *Salvia <sup>o</sup>*ffi*cinalis*. Nutmeg seeds hydrolats at the highest concentration tested (0.5 g/mL) had 56.42% and 44.19% antioxidant activity (EOH1 and EOH2, respectively).

Essential oils are known for bioactive compounds with antibacterial activity, therefore they are used as antimicrobial agents in medicine, pharmacy, cosmetology, and other fields [18]. However, di fferent essential oils a ffect microorganisms in distinct ways—some suppress gram-positive e ffects, others suppress gram-negative e ffects [19]. Also, the e ffective concentration of essential oils vary. There are various methods for determining antibacterial activity (the agar disk-di ffusion method, antimicrobial gradient method, dilution methods, and other methods) [32]. Dilution methods are the simplest methods used to determine whether the essential oil suppresses the growth of bacteria or not [23]. There are many techniques and methods used for antimicrobial activity evaluation, and therefore, it is di fficult to compare the results obtained from the di fferent studies. In our study, the EO1 essential oil (0.2%) only suppressed *Pasteurella multocida*. The essential oil EO2 with a higher quantity of sabinene, α-pinene, and limonene [22] had antimicrobial activity against three pathogens. Next to *P. multocida*, it also prevented growth *E. faecalis* of and *S. mutans*. The increased e fficiency of EO2 against pathogenic strains can be explained by the higher quantity of volatile compounds. Nurjanah et al.'s (2017) study showed (an in vitro disc di ffusion antimicrobial activity method) that *Myristica fragrans* essential oil (60% concentration was used) from Central Java inhibited the largest areas [33]. The inhibition areas were from 12.96 mm to 16.79 mm, with the control at 0 mm (*S. aureus*, *S. dysenteriae*, *S. typhi*, and *S. epidermidis*). In the essential oil used for the above-mentioned study, sabinene, α-pinene, and β-pinene quantities were the highest out of all of the chemical compounds (the concentrations were 18.82%, 16.54%, and 13.82, respectively). The essential oil EO2 investigated in this study has a similar composition, meaning it could also be e fficient against these pathogens at higher concentrations. In another study, the nutmeg essential oil with similar quantity of volatile compounds had a significant e ffect on the inhibition of the growth of *E. coli* and *S. aureus* [34]. In this study, we found that essential oil EA2 (0.5%) inhibited *E. faecalis*. This bacteria resides in infected canals of teeth and is often found in the oral cavity after tooth canal repair [35]. Repeated oral care products with chlorhexidine promotes the development of *E. faecalis* resistance [36]. Since EA2 showed activity against *E. faecalis*, nutmeg essential oil could be recommended as a safe protective component for oral care products in the future.

The investigation of human fibroblast cell culture a ffected by virus mimetic Poly I:C showed that nutmeg essential oils and hydrolats have an anti-inflammatory e ffect protecting cell viability and significantly reducing the release of cytokine IL-6. EO2 had a higher e ffect on preventing Poly I:C-induced necrosis and both EO2 and EOH2 more e fficiently protected against IL-6 release compared to preparations without aluminometasilicate EO1 and EOH1. This is most likely due to the increased amount and content of active substances (sabinene, α-pinene, and limonene) in the preparations that is a result of the use of the excipient. α-Pinene significantly decreases the LPS-induced production of IL-6, TNF-α and nitric oxide in bacterial lipopolysaccharide (LPS)-treated macrophages [37]. Sabinene from *Oenanthe crocata* essential oil significantly inhibits nitric oxide production in LPS and IFNγ-treated macrophages [38]. Limonene has a significantly decreased manifestation of inflammatory signals in rat models of ulcerative colitis via regulation of iNOS, cyclooxygenase-2 (COX-2), PGE2, and ERK [39]. However, there are not many studies about the anti-inflammatory effect of nutmeg essential oil preparations. Zhang et al. have demonstrated the anti-inflammatory activity of nutmeg oil in complete Freund's adjuvant-injected rats [16]. Their study shows that nutmeg oil is effective in inflammatory pain relief via inhibition of the COX-2 pathway and substance P release. Another in vivo study exploring carrageenan-induced paw edema in rats have also confirmed the anti-inflammatory properties of nutmeg oil [24]. To the best of our knowledge, there are no in vitro studies on virus-induced anti-inflammatory activity of nutmeg oil. Dewi et al. have found that *M. fragrans* seed ethanolic extract and pure quercetin extract from *M. fragrans* inhibited NO production and the release of inflammatory cytokines, such as TNF-<sup>α</sup>, IL-6, and IL-1β from bacterial LPS-stimulated murine macrophages (RAW 264.7) in a dose-dependent manner. The essential oil of *Monodora myristica* was found to inhibit inflammation-related lipoxygenase [40]. Because of the high content of bioactive volatile compounds, which have been widely studied and characterized by gas chromatographic techniques [41] the essential oils might be good candidates for inhalation treatment of respiratory tract infections. Most common respiratory infections are induced by respiratory viruses, such as influenza or respiratory syncytial virus [42]. As a result, we examined the anti-inflammatory efficiency of nutmeg essential oil preparations in a virus mimetic Poly I:C mediated inflammation. Fibroblasts are multifunctional cells that are responsible for support of other, more tissue-specific cell types, regeneration, wound healing, extracellular matrix production, and inflammatory response [43]. They significantly contribute to the response to infection by secreting cytokines for monocyte/macrophage attraction and their conversion to inflammatory phenotype [44]. The release of IL-6 is one of the key inflammatory signals causing activation of matrix metalloproteinases, macrophages, neutrophil production, and is also involved in autoimmune responses in the condition such as chronic arthritis, osteoporosis, and psoriasis [41,45–48].

The results of our study indicate that nutmeg essential oil preparations have anti-inflammatory properties that might be exploited further for treatment or prevention of viral inflammation-related pathologies, taking the recent emerging nanotechnological and nutraceutical approaches in the field into account [49–51]. However, to increase the applicability of these substances, more studies have to be performed analyzing the mechanism of action of the essential compounds contained in the preparations.
