**1. Introduction**

Since ancient times, *Myristica fragrans* (nutmeg) seeds have been used as a food spice, flavoring agent, a natural remedy for headaches and fever [1]. Nutmeg seeds have essential and fatty oils, resins, wax, and other components [2]. Nutmeg's essential oil has antimicrobial, antiseptic, antiparasitic, anti-inflammatory, and antioxidant properties [1,3]. The concentration of essential oil in nutmeg seeds is about 5–15% [4], and its major components are terpene hydrocarbons (sabinene, pinene, camphene, *p*-cymene, phellandrene, terpinene, limonene, and myrcene altogether make up 60% to 80% of the oil), oxygenated terpenes (linalool, geraniol, and terpineol, which make up approximately 5% to 15%) and aromatic ethers (myristicin, elemicin, safrole, eugenol, and eugenol derivatives, together constituting 15 to 20%) [5–8]. The toxicity of nutmeg seeds at high doses has been reported, mainly due to myristicin oil and elemicin, causing tachycardia, nausea, vomiting, agitation, and hallucinations. However, these effects are related to the abuse of the spice and are not observed at usual low concentrations [9]. There are many studies on the beneficial effects of nutmeg seed and various nutmeg seed extracts. One of the most prominent biological activities of the nutmeg preparations is antibacterial. Nutmeg seed lignans exert antimicrobial activity on *Bacillus subtilis*, *Staphylococcus aureus*, and *Shigella dysenteriae*[10]. Ethanol and acetone extracts of nutmeg crust have strong antibacterial activity against gram-positive bacteria *Staphylococcus aureus* [5]. Ethyl acetate extracts of flesh of the nutmeg fruit have inhibitory potential against both gram-positive and gram-negative bacteria with a minimum inhibitory concentration (MIC) ranging from 0.625 to 1.25 mg/mL [11]. Used for the preservation of sweets, nutmeg methanol extracts inhibit growth of *Staphylococcus aureus*, *Aspergillus niger*, *Saccharomyces cerevisiae*, and *Escherichia coli* at MIC between 250 and 300 mg/mL [12]. However, there are only a few studies on the biological activity of nutmeg essential oil. Takikawa et al. showed a higher antibacterial effect of essential nutmeg oil on pathogenic compared to non-pathogenic strains of *Escherichia coli* [13]. Furthermore, nutmeg essential oil decreased the growth and survival of *Yersinia enterocolitica* and *Listeria monocytogenes* in broth culture [14].

Nutmeg oil preparations are also known for their antioxidant capacity. Using the 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging assay, Piaru et al. reported a significant antioxidant activity of nutmeg oil [15]. The antioxidant properties are often related to the alleviation of inflammation. Nutmeg oil diminished chronic inflammation and pain through the inhibition of COX-2 expression and substance P release in vivo [16]. In another study, nutmeg oil suppressed reactive oxygen species (ROS) production in human neutrophils stimulated by PMA (phorbol 12-myristate 13-acetate) [17] and mildly inhibited phagocytosis in human neutrophils [18]. However, there is no published research on the effect of nutmeg seed essential oil on virus-triggered inflammatory response.

Hydrodistillation is a popular method used for the preparation of essential oils. However, hydrodistillation with excipients is not widely used—we have found just three studies applying this method so far [19–21]. Therefore, we have applied magnesium aluminometasilicate in hydrodistillation as the new excipient and have tested its effects on the nutmeg essential oil yield and its composition [22]. Aluminometasilicate is widely used as a disintegrator in the manufacturing of tablets. Furthermore, this compound is non-toxic and inexpensive, as the price is ~300 eur for 25 kg. Magnesium aluminometasilicate has significantly increased both the yield and composition of some chemical compounds (sabinene, α-pinene, and limonene). The use of the excipient also increased the essential oil yield by about 61% (hydrodistillation with water—the yield is 0.79 ± 0.04 g, using 1% excipient—1.29 ± 0.05 g; the nutmeg quantity was 15 g, the water content was 300 mL) [22].

The increased amount of active substances suggests that oil preparations with aluminometasilicate might have stronger biological activities. Therefore, in this study we compared the antioxidant, antimicrobial, and anti-inflammatory properties of *Myristica fragrans* seed essential oil preparations with and without aluminometasilicate.

#### **2. Materials and Methods**

## *2.1. Plant Material*

The dried seeds of nutmeg (*Myristica fragrans*) were from Grenada. Seeds were identified by Jurga Bernatoniene, Medical Academy, Lithuania University of Health Sciences, Kaunas, Lithuania. A voucher specimen (I 18922) was placed for storage at the Herbarium of the Department of Drug Technology and Social Pharmacy. The seeds had a characteristic odor, a strong, bitter, and spicy flavour, and they were a brown-beige color. The seeds were ground into a powder (using laboratory mill), with particles smaller than 0.5 mm. All powder samples were kept in a dark and airtight container at 20 ± 2 ◦C.

#### *2.2. Essential Oil and Hydrolat*

The essential oil from nutmeg seeds was prepared by using hydrodistillation. The modified Clevenger type apparatus was used. Two samples of essential oil were prepared: one without excipient and the other with 1% of magnesium aluminometasilicate. Each sample was prepared with 15 g of nutmeg powder and 300 mL distilled water, and 1% magnesium aluminometasilicate was used as an excipient in one of the samples. Also, hydrolat of these two essential oils was used. It was collected from Clevenger apparatus. This material was collected first, followed by the essential oil. All samples were obtained and stored in airtight bottles in the refrigerator. The hydrodistillation took 4 h.

#### *2.3. Antioxidant Activity by DPPH Radical Scavenging Assay*

Antioxidant activity of nutmeg essential oil and hydrolat were evaluated using DPPH (Sigma Aldrich, St. Louis, MO, USA) [15]. First of all, 0.1 mM 96% DPPH solution in 96% ethanol was prepared. A total of 1 mL of DPPH solution was placed in a spectrophotometer cuvette and 100 μL of ethanolic essential oil solution at concentrations ranging from 0.2% to 20% was added. For an antioxidant activity evaluation of hydrolat, the absolute hydrolat was used. 1 mL DPPH solution and nutmeg hydrolat from 0.1 mL to 1 mL were mixed in a cuvette. All samples were incubated in the dark for 20 min and absorbance was taken at 515 nm. The antioxidant activity was performed on a UV Spectrophotometer UV-1800 (Shimadzu, Kyoto, Japan). The quantity of DPPH radical scavenging activity was calculated by using this formula:

$$\text{DPPH saving} \cdot \text{efficiency} \cdot \text{effect } \%= \frac{A\_{\text{control}} - A\_{\text{sample}}}{A\_{\text{control}}} \times 100,\tag{1}$$

where *Acontrol* and *Asample* are the absorbance of the control sample (0.1 mM DPPH solution, solvent is 96% ethanol) and the experiment sample.

## *2.4. Antimicrobial Activity*

The method used for antimicrobial activity was serial dilutions in liquid medium [23]. The broth liquid medium was dispensed into test tubes to give a final volume of 10 mL (with a sample of essential oil). The medium was sterilized. The physiological solution was dispensed into 5 mL individual tubes and used for preparation of suspension of the following bacteria: *Klebsiella pneumoniae*; *Salmonella enterica* 24 SPn06; *Pseudomonas aeruginosa* 17–331; *Acinetobacter baumanni* 17–380, *Proteus mirabilis*; 6MRSA M87fox; *Enterococcus faecalis* 86; *Enterococcus faecium* 103; *Bacillus cereus* 18 01; *Streptococcus mutans* (referent); *Enterobacter cloacae*; *Citrobacter freundii*; *Staphylococcus epidermidis*; *Staphylococcus haemolyticus*; *Pasteurella multocida strains.* All bacteria were isolated from clinical material. For each bacterial culture, three tubes of Mueller Hinton broth were used (9.94 mL, 9.97 mL, and 9.98 mL each). The tubes were inoculated with 10 μL of bacterial suspension with the essential oil at concentration 0.1%, 0.2%, and 0.5%. After 48 h of incubation, each tube was inoculated with 10 μL of suspension on soy-tryptone agar (Thermo Fisher, Hampshire, UK). MIC of essential oil was evaluated based on the presence of bacterial growth (bacterial colonies growing (+)/non growing (−).

#### *2.5. Cell Culture and Treatments*

Human fibroblasts (BJ-5ta, hTERT, LGC Standards Ltd. Middlesex, UK) were grown in 75 cm<sup>2</sup> flasks in Dulbecco's Modified Eagle Medium (DMEM) with Glutamax (Thermo Fisher Scientific, Waltham, MA, USA), 10% fetal bovine serum and 100 IU/mL Penicilin/Streptomycin according to standard supplier protocol. At 70–90% confluence, the cells were detached by 0.025% Trypsin/EDTA and plated in 96 well plates at a density of 2 × 10<sup>5</sup> cells/well. A total of 24 h after plating, the cells were treated with 1 μg/mL Poly I:C to simulate viral dsRNR-induced inflammatory response. For cell culture treatments, the essential oils were dissolved in 96% ethanol at a concentration of 5% (*v*/*v*). For determination of cell viability and LD50, the solutions of essential oils and absolute hydrolats were used as a range of dilutions in cell culture medium starting from essential oil preparation to a medium *v*/*v* ratio of 1:1000 and finishing with 1:5. For the control, the same dilutions with solvent (ethanol) were performed. For anti-inflammatory activity evaluation, the solutions of essential oils at *v*/*v* dilutions of 1:100 or 1:200, or absolute hydrolats at *v*/*v* dilutions of 1:40, 1:100, and 1:200 were applied simultaneously with Poly I:C treatment.

#### *2.6. Determination of Cell Viability and Determination on LD50*

Cell viability was assessed by using double nuclear fluorescent staining with Hoechst 33342 (10 μg/mL) and propidium iodide (PI, 5 μg/mL) according to standard supplier protocol for 5 min at 37 ◦C. PI-positive nuclei indicating lost nuclear membrane integrity were considered to be necrotic. Cells were visualized under fluorescent microscope OLYMPUS IX71S1F-3, counted in fluorescent micrographs and expressed as percentage of total cell number per image. The data is presented as averages ± standard deviation. LD50 was calculated by SigmaPlot v.13 (Systat Software Inc., San Jose, CA, USA) using the equation selected by a dynamic curve fitting tool.

#### *2.7. Assessment of Interleukin-6 Concentration*

Medium collected after cell culture treatments was used to measure the concentration of pro-inflammatory cytokine interleukin-6 (IL-6) by ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA) following the standard supplier protocol. The spectrophotometric readings were performed in a plate reader Infinite 200 Pro M Nano Plex (Tecan, Mannedorf, Svizzera).

## *2.8. Statistical Analysis*

The results are presented as means of 3–7 replicates ± standard deviation. The statistical data analysis was performed by applying ANOVA with Tukey HSD post hoc test. Differences were considered statistically significant when *p* < 0.05. The data were processed using Microsoft Office Excel 2010 (Microsoft, Redmond, WA, USA) software.
