Application of Antiviral, Antioxidant and Antibacterial Glycyrrhiza glabra L., Trifolium pratense L. Extracts and Myristica fragrans Houtt. Essential Oil in Microcapsules

Viruses and bacteria can disrupt normal human functions; therefore, ways to use the beneficial properties of plants to promote health are constantly being researched. Plant materials that accumulate biologically active compounds can be used to create a new pharmaceutical form. This study aimed to investigate the biological activity of selected plant extracts and essential oil and to produce microcapsules. The main compounds in extracts and essential oil were determined using chromatographic methods, antioxidant activity was evaluated spectrophotometrically, antimicrobial activity was assessed by monitoring the growth of nine pathogens, and the antiviral effect on infected bird cells with coronavirus was evaluated. Trifolium pratense L. extract had the highest antioxidant (26.27 ± 0.31 and 638.55 ± 9.14 µg TE/g dw by the DPPH and ABTS methods, respectively) and antiviral activity (56 times decreased titre of virus). Liquorice extract expressed antibacterial activity against Gram-positive pathogens and the highest antioxidant activity using the FRAP method (675.71 ± 4.61 mg FS/g dw). Emulsion stability depended on excipients and their amount. Microcapsules with extracts and essential oil were 1.87 mm in diameter, and their diameter after swelling was increased more than two times in intestinal media, while less than 0.5 times in gastric media.


Introduction
Plant extracts and essential oils are used in many domains, including medicine, nutrition, flavouring, beverages, dyeing, repellents, fragrances, pharmaceutics, and cosmetics. Many plants have been recognized to have medicinal properties and beneficial impacts on health, e.g., antioxidant activity, digestive stimulation action, anti-inflammatory, antimicrobial, hypolipidemic, antimutagenic effects, and anticarcinogenic potential [1].
Phenolic compounds are one of the plants' most evident secondary metabolites, and their distribution is demonstrated throughout the entire metabolic process. The biological activity of plants is primarily attributed to these compounds. Polyphenols contain numerous varieties of compounds: simple flavonoids, phenolic acids, complex flavonoids, and coloured anthocyanins [2][3][4].
Botanical substances obtained from plants, such as Trifolium pratense L. flowers, Glycyrrhiza glabra L. roots, and Myristica fragrans Houtt. essential oil, which are generally

Preparation of Glycyrrhiza glabra L. Extract
Glycyrrhiza glabra L. extract was prepared by using dried and milled plant root powder. The extraction was done using 1 g ± 0.001 g of powder in 10 mL of purified water. First, the plant material was macerated in water for 4 h. After that, ultrasound-assisted extraction was performed using an ultrasound bath (frequency 38 kHz) (Grant Instruments™, XUB12 Digital, Cambridge, England). The extraction time was 30 min; the processing temperature was 40 ± 2 • C. After extraction, samples were centrifuged for 10 min at 3382 g (5500 rpm) Pharmaceutics 2023, 15, 464 4 of 20 followed by the decantation of the supernatant. The extract was filtered through the paper filter and used in the following research.

Preparation of Trifolium pratense L. Extract
Before use, clover flowers were ground to a fine powder using an Ultra Centrifugal Mill ZM 200 (Retsch, Haan, Germany). Grinding was performed at 4025 g (6000 rpm) using a 0.5 mm trapezoid hole sieve. The extraction was performed as in the previous study using excipient β-cyclodextrin (β-CD) [36].
Ultrasound-assisted extraction was performed using an ultrasound bath (38 kHz) (Grant Instruments™ XUB12 Digital, Cambridge, England). A sample of 0.3 ± 0.001 g of dried and milled flower heads was macerated in 10 mL of 50% ethanol. Additionally, 0.1 ± 0.001 g of β-CDs was added to the extraction mixture samples (10 mL) to prepare samples with CD concentrations of 1% (w/v). Ultrasound extraction time was 10 min, and the processing temperature was 40 ± 2 • C. After ultrasound processing, the samples were put in a round bottom flask and refluxed in a sand bath at 100 • C for 1 h. After that, the mixture was left to cool at a temperature of 25 ± 2 • C. The samples were centrifuged for 10 min at 3382 g (5500 rpm), followed by the decantation of the supernatant. The extract was filtered through the paper filter and used in the following research.

Essential Oil Preparation
Myristica fragrans Houtt. essential oil was prepared by modified hydrodistillation. Myristica fragrans Houtt. seed powder was mixed with magnesium aluminometasilicate, and then distilled water was added to the mixture (5:1:300). Hydrodistillation with Clevenger-type apparatus (manufacturer of apparatus Winzer Corporation, bought from Carl Roth, Drogen, Germany) and heating mantle (Pilz ® , Winkler, Germany) was carried out for 2 h. A colourless essential oil was obtained and collected in an airtight bottle. The essential oil was stored in a refrigerator at 4 • C until needed.

HPLC-PDA Conditions
High-performance liquid chromatography with diode array detectors (HPLC-PDA) was performed as described in previous work by Kazlauskaite et al. [36]. It was carried out using the Shimadzu Nexera X2 LC-30AD HPLC system (Shimadzu, Tokyo, Japan), equipped with an SPD-M20A diode array detector (DAD).
For the determination of polyphenols in plant extracts, an ACE 5 C18 250 × 4.6 mm column (Advanced Chromatography Technologies, Aberdeen, Scotland) was used. The mobile phase consisted of solvent A (acetic acid/methanol/deionised water) (1:10:89 v/v/v) and solvent B (acetic acid/methanol) (1:99 v/v). The linear gradient elution profile was as follows: 80% A/20% B at 0 min, 30% A/70% B at 30 min, and 90% A/10% B at 39 to 40 min. The flow rate was 1 mL/min, and the injection volume was 10 µL. Absorption was measured at 260 nm. Quantification of compounds was performed using reference standards.

GC-MS Conditions
Gas chromatography-mass spectrometry (GC-MS) analysis was performed as described in Matulyte et al. [37]. GC-MS was carried out using the GCMS-QP2010 system (Shimadzu, Tokyo, Japan). The column used in the process was RTX-5MS, 30 m × 0.25 mm i.d. ×0.25 µL film thickness. The flow rate of helium (99.999%, AGA Lithuania) carrier gas was set at 1.23 mL/min.
A total of 20 µL of the sample (extract or essential oil) was diluted to 1 mL with hexane (≥99%, Sigma-Aldrich, Germany). The oven temperature was maintained at 40 • C for 2 min after injection and then programmed at 3 • C/min to 210 • C, at which the column was maintained for 10 min. The split ratio was 1:10. The mass detector electron ionisation was 70 eV. Identification of volatile compounds was carried out using mass spectra library search (NIST 14).

Total Phenolic and Flavonoid Content and Antioxidant Activity
Total phenolic and flavonoid content and antioxidant activity methods ABTS, DPPH, and FRAP were carried out as described in Kazlauskaite et al. [36].

Determination of Total Phenolic Content
A total of 0.5 mL of extract was mixed with 2.5 mL of 1:9 diluted Folin-Ciocalteu's phenol reagent and 2.0 mL of 7% (w/v) sodium carbonate. Absorbance was measured at 765 nm after 1 h using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). The calibration curve was prepared using gallic acid (0-0.1 mg/g; y = 11.108; R 2 = 0.9981). The results were expressed as gallic acid equivalent per gram dry weight (mg GA/g dw).

Determination of Total Flavonoid Content
A total of 0.1 mL of extract was added to 1.0 mL 96% (v/v) of ethanol, 0.05 mL of 33% acetic acid, 0.15 mL 10% aluminium chloride, and 2.0 mL 5% hexamethylenetetramine solutions. Spectrophotometric analysis was performed after 30 min at 475 nm wavelength using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). The results were expressed as rutin equivalent per gram dry weight (RE/g dw) and calculated by the formula TFC = C · V e · F/M, where TFC is the total flavonoid content (mg RE/g dw); C is the concentration of the used standard (mg/L); V e is the volume of the used solvent (L); F is the dilution coefficient of the sample; and M is the mass of the sample (g). The calibration curve was obtained with a rutin (0-0.5 mg/g; y = 5.0867; R 2 = 0.9985). The results were expressed as rutin equivalent per gram dry weight (RU/g dw).

ABTS Radical Scavenging Activity Assay
Aqueous ABTS solution (7 mM) was mixed with potassium persulfate (2.45 mM) solution and stored in the dark for 12-16 h to produce a dark-coloured solution containing ABTS radical cation. ABTS working solution was prepared using ABTS radical solution and diluting it with water. Working solution absorbance should be regulated to 0.90 (±0.02) at 734 nm using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan).
Free radical scavenging activity was evaluated by mixing 2.0 mL of ABTS working standard with 200 µL of the test sample in the cuvette. The samples were incubated in the dark at room temperature for 30 min. The calibration curve was obtained with a Trolox (0-0.5 mg/g; y = 0.0001728x; R 2 = 0.9832). The results were expressed as Trolox equivalent per gram dry weight (TE/g dw).

DPPH Radical Scavenging Activity Assay
A total of 2.0 mL of DPPH solution (0.1 mM in ethanol) was mixed with 2.0 mL of the samples. The reaction mixture was shaken and incubated in the dark at room temperature for 30 min, and the absorbance was read at 517 nm against the blank using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). The calibration curve was obtained with a Trolox (0-0.016 mg/g; y = 0.00623x; R 2 = 0.9923). The results were expressed as Trolox equivalent per gram dry weight (TE/g dw).

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay was prepared by mixing 0.3 M acetate buffer, 10 mM TPTZ solution with 40 mM HCl, and 20 mM ferric chloride solution. A total of 10 µL of the sample was combined with 200 µL of FRAP reagent; the contents were mixed vigorously. The absorption was measured at 593 nm using a spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). The calibration curve was obtained with ferrous sulfate (0-1 mg/g; y = 2.6272; R 2 = 0.9985). The results were expressed as ferrous sulfate equivalent per gram dry weight (FS/g dw).

Antimicrobial Activity
Antimicrobial activity was determined with the diffusion method in a solid nutrient media of agar. Mueller-Hinton Agar (Mueller-Hinton II Agar, BBL, Cockeysville, MD, USA) was used.
Standard spore bacteria cultures of Bacillus cereus (ATCC 6633; soil microbiota) were grown for 7 days at 35-37 • C on Mueller-Hinton Agar. After growing the culture of spore bacteria, it was washed off the surface of the medium with a sterile physiologic solution. The prepared suspension was heated for 30 min at 70 • C and diluted with physiological saline until the spore concentration in 1 mL was between 10 × 10 6 and 100 × 10 6 .
The standard culture of the fungus Candida albicans (ATCC 10231; human microbiota) was grown for 20 to 24 h at 30 • C for 72 h on Sabouraud agar. The fungal suspension was prepared from cultivated fungal cultures in physiological saline and standardised with a McFarland standard indicator.
A 0.5 McFarland turbidity suspension of the standard bacteria was prepared. The bottom of the Petri dishes was divided into 9 segments. The technology of reference microorganisms to Mueller-Hinton agar was used to determine the antimicrobial activity of Glycyrrhiza glabra L. and Trifolium pratense L. extracts. The disk method was used to determine the antimicrobial activity of Myristica fragrans Houtt. essential oil.

Antibacterial Activity of Extracts
The chosen amount of extract was poured into a sterile Petri dish (Glycyrrhiza glabra L. extract from 1 mL to 0.0075 mL; clover from 1 mL to 0.1 mL). Then, 5 mL of Mueller-Hinton agar was added. After the solidification of the agar, suspensions of reference microorganisms were inoculated. Samples were kept in a thermostat for 20-24 h at 35 • C, then stored for 24 h at room temperature. Antimicrobial activity was evaluated. If the cultures had grown-the sample did not inhibit the growth of bacteria. If the reference culture did not grow, the sample has an antimicrobial effect against the microorganism.

Antibacterial Activity of Essential Oil
A total of 0.5 mL of bacterial suspensions were separately poured into sterile Petri dishes. After that, 5 mL of Mueller-Hinton agar was poured into the plates. After the agar solidified, an 8 mm disk was placed on top, and 30 µm of Myristica fragrans Houtt. essential oil was added. Samples were kept in a thermostat for 20-24 h at 35 • C, then stored for 24 h at room temperature. Antimicrobial activity was then evaluated: if the cultures grew around and under the disk-the sample did not inhibit the growth of bacteria. If the reference culture did not grow, the sample has an antimicrobial effect against the microorganism. incubator. Gentamycin (50 µg/mL, Gibco, TX, USA) and nystatin (100 units/mL, Gibco, TX, USA) were used to prevent microbial contamination.
The Vero-adapted Beaudette infectious bronchitis virus (IBV) strain was used. Dr. M.H. Verheije of Utrecht University in The Netherlands provided the virus. The virus stocks were prepared and stored at −80 • C in aliquots.
Determination of TCID50 of the control and the treated IBV were performed in 96-well plates (TPP, Switzerland) with Vero cells. Serial dilutions of IBV in tenfold steps were prepared. Each sample was tested in octuplicates, and the experiments were repeated twice. CPE was evaluated after 72 h. Virus titres and standard deviations were calculated using the Kärber method (Kärber 1931), and the virus reduction capacity was assessed [38].
The cytotoxicity assay was performed to determine the 50% cytotoxic concentration (CC 50 ). CC 50 was determined for each extract on Vero cells using an MTT assay [39]. First, cells were seeded at a concentration of 1 × 10 4 cells/well in a 96-well plate and grown at 37 • C for 1 day. The assay was performed in octuplicate for each extract. After 72 h, the MTT reagent (10 µL, 5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added and incubated for 4 h at 37 • C. A total of 100 µL dimethyl sulfoxide was added to each well, and the plates were placed on the shaker for 5 min. The absorbance of each well was measured at 620 nm in a microplate reader (Thermo Scientific™, Multiskan™ FC Microplate Photometer, Tokyo, Japan), and the percentage of cell survival was calculated. The relative viability in untreated cells was set as 100%. Finally, dose-response curves were plotted to calculate CC 50 that caused lysis and death of 50% of cells (Chang, 2016). The dilutions of plant extracts were prepared for screening and determination of antiviral and virucidal activity.
Testing solutions were prepared by mixing IBV (10 5.25 TCID 50/mL ) suspensions in DMEM with previously established noncytotoxic extract volumes (dilutions 1:15 and 1:30). The controls of diluents of plant preparations were used. The virus and extract mixtures were incubated at 20 • C for 1 h and then titrated as described above. Controls of cells, the virus, and extracts were included. After 72 h of incubation, the plates were examined using an inverted microscope (Leica, Germany) to detect CPE. The reduction factor was used to describe plant preparations' viral reduction potential [38]. The reduction factor ≤ 1 log 10regarded as insignificant; 1-2 log 10 -indicative/contributable; 2-4 log 10 -moderate; and >4 log 10 -high. Antiviral activity was evaluated by infection of cells after virus treatment, during virus treatment, and the treatment of cells before infection and after infection. A total of 0.05 IBV particles per cell multiplicity of infection (MOI) was used. Prepared extracts were serially diluted twofold per seven wells in DMEM and assessed for their ability to inhibit IBV replication using four mechanisms.
In the first method, IBV was treated with the diluted extract for 1 h in a separate 96-well plate and then poured onto the cells. After incubating for 1 h at 37 • C in 5% CO 2 , the mixtures were discarded, and then the cells were washed twice with PBS. After washing, 200 µL of DMEM containing 2% FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 • C in 5% CO 2 .
In the second method, the diluted extract and IBV mixtures were poured onto the cells immediately. After incubating for 1 h at 37 • C in 5% CO 2 , the mixtures were discarded, and then the cells were washed twice with PBS. After washing, 200 µL of DMEM containing 2% FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 • C in 5% CO 2 .
In the third method, the cells were inoculated with the IBV and then treated with the extract. First, the cells were inoculated with the virus and incubated for 1 h at 37 • C in 5% CO 2 . Then the unadsorbed IBV was discarded, and the cells were washed twice with PBS. After washing, the cells were treated with the diluted extracts for 1 h at 37 • C in 5% CO 2 . After washing the cells twice with PBS, 200 µL of DMEM containing 2% FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 • C in 5% CO 2 . In the fourth method, the cells were treated with extract before inoculation. First, the cells were treated with the diluted extracts for 1 h at 37 • C in 5% CO 2 . Then the cells were washed twice with PBS and inoculated with IBV. After incubation for 1 h at 37 • C in 5% CO 2 , the cells were washed twice with PBS, and 200 µL of DMEM containing 2% FBS was added. Observation by microscopy for inhibition of CPE was performed after incubation for 72 h at 37 • C in 5% CO 2 .
Every sample of the extract was tested twice in quadruplicate. The cytopathic effect (CPE) of IBV was evaluated by optical microscopy. The endpoint was the extract dilution that inhibited 100% of the CPE at two noncytotoxic concentrations.

Emulsion Preparation
First, a 4% sodium alginate solution was prepared from distilled water and alginic acid sodium salt. It was used throughout the experiment for emulsion preparation as the shell material. Emulsion with Trifolium pratense L. and Glycyrrhiza glabra L. extracts, and Myristica fragrans Houtt. essential oil were prepared as follows: solution with excipients (maltodextrin, inulin, and/ or gum Arabic) was mixed with sodium alginate solution (stirred for 15 min with a magnetic stirrer MSH-20A (Witeg, Wertheim, Germany)) and then extracts with essential oil were added. The solution was homogenised for 15 min at 5000 rpm using an IKA T18 homogeniser (IKA-Werke GmbH & Co., KG, Staufen, Germany).
The emulsion's stability was tested using a centrifuge Sigma 3-18KS (Sigma Laborzentrifugen GmbH, Osterode am Harz, Germany). The test was repeated three times using 23 • C temperature, 3000 rpm, and the duration was 5 min. The centrifugation index (CI) was calculated to evaluate emulsion stability.
where V e is the volume of the remaining emulsion after centrifugation and V i is the volume of the initial emulsion.

Particle Size and Distribution Measurements
Myristica fragrans Houtt. essential oil particles' size and distribution were assessed using Mastersizer 3000 with a Hydro EV unit (Malvern analytical Ltd., Malvern, UK). The emulsion was added dropwise in the dispersant (water) to obtain laser obscuration between 9.5% and 10.5%. The pump speed was kept constant at 2400 rpm. The refractive index used for dispersant and dispersing material was 1.330 and 1.478, respectively. The average was calculated for particle size distribution in five runs. The percentile (D10, D50, and D90) values described the formulations.

Microcapsule Preparation Using the Extrusion Method
Microcapsules were prepared by extrusion method. The medical syringe (Jiangsu Zhengkang Medical Apparatus, Yangzhou, China) and NE-1000 Programmable Single Syringe Pump (KF T technology SRL, Rozzano, Italy) were used to prepare droplets for microcapsules. The drops of emulsion were ejected from the needle into the crosslinker solution. The height from the needle to the solution surface was 10-15 cm, and the pumping speed was 3 mL/min. A solution of 5% calcium chloride was used as a crosslinker. Microcapsules were prepared by using a magnetic stirrer. Particles in the crosslinker solution were stirred for 15 min, and then the microcapsules were filtered using filter paper and washed with distilled water. Manufactured capsules were left to dry at room temperature (20 ± 2 • C) for 24 h. Dried and wet microcapsules were stored in sealed tubes until further tests.

Physical Parameters of Microcapsules 2.10.1. Size of Dry and Wet Microcapsules
The microcapsules' size was measured using a Digital Calipiper micrometre (BGS technic, Wermelskirchen, Germany). The diameter of 30 units of dried and freshly made capsules was measured, and the average was calculated.

Firmness of Microcapsules
The force of firmness was measured on freshly made microcapsules by texture analyser TA.TX.plus (Texture Technologies, Hamilton, MA, USA). For one sample, 5 units of microcapsules were taken, and the force which was required to compress 2 mm was measured using a P/100 probe.

Swelling Characteristic of Microcapsules
Dried microcapsules were swelled in gastric and intestinal media [33]. The microcapsules were weighed at successive time intervals of 0, 0.5, 1, 2, 4, and 24 h. The swollen microcapsules were removed and filtered using metal mesh and dried with a paper towel to eliminate the excess fluid. To determine the swelling index (SI), the following formula for calculation was used: where W s is the weight of swollen microcapsules at the time and W i is the dried microcapsules' weight.

Statistical Data Analysis
Data were analysed using SSPS version 20.0 (IBM Corporation, Armonk, NY, USA). All experiments were performed in triplicate. Data are expressed as mean ± standard deviation (SD). The comparisons between three different measurements were made using Friedman and Wilcoxon tests. The results were considered statistically significant at p < 0.05. For antiviral experiments, the differences between the methods and extracts were evaluated by Fisher's criteria and the Student's t-test. The data were regarded as significant when p < 0.05.

Quantification of Main Compounds in Plant Extracts and Essential Oil
The main compound with higher biological activity in Glycyrrhiza glabra L. extract is glycyrrhizic acid [40]. Glycyrrhiza glabra L. extract was made by maceration combined with ultrasound processing. The extracts yielded 349.29 ± 14.55 µg/g glycyrrhizic acid. The extract was concentrated by evaporation of water (half the weight of the extract), and the glycyrrhizin content determined after concentration was 2.07-fold higher. In the literature, there are other extraction methods to obtain glycyrrhizin. Using sonification and pressurised hot water extraction, the amount of glycyrrhizin acid was 17.21 ± 1.27 mg/g and 13.20 ± 2.37 mg/g, respectively [41]. A total of 36.4 µg/g of glycyrrhizic acid was obtained by ultrasound-assisted extraction (30:1 solvent to solute ratio, 40 • C temperature) [42]. Ultrasound alone yields lower concentrations of glycyrrhizic acid; therefore, combining it with maceration gives better results.
Trifolium pratense L. flower extract (extraction with excipient β-CD) contained 171.57 ± 12.36 µg/g of genistein and 393.23 ± 23.71 µg/g of daidzein. This amount of isoflavones is higher than the average reported in the literature. It was reported that petioles contained 0.028 mg/g of daidzein and 0.054 mg/g of genistein, and flowers contained 0.44 µg/g of genistein and daidzein, except for minimal traces were not detected [43]. Using reflux (85 • C) extraction, 0.11 ± 00.28 mg/g of daidzein and 0.10 ± 0.0053 mg/g of genistein were obtained (extrahent was methanol). These concentrations were a lot lower than those obtained in this study. Adding excipient β-CD to extraction media helps extract target compounds. β-CDs form host-guest inclusion complexes with various drugs in a solution or a solid state. The existence of a β-CD hydrophobic cavity boosts the extraction of phenolic compounds, including isoflavones [44]. Clover extract was also concentrated by evaporating water (50%), and the isoflavones concentration found in the sample were genistein 265.46 µg/g and daidzein 598.16 µg/g.

Phenols and Antioxidant Activity of Extracts and Essential Oil
Many components in extracts and essential oil, i.e., carotenoids, vitamin C, vitamin E, and phenolic compounds, contribute to the overall antioxidant activity; it is difficult to measure the total antioxidant activity based on individual active components. This is because compounds can interact with each other to produce antagonistic or synergistic effects [47]. As a result, the extracts were tested for total phenolic compounds, flavonoid content, and antioxidant activity using three different methods-ABTS, DPPH, and FRAP. The research was done using concentrated Glycyrrhiza glabra L. and Trifolium pratense L. extracts.

Total Phenolic and Flavonoid Content
The total phenolic content found in Glycyrrhiza glabra L. extract was 43.14 ± 0.06 mg GA/g dw. Higher results were obtained from the Trifolium pratense L. flowers extract-74.00 ± 0.15 mg GA/g dw (Table 1). Ethanolic Trifolium pratense L. extract had a significantly higher total phenol content compared to aqueous Glycyrrhiza glabra L. extract (p < 0.05), and the exact correlation was observed comparing total flavonoids. Varying solubility of the phenolic compounds due to different solvents could be explained by the solvent polarity, and ethanol is more efficient in extracting lower molecular weight polyphenols, especially glycosides [48,49]. The essential oil of Myristica fragrans Houtt., because of its preparation method, contained only a small amount of phenols (7.49 ± 0.04 mg GA/g dw). The main compounds found in our sample were monoterpenes (identified as 67.77%). This correlates with the results published by other researchers [10,15]. The total flavonoids determined in the essential oil was 6.84 ± 0.05 mg RU/g dw.

Antioxidant Activity of Extracts and Essential Oil
In the literature, a few antioxidant assays have been developed based on methodological differences to detect antioxidant activity in samples taken from natural sources, including extracts and essential oils. In this study, DPPH, ABTS, and FRAP methods were used to determine the antioxidant potentials of Glycyrrhiza glabra L. and Trifolium pratense L. extract, also Myristica fragrans Houtt. essential oil. Radical scavenging antioxidants are necessary for the antioxidative defence to protect cells from the injurious effects of free radicals [50]. Antioxidants can delay the progress of many chronic diseases as well as lipid peroxidation [51].
The antioxidant activity of Glycyrrhiza glabra L. and Trifolium pratense L., using the DPPH method, was similar ( Table 2). Trifolium pratense L. extract showed better antioxidant activity using the ABTS method (638.55 ± 9.14 µg TE/g dw), but Glycyrrhiza glabra L. extract had higher ferric reducing power (675.71 ± 4.61 mg FS/g dw). Studies in the literature have shown that both Trifolium pratense L. and Glycyrrhiza glabra L. are good antioxidants. Trifolium pratense L. is known to contain isoflavones (daidzein, genistein, formononetin, and biochanin A), which possess estrogenic effects, but antioxidant properties of extract can be related to quercetin, hyperoside, clovamides, and other phenolic compounds [52]. The reported main phenolic compounds in Glycyrrhiza glabra L. extract responsible for its antioxidant activity are also isoflavones, such as glabridin, hispaglabridin A, and 30hydroxy-4-O-methylglabridin [53]. Myristica fragrans Houtt. essential oil reported possessing antioxidant activity as well as Trifolium pratense L. or Glycyrrhiza glabra L. extracts. Nevertheless, the concentration of the essential oil used in this study was 1%. The isomers α-pinene and β-pinene possess antioxidant and antimicrobial activity, but the antimicrobial activity of β-pinene is more robust [17].

Antimicrobial and Antiviral Activity
The antimicrobial activity of Trifolium pratense L. and Glycyrrhiza glabra L. extract was determined using nonconcentrated extracts. Glycyrrhiza glabra L. extract had expressed antimicrobial activity against Gram-positive bacteria: Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Bacillus cereus. Additionally, it suppressed the growth of the fungus Candida albicans.
A concentration of 0.2% extract inhibited these pathogens, whereas Bacillus aureus was inhibited by a 2% Glycyrrhiza glabra L. extract solution. Glycyrrhiza glabra L. extract had no antimicrobial activity against Gram-negative bacteria: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Proteus vulgaris. The results are presented in Table 3.
The antimicrobial activity of Trifolium pratense L. extract was not expressed, but the obtained results showed that this extract inhibits gram-positive bacteria more. 0.75 mL of extract in 5 mL of agar medium (13.04% concentration of extract) inhibited the growth of Staphylococcus epidermidis and inhibited Staphylococcus aureus and Bacillus cereus weakly. The growth of other pathogens was not inhibited (Table 4).
According to the literature, the extraction solvent capability to extract active substances of the active principles has a major importance on the antibacterial and antifungal activity of the Trifolium species. Studies on the antimicrobial properties of Trifolium pratense L. included a comparison of the actions of different extracts solvents, concentration of the extract and drying conditions [19,[54][55][56]. In our study case, the concentration of isoflavones, which are responsible for antibacterial effect in Trifolium pratense L. was too low to have strong antibacterial response.
Essential oils are volatile liquids, so it is difficult to determine their effect due to evaporation. One essential oil concentration was chosen in this study, and antimicrobial activity against 9 pathogens was evaluated. Results (Table 4) showed that Myristica fragrans Houtt. Essential oil suppressed the growth of half of referenced gram-positive and half of referenced gram-negative pathogens (Staphylococcus aureus, Bacillus cereus and Escherichia coli, Klebsiella pneumoniae, respectively), and the other pathogens were not suppressed (two of gram-positive and two of gram-negative).
Observing the results obtained by other scientists and comparing it with this study, it was found that a two times higher amount of nutmeg essential oil suppressed Candida albicans, Staphylococcus aureus, Bacillus cereus, Bacillus luteus, Escherichia coli, and Pseudomonas aeruginosa [17]. In the other study, it was found that Myristica fragrans Houtt. essential oil was more effective in inhibiting the growth of S. aureus than gentamicin (sample and gentamicin (as control) concentrations were the same, and the zone of inhibition of Myristica fragrans Houtt. was 1.22% greater than the antibiotic) [57].
In the literature, it was reported that Trifolium pratense L. extract suppressed Bacillus subtilis growth. The diameter of the nongrowth zone was 20 mm, while the essential oil nongrowth zone was 12 mm. The extract had 40% higher activity against B. subtilis compared to essential oil [58]. The antibacterial activity of the essential oil against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and Bacillus cereus was also tested, and it was found that clover extract did not inhibit the growth of either pathogen [59]. Thus, it can be concluded that to increase the antibacterial activity in products, clover extract should be used instead of essential oil.
In publications about Glycyrrhiza glabra L. roots, it was revealed that an ethanolic extract of Glycyrrhiza glabra L. had a positive effect on suppressing B. cereus, B, subtilis, K. pneumoniae, and S. aureus; however, the ethanolic extract did not suppress E. faecalis growth [60]. In our study it was found that 0.01 mL of the Glycyrrhiza glabra L. extract in a 5 mL agar medium effectively suppressed E. faecalis. Table 3. Antimicrobial effect of Glycyrrhiza glabra L. extract on the growth of reference microorganism cultures.

Reference Cultures of Microorganisms
Amount of Glycyrrhiza glabra L. Extract (mL) S-inhibits the growth of reference culture of microorganisms (the antimicrobial effect was present); N-does not inhibit the growth of reference culture of microorganisms (no antimicrobial effect); ±-very weak growth. S-inhibits the growth of reference culture of microorganisms (the antimicrobial effect was present); N-does not inhibit the growth of reference culture of microorganisms (no antimicrobial effect); ±-very weak growth.

Antiviral Activity of Used Plant Extracts and Essential Oil
It was determined that the CC 50 of Trifolium pratense L. was 4.8 mg/mL and the CC 50 Glycyrrhiza glabra L. was 10.0 mg/mL for Vero cells. The diluent of Glycyrrhiza glabra L. had no virucidal effect upon IBV, whereas the diluent, ethanol, used for Trifolium pratense L. at concentrations of 8.0% ethanol (Trifolium pratense L. with 4.8 mg/mL) and 6.67% ethanol (Trifolium pratense L. with 4.0 mg/mL) had a virucidal effect ( Table 5). The diluent of the almond EO did have a positive impact on virucidal activity (p < 0.05).
Based upon cytotoxicity and control results, dilutions of 1:15 and 1:30 were chosen for comparison of the virucidal activity of extracts.
Antiviral activity was tested for the extracts of Trifolium pratense L. and Glycyrrhiza glabra L. (Table 6). It was shown that the extracts were effective against IBV and could protect the Vero cells prior to or during infection. IBV pretreatment with extracts after infection and cell pretreatment prior to infection could not inhibit 100% of the CPE. Combining these extracts together can result in a promising preparation that will have high antioxidant, antibacterial, and antiviral properties.  Table 6. Antiviral activity of plant extracts.

Virus Pre-Treatment with Extract Cell Pre-Treatment Prior to Infection Prior to Infection During Infection After Infection
Trifolium pratense L. + + --Glycyrrhiza glabra L. + + --+: had an effect; -: no effect.

Emulsion Physical Stability
Six emulsion samples were prepared, and their stability was evaluated (the emulsion's composition is shown in Table 7). Sample E3 became an inhomogeneous texture after all ingredients were mixed, and its centrifugation index was the lowest (Figure 1).
When comparing the stability of the emulsion, it was established that one excipient (maltodextrin/inulin/gum Arabic) was unable to keep the emulsions stable (CI was lower than 50%). Then, two different excipients were added to the composition, and the stability of the emulsion was increased by approximately 46% compared with E1. The highest stability of the emulsion was evaluated, and then three excipients were added. Emulsion E6 was the most stable, and its CI was 100%.
In an emulsion, particle size, distribution, and essential oil drop diameter were determined using the Mastersizer 3000 ( Figure 2). Myristica fragrans Houtt. essential oil drop diameter in the emulsion was 0.571 µm. Evaluated percentiles were measured: D10 was 0.328 ± 0.051 µm, D50 was 0.46 ± 0.03 µm, and D90 was 0.89 ± 0.094 µm. The percentiles D10, D50, and D90, indicate the size for which 10%, 50%, and 90% of the particles are equal to or less, respectively [61]. The emulsion particle size with different essential oils was 16.70-55.56 µm. The base was made from soy protein isolate and gum Arabic [62]. Studies similar to this, with Myristica fragrans Houtt. essential oil, extracts, and gum Arabic, maltodextrin, and inulin, were not found. This stable emulsion (E6) was used to prepare microcapsules by extrusion. When comparing the stability of the emulsion, it was established that one exc (maltodextrin/inulin/gum Arabic) was unable to keep the emulsions stable (CI was than 50%). Then, two different excipients were added to the composition, and the sta of the emulsion was increased by approximately 46% compared with E1. The highe bility of the emulsion was evaluated, and then three excipients were added. Emulsi was the most stable, and its CI was 100%. In an emulsion, particle size, distribution, and essential oil drop diameter we termined using the Mastersizer 3000 ( Figure 2). Myristica fragrans Houtt. essential oil diameter in the emulsion was 0.571 µm. Evaluated percentiles were measured: D1 0.328 ± 0.051 µm, D50 was 0.46 ± 0.03 µm, and D90 was 0.89 ± 0.094 µm. The perce D10, D50, and D90, indicate the size for which 10%, 50%, and 90% of the particles are to or less, respectively [61]. The emulsion particle size with different essential oil 16.70-55.56 µm. The base was made from soy protein isolate and gum Arabic [62]. St similar to this, with Myristica fragrans Houtt. essential oil, extracts, and gum A Figure 1. The emulsion stability (centrifugation index). The code of emulsion is given in Table 7.

Microcapsules' Physical Parameters
In a 5% crosslinker solution, microcapsules were formulated (Figure 3), and all microcapsules were spherical and of light brown colour. The yield of microcapsules was 68.67 ± 3.08%. The freshly prepared microcapsules' diameter was 1.87 ± 0.35 mm and soft (easily crushed between the fingers). The force of crushing (measured with a Texture analyser) was 3758.52-4135.87 g. The dried microcapsules' diameter was 0.76 ± 0.11 mm, and the device did not measure the force because the maximum force value is 6500 g; dried microcapsules were harder than this value.

Microcapsules' Physical Parameters
In a 5% crosslinker solution, microcapsules were formulated (Figure 3), and all microcapsules were spherical and of light brown colour. The yield of microcapsules was 68.67 ± 3.08%. The freshly prepared microcapsules' diameter was 1.87 ± 0.35 mm and soft (easily crushed between the fingers). The force of crushing (measured with a Texture analyser) was 3758.52-4135.87 g. The dried microcapsules' diameter was 0.76 ± 0.11 mm, and the device did not measure the force because the maximum force value is 6500 g; dried microcapsules were harder than this value.

Microcapsules' Physical Parameters
In a 5% crosslinker solution, microcapsules were formulated (Figure 3), and all microcapsules were spherical and of light brown colour. The yield of microcapsules was 68.67 ± 3.08%. The freshly prepared microcapsules' diameter was 1.87 ± 0.35 mm and soft (easily crushed between the fingers). The force of crushing (measured with a Texture analyser) was 3758.52-4135.87 g. The dried microcapsules' diameter was 0.76 ± 0.11 mm, and the device did not measure the force because the maximum force value is 6500 g; dried microcapsules were harder than this value. Extrusion technology to produce microcapsules with essential oils and extracts is rarely used, but similar research results have been found. In a study with Myristica fragrans Houtt. essential oil, when polysorbate 80 is used as an emulsifier, microcapsules of size 2.120-2.280 mm with a strength of 4333.46-5116.70 g were formed in a 2% crosslinker solution [33]. Microcapsules with 1% of rosemary essential oil and without other excipients had a diameter between 0.950 mm and 0.756 mm (non-dry and dry, respectively; only sodium alginate 4% solution was used) [25].
Microcapsules' swelling index in gastric media was from 19.74% to 13.96%, and alginate microcapsules did not swell in gastric fluid. At first, the microcapsules swelled a little, but after 30 min, the swelling index decreased. Conversely, microcapsules swelled from 25.22% to 121.59% in the intestinal medium (Figure 4). After a day, the microcapsules Extrusion technology to produce microcapsules with essential oils and extracts is rarely used, but similar research results have been found. In a study with Myristica fragrans Houtt. essential oil, when polysorbate 80 is used as an emulsifier, microcapsules of size 2.120-2.280 mm with a strength of 4333.46-5116.70 g were formed in a 2% crosslinker solution [33]. Microcapsules with 1% of rosemary essential oil and without other excipients had a diameter between 0.950 mm and 0.756 mm (non-dry and dry, respectively; only sodium alginate 4% solution was used) [25].
Microcapsules' swelling index in gastric media was from 19.74% to 13.96%, and alginate microcapsules did not swell in gastric fluid. At first, the microcapsules swelled a little, but after 30 min, the swelling index decreased. Conversely, microcapsules swelled from 25.22% to 121.59% in the intestinal medium (Figure 4). After a day, the microcapsules had lost their shape and softened. Another study found that alginate microcapsules lost weight in the gastric media (alginate, soy protein microcapsules with thyme essential oil) [26]. The maximum swelling index was reached in 40-60 min, and then the amount of microcapsules in the gastric medium decreased [63]. Another study confirmed that alginate microcapsules swell in the intestinal medium, and the swelling index could be higher than 20 times [33]. In contrast, microcapsules almost did not swell, or their amount decreased in gastric media (pH = 1-2.5) [64]. The results of other studies are in agreement with the results obtained in this study and show that sodium alginate microcapsules swell in the intestinal medium. Sodium alginate is a polymer and forms hydrogels. It is well known that a hydrogel can respond to surrounding conditions such as pH, ionic strength, temperature, and electric current. The pH sensitivity is an important factor for controlled drug release in the gastrointestinal tract, which has a variation of pH from the stomach to the intestine. Many studies support the results that sodium alginate microcapsules or gels do not swell in the intestinal medium. The pH controls the degree of dissociation of the guluronic and mannuronic acid groups (alginate molecule parts); therefore, alginate does not swell in an acidic medium [65,66]. Alginate microcapsules can be used to develop gastric insoluble pharmaceutical formulations.
temperature, and electric current. The pH sensitivity is an important factor for controlled drug release in the gastrointestinal tract, which has a variation of pH from the stomach to the intestine. Many studies support the results that sodium alginate microcapsules or gels do not swell in the intestinal medium. The pH controls the degree of dissociation of the guluronic and mannuronic acid groups (alginate molecule parts); therefore, alginate does not swell in an acidic medium [65,66]. Alginate microcapsules can be used to develop gastric insoluble pharmaceutical formulations.

Conclusions
This study determined that the chosen plant extracts and essential oil had a significant concentration of active substances. The main component in liquorice extract was glycyrrhizin acid (349.29 ± 14.55 µg/g); the red clover extract contained 171.57 ± 12.36 µg/g of genistein and 393.23 ± 23.71 µg/g of daidzein. Nutmeg essential oil had four main chemical compounds: sabinene, α-pinene, β-terpinene, and β-myrcene.
All the plant extracts possess antioxidant effects. The highest effect was determined in the red clover extract, which also was the most effective against viruses. The highest antibacterial effect was found in the liquorice extract, which was most effective against Gram-positive pathogens. Therefore, plant-derived products can be considered promising when being used as antimicrobial agents. Extruded microcapsules with essential oil and extracts could be an additional form to incorporate in other pharmaceutical forms for therapeutic benefits. Considering the obtained research results, it is planned to evaluate the release of active compounds from microcapsules in the future.

Conclusions
This study determined that the chosen plant extracts and essential oil had a significant concentration of active substances. The main component in liquorice extract was glycyrrhizin acid (349.29 ± 14.55 µg/g); the red clover extract contained 171.57 ± 12.36 µg/g of genistein and 393.23 ± 23.71 µg/g of daidzein. Nutmeg essential oil had four main chemical compounds: sabinene, α-pinene, β-terpinene, and β-myrcene.
All the plant extracts possess antioxidant effects. The highest effect was determined in the red clover extract, which also was the most effective against viruses. The highest antibacterial effect was found in the liquorice extract, which was most effective against Gram-positive pathogens. Therefore, plant-derived products can be considered promising when being used as antimicrobial agents. Extruded microcapsules with essential oil and extracts could be an additional form to incorporate in other pharmaceutical forms for therapeutic benefits. Considering the obtained research results, it is planned to evaluate the release of active compounds from microcapsules in the future.