Essential Oils from Apiaceae, Asteraceae, Cupressaceae and Lamiaceae Families Grown in Serbia: Comparative Chemical Profiling with In Vitro Antioxidant Activity

The aim of the present study was to investigate the chemical profile and antioxidant activity of essential oils obtained from the most commonly grown plant species in Serbia. Aromatic and medicinal plants from Lamiaceae (Mentha x Piperita, Ocimum basilicum, Origanum majorana, Origanum vulgare, Salvia officinalis, Satureja hortensis, Satureja montana and Thymus vulgaris), Asteraceae (Ehinacea purpurea and Matricaria chamomilla), Apiaceae (Anethum graveolens, Carum carvi, Foeniculum vulgare, Petroselinum crispum and Pimpinella anisum) and Cupressaceae (Juniperus comunis) were selected as raw material for essential oils (EOs)’ isolation. Hydrodistillation (HD) was used for the isolation of EOs while they were evaluated in terms of yield and terpenoid profiles by GC-MS. In vitro radical scavenging DPPH and ABTS+ radical activities were carried out for all EOs. Finally, a principal component analysis (PCA) was performed with the experimental results of the composition and antioxidant activity of the EOs, which showed a clear distinction between the selected plant species for the aforementioned responses. This work represents a screening tool for the selection of other EO candidates for further processing by emerging extraction techniques and the use of EOs as natural additives for meat products.


Introduction
The Republic of Serbia has rich unexplored herbal biomes with plants that are excellent raw material for the production of essential oils (EOs) and for other valuable biologically active compounds [1]. Serbia has an extreme continental climate with dry air, where plants belonging to the various families (peppermint, sage, thyme, basil, chamomile, fennel, caraway, juniper, etc.) are usually cultivated or collected in the wild.
The EOs, as a source of natural products, represent a promising substitute for synthetic active ingredients in the food, pharmaceutical, cosmetic, alternative medicine and aromatherapy industries [2]. They are commonly associated with various physiological activities, including antibacterial, antiviral, anti-inflammatory, antifungal, antimutagenic, anticarcinogenic and various antioxidant activities [3]. EOs are volatile liquids obtained from different plant parts, including flowers, roots, bark, leaves, seeds, peels, fruits, wood and herbs [3]. Usually, they consist of lipophilic and highly volatile secondary plant compounds. In general, the main constituents of EOs consist of mono-and sesqui-terpenes, which are usually in hydrocarbon or oxygenated form. The uses of EOs are highly diverse and depend on various factors such as source, quality, extraction procedure, etc. Most frequently, they are used in the production of perfumes, cosmetics, soap, shampoos or that should be helpful for industrial production while employing conventional HD. Hence, this research targeted the isolation and profiling of the pure EOs of Apiaceae, Asteraceae, Cupressaceae and Lamiaceae species grown in Serbia in order to report the influences of the different plant families on the total extraction yields, chemical compositions, bioactivity and antioxidant potential of the extracted EOs. A principal component analysis (PCA) was utilized to correlate these variables with EO samples. The aim of this was to provide a platform for the selection of EO candidates which could be applied as natural additives in various food products, particularly meat products, and nutraceuticals.

EO Yield
To ensure the intensification of mass transfer, the plant material should be crushed to an appropriate particle size, which is usually in the range of 100 µm-2 mm [19]. It is known that with a larger contact area, extraction efficiency increases. Moreover, finer particles improve the mass transfer rate from the solid to the liquid phase [20]. It is recommended that the diameter of the plant material is not larger than 2 mm and smaller than 0.5 mm (<10%). The particle size of all samples is shown in Table 1. The plant material was properly prepared, and it contained only 4.35% of particles above the established upper limit (2 mm). The fraction of fine particles was less than 0.5 mm in diameter and accounted for 8.69% of the sample. Finally, the mean particle size of the processed samples was 1.07 mm. The moisture contents of the samples are presented in Table 1. In many cases, the extraction yield improves by the moisture of the matrix, which acts as a solvent. The moisture in the plant material heats, evaporates and increases the internal pressure, causing the cell rupture and release of the solutes and resulting in a higher extraction yield [20].
Our samples showed that the obtained EO yields of the Asteraceae species were 0.29%, 0.05% and 0.01% in MC1, EP3 and EP2, respectively (Figure 1b). According to the literature, information about the genetic and environmental influences on EO yields, in similar plant parts used with HD, was available. In the study by Stanojević et al. [25], chamomile flowers collected from a plantation in the northwestern part of the Republic of Srpska, Bosnia and Herzegovina, were used for EOs' isolation. Here, the yield of the obtained dark blue colored EOs was 0.5%. Aćimovic et al. [26] compared three different tetraploid chamomile cultivars from Coka ('Zloty Lan', 'Manzana' and 'Lutea'). The EO contents ranged from 0.43 to 0.48% for all cultivars. On the other hand, in a review article by Sharifi-Rad et al. [27], it was stated that EO in Echinaceae plants contained 0.05-0.48% in fresh material and 0.1-1.25% in dried material. However, this amount may vary depending on the species. Furthermore, 1.85% for E. purpurea was reported in dry flowers and even less than 0.1% for E. angustifolia roots. These results are even higher than the values found in the research. Therefore, it could be concluded that EO in Echinaceae purpurea is found in floral heads, the amount of which depends on agroclimatic conditions. The results of EOs' yields for the Apiaceae family obtained from dried, ground seeds, located in the glands of the mericarp are shown in Figure 1a. From the graph, it can be seen that sample FV3 had the highest yield (5.80%), while the other samples (AG7, PC1, CC1 and PA1) had rather lower values (2.16%, 2.00%, 1.13% and 1.78%, respectively). As with other EOs, the literature lists numerous factors that affect their yields, e.g., origin of the seeds, maturity levels, environmental factors, etc. A recent review article by Sayed-Ahmad et al. [13] listed EO yields for different plants from Apiaceae species that were grown at different locations. Fennel, anise, dill and parsley from the Mediterranean region had the EO yields 3-6%, 2-6%, 1-4% and 2-8%, respectively, while caraway from Europe and Western Asia had 0.5-1.4% [13], which is consistent with the results from Figure 1a.
The findings for EOs' yields for the Cupressaceae family are shown in Figure 1c, for oils isolated from chopped dried fruits, as the berry fruits have elongated tubercles that serve as reservoirs for volatile oils. Here, herbal materials had similar moisture contents and particle diameters with different origins of juniper. To that end, samples were collected from three local facilities. EOs that came from the Institute for Medicinal Plant Research "Dr. Josif Pancic" had the highest yield of 1.90%, while samples from Adonis d.o.o., Sokobanja and Bilje Borča d.o.o., Borča had 1.33% and 1.52%, respectively. Our findings were in agreement with the literature data, where yields depended on the part of the plant used for HD. A review paper by Judžentienė [14] reported the main chemical compositions and EOs' yields for different organs of Lithuanian J. communis. Here, the amounts of EOs varied drastically with maturity and plant anatomy, e.g., unripe berries had 2.5-times more EOs (0.3-4.2%) than the ripe ones (~0.9%). Leaves had 0.1-0.9% of EOs, while the contents of oil in sprouts and branches were very similar and equaled 0.2 and 0.16%, respectively [14].

Chemical Profile of EOs
The chemical constituents of EOs are mainly secondary metabolites whose content and profile are strongly dependent on the developmental stage, pedoclimatic conditions, drying, storing and type of used herbal materials [28]. The analysis of chemical components identified in the EOs of the Lamiaceae species confirmed that the oil consisted of several groups of components, namely, monoterpene hydrocarbons, oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpenes. Identified compounds that constituted less than 0.1% EOs were referred to as trace components (tr). Table 2 shows the major components identified by the GC-MS in the Lamiaceae species' EOs.  A total of 104 compounds were identified in the oils and their percentage ranged from 95.38% to 100%. Lamiaceae EOs are usually characterized by two or three major constituents [29]. The EOs of all samples contained mainly oxygenated monoterpenes with percentages ranging from 35.34% to 89.82%. The main components of this terpenoid group (>10%) were menthone (31.75% in MP1, 21.02% in MP2), menthol (37.63% in MP1, 33.54% in MP2), carvone (45.61% in MP3, 47.04% in MP4), trans-anethole (16.04% in OV1), geranyl acetate (12.64% in SM3), terpinen-4-ol (30.20% in OM5), thymol (55.65% in TV6), carvacrol (61.32% in SH4), linalool (24.62% in OB7) and camphor (26.48% in SO7). The differences in the concentration of menthol and menthone in the peppermint samples are possibly due to the maturity of the leaves and/or a specific harvesting stage. For instance, menthol is abundant in leaves harvested at the flowering stage, while younger leaves harvested at the bud stage contain relatively high levels of menthone [30]. A similar composition of oregano EOs was described in the article by Radusiene et al. [31], where variations in the percentages of compounds collected from different parts of herbal material were also reported (inflorescences and leaves). Carvacrol and thymol were detected only in small amounts (~1%), which is in line with the previously reported results. In a review article by Pateiro et al. [32], it was suggested that the application of EOs (oregano, thyme) in meat products prevented biochemical and microbial deterioration. The above-mentioned essential oils showed high antioxidant activity which could be attributed to the presence of thymol and carvacrol. A GC-MS analysis of the red thyme and summer savory EOs revealed that their antioxidant protection was nearly entirely due to the phenolic components, represented by thymol and carvacrol [33]. SM3, TV6, OM5, SH4 and SO7 also contained considerable amounts of monoterpene hydrocarbons (35.23%, 36.89%, 42.89%, 37.86% and 12.21%, respectively), with the most important being p-cymene (19.24% in SM3 and 25.04% in TV6), γ-terpinene (11.63% in OM5 and 29.14% in SH4) and camphene (5.33% in SO7). Sesquiterpene hydrocarbons accounted for about 8.56% and 42.06%. They were mainly represented by β-elemene (7.46% in OB7), trans-caryophyllene (12.54%, 5.69% and 14.60% in MP3, MP4 and OV1, respectively) and germacrene-D (14.56% in OV1). Oxygenated sesquiterpene was detected in smaller percentages (12.93% and 13.57%) in two samples (OV1 and OB7). They were expressed by caryophyllene oxide (8.82% in OV1) and τ-cadinol  [34]. Similar constituents identified in the Lamiaceae species were included in the work of Ebadollahi et al. [8]. An overview of the anti-inflammatory effects of EOs by Zhao et al. [35], where a large group of plants from the Lamiaceae family are covered using in vitro and in vivo models, showed that different potential had been attributed to the varied EOs' chemical structures. In this work, the same plants from the Lamiaceae family have been studied, such as thyme, peppermint, oregano and sage, which possess oxygenated monoterpenes (menthol, menthone, carvacrol, thymol, camphor, geraniol) as the dominant constituents.
The main constituents of EOs from the Asteraceae species were terpenes (Table 3). In this case, the chemical structure of a chamomile sample was analyzed (MC1).
A total of 41 compounds were identified in the five samples. The percentages of the identified components varied from 98.71% in PA1 to 100% in the others. The predominant compound found in PA1 and FV7 belonged to the group of oxygenated monoterpenes (98.58% and 93.55%, respectively). Furthermore, trans-Anethole was detected as 96.40% in PA1 and 73.85% in FV7, and it was also responsible for the characteristic anise aroma and taste. In samples CC1 and AG7, most of the citrus aroma was derived from limonene (27.63% and 45.24%, respectively), which belonged to the group of monoterpene hydrocarbon (27.63% and 49.68%, respectively). The two previously mentioned samples contained another important flavoring and fragrance originated from carvone (70.25% and 45.90%, respectively), which belonged to the group of oxygenated monoterpenes (72.29% and 50.32%, respectively). The PC1 sample, on the other hand, had a completely different structure. Phenylpropanoids, such as myristicin, elemicin, 6-methoxyelemicin and apiole, occurred in a range from 5.53% to 35.81%. Furthermore, being extracted in these tested samples from fruit or seeds, such compounds can be also extracted from the roots.
Chizzola [39] reviewed major constituents in EOs from wild plants belonging to the European Apiaceae species grown in the Mediterranean region, including Asia Minor [39]. It was concluded in the report that, in addition to the origins of the plants, the composition of the oils in different plant organs of the same species can be quite different. Accordingly, the fruit oil of Laser trilobum was dominated by limonene and perillaldehyde, while the oil from the leaves of the same plant contained bornyl acetate as its main component. Root oils may differ from the oils of the aerial parts, for example, in Daucus carota ssp maxima, where the root contained the phenylpropanoids, dill apiole and myristicin, and the inflorescences contained mainly sabinene and terpinen-4-ol [39]. A review paper by Spinozzi et al. [40] listed a large group of plants from this family. EO is hydrodistillated from different plant parts, such as schizocarps, herbs and aerial parts, but the GC-MS profiles are relevant to the data obtained in this research. Besides their major EO constituents, this work presented the utilization of those products as botanical insecticides against mosquitos. Even if these eco-friendly insecticides show many advantages, the main issue to overcome is the low persistence of their effect, due to their volatile compounds. A chemical analysis which resulted from the detection of 40 compounds in the Cupressaceae family's EOs is shown in Table 5. This table lists the identified compounds, which accounted for 97.92% of the composition of the total extracts. Monoterpene hydrocarbons were the predominant group of compounds in all samples and were found to be between 64.96% in JC2, 69.24% in JC1 and 70.27% in JC3 of the content, followed by sesquiterpene hydrocarbons (26.04% in JC2, 23.70% in JC1 to 21.74% in JC3). In all samples, two components, α-pinene and sabinene, accounted for an average of 35.79% and 14.86%, respectively. Fourteen compounds, including limonene, β-elemene, trans-caryophyllene, γ-elemene, germacrene D and δ-cadinene, were present in moderate to high quantities (1.5-8.0%). From these analyses, it could be concluded that all samples had similar chemical compositions, and that the distributions of compounds were similar.
In the study by Rajčević et al. [41], the aim was to determine the diversity of terpene classes and common terpenes in populations of typical Juniperus communis from the northwestern Balkans (Slovenia and Croatia) grown under different climate conditions, on different substrates and at different altitudes. These samples showed the dominance of the chemotypes sabinene and α-pinene in the composition of the EOs' profiles. However, the reported EOs' contents showed much greater variability between the alpine and continental populations. Continental populations, with over 10 different compounds (including limonene, β-phellandrene, (E)-caryophyllene, β-copaene, germacrene D and germacrene B) were present in high amounts (>6.0%) [41].
The chemical structures of major constituents identified by the GC-MS analysis of the different above-mentioned families are shown in Figure 2.

In Vitro Antioxidant Activity
The in vitro antioxidant activity of EOs was evaluated by assessing their ability to bind hydrogen or scavenge radicals using DPPH and ABTS + . The study of antioxidants is a hot topic because they play an important role in preventing cellular damages caused by free radicals, leading to various degenerative diseases in humans such as cancer, heart diseases and others [42]. The changes in antioxidant capacity affected by different EOs from four families are shown in Table 6.

In Vitro Antioxidant Activity
The in vitro antioxidant activity of EOs was evaluated by assessing their ability to bind hydrogen or scavenge radicals using DPPH and ABTS + . The study of antioxidants is a hot topic because they play an important role in preventing cellular damages caused by free radicals, leading to various degenerative diseases in humans such as cancer, heart diseases and others [42]. The changes in antioxidant capacity affected by different EOs from four families are shown in Table 6.
The MC1 EO was the most effective DPPH radical scavenger (44.20 µM Trolox/g) at a tested concentration of 10 mg/mL, followed by samples from the Lamiaceae family such as TV6 (29.78 µM Trolox/g at 10 mg/mL), SH4 (23.32 µM Trolox/g at 10 mg/mL) and OB7 (44.97 µM Trolox/g at 15 mg/mL). The high antioxidant capacity of chamomile inflorescence was also reported by a review from Petronilho who attributed it to the presence of chamazulene and α-bisabolol [43].  Thymol, carvacrol, linalool and estragole were the main contributors to the antioxidant activity of the volatile extracts of thyme, summer savory and basil, as noted in a previous study [42]. The effects of the other samples were significantly different depending on the tested concentration. Accordingly, the samples of the Apiaceae species showed lower DPPH activity than the EOs of AG7 and FV7 with a value of 2.28 µM Trolox/g and 1.37 µM Trolox/g at 10 mg/mL, respectively. The antioxidant activity of the Apiaceae family was reported for extracts and EOs, with higher activity observed in extracts due to the presence of phenolic compounds, such as flavonoids and proanthocyanidins, carotenoids, etc. [13]. The study by Hajlaoui et al. [44] displayed the moderate DPPH scavenging activity of C. carvi and C. sativum EOs and their mixtures, due to the presence of γ-terpinene, carvone, linalool and p-cymene as dominant terpenes. The EOs rich in this group of monoterpenes increased the oxidative stability and shelf life of edible lipids, through their interactions with other monoterpenes, rather than single antioxidants. Samples obtained by the distillation of MP1-MP4 showed weak scavenging ability for the DPPH radicals (average 5.49 µM Trolox/g at 15 and 20 mg/mL). This was in agreement with the literature data which reported that peppermint terpenoids have modest activity in the model system [45]. Samples from the Cupressaceae species showed a good result for DPPH activity at 15 mg/mL (average 3.50 µM Trolox/g). However, the low DPPH activity of juniper berry EO may be due to the high amount of α-pinene and β-pinene, both of which were inactive in the DPPH test, as was concluded in the research paper by Emami et al. [46].
The antioxidant activity of the ABTS + assay improved dramatically for EOs derived from a large group of Lamiaceae and Cupressaceae families. This was consistent with our previous results where we also reported that peppermint EO had higher activity towards ABTS + as compared to DPPH radicals [47]. The activity of most samples from the Lamiaceae family ranged from 296.69 µM Trolox/g (MP3) to 757.98 µM Trolox/g (OB7) at a concentration of 1 mg/mL. At the same concentration, the samples from the Asteraceae family reached a high activity of 406.56 µM Trolox/g on average, similar to the samples from the Cupressaceae family, where the average activity was 308.06 µM Trolox/g. The Apiaceae family showed the lowest ABTS + radical scavenging activity: even at a ten-fold higher concentration it was still 34.44 µM Trolox/g. Similarly, Hoferl et al. [48] observed that juniper berry EO showed a significant inhibitory effect on ABTS + radicals (IC 50 10.96 µg/mL), but butylated hydroxytoluene was considerably stronger (IC 50 0.0175 µg/mL).
This work provides a preliminary study with in-depth information of the antioxidant potential of the most common EOs obtained from aromatic plants grown in Serbia. This will be used as a screening tool for the selection of further EO candidates for further processing by emerging extraction techniques such as microwave-assisted hydrodistillation and supercritical fluid extraction. Our previous studies suggested that selected EOs obtained from sage [49], winter savory [50] and wild thyme [51] could be efficiently used as natural additives with antioxidant and antimicrobial properties in various meat products [6,52]. Furthermore, coriander EO was efficiently used as a partial nitrite replacement in meat products, thus suggesting that natural extracts could diminish the concentration of potentially toxic additives in food products [53]. It could be observed that the majority of these EOs were obtained from the plant species from the Lamiaceae family. However, plants from the Apiaceae family had the highest content of EO among the investigated plant species. These EOs were already recognized with a broad spectra of applications as potent antioxidants in food, nutraceuticals and cosmetics [54]. Based on this, our further research on the application of EOs as natural additives will be focused on the Apiaceae family, particularly on dill and caraway essential oils.
In conclusion, the samples which turned out to be promising as potential antioxidants were the ones from the Asteraceae family (the most effective DPPH radical scavengers), while the samples from the Lamiaceae and Cupressaceae families showed high antioxidant activity against ABTS + radicals.

Principal Component Analysis (PCA)
A PCA was used to describe the differences between samples and to provide more information on the response variables that mainly influenced sample similarities and differences, in order to reduce the complexity of the data and address the most important features [55]. In this study, the application of the PCA on the dataset was used to analyze the antioxidant potential (DPPH and ABTS + ) of the evaluated EOs from different plant samples, as well as their yields and chemical profiles. As indicated in Table 7 and shown in Figure 3, according to the results of the PCA and Kaiser's rule, the first three PCs explained 74.2% of the total variance of the experimental data. The factor coordinates of all samples and variables are presented in Table S1 (Supplementary Materials).    The first PC (F1) was mainly associated with EOs' yield, antioxidant activity and sesquiterpenes. Samples MC1 and OB7, followed by the OV1 sample, were allocated at the positive side of F1, near oxygenated sesquiterpenes, DPPH and ABTS, thus indicating an increasing number of bioactive components such as β-elemene, τ-cadinol, α-bisabolol oxide A and α-bisabolol oxide B (sesquiterpenes) responsible for their high antioxidant potential. The negative side of F1 was related to sample FV7, which was characterized by the highest EO yield. On the other hand, according to the factor coordinates of samples and variables, based on correlations, the samples MC1, OB7 and OV1 showed the lowest EO yield. It was also observed that oxygenated monoterpenes contributed significantly to both the first and the second PCA. Therefore, samples FV7 and PA1 were strongly associated with oxygenated monoterpenes.
The second PC (F2) was mainly associated with monoterpene hydrocarbons and showed a clear difference between a group of samples from the Cuppresaceae family (JC1, JC2 and JC3) and all other samples, as they received the highest composition of monoterpene hydrocarbons, such as α-pinene, sabinene, myrcene and limonene, and a very low proportion of oxygenated monoterpenes. This chemical structure could represent those samples as promising agents against ABTS + radicals. On the other hand, a great group of samples from Apiaceae (FV7, PA1) and Lamiaceae (MP1, MP2, MP4) had dominant chemical compositions of oxygenated monoterpenes.
In addition, the third PC (F3) was associated with other chemical constituents important for the characterization of the PC1 sample, followed by the MC1 sample. The major other components were myristicin, elemicin, 6-methoxyelemicin and apiol. Since these components were mainly found in the PC1 sample, they could be a reason for the low antioxidant potential of this sample. The other plant samples were grouped, according to F3 coordinate values, indicating the small amount of other chemical constituents (<8%).

Plant Material
The plant material was collected from different places in Serbia. Some of them were grown in agricultural holdings in Bačko Novo Selo (basil, sage, fennel and dill), Kulpin (thyme) and Banatska Topola (peppermint and summer savory). Harvesting was completed by hand at the stage of full maturity, in the summer of 2019. After harvesting, the plant material was stored in paper bags at room temperature until the need for further analysis. The other plants were purchased from local institutions: the Institute for Medicinal Plant Research "Dr Josif Pancic" (peppermint, oregano, chamomile, parsley, caraway, anise and juniper); medicinal and aromatic herbs and herb-based products, Adonis d.o.o., Sokobanja (peppermint, echinacea and juniper); medicinal and aromatic herbs and herbal-based products, Bilje Borča Llc, Borča (peppermint, oregano, winter savory, echinacea and juniper); medicinal and aromatic herbs and herbal products, Geneza d.o.o., Kanjiza (marjoram). The dried plant material was grounded in a household blender (except for those that were purchased as grounded), while the particle size of the material was determined by sieve sets (CISA, Cedaceria Industrial, Barcelona, Spain). The moisture content of the plant material was analyzed using a standard procedure, i.e., by drying the plant sample at 110 • C until reaching a constant weight. All codes for the samples and characterizations of the plants are shown in Table 1.

Isolation of EO-Conventional Hydrodistillation (HD)
Clevenger's hydrodistillation was used for the isolation of EOs, i.e., slightly modified official Ph. Eur. VII Procedure [56]. Forty grams of the milled, dried plant material was placed in a round glass flask (1 L), and 400 mL of distilled water was added. Distillation was carried out for 2 h and it was performed in triplicates. The EO yield (Y) was expressed as % (v/w). Samples were collected and stored at 4 • C in dark bottles, to avoid deterioration before further analysis.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis
The chemical composition of EOs obtained by HD was analyzed by GC system (7890A, Agilent Technologies, Santa Clara, CA, USA) coupled with MS (5975C, Agilent Technologies, Santa Clara, CA, USA) and HP-5MSUI (30 m × 0.25 mm, 0.25 µm) capillary column. The EOs were dissolved in methylene chloride, and 2 µL of the properly diluted samples was added to the GC via TriPlus AS autosampler. The mobile phase was helium (>99.9997%) at a flow rate of 2 mL/min. The temperature program was as follows: the initial temperature was 45 • C (8 min), then increased to 230 • C at a rate of 8.0 • C/min and remained at this temperature for 10 min. The injector, mass transfer line and ion source temperature were 250, 200 and 220 • C, respectively. Compounds were identified using the NIST database of MS spectra and the database in the literature [57], with the final results reported as relative content (%). For additional confirmation, the linear retention indices were calculated and compared with the existing literature [57]. A quantitative analysis was performed by generating the calibration curves for the analyzed compound in the concentration range of 1-500 µg/mL.

In Vitro Antioxidant Activity
The capacity of EO samples, in terms of hydrogen donating or radical scavenging ability, was measured using the stable radical DPPH according to the published method [58] with slight modifications for lipid samples [59]. For this purpose, a methanolic solution of DPPH reagent (65 µM) was freshly prepared and adjusted to an absorbance of 0.70 (±0.02) with methanol, and 0.1 mL of the samples diluted in ethyl acetate was added to 2.9 mL of DPPH reagent. Blank samples were prepared by mixing 0.1 mL of ethyl acetate and 2.9 mL of DPPH reagent. Samples were stored at room temperature and in the dark for 60 min. Free radical scavenging measurements were performed in triplicates at a wavelength of 517 nm by UV/Vis spectrophotometer (6300 Spectrophotometer, Jenway, Staffordshire, UK). The obtained results were expressed as µM Trolox equivalents per g EO (µM Trolox/g). As is represented in Table 5, the concentrations of essential oils were 10, 15 and 20 mg/mL.
The ability of the samples to scavenge ABTS + radicals was measured using a modified method from the literature [60]. Briefly, the ABTS stock solution was freshly prepared from a mixture (1:1, v/v) of 2.45 mM potassium persulphate aqueous solution and 7 mM ABTS aqueous solution, and then allowed to stand in the dark at room temperature for 16 h. The stock solution was diluted with absolute ethanol to achieve an absorbance of 0.70 (±0.02). A volume of 0.1 mL of the properly diluted EO samples in ethyl acetate and 2.9 mL of the ABTS reagent were mixed and incubated for 5 h in the dark at room temperature. The blank samples were obtained by mixing 0.1 mL ethyl acetate and 2.9 mL ABTS reagent. Absorbance was measured at 734 nm in triplicates using a UV/Vis spectrophotometer (6300 Spectrophotometer, Jenway, Staffordshire, UK). The results were expressed as µM Trolox equivalents per g EO (µM Trolox/g), as mean ± standard deviation. As is represented in Table 5, the concentrations of essential oils were 1 and 10 mg/mL.

Statistical Analysis and PCA
All experiments were performed in triplicates. A statistical analysis was performed using the Statistica version 8 software package. A PCA calculation was performed to obtain an insight into the relationships between data obtained by the EO yield, chemical properties (compounds with abundance higher than 1%) and antioxidant activity (DPPH and ABTS assays).

Conclusions
Considering the high level of public awareness of the beneficial influences of natural bioactives for the food and pharmaceutical industries, alternative medicine and natural therapy, and in conjunction with the higher content of terpene compounds in various members of the Lamiaceae, Asteraceae, Apiaceae and Cupressaceae species, different medicinal and aromatic plants from Serbia were hydrodistilled in this work as a possible source of high-potency bioactives.
Generally, the highest EOs' yields were detected for dried, milled seeds of the Apiaceae family, which were dominated by the group of oxygenated monoterpenes (carvone and trans-anethole). Due to the high content of sesquiterpenes, dominated by α-bisabolol oxide A and α-bisabolol oxide B, the samples from the Asteraceae family were the most effective DPPH radical scavengers; however, the samples from the Lamiaceae and Cupressaceae families showed high antioxidant activity against ABTS + radicals, dominated by monoterpenes, where the main components of this terpenoid group were menthone, menthol, carvone, thymol, carvacrol and α-pinene.
This work provided in-depth information on the antioxidant potentials for the most common EOs obtained from aromatic plants commonly present in Serbia. The reported data can be used as a screening tool for further selection of EOs' candidates to be industrially exploited (microwave-assisted, supercritical fluid extractions, etc.) and processed with other types of advanced extractions. One of the industrial applications can be for the food industry, as we suggested previously for sage [49], winter savory [50], wild thyme [51] and coriander [53]. EOs from these plants can be efficiently used as natural additives with antioxidant and antimicrobial properties for various meat products [52]. From our and other available results, it can be noticed that the majority of these EOs were obtained from the Lamiaceae family. However, based on the EOs' yields, chemical profiles and in vitro antioxidant activities, further research will be focused on the Apiaceae family, particularly dill and caraway's essential oils.
at the University of Zagreb (grant number: 451-03-766/2021-14). Authors are especially grateful to Jelena Jerković, assistant professor of English language at the Faculty of Technology, University of Novi Sad, for her valuable support in language correction and editing.

Conflicts of Interest:
The authors declare no conflict of interest.