Antimicrobial, Antioxidant, and Immunomodulatory Properties of Essential Oils: A Systematic Review

Essential oils (EOs) are a mixture of natural, volatile, and aromatic compounds obtained from plants. In recent years, several studies have shown that some of their benefits can be attributed to their antimicrobial, antioxidant, anti-inflammatory, and also immunomodulatory properties. Therefore, EOs have been proposed as a natural alternative to antibiotics or for use in combination with antibiotics against multidrug-resistant bacteria in animal feed and food preservation. Most of the results come from in vitro and in vivo studies; however, very little is known about their use in clinical studies. A systematic and comprehensive literature search was conducted in PubMed, Embase®, and Scopus from December 2014 to April 2019 using different combinations of the following keywords: essential oils, volatile oils, antimicrobial, antioxidant, immunomodulation, and microbiota. Some EOs have demonstrated their efficacy against several foodborne pathogens in vitro and model food systems; namely, the inhibition of S. aureus, V. cholerae, and C. albicans has been observed. EOs have shown remarkable antioxidant activities when used at a dose range of 0.01 to 10 mg/mL in cell models, which can be attributed to their richness in phenolic compounds. Moreover, selected EOs exhibit immunomodulatory activities that have been mainly attributed to their ability to modify the secretion of cytokines.


Introduction
Foodborne-related diseases are an increasingly major public health problem worldwide [1]. Microbial contamination is one of the factors in developing foodborne diseases and food spoilage [2]. Since ancient times, different methods to preserve food for a longer period have been sought. For this reason, and thanks to the advancement of knowledge and the rapid development of new technologies, different chemical compounds have been developed, commonly known as additives, which extend the life of foods or are used as sweeteners or coloring agents [3]. however, the presence of pathogenic microorganisms continues to result in large economic losses and multiple diseases in humans [4]. On the other hand, the indiscriminate use of antibiotics in both humans and animals against pathogenic

Search Strategy
In this review, the specialized databases PubMed (US National Library of Medicine National Institutes of health), Embase ® , and Scopus were used for the literature search from December 2014 to April 2019, with the aim of restricting the search to the articles that make use of the most modern techniques, using different combinations of the following keywords: essential oils, volatile oils, antimicrobial, antioxidant, immunomodulation, and microbiota. In PubMed, we used the following search equation strategy: ("essential oils" [All Fields] OR "volatile oils" [All Fields]) AND ("immunomodulation" [All Fields] OR "drug resistance, multiple, bacterial" [All Fields] OR "immune system" [All Fields]). When we used PubMed, we included Medical Subject heading (MeSH) terms to increase the power of the search. The search equation used in Scopus was: "essential AND oils" AND immunomodulation AND drug resistance, multiple, bacterial.

Selection Criteria
Articles were organized by the antimicrobial and immunomodulatory effects of EOs; after that, two members of the team (M.V.-U. and C.G.-Ll.) extracted information about the characteristics of the studies. The information extracted from the articles included EOs, cell lines (in vitro studies), animal models (in vivo studies), doses or concentrations, routes of administration, biochemical assays, and the molecular mechanisms investigated. The quality assessment and selection were performed by two authors (M.V.-U. and C.G.-Ll.) who independently worked according to the main criteria of PICO (Population, Intervention, Comparison, Outcome) ( Table 1); in case of discrepancies, a third independent reviewer (J.P.-D.) was consulted for the final decision.

Data handling, Analyses, and Extraction
The inclusion criteria used were the following: (1) studies with EOs with antimicrobial and immunomodulatory properties in vitro and in vivo; (2) food and nutrition-related studies; and (3) studies with significant results obtained through appropriate statistical analysis. The exclusion criteria used were as follows: (1) studies written in languages other than English or Spanish; (2) the use of plant extracts or derivatives, instead of EOs; (3) review articles, conference proceedings, and editorials/letters; (4) studies without controls; and (5) agar diffusion method as the only assay for the study of the antimicrobial effect. After removing duplicates, acceptability for inclusion was evaluated based on the following: (1) reading the title and abstract; and (2) reading the full text.
We provided a narrative synthesis of the main results of the selected articles. These results were classified according to the EOs' properties: (i) antimicrobial activity, (ii) antioxidant activity, and (iii) immunomodulatory effects of EOs in cells and animals.

Study Identification and Selection
Seventy-nine relevant articles were identified, which was in agreement with our inclusion and exclusion criteria. The selected articles were grouped into the antimicrobial and antioxidant effects of the EOs, immunomodulatory effects of EOs in cell studies, and animal supplementation with EOs. We only found one article in human samples that met our eligibility criteria. We did not find any intervention studies in humans. The complete process is explained in Figure 1, which is based on a PRISMA flow chart.  Table 2 lists the selected publications and their main results of the antimicrobial effect of EOs. In total, 49 articles were identified. Forty-three articles described the effects of EOs over several bacterial strains such as E. coli, S. aureus, B. cereus, and P. aeruginosa. In six articles, the inhibition of biofilm formation by EOs was described. The dose used for the determination of minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) values ranged from 9 to 229 mg/mL, and the incubation time varied from 18 to 24 h. Table 3 depicts the articles and their main results regarding the antioxidant effects of EOs. Twenty-one studies were selected. The main methods used to evaluate the antioxidant capacity were the following: measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2′-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals scavenging activity, ferric-reducing antioxidant power (FRAP), and oxidative DNA damage protective effect induced by Fe 2+ and 2,2′azobis (2-methylpropionamidine) dihydrochloride (AAPH). EOs demonstrated ability in antioxidant assays in a concentration-dependent manner. The dose range was 0.05% to 3.2% v/w, 0.1 to 10 mg/mL, and 12.5 to 2000 µg/mL.

Immunomodulatory Activity of Essential Oils in Cells and Animals
Tables 4 and 5 describe the main effects of EOs in cellular and animal studies, respectively. Eight of the selected studies were specifically conducted in cells, whereas five out of 14 publications selected performed antimicrobial studies against different bacterial strains and immunomodulatory studies in a cellular model. Most of the studies were conducted in animal cells (nine out of 13), although studies in human cells were also performed (seven out of 14). The dose ranged from 1.25 to 1000 µg/mL, and the incubation times varied from 1 to 72 h. In the case of animal studies, six out of 10 were performed on poultry animals, while three out of 10 were performed on C57BL/6 mice. One article was performed in silver catfish. The dose administrated varied from 7.5 mg/kg to 1 kg/ton in weaned piglets.  Table 2 lists the selected publications and their main results of the antimicrobial effect of EOs. In total, 49 articles were identified. Forty-three articles described the effects of EOs over several bacterial strains such as E. coli, S. aureus, B. cereus, and P. aeruginosa. In six articles, the inhibition of biofilm formation by EOs was described. The dose used for the determination of minimum inhibitory concentration (MIC)/minimum bactericidal concentration (MBC) values ranged from 9 to 229 mg/mL, and the incubation time varied from 18 to 24 h. Table 3 depicts the articles and their main results regarding the antioxidant effects  of EOs. Twenty-one studies were selected. The main methods used to evaluate the antioxidant capacity were the following: measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radicals scavenging activity, ferric-reducing antioxidant power (FRAP), and oxidative DNA damage protective effect induced by Fe 2+ and 2,2 -azobis (2-methylpropionamidine) dihydrochloride (AAPH). EOs demonstrated ability in antioxidant assays in a concentration-dependent manner. The dose range was 0.05% to 3.2% v/w, 0.1 to 10 mg/mL, and 12.5 to 2000 µg/mL.

Standard butylhydroxytoluene
C. citriodora was higher than that of the positive control but lower than that of the standard, butylhydroxytoluene Zhao et al. [73] Fagopyrum esculentum, Fagopyrum tataricum, and Fagopyrum Cymosum  EOs treatment was able to promote LPS-stimulated splenocyte proliferation. In B16F10 cells, incubation with the EOs inhibited cell proliferation in a dose-and time-dependent fashion (IC50: 80 µg/mL). In addition, EOs treatment was also able to increase the number of apoptotic cells. EOs have no major toxic side effects on L02 cells, and even promoted cell proliferation.
In the A549 cell line, EOs promote the proliferation of cancer cells. NO production was inhibited in LPS-induced RAW264.7 cells treated with EOs at 50 and 100 µg/mL.
In addition, EOs treatment reduces the secretion of IL-6, but has no effect on TNF-α gene expression. Furthermore, EOs decreased lipid accumulation in ox-LDL-induced RAW264.7 cell, and decreased the secretion of IL-6.
Marelli et al. [  In addition, incubation with EOs inhibited the production of TNF-α, IL-1β, and IL-6, whereas it increased the release of IL-10. EOs also inhibited the secretion of NO and PGE 2 .

Discussion
There is a huge amount of different EOs from different plants around the world. Most of them have been shown to exert a well-characterized antimicrobial activity against Gram-positive and Gram-negative bacteria, but also against other microorganisms, such as yeast. The irreversible damage of the bacterial cell wall and membrane has been proposed as its main mechanisms of action. In addition, several studies revealed how EOs can inhibit biofilm formation through the inhibition of bacterial cell communication. Regarding their antioxidant and immunomodulatory properties, EOs have been shown to exert a protective effect through their radicals scavenging activity, with an inhibition percentage range of 20%-70%, and their effect against DNA oxidative damage induced by Fe 2+ . Regarding their immunomodulatory effect, EOs or their main compounds can modulate the secretion of important cytokines in a cell culture challenge with LPS. This capacity was evident in their effect in inflammatory pathways such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). It is important to highlight that a cytotoxic effect was not observed when EOs where used at low concentrations. There have been a few studies in poultry animals where EOs have a positive effect on growth parameters; however, on the gastrointestinal microbiota, EOs have a negative affect on gastrointestinal pathogen microorganisms. Their biological properties can be attributed to their complex composition with more than 300 different volatile compounds. These volatile compounds include terpenes, alcohols, phenolic acids, ethers, esters, amines, amides, ketones, and aldehydes, among other chemical components [102]. Although most of their biological actions have been related to their main components, it is important to underpin that the aforementioned properties come over the synergic effect of all the components. The results from this review indicate that EOs have important biological properties that make them suitable for use in the development of functional foods. however, in this regard, one aspect that must be considered is their strong smell, which could result in low acceptance by the consumer or modification of the organoleptic properties of the food [8].

Antimicrobial Activity of Esential Oils
In recent years, there has been a growing interest in researching and developing new antimicrobial agents from EOs due to drug resistance in foodborne bacterial enteric pathogens. Numerous publications have presented data on the antimicrobial properties of EOs [29,30].
A variety of laboratory methods can be used to evaluate the in vitro antimicrobial activity of an EO. The most well-known and basic methods are the disk diffusion and broth or agar dilution methods [103]. The lowest concentration of antimicrobial agent that completely inhibits the growth of the organism is called the minimum inhibitory concentration (MIC). The most appropriate assays for the determination of the MIC value are the dilution methods, as they offer the possibility of a precise estimation of the concentration of the tested antimicrobial agent.
The antibacterial effects based on the MIC determination of several EOs alone or in combination against different food-associated Gram-positive and Gram-negative bacteria have been described. Parsley, lovage, basil, and thyme are a few of the aromatic herbs commonly used in industry with low-cost production. Different parts of these herbs (leaves, flowers, stems, fruits, and seeds) have been used to extract EOs [60]. Parsley and lovage EOs revealed no inhibitory effects against all tested strains. Thyme EO had the highest percentage yield and antibacterial potential from all tested formulations; its use in combination with parsley, lovage, and basil EOs results in a reduction in its antibacterial activity; therefore, thyme EO should be used alone [60]. EOs of cultivated oregano (Origanum vulgare), sage (Salvia officinalis), and thyme (Thymus vulgaris) have been shown to exert a potent antimicrobial effect. Among them, the most efficient were the EOs from thyme, followed by those of oregano. With MIC values above 150 mg/mL, sage EOs did not show any antibacterial effect against the majority of the bacterial strains [38]. Three Origanum species analyzed, O. dictamnus and O. microphyllum-both endemic in Greece-and O. libanoticum, endemic in Lebanon, were evaluated, but only O. dictamnus exerted antibacterial activity [46]. Different bacterial and fungal strains have been used to determine the antibacterial effects of different Eos; these microorganisms comprise strains from Staphylococcus, Bacillus, Listeria, Helicobacter, Micrococcus, Pseudomonas, Klepsiella, Escherichia, Salmonella, Enterobacter, and Candida. EOs from Heracleum pyrenaicum subsp. orsinii, Pistacia vera L., Myrcia ovata Cambessedes, Thymus bovei, Minthostachys verticillata, Allium roseum, Petroselinum crispum, Satureja bachtiarica Bunge, Ocimum suave, Jatropha gossypifolia L., and Juniperus rigida have been shown to exert antibacterial and anti-yeast effects [34,37,41,43,47,48,51,64,[67][68][69]. One of the proposed mechanisms for those effects is the irreversible damage of the bacteria cell wall and membrane, which leads to not only a leakage of proteins but also of DNA and RNA molecules [47,48].
EOs from plants from different regions of the world have been studied. In this sense, EOs derived from Aloysia citriodora Palau, which is harvested in different regions of Morocco, showed significant antimicrobial activity against both Gram-negative and Gram-positive bacteria [52]. The EOs of Peperomia pellucida, an herbaceous plant from the Amazon region, exhibited strong antibacterial activities against six different bacteria strains [50]. Salem et al. evaluate the biological activity of the EOs derived from Corymbia citriodora leaves and Cupressus macrocarpa from Egypt. While the antibacterial activity of EO from C. citriodora leaves has MIC values ranging from 0.06 to 0.20 mg/mL, EO from C. macrocarpa branchlets showed less activity against bacterial strains [59].
In recent years, there has been a dramatic increase in resistance to antimicrobial drugs against Salmonella Enterica and Campylobacter spp. Campylobacter spp. is one of the most common causative agents of gastroenteritis in the world, whereas salmonellosis is a major foodborne disease worldwide. Bacteria can be transmitted to humans by the consumption of contaminated poultry, eggs, beef, milk, juices, fruits, and vegetables. Several studies have shown that EOs could be used as alternative therapeutics to treat antibiotic-resistant Salmonella. In this regard, Ruilopezia bracteosa EO has been described as being effective against S. aureus and E. faecalis compared with several antibiotics [26]. Similarly, Ashraf et al. studied the effect of Nigella sativa (Black seed) oil against antibiotic-resistant isolates by a well diffusion and microbroth dilution method, and they concluded that N. sativa had in vitro activity against Salmonella Enterica [27]. Chiboub et al. evaluated the biological activity of the EOs of two varieties of Foeniculum vulgare in the growth of Salmonella Enterica, and the results showed a significant antimicrobial activity [32]. Aghraz et al. showed that EOs from Cladanthus arabicus and Bubonium imbricatum contain a potent activity against the tested Salmonella strain, with MIC values between 200 and 800 µg/mL for C. arabicus and from 400 to 1600 µg/mL for B. imbricatum [25]. The evaluation of the synergistic effect of mixed EOs was also investigated. To increase the sensitivity against the Salmonella Typhimurium strain, a mixture of Thymus vulgaris L., Rosmarinus officinalis L., and Myrtus communis L was used. EOs were used in combined treatment using an experimental design methodology [36]. A mixture of 55% of T. vulgaris L. and 45% of M. communis L. EOs, respectively, can be considered for the increase of Salmonella Typhimurium sensitivity. Mutlu-Ingok et al. studied the antibacterial activities of cardamom, cumin, and dill weed EOs against Campylobacter jejuni and Campylobacter coli. The results indicated that EOs might be effective inhibitors by directly acting at the bacterial membrane integrity level [49]. It is important to highlight that EOs derived from oregano, thyme, clove, and arborvitae also showed a very strong antibacterial activity against other bacteria causing foodborne disease; therefore, they can be used as antimicrobial agents [57].
One important concern in the food industry is the presence of biofilms. Bacteria can be suspended in liquid food, usually living planktonically, although they can easily adhere to the surface of food materials and food processing equipment, forming a bacterial biofilm. Biofilms are microbial communities that are characterized by their adhesion to solid surfaces and the production of a matrix of exopolymeric substances; the matrix consists of polysaccharides, proteins, DNA, and lipids, which surround the microorganisms, proffering structural integrity and a unique biochemical profile to the biofilm [104]. Biofilms can exist on all types of surfaces in food plants ranging from plastic, glass, metal, and wood, to food products [105], resulting in food spoilage and economic losses for the producers [105]. Several studies revealed how EOs can inhibit biofilm formation [33,56,62,65,71]. Cinnamomum zeylanicum oil may be a useful approach to impair the biofilm produced by Gram-negative bacteria [33]. According to Porfirio et al., Lippia Alba EOs have a strong inhibition of S. aureus biofilm formation [56]. Likewise, EOs derived from parsley and basilic can inhibit and eradicate the mature biofilm formed by Vibrio strains on a polystyrene surface even at low concentrations. These two EOs could be used to prevent and eradicate the contamination of sea products by these strains [65]. It has been described that quorum sensing (QS), the process through which bacterial cells communicate with each other by releasing, sensing, and responding to small diffusible signal molecules [106], is involved in biofilm formation. QS has been inhibited by the EOs of several plants, such as Thymus daenensis and Satureja hortensis. Consequently, EOs act as anti-biofilm and QS inhibitor agents against bacteria [62].

Antioxidant Activity of Essential Oils
The excessive amounts of reactive oxygen species (ROS) can lead to the peroxidation of lipids, glycation/oxidation/nitration of proteins, inactivation of enzymes, DNA damage, and other alterations in the cellular organelles [107,108].
In recent years, food oxidation and food spoilage caused by microorganisms form one of the most important issues facing the food industry and consumers. Accompanied by growing consumer interest in natural food additives, the search for effective antioxidants and antibacterial agents from natural resources as alternatives to suppress food deterioration is now focused on edible plants, since they present with fewer side effects than the synthetic chemicals used in today's foods [109]. There has been an increasing realization in recent years that several plant-derived EOs may possess antioxidant, antimicrobial, anticancer, and apoptosis-inducing properties [110].
Cyperus rotundus L. is a smooth and perennial weed that is widely distributed in tropical and warmer temperate regions worldwide [77]. The antioxidant properties of the C. rotundus rhizome were determined.
An antioxidant combination effect of bay leaf, black pepper, coriander (seed and leaf), cumin, garlic, ginger, mustard, onion, and turmeric EOs was assessed by the DPPH free radical scavenging method. Only the coriander/cumin seed oil combination exhibited antioxidant activity in a synergistic interaction. Bioactive compounds responsible for this antioxidant capacity were linalool from coriander seed oil and p-coumaric acid from cumin seed oil [74]. DPPH radical scavenging activity assay, β-carotene bleaching test (BCBT), and ABTS assay were determined in Melissa officinalis and Dracocephalum moldavica EOs. Both EOs showed a strong activity in terms of the maintenance of β-carotene molecules. The ABTS radical scavenging of the EOs was dose-dependent and increased with the increase in the EOs concentration [76]. The antioxidant activity of the EO of Ruta chalepensis was tested by DPPH using Trolox as a reference compound. Percentages of inhibition for R. chalepensis collected from Jerusalem, hebron, and Jenin were 69.56%, 61.53%, and 24.12%, respectively [78]. Achillea millefolium L., Anethum graveolens L., and Carum copticum L. EOs were selected to evaluate their antioxidant properties using DPPH, FRAP, BCBT, and total phenolic content assays. A. millefolium EO had the highest antioxidant activity in all conducted assays [79]. With a similar methodology, Foeniculum vulgare, Petroselium crispum, and Lavandula officinalis EOs, six different populations of Origanum heracleoticum L. from Calabria (Italy) Eos, and Pelargonium asperum and Ormenis mixta were analyzed. Petroselium crispum had the highest phenolic content and the best antioxidant profile [80], EO samples from Bagaladi and Longobucco were the most active in DPPH and BCBT assays [81], and only Ormenis mixta EO displayed an effective antioxidant ability, as tested by DPPH assay [83].
The antioxidant properties of EOs from the fruits Dennettia tripetala G. Baker as ripe and unripe fruit oil were tested. The ripe fruit EO has shown higher antioxidant strength than unripe fruit EO and vitamin C, but a lower activity compared to BCBT. The EOs also demonstrated strong ability in terms of scavenging three other different radicals (ABTS, lipid peroxide, and nitric oxide radicals) in a concentration-dependent manner [82]. With a similar methodology, Jatropha gossypifolia L and Peperomia pellucida (L.) Kunth were tested. The EOs effectively reduced oxidants to neutral molecules in a concentration-dependent manner [50,51]. , Mentha spicata EOs, and the EO of the Pistacia vera L. variety Bronte were analyzed using DPPH assay. The highest antioxidant activity was obtained from the EO of the Kallar population [84]; in contrast, balsam fir, black spruce, white spruce, tamarack, and eastern white cedar oils again exhibited very poor antioxidant activities [85]. The antioxidant ability of the spearmint oil was 3 µg/mL, in comparison to 11.5 µg/mL for the standard compound. This interesting biological activity can be explained by the presence of the monoterpenes limonene, terpinolene, γ-terpinene, 1,8-cineole, and carvone in the EO [88]; the Pistacia vera L. variety Bronte showed little affect against the DPPH test [64].
The antioxidant properties of aerial parts of Glycyrrhiza triphylla Fisch. and CA Mey and parsley, lovage, basil, and thyme EOs were investigated with DPPH and BCBT assays. G. triphylla EO exhibited a high antioxidant activity only in terms of the DPPH radical scavenging activity [87]. Parsley and lovage had a weak antioxidant activity, whereas basil showed a moderate antioxidant activity. Finally, thyme EO showed the highest antioxidant capacity [86].
Bergamot and lemon EOs extracted from the fruit peel of several citrus varieties were analyzed to determine their antioxidant activity through a thiobarbituric acid reactive substances (TBARS) test in a fish model (sardine). Samples of sardine treated with the bergamot EO displayed greater antioxidant activity than lemon EO [75].

Immunomodulatory Activity Effects of Essential Oils in Cells and Animals
Inflammation is a complex immune response against different types of harmful factors. Pathogenic microorganisms, irritants compounds, or damaged tissue induce an acute inflammatory response that can persist for a short period of time, which is beneficial for the host. In spite of this, if resolution of the inflammation is not adequate or the stimulus persists, then it is called chronic inflammation, which predisposes the hosts to different diseases such as cancer, cardiovascular disease, neurological disease, and metabolic disorders. During a chronic inflammation response, different signaling pathways are activated, leading to the overexpression of pro-inflammatory genes and proteins such as the NF-κB transcription factor and cytokines including IL and TNF-α. This inflammation is also related to an increased release and accumulation of ROS and reactive nitrogen species (RNS). When ROS production is greater than the cellular antioxidant capacity, oxidative stress can harm lipids, proteins, and DNA [111]. In this sense, EOs are of the greatest interest because of their anti-inflammatory and antioxidant properties, which are a potential source for the development of functional foods.
In general, EOs did not produce any cytotoxic effect when they were used at low concentrations; indeed, in human blood-isolated lymphocytes from healthy donors, Pistacia vera L. EOs significantly increased cell viability [64]. however, a high dose can have a negative effect on cell viability. In the case of malignant cells, it has been described that EOs derived from Heracleum pyrenaicum subsp. orsinii inhibited cell growth, which is in agreement with the established criteria from the National Cancer Institute (NCI), whereas they showed no toxic side effects on normal MRC-5 cells [69]. In line with these results, Pituranthos tortuosus EO is able to inhibit cell proliferation in a concentration-dependent and time-dependent manner on B16F10 melanoma cancer cells, which is likely by an increased apoptotic pathway [91]. Likewise, in human colonic adenocarcinoma cancer cell lines (HT29-D4 and Caco-2 cell), Allium roseum L. EO has a growth-inhibitory effect in a dose-dependent manner, without being cytotoxic. This effect has been attributed to the presence of sulfurous compounds as the major constituents of this EO [68]. Conversely, Cirsium japonicum DC EOs could promote cell proliferation in the human pulmonary adenocarcinoma A549 cell line [92].
On the other hand, in LPS-stimulated murine macrophage RAW264.7 cells, treatment with EOs derived from Trachydium roylei, Artemisia argyi, and Chmaecyparis obtusa has been shown to inhibit the secretion of pro-inflammatory cytokines, whereas treatment with EOs derived from Trachydium roylei also increased the secretion of IL-10, which is an anti-inflammatory cytokine. Therefore, the regulation of cytokines in this cell model may be one of the mechanisms by which EOs have an anti-inflammatory effect [22,90,94,95]. In the case of Artemisia argyri EOs, the regulation of NF-κB and AP-1 translocation has been proposed as a possible mechanism for its anti-inflammatory effect. In addition, a significant phosphorylation of JAK2 and STAT1/3 was also observed, but not the activation of NF-κB and mitogen-activated protein kinase (MAPK) cascades [22]. Other important mediators in inflammation are the production of nitric oxide (NO), secretion of prostaglandin E2 (PGE2), and the production of ROS. EOs have been shown to affect the expression of inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) expression; therefore, they might affect the secretion of NO and PGE2. In line with this, Artemisia argyri and Trachydium roylei EOs have been described to alter iNOS and COX-2 gene and protein expression, and to inhibit NO and PGE2 secretion and ROS production [22,95]. In immune human cells, there are studies showing that EOs exert their anti-inflammatory effects through the regulation of cytokine secretion and ROS production [31,93].
Similarly, in C57BL/6 mice, treatment with EOs has been shown to be efficient in reducing the levels of pro-inflammatory mediators [89,94]. In the contact hypersensitivity response, treatment with Litsea cubea L. EOs was able to inhibit the immune response [89]. In one interesting article, Sutili et al. described the use of Hesperozygis ringes and Ocimun americamun in silver catfish exposed to Aeromonas hydrophila, where this EO significantly decreases the hematocrit values and increases the plasma cortisol level and complement system activity. These results indicated a potential use of EOs in the treatment of infected fish [101].
One possible use of EOs is in poultry production as a supplement in the diet to improve production and to decrease the use of antibiotics. Their use in broiler chicken has been shown to improve animal growth. One interesting point is the effect of EOs in gastrointestinal microbiota composition, where supplementation with them has been shown to exert a positive effect-decreasing the pathogenic microorganism while increasing the number of probiotic bacteria such as Lactobacillus spp. [96][97][98]. Correspondingly, in weaned piglets, EOs or their main active compounds positively modulated gastrointestinal microbiota [23,99,100]. In addition, the use of carvacrol and thymol enriched protein biosynthesis, amino acids, and lipid metabolism [100].
Owing to this, EOs could be useful to inhibit pathogenic bacteria without affecting gastrointestinal commensal bacteria. Using static batch culture systems inoculated with human feces, Thapa el al. have shown that several EO compounds selected for their effectiveness against gastrointestinal pathogen need not have a toxic outcome on commensals bacteria at concentrations that would probably suppress pathogen bacterial growth. In this regard, the relative proportion of bifidobacteria was increased, while Bacteroidetes and Clostridium clusters IV and XIVa were not significantly affected. In terms of fermentation, except for high concentrations of thymol and geraniol, the essential oil compounds had no effects [112].

Conclusions
EOs have important antimicrobial and immunomodulatory properties that make them suitable for food preservation, alternatives to antibiotics, and ingredients in functional foods. In this regard, antimicrobial activity was tested in several strains with a wide range of observed results; the inhibition of S. aureus and even V. cholerae and C. albicans has been reported. Some EOs have demonstrated their efficacy against several foodborne pathogens in vitro and model food systems, and they can be applied in foods to improve their microbiological safety; however, these aforementioned results cannot be always extrapolated. One important effect of EOs is their antioxidant properties, with a dose range between 0.01 and 10 mg/mL. The EOs have demonstrated remarkable antioxidant activities, which can also be attributed to their richness in terms of phenolic derivatives.
Concerning their immunomodulatory effect, most of the articles highlighted that EOs did not produce a cytotoxic effect when they are used at low concentrations. Their immunomodulatory activity can be attributed to their ability to modify the secretion of cytokines, which is probably through the regulation of NF-κB, but also through the MAPK signaling pathway, or through their ability to affect the cellular expression of iNOS and the secretion of prostaglandins.
Although the biological properties of EOS have been studied, there is a need for more well-designed studies, involving a normalization of dose and incubation time in cell and animal models that will allow gaining a better understanding of their biological activities and underlying mechanisms. Well-designed studies in animals and humans are compulsory to evaluate the efficacy of EOs.
Author Contributions: All authors participated in the bibliographic search, discussion, and writing of the manuscript.
Funding: Part of the research currently in progress in the authors' laboratory is funded by the company PAYMSA, S. A. through the grant #4501 managed by the Fundacion General Empresa-Universidad de Granada.