Essential Oils: Chemistry and Pharmacological Activities

In this review, we provide an overview of the current understanding of the main mechanisms of pharmacological action of essential oils and their components in various biological systems. A brief introduction on essential oil chemistry is presented to better understand the relationship of chemical aspects with the bioactivity of these products. Next, the antioxidant, anti-inflammatory, antitumor, and antimicrobial activities are discussed. The mechanisms of action against various types of viruses are also addressed. The data show that the multiplicity of pharmacological properties of essential oils occurs due to the chemical diversity in their composition and their ability to interfere with biological processes at cellular and multicellular levels via interaction with various biological targets. Therefore, these natural products can be a promising source for the development of new drugs.


Introduction
The term essential oil was created in the 16th century and refers to the theory of "Quinta essentia" proposed by the famous German-Swiss alchemist and physician Paracelsus (1493-1541), born by Theophrastus Philippus Aureolus Bombastus von Hohenheim. Paracelsus defined the role of alchemy by developing plant extracts and herbal medicines. He believed that the distillation process extracted the most significant part of the plant, namely, the "quintessence for healing" or "plant's soul", separating the "essential" constituents from the "nonessential" [1,2].
Many authors have tried to supply a definition of essential oils. According to the "Association Française de NORmalisation" [3] and to the European Pharmacopoeia (Ph. Eur.), an "essential oil" can be defined as a "product obtained from a natural raw material of plant origin, either by distillation with water or steam, or from the epicarp of Citrus sp. fruits by a mechanical process, or by "dry distillation". Essential oil is then separated from the aqueous phase by physical means" [3,4].
Plants can synthesize two kinds of oils: fixed and essential oils. Fixed oils are esters of a glycerol molecule attached to three fatty acids, also called triacylglycerols or triglycerides. Essential oils (EOs), also known as essences, volatile oils, etheric oils, or aetheroleum, are 25].
Isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphat (DMAPP) are the universal precursors of isoprene molecules [26]. In higher plants, two distinct and independent biochemical routes are involved in isoprenoid biosynthesis: th mevalonate (MVA) pathway, the first and classically acknowledged route for biosynthesi of DMAPP and IPP, and the methylerythritol 4-phosphate (MEP) pathway (non mevalonate pathway). Moreover, they may also proceed via a combination of both th MVA and the MEP routes. The MVA pathway is localized to the cytosol, whereas the MEP pathway is bound to the plastidic compartment [27,28].
We can classify the terpenes into several categories according to the number of C5 building blocks in their core structure: hemiterpenes (C5H8), monoterpenes (C10H16) sesquiterpenes (C15H24), diterpenes (C20H32), triterpenes (C30H48), tetraterpenes o carotenoids (C40H64), and polyterpenes [(C5H8)n] [29]. Among the terpenic constituents, th mono-and sesquiterpenes are the most volatile and abundant in essential oils [30,31] Figure 1 contains a general scheme for terpenoid biosynthesis. Monoterpenes (from the Latin mono, or one) are the most representative and simpl terpenes. They are produced from the assembly of two isoprene units (C10), constituting about 90% of plant essential oils and permitting a very large variety of chemical structure (around 1000 metabolites). According to their structural variation, monoterpenes can b divided into different subgroups: acyclics, bornanes, camphanes and isocamphanes fenchanes, thujanes, p-menthanes, and caranes and pinanes ( Figure 2). The variety o possibilities in which these basic skeletons can be rearranged results in amazing structura diversity observed in nature, in which p-menthane-type monoterpenes are the larges group of naturally occurring monoterpenes [21,32]. Monoterpenes (from the Latin mono, or one) are the most representative and simple terpenes. They are produced from the assembly of two isoprene units (C10), constituting about 90% of plant essential oils and permitting a very large variety of chemical structures (around 1000 metabolites). According to their structural variation, monoterpenes can be divided into different subgroups: acyclics, bornanes, camphanes and isocamphanes, fenchanes, thujanes, p-menthanes, and caranes and pinanes ( Figure 2). The variety of possibilities in which these basic skeletons can be rearranged results in amazing structural diversity observed in nature, in which p-menthane-type monoterpenes are the largest group of naturally occurring monoterpenes [21,32]. Sesquiterpenes (from the Latin sesqui, or one and a half) are built from the coupling of three isoprene molecules (C15), occurring naturally in insects and higher plants. They are the most diverse group of terpenoids and may have acyclic (linear), monocyclic, bicyclic, or tricyclic frameworks, showing many unique arrangements. Similar to monoterpenes, sesquiterpenes can also occur as hydrocarbons or contain oxygen functionality, including carboxylic acids, lactones, alcohols, aldehydes, ketones, and epoxides. Chain extension with augmentation in a number of cyclizations as well as biochemical modifications (rearrangement or oxidation) allows a good variety of structures from the sesquiterpenes ( Figure 3) [21,33]. Sesquiterpenes (from the Latin sesqui, or one and a half) are built from the coupling of three isoprene molecules (C15), occurring naturally in insects and higher plants. They are the most diverse group of terpenoids and may have acyclic (linear), monocyclic, bicyclic, or tricyclic frameworks, showing many unique arrangements. Similar to monoterpenes, sesquiterpenes can also occur as hydrocarbons or contain oxygen functionality, including carboxylic acids, lactones, alcohols, aldehydes, ketones, and epoxides. Chain extension with augmentation in a number of cyclizations as well as biochemical modifications (rearrangement or oxidation) allows a good variety of structures from the sesquiterpenes ( Figure 3) [21,33].

Phenylpropanoids
Phenylpropanoids constitute a group of organic compounds synthesized by plants via the shikimate pathway; the aromatic amino acid L-phenylalanine is their biogenetic precursor. The core of phenylpropanoids consists of a phenyl ring connected to a C3 propane moiety. They are found in the plant kingdom, but are less common than terpenes. The wonderful diversity of phenylpropanoids derives from the efficient modification of a very limited set of core structures. Aromatic compounds originated from the shikimate route ( Figure 4) comprise aldehyde, phenol, alcohol, methoxy, and methylenedioxy compounds [34,35]. Chemical structures of different essential oil constituents (mono-and sesquiterpenes, and phenylpropanoids) are depicted in  Generally, essential oils represent a small fraction of plant composition (less than 5% of vegetal dry matter), occurring in specialized secretory structures, such as secretory ducts or cavities, glandular trichomes, and oil cells [36,37]. Volatile oils can be extracted from seeds (  [38,39]. Typically, essential oils obtained from different plant organs of the same plant possess specific chemical compositions.

Phenylpropanoids
Phenylpropanoids constitute a group of organic compounds synthesized by plants via the shikimate pathway; the aromatic amino acid L-phenylalanine is their biogenetic precursor. The core of phenylpropanoids consists of a phenyl ring connected to a C3 propane moiety. They are found in the plant kingdom, but are less common than terpenes. The wonderful diversity of phenylpropanoids derives from the efficient modification of a very limited set of core structures. Aromatic compounds originated from the shikimate route ( Figure 4) comprise aldehyde, phenol, alcohol, methoxy, and methylenedioxy compounds [34,35]. Chemical structures of different essential oil constituents (mono-and sesquiterpenes, and phenylpropanoids) are depicted in

Phenylpropanoids
Phenylpropanoids constitute a group of organic compounds synthesized by plants via the shikimate pathway; the aromatic amino acid L-phenylalanine is their biogenetic precursor. The core of phenylpropanoids consists of a phenyl ring connected to a C3 propane moiety. They are found in the plant kingdom, but are less common than terpenes. The wonderful diversity of phenylpropanoids derives from the efficient modification of a very limited set of core structures. Aromatic compounds originated from the shikimate route ( Figure 4) comprise aldehyde, phenol, alcohol, methoxy, and methylenedioxy compounds [34,35]. Chemical structures of different essential oil constituents (mono-and sesquiterpenes, and phenylpropanoids) are depicted in  Generally, essential oils represent a small fraction of plant composition (less than 5% of vegetal dry matter), occurring in specialized secretory structures, such as secretory ducts or cavities, glandular trichomes, and oil cells [36,37]. Volatile oils can be extracted from seeds (  [38,39]. Typically, essential oils obtained from different plant organs of the same plant possess specific chemical compositions. Generally, essential oils represent a small fraction of plant composition (less than 5% of vegetal dry matter), occurring in specialized secretory structures, such as secretory ducts or cavities, glandular trichomes, and oil cells [36,37]. Volatile oils can be extracted from seeds (Foeniculum vulgare Mill.), flowers (Rosa abietina Gren. ex H. Christ), leaves (Mentha × piperita L.), barks (Cinnamomum cassia (L.) J. Presl), fruits (Citrus sinensis (L.) Osbeck; Spondias mombin L.), grasses (Cymbopogon citratus (DC.) Stapf.), tree blossoms (Cananga odorata (Lam.) Hook. f. & Thomson), rhizomes (Zingiber officinale Roscoe), roots (Vetiveria zizanoides (L.) Nash), woods (Juniperus virginiana L.), gums (Boswellia ameero Balf. f.), and bulbs (Allium sativum L.) [38,39]. Typically, essential oils obtained from different plant organs of the same plant possess specific chemical compositions.
Volatile oils are reasonably widespread in the plant kingdom, being rarely found in both gymnosperms and monocotyledonous angiosperms, and widely distributed in

Mechanism of Antioxidant Activity of Main Essential Oils and Their Constituents
An essential oil is a complex mixture of components, each of which can potentially contribute to its antioxidant activity. As the composition can be influenced by many factors, knowing the efficacy and the mechanism of action of each component allows us to predict the activity of the oil [44]. From a chemical point of view, antioxidant activity is defined as the ability of a given compound, present in small amounts, to protect an easily oxidizable material, such as polyunsaturated lipids, from oxidation [45]. Peroxidation is a radical-chain reaction responsible for the incorporation of O 2 into organic molecules, leading to the formation of hydroperoxides, epoxides, and other oxygenated derivatives (Scheme 1). The main chain-carrying radicals of peroxidation are alkylperoxyls (ROO • ), although other short-lived radicals can play a role in initiation (such as HO • , formed by the Fenton reaction) or in propagation (R • and RO • ) [45]. The antioxidant activity is, therefore, deeply related to the ability of a given molecule to trap ROO • radicals and is described by two independent parameters: the number of radicals trapped by the antioxidant (also known as "capacity" or stoichiometric coefficient) and the rate constant of the reaction with ROO • radicals, k inh . With respect to the chemical mechanism of peroxidation inhibition, essential oil components can be divided into three main groups: phenols, which act as radical trapping agents; highly oxidizable compounds, which enhance the termination; and 1,4-cyclohexadienes, which are able to generate the reducing HOO • radical.

Radical Trapping
Phenolic components comprise mainly carvacrol, thymol, and eugenol. Their antioxidant activity is due to the well-known ability of phenols to donate the phenolic H-atom to ROO • radicals to form resonance-stabilized phenoxyl radicals unable to propagate the oxidative chain (Scheme 1). A recent study has shown that the k inh and the stoichiometry of ROO • trapping of phenolic containing essential oils is equal to the sum of the contributions of their phenolic components [46]. 7  Peroxidation mechanism cumene (R)-limonene -terpinene p-cymene Scheme 1. Chemical mechanism explaining the ability of essential oil components to retard the peroxidation of organic compounds (RH). The two-faced reducing and oxidizing hydroperoxyl radical is evidenced in blue.

Scheme 1.
Chemical mechanism explaining the ability of essential oil components to retard the peroxidation of organic compounds (RH). The two-faced reducing and oxidizing hydroperoxyl radical is evidenced in blue.

Termination Enhancers
In specific cases, small quantities of a highly oxidizable essential oil component can slow down the autoxidation of another oxidizable substrate. The overall effect is strictly related to the nature of the substrate to be protected, as it must have a small propagation constant (k p ) associated with a low termination constant (k t ). Typical values of k p and k t of the oxidizable substrate are available in the literature [47]. Highly oxidizable essential oil components are oxidized with the substrate and, if they form peroxyl radicals able to decay with a high k t , they cause an overall decrease in the ROO • concentration (Scheme 1). An example of this mechanism has been reported by using cumene as an oxidizable substrate, that has low values of k p and k t . The diagram of cumene autoxidation rate vs. essential oil component concentration displays a typical "v" shape because, above a critical concentration, the autoxidation of the essential oil component becomes prevalent (see Figure 5) [48]. In principle, this mechanism could apply to oxidizable substrates in micellar or emulsion systems, where k p and k t are low because they are reduced by diffusion processes. Highly oxidizable essential oil components, as they are small molecules, can diffuse more easily, contributing to the decay of ROO • radicals. This mechanism may account for the antioxidant activity of essential oils having no obvious radical trapping components. idant activity is due to the well-known ability of phenols to donate the phenolic H-atom to ROO • radicals to form resonance-stabilized phenoxyl radicals unable to propagate the oxidative chain (Scheme 1). A recent study has shown that the kinh and the stoichiometry of ROO • trapping of phenolic containing essential oils is equal to the sum of the contributions of their phenolic components [46].

Termination Enhancers
In specific cases, small quantities of a highly oxidizable essential oil component can slow down the autoxidation of another oxidizable substrate. The overall effect is strictly related to the nature of the substrate to be protected, as it must have a small propagation constant (kp) associated with a low termination constant (kt). Typical values of kp and kt of the oxidizable substrate are available in the literature [47]. Highly oxidizable essential oil components are oxidized with the substrate and, if they form peroxyl radicals able to decay with a high kt, they cause an overall decrease in the ROO • concentration (Scheme 1). An example of this mechanism has been reported by using cumene as an oxidizable substrate, that has low values of kp and kt. The diagram of cumene autoxidation rate vs. essential oil component concentration displays a typical "v" shape because, above a critical concentration, the autoxidation of the essential oil component becomes prevalent (see Figure 5) [48]. In principle, this mechanism could apply to oxidizable substrates in micellar or emulsion systems, where kp and kt are low because they are reduced by diffusion processes. Highly oxidizable essential oil components, as they are small molecules, can diffuse more easily, contributing to the decay of ROO • radicals. This mechanism may account for the antioxidant activity of essential oils having no obvious radical trapping components.

Hydroperoxyl radical (HOO • ) generation
Among the highly oxidizable components, γ-terpinene (γ-T) has a unique behavior, leading to an unusual antioxidant activity. After H-atom abstraction, γ-T forms an unstable peroxyl radical which breaks down, yielding para-cymene and a hydroperoxyl (HOO • ) radical (see Scheme 1). This radical has both oxidizing and reducing activity; therefore, it

Hydroperoxyl Radical (HOO • ) Generation
Among the highly oxidizable components, γ-terpinene (γ-T) has a unique behavior, leading to an unusual antioxidant activity. After H-atom abstraction, γ-T forms an unstable peroxyl radical which breaks down, yielding para-cymene and a hydroperoxyl (HOO • ) radical (see Scheme 1). This radical has both oxidizing and reducing activity; therefore, it can donate a H-atom with very high rate constant to ROO • , to the radicals of an antioxidant [49], or to mild oxidizers such as orthoand para-quinones [50]. γ-Terpinene was shown to reduce the rate of autoxidation of methyl linoleate [51,52] and to prolong the duration of the antioxidant activity of α-tocopherol and polyphenols at low (30 • C) [53] and high (130 • C) [54] temperatures. In addition, γ-T enables the antioxidant activity of melanins, which are natural polymers rich in ortho-quinone residues [50].

Methods Suggested to Explore the Radical Trapping Activity
The antioxidant activity of essential oil components is deeply connected to the nature and the physical state of the oxidizable substrate and to their interaction with ROO • radicals. These pieces of information can hardly be obtained by using simplified assays based on the decay of stable radicals, or without the presence of an oxidizable substrate. The use of assays such as DPPH, ABTS, FRAP, and ORAC is, therefore, discouraged, despite the appeal of their simplicity and low cost [45]. More significant insights can instead be achieved by using methods relying on the autoxidation of biologically relevant substrates, such as polyunsaturated lipids (i.e., methyl linoleate, unsaturated phospholipids) in different aggregation states (solution, micelles, vesicles). The oxidation can be accelerated by using azo initiators, iron salts with peroxides, or simply by heating. Recommended methods to measure the progress of the reaction are determination of hydroperoxides or conjugated dienes, formation of malondialdehyde (thiobarbituric reactive substances, TBARS assay), O 2 consumption, β-carotene bleaching test, or consumption of fluorescent probes of lipid peroxidation such as STY-BODIPY [45]. Examples of determination of the antioxidant activity of selected essential oils are reported in Table 1. (a) only components having a concentration larger than 10% are reported.
The antiviral activities of essential oils have been attributed to the ability of essential oils and their components to interfere with viral entry through viral envelope disruption, capsid disintegration, or inhibition of viral binding to host cell receptors. Moreover, the antiviral activities of essential oils could be due to the inhibition of viral replication.
Feriotto et al. demonstrated that EOs of Thymus vulgaris L., Cymbopogon citratus (DC.) Stapf, and Rosmarinus officinalis L. bound to Tat protein of HIV and destabilized the Tat/TAR-RNA complex, which is required for HIV replication, at IC50s of 0.05-0.83 µg/mL [81]. The antiviral activity of thymol, carvacrol and other components of these essential oils has been reported [76,82,83]. Therefore, it is suggested that these components can contribute to the inhibitory action of essential oils that contain them against different viruses. Another study found that Cymbopogon nardus (L.) Rendle. EO inhibited HIV-1 reverse transcriptase with an IC50 of 1.2 mg/mL, and the activity was attributed to β-citronellol [84].
Significant numbers of studies have shown that EOs from Star Anise, Australian tea tree, oregano, Eucalyptus caesia Benth., and Mentha suaveolens Ehrh. exhibit antiviral activities against HSV-1 due to adsorption on viral particles and inhibition of viral attachment to host cells [65,76,85]. Star anise EO was found to be the most potent, with an IC50 of 1 µg/mL and a selectivity index (SI) of 160 [76]. Analysis of EOs' active ingredients identified thymol and carvacrol with antiviral activity (IC50 of 7 µM) and β-caryophyllene (IC50 of 0.25 µg/mL and SI of 140) as the most active anti-HSV-1 components [86,87]. The inhibitory activity of β-caryophyllene was also reported against the dengue-2 virus, with an IC50 of 22.5 µM and SI of 71.1 [76]. In fact, studies have shown anti-HSV-1 activities also of eugenol, limonene, β-pinene, farnesol, and p-cymene [86][87][88][89]. These findings indicate that essential oils from plants containing appreciable amounts of these components, such as β-caryophyllene, could exhibit antiviral activity, including against HSV-1.
EOs have been shown to be effective not only against enveloped viruses but also against non-enveloped viruses. An in silico study suggested that Lavandula stoechas L. essential oil could inhibit SARS-CoV-2 [90]. Osmunda regalis L. EO showed anti-coxsackie viral activity, with an IC50 of 2.24 µg/mL and SI of 789.84, which indicate its high efficacy and low toxicity [76]. It has been reported by another group that oregano EO and its major component, carvacrol, is an effective antiviral against human rotavirus [91]. The previous data (Table 2) indicate that EOs and their constituents, either individually or in combination, may serve as potential antivirals against several viral pathogens.

Antimicrobial Activity
Essential oils are recognized for their role in protecting plant structures against microorganisms. This supports the scientific hypothesis that such oils and their constituents may also present antimicrobial effects against pathogens of human interest. Most scientific investigations to evaluate the antimicrobial effects of essential oils and their constituents are conducted to determine the presence or absence of pharmacological effects and the lowest concentrations capable of inhibiting microbial growth. Research techniques, in general, seek to standardize methodological procedures and define breakpoints, and are thus recommended by research institutions, such as the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which, in many countries, are in turn recognized by regulatory agencies for drug registration. Generally, the techniques include the adoption of agar diffusion and dilution methods [95].
Pharmacological knowledge of antimicrobial activity must involve elucidating the mechanism of action of the evaluated oil and providing information concerning possible toxicological effects and drug interactions. In addition, knowledge of the molecular aspects involved in the observed effects helps to advance proposals involving possible structural changes (to discover new molecules). In this review, we highlight the main mechanisms of action of essential oils and their constituents, which involve their antibacterial, antifungal, antiviral, and antiparasitic activities.

Antibacterial Activity
Although different microbial species represent different molecular targets, the modes of action of substances with antimicrobial activity generally involve functional components of the plasma membrane, the cell wall, cell duplication, or protein synthesis [96].
Observing the effect of essential oils on bacterial morphology helps to understand possible mechanisms of action. Microscopic visualization of changes in the cell wall, the membrane, or cell shape, revealed that the essential oil of Citrus medica L. promoted action against Escherichia coli and Staphylococcus aureus, with cell wrinkling, formation of holes in the bacterial surface, or even plasma membrane ruptures [97]. Essential oils from Origanum species showed antibacterial activity against Haemophilus influenzae and Haemophilus parainfluenzae. Scanning electron microscopy analyses of the H. influenzae and H. parainfluenzae biofilms showed a decrease in the appearance of bacterial clusters due to the action of O. majorana L. essential oil [98].
Due to hydrophobicity, EOs likely act on lipids of the bacterial plasma membrane or mitochondria, functionally impairing these structures, by promoting increased proton permeability, which is measured through membrane electrical conductivity tests [99]. The essential oil of mustard showed damage to bacterial cell membranes of E. coli and Salmonella typhi with loss of cellular structures essential for the survival of the microorganism, through the occurrence of ATP depletion and decreased intracellular pH [100]. The essential oil of Origanum compactum Benth. is capable of inducing the dissipation of gradients of potassium ions, causing loss of action potential in the membrane of Pseudomonas aeruginosa [101]. Observations from scanning electron microscopy indicated that the essential oil of O. compactum Benth. promoted cell wall damage in E. coli and Bacillus subtilis [102]. The rupture of the membrane and cell wall is directly related to the decrease in ATP production at the cell membrane. Carvacrol and thymol promote a decrease in the amount of intracellular ATP and increase extracellular ATP in E. coli. This effect indicates a disrupter on the cytoplasmic membrane [103].
Additionally, the chemical diversity in the composition of essential oils increases the possibility of existing constituents that can impair the synthesis of proteins. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed by Western blotting indicated that carvacrol and p-cymene can promote a decrease in protein synthesis in E. coli [104]. Essential oils may also show mutagenic activity [105]. This effect can be analyzed in gene modulation studies [106,107]. The essential oil of Syzygium aromaticum (L.) Merrill & Perry showed action on the formation of bacterial biofilms, affecting the quorum sensing activity [108].

Antifungal Activity
Essential oils and their constituents act on fungal cell structures in a manner similar to that described for their antibacterial activity: the oils cause changes in functions that are essential for microbial survival. The main targets for these substances are involved in the maintenance of the fungal membrane and cell wall.
Changes in the permeability of the plasma membrane are largely related to the effects of essential oils involving ergosterol. The most studied mechanisms of action include both ergosterol biosynthesis modulation and direct ergosterol binding; these effects were visualized for essential oils of Anethum graveolens L. and Coriandrum sativum L., respectively [109,110]. It has not yet been fully elucidated, but the enzymes involved in the formation of ergosterol can also function as pharmacological targets.
The effect of essential oils of Anethum graveolens L. on the fungal cell membrane promotes an increase in proton pumping activity, with consequent acidification induced by the presence of glucose in the external environment. The action on the mitochondrial membrane may contribute to decreasing intracellular ATPase activity, possibly by action on mitochondrial dehydrogenases. This cellular damage can be measured by the increased production of reactive oxygen species (ROS), an important biochemical marker for the apoptosis process [111,112].
Inhibiting the formation of the fungal cell wall is another potential mechanism of action of essential oils and their constituents. In vitro experimental models, supplemented with sorbitol (an osmotic protector) in the culture medium, and in silico computational studies indicate that certain oils can act on enzymes such as delta-14-sterol reductase and 1,3-β-glucan synthase, which are involved in cell wall synthesis [113,114].
Evaluation of the effect of essential oils and their constituents on fungal micromorphology has helped augment our knowledge of their mechanisms of action. The essential oil of C. sativum caused a significant reduction in the development of hyphae, pseudohyphae, chlamydoconidia, and blastoconidia in Candida albicans [109]. The essential oil of C. sativum L. commonly has a significant amount of linalool and a smaller amount of gamma-terpinene in its chemical composition [112]. These monoterpenes have antifungal action against various strains of fungi [115,116]. Therefore, they may contribute to the antifungal activity of essential oils that contain them. Table 3 presents the principal targets involved in the activity of essential oils and their constituents against fungi and bacteria. Table 3. Main mechanisms of action of essential oils and their constituents against different microorganisms.

Antimicrobial Activity Mechanism of Action References
Antibacterial Plasmatic membrane and wall cell alteration.
[97] Change in electrical conductivity of plasma membrane. [99] Decrease in ATP production.

Antitumor Activity
The in vitro cytotoxic and in vivo antitumor effects of EOs have been widely reported. In particular, many mechanisms of action have been proposed. These include induction of apoptotic cell death that was related to the increase in the level of reactive oxygen species (ROS), suppression of the AKT/mTOR and NF-κB pathways, and activation of the MAPK pathway. For example, EO from the bulb of Allium sativum L. caused cytotoxicity in leukemia cells through the increase in ROS and induction of apoptosis and differentiation [118], while Zataria multiflora Boiss. EO increased ROS and induced apoptosis in colon cancer cells [119]. The EO from seeds of Litsea cubeba (Lour.) Pers. induced apoptotic cell death by suppressing the AKT/mTOR pathway [120]. On the other hand, the cytotoxicity of Cedrus deodara (Roxb. ex D. Don) G. Don bark EO [121] and Euphorbia intisy Drake stem EO [122] was related to the inhibition of the NF-κB pathway. Moreover, Artemisia capillaris Thunb. EO induced apoptotic cell death by activating MAPK [123]. These mechanisms of cytotoxic action are related to the chemical composition of each EO. For example, the in vivo studies carried out on perillyl alcohol showed that this monoterpene can inhibit the prenylation of specific proteins by type I and type II geranylgeranyl-protein transferases. However, it has been shown that it does not alter the farnesyl-protein transferase enzyme in NIH3T3 cells [124]. In addition, the cytotoxic action of perillyl alcohol was investigated against pancreatic cancer cells. It is suggested to inhibit the prenylation of growth-regulatory proteins, such as K-Ras and H-Ras [125]. Furthermore, studies on thymoquinone, an essential oil component found in the Nigella sativa L. plant, showed that it induces p53-independent apoptosis, as reported in tests using human osteosarcoma cells [126]. Psidium guajava L. leaf essential oil showed significant cytotoxic activity against human oral epidermal carcinoma. The main chemical constituents of this oil are limonene (38.01%) and β-caryophyllene (27.98%) [127]. Limonene has antitumor action via several mechanisms of action [13,128,129]. For example, it induces apoptosis of lung cancer cells by promoting autophagy in lung cancer cells [130]. The antitumor action of β-caryophyllene has also been reported. This compound enhances the antitumor activity of cisplatin in lung cancer cell lines via regulation of the cell cycle and apoptosis signaling molecules [131], in addition to other mechanisms of action in several tumor cell lines [128,129]. Therefore, limonene and β-caryophyllene may contribute to the antitumor activity of Psidium guajava L. leaf essential oil and other antitumor oils in which these constituents are present in significant amounts. Table 4 summarizes some cytotoxic/antitumor mechanisms related to EOs. Table 4. Some antitumor mechanisms of action have been reported for essential oils.
--induction of apoptotic cell death by activation of MAPK [123] 8. Anti-Inflammatory Activity

Inflammatory Response
Inflammation is a complex protective response against harmful exogenous stimuli or endogenous signaling, where the immune system plays a key role in eliminating the initial cause and preserving the cellular and tissue structure, which culminates in maintaining homeostasis [147]. The persistence of this response in the absence of aggressive stimuli has no biological relevance and can cause severe tissue damage. For example, chronic inflammation is associated with carcinogenesis and obesity. Therefore, there is a need for clinical intervention [148][149][150].
The triggers of the inflammatory response involve cellular stimulation with the release of mediators, such as pro-inflammatory cytokines (i.e., TNF-α, IL-1β, IL-6, and IL-8), which promote the activation of macrophages and mast cells, that potentiate this effect by activating endothelial cells, increasing vascular permeability, leakage of fluids, proteins, and influx of immune cells (polymorphonuclear) from the circulation to the inflamed site [151]. However, this response, when uncontrolled, can lead to tissue injury.
In this context, several studies have revealed biological applications of essential oils in controlling the inflammatory response, based on their use in traditional medicine [152,153]. Here, we focus on the anti-inflammatory effects and mechanisms of action of essential oils and their main constituents; details can be seen in Figure 6.

Inhibition of the Release of Mediators by Inflammatory Cells
Geranium essential oil is obtained from leaves of Pelargonium graveolens L., a plant species of the Geraniaceae family, widely known as geranium, which is native to South Africa [154]. Geranium essential oil has been used in traditional medicine for treating inflammatory skin conditions, due to its anti-inflammatory, antiseptic, antifungal, and antibacterial potential [155,156]. Using cultured mast cells (CMC) stimulated with IgE, Kobayashi and colleagues (2016) showed that geranium essential oil reduced significantly, and

Inhibition of the Release of Mediators by Inflammatory Cells
Geranium essential oil is obtained from leaves of Pelargonium graveolens L., a plant species of the Geraniaceae family, widely known as geranium, which is native to South Africa [154]. Geranium essential oil has been used in traditional medicine for treating inflammatory skin conditions, due to its anti-inflammatory, antiseptic, antifungal, and antibacterial potential [155,156]. Using cultured mast cells (CMC) stimulated with IgE, Kobayashi and colleagues (2016) showed that geranium essential oil reduced significantly, and dose-dependently, the β-hexosaminidase secretion, but did not inhibit its activity. This enzyme is released when mast cells, resident cells involved in the tissue allergic and inflammatory response, are activated and consequently degranulated [157]. Thus, this result suggests that geranium essential oil suppresses IgE-induced allergic response by inhibition of mast cell degranulation. The authors also characterized the chemical constituents of this essential oil and demonstrated the inhibitory effect of citronellol (54.6% inhibition), citronellyl formate (33.8% inhibition), and geraniol (32.2% inhibition) against mast cell degranulation. Additionally, citronellol inhibited IgE-induced TNF-α production in CMCs, a critical pro-inflammatory cytokine of inflammation, mediating edema, neutrophils recruitment, and activation of T cells to the inflamed site [158,159]. These results support the application of geranium essential oil and especially its major constituent, citronellol, in the treatment of allergic conditions. α-Phellandrene is a monoterpene found in essential oils of a variety of plants, such as Schinus molle L. [160], Monodora myristica (Gaertn.) Dunal [161], and Anethum graveolens L. [162]. Studies have demonstrated the protective effect of α-phellandrene against ifosfamide-induced hemorrhagic cystitis [163], as well as wound healing [164], antihyperalgesic, and antidepressive properties [165]. Regarding the modulatory role on the inflammatory response, a study showed that α-phellandrene inhibited leukocyte influx and a number of rolling neutrophils or adhered to vascular endothelium, associated with carrageenan-induced acute inflammation [166]. These events of inflammation begin with the release of signaling molecules, such as cytokines and chemokines, released by resistant cells and have a fundamental role in the activation of the vascular endothelium, increasing the expression of adhesion molecules [167]. In this sense, the anti-inflammatory effect of the α-phellandrene can be explained, at least in part, by the reduction of the pro-inflammatory cytokines TNF-α and IL-6. Consistent with these results, recently, Gonçalves and collaborators (2020) reported the modulatory effect of α-phellandrene on TNF-α and IL-1β secretion using an experimental model of hemorrhagic cystitis in mice [163]. In addition, the treatment with α-phellandrene reduced the number of degranulated mast cells when mesenteric tissues were incubated with compound 48/80 (a stimulator of mast cell degranulation) [166]. Thus, these results suggest that α-phellandrene promotes mast cell stabilization, reducing the release of pro-inflammatory mediators and modulating inflammatory response.
Bergapten is a furocoumarin that consists of the main component of bergamot essential oil (Citrus bergamia Risso & Poit.) [168] and Cnidium monnieri (L.) Cuss. [169], a member of the Umbelliferae family of plants. Due to its pharmacological activity, bergapten has been used for the management of skin disorders, such as psoriasis, dermatitis, mycosis, and vitiligo. Experimentally, recent studies have reported its anti-inflammatory [170] and anti-allergic [171] effects, and its prevention of osteoporosis [172]. A possible mechanism by which bergapten controls inflammation was recently reported by Adakudugu and collaborators (2020) using an animal model of colitis. Rats treated with bergapten had a reduction in macro-and microscopic colonic lesions and colon weight-to-length ratio, an indicator of colonic edema, promoted by the damaging agent acetic acid. Associated with this, bergapten decreased the number of degranulated mast cells, compared to the acetic acid group [173]. Mast cells are strongly associated with irritable bowel disease (IBD). Barbara and collaborators (2004) demonstrated the effects of these resident cells and their degranulated state: increase in histamine and tryptase significantly increased the colonic mucosa of IBS patients [174]. Thus, it is possible that bergapten promotes mast cell granule stabilization, reducing the release of pro-inflammatory mediators, which culminates in reduced colonic damage.

Interaction with Sensory Receptors
Carvacrol is a monoterpene phenol found in aromatic plants' essential oils, including those of Origanum dictamnus L., O. majorana L., Satureja montana L., and Thymus vulgaris L. [175]. The biological activities of this monoterpene include antinociceptive [176], anxiolytic [177], and anti-inflammatory [178] effects. The possible interaction of carvacrol and its semisynthetic derivative, carvacryl acetate, with membrane receptors has been studied [179,180]. The administration of carvacrol or carvacryl acetate reduced 5-fluorouracilinduced intestinal mucositis, as observed by inhibition of inflammatory (i.e., TNF-α, IL-1β, KC, MPO, NF-Kb, and COX-2) and oxidative stress (i.e., GSH and MDA) markers. Additionally, the pharmacological blocker of the TRPA1 receptor with HC-030031 reversed the protective effect of carvacrol or carvacryl acetate. The TRPA1 receptor acts as a chemosensor for cell damage signals in the lumen gastrointestinal and modulates digestive functions [181], in addition to involvement in the regulation of vascular tone by the release of vasodilator neuropeptides [182]. Additionally, results of molecular docking revealed that carvacrol binds, with a strong affinity to amino acid residues, to the active site of the TRPA1 receptor [179,180]. Together, these results strongly suggest that the anti-inflammatory effects of carvacrol and carvacryl acetate are mediated, at least in part, by its interaction with the TRPA1 receptor.
Eucalyptol is a monoterpene oxide that is the major constituent of essential oils of medicinal plants such as Eucalyptus tereticornis Sm., Eucalyptus globulus Labill. [183], and Croton rhamnifolioides Pax & K.Hoffm. [184]. This monoterpene is used as a therapeutic alternative for respiratory diseases (i.e., asthma, bronchitis, and sinusitis) and pain management [183,185]. The anti-inflammatory effect of eucalyptol and possible action mechanism was evaluated using two animal models of inflammation: (1) Complete Freund's Adjuvant (CFA)-induced paw inflammation and (2) Lipopolysaccharide (LPS)-induced pulmonary inflammation [186]. Using the first experimental model, the authors showed that eucalyptol strongly reduced paw edema, mechanical allodynia, and pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) levels. In addition, the eucalyptol administration reduced the polymorphonuclear (PMN) infiltration, primarily neutrophils, as well as pro-inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) of mice submitted to pulmonary inflammation. After elucidating the anti-inflammatory effect of eucalyptol, the authors used TRPM8 −/− mice to investigate the possible key role of the TRPM8 receptor in this effect. Eucalyptus is known to interact with TRP receptors, including TRPM8, where it has a reducing effect on visceral hypersensitivity [187]. In addition, studies have shown that the activation of the TRPM8 receptor can suppress the inflammatory response in colitis models [188] and during cold stress [189]. In this sense, Caceres and colleagues demonstrated that genetic deletion of the TRPM8 receptor completely abolished the anti-inflammatory effects of eucalyptol in both models and suggested that TRPM8 agonists can be a potential target for inflammatory conditions [186].

Suppression of the NLRP3 Inflammasome
Cinnamomum osmophloeum Kanehira is a native plant species in Taiwan, popularly known as pseudo-cinnamomum or indigenous cinnamon, used in traditional medicine as a therapeutic alternative to treat arthritis and relieve pain and fever. Several research groups have added to the study of the biological activities of essential oils from C. osmophloeum and their main compounds.
In this context, Lee and collaborators (2015) studied the key mechanism involved in the control of inflammatory response promoted by essential oil from C. osmophloeum, linalool chemotype, which also includes cinnamaldehyde as a constituent. Using an experimental model of systemic inflammatory response syndrome (SIRS)-induced endotoxin from Salmonella typhimurium, the authors reported that the essential oil reduced pro-inflammatory cytokine levels, suppressed the TLR4, MD2, and MyD88 expression in mesenteric lymph nodes (MLNs) and ileum mucosa, and reversed the activation of NF-κB in both tissues. In addition, essential oil from C. osmophloeum suppressed NLRP3, ASC, and caspase-1 expression in MLNs and ileum [190]. During the SIRS, the endotoxin interacts with TLR4, which recruits and ligates to Myd88, a signaling cascade for activation of NF-κB and, consequently, gene overexpression of pro-inflammatory mediators. In addition, TLR4 is involved in the activation of NRPL3 inflammasome, a complex of NRLP3, ASC, and pro-caspase-1, culminating in cleavage of precursors pro-IL-1β and pro-IL-18 into mature IL-1β and IL-18 [191]. Studies reported that linalool, the main constituent of essential oil from C. osmophloeum, reduces the LPS-induced lung and liver inflammation [192,193]. These results strongly suggest that the protective response promoted by essential oil from C. osmophloeum is dependent on linalool content.
Pulegone is a monoterpenic compound, serving as the major component of essential oils from plants of Mentha and Schizonepeta genera [194]. Studies reported the potential biological activity of this monoterpene, such as antispasmodic [195], analgesic [196], and reduction of atopic dermatitis [197]. The anti-inflammatory activity of pulegone was recently reported by Yang and colleagues (2019) in an animal model of LPS-induced sepsis. Pulegone ameliorated histological changes in lung tissues and reduced the levels of IL-1β, IL-5, MIP-1β, M-CSF, and GM-CSF in the serum of septic mice. Additionally, pulegone also reduced the mRNA and protein expression of ASC, NLRP3, caspase-1, and P2X7R [198]. In another study, the modulatory effects of pulegone on the NLRP3 inflammasome signaling pathway were investigated in an in vitro model of inflammation in THP-1 cells, established using LPS + ATP or the cellular toxin nigericin. Compared to LPS + ATP/nigericin group, pulegone was able to reduce the secretion of IL-1β and IL-18, as well as ROS levels. Using molecular tools (PCR analysis and immunofluorescence), the authors demonstrated that pulegone reduced the expression of NLRP3, ASC, caspase-1, and IL-1β, as well as colocalization of the NLRP3 and ASC proteins [199]. Thus, these studies reported the ability of pulegone to regulate inflammation by mechanisms that depend, at least in part, on the suppression of the NALP inflammasome.

Effects on NF-κB Signaling Pathway
Citrus aurantium L. var. amara Engl is a member of the family Rutaceae, widely distributed in Southeast Asia, especially in China, and used in the popular medicine and food industry. This species is known in the literature as a rich source of bioactive compounds, such as flavonoids [200], polysaccharides, [201], and essential oils [202]. Shen and colleagues (2017), using an in vitro model of LPS-stimulated RAW264.7 macrophages, showed that essential oil from C. aurantium suppressed the secretion of pro-inflammatory cytokines (IL-6, TNF-α, and IL-1β), NO production, and COX-2 expression. According to the authors, this potent anti-inflammatory effect promoted by essential oils is related to the inhibition of the NF-κB signaling pathway, determined by the inhibitory effect on NF-κB nuclear translocation, IκBα phosphorylation and degradation, and phosphorylation-dependent IκB kinase activation. Associated with this, the essential oil from C. aurantium also inhibited the phosphorylation of JNK and p38, which suggests an inhibitory effect on MAPK. The NF-κB signaling pathways play a pivotal role in regulating the expression of several genes involved in the initiation and development of the inflammatory response, such as proinflammatory cytokines, chemokines, and adhesion molecules [203]. The results also revealed that linalool (64.6 ± 0.04%), α-terpineol (7.61 ± 0.03%), (R)-limonene (6.15 ± 0.04%), and linalyl acetate (5.02 ± 0.03%) are major constituents of this essential oil [201]. In summary, these results suggest that essential oil from C. aurantium reduces the LPS-induced inflammatory response in RAW264.7 macrophages by suppression of MAPK/NF-κB signaling pathways, and its components can act in association or individually to induce this biological effect [201].
Eucalyptus, a genus of the family Myrtaceae, represents several species widely distributed in the world which are of importance economically and for human health. Among them, the species Eucalyptus citriodora Hook. (known as lemon-scented eucalyptus) has been reported in the literature for the extraction of essential oils and their biological potential, such as pesticidal [204], antifungal [205], analgesic, and anti-inflammatory [206].
Recently, Ho and collaborators (2020) reported the anti-inflammatory mechanism of Eucalyptus essential oil against LPS-stimulated RAW264.7 macrophages. The authors initially demonstrated that Eucalyptus essential oil inhibits LPS-induced NO production. Posteriorly, the essential oils were fractionated in eight subfractions (A-H), where fraction F presented high NO inhibitory activity, without affecting cell viability. Furthermore, it reduced the TNF-α and IL-6 levels, and the COX and NOS expression. Using NF-κB reporter cells (RAW Blue macrophages), the authors showed that NF-κB transcriptional activity was inhibited by fraction F in LPS-stimulated macrophages. This effect was confirmed by the suppressive effect of fraction F on phosphorylation levels of IKK-α and IκB-α. In addition, the mechanism of this anti-inflammatory effect was best elucidated by the demonstration of inhibited phosphorylation levels of JNK1/2, p38, and PKC by fraction F, which, when phosphorylated, promote downregulation of the NFkB signaling pathway, reducing the gene transcription essential to inflammatory response maintenance. In this study, the authors also elucidated the chemical composition of fraction F: methylsyringol (41.3%), 4-hydroxyl-benzenemethanol (24.4%), citronellic acid (12.7%), trans, cis-iridolactone (7.6%), menthol (4.6%), 2-phenyl ethyl anthranilate (2.2%), manool (1.8%), citronellyl anthranilate (1.1%), and citronellyltiglate (1.1%) [207]. To our knowledge, there are no reports of biological activities of the first three major compounds. Among the others, menthol is the most studied, and several studies have demonstrated its potential for regulating inflammation by inhibiting the synthesis/release of inflammatory mediators, such as IL-1β, COX-2, PGE2, and TNF-α in animal models of colitis [208], cutaneous carcinoma [209], and cystitis [210]. It was reported that essential oil extracted from Artemisia annua L. inhibits osteoclast differentiation. It was suggested that this action occurs via reduced TRAF6 activation and interaction on the MAPK pathway and NF-κB pathway. In addition, it probably also inhibits the expression of osteoclast-related genes. The essential oil of this plant has several volatile components, such as borneol, terpinen-4-ol, and eucalyptol. These compounds have anti-inflammatory action via different mechanisms of action [211,212]. Therefore, they can contribute to the pharmacological activity.

Regulation of the Intestinal Microbiota and Barrier Function
Using an experimental model of colitis, Zhang and colleagues reported that essential oil of Zanthoxylum bungeanum Maxim. pericarp (ZBEO) ameliorated clinical features of DSS-induced colitis, such as weight loss, disease activity index (DAI), colonic edema, and histopathological alterations. Furthermore, the MPO activity (an enzyme present in granules of PMN) and pro-inflammatory cytokines (TNF-α, IL-1β, and IL-12) levels were reduced in animals treated with ZBEO. This anti-inflammatory effect promoted by ZBEO is fundamental for the regulation of colonic inflammation, since it is known that pro-inflammatory cytokines play a critical role in inflammatory bowel disease (IBD), where they promote the signaling for polymorphonuclear migration to the inflamed site, producing oxidative stress and consequent tissue damage [213]. The authors also studied the barrier function associated with colon damage and demonstrated that ZBEO recovered the expression of TJ protein, ZO-1. Maintaining the integrity of the intestinal mucosa is crucial to prevent the entry of bacteria or other harmful agents. Thus, when there is a disorganization in the rearrangement of junctional proteins, the deep layers of the intestinal wall are reached by luminous contents, leading to a disease state, both intestinal and systemic [214]. It is known that the intestinal microbiota plays a key role in the protection of the epithelium against aggressive stimuli. In this context, the authors showed that animals treated with ZBEO modulated the intestinal bacterial composition, suppressing the E. coli and increasing Lactobacillus and Bifidobacteria levels [215]. These results suggest that ZBOE can be an important product for the pharmaceutical and nutritional industries, due to its potential to regulate microbiota and intestinal inflammation.
Thymol is a natural monoterpene phenolic compound, serving as the main constituent of the essential oils from plants of the genera Lippia and Thymus. Studies have shown the biological activity of thymol in models of depression [216] and sciatic nerve excitability [217], as well as a potential antileishmanial agent [218]. Recently, Omonijo and collaborators, using an LPS-induced inflammation model in IPEC-J2 cells, demonstrated that thymol reduced ROS production and pro-inflammatory cytokine levels altered by LPS. These results suggest that thymol, a phenolic compound, can reduce oxidative stress due to its antioxidant effect and possible modulation of inflammatory signaling pathways (i.e., MAPK/NFkB), leading to a reduction in the inflammatory response and maintenance of cell homeostasis. Additionally, the authors demonstrated that, with a reduction in the oxidative inflammatory response related to LPS, thymol also attenuated the barrier function impairment, as demonstrated by a reduced drop in the TEER and permeability, as well as increased ZO-1 and actin. These data reflect the effect of thymol in the maintenance of epithelial barrier integrity, a pivot factor that reduces the inflammation response. On the other hand, the expression of nutrient (carbohydrates, amino acids, and proteins) transporters altered by LPS incubation had not been altered by thymol treatment [219]. Thus, monoterpenes consist of potential compounds to enhance the intestinal barrier function, associated with inflammatory response and oxidative stress, reinforcing its use as a nutritional supplement. In fact, thymol comprises 32.68% of the essential oil of Lippia gracilis Schauer leaves. This oil has anti-inflammatory activity [220]. In a study carried out with the essential oil of thyme (Thymus vulgaris L.) and its major component, thymol, both showed anti-inflammatory action, evidencing the contribution of this monoterpene in the pharmacological action of the oils that contain it in their chemical composition [221].

Conclusions
The diversity of chemical compounds found in essential oils, with components in variable amounts and proportions, explains their variability of biological and pharmacological activities. The lipophilic profile of these components contributes to their ability to penetrate cells and tissues to reach biological targets and carry out the pharmacological response. In addition, the antioxidant activity of some constituents of essential oils suggests their possible action in restoring balance in pathological disorders associated with oxidative stress, including inflammatory and tumoral processes. Conversely, the bioactivity in antiinfectious screenings shows the ease with which essential oils and their constituents cross the biological membranes of infected cells and/or microorganisms and cause their death. The evidence of these biological events makes this class of natural products a promising source in the search for new drug candidates.