Clove Essential Oil (Syzygium aromaticum L. Myrtaceae): Extraction, Chemical Composition, Food Applications, and Essential Bioactivity for Human Health

Clove (Syzygium aromaticum L. Myrtaceae) is an aromatic plant widely cultivated in tropical and subtropical countries, rich in volatile compounds and antioxidants such as eugenol, β-caryophyllene, and α-humulene. Clove essential oil has received considerable interest due to its wide application in the perfume, cosmetic, health, medical, flavoring, and food industries. Clove essential oil has biological activity relevant to human health, including antimicrobial, antioxidant, and insecticidal activity. The impacts of the extraction method (hydrodistillation, steam distillation, ultrasound-assisted extraction, microwave-assisted extraction, cold pressing, and supercritical fluid extraction) on the concentration of the main volatile compounds in clove essential oil and organic clove extracts are shown. Eugenol is the major compound, accounting for at least 50%. The remaining 10–40% consists of eugenyl acetate, β-caryophyllene, and α-humulene. The main biological activities reported are summarized. Furthermore, the main applications in clove essential oil in the food industry are presented. This review presents new biological applications beneficial for human health, such as anti-inflammatory, analgesic, anesthetic, antinociceptive, and anticancer activity. This review aims to describe the effects of different methods of extracting clove essential oil on its chemical composition and food applications and the biological activities of interest to human health.

Banerjee and coworkers [27] observed the anti-inflammatory and wound healing ability of a clove oil emulsion in murine experiments. Eugenol-treated skin showed reepithelialization 20 days after the wound. This result was similar to that of a diclofenac gel and a neomycin cream currently used to control inflammation and heal wounds [27]. Other research reported that eugenol did not modify interleukin 8 (IL-8) levels in human skin cells (HaCat) but instead targeted other pro-inflammatory cytokines [27]. The inhibition of voltage-gated Na + channels modulate the analgesic effects of eugenol. Eugenol induces the activation of transient receptor potential cation channel V1 (TRPV 1 ), an effect similar to local anesthetics such as lidocaine [28].
Eugenol has shown potential anticancer activity against colon, gastric, breast, prostate, and skin cancer, as well as melanoma and leukemia [23]. Eugenol inhibits tumor proliferation and formation, increases reactive oxygen species (ROS), generates apoptosis, and has a genotoxic effect in different cancer cells [23,[29][30][31]. El-Saber Batiha, G. et al. [4] collected information related to Eugenol Pharmacokinetic and Toxicity Studies. They reported that eugenol reached plasma and blood in a half-life of 14-18 h. It also showed a cumulative effect in the treatment of neuropathic pain. Although the Food and Drug Administration (FDA) has confirmed the safety of CEO as a dietary supplement, much attention has recently been paid to its toxicity due to cytotoxic activity against human fibroblasts and endothelial cells. They also reported that eugenol showed a spermicidal effect in vitro and allergic efficacy when used in dentistry [4].

Extraction of EOs
EOs are extracted from plant feedstock by conventional methods, including cold pressing, hydro-distillation, and steam distillation. Additionally, we can include innovative techniques such as microwave-assisted hydrodistillation, microwave-assisted steam distillation, and hydrodistillation assisted by ohmic heating (Figure 2).   [19] and Hatami et al. [20]).

Conventional/Classical Extraction Methods
The conventional extraction methods are based on the distillation process by heating a plant matrix to recover EO [5]. The extraction is done by injecting steam or water, which crosses the plant matter from the bottom up and carries the volatile materials together with the water as if they were a single component. EO is immiscible in water, making it easily removable by decanting [5]. The HD and SD methods are the most extensively used for extracting EOs. These are easy to operate, have high reproducibility, and do not use organic solvents [11,19,52]. However, these methods have several drawbacks, including a long extraction time, the use of large volumes of solvent and energy, and possible thermal degradation and hydrolysis of some of the components of interest from prolonged contact with boiling water or steam. However, these compounds resulting from hydrolysis belong

Conventional/Classical Extraction Methods
The conventional extraction methods are based on the distillation process by heating a plant matrix to recover EO [5]. The extraction is done by injecting steam or water, which crosses the plant matter from the bottom up and carries the volatile materials together with the water as if they were a single component. EO is immiscible in water, making it easily removable by decanting [5]. The HD and SD methods are the most extensively used for extracting EOs. These are easy to operate, have high reproducibility, and do not use organic solvents [11,19,52]. However, these methods have several drawbacks, including a long extraction time, the use of large volumes of solvent and energy, and possible thermal degradation and hydrolysis of some of the components of interest from prolonged contact with boiling water or steam. However, these compounds resulting from hydrolysis belong to the final composition of EO [5]. It is important to note that the conventional methods of extracting essential oils have few adjustable parameters that control the selectivity of the processes and the final concentration of the essential oil [14,19].

Advanced/Innovative Extraction Methods
Advanced extraction methods, including microwave-assisted extraction (MAE), ultrasonic-assisted extraction (UAE), subcritical fluid extraction, and supercritical fluid extraction (SFE), improve extraction performance, reduce extraction time and energy consumption to obtain organic extracts [3,8,10]. These methods improve organic compound extraction yield by applying microwave or ultrasonic energy, which can destroy the cell walls of the plant matrix, allowing the compounds to flow better from the biological material [5,15,21]. Kennouche et al. [21] reported that the essential oil of Eugenia caryophyllata seed obtained by MAE and MSD contained a high percentage of eugenol (65-71%). These extracts preserved their antimicrobial and antioxidant properties. MAE reduces energy consumption, heating time, and organic extract degradation [19,21,22]. Ultrasonic waves from 20 to 100 kHz can be applied by direct contact with the sample (ultrasound system coupled with a probe) or indirectly through the walls of the sample container (ultrasonic bath). Acoustic power and wave frequencies applied in liquid media can produce the acoustic cavitation phenomenon, where the creation, expansion, and implosion of bubbles enhance the selectivity of target molecules [12,53,54].
SFE is used to selectively remove chemical compounds using a solvent in its supercritical state, typically carbon dioxide [55]. Additionally, co-solvents such as methanol, ethanol, or water change the density, viscosity, and solvation power of the supercritical solvent, promoting the extraction of specific compounds [5,14]. The SFE process reduces undesirable organic pollutants, toxins, and pesticide residues present in the biological material [5].

Effect of the Extraction Method on the Concentration of the Main Volatile Compounds of the Essential Oil and Organic Extract
It is well known that differences in the composition of EOs and organic extract depend on the species [7], the phenological stage [9], agroecological conditions [6,8,16,17], pretreatment [11,14], processing conditions [6,[10][11][12][13][14]18], and extraction method [10][11][12][13][14]. Table 2 summarizes the impact of the different extraction methods and conditions on the concentration of the main volatile compounds in cloves. The main volatile compounds obtained by the different extraction methods were similar; however, the concentration of each compound was different. The CEO obtained by the different processes was a characteristic pale yellow color. However, soxhlet extraction (SO) using ethanol could produce a brown organic extract due to impurities, waxes, and organic waste [11]. Golmakani et al. [19] reported that the extraction yield from MA HD after 60 min was similar to the final yield from HD after 240 min. Similarly, MA SD operated almost 4.8 times faster than SD [19]. The MAE reduces the extraction time from 10 to 2 h and generates an increased extraction yield by two-fold, with less severe parameters applied [11,12,14,19]. This occurs because these methods allow the extraction temperature to be reached shorter than conventional methods. However, parameters must be carefully controlled because exposure can alter the chemical composition of EOs [11,12,14,19].
The main factors in the extraction of the organic extract by SFE are particle size, temperature, pressure, and extraction time. Extraction yield increases by decreasing the crushed particle size of the clove because the diffusion paths are shorter and result in less resistance to diffusion between particles. The temperature and pressure of extraction modify the CO 2 density; thus, the extraction yield is higher due to the increased solubility of clove components. However, there is a risk that high-molecular-weight compounds (fatty acids, fatty acid methyl esters, sterols, etc.) may also be extracted in the organic extract [11,12,14].SFE offers substantial advantages over other methods in the extraction of organic extracts, including higher extraction performance, a higher percentage of active antioxidant ingredients, shorter extraction time, etc. [11,12,14].
However, SFE requires expensive equipment, highly trained operators, and high operating and maintenance costs. Likewise, the incorporation of a co-solvent, continuous operation, and CO 2 recycling could increase the cost of separation without considering the impact on the environment [14,20,55]. Hatami et al. [20] conducted an economic evaluation of obtaining a clove extract. They considered a price of USD 40.00/kg of extracted CEO, obtaining a possible annual income equivalent to USD 5.9 million, in addition to a 79% gross margin for every dollar invested, with the time for recovery of the initial investment ranging between 4 and 14 months [20].

Food Applications
In recent years, food industries have faced great challenges in producing safe foods with a longer shelf life while preserving nutritional value and sensory characteristics. The deterioration of food causes significant economic losses. It is also harmful to human health due to the toxic secondary metabolites produced. Growing consumer demand for natural alternatives to synthetic preservatives in food has made EOs a natural substitute due to their antioxidant, antibacterial, and antifungal properties [58][59][60][61][62][63]. However, the main challenge of EOs as food preservatives lies in maintaining their functional properties without changing the taste of food and increasing consumers' appetite (Table 3) [58,63]. Generally, complex food matrices require higher EO concentrations than those used in in vitro tests. For example, foods with high protein content can produce protein-phenolic EO complexes, reducing the effectiveness of EOs [58][59][60]64]. Further, the lipid fraction of food can absorb the antimicrobial agent, reducing its bactericidal action. Likewise, reducing the water content in food could hinder the transfer of antimicrobial agents to the active site in the microbial cell [58][59][60]64]. Furthermore, external factors such as storage temperature, packaging, initial concentration, application method, and the type of microorganism can interfere with the effectiveness of the EO [59,65].

Baked Food
The baked food industry emphasizes the prevention of mold growth and the maintenance of safety and nutrition [58,65,66]. The preservative methods for baked foods include modified storage atmosphere, irradiation, aseptic packaging, and preservative acids. However, the use of organic acids (propionic, benzoic, and sorbic acids) has been restricted in several countries due to their negative impact on human health [58,65,66]. Eugenol provides CEO with broad-spectrum activity against deterioration and foodborne pathogenic microorganisms such as Penicillium spp., Aspergillus spp., Escherichia coli, and Staphylococcus aureus. Adding it to baked foods can extend the shelf life without affecting the original taste, flavor, texture, appearance, or sensory acceptability [58,65,66].

Dairy Products
Consumption of dairy products such as cheese has been responsible for various outbreaks of foodborne diseases [59,71]. Ahmed et al. [59] applied approximately 1 kg of CEO per 200 liters of raw milk as an antimicrobial agent for cheese production. The CEO demonstrated significant antimicrobial action without affecting organoleptic properties during 1 month at 4 • C, showing a potential cost-effective use [59].

Processed Food
In recent years, the market for ready-to-cook processed foods (pre-cooked foods) has expanded due to lifestyle changes and the development of refrigerated distribution networks. The microbial deterioration of processed foods causes unpleasant odors, discoloration, stickiness, sediments, gases, and decreased pH, reducing the quality of food products and putting consumers' health at risk. The addition of 5% (w/w) CEO to processed foods has been shown to have a negative effect on the organoleptic properties of foods, so the focus of its application has been on its use as a flavoring component with antimicrobial and antioxidant properties [62][63][64].

Meat, Poultry, and Seafood Products
The application of CEO to animal food products reduces undesirable reactions involving the deterioration of taste, smell, color, texture, and sensory properties [60,63,67,68,71,73]. Its antimicrobial activity generates a decreased bacterial count, decreases the deamination capacity of non-protein nitrogenous compounds, and reduces hydroperoxide formation due to its antioxidant properties. Mechanisms behind the antioxidant properties of CEO include transition metal binding, inhibition of chain reactions, breakdown of hydroperoxides, and interaction with free radicals. CEO has been applied to white shrimp, salmon burgers, fish fillets, ground beef, chicken patties, and chicken breast meat during refrigerated or frozen storage [60,63,67,70,[73][74][75]. Films fortified with CEO can reduce the loss of weight, water activity, lipid oxidation, color change, and microorganism growth in foods of animal origin for up to 45 days if heat-treated and 12 days if refrigerated [61,68,70].

Vegetables
Post-harvest vegetable deterioration during transport and storage leads to significant economic losses along the supply chain [83,84,86]. The antimicrobial properties of CEO can prevent fungal spoilage in vegetables and adverse health effects, and it can serve as a potential alternative to chemical fungicides. The antimicrobial activity can be improved when it is combined with UV-C light treatment or modified packaging. These processes allow effective control of post-harvest decomposition and preserve the physicochemical quality of vegetables, prolonging their useful life without affecting organoleptic properties [83][84][85][86]. CEO is added to the wash treatment of fresh-cut vegetables as an alternative to acetic acid, sodium bicarbonate, and chlorine-based disinfectants, reducing microbi-ological hazards and extending the shelf life. Additionally, CEO wash does not impact color attributes, bioactive content, composition, or sensory attributes. Therefore, CEO application in conjunction with cold storage is an excellent ecological substitute that could be further improved for commercial applications to bring vegetables to market with better and longer-lasting post-harvest quality with greater consumer acceptance [85,86].

Packaging Materials
Recently, new biodegradable packaging materials have been developed from natural polymers (polysaccharides, lipids, proteins). Their antioxidant and antimicrobial properties can be enhanced by incorporating essential oils to extend the shelf life and reduce or inhibit foodborne pathogens [71,80]. The incorporation of EO in films for coating is aimed at modifying the functional properties, such as water vapor permeability and antimicrobial and antioxidant properties [68,[77][78][79]81]. CEO-fortified films showed antibacterial properties and growth inactivation for up to 21 days due to the penetration and destruction of the cell structure by CEO compounds [79]. The addition of CEO can modify the moisture content of packaging materials, improving the spatial distance within the film matrix, resulting in thicker films [77][78][79][80][81].
The optical properties of films affect the appearance and quality of foods. The application of a coating also reduces the rate of lipid oxidation [77,81]. In this regard, the different coloring components of CEO can change the color of the films [77,81]. Its incorporation increases opacity values due to an increase in light scattering caused by oil droplets in the film network. These reduce transparency, which represents an advantage for photosensitive food [77].
The incorporation of CEO in a film network partially replaces the stronger polymerpolymer interactions with weaker interactions (polymer-oil). This generates a more heterogeneous network and a discontinuous microstructure by a rearrangement of the polymers. Likewise, the incorporation of CEO has a plasticizing effect, decreasing the glass transition temperature and the elastic modulus of films [77][78][79][80][81]. Sarıcaoglu and Turhan [77] observed a decrease in elastic modulus and tensile strength when CEO was added to films made from mechanically deboned chicken meat protein. However, the tensile strength was kept above 3.5 MPa, a recommended value for coating film on food [77,[79][80][81]. These changes in the structure due to the incorporation of CEO also produced rougher and more porous films [77,78,81].

Biological Activities of CEO
CEO has been shown to have different health benefits, mainly due to the eugenol content. However, the other compounds have various health benefits too. The principal biological activities of CEO are shown in Table 4 [4,87].    Source: C commercial CEO, SD steam distillation, HD hydrodistillation.

Antimicrobial
CEO has shown broad-spectrum inhibitory activity against pathogens. The antibacterial mechanism has been related to the -OH groups located at the meta and ortho positions, respectively, in the main chemical composition. These functional groups can interact with the cytoplasmic membrane of microbial cells [92,96,97,99,100]. CEO can permeate through the cell membrane due to its lipophilic properties. The interaction of CEO with polysaccharides, fatty acids, and phospholipids causes loss of cellular membrane integrity, leakage of cellular contents, and interference with proton pump activity, leading to cell death [92,96,97,100,110]. CEO can inhibit Gram-negative bacteria (E. coli, Salmonella, Klebsiella pneumoniae, Erwinia carotovora, Agrobacterium, and Pseudomonas aeruginosa) and Gram-positive bacteria (S. aureus, Streptococcus, and L. monocytogenes), Aspergillus (A. flavus, A. parasiticus, and A. ochraceus), Penicillium, C. albicans, and yeast [92,96,97,100]. CEO inhibits Gram-positive bacteria to a greater extent than Gram-negative bacteria. This is attributed to a diffusible mucopeptide layer in Gram-positive bacteria that makes them susceptible to antimicrobial agents. In contrast, the complex layer of lipopolysaccharide in the outer cell membrane of Gram-negative bacteria can significantly reduce the diffusion rate of lipophilic antibacterial compounds through the cell membrane [97]. Likewise, food-related pathogens have shown greater sensitivity to CEO than probiotics and fungi [100].

Antioxidant
CEO has the antioxidant compounds eugenol, eugenyl acetate, β-caryophyllene, and α-humulene, which protect cells from free radical oxidation. Diseases such as cancer, arteriosclerosis, Alzheimer's disease, and Parkinson's disease are related to the presence of ROS compounds [110]. CEO has shown scavenging activity on radicals and inhibition of lipid peroxidation [41,93]. The hydroxyl group available in eugenol on the aromatic ring is responsible for the antioxidant activity [99]. The phenolic compounds transfer electrons or hydrogen atoms and neutralize them to free radicals, resulting in a blocked oxidative process [100].
CEO has a protective effect on biochemical changes and histopathological injuries in the kidney, liver, and brain induced by ROS. The main ROS changes inhibited were increased lipid parameters (HDL-C, TC, LDL-C, and VLDL), blood electrolyte (Na + , K + , and Cl − ) and creatinine levels in the liver, hepatic enzymes, blood urea, increased liver and kidney weight, increased serum creatinine, and decreased total protein and albumin [102]. Marmouzi et al. [99] reported that CEO antioxidant activity in three test methods was 150 mg TE/g EO for DPPH, 110 mg TE/g EO for ABTS + , and 34 mg AAE/g EO for FRAP.

Insecticidal
Insect-borne diseases are an ongoing challenge to public health. Some species are invasive urban pests, transmitting numerous pathogenic microorganisms and causing allergic reactions and asthma in young and older people. Commonly used insecticides cause significant health problems and have long-lasting adverse effects on the environment. Moreover, an increase in resistance against insecticides has been reported. Due to this, investigations have focused on developing natural insecticides based on EOs to control agricultural and urban pests [105][106][107]. However, their high volatility decreases the time during which EOs remain in the human body, so sometimes several applications are required in a day.
CEO has shown high levels of repellency and fumigant toxicity on flea, aphids, nymphal instars, mites, imported red fire ants, C. pipiens, and American and German cockroaches [103,105,106]. The oviposition-deterrent activity of CEO can be found in other mosquito species (Anopheles stephensi, An. subpictus, Ae. aegypti, C. pipiens, Ae. albopictus, Culex quinquefasciatus, and Cx. tritaeniorhynchus) [105,107]. It targets the egg stage as an oviposition deterrent and the larval stage as a larvicide against Ae. japonicus, Ae. aegypti, and Cx. quinquefasciatus. CEO has shown repellent action in the laboratory and field settings against adult Ae. aegypti, Ae. cinereus, and Ae. communis [105,107].
The primary targets of CEO and other EOs are octopamine and gamma-aminobutyric acid (GABA) receptors and transient receptor potential (TRP) channels [104]. The doseresponse ratio of CEO showed an increased mortality rate with increasing concentration [105]. CEO increased permeability activity on the cell membrane, disrupted the cytoplasmic membrane, and interacted with proteins, ATPase, histidine decarboxylase, amylase, and protease enzymes, which were also inhibited.
Lambert et al. [103] evaluated the activity of CEO against adult C. felis felis and the development of their eggs. The LC 50 was 5.70 µg/cm 2 against adult fleas and 0.30 g/cm 2 against flea eggs; however, the insecticidal activity of eugenol was three times higher [103]. Toledo et al. [104] reported that it had activity against aphids, but not against ladybugs. They reported an LC 95 of 0.17 µL/cm 2 for aphids, while the same dose only had a lethality of less than 18% for Corymbia maculata. The ladybugs that were exposed to CEO did not exhibit impaired locomotion ability. Therefore, it was concluded that the application of CEO represents an alternative to control aphid infestations [104]. Elzayyat et al. [105] evaluated the insecticidal activity against adults and larvae of Culex pipiens, and reported an LC 50 of 0.374 and 0.036%, respectively [105]. Neupane et al. [106] observed that CEO, eugenol, and eugenyl acetate applied at 4.0 mL/cm 2 provided 95, 85, and 87% mortality of German cockroaches, respectively. They also reported repellency for 30 min by applying 80% CEO. Reuss et al. [107] observed that CEO functions as an oviposition repellent and a larvicide, with an LC 50 of 17 mg/L. CEO and its main constituents are products that have low toxicity to mammals and zero residual concentration. Its application is limited to plague insect control, which is essential to prevent infestations in the environment [103].

Antiviral
CEO has shown antiviral activity against Ebola [111], influenza A virus [112], and herpes simplex virus types 1 and 2 [111]. Recent studies by de Oliveira et al. showed that eugenol derivatives could inhibit the activity of the West Nile Virus, providing a promising compound against flaviviruses such as dengue, Zika, and yellow fever [24]. Eugenol has also been studied as a possible inhibitor of the initial stage of HIV-1 infection because it can reduce virus replication. Likewise, eugenol can increase lymphocyte production; therefore, the lymphocyte proliferation capacity of eugenol may be responsible for its anti-HIV-1 activity [25].
CEO has demonstrated antiviral activity against feline calicivirus, which is used as a substitute for human norovirus. For this reason, the application of CEO in the process of washing fruits and vegetables eliminates any viral load that may exist. In addition, the application of CEO in cleaning wipes allows the decontamination of surfaces [113]. Furthermore, CEO has been shown to increase the resistance of tomato plants to tomato yellow leaf curl virus more than moroxydine hydrochloride [114].

Antinociceptive
Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs to treat inflammatory nociceptive pain. Their principal mechanism is cyclooxygenase (COX) inhibition, decreasing the prostaglandins that cause nociceptive pain. The antinociceptive and anti-inflammatory activities of eugenol are related to COX-2 inhibition and vanilloid transient receptor potential (TRPV) by high-voltage Ca 2+ current inhibition in primary afferent neurons [101]. This antinociceptive response is related to opioid, cholinergic, and α2-adrenergic receptors, but not serotoninergic receptors. The antinociceptive effect of eugenol is probably related to gamma-aminobutyric acid (GABA) receptor modulation, because eugenol administration inhibits GABA receptor currents in trigeminal ganglion neurons and inhibits GABA α1β2γ2 expressed in these neurons [28,101].

Anti-Inflammatory and Wound Healing
Oxidative stress and inflammation are near-related processes in many pathophysiological conditions such as diabetes, hypertension, and cardiovascular and neurodegenerative diseases [99]. The anti-inflammatory properties of CEO and eugenol are comparable to diclofenac gel, reducing inflammation by 60 to 20% after 3 h. Likewise, induced wounds in rats treated with CEO showed a significant contraction of more than 95% in the first 15 days. These results demonstrate that animals treated with CEO underwent similar healing to those treated with neomycin, which is currently used to control inflammation and heal wounds. Therefore, the chronic and acute side effects of synthetic antibiotics can be avoided, especially if they are given frequently [27]. CEO inhibited important antiproliferative biomarkers whose activity depends on their concentration. It decreased the levels of inflammatory biomarkers such as VCAM-1, IP-10, I-TAC, and MIG, in addition to inhibiting the tissue remodeling protein molecules collagen I, collagen III, M-CSF, and TIMP-1 [26,87,109]. The application of CEO can reduce epidermal thickness and the number of inflammatory cells expressing COX-2 without affecting COX-1. The mechanism of eugenol, as an anti-inflammatory, inhibits the expression of COX-2 and reduces the production mediators of inflammation [26,87]. Eugenol has also been reported to not alter IL-8 levels in human skin keratinocytes but to target other pro-inflammatory cytokines in pre-inflamed human dermal cells [27]. These results suggest that CEO possesses anti-inflammatory activity and favors wound healing.

Analgesic
Headaches, joint pain, toothaches, and oral hygiene issues have traditionally been treated with aromatherapy and CEO. The CEO and eugenol are safe, effective, and inexpensive analgesics, and the analgesic effect of eugenol in different pain models has been well documented [28]. Khalilzadeh et al. [28] reported that the analgesic effect of CEO is mediated by the opioidergic and cholinergic systems. The analgesia produced by CEO in acute corneal pain appears to depend on the cholinergic activity. The analgesic and local anesthetic effects of eugenol can be modulated by its inhibitory effect on voltage-gated channels (Na + and Ca 2+ ) and activation of TRPV1. The analgesic effects of CEO and eugenol are very similar to those of lidocaine. Correia et al. [88] demonstrated the analgesic efficacy of CEO in fish. When it was used in concentrations between 40 and 80 µL/L in procedures that were invasive or could cause pain, an analgesic effect in animals was reported, minimizing the effects of harmful stimuli. CEO has potential for use in painful procedures, to minimize the effects of harmful stimuli for ethical reasons, and to ensure the welfare of the animal, avoiding stress and its negative consequences [88,105,115].

Anesthetic
CEO is recognized as an anesthetic at low concentrations (50-500 µL/L) in vertebrates and invertebrates without side effects. It induces anesthesia faster, has brief reflex recovery, and shows a low mortality rate without affecting external stimulus response [88,90,91]. Recent studies showed that topical application of CEO and eugenol reduces corneal sensitivity in rats similar to lidocaine [28]. The maximum level and duration of anesthesia depending on the concentration and time of exposure, which differs between chemicals. CEO efficiently induces anesthesia in Nile tilapia, cardinal tetra, ringed cichlid, and angelfish, affecting swimming ability and balance, and decreasing the response to external stimuli until complete immobilization. Depending on the concentration of the dose, the time to achieve full anesthesia is decreased. Furthermore, there are no side effects of CEO based on the concentration and time of exposure when recovering from anesthesia [28,89,91]. CEO is an effective anesthetic for red claw crayfish and other crustaceans, including Nephrops norvegicus and grass shrimp. Induction and recovery times increase with increased crayfish size, as these are related to oxygen demand. Absorption and elimination of CEO are measured by the oxygen consumption rate, the relationship between the body and the gill surface, and the gill infusion rate. Size is inversely related to anesthetic efficacy [90]. For invasive and painful procedures, the use of CEO is recommended due to its better anesthetic effect [88].

Anticancer
The eugenol, α-humulene, and β-caryophyllene components of CEO, which have cytotoxic and antitumor activity, have been used as alternatives in the prevention and co-treatment of cancer. Some reports suggest that EOs reduce the side effects of chemotherapy, which include nausea, vomiting, loss of appetite, and weight loss [110,116,117]. The anticancer activity is mainly attributed to the antioxidant and anti-inflammatory activity, since the production of ROS specifically activates signaling pathways and contributes to the development of tumors by regulating cell proliferation, angiogenesis, and metastasis [110,116]. CEO has been tested against different cancer types, such as colon [41,93], lung [93,95], breast [41], pancreatic [41], leukemia [41,94], cervical [41], and prostate [41,93].
The anticancer properties are due to the following mechanisms: the activation of detoxifying enzymes, the destruction of DNA by oxidative stress, antimetastatic and cytotoxic activity, decreased viability, cell cycle arrest or apoptosis, the reduction of phosphate-Akt expression levels, and MMP-2 and protein leakage [87,92,95,108]. CEO has shown a low cytotoxic effect on normal cells, improving their antiproliferative activity [87,95,108].

Conclusions and Future Prospects
CEO is a food additive generally recognized as safe by the FDA. The chemical composition of CEO is directly affected by the phenological stage, agroecological conditions, pretreatment, processing conditions, and extraction methods. Innovative methods allow the selective extraction of bioactive compounds responsible for their health benefits. Eugenol, β-caryophyllene, α-humulene, and eugenyl acetate are the main volatile compounds with antioxidant, antimicrobial, anti-inflammatory, analgesic, antiviral, and anticancer properties. The CEO's antioxidant and antibacterial activities have encouraged their application in meat, poultry and seafood, vegetables, dairy products, and edible coating films in the food industry.
Even though CEO is widely consumed and applied, there are still potential areas for investigation. More studies are needed to define the roles of the main components in the various biological activities for potential application in the treatment of different diseases. In addition, it is necessary to determine whether there is synergy or antagonism among these components. Likewise, it is necessary to study the application of CEO in the food industry, mainly its use as an antioxidant or antimicrobial agent without negatively affecting the color, taste, smell, and texture of foods. Few reports were found on CEO encapsulation and its effects on the main physicochemical and biological properties. More research is still required to determine the effect of encapsulation systems on solubility, absorption, bioavailability, and shelf life by avoiding degradation (photo, oxidative, or thermal) and its effect on organoleptic properties.
Despite all the studies carried out, some properties and applications have not been thoroughly investigated. Thus, opening possibilities for investigating the effect of CEO against other diseases and its future application in industries such as pharmaceuticals, foods, cosmetics, dentistry, agriculture, and others.