**6. Synergistic and antagonistic effects of components**

When the combined effect of substances is higher than the sum of the individual effects, this is synergy; antagonism happens when a combination shows less effect compared to the individual applications [27]. Synergistic effects of some compounds, in addition to the major components in the EOs, have been shown in some studies [76–78]. Application of a certain combination of carvacrol-thymol can improve the efficacy of Eos against pathogenic micro-organisms [79].

Synergism between carvacrol and p-cymene, a very weak antimicrobial, might facilitate carvacrol's transportation into the cell by better swelling the *B. cereus* cell wall [27]. Antimicrobial activity of combination of cinnamon and clove EOs in vapor phase showed better antimicrobial with less active concentration in the vapor phase compare to liquid phase [63]. Thymol and carvacrol showed synergistic and antagonistic effects, in different combinations of cilantro, coriander, dill and eucalyptus EOs (each containing several components) and mixtures of cinnamaldehyde and eugenol, against *Staphylococcus sp*., *Micrococcus sp*., Bacillus sp. and *Enterobacter sp*. [27]. An antagonistic effect on *Bacillus cereus* was seen in rice when carvacrol and p-cymene were used with salt; high-hydrostatic pressure showed a synergistic effect in combination with thymol and carvacrol against *L. monocytogenes*. Vacuum packing in combination with oregano EOs showed a synergistic effect against *L. monocytogenes* with 2–3 log10 reduction. Similar results have been recorded when clove and coriander EOs have been used against *Aeromonas hydrophila* on vacuumpacked pork. Application of oregano EO has a synergistic effect in modified-atmosphere packaging (MAP) including 40% CO2, 30% N2 and 30% O2. The available oxygen is another factor antagonistic on EO activities; by decreasing the oxygen level, the sensitivity of micro-organisms to the EOs has been increased [27].

*Essential Oils - Bioactive Compounds, New Perspectives and Applications*

health, inter-dental hygiene and to control oral malodor [53].

**4. Effects of essential oils as antifungal agents**

species at 2.0% w/v, 300 μg/ml concentrations [59].

physiological activity [56].

production [59].

carbons [56].

**5. Mechanism of action**

toxicity of the EOs.

of vital cell contents [52]. Impairment of bacterial enzyme systems may also be a potential mechanism of action. Essential oils show bactericidal activity against oral and dental pathogenic microorganisms and can be incorporated into rinses or mouth washes for pre-procedural infection control, general improvement of oral

Some EOs demonstrate a broad range of natural fungicidal effects against post-harvest pathogens, especially because of their bioactivity in the vapor phase for storage applications [54]. However, more time is needed for vaporphase bioactivity effect, possible absorption into the food material needed to be considered [55]. Antifungal activity might be affected by the targeted fungal

The reported effective compounds against food-borne fungi, including *Aspergillus niger*; *A. flavus;* and *A. parasiticus* is carvacrol and thymol [57]. Waterdistilled EO from leaves and flowers of *Micromeria nubigena* H.B.K. (Lamiaceae) also showed anti-fungal properties [58]. Essential oils from thymol at 500 μg/ml concentration and at 1.0% and 100 μg/ml concentrations; cinnamic aldehyde and eugenol extracted from cinnamon and clove at 1.0% and 100 μg/ml concentrations also showed anti-fungal properties [59]. *Aspergillus parasiticus* growth and Aflatoxins production has been inhibited by *Thymus vulgaris* and *Citrus aurantifolia*. On the other hand *Mentha spicata* L., *Foeniculum miller*, *Azadirachta indica* A. Juss, *Conium maculatum*, and *Artemisia dracunculus* has only inhibited the fungal

growth. While *Carum carvi* L. has controlled aflatoxin production without effect on the fungal growth [34]. Linalool and methyl chavicol and vanillin extracted from sweet basil and vanilla at 2000 μg/ml concentration has the same effect on aflatoxin

Hydro-distilled EOs of stems, leaves (at vegetative and flowering stages) and flowers of *Eugenia chlorophylla* O. Berg. (Myrtaceae) and various extracts of thyme, were active against molds and yeasts [59–61], respectively. Oleoresin extracted from cinnamon and clove inhibited mycotoxin-producing *Aspergillus* and *Penicillium*

Higher levels of phenolic compounds in thyme and clove showed antifungal effects. Phenolic compounds, such as allyl isothiocyanate and citralon in mustard and lemongrass, have been more effective as volatiles [62]; however, in EOs such as marjoram oil, they have been effectively antifungal in a matrix with mainly hydro-

It has been demonstrated that the antimicrobial effects of the Eos acts by causing structural and functional damages to the bacterial cell membrane. It is also indicated that the optimum range of hydrophobicity is involved in the

Application of antimicrobials by different exposure methods, such as vapor phase compared to direct contact method, of mustard and clove EOs showed noteworthy differences [63]. The stereochemistry, lipophilicity and other factors affected the biological activity of these compounds which might be altered positively or negatively by slight modifications [64]. It has been shown that plant substances affect microbial cells by various antimicrobial mechanisms, including

**160**

Residual hydrosols after distillation of EOs from plant materials can be used as economical sources of antimicrobial components [74].

Application of nisin with carvacrol or thymol has been positively effective against *Bacillus cereus* with temperatures increasing from 8 to 30°C [27]. Application of nisin with rosemary extract enhanced the bacteriostatic and bactericidal activity of the nisin [80]. Oregano EOs, in combination with modified-atmosphere packaging, have effectively increased the shelf life of fresh chicken [72]. Antimicrobial resistance did not develop in *Yersinia enterocolitica* and *Salmonella choleraesuis* after sub-inhibitory passes with cinnamon, by direct contact or vapor phase [63]. Combination of linalool and 1, 8-cineole (1:1) created more resistance in *E. coli*, compared to application of pure linalool. Either synergism or antagonism of 1, 8-cineole and linalool derived from *Cinnamosma fragrans* could happen against Gram-negative bacteria and *Fusarium oxysporum* [81]. The synergistic effect of different components could offer a way to prevent possible off flavor caused by clove and tea tree when used to protect against *Escherichia coli* O157:H7 and minimize off flavor effects in meat products [32]. Combinations of EOs of oregano and thyme, oregano with marjoram and thyme with sage had the most effects against *Bacillus cereus*, *Pseudomonas aeruginosa*, *Escherichia coli* O157:H7 and *L. monocytogenes* [82].

## **7. Effects of essential oils as antiviral agents**

Synthetic antiviral drugs have been used for the curing of Herpes simplex virus (type I, II) that causes some of the most common viral infections in humans, and can be fatal but not all are efficacious in treating genital herpes infections. HSV-1 and HSV-2 have also developed resistance to one of these (acyclovir) mainly in immuno-compromised hosts. Essential oils are considered to cure these viral diseases because plant extracts have low toxicity as compared to synthetic antiviral drugs. Natural material is considered as potential alternative. The activity of multilamellar liposome showed better improvement by introduction of essential oils with it. Due to presence of citral and citronellal, the essential oil of *Melissa officinalis* L. can inhibit the replication of HSV-2 and the ability to replicate of HSV-1 can be suppressed by incubation with different essential oils in vitro [83]. Of these, lemongrass essential oil possessed the most potent anti-HSV-1 activity and completely inhibited viral replication after incubation for 24 h, even at a concentration of 0.1%. The virucidal activity was found at high levels in peppermint essential oil against HSV-1 and HSV-2. When the viruses were pretreated with essential oil before adsorption than its antiviral activity was confirmed. Junin virus was successfully inhibited by the essential oil of *Lippia junelliana* and *Lippia turbinate* [84]. The essential oils of eucalyptus, *Santolina insularis* and Australian tea tree showed the antiviral effects against HSV-1. These oils gave good result both before and after adsorption. The oil directly inactivated virus particles, thus preventing adsorption of virion to host cells. Isoborneol, a common monoterpene alcohol, showed dual virucidal activity against HSV-1 [85] and specifically inhibited glycosylation of viral polypeptides. The essential oils have shown good results as antiviral but unfortunately, according to our information till now there is no study about the antiviral properties against the major viruses of era such as HIV and hepatitis C viruses [86].

## **8. Effects of essential oils as preservatives**

In-food studies depend on several additional factors, which have not been tested in similar in vitro studies [87]. Spices and herbs can be used as an alternative

**163**

*Essential Oil as Antimicrobial Agents: Efficacy, Stability, and Safety Issues for Food Application*

preservative and pathogen-control method in food materials. Application of both extracts and EOs of plant-origin antimicrobials such as floral parts of *Nandina domestica* Thunb could be a potential alternative to synthetic preservatives [88]. Generally, effective EOs in decreasing order of antimicrobial activities are oregano > clove > coriander > cinnamon > thyme > mint > rosemary > mustard > cilantro/ sage [27]. However, in another study, mint showed less antimicrobial effect compared to mustard [89]. There are differences between in vitro and in-food trials of plant-origin antimicrobials, mainly because only small percentages of EOs are tolerable in food materials. Finding the most inhibitory spices and herbs depends on a number of factors such as type, effects on organoleptic properties, composition and concentration and biological properties of the antimicrobial and the target micro-organism and processing and storage conditions of the targeted food product [82, 90, 91]. In a study on blanched spinach and minced cooked beef, using clove and tea tree EOs, three and four times the MIC in in vitro studies were needed to restrict *E. coli* O157:H7 populations in the food materials [32]. Despite some positive reports in regard to application of plant-origin natural antimicrobials, two major issues are faced regarding application of plant-origin antimicrobials in food: odors created mostly by the high concentrations, and the costs of these materials [68, 69].

Many studies showed that the mutations can be prevented by the inhibition the penetration of mutagens into cells, by adding antioxidants which inactivate the free radicals produced by mutagens, also by activation of cell antioxidant enzymes and by detoxification of mutagens by activation of enzymes by using plant extracts [92]. Some antimutagenic compounds works in two ways those are by promotion of error-free DNA repair or by inhibition of error-prone DNA repair [93]. During recent years the role and reaction of reactive oxygen species (ROS) scavengers, such as glutathione, superoxide dismutase, catalase, N-acetylcystein, provitamins like retinoid, carotenoids and tocopherols, flavonoids and other polyphenols. Also the biochemistry of antimutagens has been published in various documents [94]. However, since the work of [93] on *Escherichia coli*, nobody has examined in more detail this type of antimutagenicity possibly involving interference with DNA repair via intracellular pro-oxidant reactions of the latter compounds or terpenic and phenolic compounds from aromatic plants. Natural compounds, tannic acid and apigenin, reduced the frequency induced by nitropyrenes in CHO cells [95]. *Matricaria chamomilla* essential oil inhibits SCEs (sister chromatid exchanges) induced by daunorubicin and methyl methane sulfonate in mouse bone marrow cells. The aromatic plant *Salvia officinalis* and major components thujone, 1,8-cineole, camphor and limonene inhibit UV-C-induced mutagenesis in *Salmonella typhimurium*, *Escherichia coli* and *Saccharomyces cerevisiae*. The chemical compounds extracted from plats such as α-terpinene, α-terpineol, 1,8-cineole, d-limonene, camphor, citronellal and citral modulate hepatic mono-oxygenase activity by interacting with promutagen or procarcinogen xenobiotic biotransformation [96]. In a more recent study, they showed in the same system that *Origanum compactum* essential oil and some of its sub-fractions and constituents are antimutagenic against the indirect-acting mutagen urethane and also against the direct-acting mutagen methyl methanesulfonate. It is now accepted that pro-oxidant activities can induce late apoptosis and necrosis. Pro-oxidant activities may damage cellular membranes, in particular those of mitochondria, and thus promote the release of Ca++, cytochrome C and ROS (reactive oxygen species). This leads to cell death, at least in mammalian cells, whereas yeast cells are able to

*DOI: http://dx.doi.org/10.5772/intechopen.92305*

**9. Antimutagenic properties of essential oils**

*Essential Oil as Antimicrobial Agents: Efficacy, Stability, and Safety Issues for Food Application DOI: http://dx.doi.org/10.5772/intechopen.92305*

preservative and pathogen-control method in food materials. Application of both extracts and EOs of plant-origin antimicrobials such as floral parts of *Nandina domestica* Thunb could be a potential alternative to synthetic preservatives [88]. Generally, effective EOs in decreasing order of antimicrobial activities are oregano > clove > coriander > cinnamon > thyme > mint > rosemary > mustard > cilantro/ sage [27]. However, in another study, mint showed less antimicrobial effect compared to mustard [89]. There are differences between in vitro and in-food trials of plant-origin antimicrobials, mainly because only small percentages of EOs are tolerable in food materials. Finding the most inhibitory spices and herbs depends on a number of factors such as type, effects on organoleptic properties, composition and concentration and biological properties of the antimicrobial and the target micro-organism and processing and storage conditions of the targeted food product [82, 90, 91]. In a study on blanched spinach and minced cooked beef, using clove and tea tree EOs, three and four times the MIC in in vitro studies were needed to restrict *E. coli* O157:H7 populations in the food materials [32]. Despite some positive reports in regard to application of plant-origin natural antimicrobials, two major issues are faced regarding application of plant-origin antimicrobials in food: odors created mostly by the high concentrations, and the costs of these materials [68, 69].
