**9. Antimutagenic properties of essential oils**

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

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

economical sources of antimicrobial components [74].

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

**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

Residual hydrosols after distillation of EOs from plant materials can be used as

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].

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].

**162**

survive in spite of mitochondrial damage. It has been recently demonstrated in the yeast *Saccharomyces cerevisiae* that induction of mitochondrial damage transforming Rho+ cells into Rhoo cells and the induction of apoptosis/necrosis by a combined exposure to essential oils and nuclear mutagens caused a striking reduction of the frequency of nuclear genetic events [86]. Typical mutagenic agents were used such as ultraviolet C (UV-C) radiation which forms pyrimidine dimers and 6-4 photoproducts, 8-methoxypsoralen (8-MOP) activated by ultraviolet A (UVA) radiation which forms DNA mono- and biadducts, or methyl methanesulfonate (MMS) which methylates DNA bases. The reduction in mutant frequency in the presence of essential oils was accompanied by a strong synergistic induction of cytoplasmic "petite" mutants. The anti-mutagenic effect was independent of the type of mutations, i.e., reversion, intra- or intergenic recombination. The extent of this anti-mutagenic effect depended on the mutagen and oil concentrations. However, unexpectedly, the mechanism of the decrease of mutagenicity did not depend on the type of essential oil but on the type of mutagen, thus on the type of lesions and consequently on the DNA repair or lesion avoidance system involved. In fact, after combined treatment by UVC or 8-MOP/UVA plus essential oils, the transformation of Rho+ cells into rho0 cells resulted in a decrease of the frequency of mutants accompanied by a slight resistance of the survival [97]. After UVC or 8-MOP/UVA alone, less mutants were also found in a rho0 mutant, i.e., a complete BET-induced rho0 selected by the alcaloid lycorine, than in the wild type Rho+. In that case, the reduction in mutation frequency was the same as that after the combined treatments confirming the importance of mitochondrial dysfunction for these effects [98]. The same decrease of mutant frequencies was also found in a nucleotide excision repair (NER) defective rad3 mutant after UVC/essential oil combined treatment. Thus the error-free NER repair system does not play any role in this decrease and probably not the error-free homologous recombination in the case of 8-MOP/UVA. However, as a function of survival, this additional cytotoxicity caused a notable reduction of the mutant frequencies for a same survival level. The decrease of cell survival was also accompanied by a synergistic increase of cytoplasmic petite mutants. Thus, in this case, the essential oils contributed to the elimination of the cells already affected by MMS, leading potential mutants to death by late apoptosis and necrosis [97]. The reduction by essential oils of the frequency of mutations induced by the mutagens was always accompanied by a synergistic induction of complete petite mutants. Moreover, essential oils alone or in combined treatments mainly induced necrosis rather than apoptosis. This corroborates with the fact that petite mutants were true rho0 mutants unable to perform apoptosis but only able to passively undergo necrosis, since functional mitochondria are necessary to induce apoptosis [99].
