**3. Biocompatibility of ozonated olive oil with skin**

The skin is the largest organ of the body and is the major barrier between the inside and outside of our body. It is formed of two main layers: the epidermis, a thin outer portion, and the dermis, the connective tissue layer of skin. This portion is involved in the thermoregulation process, and the resident dermal fibroblasts secrete collagen, elastin, and substances that offer support and elasticity of the skin. The epidermis is subdivided into four layers: (i) the stratum germinativum (SG) that provides the germinal cells necessary for the regeneration of epidermidis; (ii) the stratum spinosum (SS), in which the cells divided in the SG start to accumulate many desmosomes on their surface; (iii) the stratum granulosum (SGR), in which the keratinocytes accumulate dense basophilic keratohyalin granules that contain lipids, which help to form a waterproof barrier; and (iv) the stratum corneum (SC), the outermost layer, in which the cells are dead. The skin is constantly exposed to the environmental stress of pollutants or cigarette smoke, for example, so it is necessary for the wellness of the skin to preserve it from oxidative stress. To this purpose, there are a variety of antioxidants that include glutathione peroxidase, superoxide dismutase, catalases, and nonenzymatic low-molecular weight antioxidants such as vitamin E isoforms, vitamin C, glutathione (GSH), uric acid, and ubiquinol. Interestingly, the distribution of antioxidants in the SC follows a gradient with higher concentrations in deeper layers [51].

The biocompatibility of Bioxoil™ was investigated by MTT assay on fibroblast 3T3 and on keratinocytes HaCaT and compared with ozonated *dulcis* almond oils and nonozonated olive oil as controls. The MTT test values the cell metabolic activity and, in particular, measures the activity of oxidoreductase by reduction of the tetrazolium bromide dye in formazan. The *dulcis* almond oil was chosen because it is largely used in cosmetics, and as it is an oil, it is a possible vector of ozone. Data reported in **Figure 2** demonstrate the Bioxoil™ biocompatibility for both cell types. The viability of cells never significantly decreased in relation to the control and was the same as the nonozonated oil. Conversely, once ozonated, 10% of *dulcis* almond oil had a very significant negative effect on the viability of fibroblasts and keratinocytes but not when *dulcis* almond oil was nonozonated. Interestingly, the vitality test demonstrated that when these cells are exposed to a mixture of ozonated olive oil and 10% of ozonated *dulcis* almond oil (3:1), the adverse event induced by the ozonated almond oil alone was partially prevented (**Figure 2**).

#### **3.1. Biological action of ozonides**

By combining the beneficial properties of extra virgin olive oil with that of ozone, the ozonated extra virgin olive oil becomes very powerful for the topical treatment of acute and chronic skin lesions. The ozonides generated during the ozonization procedure possess many properties including a high germicidal activity on fungi, yeasts, viruses, and bacteria; activation of local microcirculation; stimulation of granulation and tissue growth; and revitalization of epithelial tissues.

of medicinal herbs. Maoz and Neeman in their research reported an antifungal action of olive oil due to the high content of oleic acid [52]. The ozonated oil increases this natural capacity, because ozone acts on microorganisms, thanks to its greater oxidizing power; in fact, it is able to break down the macromolecules that are the basis of the vital integrity of bacterial cells, fungus, protozoa, and viruses. The ozone in contact with the microorganism's lipoproteins

bactericidal activity, while final lipid oxidation products have induction activity and reactivate metabolic functions (**Figure 3**). Nagayoshi et al. [53] reported the capability of ozone to destroy Gram-positive and -negative microorganisms, and in particular, Gram-negative

O2

Powerful Properties of Ozonated Extra Virgin Olive Oil http://dx.doi.org/10.5772/intechopen.73211 237

assures bacteriostatic and

and the final products of lipid oxidation (LOP); H<sup>2</sup>

forms H2

O2

bacteria are more sensitive.

**Figure 2.** MTT assay on fibroblasts and keratinocytes.

#### *3.1.1. Germicidal activity*

The excessive consumption of antibiotics for the treatment of infectious diseases has fuelled the drug resistance phenomenon, *i.e.,* the microorganisms are resistant to antibiotics. Therefore, it is necessary to develop new molecules with antibiotic properties, preferably natural and nonsynthetic. In fact, this research is divided into the field of synthetic molecules produced by the chemical/pharmaceutical industry and the field of natural active compounds, in which plant extracts are studied also by the use of green chemistry. The plants are able to withstand fungal and bacterial infections and therefore their secondary metabolites can be applied in the field

**Figure 2.** MTT assay on fibroblasts and keratinocytes.

germinativum (SG) that provides the germinal cells necessary for the regeneration of epidermidis; (ii) the stratum spinosum (SS), in which the cells divided in the SG start to accumulate many desmosomes on their surface; (iii) the stratum granulosum (SGR), in which the keratinocytes accumulate dense basophilic keratohyalin granules that contain lipids, which help to form a waterproof barrier; and (iv) the stratum corneum (SC), the outermost layer, in which the cells are dead. The skin is constantly exposed to the environmental stress of pollutants or cigarette smoke, for example, so it is necessary for the wellness of the skin to preserve it from oxidative stress. To this purpose, there are a variety of antioxidants that include glutathione peroxidase, superoxide dismutase, catalases, and nonenzymatic low-molecular weight antioxidants such as vitamin E isoforms, vitamin C, glutathione (GSH), uric acid, and ubiquinol. Interestingly, the distribution of antioxidants in the SC follows a gradient with higher concen-

The biocompatibility of Bioxoil™ was investigated by MTT assay on fibroblast 3T3 and on keratinocytes HaCaT and compared with ozonated *dulcis* almond oils and nonozonated olive oil as controls. The MTT test values the cell metabolic activity and, in particular, measures the activity of oxidoreductase by reduction of the tetrazolium bromide dye in formazan. The *dulcis* almond oil was chosen because it is largely used in cosmetics, and as it is an oil, it is a possible vector of ozone. Data reported in **Figure 2** demonstrate the Bioxoil™ biocompatibility for both cell types. The viability of cells never significantly decreased in relation to the control and was the same as the nonozonated oil. Conversely, once ozonated, 10% of *dulcis* almond oil had a very significant negative effect on the viability of fibroblasts and keratinocytes but not when *dulcis* almond oil was nonozonated. Interestingly, the vitality test demonstrated that when these cells are exposed to a mixture of ozonated olive oil and 10% of ozonated *dulcis* almond oil (3:1), the adverse event induced by the ozonated almond oil alone

By combining the beneficial properties of extra virgin olive oil with that of ozone, the ozonated extra virgin olive oil becomes very powerful for the topical treatment of acute and chronic skin lesions. The ozonides generated during the ozonization procedure possess many properties including a high germicidal activity on fungi, yeasts, viruses, and bacteria; activation of local microcirculation; stimulation of granulation and tissue growth; and revitalization

The excessive consumption of antibiotics for the treatment of infectious diseases has fuelled the drug resistance phenomenon, *i.e.,* the microorganisms are resistant to antibiotics. Therefore, it is necessary to develop new molecules with antibiotic properties, preferably natural and nonsynthetic. In fact, this research is divided into the field of synthetic molecules produced by the chemical/pharmaceutical industry and the field of natural active compounds, in which plant extracts are studied also by the use of green chemistry. The plants are able to withstand fungal and bacterial infections and therefore their secondary metabolites can be applied in the field

trations in deeper layers [51].

236 Herbal Medicine

was partially prevented (**Figure 2**).

**3.1. Biological action of ozonides**

of epithelial tissues.

*3.1.1. Germicidal activity*

of medicinal herbs. Maoz and Neeman in their research reported an antifungal action of olive oil due to the high content of oleic acid [52]. The ozonated oil increases this natural capacity, because ozone acts on microorganisms, thanks to its greater oxidizing power; in fact, it is able to break down the macromolecules that are the basis of the vital integrity of bacterial cells, fungus, protozoa, and viruses. The ozone in contact with the microorganism's lipoproteins forms H2 O2 and the final products of lipid oxidation (LOP); H<sup>2</sup> O2 assures bacteriostatic and bactericidal activity, while final lipid oxidation products have induction activity and reactivate metabolic functions (**Figure 3**). Nagayoshi et al. [53] reported the capability of ozone to destroy Gram-positive and -negative microorganisms, and in particular, Gram-negative bacteria are more sensitive.

in agar medium was demonstrated. We defined the minimal inhibitory concentration (MIC) of 50 mg⋅ml−1 for *S. aureus* and of 25 mg⋅ml−1 for *S. epidermidis*. This study confirms that ozonated olive oil has antimicrobial properties, which can be exploited for cutaneous infections,

The ozonides easily penetrate the cell membrane and, thanks to their biological properties, stimulate skin cells and improve the tropism of the skin by promoting wound healing and repair of ulcers of various kinds; from the experiments, it was found that topical application of products based on ozonides has determined a considerable increase of fibroblasts resulting in increased production of collagen, glycosoaminoglycans, and formation of elastic and reticular fibers. The healing of skin lesions includes complex movements like tissue hemorrhage, inflammation, reepithelialization, granulation tissue, and finally remodeling. These events involve the coordination of many cell types and matrix proteins, which are important for the control of the various stages of tissue repair. Previous studies have shown that endogenous growth factors, such as fibroblast growth factor (FGF), growth factor derived from platelets (PDGF), the TGF-β factor, and vascular endothelial growth factor (VEGF) are important regulators in the healing of wounds [55]. They are released by macrophages, fibroblasts, and keratinocytes at the site of injury and participate in the regulation of re-epithelialization, formation of granulation tissue, collagen synthesis, and neovascularization [56–58]. The beneficial effects of ozone in the treatment of sores are due to the decrease of bacterial infection and the increasing oxygen tension in the wound. In the literature, it is reported that after exposure to ozone transcription factors are activated, such as NF-kB, and these are important regulators of the inflammatory response and tissue repair process [58]. In conclusion, the ozonated olive oil can accelerate acute cutaneous wound repair, by stimulation of dermal fibroblast, and the ozonized oil has shown to be effective against Gramnegative and -positive bacteria, mycetes, and viruses, so it can be used for the cure of infections.

The goal of pharmacological research has always been that of finding drugs that can cure diseases or soothe the pain that derives from them, and this research has evolved over the years in an extraordinary way both in the field of medical knowledge and scientific studies that are more and more powerful and sophisticated. Medicinal herbs, long popular in many parts of the world, are increasingly spreading in the western world and represent a large commercial market with an estimated annual growth of 25%, often replacing synthetic drugs. Among the medicinal herbs, particular interest is reserved to olive oil, called "yellow gold" by the Egyptians for its innumerable beneficial properties. Herbal trade today sees many products based on olive oil for both hygiene and personal care. In the field of oil-based products, particular interest is directed to products containing ozonated oil; these products are the result of the union of the beneficial properties of olive oil with those of ozone. Ozonated oil is the most practical, innovative, harmless, and noninvasive of the techniques of application developed in

, which displays effective disinfectant and stimulatory activities that lead

Powerful Properties of Ozonated Extra Virgin Olive Oil http://dx.doi.org/10.5772/intechopen.73211 239

by slow release of O3

*3.1.2. Biological activity*

**4. Conclusions**

to rapid healing.

**Figure 3.** Molecular mechanism of antimicrobial activity of ozonides: the oxygen internalized from ozonides reacts with the proton/protons in order to form H2 O2 that has a bacteriostatic and bactericidal activity.

**Figure 4.** Growth curves of mycete *Epidermophyton floccosum* in liquid culture medium: 1A, 2A, and 3A are the positive controls with 12.5, 25, and 50 mg/ml of nonozonized olive oil, respectively; 1B, 2B, and 3B are ozonized olive oil samples with 12.5, 25, and 50 mg/ml of ozonized olive oil, respectively; 4 is the negative control.

For virus inactivation, a higher gas dosage than that required for bacteria is necessary. The ozone oxidates and subsequently inactivates the specific viral receptors used to bind the cell wall for virus invasion [54]. We tested the antibacterial activity of Bioxoil™ on mycete *Epidermophyton floccosum*, demonstrating its efficacy in the inhibition of mycete's growth in liquid and agar medium; the bacteriostatic effect is obtained in the presence of 5 μl/ml of ozonated olive oil, and the bactericidal effect is obtained in the presence of 15 μl/ml (**Figure 4**).

The antibacterial activity of Bioxoil™ was also tested on *Staphylococcus aureus* and *Staphylococcus epidermidis*, and its efficacy in inhibiting microbes growing both in liquid medium culture and in agar medium was demonstrated. We defined the minimal inhibitory concentration (MIC) of 50 mg⋅ml−1 for *S. aureus* and of 25 mg⋅ml−1 for *S. epidermidis*. This study confirms that ozonated olive oil has antimicrobial properties, which can be exploited for cutaneous infections, by slow release of O3 , which displays effective disinfectant and stimulatory activities that lead to rapid healing.

#### *3.1.2. Biological activity*

The ozonides easily penetrate the cell membrane and, thanks to their biological properties, stimulate skin cells and improve the tropism of the skin by promoting wound healing and repair of ulcers of various kinds; from the experiments, it was found that topical application of products based on ozonides has determined a considerable increase of fibroblasts resulting in increased production of collagen, glycosoaminoglycans, and formation of elastic and reticular fibers. The healing of skin lesions includes complex movements like tissue hemorrhage, inflammation, reepithelialization, granulation tissue, and finally remodeling. These events involve the coordination of many cell types and matrix proteins, which are important for the control of the various stages of tissue repair. Previous studies have shown that endogenous growth factors, such as fibroblast growth factor (FGF), growth factor derived from platelets (PDGF), the TGF-β factor, and vascular endothelial growth factor (VEGF) are important regulators in the healing of wounds [55]. They are released by macrophages, fibroblasts, and keratinocytes at the site of injury and participate in the regulation of re-epithelialization, formation of granulation tissue, collagen synthesis, and neovascularization [56–58]. The beneficial effects of ozone in the treatment of sores are due to the decrease of bacterial infection and the increasing oxygen tension in the wound. In the literature, it is reported that after exposure to ozone transcription factors are activated, such as NF-kB, and these are important regulators of the inflammatory response and tissue repair process [58]. In conclusion, the ozonated olive oil can accelerate acute cutaneous wound repair, by stimulation of dermal fibroblast, and the ozonized oil has shown to be effective against Gramnegative and -positive bacteria, mycetes, and viruses, so it can be used for the cure of infections.
