**2. Ozone**

proliferation by blocking the cell cycle at G0/G1 and to induce apoptosis in some lines of cancer cells, as demonstrated for HL60 cells of promyelocytic human leukemia [23, 24]. However, compounds with greater biological activity are those containing the ortho-diphenol residues; it has been shown that 3,4-DHPEA and 3,4-DHPEA-EDA are more effective than p-HPEA and p-HPEA-EDA in protecting DNA from damage caused by oxidation [25]. In an *in vitro* study, by examining different virgin olive oil extracts, a chemoprotective effect was demonstrated on HL60 cell lines in relation to their composition but not to the total content of phenolic substances [26]. ROS production is also closely related to inflammatory processes in which the cyclooxygenase enzymes (COX-1 and COX-2), belonging to the oxidoreductase class, catalyze the conversion of arachidonic acid into prostaglandins. The p-HPEA-EDA, also called oleocanthal, has the ability to inhibit the activity of such enzymes and has a pharmacological effect similar to that of ibuprofen, which belongs to the class of nonsteroidal anti-inflammatory drugs [27]. It has also been shown that the consumption of olive oil may improve blood pressure regulation and cholesterol content in the blood; these events, together with the inhibition of platelet aggregation and the reduction of LDL oxidation, are important to prevent the onset of atherosclerotic plaques and, in general, cardiovascular pathologies [28, 29]. Olive oil also contains many monounsaturated fatty acids, including oleic acid, which is a key component of cellular membranes and can progressively replace polyunsaturated fatty acids. Membranes rich in monounsaturated fatty acids are more fluid and less subject to lipid peroxidation [30]. Some studies have also shown that regular intake of this food may result in a reduction in the risk of developing diabetes [31]. The therapeutic properties of olive oil include a laxative effect and stimulation of biliary function [32]. Finally, some studies on animal models have shown that the intake of olive oil can help to counteract the damage caused by epidermal ultraviolet

In recent years, in a number of fields, including cosmetics, there has been a renewed interest in materials of natural origin, particularly those of vegetable origin. Since ancient times, olive oil has been known not only for its high nutritional power but also for its cosmetic and therapeutic properties [34]. In 1971, Thiers was still pointing to its potential use in the cosmetic sector. To date, olive oil is certainly the most appreciated natural ingredient, alongside jojoba and avocado oils. The topical application of olive oil may be advised for its soothing action and its beneficial effects on eczema, surface wounds, and burns [35, 36]. In particular, the presence of phytosterols and triterpenoid compounds offers revitalizing and soothing properties for the skin. Vitamins E and A have an intense antioxidant action and have the ability to prevent irritation and aging of the skin, to help maintain its softness, smoothness, stability, and elasticity. As a result, in the cosmetic field, olive oil can be used to prevent signs of aging as a soothing emollient for dry skin and to strengthen hair [37]. Indeed, it is very often a component of lotions, lip balms, shampoos, bath oils, and massage oils. From a dermatologic point of view, olive oil has also proven to have antimicrobial activity, *in vitro*, against some positive and negative Gram and various types of fungi, including *Candida* spp. [38]. Some components of olive oil, especially certain aliphatic aldehydes, inhibit elastase activity; this enzyme is involved in the virulence process [39]. Olive oil is an important component of some

radiation [33].

232 Herbal Medicine

*1.1.2. Dermatological and cosmetic properties*

Ozone is an oxygen derivative and is known primarily for its ecological role in the Earth's balance, absorbing most of the ultraviolet radiation from the sun and preventing it from reaching humans in a harmful way. It is an unstable gas that cannot be stored; in fact, it dissolves in very short time. Ozone is totally neutral to the human body, and in fact, it does not (i) modify pH, (ii) irritate skin or mucous membranes, (iii) damage hair or clothing, (iv) interact with drugs, and (v) cause allergic reactions. This molecule has been subjected to countless studies, and in particular, its strong oxidation capacity has been tested in order to underline its disinfectant and sanitizing properties principally applied as a disinfectant of drinking and waste water [45–47]. To this purpose, the dedicated design and construction of equipment for the production of gaseous ozone for air and water purification are increasing. But research into the properties of ozone has yielded promising results in biological applications, thus confirming the ozone activity in stimulating natural cell defenses and increasing their energy availability. Indeed, since ancient times, ozone has also been used in a large number of medical indications [48, 49]. Scientific studies have shown that ozone, while being highly unstable, can be trapped inside vegetable oils. These are composed of triglycerides in which saturated and unsaturated fatty acids are present, which have the ability to retain ozone, thus allowing them to prolong their use. In addition, the greater the amount of unsaturated fats present, the greater the amount of ozone that will be retained [50]. Therefore, when extra virgin olive oil is ozonated, the produced product combines the beneficial properties of extra virgin olive oil with those of ozone. There are countless ozone-based products on the market, and in particular, in our laboratory, we have developed Bioxoil™, an ozonated extra virgin olive oil available in pharmacies. This product is exclusively made from olive cultivars from Puglia and Salento and the oil is ozonated by an innovative patented method (number: M2011A001045 titled: "Process for the ozonization of a vegetable oil") that confers quality and efficiency in various fields of application; in particular, Bioxoil™ is indicated for the treatment of acne, herpes, psoriasis, fungal infections, bed sores, and wounds in general, due to its healing and disinfectant properties (**Figure 1**).

Bioxoil™ is produced from extra virgin olive oils from two local *cultivars*, *Ogliarola* and *Cellina,* in Salento (Apulia, Italy). The ozone reacts with unsaturated compounds through the known Criegee mechanism. The quality of extra virgin oil is very important to obtain a higher grade of ozonization; for this reason, during the process, it is necessary to control different physical and chemical parameters. Of these parameters, the most important is the temperature, and in fact, during the reaction, the temperature increases provoking an alteration of antioxidant content. The oils' peroxide content and acidity value that indicate the level of hydrolytic modification and primary and secondary oxidation of oil are analyzed and reported in **Table 1**. Peroxide index (PI) and acidity index (AI) after ozonization of oils increase with respect to relative controls. In particular, *Cellina*'s oil sample has an acidity index of 0.2% and a peroxide index of 12 mmol O2 kg−1, while related ozonated oil has an AI of 1.8% and a PI of 533 mmol O2 kg−1. *Ogliarola*'s oil sample presents an acidity index of 0.3% and a peroxide index of 13 mmoli O2 kg−1, while respective ozonated oil has an AI of 1.3% and the PI increases to 677 mmoli O2 kg−1.

In our experiments, the ability of ozone to react with olive oil and in particular with the carbon-carbon double bonds present in unsaturated fatty acids was demonstrated by gas liquid chromatography (GLC). The composition of fatty acids of each olive oil and respective ozonated oil are analyzed by GLC. Data demonstrate that the amount of oleic acid decreases in both ozonized oil samples: in *Cellina*'s ozonated oil, we observed a 30% reduction, while in *Ogliarola*'s ozonized oil, the reduction was about 26% (**Table 2**). During the ozonization reaction, ozone etches mainly the double bond of acid oleic, the most abundant fatty acid in olive oil (about 80%). This explains the decrease in oleic acid and the contemporary appearance of new compounds, which are the reaction's products, among which are nonanal aldehyde and nonanoic acid, both compounds with nine carbon atoms (**Table 2**). In both cultivar ozonized oils, the composition of fatty acids changes, showing a gradual decrease in unsaturated fatty

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

acids (C 18:1, C 18:2), with a gradual increase in ozone doses.

**Table 2.** Composition of fatty acids in olive oil samples *Cellina* and *Ogliarola*.

**Sample Acidity index (AI), %** 

**(means ± SD)**

**Table 1.** Physicochemical parameters of *Ogliarola* olive oil and *Cellina* olive oil after ozonization procedure.

**Composition of fatty acids (%)** *Cellina Cellina Ogliarola Ogliarola*

Palmitic acid 12.45 11.91 11.89 11.30 Linolenic acid 5.57 2.19 4.96 2.06 Cis-oleic acid **70.27 39.82 67.00 41.25** Trans-oleic acid 8.22 6.68 7.88 7.2 Stearic acid 3.48 1.29 3.32 3.22 Nonanal 0 11.69 0 10.07 Nonanoic acid 0 1.36 0 1.98

*Ogliarola olive oil* 0.2 ± 0.03 12 ± 1.2 *Ozonated Ogliarola olive oil* 1.8 ± 0.02 533 ± 1.5 *Cellina olive oil* 0.3 ± 0.02 13 ± 1.5 *Ozonated Cellina olive oil* 1.3 ± 0.01 677 ± 1.6

**Peroxide index (PI), mmol O2 kg−1,** 

Powerful Properties of Ozonated Extra Virgin Olive Oil http://dx.doi.org/10.5772/intechopen.73211 235

**(means ± SD)**

**Control Ozonated oil Control Ozonated oil**

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

**Figure 1.** Bioxoil™ products. The Bioxoil products have different applications. Bioxoil with a red label is indicated for bed sores; the sky blue label is indicated as soothing medication; the green label is indicated for herpes labialis; the pink label is indicated for acne; and the orange tag is indicated for mycosis.


**Table 1.** Physicochemical parameters of *Ogliarola* olive oil and *Cellina* olive oil after ozonization procedure.


**Table 2.** Composition of fatty acids in olive oil samples *Cellina* and *Ogliarola*.

them to prolong their use. In addition, the greater the amount of unsaturated fats present, the greater the amount of ozone that will be retained [50]. Therefore, when extra virgin olive oil is ozonated, the produced product combines the beneficial properties of extra virgin olive oil with those of ozone. There are countless ozone-based products on the market, and in particular, in our laboratory, we have developed Bioxoil™, an ozonated extra virgin olive oil available in pharmacies. This product is exclusively made from olive cultivars from Puglia and Salento and the oil is ozonated by an innovative patented method (number: M2011A001045 titled: "Process for the ozonization of a vegetable oil") that confers quality and efficiency in various fields of application; in particular, Bioxoil™ is indicated for the treatment of acne, herpes, psoriasis, fungal infections, bed sores, and wounds in general, due to its healing and

Bioxoil™ is produced from extra virgin olive oils from two local *cultivars*, *Ogliarola* and *Cellina,* in Salento (Apulia, Italy). The ozone reacts with unsaturated compounds through the known Criegee mechanism. The quality of extra virgin oil is very important to obtain a higher grade of ozonization; for this reason, during the process, it is necessary to control different physical and chemical parameters. Of these parameters, the most important is the temperature, and in fact, during the reaction, the temperature increases provoking an alteration of antioxidant content. The oils' peroxide content and acidity value that indicate the level of hydrolytic modification and primary and secondary oxidation of oil are analyzed and reported in **Table 1**. Peroxide index (PI) and acidity index (AI) after ozonization of oils increase with respect to relative controls. In particular, *Cellina*'s oil sample has an acidity index of 0.2% and a peroxide index of 12 mmol O2 kg−1, while related ozonated oil has an AI of 1.8% and a PI of 533 mmol O2 kg−1. *Ogliarola*'s oil sample presents an acidity index of 0.3% and a peroxide index of 13 mmoli O2 kg−1, while respective ozonated oil has an AI of 1.3% and the PI increases to 677

In our experiments, the ability of ozone to react with olive oil and in particular with the carbon-carbon double bonds present in unsaturated fatty acids was demonstrated by gas liquid

**Figure 1.** Bioxoil™ products. The Bioxoil products have different applications. Bioxoil with a red label is indicated for bed sores; the sky blue label is indicated as soothing medication; the green label is indicated for herpes labialis; the pink

label is indicated for acne; and the orange tag is indicated for mycosis.

disinfectant properties (**Figure 1**).

mmoli O2 kg−1.

234 Herbal Medicine

chromatography (GLC). The composition of fatty acids of each olive oil and respective ozonated oil are analyzed by GLC. Data demonstrate that the amount of oleic acid decreases in both ozonized oil samples: in *Cellina*'s ozonated oil, we observed a 30% reduction, while in *Ogliarola*'s ozonized oil, the reduction was about 26% (**Table 2**). During the ozonization reaction, ozone etches mainly the double bond of acid oleic, the most abundant fatty acid in olive oil (about 80%). This explains the decrease in oleic acid and the contemporary appearance of new compounds, which are the reaction's products, among which are nonanal aldehyde and nonanoic acid, both compounds with nine carbon atoms (**Table 2**). In both cultivar ozonized oils, the composition of fatty acids changes, showing a gradual decrease in unsaturated fatty acids (C 18:1, C 18:2), with a gradual increase in ozone doses.
