WO2012168770A1

WO2012168770A1 - Process for ozonization of a vegetable oil

Info

Publication number: WO2012168770A1
Application number: PCT/IB2011/055394
Authority: WO
Inventors: Luciana DINI; Elvira PALMA
Original assignee: Universita' Del Salento
Priority date: 2011-06-10
Filing date: 2011-11-30
Publication date: 2012-12-13
Also published as: ITMI20111045A1

Abstract

A process for the ozonization of a vegetable oil, the product obtained with the process and the compositions that contain it.

Description

PROCESS FOR OZONIZATION OF A VEGETABLE OIL

Technical field

The present invention relates to a process for ozonization of a vegetable oil, particularly extra virgin olive oil, to an ozonized oil obtainable with said process, to a composition that comprises it, optionally further comprising vitamin A and/or E, and to said composition for use in therapeutic treatments.

Background art

The ozonization of vegetable oils is a long-known process; it is in fact disclosed in 191 1 in a first patent in the United States (US 984,722). Such document reports the ozonization of coconut oil by using medical oxygen at a temperature above 28 °C, and the process ends when a concentration of ozone of 0.5 mg in 1 ml of oil is reached. Use of the preparation thus obtained to treat nasal infections is reported. Subsequently, other patents were issued, again in the United States, regarding the ozonization of other vegetable oils, including olive oil (US 2,356,062), in which the olive oil is ozonized until it reaches a quantity of ozone equal to 1.85%. Use of the preparation thus obtained to fight skin and mucous membrane infections supported by bacteria and fungi is reported.

In 1970, Beal discloses the process of ozonization of an emulsion of water, soybean oil and pelargonaldehyde, performed in an autoclave (at a temperature between 70 and 100 °C) and under continuing agitation. The ozonization reaction is then followed by a reduction with hydrogen, and a compound that is very rich in aldehydes (i.e., ozonides) is thus obtained (US 3,504,038). In previously published documents, Otsuki and Maggiolo, too, describe the ozonization of vegetable oils emulsified with large quantities of water (US 2,862,940 and US 2,865,937).

Other patents disclose the ozonization of vegetable oils (corn, olive and sesame); in particular, US 4,451 ,480 ( 1984) discloses the ozonization of these oils to saturation, i.e., when all the double bonds of the fatty acids have broken to bind the ozone.

Subsequently, DeVillez discloses a method for ozonizing jojoba oil, in which the ozone was bubbled through a porous glass disk at a temperature of 50 °C, in this case again until saturation was reached. Use of the resulting preparation to treat acne is reported (US 4,591 ,602, 1986).

Washuettl and Viebahn also ozonize oil to saturation in order to allow longer preservation. After ozonization, the process that they patented performs an extraction in an acid environment and in the presence of a redox system, in order to separate and isolate the short-chain aldehydes from the ozonized oil. The authors indicate that the best substrate for this procedure is olive oil (US 5, 183,91 1 ).

Two Russian patents (RU2040235A, RU 2131673C 1 ) disclose the use of low-frequency ultrasound combined with the ozonization process. In this manner, the resulting product, again brought to saturation with ozone (peroxide number higher than 1000 meq), is stable for six months, keeping its antibacterial properties unchanged.

Recently, Gomez et al. developed a product formed by a mix of ozonized vegetable oils enriched with lipoic acid, vitamins, essential amino acids and other components, which is indicated for treatment of gastroduodenal ulcers and as a food supplement (WO 01/37829 Al , EP1273295 Al ).

Generally, several processes for obtaining ozonized vegetable oils are described and they all have the common characteristics that the ozonization process is performed up to oil saturation, no additives or solvents are added, and medical oxygen is used to produce the ozone. Moreover, all the ozonization process disclosed in the patents are characterized in that operations occur at ambient temperature or often at temperatures above 50 °C. The temperature increase is a spontaneous process during ozonization, because the ozonolysis reaction is exothermic, and is often used as a parameter to monitor the behavior of the process. The heat is utilized to improve the efficiency of the ozonization, because it induces a higher motility of the molecules and this increases the likelihood that the ozone molecules might collide with the fatty acid molecules, causing their lysis, which then generates ozonide formation. The most important difficulty observed in these methods is the low stability of the compounds obtained by ozonization, which does not allow long-term preservation of the product, since after a few months it loses its biological and antimicrobial properties. For this reason, in recent years some documents describe method which are supplement to ozonization, such as the use of ultrasound. Some documents describe the ozonization of vegetable oil emulsified with water; in this manner, a higher number of aldehydes is obtained. US 2006/0074129 Al ozonizes an emulsion of water and cocoa butter, obtaining a compound which is stable and rich in hydroxyaldehyde, the use of which is reported for treating several pathological conditions.

In other documents (e.g., WO2007046122), in order to accelerate the ozonization reaction, a catalyst such as iron sulfate is used which, by acting as an electron donor, reacts rapidly with the ozone, forming the anion radical of ozone 0₃ ^", which is more reactive than ozone itself against double bonds of fatty acids. However, it has been observed that in this manner the produced ozonides are extremely unstable and therefore the ozonized oil keeps for a limited period (a few days) and it has been hypothesized that the heat generated by the reaction, which is necessary for the reaction to proceed, at the same time causes the instability of the ozonides. The high temperature causes a chaotic distribution of the chains in the molecular structure of the ozonides, generating an unstable system characterized by internal tensions, which add to those caused by the steric hindrance of the ozonides. To stabilize the system, the authors use a redox system that converts the ozonides into peroxides, which are much more stable, since they have a lower steric hindrance and low internal tensions. Once the ozonization reaction has ended, alpha lipoic acid, a powerful antioxidant, is added to the product and reacts with the ozonides, leading to the formation of peroxides. In this manner a product is obtained that is reported to be stable over time.

The problem thus remains of providing a process for preparing, efficiently and without the addition of further oxidants, an ozonized derivative of a vegetable oil that is stable over time for long periods and can be used for example in a pharmaceutical formulation.

Summary of the invention

The aim of the present invention is to provide a process for ozonizing a vegetable oil that does not suffer the disadvantages of the background art.

Within this aim, an object of the present invention is to provide a composition that comprises or consists of ozonized vegetable oil that is useful as a medication.

This aim and these and other objects that will become better apparent hereinafter are achieved by means of a process for the ozonization of a vegetable oil, comprising the treatment of the vegetable oil with ozone or a gaseous mix comprising ozone in a reactor in the presence of a pulsed magnetic field and at a temperature comprised between 2 and 6 °C.

The aim and object of the invention are also achieved by means of an ozonized vegetable oil, particularly an ozonized extra virgin olive oil, that can be obtained by means of said process.

The aim and object of the invention are also achieved by means of a composition comprising the ozonized vegetable oil according to said process and optionally at least one compound selected from vitamin A, vitamin E and mixtures thereof, and by a pharmaceutical composition comprising the ozonized vegetable oil according to said process or said composition optionally comprising vitamin A and/or E and by its use as a medication.

Ways of carrying out the invention

The vegetable oil is preferably an olive oil, more preferably extra virgin olive oil and even more preferably extra virgin olive oil originating from the Salento area, particularly extra virgin olive oil of the Salento area obtained from Ogliarola leccese or Cellina di Nardo cultivars or mixtures thereof.

Vegetable oils are chemically composed of triglycerides in which saturated and unsaturated fatty acids are present and the ozone binds with the unsaturated ones; the higher the content of unsaturated fatty acids of the vegetable oil, the higher the quantity of ozone that is trapped. The reaction of the ozone with the fatty acids that are present in extra virgin olive oil leads to the formation of new molecules, such as ozonides, aldehydes and carboxylic molecules, according to the Criegee mechanism (illustrated in the following scheme).

The formation of unstable primary ozone ( 1 ) is an intermediate step of very short duration. A much more stable ozonide is obtained by settling. Molecules of fatty acids (ozonides) are thus formed in which the double bond is saturated by the tree oxygen atoms (2), trapping it and keeping it stable, making it available and ready for use for topical applications.

It has been found surprisingly that with the ozonization process of the invention, which is characterized by the application of a pulsed magnetic field and of low temperatures, it is possible to obtain a product of higher quality than the same oil subjected to ozonization (for the same time and in the same quantity) without applying the magnetic field and the low temperature.

In the case of the ozonization of olive oil, it has been found that the percentage of oleic acid in oil ozonized in the absence of magnetic field and at ambient temperature is higher than the percentage of oleic acid in olive oil ozonized with the process according to the invention, which indicates a higher efficiency of the process according to the present invention.

This means that with the ozonization procedure according to the invention, with respect to the methods of the background art, a larger number of fatty acid molecules (the most abundant of which is oleic acid) are attacked by the ozone, with consequent production of a larger quantity of ozonides.

The method according to the invention allows to obtain a product of high quality that is stable over time. The low temperatures and the magnetic field, in addition to improving the efficiency of the production process, allow to shorten processing times and consequently to lower production costs.

The product obtained with the method according to the invention is characterized by well-defined chemical parameters, which demonstrate and ensure the effectiveness of the ozonization procedure.

With this process, therefore, an ozonized oil is obtained that has the appearance of a gel with an ozone saturation between 60% and 70% and can be used directly on the skin.

The peroxide number (determined by reaction with I and titration with sodium thiosulphate 0.01 N) of the product obtained at the end of the ozonization procedure according to the present invention is comprised between 600 and 800 meq, and this indicates the high quality of the ozonized oil obtained.

Preferably, at the end of the ozonization process, the finished product is collected in containers which are kept at a temperature between 1 and 6 °C, preferably at 4 °C. The containers are preferably made of dark glass to preserve it from interactions with light and are provided with a screw cap. The ozonization process and the subsequent preservation of the product at low temperatures allows a better outcome of the transformation and higher stability during preservation.

After 12 months the peroxide number drops significantly, but nonetheless remains around 600 and 800 meq, which ensure the effectiveness of the ozonized oil in the several fields of application. The literature in fact reports that ozonized oil maintains an antimicrobial activity if it contains a peroxide number between 600 and 800 meq.

The ozonization process according to the invention can be performed inside a thermostat-controlled chamber, at a temperature between 2 and 6 °C, preferably around 4 °C.

The magnetic field applied in the method according to the invention is a magnetic field between 8 and 12 mT, preferably 10 mT, and is of the pulsed type with a frequency of 13- 130 Hz, preferably 72 Hz, generated for example by an apparatus manufactured by TWIN CEMP S.R.L.

Preferably, in the process according to the invention the ozone concentration in said gaseous mix is comprised between 8 mg/1 and 20 mg/1.

Preferably, in the process according to the invention, a flow of ozone or gaseous mix containing ozone is used which is comprised between 300 and 700 1/h.

Preferably, in the process according to the invention, the pressure inside said reactor is comprised between 0.2 and 1 bar. More preferably, the ozonization procedure is performed at 4 °C. In one aspect of the invention, the ozonization process according to the invention is applied to the extra virgin olive oil obtained from cultivars of the Salento area. The characteristics of extra virgin olive oil obtained from cultivars of the Salento area {Cellina di Nardo and Ogliarola leccese) and ozonized with the process according to the present invention, termed Bioxoil herein, have been compared with those of other ozonized oils, and it has emerged that extra virgin olive oil of the Salento area subjected to the ozonization process according to the invention provides a higher quantity of peroxides and ozonides than other extra virgin olive oils subjected to the same treatment, which gives the product advantageous characteristics.

In one embodiment, the present invention relates to the ozonized extra virgin olive oil obtained by using the process according to the invention starting from extra virgin olive oil obtained from the Salento cultivars Ogliarola leccese, Cellina di Nardo and mixtures thereof.

Use of extra virgin olive oil obtained from the cultivars Ogliarola leccese, Cellina di Nardo and mixtures thereof as raw material allows to further reduce the processing times, since using this initial product higher quantities of ozonides are produced than other extra virgin olive oils.

The best yield in the ozonization process was obtained by using extra virgin olive oil composed of 70% Cellina di Nardo oil and 30% Ogliarola leccese oil.

The Salento extra virgin olive oil used to develop the product according to the invention Bioxoil has a 98% triglyceride content and 2% of minor constituents having a biological action (polyphenols). The ozonization process according to the invention reduces considerably the triglyceride content (less than 50%) and leads to the formation of many compounds (ozonides), especially nonanal aldehyde and nonanoic acid, which contain active oxygen and are responsible for many of the biological activities of Bioxoil. In a preferred embodiment, the oil subjected to ozonization is extra virgin oil which is composed of 70% Cellina di Nardo oil and 30% Ogliarola leccese oil, the applied magnetic field is 10 mT and has a frequency of 72 Hz, the ozonization temperature is 4 °C and the preservation temperature is 4 °C.

In another aspect, the invention relates to a composition comprising a vegetable oil ozonized with the method according to the present invention, particularly an extra virgin olive oil of Salento cultivars, preferably originating from the Salento cultivars Cellina di Nardo and Ogliarola leccese, to which vitamins A and/or E are optionally added at the end of the transformation.

In one aspect, the present invention relates to a pharmaceutical composition which comprises or consists of the ozonized vegetable oil according to the invention, comprising the treatment of the vegetable oil with ozone in the presence of a pulsed magnetic field and at a temperature comprised between 2 and 6 °C.

The vegetable oil ozonized with the method according to the present invention, particularly Bioxoil, is a product for topical use having a plurality of biological properties which are due to the emollient and antioxidant properties of olive oil and to the antimicrobial properties of ozone. The results of the research conducted demonstrate that this oil or Bioxoil has a marked antimicrobial and fungicidal activity and an extremely low cytotoxocity.

The addition to the product obtained with the process according to the invention, particularly based on extra virgin olive oil and more preferably based on extra virgin olive oil obtained from Salento cultivars, of vitamins A and/or E, preferably A and E, which have known beneficial properties on the skin, provide particular characteristics and boost the therapeutic effectiveness of the product. In particular, vitamin A (retinol) helps in soothing inflammations and prevents skin aging, stimulating cell renewal, while vitamin E (tocopherol) is antioxidant, and therefore its use is also useful to further stabilize the product.

Said product thus obtained, characterized by a large peroxide number, by excellent stability over time and by a gradual and long release of ozone once it has been applied to the skin, can be not only an excellent low-cost product to be used in pharmacy and parapharmacy, but also an extremely valid alternative to ozone therapy, which is used currently in medicine for therapeutic purposes.

Clinical ozone therapy is used to treat many types of pathological conditions, from infections to ischemias to neurodegenerative disorders, and consists in injecting ozone, in gaseous form or dissolved in pyrogen-free water, intravenously/arterial ly, endopleurally, intraperitoneally, et cetera.

Ozone therapy is an excellent type of therapy that is often resolutive, even though it has many limiting factors. First of all, it cannot be performed at home, but the patient must go to the medical center, where he/she must be monitored also due to the risk of possible embolisms, it is invasive and often painful, and therapeutic cycles are composed of numerous sessions and it is also expensive.

The product according to the invention, optionally after the addition of vitamin A and/or E, preferably A and E, can be an excellent replacement of conventional ozone therapy for pathological conditions of the skin, since the patient could apply the ozonized oil himself/herself, in his/her own home, without risks and certainly reducing considerably the health costs.

Preferably, said pharmaceutical composition comprises or consists of extra virgin olive oil ozonized by means of said process.

More preferably, in said pharmaceutical composition said olive oil is extra virgin olive oil obtained from the Ogliarola leccese and Cellina di Nardo cultivars.

Even more preferably, said composition comprises an oil obtained by ozonization of extra virgin olive oil composed of 70% Cellina di Nardo oil and 30% Ogliarola leccese oil, in the presence of an applied magnetic field of 10 mT pulsed at a frequency of 72 Hz, at an ozonization temperature of 4 °C and optionally preserved at a storage temperature of 4 °C, and with the addition of vitamin A and E.

Preferably, said pharmaceutical composition is in a formulation for topical use.

In one aspect, the present invention relates to said pharmaceutical composition for use as a medication.

In one aspect, the present invention relates to said pharmaceutical composition for use in the treatment or prevention of at least one pathological condition selected from bedsores, ulcers, mycoses and onichomycoses, labial herpes, aphthae, acne, atypical dermatitides or psoriases.

The following examples illustrate in a non-limiting manner the possible methods of preparation:

EXAMPLES

Example 1

A quantity of extra virgin olive oil which is an extra virgin olive oil composed of 70% Cellina di Nardo oil and 30% Ogliarola leccese oil, equal to 100 ml, placed inside a glass cylinder wrapped with tin foil to prevent light from penetrating, is subjected to an ozonization process for 8 hours, with the application of a magnetic field with an intensity of 10 mT, pulsed with a frequency of 72 Hz at a temperature of 4 °C. Inside the cylinder, immersed in oil, a porous ceramic cylinder is provided which measured 2 cm in length and 1 cm in diameter and is connected, by means of a PVC tube with a diameter of 0.5 cm, to the ozone generator. The flow of ozonized air inside the oil is approximately 400 liters/hour and is propelled with a pressure of 0.2 bars. Such a high gas flow is adopted because this allows to reduce the concentration of the more volatile compounds when they are made to flow out of the reactor during the ozonization process. Moreover, the high flow of gas makes the distribution and diffusion of said ozone inside the oil more uniform.

The composition of the resulting ozonized product was determined by gas chromatography and is given in Table 1 by comparison with the composition of the same oil prior to ozonization.

Table 1

The acidity index, the peroxide number and the iodine number of the resulting product were determined immediately after ozonization and are given in Table 2 together with the same parameters determined after the ozonized product had been stored for 12 months at 4 °C. Table 2

Example 2 (for comparison)

Example 1 was repeated without applying the magnetic field and at a temperature of 25°C.

The composition of the resulting ozonized product was determined by gas chromatography and is given in Table 3 in comparison with the composition of the product of Example 1.

Table 3

The acidity index, the peroxide number and the iodine number of the resulting product were determined immediately after ozonization and are given in Table 4 together with the same parameters determined after the ozonized product had been stored for 6 months at 25 °C.

Table 4

The data presented in Table 3 in fact show that the percentage of oleic acid in Salento extra virgin olive oil obtained from Ogliarola leccese and Cellina di Nardo cultivars (the same one used to produce Bioxoil), ozonized at 25°C and without magnetic field, is approximately 57%, higher than Bioxoil (44.8%), indicating that the ozonization process performed at ambient temperature does not have a high efficiency. The verification of this is the percentages of the ozonides (especially nonanal and nonanoic acid) generated by ozonolysis of oleic acid, which are lower than those of Bioxoil. This means that with the ozonization process according to the invention, with respect to the methods of the background art, a larger number of fatty acid molecules (among which the most abundant is oleic acid) is attacked by the ozone, with consequent production of a larger quantity of ozonide.

Therefore, at ambient temperature the ozonization process, in the times and manners described above, and the subsequent preservation in fact do not ensure that the product acquire and keeps stable over time all the characteristics given above. It can in fact be noted that with ozonization and preservation at 25°C the chemical parameter of the product are lower, which indicates a smaller quantity of ozone trapped by the fatty acid molecules and indicates instability of the product (Table 4).

Example 3

Example 1 was repeated by using a non-Salento extra virgin olive oil, i.e., Cima di Bitonto.

The composition of the resulting ozonized product was determined by gas chromatography and is given in Table 3 in comparison with the composition of the product of Example 1. It can be noted that the Salento extra virgin olive oil subjected to the ozonization process according to the invention yields a higher quantity of peroxides and ozonides than other extra virgin olive oils subjected to the same treament, and this gives the product advantageous characteristics.

The disclosures in Italian Patent Application no. MI201 1A001045, from which this application claims priority, are incorporated herein by reference.

Claims

1. A process for the ozonization of a vegetable oil, comprising the treatment of the vegetable oil with ozone or a gaseous mix comprising ozone in a reactor in the presence of a pulsed magnetic field and at a temperature comprised between 2 and 6 °C, obtaining an ozonized vegetable oil, and optionally the preservation of the ozonized vegetable oil at a temperature between 1 and 6 °C.

2. The process according to claim 1 , wherein the pulsed magnetic field has a frequency between 13 and 130 Hz.

3. The process according to claim 1 or 2, wherein the ozone concentration in said gaseous mix is comprised between 8 mg/1 and 20 mg/1 and optionally the flow is comprised between 300 and 700 1/h, and/or the pressure inside said reactor is comprised between 0.2 and 1 bar.

4. The process according to one of the preceding claims, wherein the vegetable oil is an olive oil, preferably an extra virgin olive oil, more preferably an extra virgin olive oil of a Salento cultivar.

5. The process according to claim 4, wherein the olive oil is an extra virgin olive oil obtained from the Ogliarola leccese and Cellina di Nardo cultivars.

6. An ozonized oil obtainable by means of the process according to one of claims 1-5.

7. A composition comprising an ozonized oil according to claim 6 and optionally at least one compound selected from vitamin A, vitamin E and mixtures thereof.

8. A pharmaceutical composition comprising or consisting of an ozonized oil according to claim 6 or of the composition according to claim

7.

9. The pharmaceutical composition according to claim 8 in a formulation for topical use.

10. The oil according to one of claims 1 to 6 or the composition according to claim 8 or 9 for use in the treatment or prevention of at least one pathological condition selected from bedsores, ulcers, mycoses and onichomycoses, labial herpes, aphthae, acne, atypical dermatitides or psoriases.