Patent Description:
The clinical use of ozonized vegetable oils is broadly known in the art. The products of oxidation that are formed by reacting ozone with the fatty acids contained in vegetable oils have a germicidal, antimycotic and immunostimulating effect when the ozonized oils are applied topically.

By virtue also of their properties of stimulating tissue regeneration, ozonized vegetable oils can thus be used for the topical treatment of wounds, infected wounds in particular.

In order to facilitate the topical application of ozonized vegetable oils and prolong the presence of the product on the area to be treated, such oils are preferably marketed in the form of a gel or an extremely viscous liquid. Completely ozonized vegetable oils having a stable gel form can be simply produced by bubbling ozone inside a reactor in which liquid vegetable oil is placed in the absence of solvent, for example according to the method described in patent application <CIT>.

The peroxide value (PV) of an ozonized oil brought to complete ozonization is determined by the quantity and quality of the unsaturated fatty acids present, which give rise to different peroxidation reactions in the presence of ozone. The peroxidated products that are formed, for example peroxides, hydroperoxides and ozonides, are responsible for the wide spectrum of therapeutic action of ozonized oils.

The viscosity of the liquid, as well as the formation of a stable gel, increase with increases in the degree of ozonization of the oil, where the degree of ozonization is directly proportional to the time of contact between the ozone and the substrate. The ozonization process described in <CIT> makes it possible to obtain solely ozonized olive oil which, as a stable gel, exhibits a pre-determined peroxide value, which substantially depends on the number and type of unsaturated fatty acids contained in the oil.

There is thus a felt need in this field to have a method for ozonizing olive oil which enables the preparation of an ozonized oil that is not only in the form of a stable gel and/or has an appropriate viscosity, but whose peroxide value can also be varied without influencing the physical state of the ozonized oil.

Vegetable oil ozonization processes are known in the art which start from emulsions of oil in water. In these systems, the water is generally present in a high amount relative to the oil: from <NUM> to <NUM> times the amount of oil and solvent together in <CIT> and about <NUM> times the amount of oil in <CIT>.

However, such processes do not enable an ozonized oil in the form of a stable gel with a suitable peroxide value to be obtained.

Patent application <CIT> discloses a method for obtaining ozonized oils with a peroxide value ranging from <NUM> to <NUM> by bubbling ozone into an oil or a liquid fat to which <NUM>-<NUM> vol. % of water has been added. In the example embodiments, large amounts of water are used, <NUM> litres of water per <NUM> litres of oil, equal to about <NUM> vol. %, along with a high ratio between the gas flow rate and the volume of oil used (<NUM> litres/h of gas consisting of an oxygen/ozone mixture per <NUM> litres of oil). The peroxide value of the ozonized oils produced in the examples varies between <NUM>-<NUM> units for sunflower oil and <NUM>-<NUM> units for Theobroma oil. Furthermore, the ozonized oils produced, despite having a variable peroxide value, have a low viscosity (less than <NUM> mPa. s for ozonized sunflower oil).

<NPL>), compares ozonized olive and sunflower oils chemically and microbiologically to determine the peroxide, acidity, and iodine values and antimicrobial activity.

Patent application <CIT>, concerns the ozonolysis of unsaturated plant and/or animal oils in the presence of a participating co-reactant to form reaction products partially suitable for use in the formation of resins.

In this context, the principal object of the present invention is to propose a method for the production of ozonized oil with a controlled peroxide value, preferably in the form of a stable gel.

A further object of the present invention is to propose a method for the preparation of an ozonized oil which makes it possible to obtain the target peroxide value and/or viscosity value of the ozonized oil in a short time, using a limited amount of reagents per unit of volume of oil.

These and further objects, which will become more apparent in the course of the detailed description that follows, are achieved in accordance with the present invention, which, in a first aspect thereof, relates to a method for preparing an ozonized olive oil comprising:.

In a further embodiment, the method of the present invention comprises obtaining an ozonized olive oil which is characterized by at least one of the following properties:.

In the context of the present invention, the term "dispersion" refers to a heterogeneous system consisting of a prevalent liquid phase (dispersing phase or continuous phase) in which a liquid phase that is immiscible with the dispersing phase is dispersed (dispersed phase). The drops constituting the dispersed phase can have dimensions that are greater than or equal to approximately <NUM>-<NUM> µm. In the context of the present invention, the term "emulsion" refers to a dispersion in which the drops constituting the dispersed phase have dimensions of approximately <NUM>-<NUM> <NUM>.

In the context of the present invention, the term "W/O dispersion" or the term "W/O emulsion" refers, respectively, to a water-in-oil dispersion or emulsion, in which the oil constitutes the continuous phase and the water the dispersed phase.

In the context of the present invention, the term stable "dispersion (or emulsion)" refers to a dispersion (or emulsion) in which the drops constituting the dispersed phase do not give coalescence with the consequent formation of a continuous phase when the dispersion (or emulsion) is maintained at rest at approximately <NUM> for a time of approximately <NUM> minutes.

In the present description and in the appended claims, the term "vegetable oil" refers to a liquid mixture at <NUM> and <NUM> kPa mainly consisting of esterified lipids, generally obtained from fruits and seeds of oil-bearing plants.

In the context of the present invention, the terms "oleic acid", "linoleic acid" and more in general "fatty acid" refer, respectively, to the oleic, linoleic and fatty acids present in a vegetable oil in esterified form, preferably in the form of triglycerides. The term "free fatty acid" is used in the context of the present invention to indicate a non-esterified fatty acid.

In a first aspect, the present invention relates to a method for preparing an ozonized olive oil comprising:.

The total quantity of water added to the olive oil in steps (i) and/or (ii) is expressed as a volume percent of the total volume of the dispersion.

The olive oil can comprise oleic acid (C18:<NUM>) and linoleic acid (C18:<NUM>) in a total amount that is greater than or equal to <NUM>% by weight, wherein the total amount of oleic acid and linoleic acid is expressed as a sum of the percent amounts of the two fatty acids relative to the total amount of vegetable oils.

The olive oil can preferably be of biological origin and/or have an acid value less than or equal to approximately <NUM> mgKOH/g.

In a first embodiment, the method can comprise a step (i) of preparing a W/O dispersion comprising olive oil and the total quantity of water to be used in the method. According to this embodiment, the at least one step (ii) can be carried out using anhydrous ozone, in the absence of water vapour.

The method for preparing an ozonized olive oil comprises:.

a) can comprise adding the above-mentioned quantity of water, in the liquid phase, to the olive oil, and mixing the components with mechanical (e.g. stirrers) and/or physical (e.g. ultrasound) dispersing means normally used in the art to obtain preferably stable W/O dispersions. According to one variant, step (i. a) can be carried out by mechanically dispersing water in the vegetable oils and subsequently treating the W/O dispersion with ultrasound.

A quantity Q1 of anhydrous ozone, is subsequently bubbled into the W/O emulsion to obtain an ozonized olive oil.

According to the invention, the quantity of water Q2 is less than or equal to approximately <NUM> vol. %, preferably approximately <NUM>-<NUM> vol. %, even more preferably approximately <NUM> vol. %, wherein the quantity Q2 is expressed as a volume of water relative to the total volume of the pre-dispersion (or pre-emulsion). The Applicant has surprisingly found that if the quantity Q2 of water pre-dispersed in the at least one vegetable oil is above <NUM> vol. %, when the pre-dispersion is added to the vegetable oil there will be a coalescence of the dispersed phase with the formation of a continuous aqueous phase which prejudices the optimal completion of the ozonization reaction (step (ii.

The pre-dispersion step (i. a1) can comprise adding water to the vegetable oil using mechanical (e.g. stirrers) and/or physical (e.g. ultrasound) dispersion means normally used in the art in order to obtain preferably stable dispersions. a1) can comprise pre-dispersing the components with a mechanical stirrer at a speed of approximately <NUM>-<NUM> rpm, preferably approximately <NUM> rpm, until obtaining a stable pre-dispersion. Optionally, the pre-dispersion can be further treated with ultrasound after mechanical dispersion.

In a further embodiment, the method for preparing an ozonized olive oil can comprise a step (i) of preparing a W/O dispersion comprising olive oil and a smaller quantity of water than the total quantity of water to be used in the method. In this embodiment, the at least one step (ii) can be carried out by bubbling ozone and water vapour into the W/O dispersion or adding water in the liquid phase to the olive oil and bubbling ozone, preferably anhydrous ozone, into the dispersion, so that the total quantity of water added in step (i) and in step (ii) is approximately <NUM>-<NUM> vol.

In one embodiment of the method, step (ii) comprises bubbling a quantity Q1 of ozone into the second W/O dispersion and adding water.

The method can comprise adding water in the liquid phase to the olive oil and bubbling ozone, preferably anhydrous ozone, into the W/O dispersion. According to this embodiment, the method can comprise adding approximately <NUM>-<NUM> vol. % of the total water in step (i. a) and the remaining approximately <NUM>-<NUM> vol. % of the total water, preferably divided into <NUM>-<NUM> aliquots, more preferably aliquots of an equal amount, in step (ii.

Given the same total quantity of water added and ozone used, this embodiment enables an ozonized olive oil with a higher peroxide value to be obtained. Advantageously, furthermore, the peroxide value increases in a more regular manner as the reaction time increases when part of the water is added in step (ii. a), enabling a better control of the properties of the product, in particular the peroxide value and viscosity.

In the different embodiments of the method described above:.

Because of its great instability, ozone is generally produced in situ by means of ozone generators known in the art, from a supply gas consisting of air or pure oxygen. The main methods for producing ozone are irradiation of the gas flow with UV light and corona discharge. In the method of the invention, an ozone generator that employs corona discharge can preferably be used.

Advantageously, before the supply gas reaches the ozone generator, said gas can be conveyed to at least one drying unit to reduce the quantity of water present in the gas itself, thus producing a dried supply gas. The presence of water in the supply gas significantly reduces the capacity of an ozone generator.

In a preferred embodiment, the dried supply gas flowing to the ozone generator can be dry air with a dew point less than or equal to <NUM> approximately -<NUM>, preferably less than or equal to approximately -<NUM> and greater than or equal to approximately -<NUM>.

In one embodiment, the ozone generator supplied with dry air as described above can produce a flow of dry air with an ozone concentration of approximately <NUM>-<NUM> vol. %, preferably approximately <NUM> vol. %, relative to the total volume of dry air.

When carrying out the method comprises bubbling water vapour and ozone, the flow of ozone can be humidified downstream of the ozonizer by means of humidification devices which are in themselves known in the art. Alternatively, the ozone can be supplied to step (ii) as dry ozone and the water vapour as an additional flow on its own.

The method can be carried out in a system comprising at least one bubble column reactor of the type normally employed for gas-liquid reactions. Such reactors are in themselves known in the art and therefore they are not further described in detail.

Since the ozonization reaction of a vegetable oil is exothermal, the at least one step of bubbling ozone can preferably be carried out under conditions of cooling in order to prevent an uncontrolled temperature increase in the reaction environment from causing deterioration of the oil or triggering combustion reactions.

In the context of the present disclosure, the expression "under conditions of cooling" refers to conditions in which the reaction heat generated by the ozonization of the vegetable oils is removed from the reaction environment.

Therefore, the bubble column reactor in which the at least one step of <NUM> bubbling ozone can be carried out can comprise an external cooling compartment (or jacket), of the type known in the art, in which a cooling fluid circulates, generally water or mineral or silicone oil.

The temperature of the cooling fluid supplied to the reactor jacket can be approximately <NUM>-<NUM>, preferably approximately <NUM>°-<NUM>, the <NUM> temperature being measured at a pressure of approximately <NUM>,<NUM> kPa.

In one embodiment, the at least one step of bubbling ozone can be carried out at a temperature less than or equal to approximately <NUM>, preferably less than or equal to approximately <NUM>, more preferably approximately <NUM>-<NUM>.

The at least one step of bubbling ozone can be carried out at ambient pressure or, preferably, under vacuum, at a pressure preferably less than or equal to approximately <NUM> kPa.

The at least one step of bubbling ozone can have a variable duration.

In one embodiment, said step can be carried out until obtaining an ozonized oil in the form of a gel. In one embodiment, the at least one step of bubbling ozone can be carried out until obtaining an ozonized olive oil in the form of a stable gel, in which the change in the peroxide value ΔPV is less than or equal to approximately <NUM>, preferably <NUM>-<NUM>, where <MAT> wherein:.

In one embodiment, the at least one step of bubbling ozone can be carried out until obtaining an ozonized oil with a peroxide value PVto less than or equal to approximately <NUM> mEq O<NUM>/Kg, preferably approximately <NUM>-<NUM> mEq O<NUM>/Kg, more preferably approximately <NUM> - <NUM> mEq O<NUM>/Kg, even more preferably approximately <NUM> - <NUM> mEq O<NUM>/Kg.

The at least one step of bubbling ozone can be carried out until obtaining an ozonized oil in the form of a stable gel with a PVto as specified above.

The method according to the invention can comprise at least one step (ii) lasting approximately <NUM>-<NUM> hours, preferably approximately <NUM>-<NUM> hours, at the end of which the ozonized oil obtained can have at least one, preferably all, of the characteristics described above.

At the end of the at least one step of bubbling ozone, the flow of ozone can be interrupted and the ozonized vegetable oil removed from the reaction environment.

It is also described that the method can comprise a further step selected from:.

The method according to the invention enables an ozonized vegetable oil with a controlled viscosity and peroxide value to be obtained, wherein said oil is preferably in the form of a stable gel.

It has been surprisingly found that, by appropriately modifying the conditions under which the at least one step (ii) is carried out within the operating parameters described above, it is possible to obtain an ozonized vegetable oil in the form of a stable gel and with high values of PVto in a short time, i.e. considerably reducing the duration of the at least one step of bubbling ozone compared to the ozonization processes normally used in the art.

A further advantage of the ozonization method according to the invention lies in the fact that for the same PVto of the ozonized oil a smaller amount of reagent is used than in the ozonization processes known in the art.

In a further embodiment, the method of the present invention further comprises obtaining an ozonized olive oil,by way of the method described above, which is characterized by at least one of the following properties:.

The ozonized olive oil obtained/obtainable with the method described above has demonstrated bactericidal and/or antimycotic activity and can therefore have application in the treatment, preferably the topical treatment, of infections in humans and animals caused by bacteria and/or fungi.

Amount of oleic and linoleic acid: the percentage of fatty acids is determined by gas-liquid chromatography (GLC).

Peroxide value: iodometric titration with sodium thiosulphate of the iodine liberated by the reaction of the peroxides with potassium iodide (compliant with AOAC method No. <NUM>). Apparatus: Mettler Toledo G20 titrator provided with an internal burette and DMi147-SC combined electrode (platinum - pH) and LabX® titration software. Titrating solution: sodium thiosulphate <NUM>. 1N (EXAXOL, Italy).

Prepare a solution ("Sol. A") by mixing glacial acetic acid (#Cat. <NUM>, SIGMA Aldrich) and chloroform (#Cat. <NUM>-<NUM>, SIGMA Aldrich) in a proportion of <NUM>:<NUM> v/v, under gentle stirring. Before taking the measurement, remove the electrode from the liquid it is preserved in and wash the electrode with deionised water for a few seconds. Weigh about <NUM> of potassium iodide (#Cat. <NUM>, SIGMA Aldrich) into the beaker of the titrator; add <NUM> of "Sol. A" under stirring. Set the timer of the instrument to <NUM> minutes and start it, maintaining the solution under stirring. After <NUM>. and <NUM>" have elapsed, add <NUM> of deionised water to the solution and proceed with the automatic determination of the blank value (in the absence of the sample) with sodium thiosulphate. The blank determination makes it possible to calculate the relative error in the peroxide value due to changes in potential caused by the presence of any impurities in the reagents. The value in mmol of the titrant used for blank determination is memorised by the instrument as B[<NUM>]. To determine the peroxide value of the sample, exactly weigh about <NUM> of sample into the beaker of the titrator, recording the amount of sample weighed (p) on the instrument, add <NUM> of "Sol. A" and stir until the sample is completely dissolved. Add about <NUM> of KI to the solution to be titrated, set the timer of the instrument to <NUM> minutes and start it, maintaining the solution under stirring. After <NUM>. and <NUM>" have elapsed, add <NUM> of deionised water to the solution and proceed with the automatic titration of the iodine liberated from the sample with sodium thiosulphate. The result of the titration is positive if the instrument is able to record a minimum of <NUM> points on which to build the curve mapping the changes in potential and identify the curve inflection point. The instrument directly provides the peroxide value in meq/Kg O<NUM>. The peroxide value is calculated as the arithmetic mean of three measurements performed on the same sample. Wash the electrode with chloroform between measurements.

For examples <NUM> and <NUM>, <NUM> of a first W/O dispersion were prepared in a <NUM> beaker by dispersing a quantity Q1 of purified water (Italian Official Pharmacopeia) with suitable amounts of organic olive oil having the characteristics shown in Table <NUM>. The dispersion was mechanically stirred for approximately <NUM>. at a speed of <NUM> rpm. and a pale yellow milky W/O emulsion was obtained.

In comparative example <NUM>, the quantity of water Q2 was dispersed directly in the olive oil under mechanical stirring, without pre-mixing. This example is not according to the invention and is present for illustration purposes only.

Table <NUM> shows the quantities Q1 and Q2 of water used in the different examples. The emulsion was added to <NUM> I of the same organic olive oil, previously placed in bubble column reactor provided with a water-cooled jacket. The temperature of the cooling fluid was set at approximately <NUM>. A flow of dry air containing approximately <NUM> vol. % of ozone was bubbled into the oil at a flow rate of approximately <NUM>/h per litre of oil (about <NUM>/h of ozone per litre of oil).

The temperature of the reaction environment was kept below <NUM>°-<NUM> thanks to the cooling fluid.

Three samples of oil were taken from the reactor at the end of the reaction, <NUM> hours after the start of bubbling and the PV was determined for each sample. The PV shown in Table <NUM> was obtained as a mean of the values measured for three samples.

At the end of ozonization, the ozonized oil was in the form of an amber yellow gel.

In a sample of ozonized oil product in example <NUM>, a determination was made of the residual free water, which amounted to <NUM> at about <NUM>. This value was wholly comparable to the value obtained in the sample of the comparative example <NUM>, which showed to have a water content of <NUM> at about <NUM>.

This example is not according to the invention and is present for illustration purposes only. <NUM> of a first W/O dispersion were prepared in a <NUM> beaker by dispersing <NUM> of purified water (Italian Official Pharmacopeia) in <NUM> of organic olive oil having the characteristics shown in Table <NUM>. The dispersion was mechanically stirred for approximately <NUM>. at a speed of <NUM> rpm. and a pale yellow milky W/O emulsion was obtained.

The emulsion was added to <NUM> I of the same organic olive oil, previously placed in bubble column reactor provided with a water-cooled jacket. The temperature of the cooling fluid was set at approximately <NUM>.

A flow of dry air containing approximately <NUM> vol. % of ozone was bubbled into the oil at a flow rate of approximately <NUM>/h per litre of oil (about <NUM>/h of ozone per litre of oil).

During bubbling, coalescence of the dispersed water occurred, with the formation of bubbles that precipitated onto the bottom of the reactor. At the end of the reaction, the determination of the peroxide value obtained showed a level of approximately <NUM>, a parameter clearly indicative of a reduced and insufficient peroxidation of the fatty acids.

<NUM> of a first W/O dispersion were prepared in a <NUM> beaker by dispersing <NUM> of purified water (Italian Official Pharmacopeia) in <NUM> of organic olive oil having the characteristics shown in Table <NUM>. The dispersion was mechanically stirred for approximately <NUM>. at a speed of <NUM> rpm. and a pale yellow milky W/O emulsion was obtained.

Three samples of oil were taken from the reactor <NUM>, <NUM> and <NUM> hours after the start of the ozonization reaction and the PV was determined for each sample. The PV shown in Table <NUM> was obtained as a mean of the values measured for three samples.

At the end of the ozonization (<NUM>), the ozonized oil was in the form of an amber yellow gel.

Three samples of oil were taken from the reactor at the end of the reaction, <NUM> hours after the start of the bubbling, and a PV of approximately <NUM> mEq O<NUM>/Kg was determined as the mean of the values measured for three samples.

The product obtained was tested to verify its effectiveness in inhibiting bacterial and/or fungal proliferation in accordance with the procedures of standard UNI EN <NUM>.

The quantity of purified water (Italian Official Pharmacopeia) shown in table <NUM> was added to an olive oil in a bubble column reactor with a cooling jacket, the water being pre-emulsified in a suitable amount of oil. In examples <NUM> and <NUM>, all of the water was added at the beginning of the reaction, before starting to bubble the ozone. In examples <NUM> and <NUM>, part of the water was added before ozonization began and part once the reaction had begun, as indicated in table <NUM>. A flow of dry air containing approximately <NUM> vol. % of ozone was bubbled into the oil. The temperature of the reactor cooling fluid was set at approximately <NUM>. The temperature of the reaction environment was kept below <NUM>°-<NUM> thanks to the cooling fluid.

Samples of oil were taken from the reactor at different times during the ozonization reaction and at the end of the reaction, approximately <NUM>/<NUM> hours after the start of bubbling and the PV was determined for each sample. The change in the peroxide value of the oils of examples <NUM>-<NUM>, as a function of the reaction time, is shown in <FIG> and <FIG>.

<FIG> shows a comparison between the change in the PV, as a function of the reaction time, for examples <NUM> and <NUM>.

Claim 1:
A method for preparing ozonized olive oil comprising:
(i) preparing a dispersion comprising olive oil and <NUM>-<NUM> vol.% of water, preferably <NUM>-<NUM> vol.%, more preferably <NUM>-<NUM> vol.%, even more preferably <NUM>-<NUM> vol.%;
wherein step (i) comprises:
(i.a1) preparing a first dispersion by mixing a quantity Q2 of water and at least one vegetable oil, preferably at least one vegetable oil comprising oleic acid (C18:<NUM>) and linoleic acid (C18:<NUM>) in a total amount greater than or equal to <NUM>% by weight, thus obtaining the first dispersion, wherein the quantity of water Q2 is less than or equal to <NUM> vol.%, wherein the quantity Q2 is expressed as the volume of water relative to the total volume of the first dispersion; and
(i.a2) mixing the first dispersion and the olive oil, thus obtaining a second W/O dispersion comprising <NUM>-<NUM> vol.% of water, preferably <NUM>-<NUM> vol.%, more preferably <NUM>-<NUM> vol.%, even more preferably <NUM>-<NUM> vol.%; and
(ii) at least one step of bubbling a quantity Q1 of ozone into the second W/O dispersion and, optionally, adding water,
wherein:
- in step (ii) the water is optionally added in the liquid phase or vapour phase; and
- the total quantity of water added to the olive oil in step (i) and (ii) is <NUM>-<NUM> vol.%.