Method of isolating ozonides in ozonated vegetable oils and characterizing a mixture by applying said method

Abstract

The present invention relates to an innovative method of isolating and quantifying stable ozonides present in ozonated vegetable oils. In particular, the described method allows obtaining ozonated vegetable oils containing known amounts of specific stable ozonides. Such a method comprises, in an embodiment thereof, the following steps: transesterifying, at room temperature, at room pressure and for at least one hour, an ozonated vegetable oil using sodium methoxide in a mixture of dichloromethane and anhydrous methanol; removing dichloromethane and methanol by applying a pressure less than room pressure; by using a mixture of water and an immiscible aprotic solvent and an extraction funnel, separating the resulting mixture into an organic phase, containing an ozonide mixture, and an aqueous phase; isolating the ozonide diastereoisomer pairs from the organic phase containing the ozonide mixture by silica gel chromatography using a mixture of n-hexane / diethyl ether or any solvent of comparable polarity as eluent; separating the pure cis / trans diastereoisomers of the ozonides obtained by preparative thin layer chromatography. Possibly, the same method includes the further step of using the pure ozonide diastereoisomers obtained as standards in quantitative analytical techniques for quantifying them in ozonated vegetable oils. The present invention also relates to a formulation obtained by the application of said method. In particular, the invention relates to a mixture containing the ozonides cis-methyl 8-(5-octyl1,2,4- trioxolan-3-yl)octanoate, trans-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate, cis-3, 5-dioctyl-1,2,4-trioxolane, trans-3,5-dioctyl-1,2,4-trioxolane, cis-dimethyl 8,8'-(1,2,4-trioxolane-3,5- diyl)dioctanoate, trans-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate, said mixture being obtained by applying, under particular conditions reported in greater detail in the description, the same steps illustrated in the method of the present invention.

Description

[0001] METHOD OF ISOLATING OZONIDES IN OZONATED VEGETABLE OILS AND CHARACTERIZING A MIXTURE BY APPLYING SAID METHOD

[0002] The present invention relates to a method of isolating and quantifying ozonides in ozonated vegetable oils, and a formulation obtained by applying said method.

[0003] Ozonated vegetable oils are widely used nowadays in the field of personal care due to the several and recognized beneficial biological properties thereof, such as antimicrobial, anti¬ inflammatory, and regenerative properties. These products result from the reaction between ozone and the fatty acids present in vegetable oils, which leads to the formation of various chemical compounds including, primarily, ozonides. The scientific community agrees in supporting the thesis that the beneficial actions claimed by ozonated oils are to be assigned to oxidized substances resulting from the various ozonation processes and generally measured as peroxides.

[0004] Although ozonides, among the families of oxidized compounds, in particular ozonides defined as stable, are generally considered primarily responsible for the beneficial biological properties determined by the ozonation processes of vegetable oils, there is no definite attribution, in the prior art, of the aforesaid activities to specific ozonides, but only to the class of oxidized compounds as a whole.

[0005] Furthermore, the ozonation processes of vegetable oils result in the possibility of formation of further compounds, e.g., aldehydes, carboxylic acids, and hydroperoxides, the specific biological role of which is still the subject of research.

[0006] These gaps are primarily attributable to the absence of specific protocols which allow obtaining the isolation, complete characterization, and quantification of the compounds obtained from the ozonation processes. Therefore, despite the availability of processes for producing ozonated vegetable oils, there remains the need for a method allowing a reproducible quantitative chemical characterization of the stable ozonides present in such matrices, overcoming the non- specific approach based on the peroxide number.

[0007] Currently, in fact, the quantification of ozonides in ozonated oils is carried out by the method of determining the total peroxide number, described in Regulation (EEC) n. 2568 / 91 dd. July 11, 1991, related to the features of olive oils and olive-pomace oils, as well as to the methods pertaining thereto. However, such a method is based on an indirect, general measurement that does not distinguish between the different compounds present nor provide specific information on the stable ozonides present in vegetable oils.Therefore, despite the widespread diffusion of ozonated vegetable oils in various fields, no method known from the prior art has succeeded in overcoming these limitations, providing precise characterizations and quantifications of the ozonides obtained from the ozonolysis of vegetable oils. This has hindered a thorough scientific understanding of the biological and therapeutic role thereof, leaving broad uncertainties in the optimization and standardization of ozonide-based formulations.

[0008] Hence, the need arises to define a validated and standardized method which allows isolating and identifying the ozonides in ozonated vegetable oils.

[0009] Again, it is a technical problem to define a method which allows quantifying the ozonides in ozonated vegetable oils.

[0010] In parallel, the need arises to define a method which allows obtaining ozonated vegetable oils containing known amounts of specific stable ozonides.

[0011] Therefore, it is an object of the invention to define a method of isolating and identifying the ozonides in ozonated vegetable oils.

[0012] Therefore, it is another object of the invention to define a method of quantifying the ozonides in ozonated vegetable oils.

[0013] Furthermore, it is an object of the invention to define a method which allows obtaining, systematically, ozonated vegetable oils containing known amounts of stable ozonides and specific stable ozonides.

[0014] Again, it is an object of the invention to provide a method of isolating stable ozonides from ozonated vegetable oils, applicable to various ozonated vegetable oils, so as to obtain pure ozonide diastereoisomers usable as standards for the quantification thereof in ozonated vegetable oils through quantitative analytical techniques, and to allow the development of identifiable and reproducible formulations.

[0015] It is a further object of the invention to provide a. method of distinguishing the compounds present in ozonated vegetable oils, which allows overcoming the methods based on the peroxide number, such as Regulation (EEC) n. 2568 / 91 dd. July 11, 1991, related to the features of olive oils and olive-pomace oils, as well as to the methods pertaining thereto.

[0016] Furthermore, it is an object of the invention to provide a formulation based on the stable ozonides obtained by the method of the patent.

[0017] These and further objects are achieved by the method of isolating and characterizing stable ozonides from ozonated vegetable oils and by the use of formulations based on stable ozonides ofknown chemical composition obtained by the aforesaid method, described in the present patent application for industrial invention, in a preferred, non-limiting embodiment of further developments within the scope of the patent itself, with the aid of the accompanying drawings showing the following figure:

[0018] Fig. 1, structural formulas of stable ozonides isolated using the method of the present invention, so as to be used as reference standards for the selective quantification of the individual ozonides present in ozonated vegetable oils.

[0019] The present patent application for industrial invention relates to a method for isolating and quantifying ozonides in ozonated vegetable oils.

[0020] In particular, said method comprises the following steps:

[0021] - transesterifying, at room temperature, room pressure and for at least one hour, an ozonated vegetable oil using sodium methoxide in a mixture of dichloromethane and anhydrous methanol;

[0022] - removing dichloromethane and methanol by applying a pressure less than room pressure; - by using a mixture of water and a water- immiscible aprotic solvent and an extraction funnel, separating the resulting mixture into an organic phase, containing an ozonide mixture, and an aqueous phase;

[0023] - isolating the ozonide diastereoisomer pairs from the organic phase containing the ozonide mixture by silica gel chromatography using a mixture of n-hexane / diethyl ether or any solvent of comparable polarity as eluent;

[0024] - separating the pure cis / trans diastereoisomers of the ozonides obtained by preparative thin layer chromatography (TLC).

[0025] The present patent for industrial invention further relates to a method comprising all the steps listed above, followed by the further step of:

[0026] - using the pure ozonide diastereoisomers obtained as standards in quantitative analytical techniques such as, by way of non-limiting example, high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), UV-Vis, IR and NMR spectroscopy, and the like, for the quantification thereof in ozonated vegetable oils.

[0027] The present isolation and characterization method falls within a technical plane which is distinct and complementary with respect to the production processes of ozonated vegetable oils, enabling a level of analytical standardization not obtainable by the ozonation processes alone.In fact, the method described is a significant advancement with respect to the characterization of ozonated vegetable oils, ensuring high reproducibility of the processes and enabling the direct attribution of biological properties to the stable ozonides identified and quantified. It is a significant step forward in the standardization and quality of products resulting from ozonolysis of plant matrices, with applications in the antimicrobial, anti-inflammatory, regenerative, and antiproliferative fields.

[0028] Therefore, the suggested method allows isolating stable ozonides and then using them as standards for the quantification thereof in vegetable oils in a reproducible manner, ensuring a direct correlation between the ozonide content and the observed biological properties.

[0029] In fact, by applying the method, stable ozonides are obtained, more precisely pure ozonide diastereoisomers.

[0030] Under the same conditions, namely by applying to the same vegetable oil with the same ozonation percentage and performing the method using the same reagents and the same techniques, the same method allows obtaining the same products, comprising the same stable ozonides in the same amounts.

[0031] Therefore, once said stable ozonides have been isolated and identified and the pure ozonide diastereoisomers present in a mixture obtained by applying the described method, carried out under the defined conditions, have been quantified, it is possible to obtain the identified ozonide diastereoisomers by simply replicating the method under the same conditions.

[0032] By further modulating the ozonation times of vegetable oils, is possible to vary in a controlled manner the content of stable ozonides present in the ozonated vegetable oils, offering a flexibility that allows customizing the properties of the final product based on the different applications. In other words, by modulating the ozonation times of vegetable oils, it is possible to determine in advance the percentage of ozonides present in the ozonated vegetable oil with respect to the total ozonides potentially obtainable from the ozonation process thereof.

[0033] Such a modulation process does not modify the products obtained from the application of the method, but the concentration thereof within the mixture obtained.

[0034] Hence, by applying the described method it is possible to identify stable ozonides and then, by replicating the method under identical conditions, it is possible to obtain the same ozonides as those already identified, but at the concentration preferred from time to time, based on the specific field of use of the products.Sodium methoxide allows the ozonated fraction to be freed from glycerol, in the form of methyl esters, in a highly clean manner when compared to the saponification reaction (basic hydrolysis with sodium hydroxide). The reaction with sodium methoxide occurs in a more selective and controlled manner, optimizing the yield of the desired products. With respect to basic hydrolysis, transesterification with sodium methoxide minimizes the formation of alkaline residues and secondary soaps, simplifying the separation and disposal operations. The lower complexity in the management of by-products and the high yield of the process contribute to a greater overall cost-effectiveness. The transesterification is not aimed at the permanent modification of the stable ozonides, but at the selective removal of the triglyceride matrix, preserving the integrity of ozonides and enabling the chromatographic separation thereof.

[0035] Furthermore, said water-immiscible aprotic solvent is preferably diethyl ether. The extraction is preferably carried out with diethyl ether since it is a solvent with a low boiling point; therefore, easily removable at low temperatures. Diethyl ether is a non-participating solvent, meaning that it does not chemically interact with the ozonated compounds, preserving the integrity of the extracted fraction. Due to the low boiling point thereof, diethyl ether can be easily removed from the system by evaporation at moderate temperatures, avoiding the risk of thermal degradation of sensitive compounds.

[0036] The method described allows determining, in a specific and accurate manner, the stable ozonides derived from ozonated vegetable oils and is applicable to any ozonated vegetable oil. However, preferably, the ozonated vegetable oil is ozonated extra virgin olive oil.

[0037] In fact, extra virgin olive oil is widely used in the food, cosmetics, and pharmaceutical industries, making the results of the method immediately applicable to market contexts of great interest.

[0038] Conveniently, the vegetable oil is ozonated by bubbling, in a reactor containing the vegetable oil, an oxygen-ozone mixture obtained from pure medical oxygen subjected to electric discharge.

[0039] In particular, in the steps carried out for the ozonation of the vegetable oil, it is convenient to use the device of patent EP3900821, filed with priority on 20 / 04 / 2020 by ERBAGIL SRL.

[0040] When the method described is applied according to the preferred specifications described above, namely when:

[0041] - the transesterification is carried out using a sodium methoxide solution in dichloromethane and anhydrous methanol;- the aprotic solvent used is diethyl ether;

[0042] - the ozonated vegetable oil is ozonated extra virgin olive oil;

[0043] the stable ozonides obtained are the following:

[0044] - cis-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate,

[0045] - trans-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate

[0046] - cz,?-3,5-dioctyl-l,2,4-trioxolane,

[0047] - trans-3,5-dioctyl-1,2,4-trioxolane,

[0048] - cis-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate

[0049] - trans-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate.

[0050] The structural formula of the cis and trans isomers of the isolated ozonides obtained is shown in Figure 1.

[0051] Therefore, the present patent application for industrial invention also relates to a mixture containing the following ozonides:

[0052] -- cis-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate,

[0053] - trans-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate,

[0054] - cis-3,5-dioctyl-1,2,4-trioxolane,

[0055] - trans-3,5-dioctyl-1,2,4-trioxolane,

[0056] -- cis-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate

[0057] - trans-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate.

[0058] said mixture being obtained by applying to ozonated extra virgin olive oil a method comprising the following steps:

[0059] - transesterifying ozonated extra virgin olive oil at room temperature, room pressure and for at least one hour, using sodium methoxide in dichloromethane and anhydrous methanol;

[0060] -- removing dichloromethane and methanol by applying a pressure less than room pressure;

[0061] - by using a mixture of water and a water-immiscible aprotic solvent and an extraction funnel, separating the resulting mixture into an organic phase, containing an ozonide mixture, and an aqueous phase;

[0062] -- isolating the ozonide diastereoisomer pairs from the organic phase containing the ozonide mixture by silica gel chromatography using a mixture of n-hexane / diethyl ether or any solvent of comparable polarity as eluent;- separating the pure cis / trans diastereoisomers of the ozonides obtained by preparative thin layer chromatography;

[0063] --- using the pure ozonide diastereoisomers obtained as standards in quantitative analytical techniques for the quantification of the individual ozonides in the ozonated plant matrix.

[0064] In particular, in the described mixture, the pairs of diastereoisomers of the molecules obtained are:

[0065] - cis,trans-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate up to 55%;

[0066] - cis,trans-3,5-Dioctyl- 1,2,4-trioxolane up to 20%;

[0067] - cis, trans-dimethyl 8,8'-( 1,2,4-trioxolane-3,5-diyl)dioctanoate up to 25%.

[0068] More in particular, within each pair of diastereoisomers, the ratio of cis and trans forms is approximately 1:1.

[0069] A detailed example of the application of the described method of isolating and quantifying stable ozonides in 500 mg of ozonated extra virgin olive oil Ozoile® (stable ozonides) is provided below.

[0070] Materials and specific reagents

[0071] - 500 mg of Ozoile® (stable ozonides) were obtained by means of a patented process (EP 3900821, filed with priority on 20 / 04 / 2020 by ERBAGIL SRL) by reaction of ozone, starting from pure medical oxygen, with organic extra virgin olive oil (+OIL®, produced by ERBAGIL® TENUTA);

[0072] - 4ml of anhydrous dichloromethane at the commercially available analytical purity grade; - 12 ml of anhydrous methanol at the commercially available analytical purity grade;

[0073] - 200mg of sodium methoxide at the analytical purity grade, commercially available;

[0074] - diethyl ether at the commercially available analytical purity grade:

[0075] - silica gel chromatography column (0.063-0.2 mm) (Macherey-Nagel).

[0076] - Preparative TLC with precoated silica gel plates (Macherey-Nagel, Duren, Germany) with a film thickness of 1 mm.

[0077] - HPLC by means of 250 x 4.6 mm Hypersil ODS C18 5μm column with UV detector (230nm) using a MeOH / H₂O 1:9 mixture as eluent, flow 1.0 mL / min.

[0078] MethodTransesterifying, at room temperature, room pressure and for at least one hour, an ozonated vegetable oil using sodium methoxide in a mixture of dichloromethane and anhydrous methanol:

[0079] o adding 500 mg of Ozoile® (stable ozonides) to a reaction flask;

[0080] o dissolving with 4ml of anhydrous dichloromethane and 12 ml of anhydrous methanol,

[0081] o adding 200mg of sodium methoxide and keeping under stirring at room temperature.

[0082] o Waiting for 60 minutes for the reaction to complete.

[0083] - Removing dichloromethane and methanol by applying a pressure less than room pressure:

[0084] o removing dichloromethane and methanol using a rotary evaporator.

[0085] - By using a mixture of water and an immiscible aprotic solvent and an extraction funnel, separating the resulting mixture into an organic phase, containing an ozonide mixture, and an aqueous phase:

[0086] o dissolving the residue in diethyl ether and pouring into a separatory funnel;

[0087] o adding water and extracting with 30 ml of diethyl ether 3 times;

[0088] o recovering the organic phase and removing the solvent by applying a pressure less than room pressure using a rotary evaporator.

[0089] - Isolating the ozonide diastereoisomer pairs from the organic phase containing the ozonide mixture by silica gel chromatography using a mixture of n-hexane / diethyl ether or any solvent of comparable polarity as eluent.

[0090] o Loading the crude mixture onto the chromatographic column previously prepared using silica gel (60-120 mesh), equilibrated with n-hexane as the initial mobile phase. In particular, the column chromatography was carried out on silica gel (0.063-0.2 mm);

[0091] o carrying out the separation by gradient elution of increasing polarity with mixtures of n-hexane and diethyl ether;

[0092] o collecting the fractions containing the cis / trans pairs of diastereoisomers of the stable ozonides, and evaporating.

[0093] - Separating the pure cis / trans diastereoisomers of the ozonides obtained by preparative thin- layer chromatography:o dissolving each cis / trans pair of the obtained ozonide diastereoisomers in small amounts of dichloromethane;

[0094] o loading, by means of a Pasteur, each solution on a pre-coated silica gel plate (preparative TLC) of 1mm thickness;

[0095] o immersing the plates in chambers containing 10% n-hexane / diethyl ether eluent mixture;

[0096] o recovering the pure diastereoisomers of the stable ozonides obtained.

[0097] - Using the pure ozonide diastereoisomers obtained as standards in quantitative analytical techniques, e.g., high-performance liquid chromatography (HPLC), gas chromatography (GC), mass spectrometry (MS), UV-Vis, IR, and NMR spectroscopy, and the like, for the quantification thereof in ozonated vegetable oils:

[0098] o 2 mg of transesterified organic phase containing the ozonide mixture were solubilized in 250 μl of methanol and directly subjected to HPLC analysis. The yields of each ozonide were calculated using the pure ozonide diastereoisomers as reference standards (external standard method).

[0099] Once the listed procedures have been performed, the quantification of the ozonides is possible in the total ozonated vegetable matrix.

[0100] In particular, by operating according to the example, the following are obtained:

[0101] 15% of cis,trans-3,5-Dioctyl-1,2,4-trioxolane substantially in the ratio 1:1;

[0102] - 38% of cis,trans-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate substantially in the ratio 1:1;

[0103] - 20% of cis,trans-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate approximately in the ratio 1:1.

Claims

AMENDED CLAIMSreceived by the International Bureau on 18 May 2026 (18.05.2026).

1. Method comprising the following steps:• Transesterifying, at room temperature, at room pressure and for at least one hour, an ozonated vegetable oil using sodium methoxide in a mixture of dichloromethane and anhydrous methanol;• Removing the dichloromethane and methanol by applying a pressure lower than room pressure;• Using a mixture of water and an immiscible aprotic solvent and an extraction funnel, separating the resulting mixture into an organic phase, containing an ozonide mixture, and an aqueous phase;• Isolating the ozonide diastereoisomer pairs from the organic phase containing the ozonide mixture by silica gel chromatography using a mixture of n-hexane / diethylether or any solvent of comparable polarity as eluent;• Separating the pure cis / trans diastereoisomers of the ozonides obtained by preparative thin layer chromatography.

2. Method according to claim 1, comprising the further step of:- using the pure ozonide diastereoisomers obtained as standards in quantitative analytical techniques for quantifying the same in ozonated vegetable oils.

3. Method according to claim 1 or 2, wherein said water-immiscible aprotic solvent is preferably diethyl ether.

4. Method according to any combination of the previous claims, wherein said ozonated vegetable oil is ozonated extra virgin olive oil.

5. Method according to any combination of the previous claims, wherein the vegetable oil is ozonated by bubbling, in a reactor containing the vegetable oil, an oxygen-ozone mixture obtained from pure medical oxygen subjected to electric discharge.

6. Mixture containing the following ozonides:- cis-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate,- trans-methyl 8-(5-octyl1,2,4-trioxolan-3-yl)octanoate,- cis-3,5-dioctyl- 1,2,4-trioxolane,- trans-3,5-dioctyl- 1,2,4-trioxolane,- cis-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate,- trans-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoatewherein the diastereoisomer pairs are present in the following proportions: cis, trans-methyl 8-(5-octyll,2,4-trioxolan-3-yl)octanoate up to 55%; - cis,trans-3,5-Dioctyl-1,2,4-trioxolane up to 20%; cis, trans-dimethyl 8,8'-(1,2,4-trioxolane-3,5-diyl)dioctanoate up to 25%; and wherein, for each pair of diastereoisomers, the ratio between the cis and trans forms is approximately 1:1,said mixture being obtained by applying to ozonated extra virgin olive oil a method comprising the following steps:- Transesterifying, at room temperature, at room pressure and for at least one hour, an ozonated vegetable oil using sodium methoxide in a mixture of dichloromethane and anhydrous methanol;- Removing the dichloromethane and methanol by applying less than room pressure;- Separating the mixture resulting into an organic phase containing an ozonide mixture and an aqueous phase using a mixture of water and an aprotic solvent immiscible in water and an extraction funnel;- Isolating the ozonide diastereoisomer pairs from the organic phase containing the ozonide mixture by silica gel chromatography using a mixture of n-hexane / diethylether or any solvent of comparable polarity as eluent;- Separating the pure cis / trans diastereoisomers of the ozonides obtained by preparative TLC.

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