Highly ozonized fatty acids and methods for their production

Abstract

This invention relates to a method for producing highly ozonated fatty acids by subjecting extra virgin sunflower oil containing a high concentration of polyunsaturated fatty acids to high ozonolysis. Similarly, this invention relates to the highly ozonated fatty acids produced by said method and their cosmetic and medical uses, particularly due to their repairing effects on skin damage.

Description

[0001] This invention relates to a method for obtaining highly ozonated fatty acids by subjecting extra virgin sunflower oil containing a high concentration of polyunsaturated fatty acids to high-level ozonolysis. Similarly, this invention relates to the highly ozonated fatty acids obtained by said method and their cosmetic and medical uses, particularly for their skin damage repair effects. Therefore, this invention can be categorized in the chemical, pharmaceutical, and cosmetic fields. Background Technology

[0002] Ozonation oils themselves do not contain ozone, but they possess byproducts derived from the reaction of ozone with unsaturated compounds present in vegetable oils and fats, such as aldehydes and peroxides (hydroperoxides, ozonides, dimeroxides, polyperoxides, etc.). The mechanism of this reaction is well understood; to promote the preferential formation of some of these peroxides, the reaction must proceed under certain conditions, which are also well understood. (Ozonation in organic chemistry, Vols. 1 and 2, Philip S. Bailey, Editors: Alfred T. Blomquist and Harry H. Wasserman, Academic Press, New York, 1978).

[0003] These peroxides, primarily stable ozonides (1,2,4-trioxopentane), are responsible for a range of well-studied biological functions (antimicrobial activity, healing, cell regeneration, tissue antioxidant activity, etc.) (Martinez Sanchez, Gregorio, 2021. Scientific rationale for the medical applications of ozonized oils, update. Ozone Therapy Global Journal Vol. 11, n° 1, pp 201-237).

[0004] As described in the prior art, ozonated vegetable oils have been used to treat burns, lower extremity ulcers caused by venous insufficiency, pressure sores, acne, fistulas, and surgical wounds (see, for example, patent applications US4451480, US45916021, US984722, US2356062, US1210949).

[0005] On the other hand, cosmetic uses related to stimulating tissue renewal, cell and tissue oxygenation can be found in patent GB patent number 820,463.

[0006] Generally, ozonated oils are harmless and safe products of commercial interest. This has been demonstrated by toxicological studies (see, e.g., Revista CENIC, Vol. 26, Special No., p 105, 1995), histological and mutagenic studies (see, e.g., Revista CENIC, Biological Sciences 20 (1-2-3), 1-4 and 23, 1989), genotoxicity studies (see, e.g., Revista CENIC Biological Sciences 29 (3): 200, 1998), and teratogenicity studies (see, e.g., Proceedings of the 1st Ibero-American Congress of Ozone Applications, City of Havana, Ozone in Medicine 11, 1990; and WO 03 / 085072 A1).

[0007] Within these scopes, medical and cosmetic uses of ozonated oils have been described (see, for example, WO 01 / 37829A1 and WO 03 / 085072 A1) for rapid skin regeneration and scar formation.

[0008] In view of the medical and cosmetic benefits of the said compounds, manufacturing methods have been described, which to date allow the compounds to be obtained by, for example, ozonation of an aqueous-oil emulsion (1-50% by volume) by bubbling an ozone-oxygen mixture (ozone-air) at a concentration of 1-15% of the gas volume, using a scheme of 100 liters / hour of ozone-oxygen mixture per 500 liters of emulsion, to obtain a high peroxidation index without ozonating the unsaturated compounds to complete saturation. This favors the formation of β-hydroxy-hydroperoxides, delays polymer formation, and ensures stability, achieving the following final product stability: sunflower oil for more than one year and cocoa butter for more than 24 months (see WO 03 / 085072 A1).

[0009] Therefore, it is desirable to have this new high-ozonooxidized fatty acid matrix, which, by having a high concentration of stable ozonides, allows for improved ozonooxidized oil performance and better stability. It is also desirable to have a simple and harmless method of obtaining the stable compound, which allows for obtaining the stable compound without increasing viscosity or changing safety parameters such as high acidity index or the presence of formaldehyde. Summary of the Invention

[0010] In a first aspect, the present invention relates to a method for obtaining highly ozonated fatty acids, comprising the following steps:

[0011] i. Concentrated sunflower oil polyunsaturated fatty acid fractions to obtain a sunflower oil matrix by dry crystallization fractionation, the sunflower oil matrix having a polyunsaturated fatty acid concentration of at least 90%, while maintaining the minor fractions containing polyphenols, phytosterols, and tocopherols; and

[0012] ii. In a bubble reactor, the sunflower oil matrix from step (i) is ozonated with a gas flow of 30% to 35% ozone from pure oxygen at a temperature of 25°C to 30°C and a gas flow rate ratio of 150 L / h to 500 L / h to obtain highly ozonated fatty acids.

[0013] The method for preparing highly ozonated fatty acids of the present invention preferentially forms stable ozonides (1,2,4-trioxopentane), which are considered to have greater bioactivity than β-hydroxy-hydroperoxides. Furthermore, it achieves a high peroxidation index without ozonating unsaturated compounds to full saturation, and without requiring an emulsion, thus avoiding prior steps (i.e., the emulsion production step) as described in patent application WO 03 / 085072 A1, because it operates directly using an extra virgin sunflower oil matrix. Moreover, it surpasses the final product stability of ozonated sunflower oil and cocoa butter, as the highly ozonated fatty acids achieve a stability of 36 months. Finally, the highly ozonated fatty acids obtained by the ozonation method retain the extra virgin properties of sunflower oil, and to maintain their stability, they must be stored at a temperature not exceeding 30°C.

[0014] Therefore, by means of the method of the present invention, after linoleic acid is concentrated, high ozono-oxidized fatty acids are obtained by a simple and harmless process called dry crystallization fractionation. This process allows the saturated fatty acids and a certain percentage of oleic acid present in the matrix to be filtered out by melting point until a linoleic acid concentration of more than 90% is reached, without affecting the presence of phytosterols, polyphenols, vitamins, or the sensory characteristics of extra virgin sunflower oil with a lower melting point.

[0015] Specifically, the method of the present invention involves adding extra virgin sunflower oil to a stainless steel container maintained at -1°C for 3 hours, allowing all saturated fatty acid inclusions to reach their melting point. Subsequently, the inclusions are sieved (preferably through a 1.41 mm sieve) and poured into another stainless steel container. This allows the solid oil to be retained and the liquid oil, corresponding to unsaturated fatty acids and minor fractions (composed of polyphenols, tocopherols, and phytosterols), to be extracted. This second matrix is ​​the matrix to be ozonated.

[0016] The percentages indicated in the method steps refer to the weight and volume of the total sunflower oil matrix, respectively.

[0017] In another embodiment, the present invention relates to the method as defined above, wherein the polyunsaturated fatty acid is linoleic acid.

[0018] In another embodiment, the present invention relates to the method as defined above, wherein the sunflower oil is extra virgin sunflower oil.

[0019] In this invention, the term "highly ozonated fatty acid" refers to an extra virgin sunflower oil matrix or other plant-derived extra virgin matrix that, after a highly ozonated process, exhibits a peroxidation index greater than 200 mEq, preferably containing embedded stable ozonides (1,2,4-trioxopentane).

[0020] On the other hand, the present invention relates to extra virgin matrices that, after a high-level ozonooxidation process, still exhibit a high concentration of linoleic acid, and that the minor fractions composed of polyphenols, sterols, and tocopherols remain intact and unchanged. The latter (linoleic acid and minor fractions) exhibit a synergistic effect with the highly ozonooxidized fatty acids that enhance their medicinal and cosmetic properties.

[0021] The method of the present invention includes a dry crystallization grading step, which allows saturated fatty acids and a certain percentage of oleic acid present in the matrix to be filtered out through the melting point until a linoleic acid concentration of more than 90% is reached, without affecting the presence of phytosterols, polyphenols, vitamins, or the sensory characteristics of extra virgin sunflower oil.

[0022] The method of the present invention ozonates a matrix rich in unsaturated bonds (in this case, more than 90% linoleic acid) by using an airflow of 30-35% ozone, allowing a high peroxidation index of 200 to 1200 mEq O2 / Kg, and primarily using secondary ozonides or Criegee ozonides, thus delaying polymer formation.

[0023] On the other hand, during the entire ozonation process, which lasts for 3 hours, a 30-35% ozone stream is applied via bubbling through a sunflower oil matrix highly concentrated with linoleic acid (greater than 90%), using a gas flow (L / h) to oil volume (L) ratio between 150 and 500. This high ratio allows for a reduction in the concentration of more volatile components during ozonation, as they are entrained by the gas stream and expelled from the reactor, preferentially forming stable ozonides.

[0024] By means of the method of the present invention, the highly concentrated linoleic acid in the matrix is ​​preferably highly ozonated to a concentration between 200 and 1,200 mEq depending on the final product formulation; the reaction does not occur when the sunflower oil matrix is ​​completely saturated, so that the minor fractions of the sunflower oil matrix, such as sterols, polyphenols and tocopherols, remain unchanged during ozonation due to the affinity of ozone for linoleic acid.

[0025] In the preceding paragraph, complete saturation is understood as complete oxidation, or ozone causing the complete breakage of unsaturated bonds present in the matrix. If we ozonate the matrix of the present invention (which preferably contains 90% linoleic acid) to complete saturation, we will obtain 0% unsaturated bonds (linoleic acid) from the 90% composition after this method.

[0026] Therefore, thanks to the method of the present invention, the generation of high concentrations of stable ozonides is promoted without altering the extra virgin properties of the sunflower oil matrix or generating excess peroxides.

[0027] In the ozonation step of this invention, an ozone concentration of 30% to 35% obtained from pure oxygen (medical grade) is used to avoid the formation of toxic byproducts derived from nitrogen in the air. This avoids subsequent filtration processes that would degrade the secondary ozonides formed or alter the extra virgin properties of the sunflower oil matrix. Therefore, the method of this invention has the additional advantage of eliminating the need for a filtration step because it does not produce cytotoxic byproducts.

[0028] The reason why the method of the present invention does not produce the aforementioned cytotoxic byproducts is that the oxygen used is of medical origin rather than from the air. Furthermore, because under the saturated conditions under which it is operated, other components are not oxidized; only a portion of the major fraction, namely linoleic acid, is oxidized. Therefore, operating with a matrix having a high concentration of unsaturated bonds avoids the oxidation of all components of the matrix and the formation of toxic products.

[0029] Another aspect of the invention relates to highly ozonated fatty acids obtained by the method described above, characterized in that they contain peroxides, preferably at a concentration of 200 meq to 1200 meq, more preferably wherein the peroxide is 1,2,4-trioxopentane (Criegee ozonide), and even more preferably wherein the concentration of 1,2,4-trioxopentane (Criegee ozonide) is 200 meq to 1200 meq.

[0030] Another aspect of the invention relates to a pharmaceutical composition comprising the highly ozonated fatty acids described above and one or more pharmaceutically acceptable excipients.

[0031] Another aspect of the present invention relates to the highly ozonated fatty acids described above, which are used as pharmaceuticals.

[0032] Another aspect of the present invention relates to the use of the highly ozonated fatty acids described above in the manufacture of pharmaceuticals.

[0033] Another aspect of the invention relates to a method for treating a disease in a subject, particularly a human, comprising administering to the subject an effective amount of the hyperozonated fatty acids described above.

[0034] Another aspect of the invention relates to the highly ozonated fatty acids described above, which are used to treat and / or prevent diseases, such as: prevention and treatment of stress injuries at different stages, prevention and treatment of radiation dermatitis, and other conditions requiring repair of skin damage.

[0035] Another aspect of the present invention relates to the use of the highly ozonated fatty acids described above as cosmetics.

[0036] In another embodiment, the present invention relates to the use of the highly ozonated fatty acids described above, wherein the cosmetic use refers to skin moisturizing, restoring its elasticity and repairing photodamage.

[0037] Throughout the specification and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will become apparent to those skilled in the art, partly from the specification and partly from the practice of the invention. The following examples are provided by way of illustration and are not intended to limit the invention. Example

[0038] Example 1. Obtaining extra virgin substrate with a linoleic acid concentration greater than 90% and subsequent ozonation process for producing highly ozonated fatty acids.

[0039] Step 1. Obtain extra virgin substrate with high linoleic acid content.

[0040] 100 liters of extra virgin sunflower oil were placed in a continuously stirred reactor equipped with a cooling jacket, which allowed the temperature of the lipid matrix to be reduced to -1 degree Celsius for 3 hours. After 3 hours, the contents were passed through a No. 14 sieve (1.41 mm) to a storage container, during which solids (mainly saturated fatty acids that had reached their melting point) were extracted, and extra virgin sunflower oil with a linoleic acid content of more than 90% was obtained in the matrix storage container by gas chromatography.

[0041] Step 2. Ozonation process for producing highly ozonated fatty acids.

[0042] 500 liters of extra virgin sunflower oil with a high linoleic acid content (greater than 90%) are placed in a bubbling reactor. An ozone gas stream (ozone-oxygen) with a concentration up to 35% v / v is passed through the oil at a rate of 150 liters per hour, while the reactor temperature is maintained below 30°C. By this ozonation process, the peroxidation index of the stable ozonide (1,2,4-trioxopentane) obtained in the first hour is mainly between 200 and 400 mEq; in the second hour, the peroxidation index is between 400 and 800 mEq; and in the third hour, the peroxidation index is between 800 and 1200 mEq. The product is stored at a temperature not exceeding 30°C.

[0043] Regarding the quality of the highly ozonated fatty acids proposed in this invention, the following indicators may be used: acid index (AI), aldehyde concentration, viscosity determination, and / or peroxidation index (PI) determined using potassium iodide according to USP XXIV and BP 2000. However, given that the proposed ozonation method of this invention has an endpoint with parameters between 200 and 1200 mEq O2 / kg, and that it is a simple method, the peroxidation index (PI) and acid index (AI) are used as indicators of the progress and safety of the method, assessed hourly until a 3-hour cycle is completed.

[0044] Stability testing verified that the highly ozonated fatty acids obtained by this invention can retain their clinical and / or cosmetic properties as appropriate, with no evidence of the formation of toxic derivatives within 36 months.

[0045] Example 2. Application of highly ozonated fatty acids (AGHOZ) in Grade I and Grade II stress injuries.

[0046] The CIEC International Clinical Research Center (Centro Internacional de Estudios Clínicos) conducted an exploratory study to test the efficacy and safety of highly ozonated fatty acids (AGHOZ) in the healing of Grade I and II pressure injuries in elderly hospitalized patients in Santiago, Chile. For this purpose, the ethics committee approved the clinical protocol, trained nursing staff, and obtained informed consent or authorization as appropriate. The center implemented all standard pressure injury prevention measures (positioning, orthotics, etc.), but injuries still occurred despite these measures. A total of 6 patients were included (3 men and 3 women), aged 72 ± 2.5 years, all bedridden, with comorbidities: 83% HTN, 67% DM2, 33% renal failure, 33% organic brain injury, etc. Injuries were located at bony prominences: 80% at the sacrococcygeal region, 30% at the heel, and 10% at the elbow (2 patients had more than one pressure injury). 70% of the injuries corresponded to Grade I pressure injuries, and 30% to Grade II pressure injuries. None showed signs of active infection. All patients were frail, suffering from multiple symptoms of delayed healing, and conventional prevention and treatment of pressure injuries had failed. High ozonated fatty acids (AGHOZ) were applied twice daily, gently mixed into the dressing, as a standard measure for pressure injuries.

[0047] Daily use of highly ozonated fatty acids resulted in complete improvement of Grade I pressure injury within 10 ± 3 days and complete improvement of Grade II pressure injury within 15 ± 3 days, with no associated adverse events, and excellent tolerability and satisfaction among the responsible nurses and patients. All patients showed signs of first significant improvement within 3 to 7 days, which was considered excellent considering the study population and the condition of the patients included in the study.

Claims

1. A method for obtaining highly ozonated fatty acids, comprising the following steps: i. A concentrated polyunsaturated fatty acid fraction of sunflower oil, used to obtain a sunflower oil matrix by dry crystallization fractionation, the sunflower oil matrix having a polyunsaturated fatty acid concentration of at least 90%, while maintaining the minor fractions unchanged, the minor fractions comprising polyphenols, phytosterols, and tocopherols; and ii. In a bubble reactor, the sunflower oil matrix of step (i) is ozonated with a gas stream of 30% to 35% ozone from pure oxygen at a temperature of 25°C to 30°C and a gas flow rate of 150 L / h to 500 L / h to obtain the highly ozonated fatty acids.

2. The method according to claim 1, wherein the polyunsaturated fatty acid is linoleic acid.

3. The method according to any one of claims 1 or 2, wherein the sunflower oil is extra virgin sunflower oil.

4. A highly ozonated fatty acid, obtained by the method according to any one of claims 1 to 3, characterized in that... The highly ozonated fatty acids contain peroxides at a concentration of 200 meq to 1200 meq.

5. A pharmaceutical composition comprising the highly ozonated fatty acid according to claim 4 and one or more pharmaceutically acceptable excipients.

6. The highly ozonated fatty acid according to claim 4, which is used as a pharmaceutical.

7. The highly ozonated fatty acid according to claim 4, used for the treatment and / or prevention of diseases selected from different levels of pressure injury and different levels of radiation dermatitis.

8. The use of the highly ozonated fatty acid according to claim 4 as a cosmetic.

9. The use according to claim 8, wherein the cosmetic use refers to moisturizing the skin, restoring its elasticity, and repairing photodamage.