WO2007046122A2 - Huile ozonisee, procede d'elaboration et utilisation dans le domaine medical - Google Patents

Huile ozonisee, procede d'elaboration et utilisation dans le domaine medical Download PDF

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Publication number
WO2007046122A2
WO2007046122A2 PCT/IT2006/000736 IT2006000736W WO2007046122A2 WO 2007046122 A2 WO2007046122 A2 WO 2007046122A2 IT 2006000736 W IT2006000736 W IT 2006000736W WO 2007046122 A2 WO2007046122 A2 WO 2007046122A2
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Prior art keywords
oil
ozonised
anyone
ozonised oil
preparation
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PCT/IT2006/000736
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English (en)
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WO2007046122A3 (fr
Inventor
Orsola Modica
Maria Rosa Maietta
Gabriele Maietta
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Bioo3Pharma S.R.L.
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Priority to EP06810027A priority Critical patent/EP1937635A2/fr
Publication of WO2007046122A2 publication Critical patent/WO2007046122A2/fr
Publication of WO2007046122A3 publication Critical patent/WO2007046122A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/006—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by oxidation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J10/00—Chemical processes in general for reacting liquid with gaseous media other than in the presence of solid particles, or apparatus specially adapted therefor
    • B01J10/002—Chemical processes in general for reacting liquid with gaseous media other than in the presence of solid particles, or apparatus specially adapted therefor carried out in foam, aerosol or bubbles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J10/00—Chemical processes in general for reacting liquid with gaseous media other than in the presence of solid particles, or apparatus specially adapted therefor
    • B01J10/007—Chemical processes in general for reacting liquid with gaseous media other than in the presence of solid particles, or apparatus specially adapted therefor in the presence of catalytically active bodies, e.g. porous plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00049—Controlling or regulating processes
    • B01J2219/00051—Controlling the temperature
    • B01J2219/00054—Controlling or regulating the heat exchange system
    • B01J2219/00056—Controlling or regulating the heat exchange system involving measured parameters
    • B01J2219/00058—Temperature measurement
    • B01J2219/00063—Temperature measurement of the reactants
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L5/00—Gas handling apparatus
    • B01L5/04—Gas washing apparatus, e.g. by bubbling

Definitions

  • the present invention concerns an ozonised oil, the process for the preparation and uses thereof in medical field.
  • the present invention refers to an ozonised oil produced by a purposely provided ozonating process using ozone gas bubbling through a supporting biological matrix. Said process supplies, in a repetitive and standardised way, the same active principle, characterised by high peroxide number, high stability during the time (years) and multiplicity of therapeutic effects similar to those supplied by ozone therapy, currently used in medicine for clinical treatments.
  • the clinical ozone therapy employs ozone gas and/or ozone dissolved in apyrogenic water.
  • Therapeutic treatments involve the use, for a large range of pathologies like infective pathologies, immune dysfunctions, articular pathologies, ischemic pathologies, neurovegetative pathologies, of direct administration of ozone gas involving applications by intravenous, endo-arterial, parenteral route, endo-cavitary, end ⁇ -pleural and endo-peritoneum insufflations, intra-articular injections, intra-lesion intra-foraminal, topical applications with ozone gas.
  • therapeutic doses vary from 20 to 80 mcg/ml.
  • high doses i.e. 80 mcg/ml are used.
  • lower therapeutic gradually decreasing doses are used (from 40 mcg/ml to 20 mcg/ml).
  • low therapeutic doses (20 - 30 mcg/ml), involving more than one daily applications during a longer time period, allows to achieve better therapeutic effects than high dos,es (80 mcg/ml), involving fewer daily applications during shorter time period.
  • the ozone therapy is a therapeutic technique affording excellent and resolving outcomes for a large range of clinical pathologies, in comparison to the results obtained with traditional therapeutic treatments.
  • the ozone therapy although providing useful and resolving results, presents in the application steps operating restraints and difficulties resulting in noteworthy limitations for said technique, as follow: - the applications demand the patient to move themselves to the place of ozone production, wherein therapies are carried out;
  • the applications involve the use of invasive and often painful techniques for the patients; - possibility that during the applications emboli can be formed, whose effects often result in the death of the patient,
  • compositions including ozonised oils obtained using processes different from the present invention like for example the composition described in EP1273295 are commercially available.
  • These oils i.e. the inventive and others, although have similar IR spectra, have organoleptic characteristics not suitable for aesthetic-cosmetic applications and many pharmaceutical uses. In particular they have rancid or acid bitter smell, further are stable only for a limited time period (approximately a year), have low pH (3,5), after the application during a short time period (5-10 minutes) peroxides are developed and seem to be commercially available for rectal uses only, probably because of their organoleptic characteristics penalising for other application fields.
  • the authors of the present invention now provide a process for ozonised oil production characterised by technical foresights and selections suitable to supply a stable end product, containing high peroxide number and the ozone content therein is released gradually and continuously for a long time period (hours).
  • the ozonised oil according to the present invention exerts high functional activity and various therapeutic actions such that it performs as a particularly versatile product for various application types allowing a large range of therapeutic treatments in various clinical fields of medicine, in the trichological aesthetic-cosmetic as well in veterinary field, to be carried out.
  • an unsaturated fatty acid as for example the oleic acid present in many vegetable oils and in lipophilic environment, ozone reacts resulting in the formation of ozonides (1 ,2,4 trioxolane) and heat development.
  • ozone gas is transformed in the highly reactive ozone anion radical, under heterogenous catalytic conditions, for example using solid ferrous sulphate FeSO 4 as catalyst.
  • Ferrous sulphate (FeSO 4 ) crystalline inorganic salt, is an optimum promoter for radical reactions being an electron donor.
  • Fe 2+ (electron donor) ozone instantaneously reacts forming ozone anion radical:
  • Iron (II) compounds are oxidized by ozone (O 3 ) to iron (III) compounds, thus ozone, although powerful oxidant, is converted to strongly reactive ozone anion radical.
  • ozone although powerful oxidant, is converted to strongly reactive ozone anion radical.
  • induction step propagation step
  • interruption step interruption step
  • step ozone (O 3 ) in the presence of Fe 2+ ions, reacts instantaneously resulting in the formation of the strongly reactive ozone anion radical (O 3 "° ).
  • an appropriate and specific reaction chamber which is set forth hereafter, has been devised and realised currently and which is located and installed inside of the cavity of a porous fine grind bubbling septum after the connection of ozone gas inlet duct.
  • the system consisting of the reaction chamber and porous bubbling septum is immersed in the oil, for example linen oil, to be ozonised.
  • ozone gas produced by the ozone generator, is introduced in such a way to lick ferrous sulphate (FeSO 4 ) solid crystals previously deposited inside of the same reaction chamber.
  • FeSO 4 ferrous sulphate
  • the configuration and constitution of the reaction chamber as well as the positioning inside of the cavity of the fine grain porous septum allow to isolate the catalyst from the vegetable oil, due to the effect of the introduced ozone gas pressure inside of the reaction chamber which prevents the spreading of the linen oil within the same room. In this way the catalyst does not show any deactivation or alteration resulting from the linen oil.
  • the linen oil also, is not subjected to any contamination by the catalyst, due to the presence of the fine grain porous septum which prevents the spreading of the catalyst into the oily matrix.
  • the fir ⁇ e grain porous bubbling septum allows the fast and immediate spreading of the ozone anion radical within the oily mass, under a moderated and continuous mechanical agitation, wherein ozone escapes as microbubbles.
  • ozone gas in the presence of Fe 2+ ions, instantaneously reacts resulting in the formation of strongly reactive ozone anion radical (O 3 "° )
  • O 3 "° strongly reactive ozone anion radical
  • the above reaction occurs in lipophilic environment.
  • the propagation step occurs in the presence of ozone anion radical (0 3 "° ) which attacks the glicerol unsaturated fatty acid ester mixture.
  • ozone anion radical instantaneously attacks tri-unsatu rated fatty acid (linolenico acid:
  • the propagation step is characterised by the reaction heat development, that, with the chemical reaction progress, occurs with gradual and continuous increments during the time.
  • the interruption step occurs as the reaction between ozone and unsaturated fatty acids is completed and consequently the reaction heat does not increase any more. From above it is sufficient to measure, using a precision thermometer, the temperature values in order to detect the reaction starting, propagation and end.
  • the stabilization process of the ozonised oil has been adjusted considering the course of the reaction between ozonide and an ox-red system consisting, for example, of oxidized alpha lipoic acid and acetyl lipoate.
  • acetyl lipoate is the reaction product from the reaction between oxidised alpha lipoic acid and acetic aldehyde and that the reaction of acetyl lipoate with ozonide forms reduced alpha lipoic acid, acetic acid and unsaturated peroxide. Therefore alpha lipoic acid is free in the reduced form.
  • This oxidation occurs by means of unsaturated peroxide providing as reaction product oxidized alpha lipoic acid and peroxide.
  • Acetic acid produced from the reaction of acetyl lipoate and ozonide is not free but is bound to reduced alpha lipoic acid yielding acetic aldehyde and oxidized alpha lipoic acid. In such a way the ox-red system is restored.
  • other ox-red systems generally consisting of an A) oxidant substance and a B) reducing substance, both soluble in the ozonised oil to be treated, suitable to carry out an ox-red action against the ozonides present and suitable to restore the efficiency of the system activity, can be used.
  • Oxidised alpha lipoic acid and acetyl lipoate, at the end of the ozonization process, are added in small amounts (0.02% ⁇ 10%) to known volume of 100 ml of ozonised oil to be subjected to stabilization treatment.
  • the ozonised oil, containing the peroxides is stored in the optimum conditions, in a dark glass container equipped with screw cap and silicone sealing, in order to avoid light interactions.
  • the container with its content is stored, in optimal conditions, at room temperature and/or put in a refrigerator at temperature of 4 - 6°C, to obtain a longer safe storage left standing for 48 hours before the use.
  • said determination currently is carried out using the iodometric indirect method, described hereafter in the description, which uses sodium thiosulfate 0.1 N solution as titrant.
  • the titration in order to be completed requires several days (four days) because the ozonised oil retains remarkably tenaciously fixed peroxides therein, thereby some time must elapse so that the latter can completely be freed and this occurs in a continuous and gradual way during four days.
  • the ozonides are transformed in stable peroxides with lower steric hindrance than ozonides and are localised in an oriented and ordered way within sequential molecular layers establishing from each other electrostatic interactions due to the presence of dipoles and this justifies the remarkable tenacity according to which peroxides are fixed and maintained in the ozonised oil.
  • It is therefore a specific object of the present invention a process for the preparation of ozonised oil including the following steps: a) evolvement of ozone anion radical by heterogenous catalysis carried out by means of ozone gas licking a suitable electron donor catalyst characterised in that the redox potential thereof is lower than ozone (2.08V); - b) contacting the ozone anion radical with an oil comprising unsaturated fatty acids for the formation of ozonides avoiding the contact between the oil and catalyst; c) verification of the reaction end when temperature constant values are obtained; d) stabilisation of the ozonised oil produced at the end of step c) by means of reaction with an suitable ox-red system resulting in the peroxide formation.
  • Ozone is bubbled at a concentration from 80 to 110 mcg/ml.
  • the catalyst can be chosen from the group consisting of ferrous sulphate, powder zinc metal, tin (Sn 2+ ) salt, silver metal.
  • the oil comprising unsaturated fatty acids can be chosen from the group consisting of linen oil, ximenia oil and mixtures thereof, walnut oil, soy bean oil, wheat germ oil.
  • the ox-red system can be chosen from oxidised alpha lipoic acid and acetyl lipoate, alpha tocopherol and ascorbyl palmitate, butyl hydroxy anisole and lecithin.
  • the catalyst is in amount from 150 to 250 mg for 100 ml of oil. Any component of the ox-red system can be used in amount from 150 to 250 mg for 100 ml of oil.
  • an ozonised oil obtainable by means of the above described process constitutes a further object of the present invention.
  • the invention concerns a pharmaceutical composition comprising the ozonised oil like active principle associated with one or more pharmaceutically acceptable adjuvant and/or excipient.
  • the ozonised oil can be employed at concentration from 20 to 80 mcg/ml.
  • the ozonised oil cab be used as component for compositions in the form of emulsion, cream, suppository, clysters, pessary, capsules, powder wherein the ozonised liquid oil is adsorbed on an inert support, for example micronized silica.
  • the ozonised oil and the pharmaceutical composition according to the present invention for use in medical field represent a further object of the present invention.
  • the ozonised oil according to the present invention can be advantageously employed like carrier for drugs and/or phytotherapeutics.
  • the ozonised product exhibits synergism with specific pharmacological treatments (gentamicine, betamethasone, tetraciclin, clindamicine, erythromycin, sodium diclofenac, ketoprofen, acetyl salicylic acid, ibuprofen, nitroglycerin, phosphatidylcholine, etc) as well as with the most phytotherapeutics. In fact it enhances their activity, as a result of their greater penetration and spread and therefore it favours a greater and more effective intracellular absorption in reduced times.
  • the ozonised oil exhibits a sum synergy action.
  • the mechanism of biological action of the ozonised oil on the living tissue and, in particular, on the cell is based on the low molecular weight and on the short chain length of fatty acids saturated by atoms of oxygen as well as hydrophilic character of the molecule allowing to be blended with the cell wall causing the citosol pouring triggering the cycle of the reactions associated with the transformation of peroxides in hydrogen peroxide.
  • This reaction associated with the breakdown of the GSH molar equilibrium (reduced glutathione) - GSSG (oxidized glutathione) results in an acceleration of the pentose shunt and therefore of the glycolysis.
  • a further object of the present invention concerns the use of the ozonised oil and the pharmacological composition according to the present invention for the preparation of a medicament like germicide, antiviral, derma regenerating, anti-inflammatory, anti-phlogistic, analgesic, fungicide, immuno-stimulating medicament.
  • the ozonised oil proves to be particularly effective and resolving for the bacterial and infections, specially in all particularly chronic and the particularly external infections and caused both by circulatory defects and traumas and burns contaminated by chemio-antibiotic resistant pathogenic agents. Therefore, the use of the ozonised oil and pharmacological composition for the preparation of a medicament for the treatment of trophic ulcers, anal and vaginal abscesses, rhagades, fistulas, decubituses, phlegmon furuncles, purulent gengivitises, stomatitises, sinusitisises, vulvovaginitises, herpetic lesions, chronic osteomyelitis in immunodepressed patients, protozoa and fungal infections constitutes an object of the present invention.
  • the ozonised oil proves to be, moreover, particularly useful and effective in the derma degenerative processes enhancing in short times the regeneration of the corneous layer.
  • the ozonised oil activated biological mechanisms are developed by means of: the disinfectant action, resulting in lower bacterial loads, the vasodilatation, the iper-oxygenation, the diminution of the tissutal acidity and the edema re-absorption establish the activation of the metabolic activity.
  • the ozonised oil plays an important role in infectious pathologies deriving from a dysfunction of the insufficient immune system.
  • the ozonised oil acts on the infections supported by bacterial, viral, fungal and protozoa agents, by oxidizing any cellular component in a substantially irreversible way.
  • the ozonised oil activates the immune system through the induction of the cytokine production.
  • the virulicide action of the ozonised oil results form the peroxidation of phospholipids, binding proteins and lipoproteins of viral membranes.
  • the ozonised oil is a medicament acting through an action directed on the cellular surface, is able to penetrate and to cross the cellular membrane in order then to diffuse easy at intracellular level, and as such it is able to activate various metabolic pathways.
  • the ozonised oil due to its functional activity, allow in short times a fast and effective therapeutic action characterized by an elevated germicide, antiviral, anti-inflammatory, antiphlogistic and analgesic action to be carried out.
  • the ozonised oil containing an elevated number of present peroxides, remains unchanged for many years, in the optimal conditions involving a room temperature storage in suitable well sealed containers and protected from the light and/or a fridge storage at temperature of 4 - 6°C, for the attainment of a longer conservation.
  • the preparation is viscous at 22 0 C and very dense at 4 -6°C, manifests a typical acrid ozone smell, and has pH 4,5.
  • the concentration of the active principle to be employed can be adjusted and modulated, depending on the therapeutic dose to be executed, by means of dilution with petrolatum and/or vegetable oils and/or compatible products.
  • the ozonised oil manifests an easy adaptability for most varied requirements and high therapeutic effectiveness and through applications and/or administrations can be used in order to execute house therapies of curative and/or preventive type. It is well tolerated from the organism and it does not evidence undesired effects, employing the appropriate therapeutic doses.
  • the ozonised oil also at low dose (20 mcg/ml) displays an effective therapeutic action. At high doses (80 mcg/ml) the oil proves to be well tolerated by the organism and it does not provoke undesired effects.
  • the oil according to the present invention allows that specific aimed therapies and house operation whose modalities are quite simple and easy to be executed, to be carried out.
  • the ozonised oil proves particularly effective and resolving for treatments designed for infectious and viral symptomatologies, in dermal and mucosal regenerative processes articular inflammatory processes, dermatology, proctology, gynaecology, orthopaedics, rheumatology, urology, gastroenterology, dentistry, aesthetic cosmetic-trichological treatments and veterinary medicine.
  • Absolute contraindications do not exist for the use it of the ozonised oil, however it is necessary caution in the following conditions: existing haemorrhages since the coagulation time is increased; pregnancy, since the maternal immunity is increased and abortions can be induced; spastic conditions, since the adrenergic tone is increased; thrombocytopenia, because ozonised oil interacts with platelet activating factor; hypoglycaemia, because the glycolysis is increased, bronchospasm, because the leukotriene level is increased.
  • the ozonised oil having various functional activities, employed for therapeutic objects, is suitable to develop the following activities: bactericide; virucide; fungicide; protozocide; analgesic, anti-inflammatory, anti-phlogistic; tissue regenerating, healing; sclerosant; immuno-stimulating activities.
  • the ozonised oil due to potentiality of functional activities, physico-chemical- therapeutic characteristics, manipulation simplicity and easiness and high stability during the time (years) allows aimed therapeutic treatments for a wide range of clinical pathologies to be carried out.
  • the present invention concerns the use of the ozonised oil and pharmacological composition for the preparation of a medicament for the treatment of the gastritises, diarrhoeases, obstinate constipations, Crohn's disease, sebaceous cysts; in dermatologic field for the treatment of simplex and Zoster Herpes, contact dermatitises, chilblain, acne, mycosis, eczemas, psoriasis, hand and foot rhagades, bugs and hymenoptera bites; in angiology and phlebology for the treatment of coronary and arterial pathologies, decubitus ulcers, gangrenes, venous insufficiency, phlebopathies, diabetic ulcers, post- phlebitic ulcers; in orthopaedics for the treatment of disc-radicular conflicts, arthrosis, periarthritis, lumbar sciatica, tendinitises, strains; in rheumat
  • the present invention concerns a device for the ozonization of an unsaturated fatty acid rich oil
  • a device for the ozonization of an unsaturated fatty acid rich oil comprising an ozone feed pipe, one end of said pipe containing a catalyst for the conversion of ozone in ozone radical, said end being substantially inserted in the middle of a fine grain porous septum, said fine grain porous septum being dipped in the unsaturated fatty acid rich oil to be ozonised.
  • the device according to the invention further can comprise a temperature measuring device.
  • the catalyst is chosen from the group consisting of ferrous sulphate, powder zinc metal, tin (Sn 2+ ) salt, silver metal.
  • the fine grain porous septum is made of inert material, for example ceramic material.
  • Figure 1 shows the device according to the present invention wherein (1) it is the ozone feeding pipe, (2) is the catalyst containing end; (3) ring spacer; (4) porous septum cavity wherein the pipe end is located;
  • FIG. 1 shows IR spectra of the ozonised oil according to the present invention.
  • Figure 3 shows the microscope analysis of the number of viable cells in the non treated control.
  • Figure 4 shows the microscope analysis of the number of.viable cells in the ozonised oil treated sample at concentration of 0.057 mg/ml.
  • Figure 5 shows the microscope analysis of the number of viable cells in the ozonised oil treated sample at concentration of 0.019 mg/ml.
  • Figure 6 shows the microscope analysis of the number of viable cells in the ozonised oil treated sample at concentration of 0.057 mg/ml.
  • Figure 7 shows the microscope analysis of the number of viable cells in the ozonised oil treated sample at concentration of 0.019 mg/ml.
  • Figure 8 shows the microscope analysis of the number of viable cells (keratinocytes) in the not treated control.
  • Figure 9 shows the microscope analysis of the number of viable cells (keratinocytes) in the ozonised oil treated sample at concentration of 0.057 mg/ml.
  • Figure 10 shows the microscope analysis of the number of viable cells (keratinocytes) in the ozonised oil treated sample at concentration of 0.019 mg/ml.
  • Figure 11 shows the microscope analysis of the number of viable cells (keratinocytes) in the not ozonised oil treated sample at concentration of 0.057 mg/ml.
  • Figure 12 shows the microscope analysis of the number of viable cells (keratinocytes) in the not ozonised oil treated sample at concentration of 0.019 mg/ml.
  • reaction completion The time elapsed from the start, propagation and end of the reaction is equal to 70 minutes.
  • the end of the reaction add, under stirring, to the ozonised fluid mass 200 mg of alpha lipoic acid and subsequently 200 mg of acetyl lipoate. Continue the ozonization for additional 10 minutes.
  • the ozonised fluid mass in a dark glass bottle equipped with a screw cap with silicone sealing. The bottle with its content is stored at room temperature protected from the light and/or in fridge at temperature of 4 - 6 0 C, for the attainment of longer conservation, and left standing for 48 hours before the use.
  • figure 2 IR spectra of the obtained ozonised oil is shown.
  • the peroxide number and/or peroxide index (Ip) is the number expressing as milliequivalents of active oxygen the amount of peroxides in 1000 g of a substance, determined using the following described method: introduce 2,5 g (m) of ozonised oil to be tested in a 250 ml flask equipped with ground glass stopper; add 30 ml of glacial acetic acid; shake repeatedly and add 500 mg of potassium iodide; shake repeatedly and store the solution in the dark for 4 days assuring that the flask is well plugged.
  • the method is based on the oxidation activity of a known amount of peroxide containing sample, able to release iodine from potassium iodide (Kl) in acid environment. Iodine is titrated with known titer sodium thiosulfate (0.1 N):
  • titer i.e. the peroxide number in the ozonised oil
  • the peroxide index IP is the number expressing in milliequivalents of active oxygen the peroxide amount in 1000 grams of a substance.
  • the titer of the ozonised oil obtained after 80 minutes by the production process, is be equal to 15.335 mcg/ml.
  • the ozonised oil is used in the therapeutic treatments at the same doses as in the clinical-medical ozone therapy which uses ozone gas.
  • therapeutic doses from the lowest of 20 mcg/ml to the highest value of 80 mcg/ml are used.
  • the ozonised oil therefore, is used at doses of therapeutic use from a minimum of 20.0 mcg/ml (10.000 ppm) to a maximum of 80.0 mcg/ml (40.000 ppm).
  • the volume (ml) and/or the weight (g) of the ozonised oil can be determined.
  • the dilution of the ozonised oil can be carried out using petrolatum and/or vegetable oils and/or compatible products. To this end it is useful to pre-determine the dilution ratios which in the specific case are as follow:
  • the ozonised oil has a titer equal to 15.335 mcg/ml
  • Odour acrid, typical of ozone; - taste: metallic, slightly pungent;
  • the ozonised oil possesses a wide therapeutic potential resulting from various functional activities. In order to verify said activities several tests involving a wide range of clinical pathologies have been carried out.
  • the epithelium protecting and derma restoring process stimulating activity has been assayed using the test of the open wounds resulting from accidental cuts, or ulcers resulting from severe burns, or injuries resulting from post-surgical operations.
  • the treatment with ozonised oil at a dose of 80 mcg/ml daily has been carried out for two days, and in the following, for other two days a dose of 20 mcg/ml daily has been employed. At the end of the treatment it has been possible to observe the complete healing of the wounds.
  • the transcutaneous ⁇ applied ozonised oil is perfectly tolerated and does not interfere with the physiological reactivity of the epidermis and derma evidencing that the ozonised oil has an epithelium trophic effect favouring the regenerative processes of the wounds and burns.
  • Applications of petrolatum and/or vegetable oil diluted ozonised oil (1 :4) carried out with the simple bandaging technique, left in situ, (cute of the dorsal region) for 5 days evidenced that the transcutaneous contribution of the ozonised oil does not interfere adversely on the cute physiological responses and it does not induce side-effects.
  • the previously described method of production allows the product to be always obtained every time with the same characteristics, provided that modalities, procedures, and recommendations are observed in the methodics of the ozonised oil production process.
  • the ozonised oil produced by the above said process maintains unaltered various functional activities and unchanged chemical - physical and therapeutic characteristics as below reported: colour; density; pH; peroxide number; from one to another production process run acceptable variations, with respect to the titer value, oscillating around to +/- 3 %; a gradual and constant release of present peroxides during a time period of about 8 hours resulting in sustained therapeutic effects; a stability of the ozonised oil for long periods (years), in the optimal conditions involving that the ozonised oil is stored at room temperature in suitable well plugged and sealed containers, protected from the light, and/or put in fridge at temperature fro 4 to 6 0 C, for the attainment of a longer storage; a constant and unaltered functional activity of the ozonised oil, which exerts its own activity also after
  • the activity of the ozonised oil used at appropriate doses for therapeutic use is displayed with effective therapeutic effects and acts on a wide range of clinical pathologies.
  • the production method finally, allows repetitive and reproducible, therefore in standardized way, a reproducible final product, exerting various and particular functional activities and displaying chemical-physical-therapeutic characteristics similar to ozone gas, to be obtained.
  • the yellow brown ozonised oil is viscous at 22 0 C and very dense at 6 0 C, has an acrid ozone typical smell and pH equal to 4,5.
  • the product is very stable in the time.
  • tests aiming to verify the degradation on storage of the ozonised oil have been carried out according to the following conditions: - A (in dark bottle protected from the light and in the fridge at temperature from 4 to 6°C): - B (exposure to the light and storage at ambient temperature with thermal excursions from 15 to 35°C).
  • Condition (A) Storage of the ozonised oil protected from the light and at temperature from 4 to 6°C ;
  • Condition (B) Storage of the ozonised oil in the presence of daylight and at ambient temperature with thermal excursions from 15 to 35 0 C (table 1);
  • the evaluation of the obtained data allows to conclude that in the four performed runs the stabilized ozonised oil displays: in the first condition (A): the decay after three, six and months is about by 0.25 %, 0.5 % and 1 %, respectively; in the second condition (B): the decay after three, six and months is about by 0.1 %, 2 % and 3 %, respectively. According to these results it is possible to assess that, in the condition (A), after one year the decay of the peroxide number of the stabilised ozonised oil, stored according to the previously reported recommendations, i.e. in glass dark bottle protected from the light and in fridge at temperature from 4 1 6°C, is equal to 1 % in comparison to the initial values as determined at the production time.
  • titer peroxide number determined by the iodometric indirect method and using sodium thiosulfate 0.1 N as titrant
  • therapeutic dose from 20 to 80 mcg/ml
  • Keratinocyte is the most important epidermal cell type and it takes part in every functional aspect of skin characterization.
  • the keratinocytes used for the in vitro present tests are primary, i.e. they are taken from a biopsy of a donor's healthy skin.
  • MTT assay evaluates the cell viability in vitro after exposure to various product concentrations in comparison to not treated cells. MTT assay makes it possible to measure both the threshold dosage of tolerability of the tested product on keratinocytes and, if present, the dosage suitable to stimulate the cell growth.
  • Materials and Methods Preparations of the samples The cell cultures are treated with scalar concentrations of the test compound and controls. Standard SDS (sodium dodecyl sulfate) is used as the positive control (substance with well known cytotoxicity effects) and an internal standard with IC 50 > 5 mg/ml is the negative reference (not cytotoxic substance). The sample is tested at concentration from 0.02 to mg/ml, the negative standard at concentration from 0.16 to 5 mg/ml, SDS at concentration from 0.00005 to 0.05 mg/ml. Test execution
  • a suitable number of cells (30.000 cells/well, 28 th passage) are seeded in the wells (96 well plate, 150 ⁇ l/well of cellular suspension), when a confluence of 60-70 % has been reached, fresh medium is added with scalar dilutions of the tested product and standards. Wells containing not treated cells are negative controls. Product incubation goes on overnight (24 hrs). After medium replacement with fresh medium + MTT, cells are incubated for 3 hrs at 37°C. Then cells are washed more times to eliminate MTT solution residues. Spectrophotometer reading is carried out at 540 nm wavelength.
  • IC 50 value 50 % cell growth inhibiting concentration
  • IC 50 parameter makes it possible to evaluate the potential irritating effect according to the following scheme:
  • IC 50 ⁇ 0.5 means a strong cytotoxic/irritating effect
  • IC5 0 between 0.5 and 1.5 means a moderate cytotoxic/irritating effect
  • IC 50 > 1 ,5 means absence of any cytotoxic/irritating effect
  • IC 50 > 1 ,5 means absence of any cytotoxic/irritating effect
  • Example 3 In vitro evaluation of growth stimulating activity of ozonised oil according to the present invention through in vitro assays on keratinocyte and fibroblast cell cultures (MTT assay).
  • MTT assay can be carried out in order to evaluate in vitro the potential growth stimulating activity of a compound on keratinocytes and fibroblasts.
  • the in vitro test on skin-derived cells proves to be an experimental method suitable to provide a lot of information about the reactions which may occur in vivo.
  • Keratinocyte is the most important epidermal cell type and it takes part in every functional aspect of skin characterization.
  • the keratinocytes used for in vitro present tests are primary, i.e. they result from a biopsy of a donor's healthy skin.
  • Fibroblasts are present in derma, i.e. the cutaneous layer under epidermis, they attend to synthesize collagen and other fibers constituting the derma extracellular matrix. In these experiments primary fibroblasts, derived from human dermis, have been used.
  • MTT assay evaluates the cell viability in vitro after exposure to various product concentrations in comparison to not treated cells. MTT assay makes it possible to detect, if present, the dosage suitable to stimulate the cell growth.
  • the culture cells are treated with scalar concentrations of the tested product from 0.004 to 0.5 mg/ml (in PEG 2:1). Cell cultures
  • keratinocytes from human skin biopsies, fibroblasts, from human dermis. Cells were seeded in a homogeneous way in 96 well plates for the experiments.
  • Fibroblasts have been incubated with MEM (Minimal Essential Medium)-Sodium-Piruvate + 5% calf foetal serum (FCS).
  • a suitable number of cells (10.000 cells/well, keratinocytes 20 th passage, fibroblasts 9 th passage) are seeded in the wells (96 well plate, 150 ⁇ l/well of cellular suspension), when a confluence of 60-70 % has been reached, fresh medium containing scalar dilutions of tested product is added. Wells containing not treated cells are negative controls. Product incubation goes on for 24-72 hrs. After medium replacement with fresh medium + MTT, cells are incubated for 3 hrs at 37°C. Then cells are washed several times to eliminate MTT solution residues. The spectrophotometer reading is made at 540 nm wavelength. Table 4 shows the stimulation of the keratinocytes after 48 hours of incubation with the sample at variable concentrations.
  • the tested product proves to possess a stimulating activity of the cellular growth at concentration from 0.004 to 0.25 mg/ml.
  • Such activity is particularly meaningful at concentration from 0.125 to 0.25 mg/ml.
  • Table 5 shows the stimulation of the keratinocytes after 72 hours of incubation with the sample at variable concentration.
  • the tested product proves to possess a remarkable stimulating activity of the cellular growth at concentration from 0.031 to 0.5 mg/ml.
  • Table 6 shows the stimulation of the fibroblasts after 24 hours of incubation with the test sample at variable concentrations.
  • the tested product proves to possess a remarkable stimulating activity of the cellular growth at concentration from 0.063 to 0.5 mg/ml.
  • Example 4 Evaluation of in vitro anti-oxidant functionality of the ozonised oil according to the present invention by the study of its anti- radical action on human keratinocyte cell culture.
  • the aim of this assay is to evaluate if the tested product, at different concentration, possesses an in vitro anti-oxidant activity. For this purpose, its capacity to scavenge reactive oxygen species (ROS) and inhibit the cell death is investigated. This capacity should be useful to counteract the cutaneous cell ageing.
  • ROS reactive oxygen species
  • the in vitro test on skin-derived cells proves to be an experimental method suitable to provide a lot of information about the reactions which may occur in vivo. '
  • the keratinocytes are epidermis characteristic cells and have a key role in all the functions of the skin. In these experiments we used keratinocytes derived from biopsies of human healthy donors.
  • the first kind of test allows to evaluate whether the test compound has the capacity to scavenge ROS (Reactive Oxygen Species) by measuring in vitro the amount of cell produced ROS after exposure to induced oxidative stress, in comparison with non treated controls.
  • the second test by means of the determination of the cell viability using the MTT method, after exposure or not to the oxidative stress, allows the cell global damage to be evaluated (without and after oxidative stress) and the protection effect resulting from the test compound at different concentration.
  • the sample has been diluted at concentration from 0.001 to 0.5 mg/ml (in PEG 2:1). Suitable controls have been added to the test and Vitamin C 0.15 mg/ml being a well known antioxidant agent has been used as positive control.
  • the test substance has been diluted at final required concentration in saline. Separately, dichlorofluorescein acetate (DCA) is dissolved in suitable buffer. DCA reacts with free radicals, if present, generating a fluorescent derivative and the fluorimeter reading allows to have a quantitative value correlated to the presence of this substance in the tested cells.
  • a suitable number of cells (25000 cells/well, 29 th passage) are seeded in a 96 well plate. After an overnight pre-incubation period with the sample at different concentration, the culture medium is withdrawn from the plates and replaced with 500 ⁇ l of DCA solution. Plates are incubated at 37°C for 15' in a CO 2 thermostat. At this time DCA solution is discharged.
  • the lamp used in the experimenters is a solar light simulator with a constant emission in the UVA range from 315 to 400 nm.
  • the UVB emission is appropriately screened in order to avoid direct cell damage to cell cultures.
  • the cell containing plates are irradiated at room temperature, with an intensity of 1.7 mW/cm 2 of UVA (5 J/cm 2 ).
  • the fluorimeter reading is carried out at 485 nm as excitation wavelength and 530 nm as emission wavelength, directly on the plate (Toxicol. Letters 1997-93:47-54).
  • MTT assay Before and at the end of the UV exposure a MTT assay is perform ' ed to measure the toxic impact on the cellular energy system (mitochondria) in comparison with cells not protected from oxidative stress and cells not exposed to stress.
  • the MTT assay is simple, accurate and yields reproducible results.
  • This method developed originally by Mossman (1993), is based on (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT, having yellowish colour in solution. Mitochondrial dehydrogenase of viable cells cleaves the tetrazolium ring leading to the formation of purple water insoluble salts.
  • the crystals can be re-dissolved in acidified isopropanol and the resulting purple solution can be measured spectrophotometrically.
  • An increase/decrease in viable cell number can be evaluated as corresponding increase/decrease of formazan salt optical absorbance resulting in the quantification of the whole cytotoxic event.
  • Vitamin C 0.15 mg/ml (comparative anti-oxidant) OZONISED OIL EVALUATION OF ANTI-RADICAL ACTIVITY
  • the anti-radical activity of the sample is apparent at the tested concentration from 0.056 to 0.167 mg/ml.
  • the cellular viability has been estimated in absence of oxidative stress (UV-) and after 5 minutes of UV exposure (UV+).
  • the cellular viability of the product treated is higher than product not treated keratinocytes at the tested concentration from 0.006 to 0.5 mg/ml.
  • the ozonised oil according to the present invention possesses anti-oxidant activity at tested concentration.
  • the tested product reduces the ROS percentage in human oxidative stress subjected keratinocyte cultures.
  • the anti-radical activity of the sample is apparent at tested concentration from 0.056 to 0.167 mg/ml.
  • the protecting activity of the tested product on oxidative stress subjected keratinocytes is not determinable after UV exposure, the cellular viability of the product treated keratinocytes is not higher that of the product not treated keratinocytes.
  • Example 5 Evaluation of in vitro protecting activity of the ozonised oil according to the present invention on cell cultures after UVA exposure.
  • HaCaT cell line human immortalized keratinocyte cultures
  • MTT assay allows cell viability/death to be quantified
  • microscope analysis allows the. presence of a proper/affected cell morphology to be detected and supports the MTT assay in evaluating the cell viability a) Evaluation of cell viability The cellular viability is estimated by means of a MTT test
  • the key component is 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyl tetrazolium bromide or MTT, which is of yellowish colour in solution.
  • Mitochondrial dehydrogenase of viable cells cleaves its tetrazolium ring leading to the formation of purple water insoluble crystals.
  • the cell morphology is evaluated using fluorescence microscopy after acridine orange staining.
  • fluorescence microscopy After acridine orange staining.
  • Olympus fluorescence microscope 2OX magnification.
  • HaCaT cell line Human epidermis immortalised keratinocyte cultures
  • HaCaT cell line (HaCaT cell line) are seeded in plates and incubated overnight with the samples. On the second day the cells are transferred in microplates in PBS. After 30' UVA exposure, the cells are incubated overnight in Dulbecco medium + 2 % foetal calf serum (FCS). On the third day the cells are stained with acridine orange for observation using fluorescence microscopy.
  • - Light source the lamp used in the experiments is a solar light simulator with a constant emission in the UVA range from 320 to 400 nm.
  • - Irradiation dose The test UVA dose is enough to induce cell death phenomena in immortalized keratinocytes (HaCaT cell line) exposed without protection.
  • the cell containing microplates are irradiated for 30' at room temperature with an intensity of 3.4 mW/cm 2 of UVA (6 J/cm 2 ).
  • the cellular viability has been evaluated in absence and after 30 minutes of UVA exposure.
  • % of protection [(MTT (UVA+ Sample) - MTT (positive Control))/MTT (positive Control)]*100
  • the ozonised oil provides an elevated protecting activity (47.12- 52.27 %) on the cellular viability of immortalized keratinocytes (HaCaT) after UVA exposure (Table 13).
  • the oil according to the present invention has a remarkable keratinocyte protecting activity on UVA exposed human immortalized keratinocytes.
  • the cellular viability of the product treated keratinocytes is much higher than that of the used controls.
  • Example 6 In vitro evaluation of the antiviral activity of the ozonised oil on cell cultures.
  • NNK Newborn Swine Kidney
  • Betancur-Galvis et al. www.scielo.br/) Evaluation of the results The evaluation of the antiviral activity is expressed as reduction of the viral titer.
  • the reduction factor (FR) of the viral titer results from the ratio of the virus titer in absence and presence of the sample. The test is repeated three times in duplicate for at least 5 sample concentrations. The results are expressed as average of the data obtained in the 3 different tests (see Table 14).
  • the ozonised oil possesses a low antiviral activity, the reduction of the viral titer is higher for the Ozonised oil than Not Ozonized Oil.
  • NSK cell cultures (Newborn Swine Kidney) infected with human influenza virus strain and treated with the tested sample show a low reduction of the viral titer.
  • Example 7 In vitro evaluation of the antiviral activity of the ozonised oil on cell cultures.
  • the culture cells are treated with scalar concentrations of the tested substances.
  • Parainfluenza virus - 3 (Pl-3) SF4 strain is used. Cell cultures
  • An appropriate number of cells are seeded as monolayer in 96 well plates. After appropriate washes 100 ⁇ l/well of medium containing virus serial dilutions (1 : 10) are added. The incubation with the virus continues for 1 hour at 37°C. To infected cells 200 ⁇ l/well of serial dilutions (1 :2) of the test substance are added. The incubation continues for 4-5 days at 37 0 C. The controls are constituted of cells infected in the absence of the sample and treated and not treated infected cells. The viral titer is determined according to the Reed and Muench method.
  • the evaluation of the antiviral activity is expressed as reduction of the viral titer.
  • the reduction factor (FR) of the viral titer results from the ratio of the virus titer in absence and presence of the sample. The test is repeated three times in duplicate for at least 5 concentrations of sample. The results are expressed as average of the data obtained in the 3 different tests (Table 17).
  • OZONIZED OIL O.
  • O.N.O. OZONIZED OIL
  • O.O. Ozonised Oil
  • O.N.O. Not Ozonised Oil
  • the Ozonized oil possesses a moderate antiviral activity, the reduction of the viral titer is of log magnitude order.
  • the observed antiviral activity is higher for ozonised oil than for not ozonised oil.
  • MDBK cell cultures (Madin Darby calf kidney) infected with parainfluenza virus strain and treated with the tested sample show a moderate reduction of the viral titer.
  • Example 8 Evaluation of the antimicrobial effectiveness of a product
  • Candida albicans ATCC 10231) Test execution
  • the product is tested as such and diluted in 10 ml of medium at following concentration: 10 % - 1 % - 0.1 %.
  • the obtained solutions therefore have been inoculateD with the above reported micro-organisms.
  • After 48 hours of incubation at 37 0 C solution aliquots have been sampled.
  • appropriately diluted aliquots have been plated with the medium selective for the specific micro-organism to be titrated.
  • After 48-72 hours of incubation at 37 0 C P. aeruginosa and S. aureus colonies have been counted, after 5 days at 22-25°C C. albicans colonies, have been counted.
  • the capacity of the tested sample to normalise the microbial flora is evaluated based on the following criteria:
  • Aeruginosa Inoculum 1.2 x 10 6 1.2 x 10 B 1.2 x 10 1.2 x 10 B
  • Example 9 Evaluation in vitro of the anti-inflammatory activity of the ozonised oil on cell cultures after UVA exposure.
  • the anti-inflammatory activity of the ozonised oil has been evaluated by the analysis of cytokines synthesis in cultures of immortalized human test substance treated and UVA exposed keratinocytes (HaCaT cell line).
  • the in vitro test on cutaneous tissue derived cells is suitable to provide a lot of information about the reactions which may occur in vivo.
  • the skin is the largest human organ and its immune function attracted the attention of both immunologists and dermatopathologists.
  • epidermal cytokines can have an important role in mediating skin inflammatory and immune responses.
  • keratinocytes There are various cell types in the epidermis suitable to secrete cytokines : keratinocytes, Langerhans cells, melanocytic cells and even Merkle cells. Keratinocytes are the major source of cytokines in the epidermis and have been reported to secrete IL-1 , IL-3, IL-6, IL-8, CSF, TNF ⁇ , TGF ⁇ , TGF ⁇ and PDGF.
  • cytokine production by keratinocytes results in multiple consequences on the migration of inflammatory cells, may have systemic effects on the immune system, influences keratinocyte proliferation and differentiation processes and finally affects the production of other cytokines by keratinocytes.
  • IL-6 lnterleukin 6
  • L-8 lnterleukin 8
  • Keratinocytes express IL-6 under various conditions including UV exposure.
  • IL-6 stimulates keratinocyte proliferation and it is studied in diseases associated with epidermal hyperplasia and wound healing process.
  • IL-8 is a powerful neutrophil attractant and is produced by keratinocytes after external stimuli including contact sensitizers and irritants.
  • MTT assay allows the cell viability/death to be quantified
  • microscope analysis allows the presence of a normal/affected cell morphology to be detected and supports the MTT assay in evaluating the cell viability.
  • ELISA assay Enzyme Linked Immunosorbent Assay
  • a signal system detects antibody-antigen complexes
  • the cell viability is evaluated through a MTT assay (Mossman- 1993).
  • the key component is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide or MTT, which is of yellowish colour in solution.
  • Mitochondrial dehydrogenase of viable cells cleaves its tetrazolium ring resulting in the formation of purple crystals which are water insoluble.
  • the crystals are re-dissolved in acidified isopropanol and the resulting purple solution is measured spectrophotometrically.
  • An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of the cytotoxicity or protecting activity of the tested substance.
  • the analysis of the cell morphology has been carried out using a Nikon Eclipse E600 microscope. The pictures were taken using 2OX magnifications.
  • HaCaT cell line On the first day a suitable number of immortalized keratinocytes (HaCaT cell line) are seeded in plates and incubated overnight in Dulbecco medium + 10 % foetal calf serum (FCS). On the second day the cells are incubated overnight with the samples in Dulbecco medium + 2% FC S. On the third day the cells are transferred in microplates in PBS and UVA exposed for 25'. After irradiation, the cells are incubated in Dulbecco medium + 2 % FCS. The cytokines were determined after 2 hours, 5 hours and overnight incubation periods (recovery time). The cell viability (MTT assay - cell morphology) has been performed after overnight incubation. Experimental conditions of irradiation
  • the lamp used in the experiments is a solar light simulator with a constant emission in the UVA range from 320 to 400 nm.
  • the test UVA light dose is enough to induce cell death phenomena in immortalized keratinocytes (HaCaT cell line) exposed without protection.
  • the cell containing microplates are irradiated for 25' at room temperature with an intensity of 3.4 mW/cm 2 of UVA (6 J/cm 2 ).
  • - Radiometer the emission of the lamp has been measured using a PMA 2100 Solar Light Company (Philadelphia) model radiometer calibrated with a Beckmann 800 multimeter spectrophotometer on 2 nd March 2001 (certificate no. 03837 from Solar Light Co., Philadelphia).
  • the cytokines dosage has been carried out without and after 25 minutes of UVA exposure.
  • the measurements have been taken after a recovery time of 2, 5 and 24 hours (overnight).
  • IL-8 is synthetized also in absence of UVA exposure in immortalized keratinocytes cell cultures.
  • Such cytokine is present in Ozonised Oil treated cells in higher amount than in the controls.
  • the cellular viability has been estimated without and after 25 minutes of UVA exposure.
  • the ozonised oil according to the present invention is suitable to modulate the synthesis of IL-6 and IL-8 cytokine in UVA exposed human immortalized keratinocytes After UVA exposure Ozonized Oil is suitable to remarkably stimulate the IL-6 synthesis.
  • IL-8 levels are higher in Ozonised Oil treated cells than in controls, both in the presence and absence of irradiation..
  • the cellular viability of the product treated keratinocytes is higher than in used controls.
  • Ozonized Oil increases the cellular viability of treated and UVA exposed keratinocytes. Such an activity could be correlated to an antiinflammatory action resulting in modulation of IL-6 and IL-8 cytokine synthesis.

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Abstract

Procédé d'élaboration d'huile ozonisée selon les étapes suivantes : a) développement de radical d'anion ozone par catalyse hétérogène sous forme de léchage à l'ozone de catalyseur donneur d'électron approprié, sachant que le potentiel redox de celui-ci est inférieur au potentiel redox de l'ozone ; b) contact entre le radical considéré et une huile comprenant des acides gras insaturés pour la formation d'ozonides en évitant le contact huile/catalyseur ; c) vérification de la fin de réaction quand des valeurs de température constantes sont établies ; d) stabilisation de l'huile ozonisée produite en fin d'étape c) à travers une réaction avec un système ox-red approprié donnant la formation de peroxyde. Enfin, huile ozonisée résultante et utilisations dans le domaine médical, vétérinaire et cosmétique.
PCT/IT2006/000736 2005-10-17 2006-10-16 Huile ozonisee, procede d'elaboration et utilisation dans le domaine medical WO2007046122A2 (fr)

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ITRM20050514 ITRM20050514A1 (it) 2005-10-17 2005-10-17 Olio ozonizzato, principio attivo di elevata stabilita' a base di perossidi utilizzabile per applicazioni terapeutiche mediche e per trattamenti domiciliari similari a quelli dell'ozonoterapia, senza l'utilizzo di ozono gassoso.
ITRM2005A000514 2005-10-17

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EP1935408A1 (fr) 2006-12-15 2008-06-25 Sanipan S.r.l. Dispositif pour l'application topique d'ozone médical pour traiter des maladies de la cavité buccale.
WO2010049954A1 (fr) * 2008-10-31 2010-05-06 Lipid Pharmaceuticals Ehf. Acides gras pour utilisation comme médicament
ITFI20090118A1 (it) * 2009-05-27 2010-11-27 Italmed S R L Miscele composte di origine vegetale ad attivita' antisettica.
ITMI20110354A1 (it) * 2011-03-07 2012-09-08 Neovalis S R L Composizione a base di olio ozonizzato per uso topico
WO2012168770A1 (fr) 2011-06-10 2012-12-13 Universita' Del Salento Procédé d'ozonisation d'une huile végétale
DE102012007239A1 (de) * 2012-04-10 2013-10-10 Wolfgang Winkelmann Pharmazeutische Zusammensetzung enthaltend eine mit Sauerstoff angereicherte ungesättigte Fettsäure und ein organisches Lösungsmittel
KR101618054B1 (ko) * 2014-03-20 2016-05-09 동명대학교산학협력단 오존연고와 베타글루칸 및 히아루론산을 이용한 화장품 제조방법
KR101618055B1 (ko) * 2014-03-20 2016-05-09 동명대학교산학협력단 오존연고와 bha를 이용한 화장품 제조방법
CN107028977A (zh) * 2016-11-17 2017-08-11 陈耕 一种臭氧化油络合物及其制备方法和应用
IT201600078872A1 (it) * 2016-07-27 2018-01-27 Fb Vision S R L Olio e dispositivo per l'igiene dell'area oculare e perioculare
IT201700063112A1 (it) * 2017-06-08 2018-12-08 Project & Communications Ltd Formulazione per uso orale comprendente olio ozonizzato nella prevenzione e/o trattamento di lesioni cutanee causate dall’esposizione a radiazioni ionizzanti e/o trattamento chemioterapico.
CN110403926A (zh) * 2019-08-14 2019-11-05 中南大学湘雅三医院 一种纳米酸化脂肪酸脂在制备预防和治疗接触性皮炎药物中的应用
WO2021105638A1 (fr) 2019-11-29 2021-06-03 Demeta Utilisation des hydroperoxyalcools et de leurs dérivés comme agents antimicrobiens

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US3504038A (en) * 1966-06-24 1970-03-31 Us Agriculture Ozonization of vegetable oils in an improved aqueous medium
EP0235528A1 (fr) * 1986-03-01 1987-09-09 Dr. J. Hänsler GmbH Procédé de préparation d'huiles ozonisées stables à partir d'huiles végétales insaturées
WO2000039025A1 (fr) * 1998-12-28 2000-07-06 Daewoo Electronics Co., Ltd. Systeme de generation de radicaux
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Cited By (18)

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Publication number Priority date Publication date Assignee Title
EP1935408A1 (fr) 2006-12-15 2008-06-25 Sanipan S.r.l. Dispositif pour l'application topique d'ozone médical pour traiter des maladies de la cavité buccale.
US8372425B2 (en) 2008-10-31 2013-02-12 Lipid Pharmaceuticals Ehf. Fatty acids for use as a medicament
WO2010049954A1 (fr) * 2008-10-31 2010-05-06 Lipid Pharmaceuticals Ehf. Acides gras pour utilisation comme médicament
US9072714B2 (en) 2008-10-31 2015-07-07 Lipid Pharmaceuticals Ehf. Fatty acids for use as a medicament
ITFI20090118A1 (it) * 2009-05-27 2010-11-27 Italmed S R L Miscele composte di origine vegetale ad attivita' antisettica.
WO2012120454A1 (fr) * 2011-03-07 2012-09-13 Neovalis S.R.L. Composition à base d'huile ozonisée destinée à une utilisation topique
ITMI20110354A1 (it) * 2011-03-07 2012-09-08 Neovalis S R L Composizione a base di olio ozonizzato per uso topico
WO2012168770A1 (fr) 2011-06-10 2012-12-13 Universita' Del Salento Procédé d'ozonisation d'une huile végétale
DE102012007239A1 (de) * 2012-04-10 2013-10-10 Wolfgang Winkelmann Pharmazeutische Zusammensetzung enthaltend eine mit Sauerstoff angereicherte ungesättigte Fettsäure und ein organisches Lösungsmittel
KR101618054B1 (ko) * 2014-03-20 2016-05-09 동명대학교산학협력단 오존연고와 베타글루칸 및 히아루론산을 이용한 화장품 제조방법
KR101618055B1 (ko) * 2014-03-20 2016-05-09 동명대학교산학협력단 오존연고와 bha를 이용한 화장품 제조방법
IT201600078872A1 (it) * 2016-07-27 2018-01-27 Fb Vision S R L Olio e dispositivo per l'igiene dell'area oculare e perioculare
WO2018020456A1 (fr) * 2016-07-27 2018-02-01 Fb Vision S.R.L. Huile et dispositif pour le nettoyage de la zone oculaire et péri-oculaire
CN107028977A (zh) * 2016-11-17 2017-08-11 陈耕 一种臭氧化油络合物及其制备方法和应用
CN107028977B (zh) * 2016-11-17 2020-08-11 湖北精耕生物工程有限公司 一种臭氧化油络合物及其制备方法和应用
IT201700063112A1 (it) * 2017-06-08 2018-12-08 Project & Communications Ltd Formulazione per uso orale comprendente olio ozonizzato nella prevenzione e/o trattamento di lesioni cutanee causate dall’esposizione a radiazioni ionizzanti e/o trattamento chemioterapico.
CN110403926A (zh) * 2019-08-14 2019-11-05 中南大学湘雅三医院 一种纳米酸化脂肪酸脂在制备预防和治疗接触性皮炎药物中的应用
WO2021105638A1 (fr) 2019-11-29 2021-06-03 Demeta Utilisation des hydroperoxyalcools et de leurs dérivés comme agents antimicrobiens

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