EP1937635A2

EP1937635A2 - Ozonised oil, process for the preparation and use thereof in medical field

Info

Publication number: EP1937635A2
Application number: EP06810027A
Authority: EP
Inventors: Orsola Modica; Maria Rosa Maietta; Gabriele Maietta
Original assignee: Bioo3pharma Srl
Current assignee: Bioo3pharma Srl
Priority date: 2005-10-17
Filing date: 2006-10-16
Publication date: 2008-07-02
Also published as: WO2007046122A2; WO2007046122A3; ITRM20050514A1

Abstract

The invention relates to 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 that of ozone; 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. Further the invention relates to ozonised oil obtainable using the process and uses thereof in medical, veterinary and cosmetic fields.

Description

OZONISED OIL, PROCESS FOR THE PREPARATION AND USES THEREOF IN MEDICAL FIELD

The present invention concerns an ozonised oil, the process for the preparation and uses thereof in medical field. In particular, 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, endό-pleural and endo-peritoneum insufflations, intra-articular injections, intra-lesion intra-foraminal, topical applications with ozone gas.

For the applications, therapeutic doses vary from 20 to 80 mcg/ml. For serious pathologies for short periods (two - three days) high doses, i.e. 80 mcg/ml are used. Subsequently lower therapeutic gradually decreasing doses are used (from 40 mcg/ml to 20 mcg/ml). The use of 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, widely used in medical field, 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,

- therapeutic cycles involving a high number of sessions;

- high cost of any session; - difficulty for ozone gas manipulation;

- necessity to produce ozone gas at the time of the use, because of its insufficient stability;

- the instability of ozone gas does not allow any product and/or derivative to be available for use during a long time period. Various 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.

In the light of above therefore it is apparent the need to provide a new process and a finished product suitable to overcome the ^"3is^"advantag^"es^""of thlTpπor art?

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.

Ozone is a strong oxidant E = 2,08 V and this characteristic make it particularly reactive against compounds with double bound like unsaturated fatty acids. In the presence of 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.

In order to favour and accelerate the chemical reaction and to shift the equilibrium towards the reaction products achieving the maximum yield, ozone gas is transformed in the highly reactive ozone anion radical, under heterogenous catalytic conditions, for example using solid ferrous sulphate FeSO₄ as catalyst. Ferrous sulphate (FeSO₄), crystalline inorganic salt, is an optimum promoter for radical reactions being an electron donor. In the presence Fe²⁺ (electron donor) ozone instantaneously reacts forming ozone anion radical:

Fe ²⁺ - e^" = Fe³⁺ E⁰ _Fe3₊/Fe2₊ = 0.77 V O₃ + e^" = O₃ ^"

Iron (II) compounds are oxidized by ozone (O₃) to iron (III) compounds, thus ozone, although powerful oxidant, is converted to strongly reactive ozone anion radical. In the reaction of ozone (O₃) against the double bond of unsaturated fatty acid a process a three separate step process is presumable: induction step; propagation step; interruption step.

In the first, i.e. induction, step ozone (O₃), in the presence of Fe²⁺ ions, reacts instantaneously resulting in the formation of the strongly reactive ozone anion radical (O₃ ^"°). For said chemical reaction 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. Inside of the reaction chamber ozone gas, produced by the ozone generator, is introduced in such a way to lick ferrous sulphate (FeSO₄) solid crystals previously deposited inside of the same reaction chamber. 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 firϊe grain porous bubbling septum, moreover, 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. After the first induction step, carried out inside the reaction chamber wherein ozone gas, in the presence of Fe²⁺ ions, instantaneously reacts resulting in the formation of strongly reactive ozone anion radical (O₃ ^"°), follows the propagation step that occurs ad the porous septum/ fluid oily mass interface wherein the ozone anion radical attacks the double bond of the unsaturated fatty acids, present in the chemical composition of the linen oil to be ozonised, with formation of the ozonides and gradual and continuous development of reaction heat. The above reaction occurs in lipophilic environment. The propagation step occurs in the presence of ozone anion radical (0₃ ^"°) which attacks the glicerol unsaturated fatty acid ester mixture. In particular the ozone anion radical instantaneously attacks tri-unsatu rated fatty acid (linolenico acid:

C₁8H3₀O₂), subsequently di-unsaturated fatty acid (linoleico acid:

C₁8H3₂O₂) and finally monounsaturated fatty acid (oleic acid: Ci₈H₃₄O₂), generating ozonides and reaction heat development. The propagation step, therefore, 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 above described chemical reaction results in the formation of ozonides and development of reaction heat. Thus produce ozonised oil proves to be stable for a limited period of time (days). This behaviour, very probably, results from the development of the reaction heat favouring a chaotic distribution of the molecular chains in the molecular structure of the ozonised oil. This type of molecular structure leads to the formation of an unstable system characterised by inner tensions, in addition to those deriving from the steric hindrance of the present ozonides. In order to stabilise the ozonised oil it has been studied and realised an ox-red system suitable to transform the ozonides in peroxides having a greater stability and lesser steric hindrance. In particular, 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. In the specific situation it is to be considered that 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. In order to restore the system at the starting situation it is necessary reduced alpha lipoic acid to be oxidized. 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. Similarly 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.

Further the used doses are equivalents taking into account the stoichiometric reaction relationships of reagents.

At the end of the stabilisation process 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.

A possible explicative theory about the molecular structure of the ozonised oil produced by the present invention as a result of the stabilisation process. As an effect of the stabilisation treatment the ozonides present in the support biological matrix react with alpha lipoic acid and acetyl lipoate yielding peroxides as reaction products OR - OR C - C having a lower steric hindrance than ozonides, and they are localised in an oriented and ordered way in sequential molecular layers, establishing from each other weak electrostatic interactions resulting from the pres.ent dipoles: ⁴P - P

^" 0 -- 0

⁺C-C

O— O In this case the greater part of the forces are weak and therefore effective only within a small action range, but as a whole assumes particular importance since there are many dipoles in the molecular structure and the resulting force obtained by the addition of all the interactions between dipoles is considerable. This resultant, even if results from short range forces, acts like a long range force. In effects, the resulting of a great number of dipolar forces weakens with the distance, slower than for forces according to the distance square law. Therefore, even if initially weaker, the resultant can exceed the normal electrostatic forces. ■ The possible above exposed molecular structure is supported by the particular titration trend and times necessary to carry out the determination of the peroxide number present in the ozonised oil. 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 peroxides which became free gradually, being in a strongly acidic environment and in the presence of potassium iodide, result in the iodine development that, at the end of the reaction, is subjected to a reduction with a measured volume of known titer sodium thiosulfate, allowing therefore the determination of the peroxide number. By means of the stabilisation process 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. What above described is supported by the verifications carried out and conducted about the ozonised oil with respect to its stability. Experimental tests carried .out, widely described in the next items, allowed to verify that the ozonised oil exhibits high stability for long time periods (years). According to above it is possible to express the following considerations about the intrinsic characteristics of the active principle: the peroxides are fixed and bound with remarkable tenacity inside of the ozonised oil; the release of oily peroxides present in the ozonised oil occurs gradually and continuously in the time; in order to have the complete release of peroxides it is necessary a quite long period of time; the ozonised oil exhibits high stability for long time periods (years).

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 (Sn²⁺) 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. Moreover, the invention concerns a pharmaceutical composition comprising the ozonised oil like active principle associated with one or more pharmaceutically acceptable adjuvant and/or excipient. In the compositions according to the present invention 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.

Moreover, 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. It allow extraordinary and clearly better to achieved, when the pharmacological activity and/or functional activity of the currently used drugs and/or phytotherapeutics, respectively, are referred to, resulting in advantageous effects of costs/effectiveness in consideration of the fact that higher pharmacological doses of same drugs and greater amounts of phytotherapeutics should to be used in order to obtain equivalents results, with consequent implementations of the costs and associated side-effects.

Accordingly 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. This reaction, mediated by enzymes assigned to the defence against aggressive free radical substances like free radicals, starts from the pentose glycolysis leading to an increase of adenosintriphosphate (ATP) and therefore of the energetic content of the cells. After the application of the ozonised oil gradually and continuously during an extended time period of approximately eight hours oily peroxides are released form the active principle. For the therapeutic applications it is useful to remember that the therapeutic doses vary from 20 to 80 mcg/ml and that for serious pathologies is a good guidance to employ for short periods (two - three days) higher doses i.e. 80 mcg/ml and then progressively lower the therapeutic doses (40 mcg/ml - 20 mcg/ml). Generally it is always better to use low therapeutic doses (20 - 30 mcg/ml), involving more numerous daily applications during a long time period, allowing better therapeutic effects to be achieved, rather than high doses (80 mcg/ml), involving fewer daily applications during a short time period. 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. This involves an increment of the cellular proliferation from the endothelium, fibroblasts and keratinocytes combined with an increased synthesis of the components (fibrin, fibronectin, hyaluronic acid, condroitin sulphate, collagen I/Ill) of the interstitial matrix. 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. Moreover, 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 ⁰C 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.

Therefore, 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 rheumatology for the treatment of rheumatoid arthritis, articular rheumatisms; in urology for the treatment of infections of the urinary ways; in proctology for the treatment of proctitises, rhagades, haemorrhoid; in gynaecology for the treatment of bacterial and fungal vaginal infections, vulvovaginitises, vaginal ulcers; in dentistry for the treatment of abscesses, gum infections, stomatitises, aphtha, gum lesions; in otoralaryngology for the treatment of ear infections, cold diseases, sinusitises; in aesthetic medicine for anti-plica, breast toning, anti-stria atrophica and anti-cellulite treatments; in ophthalmology for the treatment of cornea ulcers, infections; in pneumatology for the treatment of bacterial and/or viral broncho-pulmonary infections, enphysemas; in veterinary for the treatment of pyoderma, dermatitis, post-infection arthritis, arthrosis, cystopyelitis, abscesses, infectious enterocolitis, cold - mucosal diseases, decubitus ulcers, localised mycosises, peptic ulcers, rhagades, corneal ulcers, vaginitises, mastitises, metritises.

Moreover, the present invention concerns 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. As above .reported, the catalyst is chosen from the group consisting of ferrous sulphate, powder zinc metal, tin (Sn²⁺) salt, silver metal. The fine grain porous septum is made of inert material, for example ceramic material.

Further the invention concerns the use of the ozonised oil as a cosmetic component, for example for trichological treatment. The present invention now will be described by illustrative but not limitative way, according to preferred embodiments thereof, with particular reference to the enclosed drawings, wherein:

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;

(5) porous septum; (6) ozone inlet; (7) hole for temperature measuring device; (8) ozone outlet; (9) bubbling cap. Figure 2 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.

Exemple 1 : Preparation of the ozonised oil according to the invention and analysis of the obtained product

Sample 100 ml of refined linen oil using a 100 ml metered pyrex glass cylinder and deposit it in the 250 ml glass bubbling apparatus, wherein previously a Teflon covered magnetic bar has been deposited.

Insert within the fine grain ozone diffuser 200 mg of ferrous sulphate

(FeSO₄) previously weighed using a precision balance, sensibility +/-

0.001 g. Connect the fine grain diffuser by a silicone joint tube to the end of the ozone inlet pipe. Install the precision long bulb Hg thermometer with scale from -1 O⁰C to +100°C, equipped with ground cone, in the appropriate ground room of the bubbling cap. Subsequently insert the ground conical glass stopper of the bubbling cap in the conical ground neck of the bubbling reactor. Connect with a joint silicone pipe the end of the ozone inlet glass pipe of the bubbling cap to the ozone extraction join in the ozone generator. Connect by means of silicone join tube the end of the glass outlet pipe for the bubbling cap excess ozone to the active carbon - silica gel filter. Put the thus prepared equipment on the plate of the magnetic stirrer. Connect by means of silicone join pipe the oxygen inlet join, installed in the ozone generator, to the pressure reducer (operating pressure 5 atm) mounted at the inlet of the medical grade oxygen gas cylinder. Connect to the power supply the ozone generator and magnetic stirrer and turn on. Record temperature (⁰C) and humidity (%) of the operating room.

Open the oxygen gas containing cylinder and purge three times the inner circuit of the ozone generator at operating pressure 0.5 atm, using the pressure control device installed on the same generator. After the verification that the display showing the measure of the concentration of produced ozone is set to zero, press start for the ozone production. Use to this end oxygen gas at operating pressure of 0.12 - 0.15 atm until on the display the concentration reading is 80 mcg/ml. Produced ozone will lick and penetrate into ferrous sulphate, deposited inside of the reaction chamber, then get out of the fine grain diffuser. At porous septum/oily fluid mass interface ozone (as ozone anion radical) will begin to micro-bubble through the fluid mass. Record the temperature value in ⁰C inside the fluid mass and the process start time. Concurrently turn on the magnetic stirrer and set the revolution knob at 2 rpm; this will spin the Teflon coated magnetic bar assuring a moderated, continuous and constant mixing of the entire fluid mass thus favouring the intimate contact of the oily fluid with the supplying ozone gas microbubbles, during the entire process.

Bubble ozone through the fluid mass at concentration of 80 mcg/ml recording every 5 minutes the ⁰C temperature inside of the reaction chamber (reaction start). With ozonization process in progress the recorded temperature values will increase gradually and continuously

(reaction propagation) until a not variable value remaining constant in time

(reaction completion). The time elapsed from the start, propagation and end of the reaction is equal to 70 minutes. At 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. At the end transfer 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 ⁰C, for the attainment of longer conservation, and left standing for 48 hours before the use. In figure 2 IR spectra of the obtained ozonised oil is shown.

TEMPERATURE PROFILE (⁰C) VERSUS TIME (MINUTES) DURING OZONIZATION PROCESS

The temperature values ("C) recorded every five (5) minutes using a precision Hg thermometer localized inside of the reaction chamber, during the ozonization process of 100 ml linen oil carried at room temperature 23 ⁰C and 50 % humidity arre reported:

From the value profile it is possible to point out that during 5 minutes after the start of the ozonization process the inner temperature of the system slightly exceeds ambient temperature, due to the heat development from the chemical reaction between the ozone anion radical and the double bonds of the unsaturated fatty acids in the chemical composition of the linen oil. In the following minutes and until to the 70^th minute the propagation of the reaction in object occurs with additional heat development resulting in an increment of the inner temperature whose maximum value is equal to 45⁰C. At 70^th minute the reaction ends with consequent heat development stop. Therefore the inner temperature does not increase and remains constant both at 75^th and 80^th minutes. ANALYTICAL METHOD FOR THE DETERMINATION OF THE PEROXIDE NUMBER For the determination of the peroxide number in the ozonised oil an analytical evaluation method, suitable for the chemical-physical characteristics of the ozonised oil, which is reported in the following description, as an example, without any limitation has been provided. 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. At the end of the fourth day add to the yellow brown solution 30 ml of distilled water and titer with 0.1 N sodium thiosulfate by slow addition of the titrant, with continuous shaking until the yellow colouration is nearly disappeared. Add 5 ml of previously prepared starch solution and shake the solution that will turn to a deep blue colouration, and continue the titration energetically shaking, until the blue colouration disappears (n-i, ml of 0.1 N sodium thiosulfate). Carry out a blank test in the same conditions (n₂, ml of 0.1 N sodium thiosulfate). The volume of the blank test must be lower than 0.1 ml:

100 (m - n₂) IP ₌ m

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):

O₃ + 2I^" + H₂O ==^■» O₂ + I₂ + 2OH- I₂ + 2S₂O₃ ⁼ ==^■* 2|- + S₄O₆= . The choice of the indirect iodometric method is due to the easiness of iodide oxidation to iodine and the reaction, fast and quantitative, between released iodine and thiosulfate. A few recommendations reported below are to be taken into account, since errors from the indirect iodometric method can result: 1) due to its volatility iodine can be lost during the titration, this loss is diminished by the presence of a remarkable excess of potassium iodide, that by formation of a complex with the iodine reduces the vapour pressure thereof, moreover, the iodine volatilization is remarkably reduced when the titration is carried out in flasks equipped with ground glass stopper; 2) In acidic solution, I^" ion is easily oxidized by atmospheric oxygen. In the present titration it is advisable to carry out the reduction of the oxidant in acidic solution and diminish the acidity of the solution by water dilution before the titration with sodium thiosulfate. This procedure proves to be effective because the reduced substance is not oxidized at the same rate; another system involves the addition of small amounts of Na₂CO₃ to the solution before the titration; as result CO₂ is developed constituting a blanket of inert gas above the solution. As to thiosulfate behaviour with respect to the oxidant it is to be considered that the latter is quantitatively oxidized to S₄O₆ ¹" by the iodine only in acidic solution. All the other oxidants oxidize thiosulfate to sulphate according to less or more complex pathway. The above reported analytical method involves that the solution to be titrated is conserved in the dark for four days. In the specific case the ozonised oil to be analysed keeps therein peroxides with remarkable tenacity, whereby some time period must elapse so that the same, in strongly acidic environment, can be completely released and this occurs in continuous and gradual way during four days. It is noteworthy to point out that more difficult titrations in analytical chemistry are designed to be completed at maximum during a time period of about 48 hours. The gradually released peroxides acting in strongly acid environment and in the presence of potassium iodide are responsible for the nascent iodine development that, at the end of the reaction, is subjected to subsequent reduction with a metered volume of known titer sodium thiosulfate therefore allowing the determination of the peroxide number. In conclusion it can be asserted that the course of the titration is justifiable only if the chemical-physical characteristics of the ozonised oil are considered, wherein a high number of highly stable peroxides kept with remarkable tenacity in the oily matrix and whose release occurs in continuous and gradual way in the time are present. Ad the end of the titration (disappearance of the blue colour) the number of the milliliters of sodium thiosulfate 0.1 N used is recorded and the ground glass stopper re-plugged flask is stored in the dark. The titration is completed when after 24 hours the solution remains colourless. If, on the contrary, the colour turns blue again the titration must be continued, in which case, the used millilitres of sodium thiosulfate 0.1 N are added to those previously used and so on. PEROXIDE NUMBER IN THE OZONISED OIL Ozonised oil obtained by the production process, after 80 minutes, is subjected to analytical assessment for the titer determination using the above described method. In order to determine the titer, i.e. the peroxide number in the ozonised oil, the procedures and equipments indicated in the method and the titration recorded data are used: m = weight in gram of the sample (ozonised oil); n-i = milliliter (ml) of sodium thiosulfate 0.1 N used in the titration; n₂ = milliliter of sodium thiosulfate 0.1 N used in the blank test.

The peroxide index IP is the number expressing in milliequivalents of active oxygen the peroxide amount in 1000 grams of a substance. In the specific case: where ni = ml of sodium thiosulfate 0.1 N used in the titration: (27,8 ml); ri₂ = ml of sodium thiosulfate 0.1 N used in the blank test: (0.1 ml); m = weight of the sample: (2,89 g)

IP = 100x(27,7)/2,89 = 958,0 meq O₂/1000 g = 15.335 g/ml = 7.667.500 ppm

The titer of the ozonised oil, obtained after 80 minutes by the production process, is be equal to 15.335 mcg/ml.

DILUTION OF THE OZONISED OIL

The ozonised oil is used in the therapeutic treatments at the same doses as in the clinical-medical ozone therapy which uses ozone gas. In particular in the ozone therapy 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). Consequently, after the choice of therapeutic dose and knowing the titer of the ozonised oil and, therefore, the peroxide number, it is possible to determine the volume (ml) and/or the weight (g) of the ozonised oil to be used. 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:

For example: if the ozonised oil has a titer equal to 15.335 mcg/ml, in order to prepare 100 ml of solution wherein is present a peroxide number corresponding to 65 mcg/ml, 0.42 ml and/or 0.38 g of ozonised oil are to be used, on the base of the calculations as follow: 100 gx 65 meg /ml = 6500 mcg/ml

6.500 mcg/ml: 15.335 mcg/ml = 0.42 ml 0.42 ml x 0.91 (density of the ozonised oil) = 0.38 g CHARACTERISTICS OF THE OZONISED OIL

- Odour: acrid, typical of ozone; - taste: metallic, slightly pungent;

- Colour: amber;

- Density: 0.91 g/ml;

- Peroxide Index: 15335 mcg/ml

- Alterability: very mild alterations and/or degradations of the product over a long term (years) are observed in optimal conditions requiring that the ozonised oil is conserved in suitable sealed, well plugged and protected from the light containers at room temperature and/or stored in fridge at temperature of 4 - 6°C, for the achievement of a longer conservation; - Toxicity: employing the product on the man and using therapeutic doses, i.e. from 20 mcg/ml and 80 mcg/ml in application therapeutic steps toxic effects on the treated organs or particular disturbs (to the aerial ways, lachrymation, migraine, nausea and vomit, asthma crisis) are not displayed. - Use: it can be used diluted with petrolatum and/or vegetable oils and/or compatible products;

- Tolerability: when it is used topically on the skin, using therapeutic doses from a minimum of 20 mcg/ml to a maximum of 80 mcg/ml,- oily peroxides in a gradual and continuous way are developed for an extended time period equal to 8 hours and it does not result in irritating phenomena or allergic - hyperergic modifications

- Activity: 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. Tests carried out on a wide range of clinical pathologies have shown that excellent and resolving results in short times and without side-effects are obtained applying diluted ozonised oil on anal raghades, vulvovaginitises, hand and foot rhagades, articular pains, ear inflammatory conditions, dermatitises, mycosises, acne, bugs bites, stomatitises, bronchial infections, gastroenteric infections, as well as for applications in the veterinary field and aesthetic applications. REPRODUCIBILITY OF THE METHOD OF THE OZONISED OIL PRODUCTION

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 6⁰C, for the attainment of a longer storage; a constant and unaltered functional activity of the ozonised oil, which exerts its own activity also after storage of the ozonised oil for long time periods (years).

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.

STABILITY OF THE OZONISED OIL The yellow brown ozonised oil is viscous at 22⁰C and very dense at 6⁰C, has an acrid ozone typical smell and pH equal to 4,5. The product is very stable in the time. In order to determine the stability of the ozonised oil 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). The samples have been withdrawn three, six and twelve months after the production time and subjected at the determination of the peroxide number by titration with sodium thiosulfate 0.1 N using the iodometric indirect method. Obtained results^" are reported: 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 35⁰C (table 1);

Table 1

In the first run 100 ml of linen oil have ozonised for 80' in the presence of FeSO₄, immediately 200 mg of alpha lipoic acid and 200 mg of acetyl lipoate have been added; the second run has been carried out as above with the exception that ozonization time was 120' while the same amounts of alpha lipoic acid and acetyl lipoate have been added at the end of the process; run 3 and 4 have been carried out as above with the exception that ozonization time was 180' and 240', respectively, always adding the same amounts of alpha lipoic acid and acetyl lipoate at the end of the ozonization. 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. From above it results that (according to a statistical evaluation) in order to have a 10 % general decay ten years are necessary, in the optimal conditions, i.e. complying recommendations as above indicate for its storage. A statistical lack approximation at 50 % halves the stability time of the ozonised oil at approximately five years. The ozonised oil maintains, for at least five years, nearly unchanged particular properties with respect to the functional activity and chemical - physical - therapeutic characteristics. In the condition (B) after one year the decay of the peroxide number in the ozonised product exposed to daylight and conserved in dark glass bottle at room temperature with thermal excursions from 15 to 35⁰C is equal to 3.5 % in comparison to the initial values as determined at the production time. From above it results that (according to a statistical evaluation) in order to have a 35 % general decay ten years are necessary. A statistical lack approximation at 50 % halves the stability time of the ozonised oil at approximately five years. The ozonised oil maintains, for at least five years, greatly acceptable properties with respect to the functional activities and chemical - physical - therapeutic characteristics. From above it results, also, that the (A) condition assures a greater stability, in the time, of the ozonised oil than the condition B, although this condition in any case is able to assure a quite acceptable stability in the time. As to the stability of the present product it is noteworthy to remember that a complete ozonization carried out during a time periods of 120 - 180 - 240 minutes afford a peroxide number higher than that obtained by 80 minute ozonization. An higher peroxide number offers greater assurances with respect to the decay and stability of the end product, allowing lower amounts thereof to be used in application steps.

Being known titer (peroxide number determined by the iodometric indirect method and using sodium thiosulfate 0.1 N as titrant)) of the ozonised product and therapeutic dose (from 20 to 80 mcg/ml), to be used for the specific treatment to be carried out, it is possible to determine and establish with extreme precision the amount of ozonised oil to be used.

Example 2: In vitro evaluation of cytotoxicity of the ozonised oil according to the present invention 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 IC₅₀ > 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 μl/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.

RESULTS

MTT assay results are shown in Table 2 (negative standard) and 3 (ozonised oil cytotoxicity) IC₅₀ value (50 % cell growth inhibiting concentration), means the concentration of test compound needed to inhibit cell growth by 50%.

IC₅₀ parameter makes it possible to evaluate the potential irritating effect according to the following scheme:

IC₅₀ < 0.5 means a strong cytotoxic/irritating effect IC5₀ between 0.5 and 1.5 means a moderate cytotoxic/irritating effect.

IC₅₀ > 1.5 means the absence of cytotoxic/irritating effect. Table 2

IC₅₀ > 1 ,5 means absence of any cytotoxic/irritating effect

Table 3

IC₅₀ > 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.

Sample preparation

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

We used primary cell cultures of: keratinocytes from human skin biopsies, fibroblasts, from human dermis. Cells were seeded in a homogeneous way in 96 well plates for the experiments.

Culture medium

Keratinocytes have been incubated with CEC+ in the presence of calf foetal serum 5 % (FCS). Fibroblasts have been incubated with MEM (Minimal Essential Medium)-Sodium-Piruvate + 5% calf foetal serum (FCS).

Test execution

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 μl/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.

Table 4

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.

Table 5

The tested product proves to possess a remarkable stimulating activity of the cellular growth at concentration from 0.031 to 0.5 mg/ml.

Such activity turns is particularly apparent for the highest tested concentration (0.5 mg/ml).

Table 6 shows the stimulation of the fibroblasts after 24 hours of incubation with the test sample at variable concentrations. Table 6

The tested product proves to possess a remarkable stimulating activity of the cellular growth at concentration from 0.063 to 0.5 mg/ml.

On keratinocytes the sample proves to have greater regenerating activity after 48-72 hours of incubation at concentration from 0.031 to 0.5 mg/ml

On fibroblasts the sample proves to have greater regenerating activity after 24 hours of incubation 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. 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.

We decided to perform two different tests in order to investigate the antioxidant power of the test compound. 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. Sample preparation

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.

Cell cultures

We used primary cell cultures of keratinocytes from human epidermis biopsies. For the experiments cells were seeded homogeneously in 96 well plates. ROS Determination

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 μl of DCA solution. Plates are incubated at 37°C for 15' in a CO₂ thermostat. At this time DCA solution is discharged. After an UV exposition period of 5 minutes the oxidative stress is discontinued and fluorimetric data are acquired. 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/cm² of UVA (5 J/cm²).

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).

IWTT measure of the cellular viability 06/000736

30

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. RESULTS

EVALUATION Of ANTI-RADICAL ACTIVITY AND CELLULAR VIABILITY

Negative control (C-): not treated cells

Positive control (C+): UV exposed cells without protecting treatments

Samples: UV exposed cells in the presence of protecting substances:

Vitamin C 0.15 mg/ml (comparative anti-oxidant) OZONISED OIL EVALUATION OF ANTI-RADICAL ACTIVITY

PROTECTION FROM ROS After 5 minutes of UV exposure

Results are given as fluorescence values, directly proportional to the ROS amount (table 7). Table 7

The antiradical protection afforded by the product is underestimated because of the used experimental conditions (PEG re- suspended samples)

The percentage of the substance afforded antiradical protection is calculated according to the following equation:

% of protection = [(ROS (positive control) - ROS (sample))/(ROS (positive control) - ROS (negative control))]*100 (table 8). Table 8

The anti-radical activity of the sample is apparent at the tested concentration from 0.056 to 0.167 mg/ml.

MTT TEST-CELLULAR VIABILITY

The cellular viability has been estimated in absence of oxidative stress (UV-) and after 5 minutes of UV exposure (UV+).

Absorbance values (O. D.) at 540 nm (proportional to the number of cells).

Evaluation of the cellular viability in absence of oxidative stress (Table 9).

Table 9

Without UV exposure, 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.

Evaluation of the cellular viability after 5 minutes UV exposure (Table 10). Table 10

After UV exposure, the^' cellular viability of the product treated is not higher than product not treated keratinocytes u

Summary of the acquired data and evaluation of the anti- oxidant and protecting capacities of the tested product

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.

To estimate the protecting activity of a product by means of the analysis of the cellular viability and microscope observation of tested product treated and UVA exposed human immortalized keratinocyte cultures (HaCaT cell line). The in vitro test on cutaneous tissue derived cells proves to be an experimental method suitable to provide a lot of information about the reactions which may occur in vivo.

In order to assess the protecting capacity of the tested substance we evaluated both the viability (MTT assay) and the morphology (microscope analysis) of the treated and UVA exposed immortalized keratonocytes (HaCaT cell line). MTT assay allows cell viability/death to be quantified, the 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

(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 leading to the formation of purple water insoluble crystals.

The crystals are re-dissolved in acidified isopropanol and the resulting purple solution is analysed spectrophotometrically. Increase or decrease in cell number results in a concomitant change in the amount of formed formazan, indicating the degree of the cytotoxicity or protective activity of the tested substance. b) Microscope analysis

The cell morphology is evaluated using fluorescence microscopy after acridine orange staining. For the analysis we used an Olympus fluorescence microscope, 2OX magnification. Materials and Methods

Tested samples

- OZONISED OIL: 0.019-0.057 mg/ml (O.O.) (concentrations of the test substance proved to be not toxic).

- NOT OZONISED OIL: 0.019-0.057 mg/ml (O.N.O.) - Not treated controls (C-/C+)

Cell cultures

Human epidermis immortalised keratinocyte cultures (HaCaT cell line) have been used.

Test execution On the first day a suitable number of immortalized keratinocytes

(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.

Experimental conditions for irradiation

- 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/cm² of UVA (6 J/cm²).

- 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). . EVALUATION OF THE CELLULAR VIABILITY

PROTECTION FROM UVA

The cellular viability has been evaluated in absence and after 30 minutes of UVA exposure.

Absorbance (O. D.) at 540 nm (proportional to the cell number).

O.O. = Ozonised Oil

O.N.O. = Not Ozonised Oil

C- = Negative Control

C+ = Positive Control

CELLULAR VIABILITY (UVA-) (Table 11) Table 11

In absence of UVA exposure the values of cellular viability obtained for the tested samples and the treated control do not introduce meaningful differences

CELLULAR VIABILITY (UVA+) (Table 12)

Table 12

After UVA exposure the obtained values of cellular viability for Ozonised Oil are higher than those obtained for Not Ozonised Oil and not treated control The percentage of protection afforded by test substance is calculated according to the following equation:

% 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).

Table 13 ^•

MICROSCOPE ANALYSIS - NOT TREATED CONTROL (2OX magnification)

After UVA exposure it can be observed a remarkable decrease of the number of viable cells (figure 3)

- OZONISED OIL:

(Concentration: 0.057 mg/ml; 2OX magnification) After UVA exposure the number of viable cells does not show remarkable variations (figure 4)

- OZONISED OIL:

(Concentration: 0.019 mg/ml; 2OX magnification) After UVA exposure a low decrease of the number of viable cells can be observed (figure 5)

- NOT OZONISED OIL:

(Concentration: 0.057 mg/ml; 2OX magnification) After UVA exposure a low decrease of the number of viable cells can be observed (figure 6) - NOT OZONISED OIL:

(Concentration: 0.019 mg/ml; 2OX magnification) After UVA exposure a remarkable decrease of the number of viable cells can be observed (figure 7)

SUMMARY OF THE ACQUIRED DATA AND IN VITRO EVALUATION OF THE PROTECTING CAPACITY OF THE TESTED PRODUCT ON CELL CULTURES 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.

In order to determine the antiviral activity of the treated substance the capacity of the sample to reduce the titer of a human influenza virus strain in infected cell cultures has been analysed. Materials and Methods Tested samples - OZONISED OIL - NOT OZONISED OIL The culture cells are treated with scalar concentrations of the tested substances.

Viral strain

Human influenza virus strain - A/HΛ/R 108 biotype H1 N1 is used. Cell cultures

Cell cultures of newborn swine kidney are used (NSK = Newborn Swine Kidney). Culture medium

Cellular line NSK is incubated in MEM (Minimal Essential Medium) + 10 μg/ml of tripsin + 0.5% of calf foetal serum (FCS). Test execution

An appropriate number of cells are seeded as monolayer in 96 well plates. After suitable washes 100 μl/well of medium containing virus serial dilutions (1 : 10) are added. The incubation with the virus goes on for 1 hour at 37°C. To infected cells 200 μl/well of serial dilutions (1 :2) of the tested substance are added. The incubation continues for 4-5 days at 37⁰C. 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. (Vlietinck et al. J Ethnopharmacology 46, 1995, 31-47, LA

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).

Table 14

EVALUATION OF THE ANTIVIRAL ACTIVITY

(Human Influenza)

REDUCTION OF THE VIRAL TITER

INITIAL VIRAL TITER = 10^{5 50} (LOG10=5.5)

REDUCTION OF THE VIRAL TITER INITIAL VIRAL TITER = 10^{5 50} (LOG10=5,5) Table 16

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.

In order to determine the antiviral activity of the treated substance the capacity of the sample to reduce the titer of a parainfluenza virus strain in infected cell cultures has been analysed. Materials and Methods Tested samples - OZONISED OIL - NOT OZONISED OIL

The culture cells are treated with scalar concentrations of the tested substances.

Viral strain

Parainfluenza virus - 3 (Pl-3) SF4 strain is used. Cell cultures

Cell cultures of calf kidney are used (MDBK = Madin Darby Calf Kidney).

Culture medium

Cellular line MDBK is incubated in MEM (Minimal Essential Medium) + 2% calf foetal serum (FCS). Test execution

An appropriate number of cells are seeded as monolayer in 96 well plates. After appropriate washes 100 μl/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 μl/well of serial dilutions (1 :2) of the test substance are added. The incubation continues for 4-5 days at 37⁰C. 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.

(Vlietinck et al. J Ethnopharmacology 46, 1995, 31-47, LA 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 concentrations of sample. The results are expressed as average of the data obtained in the 3 different tests (Table 17).

Table 17

EVALUATION OF THE ANTIVIRAL ACTIVITY (Parainfluence)

REDUCTION OF THE VIRAL TITER INITIAL VIRAL TITER = 10⁶-⁵⁰ (LOG10=6.5) Table 18

REDUCTION OF THE VIRAL TITER INITIAL VIRAL TITER = 10^{6 50} (LOG10=6.5) Table 19

OZONIZED OIL (OO.) vs NOT OZONIZED OIL (O.N.O.)

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

MATERIALS AND METHODS Samples

OZONISED OIL Used microbial strains

In order to estimate the antimicrobial activity of the tested product the following micro-organisms have been used: - Gram - : Pseudomonas Aeruginosa (ATCC 9027)

- Gram + : Staphylococcus aureus (ATCC 6538)

- Yeasts : 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 37⁰C solution aliquots have been sampled. Then 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 37⁰C P. aeruginosa and S. aureus colonies have been counted, after 5 days at 22-25°C C. albicans colonies, have been counted.

Results evaluation

The capacity of the tested sample to normalise the microbial flora is evaluated based on the following criteria:

1.5/3 log unit reduction at 48 hours after the inoculum: product suitable to control the microbial growth

More than 3 log unit reduction at 48 hours after the inoculum: product having bactericidal and fungicidal activity

Less than 1 ,5 log unit reduction at 48 hours after the inoculum: no activity of the product

VARIATION OF MICROBIAL FLORA

OZONISED OIL

Table 20

Sol. 100% Sol. 10% Sol. 1 % Sol. 0 .1%

Staphylococcus Inoculum 1.9 x 10⁵ 1.9 x 10⁵ 1.9 x 10⁵ 1.9 x 10⁵ aureus 48 hrs < 10 < 10 < 10 < 10

UFC/ml after the inoculum

Aeruginosa Inoculum 1.2 x 10⁶ 1.2 x 10^B 1.2 x 10 1.2 x 10^B

Pseudomonas 48 hrs < 10 < 10 < 10 < 10

UFC/ml after the inoculum

Candida Inoculum 1.8 x 10⁵ 1.8 X 10^s 1.8 x 10^s 1.8 X 10⁵ albicans 48 hrs < 10 < 10 < 10 < 10 UFC/ml after the inoculum

LOG REDUCTION OF MICROBIAL FLORA IN THE TIME

OZONISED OIL Table 21

48 Staphylococcus Aeruginosa Candida /Averages after the aureus Pseudomonas albicans Average inoculum values

Sol.100% 4.28 4.08 4.26 4.20

Sol. 10% 4.28 4.08 4.26 4.20

Sol. 1% 4.28 4.08 4.26 4.20

Sol.0.1% 4.28 4.08 4.26 4.20

Conclusions

Based on obtained results the ozonised oil has antimicrobial activity in the tested experimental conditions.

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.

IN VITRO EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF A PRODUCT ON IMMORTALIZED KERATINOCYTES AFTER UVA EXPOSURE

The skin is the largest human organ and its immune function attracted the attention of both immunologists and dermatopathologists. There is increasing evidence that epidermal cytokines can have an important role in mediating skin inflammatory and immune responses. 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, TNFα, TGFα, TGFβ and PDGF. Such 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. in order to assess the anti-inflammatory activity of the tested substance the synthesis of two cytokines has been studied: lnterleukin 6 (IL-6) and lnterleukin 8 (1L-8) in treated and UVA exposed immortalized keratinpcytes (HaCaT cell line). 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.

At the same time both the viability (MTT assay) and the morphology (microscope analysis) of the treated cell cultures have been studied. MTT assay allows the cell viability/death to be quantified, the microscope analysis allows the presence of a normal/affected cell morphology to be detected and supports the MTT assay in evaluating the cell viability. a) Evaluation of cytokines synthesis

ELISA assay (Enzyme Linked Immunosorbent Assay) allows to evaluate, by using specific antibodies, whether the tested substances are suitable to specifically stimulate/inhibit the synthesis of cytokines, using specific antibodies. This process occurs in different steps:

- primary (monoclonal) antibodies recognize and specifically bind to tested protein (antigen), detecting their presence secondary (polyclonal) antibodies recognize and bind to primary antibody-antigen complex,

A signal system detects antibody-antigen complexes

- secondary antibodies conjugated enzymes catalyse the transformation of colourless molecules to chromogenic molecules, the colorimetric reaction is directly proportional to the amount of antibodies. Results are obtained by spectrophotometry using different wavelength depending on the used immunoenzimatic system. b) Evaluation of cell viability

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. c) Microscope analysis

The analysis of the cell morphology has been carried out using a Nikon Eclipse E600 microscope. The pictures were taken using 2OX magnifications.

Materials and Methods* Tested samples

- OZONIZED OIL: 0,019-0,057 mg/ml (O.O.)

(no toxic concentrations of the tested compound). - NOT OZONIZED OIL: 0,019-0,057 mg/ml (O.N.O.)

- Not treated controls (C-/C+) Cell cultures

Immortalized keratinocytes cultures (HaCaT cell line) from human epidermis have been used. Test execution

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

- 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 doses: 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/cm² of UVA (6 J/cm²). - 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).

EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY

CYTOKINES DOSAGE

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-6 (UVA-)

O.O. = Ozonised Oil

O.N.O. = Not Ozonised Oil

C- = Negative Control

Table 22

In the absence of UVA exposure the synthesis of IL-6 in no one tested samples has been observed IL-6 (UVA+) O.O. = Ozonised Oil O.N.O. = Not Ozonised Oil C+ = Positive Control Table 23

After UVA exposure the Ozonized Oil proves to be able to stimulate the IL-6 synthesis in immortalized keratinocytes cell cultures. Such capacity for the Ozonized Oil is higher than for controls. IL-8 (UVA-) O.O. = Ozonised Oil O.N.O. = Not Ozonised Oil C- = Negative Control

Table 24

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.

IL-8 (UVA+)

O.O. = Ozonised Oil

O.N.O. = Not Ozonised Oil

C+ = Positive Control Table 25

After UVA exposure IL-8 levels are lower than those obtained without irradiation.

^' IL-8 values in Ozonised Oil treated cells are higher than in the controls.

EVALUATION OF THE CELLULAR VIABILITY

MTT ASSAY

The cellular viability has been estimated without and after 25 minutes of UVA exposure.

Absorbance values (O.D.) at 540 nm (proportional to cell number).

O.O. = Ozonised Oil

O.N.O. = Not Ozonised Oil

C- = Negative Control

C+ = Positive Control

CELLULAR VIABILITY (UVA-)

Table 26

Without UVA exposure the values of cellular viability obtained for the treated samples and not treated control are not remarkably different.

CELLULAR VIABILITY (UVA+) Table 27

After UVA exposure the obtained values of cellular viability for Ozonized oil are more higher than those obtained for Not Ozonized Oil and the not treated control.

MICROSCOPE ANALYSIS

- NOT TREATED CONTROL (2OX magnification)

After UVA exposure it is apparent a remarkable reduction of the number of viable cells (figure 8)

- OZONISED OIL:

(Concentration: 0.057 mg/ml; 2OX magnification)

After UVA exposure the number of viable cells is not remarkably modified (figure 9) - OZONISED OIL:

(Concentration: 0.019 mg/ml; 2OX magnification)

After UVA exposure the number of viable cells is not remarkably modified (figure 10)

- OIL NOT OZONISED: (Concentration: 0.057 mg/ml; 2OX magnification)

After UVA exposure a low reduction of the number of viable cells can be observed (figure 11)

- OIL NOT OZONISED:

(Concentration: 0.019 mg/ml; 2OX magnification) After UVA exposure a remarkable reduction of the number of viable cells can be observed (figure 12)

SUMMARY OF ACQUIRED DATA AND IN VITRO EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF CELL CULTURE TESTED PRODUCT 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.

Claims

1. 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 that of ozone; b) contacting the ozone anion radical with an oil comprising unsaturated fatty acids for the formation of ozonides avoiding the contact between 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 suitable ox-red system resulting in the peroxide formation.

2. Process according to anyone of preceding claims wherein ozone is introduced at a concentration from 80 to 110 mcg/ml.

3. Process according to anyone of preceding claims wherein the catalyst is chosen from the group consisting of ferrous sulphate, powder zinc metal, tin (Sn²⁺) salt, silver metal.

4. Process according to anyone of preceding claims wherein the oil comprising unsaturated fatty acids is chosen from the group consisting of linen oil, ximenia oil and mixtures thereof, walnut oil, soy bean oil, wheat germ oil.

5. Process according to anyone of preceding claims wherein the ox-red system is chosen from oxidised alpha lipoic acid and acetyl lipoate, alpha tocopherol and ascorbyl palmitate, butyl hydroxy anisole and lecithin.

6. Process according to anyone of preceding claims wherein the catalyst is used in amount from 150 to 250 mg for 100 ml of oil.

7. Process according to anyone of preceding claims wherein any component of the ox-red system is used in amount from 150 to 250 mg for 100 ml of oil.

8. Ozonised oil obtainable by means of the above described process.

9. Pharmaceutical composition comprising the ozonised oil as active principle associated with one or more pharmaceutically acceptable adjuvant and/or excipient.

10. Compositions according to claim 7 wherein the ozonised oil is at concentration from 20 to 80 mcg/ml.

11. Composition according to anyone of claims 9-10 in a form selected form the group consisting of emulsions, creams, suppositories, clysters, pessaries, capsules, powder wherein the ozonised liquid oil is adsorbed on an inert support.

12. Composition according to claim 11 wherein the inert support is micronized silica.

13. Ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for use in medical field.

14. Use of ozonised oil according to claim 8 as carrier for medicaments and/or phytotherapeutics.

15. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament as germicide, antiviral, derma regenerating, anti-inflammatory, antiphlogistic, analgesic, fungicide, immuno-stimulating medicaments. ^'

16. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for the treatment of trophic ulcers, abscesses, rhagades, anal and vaginal fistulas, decubituses, phlegmon furuncles, purulent gengivitises, stomatitises, sinusitisises, vulvovaginitises, herpetic lesions, chronic osteomyelitis in immunodepressed patients, protozoa and fungal infections.

17. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for the treatment of gastritises, diarrhoeases, obstinate constipations, Crohn's disease, sebaceous cysts.

18. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in dermatology for the treatment of simplex and Zoster Herpes, contact dermatitises, chilblain, acne, mycosis, eczemas, psoriasis, hand and foot rhagades, bugs and hymenoptera bites.

19. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in angiology and phlebology for the treatment of coronary and arterial pathologies, decubitus ulcers, gangrenes, venous insufficiency, phlebopathies, diabetic ulcers, post-phlebitic ulcers.

20. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in orthopaedics for the treatment of disc-radicular conflicts, arthrosis, periarthritis, lumbar sciatica, tendinitises, strains.

21. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in rheumatology for the treatment of rheumatoid arthritis, articular rheumatisms.

22. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in urology for the treatment of infections of the urinary ways.

23. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in proctology for the treatment of proctitises, rhagades, haemorrhoid.

24. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in gynaecology for the treatment of bacterial and fungal vaginal infections, vulvovaginitises, vaginal ulcers.

25. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in dentistry for the treatment of abscesses, gum infections, stomatitises, aphtha, gum lesions.

26. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in otolaryngology for the treatment of ear infections, cold diseases, sinusitises.

27. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in aesthetic medicine for anti-plica, breast toning, anti-stria atrophica and anti-cellulite treatments.

28. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in ophthalmology for the treatment of cornea ulcers, infections.

29. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in pneumatology for the treatment of bacterial and/or viral bronchopulmonary infections, enphysemas.

30. Use of the ozonised oil and pharmaceutical composition according to anyone of claims 8 to 13 for the preparation of a medicament for use in veterinary for the treatment of pyoderma, dermatitis, postinfection arthritis, arthrosis, cystopyelitis, abscesses, infectious enterocolitis, cold - mucosal diseases, decubitus ulcers, localised mycosises, peptic ulcers, rhagades, corneal ulcers, vaginitises, mastitises, metritises.

31. 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.

32. Device for the ozonization of an unsaturated fatty acid rich oil according to claim 31 further comprising a temperature measuring device.

33. Device according to anyone of claims 31-32 wherein the catalyst is chosen from the group consisting of ferrous sulphate, powder zinc metal, tin (Sn²⁺) salt, silver metal.

34. Device according to anyone of claims 31-33 wherein the fine grain porous septum is made of inert material.

35. Device according to claim 34 wherein the inert material is ceramic material.

36. Use of the ozonised oil according to claim 13 as a cosmetic component.

37. Use according to claim 36 for trichological treatment.