TECHNICAL FIELD The present invention refers to the field of ozonized phytotherapic compositions for tissue and bone regeneration.

STATE OF ART

Historically, the plant world has been the most important source of medicinal agents for the treatment of human and animal diseases and they have been used as prevention agents in maintaining good health. However, over the past 150 years, Western medicine has been dominated by synthetic and / or highly purified chemical agents.

However, it is increasingly recognized that medicinal plant extracts can be highly effective agents for disease prevention and treatment. This is particularly true considering the low toxicity and greatly reduced incidence of adverse effects associated with plant-based drugs compared to many synthetic or highly purified drugs. Moreover, since the plants possess a large number of pharmaceutically active agents, the obtained extracts exert their activity on various physiological processes, increasing the range of the desired therapeutic effect.

SUBSTITUTE SHEETS (RULE 26) Oral diseases are among the most painful for humans. In addition to the diseases of the rigid tissues of the oral cavity (eg dental caries), there are several pathological conditions that affect the oral mucosa and periodontal tissues. The most common conditions are gingivitis, periodontal disease, aphthous ulcers and herpes simplex lesions, as well as the oral manifestations of common vesicular-bulbous conditions such as bulbofoide, pemphigus, erythema multiforme and planctonus planus, as well as other autoimmune conditions. Other very painful conditions result from the presence of wounds in the process of healing following an operation, ulcerative lesions or bedsores.

To eliminate pain, speeding up healing, numerous phytotherapeutic formulations have been produced and are known to the state of the art. Almost all of them have been developed solely for the care of the oral cavity which narrows the choice for dermis and bone care.

For example, in US 2014/0193345 "Herbal composition for the treatment of mucosal lesions" a composition of Echinacea purpurea, Sambucus nigra and Centella asiatica is presented for the treatment of oral and anal mucosa. The study of this composition seems to be more focused on reducing pain during treatment than on healing times.

SUBSTITUTE SHEETS (RULE 26) In US 8709509 B2 "Herbal composition for the treatment of wound healing, a regenerative medicine", the phytotherapic formulation is composed of Curcuma longa, Glycyrrhiza glabara, Hamiltonia suaveolens, Tipha angustifolia and Azadirachta indica and is used for the treatment of wounds, burns, ulcers.

Both of these formulations, on the other hand, do not make use of the healing power of ozone, which speeds healing and increases the effectiveness of natural agents.

Although many pharmaceutical agents have been used in the management of oral mucosal, skin and bone lesions, many of these have been relatively ineffective, while some (particularly at systemic regimens) are associated with unacceptable adverse effects. There is therefore a need for new, effective and safe treatment modalities for many of the aforementioned diseases There is a particular need for a safe, effective and natural topical treatment.

It is evident that there are different herbal compositions reported in the state of the art, used to treat wounds, but none in the known art discusses an ozonized phytotherapic composition, resulting from the ozonization of the synergic and effective formulation of Hypericum Perforatum oil, Prunus Amigdalus Dulcis oil, Calendula Officinalis oil, Echinacea Angustifolia oil and silicon dioxide for the treatment of tissue and bone lesions.

SUBSTITUTE SHEETS (RULE 26) Summary of the Invention

The present invention relates to a composition of four suitably ozonized essential oils: Hypericum Perforatum oil, Prunus amigdalus dulcis oil, Calendula Officinalis oil, Echinacea Angustifolia oil and silicon dioxide, for the stimulation of tissue and bone cell growth based on the healing properties of the plants. The composition performs a protective action with the formation of a film that acts both chemically and mechanically on the oral cavity; the film adheres to the mucosa and produces a barrier effect that helps to relieve pain and promote healing.

The cell growth stimulating activity appears in the concentration range of the ozonized formulation object of the present invention between 0.002 and 0.170 mg / ml in the case of fibroblasts, between 0.0019 and 1.6 mg / ml in the case of osteoblasts. Fig.l and 2 show the results obtained, related to the production of fibroblasts and osteoblasts depending on the concentration of the formulation object of the invention.

This composition is explicitly presented for the prevention and mitigation of problems in different fields of medicine. In the surgical field it is applied to wounds and skin burns, in post surgery cases, in ulcerative bedsores and on skin irritations, where it has an anti-infective, antibacterial, antiviral and anti inflammatory effect. In the dental field the formulation is applied to gingival and oral mucosa wounds, and by virtue of its specific formulation in dense and viscous lipogel, it forms a protective

SUBSTITUTE SHEETS (RULE 26) film on the lesions contributing to a re-epithelialization of the gums and of the oral mucosa, keeping the microenvironment under control. It also regenerates wounds caused by mouth ulcers; aphthous ulcers; irritations and lesions of the mouth due to oral surgery, traumatic ulcers caused by orthodontic appliances or dentures. The composition is therefore indicated, as an adjunct to the therapy of irritated gums, after dental procedures, after scaling and other specific oral hygiene interventions, or in conjunction with the use of prosthetic and orthodontic appliances. In the dermatological field, the formulation object of the present invention is applied on skin irritations, psoriasis and manifestations involving redness (radiotherapy, chemical peels, and abrasions) to facilitate healing; on dermatitis, inguinal mycosis, post-exposure solar treatment, after insect bites or itching, excoriation of the skin and post-peeling; in the treatment of onychomycosis, it promotes the healing process of onycho-cryptosis, preventing their fall and flaking. The foundation of the aforementioned composition consists in the fact that the phytomedicine is particularly indicated in long-term therapies as it is generally well tolerated by the patient; in combination with synthetic drugs it is able to increase its effectiveness and reduce its side effects; it can be used as a preventive action; products based on medicinal plants allow the rapid and correct intake of a single product, instead of taking a plurality of synthetic drugs. For example, pain is one of the biggest problems which the patient fears the most.

SUBSTITUTE SHEETS (RULE 26) Traditional painkillers are excellent in their function, but they often have bothersome and possibly even dangerous side effects.

The lipophilic extracts of medicinal plants are not toxic to ingest and for this reason they are particularly suitable for treating oral lesions, aphthous ulcers and as adjuvants in post dental treatment, in inflammatory manifestations and as fast cicatrizant.

From the above, it is clear that the formulation object of the invention is rich in advantages that are even more enhanced by the ozonation of its active ingredients.

Ozone carries out various biological activities which explains its use in human and veterinary medicine. Ozone is a powerful oxidant with good antibacterial activity. It is also able to enhance the non-specific organic skin defences and to inhibit the growth and proliferation of dermatophytes during fungal dermatitis. In veterinary medicine it is used in dermatology as a therapy for superficial and deep pyoderma; in lesions of fungal, traumatic and immunological origin, and also in cases of wounds characterized by itching and inflammation. Nowadays, ozone is also used as a therapy for otitis of bacterial and fungal origin, where it performs an antalgic and anti-inflammatory activity. In orthopaedics and surgery, it is used to induce rapid healing in the case of surgical wounds, fistulas, sutures and external fixators.

SUBSTITUTE SHEETS (RULE 26) Ozonized oils are vegetable oils whose molecule contains unsaturated fatty acids, which produce ozonides when subjected to ozone (Fig.3).

The ozonides act by restoring the membrane ionic electric potential of the cells of the altered tissues and block the viral receptors killing the infected cells, therefore stopping their propagation.

It is also known that ozonides have a good activity also against protozoa and, still, they are active against nematoids, the parasites that infest a large number of animals and plants.

These considerations give an idea of the pharmacological action spectrum of these compounds.

The object of the present invention is to provide a composition for tissue and bone regeneration based on the healing properties of the plants which is both effective and has a substantially wider field of application, including a cosmetic use for anti-aging treatment.

According to the present invention this object is achieved through the effective synergy of five natural active principles and the ozonation of some of these. It has been found that the inventive composition, formulated on the basis of said measures, results in stimulating the regeneration of the epithelial tissue and of the bone cells in a surprisingly greater way than the products present on the market.

SUBSTITUTE SHEETS (RULE 26) Advantage of the formulation object of the present invention is therefore that, with it, a curative capacity substantially superior to the formulations known to the state of the art in a field of general application is obtained. In conclusion, with the composition of the present invention a curative and cosmetic composition is provided through which there are substantially faster and more effective healing processes; in particular, achieving rapid healing where this is required. The composition of the present invention is also of general application, including dermatological applications, such as for example psoriasis and dermatitis; abrasions; third-level burns; odontostomatological applications; and in particular post- surgical applications. In particular, a greater healing of the epithelial tissue is obtained, in terms of a greater healing speed and a greater regenerative capacity of the tissues, through a greater stimulation of the functions of both fibroblasts and osteoblasts.

The inventive composition is substantially advantageous also in the cosmetic field for an anti-aging treatment.

The inventive composition also has considerable application in veterinary medicine; indeed, it is particularly indicated in this field, since animals are predisposed by nature to heal themselves

SUBSTITUTE SHEETS (RULE 26) with plants, and the inventive composition is not toxic if accidentally ingested.

The inventive composition is particularly indicated in all those situations in which the aim is a rapid healing of cut wounds or ulcerative lesions of traumatic or post-surgical origin.

Therefore, the subject of the present invention is a composition for tissue and bone regeneration according to the attached independent Claim 1.

Preferred embodiments are recited in the dependent Claims

2 to 10.

Detailed description of the images

Fig. 1 is a chart indicating the % of fibroblasts as a function of the concentration of the composition. Fig. 2 is a chart indicating the % of osteoblasts as a function of the concentration of the composition.

Fig. 3 is a schematic representation of ozonide formation.

Fig. 4 shows a case of regeneration in a serious gum recession of an incisor before and after surgical intervention. Two applications per day of the Composition I object of the invention have led to a complete recovery in 60 days of a 48 years old patient. Left: before surgery, Right: after the operation.

SUBSTITUTE SHEETS (RULE 26) Fig. 5 shows a case of regeneration in a 32-year-old female patient with persistent cicatricial acne for five years. Composition II was applied daily for sixty days. As can be seen from the photos, the composition II shows an evident efficacy and a total resolution of the problem in sixty days.

Fig. 6 shows a case of regeneration in a 16-year-old male patient with persistent scar acne for five years. Composition II was applied daily for forty days. As can be seen from the photos, the composition II shows an evident efficacy and a total resolution of the problem in forty days.

Fig. 7 shows the regeneration of a fall excoriation. Two applications per day of the composition object of the invention allowed the complete recovery in twelve days.

Fig. 8 shows the regeneration of a lesion of the back of the hand with poorly vascularised necrotic tissue and uncovered bones and tendons. In fourteen days and two applications per day, a complete recovery was obtained, with the restoration of the vascularization already from the first applications.

Fig. 9 shows the regeneration of a varicose ulcer with poorly vascularised necrotic tissue on the right leg of an 82-year-old female patient. In sixty-five days and one application per day, complete recovery was achieved, with the restoration of the vascularization already from the first applications.

SUBSTITUTE SHEETS (RULE 26) Detailed description of the invention

The present invention will be fully understood on the basis of the following description given as an example, which absolutely does not limit it. The present invention is a composition for tissue and bone regeneration comprising as an active principle a formulation composed of four essential oils and silicon dioxide, in which the oils or part of them are ozonated for a greater efficacy of the product with respect to what is known in the state art; for 20 ml of formulation the Hypericum Perforatum oil is contained in the quantity between 30 and 50 mg / 20 ml; Calendula Officinalis oil is contained in a quantity between 15 and 40 mg / 20 ml; Prunus Amigdalus Dulcis oil is contained in a quantity between 15 and 25 mg / 20 ml; Echinacea Angustifolia oil is contained in a quantity between 2 and 5 mg / 20 ml; silicon dioxide contained in an amount between 2 and 10 mg / 20 ml.

For a preferred embodiment of the invention one of the oils of the formulation or a combination thereof are ozonized, since the addition of ozone to the lipophilic extracts contained in the formulation object of the present invention gives the product a high power of diffusion in the tissues and therefore reducing edemas, inflammation, pain and irritation in the treated area. Ozone molecules mixed with the oils in the formula, double the healing properties, enhancing the phy to therapeutic properties of the formulation, making it a unique product of its kind.

SUBSTITUTE SHEETS (RULE 26) The beneficial effects of ozonized lipophilic extracts are to provide greater oxygenation to the tissues, stimulating the capillary microcirculation which vascularises the wound and induce the scarring process; furthermore, ozone has anti- infective, antibacterial, antiviral and anti-inflammatory effects.

For a particular embodiment, it is contemplated that the inventive composition, in the form of a protective and healing gel, such as the Composition I, is consisting of:

TABLE 1

in which Hypericum Perforatum oil is contained in a quantity between 30 and 50 mg / 20 ml; Calendula Officinalis oil is contained in a quantity between 15 and 40 mg / 20 ml; Prunus Amigdalus Dulcis oil is contained in a quantity between 15 and 25 mg / 20 ml; Echinacea Angustifolia oil contained in an

SUBSTITUTE SHEETS (RULE 26) amount between 2 and 5 mg / 20 ml; silicon dioxide is contained in a quantity between 2 and 10 mg / 20 ml.

Hypericum Perforatum oil is ozonized with an ozone concentration between 150 and 300 meq / Kg. In this specific formulation, in addition to the formulation composed of the four oils which define the active principle of the invention, the other component that improves and characterizes the curative properties of the product claimed in the present invention is the Aloe Barbadensis juice contained in a quantity between 2 and 6 mg / 20 ml.

The main ability of the Aloe Barbadensis in the tissue repair process is to stimulate the formation of fibroblasts, particular cells of the connective tissue that contribute to generate collagen, glycosaminoglycan (elastic fibers with a reticular shape) and finally the glycoproteins that are positioned in the extracellular matter. Whenever tissue damage occurs, fibroblasts activate and induce the process of mitosis. Moreover, they are present in the scar tissue, and therefore promote tissue healing, and ultimately contribute to the production of bone cells. Fibroblasts do not have a specific shape, but are characterized by an elastic nature, just like our skin is. They are often placed along the collagen fibers and are visible with an elongated core.

Composition I is particularly effective in the treatment of deep wounds with possible exposure of bone portions. The

SUBSTITUTE SHEETS (RULE 26) ability to form a protective film allows a rapid revitalization of the wound and a rapid healing of the exposed bone portion.

Composition I has been successfully tested also in the odontostomatology field with surprising result of re- epithelialization of gums and oral mucosa. It is indicated for: treatment of injuries due to dental surgery; therapy of irritated and bleeding gums, Labial Herpes and mouth ulcers, paradentitis, peri-implantitis; as an adjuvant after oral interventions; implant operations; implant surgery; tooth extractions; scaling; and together with the use of prosthetic and orthodontic appliances.

The Composition I applied on the bone cavities of the jaw created by the void after the extraction of a molar, it was observed to strongly stimulate bone regeneration (osteoblasts) in a very short time, compacting the bone for the installation of implantology self-tapping screws. Fig. 4 shows a 48 -year-old patient with a serious recession of the incisional gingiva before and after the operation. In the photo on the right the patient was treated twice a day with Composition I, showing a total healing of the area.

For a particular embodiment, it is contemplated that the inventive composition, such as the Composition II, consists of:

SUBSTITUTE SHEETS (RULE 26) TABLE 2

For this particular form of the invention, in addition to the composition of the four essential oils which constitute the active principle of the present invention, Melaleuca Alternifolia oil is present in a quantity between 2 and 6 mg / 20 ml. Melaleuca oil has antibacterial properties, amply demonstrated by several studies. In detail, the Melaleuca oil seems to be able to exert its antibacterial action through the denaturation of the proteins of the microorganism’s cytoplasmic membrane.

Among the bacteria sensitive to the antibacterial action of the Melaleuca oil we find the Bacillus subtilis, the Enterococcus faecalis, the Escherichia coli, the Moraxella catarrhalis, the Streptococcus pyogenes and the Staphylococcus aureus. The Melaleuca enhances the disinfectant power and the healing properties of the formulation.

SUBSTITUTE SHEETS (RULE 26) The active principles of the essential oils contained in Composition II nourish and firm up the skin, making it smooth and velvety, while protecting it from ultraviolet radiation. Furthermore, Composition II protects the skin from aging, as it strongly stimulates tissue regeneration of the skin at a cellular level; it is also indicated as a strong anti-inflammatory, disinfectant and healing agent; for its healing effects, it is also suitable for the treatment of dermatitis, juvenile acne and sunburn. Fig. 5 shows the evolution of a case of a five years persistent scarring acne on a 32-year-old female patient. Composition II was applied daily for 60 days. As you can see from the photos, the composition II shows an evident efficacy and a total resolution of the problem in 60 days.

Fig. 6 shows the evolution of a case of a five years persistent scarring acne on a 16-year-old man. Composition II was applied daily for 40 days. As can be seen from the photos, the composition II shows an evident efficacy and a total resolution of the problem in 40 days.

With the same formulation, patients with facial keratosis, collagen stimulation, traumatic skin fissures of the external genital organs were treated with positive results. Composition II is also indicated for the treatment of the skin following tattoos or in the presence of mycosis.

The Composition II is also particularly suitable for the treatment of venereal diseases such as candidiasis, vulvodynia,

SUBSTITUTE SHEETS (RULE 26) genital fissures, external hemorrhoids, anal fissures, mucous reddening, drug resistant eczema and genital Lichen Sclerosus.

For a particular embodiment it is contemplated that the inventive composition, such as Composition III, consists of: TABLE 3

In Composition III the Hypericum Perforatum Oil is ozonized with an ozone concentration between 150 and 300 meq / Kg; Prunus Amigdalus Dulcis oil is ozonized with an ozone concentration between 150 and 300 meq / Kg; Calendula

Officinalis oil is ozonized with an ozone concentration between 150 and 300 meq / Kg; Echinacea Angustifolia oil is ozonized with an ozone concentration between 150 and 300 meq / Kg;

For a particular embodiment of the invention, Composition III, in the form of a protective and healing gel, has a high regenerating power, promotes tissue regeneration and complete healing of the skin, it is also indicated in all those situations that require rapid healing, such as lesions of the back of the hand with

SUBSTITUTE SHEETS (RULE 26) necrotic and poorly vascularized tissue and uncovered tendons and bones, in which curative results have been obtained already after 14 days of application (Fig. 7); facial lesion from falling, in which there have been curative results already after 12 days of application (Fig. 8).

Composition III was successfully tested also on patients aged between 80 and 90 years in serious health conditions, suffering due to their age due of slow metabolism, and multiple pathologies, including diabetes mellitus, which in conjunction with ulcerative lesions make them very difficult to cure. With Composition III the total healing of varicose ulcers was achieved in a 82-year-old patient with medullary aplasia (Fig. 9). Surprising results were also obtained with Composition III in a 10-day medication of lacerated-contused wounds reported following a road accident in a 60-year-old patient with diabetes mellitus.

In Composition III the antiseptic and regenerating activity of the active principles present in vegetable essential oils is associated with the cy to -protective activity of ozone, carrying out a surprisingly healing action, very important in promoting healthy wound healing, while limiting the risk of bacterial infections.

Local use also optimizes the on-site therapeutic activity, thus reducing systemic side effects. The Composition III is therefore suitable, as a gel for local application, for the treatment of

SUBSTITUTE SHEETS (RULE 26) wounds and burns of I and II degree of the skin, promoting tissue regeneration, for the treatment of ulcerative lesions of the skin, dermatitis, herpes zoster, psoriasis, post-surgical wounds, bedsores, (even in diabetic patients, as reported above), redness of the skin due to radiation therapy, chemical peels, sun exposure, diaper use and irritation resulting from insect bites.

Composition III has been successfully tested in animal care. Ulcerative lesions due to acute dermatosis resulting from gastro intestinal problems affecting a dog were healed in 3 days of Composition III application.

To conclude, the present invention in its embodiments is applicable and effective on many diseases. Some examples of these, absolutely non-limiting of the invention are: arthritis; bubbles; bursitis; burns, in particular chemical bums, radiation burns, X-ray burns, wind burns; keratosis; scars, particularly post-operative scars; sebaceous cysts; bruises; milk crusts; diaper dermatitis; dermabrasion; flaking of the skin; muscle pains; eczema; skin rashes, particularly allergic skin rashes; folliculitis; chilblains; impetigo; cracked lips; mycosis; urticaria; chemical peeling; irritated skin; very dry skin; rough skin; cracked skin; dull skin; scaly skin; bedsores; ulcerative sores; athlete's foot; itch; psoriasis; insect bites, in particular bee stings and flea bites; spider bites; rosacea; skin redness; sunburns; seborrhea; stretch marks; cuts; inguinal tinea; poison of ivy; oak poison; warts; blisters.

SUBSTITUTE SHEETS (RULE 26) A particular embodiment provides that the excipients of the Compositions I, II, III are those reported in Table 4:

TABLE 4

The present invention has been described and illustrated with reference to specific embodiments thereof, but it must be expressly understood that variations, additions and / or omissions can be made, without thereby departing from the scope of protection thereof, which remains defined only from the attached claims.

SUBSTITUTE SHEETS (RULE 26)