Phytological composition for the treatment of skin cancer

Abstract

The present invention relates to a phytological composition for use in the treatment and / or prevention of skin cancer, in particular melanoma and non melanoma skin cancer. The phytological composition consists of a polysaccharide of Tremella fuciformis, an extract of leaves of Vitis vinifera, an extract of leaves of Astragalus membranaceus, an extract of Medicago sativa, an extract of Fucus vesiculosus, a powder of Spirulia platensis, an extract of root of Beta Vulgaris and an extract of Polygonum cuspidatum. The present invention also relates to pharmaceutical formulations comprising the phytological composition and suitable pharmaceutically acceptable excipients.

Description

[0001] PHYTOLOGICAL COMPOSITION FOR THE TREATMENT OF SKIN CANCER FIELD OF THE INVENTION

[0002] The present invention relates to a phytological composition for use in the treatment and / or prevention of skin cancer, in particular melanoma and non melanoma skin cancer. The phytological composition consists of:

[0003] 10-20% (w / w) polysaccharide of Tremella fuciformis,

[0004] 5-15% (w / w) extract of leaves of Vitis vinifera,

[0005] 10-30% (w / w) extract of leaves of Astragalus membranaceus,

[0006] 8-15% (w / w) extract of Medicago sativa,

[0007] 4-15% (w / w) extract of Fucus vesiculosus,

[0008] 4-8% (w / w) powder of Spirulia platensis,

[0009] 8-15% (w / w) extract of root of Beta vulgaris and

[0010] 5-30% (w / w) extract of Polygonum cuspidatum.

[0011] BACKGROUND OF THE INVENTION

[0012] Melanoma is a malignant tumor which originates from the melanocytes of the skin and mucous membranes, from those which make up the skin moles or, more rarely, from the melanocytes placed in extra-cutaneous sites (eye, meninge, inner ear, etc.). Melanoma develops in successive times through various stages of progression in which it presents different clinical and histological aspects.

[0013] As it is known, melanoma is traditionally distinguished in 4 clinical varieties:

[0014] 1. superficial diffusion melanoma,

[0015] 2. malignant lentigo melanoma,

[0016] 3. acral lentiginous melanoma and

[0017] 4. nodular melanoma.

[0018] The first three lesions begin with a 'flat' diffusion on the skin surface, which represents the horizontal growth phase (flat melanoma). After a variable period of time, these tumors may develop invasive nodular components (plane / dome-shaped melanoma).

[0019] On the other hand, nodular melanoma from the first moment is a deep invasive nodule (domeshaped melanoma).

[0020] This is an important distinction that is not only academic, but also correlates well with the prognosis of the neoplasm.

[0021] In fact, among the various factors that can be taken into consideration to predict the evolution of melanoma, the maximum thickness of the tumor is currently considered of primary importance.

[0022] Very rare before puberty, melanoma affects predominantly subjects aged between 30 and 60 years and of medium-high social class.

[0023] Considered up to a few years ago as a rare neoplasm, today it shows a constant growing incidence throughout the world and numerous studies suggest that it has even doubled in the last 10 years.

[0024] Worldwide, it is estimated that cutaneous melanoma has reached 100,000 new cases per year over the last decade: an increase of about 15% over the previous decade. Cutaneous melanoma is, in particular, tens of times more frequent in European (Caucasian) strain subjects than in other ethnicities. The higher incidence rates are in fact found in the areas very sunny and inhabited by populations of Nord-European strain, with the skin particularly clear.

[0025] In European or European origin populations, the incidence of cutaneous melanoma increased between 1980 and 2000 at a rate of 4-8 per cent per year.

[0026] At the different anatomical sites, the major increase in incidence was for the melanoma of the trunk and minimal for those of the head and neck, for those of the legs the increases were more marked in the female subjects.

[0027] Although the most modest increases in mortality rates show that cutaneous melanoma has actually increased in European or European origin populations, and that the very recent reductions in mortality, especially in young people, are attributable to the first favorable results of early diagnosis.

[0028] Cutaneous melanoma has a prognosis - that is an evolution over time - strictly dependent on the thickness reached in the skin at the time of its diagnosis and removal. If melanoma is still confined to the superficial skin layers, the prognosis is generally good, with the patient healing. Conversely, if melanoma has reached the deepest layers, because it has had a long time to increase before its identification and removal, the life risks for the patient are very high. Identifying melanoma as early as possible is therefore the main weapon in trying to reduce its mortality.

[0029] Thanks, therefore, to health education campaigns to encourage people to pay attention to the "changing skin mole", now the share of melanoma discovered when the prognosis can still be favorable has reached 60-70%.

[0030] The result was the improvement of survival: in 1960 only half of the melanoma patients were still alive 5 years after the first diagnosis, while today it is about 80% of them, then 30% more. Other malignant skin tumors (carcinomas or skin epitheliomas), in addition to melanoma, include basal cells carcinomas, which, never metastasizing, thus have a "local" malignancy, and squamous cells carcinomas, fortunately more rare, because they may undergo metastatic dissemination, if diagnosed and treated late.

[0031] Also, for non-melanoma skin carcinomas, an increase in frequency has been demonstrated, most likely due to the effects of excessive solar exposure in "predisposed" subjects. In fact, these tumors are more frequent in subjects with clear skin, which do not easily tun, and their appearance is linked to the cumulative amount of sunlight absorbed by the skin during life. In fact, skin carcinomas are more frequent in those who work in the open air (sailors, farmers) and in people of advanced age.

[0032] With respect to the squamous cells carcinomas, however, it should be pointed out that the basal cells carcinomas, very frequent, about 50 times more than melanoma, do not save the adultjuvenile age (from 25-30 years on), and their onset appears not so much associated with the cumulative amount of ultraviolet radiation absorbed in life (as with the squamous cells carcinomas), but to solar bums or scalds reported, especially in childhood.

[0033] This characteristic is associated with melanoma: in fact, similarly, the risk of melanoma is associated not so much with the cumulative amount of ultraviolet radiation absorbed by the skin, but with the number of sunbums undergone, especially in childhood and adolescence.

[0034] The phytological composition of the invention comprises several natural ingredients, which may in turn comprise several phytocomplexes and has been described in the Italian patent application No. 102023000002271 and in the international patent application WO2024 / 166055.

[0035] In particular, the phytological composition described in the above-mentioned patent applications consists of the following ingredients, each in the concentration ranges by weight indicated in brackets (w / w, based on the total weight of the phytological composition):

[0036] Polysaccharide of Tremella juciformis (10-20%),

[0037] Leaf extract of Vitis vinifera (5-15%),

[0038] Root extract of Astragalus membranaceus ( 10-30%),

[0039] Extract of Medicago sativa (8-15%),

[0040] Extract of Fucus vesiculosus (4- 15%),

[0041] Powder of Spirulia platensis (4-8%),

[0042] Root extract of Beta vulgaris (8-15%) and Extract of Polygonum cuspidatum (5-30%).

[0043] The phytological composition has been described to have a regenerative, restorative and cicatrizing effect on tissue lesions of various nature, making it suitable both as a dermatological treatment of skin lesions and in particular psoriasis, and as a cosmetic. It has now been surprisingly found by the Applicant that the same phytological composition represents a remedy for the treatment and / or prevention of skin cancer, in particular melanoma and non-melanoma cancer.

[0044] DESCRIPTION OF THE INVENTION

[0045] The main object of the present invention is a phytological composition for use in the treatment and / or prevention of skin cancer, in particular melanoma and non-melanoma skin cancer. The phytological composition of the invention consists of the following ingredients, each in the concentration ranges by weight indicated in brackets (w / w, based on the total weight of the phytological composition):

[0046] Polysaccharide of Tremella juciformis (10-20%),

[0047] Leaf extract of Vitis vinifera (5-15%),

[0048] Root extract of Astragalus membranaceus ( 10-30%),

[0049] Extract of Medicago sativa (8-15%),

[0050] Extract of Fucus vesiculosus (4- 15%),

[0051] Powder of Spirulia platensis (4-8%),

[0052] Root extract of Beta vulgaris (8-15%) and

[0053] Extract of Polygonum cuspidatum extract (5-30%).

[0054] Preferably, the ingredients of the phytological composition of the invention are in the following concentration ranges by weight (w / w):

[0055] Polysaccharide of Tremella juciformis (12-16%),

[0056] Leaf extract of Vitis vinifera (8-13%),

[0057] Root extract of Astragalus membranaceus (15-25%),

[0058] Extract of Medicago sativa (8-15%),

[0059] Extract of Fucus vesiculosus (4- 15%),

[0060] Powder of Spirulia platensis (4-6%),

[0061] Root extract of Beta vulgaris (8-13%) and

[0062] Extract of Polygonum cuspidatum extract (15-25%). For the preparation of the extracts, where not indicated, the aerial parts of the plant, i.e. stem, leaves, flowers or mixtures thereof, are used.

[0063] The polysaccharide of Tremella fuciformis (CAS No. 778577-37-0) is extracted from the edible fruit bodies of the Silver Ear fungus in China. The polysaccharide comprises glucuronic acid and N-acetylglucosamine. The extracts of Tremella fuciformis can be obtained by solvent extraction from the carpophore of Tremella.

[0064] The extract of Vitis vinifera leaves (CAS No. 84929-27-1) is an extract of red grape leaves (Vitis).

[0065] The root extract of Astragalus membranaceus (CAS No. 94166-93-5) is extracted from the dried roots of the medicinal herb Astragalus membranaceus. The extracts of Astragalus can be obtained through an extraction process in the following order: Drying of Astragalus, grinding, screening and titration of the individual phytocomplexes.

[0066] The Medicago sativa extract (CAS No: 84082-36-0) is an extract of the entire Medicago sativa plant (alfalfa). The extract comprises folic acid and optionally one or more of oligosaccharides, vitamin B5 and coenzyme Q10.

[0067] The Fucus Vesiculosus extract (CAS No: 84696-13-9) is an extract of the dried thallus of the Bladderwrack, Fucus alga.

[0068] The Spirulia platensis powder (CAS No: 223751-80-2) is the powder obtained from the dried grinding preferably of the whole thallus of Arthrospira platensis, also called Spirulina platensis (spirulina).

[0069] The root extract of Beta vulgaris (CAS No: 89957-89-1 / 89957-90-4) is an extract of the roots of sugar beet, Beta vulgaris L., obtainable by drying, extraction and titration of Beta vulgaris powders or by solvent extraction. Polygonumcuspidatum extracts (CAS No. 501-36-0) can be obtained by drying and titration of powders or by solvent extraction. The Vitis vinifera leaf extracts and the Polygonum cuspidatum extracts preferably contain resveratrol.

[0070] Preferably, the phytological composition according to the present invention is formulated in a pharmaceutical formulation, more preferably for topical use. Said phytological composition is preferably present in the pharmaceutical formulation at a concentration by weight of 1-5% on the weight of the dermatological formulation; more preferably, the phytological composition is present at a concentration of 2-4%, even more preferably about 3%, on the weight of the dermatological formulation, together with suitable pharmaceutically acceptable excipients. Suitable pharmaceutically acceptable excipients include rheological additives, buffer agents, antimicrobial agents, antioxidant agents, anti-isothermal agents, antistatic agents, absorbing agents, UV absorbing agents, astringent agents, chelating agents, skin conditioning agents, preservatives, covering agents, denaturing agents, depigmenting agents, emulsifying agents, film forming agents, gelling agents, colloidal agents, hydrating agents, hydrotropic agents, binders, soothing agents, smoothing agents, opacifying agents, plasticizers, propellants, skinprotecting agents, reducing agents, refreshing agents, sebum-restoring agents, solvents, stabilizing agents, emulsifying stabilizing agents, toning agents, wetting agents, volumizing agents or combinations thereof.

[0071] Preferably, said pharmaceutically acceptable excipients comprise or consist of one or more of rheological additives, buffer agents, antimicrobial agents, antioxidant agents, emulsifying agents, hydrating agents, hydrotropic agents, smoothing agents and solvents.

[0072] The pharmaceutical formulations according to the invention are preferably aqueous formulations, more preferably comprising at least one alcohol-based solvent, at least one antimicrobial agent, at least one smoothing agent, at least one rheological additive, at least one antioxidant agent and / or at least one hydrating agent.

[0073] Particularly preferred pharmaceutically acceptable excipients which are present in the pharmaceutical formulations of the invention are: methoxymethyl butanol as an alcohol-based solvent, carbomer (or Carbopol ) as a thickening agent, propylene glycol, polyethylene glycol (PEG) compounds, such as PEG-6, PEG-32, PEG-4, including PEG-4 proline linolenate and PEG-4 proline linoleate. Preferably, the dermatological formulation of the invention further comprises one or more of: vitamins and / or provitamins, such as vitamin A, vitamin D, vitamin E and panthenol, preferably colloidal vitamins, such as colloidal vitamin D, colloidal vitamin A, inorganic elements with antimicrobial activity, such as silver, copper, zinc and other antimicrobial compounds, such as phenoxyethanol, 2-bromo-2-nitropropane-l,3-diol.

[0074] Optionally, the pharmaceutical formulation may further comprise glycosaminoglycans, such as chondroitin, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronic acid and mixtures thereof.

[0075] Preferably, the pharmaceutical formulation also comprises antioxidant and / or antimicrobial agents as active agents.

[0076] The invention therefore relates to the use of the phytological composition previously described in the treatment and / or prevention of skin cancer, in particular melanoma and non-melanoma skin cancer.

[0077] According to the present invention, other malignant tumors of the skin (skin carcinomas or epitheliomas), in addition to melanoma (or non-melanoma), include basal cell carcinomas and squamous cell carcinomas.

[0078] The formulation of said phytologic composition with specific excipients further enhances one or more of the aforementioned dermatological advantages of the phytologic composition, so as to be able to provide formulations particularly suitable for the treatment of specific dermatological disorders or tissue lesions.

[0079] Preferred formulations comprise the phytologic composition together with one or more of silver, gold, copper, silicon, zinc (preferably in colloidal form), phenoxyethanol, 2-bromo-2- nitropropane- 1,3 -diol, vitamin A, B and E (preferably in colloidal form), panthenol, propylene glycol.

[0080] The pharmaceutical formulation comprising the phytologic composition together with the following excipients is particularly suitable for the use described above, preferably in the form of a serum: colloidal silver (preferably 110 ppm) colloidal copper (preferably 20 ppm) colloidal gold (preferably 25 ppm) colloidal vitamin D (preferably 500 ppm) colloidal vitamin A (preferably 450 ppm) colloidal silicon (preferably 350 ppm) colloidal zinc (preferably 20 ppm)

[0081] MMB (3 -methyl-3 -methoxybutanol)

[0082] Phenoxyethanol bronopol (2 -bromo-2-nitro- 1,3 -propanediol) tocopherol (vitamin E)

[0083] Carbopol® panthenol propylene glycol

[0084] Aminoefaderma™ (PEG-4 proline linolenate).

[0085] Such pharmaceutical formulations comprising the phytologic composition may be in the form of ointment, lotion, cream, emulsion, paste, gel, aqueous solution, colloid, spray, plaster, serum, soaked gauze, dressing, mouthwash or a combination thereof. Formulations in the form of serum are particularly preferred.

[0086] Preferably, said pharmaceutical formulations should be administered topically, on the skin, from one to four times a day, in suitable amounts. A suitable amount is, for example, an amount that can be absorbed by massaging the formulation on the skin.

[0087] All the pharmaceutical formulation can be prepared by methods known in the pharmaceutical technique.

[0088] It should also be understood that all the combinations of preferred aspects of the phytological composition of the invention, as well as of the pharmaceutical formulation and the uses thereof, as reported above, are to be considered as described herein.

[0089] Operating examples of the present invention are given below for illustrative purposes.

[0090] EXAMPLES

[0091] Example 1

[0092] A phytological composition was prepared by mixing the following ingredients:

[0093] Example 2

[0094] A pharmaceutical formulation was prepared by mixing the phytological composition of

[0095] Example 1, at a final concentration of 3% by weight based on the weight of the formulation (w / w), with the following excipients, in water:

[0096] Example 3 - Evaluation of the anti-melanoma activity in vitro

[0097] To evaluate the anti-melanoma activity of the pharmaceutical formulation prepared in example 2 (also herein after called Anti-melanoma Formulation) human malignant melanoma cell lines (A375 purchased from the American Type Culture Collection [ATCC, Manassas, USA]) were used. Cell lines were cultured in DMEM medium (Dulbecco's modified Eagle's Medium) supplemented with 10% FBS (fetal bovine serum), 1-glutamine and antibiotics (50 JU / ml penicillin and 50 pg / ml streptomycin) and maintained in a 5% CO2 and 95% moisture incubator. For the experiments, 2* 104cells were plated in 96-well plates to study cell viability by MTT, LDH test kits, analysis of mitochondrial membrane potential by JC-1 and Crystal Violet; in addition, l * 106cells were plated on 6 wells to determine the molecular pathways involved in apoptosis and autophagy mechanism by specific ELISA kits.

[0098] On the day following sowing, the cells were placed in a serum-free white medium and stimulated with different concentrations of the Anti-melanoma Formulation for 24 hours.

[0099] After determination of cell viability by MTT, the three best concentrations were selected to perform further experiments to analyze the involved intracellular mechanisms.

[0100] In all the experiments reported below the results referred to the "control" refer to the untreated cells.

[0101] Materials and apparatus used

[0102] - Dulbecco's phosphate Buffered Saline diluted IX (PBS IX) with sterile water (Eurospital, Trieste, Italy).

[0103] - Trypsin solution - EDTA 10X (sterilely filtered, BioReagent, suitable for cell culture, 5.0 g porcine trypsin and 2 g EDTA, 4Na per liter 0.9% sodium chloride) diluted IX with PBS IX for cell rupture.

[0104] - MTT [(3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide]

[0105] - Annexin V ELISA Kit (Thermo Fisher, Milan, Italy)

[0106] - Cristal-Violet dye

[0107] - Cyclin DI ELISA Kit (MyBioSource, San Diego, CA, USA)

[0108] - Caspase 3 ELISA Kit (Thermo Fisher, Milan, Italy)

[0109] - Caspase 9 ELISA Kit (Thermo Fisher, Milan, Italy)

[0110] - LC3 Assay Kit (MyBioSource, San Diego, CA, USA)

[0111] - NFkB ELISA Kit (Cayman Chemical Company, Ann Arbour, MI, USA)

[0112] - TNFa ELISA Kit (Thermo Fisher, Milan, Italy)

[0113] - Spectrophotometer: Infinite 200 Pro MPlex, Tecan, Mahnedorf, Switzerland

[0114] Culture media and reagents were purchased from Merck Life Science, Rome, Italy, unless otherwise indicated.

[0115] MTT test

[0116] This test consists in measuring cell death, inhibition of cell growth, cell proliferation, enzyme release or reduction of vital dye (MTT, purchased by Merck Life Sciences, Milan).

[0117] In particular, the MTT test is a quantitative colorimetric assay using the vital dye 1 mg / ml MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which is reduced in the cellular mitochondria by the mitochondrial dehydrogenase enzyme of the cellular respiratory chain, in the MTT tetrazolium salt.

[0118] Crystals of formazan not soluble in water, of purple color, are formed inside the cells. These are determined with the spectrophotometer to obtain the number of active mitochondria and, therefore, the number of cells living in the sample.

[0119] The results of the MTT test on A375 after 24 hour are shown in Table 1 below.

[0120] Table 1 - Cell viability results in %

[0121] On the basis of initial results, it was seen that only the above three concentrations exerted the greatest cytotoxic effect on A375 melanoma cells, without causing excessive cytotoxicity on keratinocytes (human keratocytes, HaCat cells). Therefore, in the subsequent experiments only the concentrations 0.125 mg / mL, 0.063 mg / mL and 0.031 mg / mL were analyzed.

[0122] Annexin V

[0123] The ELISA kit for human Annexin V quantified the amount Annexin V in the supernatants of A375 cells following the manufacturer's instructions. Each well received a biotinylated detection antibody and the plate was incubated at room temperature for 1 hour. After 45 minutes of incubation with HRP-conjugated streptavidin (horseradish peroxydase), the TMB substrate solution (3,3',5,5'-tetramethylbenzidine) was added for 30 minutes and a stop solution terminated the reaction. To calculate the concentration of Annexin V, the absorbance was measured at 450 nm with a spectrometer and compared with the standard curve (0.78-50 ng / mL). The data are presented as an average of five experiments. An increase in Annexin V levels correlates with an increase in cellular apoptosis.

[0124] The results of the analysis of Annexin V after 24 hours of treatment on A375 are reported in Table 2 below.

[0125] Table 2 - Annexin V% levels

[0126] All tested concentrations showed a significant increase in comparison to control.

[0127] Cell proliferation by staining with Cristal Violet

[0128] Cells (A375) were fixed with 1% glutaraldehyde (Merck Life Science, Rome, Italy) for 15 minutes at room temperature, washed and stained with 100 pl 0.1% aqueous crystal violet (Merck Life Science, Rome, Italy) for 20 minutes at room temperature. At the end, the cells were solubilized with 100 pl of 10% acetic acid before reading the absorbance at 595 nm with a spectrophotometer. The estimated number was determined by comparing the data with the control cells normalized to TO (measurement at the start of stimulation after cell adhesion). The results were expressed as a percentage (%) with respect to the control. The data are presented as an average of five independent experiments.

[0129] The results of the analysis of the proliferation rate with Cristal Violet after 24h of treatment on A375 are reported in Table 3 below.

[0130] Table 3 - Proliferation rate %

[0131] All tested concentrations showed a significant decrease in cellular proliferation rate.

[0132] Cyclin DI

[0133] The ELISA kit for CDK1 was used according to the manufacturer's instructions. The A375 cells were lysed with lx phosphate buffered saline (PBS, Merck Life Science, Rome, Italy) cold, centrifuged at 5,000* rpm for 5 minutes and 100 pL of each sample were analyzed by reading the absorbance of the sample at 450 nm with a spectrometer.

[0134] The results were obtained by comparing the data with the standard curve (range 0.156 to 10 ng / mL) and were expressed as a percentage (%) compared to the control and as an average of five independent experiments performed in triplicate.

[0135] Cyclin DI was over-expressed in most parts of the tissues at high proliferative rate, i.e. carcinomas, hence a decrease in the levels of cyclin D 1 are correlated with an anti-proliferative activity.

[0136] The results of the analysis of cyclin D after 24 hours of treatment on A375 are reported in Table 4 below.

[0137] Table 4 - % Cyclin D levels

[0138] All concentrations showed a significant decrease in cyclin D levels compared to control. Caspase 3

[0139] The caspase 3 levels were studied in cell lysates using an ELISA kit, reading the absorbance of the sample at 450 nm with a spectrometer.

[0140] The data were obtained by comparison with a standard curve (0.039 to 2.5 ng / mL) and were expressed as mean ± SD (%) with respect to the control value (line 0).

[0141] Caspase-3 is a classical caspase involved in the implementation step which can be activated by caspases-8 and -9, two enzymes classifiable as initiators. Once activated, caspase-3 may break a quantity of protein substrates leading to cell collapse and death. Therefore, an increase in caspase levels 3 is correlated with the onset of cellular apoptosis phenomenon.

[0142] The data are presented as an average of five independent experiments.

[0143] The results of the analysis of caspase 3 at 24 hours of treatment on A375 are reported in Table 5 below.

[0144] Table 5 - % Caspase 3 levels

[0145] The two highest concentrations showed an increase in caspase levels 3 compared to control. Caspase 9

[0146] According to the guidelines provided by the manufacturer, the activation analysis of caspase 9 in the supernatant of A375 was carried out using the ELISA Human caspase 9 kit. Subsequently, 100 pl of sample was dispensed into the individual wells of a 96-well ELISA plate, followed by an incubation period at room temperature of 2 hours and then overnight at 4°C.

[0147] Five consecutive washings were followed with a wash buffer followed by the addition of 100 pl of biotinylated anti -caspase 9 in each well. After an incubation of 2 hours at room temperature, the content of each well was aspirated and washed five times before the introduction of 100 pL of streptavidin-HRP for an incubation of 1 hour. Subsequently, the streptavidin-HRP solution plate, 100 pl of chromogenic solution, was incubated in each well for 30 minutes at room temperature in the absence of light. The absorbance of each well was then determined at 450 nm using a Tecan plate reader after applying the stop solution. The data are presented as mean ± SD (%) with respect to the control sample and were obtained by interpolation with the standard curve (1.6-100 ng / mL).

[0148] The activation of the caspase-9 leads to the processing and activation of the caspase-3, -6 and - 7 which cause the cell death and therefore an increase in the caspase levels 9 is correlated with the occurrence of the cell apoptosis phenomenon. The data are presented as an average of five independent experiments. The results of the analysis of caspase 9 after 24 hours of treatment on A375 are reported in Table 6 below.

[0149] Table 6 - % Caspase 9 levels

[0150] The two highest concentrations show an increase in caspase levels 9 compared to control. From the set of data reported in this example 3, it can be seen that the Anti -melanoma Formulation showed a clear cytotoxic effect on melanoma cells.

Claims

CLAIMS1. A phytological composition consisting of:10-20% (w / w) polysaccharide of Tremella fuciformis,5-15% (w / w) extract of leaves of Vitis vinifera,10-30% (w / w) extract of leaves of Astragalus membranaceus,8-15% (w / w) extract of Medicago sativa,4-15% (w / w) extract of Fucus vesiculosus,4-8% (w / w) powder of Spirulia platensis,8-15% (w / w) extract of root of Beta vulgaris and5-30% (w / w) extract of Polygonum cuspidatum for use in the treatment and / or prevention of skin cancer, in particular melanoma and nonmelanoma skin cancer.

2. The phytological composition of claim 1, consisting of:12-16% (w / w) polysaccharide of Tremella fuciformis,8-13% (w / w) extract of leaves of Vitis vinifera,15-25% (w / w) extract of leaves of Astragalus membranaceus,8-13% (w / w) extract of Medicago sativa,8-15% (w / w) extract of Fucus vesiculosus,4-6% (w / w) powder of Spirulia platensis,8-13% (w / w) extract of root of Beta vulgaris and15-25% (w / w) extract of Polygonum cuspidatum for use in the treatment and / or prevention of skin cancer, in particular melanoma and nonmelanoma skin cancer.

3. The composition according to claim 1 or 2, wherein the non-melanoma skin cancer is basocellular or spinocellular cutaneous carcinoma.

4. The composition according to anyone of the preceding claims, wherein the use is topical use.

5. A pharmaceutical formulation comprising the phytological composition consisting of: 10-20% (w / w) polysaccharide of Tremella fuciformis,5-15% (w / w) extract of leaves of Vitis vinifera,10-30% (w / w) extract of leaves of Astragalus membranaceus,8-15% (w / w) extract of Medicago sativa,4-15% (w / w) extract of Fucus vesiculosus,4-8% (w / w) powder of Spirulia platensis,8-15% (w / w) extract of root of Beta vulgaris and5-30% (w / w) extract of Polygonum cuspidatum together with the following excipients: colloidal silver, colloidal copper, colloidal gold, colloidal Vitamina D, colloidal Vitamin A, colloidal silicon, colloidal zinc,3 -methyl-3 -methoxybutanol, phenoxyethanol,2-bromo-2-nitro-l,3-propanediol, tocoferol,Carbopol® pantenol propylene glycolPEG-4 Proline Linolenate.

6. The pharmaceutical formulation of claim 5, wherein the phytological composition is at a concentration of 1-5% in weight, on the total weight of the pharmaceutical formulation, preferably 2-4% in weight.

7. The pharmaceutical formulation of anyone of claims 5 or 6, in the form of ointment, lotion, cream, emulsion, paste, gel, aqueous solution, colloid, spray, plaster, serum, soaked gauze, dressing, mouthwash or a combination thereof, preferably in the form of serum.

8. The pharmaceutical formulation according to anyone of claims from 5 to 7, for use in the treatment and / or prevention of skin cancer, in particular melanoma and non-melanoma skin cancer.

9. La formulation according to la claim 8, wherein the non-melanoma skin cancer is basal cells or squamous cells cutaneous carcinoma.

10. The formulation according to claim 8 or 9, wherein the use is topical use.

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