Phytological composition for the treatment of non-skin inflammatory diseases

Abstract

The present invention relates to a phytological composition for topical use in the treatment of non-skin inflammatory diseases and associated chronic pain. 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 compositions comprising the phytological composition together with suitable pharmaceutically acceptable excipients.

Description

[0001] PHYTOLOGICAL COMPOSITION FOR THE TREATMENT OF NON-SKIN INFLAMMATORY DISEASES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a phytological composition for topical use in the treatment of non-skin inflammatory diseases and associated chronic pain. The phytological composition consists of:

[0004] 10-20% (w / w) polysaccharide of Tremella juciformis,

[0005] 5-15% (w / w) extract of leaves of Vitis vinifera,

[0006] 10-30% (w / w) extract of leaves of Astragalus membranaceus,

[0007] 8-15% (w / w) extract of Medicago sativa,

[0008] 4-15% (w / w) extract of Fucus vesiculosus,

[0009] 4-8% (w / w) powder of Spirulia platensis,

[0010] 8-15% (w / w) extract of root of Beta vulgaris and

[0011] 5-30% (w / w) extract of Polygonum cuspidatum.

[0012] BACKGROUND OF THE INVENTION

[0013] Chronic pain is a type of pain that persists or recurs over a period of more than three months, persists for more than one month after resolution of acute tissue damage or is associated with a lesion that does not heal. Causes include chronic diseases (e.g., neoplasms, arthritis, diabetes), lesions (e.g., disc hernia, ligament rupture), and many disorders with primary pain (e.g., neuropathic pain, fibromyalgia, chronic headache). Various drugs and psychological treatments are used for the treatment of chronic pain.

[0014] Protracted, unresolved diseases (e.g. neoplasms, rheumatoid arthritis, disc hernia) that produce continuous nociceptive stimuli can fully explain chronic pain. Alternatively, a lesion, although of slight magnitude, may lead to persistent changes (sensitization) in the nervous system, from peripheral receptors to the cerebral cortex, which may produce persistent pain in the absence of the persistence of nociceptive stimuli. With sensitization, the malaise due to a resolved disease, which would otherwise be considered as mild or just annoying, is instead perceived as significant pain.

[0015] In some cases (for example, a chronic lumbago after trauma), the triggering factor of pain is evident; in others (for example, chronic headache, atypical facial pain, chronic abdominal pain), the precipitating event is not evident or obscure. In addition, psychological factors can amplify persistent pain. Thus, chronic pain may generally appear to be disproportionate with respect to the identifiable physical processes. Chronic pain often induces or aggravates psychological problems (e.g. depression, anxiety). Distinguishing the psychological cause from the effect is often difficult, but if pain, depression and anxiety coexist, they generally intensify the overall experience of pain.

[0016] Every day, people with chronic pain consult physicians, physiotherapists and nurses of primary care. In fact, people with musculoskeletal problems - the most common cause of chronic pain - consult their general medical doctor on average five times more often than those without these conditions.

[0017] Chronic pain and musculoskeletal disorders may be debilitating, affect a person's mood, sense of well-being and quality of life.

[0018] These conditions can often coexist with other long-term conditions, such as diabetes, arthritis, cancer, heart or lung disease, and thus represent the turning point that prevents people from being able to cope with and maintain their independence.

[0019] Arthritis is a common health problem, caused by inflammation, in the world population, affecting more than 350 million people and is one of the major causes of disability. In fact, among chronic diseases in North America, for example, arthritis causes more disability than any other condition, including heart diseases, diabetes, back or spinal problems (for information on arthritis and rheumatoid arthritis see: Global RA Network. Available on: http s : / / gl ob alranetwork. org / proj ect / di sease-info / ) .

[0020] Arthritis is a generic term for over 100 different types of diseases affecting people of all ages, races, both sexes (but women develop more than men) and from children to the elderly. Rheumatoid arthritis is the most common form.

[0021] Contrary to popular belief, arthritis is not a disease of the elderly; more than three out of five people diagnosed with arthritis are less than 65 years old. It is expected that the weight of arthritis worldwide will have significant consequences in terms of health costs and loss of productivity by patients today and over the next 30 years. The annual report 2019-2020 of the British organization Versus Arthritis States, “Arthritis steals us so much: it can take away the ability to work, care for a family, to move without pain, to live independently or simply to feel ourselves. Yet arthritis is often dismissed as "only pain and discomfort" or "only a bit of wear"”. Arthritis is mistakenly seen as inevitable, insignificant or even incurable. It is not any of these things. That is why Versus Arthritis challenges the climate of tolerance, telling the world that “we will not accept that arthritis steals the foundations of life to more than 10 million people in the UK.” The related impact of arthritis pain goes far beyond the patients and people closest to them. At national level, the socio-economic burden is enormous. Not only are musculoskeletal conditions, such as OA (osteoarthritis), a major cause of lost working days. The direct effects on health-care resources should also not be underestimated.

[0022] Musculoskeletal conditions affect 18.8 million people in the UK and are the main cause of pain and disability. Overall, the health costs of osteoarthritis and rheumatoid arthritis will reach GBP 118.6 billions over the next decade. Musculoskeletal conditions account for one-fifth of all disease absences and result in the loss of approximately 28.2 million working days per year for the UK economy.

[0023] Neuropathic pain is widely recognized as one of the most difficult painful syndromes to manage and constitutes a challenge for general medical doctors and specialists in that the therapeutic results are often unsatisfactory (Van Hecke et al., Pain 2014; Torrance et al., Fam. Pract. 2007; Binder et al., Dtsch. Arztebl. Int., 2016).

[0024] Neuropathic pain is usually associated with reduced quality of life, causes suffering and disability in many patients and is an important public health problem (Freynhagen et al., BMJ 2009; Haanpaa et al., Pain 2011).

[0025] Peripheral neuropathy is a functional disorder at the level of the peripheral nervous system. The nervous system consists of two components: the central nervous system, consisting of brain and spinal cord, and the peripheral nervous system, consisting of nerves connecting the central nervous system to the muscles, skin and internal organs. The peripheral nervous system is the part which, when damaged, causes neuropathy.

[0026] Peripheral neuropathy can be determined by various triggers: diseases such as some hematological tumors, HIV, diabetes and diphtheria, exposure to toxic substances or drugs or, furthermore, hereditary causes.

[0027] Peripheral iatrogenic neuropathy means neuropathy due to the intake of molecules (drugs), the side effects of which involve the onset of peripheral nervous system disorders. Some drugs for the treatment of tumors may have this effect. Symptoms of peripheral neuropathy may take a wide variety of forms and intensities. In general, they are divided into three groups: the motor, the sensory and the vegetative symptoms.

[0028] The motor-like symptoms that can occur can be very varied: they range from the sensation of snare in the finer movements of the fingers of the hands to the reduction of force in the legs, to the sensation of fatigue in making gestures, such as walking or climbing stairs. The motor problems may be related to one or more limbs; in the case where the difficulty is with the lower limbs, it is possible for example to experience even problems in keeping the station upright or in walking.

[0029] The signs and symptoms of the sensory type are extremely varied; they generally arise slowly, can have sporadic character and are often initially underestimated. Less frequently they have continuity characteristics and hardly maintain the same intensity throughout the day. Sensory symptoms may manifest as more or less intense pain, burning, tingling, numbness and more. The neurological signs are extremely varied; in fact, changes in reflexes, more or less marked force deficiencies, increase, reduction or absence of response to sensory stimuli (hyperesthesia, hypoesthesia or anesthesia, respectively), allodynia (abnormal response to stimuli, which should not normally cause pain) and hyperalgesia can occur (excessive response to mild painful stimuli).

[0030] The severity of iatrogenic neuropathy is generally related to the administered dose and the treatment time. Peripheral neuropathy from chemotherapy is often symptomatic, especially in patients who are treated with drug combinations. Symptoms may occur even weeks or months after the administration of the drug that determines it.

[0031] Recently, it has been found that statins for hypercholesterolemia also favor the appearance of peripheral neuropathy. Of course, we are talking about long-term medication intakes.

[0032] The treatment of peripheral neuropathy resulting from drug intake is one of the most important subjects of interest in recent clinical studies.

[0033] Peripheral neuropathy is the medical condition that results from the deterioration and malfunction of the nerves of the peripheral nervous system (SNP).

[0034] Connective tissue diseases, if affecting the connective tissue surrounding the peripheral nerves, may result in a form of peripheral neuropathy.

[0035] The dermis is the intermediate layer of the skin, comprised between the hypodermis and the epidermis. Unlike the latter, from which it is separated from the basal membrane, the dermis is richly vascularized and innervated.

[0036] The dermis performs functions of mechanical and metabolic support toward the epidermis, to which it transfers nutrients and sebum, an oleaginous substance that protects the skin surface layer from bacteria and dehydration. It has an undulated shape due to the presence of dermal papillae, extroflexions, which are intended to be inserted into the ridges present in the overlying epidermal layer. This particular anatomical conformation has the purpose of increasing the adhesion between the two layers and of favoring metabolic exchanges.

[0037] From the histological point of view, the dermis is a connective tissue formed by fibrous glycoproteins immersed in a fundamental substance.

[0038] The connective tissue has a wide variety of functions that depend on the types of cells and the different classes of fibers involved.

[0039] The connective tissue, formed mainly of fibroblasts and collagen fibers, has an important role in providing a means for oxygen and nutrients to diffuse from the capillaries to the cells and carbon dioxide and waste substances to diffuse from the circulating cells again. They also allow the organs to resist the stretching and tearing forces.

[0040] Connective tissue, which forms organized structures, is an important functional component of tendons, ligaments and aponeurosis.

[0041] Therefore, the dermis performs important metabolic, immunological, thermoregulatory and sensitive functions, as well as supporting functions.

[0042] Over time, sensory neuropathy may result in skin and hair changes, joint and bone damage.

[0043] As with all tissues, even nerves, when not adequately perfused, enter a state of suffering and develop damage.

[0044] In fact, neuropathies find their genesis in cases of malabsorption of nutrients, malnutrition or the presence of toxic substances. All factors that prevent healthy cell reproduction.

[0045] 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.

[0046] In particular, 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):

[0047] Polysaccharide of Tremella juciformis (10-20%),

[0048] Leaf extract of Vitis vinifera (5-15%),

[0049] Root extract of Astragalus membranaceus ( 10-30%),

[0050] Extract of Medicago sativa (8-15%),

[0051] Extract of Fucus vesiculosus (4- 15%),

[0052] Powder of Spirulia platensis (4-8%),

[0053] Root extract of Beta vulgaris (8-15%) and Extract of Polygonum cuspidatum (5-30%).

[0054] Said phytological composition has been described to have a regenerative, healing and healing 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 topical use in the treatment of inflammatory diseases not of the skin and of the chronic pains associated therewith.

[0055] DESCRIPTION OF THE INVENTION

[0056] The main object of the present invention is a phytological composition for topical use in the treatment of inflammatory diseases not of the skin and of the chronic pains associated therewith. 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):

[0057] 10-20% (w / w) polysaccharide of Tremella Fuciformis,

[0058] 5-15% (w / w) extract of leaves of Vitis Vinifera,

[0059] 10-30% (w / w) extract of leaves of Astragalus Membranaceus,

[0060] 8-15% (w / w) extract of Medicago Sativa,

[0061] 4-15% (w / w) extract of Fucus Vesiculosus,

[0062] 4-8% (w / w) powder of Spirulia Platensis,

[0063] 8-15% (w / w) extract of root of Beta Vulgaris and

[0064] 5-30% (w / w) extract of Polygonum Cuspidatum.

[0065] Preferably, the ingredients of the phytological composition of the invention are in the following concentration ranges by weight (w / w):

[0066] 12-16% (w / w) polysaccharide of Tremella Fuciformis,

[0067] 8-13% (w / w) extract of leaves of Vitis Vinifera,

[0068] 15-25% (w / w) extract of leaves of Astragalus Membranaceus,

[0069] 8-13% (w / w) extract of Medicago Sativa,

[0070] 8-15% (w / w) extract of Fucus Vesiculosus,

[0071] 4-6% (w / w) powder of Spirulia Platensis,

[0072] 8-13% (w / w) extract of root of Beta Vulgaris and

[0073] 15-25% (w / w) extract of Polygonum Cuspidatum. 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.

[0074] 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 carpoboro di Tremella.

[0075] The extract of Vitis vinifera leaves (CAS No. 84929-27-1) is an extract of red grape leaves (Vitis).

[0076] 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.

[0077] 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.

[0078] The Fucus vesiculosus extract (CAS No: 84696-13-9) is an extract of the dried thallum of the Bladderwrack, Fucus alga.

[0079] The Spirulia platensis powder (CAS No: 223751-80-2) is the powder obtained from the dried grinding preferably of the whole thallum ofArthrospira platensis, also called Spirulina platensis (spirulina).

[0080] 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.

[0081] "[\\Q Polygonum cuspidatum extracts (CAS No. 501-36-0) can be obtained by drying and titration of powders or by solvent extraction.

[0082] Vitis vinifera leaf extracts and Polygonum cuspidatum extracts preferably contain resveratrol.

[0083] 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 agentd, solvents, stabilizing agents, emulsifying stabilizing agents, toning agents, wetting agents, volumizing agents or combinations thereof.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] Preferably, the pharmaceutical formulation also comprises antioxidant and / or antimicrobial agents as active agents.

[0089] The invention therefore relates to the phytological composition described above for topical use in the treatment of inflammatory diseases not of the skin and of the chronic pain associated therewith, in particular in the treatment of arthritis, for example rheumatoid arthritis and osteoarthritis, and neuropathies. Furthermore, the phytological composition of the present invention is also suitable for topical use in the treatment of inflammatory states caused by diabetes and cancer and chronic pain associated therewith.

[0090] The formulation of said phytological composition with specific excipients and additional components further improves one or more of the aforesaid dermatological advantages of the phytological composition, so as to be able to provide formulations particularly suitable for the treatment of specific dermatological disorders or tissue lesions. Preferred formulations include the phytological composition together with one or more of silver, gold, copper, silicon (preferably in colloidal form), phenoxyethanol, 2-bromo-2-nitropropane-l,3-diol, vitamin D (preferably in colloidal form), panthenol and propylene glycol.

[0091] The pharmaceutical formulation comprising the phytological composition together with the following components is particularly suitable for the previously described use, preferably in the form of serum: methoxymethyl butanol, carbomer, propylene glycol,

[0092] PEG-4 proline linolenate, colloidal silver (preferably 110 ppm), colloidal vitamin D (preferably 500 ppm), colloidal iron (preferably 20 ppm), colloidal copper (preferably 20 ppm), colloidal echinacea (preferably 1,000 ppm), colloidal silicon (preferably 350 ppm), hyaluronic acid (preferably 1,200 ppm) and phenoxyethanol and 2-bromo-2-nitropropane- 1,3 -diol.

[0093] 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. Particularly preferred are the formulations in the form of serum.

[0094] Preferably, said pharmaceutical formulations are to be administered topically, on the skin, from one to four times a day, in a suitable quantity. A suitable quantity is for example an amount that can be absorbed by massaging the formulation into the skin.

[0095] The entire pharmaceutical formulation can be prepared by methods known in the pharmaceutical art.

[0096] It is further to be understood that all 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.

[0097] The following are operational examples of the present invention provided for illustrative purposes. EXAMPLES

[0098] Example 1

[0099] A phytological composition was prepared by mixing the following ingredients:

[0100] Example 2 A pharmaceutical formulation was prepared by mixing the phytological composition of

[0101] Example 1, at a final concentration of 3% by weight based on the weight of the formulation

[0102] (w / w), with the following components, in water:

[0103] Example 3 - Evaluation of the in vitro antipain activity on chondrocytes

[0104] To evaluate the antipain activity of the pharmaceutical formulation prepared in Example 2 (also referred to hereinafter as the Antipain Formulation) immortalized human juvenile chondrocyte cell lines (TC28a2 purchased from the American Type Culture Collection [ATCC, Manassas, USA]) were used. Cell lines were cultured in DMEM / nutrient F-12 Ham's medium (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (FBS), 2mM L- glutamine and antibiotics (1% penicillin-streptomycin, i.e. 50 IU penicillin and 50 pg / mL streptavidin) and maintained in a 5% CO2 and 95% humidity incubator. This cell line is representative and is the most commonly used cell line for imitating joints.

[0105] For the experiments, l *104cells were plated in 96-well plates to study cell viability by MTT, ROS production and Crystal Violet; in addition, 1 * 106cells were plated on 6 wells to determine to determine ERK / MAPK activity, pro-inflammatory cytokines using a specific ELISA kit and analyze apoptotic molecular mechanisms, cartilage degradation and cartilage repair mechanisms by other specific ELISA kits.

[0106] On the day following sowing, the cells were placed in a serum-free white medium and, to mimic OA (osteoarthritis) conditions, pretreated with 10 pg / mL LPS (lipopolysaccharide, Merck Life Science, Rome, Italy) for 24 hours. At the end of the 24-hour incubation, TC28a2 cells were stimulated with different concentrations of the Antipain Formulation for 3 days.

[0107] In all the experiments reported below the results referred to the "control" refer to the untreated cells. Materials and apparatus used

[0108] -Dulbecco's phosphate Buffered Saline diluted IX (PBS IX) with sterile water (Eurospital, Trieste, Italy).

[0109] - Trypsin solution - EDTA 10X (sterilized 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 PBSIX for cell rupture.

[0110] - MTT [(3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide]

[0111] - Crystal-Violet dye

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

[0113] - SOD (superoxide dismutase) from bovine erythrocytes

[0114] - BCA assay kit (Thermofisher, Massachusetts, InstantUSA)

[0115] - LPS 10 pg / mL (lipopolysaccharide)

[0116] - ERK / MAPK ELISA Kit (InstantOne™ ELISA, Thermofisher, Massachusetts, USA)

[0117] - BAX ELISA Kit (MyBioSource, San Diego, CA, USA) 10

[0118] - Cytochrome C ELISA Kit (MyBioSource, San Diego, CA, USA)

[0119] - TNFa and IL-ip ELISA Kit (Thermo Fisher, Milan, Italy and R&D Systems, Minneapolis, MN, USA)

[0120] - MMP-1 ELISA Kit (Thermofisher, Massachusetts, USA) - ADAMTS-5 ELISA Kit (R&D Systems, Minneapolis, MN, USA) 15

[0121] - Aggrecan ELISA Kit (Thermofisher, Massachusetts, USA)

[0122] - Collagen Type II ELISA Kit (Excellent Test, Wuha

[0123] - Spectrophotometer: Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland

[0124] Culture media and reagents were purchased from Merck Life Science, Rome, Italy, unless otherwise indicated.

[0125] MTT Test

[0126] 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).

[0127] 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.

[0128] 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.

[0129] The results of the MTT test after 3 days of treatment on A375 are shown in Table 1 below.

[0130] Table 1 - Cell viability results in %

[0131] On the basis of initial results, it has been seen that the above three concentrations compensated for the cytotoxic effect of LPS on the A375 chondrocyte cells.

[0132] ROS production

[0133] This test is a semi-quantitative evaluation and measures the reactive oxygen species (ROS) produced by reduction of cytochrome C.

[0134] In particular, ROS production Test is a quantitative colorimetric test using reduction of cytochrome C by superoxide dismutase (SOD). Briefly, 30 minutes before the end of each stimulus, 10 pL of cytochrome C was added to all samples (including control) and simultaneously, in other wells, 0.1 mg / ml SOD was added (to detect the maximum production limit). The plate was incubated at 37°C in the dark for 30 min. At the end of this incubation, 100 pl was taken from each well and placed in a new plate for spectrophotometric reading. The results are expressed as mean ± SD % by moles of reduced cytochrome C for pg of sample protein (measured by the BCA assay kit) with respect to the control sample.

[0135] The results related to the production of ROS after 3 days from the treatment on the cells (TC28a2) are reported in Table 2 below.

[0136] Table 2 - ROS production in %

[0137] All tested concentrations showed a restoration of low oxidative stress or an absent oxidative stress following treatment with the Antipain Formulation. Cell proliferation by staining with Crystal Violet

[0138] Cells (TC28a2) were fixed with 1% glutaraldehyde (Merck Life Science, Rome, Italy) for 15 minutes at room temperature, washed and stained with 100 pL of 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).

[0139] The results were expressed as a percentage (%) with respect to the control. The data are presented as an average of five independent experiments.

[0140] The results of the analysis of the proliferation rate with Crystal Violet after 3 days of treatment on TC28a2 cells are reported in Table 3 below.

[0141] Table 3 - Proliferation rate %

[0142] All tested concentrations showed a significant increase in cell proliferation rate compared to LPS -treated cells.

[0143] Cell migration via ERK / MAPK

[0144] The ERK / MAPK activity was measured by InstantOneTM ELISA on TC28a2 cell lysates following the manufacturer's instructions. Briefly, 50 pL / well of TC28a2 lysed with cell lysis buffer were tested in InstantOne ELISA microplates strips after 1 hour at room temperature on a microplate stirrer with the antibody cocktail. At the end, the detection reagent was added for 20 minutes and then stopped by adding the stopping solution. The strips were measured by a spectrometer at 450 nm. The results were expressed as mean absorbance (%) with respect to the control and are indicated as an average of five independent experiments.

[0145] ERK (extracellular signal -regulated Kinase) is a Map kinase (Mitogen activated protein Kinase). MAPK pathway plays an important role in normal cells. In fact, when induced under physiological conditions, it starts to cascade phosphorylation processes of the different kinases, which ends with cell growth and differentiation.

[0146] The data are represented as an average of five independent experiments and as an increase percentage (%) compared to the control.

[0147] The results relating to the expression of ERK after 3 days of treatment on TC28a2 cells are reported in Table 4 below.

[0148] Table 4 - ERK expression %

[0149] All tested concentrations showed a significant increase in ERK expression compared to LPS treated cells.

[0150] BAX Test

[0151] According to the manufacturer's instructions, the BAX ELISA kit was used to analyze the BAX protein in TC28a2 cell lysates. 100 pL of sample or standard were added to each well, followed by incubation at 37°C for 90 min. To each well, 100 pL of biotinylated antibody was added, followed by incubation for 60 min at 37°C and cleaning, then 100 pL of SABC (Streptavidin- Biotin Complex) working solution was added for 30 min at 37°C. Subsequently, 90 pL of TMB (3,3',5,5'-Tetramethylbenzidine) substrate was added, followed by incubation at 37°C for 15- 30 min. The samples were analyzed at 450 nm by a spectrometer. The results are reported as pg / mL with respect to a standard curve (0-2,000 pg / mL) and as mean ± SD (%) with respect to the control cells (0% line).

[0152] The results were expressed as mean absorbance (%) with respect to the control and are indicated as an average of five independent experiments.

[0153] BAX (BCL2-associated-X) is a regulatory protein of cellular apoptosis. The results relating to BAX levels after 3 days of treatment on TC28a2 cells are reported in Table 5 below.

[0154] Table 5 - BAX levels %

[0155] All tested concentrations showed a significant decrease in BAX levels with respect to both LPS- treated and control-treated cells.

[0156] Cytochrome C assay

[0157] The Cytochrome-C ELISA kit was used to measure the amount of cytochrome C in TC28a2 cell lysates, following the manufacturer's instructions. An equal volume of detection solution A was added to 100 pl of each sample, followed by incubation for 45 min. at 37°C. Then, in the wells, after washing, 100 pL of detection solution B was added to the incubation wells for 45 min at 37°C. The plate was incubated at 37°C for 20 min in the dark after adding 90 pL of substrate solution to each well. A total of 50 pL of stopping solution was used to stop the reaction. The absorbance was analyzed with a spectrometer at 450 nm and the concentration was quantified in ng / mL by comparing the data with the standard curve (15.6-500 nmol / L). Cytochrome C levels are an index of the correct mechanisms of cellular respiration.

[0158] The data are presented as an average of five independent experiments and as a % percentage of the control cells.

[0159] The results relating to cytochrome C levels after 3 days of treatment on TC28a2 cells are reported in Table 6 below.

[0160] Table 6 - Cytochrome C Levels %

[0161] All tested concentrations showed a significant decrease in cytochrome C levels compared to both LPS-treated and control -treated cells.

[0162] Production of TNFa

[0163] According to the manufacturer's guidelines, an analysis of the production of TNFa (tumor Necrosis Factor-alpha) on the TC28a2 cell supernatant was carried out using the human Tumor Necrosis Factor a ELISA kit.

[0164] 100 pl of the sample was distributed in individual wells of a 96-well ELISA plate, followed by an incubation period at room temperature of 2 hours, then overnight at 4°C. This was followed by five consecutive washings using a post-incubation wash buffer and adding 100 TNFa of biotinylated anti- 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.

[0165] Subsequently, the streptavidin HRP solution plate, 100 pL of chromogenic solution, was incubated in each well for 30 min at room temperature without light. The absorbance of each well was then determined at 450 nm using a Tecan plate reader after applying the stop solution. TNFa is also an agent capable of inducing cellular apoptosis.

[0166] The data are presented as an average of five independent experiments and as a percentage (%) of the untreated control sample.

[0167] The results related to the production of TNFa after 3 days of treatment on TC28a2 cells are reported in Table 7 below.

[0168] Table 7 - TNFa levels %

[0169] All tested concentrations showed a significant decrease in TNFa production compared to both LPS-treated and control -treated cells.

[0170] Production of IL- lb

[0171] IL-ip (interleukin 1-beta) in TC28a2 cell lysates was quantified following manufacturer's instructions using an IL-ip ELISA kit. The procedure included the addition of a solution containing IL-ip to a strip of microplates (100 pL / well) incubated for 2 hours at room temperature, mixing with the IL-ip conjugate and further incubation for a further 2 hours at room temperature. Thorough washing steps were performed before and after the two incubations. Thereafter, 100 pL of substrate solution was added to generate chemiluminescence and the absorbance was measured using a 450 nm microplate reader with 570 nm correction. Quantitation of IL-ip compared the sample readings with the generated standard curve.

[0172] IL-ip is a pro-inflammatory cytokine.

[0173] The data are presented as an average of five independent experiments and as a percentage (%) of the untreated control sample.

[0174] The results related to the production of IL-ip after 3 days of treatment on TC28a2 cells are reported in Table 8 below.

[0175] Table 8 - IL-1B levels %

[0176] All tested concentrations showed a significant decrease in IL-ip production compared to both LPS-treated and control -treated cells.

[0177] MMP-1 levels

[0178] The human MMP1 ELISA kit quantified MMP1 (Matrix Metalloproteinase- 1) in TC28a2 cell supernatants following the manufacturer's instructions.

[0179] Each well received a biotinylated detection antibody and the plate was incubated at room temperature for 1 hour. After 45 minutes incubation with HRP-conjugated streptavidin, the TMB substrate solution was added for 30 minutes and a stopping solution terminated the reaction. To calculate the MMP-1 concentration, the absorbance was measured at 450 nm using a spectrometer and compared with the standard curve (0.066 to 16 ng / mL).

[0180] MMPs locally degrade the extracellular matrix allowing cells to pass therethrough. This mechanism is important for cells such as leukocytes to invade tissues damaged by inflammatory processes.

[0181] The data are presented as an average of five independent experiments and as a percentage (%) with respect to the control sample.

[0182] The results relating to the levels of MMP1 after 3 days of treatment on TC28a2 cells are reported in Table 9 below.

[0183] Table 9 - MMP1 levels %

[0184] All tested concentrations showed a significant decrease in levels of MMP 1 compared to both LPS-treated and control -treated cells.

[0185] Levels of AD AMTS-5

[0186] Human ADAMTS5 DuoSet ELISA quantified ADAMTS5 in TC28a2 cells supernatants following manufacturer's instructions. The procedure included the addition of culture medium containing ADAMTS5 to a microplates strip (100 pL / well), incubation for 2 hours at room temperature, mixing with ADAMTS5 conjugate and further incubation for a further 2 hours at room temperature. Thorough washing steps were performed before and after the two incubations. Thereafter, 100 pL of detection antibody was added and, after a 2 hour incubation, the plate was washed. Then, 100 pL of the streptavidin-HRP working dilution in each well and the plate was incubated for 20 minutes at room temperature in the dark. At the end of incubation, 100 pL of substrate solution in each well and the plate was incubated for 20 minutes at room temperature; then, 50 pL of stopping solution in each well and the plate was read at 450 nm using a spectrometer and compared with the standard curve (125 - 8,000 pg / mL).

[0187] The data are presented as an average of five independent experiments and as a percentage (%) with respect to the control sample.

[0188] The results relating to the levels of ADAMTS5 after 3 days of treatment on TC28a2 cells are reported in Table 10 below.

[0189] Table 10 - ADAMTS-5% levels

[0190] All tested concentrations showed a significant decrease in levels of ADAMTS5 compared to both LPS-treated and control -treated cells.

[0191] Aggrecan Test (PG protein)

[0192] According to the manufacturer's guidelines, an analysis of the PG protein on the TC28a2 cells supernatant was carried out using the Human Aggrecan (PG) ELISA kit. Subsequently, 50 pl of the sample and 100 pL of incubation buffer (in the appropriate wells provided for standards, controls and samples) were distributed into individual wells of a 96-well ELISA plate, followed by an incubation period at room temperature of 2 hours.

[0193] This was followed by five consecutive washings using a post-incubation wash buffer and adding 200 pL of anti-PG conjugate in each well. After an incubation of 1 hour at room temperature in a horizontal stirrer at 700 rpm, the content of each well was aspirated and washed five times before introducing 100 pL of chromogenic solution for a 15 minutes incubation in a horizontal stirrer at 700 rpm. Subsequently, the chromogenic solution plate, 200 pL of stopping solution was added in each well and the absorbance of each well was then determined at 450 nm and 490 nm using a Tecan plate reader.

[0194] The aggrecan, also known as the Cartilage-specific Proteoglycan Core protein (CPCP), is a proteoglycan (PG). Its core (core or "protein axis") is a large protein, to which long lines of glycosaminoglycans such as chondroitin sulfate and keratan sulfate are attached, to form a structure similar to a spike-shaped inflorescence. Proteoglycans are natural macromolecules capable of restoring epidermal cells and enhancing the metabolism of connective tissue components, restoring skin functions, mechanical properties and physiological appearance.

[0195] The data are presented as an average of five independent experiments and as a percentage (%) with respect to the control sample with respect to the standard curve (10-250 ng / mL).

[0196] The results relating to the PG protein levels after 3 days of treatment on TC28a2 cells are reported in Table 11 below.

[0197] Table 11 - PG Protein Levels %

[0198] All tested concentrations showed a significant increase in PG protein levels compared to both LPS-treated and control -treated cells.

[0199] Type II collagen production

[0200] According to the instructions, the Human COL2 ELISA kit was applied to verify the production of Type II collagen. After adding 100 pL of each sample to each well, the plate was incubated at 37°C for 90 min. After incubation, each well was cleaned twice with a wash buffer. The plate was incubated at 37°C for 60 min after adding 100 pL of biotin-labeled antibody solution to the wells. After incubation, each well was washed three times with wash buffer to remove the solution. After adding 100 pL HRP-streptavidin conjugate to each well, the plate was incubated at 37°C for 30 min. After washing the wells five times, 90 pl of TMB substrate was added to each well. After 10-20 min, 50 Mannedorf of stop solution was added to each well and the plate was read at 450 nm using a Tecan plate reader (Infinite 200 Pro MPlex, Tecan, Switzerland).

[0201] A curve was drawn to correlate the intensity of color (OD) with the standard concentration (46.875-3000 Pg / mL).

[0202] The results were presented as an average of five independant experiments and as a percentage (%) compared to the control.

[0203] The results related to the production of collagen II after 3 days of treatment on TC28a2 cells are reported in Table 12 below.

[0204] Table 12 - Collagen II Production %

[0205] All tested concentrations showed a significant increase in the production of collagen II both with respect to LPS-treated cells and with respect to control. From the set of data reported in this Example 3, it can be seen that the Anti-Pain Formulation showed clear anti-inflammatory, antioxidant properties and favored the synthesis of regenerating proteins and antioxidants. All these properties are very important for topical use in the treatment of inflammatory diseases not of the skin and of the chronic pain associated therewith, in particular to the treatment of arthritis, neuropathies, but also of the inflammatory states associated with diabetes and cancer.

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 and- 5-30% (w / w) extract of Polygonum cuspidatum for topical use in the treatment of non-skin inflammatory diseases and associated chronic pain, particularly in the treatment of arthritis, such as rheumatoid arthritis and osteoarthritis, and neuropathies.

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 and- 15-25% (w / w) extract of Polygonum cuspidatum for topical use in the treatment of non-skin inflammatory diseases and associated chronic pain, particularly in the treatment of arthritis, such as rheumatoid arthritis and osteoarthritis, and neuropathies.

3. The composition according to claim 1 or 2, wherein the non-skin inflammatory diseases are inflammatory states associated with diabetes and cancer.

4. A pharmaceutical formulation comprising the phytological composition consisting of:- 10-20% (w / w) polysaccharide di 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 di Beta vulgaris and- 5-30% (w / w) extract of Polygonum cuspidatum together with the following components: colloidal silver, colloidal copper, colloidal vitamin D, colloidal silicon,3 -methyl-3 -methoxybutanol, phenoxyethanol,2-bromo-2-nitro- 1 ,3 -propanediol, carbomer, propylene glycol, hyaluronic acid, colloidal Echinacea andPEG-4 Proline linolenate.

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

6. The pharmaceutical formulation of anyone of claim 4 or 5, 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.

7. The pharmaceutical formulation according to anyone of claims from 4 to 6, for topical use in the treatment of non-skin inflammatory diseases and associated chronic pain, particularly in the treatment of arthritis, such as rheumatoid arthritis and osteoarthritis, and neuropathies.

8. The formulation according to la claim 7, wherein the non-skin inflammatory diseases are inflammatory states associated with diabetes and cancer.

Images