Multicomponent mixtures and compositions containing palmitoylethanolamide and propolis extract for use in the treatment of tinnitus and peripheral neuropathies

Abstract

The present invention relates to multicomponent mixtures, to compositions containing such mixtures, and to the use thereof in the treatment of tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology). In greater detail, the present invention relates to multicomponent mixtures comprising or, alternatively, consisting of palmitoylethanolamide (PEA) and at least one ingredient selected from the group comprising or, alternatively, consisting of propolis extract (preferably with an artepillin-C titer equal to or greater than 4.0%), grape seed extract, resveratrol or mixtures thereof and, optionally, one or more of hop extract; Ginkgo Biloba extract; B group vitamins (preferably vitamin B6 and / or B12), or mixtures thereof. Furthermore, the present invention relates to compositions comprising said multicomponent mixtures. Furthermore, the present invention relates to said multicomponent mixtures and / or compositions comprising them for use in the treatment, preventive and / or curative, of tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).

Description

MULTICOMPONENT MIXTURES AND COMPOSITIONS CONTAINING PALMITOYLETHANOLAMIDEAND PROPOLIS EXTRACT FOR USE IN THE TREATMENT OF TINNITUS AND PERIPHERAL NEUROPATHIES***DESCRIPTION5 The present invention relates to mixtures, to compositions containing such mixtures, and to the use thereof in the treatment of tinnitus and peripheral neuropathies.In greater detail, the present invention relates to mixtures, to compositions containing such mixtures, and to the use thereof in the treatment of tinnitus and peripheral neuropathies, such mixtures comprising or, alternatively, consisting of palmitoylethanolamide (PEA) and at least one ingredient selected from the group comprising or,0 alternatively, consisting of a propolis extract, preferably titrated in artepillin-C, a grape seed extract, resveratrol and / or mixtures thereof.Furthermore, the present invention relates to mixtures, to compositions containing such mixtures, and to the use thereof in the treatment of tinnitus and peripheral neuropathies, such mixtures comprising or, alternatively, consisting of palmitoylethanolamide (PEA) and at least one ingredient selected from the group comprising or, alternatively, consisting of a propolis extract, preferably titrated in artepillin-C, a grape seed extract, resveratrol and / or mixtures thereof and, optionally, one or more of a hop extract; a Ginkgo Biloba extract; B group vitamins, preferably vitamin B6 and / or B12, or mixtures thereof.Finally, the present invention relates to said mixtures and / or compositions comprising them for use in the treatment, preventive and / or curative, of diseases and / or dysfunctions related to nerve malfunction and / or damage in the0 peripheral nervous system, particularly tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).BACKGROUND OF THE INVENTIONTinnitus, defined as a subjective perception of noise in the absence of an external sound, is a common hearing5 condition that affects between 10 and 15% of the adult population.The prevalence of tinnitus increases in the elderly, reaching 40%. Furthermore, about 10% of the individuals with tinnitus develop disabilities ranging from mild to severe.Tinnitus is typically associated with hearing loss, cognitive dysfunctions, depression, anxiety and sleep disorders. Therefore, the burden of tinnitus for society is substantial, reflecting both the negative impact on the quality of life0 (QoL) of patients and the high social costs due to the loss of productivity, workdays lost and expenses for treatments.Peripheral neuropathies and tinnitus seem to have a common neurological basis; in fact, from recent studies it has emerged that these disorders derive from the reorganization of the brain following damage in the sensory apparatus thereof due to a chronic inflammatory and oxidative state. 5 Document EP2475352 discloses a combination of PEA + resveratrol for oral use in capsules. EP2475352 discloses the use of PEA + resveratrol for use in neuroimmunogenic inflammation processes (page 14,

[0106] , subpar. 1.),indicating a very long list of diseases and using very generic terminology, but in this very extensive list of diseases tinnitus, craniofacial neuralgia and trigeminal neuralgia, among others, for example, are not mentioned.Document EP1844784 discloses a combination of PEA + resveratrol for oral use in tablets. EP1844784 discloses the use of PEA + resveratrol for numerous autonomic and somatic neuropathies and disorders characterized and / or sustained by a neuroimmunogenic inflammation. On page 2, paragraph

[0012] and in claim 19 of EP1844784 a long list of diseases and disorders is given. However, tinnitus, craniofacial neuralgia and trigeminal neuralgia, among others, for example, are not mentioned.Document EP 1417968 discloses a combination of resveratrol and propolis for the treatment of tumors, nothing to do with the treatment of tinnitus.The article titled “Can Propolis be a useful adjuvant...; S. Tandean et al.," discloses the use of propolis extract to treat brain and neurological damages such as, for example, Alzheimer's, ischemia, neuroinflammation and pain and oxidative stress, Parkinson's, etc. (Table 1). However, the article does not teach to repair damage to the nerves of the nervous system or to improve the myelination process.Document JP2005060242A discloses the use of a propolis extract for the treatment of a dementia-related neuropathy or a peripheral neuropathy derived from diabetes. Therefore, JP2005060242A does not teach to repair damage to the nerves of the nervous system or to improve the myelination process.However, the still incomplete understanding of the mechanisms underlying the development of tinnitus hinders the identification of effective treatments.Today there still does not exist any treatment for tinnitus, even though numerous therapeutic approaches have been tried out with variable success, most of which aim to reduce the stress related to tinnitus, without offering completely satisfactory solutions.In particular, there remains a great need to provide a solution to effectively prevent and / or treat tinnitus and / or peripheral neuropathies.Furthermore, there remains a great need to provide novel and effective solutions capable of preventing and / or treating tinnitus and peripheral neuropathies and which do not have side effects, are well tolerated by the treated subjects and are also easy to administer.The technical problem that the present invention addresses and solves is thus to provide a valid solution for effectively treating, in a preventive and / or curative manner, diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, in particular tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology), in the absence of or with reduced side effects.The Applicant, by virtue of an intense research activity, has developed and fine-tuned mixtures and compositions containing them (briefly, the mixtures and the compositions of the invention), which are effective, devoid of side effects and with high tolerability; preferably, said mixtures and compositions having been developed for use in a method of treatment, preventive and / or curative, of tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy,hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).In particular, the mixtures and / or compositions containing them of the invention, thanks to the presence of natural ingredients, in safety (i.e. without side effects and / or with high tolerability), surprisingly slow nerve malfunction and / or damage in the peripheral nervous system and have shown to be useful for treating and / or preventing tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).SUMMARY OF THE INVENTIONIn a first aspect, the present invention relates to a mixture comprising or, alternatively, consisting of: I) palmitoylethanolamide (PEA) and II) at least one ingredient selected from the group comprising or, alternatively, consisting of a propolis extract, preferably titrated in artepillin-C, a grape seed extract, resveratrol and / or mixtures thereof. Preferably, said propolis extract contains artepillin-C and has, even more preferably, an artepillin-Ctiter comprised from 2% to 20%, more preferably comprised from 5% to 15%, even more preferably comprised from 8% to 12%, for example, 4%, or 5%, or 6%, or 10%, by weight relative to the weight of the extract. This mixture of the invention is called MIX M1.Preferably, a type of palmitoylethanolamide (PEA) that can be used as component I) is a PEA having a particlesize distribution comprised from 20 to 200 mesh, preferably from 60 to 150 mesh, even more preferably from 80 to 120 mesh.Preferably, a type of palmitoylethanolamide (PEA) that can be used as component I) is a PEA having a particlesize distribution comprised from 20 to 80 mesh. For example, a PEA having CAS No. 544-31-0 and the molecular formula C18H37NO2 can be used."Mesh” numbers (from mesh materials) are Anglo-Saxon units of measurement corresponding to the number of openings per linear inch, generally used to determine the particle-size distribution of granular materials. In particular, the particle size can be defined by means of two parameters which are equivalent to each other: the mean diameter of the particles or the mesh number. The mesh number is the Anglo-Saxon unit of measurement corresponding to the number of openings per linear inch (1 inch = 25.40 mm), of the sort of sieve formed when the particles are set side by side above a surface. This means that the higher the mesh number is, the higher the filtering power will be. Exactly the opposite of microns. In fact, a 200 mesh filter filters more than a 100 mesh one, whereas a 100 micron filter filters more than a 200 micron one.Preferably, a type of propolis extract that can be used as component II), in combination with component I), is a lyophilized propolis extract titrated in artepillin-C, more preferably said extract has an artepillin-C titer comprised from 2% to 20%, more preferably comprised from 5% to 15%, even more preferably comprised from 8% to 12%, for example 4%, or 5%, or 6% or 8%, or 10% relative to the weight of the extract (measured with the HPLC method) and / or has a content of phenolic compounds comprised from 50 mg / g to 500 mg / g, preferably comprised from 100 mg / g to 400 mg / g, even more preferably comprised from 150 mg / g to 250 mg / g (measured with aspectrophotometry method).Preferably, a type of grape seed extract that can be used as component ii), in combination with component i), is a hydroalcoholic extract of grape seeds (VWs vinifera L) with an OPC (oligomeric proanthocyanidins or procyanidins) titer equal to or greater than 60% and a resveratrol titer equal to or greater than 10%. For example, said grape seed extract that can be used as component ii), in combination with component i), can be in the form of a powder with a bulk density (g / ml) comprised from 0.10 to 0.9, preferably comprised from 0.25 to 0.65 (USP<616> method); a particle size where 100% passes through 80 mesh (USP<786> method); a resveratrol content greater than 5% (HPLC method), preferably comprised from 10% to 40%, even more preferably comprised from 15% to 25%.Preferably, a type of resveratrol that can be used as component ii), in combination with component i), can be in powder form, with a resveratrol titer comprised from 70% to 99% (HPLC method), preferably comprised from 85% to 98% by weight, having a particle size where a minimum of 95% passes through 80 mesh (80 mesh screen method); a bulk density comprised from 0.1 g / ml to 0.9 g / ml, preferably comprised from 0.3 g / ml to 0.6 g / ml (CP<2020> method).In a further aspect, the present invention provides a mixture comprising or, alternatively, consisting of i) palmitoylethanolamide (PEA) and ii) at least one ingredient selected from the group comprising or, alternatively, consisting of propolis extract (preferably with an artepil lin-C titer equal to or greater than 4.0%), grape seed extract, resveratrol or mixtures thereof and which further includes iii) one or more ingredients selected from the group comprising or, alternatively, consisting of: hop extract; Ginkgo Biloba extract; B group vitamins (preferably vitamin B6 and / or B12), and / or mixtures thereof. This mixture of the invention is called MIX M2.Preferably, a type of hop extract (for example of the species Humulus lupulus L.) that can be used as component iii), in combination with said components i) and / or ii), is a dry extract titrated in flavonoids; said extract preferably has a flavonoid titer comprised from 0.1 % to 5%, more preferably comprised from 0.4% to 2.5%, even more preferably comprised from 0.8% to 1 .5%. Preferably, said extract is an extract that is obtained by extraction with solvents, for example, with an 80 / 20 volume / volume water / ethanol solution. Preferably, said hop extract is in the presence of an excipient such as corn-derived maltodextrin (20%-40%).Preferably, a type of Ginkgo Biloba extract that can be used as component iii), in combination with said components i) and / or ii), is an extract, for example a dry extract, of Ginkgo Biloba (for example of the species Ginkgo Biloba L.) that is obtained by extraction with solvents, for example, with a water / ethanol solution, for example with an 80 / 20 volume / volume water / ethanol solution.Preferably, said Ginkgo Biloba extract that can be used as component iii), in combination with said components i) and / or ii), is an extract which can have a content of Ginkgo flavone glycosides comprised from 15% to 60%, more preferably from 25% to 50%, even more preferably comprised from 30% to 40% (measured with the HPLC method) and / or a content of terpene lactones comprised from 2% to 30%, more preferably comprised from 6% to 20%, even more preferably comprised from 8% to 15% (measured with the HPLC method) and / or a content of ginkgolic acid comprised from 0.1 ppm to 5 ppm, preferably comprised from 1 ppm to 3 ppm (measured with the HPLC method). Preferably, said Ginkgo Biloba extract can further have a particle size where a minimum of 95% passes through an80 mesh screen and / or a bulk density comprised from 0.3 g / ml to 0.6 g / ml (CP<2020> and / or a loss and drying of 5% at most (5 g / 105°C / 2 hours)).Preferably, the B group vitamin that can be used as component iii), in combination with said components i) and / or ii), is preferably selected from vitamin B6 and / or B12.In a further aspect, the present invention provides a composition (composition of the invention) comprising one of the multicomponent mixtures of the invention described above (MIX M1 , MIX M2) and, optionally, an acceptable pharmaceutical and / or food grade additive and / or excipient.In a further aspect, the present invention provides a composition (composition of the invention) as defined above for use as a medicament.In a further aspect, the present invention provides multicomponent mixtures and / or compositions containing them (mixtures and / or composition of the invention) as defined above for use in the treatment, preventive and / or curative, of diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, in particular tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).The Applicant has found that the multicomponent mixtures and / or compositions containing them (mixtures and / or composition of the invention) as defined above are effective in a method of treatment, preventive and / or curative, of diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, in particular tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).Said treatment activity, preventive and / or curative, of the mixtures and / or compositions of the invention is due to the specific innovative combination of the active components (as defined in the present detailed description that follows), which imparts thereto a synergistic action / activity capable of:- increasing intestinal permeability to promote the passage thereof; and / or- correctly modulating the biological activity of Schwann cells; and / or- slowing malfunction and repairing nerve damage at the peripheral level, suggesting better effectiveness with positive effects on the whole system of regulation; and / or- inhibiting the main molecular pathways involved in demyelination, thus tending towards a correct homeostasis of neuronal cells; and / or- inhibiting the production of radicals, reducing inflammation and maintaining cell integrity, while demonstrating their safety and biological effectiveness, thus having useful application for treating, in a preventive and / or curative manner, diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, in particular tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).Furthermore, the multicomponent mixtures and / or compositions containing them (mixtures and / or composition of the invention), thanks to the presence of natural ingredients, do not have any significant adverse effects, are devoid of side effects and have high tolerability, and can be administered to a broad category of subjects.Finally, the mixtures and / or the compositions of the invention are easy to prepare and economical.These and other aims, which will become apparent from the detailed description that follows, are achieved by the multicomponent mixtures and / or compositions containing them (mixtures and / or composition of the invention) thanks to the technical characteristics claimed in the appended claims.DESCRIPTION OF THE FIGURESFigures 1A and 1 B show the results of a dose-response study of cell viability on Caco-2 cells. The results of the MTT assay are expressed as the mean ± SD (%) of 4 biological replicates, normalized versus the control. The highest curves show the best concentrations for each tested substance.Figure 2 shows the results of an analysis of viability (at the top) and of the production of oxygen radicals (at the bottom). The results of the test are expressed as the mean ± SD (%) of 4 biological replicates, normalized versus the control. The curve of MIX A (cell viability) is the highest in the first graph, whereas the one of MIX A (ROS) is the lowest in the second graph.Figure 3 shows the results of an analysis of transepithelial electrical resistance (TEER, on the left) and of permeability (Papp, on the right). The results of the test are expressed as the mean ± SD (%) of 4 biological replicates, normalized versus the control.Figure 4 shows the results of an analysis of TJ activity (Zo-1 , claudin 4, occludin). The test results are expressed as the mean ± SD (%) of 4 biological replicates, normalized versus the control. *p<0.05 vs control; #p<0.05 vs single substances.Figure 5 shows the results of an evaluation of effectiveness at the peripheral nerve level in 3D (viability, ROS production, TNF-alpha). The results are expressed as the mean ± SD (%) of 4 biological replicates, normalized versus the control. * p<0.05 vs control; # vs single substances; y p<0.05 vs GGF.Figure 6 shows the results of an evaluation of the main markers involved in peripheral nerve injury (PNI). The results are expressed as the mean ± SD (%) of 4 biological replicates, normalized versus the control. * p<0.05 vs control; # vs single substances; y p<0.05 vs GGF.Figure 7 schematically illustrates an example of a model of the gut-PNS axis, created using the Transwell® technique, where the apical portion of the insert was seeded with intestinal cells, which matured, producing microvilli; whilst a 3D reconstruction of a peripheral nerve was created in the basolateral part; it contained aligned Schwann cells encapsulated in a matrix allowing the anchorage of cells and neurons on the surface.DETAILED DESCRIPTION OF THE INVENTIONThe object of the present invention is a mixture comprising or, alternatively, consisting of: I) palmitoylethanolamide (PEA), and II) at least one propolis extract.Preferably, said mixture can, in addition, comprise at least one ingredient selected from the group comprising or, alternatively, consisting of a grape seed extract, a resveratrol and / or mixtures thereof.Furthermore, another object of the present invention is a multicomponent mixture comprising or, alternatively, consisting of:I) palmitoylethanolamide (PEA) and ii) at least one ingredient selected from the group comprising or, alternatively, consisting of propolis extract, preferably with an artepillin-C titer equal to or greater than 4.0%, grape seed extract, resveratrol or mixtures thereof.This mixture of the invention is called MIX M1 .The mixture MIX M1 of the present invention comprises I) palmitoylethanolamide (PEA).Palmitoylethanolamide or N-(2-hydroxyethyl)-hexadecanamide (herein also only "PEA”), is the ethanolamide derivative of hexadecenoic acid which is present in nature and is found in particularly abundant amounts in some foods, including egg yolks and soybeans. At room temperature and pressure, it appears as a solid and is used in therapy mainly as an anti-inflammatory, neuroprotective and pain-relieving agent. PEA is generally considered safe and devoid of adverse drug reactions (ADRs) or pharmacological interactions.In the context of the present invention, "palmitoylethanolamide'' or "PEA” is intended to refer to, and include, a palmitoylethanolamide and / or a derivative thereof from any source and in any solid physical form (for example, in the form of flakes or micronized).Preferably, a type of palmitoylethanolamide (PEA) that can be used as component I) is a PEA having a particlesize distribution comprised from 20 to 200 mesh, preferably from 60 to 150 mesh, even more preferably from 80 to 120 mesh.Preferably, a type of palmitoylethanolamide (PEA) that can be used as component I) is a PEA having a particlesize distribution comprised from 20 to 80 mesh. For example, a PEA having CAS No. 544-31-0 and the molecular formula C18H37NO2 can be used. In one embodiment, the palmitoylethanolamide is preferably in the form of PEA 80 mesh.Preferably, the palmitoylethanolamide (PEA) is present in the mixture of the invention called MIX M1 in an amount by weight, relative to the total weight of MIX M1 , in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95%.The mixture MIX M1 of the present invention comprises, in addition, II) at least one ingredient selected from the group comprising or, alternatively, consisting of propolis extract (preferably with an artepillin-C titer equal to or greater than 4.0%), grape seed extract, resveratrol or mixtures thereof, in combination with I) palmitoylethanolamide (PEA).Advantageously, the mixture of the present invention comprises or, alternatively, consists of: I) palmitoylethanolamide (PEA), and II) at least one propolis extract.The mixture that comprises or, alternatively, consists of: I) palmitoylethanolamide (PEA), and II) at least one propolis extract is more effective and better performing than a PEA + resveratrol mixture described in EP2475352 in repairing damage to the nerves of the nervous system. See, in the present description, the experimental part where the role of the Schwann cells following a nerve injury induced by means of a treatment with 200 ng / mL of GGF isdiscussed (Figure 6). Schwann cells are a type of cell of the peripheral nervous system belonging to the glia. Their main function is to cover the axons of neurons with a layer of myelin.The mixture that comprises or, alternatively, consists of: I) palmitoylethanolamide (PEA), and II) at least one propolis extract is capable of repairing damage to the nerves of the nervous system and, at the same time, restoring the myelination process thanks to the synergistic combination of PEA + propolis extract, which has made it possible to modulate various aspects tied to the progression of damage, thus succeeding in slowing down the process.Propolis is a resinous substance that honeybees collect from the buds and bark of plants. It is thus a substance of strictly plant origin, though after collecting it the bees process it by adding wax, pollens ed enzymes produced by their own bodies. Propolis extract is rich in phenolic compounds and flavonoids.In the context of the present invention, "propolis extract” is intended to refer to, and include, any hydroalcoholic extract or propolis dry extract from any source.Preferably, the propolis extract is preferably a lyophilized dry extract of Brazilian green propolis, more preferably it is a propolis extract with an artepillin-C titer equal to or greater than 4.0% (determined, for example, by means of HPLC with a Photodiode Array Detector). For example, the artepillin-C c titer can preferably be comprised from equal to or greater than 4% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract.Artepillin-C is a phenolic compound, a derivative of p-coumaric acid, and is one of the main phenolic compounds present in Brazilian green propolis.Preferably, the propolis extract (preferably, a propolis dry extract, with an artepillin-C titer comprised from equal to or greater than 4.0% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract) is present in the mixture of the invention in an amount by weight, relative to the total weight of MIX M1 , in the range from 2% to 20%, preferably from 5% to 15%, even more preferably from 10% to 12%.Grape seed extract is a well-known industrial product rich in polyphenolic compounds, mainly oligomeric proanthocyanidins and procyanidins (oligomeric repetitions of flavonoids).In the context of the present invention, "grape seed extract” is intended to refer to, and include, any grape seed extract, from any source, obtained with the methods well known to the person skilled in the art.Preferably, the grape seed extract is a hydroalcoholic extract of Vitis vinifera L. with an CPC (oligomeric proanthocyanidins and procyanidins) titer comprised from 60% to 99%, preferably comprised from 70% to 95%, by weight.Preferably, the grape seed extract is a hydroalcoholic extract of Vitis vinifera L. with an OPC (oligomeric proanthocyanidins and procyanidins) titer comprised from 60% to 99%, preferably comprised from 70% to 95%, and a resveratrol titer comprised from 5% to 45%, preferably comprised from 10% to 30%, by weight.Resveratrol is a polyphenol with a stilbenoid structure contained in micromolar concentrations in red wine and ingrape skins, as well as in some plant species such as Polygonum cuspidatum.In the context of the present invention, the term "resveratrol” is intended to refer to, and include, any source of resveratrol, trans-resveratrol or mixtures thereof. Preferably, the resveratrol is from Polygonum cuspidatum and / or in the form of trans-resveratrol.Preferably, MIX M1 comprises or, alternatively, consists of palmitoylethanolamide (PEA) and propolis extract (preferably with an artepillin-C titer comprised from equal to or greater than 4.0% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract), wherein the palmitoylethanolamide (PEA) is present in an amount by weight, relative to the total weight of MIX M1, in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95% and the propolis extract (preferably, a dry extract with an artepillin-C titer equal to or greater than 4.0%) is present in an amount by weight, relative to the total weight of MIX M1 , in the range from 2% to 20%, preferably from 5% to 15%, even more preferably from 10% to 12%.Preferably, MIX M1 comprises or, alternatively, consists of palmitoylethanolamide (PEA) and grape seed extract, wherein the palmitoylethanolamide (PEA) is present in an amount by weight, relative to the total weight of MIX M1 , in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95% and the grape seed extract is present in a complementary amount by weight, relative to the total weight of MIX M1 , to reach 100%. Preferably, MIX M1 comprises or, alternatively, consists of palmitoylethanolamide (PEA) and resveratrol, wherein the palmitoylethanolamide (PEA) is present in an amount by weight, relative to the total weight of MIX M1 , in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95% and the resveratrol is present in a complementary amount by weight, relative to the total weight of MIX M1 , to reach 100%.Preferably, MIX M1 comprises or, alternatively, consists of palmitoylethanolamide (PEA), propolis extract (preferably with an artepillin-C titer comprised from equal to or greater than 4.0% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract) and grape seed extract, wherein the palmitoylethanolamide (PEA) is present in an amount by weight, relative to the total weight of MIX M1 , in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95%; the propolis dry extract (preferably with an artepillin-C titer equal to or greater than 4.0%) is present in an amount by weight, relative to the total weight of MIX M1 in the range from 2% to 20%, preferably from 5% to 15%, even more preferably from 10% to 12%; and the grape seed extract is present in a complementary amount by weight, relative to the total weight of MIX M1 , to reach 100%.Preferably, MIX M1 comprises or, alternatively, consists of palmitoylethanolamide (PEA), propolis extract (preferably with an artepillin-C titer comprised from equal to or greater than 4.0% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract), grape seed extract and resveratrol, wherein the palmitoylethanolamide (PEA) is present in an amount by weight, relative to the total weightof MIX M1 , in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95%; the propolis dry extract (preferably with an artepillin-C titer equal to or greater than 4.0%) is present in an amount by weight, relative to the total weight of MIX M1 in the range from 2% to 20%, preferably from 5% to 15%, even more preferably from 10% to 12%; the grape seed extract and / or the resveratrol are present in a complementary amount by weight, relative to the total weight of MIX M1 , to reach 100%.A further object of the present invention is a multicomponent mixture comprising or, alternatively, consisting of: i) palmitoylethanolamide (PEA) and ii) at least one ingredient selected from the group comprising or, alternatively, consisting of propolis extract (preferably with an artepillin-C titer equal to or greater than 4.0%), grape seed extract, resveratrol or mixtures thereof, and iii) at least one ingredient selected from the group comprising or, alternatively, consisting of hop extract; Ginkgo Biloba extract; B group vitamins (preferably vitamin B6 and / or B12), or mixtures thereof.This mixture of the invention is called MIX M2.Hop extract {Humulus lupulus) has an antioxidant action thanks to its content of many prenylated aromatic compounds which can act against ROS and prevent oxidative damage to proteins and DNA.In the context of the present invention, "hop extract” is intended to refer to, and include, any hop extract, from any source, obtained with the methods well known to the person skilled in the art.Preferably, said hop extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.Ginkgo Biloba extract has demonstrated various effects in the central nervous system which can improve neuronal plasticity and the levels of neurotransmitters; for example, there have been reported effects in the protection of mitochondrial ATP synthesis in neurons in the presence of oxidative stress and in neuroprotection through anti- apoptotic activity.In the context of the present invention, “Ginkgo Biloba" is intended to refer to, and include, any Ginkgo Biloba extract, from any source, obtained with the methods well known to the person skilled in the art.Preferably, the Ginkgo Biloba extract is present in the mixture MIX M2 of the invention in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.B group vitamins, a group of water-soluble vitamins that intervene in cellular metabolism, are fundamental for the proper functioning and protection of the peripheral nervous system. In particular, vitamin B12 and vitamin B6 are important cofactors for the methylation of homocysteine and play a fundamental role in lowering the levels of this neurotoxic substance in serum. In fact, a low serum level of vitamin B and high levels of homocysteine can cause brain atrophy through oxidative stress and lead to the cognitive decline of elderly adults.In the context of the present invention, “B group vitamins” is intended to refer to, and include, one or more “B group vitamins”.In one embodiment the B group vitamins are preferably vitamin B6 and / or vitamin B12.Preferably, the B group vitamins (preferably vitamin B6 and / or B12) are present in the mixture MIX M2 of theinvention in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.Preferably, MIX M2 of the present invention comprises or, alternatively, consists of palmitoylethanolamide (PEA), propolis extract (preferably with an artepillin-C titer comprised from equal to or greater than 4.0% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract), hop extract; Ginkgo Biloba extract; and B group vitamins (preferably vitamin B6 and / or B12), wherein:- the palmitoylethanolamide (PEA) is present in an amount by weight comprised in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95%;- the propolis dry extract (preferably with an artepillin-C titer comprised from equal to or greater than 4.0% to 12% by weight; more preferably comprised from 4.5% to 10% by weight; even more preferably comprised from 5% to 8% by weight, for example 5.5%, or 6%, or 6.5%, or 7%, or 7.5% by weight, relative to the total weight of the extract) is present in an amount by weight comprised in the range from 2% to 20%, preferably from 5% to 15%, even more preferably from 10% to 12%;- the hop extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.- the Ginkgo Biloba extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.- the B group vitamins (preferably vitamin B6 and / or B12) are present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.Preferably, MIX M2 of the present invention comprises or, alternatively, consists of palmitoylethanolamide (PEA), grape seed extract (preferably with an oligomeric proanthocyanidin and / or procyanidin titer equal to or greater than 60%), hop extract; Ginkgo Biloba extract; B group vitamins (preferably vitamin B6 and / or B12), wherein:- the palmitoylethanolamide (PEA) is present in an amount by weight comprised in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95%;- grape seed extract (preferably with an oligomeric proanthocyanidin and / or procyanidin titer equal to or greater than 60%) is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the hop extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the Ginkgo Biloba extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the B group vitamins (preferably vitamin B6 and / or B12) are present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.Preferably, MIX M2 of the present invention comprises or, alternatively, consists of palmitoylethanolamide (PEA), grape seed extract (preferably with an oligomeric proanthocyanidin and / or procyanidin titer equal to or greater than60%), resveratrol (preferably with a titer equal to or greater than 10%), hop extract; Ginkgo Biloba extract; B group vitamins (preferably vitamin B6 and / or B12), wherein:- the palmitoylethanolamide (PEA) is present in an amount by weight comprised in the range from 70% to 99%, preferably from 80% to 98%, even more preferably from 85% to 95%;- the grape seed extract (preferably with an oligomeric proanthocyanidin and / or procyanidin titer equal to or greater than 60%) is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the resveratrol is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the hop extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the Ginkgo Biloba extract is present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%;- the B group vitamins (preferably vitamin B6 and / or B12) are present in an amount by weight comprised in the range from 5% to 50%, preferably from 10% to 40%, even more preferably from 15% to 25%.A further object of the present invention is a composition comprising a multicomponent mixture according to the invention (MIX M1 , MIX M2), and, optionally, at least one acceptable pharmaceutical or food grade additive and / or excipient.In one embodiment of the present invention, said composition is formulated for oral administration, preferably as a lozenge, capsule, tablet, granular powder, hard-shell capsule, mouth-dissolving granules, sachet or pill, suspensions (for example drinkable vials) or solutions (monophasic or biphasic). Alternatively, the composition is prepared extemporaneously by mixing the composition, for example, with water, milk, fruit juice or another drinkable liquid.The compositions of the invention can be pharmaceutical compositions, compositions for medical devices, dietary supplements, foods, novel foods, probiotic products or compositions for foods for special medical purposes (FSMPs).In the context of the present invention, the term "medical device" is used within the meaning according to Legislative Decree n° 46 of 24 February 1997, or in accordance with the new Medical Devices Regulation (EU) 2017 / 745 (MDR).In the context of the present invention, the term "novel food" is used within the meaning according to Regulation (EC) 258 / 97.In the context of the present invention, the expression "at least one pharmaceutical or food grade additive and / or excipient” means a substance devoid of therapeutic activity adapted for pharmaceutical use or food use. In the context of the present invention, the additives and / or excipients acceptable for pharmaceutical or food use include all the auxiliary substances known to the person skilled in the art for the preparation of compositions in solid, semisolid or liquid form, such as, for example, diluents, solvents, solubilizers, acidifiers, thickeners, sweeteners,flavor enhancers, colorants, lubricants, surfactants, preservatives, pH stabilizing buffers and mixtures thereof.The total daily doses of active ingredients provided by the composition comprising the multicomponent mixture of the invention (MIX M1 , MIX M2) are shown in Table 1 below.Table 1Unless specified otherwise, the expression composition or mixture or anything else comprising a component in an amount "comprised in a range from x to y" is used to indicate that said component can be present in the composition or mixture or extract or anything else in all the amounts present in said range, even if not specified, including the endpoints of the range.The invention is further described by the examples that follow, to be understood as given purely by way of illustration and in no way limiting the scope of protection of the invention.EXAMPLESThe following multicomponent mixtures were prepared. Example 1 (MIX M1 of the invention)able 3Example 3 (MIX M1 of the invention)The first experimental results preliminarily indicate that the multicomponent mixtures and compositions containing them (mixtures and compositions of the invention) could have useful application for treating, in a preventive and / or curative manner, diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, in particular tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss,anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).In particular, the multicomponent mixtures and compositions containing them (mixtures and compositions of the invention) are effective for treating and / or preventing the symptoms of tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology) in subjects suffering from diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system.Experimental sectionThe development of innovative treatment options requires both the study of the passage of molecules through the intestinal barrier and a thorough understanding of the cellular and molecular mechanisms involved at the level of the peripheral nervous system. For this purpose, a three-step study was conducted: a first step (1) aimed at analysing, in a dose-response study, the optimal concentration for every single component substance; a second step (2) aimed at analysing the absorption mechanisms and bioavailability underlying the passage of substances through the intestinal tract, by exploiting a 3D barrier model validated in the literature and approved by the EMA and FDA; and a third step (3) aimed at evaluating the biological activity and the beneficial effect of the novel mixtures / compositions, after intestinal absorption, directly in a 3D model of peripheral nerve regeneration.1. MATERIALS AND METHODS1. 1 Cell culturesThe human colorectal adenocarcinoma cell line Caco-2 (American Type Culture Collection, ATCC, Manassas, USA) is a widely used model of the intestinal epithelial barrier. This cell line was cultured in Dulbecco's Modified Eagle's Medium / Nutrient F-12 Ham (DMEM-F12, Merck Life Science, Rome, Italy) containing 10% fetal bovine serum (FBS, Merck Life Science, Rome, Italy), 2 mM L-glutamine and 1% penicillin-streptomycin (Merck Life Science, Rome, Italy) and kept in an incubator at 37 °C and 5% CO2. The cells used for the experiments had a passage number comprised between 26 and 32, in order to maintain the integrative properties of paracellular permeability and transport, similar to the mechanism of intestinal absorption after oral administration in humans. The rat-derived Schwann cell line, RSC-96, was obtained from the ATCC and was cultured in Dulbecco's Modified Eagle's Medium (Merck Life Science, Rome, Italy) supplemented with 10% FBS, 2 mM L-glutamine and 1 % penicillin-streptomycin, maintaining the cultures at 37 °C with 5% CO2. The RSC96 cells were sub-cultured 2-3 times a week and between 10 and 15 passages were used for the experiments.The rat neuronal cell line PC12 purchased from the ATCC was cultured in Roswell Park Memorial Institute-1640 medium (RPMI, Merck Life Science, Rome, Italy) supplemented with 2 mM glutamine, 10% horse serum (HS; Merck Life Science, Rome, Italy) and 5% FBS. The cultures were maintained in sub-confluence at 37 °C with 5% CO2 and the passage number of the cells used for the experiments was between 3 and 13. PC12 cells are one of the neuronal cell lines most widely used for in vitro screening of neuroprotective compounds. 4x106RSC96 cells and 1 x105PC12 cells were seeded in a co-culture system to reproduce 3D EngNT in vitro, in the environment of the peripheral nerve.7.2 MTT assay of cell viabilityAt the end of the experiment, an MTT assay was carried out as described in the literature to determine cell viability after the stimulations. The cells were incubated in DMEM without phenol red and 0% FBS with 1 % of the MTT probe for 2 hours at 37°C in an incubator; cell viability was then determined by measuring absorbance by means of a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 570 nm with correction at 690 nm. The results were obtained by comparing them with the control cells.1.3 ROS productionThe degree of release of the superoxide anion was used to examine the ROS produced after the stimulations. At the end of the treatment, 100 pl of cytochrome C were added into all samples (treated and untreated) and 100 pl of superoxide dismutase into another sample for 30 minutes in an incubator (all substances were from Merck Life Science, Rome, Italy). Absorbance was measured by a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland), at 550 nm, and the O2 was expressed as nanomoles of reduced cytochrome C per microgram of protein versus the control in percentage (%).1.4 Experimental model of intestinal barrierA model of an intestinal barrier was created using Caco-2 cells to analyze the passage of the substances under examination through the intestinal barrier. For this reason, the TEER values were determined with EVOM3, coupled with STX2 electrodes with sticks (World Precision Instruments, Sarasota, FL, USA); this assay was performed every 2 days for 21 days until reaching a TEER value > 400 Qcm2before stimulation, the time necessary for the formation of the cell monolayer, for cell differentiation and for the exposure of intestinal villi. On day 21 , the culture medium at the apical and basolateral levels was modified to create different pH conditions: pH around 6.5 at the apical level (acidic pH imitating the lumen of the small intestine) and pH around 7.4 at the basolateral level (neutral pH imitating human blood). The cells were maintained for 15 minutes at 37 °C and 5% CO2, after which the TEER values were measured again before the start of the experiment to verify the stabilization of the values. At the end of the stimulation interval, the medium of the basolateral environment was collected in order that it could be used to stimulate the peripheral nerve model.1.5 Analysis of intestinal permeabilityThe measurement of TEER is used to evaluate the barrier function of epithelial cells on these porous supports. This test was performed every 2 days for 21 days until reaching a TEER value > 400 Qcm2prior to stimulation. Once the threshold value was reached, the fluorescent probe, 0.04% fluorescein (Merck Life Science, Rome, Italy), was added together with the treatments and evaluated in the apical and basolateral compartments by converting the amount of total volume in relation to the surface area of the transwell (pg / cm2), according to a classic method. Briefly, the Papp (cm / s) was calculated as:Papp = dQ / dt x 1 / mO x 1 / A x VDonor dQ: amount of substance transported (nmol or pg); dt: incubation time (sec); mO: amount of substrate applied to the donor compartment (nmol or pg);A: surface area of the transwell membrane (cm2);VDonor: volume of the donor compartment (cm3).Negative controls without cells were analyzed to rule out the influence of the Transwell membrane. The fluorescence was measured with a fluorescence spectrophotometer (Infinite 200 Pro MPlex plate reader, Tecan, Mannedorf, Switzerland) at the excitation / emission wavelengths of 490 / 514 nm. The results are expressed as the proportion of the original amount that permeated through the cells.1.6 Analysis ofZO-1 activityHuman Tight Junction Protein 1 ELISA kit (MyBiosource, San Diego, CA, USA) was measured in CaCo-2 cells, following the manufacturer's instructions. Briefly, the cells were washed with cold phosphate-buffered saline 1 x (PBS 1x, Merck Life Science, Rome, Italy) and processed with two freezing / thawing cycles; the cell lysates were then centrifuged for 5 minutes at 5000xg at 4 °C. Afterwards, 100 piL of each sample were collected and incubated on the ELISA plate at 37 °C for 90 minutes; after washing, 100 pL of detection solution A were added into each well and incubated for 45 minutes at 37 °C. The wells were washed and 100 pL of detection solution B were added to the samples. After an incubation of 45 minutes, the wells were again washed and 90 pL of substrate solution were added to each well; the samples were then incubated for 20 minutes at 37 °C in darkness. Finally, 50 pL of stop solution were added and the plates were read with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at the wavelength of 450 nm. The concentration was expressed as pg / mL, comparing the data with the standard curve (range from 0 to 1000 pg / mL) and the results were expressed as a percentage (%) versus the control (0 line).1.7 Analysis ofclaudin 1 activityHuman claudin 1 was measured in the Caco-2 lysates using an ELISA kit (Cusabio Technology LLC, Huston, TX, USA), following the manufacturer's instructions. Briefly, the cells were lysed with cold PBS 1 x (Merck Life Science, Rome, Italy) and centrifuged at 1500x g for 10 minutes at 4 °C. 100 pL of each sample were then added to the ELISA plate and incubated at 37 °C for 2 hours; afterwards, the plate was washed and 100 pL of biotin antibody were added to the wells and incubated for 1 hour at 37 °C. After this time, the wells were washed and 100 pL of HRP-avidin were added into each well; the samples were incubated for 1 h at 37 °C. Subsequently, 90 pL of TMB substrate were also added to the samples and the plate was incubated for 20 minutes at 37 °C away from light. At the end, 50 pL of stop solution were used to halt the reaction and the plate was analyzed with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm. The concentration was expressed as pg / mL, comparing the data with the standard curve (range from 0 to 1000 pg / mL) and the results were expressed as a percentage (%) versus the control (0 line).1.8 Analysis ofoccludin activityThe human occludin ELISA kit (OCLN kit, MyBiosource, San Diego, CA, USA) was used to analyze the presence of occludin in the Caco-2 cell lysates, according to the manufacturer's instructions. Briefly, the Caco-2 cells were lysed with PBS 1 x (Merck Life Science, Rome, Italy), centrifuged at 1500x g for 10 minutes at 4 °C and 100 pL of each sample were transferred into the specific well prior to incubation at 37 °C for 90 minutes. The supernatantswere removed and the plate was incubated with 100 pL of detection solution A for 45 minutes at 37 °C; the wells were then washed with the washing solution and incubated with 100 pL of detection solution B for another 45 minutes. At the end of this time, 90 pL of substrate solution were added, followed by an incubation of 20 minutes at 37 °C in darkness; 50 pL of stop solution were then used to block the enzymatic reaction. The plate was analyzed with a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at the wavelength of 450 nm. The concentration is expressed in pg / mL compared to a standard curve (range from 0 to 1500 pg / mL) and the results are expressed as a percentage (%) versus the control (0 line).1.9 3D engineered neural tissue (EngNT)The 3D model of neural tissue was prepared according to what is described in the literature. The interaction between the RSC96 and PC12 cell lines is a fundamental characteristic for imitating the peripheral nerve environment in vitro, regenerating neurites and sustaining Schwann cells. Briefly, 1 mL of a solution containing 80% v / v of rat tail collagen (2 mg / mL in 0.6% acetic acid, Thermo Fischer, Milan, Italy), 10% v / v of Minimum Essential Medium (MEM, Merck Life Science, Milan, Italy), 5.8% v / v of neutralizing solution (Biosystems, Monza, Italy) and 4.2% of a suspension of Schwann cells (4x106RSC96 cells per 1 mL of gel) were added to a rectangular scaffold with dimensions of 16.4 mm x 6.5 mm x 5 mm. Each gel segment was transferred into a 24-well plate and 1 x105of PC12 cells was seeded above every segment to stabilize the co-cultures. After 1 hour at 37 °C, 1 mL of culture medium (containing DMEM supplemented with 10% FBS, 100 U / mL of penicillin and 100 pg / mL of streptomycin, all purchased from Merck Life Science, Milan, Italy) was added to each well.1.10 TNFa ELISA KitThe concentration of TNFa was determined by means of the TNFa Elisa kit (Merck Life Science, Rome, Italy), following the experimental protocol. The colorimetric intensity was measured at 450 nm by means of a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The results were calculated by generating a calibration curve (range from 24.58pg / ml to 6000pg / ml) and expressed as % versus the control.1.11 NRG1 ELISA kitThe Rat NRG1 ELISA kit (FineTest, Wuhan) was used, according to the manufacturer's instructions, in the supernatants of the cell cultures. Briefly, 100 piL of each sample were added into every well and the plate was incubated at 37 °C for 90 minutes. At the end of the incubation time, the material in every well was removed and the wells were washed twice with a wash buffer. 100 piL of working solution of biotin-labelled antibodies were added to the abovementioned wells and the plate was incubated at 37 °C for 60 minutes. At the end of the incubation, the solution in each well was removed and the wells were washed three times with the wash buffer. 100 piL of SABC working solution were then added into every well and the plate was incubated at 37 °C for 30 minutes. At the end, the wells were washed five times and 90 piL of TMB substrate were introduced into every well. After 10-20 minutes, 50 piL of stop solution were introduced into every well and the plate was immediately read at 450 nm with a plate reader (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The data were obtained and compared with the standard curve (from 0.156 to 10 ng / mL) and the results were expressed as the mean ± SD (%) versus the control (0 line) of five independent experiments carried out in triplicate.1.12 MPZero ELISA kitThe production of myelin protein zero (MPZ) was determined using an ELISA kit for rats (MyBiosource, San Diego, CA, USA) in cell lysates, according to the manufacturer's instructions. Briefly, 100 piL of each sample were added into every well and the plate was incubated at 37 °C for 2 h. At the end of the incubation time, 100 piL of biotin antibody were added into the wells and the plate was incubated at 37 °C for 60 min. At the end of the incubation, the solution in every well was removed and the wells were washed three times with a wash buffer. 100 piL of HRP- avidin solution were then added into every well and the plate was incubated at 37 °C for 1 h. At the end, the wells were washed five times and 90 piL of TMB substrate were introduced into every well. After 30 minutes at 37 °C, 50 piL of stop solution were introduced into every well and the plate was immediately read at 450 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The concentration was expressed as ng / mL compared to a standard curve (comprised between 0.06 and 18 ng / mL) and the results were reported as the mean ± SD (%) versus the control (0 line) of five independent experiments carried out in triplicate.1.13 p75 ELISA kitThe p75 ELISA kit (MyBiosource, San Diego, CA, USA) was used, according to the manufacturer's instructions, on cell lysates. Briefly, 100 piL of each sample were added into every well and the plate was incubated at 37 °C for 2 h. At the end of the incubation time, 100 piL of biotin antibody were added into the wells and the plate was incubated at 37 °C for 60 min. At the end of the incubation, the solution in every well was removed and the wells were washed three times with a wash buffer. 100 piL of HRP-avidin solution were then added into every well and the plate was incubated at 37 °C for 1 h. At the end, the wells were washed five times and 90 piL of TMB substrate were introduced into every well. After 30 minutes at 37 °C, 50 piL of stop solution were introduced into every well and the plate was immediately read at 450 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The data were obtained and compared with the standard curve (from 0.312 to 20 ng / mL) and the results were expressed as the mean ± SD (%) versus the control (0 line) of five independent experiments carried out in triplicate.1.14 ERb ELISA kitThe rat estrogen receptor beta (ERb) ELISA kit (Cloud-Clone, Houston, TX, USA) was used on cell lysates, according to the manufacturer's instructions. Briefly, 100 piL of each sample were added into every well and the plate was incubated at 37 °C for 1 h. At the end of all the reactions, 50 piL of stop solution were introduced into every well and the plate was immediately read at 450 nm with a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). The concentration was obtained and compared with a standard curve (from 0.312 to 20 ng / mL) and the results were expressed as the mean ± SD (%) versus the control of five independent experiments carried out in triplicate.1. 15 Statistical AnalysisThe reported data were obtained from at least four independent experiments carried out in triplicate for each experimental protocol and analyzed using Prism GraphPad statistical software. The results are reported as means ± SD (standard deviation) using one-way ANOVA followed by the Bonferroni post hoc test for the statistical analysis. Values of p < 0.05 were considered statistically significant.2. RESULTS2. 1 STEP 1 - Dose-response evaluation on intestinal cellsWith the aim of evaluating what the best concentration of each selected substance might be, and to prevent any cytotoxic effect, the viability of the Caco-2 cells was analysed by means of the MTT assay.Tested concentrations:• PEA 80 mesh (3ng / ml; 6ng / ml; 12ng / ml)• Propolis extract 4.5% artepillin-C (28 pig / mL; 2.8 pig / mL; 0.28 pig / mL)• Grape seed extract (25 piM; 50 piM; 100 piM)• Grape seed extract 10% resveratrol 60% OPC (referred to as 10% Resveratrol 60% OPC 25 piM; 50 piM; 100 RM)• Hop extract (30.5 mg / mL; 7.63 mg / mL; 1 .5 mg / mL)• Ginkgoo Biloba extract (300 pig / mL; 200 pig / mL; 100 pig / mL)• B group vitamins (1 mg / mL; 200 pig / mL; 100 pig / mL)As shown in Figures 1 A and 1 B, all the tested concentrations showed an increase in cell viability versus the control (p<0.05) starting from 3 h of treatment. In particular, 3 ng / ml of PEA 80 mesh, 2.8 pig / ml of propolis, 67 pig / ml of grape seed extract, 67 pig / ml of 10% resveratrol 60% OPC, 30.5 mg / mL of hop extract, 100 pig / mL of Ginkgo Biloba extract, and 1 mg / mL of vitamin B are the most effective and statistically significant concentrations (p<0.05) compared to the other tested doses at all the time intervals analyzed. Therefore, these were maintained for all the subsequent experiments.2.2 STEP 2 - Evaluation of functionality at the level of the intestinal barrierIn order to test the single substances and mixtures of the invention through the intestinal barrier, use was made of a 3D in vitro model (authorised by EMA / FDA) of the intestinal barrier, shown schematically in Figure 7. The following parameters were evaluated:• safety and toxicity;• oxidative stress;• barrier integrity;• prediction of absorption and permeability;• activity of the tight junctions.Accordingly, various experiments were carried out on the Caco-2 cell line to rule out any toxic effect or irritability and confirm the absence of oxidative stress.The following mixtures of the invention were tested:MIX A (Example 4): Hop extract 30.5 mg / mL, Propolis extract 4.5% artepillin-C 2.8 pig / mL, Ginkgo Biloba extract 100 pig / mL, Vitamin B 1 mg / mL and PEA 80 mesh 3 ng / ml.MIX B (Example 5): Hop extract 30.5 mg / mL, Grape seed extract 67 pig / ml, Ginkgo Biloba extract 100 pig / mL, Vitamin B 1 mg / mL and PEA 80 mesh 3 ng / ml.MIX C (Example 6): Hop extract 30.5 mg / mL, 10% Resveratrol 60% OPC 67 pig / ml, Ginkgo Biloba extract 100g / mL, Vitamin B 1 mg / mL and PEA 80 mesh 3 ng / ml.As shown in Figure 2, all the selected substances and mixtures maintain the intestinal environment balanced, confirming the beneficial viability data of the single substances. Furthermore, the tested mixtures (MIX A; MIX B; MIX C) increased cell viability not only compared to the control (p < 0.05), but also compared to the single substances (p < 0.05), suggesting that the substances under examination, if combined, amplify their biological effect by means of a synergistic mechanism of action. In particular, the analyses performed demonstrate that the tested substances are capable of stimulating cell viability and excluding any cytotoxic effect, thus also confirming the safety of the tested mixtures (MIX A; MIX B; MIX C) (p < 0.05). Furthermore, the tested mixtures (MIX A; MIX B; MIX C) are also capable of improving oxidative stress by reducing ROS production compared both to the single substances and to the control. Therefore, it is possible to affirm that the tested mixtures (MIX A; MIX B; MIX C) maintain cell viability without toxic effects and without inducing oxidative stress.Based on these results, it may be supposed that all the selected substances and mixtures are capable of positively influencing the intestinal barrier without side effects, with a better effect observed for MIX A (product safety) compared to the other mixtures (MIX B; MIX C).In order to evaluate and analyze the proper function of the intestinal barrier, tests were conducted to mimic the complexity of the human intestinal barrier by measuring the TEER values and TJ activity over time (2h-6h). As shown in Figure 3, the transepithelial electrical resistance (TEER) values of the Caco-2 intestinal cells are higher than the accepted threshold value for a correct activity of the intestinal barrier >400Q*cm2. The data obtained show that the intestinal metabolism has a physiological pattern. In particular, the analysis of the intestinal epithelium demonstrates that all the single substances and the tested mixtures were capable of maintaining epithelial integrity versus the control (p < 0.05), while increasing the ion flow of paracellul ar exchanges through the intestinal barrier. Once again, the best effect was obtained following stimulation with the tested mixtures (MIX A; MIX B; MIX C) (p < 0.05) during all the stimulation times, with a better effect observed with MIX A.In order to obtain further information on intestinal absorption, further investigations were carried out by analyzing the values of the Papp coefficient to measure the permeability rate and predict bioavailability. Therefore, the flow of non-electrolyte tracers (expressed as a permeability coefficient) passing through the intestinal barrier was analyzed by fluorescence analysis. In this case as well, the data shown in Figure 3 confirm a correct intestinal absorption which follows what normally occurs in a human being. In particular, the bioavailability of the tested mixtures (MIX A; MIX B; MIX C) shows to be greater compared to the single substances, with a peak of absorption at around 4h (p<0.05), and this effect is maintained, decreasing slowly over time (at 6h it is even greater than at 2h, p<0.05). Again, the best effect was observed with MIX A.The analysis of the TJs confirmed the results obtained with TEER. In particular, an analysis was made of ZO-1, which maintains and simultaneously modulates the integrity of the barrier, Claudin, the main protein forming the barrier, and Occludin, which contributes to the stabilization and optimal function of the barrier. The results shown in Figure 4 demonstrated that, with a better effect compared to the single substances (p<0.05), the tested mixtures (MIX A; MIX B; MIX C) have a positive influence on the TJs. Again, the best effect was observed with MIX A.Based on these results, one may suppose that all the selected substances and mixtures are capable of improving the rate of absorption, with a more physiological behaviour of the mixtures compared to the single substances (p<0.05), maintaining epithelial integrity and increasing the ion flow of paracellular exchanges through the intestinal epithelium. Furthermore, the data obtained from the analyses of the TJs demonstrated that the tested mixtures (MIX A; MIX B; MIX C) maintain the proper functions of the intestinal epithelium with a better effect compared to the single substances (p<0.05). For all the parameters tested, the best effect was observed with MIX A compared to the other mixtures (MIX B; MIX C).2.3 STEP 3 - Evaluation of functionality in respect of peripheral nerve injury (PNI)The product metabolized by the intestinal cells was placed directly in contact with EngNT. This technique was implemented by simulating damage to the peripheral nerve and demyelination with GGF and analyzing:• survival and mitochondrial activity (viability);• oxidative and inflammatory state;• the main pathways involved in the processes of protection of the myelin sheath and formation of neurites, such as NRG1. MPZ, p75 and ERB3.As the study must imitate peripheral nerve tissue damage in vitro, the EngNT 3D was pretreated starting from the 14th day of maturation with 200 ng / mL of glial growth factor 2 (GGF) to reproduce a robust demyelination prior to stimulation with the same agents as used previously. The results are shown in Figure 5. In this context, further experiments were conducted to analyze the effects on mitochondrial metabolism, on ROS production and on the inflammatory response. In particular, the neural tissue treated with only 200 ng / mL of GGF significantly reduced the biological activity of the nerve and increased ROS production and the inflammatory response compared to the control (p < 0.05). In contrast, both these negative conditions were significantly countered by the presence of all the substances tested individually (p<0.05). In particular, the tested mixtures (MIX A; MIX B; MIX C) were capable of improving cell viability in a statistically significant manner compared to the single substances (p<0.05) and at the same time they were also capable of reducing the oxidative stress (p<0.05) produced during the damage, thus supporting the important results previously observed for the synergistic effect of the substances. Similarly, it may be observed that GGF produced a major inflammatory state mediated by a high production of TNFor (p<0.05) compared to the untreated control which was effectively countered both by the single substances and by the tested mixtures (MIX A; MIX B; MIX C), thus supporting the slowing of the progression of damage by exploiting physiological mechanisms for repairing nerve damage.When the single substances are combined, the benefit increases thanks to the promotion of mitochondrial health (p<0.05) and to the reduction of the inflammatory response. In this case as well, better beneficial effects were observed with MIX A compared to the other tested mixtures (MIX B; MIX C).Finally, the role of Schwann cells was studied by analyzing the modulation of neuropathic pain in vitro. The mechanisms of regulation at the basis of the observed effects were analyzed on Schwann cells by mimicking nerve injury by means of treatment with 200 ng / mL of GGF (Figure 6). This damage is confirmed by the analysis of p75 and, consequently, by the inhibition of MPZ activity (p < 0.05), which shows that the myelinating cells underwentdegradation. At the same time, the condition of nerve damage (PNI) influenced the expression of neuregulin 1 (NRG1) and the activity of the epidermal receptor beta (ERb) (p < 0.05 versus the control). In contrast, all the substances tested individually reduced the damage, thus confirming their positive role in countering the demyelination process (all the markers have p < 0.05 with respect to the damage). All the tested mixtures (MIX A; MIX B; MIX C) induced greater effects compared to the control and to the single substances (p < 0.05). In fact, the tested mixtures (MIX A; MIX B; MIX C) were capable of reversing the damage by modulating the markers involved in maintaining the myelin sheath at the normal activity and improving p75 activity (p < 0.05) and MPZ activity (p < 0.05). At the same time, NRG1 and ERb activity increased in the presence of the tested mixtures (MIX A; MIX B; MIX C) compared to the single substances, supporting the hypothesis of an improvement in both these markers for improving the nerve damage and restoring the myelination process. Furthermore, there was confirmation of the active role of the tested mixtures (MIX A; MIX B; MIX C) due precisely to the synergistic combination, which enabled modulation of different aspects tied to the progression of damage, thus succeeding in slowing down the process. The best beneficial effects were observed with MIX A compared to the other mixtures (MIX B; MIX C).ConclusionsIn conclusion, based on the data obtained it is possible to highlight that:-the novel mixtures correctly modulate the biological activity of Schwann cells;-the single substances making up the mixtures are capable of increasing intestinal permeability to promote their passage;-if combined, their synergistic effect amplifies their individual effect;-the mixtures are capable of slowing malfunction and repairing nerve damage at the peripheral level, suggesting better effectiveness with positive effects on the whole system of regulation;-the mixtures inhibit the main molecular pathways involved in demyelination, tending towards a correct homeostasis of neuronal cells;-the mixtures inhibit the production of radicals, reduce inflammation and maintain cellular integrity, demonstrating their safety and biological effectiveness.

Claims

CLAIMS1. A mixture M1 comprising or, alternatively, consisting of:(I) palmitoylethanolamide (PEA), and(II) at least one propolis extract.

2. The mixture according to claim 1 , wherein said mixture, in addition, comprises at least one ingredient selected from the group comprising or, alternatively, consisting of a grape seed extract, a resveratrol and / or mixtures thereof.

3. The mixture according to claim 1 or 2, wherein said mixture M1 comprises or, alternatively, consists of:(a) palmitoylethanolamide (PEA) and a propolis extract, preferably a propolis extract with an artepillin-C titer equal to or greater than 4.0% by weight;(b) palmitoylethanolamide (PEA) and a grape seed extract;(c) palmitoylethanolamide (PEA) and a resveratrol;(d) palmitoylethanolamide (PEA), a propolis extract, preferably a propolis extract with an artepillin-C titer equal to or greater than of 4.0 %, and a grape seed extract;(e) palmitoylethanolamide (PEA), propolis extract, preferably a propolis extract with an artepillin-C titer equal to or greater than 4.0 %, grape seed extract, and resveratrol.

4. The mixture according to any one of claims 1 -3, wherein said mixture, in addition, comprises:(ill) at least one ingredient selected from the group comprising or, alternatively, consisting of a hop extract; a Ginkgo Biloba extract; B group vitamins, preferably a vitamin B6 and / or B12, or mixtures thereof.

5. The mixture according to any one of claims 1 -4, wherein said mixture comprises or, alternatively, consists of: palmitoylethanolamide (PEA); a propolis extract, preferably a propolis extract with an artepillin-C titer equal to or greater than 4.0% by weight; a hop extract; a Ginkgo Biloba extract; B group vitamins, preferably a vitamin B6 and / or B12.

6. The mixture according to any one of claims 1 -4, wherein said mixture comprises or, alternatively, consists of: palmitoylethanolamide (PEA); a grape seed extract; a hop extract; a Ginkgo Biloba extract; B group vitamins, preferably a vitamin B6 and / or B12.

7. The mixture according to any one of claims 1 -4, wherein said mixture comprises or, alternatively, consists of: palmitoylethanolamide (PEA), a grape seed extract, a resveratrol, a hop extract; a Ginkgo Biloba extract; B group vitamins, preferably a vitamin B6 and / or B12.

8. The mixture according to any one of claims 1 -7, wherein said mixture is for use as a medicament; preferably said mixture is for use as a medicament for oral use.

9. The mixture according to claim 8, wherein said mixture is for use in a method of treatment, preventive and / or curative, of diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, particularly tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology); and / or wherein said mixture is for use in a method for treating and / or preventing the symptoms of tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouthsyndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology) in subjects suffering from diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system.

10. A composition comprising a mixture according to any one of claims 1 -9 and optionally at least one acceptable pharmaceutical or food grade additive and / or excipient.

11. The composition according to claim 10, wherein said composition is for use as a medicament; preferably said composition is for use as a medicament for oral use.

12. The composition according to claim 10 or 11 , wherein said composition is for use in a method of treatment, preventive and / or curative, of diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system, particularly tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology).

13. The composition according to any one of claims 10-12, wherein said composition is for use in a method for treating and / or preventing the symptoms of tinnitus, craniofacial neuralgia, trigeminal neuralgia, Bell's palsy, hearing loss, anosmia, burning mouth syndrome, peripheral neuropathies, carpal tunnel syndrome, lumbosciatica, idiopathic pain (arthralgia, myalgia of unknown etiology) in subjects suffering from diseases and / or dysfunctions related to nerve malfunction and / or damage in the peripheral nervous system.

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