Compositions comprising gastrodin and vitamin d and uses thereof

Abstract

The present invention relates to a new association of active ingredients, useful in the prevention and repair of neuronal damage, both at central and at peripheral level. The invention also relates to the pharmaceutical and / or nutraceutical compositions that comprise the association and to their use in therapy, in particular in the protection of the functioning of the central nervous system and in the prevention and / or in the treatment of central and peripheral neuropathies.

Description

[0001] COMPOSITIONS COMPRISING GASTRODIN AND VITAMIN D AND USES THEREOF

[0002] ***

[0003] The present invention relates to a new association of active ingredients, useful in the prevention and repair of neuronal damage, both at central and at peripheral level. The invention also relates to the pharmaceutical and / or nutraceutical compositions that comprise the association and to their use in therapy, in particular in the protection of the functioning of the central nervous system and in the prevention and / or in the treatment of central and peripheral neuropathies.

[0004] The nervous system is formed of the central nervous system (CNS) and the peripheral nervous system (PNS).

[0005] The central nervous system is formed of the encephalon (brain) and the spinal cord, whereas the peripheral nervous system is formed of ganglia (neurons) and nerves (axons) that connect the information from the central nervous system to the rest of the organism.

[0006] Pathologies that damage the central and peripheral nervous system are extremely widespread and represent the leading cause of disability, with millions of deaths every year. Their increase is due in part to the ageing population and to the lack of definitive therapeutic treatments and to the low efficacy of preventive measures

[0007] Damage to the nervous system of mammals can cause serious physical damage and result in many complications that can also be lethal. Damage to the nervous system may derive, for example, from a lesion to the spinal cord or from a lesion caused by an ictus or may be the consequence of pathologies in other organs.

[0008] In addition to the pain caused by stimulation of the pain receptors, neuropathic pain can be caused by damage to or dysfunction of the peripheral or central nervous system.

[0009] Insofar as concerns the peripheral nervous system, peripheral neuropathy can result from deterioration and from consequent malfunctioning of nerves. Said deterioration may be the consequence of a lesion, a trauma or repeated stresses. However, a peripheral neuropathy is very often a consequence of an illness or a metabolic / endocrine disorder, such as diabetes for example. Peripheral neuropathy is an extremely widespread disorder among the middle-aged and elderly and it has been estimated that around 60% of diabetic patients develop damage to the peripheral nerves. Despite being a pathology that usually does not prove fatal, peripheral neuropathy, in contrast with pathologies at central level, is nonetheless very painful and often disabling.

[0010] Various substances that are useful in protecting or improving the health of the central and peripheral nervous systems are known and commercially available. These substances have also been associated with each other, in an attempt to provide a treatment that acts through different mechanisms of action on the nervous system.

[0011] However, combining different active ingredients with a reasonable expectation of obtaining a therapeutic success is not a foregone conclusion. In fact, a potential association, in addition to being effective, non-toxic and well-tolerated, must also satisfy various other requirements, including compatibility between the components, their good bio-availability and longterm tolerability. Last but not least, all the requirements for patient compliance must be satisfied, above all when it is a treatment of the chronic type. An efficient duration of action, good palatability and being simple to take are therefore further requirements that are necessary for an association of active ingredients to demonstrate itself as a valid therapeutic instrument for treatment to maintain the health of the nervous system and for treatment of neuropathic pain. It is therefore clear that there is a continual need to find new therapeutic approaches to maintain the well-being of the nervous system and to limit damage to it and possibly to assist its regeneration in the case of lesion.

[0012] The document entitled “Effects of a New Combination of Natural Extracts on Glaucoma-Related Retinal Degeneration" , FOODS, vol. 10, n° 8, 15 August 2021 (2021-08-15), on page 1885, XP093105155, describes a composition containing gastrodia and vitamin D3 combined with vitamin C, blackcurrant and lycopene for the treatment of glaucoma.

[0013] Document ON 107 625776 A (Wuxi Naomeisu Biopharmaceutical Tech Co LTD) 26.01.2018 belongs to the field of sleep improvement and relates, in particular, to magnesium-based compositions to improve sleep. The compositions may contain magnesium threonate, in addition to vitamin C, vitamin D and theanine, such as L-theanine, to improve the quality of sleep.

[0014] Following an intensive research activity, the Applicant has been able to develop a new association of active ingredients that is capable of responding to the still unsatisfied needs in the field of well-being of the nervous system and curative and preventive treatment of neuropathic pain.

[0015] In particular, the Applicant has observed that the association of gastrodin and vitamins of the D group possesses an interesting activity of prevention and repair of damage to the nervous system and that, thanks to an anti-inflammatory and antioxidant mechanism of action, is capable of stimulating the endogenous production of neurotrophic factors.

[0016] According to one of its aspects, the object of the invention is an association that consists of gastrodin or a derivativethereof and at least one vitamin of the D group or a derivative thereof.

[0017] According to another of its aspects, the object of the invention is an association that comprises gastrodin or a derivative thereof and at least one vitamin of the D group or a derivative thereof.

[0018] Gastrodia elata belongs to the Orchidaceae family. The part of the plant that performs an activity is its rhizome (Rhizoma Gastrodiae), from which it is possible to isolate, for example, phenols, polysaccharides, organic acids and steroids, and gastrodin, which is one of the phenolic glycosides. Each of these categories of compounds may contain, inside them, more specific compounds or specific chemical entities, such as gastrodin, for example.

[0019] Gastrodia extract, preferably Gastrodia elata extract, is therefore intended as encompassing an extract comprising or consisting of one or more of said categories of isolated compounds, each of which may be present in said extract in a quantity variable from zero to 100% by weight, with respect to the weight of the extract. For example, said extract may comprise or consist of gastrodin, or may comprise or consist of a mixture of said specific compounds or specific chemical entities belonging to one or more of said categories of compounds. We point out that, on the market, it is an established custom to refer to a Gastrodia "extract”, preferably a Gastrodia elata extract, with the term "Gastrodin 10: 1 extract”. In practice, even if the word gastrodin refers to a specific compound isolated from the rhizome of the plant (Gastrodin, from Gastrodia alata >98%; (2R,3S,4S,5R,6S)-2-Hydroxymethyl-6-(4-hydroxymethyl-phenoxy)-tetrahydro-pyran-3,4,5-triol, 4- p-D-glucopyranosyloxybenzyl alcohol, 4-Hydroxybenzyl alcohol 4-O-p-D-glucoside, CAS 62499-27-8), on the market the word gastrodin is also used to indicate a 10: 1 extract or a 5: 1 extract , which contains not only gastrodin as such, but also other specific compounds.

[0020] In the context of the present invention, gastrodia extract is intended as encompassing all types of extracts, for example 20:1 or 15:1 or 10:1 or 5:1 containing gastrodin, but also gastrodin as such, without any limitation.

[0021] As mentioned above, the Gastrodia extracts, preferably Gastrodia elata extracts, contain active ingredients (specific compounds or specific chemical entities) that include not only molecular compounds of small dimensions, such as, for example, gastrodin, or 4-hydroxybenzyl alcohol-4-O-p-D-glucopyranoside; [CAS RN: 62499-27-8], of formula (I): which is the active ingredient of Gastrodia Elata that has been studied the most, Parishin, phenolic compounds, 4- hydroxybenzyl alcohol and p-sitosterol, but also active polysaccharide macromolecules.

[0022] In the context of the present invention, the term “a Gastrodia elata extract” is intended as referring to, and including, any extract (and / or mixtures of extracts) of Gastrodia elata, obtained, from any source, with methods and equipment that are well-known to a person skilled in the art.

[0023] Preferably, the Gastrodia elata extract may, for example, be a Gastrodia elata 10:1 extract, i.e. standardised to contain, for example, a concentration 10 times higher than the concentration of active substances present in the Gastrodia elata tuber.

[0024] For example, the Gastrodia elata extract may be a Gastrodia elata 5:1 extract, or also a 3:1 extract.

[0025] In the context of the present invention, a 10:1 extract means the ratio [drug:extract] (the active ingredients contained in 10 grams of plant correspond with 1 gram of extract).

[0026] Said Gastrodia elata extract, preferably a dry Gastrodia elata extract, may be obtained by means of extraction with an extraction solvent, for example water or a water / alcohol solution (such as ethyl alcohol or ethanol), at a temperature, for example, comprised between 20°C and 70°C. Subsequently, a powder is preferably obtained through drying or through nebulising with a Spray-Drying system to give a nebulised dry extract, in which, for example, the extract is nebulized in a steam stream at high temperature. The brief contact time between the extract and the steam causes instantaneous drying without damaging the active ingredients. Highly concentrated extracts are obtained.

[0027] Preferably, the Gastrodia elata extract, for example a dry Gastrodia elata extract, with a 10:1 drug:extract ratio, may have a phytochemical profile of the type:

[0028] - a total polysaccharides content comprised between 40% and 90%, preferably comprised between 50% and 80%, more preferably comprised between 60% and 70%; for example, alpha-glucans and / or maltodextrins may be present in a quantity from higher than zero to lower than 10%, preferably from higher than 0.1% to lower than 7.5%, more preferably from higher than 2% to lower than 5%;

[0029] - a triterpenes content comprised between 0.5% and 5%, preferably comprised between 1 % and 4%, more preferably comprised between 1 .5% and 3%;

[0030] - a total polyphenols content comprised between 0.05% and 3%, preferably comprised between 0.1 % and 2%, more preferably comprised between 0.2% and 1%.

[0031] Furthermore, lignans, for example, may also be present in said extract, in a quantity comprised between higher than zero and lower than 0 01 %, for example, lower than 0.05%, and also triterpenes in a quantity comprised between higher than zero and lower than 0.01 %, for example, lower than 0.05% Preferably, said triterpenes and polyphenols may have a low molecular weight and be attributable to the class of phenolic glycosides.

[0032] As a further example, the Gastrodia elata extract may be a Gastrodia elata extract from the rhizome or root, prepared, for example, through extraction with water and ethanol and having a polysaccharides content (measured with the UV Phenol Sulfuric Acid, 490 nm method) from 50% to 90%, preferably from 60% to 80%; a particle size of 80 mesh (Min. 95% through 80 mesh) and a bulk density CP <2020> comprised between 0.3 g / ml and 0.6 g / ml.

[0033] As a further example, the Gastrodia elata extract may be a Gastrodia elata extract rhizome P.E. 10:1 (dry extract in the presence of dextrin, as an excipient) and having a polysaccharides content (measured with the UV Phenol Sulfuric Acid, 490 nm method) from 10% to 40%, preferably from 15% to 20%; a particle size of 100 mesh (100% through 100 mesh), a solubility in water of 100% at a temperature of 25°C and a tap density comprised between 0.35 and 0.55 g / cm3.

[0034] The gastrodin, 4-(hydroxymethyl)phenyl (3-D-glucopyranoside, with formula (I) (CAS RN. 62499-27-8) is the active ingredient contained in the Gastrodia Elata that is a member of the Orchidaceae family and is traditionally used for treatment of cerebrovascular damage. Its activity is based on the increase in activity of the antioxidant enzymes and on the reduction of oxidative stress which, together with the caspase inhibition, leads to attenuation of the apoptosis mechanism. Gastrodin may also diminish the production of IL-6 and TNF-a, significantly reducing the expression of i NOS, with a consequent considerable anti-inflammatory effect. Lastly, it has an extremely powerful antioxidant effect, reducing the production of ROS and the oxidative stress in various anatomical areas.

[0035] Gastrodin derivatives include, for example, the ester derivatives.

[0036] The vitamins of the D group that are useful in the association of the invention are the following: vitamin D1 compound consisting in 1 :1 parts ergocalciferol and luministerol, vitamin D2 ergocalciferol, vitamin D3 cholecalciferol, vitamin D4 dihydroergocalciferol and vitamin D5 sitocalciferol they are useful for the well-being of the skeletal system and teeth, and also exert functions at cerebral and immune system level.

[0037] In the context of the present invention, the term association or formulation or mixture may be used interchangeably with each other, as they have the same meaning here. The word composition, on the other hand, refers to an association or a formulation or a mixture, together with one or more pharmaceutically acceptable or food-grade excipients and / or carriers. For example, the term association (or formulation or mixture) may refer to gastrodia, or its derivatives, in the presence of at least one vitamin of the D group and nothing else. In this case, the association consists of gastrodia, or its derivatives, and at least one vitamin of the D group.

[0038] For example, the term association (or formulation or mixture) may refer to gastrodia or its derivatives in the presence of at least one vitamin of the D group and at least one other component, preferably at least one other active component. In this case, the association comprises gastrodia, or its derivatives, at least one vitamin of the D group and at least one other component.

[0039] Preferably, the association of the invention consists of gastrodin and / or a derivative thereofand at least one vitamin of the D group and / or derivative thereof. Preferably, the association of the invention comprises gastrodin and / or a derivative thereof and at least one vitamin of the

[0040] D group and / or derivative thereof.

[0041] Preferably, the association of the invention consists of gastrodin and at least vitamin D2 and / or vitamin D3

[0042] Preferably, the association of the invention comprises gastrodin and / or a derivative thereof and at least vitamin D2 and / or vitamin D3 and / or derivative thereof.

[0043] According to one of its aspects, the object of the invention is a pharmaceutical and / or nutraceutical composition that comprises, of alternatively consists of, the association of the invention together with at least one pharmaceutically acceptable excipient and / or carrier.

[0044] According to an embodiment, the composition of the invention comprises gastrodin and at least vitamin D2 and / or vitamin D3, together with at least one pharmaceutically acceptable excipient and / or carrier.

[0045] More preferably, the composition of the invention comprises gastrodin and vitamin D3, together with at least one pharmaceutically acceptable excipient and / or carrier.

[0046] The composition of the invention is a composition for oral use, preferably a composition in the solid state, preferably formulated in dosing units. ‘‘Solid state" means that the composition may exist in the form of granules or powder. The granular or powder compositions are mixed with pharmacologically acceptable additives and excipients to provide an end product such as, for example, a dietary supplement, a medical device or a pharmaceutical composition. The end product may be in pharmaceutical dosing units, such as, for example, granulate in a sachet, tablet, gel or capsule.

[0047] The tablets may have different shapes among the known ones in the field of pharmaceutical shapes, such as, for example, a cylindrical or spheroidal shape. The tablets may be coated or filmed with one or more layers of coating or film capable of passing through the gastric barrier, according to the known methods.

[0048] The capsules in gel may be formed of hard gelatine or soft gelatine or soft gel.

[0049] Preferably, the oral composition of the invention is a solid or semi-solid (gel) composition, as described above. However, if desired or necessary, the composition may be formulated in liquid form, for example through dissolving or suspension in water.

[0050] The solid compositions of the invention may contain, as said, conventional physiologically acceptable excipients or carriers, such as diluents, bulking agents, binding agents, disaggregators, substances that facilitate sliding, lubricants, etc. Nonlimiting examples of suitable carriers and excipients are described in “Remington: The Science and Practice of Pharmacy, 21stEd., Lippincott, Williams & Wilkins”. The compositions of the invention may, for example, include derivatives of cellulose, glucose, lactose, sucrose, gelatine, malt, rice, flower, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, glycol, water, ethanol, and similar. The composition may also contain pH-buffering reagents and wetting agents or emulsifiers.

[0051] In addition to the components described above, if desired or necessary, the composition of the invention may comprise further active components, for example components capable of assisting the active ingredients in performance of the action in the organism of the treated subject, provided that said further components are physically and chemically compatible with the active ingredients of the composition. Examples of further components are vitamins of the B or E group, or hyaluronic acid.

[0052] In one embodiment, the composition is a capsule.

[0053] In one embodiment, the composition is a tablet. In one embodiment, the composition is a gel for oral use.

[0054] In one embodiment, the composition is a granulate.

[0055] The composition of the invention, preferably formulated in dosing units, may be administered one or more times per day.

[0056] The composition of the invention preferably comprises: from 10 to 1000 mg, preferably from 50 to 800 mg of gastrodin and from 5 g to 200 pg, preferably from 10 to 100 pg, of vitamin D, preferably D2 and / or D3.

[0057] The composition of the invention preferably comprises: from 100 to 400 mg of gastrodin, preferably from 200 to 300 mg; and from 10 to 50 pg of at least one vitamin of the D group, preferably from 25 to 50 pg; together with one or more pharmaceutically acceptable excipients and / or carriers.

[0058] More preferably, the composition of the invention comprises from 200 to 300 mg of gastrodin, preferably from 200 to 250 mg; and from 20 to 50 pg of vitamin D3, preferably from 25 to 35 pg; together with one or more pharmaceutically acceptable excipients and / or carriers.

[0059] The compositions of the invention may be prepared by mixing of the individual components, and of any conventional excipients and / or carriers.

[0060] The daily dose depends on the state of health of the patient, on weight, on gender and on age. In general, the compositions of the invention are well-tolerated and may be administered one or two times a day, even for a prolonged period of time. As will be shown in the Experimental Section that follows and by the figures that illustrate the results of the experimental assays conducted, the association of the invention was demonstrated to be particularly effective in the treatment of disorders of the nervous system, in maintaining the state of health and in regeneration of the central and peripheral nervous system

[0061] In particular, the association, in addition to being non-toxic and well-tolerated, has shown an activity greatly superior to that of the individual components, exerting a powerful synergistic effect.

[0062] Furthermore, the association has shown itself to be much more effective than the association of vitamin D with 6-shogaol, which is a compound extracted from ginger (Zingiber officinale Roscoe) well-known for its powerful neuroprotective activity. The association and the composition of the invention are therefore useful in maintaining the well-being and health of the nervous system and in preventing and / or treating the pathologies associated with it, as well as in preventing and / or treating neuropathic pain.

[0063] According to another one of its aspects, the object of the invention is the association and / or the composition of the invention in therapy, in particular, in the prevention and repair of damage to the central and peripheral nervous system and in preventing and / or treating the pathologies associated with it, in the protection of functioning of the nervous system and in the prevention and / or treatment of central and peripheral neuropathies, in preventing and / or treating neuropathic pain and stimulating the endogenous production of neurotrophic factors.

[0064] According to another one of its aspects, the object of the invention is a method for the prevention and repair of damage to the central and peripheral nervous system and for preventing and / or treating the pathologies associated with it, for the protection of functioning of the nervous system and for the prevention and / or treatment of central and peripheral neuropathies, as well as for preventing and / or treating neuropathic pain and stimulating the endogenous production of neurotrophic factors, which comprises administering, to a subject in need thereof, the association and / or the composition of the invention, preferably in a dose effective for the purpose.

[0065] An object of the present invention is a pharmaceutical and / or nutraceutical association A1 that consists of gastrodin or a derivative thereof and at least one vitamin of the D group, preferably at least vitamin D3 or a derivative thereof. Preferably, said association A1 consists of gastrodin and at least vitamin D2 and / or vitamin D3. Preferably, said association A1 consists of gastrodin and vitamin D3. Preferably, said association A1 comprises the gastrodin that is present in a Gastrodia elata extract; preferably, said Gastrodia elata extract comprises or consists of gastrodin.

[0066] An object of the present invention is a pharmaceutical or nutraceutical composition 01 that comprises the association A1 together with one or more pharmaceutically acceptable excipients and / or carriers. Preferably, said composition 01 comprises or, alternatively, consists of: from 100 to 400 mg of gastrodin, preferably from 200 to 300 mg; and from 10 to 50 pg of at least one vitamin of the D group, preferably from 25 to 50 pg, preferably vitamin D3; together with one or more pharmaceutically acceptable excipients and / or carriers. Preferably, said composition C1 comprises or, alternatively, consists of: from 200 to 300 mg of gastrodin, preferably from 200 to 250 mg; and from 20 to 50 pg of vitamin D3, preferably from 25 to 35 pg; together with one or more pharmaceutically acceptable excipients and / or carriers. Preferably, said composition C1 is for oral use, preferably formulated in the solid state, more preferably in dosing units. Preferably, said composition C1 of claims 5 to 8, is characterised in that it is a granulate, a tablet, a gel or a capsule.

[0067] An object of the present invention is an association A1 or a composition C1 for use in therapy. Preferably, said association A1 or said composition C1 is for use in a method for protection of the functioning of peripheral nerves, prevention and / or treatment of peripheral neuropathies, and for use in a method to prevent and / or to treat neuropathic pain.

[0068] An object of the present invention is a use of said association A1 or said composition C1 to maintain the well-being and health of the peripheral nervous system of a subject.

[0069] An object of the present invention is a method to maintain the well-being and health of the peripheral nervous system that provides for administration of said association A1 or said composition C1 to a subject.

[0070] An object of the present invention is an association A2 that consists of or comprises a gastrodin or a derivative thereof, and at least one vitamin of the D group, preferably at least vitamin D3, or a derivative thereof, for use in a treatment method: I) of pathologies or disorders of the peripheral nervous system; II) in protection of the functioning of peripheral nerves; III) in prevention and / or treatment of peripheral neuropathies, and IV) to prevent and / or treat neuropathic pain. Preferably, said association A2 that comprises said vitamin of the D group is at least vitamin D2 and / or vitamin D3. Preferably, said association A2 comprises said gastrodin that is present in a Gastrodia elata extract; preferably, said Gastrodia elata extract comprises or consists of gastrodin.

[0071] An object of the present invention is a pharmaceutical or nutraceutical composition C2 that comprises said association A2, together with one or more pharmaceutically acceptable excipients and / or carriers. Preferably, said composition C2 comprises or, alternatively, consists of: from 100 to 400 mg of gastrodin, preferably from 200 to 300 mg; and from 10 to 50 pg of at least one vitamin of the D group, preferably from 25 to 50 pg, preferably vitamin D3; together with one or more pharmaceutically acceptable excipients and / or carriers. Preferably, said composition comprises or, alternatively, consists of: from 200 to 300 mg of gastrodin, preferably from 200 to 250 mg; and from 20 to 50 pg of vitamin D3, preferably from 25 to 35 pg; together with one or more pharmaceutically acceptable excipients and / or carriers. Preferably, said composition C2 is for oral use, preferably said composition C2 is formulated in the solid state, more preferably in dosing

[0072] RECTIFIED SHEET (RULE 91) ISA / EP units Preferably, said composition C2 is a granulate, a tablet, a gel or a capsule. Preferably, said composition C2 is for use in a treatment method: I) of pathologies or disorders of the peripheral nervous system; II) in protection of the functioning of peripheral nerves; III) in prevention and / or treatment of peripheral neuropathies, and IV) to prevent and / or treat neuropathic pain.

[0073] Embodiments of the compositions of the invention are reported in the Experimental Section that follows, purely by way of illustration and not limiting in any way.

[0074] Brief description of the Figures

[0075] Figure 1 shows an assay on cell viability of the intestinal epithelium over time.

[0076] Figure 2 shows the evaluation of ROS production in the intestinal epithelium.

[0077] Figure 3 shows the evaluation of transepithelial resistance.

[0078] Figure 4 shows the permeability analysis.

[0079] Figure 5 shows the plasma bioavailability.

[0080] Figure 6 shows the analysis of the activity of Zo-1; claudin and occludin.

[0081] Figure 7 shows the evaluation of cell viability and ROS production in terms of inhibition for reduction of the oxidative stress correlated with degeneration.

[0082] Figure 8 shows the evaluation of production of TNFa and IL-1 b.

[0083] Figure 9 shows the evaluation of the activity of APP, pTAU and SIRT1.

[0084] Figure 10 shows the stimulation of endogenous production of BDNF.

[0085] Figure 11 shows the evaluation of the activity of GABA and NGF.

[0086] Experimental Section

[0087] Example 1

[0088] Composition in tablet form containing

[0089] 200 mg of gastrodin

[0090] 25 pig of vitamin D3

[0091] Example 2

[0092] Composition in tablet form containing

[0093] 400 mg of gastrodin

[0094] 50 pig of vitamin D3

[0095] Example 3

[0096] The activity of the association of the invention was evaluated on the basis of various experimental assays.

[0097] In particular, absorption and bioavailability were evaluated, through the use of a 3D intestinal system, and the following tests were conducted:

[0098] Analysis of absorption at small intestine level from 1 h to 24h

[0099] Analysis of toxicity (product safety)

[0100] Analysis of ROS production

[0101] Absorption and bioavailability (TEER and Papp analysis)

[0102] Analysis of tight junctions (TJ) by means of a kit

[0103] Statistical analysis of the data (5 independent experiments conducted in triplicate) The activity at CNS level was also evaluated, through the use of a cerebral organoid and the following tests: Treatment for 24h to study the effect on CNS

[0104] Analysis of toxicity (product safety)

[0105] Analysis of ROS production and the inflammatory situation

[0106] Analysis of the principal markers involved in the processes of protection against neuronal damage (APP, pTAU and SIRT1)

[0107] Analysis of endogenous production of BDNF with formation of microvesicles

[0108] Statistical analysis of the data (5 independent experiments conducted in triplicate)

[0109] Lastly, the activity at CNS level was also evaluated, through the use of a 3D model formed of Schwann cells and motor neurons and the following tests:

[0110] Treatment for 24h to study the effect on PNS

[0111] Analysis of the principal markers involved in the processes of protection of the myelin sheath and in the formation of neurites (NGF and GABA)

[0112] Statistical analysis of the data (5 independent experiments conducted in triplicate)

[0113] Toxicity

[0114] The cell viability of the intestinal epithelium over time was evaluated to exclude toxicity with the following tests and results (Figure 1 , the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. For all the forms tested, the results obtained between 1h and 5h are statistically significant on the control (p<0.05)).

[0115] An analysis was conducted on the individual and combined substances over time (from 1 h to 24h) to determine differences in the mitochondrial metabolism. Among the substances examined, it was observed that they all induce a high cell viability with respect to the untreated control (p<0.05). All the forms, single and combined, showed a similar trend over time, with a peak at around 4 hours, which is maintained over time. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Shagaol+Vit D3 (p<0.05).

[0116] ROS production

[0117] Evaluation of ROS production in the intestinal epithelium overtime to exclude irritability, confirming the safety of the product (Figure 2, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. For all the forms tested, the results obtained between 1h and 5h are statistically significant on the control (p<0.05)).

[0118] An analysis was conducted on the individual and combined substances over time (from 1 h to 24h) to determine any ROS production. Among the substances examined, it was observed that the individual substances produce ROS within the physiological range in respect of the tissue tropism (p<0.05). All the combined forms showed a similar trend over time, with a time-dependent increase in ROS production. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Shagaol+Vit D3 (p<0.05). This data confirms the synergistic antioxidant effect of Gastrodin and VD

[0119] TEER

[0120] Evaluation of transepithelial resistance to determine whether the integrity of the intestinal tissue is maintained (Figure 3, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. For all the forms tested, the results obtained between 1 h and 5h are statistically significant on the control (p<0.05)) .

[0121] An analysis was conducted on the individual and combined substances overtime (from 1h to 24h) to determine the integrity of the absorption mechanisms The analysis supported absorption also in terms of the permeation kinetics of the intestinal wall. It was observed that gastrodin combined with VD is capable of improving absorption, resulting in higher values with respect to the other combinations tested. The peak at 4h confirms the data obtained previously, although an increase is already observable after 2h of stimulation. Among the combined forms, substantial differences were observed between Gastrod in+Vit D3 with respect to 6-Sh ag aol+Vit D3 (p<0.05).

[0122] Permeability analysis

[0123] Figure 4 shows the permeability analysis.

[0124] The values derive from application of the formula for calculation of permeability

[0125] Jmax [C] / (Kt + [C])

[0126] 0: initial concentration of the probe

[0127] Jmax: passage of fluorescence.

[0128] Kt: Michaelis-Menten constant

[0129] Plasma bioavailability

[0130] Figure 5 shows the plasma bioavailability.

[0131] The values derive from application of the formula for calculation of the Papp the Papp values <0.2x10'6cm / s indicate a very low absorption with bioavailability <1% the data comprised between 0.2x10'6and 2x106cm / s a bioavailability between 1 and 90%.

[0132] Tight junctions

[0133] An analysis was conducted of the activity of Zo-1 (mediates adhesion); claudin (maintains the structure) and occludin (contributes to stabilisation) (Figure 6, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. All the substances p<0 05 vs control; ** p<0.05 vs individual substances; the bars p<0 05 vs 6- Shagaol+VD).

[0134] The test is decisive for integrity of the barrier and its functioning (avoid irritability or conditions that could lead to leaky bowel syndrome). The analysis of Zo-1 , claudin and occludin confirmed the active role of the TJ in absorption of the compounds. The analysis was conducted at 6h from the end of stimulation, to check that there were no alterations in the intestinal structures. The results showed that the combination of Gastrodin+VD has a greater influence on the TJs. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Shagaol+Vit D3 (p<0.05).

[0135] Toxicity at CNS level

[0136] Evaluation of cell viability and ROS production in terms of inhibition for reduction of the oxidative stress correlated with degeneration (safety and antioxidant effect) (Figure 7, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. All the substances p<0.05 vs control; ** p<0.05 vs individual substances; the bars p<0.05 vs 6-Shagaol+VD).

[0137] An analysis was conducted on viability and ROS production of the different formulations that passed through the intestinal barrier after 24h of treatment. All the combinations tested increased cell viability and reduced ROS production, allowing exclusion of toxicity in their use at cerebral level after intestinal metabolism. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Shagaol+Vit D3 (p<0.05).

[0138] Inflammation at CNS level Evaluation of the production of TNFa and IL-1 b to establish the anti-inflammatory capacity (Figure 8, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. All the substances p<0.05 vs control; ** p<0.05 vs individual substances; the bars p<0.05 vs 6-Shagaol+VD).

[0139] An analysis was conducted on activation of the transcription factors and the pro-inflammatory cytokines probably due to administration of the different formulations that passed through the intestinal barrier after 24h of treatment. All the combinations tested reduced (p<0.05 vs control) the inflammatory markers, allowing exclusion of activation of inflammatory processes in their use at cerebral level after intestinal metabolism. The gastrodin significantly reduced inflammation, having an excellent anti-inflammatory activity. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Shagaol+Vit D3 (p<0.05).

[0140] CNS biological markers

[0141] Evaluation of the activity of APP, pTAU and SIRT1 to maintain cerebral homoeostasis (Figure 9, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. All the substances p<0.05 vs control; ** p<0.05 vs individual substances; the bars p<0.05 vs 6-Shagaol+VD).

[0142] An analysis was conducted on the biological activity of the different formulations that passed through the intestinal barrier after 24h of treatment. SIRT1 performs a key role in cerebral ageing, in neuroprotection against oxidative stress, in neuroinflammation and in ischemic damage, in central control of energy homeostasis, in modulation of the circadian cycle and in the neuroendocrine functions. APP is a transmembrane protein involved in the destruction of synapses, neuroplasticity and ionic transport. pTAU is a protein associated with the dysfunction of microtubules and axonal transport. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6- Shagaol+Vit D3 (p<0.05).

[0143] BDNF production

[0144] Stimolation of the endogenous production of BDNF (Figure 10, the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. All the substances p<0.05 vs control; ** p<0.05 vs individual substances; the bars p<0.05 vs 6-Shagaol+VD)

[0145] An analysis was conducted on endogenous BDNF production by the different formulations that passed through the intestinal barrier after 24h of treatment. All the combinations tested increased BDNF production, allowing neuronal activity to be stimulated thanks to their use at astrocyte level. Gastrodin showed a higher endogenous BDNF production with respect to the other concentrations tested. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Sh ag aol+Vit D3 (p<0.05).

[0146] PNS biological markers

[0147] Evaluation of the activity of GABA and NGF to maintain PNS homoeostasis (Figure 11 , the data shown is expressed as average + / - SD of 5 independent experiments conducted in triplicate. All the substances p<0.05 vs control; ** p<0.05 vs individual substances; the bars p<0.05 vs 6-Shagaol+VD).

[0148] NGF and GABA have a key role in establishing myelination and the formation of compact myelin; they also stimulate neuronal repair in post-trauma oxidative stress. Gastrodin considerably induced the activity of these two markers, having a good biological activity at PNS level. Among the combined forms, substantial differences were observed between Gastrodin+Vit D3 with respect to 6-Sh ag aol+Vit D3 (p<0.05).

Claims

CLAIMS1 . A pharmaceutical and / or nutraceutical association that consists of gastrodin or a derivative thereof and at least one vitamin of the D group, preferably at least vitamin D3 or a derivative thereof2. The association according to claim 1, that consists of gastrodin and at least vitamin D2 and / or vitamin D3.

3. The association according to claim 1, that consists of gastrodin and vitamin D3.

4. The association according to any one of the preceding claims, wherein said gastrodin is present in a Gastrodia elata extract; preferably, said Gastrodia elata extract comprises or consists of gastrodin5. A pharmaceutical and / or nutraceutical composition that comprises the association of any one of claims 1 to 4, together with one or more pharmaceutically acceptable excipients and / or carriers.

6. The composition according to claim 5, which comprises, or alternatively consists of from 100 to 400 mg of gastrodin, preferably from 200 to 300 mg; and from 10 to 50 g of at least one vitamin of the D group, preferably from 25 to 50 pg, preferably vitamin D3; together with one or more pharmaceutically acceptable excipients and / or carriers.

7. The composition according to claim 4 or 5, which comprises, or alternatively consists of from 200 to 300 mg of gastrodin, preferably from 200 to 250 mg; and from 20 to 50 pg of vitamin D3, preferably from 25 to 35 pg; together with one or more pharmaceutically acceptable excipients and / or carriers.

8. The composition according to any one of claims 5 to 7, characterised in that it is for oral use, preferably formulated in the solid state, more preferably in dosing units.

9. The composition according to any one of claims 5 to 8, characterised in that it is a granulate, a tablet, a gel or a capsule.

10. The association according to any one of claims 1 to 4 or the composition according to any one of claims 5 to 9, for use in therapy.11 . The association or composition for the use according to claim 10, wherein said association or composition is for use in a method for protection of the functioning of peripheral nerves, the prevention and / or treatment of peripheral neuropathies, and for use in a method to prevent and / or to treat neuropathic pain.

12. Use of an association according to any one of claims 1-4 or of a composition according to any one of claims 5-9 to maintain the well-being and health of the peripheral nervous system of a subject.

13. A method to maintain the well-being and health of the peripheral nervous system that provides for the administration of an association according to any one of claims 1-4 or of a composition according to any one of claims 5-9, to a subject.

14. A pharmaceutical and / or nutraceutical association that consists of or comprises a gastrodin or one of its derivatives, and at least one vitamin of the D group, preferably at least vitamin D3, or a derivative thereof, for use in a treatment method:I) of pathologies or disorders of the peripheral nervous system;II) in protection of the functioning of peripheral nerves;III) in prevention and / or treatment of peripheral neuropathies, andRECTIFIED SHEET (RULE 91) ISA / EPIV) to prevent and / or treat neuropathic pain.

15. The association according to claim 14, wherein said vitamin of the D group is at least vitamin D2 and / or vitamin D3.

16. The association according to claim 14 or 15, wherein said gastrodin is present in a Gastrodia elata extract; preferably, said Gastrodia elata extract comprises or consists of gastrodin.

17. A pharmaceutical and / or nutraceutical composition that comprises the association according to any one of claims 14-16, together with one or more pharmaceutically acceptable excipients and / or carriers.

18. The composition according to claim 17, which comprises, or, alternatively, consists of: from 100 to 400 mg of gastrodin, preferably from 200 to 300 mg; and from 10 to 50 pig of at least one vitamin of the D group, preferably from 25 to 50 pg, preferably vitamin D3; together with one or more pharmaceutically acceptable excipients and / or carriers.

19. The composition according to claim 17 or 18, which comprises or, alternatively, consists of: from 200 to 300 mg of gastrodin, preferably from 200 to 250 mg; and from 20 to 50 pg of vitamin D3, preferably from 25 to 35 pg; together with one or more pharmaceutically acceptable excipients and / or carriers.

20. The composition according to any one of claims 17-19, wherein said composition is for oral use, preferably said composition is formulated in the solid state, more preferably in dosing units.

21. The composition according to any one of claims 17-20, wherein said composition is a granulate, a tablet, a gel or a capsule22. The composition according to any one of claims 17-21 , wherein said composition is for use in a treatment method:I) of pathologies or disorders of the peripheral nervous system;II) in protection of the functioning of peripheral nerves;III) in prevention and / or treatment of peripheral neuropathies, andIV) to prevent and / or treat neuropathic pain.RECTIFIED SHEET (RULE 91) ISA / EP

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