"MULTICOMPONENT COMPOSITION COMPRISING EPIGALLOCATECHIN GALLATE AND SAFFRON DRY EXTRACT, AND ITS USE IN THE PREVENTION AND TREATMENT OF PARKINSON'S DISEASE."

The present invention relates to multicomponent compositions comprising an epigallocatechin gallate (EGCG) and/or an extract of Camellia sinensis, titrated in EGCG epigallocatechin gallate, an extract of Crocus sativus, and optionally, a lipoic acid or a pharmaceutical or food grade salt thereof, and optionally, at least one omega-3 fatty acid, said mixture being capable of treating, preventively and/or curatively, Parkinson's disease, particularly with a focus on non-motor symptoms of both the prodromal and full-blown stages of the disease.

Parkinson's disease (PD) is the second-largest neurodegenerative disease in the world, affecting 100 to 200 people per 100,000, and is characterized by a rapid incidence above age 60. Generally, it is diagnosed on average at age 70, when the disease is well established and neurodegeneration has been in progress for some time. Parkinson's disease is an extremely complex disease that includes neuropsychiatric and non-motor manifestations in addition to motor neuron degeneration and death.

The cardinal symptomatic features of Parkinson's are tremor (resting tremor), bradykinesia and rigidity. In addition, neuropsychiatric symptoms such as depression, hallucinations, and cognitive malaise also occur concurrently with the motor symptoms.

Parkinson's disease is also characterized by a rather long prodromal period, which can begin up to 20 years before diagnosis. This early phase of the disease is mainly characterized by the onset of various non-motor symptoms including hyposmia, RBD (rapid eye movement sleep behavior disorder), constipation (due to altered bowel motility, worsened later by medication use), urinary disorders, sleep disorders (difficulty in falling asleep and staying asleep), mood disorders (apathy, anger, moodiness, sadness, pessimism, anxiety), and leg pain of both muscular and neuropathic origin. All these symptoms are perceived by patients to be the most disabling in the early period of the disease.

In addition, recognizing the prodromal state is crucial for undertaking neuroprotective therapies at a time when degeneration is not yet such that it causes classic motor symptoms.

Thus, the above non-motor symptoms constitute a very important plethora of symptoms in the management of the parkinsonian patient.

Currently, in order to treat non-motor symptoms, parkinsonian patients are treated, in the prodromal phase or in the full-blown phase, with antidepressant drugs, benzodiazepines, osmotic laxatives to reduce constipation, and/or sleeping pills to conciliate sleep. The administration of multiple drugs or substances to treat the parkinsonian patient's multiple symptoms is not always possible or easily accomplished. Consequently, there remains a high need in the marketplace for products, which on the one hand support therapies currently in use for the treatment of motor symptoms (e.g., dopamine based treatments), and on the other hand treat or support, preventively and/or curatively, therapies currently in use for non-motor symptoms.

The present invention, following intensive research and development carried out by the Applicant, addresses and solves the aforementioned technical problem by providing a product for the treatment, preventive and/or curative, of Parkinson's disease, particularly of non-motor symptoms in both the prodromal phase and the full-blown phase and/or to support the treatment of motor and non-motor symptoms in the full-blown phase.

Specifically, the present invention provides a product with strong antioxidant action, considering that oxidative stress is the primary cause of neuronal degeneration (and thus of motor symptoms), and comprising active ingredients that can alleviate non-motor symptoms, such as peripheral neuropathic pain, constipation, sleep disorders, and/or mood disorders.

For this purpose, the present invention provides multi-component mixtures or compositions (mixtures or compositions of the invention) comprising, (b) an epigallocatechin gallate (EGCG) and/or an extract of Camellia sinensis (L.) (green tea) titrated in epigallocatechin gallate (EGCG), (c) an extract of Crocus sativus, optionally, (a) a lipoic acid or a salt thereof, optionally (d) at least one omega-3 fatty acid (EPA- DHA), and optionally, (e) at least one vitamin, and/or (f) at least one strain of bacteria belonging to the genus Bifidobacterium or Lactobacillus, and/or (g) at least one prebiotic, as set forth in this description and in the attached claims.

Said preventive and/or curative treatment activity of the mixtures or compositions of the invention is due to the specific and innovative combination of the active components (as defined in this description) in that each component acts through complementary and/or synergistic and/or cooperative mechanisms of action (e.g., with antioxidant, anti-inflammatory, mood-modulating, or gut-regulating activity), making the mixtures and compositions of the invention particularly effective in their action.

For example, lipoic acid has demonstrated anti-inflammatory and antioxidant action at the level of the central nervous system in both in vitro and in vivo studies. In addition, in clinical studies, the use of a supplement comprising lipoic acid has shown efficacy in the treatment of peripheral neuropathy, particularly peripheral neuropathy caused by long-term use of L-dopa.

Green tea extract is also known for its strong antioxidant action, and in several observational studies its consumption has been associated with a reduced risk of Parkinson's disease onset. In particular, epigallocatechin gallate has been found to be effective in in vivo/ex-vivo studies in reducing neuronal death by apoptosis, as it is able to interfere with pathways that regulate the cell cycle, such as mTOR/p- AKT/P-GSK3p. Crocus sativus extract has properties of mood modulation (maintenance of normal mood) and improvement of sleep quality.

The use of omega-3 fatty acids, especially DHA, is known to be associated with the maintenance of normal brain function. In particular, DHA is able to repair brain damage by significantly increasing the amount of TH-positive neurons at the level of the gray substantia in animal models of Parkinson's disease. The administration of omega-3 fatty acids in Parkinson's patients appears to act on an improvement of depressive symptoms.

Finally, one of the most frequent and troublesome non-motor symptoms of Parkinson's disease is constipation, which is due to both improper bowel motility and drug treatment of the disease. Daily treatment with probiotics helps to resolve the intestinal issue in terms of the ease of complete emptying of the intestines. In particular, Lactobacillus rhamnosus LR06 and Lactobacillus plantarum LP01 strains have been shown to totally restore the integrity of the intestinal membrane and actively defend the host from pathogenic infection by preventing the growth of pathogens such as Escherichia coli and Klebsiella pneumoniae.

The mixtures and compositions of the invention, showing a high safety profile, are easy to prepare and cost-effective.

These purposes, and others that will become clear from the detailed description that follows, are achieved by the mixtures and compositions of the present invention because of the technical features in the description and the attached claims.

DRAWINGS

Figure 1 shows the results of the dose-response study of cell viability on N27 cells related to the compounds EGCG (from Camellia sinensis extract, referred to as "green tea") (a), decosahexanoic acid (DHA) (b) and Crocus sativus extract (referred to as "saffron") (c) at different concentrations (between 200 and 25, e.g., 50 piM or 100 piM). The lipoic acid assay was replicated based on a previously published study, and was maintained in all subsequent tests (Molinari, Claudio, et al. "Role of combined lipoic acid and vitamin D3 on astrocytes as a way to prevent brain aging by induced oxidative stress and iron accumulation." Oxidative Medicine and Cellular Longevity 2019 (2019). MTT test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Asterisks above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; **p<0.05 vs other concentrations; * ** p<0.05 vs both (control and other concentrations). All concentrations are expressed as piM.

Figure 2 shows the results of permeability analysis by the experimental blood-brain barrier (BBB) model. The test results are expressed as the mean ± SD (%) of 5 biological replicates normalized against the control, the p-value, calculated according to statistical analysis, is in all cases <0.05 against the control. All concentrations are expressed as piM. Figure 3 shows data related to the validation of the Parkinson's disease (PD) model using 6-OHDA (6- hydroxydopamine) at different concentrations (between 200 and 25 piM) and successive times (1 and 6 hours (a), 12 and 24 hours (b)). MTT test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Asterisks above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control. All concentrations are expressed as pi M.

Figure 4 shows data related to the evaluation of neuroprotective effects under PD condition, specifically with respect to the measurement of cell viability by analysis of mitochondrial metabolism. MTT test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Symbols above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; ** p<0.05 vs 6-OHDA; * ** p<0.05 vs both (control and 6-OHDA); lines p<0.05 vs mixture 1 (MIX 1); # p<0.05 vs mixture 3 (MIX 3). All concentrations are expressed as pi M.

Figure 5 shows data related to the evaluation of neuroprotective effects under PD condition, specifically with respect to the measurement of ROS (reactive oxigen species) production. ROS production test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Symbols above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; **p<0.05 vs 6-OHDA; * ** p<0.05 vs both (control and 6-OHDA); # p<0.05 vs single substances; lines p<0.05 vs mixture 1 (MIX 1); p<0.05 vs mixture 3 (MIX 3). All concentrations are expressed as pi M. Figure 6 shows data related to the evaluation of neuroprotective effects under PD condition, particularly with respect to lipid peroxidation activity. Lipid peroxidation test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Symbols above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; **p<0.05 vs 6-OHDA; * ** p<0.05 vs both (control and 6-OHDA); # p<0.05 vs single substances; lines p<0.05 vs mixture 1 (MIX 1); p<0.05 vs mixture 3 (MIX 3). All concentrations are expressed as pi M.

Figure 7 shows data related to the evaluation of neuroprotective effects under PD condition; specifically with respect to nitric oxide (NO) production. NO production test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Symbols above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; **p<0.05 vs 6-OHDA; * ** p<0.05 vs both (control and 6-OHDA); # p<0.05 vs single substances; lines p<0.05 vs mixture 1 (MIX 1); p<0.05 vs mixture 3 (MIX 3). All concentrations are expressed as pi M.

Figure 8 shows data related to the evaluation of neuroprotective effects under PD condition, specifically with respect to the measurement of inflammatory markers TFNo (a), IL1b (b) and IL6 (c). TNFo and Interleukins production test results are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Symbols above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; **p<0.05 vs 6-OHDA; * ** p<0.05 vs both (control and 6-OHDA); # p<0.05 vs single substances; lines p<0.05 vs mixture 1 (MIX 1); p<0.05 vs mixture 3 (MIX 3). All concentrations are expressed as piM.

Figure 9 shows data related to the evaluation of neuroprotective effects under PD condition, specifically with respect to PINK1 (a) and Parkin (b) activity. The results of PINK1 and Parkin activity assay are expressed as mean ± SD (%) of 5 biological replicates normalized against the control. Symbols above histogram bars indicate p-values calculated according to statistical analysis, specifically: * p<0.05 vs control; **p<0.05 vs 6-OHDA; * ** p<0.05 vs both (control and 6-OHDA); # p<0.05 vs single substances; lines p<0.05 vs mixture 1 (MIX 1); p<0.05 vs mixture 3 (MIX 3). All concentrations are expressed as pi M.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a mixture (in short, mixture of the invention) comprising or alternatively consisting of (b) an epigallocatechin gallate (EGCG) and/or an extract of Camellia sinensis (L.) (or green tea extract), (c) an extract of Crocus sativus (L), optionally, (a) a lipoic acid, optionally, (d) at least one omega-3 fatty acid, and optionally, (e) at least one vitamin (a vitamin D, a vitamin E, and/or at least one B group vitamin), and/or (f) at least one strain of bacteria belonging to the genus Bifidobacterium and/or Lactobacillus, and/or (g) at least one prebiotic.

A second aspect of the present invention relates to a composition (in short, composition of the invention) comprising said mixture of the invention and at least one pharmaceutical or food grade acceptable additive and/or excipient.

A third aspect of the present invention relates to said mixture or composition of the invention containing said mixture, for use in a method of treatment, preventive and/or curative, of Parkinson's disease (PD), particularly of non-motor symptoms in both the prodromal phase and the full-blown phase and/or to support the treatment of motor and non-motor symptoms in a subject in need thereof.

A fourth aspect of the present invention relates to a method of treatment, preventive and/or curative, of Parkinson's disease (PD), specifically a method of treatment of non-motor symptoms in both the prodromal phase and the full-blown phase and/or to support the treatment of motor and non-motor symptoms, in a subject in need thereof, by administering to said subject a therapeutically effective amount of the mixture or composition of the invention, containing said mixture.

A fifth aspect of the present invention relates to the use of said mixture, or composition of the invention containing said mixture, to reduce oxidative stress conditions in healthy subjects.

DETAILED DESCRIPTION OF THE INVENTION

It is an object of the present invention a mixture comprising or alternatively consisting of: (b) an epigallocatechin gallate (EGCG) and/or a green tea extract titrated in EGCG, (c) an extract of Crocus sativus (L.) (e.g., commercial product Affron® or Satiereal®), optionally, (a) a lipoic acid or a pharmaceutical or food grade salt thereof, and optionally (d) at least one omega-3 fatty acid or a pharmaceutical or food grade salt thereof or a precursor thereof (preferably EPA-DHA).

In the context of the present invention, the terms "titrated in" and "comprising" are synonymous terms, used interchangeably when referring to an extract of a plant species (botanical) and a component thereof.

Lipoic acid (or alpha-lipoic acid or (R)(+)alpha-lipoic acid, IUPAC name (R)-5-(1,2-dithiolane-3- yl)pentanoic acid) is a small amphipathic molecule (empirical formula C8H14O2S2); example of CAS no. 1077-28-7. It exists in nature in two forms, as a cyclic disulfide (oxidized form) or as an open chain under the name dihydrolipoic acid, showing two sulfhydryl groups at positions 6 and 8; the two forms are easily interconverted by redox reactions.

Epigallocatechin gallate (EGCG for short) is a type of catechin (example of CAS no. 989-51-5) abundant in tea, particularly in green tea (e.g., Camellia sinensis (L.)), with high antioxidant properties.

The mixture according to the present invention comprises (b) and (c), as described in the present context.

In an embodiment, the mixture of the present invention comprises (a), (b), and (c), as described in the present context.

In an embodiment, the mixture of the present invention comprises (a), (b), (c) and (d), as described in the present context.

Said (b) epigallocatechin gallate (EGCG) may be an extract of a tea plant (e.g., Camellia sinensis) comprising EGCG, preferably an extract (or dry extract) of Camellia sinensis (L.) Kuntze titrated in EGCG at a percentage by weight in the range from 5% or 10% to 95% with respect to the total weight of the extract (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%), preferably from 20% to 45% (e.g., about 40%).

Said extract of Camellia sinensis comprising EGCG usable in the context of the present invention may advantageously be an extract obtained and titrated according to standard procedures known to a person skilled in the art and/or reported in the literature, e.g., a dry extract of Camellia sinensis leaves with hydroalcoholic extraction solvent (e.g., ethanol/water) with titration in EGCG by spectroscopic method, e.g., HPLC.

"Dry extract" in the context of the present invention is understood to mean an extract in a powder form having a water content by weight percentage from 0.05% to 15% (e.g., 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, or 14%), preferably from 0.05% to 10% or from 0.05% to 5%.

Said (c) extract of Crocus sativus (L.) or saffron (dry saffron extract) used in the context of the present invention is preferably a dry extract of Crocus sativus stigmas. The Crocus sativus extract used in the present invention comprises (is titrated in) safranal, preferably in a weight percentage of at least 2% or greater (titration performed in UV-visible) with respect to the total weight of the extract (e.g., from 2% to 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, or 50%; preferably from 2% to 10%). Preferably, said (c) extract of Crocus sativus (L.) or saffron (dry extract of saffron) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

Crocus sativus extract used in the present invention may comprise (be titrated in) safranal and additional components such as crocin and/or picrocrocin.

In a preferred embodiment, in the mixture of the present invention (comprising (a), (b), (c), (d), and optionally (e) and/or (f) and/or (g)), said (c) extract of Crocus sativus is an extract of Crocus sativus (or dry extract of stigmas) titrated in Lepticrosalides® (or mixture comprising safranal crocin, and picrocrocin) from 0.5% to 15% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, preferably about 3.5%) and titrated in safranal from 0.5% to 10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%, preferably about 2%), wherein said percentages are weight percentages with respect to the total weight of Crocus sativus extract. Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

A preferred example of said (c) extract of Crocus sativus is an extract (or dry extract of stigmas) titrated in Lepticrosalides® (comprising safranal, crocin and picrocrocrocin) to at least 3.5%, and in safranal from 0.1% to 5%, or to at least 2%.

An example of a commercial product of Crocus sativus extract that can be used in the context of the present invention is the Affron-labeled product® having the technical characteristics given in the examples, such as Lepticrosalide (HPLC-DAD method) greater than 3.5%; bulk density (Eur. Pharm. (2.9.34)/USP, 616)) greater than 0.3 g/ml; particle size (Eur. Pharm. (2.9.12)/USP34, 786)) 240 pim/60 mesh.

The three main omega-3 fatty acids are alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).

Preferably, said (d) at least one omega-3 fatty acid used in the context of the present invention comprises or, alternatively, consists of eicosapentaenoic acid (EPA) and/or docosahexaenoic acid (DHA) or salts thereof, EPA-DHA for short.

Said (d) at least one omega-3 fatty acid used in the context of the present invention may be a derivative or precursor (of the synthesis) of said omega-3 fatty acid.

The mixture of the present invention may comprise or alternatively consist of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g. about 30%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., commercial product Affron®), (d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA), and (e) at least one vitamin selected from the group comprising or alternatively consisting of: (e.1) a vitamin D (or D3), (e.2) a vitamin E, (e.3) at least one B group vitamin (such as B1, B2, B3, B5, B6, B9 and/or B12) and a mixture thereof (such as (e.i) and (e.2) and (e.3)). Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

For example, said mixture of the present invention comprises or alternatively consists of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g., about 40%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., Affron®), (d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA), (e.1) a vitamin D (or D3), (e.2) a vitamin E, and (d.3) at least one B group vitamin selected from B1, B2, B3, B5, B6, B9, B12, and a mixture thereof (preferably a mixture thereof, such as B1, B2, B3, B5, B6, B9, and B12). Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage ranging from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

The mixture of the present invention may comprise or alternatively consist of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g., about 30%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., Affron®), (d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA), (f) at least one strain of bacteria belonging to the genus Bifidobacterium or Lactobacillus, preferably belonging to the species Lactobacillus rhamnosus and/or Lactobacillus plantarum, more preferably Lactobacillus plantarum LP01 (LMG P-21021) and/or Lactobacillus rhamnosus LR06 (DSM 21981); and optionally, (g) at least one prebiotic selected from the group comprising or alternatively consisting of: an inulin, a fructo-oligosaccharide (FCS), a galacto-oligosaccharide (GOS), a xylitol-oligosaccharide (XOS), preferably inulin. Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

For example, said mixture of the present invention comprises or alternatively consists of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g., about 30%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., Affron®), (d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA), (f) Lactobacillus plantarum LP01 (LMG P-21021) and/or Lactobacillus rhamnosus LR06 (DSM 21981), and optionally, (g) at least one prebiotic, preferably an inulin. Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

The mixture of the present invention may comprise or alternatively consist of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g., about 30%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., Affron®), (d) at least one omega-3 fatty acid or a salt therteof (preferably EPA-DHA), (e) at least one vitamin selected from the group comprising or, alternatively, consisting of: (e.1) a vitamin D (or D3), (e.2) a vitamin E, (d.3) at least one B group vitamin (such as B1, B2, B3, B5, B6, B9 and/or B12) and a mixture thereof (such as (e.i) and (e.2) and (e.3)), (f) at least one strain of bacteria belonging to the genus Bifidobacterium or Lactobacillus, preferably belonging to the species Lactobacillus rhamnosus and/or Lactobacillus plantarum, more preferably Lactobacillus plantarum LP01 (LMG P-21021) and/or Lactobacillus rhamnosus LR06 (DSM 21981); and optionally, (g) at least one prebiotic selected from the group comprising or alternatively consisting of: an inulin, a fructo-oligosaccharide (FOS), a galacto-oligosaccharide (GOS), a xylitol-oligosaccharide (XOS), preferably inulin. Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

For example, said mixture of the present invention comprises or alternatively consists of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g., about 30%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., Affron®), (d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA), (e.1) a vitamin D (or D3), (e.2) a vitamin E, (e.3) at least one B group vitamin selected from B1, B2, B3, B5, B6, B9, B12, and a mixture thereof (preferably a mixture of B1, B2, B3, B5, B6, B9, and B12), (f) Lactobacillus plantarum LP01 (LMG P-21021) and/or Lactobacillus rhamnosus LR06 (DSM 21981); and optionally, (g) at least one prebiotic, preferably an inulin. Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV- visible, e.g., at 330 nm).

According to a preferred example, said mixture of the present invention comprises or alternatively consists of: (a) a lipoic acid or a salt thereof, (b) an epigallocatechin gallate (EGCG) or a green tea extract (e.g., Camellia sinensis) titrated in EGCG (e.g., about 30%), (c) Crocus sativus extract, preferably titrated in safranal from 2% to 10% (e.g., Affron®), (d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA), (e.1) a vitamin D (or D3), (e.2) a vitamin E, (e.3) at least one B group vitamin selected from B1, B2, B3, B5, B6, B9, B12, and a mixture thereof (preferably a mixture of B1, B2, B3, B5, B6, B9, and B12), (f) Lactobacillus plantarum LP01 (LMG P-21021) and Lactobacillus rhamnosus LR06 (DSM 21981); and (g) an inulin. Preferably, said (c) extract of Crocus sativus (L.) or saffron (saffron dry extract) used in the context of the present invention comprises (is titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3% (titration performed in UV-visible, e.g., at 330 nm).

Examples of the mixture of the present invention comprise a mixture comprising: (b), (c), or alternatively,

(a), (b), (c), or alternatively,

(a), (b), (c), (d), (e.1), and (e.2) or, alternatively, (a), (b), (c), (d), (e.1), and (e.3) or, alternatively, (a), (b),

(c), (d), (e.1), (e.2), and (e.3), or, alternatively, (a), (b), (c), (d), and (f) or, alternatively, (a), (b), (c), (d), (e.1), and (f) or, alternatively, (a), (b), (c), (d), (e.1), (e.2) and (f) or, alternatively, (a), (b), (c), (d), (e.1), (e.3) and (f) or, alternatively, (a), (b), (c), (d), (e.1), (e.2), (e.3) and (f) or, alternatively, (a), (b), (c), (d), (f) and (g) or, alternatively, (a), (b), (c), (d), (e.1), (f) and (g) or, alternatively, (a), (b), (c), (d), (e.1), (e.2), (f) and (g) or, alternatively, (a), (b), (c), (d), (e.1), (e.3), (f) and (g) or, alternatively, (a), (b), (c), (d), (e.1), (e.2), (e.3), (f) and (g); wherein (a), (b), (c), (d), (e.1), (e.2), (e.3), (f) and (g) are the active components as defined in the present description.

The mixture or composition of the invention is advantageously formulated for oral (or sublingual) administration.

The dosage form of the mixture or composition of the invention may be a solid form, such as tablet, chewable tablet, effervescent tablet, multi-layered tablet (e.g., time-release), capsule, lozenge, granules or powder (granules or powder to be dissolved in water or orosoluble granules or powder), or a semi-solid form, such as soft-gel, or a liquid form, such as solution, suspension, dispersion, emulsion or syrup; preferably the composition of the invention is in a solid form for oral use, more preferably in tablet or powder/granule form to be dispensed in sachets to be dissolved in water.

According to an aspect of the present invention, the composition of the invention (comprising (b), (c), optionally (a), and optionally (d), and/or (e) and/or (f), according to any one of the described embodiments or aspects) can be formulated in a solid form of multilayer tablet or multilayer tablet with a differentiallyrelease of the multicomponents (two- or three-layer tablet).

The amount and weight ratios of the various components and additives and/or excipients in each layer of said multilayer formulation can be determined and varied by the person skilled in the art in order to achieve the desired release timing, according to known methodologies from the solid oral formulation industry.

The mixture or composition of the invention may advantageously comprise per "dosage unit" in solid form (e.g. of tablet or powder/granules in sachet), the following amounts:

(b) epigallocatechin gallate (e.g., from Camellia sinensis extract) from 50 mg to 1000 mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably from 250 mg to 350 mg or up to 700 mg in the case of powder/granules (e.g., about 200, or 250, or 280, or 300 mg per tablet or from 200 mg to 600 mg per powder/granule sachet) e.g., 250, or 280, or 300, or 350; and (c) Crocus sativus extract, preferably titrated in safranal from 0.1 % to 10%, preferably from 0.2% to 10%, more preferably from 2% to 10% (e.g. Affron®) from 1 mg to 100 mg (e.g., 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 90 mg), preferably from 20 mg to 30 mg or up to 60 mg in the case of powder/granules (e.g., about from 20 to 30 mg per tablet or from 20 mg to 50 mg per powder/granule sachet).

The mixture or composition of the invention may advantageously comprise per "dosage unit" in solid form (e.g. of tablet or powder/granules in sachet), the following amounts:

(b) epigallocatechin gallate (e.g., from Camellia sinensis extract) from 50 mg to 1000 mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably from 250 mg to 350 mg or up to 700 mg in the case of powder/granules (e.g., about 200, or 250, or 280, or 300 mg per tablet or from 200 mg to 600 mg per powder/granule sachet) e.g., 250, or 280, or 300, or 350; and

(c) Crocus sativus extract, preferably titrated in safranal from 0.1 % to 10%, preferably from 0.2% to 10%, more preferably from 2% to 10% (e.g. Affron®) from 1 mg to 100 mg (e.g., 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 90 mg), preferably from 20 mg to 30 mg or up to 60 mg in the case of powder/granules (e.g., about from 20 to 30 mg per tablet or from 20 mg to 50 mg per powder/granule sachet); and

(a) lipoic acid or a salt thereof from 50 mg to 1000 mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably from 250 mg to 350 mg or up to 700 mg in the case of powder/granules (e.g., about 300 mg per tablet or from 300 mg to 600 mg per powder/granule sachet).

The mixture or composition of the invention may advantageously comprise per "dosage unit" in solid form (e.g. of tablet or powder/granules in sachet), the following amounts:

(a) lipoic acid or a salt thereof from 50 mg to 1000 mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably from 250 mg to 350 mg or up to 700 mg in the case of powder/granules (e.g., about 300 mg per tablet or from 300 mg to 600 mg per powder/granule sachet); and

(b) epigallocatechin gallate (e.g., from Camellia sinensis extract) from 50 mg to 1000 mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably from 250 mg to 350 mg or up to 700 mg in the case of powder/granules (e.g., about 200, or 250, or 280, or 300 mg per tablet or from 200 mg to 600 mg per powder/granule sachet) e.g., 250, or 280, or 300, or 350; and

(c) Crocus sativus extract, preferably titrated in safranal from 0.1 % to 10%, preferably from 0.2% to 10%, more preferably from 2% to 10% (e.g. Affron®) from 1 mg to 100 mg (e.g., 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, or 90 mg), preferably from 20 mg to 30 mg or up to 60 mg in the case of powder/granules (e.g., about from 20 to 30 mg per tablet or from 20 mg to 50 mg per powder/granule sachet); and

(d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA) from 50 mg to 1000 mg (e.g., 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg), preferably from 200 mg to 300 mg or up to 600 mg in case of powder/granules (e.g., about 250 mg per tablet or from 250 mg to 500 mg per powder/granule sachet); and, optionally,

(e.1) a vitamin D according to the RDIs (Reference Daily Intake), such as in an amount from 0.5 pig to 50 pig (e.g., 1 pig, 10 pig, 15 pig, 20 pig, 25 pig, 30 pig, 35 pig, 40 pig, or 45 pig), preferably from 20 pig to 50 pig (e.g., about 15 pig or 300% RDI, Reference Daily Intake);

(e.1) a vitamin E according to RDIs, such as in an amount from 300 IU to 500 IU or from 5 mg to 60 mg (e.g., about 400 IU), where 1 IU (IU: International Units) is the biological equivalent of about 0.667 mg of D-o-tocopherol (2/3 of mg), or 0.45 mg or 50 mg of DL-o-tocopherol (or tocopheryl) acetate, e.g., 50 mg of DL-alpha-tocopheryl acetate (about 50 IU of vitamin E); and/or

(e.3) at least one B group vitamin or a mixture thereof according to the RDIs; and/or

(e) one strain of bacteria belonging to the genus Bifidobacterium or Lactobacillus (preferably Lactobacillus plantarum LP01 (LMG P-21021) and/or Lactobacillus rhamnosus LR06 (DSM 21981)) in an amount for each strain of bacteria ranging from 10 ⁷ CFU to 10 ¹² CFU, preferably from 1x10 ⁹ CFU to 5x10 ¹⁰ CFU, more preferably about 1-2 x 10 ⁹ CFU for each strain of bacteria (CFU: Colony Forming Units); and/or

(f) inulin in an amount from 10 mg to 2500 mg, preferably from 25 mg to 2000 mg, even more preferably from 400 mg to 1500 mg (e.g., about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 800 mg, or 1200 mg), preferably about 1000 mg.

One or more B group vitamins may be included in a "dosage unit" (such as a tablet or sachet comprising powders or granules) of the mixtures or compositions of the present invention in the following amounts:

- Vitamin B1 : 0.05 mg - 50 mg, e.g., 0.1 mg, 0.5 mg, 1 mg, 1.5 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, or 25 mg, 30 mg, or 40 mg (preferably about 1.60-1.70 mg - or 150% RDI); and/or

- Vitamin B2: 0.05 mg - 50 mg, e.g. 0.1 mg, 0.5 mg, 1 mg, 1.5 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg or 40 mg (preferably 2.0-2.2 mg - 150%(??) RDI); and/or Vitamin B3: 1 mg - 100 mg (preferably 23-25 mg - or 150% RDI); and/or

- Vitamin B5: 0.5 mg - 50 mg, e.g., 1 mg, 2 mg, 4 mg, 5 mg, 7 mg, 8 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg or 40 mg (preferably 8-10 mg - or 150% RDI); and/or

- Vitamin B6: 0.05 mg - 50 mg, e.g., 0.1 mg, 0.5 mg, 1 mg, 1.5 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, or 40 mg (preferably 2.0-2.2 mg - or 150% RDI); and/or - Vitamin B9: 10 ug - 1000 ug, e.g., 50 ug, 100 ug, 150 ug, 200 ug, 250 ug, 300 ug, 400 ug, 500 ug, 600 ug, 700 ug, 800 ug, or 900 ug (preferably about 300 ug - or 150% RDI); and/or

- Vitamin B12: 0.1 ug - 50 ug, e.g. 0.5 ug, 1 ug, 1.5 ug, 2 ug, 2.5 ug, 3 ug, 4 ug, 5 ug, 10 ug, 20 ug, 30 ug, or 40 ug (preferably 3.7-3.8 ug - or 150% RDI).

According to a preferred example, the mixture or composition of the invention, preferably in a solid form (e.g., tablet or powder/granules in sachet), comprises the following amounts per "dosage unit."

(a) lipoic acid or a salt thereof from 250 mg to 350 mg or up to 700 in case of powder/granules;

(b) epigallocatechin gallate (e.g., from Camellia sinensis extract) from 250 mg to 350 mg or up to 700 in the case of powder/granules, e.g., 280 mg per sachet;

(c) Crocus sativus extract, preferably titrated in safranal from 0.1% to 10%, preferably from 0.2% to 10%, more preferably from 2% to 10% (e.g., Affron®) from 20 mg to 35 mg or up to 50 in case of powder/granules;

(d) at least one omega-3 fatty acid or a salt thereof (preferably EPA-DHA) from 200 mg to 300 mg or up to 600 in case of powder/granules;

(e.1) a vitamin D from 10 pig to 20 pig (e.g., about 15 ug (300% RDI));

(e.2) a vitamin E from 300 IU to 500 IU pig (e.g., about 400 IU (50 mg; 416.67%);

(e.3) at least one B group vitamin or a mixture thereof according to RDIs;

(e) Lactobacillus plantarum LP01 (LMG P-21021) and/or Lactobacillus rhamnosus LR06 (DSM 21981) in an amount for each strain of bacteria ranging from 1 x10 ⁹ CFU to 5x10 ⁹ CFU; and

(f) an inulin from 50 mg to 1500 mg or about 1000 mg in case of powder/granules.

Said "dosage unit" (e.g., tablet or powder or granules sachet) of the composition of the invention may be administered to a subject in need thereof in the 24-hour interval 2, 3, or 4 times at intervals from 4 hours to 12 hours apart, depending on the type of dosage form and the needs of the subject. Preferably, said "dosage unit" of the composition of the invention is administered to a subject in need thereof once or twice a day (e.g., away from meals), preferably once a day.

It is an object of the present invention, the mixture or composition of the invention (comprising (b), (c), optionally (a), optionally (d), and optionally (e), and/or (f) and/or (g), according to any one of the described embodiment or aspects) for use as a medicament.

It is an object of the present invention, the mixture or composition of the invention (comprising (b), (c), optionally (a), optionally (d), and optionally (e), and/or (f) and/or (g), according to any one of the described embodiments or aspects) for use in a method of treatment, preventive and/or curative, of Parkinson's disease in both the prodromal phase and the full-blown phase to support the treatment of motor symptoms and/or non-motor symptoms, in a subject in need thereof, by administering to said subject a therapeutically effective amount of the mixture or composition of the present invention.

The mixture or composition of the present invention (comprising (b), (c), optionally (a), optionally (d), and optionally (e), and/or (f) and/or (g), according to any one of the described embodiments or aspects) is effective, for example, for use in a method of treatment, preventive and/or curative, of non-motor symptoms of Parkinson's disease, particularly non-motor symptoms of the prodromal phase of Parkinson's disease and/or non-motor symptoms of the full-blown phase of Parkinson's disease.

The mixture or composition of the present invention (comprising (b), (c), optionally (a), optionally (d), and optionally (e), and/or (f) and/or (g), according to any one of the described embodiments or aspects) is effective, for example, for use in a method of treatment, preventive and/or curative, of the motor symptoms of Parkinson's disease, preferably as an adjunct to a therapy for the treatment of Parkinson's disease.

Said motor symptoms can be selected from the group comprising or alternatively consisting of: tremor or resting tremor, bradykinesia, and rigidity.

Said non-motor symptoms can be selected from the group comprising or alternatively consisting of: sleep disorders (difficulty in falling asleep and staying asleep), mood disorders (apathy, anger, moodiness, sadness, pessimism, anxiety), peripheral neuropathic pain (particularly of the legs), muscle pain (particularly of the legs), bowel constipation, urinary disorders, hyposmia (decreased ability to perceive all or part of odors), RBD (rapid eye movement (REM) sleep behavior disorder).

The mixtures or compositions of the present invention may be for use in a method of treatment of Parkinson's disease (as defined in the present description) when administered to a subject as the sole therapy or when administered as an adjunctive therapy to at least one other therapy or composition capable of treating Parkinson's disease (motor symptoms and/or non-motor symptoms, e.g., a dopamine drug therapy).

For clarity, in order to achieve the purpose of the present invention, the active compounds of the mixture or composition of the present invention ((b), (c), optionally (a), optionally (d), and optionally (e), and/or (f) and/or (g)) can also be administered separately or in groups (preferably in a time interval of 30 minutes up to 2-3 hours) and in any order.

Said at least one additive and/or excipient of pharmaceutical or food grade, included in the composition of the invention together with the mixture of (a), (b), (c), (d), and optionally (e) and/or (f) and/or (g), consists of a substance without therapeutic activity suitable for pharmaceutical or food use selected from auxiliary substances known to the person skilled in the art such as, e.g., diluents, solvents (including water, glycerin, ethyl alcohol), solubilizers, thickeners, sweeteners, flavoring agents, dyes, lubricants, surfactants, antimicrobials, antioxidants, preservatives, pH-stabilizing buffers, and mixtures thereof. Non-limiting examples of such substances are phosphate buffers (e.g., dicalcium phosphate), stearate of an alkaline or alkaline earth metal (e.g., magnesium), silicon dioxide, mono- and diglycerides of fatty acids, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, starch or corn starch, natural or artificial flavorings (e.g., iron oxides).

Said composition of the invention can be a pharmaceutical composition, a medical device composition (Medical Device Regulation (EU) 2017/745 (MDR)), a dietary supplement and/or a food for special medical purposes (FSMP).

The term "subject(s)" in the context of the present invention refers to mammals (animal and human), preferably human subjects.

The term "therapeutically effective amount" refers to the amount of mixture or compound or formulation that elicits biological or medicinal response in a tissue, system, or subject that is researched and defined by an expert in the field.

Unless otherwise specified, the expression mixture or composition comprising a component in an amount "within a range from x to y" means that said component may be present in the composition in all amounts present in said range, even if not made explicit, extremes of the range included.

Examples of active components that can be used in the context of the present invention are given below. An example of (a) lipoic acid extract usable in the present invention may be that marketed under the name M.A.T.R.I.S.

An example of (b) green tea extract comprising EGCG usable in the present invention is the commercial product "Green tea 95% polyphenols I 65% catechins" produced by EPO Istituto Farmochimico Fitoterapico S.r.l. Italy (CAS No.: 84650-60-2) by extraction of Camellia sinensis leaves with extraction solvent: ethanol 70 I water 30 and having the following characteristics: excipients: maltodextrin; particle size not less than 90% through 300 microns; Titer: catechins (HPLC met.) >= 65.0 % w/w, epigallocatechin gallate (EGCG): >= 30.0 % w/w, caffeine: <= 5.0 % w/w, epicatechin gallate (ECG): >= 8.0 % w/w; Titer: total polyphenols (spectrophotometric met. Folin - ciocalteau)>= 95.0 % w/w; Relative density: 350 - 650 g/l; Loss on drying: <= 5.0 % w/w; pH: 3.0 - 6.0.

An example of (c) Crocus sativus extract usable in the present invention (e.g., comprising safranal at least 2% w/w or higher) is the product with trade name Affron® (trade mark), as the dry extract of stigmas of Crocus sativus L. titrated to about 3.5% (minimum titer) in Lepticrosalides® (trade mark for a compound comprising at least safranal, crocin, picrocrocrocin) and about 2% safranal (percentages w/w of total extract); titer standardized by HPLC. Another example of (c) extract of Crocus sativus usable in the present invention (e.g., comprising (titrated in) safranal, in a weight percentage from 0.1% to 10%, preferably from 0.2% to 5%, e.g., 0.3%-titration performed in UV-visible, e.g., at 330 nm) is the product with trade name Satiereal®.

One example of at least one omega-3 fatty acid (preferably EPA-DHA) that can be used in the present invention is represented by the products already commercially available to the public. An example of vitamin D usable in the present invention is a vitamin D3 having CAS No. 67-97-0, min. 90,000 IU vitamin D3/g (equivalent to 2250 pig cholecalciferol/g), density (bulk density) ~0.6 g/mL.

An example of a vitamin E (or tocopherol or compound comprising tocopherol) usable in the present invention is a commercially available and publicly available vitamin E.

An example of vitamin B1 usable in the present invention is a vitamin B1 (thiamine chlorohydrate) with a purity of 98.5-101.0% w/w (Ph.Eur. method) or 98.0-102.0% w/w (USP method), pH 2.7-3.3 (Ph.Eur.) or 2.7-3.4 (USP), water max. 5.0% w/w (Ph.Eur, USP), sulfated ash max. 0.1% w/w (Ph.Eur.), residues on ignition max. 0.2% w/w (USP), residual solvents (USP): methanol max. 0.3% w/w and ethanol max. 0.5%.

An example of vitamin B2 usable in the present invention is a vitamin B2 (riboflavin high flow 100 (HF)) having CAS No. 83-88-5.

An example of vitamin B3 usable in the present invention is vitamin B3 (niacinamide food grade FCC) having CAS No. 98-92-0 purity 99.0-101.0% w/w (HPLC), obtained from 3-cyanopyridine as starting material by the following steps: hydrolysis of 3-cyanopyridine on bio-catalyst on fixed bed to obtain crude niacinamide solution, purification on activated carbon on fixed bed, nanofiltration, evaporation, and lyophilization (spray drying).

An example of vitamin B5 that can be used in the present invention is a calcium D-pantothenate having CAS No. 137-08-6.

An example of vitamin B6 usable in the present invention is a vitamin B6 (pyridoxine chlorohydrate food grade) purity 99.0-101.0% w/w (Ph.Eur. method) or 98.0-102.0% w/w (USP method), pH 2.4~3.0(Ph.Eur.), chlorine (in anhydrous compound) 16.9%~17.6% w/w (USP), sulfated ash max. 0.1% w/w (Ph.Eur.), drying loss max. 0.5% w/w (Ph.Eur.), residual solvents (Ph.Eur.): ethanol max. 0.5%.

An example of vitamin B9 that can be used in the present invention is a vitamin B9 (folic acid) having CAS No. 59-30-3 and molecular weight 441.40, IR identification, Residue on ignition <0.30 % w/w, specific absorbance ratio 256/365 nm 2.80-3.00, water <8.50% w/w, titer (on anhydrous) 95.00-102.00 % w/w.

An example of vitamin B12 that can be used in the present invention is a vitamin B12 (Dry vitamin B12 0.1% GFP) having CAS No. 68-19-9.

An example of inulin that can be used in the present invention (example of CAS No. 9005-80-5; saccharidic polymer with molecular weight of about 5000 Da) is an inulin extracted from chicory roots comprising: inulin minimum 90% w/w, fructose + glucose + sucroester maximum 10% w/w, average chain length 8-13 monomers, ash maximum 0.2% w/w, pH about 6(±1), density (tapped density) about 700 (±100) g/L.

EXPERIMENTAL PART

1. EXAMPLES OF COMPOSITIONS Table 1 shows an example embodiment of the composition of the invention comprising (a), (b), (c) and (d) formulated for oral use in solid form (in short, dose).

Table 1

Table 1A shows an example embodiment of the composition of the invention comprising (a), (b), (c) and (d) formulated for oral use in solid form of granules/powder packaged in sachet (in short, dose).

Table 1A

Table 2 shows an example embodiment of the composition of the invention comprising (a), (b), (c), (d), (e), (f) and (g) formulated for oral use in solid form of tablet (in short, dose).

Table 2

Witamin B1 1-2 mg, Vitamin B2 1-3 mg, Vitamin B3 20-30 mg, Vitamin B5 5-15 mg, Vitamin B6 1-3 mg, Vitamin B9 250-350 pg, Vitamin B12 2-6 pg.

< ^b >example of additives/excipients: Mg stearate, Si dioxide, dicalcium phosphate, mono and diglycerides of fatty acids, microcrystalline cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, flavoring, and/or corn starch.

Table 2A shows an example embodiment of the composition of the invention comprising (a), (b), (c), (d), (e), (f) and (g) formulated for oral use in solid form of granules/powder packaged in sachets (in short, dose).

Table 2A 2. In vitro TEST

2.1 Materials and methods

2. 1. 1 Cell cultures N27 mesencephalic dopaminergic cells were grown in Roswell Park Memorial Institute medium (RPMI, Merck, Milan, Italy) supplemented with 10% fetal bovine serum (FBS; Merck, Milan, Italy), 1% L-glutamine, penicillin (10 U/ml) and streptomycin (10 U/ml; GIBCO). Cells were seeded at a density of 0.5 x 10 ⁶ in a 75 cm ² flask (Corning, New York, NY) and incubated at 37 °C in 5% CO ₂ under 95% saturable humidity condition [Selvakumar, G. P. et al. "Glia maturation factor-dependent inhibition of mitochondrial PGC-1o triggers oxidative stress-mediated apoptosis in N27 rat dopaminergic neuronal cells" Molecular neurobiology 55-9 (2018), 7132-7152],

The astrocytic cell line CCF-STTG1 was grown in flask in RPMI medium (Merck, Milan, Italy) supplemented with 10% FBS (Merck, Milan, Italy) with the addition of Hepes 2 mM (Merck, Milan, Italy), L- glutamine 2 mM (Merck, Milan, Italy) and 1% penicillin-streptomycin (P/S, Merck, Milan, Italy) and maintained in a 37°C incubator with 95% humidity and 5% CO ₂ [Jogalekar, M. P. et al. "Total RNA isolation from separately established monolayer and hydrogel cultures of human glioblastoma cell line" Bio-protocol 9- 4 (2019), e3305-e3305],

Huvec cells, were cultured in 0.1% gelatin-coated flask and grown with Endothelial Growth Medium-2 (EGM-2) medium containing 2% fetal bovine serum (FBS), 0.04% hydrocortisone, 0.4% hFGF-B, 0.1% VEGF, 0.1% R3-IGF-1, 0.1% ascorbic acid, 0.1% hEGF, 0.1% GA-1000, 0.1% heparin (all produced by Lonza, Walkersville, MD, USA) and maintained at 37 °C and 5% CO ₂ as previously described [Molinari, C. et al. "The role of BDNF on aging-modulation markers" Brain Sciences 10-5 (2020), 285], For all experiments, passage from 3 to 6 cells were used.

2. 1.2 MTT cell viability assay

At the end of the experiment, MTT assay was performed as described in the literature [Uberti, F. et al. "Iron absorption from three commercially available supplements in gastrointestinal cell lines" Nutrients 9-9 (2017), 1008] to determine cell viability after stimulations. Cells were incubated in DMEM without Red phenol 0% FBS with 1% of the MTT probe for 2 hours at 37°C in an incubator, then, cell viability was determined by measuring the absorbance through 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 control cells (100% viable).

2. 1.3 Experimental blood-brain barrier (BBB) model.

Astrocytes were co-cultured with endothelial cells (HUVECs) according to methods reported in the literature [Molinari, C. et al. "The role of BDNF on aging-modulation markers" Brain Sciences 10-5 (2020), 285], Briefly, to create the BBB, 4 x 10 ⁴ astrocytes/cm ² were seeded on the basolateral side of 6.5-mm inverted Transwell® inserts (Corning Costar, Sigma-Aldrich, Milan, Italy) and allowed to adhere for 4 h. Transwell® were then placed in the correct orientation and the cells were allowed to grow for 48 h. Next, 1 x 10 ⁵ HUVEC cells/cm ² were seeded in the apical compartment and the inserts were then placed in a 24- well plate. After 7 days of culture, Transwell® were processed and permeability studies were performed [Kulczar, C. et al. "Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model" Journal of Pharmacy and Pharmacology 69-12 (2017), 1684-1696], To understand the ability of the tested substances to cross the blood-brain barrier, the medium in the lower compartment of the Transwells insert® was taken to assess the uptake rate of all substances, both singly and in combination, using a fluorescent probe.

2. 1.4 ROS production

The release rate of superoxide anion was used to examine ROS produced after stimulations [Molinari, C. et al. "Role of combined lipoic acid and vitamin D3 on astrocytes as a way to prevent brain aging by induced oxidative stress and iron accumulation" Oxidative Medicine and Cellular Longevity February (2019)]. At the end of treatment, 100 pl of cytochrome C and, in another sample, 100 pl of superoxide dismutase were added to all samples (treated and untreated) for 30 min in an incubator (all substances were from Sigma-Aldrich). Absorbance was measured by a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland), at 550 nm, and the O ₂ was expressed as nanomoles (nM) of reduced cytochrome C per microgram (pig) of protein with respect to the control, as percentage (%).

2. 1.5 Lipid peroxidation measurement kit

Lipid peroxidation in cells was estimated using the thiobarbituric acid assay (TBARS) [Han, F. et al. "Interleukin-6 promotes ferroptosis in bronchial epithelial cells by inducing reactive oxygen speciesdependent lipid peroxidation and disrupting iron homeostasis" Bioengineered 12-1 (2021), 5279-5288], Briefly, 10Opi of sample or standard was added into a 5ml vial and then 10Opi of SDS solution was added. In each vial, 4ml of dye reagent was added and boiled for 1 hour. At the end of incubation, they were incubated for 10 min on ice, centrifuged for 10 min at 1600 x g at 4°C, and then 150 pl for each sample was added to the wells of the 96 plate and read for absorbance at 530-540 nm (Infinite 200 Pro MPlex spectrometer, Tecan, Mannedorf, Switzerland).

2. 1.6 NO production

To quantify NO production, a kit was used to assess its production after various stimuli, following the manufacturer's instructions [Lattuada, D. et al. "Fimbrial cells exposure to catalytic iron mimics carcinogenic changes" International Journal of Gynecologic Cancer 25-3 (2015)]. The absorbance of the samples was read by a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at a wavelength between 520 nm and 550 nm. Results were expressed as a normalized percentage (%) with respect to untreated samples, referenced to the standard curve, generated with standard nitrate. 2.1.7 ELISA kit for TNFa

TNFa concentration was determined by TNFa ELISA kit (Merck Life Science, Rome, Italy) following the experimental protocol [Molinari, C. et al. "Preventing c2c12 muscle cells damage by combining magnesium and potassium with vitamin D3 and curcumin" Journal of traditional and complementary medicine 11-6 (2021), 532-544], Colorimetric intensity was measured at 450 nm by spectrophotometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland). Results were calculated by generating a calibration curve (range from 24.58 pg/ml to 6000 pg/ml) and expressed as % relative to control.

2.1.8 ELISA kit for IL-1

Quantification of IL-1p was performed with the IL-1p ELISA kit (R&D systems, MN) according to the manufacturer's instructions [Uberti, F. et al. "A combination of a-lipoic acid (ALA) and palmitoylethanolamide (PEA) blocks endotoxin-induced oxidative stress and cytokine storm: A possible intervention for COVID-19" Journal of Dietary Supplements (2021), 1-23], Absorbance was determined with a microplate reader at 450 nm with correction at 570 nm. IL-1p was quantified by relating sample readings to the generated standard curve (range from 12.5 pg/ml to 800 pg/ml) and expressed as % relative to control.

2.1.9 ELISA Kit IL-6

IL-6 concentration in basolateral co-culture media was analyzed with the IL-6 ELISA kit (eBioscience, USA) [Huang, K. et al. "Effect of PM2.5 on invasion and proliferation of HeLa cells and the expression of inflammatory cytokines IL1 and IL6" Oncology Letters 16-6 (2018), 7068-7073], The plate was measured at a wavelength of 450 nm, and the data were analyzed by relating the sample readings to the generated standard curve (range from 3.1 pg/ml to 300 pg/ml) and expressed as % relative to control.

2.1.10 PINK1 ELISA Kit

PINK1 activity was determined in cell lysates using an ELISA kit (PINK1 ELISA kit, MyBiosource, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, cells were lysed with cold 1x PBS and centrifuged at 1500* g for 10 min at 4 °C. A quantity of 100 pl of each sample was added to a well and incubated at 37 °C for 90 min; then, the contents were removed and, to each well, 100 pl of detection solution (a) was added and incubated for 45 min at 37 °C. After this time had elapsed, the wells were washed and 100 pl of detection solution (b) was added to each well and then incubated for 45 min. Then, 90 pl of substrate solution was also added to each well and the plate was incubated for 20 min at 37 °C in the dark. A quantity of 50 pl of stop solution was used to stop the reaction, and the absorbance was analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm, and the concentration is expressed in ng/ml by comparing the data with the standard curve (range 0.625 ng/ml-20 ng/ml).

2.1.11 Parkin ELISA kit

Parkin activity was determined in cell lysates using an ELISA kit (Parkin ELISA kit, MyBiosource, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, 100 pl of each sample was added and incubated at 37 °C for 90 min; then, the material was removed and 100 pl of detection solution (a) was added and incubated for 45 min at 37 °C. After this time had elapsed, the wells were washed and 100 pl of detection solution (b) was added to each well and then incubated for 45 min.

Then, 90 pl of substrate solution was also added to each well, and the plate was incubated for 20 min at 37 °C in the dark. An amount of 50 pl of stop solution was used to stop the reaction, and the absorbance was analyzed by a spectrometer (Infinite 200 Pro MPlex, Tecan, Mannedorf, Switzerland) at 450 nm, and the concentration is expressed in ng/ml by comparing the data with the standard curve (range 0.625 ng/ml-20 ng/ml).

2. 1. 12 Statistical analysis

Data reported were obtained from at least four independent experiments performed in triplicate for each experimental protocol, and analyzed using Prism GraphPad statistical software. Results are reported as means ± SD (standard deviation) using one-way ANOVA, followed by Bonferroni post hoc test for statistical analysis. Values of p < 0.05 were considered statistically significant.

2.2 Results

The tests were carried out by treating the cell cultures with the single substances, lipoic acid, EGCG (from Camellia sinensis extract, referred to as "green tea"), decosahexanoic acid (DHA) and Crocus sativus extract (referred to as "saffron"), or with three different mixtures made up as follows:

- mixture 1 (MIX 1): lipoic acid 50piM + green tea 100piM + DHA 25piM + saffron 25piM;

- mixture 2 (MIX 2): green tea 100piM+ saffron 25piM;

- mixture 3 (MIX 3): green tea 100piM + DHA 25piM + saffron 25piM.

2.2.1 Dose-response assessment

In order to assess what might be the best concentration of each selected substance, and to prevent any cytotoxic effect, the cell viability of N27 cells was analyzed by MTT assay. As shown in Figure 1, all concentrations tested showed an increase in cell viability compared with the control (p<0.05). In particular, a greater increase was observed in cells treated with EGCG (from Camellia sinensis extract, referred to as "green tea" in the figures) in 100pim concentration, which was able to induce an increase in viability of 48% compared to 200pm, 68% compared to 50pm and 82% compared to 25pm (p<0.05) (Figure 1a). In contrast, with regard to DHA, although all tested concentrations induced no cytotoxic effect (p<0.05 compared with control), the most effective concentration was 25pm which increased cell viability by 16%, 38% and 90% compared with the other tested concentrations (150pm, 100pm and 50pm respectively, p<0.05) (Figure 1b). Crocus sativus extract (referred to as "saffron" in the figures) was also able to increase cell viability compared to the control (p<0.05); in particular, the concentration that proved to be most effective was 25pm, which was able to increase viability by 78%, 34% and 25% compared to the 200pm, 100pm and 50pm concentration respectively (p<0.05) (Figure 1c). Finally, the best dosage of lipoic acid (indicated as "Lipo" in the figures) was derived from the study Molinari, C. et al. "Role of combined lipoic acid and vitamin D3 on astrocytes as a way to prevent brain aging by induced oxidative stress and iron accumulation" Oxidative Medicine and Cellular Longevity February (2019).

2.2.2 Assessment of permeability through an in vitro blood-brain barrier (BBB) model

The BBB regulates the passage of water, some gases, and some fat-soluble molecules by passive diffusion, but also the selective transport of molecules crucial for neuronal function, or of drugs and active ingredients with neuroprotective function; for this reason, it was essential to assess the rate of uptake of all test substances. As observed in Figure 2, in the time interval between 4h and 24h, the BBB tests showed that all test substances increased the permeability of the blood-brain barrier, peaking at 12h and maintaining their effect up to 24h (p<0.05). The combined effect of the tested substances, and especially in the case of MIX 1 (solid line with filled square indicators) and MIX 3 (solid line with filled triangular indicators), was able to amplify permeability across the BBB, confirming the data obtained previously about the correct concentration to be used in the new formulation, and suggesting that a correct treatment dosage might be to be considered as "to be administered once a day."

2.2.3 Validation of the Parkinson's disease (PD) model.

In order to verify the beneficial effect exerted by lipoic acid, EGCG (from Camellia sinensis extract, referred to as "green tea"), DHA and Crocus sativus extract (referred to as "saffron") in a pathological context, it was necessary to mimic dopamine depletion in vitro. Therefore, experiments were conducted using 6-OHDA as a damage inducer, and from the results obtained in a dose-response time-dependent study, it was possible to confirm the damaging effects of 6-OHDA as a Parkinson's inducer. As can be seen from Figure 3, cells were stimulated with different concentrations of 6-OHDA in a range from 200pm to 25pm for a treatment duration from 1 hour to 24 hours (1 and 6 hours Figure 3a; 12 and 24 hours Figure 3b), and although all concentrations are able to induce a damaging effect, only 150pm was able to induce damage similar to what occurs in PD while maintaining a stable effect even at 24 hours. For this reason, 6-OHDA 150pm was used in subsequent experiments to mimic in vitro the molecular mechanisms of alteration attributable to PD.

2.2.4 Evaluation of neuroprotective effects under PD condition

This study aimed to investigate whether the compounds of interest, lipoic acid, EGCG (from Camellia sinensis extract, referred to as "green tea"), DHA and Crocus sativus extract (referred to as "saffron"), and mixtures thereof, were able to stimulate the recovery of biological activities of degenerating neurons. Analysis of mitochondrial metabolism (data shown in Figure 4) shows that 6-OHDA produces cellular damage compared with the untreated control (p<0.05) and that the test substances are able to convert this situation back to a physiological condition. This effect is amplified when the substances are combined together, particularly when combined all four (MIX 1). Mixture 1, in fact, was shown to induce an increase in mitochondrial well-being of 15%, 25%, 17%, and 22% compared to lipoic acid alone in 50pm concentration, "green tea" alone in 100pm concentration, DHA alone in 25pm concentration, and "saffron" alone in 25pm concentration (p<0.05), respectively. All these data suggest that the individual compounds, their combinations, and, in particular, the combination of all four (MIX 1) are able to exert a neuroprotective effect against damage caused by PD.

Since oxidative stress represents one of the main pathogenetic mechanisms of neuronal death, as it causes damage to neurons in the Central Nervous System (CNS) by leading to hyperproduction of free radicals due to increased oxidative metabolism of dopamine, further tests were conducted to assess oxidative stress levels under PD conditions.

The data shown in Figure 5, demonstrate that while PD increases the levels of oxidative stress, the compounds of interest are able to inhibit ROS production (p<0.05) and, especially when combined, they are able to have a stimulatory effect, likely acting on the antioxidant capacities of neuronal cells deputed to restoring homeostasis. These data suggest, therefore, that both the individual compounds and, in particular, their combination and, even more particularly, the mixture 1 that includes all four of them, are capable of exerting a neuroprotective effect against 6-OHDA-generated damage.

In addition, the increase in oxygen free radicals due to the PD condition increases the production of MDA (malonyldialdehyde), causing lipid peroxidation.

The data shown in Figure 6 demonstrate that lipid peroxidation, increased by the presence of 6-OHDA, a model of PD, is reduced following treatment with the substances of interest compared with untreated cells (control) and, more importantly, compared with cells treated with 6-OHDA (p<0.05). Furthermore, the combination of the substances tested, and especially Mixture 1 (MIX 1), that includes all four of them, was able to amplify the neuroprotective effect, reducing MDA production by 3-fold compared with 6-OHDA (p<0.05), suggesting an active role of this new combination in slowing down the disease. Analysis of nitric oxide (NO) production also confirms a negative role of 6-OHDA in neurodegenerative processes, as it is capable of causing the formation of highly reactive species that kill dopaminergic neurons and activated glial cells. Therefore, the inhibition of nitric oxide production by the tested substances, as demonstrated in Figure 7, confirms their potential usefulness for the treatment of Parkinson's disease (p<0.05). Again, their combination, and particularly mixture 1 (MIX 1), enhances their effect, confirming that their synergistic effect plays a key role in modulating Parkinson's disease.

Much scientific evidence accurately indicates that there is a link between inflammation and neurotoxicity identified in Parkinson's disease; therefore, to confirm the assessments made in previous studies, the main inflammatory markers involved in PD processes were also analyzed. As shown in Figure 8, the analysis of TNFo and the cytokines it produces reveals how, following the induction of PD determined by 6-OHDA, the inflammatory picture increases, increasing the production of pro-inflammatory factors such as IL1b and IL6 (p<0.05). Despite this, the substances tested were shown to reduce the production of TNFo (Figure 8a), IL1b (Figure 8b) and IL6 (Figure 8c), compared with the untreated control and compared, especially, with 6-OHDA (p<0.05). Again, especially the mixture of all compounds of interest (MIX 1) is able to amplify the beneficial effect (p<0.05) by bringing the production of inflammatory markers back to threshold levels, suggesting the potent anti-inflammatory role exerted by the new formulation during PD.

Finally, we wanted to analyze the PINK1/Parkin pathway, which is the main molecular mechanism involved during PD. Normally, when PINK1 recognizes defective mitochondria in cells, it activates Parkin, which "cleans" the cell of defective mitochondria, protecting it. As such, research has shown that increased dopamine production leads to an imbalance in mitochondrial metabolism, activating the PINK1/Parkin pathway. Confirming these data, the results obtained from the present studies, shown in Figure 9a for PINK1 and Figure 9b for Parkina, demonstrate the activation of this molecular mechanism following stimulation with 6-OHDA (p<0.05); on the other hand, this situation is down-regulated following the administration of the substances under investigation (p<0.05), which, especially when combined all together (MIX 1), increase the beneficial effect, thanks to their synergistic activity, returning the cells to a condition close to that of the control.

2.3 Conclusion.

In conclusion, from the obtained data shown in Figures 1-9, it can be demonstrated that the compounds of interest, lipoic acid, EGCG (from extract of Camelilla sinensis, referred to as "green tea"), DHA and extract of Crocus sativus (referred to as "saffron"), are able to play a positive role in the treatment of Parkinson's disease.

Notably, these substances are also able to increase the BBB permeability to promote their passage. Moreover, when combined together, and particularly when combined all four together (MIX 1), the positive effect on all studied neuroprotective and molecular parameters is found to be increased compared to their effect when taken individually.

The combination of the analyzed substances is able to maintain proper mitochondrial metabolism, modulate antioxidant and anti-inflammatory effects that, in PD, are impaired, as well as inhibit the main molecular pathways involved during Parkinson's disease by maintaining proper neuronal cell homeostasis.