WO2022029736A1

WO2022029736A1 - Multi -component composition comprising ganoderma lucidum extract, panax extract, vitamins, zinc and bacterial strains and use thereof in the prevention and treatment of flu symptoms and in the increase in immune defences

Info

Publication number: WO2022029736A1
Application number: PCT/IB2021/057324
Authority: WO
Inventors: Valeria CURTI; Rita Paola PETRELLI
Original assignee: Kolinpharma S.P.A.
Priority date: 2020-08-07
Filing date: 2021-08-09
Publication date: 2022-02-10
Also published as: CN116348124A; IT202000019789A1

Abstract

The present invention relates to multicomponent compositions comprising plant extracts, vitamins, minerals, and optionally at least one bacterial strain and a prebiotic, capable of stimulating and/or strengthening the immune system and of preventively and/or curatively treating flu symptoms. Furthermore, the present invention relates to an innovative formulation for oral use in a differentiated release multilayer solid form of said multicomponent compositions of the invention.

Description

DESCRIPTION of the invention having the title:

“MULTI-COMPONENT COMPOSITION COMPRISING GANODERMA LUCIDUM EXTRACT, PANAX EXTRACT, VITAMINS, ZINC AND BACTERIAL STRAINS AND USE THEREOF IN THE PREVENTION AND TREATMENT OF FLU SYMPTOMS AND IN THE INCREASE IN IMMUNE DEFENCES”.

The present invention relates to multi-component compositions comprising plant extracts, vitamins, minerals, and optionally at least one bacterial strain and a prebiotic, capable of stimulating and/or strengthening the immune system and preventively and/or curatively treating flu symptoms, and/or diseases of the respiratory tract, decreasing the duration, severity and frequency thereof. Furthermore, the present invention relates to an innovative formulation of said composition of the invention for oral use in a differentiated release multilayer solid form of the multi-components.

Flu syndrome is a public health problem with a significant impact from the epidemiological, clinical and economic point of view. Flu is actually the first cause of absence from work, resulting in about 10% of all absences.

The frequency with which the flu symptoms arise, whether caused by bacterial or viral infection, although significantly different from season to season, is on average around 9% (range: 4-15%) of the general population, each year, while in the 0-14 age group, which is the most affected one, the incidence is on average about 26% (12-40%).

The typical symptoms are those affecting the respiratory system that arise suddenly, after a generally quite short incubation (about 1-2 days), and usually persist for 3-4 days, but they can last for one or two weeks.

The therapies that can be used are different and vary depending on the aetiological agent that causes the flu syndrome. Generally speaking, an effective approach is to strengthen the immune system of the organism in order to combat the onset of diseases of the respiratory tract, particularly those of the upper airways. Such approach may be either preventive (treatment before the onset of flu symptoms) or curative (treatment upon the onset of the first flu symptoms). Strengthening the immune system and preventing the onset or evolution of a flu syndrome is important, given that an improperly treated infection of the respiratory tract can cause the onset of bacterial comorbidities, forcing the use of antibiotics and therefore contributing to the emergence of antibiotic resistance.

The technical problem addressed and solved by the present invention lies in providing effective and side effect-free compositions for use in a method for the preventive and/or curative treatment of flu symptoms, such as for example those due to infections of the respiratory system, in particular of the upper airways. Furthermore, the present invention addresses and solves the technical problem of providing compositions for the prevention and treatment of flu conditions that allow the subject in need to minimise or avoid taking pharmacological therapies, in particular treatment with antibiotics. Lastly, in the context of the aforementioned technical problem, the present invention addresses and solves the technical problem of providing in a single formulation and in a single dose form the substances useful for the prevention and/or curative treatment of flu syndromes and related symptoms, thus allowing the presence of various kinds of active components (for example, plant extracts, vitamins, minerals, probiotics and/or prebiotics) with the aim of maximising the effectiveness of the product, ensuring a better absorption of the components, as well as avoiding possible gastric side effects of some substances, such as for example the extract of a plant of the genus Panax, preferably Panax ginseng.

In the context of the present invention, the expression "flu symptoms” is used to indicate muscle and joint pain, tiredness/fatigue, cough, fever, headache, sore throat, nasal congestion, shivers.

In the context of the present invention, the expression "respiratory system” is used to indicate the set of organs and structures which allow the gaseous exchanges between the surrounding environment (filled with oxygen) and the human organism (whose blood is loaded with carbon dioxide); specifically, the organs part of such apparatus are: nose, pharynx, larynx, trachea, lungs, pleura, bronchi and bronchioles. In the context of the present invention, the expression "upper respiratory system” is used to indicate: the nasal cavity, paranasal sinus, oral cavity, pharynx, epiglottis and larynx.

In order to overcome said technical problems, following an intense research phase, the Applicant provides multicomponent compositions (compositions of the invention) comprising (a) an extract of Ganoderma lucidum titrated in polysaccharides, (b) an extract of a plant of the genus Panax titrated in ginsenosides, (c) at least one vitamin, (d) zinc, and, optionally, (e) a bacterial strain belonging to the genus Bifidobacterium or Lactobacillus and (f) a prebiotic, as reported in the present description and in the attached claims.

Said compositions of the invention, when administered to a subject in need, are capable of stimulating and strengthening the immune system and, as a result, preventing and/or curing flu symptoms, such as for example symptoms and/or disorders of the respiratory system, in particular of the upper respiratory system, effectively, rapidly and without side effects.

Said preventive and/or curative treatment activity of the compositions of the invention is due to the specific and innovative combination of the active components (from (a) to (f), as defined in the present description) given that, although each component has an immunomodulatory and/or immunostimulatory activity, each component acts by means of a different mechanism of action, making the composition of the invention particularly effective in the action thereof. Furthermore, the active components of the composition of the invention stimulate the immune system and act on physiological mechanisms in a complementary and/or synergistic manner, such to make the compositions of the invention not only effective in the treatment of flu symptoms and/or disorders of the respiratory system, but also capable of treating a wide spectrum of said symptoms and/or disorders.

For example, the fungus Ganoderma lucidum (or the extract thereof) is capable of stimulating the immune system cells (NK, T cells, dendritic cells, macrophages and lymphocytes) to produce and release cytokines and interleukins. The extract of Panax (e.g. Panax Ginseng) is capable of decreasing the duration, severity and frequency of flu symptoms and it can effectively reduce the incidence of flu-like diseases. Vitamin C contributes to the normal function of the immune system and to the maintenance thereof during and after an intense physical effort, it facilitates the reduction of tiredness and fatigue, and it also contributes to normal energy metabolism and to the protection of cells against oxidative stress. Daily consumption of vitamin D has preventive efficacy in reducing the risk of developing infections of the respiratory tract. The group B vitamin complex plays a crucial role in supporting the immune system and the nervous system, it participates in many biological processes and it acts as a cofactor in key enzymatic reactions for the energy metabolism of the cell. A deficiency in this vitamin complex is associated with an increase in levels of inflammation, caused by a high and unbalanced release of proinflammatory cytokines, such as TNF-o, IL-6 and IL-1p. In particular, vitamin B6 affects both the innate and adaptive immune response; a deficiency in this vitamin causes both an inhibition of the release of cytokines and chemokines and a decrease in the proliferation, activation, response and activity of T lymphocytes. Similarly, a deficiency in vitamin B12 is associated with an immunodeficiency caused by non-responsive T lymphocytes, a generalised hypogammaglobulinemia, and an unbalanced proinflammatory cytokine release profile. Among the effects of the vitamins of group B, the following are the most important: vitamin B1 contributes to the normal psychological function, to the normal functioning of the nervous system and to the normal energy metabolism; vitamins B2 and B3 contribute to the maintenance of the normal mucous membranes, they contribute to the protection of cells against oxidative stress and to the reduction of tiredness and fatigue; vitamin B5 contributes to the reduction of tiredness and fatigue and to the maintenance of normal mental performance; vitamins B6 and B12 contribute to the well-being of the immune system, to the reduction of tiredness and fatigue and to the normal psychological function. Zinc contributes to the normal function of the immune system (correct immune response of both innate and adaptive type), to the protection of cells against oxidative stress and it has a direct antiviral action. Various probiotic bacterial strains of the genus Bifidobacterium, such as for example the strain Bifidobacterium lactis BL-04®, are beneficial to the health of the host thanks, but not only, to an immunomodulatory activity (immunobiotic bacterial strain) and to the ability to promote the equilibrium of the gut flora; in particular, the strain Bifidobacterium Lactis BL-04® is capable of modulating the innate response of the host at the nasal level and affect the replication of flu viruses. Lastly, inulin, as prebiotic, stimulates the development and the metabolic action of some bacteria that colonise the colon, especially bifidobacteria and lactobacilli, supporting the health of the gut microbiota; furthermore, inulin acts on the immune system altering the concentration of bacterial lactic acid and, as a result, stimulating (indirectly) the action of T cells, NK cells and macrophages against pathogens.

Furthermore, in order to overcome the aforementioned technical problems, the Applicant devised a formulation for oral use in solid form of differentiated release multilayer tablet of the compositions of the invention (in short, formulation of the invention). Said formulation is structured so as to comprise two or three or four different layers, each layer comprising different active compounds, and each layer having a variable release time of said active compounds (for example, a rapid release, an intermediate release and/or a prolonged release) into the gastrointestinal tract. Said differentiated release formulation allows a better intestinal absorption of each active compound of the composition and, as a result, a greater therapeutic efficacy thereof given that: 1) when formulating the components, the preferential absorption sites of each substance at the level of the gastrointestinal tract were taken into account, so that the substances are released predominantly in the preferential tract 2) it prolongs the efficacy of the formulation, 3) it provides greater tolerability of a multicomponent composition by the organism, 4) it eliminates possible gastric side effects.

As a matter of fact, a differentiated release formulation of a multicomponent composition allows to dose a pool of active compounds to a subject in a single administration, which is easier for the subject, but at the same time, not to overload the organism of the subject with endogenous substances and not to have interference in the gastrointestinal absorption of said active compounds and/or interference between the mechanisms of action thereof.

Showing a high safety profile, the compositions of the invention can be used by a broad category of subjects, such as adults, the elderly and sportsmen and sportswomen. The mixtures and compositions and formulations of the invention are easy to prepare and cost-effective.

These and other objects which will be apparent from the detailed description that follows are achieved by the mixtures, compositions and formulations of the present invention thanks to the technical characteristics present in description and in the attached claims. DETAILED DESCRIPTION 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:

(a) an extract of Ganoderma lucidunr,

(b) an extract of a plant of the genus Panax,

(c) at least one vitamin;

(d) zinc; and, optionally,

(e) at least one bacterial strain belonging to the genus Bifidobacterium or Lactobacillus (probiotic strain); and/or

(f) at least one prebiotic.

In a preferred embodiment of the present invention, said mixture comprises or, alternatively, consists of:

(a) an extract of Ganoderma lucidum titrated in polysaccharides (% w/w of polysaccharides: from 5% to 60% or from 20% to 40% or from 25% to 35%);

(b) an extract of a plant of the genus Panax titrated in ginsenosides (% w/w of ginsenosides: from 5% to 50% or from 10% to 30% or from 15% to 25%), wherein said plant of the genus Panax, is selected from the group comprising or, alternatively, consisting of: Panax ginseng, Panax notoginseng, Panax pseudoginseng, Panax guinguefolium and mixtures thereof, preferably Panax ginseng,

(c) at least one vitamin selected from the group comprising or, alternatively consisting of: (c.1) vitamin C, (c.2) vitamin D, (c.3) at least one vitamin of group B (such as B1, B2, B3, B5, B6, B9 and/or B12) and a mixture thereof, preferably said (c) comprises or, alternatively, consists of (c.1), (c.2) and (c.3);

(d) zinc selected from the group comprising or, alternatively consisting of: zinc oxide, zinc bisglycinate and zinc sulfate, preferably zinc oxide; and, optionally,

(e) at least one bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably belonging to the species selected from the group comprising or, alternatively, consisting of Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus rhamnosus, Lactobacillus helveticus, more preferably selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Bifidobacterium breve BR-03 (DSM 16604), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS 116411); and optionally,

(f) at least one prebiotic selected from the group comprising or, alternatively, consisting of: an inulin, a fructo-oligosaccharide (FOS), a galacto-oligosaccharide (GOS), a xylo-oligosaccharide (XOS), more preferably inulin. Preferably, said mixture of the invention comprises (a), (b), (c), (d) and (e), even more preferably said mixture of the invention comprises (a), (b), (c), (d), (e) and (f), wherein (a), (b), (c), (d), (e) and (f) are the active components as defined in the present description.

In the context of the present invention the terms "titrated in” and "comprising” are synonyms and used interchangeably.

A second aspect of the present invention relates to a composition (in short, composition of the invention) comprising: said mixture of the invention comprising or, alternatively, consisting of (a), (b), (c), (d), and, optionally, (e) and/or (f) (according to any one of the described embodiments or aspects), and said composition further comprises at least one acceptable pharmaceutical or food grade additive and/or excipient.

A third aspect of the present invention relates to a formulation (in short, formulation of the invention) in solid form of a multilayer tablet (with 2 or 3 or 4 layers, preferably with 3 layers) with differentiated release comprising the mixture or composition of the invention comprising or, alternatively, consisting of (a), (b), (c), (d), and optionally (e) and/or (f), according to any one of the described embodiments or aspects.

A fourth aspect of the present invention relates to said mixture or composition or formulation of the invention for use in a preventive and/or curative method as defined in the context of the present description.

Ganoderma lucidum (Curtis) P. Karst, (synonyms: Reishi, Ling zhi) is a saprophytic and sporigenic fungus originating in China and Japan, used in oriental medicine for various therapeutic purposes (Scientific classification: Fungus kingdom, Basidiomycota division, Agaricomycetes class, Polyporales order, Ganodermataceae family).

In the mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), and optionally (e) and/or (f), according to any one of the described embodiments or aspects), preferably said (a) extract of Ganoderma lucidum comprises polysaccharides in a percentage by weight comprised in a range from 5% to 60% (for example, 10%, 45% or 50%), preferably from 20% to 40% (for example 22%, 24%, 36% or 38%), more preferably from 25% to 35% (for example 26%, 28%, 30%, 32% or 34%), with respect to the total weight of (a). Said polysaccharides comprise or consist of linear or branched polysaccharides having an average molecular weight comprised from 10 kDa (KiloDaltons) to 10,000 kDa, preferably from 10 kDa to 1,000 kDa (for example, 100 kDa, 200 kDa, 500 kDa, or 800 kDa). The polysaccharide content in the extract of Ganoderma lucidum may be determined by means of titration methods (for example titration by means of UV technique) and standard equipment known to the person skilled in the art.

The extract of (a) Ganoderma lucidum comprising polysaccharides used in the present invention is preferably obtained by extracting the fungus (or parts of the fungus) according to the methods and equipment known to the person skilled in the art. For example, the extract of (a) may be obtained by means of a step for extracting with hydroalcoholic solvent (for example water-ethanol mixture) to obtain an extraction liquid. Said extraction liquid is subsequently dried (for example, by means of spray drying or techniques known to the person skilled in the art) and the obtained dry extract, crushed and sieved to obtain a fine powder, which is lastly mixed to obtain the extract of Ganoderma lucidum comprising polysaccharides.

Panax L. (Linnaeus), also known as Ginseng, is a genus of plant originating in the eastern mountainous regions, between China and Korea, known for the tonic-adaptogenic properties of its roots (Scientific classification: Plantae kingdom, Magnoliophyta division, Magnoliopsida class, Asteridae subclass, Apiales order, Araliaceae family, Aralioidae subfamily). In the mixture or composition of the present invention, as the plant of the genus Panax to obtain said extract (b) there may be used a plant belonging to a species selected from the group comprising or, alternatively, consisting of: Panax ginseng {Panax ginseng Meyer), Panax notoginseng {Panax notoginseng Chen.), Panax pseudoginseng {Panax pseudoginseng Wall.), Panax guinguefolium {Panax guinguefolium L.) and mixtures thereof.

In the mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), and, optionally, (e) and/or (f), according to any one of the described embodiments or aspects), preferably said (b) extract of a Panax plant, preferably Panax ginseng, comprises ginsenosides in a percentage by weight comprised in a range from 5% to 50% (for example, 8%, 40% or 45%), preferably from 10% to 30% (for example 12%, 14%, 26% or 28%), more preferably from 15% to 25% (for example 16%, 18%, 20%, 22% or 24%), with respect to the total weight of (b). The ginsenoside content in the extract of a Panax plant, preferably Panax ginseng, may be determined by means of titration methods and standard equipment known to the person skilled in the art; for example by means of HPLC or HPTLC with respect to the reference standard. The extract of (b) a plant of the genus Panax, preferably Panax ginseng, comprising ginsenosides used in the present invention is preferably obtained by extracting roots according to methods known to the person skilled in the art. For example, the extract of (b) may be obtained from the roots of the plant by means of a step for extracting with hydroalcoholic solvent (for example water-ethanol in an about 8:1 w/w ratio) to obtain an extraction liquid. Said extraction liquid is subsequently dried (for example, by means of spray drying or techniques known to the person skilled in the art) and the extract obtained, crushed and sieved to obtain a fine powder, which is lastly mixed to obtain the extract of Panax, preferably Panax ginseng. The mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), and, optionally, (e) and/or (f), according to any one of the described embodiments or aspects), may comprise said (a) Ganoderma lucidum extract comprising polysaccharides in a percentage by weight comprised from 5% to 60% with respect to the total weight of (a) and said (b) extract of a Panax plant, preferably Panax ginseng, comprising ginsenosides in a percentage by weight comprised from 5% to 50% with respect to the total weight of (b), preferably (a) from 20% to 40% and (b) from 10% to 30%, more preferably (a) from 25% to 35% (for example 30%) and (b) from 15% to 25% (e.g. 20%).

In the mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), and, optionally, (e) and/or (f), according to any one of the described embodiments or aspects), preferably said (c) at least one vitamin comprises or, alternatively, consists of: (c.1) vitamin C (such as ascorbic acid or ascorbate), (c.2) vitamin D, preferably vitamin D3 (cholecalciferol), and (c.3) vitamins of group B comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9 (synonym: folic acid), and (c.3-vii) vitamin B12.

In the mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), and, optionally,

(e) and/or (f), according to any one of the described embodiments or aspects), preferably said (d) zinc (elemental zinc) is understood to be zinc oxide (ZnO, example of CAS no. 1314-13-2), zinc bisglycinate (form of chelated zinc, surrounded by amino acids; of synthetic origin), or zinc sulfate (ZnSO4, example of CAS no. 7733-02-0).

In the mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), (e) and optionally

(f), according to any one of the described embodiments or aspects), said (e) at least one bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably belonging to the species selected from the group comprising or, alternatively, consisting of Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus rhamnosus, Lactobacillus helveticus, more preferably it is selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (deposit number SD5219; also known as DGCC2908 and RB 4825) (Danisco), Bifidobacterium breve BR-03 (deposit number DSM 16604) (Probiotical), Lactobacillus rhamnosus crl1505 (Sacco), Lactobacillus rhamnosus GG (deposit number ATCC 53103) (Hansen), Lactobacillus helveticus Lafti® L-10 (deposit number CBS 116411) (Lallemand), and a mixture thereof, or, alternatively, from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Bifidobacterium breve BR-03 (DSM 16604), Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS 116411), and a mixture thereof (ATCC, abbreviation of American Type Culture Collection,' DMS: abbreviation of Das Leibniz-lnstitut Deutsche Sammlung von Mikroorganismen und Zellkulturem, CBS: abbreviation of Centraalbureau voor Schimmelcultures). Said (e) bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably selected from the strains Bifidobacterium lactis BL-04® (SD5219) Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), may be present in the mixture or composition or formulation of the invention in form of live and viable strain (synonym: probiotic strain) or inactivated strain (for example, inactivated by means of tyndallization or gamma irradiation or sonication) or derivative of the probiotic strain.

In the context of the present invention, the expression "derivative” of the probiotic strain is used to indicate a parabiotic or a postbiotic, such as for example the lysates or homogenates of the bacterial strain, the extracts or the parietal fraction of the bacterial strain, the metabolites or metabolic bioproducts or exopolysaccharides (EPS) generated by the bacterial strain and/or any other product derived from bacterial strain known to the person skilled in the art. Said derivatives are obtained according to methods and equipment known to the person skilled in the art. Preferably, the expression "derivative” of the bacterial strain is used to indicate: the lysates or homogenates of the bacterial strain, the extracts or parietal fraction of the bacterial strain.

In the mixture or composition or formulation of the invention (comprising (a), (b), (c), (d), (e) and (f), according to any one of the described embodiments or aspects), preferably said (f) prebiotic is selected from the group comprising or, alternatively, consisting of: an inulin, a fructo-oligosaccharide (FOS), a galacto-oligosaccharide (GOS), a xylo-oligosaccharide (XOS), and a mixture thereof; preferably said (f) prebiotic comprises or, alternatively, consists of an inulin (polysaccharide of plant nature, example of CAS no. 9005-80-5, chemical formula CenHwn^Osn+i).

Said mixture of the invention (comprised in the composition or formulation of the invention) may comprise or, alternatively, consist of: said (a) extract of Ganoderma lucidum titrated in polysaccharides (% w/w of polysaccharides: from 5% to 60% or from 20% to 40% or from 25% to 35%); said (b) said extract of a Panax ginseng plant titrated in ginsenosides (% w/w of ginsenosides: from 5% to 50% or from 10% to 30% or from 15% to 25%); said (c) at least one vitamin comprising or, alternatively consisting of: (c.1) vitamin C, (c.2) vitamin D, and (c.3) at least one vitamin of group B (such as B1, B2, B3, B5, B6, B9 and/or B12); (d) zinc (elemental zinc), preferably wherein said zinc is selected from the group comprising or, alternatively, consisting of: zinc oxide, zinc bisglycinate and zinc sulfate, more preferably zinc oxide.

Said mixture of the invention (comprised in the composition or formulation of the invention) may comprise or, alternatively, consist of: said (a) extract of Ganoderma lucidum titrated in polysaccharides (% w/w of polysaccharides: from 5% to 60% or from 20% to 40% or from 25% to 35%); said (b) said extract of a Panax ginseng plant titrated in ginsenosides (% w/w of ginsenosides: from 5% to 50% or from 10% to 30% or from 15% to 25%); said (c) at least one vitamin comprising or, alternatively consisting of: (c.1) vitamin C, (c.2) vitamin D, and (c.3) at least one vitamin of group B (such as B1, B2, B3, B5, B6, B9 and/or B12); (d) zinc (elemental zinc), preferably wherein said zinc is selected from the group comprising or, alternatively, consisting: zinc oxide, zinc bisglycinate and zinc sulfate, more preferably zinc oxide; said (e) strain selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Bifidobacterium breve BR-03 (DSM 16604), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114) (live and viable strain (probiotic) or inactivated strain); and, optionally, said (f) prebiotic, preferably inulin.

Said mixture of the invention (comprised in the composition or formulation of the invention) may comprise or, alternatively, consist of: (a) an extract of Ganoderma lucidum titrated in polysaccharides (% w/w of polysaccharides: from 5% to 60% or from 20% to 40% or from 25% to 35%); said (b) extract of a Panax ginseng plant titrated in ginsenosides (%w/w of ginsenosides: from 5% to 50% or from 10% to 30% or from 15% to 25%); said (c.1) vitamin C, (c.2) vitamin D, preferably vitamin D3, (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12; said (d) zinc (elemental zinc), preferably zinc oxide; said (e) strain selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Bifidobacterium breve BR-03 (DSM 16604), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114) (live and viable strain (probiotic) or inactivated strain); and, optionally, said (f) prebiotic, preferably inulin.

Said mixture of the invention (comprised in the composition or formulation of the invention) may comprise or, alternatively, consist of: (a) an extract of Ganoderma lucidum titrated in polysaccharides(% w/w of polysaccharides: from 5% to 60% or from 20% to 40% or from 25% to 35%); said (b) extract of a plant of the genus Panax titrated in ginsenosides (% w/w of ginsenosides: from 5% to 50% or from 10% to 30% or from 15% to 25%), wherein said plant of the genus Panax, is selected from the group comprising or, alternatively, consisting of: Panax ginseng, Panax notoginseng, Panax pseudoginseng, Panax guinguefolium and mixtures thereof, preferably Panax ginseng, said (c.1) vitamin C, (c.2) vitamin D, preferably vitamin D3, (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12; said (d) zinc (elemental zinc), preferably zinc oxide; said (e) said (e) strain Bifidobacterium lactis BL-04® SD5219 (live and viable strain (probiotic) or inactivated strain); and, optionally, said (f) prebiotic, preferably inulin.

Preferably, said mixture of the invention (comprised in the composition or formulation of the invention) comprises or, alternatively, consists of: said (a) extract of Ganoderma lucidum comprising polysaccharides from 5% to 60% by weight, said (b) extract of a Panax ginseng plant comprising ginsenosides from 5% to 50% by weight, preferably (a) from 20% to 40% and (b) from 10% to 30%, more preferably (a) from 25% to 35% and (b) from 15% to 25%; said (c.1) vitamin C, (c.2) vitamin D, preferably vitamin D3, (c.3-i) vitamin B1 , (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12, (d) zinc, preferably zinc oxide, said (e) strain Bifidobacterium lactis BL-04® SD5219 (live and viable strain (probiotic) or inactivated strain), and said (f) inulin.

According to a preferred aspect, the composition of the invention is formulated for oral (or sublingual) administration.

The dosage form of the composition of the invention may be a solid form, such as tablet, chewable tablet, capsule, lozenge, granules or powder (granules or powder to be dissolved in water or mouth dissolvable), 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 solid form for oral use, more preferably in tablet form.

According to an aspect of the present invention, the composition of the invention (comprising (a), (b), (c), (d), and optionally (e) and/or (f), according to any one of the described embodiments or aspects) is formulated in solid form of a differentiated release multilayer tablet (in short, formulation of the invention). Said formulation of the invention, for example two-layer formulation, may comprise:

(I) a quick release layer comprising said (a) Ganoderma lucidum extract comprising polysaccharides (% w/w of polysaccharides: 5% to 60% or from 20% to 40% or from 25% to 35%), said (c) at least one vitamin, preferably (c.2) vitamin D and/or (c.3) at least one vitamin of group B, and said (d) zinc (for example, zinc oxide); wherein said quick release layer (I) disintegrates and releases (a), (c) and (d) in the intestinal tract over a period of time comprised from 30 minutes to 120 minutes subsequent to the administration of the composition to a subject, preferably comprised from 60 minutes to 120 minutes, more preferably comprised from 75 minutes to 105 minutes; and

(III) a prolonged release layer comprising said (b) extract of a plant of the genus Panax, preferably Panax ginseng, comprising ginsenosides (% w/w of ginsenosides: from 5% to 50% or from 10% to 30% or from 15% to 25%) and, optionally, (c.1) vitamin C, wherein said (III) prolonged release layer disintegrates and releases (b) (and, optionally, (c.1)) in the intestinal tract over a period of time comprised from 180 minutes to 270 minutes subsequent to the administration of the composition to said subject, preferably comprised from 180 minutes to 240 minutes, more preferably comprised from 180 minutes to 210 minutes.

Said formulation of the invention may comprise:

(II) an intermediate release layer comprising said (e) bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably the strain Bifidobacterium lactis BL-04® SD5219, and said (f) prebiotic, preferably an inulin, wherein said (II) intermediate release layer disintegrates and releases (e) and (f) in the intestinal tract over a period of time comprised from 120 minutes to 180 minutes subsequent to the administration of the composition to said subject, preferably comprised from 120 minutes to 160 minutes, more preferably comprised from 120 minutes to 140 minutes; and

According to a preferred embodiment, said differentiated release multilayer tablet of the invention comprises three layers, wherein said (I) quick release layer comprises (a), (d), (c.2) and (c.3), preferably wherein said (c.3) at least one vitamin of group B is selected from the group comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5 (c.3-v), vitamin B6, (c.3-vi) vitamin B9, (c.3-vii) vitamin B12, and a mixture thereof (preferably said (c.3) is a mixture comprising (c.3-i), (c.3-ii), (c.3-iii), (c.3-iv), (c.3-v), (c.3-vi) and (c.3-vii)); said (II) intermediate release layer comprises (e) and (f); and said (III) prolonged release layer comprises (b) and (c.1 ).

For example, the formulation of the invention of a differentiated release multilayer (triple-layer) tablet advantageously comprises:

- said quick release layer (I) comprising or, alternatively, consisting of: the active compounds (a), (d) and, optionally, (c.2) and/or (c.3), and additives and/or excipients of layer (I) selected from the group comprising or, alternatively, consisting of: alkali or alkali-earth metal stearate (for example of magnesium), silicon dioxide, mono- and diglycerides of fatty acids, microcrystalline cellulose, hydroxypropyl methylcellulose, natural or artificial flavours and mixtures thereof;

- said intermediate release layer (II) comprising or, alternatively, consisting of: active compounds (e) and, optionally, (f) and additives and/or excipients of layer (II) selected from the group comprising or, alternatively, consisting of: phosphate buffer (for example dicalcium phosphate), alkali or alkali-earth metal stearate (for example of magnesium), silicon dioxide, microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, starch or corn starch, and mixtures thereof;

- said prolonged release layer (III) comprising or, alternatively, consisting of: the active compound (b) and, optionally, (c.1) and additives and/or excipients of layer (III) selected from the group comprising or, alternatively, consisting of: alkali or alkali-earth metal stearate (for example of magnesium), silicon dioxide, microcrystalline cellulose, hydroxypropyl methylcellulose, and mixtures thereof.

It is understood that the amount and the by weight ratios between the various additives and/or excipients of each layer of said multilayer formulation (for example, layer (I), (II) and/or (III)) will be determined and varied by the person skilled in the art in order to obtain the desired release timing, according to known methods of the field of solid formulations for oral use.

Said (b) extract of a plant of the genus Panax, preferably Panax ginseng, is advantageously inserted into the prolonged release layer (III) given that it allows to avoid release at the gastric level, which could cause gastric side effects in hypersensitive subjects, and be absorbed mainly at the intestinal level.

Furthermore, the probiotic bacterial strain (viable or inactivated) (e) and inulin (f) are advantageously inserted into the intermediate release layer (II) given that it allows to isolate them in a dedicated layer, hence the viability of the probiotic bacterial strain is not affected by the presence of other substances such as, for example, extracts of plant origin. The bacterial strain present in layer (II) is a gastro-resistant strain, therefore the timing of gastro-intestinal release does not affect the viability thereof.

Said differentiated release three-layer tablet formulation of the invention may comprise said intermediate release layer (II) in a position between (between) said quick release layer (I) and said prolonged release layer (III), wherein said layers (I), (II) and (III) are characterised according to any one of the described embodiments.

The mixture or composition or formulation of the invention may advantageously comprise, for "daily dose units”, preferably in solid form (such as, for example, a differentiated release two-layer or three-layer tablet), the following amounts: (a) a Ganoderma lucidum extract titrated in polysaccharides in a percentage by weight from 5% to 60% or from 20% to 40% or from 25% to 35% (e.g. dry extract, 30% polysaccharides), in an amount comprised from 10 mg to 500 mg, preferably comprised from 50 mg to 300 mg, more preferably comprised from 100 mg to 200 mg (for example, about 150 mg of which 45 mg of polysaccharides);

(b) an extract of a plant of the genus Panax, preferably Panax ginseng, titrated in ginsenosides in a percentage by weight comprised from 5% to 50% or from 10% to 30% or from 15% to 25% (e.g. dry root extract, 20% ginsenosides) in an amount comprised from 1 mg to 300 mg, preferably comprised from 10 mg to 150 mg, more preferably comprised from 20 mg to 75 mg (for example, about 45 mg of which 9 mg of ginsenosides);

(c) at least one vitamin according to the nutrient reference value pursuant to Reg. EU 1169/2011 (in short, NRV), preferably wherein said vitamins are selected from vitamin C, vitamin D, a vitamin of group B and a mixture thereof;

(d) zinc (for example zinc oxide) in an amount as elemental zinc comprised from 1 mg to 30 mg, preferably comprised from 5 mg to 30 mg, more preferably comprised from 5 mg to 15 mg (e.g. 6mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg or 14 mg; 15 mg/day is the maximum daily dose for adult subject currently admitted in Italy); and, optionally, (e) a bacterial strain belonging to the genus Bifidobacterium or Lactobacillus (preferably selected from the strains Bifidobacterium Lactis BL-04® SD5219, Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), or Lactobacillus helveticus Lafti® L-10 (CBS116114)) in an amount comprised from 10⁷ CFUs to 10¹² CFUs, preferably from 10⁹ CFUs to 10¹⁰ CFUs, more preferably about 2 x 10⁹ CFUs (CFUs: Colony Forming Units); and, optionally, (f) inulin in an amount comprised from 10 mg to 500 mg, preferably comprised from 10 mg to 350 mg, more preferably comprised from 50 mg to 150 mg (for example, about 60 mg, 70 mg, 80 mg, 90 mg or 100 mg, preferably about 100 mg).

(c.1) vitamin C in an amount comprised from 10 mg to 500 mg, preferably comprised from 50 mg to 300 mg, more preferably comprised from 100 mg to 200 mg (for example about 150-160 mg and % NRVs 200); (c.2) vitamin D in an amount comprised from 0.5 pig to 100 pig, preferably comprised from 2 pig to 50 pig, more preferably comprised from 5 pig to 30 pig (for example about 18-20 pig and % NRVs 360); (c.3) vitamins of group B comprising or, alternatively, consisting of: (c.3-i) vitamin B1 in an amount comprised from 0.05 mg to 30 mg, preferably comprised from 0.1 mg to 10 mg, more preferably comprised from 0.5 mg to 3 mg (for example about 2.2 mg and % NRVs 200); (c.3-ii) vitamin B2 in an amount comprised from 0.05 mg to 35 mg, preferably comprised from 0.1 mg to 10 mg, more preferably comprised from 1 mg to 3.5 mg (for example about 2.8 mg and % NRVs 200); (c.3-iii) vitamin B3 in an amount comprised from 1 mg to 100 mg, preferably comprised from 10 mg to 50 mg, more preferably comprised from 20 mg to 30 mg (for example about 24-25 mg and % NRVs 150); (c.3-iv) vitamin B5 in an amount comprised from 0.5 mg to 50 mg, preferably comprised from 1 mg to 30 mg, more preferably comprised from 5 mg to 15 mg (for example about 9-10 mg and % NRVs 150); (c.3-v) vitamin B6 in an amount comprised from 0.05 mg to 50 mg, preferably comprised from 0.1 mg to 10 mg, more preferably comprised from 1 mg to 3.5 mg (for example about 2.8 mg and % NRVs 200); (c.3-vi) vitamin B9 in an amount comprised from 10 pig to 1000 pig, preferably comprised from 50 pig to 750 pig, more preferably comprised from 100 pig to 500 pig (for example about 400 pig and % NRVs 200), and (c.3-vii) vitamin B12 in an amount comprised from 0.1 pig to 50 pig, preferably comprised from 0.5 pig to 15 pig, more preferably comprised from 1 pig to 7 pig (for example about 5 pig and % NRVs 200);

(d) zinc, preferably in form of zinc oxide, in an amount as elemental zinc comprised from 1 mg to 40 mg, preferably comprised from 1 mg to 30 mg, more preferably comprised from 5 mg to 15 mg (e.g. 15 mg); and, optionally, (e) a bacterial strain belonging to the genus Bifidobacterium or Lactobacillus (preferably selected from the strains Bifidobacterium Lactis BL-04® SD5219, Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), or Lactobacillus helveticus Lafti® L-10 (CBS116114)) in an amount comprised from 10⁷ CFUs to 10¹² CFUs, preferably from 10⁹ CFUs to 10¹⁰ CFUs, more preferably about 2 x 10⁹ CFUs (CFUs: Colony Forming Units); and, optionally, (f) inulin in an amount comprised from 10 mg to 500 mg, preferably comprised from 10 mg to 350 mg, more preferably comprised from 50 mg to 150 mg (for example about 100 mg).

Said "daily dose unit” of the composition of the invention may be administered to a subject in need over the 24-hour interval by means of a single dose or divided into 2, 3 or 4 doses at 4-hour to 12-hour intervals, depending on the type of dosage form and the needs of the subject. Preferably, said "daily dose unit” of the composition of the invention is administered to a subject in need once a day (for example, away from meals) in the form of two-layer or three-layer tablet according to a described embodiment of the formulation of the present invention.

For example, a differentiated release two-layer tablet, as formulation of the composition of the invention (equivalent to a daily dose), comprises the following amounts per layer: - said (I) quick release layer comprising: (a) a Ganoderma lucidum extract titrated in polysaccharides in a percentage by weight from 5% to 60% or from 20% to 40% or from 25% to 35% (e.g. dry extract, about 30% polysaccharides), in an amount comprised from 10 mg to 500 mg, preferably comprised from 50 mg to 300 mg, more preferably comprised from 100 mg to 200 mg (for example about 150 mg of which 45 mg of polysaccharides);

(d) zinc, preferably in form of zinc oxide, in an amount as elemental zinc comprised from 1 mg to 40 mg, preferably comprised from 1 mg to 30 mg, more preferably comprised from 5 mg to 15 mg (e.g. about 15 mg);

(c) at least one vitamin, preferably selected from (c.2) vitamin D according to the NRVs and/or (c.3) vitamins of group B according to the NRVs; and

- said (III) prolonged release layer comprising: (b) an extract of a plant of the genus Panax, preferably Panax ginseng, titrated in ginsenosides in a percentage by weight comprised from 5% to 50% or from 10% to 30% or from 15% to 25% (e.g. dry root extract, about 20% ginsenosides) in an amount comprised from 1 mg to 300 mg, preferably comprised from 10 mg to 150 mg, more preferably comprised from 20 mg to 75 mg (for example about 45 mg of which 9 mg of ginsenosides); and, optionally, (c.1) vitamin C according to the NRVs.

For example, a differentiated release three-layer tablet, as formulation of the composition of the invention (equivalent to a daily dose), comprises the following amounts per layer:

- said (I) quick release layer comprising: (a) a Ganoderma lucidum extract titrated in polysaccharides in a percentage by weight from 5% to 60% or from 20% to 40% or from 25% to 35% (e.g. dry extract, about 30% polysaccharides), in an amount comprised from 10 mg to 500 mg, preferably comprised from 50 mg to 300 mg, more preferably comprised from 100 mg to 200 mg (for example about 150 mg of which 45 mg of polysaccharides);

- said (II) intermediate release layer comprising: (e) a bacterial strain belonging to the genus Bifidobacterium or Lactobacillus (preferably selected from the strains Bifidobacterium Lactis BL-04® SD5219, Lactobacillus rhamnosus crl 1505, Lactobacillus rhamnosus GG (ATCC 53103), or Lactobacillus helveticus Lafti® L-10 (CBS116114)) in an amount comprised from 10⁷ CFUs to 10¹² CFUs, preferably from 10⁹ CFUs to 10¹⁰ CFUs, more preferably about 2 x 10⁹ CFUs; and, optionally, (f) inulin in an amount comprised from 10 mg to 500 mg, preferably comprised from 10 mg to 350 mg, more preferably comprised from 50 mg to 150 mg (for example about 100 mg); and

According to a preferred example, a differentiated release three-layer tablet, as formulation of the composition of the invention (equivalent to a daily dose), comprises the following amounts per layer:

(c.2) vitamin D in an amount comprised from 0.5 pig to 100 pig, preferably comprised from 2 pig to 50 pig, more preferably comprised from 5 pig to 30 pig (for example about 18-20 pig and % NRVs 360); (c.3) vitamins of group B comprising or, alternatively, consisting of: (c.3-i) vitamin B1 in an amount comprised from 0.05 mg to 30 mg, preferably comprised from 0.1 mg to 10 mg, more preferably comprised from 0.5 mg to 3 mg (for example about 2.2 mg and % NRVs 200); (c.3-ii) vitamin B2 in an amount comprised from 0.05 mg to 35 mg, preferably comprised from 0.1 mg to 10 mg, more preferably comprised from 1 mg to 3.5 mg (for example about 2.8 mg and % NRVs 200); (c.3-iii) vitamin B3 in an amount comprised from 1 mg to 100 mg, preferably comprised from 10 mg to 50 mg, more preferably comprised from 20 mg to 30 mg (for example about 24-25 mg and % NRVs 150); (c.3-iv) vitamin B5 in an amount comprised from 0.5 mg to 50 mg, preferably comprised from 1 mg to 30 mg, more preferably comprised from 5 mg to 15 mg (for example about 9-10 mg and % NRVs 150); (c.3-v) vitamin B6 in an amount comprised from 0.05 mg to 50 mg, preferably comprised from 0.1 mg to 10 mg, more preferably comprised from 1 mg to 3.5 mg (for example about 2.8 mg and % NRVs 200); (c.3-vi) vitamin B9 in an amount comprised from 10 pig to 1000 pig, preferably comprised from 50 pig to 750 pig, more preferably comprised from 100 pig to 500 pig (for example about 400 pig and % NRVs 200), and (c.3-vii) vitamin B12 in an amount comprised from 0.1 pig to 50 pig, preferably comprised from 0.5 pg to 15 pg, more preferably comprised from 1 pg to 7 pg (for example about 5 pg and % NRVs 200); and

- said (II) intermediate release layer comprising: (e) a bacterial strain belonging to the genus Bifidobacterium or Lactobacillus (preferably selected from the strains Bifidobacterium Lactis BL-04® SD5219, Lactobacillus rhamnosus crl 1505, Lactobacillus rhamnosus GG (ATCC 53103), or Lactobacillus helveticus Lafti® L-10 (CBS116114)) in an amount comprised from 10⁷ CFUs to 10¹² CFUs, preferably from 10⁹ CFUs to 10¹⁰ CFUs, more preferably about 2 x 10⁹ CFUs; and (f) inulin in an amount comprised from 10 mg to 500 mg, preferably comprised from 10 mg to 350 mg, more preferably comprised from 50 mg to 150 mg (for example about 100 mg); and

- said (III) prolonged release layer comprising: (b) an extract of a plant of the genus Panax, preferably Panax ginseng, titrated in ginsenosides in a percentage by weight comprised from 5% to 50% or from 10% to 30% or from 15% to 25% (e.g. dry root extract, about 20% ginsenosides) in an amount comprised from 1 mg to 300 mg, preferably comprised from 10 mg to 150 mg, more preferably comprised from 20 mg to 75 mg (for example about 45 mg of which 9 mg of ginsenosides);

(c.1) vitamin C in an amount comprised from 10 mg to 500 mg, preferably comprised from 50 mg to 300 mg, more preferably comprised from 100 mg to 200 mg (for example about 150-160 mg and % NRVs 200).

According to a further preferred example, a differentiated release three-layer tablet, as formulation of the composition of the invention (equivalent to a daily dose), comprises the following amounts per layer:

- said (I) quick release layer comprising: (a) a Ganoderma lucidum extract titrated in polysaccharides in a percentage by weight from 25% to 35% (e.g. dry extract, about 30% polysaccharides), in an amount comprised from 100 mg to 200 mg (for example about 150 mg of which 45 mg of polysaccharides);

(c.2) vitamin D in an amount comprised from 5 pig to 30 pig (for example about 18-20 pig and % NRVs 360); (c.3) vitamins of group B comprising or, alternatively, consisting of: (c.3-i) vitamin B1 in an amount comprised from 0.5 mg to 3 mg (for example about 2.2 mg and % NRVs 200); (c.3-ii) vitamin B2 in an amount comprised from 1 mg to 3.5 mg (for example about 2.8 mg and % NRVs 200); (c.3-iii) vitamin B3 in an amount comprised from 20 mg to 30 mg (for example about 24-25 mg and % NRVs 150); (c.3-iv) vitamin B5 in an amount comprised from 5 mg to 15 mg (for example about 9-10 mg and % NRVs 150); (c.3-v) vitamin B6 in an amount comprised from 1 mg to 3.5 mg (for example about 2.8 mg and % NRVs 200); (c.3-vi) vitamin B9 in an amount comprised from 100 pig to 500 pig (for example about 400 pig and % NRVs 200), and (c.3-vii) vitamin B12 in an amount comprised from 1 pig to 7 pig (for example about 5 pig and % NRVs 200);

(d) zinc, preferably in the form of zinc oxide, in an amount of elemental zinc comprised from 5 mg to 15 mg (for example about 15 mg); and - said (II) intermediate release layer comprising: (e) a bacterial strain selected from the strains Bifidobacterium Lactis BL-04® SD5219, Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), or Lactobacillus helveticus Lafti® L-10 (CBS116114), preferably Bifidobacterium Lactis BL- 04® SD5219, in an amount of about 2 x 10⁹ CFUs; and (f) inulin in an amount comprised from 50 mg to 150 mg (for example about 100 mg); and

- said (III) prolonged release layer comprising: (b) a Panax ginseng extract titrated in ginsenosides in a percentage by weight comprised from 15% to 25% (e.g. dry root extract, about 20% ginsenosides) in an amount comprised from 20 mg to 75 mg (for example about 45 mg of which 9 mg of ginsenosides);

(c.1) vitamin C in an amount comprised from 100 mg to 200 mg (for example about 150-160 mg and % NRVs 200).

Said differentiated release three-layer tablet may have a thickness of from 10 mm to 20 mm.

Forming an object of the present invention is the mixture or composition of the invention (comprising (a), (b), (c), (d), and, optionally, (e) and/or (f), according to any one of the described embodiments or aspects) and/or the differentiated release multilayer formulation thereof according to the invention for use as medicament.

Forming an object of the present invention is the mixture or composition of the invention (comprising (a), (b), (c), (d), and, optionally, (e) and/or (f), according to any one of the described embodiments or aspects) and/or the formulation thereof in a differentiated release multilayer solid form according to the invention for use in a method for the preventive and/or curative treatment of flu symptoms and/or infection of the respiratory tract, preferably infection of the upper respiratory tract, in a subject in need, by administering a therapeutically effective amount of the mixture or composition or formulation of the present invention to said subject.

Said flu symptoms may be selected from the group comprising or, alternatively, consisting of: muscle and joint pain, asthenia/fatigue, cough, increased respiratory secretions, fever, headache, sore throat (pharyngo-laryngodynia), nasal congestion, congestion of the respiratory tract, dyspnoea, shivers.

Said infection of the respiratory tract, of bacterial and/or viral origin, may be selected from the group comprising or, alternatively, consisting of: rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia.

The mixture or composition or formulation of the invention may be for use in a method for the preventive treatment of flu symptoms (as defined above) to avoid the onset of said flu syndromes or to attenuate the intensity of said symptoms if they occur.

The mixture or composition or formulation of the invention may be for use in a method for the curative treatment of flu symptoms (as defined above) both when administered to a subject as the only therapy capable of treating said fly symptoms, either when administered as an adjuvant of at least one other therapy or composition capable of treating said flu symptoms.

For the sake of clarity, in order to achieve the object of the present invention, the active compounds of the mixture or composition of the present invention ((a), (b), (c.1), (c.2), (c.3), (d), and, optionally, (e) and/or (f)) may also be administered separately or in groups (preferably in a time interval of 30 minutes-2-3 hours) and in any order.

Said at least one pharmaceutical or food grade additive and/or excipient, comprised in the composition of the invention together with the mixture of (a), (b), (c), (d), and, optionally, (e) and/or (f), consists of a substance devoid of therapeutic activity suitable for pharmaceutical or food use selected from ancillary substances known to the person skilled in the art such as, for example, diluents, solvents (including water, glycerine, ethyl alcohol), solubilisers, thickeners, sweeteners, flavour enhancement agents, colorants, lubricants, surfactants, antimicrobials, antioxidants, preservatives, pH stabilising buffers and mixtures thereof. Non-limiting examples of such substances are phosphate buffers (for example, dicalcium phosphate), alkali or alkali-earth metal stearate (for example of magnesium), silicon dioxide, mono- and diglycerides of fatty acids, microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, starch or corn starch, natural or artificial flavours (for example, iron oxides).

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

In the context of the present invention, the expression "subject/s” is used to indicate mammals (animals and humans), preferably human subjects.

The expression "therapeutically effective amount” is used to indicate the amount of mixture or compound or formulation which elicits the biological or medicinal response in a tissue, system or subject which is sought and defined by a person skilled in the art.

Unless otherwise specified, the expression mixture or composition or formulation comprises a component in an amount "comprised in a range from x to y” is used to indicate that said component can be present in the composition in all the amounts present in said range, even though not specified, extremes of the range comprised.

Examples of active components that can be used in the context of the present invention are reported below. An example of Panax ginseng extract (Meyer) that can be used in the composition of the present invention is a Panax ginseng root extract (water:ethanol extraction solvent =8:1), HPTLC identification, comprising 10% w/w of maltodextrin, 20.0% of ginsenosides (HPLC), polycyclic aromatic hydrocarbons (PAHs) lower than 50 ppb (GC-MS), benzo(a) pyrene <10 jjg/kgm, sieve analysis: 100% through 80 mesh (USP39 <786>), water (KF) <5% (Eur.Ph.7.0. [2.5.12]), total ash <5% (Eur.Ph.7.0. [2.4.16]).

An example of Ganoderma lucidum extract (Curtis) P. Karst that can be used in the composition of the present invention is a Ganoderma lucidum extract harvested by hand, washed, dried, cut and extracted with water and then 90% ethanol (v/v), wherein said extract comprises about 5% of maltodextrin, residual solvents: ethanol <5000ppm, benzo(a)pyrene ppb <10 and PAHs 4 ppb <50.

An example of zinc oxide that can be used in the composition of the present invention is zinc oxide 99.7% FU (INCI name Cl 77947, CAS no. 1314-13-2) obtained by means of the following steps: melting of zinc ingot in a furnace to obtain liquid zinc, evaporation of liquid zinc in the melting furnace to obtain gaseous zinc, air oxidation to obtain zinc oxide.

An example of vitamin C that can be used in the composition of the present invention is a coated ascorbic acid having an ascorbic acid content: VC-90: 89.0%~91.0%, VC-93: 92.0%~94.0%, VC-95: 94.0%~96.0%, VC-97: 96.0%~98.0% (97.5%-99.3%, VC-99: 98.0%~100.0%; and a loss on drying: VC-90: <0.40%, VC- 93: <0.20%, VC-95: <0.20%, VC-97: <0.20%, VC-99: <0.20%.

An example of vitamin D that can be used in the composition of 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 vitamin B1 that can be used in the composition of the present invention is a vitamin B1 (thiamine chlorhydrate) purity 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.), residue at 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 that can be used in the composition of the present invention is a vitamin B2 (riboflavin high flow 100 (HF)) having CAS no. 83-88-5.

An example of vitamin B3 that can be used in the composition of 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 means of the following steps: hydrolysis of 3-cyanopyridine on biocatalyst on fixed bed to obtain a solution of raw niacinamide, purification on activated carbon on fixed bed, nanofiltration, evaporation and freeze-drying (spray drying).

An example of vitamin B5 that can be used in the composition of the present invention is a D-calcium pantothenate having CAS no. 137-08-6. An example of vitamin B6 that can be used in the composition of the present invention is a vitamin B6 (pyridoxine chlorhydrate 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.), loss on drying 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 composition of the present invention is a vitamin B9 (folic acid) having CAS no. 59-30-3 and molecular weight 441.40, IR identification, Residue at ignition <0.30% w/w, specific absorbance ratio 256/365 nm 2.80-3.00, water <8.50% w/w, titre (on anhydrous) 95.00-102.00% w/w.

An example of vitamin B12 that can be used in the composition of the present invention is a vitamin B12 (Dry vitamin B120.1% GFP) having CAS no. 68-19-9.

Inulin (example of CAS no. 9005-80-5) is a glucose polymer with a molecular weight lower than starch (about 5000 Da), poorly soluble in water. It is the p-D-fructose polymer, in which monomers are bonded with p-2, 1 -glycosidic bonds.

An example of inulin that can be used in the composition of the present invention is an inulin extracted from chicory roots comprising: minimum inulin 90% w/w, fructose + glucose + sucrester maximum 10% w/w, average length of the chain 8-13 monomers, maximum ash 0.2% w/w, pH about. 6(±1), density (tapped density) about. 700 (±100) g/L.

A method for titrating the polysaccharides contained in said Ganoderma lucidum extract by means of spectrophotometry, that can be used in the context of the present invention, is carried out according to the following procedure.

(I) Equipment: spectrometer 721 (or other model);

(II) Reagents:

1. glucose, standard solution (accurately weigh 10 mg of standard glucose to a constant dry weight at 105°C, in a 50 ml Erlenmeyer flask, add distilled water).

2. phenol, test solution (weigh 100 g of phenol, add 0.1 g of aluminium 0.05 g of sodium bicarbonate, distil and collect the distillate at 182°C. Weigh 10 g and add 150 g of distilled water in brown bottles).

(III) Process:

1) Preparation of the standard curve: pipette 0.00ml, 0.05ml, 0.1ml, 0.15ml, 0.2ml or 0.3ml of standard glucose solution into various test tubes, add distilled water to all up to volume 2 ml, then add Pheno 1.0 ml, stir, add 5.0 ml of concentrated sulfuric acid, stir, and allow to stand for 5 minutes, heat up to boiling for 15 minutes, and allow to cool at room temperature. Test A at 490 nm and draw the standard curve.

2) Preparation of the samples: Accurately weigh 0.2 g of samples into the round-bottom flask, add 100 ml of 80% ethanol, backflow 1 hour, filter, use washing residues with ethanol 80% (10 ml * 3). Transfer to a round-bottom flask using filter paper, add 100 ml distilled water, heat 1 hour, filter, use hot water to wash residues (10 ml * 3). Lotion and Filtrate placed together, after cooling, placed in a 250 ml Erlenmeyer flask, diluted until a reserve is obtained.

3) Test the polysaccharides in the samples: take a portion of the sample solution, add distilled water up to a volume of 2 ml, then add phenol 1.0 ml, stir, add 5 ml of concentrated sulfuric acid, stir, then allow to stand for 5 minutes, warm to boiling for 15 minutes, and allow to cool to room temperature. Test A at 490 nm.

(IV) Calculation

Cu%= (Cs%xAuxWs) I (WuxAs)

Cu: polysaccharide content in samples

Cs: polysaccharide content in standard

As: standard absorption (Amax)

Au: sample absorption (Amax)

Wu: sample weight (mg)

Ws: standard weight (mg)

DESCRIPTION OF THE FIGURES

Figure 1: shows the percent inhibition of the release of IL-6 in Post-Mix2-C2, Post-PG C2 and Post-GL C2 samples.

Figure 2: shows the percent inhibition of the release of IL-6 in Post-Mix4-C4, Post-PG C4, Post-GL C4, Post-Vit C 04 and Post-Zn 04 samples.

Figure 3: Percent inhibition of the release of TNF-o in the Post-treatment Mix4-C4, PG-04, GL-04, Vit C- 04 and Z-04 samples.

Figure 4: Percent inhibition of the release of TNF-o in the Post treatment Mix2-C2, PG-01 and GL-01 samples.

EXPERIMENTAL PART

Table 1 reports an exemplifying embodiment of the composition of the invention comprising (a), (b), (c) and (d) formulated for oral use in solid form of differentiated release two-layer tablet, including a first quick release layer (layer I) and a second prolonged release layer (layer III).

Table 1. (% weight/weight); *Vitamin B1 0.5-3 mg, Vitamin B2 1-3.5 mg, Vitamin B3 10-40 mg, Vitamin B5 3-15 mg, Vitamin B6 1-3.5 mg, Vitamin B9 100-500 ug, Vitamin B12 1-7 ug.

Tables 2-4 report exemplifying embodiments of the composition of the invention comprising (a), (b), (c), (d), (e) and (f) formulated for oral use in solid form of differentiated release three-layer tablet, including a first quick release layer (layer I), a second intermediate release layer (layer II) and a third prolonged release layer (layer III).

In this example, layer (II) is arranged between layer (I) and layer (III).

Table 2. (% weight/weight); *Vitamin B1 0.5-3 mg, Vitamin B2 1-3.5 mg, Vitamin B3 10-40 mg, Vitamin B5 3-15 mg, Vitamin B6 1-3.5 mg, Vitamin B9 100-500 ug, Vitamin B12 1-7 ug.

Table 3. (% weight/weight); *Vitamin B1 0.5-3 mg, Vitamin B2 1-3.5 mg, Vitamin B320-30 mg, Vitamin B5 3-15 mg, Vitamin B6 1-3.5 mg, Vitamin B9 100-500 ug, Vitamin B12 1-7 ug. <^a>Mg stearate, Si dioxide, mono- and diglycerides of fatty acids, microcrystalline cellulose, hydroxypropyl methylcellulose, flavours; <^b>dicalcium phosphate, Mg stearate, Si dioxide, microcrystalline cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and corn starch. <^c>Mg stearate, Si dioxide, microcrystalline cellulose and hydroxypropyl methylcellulose.

Table 4. * mg/tablet: is used to indicate mg of a commercial compound comprising or, alternatively, consisting of the listed ingredient.

Disintegration tests or dissolution tests of the solid formulations according to the invention (for example two-layer or three-layer tablets according to Tables 1-4) can be carried out in order to evaluate the ability of said solid formulations to release the active components at different times and/or at different pHs.

Embodiments FRn of the present invention are reported below.

FR1. A composition comprising a mixture comprising, or alternatively, consisting of: (a) an extract of Ganoderma lucidum comprising polysaccharides,

(b) an extract of a plant of the genus Panax comprising ginsenosides, wherein said plant is selected from the group comprising or, alternatively, consisting of: Panax ginseng, Panax notoginseng, Panax pseudoginseng, Panax guinguefolium and mixtures thereof,

(c) at least one vitamin selected from the group comprising or, alternatively, consisting of: (c.1) vitamin C, (C.2) vitamin D, (C.3) at least one vitamin of group B, and a mixture thereof, and

(d) zinc, and, optionally, said composition further comprises at least one food or pharmaceutical grade additive and/or excipient.

FR2. The composition according to FR1, wherein said composition further comprises

(e) at least one bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably wherein said strain belongs to species selected from the group comprising or, alternatively, consisting of Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus rhamnosus, Lactobacillus helveticus,' and, optionally, (f) at least one prebiotic, preferably wherein said prebiotic is selected from the group comprising or, alternatively, consisting of: an inulin, a fructo-oligosaccharide (FOS), a galactooligosaccharide (GOS), a xylo-oligosaccharide (XOS), and a mixture thereof, more preferably inulin.

FR3. The composition according to FR1 or FR2, wherein said (e) bacterial strain is selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), and a mixture thereof.

FR4. The composition according to any one of FR1-3, wherein said (b) extract of a plant of the genus Panax consists of a plant of the species Panax ginseng (Meyer).

FR5. The composition according to any one of FR1-4, wherein said (a) extract of Ganoderma lucidum comprises polysaccharides in a percentage by weight comprised in a range from 5% to 60%, preferably from 20% to 40%, more preferably from 25% to 35%, with respect to the total weight of said (a); and/or wherein said (b) extract of a plant of the genus Panax, preferably Panax ginseng, comprises ginsenosides in a percentage by weight comprised in a range from 5% to 50%, preferably from 10% to 30%, more preferably from 15% to 25%, with respect to the total weight of said (b).

FR6. The composition according to any one of FR1-5, wherein said (c) at least one vitamin comprises or, alternatively, consists of: (c.1) vitamin C, (c.2) vitamin D, and (c.3) at least one vitamin of group B; preferably wherein said (c.3) at least one vitamin of group B is selected from the group comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, (c.3-vii) vitamin B12, and a mixture thereof; more preferably said (c.3) at least one vitamin of group B comprises or, alternatively, consists of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12.

FR7. The composition according to any one of FR1-6, wherein said mixture comprises or, alternatively, consists of:

- said (a) extract of Ganoderma lucidum comprising polysaccharides in a percentage by weight comprised in a range from 5% to 60%, preferably from 20% to 40%, more preferably from 25% to 35%, with respect to the total weight of (a);

- said (b) extract of Panax ginseng comprising ginsenosides in a percentage by weight comprised in a range from 5% to 50%, preferably from 10% to 30%, more preferably from 15% to 25%, with respect to the total weight of (b);

- said (c.1) vitamin C,

- said (c.2) vitamin D,

- said (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3- vi) vitamin B9, (c.3-vii) vitamin B12,

- said (d) zinc, preferably zinc oxide,

- said (e) strain is selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), and a mixture thereof, preferably Bifidobacterium lactis BL-04® (SD5219) and

- said (f) inulin.

FR8. A formulation in solid form of differentiated release multilayer tablet, wherein said formulation comprises the mixture or composition according to any one of FR1-7, and wherein said formulation comprises:

(I) a quick release layer comprising

- said (a) extract of Ganoderma lucidum comprising polysaccharides,

- said (c) at least one vitamin, preferably selected from the group comprising or, alternatively, consisting of: (c.2) vitamin D, (c.3) at least one vitamin of group B, and a mixture thereof, and

- said (d) zinc, wherein said (I) quick release layer disintegrates in the intestinal tract, releasing (a), (c) and (d), over a period of time comprised from 30 minutes to 120 minutes subsequent to the administration of the composition to a subject, preferably comprised from 60 minutes to 120 minutes, more preferably comprised from 75 minutes to 105 minutes;

(III) a prolonged release layer comprising

- said (b) extract of a plant of the genus Panax, preferably Panax ginseng, comprising ginsenosides, wherein said (III) prolonged release layer disintegrates in the intestinal tract, releasing (b), over a period of time comprised from 180 minutes to 270 minutes subsequent to the administration of the composition to said subject, preferably comprised from 180 minutes to 240 minutes, more preferably comprised from 180 minutes to 210 minutes.

FR9. The formulation in solid form of differentiated release multilayer tablet according to FR8, wherein said formulation further comprises

(II) an intermediate release layer comprising

- said (e) bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), and a mixture thereof, more preferably Bifidobacterium lactis BL-04® (SD5219); and, optionally,

- said (f) prebiotic, preferably an inulin; wherein said (II) intermediate release layer disintegrates in the intestinal tract, releasing (e) and (f), over a period of time comprised from 120 minutes to 180 minutes subsequent to the administration of the composition to said subject, preferably comprised from 120 minutes to 160 minutes, more preferably comprised from 120 minutes to 140 minutes.

FR10. A formulation according to FR8 or FR9, wherein said (I) quick release layer further comprises said (c.2) vitamin D, and said (c.3) at least one vitamin of group B; preferably wherein said (c.3) at least one vitamin of group B is selected from the group comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5 (c.3-v), vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12, and a mixture thereof; and wherein said (III) prolonged release layer further comprising said (c.1) vitamin C.

FR11. The composition according to any one of FR1-7 or formulation according to FR8-10, for use in a method for the preventive and/or curative treatment of at least one flu symptom and/or of a respiratory tract infection; preferably for use in a method for the treatment of: muscle and joint pain, asthenia/fatigue, cough, increased respiratory secretions, fever, headache, sore throat (pharyngo-laryngodynia), nasal congestion, respiratory tract congestion, dyspnoea, shivers, rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia.

EXPERIMENTAL PART

Introduction

The following work has the purpose of evaluating the anti-inflammatory activity of substances admitted in dietary supplements by means of in vitro assay on a THP-1 cell line and subsequent evaluation of inflammation markers.

In particular, the following markers will be evaluated:

IL-6 and TNF-o.

The substances for the present work will be:

• Panax ginseng C. A. Mey.,

• Ganoderma lucidum,

• Vitamin C,

• Zinc.

The experimental plan provides for:

1) Solubility tests in water and in the growth medium of the THP-1 cells;

2) Determination of non-cytotoxic concentrations of substances on THP-1 cells tested individually and combined:

Panax ginseng C. A. Mey.

Ganoderma lucidum

Vitamin C

Zinc

Ganoderma lucidum + Panax ginseng + Vitamin C + Zinc (Mix4)

Ganoderma lucidum + Panax ginseng (Mix2)

3) In vitro efficacy study of individual and combined substances (Mix4 and Mix2):

Panax ginseng C. A. Mey.

Ganoderma lucidum

Vitamin C

Zinc

Ganoderma lucidum + Panax ginseng + Vitamin C + Zinc (Mix-4)

Ganoderma lucidum + Panax ginseng (Mix-2) The in vitro efficacy evaluation will be carried out using the substances of interest individually and combined as post-treatment of the cells with respect to the inflammatory stimulus induced with LPS and determining the concentration of the aforementioned markers in the supernatant.

Materials

Human monocytic leukaemia cells, THP-1 (ECACC 88081201);

RPMI Medium 1640 (1X) + L-Glutamine (1X), Gibco by life technologies;

Fetal bovine serum (FBS), Gibco by Life technologies;

Penicillin-Streptomycin, Gibco by Life Technologies;

Lipopolysaccharides of Escherichia coll (LPS), Sigma-Aldrich;

Panax ginseng extract, Ganoderma lucidum extract, Vitamin C, Zinc

Human TNF-o ELISA Kit (3455-HP-2), Mabtech Inc.

Human IL-6 ELISA Kit (3445-1 HP-2), Mabtech Inc.

Methods

THP-1 cell line

The American Type Culture Collection THP-1 cell line was cultured in RPMI 1640 growth medium supplemented with 10% foetal bovine serum (FBS), 100 U/mL of penicillin and 100 pg/mL of streptomycin (complete medium). The cells were maintained at 37 °C in a 5 % CO2 incubator and the medium was replaced every 48 hours.

CELL VIABILITY TEST (MTT TEST)

The samples were tested for individual and combined cytotoxicity evaluation (Mix4 and Mix2).

For the Panax ginseng, Ganoderma lucidum, vitamin C samples, 10 mg of powder were weighed in a 15 mL test tube and resuspended in 10 mL of bidistilled water in order to obtain 1 mg/mL stock solutions.

For zinc, on the other hand, 10 mg of powder were weighed in a 50 mL test tube and resuspended in 20 mL of water to obtain a final solution of 0.5 mg/mL.

After solubilisation, the solutions were filtered (filters with porosity equal to 0.22 pm) and divided into aliquots in 2 ml microtubes. All samples were preserved at -20 °C before use. The MTT viability test for THP-1 cells was prepared by seeding 5 x 104 cells per well in a 96-well plate at cell passage 8 (p8).

For the Ganoderma lucidum, Panax ginseng and vitamin C sample, the cells were treated in the 1000 - 0.976 pig/mL concentration range, by preparing serial dilutions starting from the 1000 pig/mL stock solution, while for Zinc, concentrations between 500 and 0.625 pig/mL were analysed. Cells which were not treated with the samples and which represent 100% of cell viability were used as control.

10 piL of an MTT solution at a concentration of 1 mg/mL were added after 24 hours of incubation. After 3 hours, the content of each well was collected and placed in a 1.5 mL microtube and centrifuged (1000 rpm, 5 min, 25°C). The cell pellet was solubilised in 100 piL DMSO and cell viability was verified by reading absorbance at a 570 nm wavelength using an Envision microplate reader (Perkin Elmer, Waltham, Massachusetts, United States).

Lastly, the cytotoxicity of the combined samples was evaluated:

Mix-4 = Panax ginseng, Ganoderma lucidum, vitamin C, zinc

Mix-2 = Panax ginseng and Ganoderma lucidum.

Mix4 was tested on THP-1 cells at the concentrations reported in Table 5, Mix2 was tested for the combinations reported in table 6.

*acronyms used in statistical analysis

Table 5: Concentrations of the components of Mix4 used

*acronyms used in statistical analysis

Table 6: Concentrations of the components of Mix2 used

IN VITRO EFFICACY EVALUATION OF INDIVIDUAL AND COMBINED SAMPLES (MIX2 AND MIX4)

The evaluation was conducted by treating cells with individual and combined active components, administered as a post-treatment with respect to the induction of the inflammatory condition caused by LPS.

Cells in which stimulus was induced with LPS, but not treated with the individual and combined samples, were used as positive control. INFLAMMATION OF THP-1 CELLS AND POST-TREATMENT

The THP-1 cells at passage 12 were transferred to each well in a 24-well plate (24-multi-well): for each well, 106 cells were incubated in 0.9 mL of medium containing 50 ng/mL LPS for 3 hours, to which treatments with the samples were subsequently added. All treatments were prepared in 10X concentrated solutions starting from 1 mg/mL stock solutions.

3 solutions with decreasing concentrations equal to 625 pg/mL, 312 pg/mL and 78.1 pg/mL were prepared from the Panax ginseng, Ganoderma lucidum and vitamin C samples.

For zinc, two solutions with decreasing concentrations equal to 12.5 pg/mL and 31.25 pg/mL were prepared.

The specific concentrations of the active components present in Mix-4 are reported in Table 5. As regards Mix-2, the concentrations used are reported in Table 6.

The treatment after induction of inflammatory stimulus lasted 24 hours. At the end of the experiment all the samples were collected in 1.5 mL microtubes and centrifuged (2000 rpm for 5 min, 25°C) to enhance the sedimentation of cell pellets.

The supernatants were collected and stored at - 20 °C until analysis by means of ELISA assay.

EVALUATION OF LEVELS OF PRO-INFLAMMATORY CYTOKINES BY MEANS OF ELISA ASSAY Levels of IL-6 and TNFo in the supernatants of THP-1 cell cultures were evaluated. All reagents present in the ELISA kits were brought to room temperature before use. The samples were diluted where necessary; standards at known concentration and blanks for processing calibration lines were prepared. The samples and standards were incubated together with the primary antibody and subsequently with the secondary antibody in the multi-well plates. Lastly, the substrate of the peroxidase enzyme conjugated to the secondary antibody was added to develop a coloured product whose absorbance, being directly proportional to the concentration of IL-6 and TNFo present in the samples, was evaluated at the 450 nm wavelength.

RESULTS

Determination of non-cytotoxic concentrations of samples 1- 4 and of Mix2 and Mix4.

The concentration range tested for each sample (see Methods) allowed to evaluate the maximum non- cytotoxic concentration.

In detail:

For Ganoderma lucidum, the maximum non-cytotoxic concentration was equal to 62.5 g/mL,

1) For Panax ginseng the maximum non-cytotoxic concentration is equal to 62.5 g/mL,

2) For Vitamin C the maximum non-cytotoxic concentration is equal to 62.5 g/mL,

3) For Zinc the maximum non-cytotoxic concentration could not be calculated. Therefore, cell viability was also evaluated after treatment with the combination of Mixes at different concentrations.

The doses reported in Tables 5 and 6 were non-cytotoxic for the THP-1 cell line.

*acronyms used in statistical analysis

Table 5: Concentrations of the components of Mix4 used

*acronyms used in statistical analysis

Table 6: Concentrations of the components of Mix2 used

EVALUATION OF THE ANTI-INFLAMMATORY EFFICACY OF SAMPLES 1-4 INDIVIDUALLY OR

COMBINED WITH IL-6

Table 7 (A and B) reports the absorbance values, as the mean value of three determinations detected in the supernatants of THP-1 cells treated with LPS alone (positive control) or treated with the samples and the combination thereof in mixture (M 1x2 and Mix4) at different concentrations (shown in table 7 (A and B)) under LPS treatment condition (POST-treatment).

Table 7A

Table 7B

Table 7 (A and B): Mean absorbance value ± SD determined in the various samples.

Table 8 reports the concentrations of IL-6 expressed in pg/mL, as the mean value of three determinations detected in the supernatants of THP-1 cells treated with LPS alone (positive control) or treated with the samples and the combination thereof in mixture (Mix2 and Mix4) at different concentrations (shown in table 7 (A and B) and 8) under LPS treatment condition (POST-treatment).

From the analysis reported in table 8 it is observed that treatment with LPS induces the production of IL-6 (positive control) and that in some cases the samples tested at different concentrations induce an inhibition of the release of interleukin, while in other cases it stimulates the release thereof (negative percent inhibition).

Table 8: Sample ID, concentrations (pg/mL) of IL-6, % of IL-6 released and % inhibition of IL-6.

ND*: data not determined because it is outside the linearity range and therefore the determination in g/mL is inaccurate.

In order to better understand the data reported above, it was decided to compare the activities of the individuals and combinations, expressing the result in percent inhibition of the release of the interleukin.

POST-TREATMENT OF CELLS WITH SAMPLES 1-4 INDIVIDUALLY AND COMBINED AFTER THE TREATMENT OF CELLS WITH THE INFLAMMATORY AGENT LPS

Comparing the percent inhibition of the release of IL-6 induced in post-treatment by Panax ginseng and Ganoderma lucidum and the combination thereof at the concentration of 02 (31 pig/ml) (figure 8), it is observed that the two individual active components at this concentration stimulate the release of IL-6 with respect to the control and therefore they show a negative percent inhibition (given that they do not inhibit, but stimulate release), on the contrary the combination thereof (Post-Mix2-C2) causes an inhibition of the release, reversing the trend, and thus demonstrating synergistic anti-inflammatory action (as shown in Figure 1).

The same operation was carried out taking into account Mix4-C4 and the activities of the individual samples that form it (see Figure 2, which shows a positive and higher percent inhibition of Mix4-C4, with respect to the activities of the individual substances at the same concentration).

TNF-ALPHA

Table 9 shows TNF-o values expressed as mean value (pg/mL), minimum and maximum standard deviation determined in the supernatants of cells treated with post-treatment protocol (POST) with the substances, in individual and in mix according to the scheme reported in tables 5 and 6.

Table 9: Observed TNF-a va ues (mean ± minimum and maximum standard deviation) in 4 comparisons between mix and individual active components in the POST protocol in each of the 4 concentrations tested, n* = number of replicates. The data were processed according to the random intercept linear mixed model (LMM). The statistical analysis showed a statistically significant difference p < 0.001 between Mix4-C4 and the individual components at the corresponding concentrations 04, as shown by the statistical parameters reported in tables 10 and 11.

Table 10: Results of LMMs for TNF values in 4 comparisons between Mix4-C4 and individual active components in the POST protocol.

Table 11: Results of LMMs for TNF -a va ues in 4 comparisons between Mix4-C4 and individual reagents in the POST protocol.

In order to better understand the data reported above, it was decided to express the result in percent inhibition of release of TNF-o (Table 12, figure 3). Comparing the percent inhibition of the release of TNF- o induced in post-treatment by Panax ginseng, Ganoderma lucidum, Vitamin C and Zinc and by the combination thereof at the concentration of C4 (see figure 3), It is observed that the two individual active components stimulate the release of TNF-o with respect to the control and therefore they show a negative percent inhibition (given that they do not inhibit, but stimulate release), on the contrary the combination thereof (Mix4-C4) causes an inhibition of the release, reversing the trend, and thus demonstrating synergistic anti-inflammatory action.

Table 12: Percent release of TNF-o and percent inhibition of the release of TNF-o in the Mix4-C4 samples and individual components.

The LLM statistical analysis showed a statistically significant difference between the Mix2-C2 and the individual components GL and PG at the concentrations of 01 as reported in table 13.

Table 13: Results of LMMs for TNF-o values in 2 comparisons between Mix2-C2 and individual compounds GL-C1 and PG-C1 in the POST protocol.

Table 14 shows the concentrations of TNF-o (pg/mL) determined in the control, in the individual samples PG-C1, GL-C1 and in the Mix2-C2. Also in this case, in order to better understand the result, it was decided to express the datum obtained in a percent release ratio of TNF-o and percent inhibition of the release of the marker.

Table 14: Sample D, mean ± standard deviation of the concentrations of TNF-o(pgZmL) determined in the control, in the individual samples PG-C1, GL-C1 and in the Mix2-C2, percent release of TNF-o and percent inhibition of the release of TNF-o.

As observable from the statistical analysis (table 13) and from the percent inhibition (table 14 and figure 4), Mix2-C2 (consisting of PG 31 pg/mL and GL 31 pg/mL) has a statistically greater efficacy in reducing the release of TNF-o after stimulation with LPS, when compared to the individual bioactive components at a double concentration (PG-C1 = 62 pg/mL and GL-C1 62 pg/mL). However, the statistically significant difference recorded above does not allow, in our opinion, to demonstrate positive synergism. As a matter of fact, a positive synergism could be presumed only if PG and GL were known to act in inhibiting the release of TNF-o with the same mechanism of action. As a matter of fact, were the mechanism of action of the two extracts known to be the same, the mixture Mix2-C2 (consisting of PG 31 pg/mL and GL 31 pg/mL) could be compared with PG-C1 = 62 pg/mL and GL-C1 62 pg/mL. However, nothing is known about the mechanisms of action of PG and GL and therefore such presumption is not corroborated by current knowledge.

CONCLUSIONS

In conclusion, the present report reports the effect - on the release of cytokines IL-6 and TNF-o - of 4 samples and the combination thereof {Panax ginseng C. A. Mey., Ganoderma lucidum, Vitamin C, Zinc, Panax ginseng + Ganoderma lucidum + Vitamin 0 + Zinc (Mix-4) and Panax ginseng + Ganoderma lucidum (Mix-2) tested at various non-cytotoxic concentrations in THP-1 cells, after treatment with LPS. The results on IL-6 obtained indicate the presence of a synergism in the action inhibiting the release of IL- 6 by two of the Mixes considered with respect to the substances tested individually. In particular:

The combination Post-Mix2-C2 (consisting of Panax ginseng and Ganoderma lucidum at the concentration of 31 pig/ml) inhibits the release of IL-6 unlike the two individually tested components, which, on the contrary, enhance the release of cytokine. As a matter of fact, while Panax ginseng and Ganoderma lucidum at the concentration of 31 pig/ml stimulate the release of IL-6, the combination thereof (Post-Mix2-C2) causes an inhibition of the release of IL-6.

The combination Post-Mix4-C4 (consisting of 1.953 pig/ml of Panax ginseng, 1.953 pig/ml of Ganoderma lucidum, 1.953 pig/ml of Vitamin C and 0.3125 pig/ml of Zinc) inhibits the release of IL-6, unlike the individual tested components which either only minimally inhibit the release of IL-6 or even enhance the release of cytokine. It can therefore be concluded that in both the cases reported above (Mix2-C2 and Mix4-C4) in posttreatment, the percent inhibition of the release of IL-6 is higher than the sum of the percent inhibition of the components of the mixture, tested at the same concentrations.

As regards TNF-o and the results obtained following the post-treatment of the cells with the bioactive components alone and combined, in this case, the comparison was carried out between the mixtures and the individual substances, and the statistical analysis was conducted by the Proprietor.

The Post-Mix4-C4 (consisting of 1.953 pig/ml of Panax ginseng, 1.953 pig/ml of Ganoderma lucidum, 1.953 pig/ml of Vitamin C and 0.3125 pig/ml of Zinc) showed to be capable of inhibiting the release of TNF-o synergistically with respect to the individual active components that form it, which on the contrary, showed a pro-inflammatory effect given that they promote the release of cytokine. The synergistic effect is given by the fact that the Mix in question is capable of inhibiting the release of TNF-o to a greater extent with respect to the sum of the individual active components.

As regards Post-Mix2-C2 (consisting of 31 pig/ml di Panax ginseng and 31 pig/ml of Ganoderma lucidum) we can conclude that it shows a greater anti-inflammatory activity with respect to the individual active components that form it taken at the concentration of 62 pig/ml. This means that a halved dose of the individual active components combined in the Mix is more effective with respect to the treatment with the individual active components at a double concentration. However, it is deemed that a synergistic effect cannot be demonstrated in this case.

Embodiments of the present invention FRRn are reported below.

FRR1 . A mixture comprising, or alternatively, consisting of:

(a) an extract of Ganoderma lucidum comprising polysaccharides, and

(b) an extract of a plant of the genus Panax comprising ginsenosides, wherein said plant is selected from the group comprising or, alternatively, consisting of: Panax ginseng, Panax notoginseng, Panax pseudoginseng, Panax guinguefolium and mixtures thereof, wherein said mixture is for use in a method for the preventive and/or curative treatment of at least one flu symptom and/or an infection of the respiratory tract, and for use in a treatment method to increase the immune defences.

FRR2. The mixture for use according to FRR1, wherein said mixture is for use in a method for the treatment of: muscle and joint pain, asthenia/fatigue, cough, increased respiratory secretions, fever, headache, sore throat (pharyngo-laryngodynia), nasal congestion, congestion of the respiratory tract, dyspnoea, shivers, rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia.

FRR3. The mixture for use according to FRR1 or FRR2, wherein said mixture has an anti-inflammatory activity.

FRR4. The mixture according to any one of FRR1-3, wherein said (a) extract of Ganoderma lucidum comprises polysaccharides in a percentage by weight comprised in a range from 5% to 60%, preferably from 20% to 40%, more preferably from 25% to 35%, with respect to the total weight of said (a).

FRR5. The mixture according to any one of FRR1-4, wherein said (b) extract of a plant of the genus Panax consists of a plant of the species Panax ginseng (Meyer); preferably said (b) extract of a plant of the genus Panax is preferably Panax ginseng and it comprises ginsenosides in a percentage by weight comprised in a range from 5% to 50%, preferably from 10% to 30%, more preferably from 15% to 25%, with respect to the total weight of said (b).

FRR6. The mixture according to any one of FRR1-5, wherein said mixture further comprises (c) at least one vitamin selected from the group comprising or, alternatively, consisting of: (c.1) vitamin C, (c.2) vitamin D, (c.3) at least one vitamin of group B, and a mixture thereof.

FRR7. The mixture according to any one of FRR1-6, wherein said mixture comprises (c) at least one vitamin comprising or, alternatively, consisting of: (c.1) vitamin C, (c.2) vitamin D, and (c.3) at least one vitamin of group B; preferably wherein said (c.3) at least one vitamin of group B is selected from the group comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, (c.3-vii) vitamin B12, and a mixture thereof; more preferably said (c.3) at least one vitamin of group B comprises or, alternatively, consists of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12.

FRR8. The mixture according to any one of FRR1-7, wherein said mixture further comprises (d) zinc, preferably said zinc is a zinc oxide.

FRR9. The mixture according to any one of FRR1-8, wherein said mixture further comprises (e) at least one bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably wherein said strain belongs to species selected from the group comprising or, alternatively, consisting of Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus rhamnosus, Lactobacillus helveticus,' and, optionally, (f) at least one prebiotic, preferably wherein said prebiotic is selected from the group comprising or, alternatively, consisting of: an inulin, a fructo-oligosaccharide (FOS), a galactooligosaccharide (GOS), a xylo-oligosaccharide (XOS), and a mixture thereof, more preferably inulin.

FRR10. The mixture according to any one of FRR1-9, wherein said (e) bacterial strain is selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), and a mixture thereof.

FRR11. The mixture according to any one of FRR1-10, wherein said mixture comprises or, alternatively, consists of:

- said (c.1) vitamin C,

- said (c.2) vitamin D,

- said (d) zinc, preferably zinc oxide,

- said (f) inulin.

FRR12. A composition comprising a mixture according to any one of FRR1-11 and, optionally, said composition further comprises at least one food or pharmaceutical grade additive and/or excipient.

FRR13. A formulation in solid form of differentiated release multilayer tablet, wherein said formulation comprises the mixture according to any one of FRR1-11, or the composition according to FRR12, and wherein said formulation comprises:

(I) a quick release layer comprising

- said (a) extract of Ganoderma lucidum comprising polysaccharides,

(III) a prolonged release layer comprising

FRR14. The formulation in solid form of differentiated release multilayer tablet according to FRR13, wherein said formulation further comprises

(II) an intermediate release layer comprising

FRR15. The formulation according to FRR13 or FRR14, wherein said (I) quick release layer further comprises said (c.2) vitamin D, and said (c.3) at least one vitamin of group B; preferably wherein said (c.3) at least one vitamin of group B is selected from the group comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5 (c.3-v), vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12, and a mixture thereof; and wherein said (III) prolonged release layer further comprising said (c.1 ) vitamin C.

FRR16. The composition according to FRR12 or the formulation according to any one of FRR13-15 for use in a method for the preventive and/or curative treatment of at least one flu symptom and/or of an infection of the respiratory tract; preferably for use in a method for the treatment of: muscle and joint pain, asthenia/fatigue, cough, increased respiratory secretions, fever, headache, sore throat (pharyngo- laryngodynia), nasal congestion, congestion of the respiratory tract, dyspnoea, shivers, rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia.

Claims

1. A mixture comprising or, alternatively, consisting of:

(a) an extract of Ganoderma lucidum comprising polysaccharides, and

2. The mixture for use according to claim 1, wherein said mixture is for use in a method for the treatment of: muscle and joint pain, asthenia/fatigue, cough, increased respiratory secretions, fever, headache, sore throat (pharyngo-laryngodynia), nasal congestion, congestion of the respiratory tract, dyspnoea, shivers, rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia.

3. The mixture for use according to claim 1 or 2, wherein said mixture has an anti-inflammatory activity.

4. The mixture according to any one of claims 1-3, wherein said (a) extract of Ganoderma lucidum comprises polysaccharides in a percentage by weight comprised in a range from 5% to 60%, preferably from 20% to 40%, more preferably from 25% to 35%, with respect to the total weight of said (a).

5. The mixture according to any one of claims 1-4, wherein said (b) extract of a plant of the genus Panax consists of a plant of the species Panax ginseng (Meyer); preferably said (b) extract of a plant of the genus Panax is preferably Panax ginseng and it comprises ginsenosides in a percentage by weight comprised in a range from 5% to 50%, preferably from 10% to 30%, more preferably from 15% to 25%, with respect to the total weight of said (b).

6. The mixture according to any one of claims 1-5, wherein said mixture further comprises (c) at least one vitamin selected from the group comprising or, alternatively, consisting of: (c.1) vitamin C, (c.2) vitamin D, (c.3) at least one vitamin of group B, and a mixture thereof.

7. The mixture according to any one of claims 1-6, wherein said mixture comprises (c) at least one vitamin comprising or, alternatively, consisting of: (c.1) vitamin C, (c.2) vitamin D, and (c.3) at least one vitamin of group B; preferably wherein said (c.3) at least one vitamin of group B is selected from the group

46 comprising or, alternatively, consisting of: (c.3-i) vitamin B1 , (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, (c.3-vii) vitamin B12, and a mixture thereof; more preferably said (c.3) at least one vitamin of group B comprises or, alternatively, consists of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5, (c.3-v) vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12.

8. The mixture according to any one of claims 1-7, wherein said mixture further comprises (d) zinc, preferably said zinc is a zinc oxide.

9. The mixture according to any one of claims 1-8, wherein said mixture further comprises (e) at least one bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably wherein said strain belongs to the species selected from the group comprising or, alternatively, consisting of Bifidobacterium lactis, Bifidobacterium breve, Lactobacillus rhamnosus, Lactobacillus helveticus, and, optionally, (f) at least one prebiotic, preferably wherein said prebiotic is selected from the group comprising or, alternatively, consisting of: an inulin, a fructo-oligosaccharide (FOS), a galactooligosaccharide (GOS), a xylo-oligosaccharide (XOS), and a mixture thereof, more preferably inulin.

10. The mixture according to any one of claims 1-9, wherein said (e) bacterial strain is selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), and a mixture thereof.

11. The mixture according to any one of claims 1-10, wherein said mixture comprises or, alternatively, consists of:

- said (c.1) vitamin C,

- said (c.2) vitamin D,

- said (d) zinc, preferably zinc oxide,

47 - said (e) strain is selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus crl1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10 (CBS116114), and a mixture thereof, preferably Bifidobacterium lactis BL-04® (SD5219) and

- said (f) inulin.

12. A composition comprising a mixture according to any one of claims 1-11 and, optionally, said composition further comprises at least one food or pharmaceutical grade additive and/or excipient.

13. A formulation in solid form of differentiated release multilayer tablet, wherein said formulation comprises the mixture according to any one of claims 1- 11 or the composition according to claim 12, and wherein said formulation comprises:

(I) a quick release layer comprising

- said (a) extract of Ganoderma lucidum comprising polysaccharides,

(III) a prolonged release layer comprising

14. The formulation in solid form of differentiated release multilayer tablet according to claim 13, wherein said formulation further comprises

(II) an intermediate release layer comprising

- said (e) bacterial strain belonging to the genus Bifidobacterium or Lactobacillus, preferably selected from the group comprising or, alternatively, consisting of: Bifidobacterium lactis BL-04® (SD5219), Lactobacillus rhamnosus cr!1505, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus helveticus Lafti® L-10

48 (CBS116114), and a mixture thereof, more preferably Bifidobacterium lactis BL-04® (SD5219); and, optionally,

15. The formulation according to claim 13 or 14, wherein said (I) quick release layer further comprises said (c.2) vitamin D, and said (c.3) at least one vitamin of group B; preferably wherein said (c.3) at least one vitamin of group B is selected from the group comprising or, alternatively, consisting of: (c.3-i) vitamin B1, (c.3-ii) vitamin B2, (c.3-iii) vitamin B3, (c.3-iv) vitamin B5 (c.3-v), vitamin B6, (c.3-vi) vitamin B9, and (c.3-vii) vitamin B12, and a mixture thereof; and wherein said (III) prolonged release layer further comprising said (c.1) vitamin C.

16. The composition according to claim 12 or the formulation according to any one of claims 13-15 for use in a method for the preventive and/or curative treatment of at least one flu symptom and/or of an infection of the respiratory tract; preferably for use in a method for the treatment of: muscle and joint pain, asthenia/fatigue, cough, increased respiratory secretions, fever, headache, sore throat (pharyngo- laryngodynia), nasal congestion, congestion of the respiratory tract, dyspnoea, shivers, rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis, tracheitis, bronchitis, bronchiolitis, pneumonia.