Chemical composition based on propolis extract

Abstract

The invention relates to a method for inducing the trans-phenotyping of inactive anti-inflammatory M2c macrophages to active pro-inflammatory M1 macrophages, in an anti-inflammatory cellular context, using a chemical composition comprising an extract of brown propolis of natural and standardized origin, said extract of brown propolis comprising at least, as a mixture, a molecule A of the phenolic acid family, a molecule B of the flavanone family, a molecule C of the flavone family, a molecule D of the dihydroflavonol family and a molecule E of the flavonol family.

Description

Chemical composition based on Propolis extract

[0001] The present invention relates to a chemical composition with immunomodulatory activity comprising, as active ingredient, a propolis extract, of natural origin and standardized, which comprises at least a mixture of five molecules belonging respectively to the family of phenolic acids, flavanones, flavones, dihydroflavonoles and flavonoles.

[0002] The invention also relates to the use of this composition, as a medicament, for the prevention or treatment, in particular by modulation of the defenses of the immune system, of bacterial infections, viral infections, fungal infections or tumor conditions generating an inflammatory immune response attenuating the innate defense system.

[0003] The invention also relates to the use of this composition as a food supplement for the prevention of the above-mentioned infections.

[0004] The present invention also relates to the use of such a chemical composition as an ingredient for inducing the trans-phenotyping of inactive anti-inflammatory M2 macrophages, in particular of the “M2c” type, into active pro-inflammatory macrophages, of the “M1” type.

[0005] Currently, the treatment or prevention of the onset of pathologies involving an inflammatory immune response is mainly based on the use of anti-inflammatory agents comprising a chemically synthesized active substance.

[0006] However, there are major drawbacks associated with the use of these known agents, including:

[0007] - the harmful side effects they can generate on the human body, such as stomach aches, headaches,

[0008] - the lack of precision of action on the targets involved in the anti-inflammatory mechanism, with the risk of weakening the active immune system to fight against the pathology,

[0009] – the need to have, for their galenic formulation, a multitude of excipients, inactive against the pathology, these excipients being essential to preserve the chemical integrity of the synthetic active substance, to allow its diffusion and bioavailability in the body and to guarantee its effect on the pathology, in particular by a topical action.

[0010] In order to partially limit side effects and overcome the drawbacks encountered with chemically synthesized elements, users are increasingly turning to agents of natural origin. These are derived from natural products and have the advantage of minimizing toxicity for the organism being treated.

[0011] The use of propolis as a natural ingredient for its activity against inflammatory disease is known.

[0012] Propolis is a natural resinous, gummy and balsamic substance present on certain parts of plants, which is collected by bees, then transformed by the contribution of their salivary and wax secretions, before being used in the hive as a construction material (sealing cracks, fixing, sealing the hive) and protection against the development of microorganisms.

[0013] Raw propolis can be recovered by beekeepers for various uses such as pharmaceutical, cosmetic or even agri-food use.

[0014] The chemical composition and color of propolis extract vary depending on the geographical origin, the botanical genus and the part of the plant taken, the harvesting period by the bees, the species of bee, the harvesting method by the beekeeper and the propolis extraction method.

[0015] This variability and diversity of composition of natural propolis represents a major risk to guarantee a reproducible and homogeneous activity to fight against an infection by all types of pathogens, or, to act on a tumor condition. In particular, this variability of composition of natural propolis makes its involvement in the inflammatory response random.

[0016] Thus, to date, there is no propolis-based composition with a homogeneous and reproducible effect acting on the inflammatory reaction mechanism, which is non-toxic, of natural origin, with standard constituents and whose effectiveness is equivalent to commonly used anti-inflammatory agents.

[0017] The objective of the present invention is to propose a composition based on propolis extract of natural and standardized origin, with a controlled molecular composition of active substance, overcoming the aforementioned drawbacks of the state of the art and providing a solution making it possible to guarantee an immuno-modulation activity of the inflammatory mechanism.

[0018] The present invention relates to a chemical composition with immunomodulatory activity, inducing in particular the trans-phenotyping of inactive anti-inflammatory macrophages, of type M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context, which comprises, as active substance, a propolis extract of natural and standardized origin comprising at least, in a mixture, a molecule A from the family of phenolic acids, a molecule B from the family of flavanones, a molecule C from the family of flavones, a molecule D from the family of dihydroflavonoles and a molecule E from the family of flavonoles.

[0019] The present invention also relates to the use of such a chemical composition comprising, as active substance, a brown propolis extract of natural origin and standardized as an agent inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context.

[0020] The present invention also relates to a method for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages M1, in an anti-inflammatory cellular context, using such a chemical composition comprising a brown propolis extract of natural origin and standardized.

[0021] The invention also relates to a method for stimulating and / or strengthening the immune system of a subject, in particular his trained immunity, said method comprising the administration, of a therapeutically effective amount, of such a chemical composition comprising a brown propolis extract of natural origin and standardized.

[0022] The present invention also relates in particular to a method for preventing the occurrence or treating, in a subject, a disease physiologically inducing the trans-phenotyping of M1 macrophages into M2 macrophages as a pathological phenomenon, said method comprising the administration of a therapeutically effective amount of such a chemical composition comprising a standardized natural brown propolis extract.

[0023] For the purposes of the invention, the term "disease physiologically inducing the trans-phenotyping of M1 macrophages into M2 macrophages as a phenomenon" means a condition which, in an in vivo context, in response to a stimuli (infection by a pathogen or external environmental stimuli such as overexposure to a UV molecule, radiation) will cause and induce the transphenotyping of M1 macrophages into M2. For example, this may refer to a disease due to an infection by a virus or a bacterium whose development strategy is to physiologically induce, in vivo in the host, the trans-phenotyping of M1 into M2 macrophages.

[0024] In the present invention, "propolis", "propolis extract" or "standardized propolis extract" means propolis derived from a plant belonging to the families of Salicaceae including the genus PopulusL. spp represents the majority species source for harvesting (poplar) andSalixL. spp (willows), Betulaceae dontBetulaL. spp (birches) etAlnusMill. spp (aulnes), Fagaceae dontQuercusL. spp (oaks),CastoneaMill. spp (chestnuts) etFagusL. spp (beech), Ulmaceae dontUlmusL. spp (ormes), Oleaceae dontFraxinusTourn. ex L. spp (strawberries), Sapindaceae dontAesculusL. spp (marronniers d'Inde), Pinaceae dontPiceaA.Dietr. spp (epicéas),PinusL. spp (pins) etAbiesMill. spp (sapins), Araucariaceae dontAraucariaJuss. spp (araucarias), Cupressaceae dontJuniperusL. spp (cedar), Anacardiaceae dontPistaciaL. spp (pistachiers), Rutaceae dontRutaL. spp (rue des jardins), Myrtaceae dontEucalyptusL'Hér. Spp.

[0025] The propolis according to the invention is of natural origin, that is to say that it is collected, in all seasons, by bees of the genus Apismellifera on the buds, leaves, stems, bark or any aerial parts of the plant producing resinous exudates.

[0026] Preferably, in the present invention, the term "propolis", "propolis extract" or "standardized propolis extract" means a propolis derived from a plant belonging to the family of the poplar genus (called Populus spp, called brown propolis), geographically present in Europe, Asia and South America.

[0027] According to the invention, the propolis extract consists of brown propolis, derived from a part of a plant belonging to the poplar family of the genus Populusspp, such as Populusnigra L., Populusalba L., or Populustremula L.

[0028] To obtain the brown propolis of the invention, bees collect the resin from leaves or leaf buds.

[0029] According to the invention, the chemical composition comprises a propolis extract of natural and standardized origin.

[0030] The propolis extract of the composition of the invention is obtained by a standardized process of the dynamic multi-extraction method type in which:

[0031] – raw propolis is taken from the hive,

[0032] –a first extraction is carried out in an aqueous medium on the raw propolis collected in order to remove the beeswax and impurities (bee residue, wood),

[0033] – a series of three extractions is carried out on the residue of the aqueous extraction obtained previously with a hydro-alcoholic solvent consisting of a water / ethanol mixture, increasing the extraction temperature and the alcohol concentration with each successive extraction.

[0034] After the series of three extractions with a hydro-alcoholic solvent, the brown propolis extract used in the invention is obtained, which has the specific effect of inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context through the synergistic action of molecules A to E.

[0035] In this process, the ratio between the crude propolis residue extracted and the hydroalcoholic solvent is between 0.1:1 and 1:1, with extraction times between 4 and 36 hours.

[0036] The implementation of this standardized process makes it possible to obtain a propolis extract, in particular brown propolis, of natural origin and standardized systematically comprising at least, in a mixture, one molecule A from the phenolic acid family, one molecule B from the flavanone family, one molecule C from the flavone family, one molecule D from the dihydroflavonole family and one molecule E from the flavonole family, this regardless of the species of the plant genus of the Populusspp type, the part of the plant used to harvest the resin (either the buds or the leaves), the date of collection by the beekeeper and the season.

[0037] According to a preferred embodiment of the invention, the chemical composition of the invention further comprises gum arabic, sucrose and silicon dioxide, as a carrier for the propolis extract.

[0038] Preferably, the chemical composition comprises, in mass percentage relative to its total mass, between 40% and 60% of gum arabic, less than 10% of sucrose and at most 3% of silicon dioxide as a carrier in mixture with between 30% and 50% of propolis extract.

[0039] According to other embodiments, the chemical composition of the invention may comprise other types of carrier for the propolis extract such as, for example, propylene glycol; soybean seed oil; sunflower seed oil or a mixture of water and ethanol.

[0040] For example, the carrier of the propolis extract may comprise a mixture of glycerin and water, with or without heat-treated liquid sucrose, the latter being, for example, that marketed under the brand name FruitMax® Brown 1203 WS.

[0041] In another example, the propolis extract carrier may also comprise a mixture of maltodextrin, silicon dioxide and heat-treated liquid sucrose.

[0042] According to the invention and in all cases, the support is inert with respect to the propolis extract of natural and standardized origin obtained by the aforementioned process, it does not modify in any way its activity and its effects in the inflammatory mechanism. More particularly, the support is inert with respect to the mixture of molecules A to E of the propolis extract, the support does not prevent in any way the immuno-modulatory activity of the composition.

[0043] Preferably, for its immunomodulatory activity of trans-phenotyping M2 macrophages, in particular of the M2C type, into M1 macrophages in an anti-inflammatory context, the chemical composition comprises, in mass percentage relative to its total dry mass, between 1.3% and 1.8% of molecule A from the phenolic acid family, between 0.6% and 1.0% of molecule B from the flavanone family, between 1.4% and 2.8% of molecule C from the flavone family, between 2.4% and 3.3% of molecule D from the dihydroflavonole family and between 1.1% and 1.7% of molecule E from the flavonole family.

[0044] Through the examples below, it has been demonstrated that these proportions of molecules are sufficient and allow, in an anti-inflammatory context, significant immunomodulation activity by trans-phenotyping of M2C macrophages in M1.

[0045] Advantageously, within the chemical composition used in the invention:

[0046] - the family of phenolic acids, has a basic carbon chain of formula IA below of the cinnamic acid type or its derivatives such as o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid;

[0047] AI formula:

[0048] or; of formula IB below of benzoic acid type and its IB derivatives such as m-hydroxy benzoic acid, p-hydroxy benzoic acid, protacatechic acid, vanillic acid, syringic acid:

[0049] formula IB: the flavanone family has a basic carbon chain of formula II below: the flavone family has a basic carbon chain of formula III below: the dihydroflavonole family has a basic carbon chain of formula IV below: the flavonol family has a basic carbon chain of formula V below: .

[0050] According to the invention, within the chemical composition, the molecules belonging to the aforementioned families, which are present in the propolis extract, interact together and make it possible to obtain the immunomodulatory activity of the composition of the invention.

[0051] The expression "immunomodulatory activity" refers, in particular, to the capacity of the propolis extract to participate in the induction of trans-phenotyping of macrophages towards one or other of the possible macrophage phenotypes.

[0052] According to the invention, the brown propolis extract of natural and standardized origin is obtained by carrying out the standardized protocol comprising the following successive steps:

[0053] – a) raw propolis is taken from the hive,

[0054] – b) the beeswax and impurities are removed from the raw propolis by aqueous extraction, so as to recover only the resinous, balsamic component of the raw propolis in pasty form,

[0055] – c) three successive hydro-alcoholic extractions are carried out on the residue of the previous extraction, for example with the propolis removed from the beeswax, then on the extraction residues obtained, with a water / ethanol extraction mixture,

[0056] – d) the extract of brown propolis of natural origin and standardized is recovered,

[0057] – e) optionally, the propolis extract from step d) is spray-dried so as to obtain a dry extract of brown propolis, in powder form, preferably the dry extract of brown propolis having a final humidity of less than 10%.

[0058] According to the invention, the final humidity level of the dry extract of brown propolis is evaluated by any techniques known to those skilled in the art.

[0059] In the present invention, the expression "standardized natural brown propolis extract" refers to an extract obtained by the aforementioned standardized process in accordance, for example, with the protocol indicated in Example 1 below.

[0060] Advantageously, the use of a chemical composition, comprising a brown propolis extract obtained by the aforementioned standardized protocol, which comprises the molecules belonging to the aforementioned families A to E, generates a synergistic action of the molecules at the origin of an immuno-modulatory activity of induction of the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of types M1, in an anti-inflammatory cellular context.

[0061] For example, the following molecules will be cited as molecule A of the phenolic acid family: caffeic acid, vanillic acid, p-coumaric acid, ferulic acid, 3,4 dimethyl caffeic acid, isoferulic acid, trans-cinnamic acid, cinnamylidene acetic acid, (caffeic acid isoprenyl ester) prenyl caffeate ester - C 14 H 14 O4, (caffeic acid benzyl ester) benzyl caffeate ester - C 16 H 14 O4, (caffeic acid phenethyl ester) phenylethyl caffeate ester (CAPE) - C 17 H 16 O4, (caffeic acid cinnamyl ester) cinnamyl caffeate ester - C 18 H 16 O 4, (coumaric acid cinnamyl ester) cinnamyl p-coumarate ester .

[0062] As an example, pinocembrin is a B molecule from the flavanone family.

[0063] For example, the following molecules from the flavone family are mentioned as molecule C: 5-methyl chrysin ester, apigenin, 5-methyl luteolin ester, chrysin, methoxychrysin, Tectochrysin.

[0064] For example, the following molecules are cite as molecule D of the dihydroflavonole family: 5 methyl pinobanksin, pinobanksin, pinobanksin-3-O-acetate, pinobanksin-3-O-propionate, pinobanksin derivatives.

[0065] For example, the following molecules are included as molecule E of the flavonol family: quercetin, 3 methyl quercetin, kaempferol, Isorhamnetin, 5,7-dimethyl quercetin, 5-methyl galangin, rhamnetin, 7-methyl quercetin, galangin.

[0066] According to a preferred embodiment of the chemical composition of the invention, exhibiting significant immunomodulatory activity, the propolis extract, in particular brown propolis, comprises at least, in a mixture,: as molecule A of caffeic acid of formula below:

[0067] as molecule B of pinocembrin with the formula below:

[0068] as molecule C of chrysin with the formula below:

[0069] as molecule D of pinobanskin with the formula below:

[0070] and as molecule E of galangin with the formula below: .

[0071] According to this specific embodiment, caffeic acid, pinocembrin, chrysin, pinobanskin and galangin present in a mixture in the standardized brown propolis extract of natural origin, obtained by the standardized protocol, interact synergistically and are responsible for the immuno-modulatory activity of the chemical composition of the invention.

[0072] More specifically, caffeic acid, pinocembrin, chrysin, pinobanskin and galangin, present in a mixture in brown propolis extract, induce the activation of macrophages and their trans-phenotyping from an M2 phenotype, notably M2C, to an M1 phenotype, in an anti-inflammatory cellular context, that is to say with an environment comprising anti-inflammatory cytokines.

[0073] According to the invention, the chemical composition is in a liquid or solid form, in powder, solution, gel, emulsion, cream, capsule or tablet, for use in vitro, or in vivo by a living being.

[0074] For the purposes of the invention, the expression "in vivo use" means use of the chemical composition on a living being, whose organism is complete and in its natural living environment.

[0075] For the purposes of the invention, the expression "in vitro use" means a use of the chemical composition, outside the complete living organism, in an artificial environment, in particular in a laboratory, which corresponds to all experimental activities carried out on cells, tissues, organs or other components of a living being outside their natural living context.

[0076] According to the invention, the chemical composition is in a pharmaceutically and / or dermatologically acceptable galenic form, by the human or animal organism, for in vivo administration.

[0077] For the purposes of the invention, the “galenic form” corresponds to the form in which the composition is presented (cream, liquid, tablet) which is specially designed for the route of administration for which the composition is intended (for example: cutaneous route or oral route).

[0078] “Pharmaceutically acceptable” means that which is useful in the preparation of the composition and which is safe, stable, non-toxic biologically, nor otherwise undesirable and which is acceptable for human or animal pharmaceutical use, including via in vivo administration.

[0079] For the purposes of the invention, “pharmaceutically acceptable” dosage forms also specifically include “nutritionally acceptable” dosage forms, i.e. the dosage form can be used as food or “food supplement” and allows, after administration, a transformation in the living organism, which involves a set of physiological and metabolic phenomena allowing the production of vital energy and promoting the proper functioning of the immune system.

[0080] "Dermatologically acceptable" means compatibility with the skin, i.e. anything that is useful in the preparation of the composition and that is non-toxic, following cutaneous administration to humans or animals, in particular by topical application. In other words, this corresponds to anything that can be applied to the skin and / or mucous membranes, without risk of causing a reaction such as, for example, an allergic or inflammatory reaction.

[0081] According to the invention, the galenic form of the chemical composition allows one of the following routes of administration: oral route, intravenous route, intramuscular route, cutaneous, subcutaneous or transdermal route, sublingual route, ocular route, auricular route, nasal route.

[0082] Preferably, for in vivo administration by the human or animal body, the oral route or the cutaneous route is preferred.

[0083] The galenic form of the chemical composition allows, for example, an action extended to the entire organism (systemic).

[0084] Preferably, the galenic form of the chemical composition allows a local (topical) action targeting the area of ​​the inflammatory reaction, preferably targeting the area of ​​the anti-inflammatory cellular context where the macrophages of the immune system intervene.

[0085] More specifically, the present invention relates to a method for inducing trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages M1, in an anti-inflammatory cellular context, using a chemical composition of the invention as previously described; said composition comprising a brown propolis extract of natural origin and standardized, obtained by the aforementioned standardized protocol, said brown propolis extract comprising at least, in a mixture, a molecule A from the phenolic acid family, a molecule B from the flavanone family, a molecule C from the flavone family, a molecule D from the dihydroflavonole family and a molecule E from the flavonole family, as an inducer of trans-phenotyping of macrophages.

[0086] According to another feature of the aforementioned method of the invention, in an anti-inflammatory cellular context, in particular of the in vivo or in vitro type, said chemical composition comprising the extract of brown propolis of natural and standardized origin is in contact with the macrophages.

[0087] For the purposes of the invention, the term "in contact with macrophages" means that the molecules A to E of the brown propolis extract of natural origin and standardized in the composition of the invention are in a physiological cellular environment in vivo, or a cellular environment in vitro, sufficiently close to the environment of the macrophages, to allow the induction of their trans-phenotyping.

[0088] According to another feature of the invention, said chemical composition is in a food product, preferably a food supplement, or in a cosmetic product, or even in an agrochemical product.

[0089] Another aspect of the present invention relates to the use of the chemical composition of the invention in the preparation of a cosmetic product, a food product, preferably a food supplement, or an agrochemical product.

[0090] The term "cosmetic product" means any substance or mixture intended to be placed in contact with the superficial parts of the human body (epidermis, hair and capillary systems, nails, lips and external genital organs) or with the teeth and oral mucous membranes with a view, exclusively or mainly, to cleaning them, perfuming them, changing their appearance, protecting them, keeping them in good condition or correcting body odors.

[0091] "Food supplements" means all foodstuffs intended to supplement a normal diet, for humans or animals, and which constitute a concentrated source of nutrients or other substances having a nutritional or physiological effect.

[0092] The present invention therefore also relates to a food supplement, which comprises, or consists of, a chemical composition according to the invention.

[0093] According to another aspect, the invention also relates to a chemical composition according to the invention, comprising a brown propolis extract of natural origin and standardized, for use as a food supplement, in particular for use as a food supplement stimulating or strengthening the immune system; preferably for use as a food supplement for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of types M1, in an anti-inflammatory cellular context, in particular an anti-inflammatory cellular context in vivo.

[0094] Another aspect of the present invention relates to the chemical composition comprising a naturally occurring and standardized brown propolis extract of the invention as a medicament.

[0095] The invention also relates to the use of such a chemical composition as an active ingredient in a drug formulation.

[0096] Specifically, the chemical composition of the invention is for use as a medicament for stimulating or strengthening the immune system.

[0097] The chemical composition of the invention is also for use as a medicament for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages M1, in an anti-inflammatory cellular context, in particular an anti-inflammatory cellular context in vivo or in vitro.

[0098] In particular, the composition of the invention is intended to be used in the prevention or treatment of diseases physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages, or, for use in a therapeutic method for preventing or treating these diseases, or for use as an active ingredient in a drug formulation intended to prevent or treat these diseases.

[0099] Indeed, it is known that for certain diseases, of viral or bacterial origin, pathogens use as an infectious strategy the trans-phenotyping of M1 macrophages into M2 in order to weaken the host's immune system. In other words, the manifestation of the disease results in a pathological and physiological phenomenon of trans-phenotyping of M1 macrophages into M2 macrophages.

[0100] Thus, the invention, in particular the use of the chemical composition comprising a brown propolis extract of natural and standardized origin, makes it possible to counterbalance this pathological phenomenon, at the origin of the viral or bacterial disease, by induction of the "reverse" trans-phenotyping of M2 macrophages, in particular M2C into M1 macrophages. More specifically, in an anti-inflammatory cellular context, generated by the pathogen to weaken the immune system, the effect of the infectious strategy of these pathogens responsible for the symptoms of the disease will be attenuated. Thus, the invention makes it possible to prevent or treat a disease by modulating the immune response by specific trans-phenotyping of macrophages.

[0101] In other words, the invention is advantageously intended to be used or implemented within the framework of a therapeutic method for preventing or treating diseases generated by pathogens promoting the trans-phenotyping of M1 macrophages into M2 to establish their infection.

[0102] More particularly, the invention also relates to the use of the chemical composition of the invention in the prevention or treatment of diseases of the inflammatory system, bacterial, viral or fungal infections and tumor diseases.

[0103] More specifically, the composition of the invention is intended for use in the prevention or treatment of diseases of the inflammatory system, bacterial, viral or fungal infections and tumor conditions; or for use in a therapeutic method for preventing or treating these diseases; or as an active ingredient in a drug formulation intended to prevent or treat these diseases.

[0104] Examples of diseases include cancer, including breast, pancreatic or colorectal cancer, as well as all tumors infiltrated by tumor-associated macrophages, upper respiratory tract infections, infections by parasites of the Helminthes genus, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, allergies.

[0105] For example, infections by parasites of the genus Helminthes include infection by Schistosomamansoni, Plasmodiumspp, Schistosomaspp, Leishmaniaspp, Toxoplasmagondi, Trypanosomacruzi, Echinococcusspp, Trichinellaspiralis

[0106] These parasites are well known to polarize macrophages into M2 macrophages, their presence and infection triggering the production of cytokines such as IL-4, IL-10, and IL-13, which are the main inducers of macrophage polarization into M2. It is notably known that Schistosoma mansoni

[0107] induces an M2 response to limit tissue damage due to parasite eggs.

[0108] For example, chronic and intracellular bacterial infections include those related to Mycobacteriumtuberculosis, Mycobacteriumleprae, Helicobacterpilori, Escherichiacoli, Boreliaburgdorferi, Staphyloccocusaureus, Porphyromonasgingivalis. It is known that in chronic forms of tuberculosis infection with Mycobacteriumtuberculosis or in the lepromatous form of leprosy with Mycobacteriumleprae, an immune response with M2 macrophages is predominant.

[0109] For example, diseases caused by viral infection include those related to Epstein-Barr (EBV), Cytomegalovirus (CMV), Influenza virus, SARS-Cov2 (COVID-19), Hepatitis B (HBV), Hepatitis C (HCV), Herpes simplex virus (HSV), Human immunodeficiency virus (HIV), Varicella zoster (VZV), Measles virus (Measles virus). In particular, it is known that infection by HIV, HCV and HSV causes macrophages to be polarized towards an M2 phenotype contributing to immunosuppression and persistence of the virus in vivo.

[0110] Examples of fungal infections include diseases linked to Candida albicans or Aspergillus spp, for which the induction of an immune response in vivo, with M2 macrophages, in response to infection, in particular to promote tissue repair, has been demonstrated.

[0111] Metabolic diseases include obesity, metabolic syndromes, type 2 diabetes, and chronic inflammatory bowel diseases.

[0112] Examples of fibrosis include idiopathic pulmonary fibrosis, hepatic fibrosis and renal fibrosis.

[0113] Examples of cardiovascular disease include atherosclerosis and heart failure.

[0114] Autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus (SLE). Allergies include asthma and atopic dermatitis.

[0115] According to another particular aspect, the invention also relates to the use of the chemical composition of the invention, as an ingredient for inducing the trans-phenotyping of inactive anti-inflammatory macrophages of type M2 into active pro-inflammatory macrophages of type M1 in an anti-inflammatory cellular context.

[0116] In other words, the invention relates to the use of a chemical composition comprising a brown propolis extract of natural origin and standardized of the invention, in the treatment of a disease physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages, such as for example diseases of the inflammatory system, bacterial, viral or fungal infections and tumor conditions, in particular cancer, including breast cancer, pancreatic cancer or colorectal cancer, as well as all tumors infiltrated by macrophages associated with tumors, upper respiratory tract infections, infections by parasites of the genus Helminthes, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, and allergies.

[0117] Thus, according to the invention, the chemical composition comprising the extract of brown propolis of natural origin and standardized, obtained by the aforementioned protocol, is intended to be used as a medicament whose function is to induce the trans-phenotyping of inactive anti-inflammatory macrophages, M2, in particular M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context.

[0118] More specifically, the brown propolis extract, obtained according to the aforementioned protocol, in particular the aforementioned molecules A to E through their synergistic action are responsible for the induction function of trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of types M1, in an anti-inflammatory cellular context.

[0119] Macrophages are cells involved in innate and adaptive immune responses that allow pathogen elimination. Depending on their environment and the signals they detect, macrophages adapt their functions in different ways, a process known as "polarization."

[0120] We distinguish between "classical" M1 type polarization, whose activity is pro-inflammatory, and "alternative" M2 type polarization, which participates in the regulation of inflammation and the phenomenon of tissue repair.

[0121] M1 macrophages, described as pro-inflammatory, are important in direct host defense against pathogens, mediated by phagocytosis, secretion of pro-inflammatory cytokines and microbicidal molecules.

[0122] For example, macrophages are known to polarize into M1 macrophages after induction by interferon gamma (IFNg) and secrete several cytokines such as interleukins IL12p70, IL12p40, IL-6 or IL-23, tumor necrosis factor TNFα and Arginase.

[0123] M2 macrophages, described as regulators, act in the resolution phase of inflammation and the repair of damaged tissues. A distinction is made between the M2a and M2c phenotypes. The M2a phenotype, induced for example by interleukin IL-4, secretes mainly Arginase, TARC and interleukin IL1b. The M2c phenotype is induced by interleukin IL-10.

[0124] In the body, the immune response presents itself as a state of balance between an anti-inflammatory phenotype and an inflammatory phenotype.

[0125] In the presence of a pathogen, specific biomarkers are produced and detected by macrophages. Depending on the nature and presentation of these biomarkers, trans-phenotyping of macrophages to the M1 or M2 state occurs.

[0126] In the macrophage environment, the presence of these biomarkers disrupts the equilibrium state of the anti-inflammatory phenotype towards an inflammatory phenotype and vice versa, the polarization of macrophages occurs. Consequently, trans-phenotyping is a phenomenon that strengthens the immune response during an infection.

[0127] Advantageously, the chemical composition of the invention is used as an ingredient inducing the phenomenon of trans-phenotyping of macrophages. More particularly, the propolis extract, in particular brown propolis, of natural origin and standardized, of the composition of the invention, in particular the synergistic action of molecules A to E, has the effect of inducing the trans-phenotyping of M2 macrophages, in particular M2C, towards an M1 phenotype in an anti-inflammatory cellular context, and thus allowing immuno-modulation of the inflammatory response in the event of infection by a pathogen or tumor disease.

[0128] Thus, according to the invention, the administration, in a subject, of a therapeutically effective quantity of a chemical composition according to the invention, within the framework of a method for stimulating and / or strengthening the immune system or in a method for preventing the appearance or treating a disease physiologically inducing the trans-phenotyping of M1 macrophages into M2 macrophages as a pathological phenomenon, is beneficial for the state of health of the subject.

[0129] “Therapeutically effective amount” means the amount of chemical composition sufficient or effective to be administered to prevent, treat (stop, prevent progression of, inhibit, diminish or slow down) the disease, including the symptoms and diseases resulting therefrom, necessary to stimulate and / or strengthen the immune system of a subject.

[0130] The methods of the invention are to be applied to subjects suffering from a disease of the inflammatory system, a bacterial, viral or fungal infection and / or a tumor condition, in particular a cancer including breast cancer, pancreatic cancer or colorectal cancer, as well as all tumors infiltrated by macrophages associated with tumors, an infection of the upper respiratory tract, infections by parasites of the Helminthes genus, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, a cardiovascular disease, an autoimmune disease, and allergies.

[0131] According to a particular embodiment of the methods of the invention, the therapeutically effective quantity of chemical composition to be administered to a subject is 400 mg / day, this therapeutically effective quantity of chemical composition is ensured with a morning and evening administration dosage of a chemical composition of 200 mg comprising, in mass percentage relative to its total mass, between 30 and 50%, preferably 25.5% of dry extract of brown propolis; between 40 and 70%, preferably 63.8% of gum arabic; between 1 and <10%, preferably 9.6% of sucrose and maximum 3%, preferably 1.1% of silicon dioxide.

[0132] Preferably, said chemical composition of 200 mg is in a galenic form intended for oral administration, advantageously in the form of a tablet or a capsule.

[0133] Other characteristics and advantages of the invention will emerge from the detailed description which follows of the non-limiting embodiments of the invention, with reference to the appended figures, in which:

[0134] shows the summary of the steps of the experimental protocol allowing to show the impact of the composition on the polarization of macrophages,

[0135] shows the polygon of the expression of the biomarkers CD14, CD64, CD11a, CD86, detected by flow cytometry, of different populations of macrophages presenting different phenotypes induced or not by cytokines (M1=INFg, M2a=IL4, M2c=IL10, M0=medium alone)

[0136] shows the polygons of the expression of the biomarkers CD14, CD64, CD11a, CD86, detected by flow cytometry, of a population of macrophages, in an anti-inflammatory context, after induction by IL10, in the presence of a chemical composition of the invention of type ESIT 12, or individual molecules, namely: galangin, chrysin, pinocembrin, pinobanskin and caffeic acid at a weighted concentration

[0137] shows the hierarchical clustering of the results obtained for ESIT 12, ESIT 12r as well as the individual molecules purified at a weighted concentration galangin, chrysin, pinocembrin, pinobanskin and caffeic acid, in anti-inflammatory condition (induction to IL10 = macrophage M2c) for the 6 donors taking into account all the cytokine and membrane markers.

[0138] [Fig 4A] shows the expression of the cytokine TNF-α in the presence of ESIT 12, ESIT 12r and in the presence of the individual molecules: galangin, chrysin, pinocembrin, pinobanskin and caffeic acid at a weighted concentration, in anti-inflammatory condition with M2c macrophages, after induction with IL-10,

[0139] [Fig 4B] shows the expression of the cytokine IL-6 in the presence of ESIT 12, ESIT 12r and in the presence of the individual molecules galangin, chrysin, pinocembrin, pinobanskin and caffeic acid at a weighted concentration, in anti-inflammatory condition with M2c macrophages, after induction with IL-10,

[0140] [Fig 4C] shows the expression of the cytokine IL-10 in the presence of ESIT 12, ESIT 12r and in the presence of the individual molecules galangin, chrysin, pinocembrin, pinobanskin and caffeic acid at a weighted concentration, in anti-inflammatory condition with M2c macrophages, after induction with IL-10,

[0141] shows the survival curve of a panel of patients indicating the number of patients who did not contract the symptoms of an infectious disease over 84 days, in a patient population with administration of a chemical composition according to the invention compared to a population administering a placebo chemical composition without the brown propolis extract.

[0142] Aspects of the invention will be better understood by reading the examples, given for informational purposes only and without limitation, and the test results which follow.

[0143] Example 1: Process for preparing a propolis extract of natural origin and standardized according to the invention

[0144] The propolis extract of natural origin and standardized with the chemical composition of the invention is obtained by carrying out a dynamic multi-extraction method, comprising the following successive steps:

[0145] – a) raw propolis, in particular raw brown propolis, is taken from the hive,

[0146] – b) the beeswax and impurities are removed from the raw propolis by aqueous extraction, so as to recover only the resinous, balsamic component of the raw propolis in pasty form,

[0147] – c) three successive hydro-alcoholic extractions are carried out on the residue of the previous extraction, for example with the propolis removed from the beeswax, then on the extraction residues obtained, with a water / ethanol extraction mixture,

[0148] – d) the propolis extract, in particular the brown propolis extract, of natural origin and standardized with the chemical composition of the invention, is recovered,

[0149] – e) optionally, the propolis extract from step d) is spray-dried so as to obtain it in the form of a powder, for example the drying is carried out by atomization at 180°C, so as to obtain a dry propolis extract in the form of a powder having a final humidity of less than 10%.

[0150] For step b), the separation of the raw propolis, beeswax and impurities is carried out by aqueous extraction, 100% water at approximately 80°C for approximately 10 hours under ultrasound, followed by a cooling step to approximately 8°C and a filtration step on a filter of around 30µm.

[0151] For step c), each of the extractions with the hydro-alcoholic water / ethanol mixture is carried out on the residue of the previous extraction, increasing the alcohol content of the water / ethanol extraction mixture and increasing the extraction temperature. In addition, each of the extractions carried out ends with a cooling step at 15°C, a filtration step with a 30 to 50µm filter and a concentration step by evaporation to eliminate the alcohol residues, so as to obtain a propolis extraction residue as concentrated as possible in active ingredient.

[0152] Preferably, for step c):

[0153] –the first extraction, on raw propolis, is carried out with a water / ethanol mixture between 35 and 40 degrees of alcohol at an extraction temperature between 40 and 60°C,

[0154] - the second extraction, on the propolis residue from the first extraction, is carried out with a water / ethanol mixture between 55 and 60 degrees of alcohol at an extraction temperature between 40 and 80°C,

[0155] - the third extraction, on the propolis residue from the second extraction, is carried out with a water / ethanol mixture between 70 and 80 degrees of alcohol at an extraction temperature between 60 and 80°C.

[0156] For step c), the ratio between the propolis extraction residue and the extraction mixture is between 0.1:1 and 1:1.

[0157] For step e), spray drying is carried out at minimum temperatures between 65°C and 85°C, preferably at a minimum temperature of 65°C and at maximum temperatures between 175°C and 185°C, preferably at 185°C so as to preserve the pharmaceutical qualities of the propolis extract and to maintain its action.

[0158] For the series of three extractions, the time to complete an extraction is between 4 and 36 hours.

[0159] At the end of the third extraction, the brown propolis extract of natural origin and standardized according to the invention is obtained, in the form of a hydro-alcoholic solution of brown propolis comprising at least in mixture, one molecule A from the phenolic acid family, one molecule B from the flavanone family, one molecule C from the flavone family, one molecule D from the dihydroflavonole family and one molecule E from the flavonole family.

[0160] Example 2: Chemical composition according to the invention with different supports

[0161] Table 1 below shows examples of chemical compositions in liquid or solid powder form according to the invention. Each of these chemical compositions comprises different supports mixed with the standardized natural propolis extract, in powder form, or in liquid form, obtained by the process of Example 1.

[0162] To manufacture these chemical compositions according to the invention, the standardized natural propolis extracts in powder form are obtained after carrying out step e) of drying of the process of example 1. In particular, step e) consists of spray drying carried out at a minimum temperature of 65°C to obtain the natural and standardized dry extract of brown propolis.

[0163] In the same way, the standardized natural propolis extracts in liquid form are obtained after carrying out step c) and recovered in step d) of the process of example 1.

[0164] The percentage ranges are expressed in mass relative to the total mass of the chemical composition, which corresponds to a value of 100%.

[0165] Table 1: CompositionComponents%CompositionComponents%1Propolis extract4-156Propolis extract8-50Ethanol65-83Gum arabic40-85Water7-24Sucrose<102Propolis extract70-75Silicon dioxide≤3Ethanol21-257Propolis extract10-35Water3-5Maltodextrin58-823Propolis extract3-10Heat-treated liquid sucrose4-5Glycerin85-94Silicon dioxide≤3Water3-58Propolis extract13-204Propolis extract1-3Propylene glycol80-90Glycerin92-969Propolis extract10-15Water1-3Soybean oil85-905Propolis extract2-310Extract Propolis10-15Glycerin85-90Sunflower seed oil85-90Water7-12Heat-treated liquid sucrose1-1.5

[0166] Example 3: Chemical composition of the invention

[0167] Chemical composition according to the invention, in powder form, named ESIT12, comprising, by mass relative to its total dry mass: 25.5% of a dry extract of brown propolis, 63.8% of gum arabic, 9.6% of sucrose and 1.1% of silicon dioxide.

[0168] The dry propolis extract is a dry extract of brown propolis of natural origin and standardized obtained by the protocol of example 1, after carrying out step e) of drying by atomization at a temperature of 65°C.

[0169] The biochemical analysis of several dry extracts of brown propolis, obtained by the protocol as mentioned above, was carried out by any technique known to those skilled in the art, for example high-performance liquid chromatography in reverse phase by ultraviolet-visible absorption spectroscopy and mass spectrometry. This analysis demonstrated that 100% of the brown propolis extracts comprise at least one molecule A from the family of phenolic acids, in particular caffeic acid; one molecule B from the family of flavanones, in particular pinocembrin; one molecule C from the family of flavones, in particular chrysin; one molecule D from the family of dihydroflavonoles, in particular pinobanskin and one molecule E from the family of flavonoles in particular galangin.

[0170] In the present invention, the chemical composition of ESIT 12 of example 3 is obtained by a protocol which consists of mixing said aforementioned dry extract of brown propolis, with the powders of gum arabic, sucrose and silicon dioxide, in the aforementioned mass proportions.

[0171] A biochemical analysis of the compounds of several ESIT 12 compositions obtained by implementing the aforementioned protocol was carried out by any standard technique of a person skilled in the art, in particular that mentioned above. This analysis made it possible to show that 100% of the “ESIT 12” chemical compositions obtained according to the methods of this example 3, comprise, in mass percentage relative to their total dry mass, between 1.3% and 1.8% of molecule A from the family of phenolic acids, including caffeic acid; between 0.6% and 1.0% of molecule B from the family of flavanones, including pinocembrin; between 1.4% and 2.8% of molecule C from the family of flavones, including chrysin; between 2.4% and 3.3% of molecule D from the dihydroflavonole family including pinobanskin and between 1.1% and 1.7% of molecule E from the flavonole family including galangin.

[0172] Example 4: Impact of the chemical composition of the invention on the trans-phenotyping of macrophages

[0173] It is known that an in vitro culture of macrophages in the basal M0 state polarizes towards a specific phenotype, after induction in the presence of differentiation cytokines.

[0174] Thus, for an in vitro culture of macrophages, it is known:

[0175] – that in the presence of interleukin 10 called “IL10”, macrophages polarize towards an M2c phenotype, which is representative of an anti-inflammatory context,

[0176] – that in the presence of gamma interferon known as “IFNg”, macrophages polarize towards an M1 phenotype,

[0177] – that in the presence, at intervals of a few days, of IL10 then of IFNg, it is possible to visualize a trans-phenotyping of macrophages from an M2c phenotype to an M1 phenotype,

[0178] – that Lipopolysaccharide called “LPS”, as a bacterial membrane constituent, activates macrophages, in order to allow their increased polarization towards a specific phenotype and the secretion of cytokines in their culture environment.

[0179] The impact of a chemical composition according to Example 3, named “ESIT12” on macrophage polarization was carried out in vitro on primary human macrophages obtained from peripheral mononuclear cells (PBMC) from six donors D1 to D6.

[0180] Schematically shows the experimental protocol carried out to visualize the impact of the chemical composition of the invention on the trans-phenotyping of macrophages.

[0181] PBMCs are isolated from blood samples and then placed in culture medium with a density of 600,000 adherent cells / well for 2 days before being placed in contact with the extracts to be tested.

[0182] To visualize the impact of propolis extracts in the trans-phenotyping of macrophages, macrophages are incubated for 9 days at 37°C and 5% CO2, with:

[0183] – either the chemical composition according to example 3 called “ESIT 12”,

[0184] – either a solution comprising one of the molecules A to E purified at a weighted concentration,

[0185] – either with a solution comprising all the purified molecules representative of the propolis extract.

[0186] At 6 days, the culture medium is renewed or supplemented with cytokine inducing trans-phenotyping of macrophages.

[0187] At 8 days, LPS was added to all culture wells, so as to have increased macrophage activity and cytokine secretion into the culture environment depending on the phenotype of polarized macrophages.

[0188] After 9 days of culture, the cells are detached and then dissociated to carry out multiparametric immunolabeling with acquisition of fluorescence signals by flow cytometry.

[0189] The markers used in flow cytometry to identify the different macrophage populations are described in Table 2 below:

[0190] Table 2:MarkersM0M1M2aM2CD14+ / -+ / --+++CD16++ / --++CD11a+++++++++CD64++++-+++HLA-DR+++++++++ / -CD86+++++-CD200R--+++-

[0191] The combination of CD14 / CD64 / CD11a / CD86 marker expression was retained to distinguish and discriminate between different macrophage phenotypes.

[0192] The polygon of the compound of these 4 markers is used as a basis for comparing the impact of the chemical composition of the invention and the purified molecules on macrophage polarization.

[0193] On the 4-marker polygons, the points correspond to the fluorescence medians of the 4 CD14 / CD64 / CD11a / CD86 markers and the circles at the distance to the average of the fluorescence signals detected for the population of cells detached after 9 days of culture.

[0194] After 9 days of culture, a dosage of 13 cytokines is also carried out on the recovered culture supernatant.

[0195] The 13 cytokines detected by flow cytometry in the supernatant to discriminate the secretomes of the different macrophage phenotypes are identified in Table 3 belowMarkersMoM1M2aM2cIl12-p70-+--TNF-α++++IL-6+++-IL-4--NA-IL-10+--NAIL-1b++++Arginase++++TARC++++IL1-RA++++IL12-p40+++-IL-23-+--IFN-g-NA--IP-10++++

[0196] In Table 3: The indication “+” indicates that the cytokine / marker was detected by its fluorescence signal in flow cytometry in the culture supernatant. The indication “-” indicates that the fluorescence signal corresponding to the marker was not detected in the supernatant. The indication “NA” indicates that the fluorescence signal corresponding to the cytokine / marker was not analyzed because it was already present in the culture medium.

[0197] The results show that the secretome of different macrophage populations is as follows:

[0198] – M0 macrophages, in the basal state, only in the culture medium, secrete several cytokines but at a relatively low level,

[0199] – macrophages of M1 phenotype, after induction with IFNg in the medium, differentially secrete at M0: IL12-p70; IL12-p40; TNF-α, IL-6, IL-23 and Arginase,

[0200] – macrophages of M2c phenotype, after induction with IL-10, repress the expression of a majority of cytokines, notably IL-6 and TNF-α.

[0201] Macrophage activation controls IFNg / IL10 are used to capture M1 / M2c phenotypes respectively.

[0202] The purified extracts and molecules are incubated after differentiation cytokines to verify their ability to induce trans-phenotyping of macrophages.

[0203] More specifically in the experimental protocol, after 2 days of growth of PBMCs at a basal M0 state, 20 ng / ml of IL-10 is added to all culture wells in order to induce the polarization of macrophages towards an M2c phenotype. The addition of IL-10 in the macrophage culture medium makes it possible to simulate in vitro an anti-inflammatory cytokine environment of macrophages. Thus, in this anti-inflammatory context induced by IL-10 the macrophages will be M2c.

[0204] Then, on day 6, the following elements are added to different wells:

[0205] – 7 µmol / L of the chemical composition of the invention according to example 3 called “ESIT12”,

[0206] – 7 µmol / L of a solution called “ESIT 12r”, comprising in mass percentage relative to its total mass, between 1.3% and 1.8% of caffeic acid, between 0.6% and 1.0% of pinocembrin; between 1.4% and 2.8% of chrysin; between 2.4% and 3.3% of pinobanskin and between 1.1% and 1.7% of galangin molecule E, that is to say in the same proportions and concentrations where they are present in the composition of ESIT 12,

[0207] – 7 µmol / L of a solution of purified galangin molecules called “Galanr”,

[0208] - 7 µmol / L of a solution of purified chrysin molecules called “Chrysr”,

[0209] - 7 µmol / L of a solution of purified pinocembrin molecules called “PinoCr”,

[0210] - 7 µmol / L of a solution of purified pinobanskin molecules called “PinoBr”,

[0211] - 7 µmol / L of a solution of purified caffeic acid molecules called “CafAcr”.

[0212] A control well for the differentiation of cells into M1 is made by adding, on day 2, 20 ng / mL IFNg.

[0213] A control well for the differentiation of cells into M2c is made by adding, on day 2, 20 ng / mL of IL10.

[0214] A control well for trans-phenotyping of macrophages from M2c to M1 is performed by adding 20 ng / mL of IL 10 on day 2, then at 6 days 20 ng / mL of IFNg.

[0215] The results are shown on the 4-marker polygons, in which the arrows represent the fluorescence signals detected by flow cytometry for the cell population detached after 9 days and placed in the presence of the different extracts and solution.

[0216] With this, we observe that in an anti-inflammatory context, created in vitro after induction with IL-10, the macrophages of M2c profile, initially present in culture, return mainly to an M0 / M1 phenotype in the presence of ESIT 12 of the invention and the purified molecules at weighted concentration.

[0217] Thus, the ESIT 12 composition of the invention exhibits immunomodulatory activity, from trans-phenotyping of M2c macrophages to an M0 / M1 phenotype, in an anti-inflammatory context induced by IL-10.

[0218] These results show that the chemical composition of the invention therefore participates in regulating the inflammatory mechanism, by acting on the trans-phenotyping of macrophages.

[0219] Lacompile the results of measurements of the intensity of the fluorescence signals obtained by flow cytometry analysis for each of the markers (IP10, IL12 p40, CD86, CD16, CD14, CD64, CD200r, CD11a, HLA-DR, IL6, IL23, IL4, IL1b, IFNg, IL12 p70, TNF-α, Arginase, TARC, IL1RA) of the supernatants of the cultures from each of the donors D1, D2, D3, D4, D5 and D6, in anti-inflammatory conditions (after induction with IL-10) after 9 days of culture, brought into contact with the extracts and activated with LPS.

[0220] Bioinformatics analysis of the intensity of these signals allows the results to be grouped in the form of a hierarchical cluster by intensity and nature of the fluorescence signals detected by flow cytometry. Groupings by signals and fluorescent intensity called hierarchical "clusters" are created for ESIT 12, ESIT12r as well as the weighted purified molecules, this by analogy of the signals detected for the controls in particular by comparison with the M2c to M1 trans-phenotyping control (IL-10 induction then IFNg).

[0221] Thus, it is shown, by a hierarchical grouping of the intensity of the fluorescence signals for each marker and each donor population, that ESIT 12, and similarly ESIT 12r, make it possible to recreate a phenotype similar to the M1 phenotype from an M2c macrophage (cluster A).

[0222] Lamontre also shows that the molecules alone, independently of each other, are not sufficient to allow trans-phenotyping of macrophages from M2c to M1 as evidenced by cluster B.

[0223] Only the combination of the 5 concentration-weighted molecules (CafAcr, Chrysr, PinoCr, PinoBr and Galanr) combined under ESIT12r allows for a compilation of fluorescence signals comparable to the M1 control.

[0224] Lamontre therefore, on the basis of 13 cytokines, whose fluorescence intensity is detected in the supernatant, that only the combination of the 5 molecules at weighted concentration allows the trans-phenotyping of M2c to M1 in an anti-inflammatory context. A synergy between molecules A to E of the brown propolis extract, in particular between caffeic acid, pinocembrin; chrysin; pinobanskin and galangin in the indicated proportions, is essential for the chemical composition of the invention to exhibit an immuno-modulatory activity of trans-phenotyping of macrophages from an M2c to M1 phenotype in an anti-inflammatory cellular context, in vitro.

[0225] For example, Figures 4A, 4B and 4C show, respectively, the fluorescence intensity of TNFα, IL-6 and IL-10 markers within cell populations and the dosage of cytokines excreted in the cell culture supernatant after 9 days of culture, IL10 induction and LPS overactivation.

[0226] The chemical composition of the invention ESIT 12 and the ESIT 12r solution with molecules A to E acting in synergy, in the indicated proportions, each secrete the pro-inflammatory cytokines markers of inflammation, namely TNF-α and IL-6, while the IL-10 of the secretome is low. ESIT 12 and ESIT12r, in anti-inflammatory condition induced by IL-10 allow the macrophage to polarize into M1 and to regain a pro-inflammatory cytokine producing function.

[0227] It should be noted that taken individually, looking at the dosage of TNFα and IL-6, the overall effect of the purified molecules is similar to that of the chemical composition of the invention ESIT 12 or the solution of the molecules in a mixture at weighted concentration ESIT12r. This is why an analysis by individual marker is not sufficient to report the trans-phenotyping of macrophages from M2c to M1, only a multiparametric analysis of the fluorescence intensity of several markers (cluster of the) really allows to show the impact and the synergy of the molecules in a mixture on the trans-phenotyping of macrophages.

[0228] The measurement of the fluorescence intensity of the cytokine markers by flow cytometry shows that in anti-inflammatory conditions, the chemical composition of the invention ESIT 12, in particular all of these molecules A to E acting in synergy, plays a role of immunostimulant of macrophages, synonymous with an immuno-modulatory activity.

[0229] These results show that the chemical composition of the invention ESIT 12, obtained according to example 3, by the presence of a brown propolis extract of natural origin and standardized comprising the 5 molecules A to E acting in synergy, in an anti-inflammatory context, allows:

[0230] – to restore the production of pro-inflammatory cytokines such as TNF-α and IL-6, thus promoting the recruitment of immune cells to the site of inflammation,

[0231] - the M2c macrophage to polarize towards an M0 / M1 phenotype.

[0232] Example 5: Impact of the administration of the chemical composition of the invention on the probability of a subject developing an infection

[0233] A clinical study of the impact of administering a chemical composition of the invention was conducted on 309 adult subjects for 84 days, the results of this study are indicated in the.

[0234] Subjects were adults between 25 and 69 years of age, not subjected to antibiotics in the 12 weeks preceding the study and to anti-inflammatory or immunological drugs in the 4 weeks preceding the study. Test subjects were recruited healthy.

[0235] Test subjects administered either the chemical composition of the invention or its placebo equivalent devoid of brown propolis extract.

[0236] The chemical composition administered in the clinical study is in the form of a 200 mg capsule

[0237] The 200 mg capsule of the chemical composition of the invention tested in the study includes:

[0238] - between 30 and 50%, preferably 25.5% of dry extract of brown propolis obtained according to the process indicated in example 3,

[0239] - between 40 and 70%, preferably 63.8% of gum arabic;

[0240] - between 1 and <10%, preferably 9.6% of sucrose and,

[0241] - maximum 3%, preferably 1.1% of silicon dioxide.

[0242] The subjects tested with the chemical composition administered each day an effective quantity of 400 mg of chemical composition of the invention, corresponding to an extract of brown propolis of natural and standardized origin obtained according to the protocol of example 3 comprising at least 48 mg of polyphenols.

[0243] More specifically, for the study, the test subjects absorbed orally, each day, one 200 mg capsule in the morning and one 200 mg capsule in the evening (i.e. a dosage of 400 mg / day). These 200 mg capsules corresponded to the chemical composition of the invention as mentioned above or to its placebo equivalent devoid of 30 to 50% of dry extract of brown propolis. In the placebo, the percentage of propolis extract was replaced by the support gum arabic, sucrose, silicon dioxide with a mixture in the same proportions.

[0244] Of the 309 subjects, 153 subjects received the chemical composition of the invention as described above and 156 subjects received a placebo chemical composition.

[0245] The placebo chemical composition corresponds to the chemical composition of the invention without brown propolis extract.

[0246] Over 84 days, each subject was monitored for symptoms of contracting or not contracting a significant upper respiratory tract infection.

[0247] Symptoms of infection were diagnosed based on subjects' responses to an upper respiratory symptom assessment questionnaire and a score determined to be indicative of an upper respiratory infection.

[0248] Specifically, the severity of the following symptoms was assessed on a scale of 0 (no symptoms) to 7 (severe symptoms): runny nose; blocked nose, sneezing, sore throat, irritated throat, cough, hoarseness, head congestion, chest congestion, fatigue, headache, body aches, fever.

[0249] The impact of existing symptoms and how they interfere with the subject's daily activity was also rated on a scale of 0 (not at all) to 7 (severe interference) with respect to the following activities: clear thinking, quality of sleep, easy breathing, ability to walk, climb stairs or exercise, performing daily tasks, working outside and inside the home, interacting with others and personal life. All significant criteria of symptom severity and their impact, assessed for each subject, result in a score. Based on a determined score, the subject will be diagnosed as having a significant infection, including an upper respiratory tract infection.

[0250] As soon as a subject presents symptoms of a significant infection, he is eliminated from the survival curve and considered as a subject who did not "survive" the onset of an infection.

[0251] During the study, 5 subjects administering the chemical composition of the invention and 8 subjects administering the placebo did not return to one of the visits; they are considered to have left the study.

[0252] Ladonne the survival curve of each population of subjects treated with the chemical composition of the invention or its placebo equivalent devoid of brown propolis extract. This curve indicates the probability of a subject not contracting an infection, following the administration of a chemical composition of the invention or a placebo over 84 days, according to the daily dosage indicated previously.

[0253] The results of the survival curve show that the risk of contracting an infection, particularly of the upper respiratory tract, is reduced by 31% in subjects who have administered in vivo the chemical composition of the invention compared to subjects who have administered the placebo.

[0254] Thus, the administration of the chemical composition of the invention in a method of preventing and / or treating upper respiratory tract infections makes it possible to reduce the risks of developing an infection in subjects already presenting as symptoms a sore throat, dull dysphonia or swelling and redness of the throat.

[0255] This study on the administration of the chemical composition of the invention clearly shows that the immunomodulatory activity of trans-phenotyping inactive anti-inflammatory macrophages, type M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context observed in vitro, is also functional in vivo to treat or prevent upper respiratory tract diseases.

[0256] Based on this same 84-day study, the interferon-gamma / interleukin 4 (IFN-ʏ / IL4) ratio in a blood sample taken from subjects with placebo or having administered the composition of the invention with propolis extract was evaluated by any method known to those skilled in the art.

[0257] The IFN-ʏ / IL4 ratio allows us to assess the balance between the immune responses of the adaptive immune system, mainly involved in defense against intracellular infections by pathogens such as bacteria and viruses; and the innate immune system, mainly involved in defense against allergies or parasites. A stable IFN-ʏ and IL4 ratio over time indicates the absence of an immune response.

[0258] A modification of the IFN-ʏ / IL4 ratio allows a pre-orientation of the polarization of macrophages towards an immune response called TH1 which notably gives rise to macrophages of the M1 phenotype, which during an illness of any nature allows the innate immune system to exert an inflammatory response capable of reacting in defense where these illnesses tend to weaken the immune response by polarizing the macrophages according to an M2 phenotype.

[0259] The IFN-ʏ / IL4 ratio also serves as a specific marker of trained immunity. Trained immunity refers to the ability of the immune system to respond more effectively to infections after prior exposure to pathogens, even if this exposure does not necessarily result in infection. Trained immunity is a long-term functional change in cells of the innate immune system that leads to an altered response to a second, unrelated challenge / infection. Trained immunity is known to be mediated by epigenetic modifications.

[0260] In the placebo group, the IFN-ʏ / IL4 ratio did not change significantly (+27%, with a p-value of 0.98), whereas it changed significantly in the propolis extract group (+293%, with a p-value of <0.05).

[0261] Over the same 84-day period, the inflammatory state was assessed with the interleukin 6 / interleukin 10 (IL-6 / IL-10) ratio. The non-variation of the IL6 / IL10 ratio demonstrates that neither the pro-inflammatory nor the anti-inflammatory state are at this stage with polarized macrophages, the innate immune system is on alert, this is called trained immunity.

[0262] The IL-6 / IL-10 ratio therefore serves as a specific marker of trained immunity. Trained immunity refers to the ability of the immune system to respond more effectively to infections after prior exposure to pathogens, even if this exposure does not necessarily result in infection. Trained immunity is a long-term functional change in the cells of the innate immune system that leads to an altered response to a second, unrelated challenge / infection. Trained immunity is known to be mediated by epigenetic modifications.

[0263] During the study, the IL-6 / IL-10 ratio showed no imbalance or difference between subjects with placebo (-4%, with a p value of 0.81) and subjects with the chemical composition of the invention (-7%, with a p value of 0.74).

[0264] Based on measurements of the IFN-ʏ / IL4 and IL-6 / IL-10 ratio, measured in a population having administered the placebo or the chemical composition of the invention, it is observed that the immune system has not modified its “inflammatory state” (IL-6 / IL-10 ratio) but in the population with the propolis extract composition of the invention, the immune system is in a state of training which makes it more reactive in M1 polarization of macrophages in the event of infection.

[0265] Example 6: Bioavailability and pharmacokinetic study of the chemical composition of the invention

[0266] A bioavailability and pharmacokinetic study of the chemical composition used in example 5 was carried out to demonstrate that the metabolism of phenolic compounds does not cause any major modification, in particular the glucuronidation of the native forms of molecules A to E which are known to allow better solubilization in vivo before renal elimination.

[0267] For the study, after administration to a patient, molecules A to E were searched for and measured in the blood or urine, then their metabolic form determined by any technique known to those skilled in the art. …..

[0268] This study has demonstrated that, after administration to a subject of the chemical composition of the invention, the metabolized form in the blood of molecules A to E of the brown propolis extract of this chemical composition is identical to their forms in the chemical composition.

[0269] Indeed, after administration by a patient, in the blood the molecules A to E, in particular caffeic acid, pinocembrin; chrysin; pinobanskin and galangin, are present, associated with a glucuronide, but in a form of molecular structure identical to that present in the chemical composition of the invention.

[0270] The data from this study also show that in a dynamic in vivo environment of the blood circulation, the maximum concentration of molecules A to E reaches approximately 1.5 µM, which is comparable to the concentration of approximately 7.5 µM obtained with static in vitro tests on macrophages.

[0271] This pharmacokinetic study allows to show the presence and the implication of molecules A to E, acting in synergy, in the mechanism of action in vivo to have an immuno-modulation activity by trans-phenotyping of inactive anti-inflammatory macrophages, of type M2C, in active pro-inflammatory macrophages of type M1.

[0272] All of these results show that the chemical composition of the invention has an immunomodulatory activity which makes it possible to strengthen the activity of the immune system, in particular in an anti-inflammatory context, notably by modulating macrophages of the M2c phenotype towards an M1 phenotype producing cytokines.

[0273] These results show that a chemical composition according to the invention comprising a propolis extract, obtained by the aforementioned process guaranteeing the presence of molecules A to E, in the indicated proportions, has an immunomodulatory activity in the inflammatory mechanism which makes it possible to strengthen the immune system in the event of infection by a pathogen or tumor disease. The immunomodulatory activity consisting of a trans-phenotyping mechanism of inactive anti-inflammatory macrophages, of type M2C, into active pro-inflammatory macrophages of types M1, in an anti-inflammatory cellular context

[0274] The chemical composition of the invention presents no toxicity to the human or animal organism and has the advantage of being of natural origin.

[0275] In addition, the synergistic action of molecules A to E of the brown propolis extract contained in the chemical composition of the invention makes it possible to reduce pathological signs in the event of infection and to strengthen the immune activity mediated by macrophages.

[0276] Accordingly, the chemical composition of the invention is a good clinical candidate for preventing and / or treating conditions inducing an adverse inflammatory response of the immune system.

Claims

Method for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages M1, in an anti-inflammatory cellular context, using a chemical composition comprising a brown propolis extract of natural origin and standardized, said brown propolis extract comprising at least, in a mixture, a molecule A from the phenolic acid family, a molecule B from the flavanone family, a molecule C from the flavone family, a molecule D from the dihydroflavonole family and a molecule E from the flavonole family. Method for inducing trans-phenotyping of macrophages, according to claim 1, characterized in that the brown propolis extract consists of brown propolis, derived from a part of a plant belonging to the poplar family of the genus Populusspp, such as Populusnigra L., Populusalba L., or Populustremula L. A method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that said chemical composition further comprises gum arabic, sucrose and silicon dioxide. Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that said chemical composition comprises, in mass percentage relative to its total dry mass, between 1.3% and 1.8% of molecule A from the phenolic acid family, between 0.6% and 1.0% of molecule B from the flavanone family, between 1.4% and 2.8% of molecule C from the flavone family, between 2.4% and 3.3% of molecule D from the dihydroflavonole family and between 1.1% and 1.7% of molecule E from the flavonole family. Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that:the family of phenolic acids corresponds to a cinnamic acid or its derivatives, such as o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid;or corresponds to a benzoic acid and its derivatives, such as m-hydroxybenzoic acid, p-hydroxybenzoic acid, protacatechic acid, vanillic acid, syringic acid:the family of flavanones has a basic carbon chain of formula II below: the flavone family has a basic carbon chain of formula III below: the dihydroflavonole family has a basic carbon chain of formula IV below: the flavonol family has a basic carbon chain of formula V below: . Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that the brown propolis extract comprises at least, in a mixture,: as molecule A of caffeic acid of formula below: as molecule B of pinocembrin with the formula below: as molecule C of chrysin with the formula below: as molecule D of pinobanskin with the formula below: and as molecule E of galangin with the formula below: . Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that the standardized natural brown propolis extract is obtained by carrying out the following successive steps:– a) the raw propolis is taken from the hive,– b) the beeswax and impurities are removed from the raw propolis by aqueous extraction, so as to recover only the resinous, balsamic component of the raw propolis in pasty form,– c) three successive hydro-alcoholic extractions are carried out on the residue from the previous extraction, for example with the propolis removed from the beeswax, then on the extraction residues obtained, with a water / ethanol extraction mixture,– d) the standardized natural brown propolis extract is recovered,– e) optionally, the propolis extract from step d) is spray-dried so as to obtain a dry brown propolis extract,in powder form, preferably the dry extract of brown propolis with a final humidity of less than 10%., Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that said chemical composition is in a liquid or solid form, in powder, solution, gel, emulsion, cream, capsule or tablet. Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that, in an anti-inflammatory cellular context, in particular of the in vivo or in vitro type, said chemical composition comprising the extract of brown propolis of natural origin and standardized is in contact with the macrophages. Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that said chemical composition is in a food product, preferably a food supplement, or in a cosmetic product. Method for inducing trans-phenotyping of macrophages, according to any one of the preceding claims, characterized in that said chemical composition is in a pharmaceutically and / or dermatologically acceptable galenic form for the human or animal organism, for in vivo administration. Food supplement, characterized in that said food supplement comprises, or consists of, a chemical composition according to any one of claims 1 to 11, comprising an extract of brown propolis of natural origin and standardized. Chemical composition according to any one of claims 1 to 11, comprising a standardized natural brown propolis extract, for use as a food supplement. Chemical composition according to the preceding claim, for its use as a food supplement stimulating and / or strengthening the immune system. Chemical composition according to any one of claims 13 or 14, for its use as a food supplement for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context, in particular an anti-inflammatory cellular context in vivo. Chemical composition according to any one of claims 1 to 11, comprising a standardized natural brown propolis extract, for use as a medicament. Chemical composition according to the preceding claim, for its use as a medicament stimulating and / or strengthening the immune system. Chemical composition according to any one of claims 16 or 17, for its use as a medicament for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages M1, in an anti-inflammatory cellular context, in particular an anti-inflammatory cellular context in vivo or in vitro. Chemical composition according to any one of claims 16 to 17, comprising a brown propolis extract of natural origin and standardized, intended to be used in the prevention or treatment of diseases physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages. Chemical composition according to the preceding claim, comprising a brown propolis extract of natural origin and standardized, intended for use in the prevention or treatment of diseases of the inflammatory system, bacterial, viral or fungal infections and tumor conditions. Chemical composition according to the preceding claim, comprising a brown propolis extract of natural origin and standardized, intended for use in the prevention or treatment of cancer including breast, pancreatic or colorectal cancer, as well as all tumors infiltrated by tumor-associated macrophages, upper respiratory tract infections, infections by parasites of the Helminthes genus, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, and allergies. Chemical composition according to any one of claims 16 to 17, comprising a brown propolis extract of natural origin and standardized, for use in a therapeutic method for preventing or treating diseases, physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages. Chemical composition according to the preceding claim, comprising a brown propolis extract of natural origin and standardized, for use in a therapeutic method for preventing or treating diseases of the inflammatory system, bacterial, viral or fungal infections and tumor conditions. Chemical composition according to the preceding claim, for use in a therapeutic method for preventing or treating cancer including breast, pancreatic or colorectal cancer, as well as all tumors infiltrated by tumor-associated macrophages, upper respiratory tract infections, infections by parasites of the genus Helminthes, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, and allergies. Use of a chemical composition comprising a brown propolis extract of natural origin and standardized according to any one of claims 1 to 11, as an agent for inducing the trans-phenotyping of inactive anti-inflammatory macrophages M2, in particular M2C, into active pro-inflammatory macrophages of type M1, in an anti-inflammatory cellular context. Use of a chemical composition, according to any one of claims 1 to 11, as an active ingredient in a food supplement formulation; in particular in a food supplement formulation intended to prevent diseases physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages; preferably in a food supplement formulation intended to prevent diseases of the inflammatory system, bacterial, viral or fungal infections and tumoral conditions;in particular in a formulation of a food supplement intended to prevent cancer, including breast, pancreatic or colorectal cancer, as well as all tumors infiltrated by tumor-associated macrophages, upper respiratory tract infections, infections by parasites of the genus Helminthes, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, and allergies.; Use of a chemical composition, according to any one of claims 1 to 11, as an active ingredient in a drug formulation. Use of a chemical composition, according to the preceding claim, as an active ingredient in a formulation of a drug intended to prevent or treat diseases physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages. Use of a chemical composition, according to the preceding claim, as an active ingredient in a drug formulation intended to prevent or treat diseases of the inflammatory system, bacterial, viral or fungal infections and tumor conditions. Use of a chemical composition, according to the preceding claim, as an active ingredient in a formulation of a drug intended to prevent or treat cancer, including breast, pancreatic or colorectal cancer, as well as all tumors infiltrated by tumor-associated macrophages, upper respiratory tract infections, infections by parasites of the Helminthes genus, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, and allergies. Use of a chemical composition comprising a brown propolis extract of natural origin and standardized according to any one of claims 1 to 11, in the treatment of a disease physiologically inducing, as a pathological phenomenon, the trans-phenotyping of M1 macrophages into M2 macrophages, such as for example diseases of the inflammatory system, bacterial, viral or fungal infections and tumor conditions, in particular cancer, including breast cancer, pancreatic cancer or colorectal cancer, as well as all tumors infiltrated by macrophages associated with tumors, upper respiratory tract infections, infections by parasites of the helminth genus, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, cardiovascular diseases, autoimmune diseases, and allergies. A method for stimulating and / or strengthening the immune system of a subject, in particular his trained immunity, said method comprising the administration, of a therapeutically effective amount, of a chemical composition according to any one of claims 1 to 24. A method for preventing the occurrence or treating, in a subject, a disease physiologically inducing the trans-phenotyping of M1 macrophages into M2 macrophages as a pathological phenomenon, said method comprising administering a therapeutically effective amount of a chemical composition according to any one of claims 1 to 24. Method according to claim 32 or 33, characterized in that the administration of said chemical composition is carried out orally, or cutaneously, or even intravenously, intramuscularly, subcutaneously or transdermally, sublingually, ocularly, auricularly or nasally. Method according to the preceding claim, characterized in that the subject is suffering from a disease of the inflammatory system, a bacterial, viral or fungal infection and / or a tumor condition, in particular a cancer including breast cancer, pancreatic cancer or colorectal cancer, as well as all tumors infiltrated by macrophages associated with tumors, an infection of the upper respiratory tract, infections by parasites of the Helminthes genus, infections by chronic and intracellular bacteria, infections by fungi, metabolic diseases, fibrosis, a cardiovascular disease, an autoimmune disease, and allergies. Method according to any one of claims 32 to 35, characterized in that the therapeutically effective quantity of the chemical composition is 400 mg / day, provided with a morning and evening administration dosage of a chemical composition of 200 mg, said chemical composition of 200 mg comprising, in mass percentage relative to its total mass, between 30 and 50%, preferably 25.5% of dry extract of brown propolis; between 40 and 70%, preferably 63.8% of gum arabic; between 1 and <10%, preferably 9.6% of sucrose and at most 3%, preferably 1.1% of silicon dioxide.

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