Chemical composition based on Propolis extract

Abstract

A chemical composition with immunomodulatory activity comprising, as the active substance, a standardized, natural propolis extract 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 dihydroflavonol family, and one molecule E from the flavonol family.

Description

Title of the invention: Chemical composition based on Propolis extract

[0001] The present invention relates to a chemical composition with immunomodulatory activity comprising, as active principle, 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 of bacterial infections, viral infections, fungal infections or tumor conditions generating an inflammatory immune response.

[0003] The present invention also relates to the use of such a chemical composition as an ingredient inducing the trans-phenotyping of inactive anti-inflammatory macrophages, of the "M2c" type, into active pro-inflammatory macrophages, of the "M1" type.

[0004] 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.

[0005] Nevertheless, there are major drawbacks associated with the use of these known agents, including:

[0006] - the harmful side effects that they can generate on the human body, such as stomach aches, headaches,

[0007] - the lack of precision of action on the targets involved in the anti-mechanism inflammatory, with the risk of weakening the active immune system to fight against the pathology,

[0008] - 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.

[0009] In order to partially limit the side effects and overcome the disadvantages 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 to be treated.

[0010] The use of propolis as a natural ingredient for its activity against inflammatory disease is known.

[0011] Propolis refers to 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.

[0012] Raw propolis can be recovered by beekeepers for various uses such as pharmaceutical, cosmetic or even agri-food use.

[0013] 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 period of harvest by the bees, the species of bee, the method of harvest by the beekeeper and the method of extraction of the propolis.

[0014] This diversity of composition of natural propolis represents a major obstacle to guaranteeing a homogeneous activity to fight against an infection by all types of pathogens, or against a tumor condition. In particular, this diversity of composition of natural propolis makes its involvement in the inflammatory response random.

[0015] Thus, to date, there is no propolis-based composition with a homogeneous effect acting in 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.

[0016] The objective of the present invention is to propose a composition based on natural and standardized propolis extract, 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.

[0017] The subject of the present invention is a chemical composition with immunomodulatory activity comprising, as active substance, a propolis extract of natural origin and standardized 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 dihydro-flavonole family and one molecule E from the flavonole family.

[0018] In the present invention, "propolis", "propolis extract" or "standardized propolis extract", means propolis derived from a plant belonging to the families of Salicaceae of which the genus Populus L. spp represents the main source species (Salicaceae) L. spp (willows), Betulaceae including Betula L. spp (birches) and Alnus Mill. spp (aulnes), Fagaceae including Quercus L. spp (oaks), Castanea Mill. spp (chestnut trees) and Fagus L. spp (beech trees), Ulmaceae including Ulmus L. spp (elms), Oleaceae including Fraxinus Toum. ex L. spp (ash), Sapindaceae including Aesculus L. spp (Indian chestnuts), Pinaceae including Picea A.Dietr. spp (epicéas), Pinus L. spp (pins) and Abies Mill. spp (sapins), Araucariaceae including Araucaria Juss. spp (araucarias), Cupressaceae including Juniperus L. spp (cedar), Anacardiaceae including Pistacia L. spp (pistachios), Rutaceae including Ruta L. spp (garden street), Myrtaceae including Eucalyptus L'Hér. Spp.

[0019] 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 Apis mellifera on the buds, leaves, stems, bark or any aerial parts of the plant producing resinous exudates.

[0020] 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 (known as Populus spp, known as brown propolis), geographically present in Europe, Asia and South America.

[0021] Advantageously in the chemical composition of the invention, the propolis extract consists of brown propolis, derived from a part of a plant belonging to the poplar family of the genus Populus spp, such as Populus nigra L., Populus alba L., or Populus tremula L.

[0022] To obtain the brown propolis of the invention, bees harvest the resin from leaves or leaf buds.

[0023] According to the invention, the chemical composition comprises a propolis extract of natural and standardized origin.

[0024] The propolis extract of the composition of the invention is obtained by a standardized process of the dynamic multi-extraction method type in which:

[0025] - raw propolis is taken from the hive,

[0026] - a first extraction is carried out in an aqueous medium on the raw propolis taken in order to remove the beeswax and impurities (bee residue, wood),

[0027] - a series of tests are carried out on the residue of the aqueous extraction, obtained previously: three extractions with a hydro-alcoholic solvent consisting of a water / ethanol mixture, increasing, with each successive extraction, the extraction temperature and the alcohol concentration.

[0028] 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.

[0029] The implementation of this process makes it possible to obtain a propolis extract of natural and standardized origin systematically comprising at least, in a mixture, one molecule A from the family of phenolic acids, one molecule B from the family of flavanones, a C molecule from the flavone family, a D molecule from the dihydroflavonole family and an E molecule from the flavonole family, regardless of the species of the plant genus Populus spp, 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.

[0030] According to a preferred embodiment, the chemical composition of the invention further comprises gum arabic, sucrose and silicon dioxide, as a carrier for the propolis extract.

[0031] Preferably, the chemical composition comprises, as a percentage by mass 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 a mixture with between 30% and 50% of propolis extract.

[0032] 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.

[0033] For example, the support for 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.

[0034] According to another example, the carrier of the propolis extract may also comprise a mixture of maltodextrin, silicon dioxide and heat-treated liquid sucrose.

[0035] 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.

[0036] Preferably, for its immunomodulatory activity, 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.

[0037] Advantageously, within the chemical composition of the invention:

[0038] - the family of phenolic acids, has a basic carbon chain of formula IA below of cinnamic acid type or its derivatives such as o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, acid

[0039] sinapic; AI formula:

[0040]

[0041] 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: formula IB: the flavanone family has a basic carbon chain of formula II below: FUvnnone: the flavone family has a basic carbon chain of formula III below: o Fiavone the dihydroflavonole family has a basic carbon chain of formula IV below: OH ISî hy th'ofl a vwao t the flavonol family has a basic carbon chain of formula V below:

[0042] 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.

[0043] The expression "immunomodulatory activity" is understood to mean, in particular, 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.

[0044] As an 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 - CM HM O4, (caffeic acid benzyl ester) benzyl caffeate ester - C16 HM O4, (caffeic acid phenethyl ester) phenylethyl caffeate ester (CAPE) - C17 Hi6 O4, (caffeic acid cinnamyl ester) cinnamyl caffeate ester - C18 Hi6 O4j (coumaric acid cinnamyl ester) cinnamyl p-coumarate ester.

[0045] As an example, pinocembrin is a molecule B from the flavanone family.

[0046] As an example, the following molecules will be cited as molecule C of the flavone family: 5-methyl chrysin ester, apigenin, 5-methyl luteolin ester, chrysin, methoxychrysin, Tectochrysin.

[0047] As an example, the following molecules will be cited as molecule D of the dihydroflavonole family: 5 methyl pinobanksin, pinobanksin, pinobanksin-3-O-acetate, pinobanksin-3-O-propionate, pinobanksin derivatives.

[0048] As an example, the following molecules will be cited 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.

[0049] According to a preferred embodiment of the chemical composition of the invention, exhibiting significant immunomodulatory activity, the propolis extract comprises at least, as a mixture: as molecule A of caffeic acid with the formula below:

[0050] as molecule B of pinocembrin of formula below:

[0051] as molecule C of chrysin of formula below:

[0052] as molecule D of pinobanskin of formula below:

[0053] and as molecule E of galangin of formula below:

[0054] According to this specific embodiment, caffeic acid, pinocembrin, chrysin, pinobanskin and galangin present in a mixture in the standardized and naturally occurring propolis extract, interact synergistically and are res responsible for the immunomodulatory activity of the chemical composition of the invention.

[0055] More specifically, caffeic acid, pinocembrin, chrysin, pinobanskin and galangin present in a mixture in the propolis extract induce the activation of macrophages and their trans-phenotyping from an M2c phenotype to an Ml phenotype, in an environmental context with anti-inflammatory cytokines.

[0056] According to the invention, the chemical composition is in a liquid or solid form, in powder, solution, gel, emulsion, cream, capsule or tablet.

[0057] According to the invention, the chemical composition is in a pharmaceutical and / or cosmetically acceptable galenic form for the human or animal body.

[0058] 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.

[0059] The galenic form of the chemical composition allows, for example, an action extended to the entire organism (systemic)

[0060] Preferably, the galenic form of the chemical composition allows a local (topical) action targeting the area of ​​the inflammatory reaction.

[0061] 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.

[0062] 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, modifying their appearance, protecting them, keeping them in good condition or correcting body odors.

[0063] “Food supplements” means all foodstuffs intended to supplement a normal diet and which constitute a concentrated source of nutrients or other substances having a nutritional or physiological effect.

[0064] Another aspect of the present invention relates to the use of the chemical composition of the invention as a medicament.

[0065] 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 conditions.

[0066] According to another particular aspect, the invention also relates to the use of the chemical composition of the invention, as an ingredient for inducing trans-phenotyping of inactive anti-inflammatory macrophages of type M2C into active pro-inflammatory macrophages of type M1 in an anti-inflammatory cellular context.

[0067] Macrophages are cells involved in innate and adaptive immune responses that allow pathogen elimination. Depending on their environment and the signals detected, macrophages adapt their functions in different ways, this is called "polarization".

[0068] We distinguish between the “classical” polarization of type M1, whose activity is pro-inflammatory, and the “alternative” polarization of type M2, which participates in the regulation of inflammation and the phenomenon of tissue repair.

[0069] Ml macrophages, described as pro-inflammatory, are important in the direct defense of the host against pathogens, mediated by phagocytosis, secretion of pro-inflammatory cytokines and microbicidal molecules.

[0070] For example, it is known that macrophages polarize into the form of macrophage Ml after induction by interferon gamma (IFNg) and secrete several cytokines such as interleukins IL12p70, IL12p40, IL-6 or IL-23, tumor necrosis factor TNFa and Arginase.

[0071] 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 phenotype and the M2c phenotype. The M2a phenotype, induced for example by interleukin IL-4, secretes mainly Arginase, TARC and interleukin IL-1b. The M2c phenotype is induced by interleukin IL-10.

[0072] In the body, the immune response is presented in the form of a state of balance between an anti-inflammatory phenotype and an inflammatory phenotype.

[0073] 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.

[0074] In the environment of macrophages, 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 takes place. Consequently, trans-phenotyping is a phenomenon that strengthens the immune response during an infection.

[0075] 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, 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 trans-phenotyping of macrophages by allowing immuno-mo- dulation of the inflammatory response in case of infection by a pathogen or tumor disease.

[0076] 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:

[0077] [Fig. 1] the summary of the steps of the experimental protocol allowing to show the impact of the composition on the polarization of macrophages,

[0078] [Fig.2A] 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 (Ml=INFg, M2a=IL4, M2c=IL10, M0=medium alone)

[0079] [Fig.2B] 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

[0080] [Fig.3] 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 IL 10 = macrophage M2c) for the 6 donors taking into account all the cytokine and membrane markers.

[0081] [Fig 4A] Expression of the cytokine TNF-a 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,

[0082] [Fig 4B] 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,

[0083] [Fig 4C] 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

[0084] The aspects of the invention will be better understood by reading the examples, given for information purposes only and without limitation, and the test results which follow.

[0085] Example 1: Process for preparing a propolis extract of natural origin and standardized according to the invention

[0086] 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:

[0087] - a) raw propolis is taken from the hive

[0088] - b) the beeswax and impurities are removed from the raw propolis by an ex aqueous traction, so as to recover only the resinous, balsamic component of the raw propolis in pasty form,

[0089] - c) three successive hydro-alcoholic extractions are carried out on the residue of the previous extraction, for example with propolis removed from the beeswax and then on the extraction residues obtained, with a water / ethanol extraction mixture,

[0090] - d) the propolis extract of natural origin and standardized from the com is recovered chemical position of the invention,

[0091] - e) optionally, the propolis extract from step d) 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 extract of propolis in the form of a powder having a final humidity of less than 10%.

[0092] For step b), the separation of the raw propolis, the beeswax and the impurities is carried out by an aqueous extraction, 100% water at approximately 80°C for approximately 100 minutes under ultrasound, followed by a cooling step to approximately 8°C and a filtration step on a filter of the order of 30 μm.

[0093] For step c), each of the extractions with the water / ethanol hydro-alcoholic mixture is carried out on the residue of the previous extraction, by increasing the alcohol content of the water / ethanol extraction mixture and by 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 50 pm 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.

[0094] Preferably, for step c):

[0095] - 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,

[0096] - the second extraction, on the propolis residue from the first extraction, is made with a water / ethanol mixture between 55 and 60 degrees of alcohol at an extraction temperature between 40 and 80°C,

[0097] - the third extraction, on the propolis residue from the second extraction, is made with a water / ethanol mixture between 70 and 80 degrees of alcohol at an extraction temperature between 60 and 80°C.

[0098] For step c), the ratio between the propolis extraction residue and the mixture extraction ratio is between 0.1:1 and 1:1.

[0099] For step e), the 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 retain its action.

[0100] For the series of three extractions, the time taken to complete an extraction is between 4h and 36h.

[0101] At the end of the third extraction, the propolis extract of natural origin and standardized is obtained, in the form of a hydro-alcoholic solution of propolis comprising at least in mixture, one molecule A from the family of phenolic acids, one molecule B from the family of flavanones, one molecule C from the family of flavones, one molecule D from the family of dihydroflavonoles and one molecule E from the family of flavonoles.

[0102] Example 2: Chemical composition according to the invention with different supports

[0103] Table 1 below shows examples of chemical composition according to the invention comprising different support in mixture with the standardized natural propolis extract in powder form and in liquid form, obtained by the process of Example 1.

[0104] The percentage ranges are expressed in mass relative to the total mass of the chemical composition, which corresponds to a value of 100%.

[0105] Table 1: Composition Components % Compositio n Components % 1 Propolis extract 4-15 6 Propolis extract 8-50 Ethanol 65-83 Gum arabic 40-85 water 7-24 Sucrose <10 2 Propolis extract 70-75 Silicon dioxide <3 Ethanol 21-25 7 Propolis extract 10-35 water 3-5 maltodextrin 58-82 3 Propolis extract 3-10 Heat-treated liquid sucrose 4-5 Glycerin 85-94 Silicon dioxide <3 water 3-5 8 Propolis extract 13-20 4 Propolis extract 1-3 Propylene glycol 80-90 Glycerin 92-96 9 Propolis extract 10-15 water 1-3 Soybean oil 85-90 5 Propolis extract 2-3 10 Propolis extract 10-15 Glycerin 85-90 Sunflower seed oil 85-90 Water 7-12 Heat-treated liquid sucrose 1-1.5

[0106] Example 3: Chemical composition of the invention

[0107] 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 propolis, 63.8% gum arabic, 9.6% sucrose and 1.1% silicon dioxide.

[0108] Example 4: Impact of the chemical composition of the invention on the trans-phenotyping of macrophages

[0109] It is known that an in vitro culture of macrophage in the basal MO state polarizes towards a specific phenotype, after induction in the presence of differentiation cytokines.

[0110] Thus, for an in vitro culture of macrophages, it is known:

[0111] - that in the presence of interleukin 10 called “IL10”, macrophages polarize towards an M2c phenotype, which is representative of an anti-inflammatory context,

[0112] - that in the presence of gamma interferon called "IFNg", macrophages polarize towards an Ml phenotype,

[0113] - 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 Ml phenotype,

[0114] - that the Lipopolysaccharide called “LPS”, as a membrane constituent bacterial, activates macrophages, to allow their increased polarization towards a specific phenotype and the secretion of cytokines into their culture environment.

[0115] The impact of a chemical composition according to Example 3, named “ESIT12” on the polarization of macrophages was carried out in vitro on primary human macrophages obtained from peripheral mononuclear cells (PBMC) from six donors DI to D6.

[0116] [Fig. 1] schematically shows the experimental protocol carried out to visualize the impact of the chemical composition of the invention on the trans-phenotyping of macrophages.

[0117] PBMCs are isolated from blood samples, then placed in culture medium with a density of 600,000 adherent cells / well, for 2 days before being brought into contact with the extracts to be tested.

[0118] To visualize the impact of propolis extracts in the trans-phenotyping of macrophages, the macrophages are incubated for 9 days at 37°C and 5% CO2, with:

[0119] - either the chemical composition according to example 3 called “ESIT 12”,

[0120] - either a solution comprising one of the molecules A to E purified at a concentration weighted,

[0121] - either with a solution comprising all of the purified molecules represented indications of propolis extract.

[0122] At 6 days, the culture medium is renewed or supplemented with cytokine inducing trans-phenotyping of macrophages.

[0123] At 8 days, LPS is added to all culture wells, so as to have increased macrophage activity and cytokine secretion into the environment of culture according to the phenotype of polarized macrophages.

[0124] After 9 days of culture, the cells are detached and then dissociated to carry out multiparametric immuno-labeling with acquisition of fluorescence signals by flow cytometry.

[0125] The markers used in flow cytometry to identify the different macrophage populations are described in Table 2 below:

[0126] Table 2: Markers MO Ml M2a M2 CD 14 + / - + / - - +++ CD16 + + / - - ++ CDlla ++ +++ ++ ++ CD64 + +++ - +++ HLA-DR ++ +++ +++ + / - CD86 + + +++ - CD200R - - +++ -

[0127] The combination of the expression of the markers CD14 / CD64 / CD11a / CD86 was retained to distinguish and discriminate the different phenotypes of macrophages.

[0128] The polygon of [Fig.2A] composed 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 the polarization of macrophages.

[0129] On the 4-marker polygons, the points correspond to the fluorescence medians of the 4 CD14 / CD64 / CD11a / CD86 markers and the circles correspond to the average of the fluorescence signals detected for the population of cells detached after 9 days of culture.

[0130] After 9 days of culture also, a dosage of 13 cytokines is carried out on the recovered culture supernatant.

[0131] The 13 cytokines detected by flow cytometry in the supernatant to discriminate the secretomes of the different macrophage phenotypes are identified in Table 3 below. Markers Mo Ml M2a M2c I112-p70 - + - - TNF-a + + + + IL-6 + + + - IL-4 - - NA - IL-10 + - - NA IL-lb + + + + Arginase + + + + TARC + + + + IL1-RA + + + + IL12-p40 + + + - IL-23 - + - - IFN-g - NA - - IP-10 + + + +

[0132] 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 markers 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.

[0133] The results show that the secretome of the different macrophage populations is as follows:

[0134] - MO macrophages, in the basal state, only in the culture medium, secrete several cytokines but at a relatively low level,

[0135] - macrophages of phenotype Ml, after induction with IFNg in the medium, differentially secrete at the MO: IL12-p70; IL12-p40; TNF-a, IL-6, IL-23 and Arginase,

[0136] - macrophages of M2c phenotype, after induction with IL-10 repress the expression of a majority of cytokines, notably IL-6 and TNF-a.

[0137] The IFNg / IL10 macrophage activation controls are used to capture the M1 / M2c phenotypes respectively.

[0138] The purified extracts and molecules are incubated after the differentiation cytokines in order to verify their capacity to induce trans-phenotyping of macrophages.

[0139] More specifically in the experimental protocol, after 2 days of growth of PBMCs in a basal MO 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 environment anti-inflammatory cytokine activity of macrophages. Thus, in this anti-inflammatory context induced by IL-10, macrophages will be M2c.

[0140] Then, on day 6, the following elements are added to different wells:

[0141] - 7 pmol / L of the chemical composition of the invention according to example 3 said "ESIT12",

[0142] - 7 pmol / L of a solution called “ESIT 12r”, comprising galangin, chrysin, pino- cembrine, pinobanskin and caffeic acid in the same proportions and concentrations as they are present in ESIT 12,

[0143] - 7 pmol / L of a solution of purified galangin molecules called “Galanr”,

[0144] - 7 pmol / L of a solution of purified chrysin molecules called “Chrysr”,

[0145] - 7 pmol / L of a solution of purified pinocembrin molecules called “PinoCr”,

[0146] - 7 pmol / L of a solution of purified pinobanskine molecules called “PinoBr”,

[0147] - 7 pmol / L of a solution of purified caffeic acid molecules called “CafAcr”.

[0148] A control well for the differentiation of cells into Ml is produced by adding, on day 2, 20 ng / mL IFNg.

[0149] A control well for the differentiation of cells into M2c is produced by adding, on day 2, 20 ng / mL dTL10.

[0150] A control well for the trans-phenotyping of macrophages from M2c to Ml is carried out by adding 20 ng / mL of IL 10 on day 2, then at 6 days 20 ng / mL of IFNg.

[0151] The results are shown on the 4-marker polygons of [Fig.2B], in which the arrows represent the fluorescence signals detected by flow cytometry for the population of cells detached after 9 days and placed in the presence of the different extracts and solution.

[0152] With [Fig.2B], we see that in an anti-inflammatory context, created in vitro after induction at 1TL-10, the macrophages of profile M2c, 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.

[0153] Thus, the ESIT 12 composition of the invention exhibits immunomodulatory activity, transphenotyping M2c macrophages to an M0 / M1 phenotype, in an anti-inflammatory context induced by IL-10.

[0154] 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.

[0155] [Fig.3] compiles the results of measurements of the intensity of the fluorescence signals obtained by flow cytometry analysis for each of the markers (IP 10, IL12 p40, CD86, CD16, CD14, CD64, CD200r, CD11a, HLA-DR, IL6, IL23, IL4, IL1b, IFNg, IL12 p70, TNF-a, 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, placed in contact with the extracts and activated with LPS.

[0156] Bioinformatics analysis of the intensity of these signals makes it possible to group the results in the form of a cluster hierarchically organized 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 Ml trans-phenotyping control (IL-10 induction then IFNg).

[0157] Thus, [Fig.3] shows, 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 Ml phenotype from an M2c macrophage (cluster A [Fig.3]).

[0158] [Fig.3] also shows that the molecules alone, independently of each other, are not sufficient to allow trans-phenotyping of macrophages from M2c to Ml as evidenced by cluster B.

[0159] 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 ML control.

[0160] [Fig.3] therefore shows, 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 Ml in an anti-inflammatory context. A synergy between molecules A to E of the propolis extract is essential for the chemical composition of the invention to exhibit immunomodulatory activity.

[0161] For example, Figures 4A, 4B and 4C respectively show the fluorescence intensity of the TNFa, IL-6 and IL-10 markers within the cell populations and dosage of cytokines excreted in the cell culture supernatant after 9 days of culture, induction with IL10 and over-activation with LPS.

[0162] The chemical composition of the invention ESIT 12 and the ESIT 12r solution with molecules A to E acting in synergy, each secrete the pro-inflammatory cytokines markers of inflammation, namely TNF-a and IL-6, while 1TL-10 of the secretome is low. ESIT 12 and ESIT12r, in anti-inflammatory condition induced by IL-10 allow the macrophage to polarize into Ml and to regain a pro-inflammatory cytokine producing function.

[0163] It should be noted that taken individually, looking at the dosage of TNFa and IL-6, the overall effect of the purified molecules is similar to that of the chemical composition of the invention ESIT 12 or of the solution of the molecules in a mixture at concentration weighted ESIT12r. This is why an analysis by individual marker is not sufficient to report the trans-phenotyping of macrophages from M2c to Ml, only a multiparametric analysis of the fluorescence intensity of several markers (cluster of [Fig.3]) really allows to show the impact and the synergy of the molecules in mixture on the trans-phenotyping of macrophages.

[0164] 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.

[0165] These results show that the chemical composition ESIT 12 of the invention, by the presence of a propolis extract comprising the 5 molecules A to E acting in synergy, in an anti-inflammatory context, allows:

[0166] - to restore the production of pro-inflammatory cytokines such as TNF-a and 1TL-6 promoting the recruitment of immune cells to the site of inflammation,

[0167] - to the M2c macrophage to polarize towards an M0 / M1 phenotype.

[0168] 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, in particular by modulating macrophages of the M2c phenotype towards a cytokine-producing Ml phenotype.

[0169] 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, has an immuno-modulatory 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.

[0170] The chemical composition of the invention presents no toxicity for the human or animal organism and has the advantage of being of natural origin.

[0171] In addition, the synergistic action of molecules A to E of the 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.

[0172] Consequently, the chemical composition of the invention is a good clinical candidate for treating diseases inducing an inflammatory response of the immune system.

Claims

Claims

1. Chemical composition with immunomodulatory activity comprising, as active substance, a propolis extract of natural origin and standardized 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.

2. Chemical composition, according to claim 1, characterized in that the propolis extract consists of brown propolis, derived from a part of a plant belonging to the poplar family of the genus Populus spp, such as Populus nigra L., Populus alba L., or Populus tremula L.

3. Chemical composition according to any one of the preceding claims, characterized in that it further comprises gum arabic, sucrose and silicon dioxide.

4. Chemical composition according to any one of the preceding claims, characterized in that it 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.

5. Chemical composition according to any one of the preceding claims, characterized in that: - the family of phenolic acids corresponding 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 corresponding 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: Flavanone The flavone family has a basic carbon chain of formula III below: the dihydroflavonole family has a basic carbon chain of formula IV below: O sby <1 av » ss $ F the va « <si> the flavonol family has a basic carbon chain of formula V below: OH Oh Flavtmol

6. Chemical composition, according to any one of the preceding claims preceding, characterized in that the propolis extract comprises at least, as a mixture: as molecule A of caffeic acid with the formula below: a. as molecule B of pinocembrin with the formula below: a. as molecule C of chrysin with the formula below: OH a. as molecule D of pinobanskin with the formula below: a. and as molecule E of galangin with the formula below:

7. Chemical composition according to any one of the preceding claims, characterized in that it is in a liquid or solid form, in powder, solution, gel, emulsion, cream, capsule or tablet.

8. Chemical composition, according to any one of the preceding claims, for use in a food product, preferably a food supplement, or an agrochemical product.

9. Chemical composition, according to any one of claims 1 to 7, for use as a medicament.

10. A chemical composition, according to any one of claims 1 to 7 or claim 9, for use in the prevention or treatment of diseases of the inflammatory system, bacterial, viral or fungal infections and tumor disorders.

11. Chemical composition, according to any one of claims 1 to 10, for its use as an ingredient for inducing 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.< / si>

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