WO2023144289A1

WO2023144289A1 - Use of a mixture of flavanols to increase the concentration of the active form of vitamin d, calcitriol

Info

Publication number: WO2023144289A1
Application number: PCT/EP2023/051978
Authority: WO
Inventors: David Gaudout; Stéphane REY; Benoit Lemaire; Line POURTAU
Original assignee: Activ'inside
Priority date: 2022-01-28
Filing date: 2023-01-27
Publication date: 2023-08-03
Also published as: FR3132221A1

Abstract

The invention relates to the use of a mixture of flavanols, but also to a composition comprising said mixture, for the purpose of increasing the concentration of the active form of vitamin D, namely calcitriol, in humans or animals. In particular, the mixture of flavanols consists of low molecular weight flavanols.

Description

USE OF A MIXTURE OF FLAVANOLS TO INCREASE THE CONCENTRATION OF THE ACTIVE FORM OF VITAMIN D, CALCITRIOL.

[0001] Technical area

The invention relates to the use of a mixture of flavanols, but also of a composition comprising it, in order to increase the concentration of the active form of vitamin D, calcitriol, in humans or 'animal. In particular, the mixture of flavanols consists of low molecular weight flavanols.

[0003] State of the art

[0004] Vitamin D is a fat-soluble vitamin essential to the proper functioning of our body. Its main function is to increase the concentrations of calcium and phosphorus in the blood. Maintaining a sufficient level of calcium in the blood ensures optimal mineralization of tissues, in particular bones, cartilage and teeth; effective muscle contraction; good nerve transmission; and adequate coagulation. It is also involved in hormonal regulation, the differentiation and activity of immune system cells and the differentiation of certain skin cells.

[0005] Unlike other vitamins which are provided exclusively by food, vitamin D has a dual origin, exogenous, corresponding to food intake but also endogenous, resulting from neosynthesis occurring in the epidermis.

[0006] Endogenously, vitamin D begins to be synthesized from 7-dehydrocholesterol (7-DHC) in the keratinocytes, in the skin, which by the action of ultraviolet B, called UVB, is transformed into cholecalciferol ( Vitamin D3). Subsequently, in the liver, the microsomal or mitochondrial 25-hydroxylase enzyme (CYP27A1) converts vitamin D3 into calcifediole (or 25-hydroxyvitamin D (25(OH)D) then 25-hydroxyvitamin D-la -mitochondrial hydroxylase (laOHase, CYP27B1) in the kidneys will convert calcifediole into 1,25-dihydroxyvitamin D (1,25(OH)2D3 (or calcitriol) which is the biologically active metabolite of vitamin D.

[0007] At the blood level, the level of 25-hydroxy-vitamin D3 is the best biomarker of the vitamin D status of individuals. When the serum level of 25(OH)D is above 30 ng/ml (75 nmol/L), the vitamin D status can be considered optimal. We can then distinguish between insufficiency, defined by a level of 25(OH)D between 11 and 30 ng/ml, and deficiency, defined by a level below 11 ng/ml (25 nmol/L).

[0008] Vitamin D deficiency is a frequent problem in our modern societies and unfortunately under-diagnosed. Globally, it is estimated that one billion people are affected. For example, in healthy older people, the prevalence of vitamin D deficiency is 50%, and 80% in very old people with a history of hip fracture.

[0009] To ensure that the body receives a sufficient supply, it is therefore advisable, on the one hand, to expose oneself to the sun, given that exposure for 15 to 20 minutes at the end of the morning or in the afternoon midday makes it possible to cover the daily needs in vitamin D and on the other hand to have a balanced and varied food in order to optimize the exogenous contributions. Indeed, vitamin D3 can be found in oily fish, egg yolk or butter.

[0010] However, the main source of vitamin D3 remains endogenous synthesis which takes place in the epidermis. However, exposure to the sun and therefore to UVB strongly depends on the season, the behavior of each individual (clothing, sedentary lifestyle or not), the composition of the skin, and age.

[0011] Also, for people suffering from a vitamin D deficiency, or even a deficiency, it is not always possible to expose themselves long enough to restore an optimal vitamin D concentration. In addition, intake through food alone does not satisfy all daily needs.

[0012] Vitamin D3 supplementation via high-dose vials taken monthly or quarterly is possible. However, these do not make it possible to obtain a regular concentration of vitamin D in the body.

[0013] There is therefore a need for new solutions making it possible to remedy and/or prevent and/or treat a vitamin D deficiency or insufficiency or even a vitamin D deficiency; and thus maintain in the body an optimal concentration of the active form of vitamin D, ca Icitriol.

[0014] To meet this need, the invention proposes polyphenol supplementation, in particular the supply of a mixture of flavanols which makes it possible to promote the active form of vitamin D in humans or animals and thus cover daily needs.

[0015] Summary of the Invention

[0016] Polyphenols are secondary metabolites produced by plants for their defense against external attacks (parasites, microorganisms, UV rays, etc.).

[0017] Widespread in food, they are responsible for both aroma and color and are essentially known for their antioxidant properties. Polyphenols can be divided into two main groups, flavonoids (2/3) and non-flavonoids (1/3). Among the flavonoids, we can differentiate between flavonols, flavanols, flavones, chalcones and anthocyanins.

[0018] Quercetin is the main flavonol in food, found in fruits and vegetables. Anthocyanins are pigments responsible for the red or blue color of fruits. Among the flavones, flavanones are mainly found in citrus fruits, while flavanols (flavan-3-ols), in particular catechins, epicatechins, epicatechin 3 gallate, are found in tea, grapes (Vitis vinifera, but also Vitis labrusca), wine, or chocolate. Catechins and epicatechins can be more or less polymerized to form oligomers called oligoprocyanidins (OPC). The oligomers have a degree of polymerization ranging from 2 to 10, and the polymers a degree of polymerization greater than 10.

[0019] While the interest of polyphenols as antioxidants, and more particularly the use of flavanols, no longer needs to be demonstrated, only their interactions with vitamin C are described.

[0020] However, surprisingly, the inventors have discovered that flavanols and more particularly low molecular weight flavanols make it possible to promote and thus increase the concentration of the active form of vitamin D in humans or animal.

[0021] The inventors have in particular demonstrated on HACAT cells, a non-cancerous line of human keratinocytes, comprising all the enzymes necessary for the synthesis of vitamin D, and in the presence of a mixture rich in flavanol monomers, an increase calcitriol, the active form of vitamin D, even in the absence of UVB exposure.

Thus, the invention relates to the use of polyphenols, in particular of a mixture of flavanols in which the flavanols have a low molecular weight, that is a degree of polymerization comprised between 1 and 10, to increase the concentration of the active form of vitamin D, calcitriol.

[0023] The mixture of flavanols can be of any origin, natural or not. In particular, the latter can be obtained from at least one plant species such as vines, grapes, tea, cocoa, maritime pine, red fruits, fruits with drupes, or nuts, as well as from a mixture of said plant species, in particular the mixture can be obtained from at least two, three, four, five, six, seven or the combination of the eight species described above.

Advantageously, the mixture of flavanols according to the invention is integrated into a composition. The composition can take the form of a food, a drink, a food supplement, a cosmetic product or a drug. Said composition thus aims to increase the concentration of the active form of vitamin D, and therefore, preferentially, to remedy, promote, prevent or treat an insufficiency, or even a vitamin D deficiency.

Also, according to one aspect, the invention relates to a mixture of low molecular weight flavanols or of a composition comprising it for its use to increase the concentration of the active form of vitamin D, preferably prevention, and/ or the treatment of vitamin D insufficiency, deficiency or deficiency and/or the maintenance of vitamin D levels in humans or animals. According to a variant, the invention also relates to the use of a mixture of low molecular weight flavanols or of a composition comprising it for maintaining a vitamin D content in healthy humans or animals.

When the composition is in the form of a cosmetic product, it comprises cosmetically acceptable excipients. When the composition is in the form of a food, drink or food supplement, it comprises acceptable nutrient excipients. Such compositions make it possible to provide a sufficient intake to maintain the necessary daily intakes of vitamin D. Preferably, they make it possible to overcome and/or remedy, prevent, treat vitamin D insufficiency in humans or animals, in particular healthy man or animal.

When the composition is in the form of a drug, it comprises pharmaceutically acceptable excipients. Said composition then preferentially makes it possible to prevent and/or treat vitamin D deficiencies and the associated diseases, for example rickets, osteomalacia, etc., but also to strengthen the immune system.

Also, according to a last aspect, the invention also relates to the mixture of flavanols according to the invention or the composition comprising it for its use in the prevention, and/or the treatment of a vitamin D deficiency and/or the maintenance of a vitamin D level in humans or animals.

Other characteristics and advantages will emerge from the detailed description of the invention, the examples and the figures which follow.

[0030] Brief description of the Figures

Figure 1 shows the effect of the mixture according to the invention on the production of active vitamin D, without UVB stimulation. Data are represented as means ±SEM. The mixture according to Example 1 (Invention) and according to Example 2 (Outside the Invention) were tested at 50 mg/ml. Mixture 3 (Invention) consists of a mixture according to example 1 tested at 50 mg/ml and example 2 tested at 50 mg/ml. **p<0.01, ***p<0.001.

Figure 2 shows the synergistic effect of the mixture according to Example 4 (Invention) at 50mg/ml in combination with cholecalciferol at 10pM and 25pM (respectively panel A and B) on the production of active vitamin D, calcitriol in in vitro cellular model of keratinocytes. Data are represented as mean ±SEM. AT.

[0033] Detailed description of the invention

[0034] Definition

[0035] By “mixture of flavanols” within the meaning of the invention, is meant at least one flavanol, preferably several flavanols. The mixture may comprise flavanol monomers, flavanol oligomers, optionally flavanol polymers. Preferably, the mixture is rich in flavanol monomers and in oligomers, that is to say that it comprises at least 40% of low molecular weight flavanols, by weight of the total weight of flavanol. By way of example, the oligomers can be oligomers of procyanidins or proanthocyanidins. The mixture can be obtained from any origin, preferably from plants rich in low molecular weight flavanols. In the context of the invention, said mixture of flavanols constitutes an ingredient when it is integrated into a nutritional composition or an active principle when it is integrated into a therapeutic or cosmetic composition.

By “low molecular weight flavanols” within the meaning of the invention, is meant flavanols having a degree of polymerization (DP) of less than 10, i.e. a degree of polymerization (DP) of between 1 and 10 (DPI to DP 10).

[0037] By “vitamin D deficiency” within the meaning of the invention, is meant an organism having a low concentration of vitamin D, in particular a concentration of vitamin D between 11 and 30 nanograms per milliliter of blood. Such a concentration is found in people with low exposure to the sun and/or an unsuitable diet.

[0038] By “vitamin D deficiency” within the meaning of the invention, is meant an organism having a very low concentration of vitamin D, in particular a concentration of vitamin D of less than 11 nanograms per milliliter of blood. Unlike vitamin D insufficiency, such a concentration is likely to promote the appearance of pathologies in humans or animals, in particular bone diseases, such as rickets in children or osteomalacia. in adults.

By “acceptable excipient” within the meaning of the invention, is meant an excipient suitable for the use of the composition according to the invention. The addition of an excipient can give the finished product physical properties, for example consistency; and taste properties.

By “x g/100g” within the meaning of the invention, is meant x gram of low molecular weight flavanols, for example of DP between 1 and 10 or between 1 and 5 or only flavanol monomers per 100g of mixture of flavanols.

[0041] Mixture of flavanols according to the invention

The present invention therefore relates to a mixture of specific flavanols, of low molecular weight to increase, promote the concentration of the active form of vitamin D, calcitriol, in humans or animals.

In particular, the invention relates to the use of a mixture of flavanols to increase the concentration of the active form of vitamin D, calcitriol, said flavanols included in the mixture having a degree of polymerization between 1 and 10. The invention also relates to a mixture of flavanols having a degree of polymerization of between 1 and 10 for its use to increase the concentration of the active form of vitamin D, calcitriol.

The degree of polymerization (DP) defines the length of a polymer chain and this is directly proportional to the molar mass of the polymer. The degree of polymerization is the number of monomer units (repeating units) that make up this chain.

[0045] In the context of the invention, the mixture of flavanols can comprise monomers, oligomers and optionally polymers of flavanols. An oligomer comprises a small number of monomer units, for example between 2 and 10, whereas a polymer comprises at least 10 monomer units. Preferably, the mixture of flavanols according to the invention mainly comprises monomers and oligomers, and optionally polymers of flavanols.

The mixture of flavanols can be of any origin, natural or not. Preferably, the mixture of flavanols is obtained from a plant species comprising flavanols such as vines, grapes, tea, cocoa, maritime pine, red fruits, drupe fruits, nuts, etc., as well as mixtures thereof. Flavanols can be obtained from leaves, fruits, skins, seeds, bark, wood, etc. Preferably, the species are chosen from Vitis (vinifera, labrusca), Camellia sinensis, Theobroma cacao, Pinus pinaster, Ribes nigrum, fragaria vesca, Vaccinium (angustifolium, myrtillus, macrocarpon, oxycoccos, corymosum), Punica granatum, Jugions L, Pistacia vera L., Prunus du Ids, Cory lus avellana, Malus domestica, Prunus (domestica, armeniaca), Hippophae rhamnoides and their combinations. Preferably, the mixture of flavanols is obtained from Vitis vinifera or Vitis labrusca.

According to a particular embodiment, the mixture of flavanols comprises at least 40% of flavanols having a degree of polymerization of between 1 and 10, by weight of the total weight of the mixture of flavanols, preferably said mixture mainly comprises flavanols having a degree of polymerization of between 1 and 10, that is to say that the quantity of flavanols having a degree of polymerization of between 1 and 10 in the mixture is strictly greater than 50%, by weight, of the total weight of the mixture of flavanols.

Thus, the flavanols whose degree of polymerization (DP) is between 1 and 10 (DPI to DP 10) preferably represent at least 40 g/100 g, more preferably at least 50 g/100 g of the mixture of flavanols.

The effect of the mixture of flavanols is further improved, when the latter preferably comprises flavanols whose degree of polymerization is between 1 and 5, more preferably they represent at least 40% of flavanols, by weight of the weight total of the mixture of flavanols, more preferably at least 50% by weight of the total weight of the mixture of flavanols. Also, they represent at least 40g/100g of the mixture of flavanols, even more preferably at least 50g/100g of the mixture of flavanols.

According to an even more preferred embodiment, the mixture comprises flavanols having a degree of polymerization of between 1 and 2. Preferably, the flavanols having a degree of polymerization of between 1 and 2 represent at least 30% of flavanols, by weight of the total weight of the mixture of flavanols, more preferably at least 40% by weight of the total weight of the mixture of flavanols. Also, they represent at least 30g/100g, more preferably at least 40g/100g of the mixture of flavanols.

Even more preferably, the mixture comprises flavanols having a degree of polymerization equal to 1 (monomers). Preferably these flavanols having a degree of polymerization equal to 1 (monomers) represent in the mixture of flavanols at least 15% of flavanols, by weight of the total weight of the mixture of flavanols, more preferably at least 20% by weight of the total weight of the mixture of flavanols. Also, they represent at least 15g/100g of the mixture of flavanols, more preferably at least 20g/100g of the mixture of flavanols.

Also, the greater the quantity of low molecular weight flavanols (DP between 1 and 10) in the mixture, preferably at least 40%, by weight of the total weight of the mixture, the more the mixture is capable of increasing the concentration of calcitriol in the body of humans or animals.

In the context of the invention, the mixture of flavanols can be integrated into a composition and then comprises at least one acceptable excipient. The composition is thus rich in flavanols, more particularly in low molecular weight flavanols (DP less than or equal to 10), it may consist exclusively of the mixture of molecules and an acceptable excipient, that is to say of the mixture of flavanols according to the invention and of the excipient, or comprise other constituents.

Preferably, in addition to the mixture of flavanols, the composition according to the invention comprises other constituents, in particular vitamins and/or excipients or coating agents, such as maltodextrin, microcrystalline cellulose , cyclodextrins, starch, soluble or insoluble fibers.

When the composition comprises at least one vitamin, the vitamin is chosen from water-soluble and fat-soluble vitamins, preferably the vitamin is vitamin D and/or a precursor of vitamin D.

Vitamin D is present essentially in the form of two compounds, ergocalciferol or vitamin D2, present in food of plant origin (cereals but also mushrooms, yeasts); and cholecalciferol or vitamin D3, produced by the skin under the action of ultraviolet rays but also present in foods of animal origin (fatty fish, fortified milk foods). Vitamins D2 and D3 are particularly used in the prevention and curative treatment of rickets. The main derivatives of vitamin come mainly from vitamin D3, of endogenous origin.

[0057] In fact, there are few dietary sources of ergocalciferol. This vitamin D, of plant origin, is mainly found in mushrooms. For example, Shiitake (Lentinus edodes) naturally has a high concentration of pro-vitamin D2. Dried in the sun and therefore subjected to ultraviolet radiation, the pro-vitamins D2 it contains are transformed into previtamins D2, which are also quickly transformed into vitamins D2, which then offers a significant contribution of ergocalciferol.

On the other hand, vitamin D3, of animal origin, is present in many foods, such as fatty fish, egg yolk or butter.

Also, the vitamin is preferably chosen from vitamin in the D2 form (ergocalciferol) and vitamin in the D3 form (cholecalciferol); and/or the vitamin D precursor is chosen from 7-DHC or 7-dehydrocholesterol (cutaneous provitamin D), previtamin D3, and 25-hydroxyvitamin D (25(OH)D).

According to the invention, the composition or the mixture of flavanols can also be encapsulated in a food support or not. Once encapsulated, the composition can be in the form of a powder, capsule, tablet, capsule, solution, suspension, emulsion, cream or chewing gum.

[0061] Thus, the composition or the mixture of flavanols can be encapsulated (e) or microencapsulated (e) in a food carrier chosen from, without this list being exhaustive, a maltodextrin, or a cyclodextrin, a gum arabic, a hydrogenated oil, a non-hydrogenated oil, a wax, an alginate, starches, proteins.

The composition can be in the form of a nutritional, cosmetic or pharmaceutical composition.

[0063] Indeed, the composition is intended for various applications, curative, preventive or not. When the composition is neither preventive nor curative, it is intended to be used as a nutritional or cosmetic product and is intended to improve or maintain, promote the amount of calcitriol in a healthy person. By “healthy person”, we mean a person who does not suffer from vitamin D insufficiency, deficiency or deficiency.

In the context of the invention, the composition can be administered orally or topically. By way of example, it can be administered orally as a food product, food supplement, drink, or drug; or topically as a cosmetic product. The nutritional composition can be in the form of a food product, food supplement or drink. By way of example, the nutritional composition can take the form of bars, dairy products, powders to be swallowed or rehydrated, gels, jams, sweets, carbonated or non-carbonated drinks, dry drinks to be rehydrated, compotes, gummies or chewing gums .

The cosmetic composition can be in the form of tablets, capsules, gel capsules, powders, sticks, chewing gums, gummies, solutions, microcapsules, suspensions, emulsions, lotions, gels, creams, sprays, or patches.

The pharmaceutical composition can also be in different galenic forms, such as in the form of tablets, capsules, sticks, powders, solutions, suspensions, capsules.

In the context of the invention, it may also be a nutritional or therapeutic composition intended for animals. The term "animal" means any animal that can receive a nutritional or therapeutic agent according to the invention, for example but not limited to a pet, or a farm animal such as a poultry, a pig, a ruminant, a goat, or even a mouse.

The composition can be in the form, for example, of dry foods, such as croquettes (extruded, co-extruded or freeze-dried), sweets (or treats), snacks, moist or semi-moist foods such as pieces in sauce, pieces in jelly, drinks, or food supplements. Preferably, the agent is integrated into dry foods such as croquettes or granules.

[0070] In the case of a medicament intended for animals, or a veterinary product, the composition can be in several forms, for example tablets, capsules, sprays, liquids administered by drops .

Advantageously, the mixture of flavanols intended for animals can be integrated into a composition, in particular into a nutritional or therapeutic composition, by inclusion, that is to say by adding it to the mass of the composition, for example by impregnation or mixing, or by coating, that is to say by applying it to the surface of the composition, by spraying or by dusting, for example by mixing it beforehand with one or more ingredients such as at least one palatability factor.

Thus, according to one object, the present invention relates to the use of a mixture of flavanols according to any one of the embodiments or of a composition the comprising for increasing, promoting, maintaining or further promoting the concentration of the active form of vitamin D, calcitriol in humans or animals, in particular healthy humans or animals.

According to another object, the present invention relates to the mixture of flavanols according to any one of the embodiments or of a composition as described above for its use to increase the concentration of the active form of vitamin D , calcitriol, in humans or animals, characterized in that the mixture comprises flavanols whose degree of polymerization is between 1 and 10.

According to another particular subject, the present invention relates to the mixture of flavanols according to any one of the embodiments or of a composition as described above for its use in the prevention and/or treatment of a vitamin D deficiency or insufficiency or deficiency in humans or animals.

[0075] Process

The mixture of flavanols can be of any origin. However, it is preferably obtained from plants.

[0077] Also, a process adapted from a grape extract (preferably seed with or without film), tea, cocoa, red fruits, fruit with drupes, maritime pine, skin of nuts such as hazelnut, rich in low molecular weight flavanols is a process comprising the following steps: a. Grinding of the material (except for pips or fruit pits), b. extraction with water and/or ethanol, c. separation of the water and/or ethanol solution from the solid matter, for example by filtration on a vibrating screen, and/or decantation; d. evaporation of the ethanol and of the water by vacuum evaporation at a temperature preferably below 60° C. and at a pressure below 100 mbars; e. separation of low molecular weight flavanol monomers and oligomers (DP<10) and flavanol polymers (DP>10) ₁ f. drying the extracts obtained in step e. by atomization, vacuum oven or freeze-drying with or without a support such as a maltodextrin or acacia gum.

The water and/or ethanol extraction step can be implemented with a quantity of solvent of between 30% v/v and 96% v/v, for a hydroalcoholic solution, i.e. between 2 and 10 times the mass of initial material obtained in step a. The duration of the extraction can be between 30 minutes and 24 hours. The extraction temperature is between 20° C. and 95° C. when the extraction is carried out at atmospheric pressure, and from 100° C. to 150° C. when the extraction is carried out under a pressure of between 1 bar and 150 bars. The raw materials used can be in dry, fresh or whole frozen forms.

The step of separating the monomers and oligomers of low molecular weight flavanols (DP<10) and the polymers of flavanols (DP>10) can be implemented by membrane separation of the previously desolventized extract so as to preferentially select the monomers (degree of polymerization of 1) and the proanthocyanidin oligomers (having a degree of polymerization between 2 and 10 inclusive) and to separate the flavanol polymers (DP>10), which makes it possible to obtain a characterized extract by a flavanol polymer content of less than 50g/100g of flavanols. This step can be carried out using a filtration membrane having a cut-off threshold of less than 15,000 daltons and more preferably less than 3,000 daltons.

According to a variant, the step of separating the monomers and oligomers of low molecular weight flavanols (DP<10) and the polymers of flavanols (DP>10) can be implemented by a liquid/liquid separation step. with solvents known to those skilled in the art such as ethyl acetate.

According to another variant and regardless of the embodiments described above, before the drying step f. and after step e, the process may comprise an additional purification operation of the chromatographic type comprising the following steps:

- loading on a resin of the solutions of extracts mixed or not,

- rinsing the resin with water,

- application of an eluent water/ethanol solution on the resin,

- recovery of the purified eluate,

- evaporation of the ethanol of said eluate,

- concentration of said eluate, and

- drying of said purified aqueous extract.

The invention is now illustrated by non-limiting examples of compositions according to the invention and by results.

[0083] Examples Example 1 according to the invention.

A mixture of flavanols according to the invention is obtained from grape seeds according to the following process: a. Water extraction, 40 g of raisin seeds mixed with 200 g of water at a temperature of 85°C for 2 hours, b. Solid/liquid separation of the liquid seed extract by filtration through a vibrating sieve c. Concentration of the aqueous extract under vacuum at a temperature preferably of 55° C. and at a pressure of less than 90 mbar; d. Membrane separation by ultrafiltration with a cut-off threshold of 5000 Daltons with recovery of the ultrafiltration permeate; the ultrafiltration retentate will constitute the composition of Example 2 e. Purification of the permeate on an adsorption chromatographic column comprising the following steps: i. loading on an adsorption resin; ii. rinsing the resin with water; iii. elution using a water/ethanol (80% V/V) eluent solution on the resin, iv. recovery of the purified eluate; v. vacuum evaporation at a temperature of 55° C. (pressure 90 mbar) of ethanol and concentration of said eluate at 50% dry matter, f. Drying of the purified extract obtained in step e. by atomization and obtaining one gram of extract from example 1.

The mixture according to Example 1 mainly comprises flavanols having a DP of less than 10, as described in Table 1 below.

[0087] Example 2 outside the invention.

A 2 ^nd example of flavanol mixture is obtained from Example 1 from the fraction corresponding to the membrane separation filtration retentate according to the following process: a. Water extraction of 40 g of raisin seeds mixed with 200 g of water at a temperature of 85° C. for 2 hours; b. Solid/liquid separation of the liquid seed extract by filtration on a vibrating sieve; vs. Concentration of the aqueous extract under vacuum at a temperature preferably of 55° C. and at a pressure of less than 90 mbar; d. Membrane separation by ultrafiltration with a cut-off threshold of 5000 Daltons with recovery of the ultrafiltration retentate; e. Drying of the purified extract by atomization and obtaining 3 grams of extract from example 2.

The mixture according to Example 2 mainly comprises flavanols having a DP greater than 10, as described in Table 1 below.

Example 3 according to the invention.

A 3 ^rd extract of flavanol mixture is obtained from example 1 and from example 2, in particular, example 3 consists in mixing 50% of example 1 and 50% of the example 2.

The mixture according to Example 3 comprises at least 40% of flavanols having a DP of less than 10, as described in Table 1 below.

Example 4 according to the invention.

A mixture of flavanols according to the invention is obtained from green tea leaf according to the following process: a. Extraction with water, 17.5 g of green tea leaf in 100 ml of water at a temperature of 85°C for 2 hours; b. Solid/liquid separation of liquid tea leaf extract by filtration c. Purification of the aqueous extract by chromatography on adsorption resin with elution with ethanol using an 80% v/v solution; d. Evapoconcentration of the ethanol under vacuum and concentration of the aqueous extract at a temperature preferably of 60° C. and at a pressure of less than 90 m bars; e. Pasteurization of the concentrated aqueous extract at a temperature of 85°C for

10 minutes. f. Drying of the purified extract by atomization and obtaining one gram of extract from Example 4.

The mixture according to Example 4 comprises at least 40% of flavanols having a DP of less than 10, as described in Table 1 below.

Characterization of flavanol mixtures according to examples 1 to 4. The flavanol mixtures of Examples 1 to 4 are then analyzed according to methods known to those skilled in the art by HPLC to determine the polymers having a degree of polymerization of between 1 and 10. The total flavanol concentrations are determined by spectrophotometric analyzes according to the method of Folin Ciocalteu. The polymer concentrations (DP>10) are determined by differences in the total flavanols of the flavanols with a DP between 1 and 10.

The results are presented in Tables 1 to 2 below.

[0099] [Table 1]

[0100] [Table 2]

* g/100g of dry matter

** % of total flavanols

0101] The effectiveness of the mixtures of flavanols (Examples 1 to 4) described above was then studied.

[0102] Test 1 - Evaluation of the effectiveness of the composition as a promoter (of the conversion) of vitamin D into its active form, calcitriol according to the invention

The objective of this test is to measure the potential of the mixture according to the invention to increase the concentration of calcitriol (active form of vitamin D) in an in vitro cellular model of keratinocytes.

To do this, HACAT cells which have the standard characteristics of normal human keratinocytes were cultured in T75 flasks in DMEM 4.5 g glucose medium supplemented with 10% decomplemented FCS and antibiotics (penicillin (100 IU/ml) and streptavidin (100 µg/ml)). For the purposes of the study, the cells were then cultured at a rate of 5× ^{10 4} cells per well in a 24-well plate in an oven at 37° C., with 5% CO2 and 95% humidity. A mixture according to the invention (Example 1), a mixture outside the invention (Example 2) and another mixture according to the invention (Example 3) were then incubated with the keratinocytes for 24 hours at a concentration of 50 mg/ml. The culture medium was then changed to DMEM with 10% FCS and then the cells were returned to the oven at 37° C. for 12 h. At the end of these 12 hours, the medium was removed and immediately frozen at -20°C. In parallel, the cells were lysed in RIPA buffer in the presence of protease inhibitors and then frozen for subsequent protein assay. Calcitriol concentrations were measured by a sandwich enzyme-linked immunoassay (ELISA). ELISA plates were pre-treated with an anti-vitamin D antibody according to the supplier's recommendations.

The proteins were assayed using a BCA kit in order to standardize the results of the ELISA test on the protein concentration of each well.

The significance of the results was determined by an analysis of variance (ANOVA) followed by a multiple comparison test (Holm-Sidak). A p value <0.05 is considered significant.

The results are shown in Figure 1. In the absence of UVB exposure, the mixtures according to the invention induce a significant production of active vitamin D whereas the mixture outside the invention does not, and this compared to the control.

These results show that the mixture according to the invention makes it possible to overcome and/or remedy a vitamin D deficiency in humans or animals in the absence of optimal UVB exposure.

[0109] Test 2 - Evaluation of the synergistic effect of the composition according to the invention and of cholecalciferol on the production (conversion) of vitamin D into its active form

The objective of this test is to measure the potential of the mixture according to the invention in combination with cholecalciferol to increase the concentration of calcitriol in an in vitro cellular model of keratinocytes.

To do this, the same methodology as for test 1 was used on the HACAT cells. A mixture according to the invention (example 4) at a concentration of 50mg/ml, cholecalciferol at a concentration of 1OpM or 25pM, or a mixture according to the invention (example 4) (50mg/ml) combined with cholecalciferol (10pM or 25 µM) were then incubated with the keratinocytes. Calcitriol concentrations were measured by sandwich enzyme-linked immunoassay (ELISA) and are expressed as pg/ml/g protein. The significance of the results was determined by a two-factor analysis of variance (ANOVA) where factor 1 was the treatment with the mixture according to the invention and factor 2 was the treatment with cholecalciferol. A p value <0.05 is considered significant.

The results are presented in Figure 2. The results show that there is a significant interaction between the mixture according to the invention and cholecalciferol at 1OpM (p=0.0019) (Figure 2A) and 25pM (p=0.0029) (Figure 2B). Indeed, the production of calcitriol is higher in the presence of the mixture according to the invention associated with cholecalciferol (whether at 10 pM or 25 pM) than in the presence of cholecalciferol or the mixture according to the invention alone, which itself had already a significant effect compared to the control. This production is also higher than the productions in the presence of cholecalciferol and of the mixture according to the invention combined. These results therefore demonstrate a synergistic effect between the composition according to the invention and cholecalciferol.

Example 5 of nutritional composition according to the invention

A ^5th example of composition according to the invention comprises 120 mg of a mixture obtained from Vitis vinifera according to example 1 and 5 pg of vitamin D3 (cholecalciferol) (i.e. 200UI) per capsule. Example 5 is obtained by mixing the constituents mentioned above with acacia fiber as bulking agent and then packaged in 1 capsule under conventional manufacturing conditions known to those skilled in the art.

[0116] Example 6 of nutritional composition according to the invention

A ^6th example of a composition according to the invention is a solution comprising 20 ml of virgin rapeseed oil, 1000 mg of green tea extract containing 55% of flavanols, the degrees of polymerization of which are less than 10 per 100g of flavanols, 3 mg of vitamin K2, 1 mg of vitamin D3, and antioxidants (extracts rich in tocopherols). The composition according to Example 6 is obtained by mixing the constituents then packaged in a bottle under conventional manufacturing conditions known to those skilled in the art.

[0118] Example 7 of cosmetic composition according to the invention

A ^7th example of composition according to the invention comprises 800 mg of the mixture according to Example 1; 700 mg of preservative and biosaccharide; 1500 mg of perfumes; 97 g of fatty and aqueous phases the fatty phase consisting of an emulsifier and an emollient, the aqueous phase consisting of water combined with a pidolate. The composition according to Example 7 is obtained by mixing the constituents then packaged in a pot under conventional manufacturing conditions known to those skilled in the art.

[0120] Example 8 of food composition according to the invention

Example 8 is a formulation in the form of yoghurt having the following characteristics:

- Serving size: 100g

- Dairy protein: 4.8g

- Lipids: 2.8g

- Carbohydrates: 6.7g

- Mixture according to Example 3: 250 mg

- Vitamin D (pg): 15

- Provitamin A (mg): 1

- Vitamin E (mg): 10

- Vitamin B9 (pg): 250

- Vitamin B12 (pg): 4

- Vitamin C (mg): 80

- Zinc (mg): 5. [0122] Example 9: Composition in the form of a medicament

Example 9 is a medicinal formulation, in the form of a tablet comprising the mixture according to example 1: 1000 mg, the following excipients: sodium carboxymethyl starch, microcrystalline cellulose, gelatin, talc; the following excipients and film-coating: magnesium stearate; the following dyes: titanium dioxide, yellow iron oxide, red iron oxide; and the following coating: glycerol, sodium lauryl sulphate, macrogol 6000, hypromellose. The composition according to Example 9 is obtained by mixing the constituents and then the tablet is made up according to the standard manufacturing conditions known to those skilled in the art.

[0123] Example 10 Food supplement of the gummies type [0124] Example 10 is a food supplement, in the form of gummies comprising the mixture according to example 1: 10 g, pectin 10 g, trisodium citrate: 1 g, sorbitol: 150 g , water 150g, and citric acid: 2g.

The composition according to Example 10 is obtained by mixing the constituents and then the gummies are manufactured according to the standard manufacturing conditions known to those skilled in the art.

Claims

[Claim 1] Mixture of flavanols for its use to increase the concentration of the active form of vitamin D, calcitriol, in humans or animals, characterized in that the mixture comprises flavanols whose degree of polymerization is between 1 and 10.

[Claim 2] Mixture of flavanols for its use according to the preceding claim, characterized in that the flavanols whose degree of polymerization is between 1 and 10 represent at least 40g/100g of the mixture of flavanols.

[Claim 3] Mixture of flavanols for its use according to one of the preceding claims, characterized in that the flavanols whose degree of polymerization is between 1 and 5 represent at least 40 g/100 g of the mixture of flavanols.

[Claim 4] Mixture of flavanols for its use according to one of the preceding claims, characterized in that the flavanols whose degree of polymerization is between 1 and 2 represent at least 30 g/100 g of the mixture of flavanols.

[Claim 5] Mixture of flavanols for its use according to one of the preceding claims, characterized in that the flavanols whose degree of polymerization is equal to 1 represent at least 15 g/100 g of the mixture of flavanols.

[Claim 6] Mixture of flavanols for its use according to one of the preceding claims, characterized in that the mixture of flavanols is obtained from at least one plant species chosen from vines, grapes, tea, chocolate , maritime pine, red fruits, fruit with drupes, nuts, and their mixtures.

[Claim 7] Composition for its use to increase the concentration of the active form of vitamin D, calcitriol, in humans or animals, said composition comprises a mixture of flavanols according to one of the preceding claims, and in least one acceptable excipient.

[Claim 8] Composition for its use according to the preceding claim, characterized in that the composition or the mixture of flavanols is encapsulated in a food support.

[Claim 9] Composition for its use according to one of Claims 7 or 8, characterized in that the composition also comprises at least one vitamin.

[Claim 10] Composition for its use according to the preceding claim, characterized in that the vitamin is vitamin D and/or the precursors of vitamin D.

[Claim 11] Composition for its use according to the preceding claim, characterized in that the vitamin D is chosen from vitamin in the D2 form (ergocalciferol) and vitamin in the D3 form (cholecalciferol); and/or the vitamin D precursors are chosen from 7-DHC (cutaneous provitamin D) and previtamin D3.

[Claim 12] Composition for its use according to one of Claims 7 to 11, characterized in that the composition is in a form chosen from a food supplement, a foodstuff, a drink or a cosmetic product.

[Claim 13] Composition for its use according to one of Claims 7 to 12, characterized in that the composition is in the form of a tablet, capsule, stick, chewing gum, gummies, gel, powder, capsule, solution, suspension , emulsion, spray, lotion, cream, or patch.

[Claim 14] Mixture of flavanols according to one of Claims 1 to 6, or composition according to one of Claims 7 to 13, for its use in the prevention and/or treatment of a deficiency or insufficiency or vitamin D deficiency in humans or animals.

[Claim 15] Non-therapeutic use of a mixture of flavanols according to one of Claims 1 to 6, or of a composition according to one of Claims 7 to 13, to increase the concentration of the active form of vitamin D , calcitriol, in humans or healthy animals.