FR3132221A1 - Use of a mixture of flavanols to increase the concentration of the active form of vitamin D, calcitriol. - Google Patents

Abstract

The present 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 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.

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 animals. In particular, the flavanol mixture consists of low molecular weight flavanols.

State of the art

Vitamin D is a fat-soluble vitamin essential for the proper functioning of our body. Its main function is to increase the concentrations of calcium and phosphorus in the blood. Maintaining sufficient calcium levels in the blood ensures optimal mineralization of tissues, particularly 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.

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

Endogenously, vitamin D begins to be synthesized, from 7-dehydrocholesterol (7-DHC) at the level of keratinocytes, in the skin, which by the action of ultraviolet B rays called UVB is transformed into cholecalciferol (Vitamin D3) . Subsequently, in the liver, the microsomal or mitochondrial 25-hydroxylase enzyme (CYP27A1) will convert vitamin D3 into calcifediole (or 25-hydroxyvitamin D (25(OH)D) then 25-hydroxyvitamin D-1α -mitochondrial hydroxylase (1αOHase, 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.

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 25(OH)D level is greater than 30 ng/ml (75 nmol/L), the vitamin D status can be described as optimal. We can then distinguish between insufficiency, defined by a 25(OH)D level between 11 and 30 ng/ml, and deficiency, defined by a level below 11 ng/ml (25 nmol/L).

Vitamin D deficiency is a frequent problem in our modern societies and unfortunately underdiagnosed. Globally, it is estimated that one billion people are affected. For example, among healthy elderly people, the prevalence of vitamin D deficiency is 50%, and 80% among very elderly people with a history of hip fracture.

To ensure the body has 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 allows cover daily vitamin D needs and on the other hand have a balanced and varied diet in order to optimize exogenous intake. In fact, we can find vitamin D ₃ in fatty fish, egg yolk or butter.

However, the main source of vitamin D ₃ 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 (dress, sedentary lifestyle or not), the composition of the skin, and age.

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

Vitamin D ₃ supplementation via high-dose ampoules taken monthly or quarterly is possible. However, these do not make it possible to obtain a regular concentration of vitamin D in the body.

There is therefore a need for new solutions 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, calcitriol.

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

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

Widely distributed 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.

Quercetin is the majority flavonol in the diet, 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), notably 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). Oligomers have a degree of polymerization ranging from 2 to 10, and polymers have a degree of polymerization greater than 10.

If the interest of polyphenols as an antioxidant agent, and more particularly the use of flavanols, is no longer in doubt, only their interactions with vitamin C are described.

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 animals.

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

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

The mixture of flavanols can be of any origin, natural or not. In particular, this can be obtained from at least one plant species such as vines, grapes, tea, cocoa, maritime pine, red fruits, drupe fruits, or nuts. .

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 medicine. Said composition thus aims to increase the concentration of the active form of vitamin D, and therefore remedy, promote, prevent or treat an insufficiency, or even a deficiency, in vitamin D.

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, a drink or a food supplement, it comprises acceptable nutritional excipients. Such compositions make it possible to provide a sufficient intake to maintain the necessary daily intake of vitamin D. Preferably, they make it possible to overcome and/or remedy, prevent, treat vitamin D insufficiency in humans or animals.

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

Also, according to a final 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 treatment of a vitamin D deficiency and/or the maintenance of vitamin D content in humans or animals.

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

Brief description of the Figures

represents 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 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.

Detailed description of the invention

Definition

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 oligomers, that is to say it comprises at least 40% of low molecular weight flavanols, by weight of the total weight of flavanol. For example, the oligomers may be oligomers of procyanidins or proanthocyanidins. The mixture can be obtained from all origins, 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) less than 10, i.e. a degree of polymerization (DP) of between 1 and 10 (DP1 to DP 10) .

By “vitamin D insufficiency” 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 healthy people with low exposure to the sun and/or an unsuitable diet.

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 less than 11 nanograms per milliliter of blood. Unlike vitamin D insufficiency, such a concentration causes 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, we mean x gram of low molecular weight flavanols, for example DP of between 1 and 10 or between 1 and 5 or only flavanol monomers per 100g of mixture of flavanols. .

Mixture of flavanols according to the invention

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

In particular, the subject of the invention is 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 of between 1 and 10.

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) constituting this chain.

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

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 vine, grape, tea, cocoa, maritime pine, red fruits, drupe fruits, nuts, etc. 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 , Juglans L, Pistacia vera L., Prunus dulcis , Corylus avellana , Malus domestica , Prunus ( domestica , armeniaca ) , Hippophae rhamnoides . 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 between 1 and 10, that is to say that the quantity of flavanols having a degree of polymerization 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 (DP1 to DP 10) preferably represent at least 40g/100g, more preferably at least 50g/100g of the mixture of flavanols.

The effect of the mixture of flavanols is further improved when it 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 total weight 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 between 1 and 2. Preferably, the flavanols having a degree of polymerization 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 human or animal body.

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 can 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 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 fiber.

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 mainly in the form of two compounds, ergocalciferol or vitamin D2, present in foods 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 (oily fish, enriched milk foods). Vitamins D2 and D3 are used in particular in the prevention and curative treatment of rickets. The main vitamin derivatives mainly come from vitamin D3, of endogenous origin.

Indeed, there are few food 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 in ergocalciferol.

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

Also, the vitamin is preferentially chosen from vitamin in form D2 (ergocalciferol) and vitamin in form D3 (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 mixture of flavanols can also be encapsulated in a food support or not. Once encapsulated, the composition can be in the form of powder, capsule, tablet, capsule, solution, suspension, emulsion, cream or chewing gum.

Thus, the composition or mixture of flavanols can be encapsulated or microencapsulated 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 may be in the form of a nutritional, cosmetic, or pharmaceutical composition.

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

In the context of the invention, the composition can be administered orally or topically. For example, it can be administered orally as a food product, food supplement, drink, or medicine; or topically as a cosmetic product.

The nutritional composition can be in the form of a food product, food supplement, drink. For example, the nutritional composition can take the form of bars, dairy products, powders to swallow or rehydrate, gels, jams, candies, carbonated or non-carbonated drinks, dry drinks to rehydrate, compotes, gummies or chewing gum. .

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

The pharmaceutical composition can also be presented in different dosage forms, such as in the form of tablet, capsule, stick, powder, solution, 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 capable of receiving a nutritional or therapeutic agent according to the invention, for example but not limited to a pet animal, or a livestock 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 kibbles (extruded, co-extruded or freeze-dried), candies (or treats), snacks, moist or semi-moist foods such as pieces in sauce, jelly pieces, drinks, or even food supplements. Preferably, the agent is integrated into dry foods such as kibble or pellets.

When it is a medication intended for animals, or a veterinary product, the composition can be in several forms, such as 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 by example by impregnation or mixing, or by coating, that is to say by applying it to the surface of the composition, by spraying or sprinkling, for example by mixing it beforehand with one or more ingredients such as at least one palatability factor.

Thus, the present invention relates to the use of a mixture of flavanols according to any one of the embodiments or of a composition comprising it to increase, promote, maintain or even promote the concentration of the active form of the vitamin D, calcitriol in humans or animals.

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

Process

The mixture of flavanols can be of all origins. However, it is preferentially obtained from plants.

1. Broyage de la matière (sauf pour les pépins ou les noyaux de fruits),
2. extraction à l’eau et/ou à l’éthanol,
3. séparation de la solution d’eau et/ou éthanol de la matière solide, par exemple par filtration sur un tamis vibrant, et/ou décantation ;
4. évaporation de l’éthanol et de l’eau par évaporation sous-vide à une température préférentiellement inférieure à 60°C et à une pression inférieure à 100mbars ;
5. séparation des monomères et oligomères de flavanols de faible poids molécules (DP<10) et des polymères de flavanols (DP>10),
6. séchage des extraits obtenus à l’étape e. par atomisation, étuve sous vide ou lyophilisation avec ou sans support comme une maltodextrine ou de la gomme d’acacia.

Also, a process adapted from an extract of grape (preferably seed with or without skin), tea, cocoa, red fruits, fruit with drupes, maritime pine, shell of nut fruits such as that hazelnut, rich in low molecular weight flavanols, is a process comprising the following steps:

1. Grinding of the material (except for fruit seeds or stones),
2. extraction with water and/or ethanol,
3. separation of the water and/or ethanol solution from the solid material, for example by filtration on a vibrating sieve, and/or decantation;
4. evaporation of ethanol and water by vacuum evaporation at a temperature preferably lower than 60°C and at a pressure lower than 100mbar;
5. separation of low molecular weight flavanol monomers and oligomers (DP<10) and flavanol polymers (DP>10) ,
6. drying of the extracts obtained in step e. by atomization, vacuum drying or freeze-drying with or without a support such as maltodextrin or acacia gum.

The extraction step with water and/or ethanol can be carried out with a quantity of solvent 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 pressure between 1 bar and 150 bars. The raw materials used can be in dry, fresh, or whole frozen forms.

The step of separating monomers and oligomers of low molecular weight flavanols (DP<10) and flavanol polymers (DP>10) can be carried out by membrane separation of the previously desolvented extract so as to preferentially select the monomers (degree of polymerization of 1) and proanthocyanidin oligomers (having a degree of polymerization of between 2 and 10 inclusive) and to separate the flavanol polymers (DP>10), which makes it possible to obtain an extract characterized by a content in flavanol polymers 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 3000 daltons.

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

• chargement sur une résine des solutions d’extraits mélangées ou non,
• rinçage de la résine avec de l’eau,
• application d’une solution éluante eau/éthanol sur la résine,
• récupération de l’éluat purifié,
• évaporation de l’éthanol dudit éluât,
• concentration dudit éluât, et
• séchage dudit extrait aqueux purifié.

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

• loading onto a resin extract solutions mixed or not,
• rinsing the resin with water,
• application of a water/ethanol eluent solution to the resin,
• recovery of the purified eluate,
• evaporation of the ethanol from 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.

Examples

Example 1 according to the invention.

1. Extraction à l’eau, 40 g de pépin de raisins secs mélangés à 200g d’eau à une température de 85°C pendant 2 heures,
2. Séparation solide/liquide de l’extrait liquide des pépins par filtration sur un tamis vibrant
3. Concentration de l’extrait aqueux sous-vide à une température préférentiellement de 55°C et à une pression inférieure à 90mbars ;
4. Séparation membranaire par ultrafiltration avec un seuil de coupure de 5000 Daltons avec récupération du perméat d’ultrafiltration ; le rétentat d’ultrafiltration constituera la composition de l’exemple 2
5. Purification du perméat sur une colonne chromatographique d’adsorption comprenant les étapes suivantes :
   1. chargement sur une résine d’adsorption ;
   2. rinçage de la résine avec de l’eau ;
   3. élution à l’aide d’une solution éluante eau/éthanol (80%V/V) sur la résine,
   4. récupération de l’éluat purifié ;
   5. évaporation sous vide à une température de 55°C pression 90 mbars) de l’éthanol et concentration dudit éluât à 50% matière sèche,
6. Séchage de l’extrait purifié obtenu à l’étape e. par atomisation et obtention d’un gramme d’extrait de l’exemple 1.

A mixture of flavanols according to the invention is obtained from grape seed according to the following process:

1. Extraction with water, 40 g of raisin seeds mixed with 200g of water at a temperature of 85°C for 2 hours,
2. Solid/liquid separation of the liquid extract of the seeds by filtration on a vibrating sieve
3. Concentration of the aqueous extract under vacuum at a temperature preferably of 55°C and at a pressure less than 90mbar;
4. 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
5. Purification of the permeate on a chromatographic adsorption column comprising the following steps:
   1. loading onto an adsorption resin;
   2. rinsing the resin with water;
   3. elution using a water/ethanol eluent solution (80% V/V) on the resin,
   4. recovery of the purified eluate;
   5. vacuum evaporation at a temperature of 55°C pressure 90 mbar) of the ethanol and concentration of said eluate at 50% dry matter,
6. 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 less than 10, as described in Table 1 below.

Example 2 outside the invention.

1. Extraction à l’eau de 40 g de pépin de raisins secs mélangés à 200g d’eau à une température de 85°C pendant 2 heures ;
2. Séparation solide/liquide de l’extrait liquide des pépins par filtration sur un tamis vibrant ;
3. Concentration de l’extrait aqueux sous-vide à une température préférentiellement de 55°C et à une pression inférieure à 90mbars ;
4. Séparation membranaire par ultrafiltration avec un seuil de coupure de 5000 Daltons avec récupération du rétentat d’ultrafiltration ;
5. Séchage de l’extrait purifié par atomisation et obtention de 3 grammes d’extrait de l’exemple 2.

A ^2nd example of ^a mixture of flavanols is obtained from Example 1 from the fraction corresponding to the membrane separation filtration retentate according to the following process:

1. Water extraction of 40 g of raisin seeds mixed with 200g of water at a temperature of 85°C for 2 hours;
2. Solid/liquid separation of the liquid extract of the seeds by filtration on a vibrating sieve;
3. Concentration of the aqueous extract under vacuum at a temperature preferably of 55°C and at a pressure less than 90mbar;
4. Membrane separation by ultrafiltration with a cut-off threshold of 5000 Daltons with recovery of the ultrafiltration retentate;
5. Drying of the purified extract by spraying 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' invention.

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

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

Characterization of mixtures of flavanols according to Examples 1 to 3.

The mixtures of flavanols of Examples 1 to 3 are then analyzed according to methods known to those skilled in the art by HPLC to determine the polymers having a degree of polymerization between 1 and 10. The total flavanol concentrations are determined by analyzes spectrophotometric according to the method of Folin Ciocalteu. The polymer concentrations (DP>10) are determined by differences in total flavanols from DP flavanols between 1 and 10.

The results are presented in Tables 1 to 2 below.

Total polyphenols (Flavanols) (Folin) DP1
HPLC DP2 to DP10 HPLC DP1 to DP10
HPLC Flavanol polymers >DP10 Example 1 – Invention g/100g DM 98.9 31.8 33.1 64.9. 34.0 % of flavanols 100% 32.2% 33.4% 65.6% 34.4% Example 2 – Outside of Invention g/100g DM 68.6 0.9 8.7 9.5 59.1 % of flavanols 100% 1.3% 13.8% 13.9% 86.1% Example 3 – Invention g/100g DM 83.8 16.4 21.3 37.7 46.1 % of flavanols 100% 19.6% 25.4% 44.4% 55.6%

Total flavanols (Folin) DP1 (HPLC) DP2 (HPLC) DP3 (HPLC) DP4 (HPLC) DP5 (HPLC) DP6 (HPLC) DP7 (HPLC) DP8 (HPLC) DP9 (HPLC) DP10 (HPLC) Example 1 * 98.9 31.76 18.76 7.80 3.88 1.69 0.98 ** 100% 32.1% 19.0% 7.9% 3.9% 1.7% 1.0% 0.0% 0.0% 0.0% 0.0% Example 2 * 68.6 0.88 2.49 1.76 1.61 1.03 0.71 0.44 0.30 0.20 0.11 ** 100% 1.3% 3.6% 2.6% 2.3% 1.5% 1.0% 0.6% 0.4% 0.3% 0.2% Example 3 * 83.75 16.32 10.63 4.78 2.75 1.36 0.85 0.22 0.15 0.10 0.06 ** 100% 19.5% 12.7% 5.7% 3.3% 1.6% 1.0% 0.3% 0.2% 0.1% 0.1% * g/100g of dry matter **% of total flavanols
Total flavanols (Folin) DP1 to 10 (HPLC) DP1 to 2 (HPLC) DP1 to 3 (HPLC) DP1 to 4 (HPLC) DP1 to 5 (HPLC) Polymers [Total flavanols -DP(1 to 10)] Example 1 * 98.9 64.87 50.52 58.32 62.20 63.89 34.03 ** 100% 65.6% 51.1% 59.0% 62.9% 64.6% 34.4% Example 2 * 68.6 9.53 3.37 5.13 6.74 7.77 59.07 ** 100% 13.9% 4.9% 7.5% 9.8% 11.3% 86.1% Example 3 * 83.75 37.20 26.95 31.73 34.47 35.83 46.55 ** 100% 44.4% 32.2% 37.9% 41.2% 42.8% 55.6%

The effectiveness of the flavanol mixtures (Examples 1 to 3) described above was then studied.

Essay 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 4.5g glucose medium supplemented with 10% decomplemented FBS 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 5x10 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 with DMEM with 10% FCS and then the cells were returned to the oven at 37°C for 12 hours. At the end of these 12 hours, the medium was collected and immediately frozen at -20°C. At the same time, 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.

Proteins were measured using a BCA kit to normalize the ELISA test results to 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 presented in the . In the absence of exposure to UVB, 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 vitamin D insufficiency in humans or animals in the absence of optimal UVB exposure.

Example 4 of nutritional composition according to the invention

A ^4th example of composition according to the invention comprises 120 mg of a mixture obtained from Vitis vinifera according to example 1 and 5 µg of vitamin D3 (cholecalciferol) (i.e. 200IU) per capsule. Example 4 is obtained by mixing the constituents mentioned above with acacia fiber as bulking agent then packaged in 1 capsule under standard manufacturing conditions known to those skilled in the art.

Example 5 of nutritional composition according to the invention

A ^5th example of composition according to the invention is a solution comprising 20 ml of virgin rapeseed oil, 1,000 mg of green tea extract containing 55% of flavanols whose degrees of polymerization 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 5 is obtained by mixing the constituents then packaged in a bottle under standard manufacturing conditions known to those skilled in the art.

Example 6 of cosmetic composition according to the invention

A ^6th 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 associated with a pidolate. The composition according to Example 6 is obtained by mixing the constituents then packaged in a pot under standard manufacturing conditions known to those skilled in the art.

Example 7 of food composition according to the invention

• Portion : 100g
• Protéines laitières : 4.8g
• Lipides : 2.8g
• Glucides : 6.7g
• Mélange selon l’exemple 3 : 250 mg
• Vitamine D (μg) : 15
• Provitamine A (mg) : 1
• Vitamine E (mg) : 10
• Vitamine B9 (μg) : 250
• Vitamine B12 (μg) : 4
• Vitamine C (mg) : 80
• Zinc (mg) : 5.

Example 7 is a formulation in the form of yogurt having the following characteristics:

• Serving size: 100g
• Dairy proteins: 4.8g
• Lipids: 2.8g
• Carbohydrates: 6.7g
• Mixture according to example 3: 250 mg
• Vitamin D (μg): 15
• Provitamin A (mg): 1
• Vitamin E (mg): 10
• Vitamin B9 (μg): 250
• Vitamin B12 (μg): 4
• Vitamin C (mg): 80
• Zinc (mg): 5.

Example 8: Composition in drug form
Example 8 is a medicinal formulation, in tablet form comprising the mixture according to Example 1: 1,000 mg, the following excipients: sodium carboxymethyl starch, microcrystalline cellulose, gelatin, talc; the following excipients and coating: magnesium stearate; the following dyes: titanium dioxide, iron oxide yellow, iron oxide red; and the following coating: glycerol, sodium lauryl sulfate, macrogol 6000, hypromellose. The composition according to Example 8 is obtained by mixing the constituents then the tablet is formed according to the standard manufacturing conditions known to those skilled in the art.

Example 9 Food supplement type gummies

Example 9 is a food supplement, in the form of gummies comprising the mixture according to example 1: 10 g, pectin 10g, trisodium citrate: 1g, sorbitol: 150g, water 150g, and citric acid: 2g.

The composition according to Example 9 is obtained by mixing the constituents then the gummies are manufactured according to the standard manufacturing conditions known to those skilled in the art.

Claims (14)

Use of a mixture of flavanols to increase the concentration of the active form of vitamin D, calcitriol, in humans or healthy animals, characterized in that the mixture comprises flavanols whose degree of polymerization is between 1 and 10. 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. 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 40g/100g of the mixture of flavanols. 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 30g/100g of the mixture of flavanols. Use according to one of the preceding claims, characterized in that the flavanols whose degree of polymerization is equal to 1 represent at least 15g/100g of the mixture of flavanols. 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, drupe fruits and nuts. Use according to one of the preceding claims, characterized in that the mixture of flavanols is included in a composition, said composition comprising at least one acceptable excipient. Use according to one of the preceding claims, characterized in that the composition or mixture of flavanols is encapsulated in a food support. Use according to one of claims 7 or 8, characterized in that the composition also comprises at least one vitamin. Use according to the preceding claim, characterized in that the vitamin is vitamin D and/or vitamin D precursors. Use according to the preceding claim, characterized in that vitamin D is chosen from vitamin in the form D2 (ergocalciferol) and vitamin in the form D3 (cholecalciferol); and/or the vitamin D precursors are chosen from 7-DHC (cutaneous provitamin D) and previtamin D3. 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, a cosmetic product. Use according to one of claims 7 to 12, characterized in that the composition is in the form of tablet, capsule, stick, chewing gum, gummies, gel, powder, capsule, solution, suspension, emulsion, spray, lotion, cream, or patch. Mixture of flavanols comprising flavanols whose degree of polymerization is between 1 and 10, for its use in the prevention and/or treatment of vitamin D deficiency in humans or animals.