WO2020074134A1 - Polyphenol extract from fruits of the date palm phoenix dactylifera for its nutraceutical use - Google Patents

Abstract

The present invention relates to a purified extract concentrated in polyphenolic compounds obtained from the fruit of the date palm Phoenix dactylifera in the preparation of a nutraceutical composition, in particular useful in the prevention of pathologies related to oxidation.

Description

 PHYENIX DACTYLIFERA POLYPHENOL EXTRACT FROM THE FRUITS OF THE DATE PALM FOR ITS NUTRACEUTICAL USE

TECHNICAL AREA

 The present invention relates to the use in the nutraceutical field of a purified extract concentrated in polyphenolic compounds obtained from the fruit of the date palm Phoenix dactylifera.

 For the sake of well-being and health, research and industrial innovation are now focusing on the use of natural substances, both in the field of nutrition and in those of cosmetics, nutraceuticals or from the pharmacy.

 Several phytochemical studies have shown that the bioactivity of plant extracts lies mainly in their richness in antioxidants. Indeed, current research on antioxidants and free radicals have confirmed that plant species rich in antioxidants play an essential role in the prevention of certain cardiovascular diseases, cancers and neurodegenerative diseases. For example, excellent capacities to inhibit oxidative reactions have been demonstrated for extracts of rosemary, sage, thyme, oregano, cloves and turmeric (Cuvelier et al. 1992 and 1996). Since then, research on natural antioxidants has aroused great interest and has been the subject of several scientific studies (Bucic-Kojic et al. 2011).

 Polyphenols are antioxidant compounds specific to the plant kingdom, and endowed with various biological activities demonstrated by numerous studies described in the scientific literature. Mention will be made in particular, in addition to their antioxidant activity, of anti-inflammatory, antimicrobial, antiviral or even anticancer activities (Pathak D et al. 1991) (Pincemail et al. 2004) (Skerget, M et al. 2005), (Catherine Rice- Evans et al. 1997), (Meishiang Jang et al. 1997), (Tipoe Geaorge L. et al. 2007).

 Polyphenols are present in all the organs of the plant. The fundamental structural element which characterizes them is the presence of at least one benzene nucleus, with at least one hydroxyl group, free or engaged in another chemical function such as ether, ester, heteroside. Phenolic compounds thus have a very wide variety of structures. Thus, they are divided into different families:

 phenolic acids, such as derivatives of hydroxybenzoic acid and derivatives of cinnamic acid,

flavonoids such as flavan-3-ols, including catechins and tannins, and flavones, including flavonols. Because of the interest represented by phenolic compounds, the purification and characterization of these compounds constitute a fundamental line of research today.

 The invention, which is part of the development of natural substances, thus relates to an extract of polyphenols from the fruit of the date palm Phoenix dactylifera as an active ingredient in the nutraceutical field, in particular incorporated into a composition nutraceutical.

 Dates can be considered an interesting source of polyphenols. In addition, a large part of the date production is processed into by-products, such as jams, juices and syrups. Consequently, a large quantity of date stones and non-marketed fruits is produced as waste which can be recovered on the basis of its high content of bioactive compounds and dietary fibers (Al-Larsi et al., 2008; Al-Shahib and Marshall, 2003).

 The inventors have thus, in the course of their research, discovered that extracts of date polyphenols have interesting nutraceutical properties which have never been described until now.

STATEMENT OF THE INVENTION

 One of the aims of the present invention is thus to propose an extract of polyphenols from fruits of the date palm Phoenix dactylifera for the preparation of a nutraceutical composition.

 According to the invention, the extract contains, by weight relative to the total weight of the dry purified extract, up to 80% of polyphenolic compounds.

 Advantageously, the extract contains at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to the total weight of polyphenols

 The tannins condensed in the extract according to the invention are oligomers or polymers of proanthocyanidins.

 Advantageously, the condensed tannins have an average degree of polymerization of less than 30, preferably less than 27, more preferably less than 20.

As the examples which follow will demonstrate, the extracts according to the invention exhibit, on the one hand, very high ORAC index values, representative of a high antioxidant capacity, and, on the other hand, the capacity to regulate the expression of genes encoding the synthesis of many proteins involved in biological mechanisms in the skin. Thus, the invention also relates to an extract of polyphenols from the fruit of the date palm Phoenix dactylifera for its use in maintaining eye health, vision, cardiovascular health, functioning of the immune system, blood circulation, brain activity, slowing brain aging, prevention and care of joint pain or inflammation, the proper functioning of the digestive system, including intestinal flora, prevention or treatment of the syndrome metabolic, prevention of chronic diseases, its use against stress, maintenance of health and appearance of the skin.

 Advantageously, the polyphenol extract is obtained from the stone, the skin and / or the pulp of the fruit.

 According to a preferred embodiment of the invention, the polyphenol extract is obtained using a process comprising the steps:

 obtaining and preparing the plant material,

 solid-liquid extraction using a polar solvent and recovery of an extract containing polyphenolic compounds,

 of concentration of the polyphenolic compounds in the extract, of purification of the polyphenolic compounds of the extract by a method of solid phase chromatography,

 recovery of a purified extract concentrated in polyphenolic compounds.

The invention also relates to a nutraceutical composition comprising, in a nutraceutically acceptable medium, an extract of polyphenols from the fruit of the date palm Phoenix dactylifera.

 In such a composition, the extract is as described above.

 According to the invention, the nutraceutical composition is in the form of a composition for enteral administration, such as a food, a drink, a capsule, a capsule, a tablet, a powder or a liquid solution.

 Advantageously, the composition comprises at least 0.01% of said polyphenol extract.

The invention also relates to a nutraceutical composition as defined above, for its use in maintaining eye health, vision, cardiovascular health, the functioning of the immune system, blood circulation, activity, slowing aging brain, prevention and care of joint pain or inflammation, the proper functioning of the digestive system, especially of the intestinal flora, prevention or treatment of metabolic syndrome, prevention of chronic diseases, its use against stress , maintaining the health and appearance of the skin.

DETAILED DESCRIPTION OF THE INVENTION

Obtaining and preparing the plant material:

Plant matter:

 The starting plant material is the fruit of the date palm Phoenix dactylifera.

 The date fruit includes four stages of maturity: Hababouk, Blah (Kimri), Besser (Khalal) and Tamar. The selection of the fruit according to one of its stages, and according to the tissues, makes it possible to obtain different results in terms of polyphenol content in the dry extract purified at the end of the process.

 Once the fruit has been selected, the tissues, that is to say the core, the skin and the pulp of the fruit, are separated from each other, crushed, for example in a controlled atmosphere, reduced to powder and then the powder is dried. under vacuum.

Tissue separation includes isolation of the stone, skin and pulp from the fruit.

 Preferably, the stage of obtaining and preparing the plant material includes the selection of a fruit according to its stage of maturity. Solid-liquid extraction using an apolar solvent:

 According to one embodiment, but not necessarily, the method comprises, prior to the solid-liquid extraction step using a polar solvent, a solid-liquid extraction using a non-polar solvent, such as hexane, and recovering the residue. This extraction step allows the separation of apolar compounds, such as lipids, chlorophylls and carotenoids, from polar compounds including polyphenols. The extraction residue is kept for the implementation of the next step of the process. The liquid phase containing the apolar compounds can be used elsewhere.

Solid-liquid extraction using a polar solvent: This extraction step is carried out successively to the extraction step with non-polar solvent. It allows the extraction and recovery in the polar solvent of an extract containing the polyphenols.

 Advantageously, the polar solvent of the solid-liquid extraction is a mixture of ethanol, water and acetic acid, preferably in a ratio of 50 to 80 ethanol / l to 10 acetic acid / qs in water. For example, you can use a ratio of 80: 19: l.

 Alternatively, the solvent is a mixture of acetone, water and acetic acid, in a ratio of 50 to 80 in acetone, between 1 and 10 acetic acid, qs in water. For example, you can use a ratio of 50: 49: 1.

 Thus, the extraction in polar solvent is carried out on the residue obtained after extraction in non-polar solvent. The extract obtained is filtered by frontal filtration with filter aid. Concentration of polyphenolic compounds in the extract:

 The polyphenols are concentrated in the extract by evaporation of the polar solvent under vacuum.

 Evaporation is carried out by taking the precaution to avoid concentrating acetic acid capable of degrading phenolic compounds. To do this, several stages of evaporation are advantageously carried out, by adding water between each stage.

 The evaporation step is very important in order to avoid that the polyphenols are not elected by the column, during the next step. This evaporation step replaces the solvents used with osmosis or softened water. This also makes it possible to avoid an excessive concentration of acetic acid which can degrade the phenolic molecules. If this step is not carried out, the polyphenols will not be eluted by the column and the fraction will be lost.

 The preparation of the extract by filtration just before concentration is another important step in the process according to the invention. This step avoids clogging of the separation system.

 Various tests show that the following purification protocol cannot be applicable without carrying out these two stages of evaporation and filtration.

Purification of the polyphenolic compounds of the extract by a solid phase chromatography method: The solid phase purification is carried out by reverse phase chromatography or by ion exchange.

 Preferably, this step is carried out by adsorption of the polyphenols on a solid support included in a cartridge or a column of silica grafted with C18 hydrocarbon chains or of nonionic ion exchange resin, the elution of the polyphenols using 'a polar eluent and the recovery of a fraction enriched in polyphenols.

 In particular, the cartridges used are the grafted silica gel cartridge C18 and sold under the brand Sep-Pack C18 by the company Waters or the resin cartridge sold under the brand Amberlite FPX 66 by the company Rohm & Haas France SAS.

 According to one embodiment of the invention, the eluent is composed of a mixture of ethanol, water and acetic acid in a ratio of between 50 and 80 ethanol, 1 and 10 acetic acid , qs in water. For example, it can be a 50: 49: l ratio.

Advantageously, following elution, a step of washing with ethanol can be carried out so as to remove the very hydrophobic compounds which can still be fixed.

 Recovery of a purified extract concentrated in polyphenolic compounds:

The purified fraction obtained after elution in the previous step is recovered and concentrated by evaporation. Advantageously, this fraction is then dried.

 To do this, evaporation is carried out by taking the precaution to avoid concentrating the acetic acid liable to degrade the phenolic compounds. To do this, several evaporation steps are advantageously carried out, by adding water between each step, in the same way as that which was described during the previous concentration step.

The purified polyphenol extract obtained by the process described above comprises, by weight relative to the total weight of the dry purified extract, up to 80% of polyphenolic compounds.

 In addition, the extract contains at least 95%, preferably at least 99% of tannins, by weight relative to the total weight of polyphenols.

DETAILED DESCRIPTION OF EMBODIMENTS The characteristics of the invention mentioned above, as well as others, will appear more clearly on reading the following description of exemplary embodiments, said description being made in relation to the accompanying drawings, among which:

 The [Fig. 1] is a graphic representation of the steps of a method according to an embodiment of the invention, and

 The [Fig. 2] and [Fig. 3] represent bar diagrams indicating the purification yields obtained for different embodiments of the method according to the invention.

Although different varieties of dates have been used in the context of the invention, the following examples illustrate the results obtained from the variety deglet nour (VI) and Medjool (V2). The dates were selected according to the following four stages of maturity:

 - Hababouk (stage 1),

 - Blah (Kimri) (stage 2),

 - Besser (Khalal) (stage 3),

 - Tamar (stage 4).

 The fruits were calibrated manually and, for each sample, two lots were made up for the analysis of polyphenols. The tissues, that is, the skin, pulp and stone, were separated from each other and then frozen with liquid nitrogen to prevent oxidation. After a grinding operation, the fabrics are dried.

It should be noted that at stage 1 of ripening, the fruit was studied entirely without separation of the tissues because the nucleus is not yet formed.

 Powder samples are stored in a dark place in a desiccator, containing phosphorus pentoxide to prevent moisture pickup. The samples are weighed before and after preparation to determine the percentage of dry matter (% DM) and the average mass of a fruit.

Examples of methods for obtaining polyphenol extracts from

 Phoenix dactxlifera

Example 1: obtaining a witness Control batches are produced from the tissues of the fruits reduced simply to powder, without implementing the method according to the invention.

Example 2: Obtaining Purified Extracts of Polyphenols

Successive solid-liquid extractions are carried out from the samples of the plant material prepared in powder form: a first extraction using a nonpolar solvent then a second extraction using a polar solvent.

 Solid-liquid extraction by non-polar solvent: 10 g of sample in powder form are brought into contact with 80 ml of hexane for 15 min., With stirring, then filtered, during a first extraction. The extract obtained, called Hl, is set aside, and the residue obtained is subjected to a second extraction by contacting with 80 ml of hexane for 15 min., With stirring and then filtered. The extract obtained, named H2, is set aside, and the residue obtained is recovered and then dried under nitrogen for 1 hour.

 Solid-liquid extraction by polar solvent: the polar solvent is composed of ethanol / water / acetic acid in a ratio of 80: 19: l (V / V / V) (hydro alcoholic solvent).

 The dry residue from the previous extraction is brought into contact with 30 ml of polar solvent for 15 min., With stirring, then filtered, during a first extraction. The extract obtained, named El, is kept, and the residue obtained is subjected to a second extraction by contacting with 30 ml of the same solvent for 15 min., With stirring and then filtered. The extract obtained, named E2, is stored and the residue obtained is recovered and subjected to a third extraction by contacting with 30 ml of the same solvent for 15 min., With stirring and then filtered. The extract obtained, named E3, is kept.

The extracts E1, E2 and E3 are pooled.

 The extract obtained is filtered by frontal filtration with filter aid.

Concentration:

The hydro-ethanolic extracts are evaporated at a temperature of 48 ° C, taking the precaution of adding twice a volume of water between each evaporation step, resulting in the reduction of half the volume. This makes it possible to avoid too high a concentration of acetic acid liable to degrade the phenolic molecules. The last stage of evaporation is stopped when the volume reaches about half. Purification:

 The extracts stored at 2 ° C under argon contain a significant deposit which can clog the sinter of the SPE cartridge. Centrifugation at 4500 rpm for 10 min at 4 ° C is carried out in order to recover the supernatant which will be used for the Sep Pack C18 cartridge (sold by the company Waters) for the implementation of a solid phase extraction ( SPE).

 SPE cartridges containing 5 g of Cl 8 gel are conditioned with ethanol (20 ml) and then equilibrated with acidified water (40 ml) before the centrifuged extracts (40 ml) from each sample are deposited. Rinsing is carried out with 40 ml of acidified water (2% acetic acid), then the retained fraction is eluted with 40 ml of ethanol / water / acetic acid mixture (50: 49: 1; V / V / V ). The column is then washed with 30 ml of ethanol to remove the very hydrophobic polyphenols which can still be fixed. Concentration and recovery of a purified fraction of polyphenols

 Evaporation of the extracts obtained after purification on the cartridges is carried out at a temperature of 48 ° C, taking the precaution of adding twice a volume of water between each evaporation step resulting in the reduction of half the volume. This avoids too high a concentration of acetic acid which can degrade phenolic molecules. The last stage of evaporation is stopped when the volume reaches about half. The aqueous suspensions obtained are put into the form of dry powders.

 The samples are then ready for characterization by HPLC with and without prior phloroglucinolysis.

Example 3:

 Purified polyphenol extracts are obtained using a process identical to that described in Example 2 except that the purification step is carried out using an FPX 66 ion exchange column ( Rohm & Haas France SAS).

Example 4:

Purified polyphenol extracts are obtained using the same process as described in Example 2 except that the extraction step using the polar solvent is carried out using a solvent composed of acetone / water / acetic acid in a ratio of 50: 49: l (V / V / V) (hydro-acetonic solvent). Example 5:

 Purified polyphenol extracts are obtained using the same process as described in Example 4, except that the purification step is carried out using an FPX 66 ion exchange column ( Rohm & Haas France SAS).

Analysis and structural characterization of the polyphenols in the control powders (Example 1) and of the purified polyphenolic extracts (Examples 2, 3, 4, 5):

Procedure

 For each phenolic date extract (e.g. 2 to 5) or each sample of date powder (e.g. l), an aliquot (of the order of 3 mg, weighed precisely to the nearest 0.01 mg) is placed in an Eppendorf. 1200 μL of 1% acetic acid diluted in methanol are added. The mixture is dispersed by passage through an ultrasonic bath for approximately 15 min and the supernatant is filtered using a teflon filter (0.45 μm of porosity and 13 mm in diameter). Under these conditions, the extracts are soluble and directly injectable in HPLC by LC-ESI / MS.

Acid hydrolysis: the acid hydrolysis method was used to confirm the identification of the conjugated aglycones of flavonols or flavones present in the samples. One milliliter of the methanoic date extract was mixed with 1 ml of HCl (2N), the mixture was heated at 90 ^° C. for 30 min in closed glass flasks. Neutralization was carried out by adding 1 ml of NaOH (2N) (Bloor, 2001). In order to obtain sufficient fragmentation of the aglycone molecular ions, the MS / MS collision energy was set at 50% (arbitrary units).

 Equipment

The separation of the compounds is carried out on an HPLC chain consisting of a SCM1000 degassing system (ThermoQuest, San José, CA, USA), an automatic injection system (ThermoFinnigan, San José, CA, USA), a 1100 binary pump Series (Agilent Technologies, Palo Alto, CA, USA), and a Spectra System UV6000LP diode array detector (ThermoFinnigan, San José, CA, USA). The LCQ Deçà ion trap mass spectrometer (ThermoFinnigan San José, CA, USA) is equipped with an Electrospray ionization source (EIS). All of the effluent from the diode array detector is injected into the mass spectrometer. The injected samples (5 μL) are separated on a Zorbax Eclipse XDB-C18 column (2.1 mm × 150 mm, 3.5 μm, Agilent Technologies) where the temperature is maintained at 30 ° C. Analysis conditions

 The analysis conditions are as follows: eluent A is formic acid (Fischer scientific, Loughborough, UK) diluted to 0.1% in ultrapure water, and eluent B is formic acid at 0 , 1% diluted in acetonitrile. The gradient applied with a flow rate of 0.2 mL / min is: initial state, 3% B; 0-5 min, 9% B, linear; 5-15 min, 16% B, linear; 15-45 min, 50% B, linear; then a rinse and a return to the initial conditions.

 II.4.5 SIE, MS and MSn parameters

 The dinitrogen (N2) is used to vaporize the sample in the form of droplets and to dry them in order to obtain the pseudo-molecular ions.

 The analyzes are carried out in negative mode with the following parameters: voltage 5 kV, the main N2 gas is at 67 u.a. the auxiliary N2 gas at 5 u.a and the temperature of the capillary is fixed at 240 ° C. Helium is used as a stabilization, focusing and collision gas to fragment the ions into MSn. These ions are then converted into peaks of different intensities which can be observed on the chromatograms. The collision energy is set at 35% for the fragmentation in MSn.

 The spectra of the HPLC-MS are acquired in "Full Scan", on the whole range of masses (m / z) going from 50 to 2000. The whole of the data is then collected and treated by the software Xcalibur version 1.2.

Results:

 It should be noted that at the first stage of maturation (Hababouk), the size of the fruits does not allow the three fabrics to be considered separately. The results below are obtained by analysis by HP LC in reverse phase with UV-visible detection of methanolic extracts of the powders of the tissues considered and depolymerization by phloroglucinolysis. It is therefore possible to distinguish between the terminal catechin units and the extension catechin units by also giving access to the calculation of mean polymerization degree (DPn) of flavan-3ols.

The quantification was carried out by plotting the result of the integration of the different chromatographic peaks in the equation of a calibration line obtained with the respective standards of each phenolic class. Quantification of the four most important phenolic classes of the date, namely flavan-3-ols, flavones, flavonols and hydroxycinnamic acids, has been carried out. For the phenolic class flavan-3-ols, a distinction was made between monomeric catechins, oligomeric procyanidins up to the pentamer and the rest of the procyanidins (condensed polymeric tannins) accessible by dosage via phloroglucinolysis. 1) Qualitative analysis and quantification of the total polyphenols in the powders of Example 1

 Quantification of total polyphenols:

 The total amounts of polyphenols (in g / kg of DM), depending on the variety and the stage of ripening of the date fruits according to Example 1, are shown in Table 1.

 Table 1:

Quantification of fiavan-3-ols (condensed tannins) in total polyphenols:

The percentages of tannins condensed in total polyphenols as a function of the variety and stage of ripening of the fruits according to Example 1 are shown in Table 2.

 Table 2:

Degree of polymerization (DPn) of flavan-3-ols (condensed tannins)

 The degrees of polymerization as a function of the variety and of the stage of maturation of the date fruits according to Example 1 are indicated in Table 3.

Table 3

The results show the richness of the samples studied in polyphenols (Table 1). By comparison with some fruits, we observe that the date fruit has the highest polyphenol content by a value that exceeds 25 g / kg of fresh matter. On the other hand, in the apple, the polyphenol contents do not exceed 7 g / kg (Sanoner et al., 1999). In grapes, total polyphenols vary between 0.5 and 5.7 g / kg of fresh matter (Nile et al., 2013). The pear has a polyphenol content which does not exceed 2 g / kg of fresh matter (Galvis Sanchez et al., 2003). The analyzes show that the polyphenol contents decrease from stage 1 to stage 2, then a slight decrease during maturation where oxidation plays a primordial role for this decrease in the total contents of polyphenols. The characterization of phenolic compounds shows that the majority class of date polyphenols in the various tissues is that of flavan-3-ols (tannins) characterized by the presence of monomeric catechins and oligomeric procyanidins (Table 2). The other phenolic classes are present in date powders in a minority way such as hydroxycinnamic acids (caféoylshikimic acid) or flavonols and flavones.

 The results also show that in stage 1 flavan-3-ols have very high degrees of polymerization. The lowest DPn are found in the stage 4 samples of maturation (Table 3).

2) Qualitative analysis and quantification of the polyphenols in the extracts from Examples 2, 3, 4 and 5

 Many parameters can influence the solid-liquid extraction and polyphenol purification operations, such as the volumes of solvents, the extraction times and the temperature. It is important to select them carefully to ensure efficient extraction. Extraction is one of the first steps to isolate phenolic compounds. An efficient extraction procedure optimizes the extraction yields and ensures that the stability of the polyphenols is maintained. In addition, the nature of the separation and purification system and the eluents used can change the mass yields of the purified products.

 Examples 2 to 5 illustrate the selection work carried out with regard to the choice of extraction solvents and of the purification system.

 As a reminder, according to Example 2, the extracts are obtained after an extraction using a hydro-alcoholic polar solvent composed of ethanol / water / acetic acid and a purification carried out on a column in reverse phase (Sep Pack Cl 8).

 According to Example 3, the extracts are obtained after an extraction using a hydroalcoholic polar solvent of ethanol / water / acetic acid and a purification carried out on an FPX 66 column.

 According to Example 4, the extracts are obtained after an extraction using a hydro-acetonic polar solvent composed of acetone / water / acetic acid and a purification carried out on a column in reverse phase (Sep Pack Cl 8)

 According to Example 5, the extracts are obtained after an extraction using a polar hydro-acetonic solvent composed of acetone / water / acetic acid and a purification carried out on an FPX 66 column.

Purification yields Figs. 2 and 3 indicate, respectively, the yields (in g / kg) for the hydro-alcoholic extracts obtained by purification on a Sep Pack C18 or FPX 66 column (Fig. 2), and for the hydro-acetonic extracts obtained by purification on a column Sep Pack Cl 8 or FPX 66 (Fig. 3).

In these figures are indicated the varieties and stages of maturation: VI: variety 1; V2: variety 2; 1, 2, 3, 4: maturation stage.

 The results indicate that the purification by the FPX 66® column gives the best mass yields by comparison with the Sep Pack Cl 8® column. Likewise, the results indicate that a better yield is obtained using the acetone / water / acetic acid solvent compared with the ethanol / water / acetic acid solvent.

Quantification of total polyphenols:

The total amounts of polyphenols (in g / kg of DM), as a function of the variety and the stage of ripening of the date fruits according to Examples 2 to 5, are indicated in Table 4.

Table 4

By comparison with the amounts of polyphenols observed for Example 1, the results reported in Table 4 indicate that the two purification columns are effective in purifying the polyphenols.

By comparison of Examples 2 and 3, the results indicate that by using the ethanol / water / acetic acid mixture as extraction solvent, the FPX 66® purification column makes it possible to obtain a larger amount of polyphenols (e.g. 3 ).

 By comparison of Examples 3 and 5, the results indicate that by using the FPX 66® purification column, the acetone / water / acetic acid mixture makes it possible to obtain a greater amount of polyphenols.

 For all the examples, the highest polyphenol contents are found in stage 1 of maturation. At this stage, the extracts contain between 66 and 80% polyphenols. In stages 2 to 4 of maturation, it is also always the core that contains the highest content of polyphenols, compared to other tissues. For the same fabric, there is a slight decrease in the polyphenol content from stage 1 to stage 4.

 By comparison, between the extraction solvents using hydro-alcoholic and hydro-acetonic mixtures, the most effective solvent for purification is acetone, given its capacity to extract highly polymerized molecules (Example 2 versus Example 4 and Example 3 versus example 5).

Quantification of flavan-3-ols (condensed tannins) in total polyphenols: The percentages of flavan-3-ols in total polyphenols as a function of the variety and the stage of maturation of date fruits according to Examples 2 to 5 are indicated in Table 5. Table 5:

The results show that the class of flavan-3-ols (tannins) is the majority phenolic class in date extracts and that this class is mainly in the form of condensed tannins. The percentages of the latter vary between 55 and more than 99%, which makes the other phenolic classes such as hydroxycinnamic acids, flavonols and flavones minority in the purified fractions.

 The highest concentration of flavan-3-ols is found in the nuclei, whatever the stage of maturation and the plant variety. Indeed, whatever the examples, the results indicate that more than 99% of the polyphenols in the nuclei are tannins. The highest proportions of tannins are found in stages 3 and 4.

 Comparison of these results with other fruits shows that the tannins in the dates are specific.

 In cider apples, the percentages of flavan-3-ols range from 49 to 86% (Guyot et al., 2003). On the other hand, in the pulp of apples at the table the percentage is between 68 and 81% and in the skins it varies between 72 and 86% (Guyot et al., 2002).

The percentages of flavan-3-ols in pears are lower compared to apples or our fruit which is the date. The results of previous work show that the values of the percentages of flavan-3-ols in pears vary between 1% and 31% which is the value found in the coscia variety (Galvis-Sanchez et al., 2003).

 Buendia and his collaborators studied several varieties of strawberry and according to this study the percentages of flavan-3-ols in the polyphenols measured vary between 32% and 72% (Buendia et al., 2010).

 Studies have been carried out on grape seeds, the results show that tannins represent percentages which vary between 27 and 34% of total polyphenols (Bozan et al., 2008).

 According to previous work, dates can be considered among the fruits richest in polyphenols where the tannins represent the majority compounds.

 Furthermore, the comparison of Examples 3 and 5 shows that the FPX 66 food column is more effective in purifying fractions richer in polyphenols and mainly composed of tannins. In fact, the percentages of flavan-3-ols (tannins) in the total polyphenols dosed in the hydro-acetone extracts purified by the food column FPX 66 are very high (Example 5). The values exceed 95% in extracts of pulps and skins and exceed 99% in extracts of pits.

 Polymerization levels (DPn) of flavan-3-ols (condensed tannins)

 The average degrees of polymerization (DPn) of the flavan-3-ols in the extracts of Examples 2,3,4 and 5 are indicated in Tables 6, 7, 8 and 9, respectively.

Table 6

Table 7:

Table 8

Table 9

 The values show that the flavan-3-ols have higher degrees of polymerization in stage 1 compared to the other stages of maturation. The lowest DPn are found in the extracts of stage 4. Generally, the extracts of nuclei have the highest DPn in examples 4 and 5.

 It is interesting to note that in the extracts purified by the hydroacetonic mixture, the DPn of the flavan-3-ols are slightly higher than those purified by the hydroalcoholic mixture (example 2 versus example 4; example 3 versus example 5). Similarly, in the extracts purified by the FPX 66 column, the DPn of the flavan-3-ols are slightly higher than those purified by the Cl 8 column (example 2 versus example 3; example 4 versus example 5).

 Finally, it is interesting to note that the DPn of flavan-3-ols or especially the tannins decrease during maturation. One hypothesis is that at the mature stage the high DPn tannins may be involved in oxidation mechanisms or that these latter have formed complexes with the parietal compounds. These chemical transformations can make the tannins difficult to extract, which leads to a reduction in DPn.

 Compared with the DPn obtained in Example 1, the DPn of the purified extracts are on average lower.

Determination of total nolvnhenols in extracts by the Folin-Ciocalteu method and estimation of the quantity of oxidized polyphenols

The objective of this paragraph is the determination of the total polyphenols in the purified extracts as well as the evaluation of the quantity of the oxidized polyphenols. This assay is carried out using the Folin-Ciocalteu reagent. The approach was used for the characterization of oxidation products. A subtraction is carried out between the total of polyphenols in mg equivalent of gallic acid and the total of polyphenols assayed by chromatography in g / kg. The difference corresponds to the polyphenols in non-native form (Table 9 for Examples 2 and 3; Table 10 for Examples 4 and 5).

Table 9

SUBSTITUTE SHEET (RULE 26)

i, 2, 3, 4: maturation stages

Pe: skin; Pu: pulp; N: nuclei

VI: variety 1; V2: variety 2

Table 10

SUBSTITUTE SHEET (RULE 26)

i, 2, 3, 4: maturation stages

Pe: skin; Pu: pulp; N: nuclei

VI: variety; V2: variety 2

The results show that the percentages of oxidized polyphenols increase as a function of maturation with very little difference between stage 1 and 2 of maturation. At the last stage of maturation (tamar), the percentages of oxidized polyphenols increase remarkably with high values in the consumable parts of the fruit (skin + pulp). One hypothesis is that during maturation, polyphenols undergo oxidation mechanisms or complexations with parietal compounds, which results in a modification of extractability and a decrease in the quantities of native polyphenols.

 The comparison between the two purification columns shows that the FPX66 column is more adequate when the extraction solvent is the hydro-alcoholic mixture while the SEP Pack Cl 8 column is more adequate when the extraction solvent is the hydro-alcoholic mixture acetonic.

Example 6:

 By way of comparison, the process according to the invention was carried out using water acidified with 1% acetic acid as the solvent for the polar extraction. The results show that this solvent gives low values in purified phenolic fractions. The mass powder yields of the phenolic extracts obtained are quite low compared to the results found by hydro-alcoholic and hydro-acetonic mixtures. The results show that the contents of phenolic compounds determined by high pressure liquid chromatography (HPLC) or by the method of estimation of total polyphenols by the Folin-Ciocalteu reagent are generally low.

Examples of applications of polyphenol extracts from Phoenix dactylifera fruits

FIRE I LLE DE REM PLACEM ENT (REG 26) Example 7: Evaluation of antioxidant power by the ORAC method (Oxygen Radical Absorbance Capacity)

The ORAC index makes it possible to assess the antioxidant capacity of a food. It is calculated by means of a test which has the same name, and which is well known to those skilled in the art. The ORAC test is based on the oxidation of a fluorescent probe via a transfer of hydrogen atoms by free radicals, which are often peroxy radicals, but can also be hydroxyl radicals. These free radicals are produced by a generator. During the experiment, free radicals damage the probe and therefore decrease the intensity of the fluorescence.

 Preparation of hydrophilic solutions

 A stock solution of fluorescein or working FlNa at 1 mM (which can be stored in the freezer) is prepared immediately in a phosphate buffer solution at 75 mM and pH 7.4. The resulting 1 mM daughter and 30 nM granddaughter solutions are also produced in the phosphate buffer solution.

 The solution of 2,2’-azobis (2-methyl-propionamidine) dihydrochloride or AAPH is freshly prepared before injection into a hemolysis tube by depositing 42.4 mg of powder in 1 ml of phosphate buffer solution.

 Finally, a stock solution buffered with Trolox® at 1 mM is produced.

 Preparation of amphiphilic / lipophilic solutions

 A solution of b-cyclodextrinmethylated (RMCD) (7% (w / v)), and a solution of Trolox® are prepared in an acetone / water mixture at 50% (v / v). A daughter solution of Trolox® of 100 pM at 20% (v / v) is produced in the RMCD solution. The mixture is then stirred for 1 h at room temperature, and protected from light for the drawing of the calibration line. These proportions will then be kept for studying the extracts. The working solutions of FlNa and AAPH are produced according to the method described above.

 Procedure

The ORAC test is applied to extracts having a hydrophilic character by following the method developed by Ou et al. (2001). There is a variant which consists in introducing b-cyclodextrinemethylated to allow the solubilization of lipophilic antioxidants in aqueous solution by formation of inclusion complexes. The test consists in mixing, directly in the glass tanks, 200 μl of diluted extract or methanol with 2000 μl of FlNa solution. The tanks are then placed in the sample changer with mechanical stirring at 37 ° C. Two hundred µl of AAPH solution is then added to the medium to trigger the generation of free radicals. A fluorescence measurement (/.excitation = 485 nm and /.emission = 520 nm) is carried out every minute for 30 min. Calibration line, and expression of results

 Each sample concentration generates a different kinetic curve. Thus, the areas under the curves of each sample reflecting the reaction kinetics are calculated with the following formula: AUC = 1 + fl / fO + f2 / f0 + ... + f29 / f0 + f30 / f0

 With fO = the initial fluorescence read at 0 min and fi = the fluorescence read at time i

The result is then expressed in net area (AUCnette) which is obtained with the following formula: AUC NET = AUC sample - AUC white

 It is therefore possible to draw a calibration line for the shape from solutions of Trolox® at different concentrations. It has, then, for equation:

 AUC net = a x [Trolox®] + b

 "A" represents the slope and "b" the ordinate at the origin of the linear regression.

 The calculation of the antioxidant activity of an extract of a given concentration is also based on the determination of its net AUC. By plotting this AUCnet on the Trolox® calibration line, the result can finally be expressed in pmol of Trolox® equivalent per gram of dry extract and per gram of dry matter.

Results:

 The ORAC index is expressed in pmol Trolox / g of sample, that is to say the micromole of Trolox equivalent per gram of sample. The larger the number, the more antioxidant the sample.

 The results show that the ORAC values found in date powders (Example 1) or the purified phenolic extracts according to the invention (Examples 2 to 5) are very high compared to the marketed products which served as a reference, in particular a dry extract. grape or pomegranate seeds.

 By comparison between the ORAC values in the different stages of maturation, it is noted that the values are very high at stage 2, which is logical given the high content of polyphenols and specifically in condensed tannins, at this stage of maturation. Indeed, Table 4 indicates that it is in stage 1 of maturation that the polyphenol contents are the highest for all of examples 2 to 5.

In addition, the nuclei have the strongest antioxidant activities, which validates the hypothesis that the antioxidant activity in the purified extracts is proportional to the tannin content that exists in these extracts. The percentages of tannins in the polyphenols of the extracts from the nuclei of Examples 2 to 5 in fact exceed 98%, or even 99%, as shown in Table 5, and the nuclei have the highest ORAC activities (Table 11).

 Table 11

EXAMPLE 8 Transcriptomic Study on DNA Chips of Extracts of Date Polyphenols Applied Systemically in a Culture Medium of Human Keratinocytes.

 Goal :

 The objective was to study the in vitro effects of extracts according to the invention on the expression of genes in human primary keratinocytes NHEKs.

 Preparation:

 The study was carried out on NHEKs (Normal Human Epidermal Keratinocytes) keratinocytes cultivated in a monolayer in Epilife medium containing HKGS (Human Keratinocyte Growth Supplement) and gentamycin.

 The extracts involved in the study were extracts of polyphenols obtained from nuclei (TN), on the one hand, and extracts of polyphenols obtained from consumable skin and pulp (TC) tissues, on the other hand, using a method as described in examples 2 to 5.

 A cytotoxicity study was carried out to determine the optimal dose to apply for the transcriptomic analysis. A concentration of 0.0008% to be applied to the NHEKs keratinocytes has been determined.

 The extracts were applied at this concentration in the culture medium for NHEKs keratinocytes, for 24 h. Gene expression changes have been studied using the DNA microarray technique (GeneChip Human Gene 2.0 ST, Affymetrix).

At the end of the treatments, the populations of total RNAs were extracted. The RNAs were quantified by spectrophotometry to verify that the sample is pure and free from contamination by proteins. Then their integrity was checked by capillary electrophoresis (for triples of each condition; n = 3). Amplification of total RNA was carried out from 50 ng of sample, using Ribo-SPIA technology (Ovation Pico WTA System V2, NuGEN, 3302-12) and the amplified samples were purified using "Agencourt RNA Clean up XP Beads" (Agencourt - Beckam Coulter Genomics, A29168). For each amplified cDNA, the fragmentation and labeling with biotin was carried out from 5 μg with the “NuGEN Encore Biotin” module (NuGEN, 4200-12).

 Hybridization on chips:

 Hybridization was carried out on "GeneChip Human Gene 2.0 ST arrays" (Affymetrix, 902112). Hybridization, washes and labeling were carried out according to the procedure recommended by Affymetrix. The hybridization cocktail was produced using the “Affymetrix Genechip Expression 3 'Amplification Reagant Hybridization Controls” (Affymetrix, 900454) and the “Hybridization Module of GeneChip Hybridization” module, as well as the “Wash and Stain” kit (Affymetrix, 900720), and mixed with the amplified cDNA samples. Hybridization was carried out during 18 hours in the "GeneChip Hybridization Oven 640" (Affymetrix, 800139). The arrays were washed and marked in the “GeneChip Fluidics Station 450” (Affymetrix, 00-0079) and the scan with the “GeneChip Scanner 3000” (Affymetrix).

 Data processing :

 The analysis of the raw data was carried out using the R program (v3.2.3) and the oligo-package (vl.34.2) from the Bioconductor project (v3.2) corresponding to the latest version provided by Affymetrix, built at from version 19 of the human genome (UCSC Human genome 19)), and the RMA method described by Irizarri et al. in order to guide and carry out the pre-treatments as well as the annotations.

 Results:

 The genes have been grouped by themes and by groups according to functional themes. These groups are listed in Table 12 below. The p-value by group is indicated for the TN and TC extracts.

 Groups that are very highly significantly regulated have a p-value <0.001, those that are highly significantly regulated have a p-value such as 0.00l <p-value <0.0l, genes so regulated significant have a p-value such as 0.0l <p-value <0.05.

 Table 12

For the two extracts TC and TN (placed at the same concentration of 0.0008%), many groups are significantly regulated. The p-value calculated for certain groups with the TN extract is lower than with the TC extract, which corresponds to an increase in significance. This is the case, for example, for the group "Wound healing", "Epidermal Biology", "Terminal differentiation and" Epidermal Differentiation Complex ". It appears that the effects obtained with the TN extract are greater than those obtained with the TC extract, at least for certain groups.

 When the analysis is carried out at the level of genes belonging to these groups, it appears that, for the majority of these, the TN extract regulates more genes and more importantly than the TC extract. For example, in the "Wound healing" group, the TC extract significantly increases and decreases 52 and 45 genes, respectively. The TN extract, on the other hand, induces 22 more genes (76 genes) for a similar effect on down-regulated genes (46 genes). A second example concerns the “Epidermal differentiation complex” group (13 up-regulated genes and 4 down-regulated genes with TC; 27 up-regulated genes and no down-regulation with TN).

 This more pronounced effect is also clearly visible from the number of genes that are significantly globally regulated after treatment of the cells with the extracts: 5,253 genes with a p-value <0.05 for the TN extract and 5,220 genes with a p-value < 0.05 for the TC extract.

 In general, numerous variations in gene expression have been observed within keratinocytes. In what follows, the analysis continues in more detail for certain genes representative of particularly interesting groups.

These genes are represented by their symbol with, in brackets, 2 digits, namely 1 efold change and the p-value: ex. SYMB (Fc - p-value). Differentiation group of the epidermis and corneal envelope

 Differentiation and barrier function of the epidermis are mechanisms directly involved in the homeostasis of epidermal tissue. The skin is an organ that sits at the interface between the body and the environment and offers a physical barrier against damage such as chemicals, pathogens, UV light or even dehydration. This protective function depends largely on the horny envelope, formed from the differentiation of keratinocytes. This differentiation process is complex and involves the sequential expression of many genes.

 Some of the genes whose expression is induced by the TN extract are presented below.

 Thus the TN extract induces the gene expression of members of the EDC (epidermal differentiation complex) such as SPRRlA (4.5-1.8E-12), SPRRlB (7.8-5.4lE- 09), SPRR2C (l.5 - 0.076-NS), SPRR2E (l.5-0.07l-NS), SPRR2F (l.38-0.039),

SPRR2G (l.96-0.003), SPRR3 (l.9-2.34-05), SPRR4 (2.0- 0.00061), Sl00A8 (4.6- 1.36E-08), Sl00A9 (3.02- 0.00025), Sl00Al0 (l.2- 0.017), Sl00Al2 (2.32- 0.00013), LCE1A (2.5-0.03), LCE1B (1.8- 0.03), LCE2C (2.5-0.00l6), LCE3B (1.4- 0.033), LCE3D (2.5- 0.004), LCE3E (2.03- 0.026), LCE6A (l.7- 0.023), as well as genes coding for involucrine (IVL (5.7- 7.31E-11), filaggrin (FLG (1.8- 0.00082)), filaggrin 2 (FLG2 (2.1 - 8.8E-5)), caspase-14 (CASP14 (8.8-1.3E-10)), matriptase (ST14 (1.5- 0.002)), peptidoglycan recognition protein 4

(PGLYRP) (4l.6- 0.0006)), trichohyalin like 1 (TCHHLl) (l.2- 0.04)), KPRP (2.82- 0.00013), periplakin (PPL) (l.85-5.67E-5 )), transglutaminase 1 (TGM1) (2.8- 6.3E-9)), keratins 1 (KRT1) (5.3-2.3E-6)), 2 (KRT2) (1.8-0.017)), 4 (KRT4) (1.4-0.022)), 5 (KRT5) (1.2-0.41), 6A (KRT6A (1.5- 0.006)), 10 (KRT10) (12.4- 7.46E-10)), 14 (KRT14) (1.3- 0.0012 )) and 15 (KRT15) (2.4-8.3E-7)) and the genes DSG1 (19.7-1.9E-16), DSG3 (2.1- 3.3E-8), DSG4 (1.2- 0.07), DSC1 (15.4- 1.4E-14), DSC2 (2.7- 7.3E-10), DSC3 (1.7-1.3E-5) and DSP (2.4- 8.3E-9). The genes coding for key transcription factors such as KLF4 (1.9-4.1E-5), DLX3 (3.l-2.77-6), GRHL3 (4.5-3.6E-7), ARNT (1.2-0.03), TP63 (L4- 0.002), and TP73 (l.5- 0.0011) are also remarkably induced by the TN extract, which demonstrates that it acts on the different actors involved in the differentiation of the epidermis, and this, upstream in the regulatory process. It is important to note that the DSG1 and DSC1 genes are remarkably regulated by the TN extract, with increases of more than 15 times.

Some of the genes whose expression is induced by the TC extract are presented below. The TC extract also induces the gene expression of EDC components such as SPRRlA (2.4- 6.4E-9), SPRR2C (2.2-0.002), SPRRlB (l.3-0.02), SPRR3 (l.6- 0.00l), Sl00A8 (l.5- 0.01), Sl00P (2.4- 6.4E-9), LCE1A (2.2- 0.059 - NS), LCElC (3.5- 3.4E- 8), LCE2B (3.2-1.8E-10 ), LCE1F (2.7-0.0014), LCE1B (1.6-0.07 -NS), LCE3B (1.4- 0.05), as well as genes encoding involucrine (IVL) (2.2- 5.7E-06), filaggrin (FLG ) (1.5-0.006)), filaggrin 2 (FLG2) (1.5- 0.02)), loricrin (LOR) (2.8-0.006)), KPRP (1.7-0.02), periplakin (PPL) (1.4-0.02) )), transglutaminase 1 (TGM1) (1.3-0.008)), keratins 1 (KRT1) (2.2- 0.004)) and 10 (KRT10) (2.3-3, 24E- 05) and the DSG1 genes (3.3-1.8 E-10), DSG3 (1.2- 0.048), DSG4 (1.4- 0.005), DSC1 (2.4- 1.6E-7), DSC2 (1.2-0.013) and DSP (1.5- 4.6E-5).

 These regulations by TC and TN extracts indicate a strengthening of the corneal envelope and therefore of the barrier function of the epidermis.

Anti-microbial defense group

 Anti-microbial defenses participate in the barrier function of the epidermis and intervene on multiple levels. Defense peptides and proteins exhibit broad spectrum destruction activity against bacteria, viruses, fungi and several parasites. They will therefore intervene, for example, following a physical injury, during the restoration of the skin barrier and to maintain or restore homeostasis, but also to maintain the skin microbiome.

 The TC extract stimulates, in particular, the expression of several genes involved in antimicrobial defenses including the main peptides, defensins beta 1 (DEFBl) (l.6 - 0.014)) and beta 4A (DEFB4A) (l.4 - 0.035)) and the genes (DEFBl0) (l.4 - 0.0033)), (DEFB5A) (l.3 - 0.07)), (DEFBl28) (l.3 - 0.034)), (DEFBl27) (l.3 - 0.034)).

 The TN extract stimulates, in particular, the expression of defensin beta 1 (DEFBl) (2.l - 9.E-5)) the gene (DEFBl25) (l.3 - 0.045)).

 TN and TC extracts can improve and strengthen the antimicrobial arsenal, cell defense and barrier function against environmental aggressions and pathogens.

 Flaking group

 Exfoliation gradually removes the surface layers of the epidermis in the form of small strips.

For the TN and TC extracts the highly significant data on the induction of key genes involved in the formation of the corneal envelope and members of the epidermal differentiation complex are interestingly supplemented by an increase in the expression of genes involved in the flaking. There is, for the TC extract, an increase in the expression of the SLPI gene (1.4-0.03) and of the genes coding for cathepsin V (CTSV) (2.1-1.5E-05)), peptidases 5 and 7 linked to the kallikrein protease (KLK5) (1.3- 0.04) and KLK7 (1.7-5.7E-05)), the Kazal serine protease type 5 (SPINK5) (2.1- 6.7E-05)), also called LEKTI for Lympho-epithelial Kazal-type-related inhibitor), corneodesmosine (CDSN) (1.5- 0.005)), cystatin M / E (CTS6) (1.9-0.001)) and corneodesmosine (CDSN) (1.5- 0.005)) .

 There is, for the TN extract, an increase in the expression of the SLPI gene (3.0, 1.5E-6) and of the genes coding for cathepsin V (CTSV) (5.1-4.5E-10)), peptidases 5 and 7 linked to kallikrein protease (KLK5) (2.8-2.5E-7) and KLK7 (3.6- 9.9E-10)), Kazal type 5 serine protease (SPINK5) (2.8-1.03E-06), corneodesmosine (CDSN) (2.85-5.7E-7)), cystatin M / E (CTS6) (3.3-1.01E-6)) and cathepsin D (CTSD) (2.4-2.5E-5)).

 These inductions of genes coding, among others, for proteases indicate a controlled activation of the desquamation process by the TN and TC extracts. A balance between the strengthening of the corneal envelope and the desquamation promotes epidermal renewal.

 TN and TC extracts thus promote epidermal differentiation, strengthen the barrier function, while ensuring good desquamation. They bring a significant benefit on the protection of the first layer of the skin against the environment and dehydration and promote epidermal renewal in order to remove debris, damage or dead cells. This notably helps to counter environmental pollution and revive the radiance of the complexion.

Healing group:

 Many genes involved in the complex process of wound healing have their expression modulated by TN and TC extracts.

 Genes of the inflammatory phase:

 The TN extract makes it possible to induce the expression of the gene coding for MMP9 (3.6- 6.8E-10), of the gene coding for phospholipase A2 group X (PLA2G10) (5.3-1.5E-07).

The TC extract also makes it possible to induce the expression of the gene coding for MMP9 (1.53 - 4E-04) and PLA2G10 (2.6- 8.4E-05). It also induces the gene coding for MMR2 (1.3- 0.04).

The two extracts therefore have a beneficial effect on the initiation of several processes associated with wound healing such as cell migration and remodeling of CEM. Genes of the granulation phase and the proliferative phase:

 The TC extract down-regulates many genes playing an essential role in angiogenesis, such as CXCLl (-l.5- 0.014) and CXCL5 (-L8- 4.3E-05) (CXC motif chemokine ligand 1 and 5), ILlB (-l.9- 0.002) (C interleukin 1 beta), CSF3 (-l.5- 0.0083) (GCSF - colony stimulating factor 3), CTGF (-l.3- 0.022) (connective tissue growth factor), TGFA (-l.3- 0.04) (transforming growth factor alpha), TGFBl (-l.4- 0.033) (transforming growth factor betal), VEGFA (-l.6l- 0.0015) (vascular endothelial growth factor A).

 The TC extract overexpresses the ACTA2 gene (l.6- 0.028) (“actin alpha 2 smooth muscle”), the MUH9 (1.5-0.0005), MUH10 (1.6, 0.0011) genes, the ENAH genes (l.4-0.00 l), SVIL (1.4-0.002), WASFl (l.4- 0.004), RHOB (l.3- 0.006), MYL9 (l.3- 0.01), ROCKl (l.3-O.Ol), RDN3 ( l.2-0.04), EZR (l.2- 0.03), EGF ("epidermal growth factor") (1.3- 0.017) and FGF2 ("fibroblast growth factor 2") (1.5- 0.005).

 The TN extract down-regulate many genes playing an essential role in angiogenesis, such as CXCLl (-l.6-0.005) and CXCL5 (-2.9-2.6E-08) (CXC motif chemokine ligand 1 and 5), ILlB (-3.3- 7.3E-6) (C interleukin 1 beta), CTGF (-l.8- 3.3E-5) (connective tissue growth factor), TGFA (-l.7- 0.00017) (transforming growth factor alpha), VEGFA (-l.6-0.00l2) (vascular endothelial growth factor A).

 The extract TC overexpresses the genes ENAH (l.7-l.8E-5), SVIL (L6- 7.6E-5), RHOB (1.4- 0.003), MYL9 (L7- 3.7E-5), EZR (l .3- 0.01) and SERPINFl (l.4- 0.003) (serpine- 1).

 Due to the induction of the expression of genes involved in re-epithelialization or serpin-1, combined with a negative regulatory effect of the extracts on key genes controlling angiogenesis, the extracts promote the initiation of biological mechanisms associated with wound healing, mainly at the granulation phase, such as cell migration and proliferation, re-epithelialization and remodeling of the ECM while avoiding exacerbated neo-vascularization which would cause undesirable redness.

Maturation and tissue remodeling phase

TN and TC extracts induce a significant overexpression of the gene coding for cathepsin K, a cysteine protease possessing collagenase and elastase activities involved in the renewal and homeostasis of the extracellular matrix (ECM). The values are as follows: CTSK (l.5- 0.02) for TN and CTSK (l.4- 0.04) for TC. Applied in a healing process, TN and TC extracts can therefore facilitate the reshaping of the extra cellular matrix and prevent the appearance of prominent and unsightly scars. Chemokines

 The TN extract allows the induction of genes coding for CXCL10 (l.8-0.0008), a "chemokine C-X-C motif chemokine ligand" involved in the proliferation and maturation phase during healing. On the other hand, the genes coding for CXCLl (- 1.6 - 0.005) CXCL5 (-3 - 2.6E-08), whose proteins are involved in the 1st phase of inflammation, are downregulated. Furthermore, a gene remarkably overexpressed by the TN extract is CXCL14 (32 .1 - 8.1E -15).

 The TC extract allows the induction of the genes coding for CXCL8, CXCL10 and CXCL11, "C-X-C chemokine motif chemokine ligand" involved in the proliferation and maturation phase (CXCL10 and CXCL11) during healing. On the other hand, the genes coding for CXCLl (-l.5- 0.01), CXCL5 (-l.8- 4.3 E-05), whose proteins are involved in the 1st phase of inflammation, are negatively regulated by this TC extract . The TC extract also remarkably overexpresses the CXCL14 gene (20.9 - 6.10E-14).

TC and TN extracts will therefore promote the different key phases of wound healing, while modulating angiogenesis and limiting inflammation, in order to limit redness in particular.

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Claims

1. Extract of polyphenols from the fruit of the date palm Phoenix dactylifera for the preparation of a nutraceutical composition.

2. Polyphenol extract according to claim 1, characterized in that it contains by weight relative to the total weight of the dry purified extract, up to 80% of polyphenols.

3. Polyphenol extract according to claim 1 or 2, characterized in that it contains at least 95%, more preferably at least 97%, even more preferably at least 99% of condensed tannins, by weight relative to the total weight of polyphenols.

4. Polyphenol extract according to claim 3, characterized in that the tannins have an average degree of polymerization of less than 30, preferably less than 27, more preferably less than 20.

5. Extract according to one of claims 1 to 4, characterized in that it is obtained from the stone, the skin and / or the pulp of the fruit.

6. Extract of polyphenols from the fruit of the date palm Phoenix dactylifera for its use in the maintenance of eye health, vision, cardiovascular health, functioning of the immune system, blood circulation, brain activity, slowing down of brain aging, prevention and care of joint pain or inflammation-related pain, proper functioning of the digestive system, especially of the intestinal flora, prevention or treatment of metabolic syndrome, disease prevention chronic, its use against stress, maintaining health and appearance of the skin, tissue healing.

7. Extract according to claim 1 to 5 or 6, characterized in that it is obtained using a process comprising the steps:

 obtaining and preparing the plant material,

 solid-liquid extraction using a polar solvent and recovery of an extract containing polyphenolic compounds,

concentration of polyphenolic compounds in the extract, purifying the polyphenolic compounds of the extract by a solid phase chromatography method,

 recovery of a purified extract concentrated in polyphenolic compounds.

8. Nutraceutical composition comprising an extract of polyphenols from the fruit of the date palm Phoenix dactylifera.

9. nutraceutical composition according to claim 8, characterized in that the extract contains by weight relative to the total weight of the dry purified extract, up to 80% of polyphenols.

10. nutraceutical composition according to claim 8 or 9, characterized in that the extract contains at least 95%, more preferably at least 97%, even more preferably at least 99% of tannins, by weight relative to the total weight of polyphenols .

11. nutraceutical composition according to claim 10, characterized in that the tannins have an average degree of polymerization less than 30, preferably less than 27, more preferably less than 20.

12. nutraceutical composition according to one of claims 8 to 11, characterized in that it comprises at least 0.01% of said polyphenol extract.

13. nutraceutical composition according to one of claims 8 to 12, characterized in that it is in the form of a composition for enteral administration, such as a food, a drink, a capsule, a capsule, a tablet, powder or liquid solution.

14. nutraceutical composition as defined in one of claims 8 to 11, for its use in maintaining eye health, vision, cardiovascular health, functioning of the immune system, circulation blood, brain activity, slowing brain aging, prevention and care of joint pain or inflammation, the proper functioning of the digestive system, including intestinal flora, prevention or treatment of metabolic syndrome , prevention of chronic diseases, its use against stress, maintenance of health and appearance of the skin.