Extract of the Cybele variety of Punica granata, and its use in cosmetics.

Abstract

The present invention relates to the cosmetic and / or dermatological use of an extract of the cubere variety of Punica granata, or a composition containing said extract.

Description

Technical Field

[0001] The present invention relates to an extract of the Cybele variety (var. Cybele) of Punica granatum, and a composition containing the extract. The present invention also relates to the cosmetic and / or dermatological use of the extract or the composition for skin care, particularly for combating skin aging.

Background Art

[0002] Pomegranate plants belonging to the species Punica granatum are shrubs with an average height of 3 to 6 m and are classified in the family Lythraceae.

[0003] Its geographical range is very wide. Pomegranate plants are native to countries ranging from Turkey to the Caucasus and from East Turkmenistan to Iran, Afghanistan and Pakistan. Pomegranate plants, particularly their fruits, have been cultivated for centuries throughout the Mediterranean and the Middle East.

[0004] Pomegranates are spherical berries with a hard outer skin that, when ripe (i.e., from September to October), varies from yellow to orange-red or even from purple-brown to black depending on the variety. The fruit bears the remains of the flower calyx, forming a serrated crown-like shape that gives it a unique and easily identifiable shape. Pomegranates consist of 6 to 12 whitish membranous compartments with a bitter taste, which contain many triangular seeds called arils. These seeds have a fleshy and gelatinous mesocarp, which is usually sweet and sour and is the edible part of the fruit.

[0005] There are over 1200 different varieties of pomegranate plants worldwide. They are particularly distinguished by, among other things, the acidity and diameter of the fruit, the color of the outer skin, the number of seeds and the ripening rate. For example, mention may be made of Wonderful, a red pomegranate from Israel, which is famous for its sweet fruit. Some varieties of pomegranate plants are established in the warm winter regions of central Europe, particularly in France, and are particularly found in the southern half of the country.

[0006] The phytochemical composition of the red-fruited variety *Punica granata* is well documented, allowing for the emphasis on dominant families such as hydroxycinnamic acid, and more specifically, caffeic acid, chlorogenic acid, ferulic acid, quinic acid, and p-coumaric acid, which are present in significant amounts in the fruit peel; flavonoids of the flavonol type (luteolin, quercetin, apigenin, naringenin, and isorhamnetin glycosides), which are also abundant in the peel; or hydrolyzable tannins of the ellagitanin type (punicalagin) and triterpene sapogenin (ursolic acid), which are present in the seeds, peel, and leaves.

[0007] Considering their specific and well-known phytochemical compositions, the red-fruited varieties of Punica granata are suitable for use in cosmetic formulations or nutritional supplements.

[0008] While fruit is the most commonly used source of extract, specific parts of the fruit, namely the peel or seeds, are primarily utilized, requiring additional separation steps that directly impact the extraction cost.

[0009] For example, references FR3031901, WO2021 / 064034, or WO2021 / 209593 describe, in particular, extracts of red berries of Punica granata for the production of oral compositions or nutritional supplements, sunscreens, or for combating acne, and their cosmetic use. It should be noted that sebaceous gland cells contribute to the pathophysiology of acne through their role in sebum production. In fact, (i) seborrhea appears to worsen the quality of sebum, particularly due to vitamin E deficiency. Components of sebum, more specifically squalene, are oxidized. Oxidized lipids are known for their pro-inflammatory and cytotoxic properties, and (ii) hyperseborrheic dermatitis is excessive sebum production or excess sebum that causes skin problems such as acne. In particular, hyperseborrheic dermatitis contributes to the local inflammatory response by contributing to the proliferation of Propionibacterium acnes. The extracts described in the aforementioned references FR3031901, WO2021 / 064034, or WO2021 / 209593 require concentration at different stages of specific phytochemical families, or even purification, to increase their efficacy. Therefore, these processes necessitate additional production steps that directly impact energy consumption and thus the overall production cost.

[0010] Some varieties, intended for ornamental purposes, produce smaller, generally more acidic and bitter fruits, making them unsuitable for consumption.

[0011] The Cuvée variety of purple pomegranate, or the Cuvée variety of Punica granata, originates from one of these varieties that is unsuitable for consumption. The skin of this Cuvée variety is a rare purplish, almost black color. However, unlike other varieties called black, such as Asmar or Nera del Salento, which have sweet fruit containing a red aril, the fruit of the Cuvée variety has a white to very pale pink aril and is bitter and extremely acidic, making them unsuitable for consumption.

[0012] This Cybele variety has not been the subject of any scientific publications, particularly regarding its genetic and phytochemical characteristics.

[0013] The Cybele variety of Punica granata is an ornamental variety, but it also has agricultural advantages, being effective in repelling pests and allowing cultivation without the use of insecticides. This is a major advantage for biodynamic or co-culture production.

[0014] The pomegranate (Punica granatum) has a long and rich history of traditional and ethnic uses, dating back to ancient Egypt. While pomegranates are particularly described in traditional Chinese and Ayurvedic medicine, they are also found in Persian and Greek-Arabian medical systems.

[0015] All parts of the pomegranate plant are recognized for their anthelmintic properties, particularly their effectiveness against tapeworms, and for their therapeutic properties in treating oral diseases, stomach aches, and ulcers, such as treating inflammation, bleeding gums, or removing plaque. A few uses, mentioned very rarely, refer to skin conditions. In fact, traditional Chinese medicine describes the fruit as useful for treating boils or bedsores.

[0016] Studies based on different cell or tissue models confirm the biological properties of different parts of the pomegranate plant. Examples include antioxidant properties, primarily provided by their polyphenolic compound (flavonoid, tannin, anthocyanin) content; anti-inflammatory, antibacterial, anti-acne, wound-healing, and anti-aging properties; or depigmentation activity.

[0017] There are many cosmetics, especially for skincare, that contain pomegranate extract. However, these extracts primarily originate from pomegranate varieties that have edible fruit, particularly due to the abundance of these red varieties and their specific and well-described phytochemical composition, such as the Wonderful variety of Punica granata. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] FR3031901 [Patent Document 2] WO2021 / 064034 [Patent Document 3] WO2021 / 209593 [Patent Document 4] FR3036618 [Overview of the project] [Problems that the invention aims to solve]

[0019] Therefore, there is a clear need for an extract of Punica granata that is at least equivalent to, or even better than, the Punica granata extract of prior art, in terms of its cosmetic properties, particularly for effectively combating skin aging. This extract is free from both toxicity and irritation, effective for cosmetic skincare, and is cheaper to produce than the production process of prior art, with lower energy intensity and water consumption (i.e., an eco-design approach). This extract, especially as a substitute for existing extracts, is clearly needed. [Means for solving the problem]

[0020] To our surprise and delight, the applicant was able to identify a specific variety of Punica granatatum that is perfectly suitable for cosmetic and / or dermatological use on human skin and that meets the needs detailed above.

[0021] In particular, the applicant has identified the species *Punica granata* 'Cybele', whose fruit is not edible and, as mentioned above, is an effective substitute for *Punica granata* fruit extract, especially that derived from edible varieties such as *Punica granata* 'Wonderful'.

[0022] The applicant has carried out variety comparison studies, which have made it possible to demonstrate the genetic diversity between the Wonderful variety of Punica granatum and the Cubery variety of Punica granatum (see Example 1).

[0023] The phytochemical composition of the Cubery variety of purple pomegranate is different from that of other red fruit varieties of Punica granatum, such as the Wonderful variety of Punica granatum. Indeed, the applicant has carried out phytochemical composition comparison studies, which have made it possible to demonstrate the phytochemical diversity between the fruits of the Wonderful variety of Punica granatum and the Cubery variety of Punica granatum (see Example 4).

[0024] Therefore, the first subject of the present invention relates to the cosmetic, i.e., non-therapeutic, and topical use of an extract of the Cubery variety of Punica granatum or of a composition comprising said extract.

[0025] The present invention proposes various advantages, particularly the provision of specific biological properties related to the activity of the extract of the present invention, i.e., antioxidant, anti-inflammatory, anti-aging, antibacterial, wound healing, depigmenting, sebum regulating or anti-acne cosmetic activity. Furthermore, the use of the Cubery variety of Punica granatum makes it possible not to use varieties of Punica granatum that produce edible fruits, such as the Wonderful variety of Punica granatum.

[0026] In the context of the present invention, the expression "whole fruit" denotes an aggregate consisting of the pericarp (outer skin), the remaining calyx, the membranous locules, the pulp and the seeds (arils).

[0027] In the context of the present invention, the expression "extraction solvent" denotes the solvent used during the solid-liquid extraction process.

[0028] Advantageously, the cosmetic use of an extract of the Cubery variety of Punica granatum according to the present invention or of a composition comprising said extract has, individually or in combination, the following additional technical characteristics: - Its use as a cosmetic is to combat skin aging. - Its use as a cosmetic is to stimulate the radiance of the complexion. - Its use as a cosmetic is to soothe the skin. - Its use as a cosmetic is to combat skin sagging. - For cosmetic use, the purpose is to counteract the appearance of wrinkles and / or fine lines. - For cosmetic use, it is intended to combat wrinkles and / or fine lines. - Its use in cosmetics is as an antioxidant. - Its use in cosmetics is as an anti-inflammatory agent. - For cosmetic use, it is used as a sebum regulating factor / sebum regulator / a substance that improves the sebum regulation of the skin. - Its use in cosmetics is to reduce the size of skin pores. - Cosmetic use is for healthy skin and / or acne-prone skin, i.e., for subjects with healthy skin. For the purposes of this invention, acne-prone skin should be distinguished from acne-affected skin (i.e., skin with recognized inflammation). Acne-prone skin is generally oily due to excessive sebum secretion, but is healthy skin that does not exhibit inflammation and / or excessive proliferation of commensalism or pathogenic bacterial populations. - For cosmetic use, use is intended for healthy skin and / or oily skin (skin with a shiny, lustrous appearance) and / or combination skin and / or skin with non-inflammatory defects (e.g., blackheads, acne, comedones). For the purposes of the present invention, oily or combination skin and / or skin with non-inflammatory defects is not diseased skin. - For cosmetic use, it is intended for oily hair. - An extract of the Cybele variety of Punica granata can be obtained by a solid-liquid extraction process using an extraction solvent, followed by a second step of solid-liquid separation, and finally a third step of recovering the liquid phase. - The extraction solvent (or extraction solvent) used in the solid-liquid extraction process is selected from the following group of substances and used alone or in mixtures: water; glycols such as 1,3-propanediol, 1,2-propanediol, and 1,3-butanediol; polyols such as glycerol; alcohols such as ethanol; and natural deep eutectic solvents (NADES) such as those described in FR3036618, particularly fructose / glycerol / water NADES (indicated as NADES FGE115) in a molar ratio of 1:1:5; advantageously, a mixture of propanediol and water; a mixture of ethanol and water; and a mixture of fructose, glycerol and water; preferably a mixture of propanediol and water. - The mass ratio of the propanediol and water mixture is between 10 / 90 and 90 / 10, and preferably 60 / 40. - The mass ratio of the ethanol and water mixture is between 10 / 90 and 90 / 10, and preferably 60 / 40. - The mass ratio of the fructose, glycerol, and water mixture during the extraction process is between 55 / 28 / 17 and 45 / 23 / 32, preferably 50 / 25 / 25. - The mass ratio of crushed Punica granata cuvelet variety / solvent is between 1:99 and 50:50, preferably between 2:98 and 15:85. - The extract of the Punica granata 'Cuvelet' variety used in accordance with the present invention is an extract obtained from mature or unripe fruit, preferably from mature fruit. - The extract of the Punica granata 'Cuvelet' variety used in accordance with the present invention is an extract obtained from the whole fruit, the fruit peel, the fruit seeds, the bark, the roots, the flowers, the petioles, and / or the leaves, and is advantageously a whole fruit extract. - The plant parts of the Punica granata 'Cuvelet' variety used to obtain the extract used in the present invention are fresh, frozen, dried, whole, chopped and / or crushed. - The extract of the Cybele variety of Punica granata used in accordance with the present invention has a phytochemical composition / total polyphenol content of 0.5 g / 100 g or more, preferably between 0.8 and 1.0 g / 100 g. - The extract of the Cybele variety of Punica granata used in accordance with the present invention has a phytochemical composition / α- and β-punicalagin content of 0.3 g / 100 g or more, preferably between 0.4 and 0.6 g / 100 g. - The extract of the cuvelet variety of Punica granata used in accordance with the present invention has a phytochemical composition / ellagic acid content of 30 mg / kg or more, preferably between 100 and 150 mg / kg. - The extract of the Cybele variety of Punica granata used in accordance with the present invention has a phytochemical composition / total anthocyanin content of 3 mg / kg or more, preferably between 4 and 9 mg / kg. - The extract of the Cybele variety of Punica granata used in accordance with the present invention has a phytochemical composition / myrtilin content of 1 mg / kg or more, preferably between 2 and 4 mg / kg. - The extract of the Cybele variety of Punica granata used in accordance with the present invention has a phytochemical composition / chromatin content of 1 mg / kg or more, preferably between 1.5 and 3 mg / kg. - The extract of the Cuvelet variety of Punica granata used in accordance with the present invention has a phytochemical composition / anthocyanin content other than kuromarin and myrtilin of 0.5 mg / kg or more, preferably between 1.0 and 2.5 mg / kg.

[0029] Another subject of the present invention relates to an extract of the Cuvelet variety of Punica granata, which can be obtained by a solid-liquid extraction process from ground Punica granata 'Cuvelet' using an extraction solvent, followed by a second step of solid-liquid separation and finally a third step of recovering the liquid phase, characterized in that the extraction solvent is selected from the group consisting of a mixture of propanediol and water, a mixture of ethanol and water, and a mixture of fructose, glycerol and water.

[0030] Advantageously, the extracts of the Punica granata 'Cuvée' variety of the present invention, individually or in combination, have the following additional technical features: - The extraction solvent according to the present invention is liquid at room temperature, which facilitates the implementation of the process leading to the extract of the present invention. - Solid-liquid extraction can be carried out by various techniques known to those skilled in the art in the field, for example, maceration, re-maceration, hot maceration, dynamic maceration, decoction, fluid-bed extraction, microwave-assisted extraction, ultrasonic-assisted extraction, countercurrent extraction, leaching, re-leaching, washing, extraction under reduced pressure, and diacorization. - The mass ratio of plant portion / extraction solvent for the Punica granata 'Cuvelet' variety applied to the extraction process is between 1:99 and 50:50, preferably between 2:98 and 15:85. - The extraction process is carried out at a temperature between 2 and 100°C, preferably between 20 and 80°C. - The extraction process can be maintained for several minutes to several days. - The solid-liquid extraction process is carried out with stirring and / or under a nitrogen atmosphere to optimize the extraction of active compounds while protecting them from oxidation by oxygen in the atmosphere. - In order to recover the liquid phase (or solid-liquid separation filtrate) containing the active material, a solid-liquid separation step is performed following a solid-liquid extraction step. - The separation process may be carried out by any technique known to those skilled in the art in question, particularly by dehydration, pressing, rotation, centrifugal separation, or filtration. - A concentration step is performed following the liquid-solid separation step, making it possible to obtain a concentrate in liquid or semi-solid form depending on the concentration factor. - The concentration process is carried out by evaporation or reverse osmosis under reduced pressure. - The solid-liquid separation filtrate or concentrate undergoes one or more clarification steps. - The clarification process implements any kind of vertical and / or tangential filtration known to those skilled in the art in the subject matter. - A fractionation step is performed following a liquid-solid separation or concentration or clarification step to obtain fractions of one or more phytochemical families in liquid or semi-solid form depending on the concentration factor. - The separation process may be carried out by any technique known to those skilled in the art in question, in particular by chromatography under reduced pressure, ultrafiltration or nanofiltration, and / or - The method of the present invention may include a step of sterilization filtration. - The sterilization filtration step is performed by filtering the product through a filter containing pores with a diameter of 0.22 μm.

[0031] Another subject of the present invention relates to a composition comprising an extract of the Cuvelet variety of Punica granata as described above.

[0032] Advantageously, compositions according to the present invention are cosmetic and / or dermatological compositions.

[0033] Advantageously, the compositions of the present invention, individually or in combination, have the following additional technical features: - The extract of the Punica granata 'Cuvelet' variety according to the present invention shall constitute between 0.1% and 20% by mass of the composition, preferably between 0.5% and 5% by mass. - The composition of the present invention is a skincare composition, particularly a composition for combating skin aging. - The composition of the present invention is an anti-aging composition. - The composition of the present invention is a composition that improves the regulation of sebum in the skin. - The compositions of the present invention may be in any of the following forms commonly used for rinse-off or non-rinse topical application, for example, in an anhydrous form, such as an oil-in-water emulsion, water-in-oil emulsion, multiple emulsion, silicone emulsion, microemulsion, nanoemulsion, gel, aqueous solution, or water-alcohol solution. - The compositions of the present invention may be fluid to varying degrees and may be in the form of white or colored creams, ointments, milks, lotions, serums, gels, cleansing bases, or sticks. They may be used in a variety of treatments for the skin, lips, and hair, including the scalp, particularly for protecting and / or caring for the skin, lips, and / or hair, and / or for makeup application to the skin and / or lips. - The compositions of the present invention may contain additives commonly used in the fields of cosmetics and dermatology, such as fats, detergents and / or conditioning surfactants, emulsifiers and co-emulsifiers, hydrophilic or lipophilic gelling agents, preservatives, antioxidants, solvents, exfoliants, fragrances, fillers, hydrophilic and lipophilic shielding agents, dyes, acidic or basic neutralizing agents, penetration enhancers, and polymers. These types of additives are well known to those skilled in the art. - The amounts of the various additives contained in the composition of the present invention are known to those skilled in the art in the subject, and the sum of the amounts of the additives represents 0.01% to 99.99% of the total mass of the composition. Suitable fats include mineral oil, animal-derived oils such as lanolin, vegetable oils, synthetic oils such as isopropyl myristate, octyldodecanol, isostearyl isostearate, decyl oleate, and isopropyl palmitate, and silicone oils such as cyclomethicone and dimethicone. Fatty materials such as aliphatic alcohols, fatty acids, waxes, and gums, and especially silicone elastomers, can also be used. - Suitable detergent and / or conditioning surfactants include nonionic, anionic, cationic, or amphoteric surfactants, and mixtures thereof, such as alkyl sulfates, alkyl ether sulfates such as sodium lauryl ether sulfate, alkyl betaines such as cocamidopropyl betaine, or quaternary ammonium salts. Suitable emulsifiers and co-emulsifiers include, for example, polyglycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, oxyethylene-sorbitan (sprbitan) fatty acid esters, aliphatic alcohol PEG ethers, glycerol fatty acid esters, alkyl sulfates, alkyl ether sulfates, alkyl phosphates, alkyl polyglucosides, alkyl polypentosides, and dimethicone copolyols. Suitable hydrophilic gelling agents include, for example, carboxyvinyl polymers, acrylic copolymers (carbomers) such as acrylate / alkyl acrylate copolymers, polysaccharides such as polyacrylamide, xanthan gum, and guar gum, natural gums such as cellulose gum and its derivatives, starch and its derivatives, clay, and 2-acrylamido-2-methylpropanoic acid copolymer. Suitable lipophilic gelling agents include, for example, modified clays such as bentonite, metal salts of fatty acids, hydrophobic silica, ethylcellulose, dextrose esters, hydrogenated castor oil and its derivatives, polyurethanes, and magnesium salts. Suitable preservatives include, for example, benzoic acid, sorbic acid, propionic acid, salicylic acid, and dehydroacetic acid and their salts, benzyl alcohol, ethylhexylglycerol, parabens, their salts and esters, triclosan, imidazolidinyl urea, phenoxyethanol, DMDM ​​hydantoin, diazolidinyl urea, and chlorphenesin. Suitable antioxidants include, for example, chelating agents such as EDTA and its salts, sodium metabisulfite, salicylic acid, ascorbic acid and citric acid and their salts, sodium tartrate, sodium gluconate, carotenoids, and tocopherol. - Suitable solvents (other than extraction solvents) that can be used in the composition include, for example, water, ethanol, glycerol, propylene glycol, propanediol, butylene glycol, and sorbitol. - Suitable exfoliating agents according to the present invention include, for example, chemical exfoliating agents such as AHA, and physical exfoliating agents such as natural or synthetic powders. - Suitable fillers according to the present invention include, for example, organic powders such as talc, kaolin, mica, serecite, magnesium carbonate, aluminum silicate, magnesium silicate, and nylon. Suitable dyes according to the present invention include, for example, lipophilic dyes, hydrophilic dyes, pigments, and pearlescent agents commonly used in cosmetic or dermatological compositions, as well as mixtures thereof. Suitable neutralizing agents include basic agents such as sodium hydroxide, triethanolamine, aminomethylpropanol, and potassium hydroxide, and acidic agents such as citric acid and lactic acid. Suitable pro-penetrants include, for example, alcohols and glycols (ethanol, propylene glycol), ethoxydiglycol, alcohols and fatty acids (oleic acid), fatty acid esters, and dimethyl isosorbide. - The composition of the present invention may also further contain one or more activators other than the extract of the Cuvelet variety of Punica granata according to the present invention. - Suitable activators according to the present invention are selected from anti-free radicals or more generally antioxidants, whitening agents, colorants, emollients, moisturizers, anti-seborrheic agents, anti-inflammatory agents, anti-acne agents, keratolytic and / or desquamating agents, anti-wrinkle and astringent agents, dehydrating agents, anti-irritants, sedatives, vitamins and mixtures thereof, mattifying agents, anti-aging activators such as retinol, wound healing agents, preservatives, and essential oils.

[0034] Another subject of the present invention relates to an extract of the cuveré variety of Punica granata as described above, or a composition containing said extract, for use as a pharmaceutical, advantageously as a pharmaceutical for dermatological use, and preferably as a topical pharmaceutical for dermatological use.

[0035] Another subject of the present invention relates to an extract of the cuveré variety of Punica granata, or a composition containing said extract, for dermatological use in the prevention and treatment of inflammatory conditions.

[0036] Another subject of the present invention relates to an extract of the cuvelet variety of Punica granata as described above, or a composition containing said extract, for use as an antioxidant.

[0037] Another subject of the present invention relates to an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for use as an anti-inflammatory agent.

[0038] Another subject of the present invention relates to an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for use as an antimicrobial agent.

[0039] Another subject of the present invention relates to an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for use as an anti-acne agent.

[0040] Another subject of the present invention relates to the use, advantageously cosmetic and / or non-therapeutic use, of an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, as a sebum regulator for skin (or for regulating sebum production in the skin), advantageously for acne-prone skin and / or oily skin and / or combination skin and / or skin with non-inflammatory defects.

[0041] Another subject of the present invention relates to the use, advantageously cosmetic and / or non-therapeutic use, of an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for preventing and / or reducing excessive seborrhea of ​​the skin, preferably of acne-prone skin.

[0042] Another subject of the present invention relates to the use, advantageously cosmetic and / or non-therapeutic use, of an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for improving and / or correcting the size of skin pores, advantageously for acne-prone skin and / or oily skin and / or combination skin and / or skin with non-inflammatory defects.

[0043] Another subject of the present invention relates to the use, advantageously for cosmetic and / or non-therapeutic use, of an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, as a sebum regulator for hair, advantageously for oily hair.

[0044] Another subject of the present invention relates to an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for use as a wound healing agent.

[0045] Another subject of the present invention relates to an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for use as an anti-aging agent.

[0046] Another subject of the present invention relates to an extract of the cubere variety of Punica granata as described above, or a composition containing said extract, for use as a decolorizing agent.

[0047] Another subject of the present invention relates to the use of extracts of the cubere variety of Punica granata as described above, or compositions containing such extracts, as anti-aging or depigmenting agents, advantageously for cosmetic and / or non-therapeutic use.

[0048] Another subject of the present invention relates to a method for combating skin aging, comprising topical application of an extract of the cuverée variety of Punica granata as described above to the skin and / or mucous membranes and / or skin appendages.

[0049] A method by which the present invention can be carried out, and whose advantages are expected to become clearer from the following exemplary embodiments, is described with the aid of the accompanying figures, as non-limiting indicators. [Brief explanation of the drawing]

[0050] [Figure 1] This graph shows the evaluation of the antioxidant activity of whole fruit extracts from the Wonderful variety and the Cybele variety of Punica granata. [Figure 2] This graph shows the evaluation of the effects of whole fruit extracts from the Wonderful and Cybele varieties of Punica granata on IL-6 secretion. [Figure 3] This graph shows the evaluation of the effects of whole fruit extracts from the Wonderful and Cybele varieties of Punica granata on IL-8 secretion. [Modes for carrying out the invention] [Examples]

[0051] (Example 1) A study on the genetic diversity between pomegranate varieties Cybele and Wonderful. A comparative study of varieties was conducted using whole fruit samples from the Wonderful variety and the Cybele variety of Punica granata.

[0052] The microsatellite gene markers (or short sequence repeats, SSRs) used during the analysis allow for the characterization of varieties or ecotypes of the same species (i.e., varieties closely related to their ecological environment). These specific markers enable differentiation between varieties by targeting species-specific genetic regions while possessing gene patterns that are repeated a specific number of times depending on the variety or ecotype.

[0053] From approximately 10 microsatellites identified through bibliographic references, four SSR markers (PG-2, 3, 4, and 6) were selected for the species Punica granatum based on different criteria specific to genetic analysis (i.e., heterozygosity percentage, usage conditions, and target gene fragment size).

[0054] The PG-2, 4, and 6 markers are not suitable for identification: the size of the markers in base pairs is identical for both varieties. In contrast, the PG-3 marker has a significant difference of three base pairs (bp): PG-3 is 107 bp in size for the 'Cybele' variety and 104 bp in size for the 'Wonderful' variety. This difference present in the genome of the Cybele variety is not dependent on the growth stage and is not the result of environmental changes or modifications due to cultivation methods. The presence of extra copies of the PG-3 marker compared to the Wonderful variety clearly demonstrates the genetic difference between the Cybele and Wonderful varieties.

[0055] This comparative study demonstrates the genetic diversity between the Wonderful and Cybele varieties of Punica granata.

[0056] (Example 2) Preparation of fruit extracts from the Cuvere and Wonderful varieties of Punica granata The whole fresh fruits (i.e., pomegranates) of the Punica granata 'Cuvelet' variety (indicated as purple pomegranate) and the Punica granata 'Wonderful' variety (indicated as red pomegranate) from Example 1 were harvested, sliced, dried in a ventilated oven at 70°C, and then ground using a knife mill to obtain a powder with an average particle size.

[0057] Three extraction processes using three different extraction solvents were applied to each of the crushed and dried pomegranates to obtain the six extracts identified in Table 1 below.

[0058] [Table 1]

[0059] Process 1: PDo / Water, Extracts 1A and 1B Using a solvent propanediol / water (indicated as PDo / water) with a composition of 60 / 40 by mass, pulverized and dried pomegranates were extracted at 70°C for 3 hours under continuous mechanical stirring at a pulverized material / solvent mass ratio of 5:95. The extract and plant press cake were then separated by filtration on a 200 μm mesh nylon cloth. The crude extract was then filtered under pressure on a cellulose plate, and subsequently on a cellulose acetate membrane with a cutoff threshold of 0.2 μm.

[0060] Process 2: NADES FGE115, Extracts 2A and 2B Using a fructose / glycerol / water NADES solvent (labeled FGE115) with a composition of 50 / 25 / 25 by mass, crushed and dried pomegranates are extracted at 70°C for 3 hours under continuous mechanical stirring in a crushed material / solvent mass ratio of 5:95. The extract and plant press cake are separated by filtration on a 200 μm mesh nylon cloth. The crude extract is then filtered under pressure on a cellulose plate, and subsequently on a cellulose acetate membrane with a cutoff threshold of 0.2 μm.

[0061] Process 3: EtOH / water, extracts 3A and 3B Using a solvent ethanol / water (indicated as EtOH / water) with a composition of 60 / 40 by mass, pulverized and dried pomegranates are extracted at 70°C for 3 hours under continuous mechanical stirring at a pulverized material / solvent mass ratio of 5:95. The extract and plant press cake are separated by filtration on a 200 μm mesh nylon cloth. The crude extract is then filtered under pressure on a cellulose plate, and subsequently on a cellulose acetate membrane with a cutoff threshold of 0.2 μm. The dry extract concentration of the liquid extract is determined by drying the sample at 105°C. If necessary, the ethanol in the extract may be replaced with a glycol such as propanediol to facilitate its use in as many different cosmetic compositions as possible. For this purpose, the aqueous ethanol extract is concentrated under reduced pressure until a soft extract is obtained. The soft extract is then completely dissolved in the aqueous glycolic acid solvent (propanediol / water 60 / 40 w / w) from Process 1 at the same dry extract concentration as the dry extract determined for the aqueous ethanol extract. Next, the glycolic acid aqueous solution is filtered under pressure on a cellulose plate, and then on a cellulose acetate membrane having a cutoff threshold of 0.2 μm.

[0062] (Example 3) Physicochemical characterization of purple and red pomegranate extracts The visual appearance, L*a*b* color parameters, and dry extract concentrations of the six extracts obtained in Example 2 were determined according to the following method.

[0063] The visual appearance of a pure extract placed in a colorless, transparent glass tube with a diameter of 1 cm will be determined.

[0064] The L*a*b* color index of pure extracts is measured using a square PMMA plastic cuvette (Konica Minolta instrument) with a 10mm optical path length, a 10° observer, and a D65 light source.

[0065] For a 3g pure extract test sample, the dried extract is determined by halogen drying (Mettler Toledo instrument) by applying a drying program at 125°C using a preliminary plateau (180°C-5 min) aligned with the stopping criteria of 3. This determination is not technically feasible for the NADES FGE115 extract, considering the non-volatility of the solvent.

[0066] The results are shown in Table 2 below.

[0067] [Table 2]

[0068] These results indicate that purple and red pomegranate extracts have different physicochemical characteristics from the same process. Differences in characteristics are also observed depending on the extraction solvent for the same variety of pomegranate. In particular, purple pomegranate extract has a predominantly red hue, while red pomegranate extract has a predominantly orangey-yellow hue.

[0069] (Example 4) Characterization of the phytochemical composition of purple and red pomegranate extracts For the six extracts obtained in Example 2, the total polyphenol content was determined, and qualitative and quantitative analyses of ellagitannins, ellagic acid, and anthocyanins were also performed according to the following method.

[0070] The extract is reacted with iron(III) chloride and potassium hexacyanoferrate(III) using the Prussian blue method, and the total polyphenol concentration in the extract is measured by absorbance analysis at 725 nm (Mettler Toledo UV-Vis spectrophotometer). The calibration range is prepared using punicalazine standards (Sigma, α- and β-compound). The total polyphenol concentration, expressed as punicalazine equivalents, is determined in grams per 100 g of extract.

[0071] The presence of α- and β-punicalagin (ellagitannin) and ellagic acid in the extract, as well as their quantification if detected, will be investigated using HPLC-UV at a wavelength of 255 nm (Shimadzu Corporation equipment, Symmetry Shield RP18 column). Two calibration curves will be generated using punicalagin standards (Sigma, α- and β- mixture) and ellagic acid standards. The concentrations of α- and β-punicalagin will be expressed in grams per 100 g of extract, and the concentration of ellagic acid will be expressed in mg / kg of extract.

[0072] The presence and quantification of anthocyanins in the extract will be investigated using HPLC-DAD (Shimadzu Corporation equipment, Kinetex 40A column). Chromanin will be detected and quantified at a wavelength of 515 nm according to a calibration curve generated using the corresponding standard molecule. Mirtilin will be detected and quantified at a wavelength of 525 nm according to a calibration curve generated using the corresponding standard molecule. Other anthocyanins detected by their UV-Vis absorption spectra will be quantified by comparing them to the chromatin calibration curve at 515 nm. The concentrations of chromatin, mirtilin, other anthocyanins, and total anthocyanins (sum of the three concentrations) will be expressed in mg / kg of extract.

[0073] The complete results are reported in Table 3 below (NQ: Not quantifiable).

[0074] [Table 3]

[0075] The concentrations of total polyphenols, α- and β-punicalagin, and ellagic acid are significantly higher in all purple pomegranate extracts according to the present invention than in red pomegranate extracts.

[0076] The results also show significant differences in anthocyanin composition between the purple and red pomegranate extracts. The total anthocyanin concentration appears to be 3 to 4 times higher in the purple pomegranate extract than in the red pomegranate extract. Myrtilin and chromanin are dominant anthocyanins in the purple pomegranate extract according to the present invention, while they are not observed in the red pomegranate extract, where these compounds appear only in trace amounts. In the red pomegranate extract, the proportion of other anthocyanins is dominant. The dominance of two specific anthocyanins, myrtilin and chromanin, in the purple pomegranate extract explains the difference in extract color observed in Example 3.

[0077] For each of the two pomegranate varieties, the PDo / water solvent allows for the acquisition of the highest concentrations of total polyphenols, α- and β-punicalagin, ellagic acid, and total anthocyanins. Two other extraction solvents (i.e., FGE115 and EtOH / water) also allow for the extraction of these families, but yields vary depending on the molecule.

[0078] (Example 5) Evaluation of the antioxidant activity of purple and red pomegranate extracts The antioxidant activity of the six extracts obtained in Example 2 was studied according to the following method, which allows for the measurement of anti-free radical activity.

[0079] 3.1. Measurement Principle 1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a free radical that is stable at room temperature, has a dark purple color in solution in ethanol, and absorbs wavelengths between 510 and 530 nm.

[0080] Due to its structure, DPPH can be reduced by accepting hydrogen atoms from other compounds. This reduction is accompanied by the disappearance of the purple color of the solution, and therefore a decrease in absorbance directly proportional to the number of atoms accepted.

[0081] 3.2. Experimental Protocol Add the product to be tested to an ethanolic solution of DPPH and incubate in the dark at room temperature (18-25°C) for 30 minutes. Then, take an absorbance reading at 518 nm using a spectrophotometer. Prepare an experimental blank using ethanol solution alone and add the product to be tested to it. Subtract the blank value from the experimental value to avoid any interference related to the actual color of the product.

[0082] 3.3.Results The results are described in Table 4 and Figure 1 below.

[0083] [Table 4]

[0084] 3.4. Conclusion The experimental results show that all samples possess antioxidant activity starting from a concentration of 0.01%.

[0085] The results also show that all of the purple pomegranate extracts (i.e., extracts 1-3A) have higher antioxidant activity than all of the corresponding red pomegranate extracts (i.e., extracts 1-3B). These results indicate that purple pomegranate extracts have higher antioxidant activity than red pomegranate extracts, regardless of the extraction solvent and process used.

[0086] (Example 6) Evaluation of the anti-inflammatory activity of purple and red pomegranate extracts. The anti-inflammatory activity (reduction of IL-6 and IL-8 synthesis) of the six purple and red pomegranate extracts described in Example 2 was studied.

[0087] It is known to those skilled in the art that, in order to demonstrate the anti-inflammatory effects on the skin, models are used that allow assays of pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEKs). Indeed, under the influence of physical or chemical pro-inflammatory stimuli, NHEKs secrete several pro-inflammatory cytokines, particularly interleukins 6 and 8 (IL-6 and IL-8), which play a significant role in the acute phase of tissue inflammation.

[0088] 4.1. Measurement Principle The assay is performed using NHEK monolayer culture. Cytokines IL-6 and IL-8 are assayed in the culture supernatant of pre-incubated NHEK in the presence of the product of test, and then the culture is stimulated for 24 hours with a mixture of interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) in the presence of the product of test.

[0089] 4.2. Experimental Protocol Next, two assay methods are used: an ELISA assay and a flow cytometry assay.

[0090] For the ELISA assay, use the Biolegend LEGEND MAX® kit from Biolegend, following the manufacturer's instructions for use.

[0091] For the flow cytometry assay, use the Biolegend LEGENDplex® Human Thrombosis Panel (3plex) kit from Biolegend, following the manufacturer's instructions for use.

[0092] 4.3.Results The results of IL-6 and IL-8 secretion are shown in Tables 5-6 and Figures 2-3, respectively.

[0093] [Table 5]

[0094] [Table 6]

[0095] 4.4. Conclusion The results obtained using the two techniques (ELISA and flow cytometry) show the same trend.

[0096] Three extracts obtained from two varieties of Punica granatum exhibit potent anti-inflammatory effects at concentrations between 0.001 and 0.005% in a model of NHEK stimulated by pro-inflammatory stress.

[0097] At the same concentration, purple pomegranate extract exhibits stronger anti-inflammatory activity than that of the corresponding red pomegranate extract with respect to the two pro-inflammatory cytokines evaluated, namely IL-6 and IL-8.

[0098] These results indicate that purple pomegranate extract has higher anti-inflammatory activity than red pomegranate extract, regardless of the extraction solvent and process.

[0099] (Example 7) Evaluation of the sebum-regulating activity of purple and red pomegranate extracts. Two purple and red pomegranate extracts, prepared using Process 1 (PDo / water) as described in Example 2, were evaluated for their sebum-regulating ability.

[0100] To demonstrate the sebum-regulating effects of extracts 1A (purple pomegranate) and 1B (red pomegranate) in the skin, sebaceous gland cells, which are specialized cells that produce lipids that form sebum in the skin, were used in in vitro culture.

[0101] 7.1. Experimental Protocol Normal human sebaceous gland cells were cultured in vitro in a monolayer (in the presence of 50 μM linoleic acid). The cells were treated with 0.1% and 0.2% purple pomegranate extract (extract 1A) and red pomegranate extract (extract 1B) for 5 days. To verify the sebum-regulating effect of extracts 1A and 1B, epigallocatechin gallate / EGCG (100 μM), which inhibits lipid synthesis in sebaceous gland cells, was used. The sebaceous gland cells were then fixed using Antigenfix products and prepared for lipid assays.

[0102] 7.2. Lipid Assay Sebaceous gland cells are incubated for 30 minutes with the BODIPY® fluorescent probe, which stains lipids fluorescent green. Lipids are then visualized by fluorescence (excitation 493 / emission 503) and quantified by image analysis using a Citation 5 multimode plate reader (Agilent Biotek). The nuclei of the sebaceous gland cells are stained with DAPI to normalize the amount of lipid per cell.

[0103] 7.3.Results The results of the lipid assay are shown in Figure 4 and Table 7.

[0104] [Table 7]

[0105] 7.4. Conclusion The amount of lipids assayed in sebaceous gland cells treated with extract 1A (purple pomegranate) was significantly lower than that assayed in untreated sebaceous gland cells. In other words, extract 1A has the property of regulating sebum by reducing lipid synthesis in sebaceous gland cells. The sebum-regulating effect of extract 1A is stronger at 0.2% than at 0.1%.

[0106] The amount of lipids assayed in sebaceous gland cells treated with extract 1B (red pomegranate) was equivalent to the amount assayed in untreated sebaceous gland cells. In other words, extract 1B does not possess sebum-regulating properties. These results indicate that the two varieties of Punica granata differ in terms of their sebum-regulating activity, and that only purple pomegranate has the ability to reduce lipid synthesis in normal human sebaceous gland cells. In other words, this data clearly demonstrates the reduction in sebum production by the extract of the Punica granata cybele variety (i.e., purple pomegranate) of the present invention, and therefore, it has the ability to prevent excessive seborrhea in healthy skin, especially in oily or even combination skin prone to acne.

Claims

1. Topical cosmetic use of an extract of the cubere variety of Punica granata, or a composition containing said extract.

2. The use according to claim 1 for combating skin aging.

3. The use according to claim 1 or claim 2 for stimulating the radiance of the complexion.

4. Use according to any one of claims 1 to 3 for soothing the skin.

5. The use according to claim 1 for improving the regulation of sebum production in the skin.

6. The use according to any one of claims 1 to 5, characterized in that an extract of the cubere variety of Punica granata is obtained by a solid-liquid extraction process using an extraction solvent, followed by a second step of solid-liquid separation and finally a third step of recovering the liquid phase, wherein the extraction solvent is a mixture of propanediol and water.

7. An extract of the Punica granata 'Cuvelet' variety, which can be obtained by a solid-liquid extraction process from pulverized Punica granata 'Cuvelet' variety using an extraction solvent, followed by a second step of solid-liquid separation and finally a third step of recovering the liquid phase, characterized in that the extraction solvent is a mixture of propanediol and water.

8. The extract according to claim 7, characterized in that it is an extract of the whole fruit, preferably a crushed and dried whole fruit.

9. The extract according to claim 7 or 8, characterized in that the extraction uses a mass ratio of ground Punica granata 'Cuvelet' variety / solvent between 1:99 and 50:50, preferably between 2:98 and 15:

85.

10. Its phytochemical composition is, - The total polyphenol content is 0.5g / 100g or more, and / or - Miltilin is 1 mg / kg or more, and / or - Chromanin is 1 mg / kg or more. The extract according to any one of claims 7 to 9, characterized in that

11. A composition comprising the extract according to any one of claims 7 to 10.

12. An extract according to claim 7 or 10, or a composition according to claim 11, which is advantageous as a pharmaceutical, particularly for use as a pharmaceutical for dermatological use.

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