WO2024256768A1 - Extract of the cybele variety of punica granatum, and uses thereof in cosmetics - Google Patents

Abstract

The invention relates to the cosmetic and/or dermatological use of an extract of the Cybele variety of Punica granatum, or to a composition comprising said extract.

Description

Description

Title of the invention: Extract of Punica granatum variety Cybèle and its uses in cosmetics

Field of invention

[0001] The present invention relates to an extract of Punica granatum variety Cybele and a cosmetic composition comprising said extract. The invention also relates to the cosmetic and/or dermatological use of said extract or said composition for skin care, in particular for combating skin aging.

State of the art

[0002] The pomegranate tree, belonging to the species Punica granatum, is a shrub measuring on average 3 to 6m in height and classified in the Lythraceae family.

[0003] Its geographical distribution area is very wide. The countries of origin of the pomegranate tree extend from Turkey to the Caucasus, from Turkmenistan to Iran, Afghanistan and Pakistan. Throughout the Mediterranean region and in the Near East, the pomegranate tree has been cultivated for centuries, particularly for its fruit, the pomegranates.

[0004] Pomegranates are globose berries with a leathery pericarp which, when ripe (i.e., from September to October), turns from yellow to red-orange or even purplish to black depending on the variety. The fruit is topped with the remains of the flower calyx, forming a serrated crown and giving it an atypical shape that is easily identifiable. The pomegranate is made up of 6 to 12 whitish membranous cells with a bitter taste containing numerous triangular seeds called arils. These seeds have a fleshy and gelatinous mesocarp, generally acidic and sweet, representing the edible part of the fruit.

[0005] There are over 1200 different varieties of pomegranate in the world. They are distinguished in particular with regard to the fruit by the acidity and diameter, the color of the pericarp, the number of seeds and the precocity. We can cite for example the variety Wonderful, red pomegranate from Israel, known for its sweet and sugary fruits. Some varieties of pomegranate have been planted in the mild winter regions of central Europe, in particular in France where it is found in particular in the regions of the southern half.

[0006] The phytochemical composition of varieties producing red fruits of the species Punica granatum is well documented and allows to highlight the majority families such as hydroxycinnamic acids and more particularly caffeic, chlorogenic, ferulic, quinic and p-coumaric acids very present in the pericarp of the fruit; flavonoids of the flavone and flavonol type (luteolin glycosides, quercetin, apigenin, naringenin and isorhamnetin) also abundant in the pericarp; or even hydrolysable tannins of the ellagitannin type (punicalagins) and triterpenic sapogenins (ursolic acid) present in the seeds, the pericarp and the leaves.

[0007] Given their particular and well-known phytochemical composition, Punica granatum varieties producing red fruits are preferred in cosmetic formulations or food supplements, etc. [0008] The fruit constitutes the most commonly used source of extraction, but it is above all a specific part which is exploited, namely the peel or the seed, which requires additional separation steps which have direct effects on the cost of extraction.

[0009] For example, documents FR 3 031 901, WO 2021/064034 or WO 2021/209593 describe extracts of Punica granatum producing red fruits and their cosmetic uses, in particular for the production of oral compositions or food supplements, sun prevention or to combat acne. It should be noted that sebocytes participate in the pathophysiology of acne due to their role in producing sebum. Indeed, it appears that (i) dysseborrhea makes the sebum of poor quality due to a deficiency in vitamin E, in particular. The elements of sebum, and more particularly squalene, are oxidized. Oxidized lipids are known for their pro-inflammatory and cytotoxic properties (ii) hyperseborrhea is an overproduction of sebum or excess sebum that induces skin problems such as acne. Hyperseborrhea contributes in particular to the proliferation of Cutibacterium acnes which contributes to the local inflammatory reaction. The extracts described by the documents FR 3 031 901, WO 2021/064034 or WO 2021/209593, mentioned above, require different phases of concentration, or even purification of particular phytochemical families in order to increase their effectiveness. These processes therefore require additional manufacturing steps which have direct effects on energy consumption and therefore on the overall production cost.

[0010] Some varieties considered ornamental bear smaller fruits, and generally rather acidic and bitter, which makes them unfit for consumption.

[0011] It is from this type of unfit for consumption varieties that the purple pomegranates of the Cybele variety or Punica granatum variety Cybele (var. Cybele) come. The skin of this Cybele variety is of a rare, purplish, almost black color. However, unlike other so-called black varieties, such as the Asmar or Nera del Salento varieties whose fruits with red arils are sweet and sugary, the fruit of the Cybele variety has arils of white to very pale pink color, with a bitter and extremely acidic flavor, making them unfit for consumption.

[0012] This Cybèle variety has not been the subject of any scientific publication, particularly concerning its genetic and phytochemical characteristics.

[0013] Punica granatum var. Cybele is an ornamental variety, which also has agronomic advantages: it is effective in repelling insect pests and allows cultivation without the use of pesticides. It is a major asset for biodynamic or co-culture production.

[0014] The species Punica granatum has a long history of traditional and popular uses dating back to ancient Egypt. The pomegranate is particularly described in traditional Chinese and Ayurvedic medicine, but it is also present in the Persian and Greco-Arabic medical systems.

[0015] All parts of the pomegranate tree are described as having therapeutic properties such as vermifuge properties, particularly recognized as effective against tapeworm; in the treatment of oral conditions, stomach aches, ulcers, to treat inflammations, bleeding gums or even to eliminate dental plaque. Some applications, more rarely cited, refer to skin conditions. Traditional Chinese medicine, in fact, describes the fruit as useful for treating furunculosis or bedsores.

[0016] Studies based on different cellular or tissue models confirm the biological properties of different parts of the pomegranate tree. For example, we can cite antioxidant properties mainly carried by its content of polyphenolic compounds (flavonoids, tannins, anthocyanins); anti-inflammatory, anti-bacterial, anti-acne, healing, anti-aging properties or even a depigmenting activity.

[0017] There are many cosmetic products, particularly for skin care, containing pomegranate extracts. However, these extracts are mainly derived from pomegranate varieties whose fruit is edible, particularly due to the abundance of these red varieties and their specific and well-described phytochemical composition, such as Punica granatum var. Wonderful.

[0018] There therefore remains a clear need for a Punica granatum extract, in particular an alternative to existing extracts having cosmetic properties that are at least equivalent, or even improved, compared to the Punica granatum extracts of the state of the art, in particular to effectively combat skin ageing, which is free of toxic or irritant effects, effective for cosmetic skin care and the production of which is less costly, less energy-intensive and less water-consuming (e.g., eco-design approach) compared to the manufacturing processes of the state of the art.

Disclosure of the invention

[0019] The Applicant was able to identify, unexpectedly and surprisingly, a particular variety of Punica granatum, perfectly suited to cosmetic and/or dermatological use on human skin, and which meets the needs detailed above.

[0020] In particular, it has identified the species Punica granatum variety Cybele whose fruits are not edible and which, as mentioned above, is an effective alternative to Punica granatum fruit extracts, in particular those from edible varieties such as Punica granatum var. Wonderful.

[0021] The Applicant has conducted varietal comparison studies which have made it possible to highlight a genetic divergence between Punica granatum var. Wonderful and Punica granatum var. Cybèle (see example 1).

[0022] The phytochemical composition of the purple pomegranate variety Cybele differs from that of other red-fruited varieties of Punica granatum such as Punica granatum var. Wonderful. The Applicant has in fact conducted studies comparing the phytochemical composition, which have made it possible to highlight phytochemical divergences between the fruits of Punica granatum var. Wonderful and Punica granatum var. Cybele (see example 4).

[0023] Thus, a first object of the invention relates to a cosmetic, i.e. non-therapeutic, and topical use of an extract of Punica granatum var. Cybele or of a composition comprising said extract. [0024] The present invention offers various advantages, in particular the fact of having particular biological properties linked to the activities of the extract of the invention, namely antioxidant, anti-inflammatory, anti-aging, antibacterial, healing, depigmenting, sebum-regulating or even anti-acne cosmetic activities. In addition, the use of Punica granatum var. Cybèle makes it possible not to divert the varieties of Punica granatum producing edible fruits, for example Punica granatum var. Wonderful.

[0025] In the context of the invention, the expression “whole fruit” designates the whole consisting of the pericarp (rind), the rest of the calyx, the membranous lodges, the pulp and the seeds (arils).

[0026] In the context of the invention, the expression “extraction solvent” designates the solvent used during the solid/liquid extraction step.

[0027] Advantageously, the cosmetic use of the extract of Punica granatum var. Cybele or of a composition comprising said extract according to the invention has the following additional technical characteristics, taken alone or in combination:

- cosmetic use is to combat skin aging;

- cosmetic use is to stimulate the radiance of the complexion;

- cosmetic use is to soothe the skin;

- cosmetic use is to combat sagging skin;

- cosmetic use is to combat the appearance of wrinkles and/or fine lines;

- cosmetic use is to combat wrinkles and/or fine lines;

- cosmetic use is as an antioxidant;

- cosmetic use is as an anti-inflammatory;

- cosmetic use is as a sebum regulator/sebum regulating agent/sebum regulation improving agent of the skin;

- cosmetic use is to reduce the size of skin pores;

- the cosmetic use is on healthy skin and/or on acne-prone skin, i.e. on a subject with healthy skin. For the purposes of the invention, acne-prone skin is to be distinguished from acne-prone skin, which is pathological skin (i.e. proven inflammation). Acne-prone skin is healthy skin that is generally oily due to excessive sebum secretion but which does not exhibit inflammation and/or excessive proliferation of commensal or pathogenic bacterial populations;

- the cosmetic use is on healthy skin and/or on oily skin (shiny and shiny appearance) and/or combination skin and/or skin with non-inflammatory imperfections (e.g. blackheads, pimples, comedones). For the purposes of the invention, oily, combination skin and/or skin with non-inflammatory imperfections is not pathological skin;

- cosmetic use is on oily hair;

- the extract of Punica granatum var. Cybèle is likely to be obtained by a solid/liquid extraction process using an extraction solvent, followed by a second solid/liquid separation step and finally a third step of recovery of the liquid phase;

- the extraction solvent used in the solid/liquid extraction process (or extraction solvent) is selected from the following groups of substances used alone or in mixture: water; glycols such as 1.3-propanediol, 1,2-propanediol, 1.3-butanediol; polyols such as glycerin; alcohols such as ethanol; and Natural Deep Eutectic Solvents (NaDES) such as those described in FR3036618, in particular NaDES Fructose/Glycerin/Water in molar proportions 1:1:5 (denoted NaDES FGE115); advantageously consisting of a mixture of propanediol and water; a mixture of ethanol and water; and a mixture of fructose, glycerin and water; preferably a mixture of propanediol and water

- the mass ratio of the mixture of propanediol and water is between 10/90 and 90/10, advantageously 60/40;

- the mass ratio of the mixture of ethanol and water is between 10/90 and 90/10, advantageously 60/40;

- the mass ratio of the mixture of fructose, glycerin and water during the extraction step is between 55/28/17 and 45/23/32, advantageously 50/25/25;

- the mass ratio of the ground Punica granatum var. Cybèle/solvent is between 1:99 and 50:50, advantageously between 2:98 and 15:85;

- the extract of Punica granatum var. Cybele used according to the invention is an extract obtained from the mature or immature fruit, advantageously from the mature fruit;

- the extract of Punica granatum var. Cybele used according to the invention is an extract obtained from the whole fruit, fruit peel, fruit seed, trunk bark, roots, flowers, leaf stems, and/or leaves, advantageously a whole fruit extract;

- the part of the Punica granatum var. Cybele plant used to obtain an extract used according to the invention is fresh, frozen, dried, whole, cut and/or crushed;

- the extract of Punica granatum var. Cybele used according to the invention has a phytochemical composition/total polyphenol content greater than or equal to 0.5 g/100g; advantageously between 0.8 and 1.0 g/100g;

- the extract of Punica granatum var. Cybele used according to the invention has a phytochemical composition/punicalagins a and P content greater than or equal to 0.3 g/100g; advantageously between 0.4 and 0.6 g/100g;

- the extract of Punica granatum var. Cybele used according to the invention has a phytochemical composition/ellagic acid content greater than or equal to 30 mg/kg, advantageously between 100 and 150 mg/kg;

- the extract of Punica granatum var. Cybèle used according to the invention has a phytochemical composition/total anthocyanin content greater than or equal to 3 mg/kg, advantageously between 4 and 9 mg/kg;

- the extract of Punica granatum var. Cybele used according to the invention has a phytochemical composition/myrtillin content greater than or equal to 1 mg/kg; advantageously between 2 and 4 mg/kg;

- the extract of Punica granatum var. Cybele used according to the invention has a phytochemical composition/kuromanin content greater than or equal to 1 mg/kg; advantageously between 1.5 and 3 mg/kg;

- the extract of Punica granatum var. Cybele used according to the invention has a higher phytochemical composition/content of anthocyanins other than kuromarin and myrtillin or equal to 0.5 mg/kg; advantageously between 1.0 and 2.5 mg/kg.;

[0028] Another subject of the present invention relates to an extract of Punica granatum var. Cybele obtainable by a solid/liquid extraction process of ground Punica granatum var. Cybele using an extraction solvent, followed by a second solid/liquid separation step and finally a third liquid phase recovery step, 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, glycerin and water.

[0029] Advantageously, the extract of Punica granatum var. Cybele of the invention has the following additional technical characteristics, taken alone or in combination:

- the extraction solvent according to the invention is liquid at room temperature, which facilitates the implementation of the process leading to the extract of the invention;

- the solid/liquid extraction can be carried out by different techniques known in the field considered by the person skilled in the art, such as maceration, remaceration, digestion, dynamic maceration, decoction, fluid bed extraction, microwave-assisted extraction, ultrasound-assisted extraction, counter-current extraction, percolation, re-percolation, leaching, extraction under reduced pressure, diacolation;

- the mass ratio of plant part(s) of Punica granatum var. Cybèle /extraction solvent applied for the extraction step is between 1:99 and 50:50, advantageously between 2:98 and 15:85;

- the extraction step is carried out at a temperature between 2 and 100°C, advantageously between 20 and 80°C;

- the extraction step can be maintained for a few minutes to several days;

- the solid/liquid extraction step is carried out with stirring and/or under a nitrogen atmosphere so as to optimize the extraction of the active compounds while protecting these compounds from oxidation by atmospheric oxygen;

- the solid/liquid extraction step is followed by a solid/liquid separation step, to recover the liquid phase (or solid/liquid separation filtrate), containing the active material;

- the separation step can be carried out by any technique known in the field considered by the person skilled in the art, in particular draining, pressing, spinning, centrifugation or filtration;

- the liquid/solid separation step can be followed by a concentration step, which makes it possible to obtain a concentrate, in liquid or semi-solid form depending on the concentration factor;

- the concentration step is carried out by evaporation under reduced pressure or reverse osmosis;

- the solid/liquid separation filtrate or concentrate additionally undergoes one or more clarification steps;

- the clarification step implements any type of frontal and/or tangential filtration known in the field considered by the person skilled in the art;

- the liquid/solid separation or concentration or clarification step can be followed of a fractionation step to obtain a fraction of one or more phytochemical families, in liquid or semi-solid form depending on the concentration factor;

- the fractionation step can be carried out by any technique known in the field considered by the person skilled in the art, in particular by chromatography under reduced pressure, ultrafiltration or nanofiltration; and/or

- the method of the invention may comprise a sterilizing filtration step;

- the sterilizing filtration step is carried out by filtering the product through a filter comprising pores with a diameter of 0.22 μm.

[0030] Another object of the present invention relates to a composition comprising the extract of Punica granatum var. Cybele as described above.

[0031] Advantageously, the composition according to the invention is a cosmetic and/or dermatological composition.

[0032] Advantageously, the composition of the invention has the following additional technical characteristics, taken alone or in combination:

- the extract of Punica granatum var. Cybèle according to the invention represents between 0.1% and 20% by weight of the composition, advantageously between 0.5% and 5%;

- the composition of the invention is a skin care composition, in particular a composition for combating skin aging;

- the composition of the invention is an anti-aging composition;

- the composition of the invention is a composition improving the sebum regulation of the skin;

- the composition of the invention may be presented in all the galenic forms normally used for a rinsed or non-rinsed topical application, for example in anhydrous form, in the form of an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion, a silicone emulsion, a microemulsion, a nanoemulsion, a gel, an aqueous solution or a hydroalcoholic solution;

- the composition of the invention may be more or less fluid and may be in the form of a white or colored cream, an ointment, a milk, a lotion, a serum, a gel, a washing base, or a stick. It may be used in many treatments of the skin, lips and hair, including the scalp, in particular to protect and/or care for the skin, lips and/or hair, and/or to make up the skin and/or lips;

- the composition of the invention may contain excipients usually used in the cosmetic and dermatological fields, such as fatty substances, detergent and/or conditioning surfactants, emulsifiers and co-emulsifiers, hydrophilic or lipophilic gelling agents, preservatives, antioxidants, solvents, exfoliating agents, perfumes, fillers, hydrophilic and lipophilic filters, coloring materials, acidic or basic neutralizing agents, pro-penetrating agents, and polymers. These types of excipients are all well known to those skilled in the art;

- the quantities of the various excipients included in the composition of the invention are those known in the field considered by those skilled in the art, and the sum of the quantities of excipients represents 0.01% to 99.99% of the total weight of the composition; - suitable fats include mineral oils, oils of animal origin 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 alcohols, fatty acids, waxes and gums, and in particular silicone elastomers, may be used as fats;

- as suitable detergent and/or conditioning surfactants, mention may be made of nonionic, anionic, cationic or amphoteric surfactants, and mixtures thereof, such as for example alkyl sulfates, alkyl ether sulfates such as sodium lauryl ether sulfate, alkyl betaines such as cocamidopropyl betaine, or quaternary ammonium salts;

- as suitable emulsifiers and co-emulsifiers, mention may be made, for example, of polyglycerol fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, oxyethylenated sorbitan fatty acid esters, fatty alcohol PEG ethers, glycerol fatty acid esters, alkyl sulfates, alkyl ether sulfates, alkyl phosphates, alkyl polyglucosides, alkyl polypentosides, dimethicone copolyols;

- suitable hydrophilic gelling agents include, for example, carboxyvinyl polymers, acrylic copolymers (carbomers) such as acrylate/alkylacrylate copolymers, polyacrylamides, polysaccharides such as xanthan gum, guar gum, natural gums such as cellulose gum and derivatives, starches and derivatives thereof, clays and 2-acrylamido-2-methylpropane acid copolymers;

- suitable lipophilic gelling agents include, for example, modified clays such as bentones, 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, sorbic, propionic, salicylic, dehydroacetic acids and their salts, benzyl alcohol, ethylhexylglycerin, parabens, their salts and esters, triclosan, imidazolidinyl urea, phenoxyethanol, DMDM hydantoin, diazolidinyl urea, chlorphenesin;

- suitable antioxidants include, for example, chelating agents such as EDTA and its salts, sodium metabisulfite, salicylic, ascorbic and citric acids and their salts, sodium tartrate, sodium gluconate, carotenoids and tocopherols;

- as solvents which can be used in the composition (separate from the extraction solvent), we can cite for example water, ethanol, glycerin, propylene glycol, propanediol, butylene glycol, sorbitol;

- as suitable exfoliating agents according to the invention, mention may be made, for example, of chemical exfoliants such as AHAs, and physical exfoliants such as natural or synthetic powders;

- as suitable fillers according to the invention, we can cite for example talc, kaolin, mica, serecite, magnesium carbonate, aluminum silicate, magnesium silicate, organic powders such as nylon;

- as suitable dyes according to the invention, mention may be made, for example, of lipophilic dyes, hydrophilic dyes, pigments and nacres usually used in cosmetic or dermatological compositions, and their mixtures;

- suitable neutralising agents include basic agents such as, for example, sodium hydroxide, triethanolamine, aminomethylpropanol and potassium hydroxide, and acidic agents such as, for example, citric acid and lactic acid;

- suitable pro-penetrating agents include, for example, alcohols and glycols (ethanol, propylene glycol), ethoxydiglycol, alcohols and fatty acids (oleic acid), fatty acid esters, dimethyl isosorbide;

- the composition of the invention may also contain in addition one or more other active ingredients than the extract of Punica granatum var. Cybèle according to the invention;

- the appropriate active agent according to the invention is chosen from anti-radicals or more generally antioxidants; whiteners; pigmentants; emollients; moisturizers; anti-seborrheic agents; anti-inflammatories; anti-acne agents; keratolytic and/or desquamating agents; anti-wrinkle and tightening agents; draining agents; anti-irritant agents; soothing agents; vitamins and their mixtures; notifying agents; anti-aging active agents such as retinol; healing agents; antiseptics and essential oils.

[0033] Another subject of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as a medicament, advantageously as a medicament for dermatological use, preferably as a topical medicament for dermatological use.

[0034] Another subject of the present invention relates to an extract of Punica granatum var. Cybele or a composition comprising said extract for dermatological use in the prevention and treatment of inflammatory conditions.

[0035] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as an antioxidant.

[0036] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as an anti-inflammatory.

[0037] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as an antibacterial.

[0038] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as an anti-acne agent.

[0039] Another object of the present invention relates to the use, advantageously the cosmetic and/or non-therapeutic use, of an extract of Punica granatum var. Cybele as described above or a composition comprising said extract as an agent skin sebum regulator (or regulator of skin sebum production), advantageously for acne-prone skin and/or oily skin and/or combination skin and/or skin with non-inflammatory imperfections.

[0040] Another subject of the present invention relates to the use, advantageously the cosmetic and/or non-therapeutic use, of an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for preventing and/or reducing hyperseborrhea of the skin, preferably acne-prone skin.

[0041] Another subject of the present invention relates to the use, advantageously the cosmetic and/or non-therapeutic use, of an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for improving and/or correcting the size of the pores of the skin, advantageously of acne-prone skin and/or oily skin and/or combination skin and/or skin with non-inflammatory imperfections.

[0042] Another subject of the present invention relates to the use, advantageously the cosmetic and/or non-therapeutic use, of an extract of Punica granatum var. Cybele as described above or a composition comprising said extract as a sebum-regulating agent for hair, advantageously oily hair.

[0043] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as a healing agent.

[0044] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as an anti-aging agent.

[0045] Another object of the present invention relates to an extract of Punica granatum var. Cybele as described above or a composition comprising said extract for use as a depigmenting agent.

[0046] Another object of the present invention relates to the use, advantageously the cosmetic and/or non-therapeutic use, of an extract of Punica granatum var. Cybele as described above or a composition comprising said extract as an anti-aging or depigmenting agent.

[0047] Another subject of the present invention relates to a method for combating skin aging, the method comprising the topical application to the skin and/or mucous membranes and/or appendages of an extract of Punica granatum var. Cybele as described above.

[0048] The manner in which the invention can be implemented and the advantages which result therefrom will emerge more clearly from the following examples of implementation, given for information purposes and without limitation, in support of the appended figures.

Brief description of the drawings

[Fig 1] Evaluation of the antioxidant activity of whole fruit extracts of Punica granatum var. Wonderful and Punica granatum var. Cybèle.

[Fig 2] Evaluation of the effect of whole fruit extracts of Punica granatum var. Wonderful and Punica granatum var. Cybele on IL-6 secretion.

[Fig 3] Evaluation of the effect of whole fruit extracts of Punica granatum var. Wonderful and Punica granatum var. Cybèle on IL-8 secretion.

Examples

[0049] Example 1 - Study of the genetic divergence between the pomegranate varieties Cybele and Wonderful

[0050] Varietal comparison studies were carried out on whole fruit samples of Punica granatum var. Wonderful and Punica granatum var. Cybèle.

[0051] The microsatellite genetic markers (or Short Sequence Repeat or SSR) used during the analysis allow the characterization of variety or ecotype (i.e., variety closely linked to its ecological environment) of the same species. These specific markers make it possible to differentiate varieties by targeting the genetic zones specific to each species while presenting a genetic motif repeated a certain number of times depending on the variety or ecotype.

[0052] For the species Punica granatum, out of the ten microsatellites identified through bibliographic references, 4 SSR markers (PG-2, 3, 4 and 6) were chosen on the basis of different criteria specific to genetic analysis, i.e., the percentage of heterozygosity, the conditions of use and the size of the targeted genetic fragment).

[0053] Markers PG-2, 4 and 6 are not differentiating: the size of the markers in base pairs is identical for the 2 varieties. On the other hand, marker PG-3 shows a significant difference of 3 base pairs (bp): PG-3 has a size of 107bp for the variety “Cybèle”, and a size of 104bp for the variety Wonderful. This difference present in the genome of the variety Cybèle is not a function of the growth stage, nor the result of a modification by an environmental change or by a cultivation method. The presence of a supernumerary copy of marker PG-3 compared to the variety Wonderful clearly demonstrates the genetic difference between the varieties Cybèle and Wonderful.

[0054] This varietal comparison study highlights a genetic divergence between the varieties Punica granatum var. Wonderful and Punica granatum var. Cybèle.

[0055] Example 2 - Preparation of fruit extracts of Punica granatum var. Cybele and var. Wonderful

[0056] Fresh whole fruits (i.e., pomegranate) of the varieties Punica granatum var. Cybele (denoted purple pomegranate) and Punica granatum var. Wonderful (denoted red pomegranate) of Example 1 were harvested, sliced and then dried in a ventilated oven at 70°C before being ground using a knife mill to obtain a powder of medium particle size.

[0057] 3 extraction processes using 3 different extraction solvents were applied to each dried pomegranate grind to obtain 6 extracts identified in Table 1 below.

[Table 1]

[0058] Method 1: PDo/Water. extracts IA and IB

The dried pomegranate ground material is extracted with the solvent Propanediol/Water with a mass composition of 60/40 (denoted PDo/Water), according to a mass ratio of ground material/solvent of 5:95 for 3 hours at 70°C with continuous mechanical stirring. The extract and the vegetable cake are then separated by filtration on a 200um mesh nylon cloth. The crude extract is then filtered under pressure on cellulose plates then on a cellulose acetate membrane with a cut-off threshold of 0.2iim.

[0059] Method 2: NaDES FGE115, extracts 2A and 2B

The dried pomegranate ground material is extracted by the NaDES solvent Fructose/Glycerin/Water with a mass composition of 50/25/25 (denoted FGE115), according to a mass ratio of ground material/solvent of 5:95 for 3 hours at 70°C with continuous mechanical stirring. The extract and the vegetable cake are then separated by filtration on a 200 um mesh nylon cloth. The crude extract is then filtered under pressure on cellulose plates then on a cellulose acetate membrane with a cut-off threshold of 0.2 um.

[0060] Method 3: EtOH/Water, extracts 3A and 3B

The dried pomegranate ground material is extracted with the Ethanol/Water solvent with a mass composition of 60/40 (denoted EtOH/Water), according to a ground material/solvent mass ratio of 5:95 for 3 hours at 70°C with continuous mechanical stirring. The extract and the plant cake are then separated by filtration on a 200um mesh nylon cloth. The crude extract is then filtered under pressure on cellulose plates and then on a cellulose acetate membrane with a cut-off threshold of 0.2um. The dry extract concentration of the liquid extract is determined on a sample by drying at 105°C. Optionally, the ethanol in the extract can be substituted by a glycol, such as propanediol, in order to facilitate its use in the greatest number of cosmetic compositions. For this, the hydro-ethanolic extract is then concentrated under reduced pressure until a soft extract is obtained. The soft extract is then completely solubilized in the hydro-glycolic solvent of process 1 (Propanediol/Water 60/40 m/m), at a dry extract concentration identical to the dry extract determined on the hydro-ethanolic extract. The hydro-glycolic solution is then filtered under pressure on cellulose plates then on a cellulose acetate membrane at a cut-off threshold of 0.2 pm.

[0061] Example 3: Physicochemical characterization of purple and red pomegranate extracts

[0062] The visual appearance, the L*a*b* color parameters and the dry extract concentration of the 6 extracts obtained in example 2 are determined according to the following methods.

[0063] The visual appearance is determined for the pure extract placed in a colorless and transparent glass tube with a diameter of 1 cm.

[0064] The L*a*b* color indices are measured on the pure extract in a square PMMA plastic tank with a 10 mm optical path, with the 10° observer and the D65 illuminant (Konica Minolta equipment). [0065] The dry extract is determined by halogen drying by applying a drying program at 125°C with preliminary stage (180°C - 5min) for a stopping criterion of 3, for a test sample of 3g of pure extract (Mettler Toledo equipment). This determination is not technically feasible for NaDES FGE115 extracts, given the non-volatility of the solvent.

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

[Table 2]

[0067] These results show that for the same process the physicochemical characteristics of the purple and red pomegranate extracts are different. For the same variety of pomegranate, differences in characteristics depending on the extraction solvent are also observed. In particular, the color of the purple pomegranate extracts has a dominant red tint, while that of the red pomegranate extracts has a dominant orange-yellow tint.

[0068] Example 4: Characterization of the phytochemical composition of purple and red pomegranate extracts

[0069] The dosage of total polyphenols as well as the qualitative and quantitative analysis of ellagitannins, ellagic acid and anthocyanins are carried out for the 6 extracts obtained in example 2 according to the following methods.

[0070] The concentration of total polyphenols in the extracts is measured according to the Prussian blue method by absorption spectrophotometry at 725 nm after reaction of the extract with iron III chloride and potassium hexacyanoferrate III (Mettler Toledo UV-visible spectrophotometer). A calibration range is carried out with the punicalagin standard (Sigma, mixture a and P). The concentration of total polyphenols expressed in punicalagin equivalent is determined in g per 100 g of extract.

[0071] The search for the presence of punicalagins a and P (ellagitannins) and ellagic acid in the extracts as well as their quantification, if they are detected, are carried out by HPLC-UV at the wavelength of 255 nm (Shimadzu equipment, SymmetryShield RP 18 column). Two calibration lines are produced with the punicalagin standard (Sigma, mixture a and P) and the ellagic acid standard. The concentration of punicalagins a and P is expressed in g per 100 g of extract, and the concentration of ellagic acid in mg/kg of extract.

[0072] The search for the presence of anthocyanins and their quantification in the extracts is carried out by HPLC-DAD (Shimadzu equipment, Kinetex 40A column). Kuromanin is detected and quantified at the wavelength of 515 nm, according to a calibration line carried out with the corresponding standard molecule. Myrtillin is detected and quantified at the wavelength of 525nm, according to a calibration line carried out with the corresponding standard molecule. The other anthocyanins detected with respect to their UV-visible absorption spectrum are quantified relative to the calibration line of kuromanin at 515nm. The concentrations of kuromanin, myrtillin, other anthocyanins and total anthocyanins (sum of the 3 previous concentrations) are expressed in mg/kg of extract.

[0073] All the results are shown in Table 3 below (NQ: not quantifiable).

[Table 3]

0074] The concentration of total polyphenols, punicalagins a and P and ellagic acid is significantly higher in all purple pomegranate extracts according to the invention compared to that of red pomegranate extracts.

[0075] The results also show significant differences in the composition of anthocyanins between the purple and red pomegranate extracts. It appears that the concentration of total anthocyanins is 3 to 4 times higher in the purple pomegranate extracts than in the red pomegranate extracts. Myrtillin and kuromanin are the predominant anthocyanins in the purple pomegranate extracts according to the invention, which is not observed in the red pomegranate extracts in which these compounds appear in trace amounts. In the red pomegranate extracts, the proportion of other anthocyanins is predominant. The predominance of 2 particular anthocyanins, namely myrtillin and kuromanin, in the purple pomegranate extracts explains the differences in color of the extracts observed in Example 3.

[0076] For each of the 2 varieties of pomegranate, the PDo/Water solvent allows to obtain the highest concentration of total polyphenols, punicalagins a and P and ellagic acid and total anthocyanins. The 2 other extraction solvents (e.g., FGE115 and EtOH/Water) also allow to extract these families but with different quantitative performances depending on the molecules.

[0077] Example 5: Evaluation of the antioxidant activity of purple and red pomegranate extracts

[0078] The antioxidant activity was studied for the 6 extracts obtained in example 2 according to the following method which allows the anti-radical activity to be measured.

[0079] 3.1. Principle of measurement

[0080] 1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a free radical, stable at room temperature, which has a dark violet color in solution in ethanol and absorbs in the wavelengths between 510 and 530 nm.

[0081] Due to its structure, DPPH can be reduced by accepting a hydrogen atom from other compounds. This reduction is accompanied by a disappearance of the violet color of the solution and therefore a drop in absorbance directly proportional to the number of atoms captured.

[0082] 3.2, Experimental protocol

[0083] The products to be tested are added to an ethanolic solution of DPPH, incubated for 30 minutes at room temperature (18-25°C) away from light, then an absorbance reading is taken at 518 nm using a spectrophotometer. An experimental blank is carried out using an ethanol solution alone to which the products to be tested are added. The blank values are deduced from the experimental values to avoid any possible interference linked to the specific color of the products.

[0084] 3.3. Results

[0085] The results are described in Table 4 below and Figure 1.

[Table 4]

0086] 3.4, Conclusion

[0087] The experimental results show that all the samples tested have antioxidant activity from a concentration of 0.01%.

[0088] The results also show that all purple pomegranate extracts (e.g., extracts 1-3A) have a higher antioxidant efficacy than the corresponding red pomegranate extracts (e.g., extracts 1-3B). These results show that, regardless of the extraction solvent and the process used, purple pomegranate extracts have a higher antioxidant activity than red pomegranate extracts.

[0089] Example 6: Evaluation of the anti-inflammatory activity of purple and red pomegranate extracts

[0090] The anti-inflammatory activity (reduction in the synthesis of IL-6 and IL-8) of the 6 purple and red pomegranate extracts described in Example 2 was studied.

[0091] To demonstrate an anti-inflammatory effect on the skin, it is known to those skilled in the art to use a model allowing the measurement of pro-inflammatory cytokines in normal human epidermal keratinocytes (NHEKs). Indeed, under the effect of a physical or chemical pro-inflammatory stimulus, NHEKs secrete several pro-inflammatory cytokines, in particular interleukins 6 and 8 (IL-6 and -8) which play a major role in the acute phase of tissue inflammation.

[0092] 4, 1, Principle of measurement

[0093] The assay is performed using monolayer cultures of KEHN. The cytokines IL-6 and IL-8 are assayed in the supernatants of KEHN cultures pre-incubated in the presence of the test products before being stimulated for 24 hours by a mixture of interferon gamma (IFN-y) and tumor necrosis factor alpha (T Fa) in the presence of the test products. [0094] 4.2, Experimental protocol

[0095] Two assay methods are then used: an ELISA assay and a flow cytometry assay.

[0096] For ELISA assay, LEGEND MAX™ kits from Biolegend are used following the manufacturer's instructions.

[0097] For flow cytometry assay, the LEGENDplex™ Human Thrombosis Panel (3-plex) kit from Biolegend is used following the manufacturer's instructions.

[0098] 4.3. Results

[0099] The results of IL-6 and IL-8 secretion are shown in Tables 5-6 and Figures 2-3, respectively.

[Table 6]

[0100] 4.4. Conclusion [Table 5]

[Table 6]

[0100] 4.4. Conclusion

[0101] The results obtained by the two techniques (ELISA and flow cytometry) show identical trends.

[0102] The three types of extracts obtained with the two varieties of Punica granatum show powerful anti-inflammatory effects in the model of KEHN stimulated by proinflammatory stress for concentrations between 0.001 and 0.005%.

[0103] At identical concentrations, purple pomegranate extracts show more potent anti-inflammatory activity than the corresponding red pomegranate extracts against the two pro-inflammatory cytokines evaluated, namely IL-6 and -8.

[0104] These results show that, regardless of the extraction solvent and the process, purple pomegranate extracts have superior anti-inflammatory activity to red pomegranate extracts.

[0105] Example 7: Evaluation of the sebum-regulating activity of purple and red pomegranate extracts

[0106] The 2 purple and red pomegranate extracts prepared with method 1 (PDo/Water), as described in example 2, were evaluated for their sebum-regulating capacity.

[0107] To demonstrate the sebum-regulating effect of IA (purple pomegranate) and IB (red pomegranate) extracts on the skin, sebocytes, which are the specialized cells producing the lipids constituting the skin's sebum, were used in in vitro culture.

[0108] 7.1. Experimental protocol

[0109] Normal human sebocytes are cultured in vitro in monolayer (in the presence of 50uM linoleic acid). The cells are treated for 5 days with purple (extract IA) and red (extract IB) pomegranate extracts at 0.1% and 0.2%. To validate the sebum-regulating effect of extracts 1A-1B, epigallocatechin gallate/EGCG (100pM) was used, which inhibits lipid synthesis in sebocytes. The sebocytes are then fixed with the product Antigenfix and prepared for lipid assay.

[0110] 7.2, Lipid dosage

[OR I] Sebocytes are incubated for 30 minutes with the BODIPY™ fluorescent probe, which stains lipids in fluorescent green. Lipids are then visualized by fluorescence (Ex493/Em503) and then quantified by image analysis with the Cytation 5 multimode plate reader (Agilent Biotek). Sebocyte cell nuclei are labeled with DAPI to normalize the amount of lipids per cell.

[0112] 7.3. Results

[0113] The results of the lipid assay are illustrated in Figure 4 and Table 7. [0114] [Table ?]

[0115] 7.4, Conclusion

[0116] The amounts of lipids measured in sebocytes treated with extract IA (purple pomegranate) are significantly lower than those measured in untreated sebocytes. In other words, extract 1 A has sebum-regulating properties by reducing lipid synthesis in sebocytes. The sebum-regulating effect of extract IA is stronger at 0.2% than at 0.1%.

[0117] The quantities of lipids measured in sebocytes treated with the IB extract (red pomegranate) are equivalent to the quantities measured in untreated sebocytes. In other words, the IB extract does not have sebum-regulating properties.

These results show that the two varieties of Punica granatum are different on the criterion of a sebum-regulating activity; only the purple pomegranate has the capacity to decrease the synthesis of lipids in normal human sebocytes. In other words, these data clearly demonstrate a decrease in sebum production with the extract of Punica granatum var. Cybele (i.e. purple pomegranate) of the invention, which therefore has the capacity to prevent hyperseborrhea on healthy skin, in particular acne-prone, oily or combination skin.

Claims

Claims [Claim 1] Topical cosmetic use of an extract of Pimica granatum var. Cybele or of a composition comprising said extract. [Claim 2] Use according to claim 1, for combating skin aging. [Claim 3] Use according to one of claims 1 or 2, to stimulate the radiance of the complexion. [Claim 4] Use according to one of claims 1 to 3, for soothing the skin. [Claim 5] Use according to claim 1, for improving the sebum regulation of the skin. [Claim 6] Use according to one of claims 1 to 5, characterized in that the extract of Punica granatum var. Cybele is capable of being 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 recovery of the liquid phase, characterized in that said extraction solvent is a mixture of propanediol and water. [Claim 7] Extract of Punica granatum var. Cybele obtainable by a solid/liquid extraction process of ground Punica granatum var. Cybele using an extraction solvent, followed by a second solid/liquid separation step and finally a third step of recovery of the liquid phase, characterized in that said extraction solvent is a mixture of propanediol and water. [Claim 8] Extract according to claim 7, characterized in that it is whole fruit, advantageously whole crushed dried fruit. [Claim 9] Extract according to one of claims 7 or 8, characterized in that the extraction uses a mass ratio of ground Punica granatum var. Cybèle/solvent of between 1:99 and 50:50, advantageously between 2:98 and 15:85. [Claim 10] Extract according to one of claims 7 to 9, characterized in that its phytochemical composition: - in total polyphenols is greater than or equal to 0.5 g/100g; and/or - in myrtillin is greater than or equal to 1 mg/kg; and/or - in kuromanine is greater than or equal to 1 mg/kg. [Claim 11] Composition comprising the extract according to one of claims 7 to 10. [Claim 12] Extract according to one of claims 7 or 10 or composition according to claim 11 for use as a medicament, advantageously as a medicament for dermatological use.