FR3110419A1 - Moringa peregrina seed extract rich in 2,5-diformylfuran, process for obtaining it and its use in cosmetic compositions - Google Patents

Abstract

The invention relates to an extract of Moringa peregrina seeds rich in the compound 2,5-diformylfuran, more specifically an extract from the cake of said seeds and a process for extracting the extract. It also relates to cosmetic or nutricosmetic compositions comprising said extract and the use of said compositions for improving the appearance of the skin, mucous membranes or appendages, for relaxing, soothing and de-stressing the skin and preventing and/or combating skin signs of aging and/or photo-aging and preventing age spots.

Description

Moringaperegrina seed extract rich in 2,5-diformylfuran, process for obtaining it and its use in cosmetic compositions

The invention relates to the field of cosmetics and nutricosmetics and more particularly that of the active ingredients used in the formulation of compositions for skin care. The invention relates to an extract of the seeds of Moringa peregrina rich in the compound 2,5-diformylfuran (DFF). The invention also relates to the process for obtaining a particular extract from the seeds of Moringa peregrina , the cosmetic compositions comprising such extracts, and finally the cosmetic or nutricosmetic use of such compositions for the care of the skin, the scalp and appendages.

Technology background

The Moringaceae constitute a mono-generic family (a single genus, Moringa Adans), an element of the Saharo-Sindian flora, made up of between twelve and fourteen species according to the authors, distributed from East Africa to Asia. The genus is classically divided into 3 sections which are, however, not confirmed as monophyletic by phylogenetic analyses. The latter have instead highlighted clades focused on certain morphological characters: pachycaules (“bottle trees”); tuberous ("tuberous trees") and neither pachycaules - nor tuberous ("slender trees"). The species Moringa peregrina (Forssk.) Fiori, belongs to the third group. The few genetic studies on the genus or the family confirm the reality of the species compared to the other species in the genus, in particular vis-à-vis the Indian Moringa, Moringa oleifera Lam. (see in particular the articles: Olson Mr. E . 2002, Combining Data from DNA Sequences and Morphology for a Phylogeny of Moringaceae (Brassicales), Systematic Botany 27(1): 55-73; Hassanein AT. Mr. A. and AL-SOQEE, AA, 2018, Morphological and genetic diversity of Moringa oleifera and Moringa peregrina genotypes, Horticulture, Environment and Biotechnology 59(2): 251-261). A recent article on Moringa peregrina sampled from different localities in Saudi Arabia concluded, using ITS markers, that the species is genetically stable ( AT there klabi AT., 2015, Genetic diversity of Moringa peregrina species in Saudi Arabia with ITS sequences, Saudi Journal of Biological Sciences 22: 186-190) with however a high level of intra-population genetic variation.

The Moringa peregrina species is found in the rocky environments of Yemen, Oman, and Saudi Arabia, East Africa, Sudan, Ethiopia, Eritrea, Somalia, and Djibouti. Its presence in Iran seems limited to the south-eastern provinces but needs to be confirmed ( PROTA14 = Munyanziza E. and Yongabi KA, Vegetable oils /Oléagineux , Moringa peregrina (Forssk.) Fiori, http://database.prota.org/protahtml/moringa peregrina_fr.htm, accessed 23/10/2019). In the Levant and in Egypt, the species now represents only rare dispersed, relict sites (with the exception of a few altitudinal stands), mainly in sectors of the Sudanian area. Moringa peregrina is also now considered rare and endangered in Sudan and Yemen. Moringa peregrina occupies compared to other species of its clade the most arid and inhospitable habitats. It is apparently more drought resistant than Moringa oleifera which is planted commercially on a large scale in tropical and subtropical areas. Recent work has shown that the size and the size of the seeds have a favorable impact on the germination time and the rate and rapidity of the growth of young individuals ( Gomaa NH and Pico F.X ., 2011, Seed germination, seedling traits, and seed bank of the tree Moringa peregrina (Moringaceae) in a hyper-arid environment , American Journal of Botany 98(6): 1024-1030), indicating an adjustment in the allocation of resources on seed quality rather than number, allowing Moringa peregrina to reproduce effectively in extreme abiotic (hyperarid) environments. Moringa peregrina seeds have a thicker central mesotesta, in terms of cell layer, than those of Moringa oleifera .
There are a few historical records that tend to indicate that Moringa peregrina oil was actively traded in early Islam in the AlUla region ( Naseef , AAS, 1995, A l-' Ulā , A study of Cultural and Social Heritage ). The oil produced locally from Moringa peregrina is now mainly intended for self-consumption or local markets. In Saudi Arabia, the leaves were traditionally used in decoction for internal use to treat diabetes, colon diseases, eye diseases and anemia ( Abdel Kader Mr. S., Hazazi AT. Mr. AT., Elmakki OA and Alqasoumi IF, 2018, A survey on the traditional plants used in Al Kobah village, Saudi Pharmaceutical Journal 26(6): 817-821) and as a diuretic, rubefacient and astringent ( Aqeel AA Mr. , Tariq Mr. , Mossa JS, Al-Yahya Mr. A. and Al- Said Mr. S., 1984, Plants used in Arabia Folk medicine, Report submitted to Saudi Arabian National Center for Science and Technology , Riyadh, Saudi Arabia). In Oman, the oil, extracted by women at the end of summer, is used against migraines, fever, burns, lacerations and fractures, constipation and stomach pains, against muscular pains and against capillary dryness, against the pains of childbirth ( Ghazanfar SA, 1994, Handbook of Arabian Medicinal Plan ts, ^1st ed., CRC Press, Boca Raton, Ann Harbor, su; Ghazanfar SA, 1998, Plants of Economic Importance, cap. 15 in Ghazanfar , SA and Fisher, M . (ed.) Vegetation of the Arabian Peninsula . Geobotany 25, p. 241-264, Kluwer Academic Publishers, table 11.1, p. 247 and 11.7 p. 251). It was also used in perfume compositions ( Ghazanfar SA, 1998, p. 259) and in Oman and Yemen as a facial lotion ( Ghazanfar SA and Rechinger B., 1996, Two multi-urpose seed oils form Oman. Plants for Food and Medicine. Paper presented at the joint meeting of the Society for Economic Botany and International Society for Ethnopharmacology , 1-7 July 1996, London).

Extracts from the seed of Moringa oleifera are known in the cosmetics field. For example, document FR296879 discloses an extract of whole seeds (with integuments) of Moringa oleifera containing oil (including triglycerides, fatty acids and polar lipids) and polyphenols and its use in cosmetic compositions to combat skin aging. . In this document, it is the apolar part of the seed of Moringa oleifera which appears to be active, and more particularly the oily part. It is also known from document FR2776519 that protein extracts of Moringa oleifera seeds, known for their clarifying effects on turbid waters, have a softening, physiological conditioning, moisturizing, restructuring, repairing effect on the skin and mucous membranes and as anti-pollution active. In this document, the active principles are proteins of molecular weight between 6500 and 8800 Da which are obtained by aqueous extraction on the cake of Moringa oleifera . The document FR3076460 is also known, which concerns the use of a protein extract of ungerminated and de-oiled seed of Moringa oleifera for the treatment of sensitive, sensitized, reactive, fragile and/or weakened skin and/or mucous membranes and/or in the treatment and/or prevention of erythema, in particular diaper rash. In this document, the extraction process makes it possible to obtain a major fraction of proteins whose molecular weights are approximately 8800 Da. The use of an extract of whole germinated seed of Moringa oleifera in cosmetics, in particular obtained by extraction using a supercritical fluid, is also known from document KR20130088224. This process makes it possible to isolate apolar carotenoids and amino acids which are described as active for a whitening cosmetic use. All the aforementioned documents relate to the use of the Moringa oleifera species, none describes or suggests the use in the cosmetics field of the Moringa peregrina species .

More specifically for the Moringa peregrina species, certain phenolic compounds and flavonoids obtained from the leaf or whole seed of Moringa peregrina are known to exhibit antioxidant activity ( Al- Dabbas M., 2017, Antioxidant activity of different extracts from the aerial part of Moringa peregrina (Forssk.)Fiori, from Jordan, Pakistan Journal of Pharmaceutical Sciences , 30(6): 2151-2157). These compounds are extracted with solvents such as methanol, ethyl acetate or hexane from the leaves or whole seeds. It appears that the leaves contain the most active compounds.

Thus, depending on the species used in the genus Moringa , it is observed that depending on the part of the plants (leaf, seed), the part of the seeds (whole or not, shelled or not) and the extraction process implemented, in particular the choice of solvent, the extracted molecules turn out to be different. However, the composition of the extract conditions the biological activity and consequently the cosmetic efficacy.

Considering the foregoing, a problem which the invention proposes to solve is to develop new products based on an extract of the species M. peregrina of the genus Moringa which can be used in cosmetics and are easy to implement.

As a result, the Applicant has demonstrated a new extract obtained from the seeds and more specifically from the cake of the seeds of the Moringa peregrina species , which in particular has a relaxing and anti-stress activity for the skin, an anti- aging as well as a preventive activity against age spots. The extract according to the invention is rich in 2,5-diformylfuran (DFF), a compound also called 2,5-furandicarboxaldehyde. The extract is obtained specifically from the seeds or more specifically from the unshelled seed cake of Moringa peregrina , in particular by alcoholic extraction. The extract according to the invention is new for two reasons in the field of cosmetics compared to the extracts of the prior art, on the one hand by the specific species of origin used and on the other hand by its content in particular compounds.

Summary

The first subject of the invention is an extract of the seeds of Moringa peregrina rich in the compound 2,5-diformylfuran. The compound 2,5-diformylfuran is a rare carbohydrate compound in the plant kingdom and synthesized from furfural via the intermediate synthesis of 5-hydroxymethylfurfural.

Thanks to its characteristic, a concentration rich in 2,5-diformylfuran, the extract according to the invention is unique in the genus Moringaceae. It will be demonstrated that the Moringa peregrina species has a particular molecular profile that is different from the other species of the genus, in particular from the Moringa oleifera species, which the Applicant has been able to demonstrate.

The second subject of the invention is a process for obtaining an extract of the seeds of Moringa peregrina according to the invention, comprising the following steps according to which:
a) the unshelled Moringa peregrina seeds are collected and dried to obtain an internal humidity level of less than 8%;
b) optionally pressing the dried seeds so as to separate the oil from the rest of the seed to obtain the cake,
c) the dried seed obtained in step a) or the cake obtained in step b) is ground,
d) the ground material obtained in step c) is dispersed in a proportion of 5% to 50% by weight of solid matter relative to the total weight used in a predominantly alcoholic solvent, the alcohol being chosen from ethanol or methanol optionally with a co-solvent of the polyol or subcritical water type, in a proportion of 80 to 100% by weight of alcohol relative to the total weight of the solvent;
e) a solid-liquid extraction is carried out, with stirring, at a temperature of between 4 and 50° C. for a period of between 10 minutes and 4 hours,
f) the liquid and solid phases are separated so as to eliminate the solid phase and recover a liquid extract of seed or cake of Moringa peregrina , and
g) optionally, when the alcohol is ethanol, the liquid Moringa peregrina extract obtained is dried so as to obtain a solid Moringa peregrina extract.

A third subject of the invention is a cosmetic or nutricosmetic composition comprising, as active agent, an effective amount of a Moringa peregrina seed extract according to the invention and a physiologically acceptable excipient.

Finally, a fourth subject of the invention is the cosmetic or nutricosmetic use of a composition according to one of the invention, to improve the appearance of the skin, mucous membranes or appendages, to relax, soothe and de-stress the skin and prevent and/or fight against the cutaneous signs of aging and/or photo-ageing, to prevent age spots.

Brief description of figures

The invention will be better understood, and other aims, details, characteristics and advantages thereof will appear more clearly during the following description of several particular embodiments of the invention, given solely by way of illustration and not limitation. , with reference to the accompanying drawings.

In this description, unless otherwise specified, it is understood that when an interval is given, it includes the upper and lower limits of said interval.

In the present invention, the following abbreviations mean:
- MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT test is a rapid method for live cell count)
- SDS: Sodium Dodecyl Sulfate
- PBS: Phosphate Buffer Saline
- ELISA: Enzyme-Linked Immunosorbent Assay
- PCR: Polymerase Chain Reaction
- ANOVA: Analysis Of Variance
- MSH: Melanocyte Stimulating Hormone

In the present invention, the following terms mean:
- “extract rich in 2,5-diformylfuran compound” an extract containing a greater quantity of 2,5-diformylfuran compound than the other identified ingredients, ie a quantity greater than 50% relative to the dry matter of the total extract.
- "effective amount" the necessary amount of active molecules to obtain the desired result, namely, to allow in particular to obtain protection of the extracellular matrix of the skin.
- “topical application”, the fact of applying or spreading the active principle according to the invention, or a composition containing it, on the surface of the skin, of a mucous membrane or of the appendages.
- "physiologically acceptable" which is suitable for topical use, in contact with human skin, or for use by other routes of administration, for example the oral route or injection into the skin, without risk of toxicity, incompatibility, instability, allergic response.
- “cake”, the oil-free part of the seed after pressing. This is the solid residue from the extraction of the oil from the seeds. It is a co-product of trituration, an oil production process. It generally represents 50 to 75% of the mass of the seeds.
- "unshelled seeds", the fact that the shell, or pericarp, of the harvested seeds is maintained around the seeds.
- "predominantly alcoholic solvent", the fact that the solvent of the alcoholic type may comprise a co-solvent having sufficient characteristics to extract the active principle, knowing that pure 96° ethanol appears to be the most suitable alcoholic solvent.
- "approximately" a margin more or less of 10% to 20% in relation to the information given.
- “active molecule”, also “active principle”, the 2,5-diformylfuran molecule extracted according to the process of the invention from the seeds of Moringa peregrina . This molecule is responsible for the biological activities described in the present invention.
- “active agent” a sufficient quantity of an extract according to the invention to obtain the biological activities described. Depending on whether the extract is liquid or dried, concentrated or not, the amounts of the active agent can vary in the proportions of 0.002 to 20% by weight relative to the total weight of the composition.
- "signs of skin aging", any changes in the external appearance of the skin and appendages due to aging such as, for example, wrinkles and fine lines, withered skin, flabby skin, thin skin, lack of elasticity and/or skin tone, dull, lackluster skin or patches of skin pigmentation, hair discoloration or spots on the nails, but also any internal changes in the skin that do not consistently manifest by a modified external appearance such as, for example, any internal degradation of the skin following exposure to ultraviolet (UV) radiation.

The invention relates firstly to an extract of Moringa peregrina seeds rich in the 2,5-diformylfuran compound. The 2,5-diformylfuran molecule, also called 2,5-furandicarboxaldehyde, has never been characterized in an extract from the seeds of species of the genus Moringaceae. The Moringa peregrina species grows in very arid climates. Thus, its ability to resist drought has enabled it to acquire unique special characteristics, which the Applicant has been able to identify by implementing an extraction process adapted to whole seeds or preferentially to seed cake. .

In the context of the present invention, the plant part chosen is the seed of Moringa peregrina . It is known that the seeds of Moringa peregrina are used for the extraction of their oil called peregrina oil (INCI name "Moringa peregrina seed oil"), used regionally for self-consumption or in various traditional medicinal indications. The cake obtained after the seed has been de-oiled is a waste currently used for animal feed in particular.

According to a preferred embodiment, the extract according to the invention is obtained from unshelled whole seeds or from the cake of unshelled seeds of Moringa peregrina .

Preferably, the extract according to the invention is obtained from the cake of the unshelled seeds of Moringa peregrina after extraction of the peregrina oil (INCI name “Moringa peregrina seed oil”).

In one embodiment, the extract according to the invention is obtained by solid-liquid extraction of whole seeds or seed cake, with stirring, in a proportion of 5% to 50% by weight of solid matter relative to the weight total implemented in a predominantly alcoholic solvent, the alcohol being chosen from ethanol or methanol with optionally a co-solvent of the polyol or subcritical water type, in a proportion of 70 to 100% by weight of alcohol relative to the total weight of the solvent, at a temperature of between 4 and 50°C for a period of between 10 minutes and 4 hours, and by separation of the liquid and solid phases so as to eliminate the solid phase and recover a liquid extract of the seed of Moringa peregrina .

It should be noted that the active principle of the extract according to the invention, namely 2,5-diformylfuran, is a molecule of mixed polarity exhibiting a certain fragility. Thus, the extract obtained from the whole unshelled seed will have to be selectively extracted to obtain a high concentration of active ingredients, using the appropriate solvents and co-solvents and a temperature not exceeding 50° vs.

The cosolvents can be, for example, glycol ethers (mono- or di-propylene glycol, propanediol, and other propylene glycol derivatives, ethylene or diethylene glycol derivatives), glycerol, dimethyl ether isosorbide, methyl or ethyl or propyl esters of fatty acids; dicraprylyl carbonate, dicaprylyl ether, alkyl acetate or propionate, acetone, methyl or ethyl ketone, monoterpenes such as alpha-pinene or limonene. The proportions of these co-solvents can be mixed with the primary solvent (eg Ethanol) from 0 to 30% (V/V).

The extraction conditions can be under atmospheric pressure or under vacuum or under an inert atmosphere, but preferably in the dark at a temperature between 10 and 50°C, and more preferably at a temperature between 16 and 30°C.

In an even more preferred embodiment according to the invention, the extract is obtained from the cake of the unshelled seeds by solid-liquid extraction, with stirring, in a proportion of approximately 25% by weight of solid matter relative to the weight total implemented in a pure alcoholic solvent of 96° ethanol, at a temperature between 16 and 30° C. for a period of approximately 2 hours.

In yet another embodiment, the liquid extract obtained is dried so as to obtain a dry extract of Moringa peregrina seed cake containing more than 50% by weight of 2,5-diformylfuran relative to the total weight of the material dried.

The dry extract of Moringa peregrina seed cake contains more precisely about 55% by weight of 2,5-diformylfuran, 2.5% of furfural, 1.2% of isopropyl myristate, 4.7% of palmitic acid, 11.1% of oleic acid and 25.8% triglycerides relative to the total weight of dry matter.

The second subject of the invention is a process for obtaining an extract of Moringa peregrina seeds according to the invention comprising the following steps according to which:
a) the unshelled Moringa peregrina seeds are collected and dried to obtain an internal humidity level of less than 8%;
b) optionally pressing the dried seeds so as to separate the oil from the rest of the seed to obtain the cake,
c) the dried seed obtained in step a) or the cake obtained in step b) is ground,
d) the ground material obtained in step c) is dispersed in a proportion of 5% to 50% by weight of solid matter relative to the total weight used in a predominantly alcoholic solvent, the alcohol being chosen from ethanol or methanol optionally with a co-solvent of the polyol or subcritical water type, in a proportion of 80 to 100% by weight of alcohol relative to the total weight of the solvent;
e) a solid-liquid extraction is carried out, with stirring, at a temperature of between 4 and 50° C. for a period of between 10 minutes and 4 hours,
f) the liquid and solid phases are separated so as to eliminate the solid phase and recover a liquid extract of seed or cake of Moringa peregrina , and
g) optionally, when the alcohol is ethanol, the liquid Moringa peregrina extract obtained is dried so as to obtain a solid Moringa peregrina extract.

The unshelled seeds are collected, i.e. the shell of which is kept when the fruit matures and preferably when the pod is at the start of dehiscence.

The seeds are dried to obtain an internal humidity rate of less than 8% and preferably around 6%.

If necessary, the dried seeds are then ground extemporaneously by cold pressing which makes it possible to mechanically separate the peregrina oil (INCI name “Moringa peregrina seed oil”) from the rest of the compressed seed, namely the cake.

The seed or meal is then crushed mechanically with any type of mechanical crusher such as a hammer, flail, knife or crushing/shredding system.

The extraction is advantageously always carried out with stirring, thus allowing dispersion and homogenization of the solid in the liquid, improving the diffusion of the solute in the solvent.

To mainly extract the 2,5-diformylfuran compound of interest, an alcoholic solvent of 96° ethanol type will be preferred, but methanol can also be used with a co-solvent of polyol type or subcritical water. At the end of the extraction, the vegetable matter, residual and depleted in the compound of interest, is advantageously separated from the liquid phase by clarifying filtration. A liquid extract will advantageously be obtained from the seeds or from the cake of the seeds of Moringa peregrina comprising respectively 0.1% and 0.6% of 2,5-diformylfuran by weight of the total weight of the liquid extract.

In a preferred embodiment according to the invention, the process for obtaining a seed extract is obtained from the cake of unshelled seeds by solid-liquid extraction, with stirring, in a proportion of 25% by weight of solid matter relative to the total weight used in the pure alcoholic solvent of 96° ethanol, at a temperature between 16 and 30° C. for a period of approximately 2 hours. In this embodiment, the Moringa peregrina seeds are dried to obtain an internal humidity level of less than 8% and preferably around 6%, then pressed with a mechanical screw press, so as to separate the oil from the rest of the seed to obtain virgin oil on the one hand and cake on the other. The cake is then isolated in the form of pre-cut sausages.

In one embodiment of the method for obtaining according to the invention, the extract ofmoringa peregrinaliquid obtained is purified by distillation, microfiltration, ultrafiltration and/or nanofiltration to concentrate the extract in the 2,5-diformylfuran compound of interest with respect to the organic matter also extracted, in particular the rest of the matter extracted such as fatty substances and derivatives also extracted. These purification steps allow to concentrate the compound of interest at the expense of other extracted compounds as mentioned as well as the solvent.

In another embodiment of the method for obtaining according to the invention, the liquid extract obtained is dried so as to obtain a dry extract of the cake of Moringa peregrina seeds containing more than 50% by weight of compound 2,5- diformylfuran of interest relative to the total weight of dry matter extracted.

According to an advantageous embodiment of the invention, when the solvent is ethanol, the liquid Moringa peregrina seed extract obtained is preferably dried, for example by atomization, freeze-drying or zeodration so as to obtain a Moringa seed extract solid peregrina , the ethanol being evaporated. The drying can be carried out in the presence of a support such as maltodextrin, organic support such as maltodextrin, cyclodextrin or inulin, or in the presence of an inorganic support such as phyllosilicate, magnesium silicate or carbonate and its salts.

The invention also relates to the extract of the seeds of Moringa peregrina capable of being obtained by the process for obtaining according to the invention.

A third subject of the invention is a cosmetic or nutracosmetic composition comprising, as active agent, an effective amount of an extract of seeds of Moringa peregrina according to the invention, and a physiologically acceptable excipient.

The composition according to the invention can be formulated in the form of various preparations suitable for topical administration or for oral administration.
According to a first variant, the various preparations are suitable for topical administration and include creams, oil-in-water and water-in-oil emulsions, milks, ointments, lotions, oils, balms, aqueous or hydro - alcoholic or glycolic, serums, powders, patches, sprays or any other product for external application such as, for example, medical devices or cosmetic-textile products.
According to a second variant, the different preparations are suitable for oral administration; the plant extract comprising the active compound 2,5-diformulfuran being able to enter either into a food composition or into a food supplement. The food supplement may be in the form of capsules or soft gelatin or vegetable capsules within the scope of the present invention. Said food supplement can then contain from 0.01 to 100% by weight of the plant extract.

In the context of food use, for nutritional or cosmetic purposes (cosmeto-food or nutricosmetics), the composition will advantageously be formulated in the form of a preparation suitable for oral administration. It may not include an excipient and may consist entirely of the plant extract comprising the active compound 2,5-diformylfuran.

According to a preferred embodiment, the compositions according to the invention are intended more particularly for topical administration. These compositions must therefore contain a cosmetically acceptable medium, that is to say compatible with the skin and the integuments, and cover all cosmetic forms. These compositions may in particular be in the form of creams, oil-in-water or water-in-oil emulsions or multiple emulsions, serums, solutions, suspensions, gels, milks, lotions, sticks or even powders, and suitable for application on the skin, lips and/or appendages. These compositions include the excipients necessary for their formulation, such as solvents, emollients, thickeners, diluents, surfactants, antioxidants, bioactive agents, dyes, preservatives, fragrances. They can be used as a care product and/or as a make-up product for the skin.

The composition according to the invention may in particular consist of a composition for hair care, and in particular a shampoo, a conditioner, a treatment lotion, a styling cream or gel, a restructuring lotion for the hair, a mask, etc. The cosmetic composition according to the invention can be used in particular in treatments implementing an application which may or may not be followed by rinsing, or else in the form of a shampoo. The composition according to the invention can advantageously be used in anti-dandruff care. It may also be in the form of a dye or a mascara to be applied with a brush or a comb, in particular on the eyelashes, the eyebrows or the hair.

The compositions according to the invention further comprise any additive commonly used in the field of application envisaged as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, cosmetic or pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

The INCI Dictionary & Handbook ("International Nomenclature of Cosmetic Ingredients (13th Ed. 2010) published by the Personal Care Products Council, Inc.", Washington, D.C.) describes a wide variety, without limitation, of cosmetic and pharmaceutical ingredients commonly used in the skin care industry, which are suitable for use as additional ingredients in the compositions according to the present invention.

In all cases, those skilled in the art will ensure that these adjuvants and their proportions are chosen in such a way as not to harm the desired advantageous properties of the composition according to the invention.

According to an advantageous embodiment of the invention, the quantity or content of plant extract in the composition according to the invention is from 0.002 to 20% by weight, and in particular from 0.2 to 10% by weight relative to the total weight of the composition.

Finally, the fourth object of the invention is the cosmetic or nutricosmetic use of a composition according to the invention for improving the appearance of the skin, mucous membranes and appendages, for relaxing, soothing and de-stressing the skin and preventing and/or or to fight against the cutaneous signs of aging and/or photo-aging and prevent age spots.

According to one embodiment, the purpose of the use according to the invention is more particularly to relax, soothe and de-stress the skin and to fight against the signs of skin ageing.

According to another embodiment, the purpose of the use according to the invention is to prevent the appearance of aging spots.

Although the invention has been described in connection with several particular embodiments, it is obvious that it is in no way limited thereto and that it includes all the technical equivalents of the means described as well as their combinations if these fall within the scope of the invention.

The use of the verb "to comprise", "to understand" or "to include" and of its conjugated forms does not exclude the presence of other elements or other steps than those set out in a claim.

In the claims, any reference sign in parentheses cannot be interpreted as a limitation of the claim.

Examples

Example 1: Preparation of a plant extract from the cake of moringa peregrina

Seeds of Moringa peregrina (Forssk.) Fiori were dried to obtain an internal humidity rate of less than 8% and preferably around 6%, then pressed with a mechanical screw press, so as to separate the oil from the rest of the seed to obtain virgin oil on the one hand and cake on the other. The cake is then isolated in the form of sausages pre-cut into 1 to 2 cm pieces. From the cake, maceration and extraction are carried out with 96° ethanol preheated for 10 min at 55° C. in the ratio 25/75% (m/m), sheared with a mixer for 15 min then left to stir at the propeller for 2 hours at a temperature between 16°C and 30°C. A Büchner filter is then filtered under vacuum and a pale yellow filtrate containing 1.15% of dry matter is obtained. The liquid extract obtained is subsequently called “peregrina extract according to the invention ” or “peregrina extract” .

This peregrina extract according to the invention contains 1.15% of dry matter comprising itself (results expressed in relation to the dry matter (DM):

Active compounds MS total compounds % Furfural 2.528 57.18 2,5-Furandicarboxaldehyde 54.66 Fats of plant origin Isopropyl myristate 1.175 42.812 palmitic acid 4.713 Oleic acid 11.093 Trigycerides 25.831 The dry extract described above is obtained by a gravimetric method based on the mass before and after evaporation present in the liquid extract.
2,5-Furandicarboxaldehyde or 2,5-diformylfuran is the active principle of this extract. It was assayed by chromatographic method, and more precisely by gas chromatography coupled with a flame ionization detector.
Furfural was dosed using an identical method
Isopropyl Myristate was assayed using an identical method
Palmitic and oleic acid were assayed using an identical method
Triglycerides were isolated by ultracentrifugation.

Example 2: E effect of extract peregrina according to the invention as antioxidant

The objective of this study is to evaluate the modulation of the antioxidant activity by the peregrina extract in an acellular in vitro colorimetric model using the radical DPPH (2,2-diphenyl-1-picrylhydrazyl) as well as to the reference antioxidant, ascorbic acid. The method used is called inhibition. It is based on the degradation of the oxidizing radical DPPH, with a violet color absorbing at 540 nm, by a reference antioxidant, ascorbic acid. This reaction serves as a positive control and leads to the formation of the DPPH compound which is colorless or even light yellow. The peregrina extract according to the invention and the “Ascorbic Acid” reference product are brought into contact with the DPPH solution for 30 min at 40°C. The antioxidant activity is then evaluated by measuring absorbance at 540 nm. The modulation of this activity is expressed as a percentage of stimulation of the antioxidant activity by the active ingredient tested, with the maximum antioxidant activity obtained in the presence of ascorbic acid (T+) as a reference.

Protocol: A DPPH solution is incubated for 30 minutes at 40°C, in the absence (control), in the presence of the peregrina extract according to the invention (T+) and at decreasing concentrations of the sample to be tested. At the end of the incubation period, the antioxidant activity in the presence of the reference product and in the presence or absence of the peregrina extract was revealed by staining after 30 minutes at 40°C. It was thus evaluated by measuring the absorbance of the reaction medium at 540 nm. For each concentration tested, the modulation of the antioxidant activity by the product tested is calculated according to the following formula.

Percentage of modulation of the antioxidant activity = 100 x [(DO540 Control – DO540 Product to be tested) / DO540 Reference product].

If the result is negative, the product to be tested will be considered as oxidizing; if the result is positive, the percentage will be expressed as stimulation of the anti-radical activity. The results obtained are given below.

DPPH inhibition Peregrina extract according to the invention 2% 36 1.0% 23 0.1% 5

Conclusion: the peregrina extract according to the invention is capable of protecting against free radicals, it has significant antioxidant properties from a concentration of 1%.

Example 3: Effect of extract peregrina according to the invention as inhibitor of m et al I oproteases

The objective of this study is to evaluate the modulation of the anti-metalloprotease activity by the peregrina extract according to the invention in an acellular in v i t r o model using a type I collagenase and a hyaluronidase, a substrate complex and a chromophore, Ninhydrin. A buffered solution of collagenase type I and hyaluronidase reacts with a specific substrate complex and converts it to form a compound capable of activating a chromophore by incubation at 80°C for 15 min. The activity of collagenase and hyaluronidase can thus be evaluated by measuring absorbance at 565 nm. The sample is brought into contact with the solution of collagenase and hyaluronidase at the same time as the substrate complex of the enzymes at 37° C. for 5 minutes. The substrate transformed by the enzymes is capable of activating the chromophore by incubation at 80° C. for 15 minutes. The collagenase and hyaluronidase activity in the presence/absence of the sample is then evaluated by measuring the absorbance at 565 nm. The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of collagenase and hyaluronidase in the absence of active ingredient, that is to say only in the presence of the substrate of the enzyme.

Protocol: A solution of type I collagenase and hyaluronidase enzymes is incubated in its substrate, for 5 minutes, in the absence or presence of the peregrina extract according to the invention tested. The solutions are then brought into contact with the chromogen, Ninhydrin, before incubation for 15 minutes at 80°C. At the end of the incubation period, the activity of collagenase and hyaluronidase enzymes with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 565 nm. For each concentration tested, the modulation of the activity of collagenase and hyaluronidase enzymes by the product under test is calculated according to the following formula.

Percentage of modulation of collagenase/hyaluronidase enzyme activity = 100 x [(DO product under test or reference – DO collagenase/hyaluronidase alone)/OD collagenase/hyaluronidase alone].

If the result is negative, the percentage is expressed as enzyme inhibition; if the result is positive, the percentage is expressed as enzyme activation. The results of the inhibition of metaloproteases by the peregrina extract according to the invention are given below.

Inhibition versus Control (%)
Peregrina extract according to the invention 1% 87 0.5% 87 0.1% 88 0.01% 62

Conclusion: The peregrina extract according to the invention generates a strong inhibition of metaloproteases (collagenase / hyaluronidase) of 62% at very low levels of 0.01%. The peregrina extract according to the invention is capable of strongly inhibiting these metaloproteases and has good potential for protecting the extracellular matrix of the skin with great efficiency and, through this inhibition, it shows an anti-aging effect.

Example 4 : Effect of extract peregrina according to the invention for inhibit st enzymes Hi stones Disabled tylases ( HDACs ) And Sirtuin I

The objective of this study is to demonstrate the inhibitory activity of the peregrina extract according to the invention on the HDACs and Sirtuins I enzymes. A buffered solution of HDACs & Sirtuins I reacts with a substrate for 20 minutes at 37°C and transforms it to form a compound which is colored in the presence of a developer after incubation at 37° C. for 10 minutes. The maximum deacetylation activity of Sirtuins can thus be evaluated by measuring the absorbance at 405 nm. The peregrina extract according to the invention or the reference product “Trichostatin A (STA) inhibitor 1 μM” are brought into contact with the solution of Sirtuins at the same time as the substrate of the enzyme for 20 minutes at 37° C., the substrate transformed by the enzyme is colored by the addition of a developer. The deacetylating activity of HDACs and Sirtuin I in the presence of the active ingredient is then evaluated by measuring the absorbance at 405 nm. The modulation of this activity is expressed as a percentage of inhibition or activation of the maximum activity of HDACs & Sirtuin I in the absence of active ingredient, that is to say only in the presence of the substrate of HDACs & Sirtuin I enzymes. .

Protocol: A solution of Sirtuins enzymes is incubated in its substrate for 20 min in the absence (control) or presence of the reference product, or of increasing concentrations of the products under test. The peregrina extract according to the invention is tested at the following concentrations: 2%; 1%; 0.1% (V/V). At the end of the incubation period, the activity of the Sirtuins enzymes with and without test or reference product was revealed by staining using a developer solution (10 min at 37°C) and evaluated by measuring the absorbance of the reaction media at 405 nm. For each concentration tested, the modulation of the deacetylating activity of the enzymes Histones Deacetysases and Sirtuins I by the product under test is calculated according to the following formula.

Percentage of modulation of the activity of the Sirtuin enzymes = 100×[(DO _{4 0 5} trial or reference product) – (DO _{4 0 5} HDACs & Sirtuin I alone)]/OD _{4 0 5} Sirtuin alone.

If the result is negative, the percentage is expressed as inhibition of the enzymatic reaction; if the result is positive, the percentage is expressed as activation of the enzymatic reaction. The results of the inhibition of Histones Deacetylases (HDACs) enzymes are given below.

Peregrina extract according to the invention
percentage
Inhibition versus Control (%) 2% 20 1% ns 0.10% -18

Conclusion: At 2% the peregrina extract according to the invention shows a significant inhibition of HDACs; this inhibition means the ability to promote the self-protection of skin cells against genetic drift, especially related to the aging process. Thus the extract appears to be useful against one of the most common genetic drifts on the surface of the skin, namely fibrosis which is manifested by the appearance of a “button” of flesh (fibrotic protuberance). The extract can advantageously interfere with the phenomena of fibrosis on the surface of the skin and thus prevent the aging of the skin.

Example 5: Effect of extract peregrina according to the invention to modulate the anti-inflammatory activity of the enzyme phospholypase-A2.

The objective of this study is to evaluate the modulation of the anti-inflammatory activity of the enzyme phospholipase A2 by one or more samples in an acellular In Vitro model using the "SPLA2 (type V) Inhibitor" analysis kit. Screening Assay Kit”. Phospholipase A2 is a key enzyme upstream of the inflammatory process triggered by the arachidonic cascade. A buffered solution of Phospholipase A2 reacts with a specific substrate, diheptanoyl thio-PC, and converts it to form a compound which binds to a chromogen, DTNB under stirring at room temperature. The activity of phospholipase A2 can thus be evaluated by measuring the absorbance at 413 nm. The peregrina extract according to the invention or the inhibitor reference product "Thioetheramide-PC" are brought into contact with the solution of Phospholipase A2 at the same time as the substrate of the enzyme. The substrate transformed by the enzyme is stained using the chromogen DTNB by stirring at room temperature. The activity of the peregrina extract according to the invention or of the reference product is then evaluated by measuring the absorbance at 413 nm. The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Phospholipase A2 in the absence of active ingredient, that is to say only in the presence of the substrate of the enzyme (diheptanoyl thio- pc).

Protocol: A solution of Phospholipase A2 enzyme is incubated in its substrate, diheptanoyl thio-PC, in the absence or presence of the reference inhibitor and the peregrina extract according to the invention, tested at the following concentrations: 2%; 1%; 0.1% (V/V), then the chromogen DTNB is incorporated before incubation for 15 minutes at 25°C. At the end of the incubation period, the activity of the Phospholipase A2 enzyme with and without test or reference product is evaluated by measuring the absorbance of the reaction media at 413 nm. For each concentration tested, the modulation of the activity of the Phospholipase A2 enzyme by the product under test is calculated according to the following formula.

Percentage modulation of the activity of the Phospholipase A2 enzyme = 100 x [(OD _{40 5} test or reference product – OD _{4 0 5} sPLA2 alone) / OD _{4 0 5} sPLA2 alone].

If the result is negative, the percentage is expressed as enzyme inhibition; if the result is positive, the percentage is expressed as enzyme activation. The results of modulation of the anti-inflammatory activity of the enzyme phospholypase-A2 are given below.

Inhibition Versus
Control (%) Peregrina extract according to the invention 2% 19 1% 16 0.10% 11

Conclusion: The peregrina extract according to the invention generates a slight stable inhibition of PLA2 from a dose of 0.1% but more preferably at 1 or 2%. This means that the peregrina extract according to the invention has the ability to reduce the arachidonic cascade / inflammation cascade very upstream; this extract thus has a good soothing or relaxing potential on the skin.

Example 6 : Effect of extract peregrina according to the invention For inhibit the action of endothelin 1

The objective is to assay type 1 endothelin in human microvascular endothelial cells after exposure for 24 hours to the peregrina extract according to the invention.

Protocol: Human microvascular endothelial cells were supplied by PELOBiotech and cultured in 96-well plates according to the supplier's production procedures. This involves letting the extracts act at different concentrations on the endothelial cells at 80% confluence for 24 hours, then quantifying endothelin 1 using the ELISA PicoKine (EDN1) kit in the cell supernatants. A viability test is carried out beforehand to define the non-toxic doses to be used when assaying endothelin 1. The negative control is carried out using cells in culture medium without treatment. The positive control in the viability test is 0.5% SDS. All the conditions are prepared in culture media, and the cells are then incubated at 36.5°C / 5% CO2 for 24 hours.
a) Application of the solutions to be tested on the endothelial cells:
The products to be tested are brought into contact with the endothelial cells in sub-confluence in the 96-well plates. For each concentration, the test is carried out on 3 wells. The plates are incubated for 24 hours ± 1 hour at 36.5°C / 5% CO2.
b) Viability test:
The cell viability is evaluated by the MTT method on the cells after incubation with the products. After 24 hours of incubation, the supernatants are recovered and stored at -20° C. for the assays. The wells are then rinsed once with 200 μL of PBS. 50 μL of 0.5 mg/ml MTT solution are added to each well: incubation for 3 hours at 36.5° C./5% CO2. In each well 100 μL of isopropanol are added. After homogenization, a reading of the absorbance is carried out at 550 nm. For each condition, the ratio of the mean of the optical densities of the cells to the mean of the optical densities of the negative controls will determine the viability rate.
c) Dosage of endothelin 1:
The assay is carried out using the ELISA kit.

Concentrated extract
Cell growth versus control (%)
Endothelin 1 versus control (%) Endothelin 1 versus control ( pg/ml) Peregrina extract according to the invention 5% -22.31 -53.21 -71.8 1% 0 -11.61 -15.66 0.10%
-5.77 -25.11
-33.88

Conclusion: The viability test carried out at the end of treatment did not show any toxic effect for the concentrations tested. Endothelin 1 is measured in cell supernatants at non-toxic concentrations. The amount of endothelin 1 for each condition is assayed using the ELISA kit. For the cells of the negative control, the values are of the order of 134.94 pg/ml. For the cells treated with different concentrations of extracts, the values are from 63.14 pg/ml (with 5% of the extract according to the invention) to 101.06 pg/ml (with 0.1% of the extract according to the invention) , which shows very significant inhibitions from 0.1% of the extract according to the invention with approximately 25% inhibition of type 1 endothelin production and up to 53% inhibition with 5% of the extract according to the 'invention.

Example 7 : Effect of extract peregrina according to the invention For stimulate the activity of telomerase

Endothelin is the most potent vasoconstrictor known in the human body. On the other hand, the decrease in endothelin is also known to create a vasodilating effect [ H irata Y et al. , 1988, Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells, B ioch e mical and B ioph y s ical R e s earch C om m u n unication 154: 3, p. 868-875,] [ S halinkumar P et al . , 2018, H2S Mediates the Vasodilator Effect of Endothelin-1 in the Cerebral Circulation. A merican J ournal of P y siology. H e a r t and C i r culatory P h ysioliogy 315, p. 1759-1764].

Telomeres are protective DNA-protein complexes located at the end of linear chromosomes that promote chromosomal stability. Telomere shortness in humans is emerging as a prognostic marker for disease risk and progression, and premature mortality in many types of cancers, including breast, prostate, colon, bladder , head and neck, lung and kidney cells [ Ornish D., 2008, Increased Telomerase Activity and Comprehensive Lifestyle Changes: a Pilot Study, Lancet Oncology 9, p. 1048-1057]. Telomere shortening is counteracted by the cellular enzyme telomerase.

The objective of the study is to evaluate the effect of the compound named “Peregrina extract according to the invention” on telomerase activity in a model composed of adult human keratinocytes at low passage level in monolayer culture.

Protocol: Human keratinocytes were obtained from a 49-year-old donor. To perform the experiments, keratinocytes were used at a low passage level (i.e., passage #2 of cell isolation). Cells were cultured in a monolayer until they reached approximately 75% confluence before being used in the experiment.
Reference Product: FK228 at 100 ng/ml was used as the reference inducer of telomerase1 activity.
Incubation protocol: The cells were incubated for 24 hours in the absence (control) or in the presence of reference product or of increasing concentrations of test compounds such as the peregrina extract according to the invention at: 0.5; 1 and 5% (v/v).
The peregrina extract according to the invention is diluted directly in the incubation medium to reach the different concentrations described above.
Assessment of effects:
- Protein measurement
At the end of the incubation period, the total cell proteins were extracted from the cells and measured using a spectro-colorimetric method (Bradford method). This measurement makes it possible to determine the exact volume of extract to be used during the measurement of telomerase activity, in order to maintain the same quantity of proteins (containing telomerase) for all the conditions tested at the level of the PCR step.
- Measurement of telomerase activity
At the end of the incubation period, telomerase was extracted from the cells and its activity was determined using a specific and sensitive kit. The principle of the telomerase kit is to measure telomerase activity coupling a PCR step (in which telomerase works on its elongation activity) with an ELISA step allowing the semi-quantitative determination of amounts of telomerase elongation product .
- Statistics
Results are expressed in arbitrary units for telomerase activity level (mean ± SD). The level of significance between "vehicle" and "reference product" was evaluated using a Student's t test (*: p <0.05). The level of significance between "control" and "test compound" was assessed independently for each product by one-way analysis of variance (one-way ANOVA) followed by Holm-Sidak test (*: p<0.05 ).
The peregrina extract according to the invention, tested at 0.5% and 1% (v/v), did not significantly modulate the activity of telomerase compared to “Control”. Tested at 5% (v/v), the peregrina extract according to the invention significantly increased the activity of telomerase by 18.9% (p<0.001), in comparison with “Control”.
The reference product, called “FK228”, tested at 100 ng/ml, significantly increased telomerase activity by 28.0% (p <0.01). This result was expected and validates the experiment. The results of stimulation of telomerase activity are given below.

Extract strength Cell growth versus control (%) Telomerase activity versus control (%) Peregrina extract according to the invention 5.00% +5.8 +18.90 1.00% That. -2.2 +4.00 0.50% -2.6 +2.30

Conclusion the peregrina extract according to the invention, tested at 0.5% and 1% (v/v), did not significantly modulate the activity of telomerase compared to "Control". Tested at 5% (v/v), the peregrina extract according to the invention significantly increased the activity of telomerase by 18.9% (p<0.001), in comparison with "Control". The extract according to the invention acts directly on this enzymatic pathway which builds protective telomeres at the ends of chromosomes and slows down the natural aging of genetic material. The extract according to the invention therefore makes it possible to produce an anti-aging effect on the chromosomes.

Examples 2 to 7 demonstrate that the peregrina extract according to the invention has the profile of a good skin protector, which gives it anti-ageing, anti-stress and relaxing properties.

Example 8 : Effect of extract peregrina according to the invention For stimulate tyrosinase activity

The objective of this study is to evaluate the activity on the Tyrosinase enzyme of the peregrina extract according to the invention in an acellular in vitro model using a tyrosinase enzyme of fungal origin (Sigma Aldrich ref. T3824), to its substrate L-Tyrosine (Sigma Aldrich ref. T3754) and a reference inhibitor hydroquinone (Sigma Aldrich ref. H17902, Inhibitor=2.5 mM Hydroquinone). A buffered solution of Tyrosinase reacts with a 2.5 mM Tyrosine L substrate for 60 minutes at 23°C and converts it to form a colored compound. The maximum activity of Tyrosinase can thus be evaluated by measuring the absorbance at 475 nm. The peregrina extract according to the invention or the "Hydroquinone" reference product are brought into contact with the Tyrosinase solution at the same time as the enzyme substrate for 60 minutes at 23° C., the substrate transformed by the enzyme is naturally colored. Tyrosinase activity in the presence of the active ingredient is then evaluated by measuring the absorbance at 475 nm. The modulation of this activity is expressed as a percentage of inhibition or activation of the maximum activity of Tyrosinase in the absence of active ingredient, that is to say only in the presence of the substrate of the enzyme (L Tyrosine) .

Protocol: A Tyrosinase enzyme solution is incubated in its L Tyrosine substrate for 60 minutes in the absence (control) or presence of the reference product, or of increasing concentrations of the peregrina extract according to the invention/concentrations; 2%; 1%; 0.1% (V/V). At the end of the incubation period, the activity of the Tyrosinase enzyme with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 475 nm. For each concentration tested, the modulation of the activity of the enzyme Tyrosinase by the product under test is calculated according to the following formula: Percentage of modulation of the activity of the enzyme Tyrosinase = 100 x [(DO475 produced at reference) – (DO475 Tyrosinase alone)] /DO475 Tyrosinase alone.

If the result is negative, the percentage is expressed as enzyme inhibition; if the result is positive, the percentage is expressed as enzyme activation. The stimulation results on tyrosinase activity are given below.

Activating Versus
Control (%) Extract peregrina
according to the invention 2% 65 1% 37 0.10% 8

Conclusion: The peregrina extract according to the invention is capable of lowering the level of basal activity of tyrosinase, which makes it possible to indicate that this extract has the capacity to increase one of the natural protections of the skin, the protection against ultraviolet rays.

Example 9 : Effect of extract peregrina according to the invention For inhibit melanin production

The objective of this study on human cell cultures is to bring together all the data used, as well as the results obtained, for carrying out the melanin modulation test on human melanocytes after exposure for 5 days to the peregrina extract. according to the invention.
Protocol: Human melanocytes are cultured in 96- and 24-well plates.
This involves allowing the peregrina extract according to the invention to act at concentrations of 5%; 2%; 1% and 0.1% on confluent melanocytes for 5 days. A pre-test of viability with MTT after 24 hours makes it possible to evaluate the cytotoxicity and to choose the concentrations for the melanin modulation test. This modulation is evaluated by measuring the melanin in the cell lysates after 5 days of exposure to the extracts. The negative control is carried out using cells in culture medium without treatment. The positive control for the viability test is 0.5% SDS. For the melanin modulation test, media with and without α–MSH are used as negative controls.
All the conditions are prepared in culture media, and the cells are then incubated at 36.5°C / 5% CO2 for 24 hours for the cytotoxicity test and 5 days for the melanin assay.
a) Application of the solutions to be tested on the melanocytes: The concentrations to be tested are brought into contact with the confluent melanocytes in the 96 (cytotoxicity test) and 24 (melanin assay) well plates. For each concentration, the test is carried out on 3 wells. The plates are incubated for 24 ± 1 hours and 5 days at 36.5°C / 5% CO2.
b) Viability test: The cell viability is evaluated by the MTT method on the cells after 24 hours of incubation with the products. After 24 hours of incubation, the planned wells are rinsed once with 200 μL of PBS. In each well, 50 μl of MTT solution at 0.5 mg/ml are added and the incubation is carried out for 3 hours at 36.5°C / 5% CO2. In each well 150 μl of isopropanol are added. After homogenization, a reading of the absorbance is carried out at 550 nm. For each condition, the ratio of the mean of the optical densities of the cells to the mean of the optical densities of the negative controls will determine the viability rate.
A viability cut-off value of 70% relative to the negative control value is used to classify the substances tested as cytotoxic or non-cytotoxic. A “non-cytotoxic” classification is given on the resultsin vitrofor a viability > 70% and a “cytotoxic” classification for a viability ≤ 70%.
The 5% concentration of the extract according to the invention was shown to be cytotoxic at 5% under the test conditions. The 2%, 1% and 0.1% concentrations are therefore used for the melanin modulation test. The amount of melanin present in the cells is assayed after cell lysis. The results on the inhibition of melanin production are given below.

Concentrated extract

Cell growth versus control (%)

Melanin inhibition versus control (%)

Melanin content
( µg/ml) Peregrina extract according to the invention 2% -3.41 +16.50 152 1.0% +13.46 +65.90 62 0.10%
+13.29
+71.40
52

Conclusion: the peregrina extract according to the invention inhibits the production of melanin in cellulo , which gives it a protective property for the skin. This inhibition also shows a relaxing effect on the melanocytes because the basal level is lower than the control (absence of extract according to the invention in the culture medium); we recall that the production of melanin is a response to cellular stress. Thus, the peregrina extract according to the invention demonstrates the ability to prevent aging spots.

Example 10 : Analytical characterization of I' extract peregrina according to the invention versus I' extract oh leifera with A process extraction identical
Based on cake ofmoringa peregrinaand that ofmoringa oleifera, the extraction process according to the invention described in Example 1 was applied. The comparative composition of the extracted ingredients on a dry matter basis is given below. Compounds Oleifera (%) Peregrina (%) Acetic acid 1.431 - 1-hydroxy-2-propanone 1.962 - Furfural 6.140 2.528 2,5-Furandicarboxaldehyde (DFF) 0.823 55.660 Isopropyl myristate 0.128 1.175 Methyl palmitate 0.230 _ palmitic acid 10.356 4.713 Ethyl Palmitate 0.399 _ Methyl linoleate 0.428 _ Methyl oleate 2.443 _ Oleic acid 47.844 11.093 Ethyl oleate 2.675 _ Stearic acid 5.064 _ Bis 2-ethyl hexyl hexanedioate 1.688 _ Methyl 7-oxo-dehydroabietate 0.327 _ 2-ethyl-hexyl 1,4-terephthalate 0.655 _ Total 84.317 74.169 It appears that the two extracts have a very different molecular profile. The extract frommoringa oleiferacontains less than 1% DFF while the extract ofmoringa peregrinacontains more than 50%.

Example 11 : Comparative test with moringa oleifera For test in tube collagenase

The objective of this study is to evaluate the modulation of the anti-Metalloprotease activity by the peregrina extract according to the invention in an acellular in vitro model using a type I collagenase and a hyaluronidase, a substrate complex and a chromophore, Ninhydrin. A buffered solution of collagenase type I and hyaluronidase reacts with a specific substrate complex and transforms it to form a compound capable of activating a chromophore by incubation at 80°C for 15 min. The activity of collagenase and hyaluronidase can thus be evaluated by measuring absorbance at 565 nm. The sample is brought into contact with the solution of Collagenase and Hyaluronidase at the same time as the substrate complex of the enzymes at 37° C. for 5 minutes. The substrate transformed by the enzymes is capable of activating the chromophore by incubation at 80° C. for 15 minutes. The Collagenase and Hyaluronidase activity in the presence/absence of the sample is then evaluated by measuring the absorbance at 565 nm. The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Collagenase and Hyaluronidase in the absence of active ingredient, that is to say only in the presence of the substrate of the enzyme.

Protocol: A solution of collagenase type I and hyaluronidase enzymes is incubated in its substrate, for 5 minutes, in the absence or presence of the peregrina extract according to the invention tested. The solutions are then brought into contact with the chromogen, Ninhydrin, before incubation for 15 minutes at 80°C. At the end of the incubation period, the activity of the Collagenase and Hyaluronidase enzymes with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 565 nm. For each concentration tested, the modulation of the activity of the Collagenase and Hyaluronidase enzymes by the product under test is calculated according to the following formula.

Percentage of modulation of the activity of the Collagenase/Hyaluronidase enzymes = 100 x [(OD test or reference product – DO Collagenase/Hyaluronidase alone)/OD Collagenase/Hyaluronidase alone].

If the result is negative, the percentage is expressed as enzyme inhibition; if the result is positive, the percentage is expressed as enzyme activation. The result of Metalloprotease inhibition is given below.

Inhibition versus Control (%)
Peregrina extract according to the invention 1% 87 0.5% 87 0.1% 88 0.01% 62

Conclusion: The peregrina extract according to the invention generates a strong inhibition of Metalloproteases (Collagen / Hyaluronidase). It is capable of inhibiting these Metalloproteases by 88% from a concentration of 0.1% and has a good potential to protect the extra-cellular matrix of the skin with great efficiency and, through this inhibition, it shows an anti-aging effect.

To be compared with the extract according to the patent FR2946879 of Pierre Fabre of which here are the results according to the same test.

Inhibition versus Control (%)
Moringa oleifera extract according to Pierre Fabre patent 1% Concentration not compatible with test system 0.5% 4 0.1% 24 0.01% 42

Conclusion the extract according to the Pierre Fabre patent shows a slight inhibitory action on collagenase activity in inverse dose-dependence with a peak inhibition of 42% at all concentrations combined against a peak inhibition of 100% for the extract peregrina according to the invention.

The anti-aging activity on this parameter appears different and new compared to the effects observed with the extract according to the Pierre Fabre patent.

Example 12: Comparative tests p for anti-stress activity (by inhibition of PLA2).

For the anti-stress activity by inhibition of PLA2 on the skin which provides a soothing/anti-stress orientation with an anti-aging effect, two additional PLA2 in-tubo tests were carried out; one with the process according to the invention carried out on oleifera cake (extract made identically to that on peregrina in the process according to the invention), one with the extract corresponding to document FR2946879 from Pierre Fabre, another again corresponding to patent FR3076460 from BASF Beauty Care Solutions with the product Purisoft® and finally a last one with document FR2825267 from Chuun & Thurot.

The objective of this study is to evaluate the modulation of the anti-inflammatory activity of the enzyme phospholipase A2 by one or more samples in an acellular In Vitro model using the "SPLA2 (type V) Inhibitor" analysis kit. Screening Assay Kit”.

A buffered solution of Phospholipase A2 reacts with a specific substrate, diheptanoyl thio-PC, and converts it to form a compound which binds to a chromogen, DTNB under stirring at room temperature. The activity of phospholipase A2 can thus be evaluated by measuring the absorbance at 413 nm.

The “Peregrina extract according to the invention” products or the inhibitor reference product “Thioetheramide-PC” are brought into contact with the Phospholipase A2 solution at the same time as the enzyme substrate. The substrate transformed by the enzyme is stained using the chromogen DTNB by stirring at room temperature. The activity of Phospholipase A2 in the presence/absence of the product “Peregrina extract according to the invention” or of the reference product is then evaluated by measuring the absorbance at 413 nm.

The modulation of this activity is expressed as a percentage of inhibition or activation of the activity of Phospholipase A2 in the absence of active ingredient, that is to say only in the presence of the substrate of the enzyme (diheptanoyl thio- pc).

The "Thioetheramide-PC" inhibitor at a concentration of 1mg in 100µl is the reference product (active control) in this study, the latter inhibiting the activity of PLA2 by 93%, which validates the test.

A solution of Phospholipase A2 enzyme is incubated in its substrate, diheptanoyl thio-PC, in the absence or presence of the reference inhibitor and of the product "Peregrina extract according to the invention" tested, then the chromogen DTNB is incorporated before incubation of 15 minutes at 25°C.

At the end of the incubation period, the activity of the Phospholipase A2 enzyme with and without test or reference product was evaluated by measuring the absorbance of the reaction media at 413 nm. For each concentration tested, the modulation of the activity of the enzyme Phospholipase A2 by the product under test is calculated according to the following formula:

Percentage modulation of Phospholipase A2 enzyme activity = 100 x [(DO405 test or reference product – DO405 sPLA2 alone) / DO405 sPLA2 alone]

If the result is negative, the percentage is expressed as enzyme inhibition; if the result is positive, the percentage is expressed as enzyme activation.

Peregrina extract according to the invention 1% 19 0.1% 16 0.01% 11 The constancy and the substantially dose-dependent activity of this peregrina extract according to the invention on the inhibition of PLA2 highlight a soothing activity by reducing the intensity upstream of the arachidonic cascade. Such a soothing action very upstream of the arachidonic cascade reduces the impact of basal physiological stress. The peregrina extract according to the invention is therefore an anti-stress.

With the protocol extract from patent FR2946879 of Pierre Fabre, we obtain the following results.

Excerpt from Pierre Fabre 1% 0 0.1% 16 0.01% 8 At 1%, which corresponds to its minimum use dosage, the extract shows no activity. At dilution an activity appears but the lack of dose-dependent effect does not allow to validate a specific and reliable action of the extract on the inhibition of this enzyme.

With the protocol extract from patent FR3076460 of BASF Beauty Care Solutions with the product Purisoft® LS9726, the following results are obtained.

Extract BASF
Purisoft LS9726 1% 10 0.1% 12 0.01% 6 This extract shows a slight inhibitory action which does not reach the maximum observed at 1% with the peregrina extract according to the invention, namely 19% inhibition for the extract according to the invention against 10% inhibition at 1% for this extract.

With the protocol extract from patent FR2825267 of Chuun & Thurot we observe the following results.

Excerpt from Chuun & Thurot 1% 0 0.1% 0 0.01% 0 This extract does not show any inhibitory action on this enzyme.

With the protocol extract resulting from the process according to the present invention but applied to the cake of Moringa oleifera instead of the cake of Moringa peregrina , we observe the following results.

Moringa oleifera extract 1% Not dosable 0.1% 0 0.01% 6 This extract does not show any significant or stable inhibitory action on this enzyme.

Example 13 : Formulation of a make-up product

Ingredients % Water QSP Caprylic / Capric triglyceride 19.0000 Senegal Acacia gum 7.0000 Charcoal 6.0000 Glycerin 5.0000 Propanediol 5.0000 Bentonite 3.1500 Cetearyl glucoside 3.0000 Cetearyl alcohol 3.0000 Benzylalcohol 1.0000 Peregrina extract according to invention 2.0000 Cellulose Gum 0.8000 Xanthan gum 0.1750 Citric acid 0.1750

Example 14: Formulation of a washing product

Ingredients % Water QSP Sodium coco-sulfate 5.0000 Sodium cocoyl isethionate 4.0000 Bentonite 3.7800 Caprylic / Capric triglyceride 2.0000 Peregrina extract according to the invention 1.0000 Gluconolactone 0.7500 Sodium benzoate 0.5450 Scent 0.5000 Xanthan gum 0.2700 Sodium stearoyl glutamate 0.2250 Citric acid 0.2250 Calcium gluconate 0.0050

Example 15: formulation of a care product (anti-aging anti-stress cream)

Ingredient % Water QSP Caprylic / Capric triglyceride 18.0000 Bentonite 4.2000 Cetearyl alcohol 1.5000 Peregrina extract according to invention 5.0000 Gluconolactone 0.7500 Sodium benzoate 0.5450 Xanthan gum 0.5000 Scent 0.5000 Sodium stearoyl glutamate 0.2500 Citric acid 0.2500 Calcium gluconate 0.0050

Example 16: oral formulation for nutrition icosmetics

A 1 g tablet for soothing/anti-stress activity comprises: 3% dry extract according to the invention (containing 0.6% of 2,5-diformylfuran on an inulin carrier) + 47% of calcium carbonate containing 200 IU of vitamin D + 25% magnesium gluconate + 22% inulin + 3% magnesium stearate.

Claims (12)

Moringa peregrina seed extract, characterized in that it is obtained by solid-liquid extraction of whole seeds or seed cake, with stirring, in a proportion of 5% to 50% by weight of solid matter relative to the weight total implemented in a predominantly alcoholic solvent, the alcohol being chosen from ethanol or methanol with optionally a co-solvent of the polyol or subcritical water type, in a proportion of 70 to 100% by weight of alcohol relative to the total weight of the solvent, at a temperature of between 4 and 50°C for a period of between 10 minutes and 4 hours, and by separation of the liquid and solid phases so as to eliminate the solid phase and recover a liquid extract of the seed of Moringa peregrina , said extract being rich in the compound 2,5-diformylfuran. Extract according to Claim 1, characterized in that it is obtained from unhulled whole seeds or from the cake of unhulled seeds of Moringa peregrina . Extract according to Claim 2, characterized in that it is obtained from the cake of the unshelled seeds. Extract according to claim 3, characterized in that it is obtained from the unshelled seed cake by solid-liquid extraction, with stirring, in a proportion of approximately 25% by weight of solid matter relative to the total weight used. in a pure alcoholic solvent of 96° ethanol, at a temperature between 16 and 30° C. for a period of approximately 2 hours. Extract according to Claim 4, characterized in that the liquid extract obtained is dried so as to obtain a dry extract of the cake of Moringa peregrina seeds containing more than 50% by weight of 2,5-diformylfuran relative to the total weight of the dry matter. Process for obtaining a Moringa peregrina seed extract according to one of Claims 1 to 5, characterized in that it comprises the following steps according to which:
a) the unshelled Moringa peregrina seeds are collected and dried to obtain an internal humidity level of less than 8%;
b) optionally pressing the dried seeds so as to separate the oil from the rest of the seed to obtain the cake,
c) the dried seed obtained in step a) or the cake obtained in step b) is ground,
d) the ground material obtained in step c) is dispersed in a proportion of 5% to 50% by weight of solid matter relative to the total weight used in a predominantly alcoholic solvent, the alcohol being chosen from ethanol or methanol optionally with a co-solvent of the polyol or subcritical water type, in a proportion of 80 to 100% by weight of alcohol relative to the total weight of the solvent;
e) a solid-liquid extraction is carried out, with stirring, at a temperature of between 4 and 50° C. for a period of between 10 minutes and 4 hours,
f) the liquid and solid phases are separated so as to eliminate the solid phase and recover a liquid extract of seed or cake of Moringa peregrina , and
g) optionally, when the alcohol is ethanol, the liquid Moringa peregrina extract obtained is dried so as to obtain a solid Moringa peregrina extract. Process according to Claim 6, characterized in that it is obtained from the cake of unshelled seeds by solid-liquid extraction, with stirring, in a proportion of 25% by weight of solid matter relative to the total weight put in work in the pure alcoholic solvent ethanol 96°, at a temperature between 16 and 30°C for a period of approximately 2 hours. Process according to Claim 7, characterized in that the liquid Moringa peregrina extract obtained is purified by distillation, microfiltration, ultrafiltration and/or nanofiltration in order to concentrate the extract in 2,5-diformylfuran with respect to the organic matter also extracted. Cosmetic or nutricosmetic composition, characterized in that it comprises, as active agent, an effective amount of a Moringa peregrina seed extract according to one of Claims 1 to 5, and a physiologically acceptable excipient. Composition according to Claim 9, characterized in that it is a cosmetic composition formulated to be applied topically to the skin and in that the Moringa peregrina seed extract is present in the composition at a concentration of 0.002 to 20% by weight relative to the total weight of the composition, preferably from 0.2 to 10%. Composition according to Claim 9, characterized in that it is a nutricosmetic composition formulated to be ingested and in that the Moringa peregrina seed extract is present in the composition at a concentration of 0.002 to 40% by weight relative to the total weight of the composition, preferably from 0.2 to 20%. Cosmetic or nutricosmetic use of a composition according to one of Claims 9 to 11, for improving the appearance of the skin, mucous membranes or appendages, for relaxing, soothing and de-stressing the skin and preventing and/or combating skin signs of aging and/or photo-aging and preventing age spots.