HK40069900B

HK40069900B - Moringa peregrina seed extract rich in 2,5-diformylfuran, method for obtaining same and use thereof in cosmetic composition

Info

Publication number: HK40069900B
Application number: HK62022059158.5A
Authority: HK
Inventors: 伊丽莎白·多迪内; 文森特·布尔热托
Original assignee: 法国生态农业发展联合署
Priority date: 2020-05-21
Filing date: 2021-05-21
Publication date: 2023-08-04

Description

Moringa oleifera seed extract enriched in 2, 5-diformylfuran, method for obtaining same and use thereof in cosmetic compositions

Technical Field

The present invention relates to the field of cosmetics and nutritional cosmetics, and more particularly to the field of active ingredients included in skin care composition formulations. The present invention relates to seed extracts of Moringa oleifera (Moringa peregrina) enriched in the compound 2, 5-Diformylfuran (DFF). The invention also relates to a method for obtaining a specific extract of seeds of moringa oleifera, to a cosmetic composition comprising such an extract, and finally to the cosmetic or nutraceutical cosmetic use of such a composition for caring for the skin, scalp and body covers.

Background

Moringaceae (Moringaceae) is a single genus (only one genus, moringa adans), an element of the Saharo-sindiin flora, which, according to the authors, consists of 12 to 14 species, distributed in eastern africa to asia. The genus is conventionally divided into three parts, however, these three parts are not confirmed to be monoclinic by phylogenetic analysis. The analysis has shown to some extent branches centered on certain morphological features: pachlcaus ("bottle trees)"); "tuber trees" and those that are neither bottle trees nor tuber trees ("slender trees"). The species moringa oleifera (Moringa peregrina (forsk.) Fiori) belongs to the third group. Sparse genetic studies of this genus or this family confirm the realistic presence of this species relative to other species of this genus, especially with respect to the Indian Moringa genus (Indian Moringa), moringa oleifera (Moringa oleifeera lam.) (see, inter alia, articles: olson, M.E.2002, combining Data from DNA Sequences and Morphology for a Phylogeny of Moringaceae (brassicas), systematic Botany (1): 55-73;Hassanein,A.M.A.and Al-Soqee, A.A.,2018,Morphological and genetic diversity of Moringa oleifera and Moringa peregrina genotypes,Horticulture,Environment and Biotechnology 59 (2): 251-261). Recent articles on moringa sampled at different locations of saudi arabia have concluded that this species has genetic stability (Alaklabi, a.,2015,Genetic diversity of Moringa peregrina species in Saudi Arabia with ITS sequences,Saudi Journal of Biological Sciences 22:186-190) but has a high level of intra-population genetic variation through the use of ITS markers.

The species arabinoga is found in the rock environment of also portal, amann, sauter arabia, eastern africa, sudan, russian, iritrelia, sumatriy and gibbs. Its presence in iran appears to be limited to southeast provinces, but this requires confirmation (prota14=munylanza e.and Yongabi k.a., vegetable oil s/Oleaginous plants, moringa peregrina (forssk.) Fiori, http:// database.prota.org/PROTA html/moringa peregrina _fr.htm, access to 10/23/2019). In the middle east and in Egypt, this species is now represented only by rare scattered residual stations (except for a few populations at high altitudes), mainly in the region of Sudan. Arabic moringa is also considered rare and endangered in Sudan and also in the gate today. The arabinogalactan occupies the most dry and severely conditioned habitat relative to other species from which it branches. It is clearly more drought tolerant than moringa grown commercially on a large scale in tropical and subtropical areas. Recent studies have shown that the size and circumference (girth) of the seeds have a favourable effect on germination time as well as on the growth rate and speed of young individuals (Gomaa N.H. and Pic F.X.,2011,Seed germination,seedling traits,and seed bank of the tree Moringa peregrina (Morigaceae) in a hyper-area environment, american Journal of Botany (6): 1024-1030), indicating that resource allocation is regulated with respect to seed quality rather than quantity, which enables efficient propagation of Arabica in extreme (ultra-dry) abiotic environments. The seeds of the arabinogalactan tree have a central mesocarp (mesotesta) thicker than those of the moringa oleifera in terms of the cell layer.

There are some historical reports that tend to suggest that Arabian moringa oil was actively traded in Euler (Al-Ula) regions early in Islam (Naseef, A.A.S.,1995, al-' Ul ā, A study of Cultural and Social Heritage). Locally produced oil from moringa oleifera is today mainly designated for personal consumption or for the local market. In saudi arabia, leaves have traditionally been used as an internal decoction for the treatment of diabetes, intestinal diseases, ocular diseases and anaemia (Abdel-Kader, m.s., hazazi a.m. a., elmakki o.a. and Alqasoumi S.I.,2018,A survey on the traditional plants used in Al Kobah village,Saudi Pharmaceutical Journal 26 (6): 817-821) and as diuretics, reddening agents and astringents (Aqeel a.m., tarq m., mossa j.s., al-Yahya m.a. and Al-Said m.s.,1984, "Plants used in Arabian Folk medicine", report submitted to Saudi Arabian National Centre for Science and Technology Riyadh, saudi Arabidopsis). In amann, oil extracted from female in summer is used for treating migraine, fever, burn, laceration and fracture, constipation and gastralgia, and for treating muscle pain, dry hair and pain in gustifar (Ghazanfar S.A.,1994,Handbook of Arabian Medicinal Plants,1) ^st ed. CRC Press, boca Raton, ann Arbor, U.S.; ghazanfar S.A.,1998,Plants of Economic Importance,cap.15,in Ghazanfar,S.A.and Fisher,M (ed.) Vegetation of the Arabian pennisula geobiotan 25 pages 241-264,Kluwer Academic Publishers,table 11.1,page 247and 11.7page 251). It is also used in fragrance compositions (Ghazanfar s.a.,1998, page 259) and as a facial lotion in the aman and also in the gate (Ghazanfar s.a. and rehinger b.,1996, two multi-purpose seed oils from oman.plants for Food and medicine. Paper is presented in the conference (the joint meeting of the Society for Economic Botany and International Society for Ethnopharmacology) of the society of economic botanicals and traditional pharmacological international in london, 7 months 1-7 days 1996).

Extracts derived from moringa seeds are known in the cosmetic arts. For example, FR 296 879 discloses a moringa whole seed (with skin) extract comprising oil (including triglycerides, fatty acids and polar lipids) and polyphenols and its use in cosmetic compositions for combating skin ageing. In said document, the non-polar part of the moringa seed appears to be active, and more particularly the oily part. Protein extracts of moringa seed are also known from FR 2 776 519 to have a cleansing effect on turbid water, to have a softening, physiological conditioning, moisturizing, reorganizing and repairing effect, and to have an effect on the skin and mucous membranes as anti-pollution active agents. In said document, the active ingredients are proteins having a molecular weight of 6500 to 8800Da, which are obtained by aqueous extraction of moringa oleifera cake. FR 3 076 460 is also known, which relates to the use of protein extracts of ungerminated and deoiled moringa seeds for the treatment of sensitive, sensitized, reactive, vulnerable and/or embrittled skin and/or mucous membranes and/or for the treatment and/or prevention of erythema, in particular diaper rash in infants. In said document, the extraction method is capable of producing a main component (fraction) of proteins having a molecular weight of about 8800 Da. KR2013/0088224 also discloses the use of germinated whole moringa seed extracts in cosmetics, in particular extracts obtained by extraction with supercritical fluids. The process makes it possible to isolate nonpolar amino acids and carotenoids, which are described as active agents for bleaching cosmetic applications. All of the above documents relate to the use of the species moringa oleifera; none of them describe the use of the species Moringa oleifera in the cosmetic field. XP055753955,2011 to Kolheil et al discloses the extraction of bioactive polyphenol compounds, tannins, flavonoids, saponins, unsaturated sterols and/or triterpenes from intact seeds of Moringa oleifera with ethanol. The obtained extract is stored at 4deg.C and has antioxidant effect. XP055753970,2018 of Abbas Alba et al discloses ethanol extraction of whole seeds of Moringa oleifera for three or more days. The filtrate was concentrated under reduced pressure at a temperature of 45 to 50 ℃. Abou-Hashem et al XP055754018,2019 discloses ethanol extraction of whole seeds of Moringa oleifera for 3X 72 hours. The extract obtained is then filtered and concentrated in a rotary evaporator at a temperature of about 45 ℃. XP055754048,2015 by Majali Ibarahim et al discloses ethanol extraction of intact seeds of Moringa oleifera with stirring for 30 minutes followed by precipitation within 72 hours. None of the above documents discloses ethanol extraction of a non-dehulled moringa seed cake.

More particularly, for the species Moringa oleifera, certain phenolic and flavonoid compounds obtained from the leaves or whole seeds of the Moringa oleifera are known to have 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 from leaves or whole seeds with solvents such as methanol, ethyl acetate or hexane. It appears that the leaves comprise the greatest amount of active compound.

Thus, depending on the species used in the moringa genus, it was observed that the extracted molecules were different depending on the plant part (leaves or seeds), the seed part (whole seeds or otherwise, dehulled or not) and the extraction method performed, in particular the solvent selection. Nowadays, the composition of the extract influences the biological activity and thus the efficacy of the cosmetic.

In view of the foregoing, one problem addressed by the present invention is to develop a novel product that is useful in cosmetics and easy to use based on an extract of the species Moringa oleifera.

Thus, the applicant has shown novel extracts obtained from seeds of the species peppery, and more particularly from cakes of seeds of the species peppery, which show in particular relaxing and anti-stress activity on the skin, anti-aging activity and also preventive activity on senile plaques. The extract according to the invention is enriched in 2, 5-Diformylfuran (DFF), also known as 2, 5-furandicarboxaldehyde. The extract is obtained in particular from seeds of the peppery tree, or more in particular from cakes of non-dehulled seeds of the peppery tree, in particular via an alcohol extraction. The extract according to the invention is novel in two respects in the cosmetic field with respect to the extracts of the prior art, firstly because of the particular source species used and secondly because of its specific compound content.

By government intergovernment agreement between the government of the france republic and the saute arabino-kingdom at 4.10.2018, the applicant, the france elaola development agency (AgenceThe Pour Le D' AIUla) (AFALULA) and the elaola royalty committee (Commission Royale Pour AlUlA) (RCU) have, inter alia, the ability to develop a combination of sustainable agriculture and local economy, especially to produce native products locally from native plants, and to protect the biodiversity and rights of the sauter arabia Wang Guoai-mole aola (AlUla) region. Saudi arabian kingdom became a member of the ancient house Protocol (Nagoya Protocol) since 10/8 of 2020. In drafting this patent, regulations concerning the "famous ancient house protocol" will be incorporated into the prosecution of the relevant aspects of local law. Thus, at this stage, sauter AThe kingdom of Rubia has no specific requirements for this patent application and the Ming Guo House protocol. Therefore, on the date of filing the patent application, there is no requirement for obtaining a compliance certificate of genetic resources.

Disclosure of Invention

The first subject of the present invention is an extract of seeds of Moringa oleifera enriched in the compound 2, 5-diformylfuran. The compound 2, 5-diformylfuran is a compound of sugar nature, which is rare in the plant kingdom, and which is synthesized from furfural via 5-hydroxymethylfurfural intermediate synthesis.

The extract according to the invention is unique in the genus moringa due to its specific characteristics of high concentration of 2, 5-diformylfuran. It will be demonstrated that the species arabinogalactan has a specific molecular profile that is different from those known from other species of this genus, in particular from species of moringa that applicant has sought to demonstrate.

A second subject of the present invention is a process for obtaining an extract of seeds of moringa oleifera according to the present invention, comprising the steps of:

a) Collecting and drying the non-shelled arabinogalactan seeds to obtain an internal moisture content of less than 8%,

b) Squeezing the dried seeds to separate the oil from the remainder of the seeds to obtain a cake,

c) Milling the cake obtained in step b),

d) Dispersing the milled material obtained in step c) in a solvent which is predominantly an alcohol selected from ethanol or methanol, optionally together with co-solvents such as polyols or subcritical water, in a proportion of about 25% by weight of solid material relative to the total weight used, in a proportion of 80% to 100% by weight of alcohol relative to the total weight of the solvent;

e) Solid-liquid extraction is carried out with stirring at a temperature of 16 to 30 ℃ for a period of about 2 hours,

f) Separating the liquid phase from the solid phase to remove the solid phase and recover a liquid moringa oleifera cake extract, and

g) Optionally, drying the obtained liquid arabinogalactan extract to obtain a solid arabinogalactan extract.

A third subject of the present invention is a cosmetic or nutraceutical cosmetic composition comprising as active agent an effective amount of an extract of seeds of moringa oleifera according to the present invention, together with a physiologically acceptable excipient.

Finally, a fourth subject of the present invention is the cosmetic or nutraceutical cosmetic use of the composition according to the invention for improving the appearance of skin, mucous membranes or integuments, for relaxing, soothing and pressing the skin, and for preventing and/or combating the signs of aging and/or photoaging of the skin, and for preventing senile plaques.

Drawings

The present invention will be better understood from the following description of several specific embodiments thereof, and further objects, details, features and advantages thereof will be more clearly apparent from the description, which is given by way of illustration and not of limitation.

Detailed Description

In this description, unless otherwise indicated, it is to be understood that when a range is given, it includes both the upper and lower limits of the range.

In the present invention, the following abbreviations have the meanings given below:

MTT:3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT assay is a rapid method for counting living cells)

SDS: sodium dodecyl sulfate

PBS: phosphate buffered saline

-ELISA: ELISA (enzyme-linked immunosorbent assay)

-PCR: polymerase chain reaction

-ANOVA: analysis of variance

MSH: melanocyte stimulating hormone

In the present invention, the following definitions apply:

"extract enriched in the compound 2, 5-diformylfuran": an extract comprising the compound 2, 5-diformylfuran in an amount greater than the amount of the other identified ingredients, i.e. greater than 50% relative to the dry matter of the total extract.

"effective amount": the amount of active molecules necessary to obtain the desired result, i.e. to enable significant protection of the skin extracellular matrix.

- "topical administration": the active ingredient according to the invention or the composition comprising it is applied or spread onto the surface of the skin, mucous membranes or integuments.

- "physiologically acceptable": suitable for topical use, contact with human skin or for use via other routes of administration, such as oral or by injection into the skin, without any risk of toxicity, incompatibility, instability or allergic response.

- "cake": and (3) an oil-removed part after the seed is pressed. It is a solid residue of oil extracted from seeds. It is a byproduct of the milling operation, the process used to make oil. It generally represents 50% to 75% of the seed mass.

- "non-shelled seed": meaning that the shell or pericarp of the harvested seed remains around the seed.

"when fruit ripens": meaning that the fruit ripens, preferably when the pod begins to split and change from dark beige to brown, and when the lower quarter of the pod turns 180 ° causing the valve (valves) to open.

"solvent mainly of alcohol": the solvent, meaning an alcohol type, may include a co-solvent having sufficient characteristics to extract the active ingredient, considering that ethanol of 96 ° purity appears to be the most suitable alcohol solvent.

- "about": the amplitude of 10% to 20% is added or subtracted with respect to a given information (duration, percentage, etc.).

"active molecule", also called "active ingredient": 2, 5-diformylfuran molecules extracted from seeds of Moringa oleifera according to the method of the present invention. This molecule is responsible for the biological activity described in the present invention.

- "active agent": an extract according to the invention in an amount sufficient to obtain said biological activity. Depending on whether the extract is liquid or dry, and concentrated or otherwise, the amount of active agent may vary in a proportion of 0.002% to 20% by weight relative to the total weight of the composition.

"sign of skin aging": the external appearance of skin and integuments is due to any change in aging, such as wrinkles and fine lines, skin sagging, skin thinning, skin lack of elasticity and/or complexion, darkness, matt skin or pigmentation spots on skin, hair discoloration or nail stains, and also any internal changes in skin that are not systematically reflected by the changed external appearance, such as any internal degradation of skin after exposure to Ultraviolet (UV) radiation.

The first subject of the present invention relates to an extract of seeds of Moringa oleifera enriched in the compound 2, 5-diformylfuran. The 2, 5-diformylfuran molecule, also known as 2, 5-furandicarboxaldehyde, has never been characterized by seed extracts of moringa species. The species arabinogalactan grows in very arid climates. Thus, its ability to withstand drought enables it to acquire unique features that the applicant has been able to identify via the use of extraction methods suitable for whole seeds or preferably for seed cakes.

In the context of the present invention, the selected plant part is the seeds of Moringa oleifera. It is known to use arabinogalactan seeds for extracting an oil called arabinogalactan (peregrina) oil (INCI name: arabinogalactan seed oil), which is used geographically for personal consumption or various traditional medical indications. The cake obtained after de-oiling of seeds is a waste product, which is currently used in particular for animal feed.

According to a first object of the present invention, the extract of moringa oleifera is obtained by: the non-dehulled seed cake is solid-liquid extracted in a solvent, mainly an alcohol, optionally together with a co-solvent such as a polyol or subcritical water, in a proportion of about 25% by weight of solid matter relative to the total weight used, selected from ethanol or methanol, in a proportion of 70% to 100% by weight relative to the total weight of the solvent, in a solvent, mainly an alcohol, at a temperature of 16 to 30 ℃ for a period of about 2 hours, and the liquid phase and the solid phase are separated to remove the solid phase and recover a liquid extract of the seeds of moringa oleifera, said extract being rich in the compound 2, 5-diformylfuran. Preferably, the extract according to the present invention is obtained from the harvested seed cake after extraction of the arabinogalactan oil (INCI name: arabinogalactan seed oil) when the arabinogalactan fruit is ripe.

It should be noted that the active ingredient of the extract according to the invention, namely 2, 5-diformylfuran, is a mixed polar molecule with a certain friability. Thus, the extract obtained from intact non-dehulled seeds should undergo selective extraction using suitable solvents and cosolvents and temperatures not exceeding 30 ℃ to obtain high concentrations of active ingredient.

The co-solvent may be, for example, glycol ether (monopropylene glycol or dipropylene glycol, propylene glycol and other propylene glycol derivatives, ethylene glycol or diethylene glycol derivatives) glycerol, dimethyl ether isosorbide, methyl or ethyl esters or propyl esters of fatty acids; dioctyl carbonate, di Xin Xianmi, alkyl acetate or propionate, acetone, methyl or ethyl ketone and monoterpenes such as alpha-pinene or limonene. These co-solvents may be mixed with a main solvent (e.g., ethanol or methanol) in a ratio of 0 to 30% (V/V).

The extraction conditions may be under atmospheric pressure or under vacuum or under an inert atmosphere, but are preferably in the dark at a temperature of 16 to 30 ℃.

In a preferred embodiment according to the invention, the extract is obtained from a non-dehulled seed cake by solid-liquid extraction with 96 ° ethanol as alcoholic solvent.

In yet another embodiment, the obtained liquid extract is dried to obtain a dry extract of said arabinogalactan seed cake comprising more than 50% by weight of 2, 5-diformylfuran relative to the total weight of dry matter.

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

A second subject of the present invention is a process for obtaining an extract of a seed cake of moringa oleifera according to the present invention, comprising the steps of:

c) Milling the cake obtained in step b),

d) Dispersing the milled material obtained in step c) in a solvent which is predominantly an alcohol selected from ethanol or methanol, optionally together with co-solvents such as polyols or subcritical water, in a proportion of about 25% by weight of solid material relative to the total weight used, in a proportion of 70% to 100% by weight of alcohol relative to the total weight of the solvent;

g) Optionally, when the alcohol is ethanol, drying the obtained liquid arabinogalactan extract to obtain a solid arabinogalactan extract.

In a preferred embodiment, the non-dehulled seeds are collected, i.e. the shells of the seeds remain, when the fruit ripens and preferably when the pod begins to split.

In a preferred embodiment, the seeds are dried to obtain an internal moisture content of about 6%, preferably in a ventilated cage protected from light, preferably in an outdoor shade.

The dried seeds were then immediately milled under cold pressing, which allowed the mechanical separation of the arabinogalactan oil (INCI name: arabinogalactan seed oil) from the remainder of the compressed seeds (i.e., cake).

The cake is then mechanically milled using any type of mechanical mill such as a hammer mill, flail mill, knife mill or crushing/shredding mill.

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

For the main extraction of the compound of interest 2, 5-diformylfuran, an alcoholic solvent such as 96 ° ethanol will be preferred, but methanol may also be used, together with a co-solvent such as a polyol or subcritical water. At the end of the extraction, the residual plant material depleted in the compound of interest is advantageously separated from the liquid phase by clarification filtration. Even more preferably, the solvent is 96 ° ethanol. It would be advantageous to obtain a liquid extract from a seed cake of moringa oleifera comprising from 0.5% to 1.6% of dry matter consisting of: at least 50% of 2, 5-diformylfuran, corresponding to about 0.25% to 0.8% by weight of the total weight of the liquid extract.

In one embodiment of the production process according to the invention, the liquid arabinogalactan extract obtained is purified by distillation, microfiltration, ultrafiltration and/or nanofiltration to concentrate the compound of interest 2, 5-diformylfuran of the extract with respect to the organic material still extracted, in particular with respect to the remaining part of the extracted material, such as fatty substances and derivatives still extracted. These purification steps enable concentration of the compound of interest at the cost of other extracted compounds as mentioned and also solvents.

In another embodiment of the production process according to the invention, the liquid extract obtained is dried to obtain a dry extract of a seed cake of moringa oleifera comprising more than 50% by weight of the compound of interest 2, 5-diformylfuran with respect to the total weight of extracted dry matter.

According to an advantageous embodiment of the invention, when the solvent is ethanol, the liquid extract of seeds of moringa oleifera obtained is preferably dried, for example by nebulization, lyophilization or zeodration to obtain a solid extract of seed cake of moringa oleifera, the ethanol having been evaporated. The drying may be carried out in the presence of an organic carrier such as maltodextrin, cyclodextrin or inulin, or in the presence of a mineral carrier such as a layered silicate, magnesium silicate or carbonate and salts thereof.

The invention also relates to an extract of seeds of Moringa oleifera obtainable via the production process according to the invention.

The composition according to the present invention may be formulated in various formulations suitable for topical or oral administration.

According to a first variant, the various formulations are suitable for topical administration and include creams, oil-in-water and water-in-oil emulsions, ointments, lotions, oils, balms, aqueous or aqueous alcohol or ethanol solutions, slurries, powders, patches, sprays or any other product for external application, for example medical devices or cosmetic textile products.

According to a second variant, the various formulations are suitable for oral administration; plant extracts comprising the active compound 2, 5-diformylfuran, which may be included in a food composition or food supplement. In the context of the present invention, the food supplement may be in the form of a hard or soft gelatin or vegetable capsule. The food supplement may then comprise from 0.01% to 100% by weight of the plant extract. More preferably, the amount of plant extract is from 0.02% to 40% by weight, and in particular from 0.2% to 20% by weight, relative to the total weight of the composition.

In the context of food use, the composition is advantageously formulated for nutritional or cosmetic (cosmetic food or nutritional cosmetic) purposes in a formulation suitable for oral administration. It may not include excipients and may consist entirely of a plant extract comprising the active compound 2, 5-diformylfuran.

According to a preferred embodiment, the composition according to the invention is more particularly intended for topical administration. Thus, these compositions must contain a cosmetically acceptable medium, i.e., a medium compatible with the skin and body, and encompass all cosmetic forms. These compositions may in particular be in the form: a cream, oil-in-water or water-in-oil emulsion or a multiple emulsion, slurry, solution, suspension, gel, emulsion, lotion, stick or even powder, and may be suitable for application to the skin, lips and/or integument. These compositions include excipients necessary for their formulation, such as solvents, emollients, thickeners, diluents, surfactants, antioxidants, bioactive agents, dyes, preservatives and fragrances. They can be used as skin care products and/or skin cosmetic products.

The compositions according to the invention may in particular consist of hair care compositions and are in particular shampoos, hair conditioners, therapeutic lotions, styling creams or gels, hair conditioning lotions, masks and the like. The cosmetic composition according to the invention may be used in particular in therapy involving application which may or may not be followed by rinsing, or alternatively in the form of a shampoo. The composition according to the invention can be advantageously used in anti-dandruff treatment. It can also be in the form of a dye or mascara, applied with a brush or comb, in particular on the eyelashes, eyebrows or hair.

The compositions according to the invention also comprise any additives commonly used in the field of application envisaged and also adjuvants required for their formulation, such as solvents, thickeners, diluents, antioxidants, dyes, sunscreens, self-tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, cosmetic or pharmaceutical active agents, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

The INCI dictionary and handbook ("cosmetic ingredients international designation (International Nomenclature of Cosmetic Ingredient)" (13 th edition, 2010), published by Personal Care Products Council inc., washington, d.c.) describe a wide variety of cosmetic and pharmaceutical ingredients commonly used in the skin care industry, but are not limited thereto, as additional ingredients in compositions according to the present invention.

In any event, the skilled artisan will take care to ensure that these adjuvants and their proportions are selected so that the desired beneficial properties of the composition according to the invention are not adversely affected.

According to an advantageous embodiment of the invention, the amount of plant extract in the composition according to the invention is from 0.002% to 20% by weight, and in particular from 0.001% to 10% by weight, relative to the total weight of the composition.

Finally, a fourth subject of the present invention is the cosmetic or nutraceutical cosmetic use of the composition according to the invention for improving the appearance of skin, mucous membranes and integuments, for relaxing, soothing and pressing the skin, and for preventing and/or combating the signs of aging and/or photoaging of the skin, and for preventing senile plaques.

According to one embodiment, the purpose of the use according to the invention is more particularly to relax, relax and relax the skin and to combat signs of skin aging.

According to another embodiment, the purpose of the use according to the invention is to prevent the appearance of senile plaques.

While the invention has been described in connection with several specific embodiments, it will be clear that the invention is not so limited in any way, and that it encompasses all technical equivalents of the described means and also combinations thereof if they fall within the context of the invention.

Use of the verb "to comprise" or "include" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Examples

Example 1: preparation of plant extracts from arabinogalactan cake

When the fruit ripens, the harvested, non-dehulled peppery seed is dried to obtain an internal moisture content of less than 8% and preferably about 6%, and then pressed with a mechanical headless screw press to separate the oil from the rest of the seed, so as to obtain, on the one hand, a pre-pressed oil and, on the other hand, a cake. The cake was then separated into the form of pre-cut rolls in 1 to 2cm pieces. The cake was preheated at 55℃for 10 minutes, impregnated and extracted with 96℃ethanol at 25%/75% (m/m) ratio for 10 minutes at 55℃the mixture was sheared with a blender for 15 minutes and then stirred by an impeller at a temperature of 16℃to 30℃for 2 hours. The product was then filtered under vacuum through a Buchner funnel to obtain a pale yellow filtrate containing 1.15% dry matter. The obtained liquid extract is hereinafter referred to as "the arabinogalactan extract according to the present invention" or "the arabinogalactan extract" or "the arabinogalactan cake extract". This liquid extract was then used for various efficacy tests.

This extract of moringa oleifera according to the invention comprises 1.15% dry matter, itself including (relative to the results expressed on Dry Matter (DM):

TABLE 1

The dry extracts described above are obtained via 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 ingredient of this extract. It is determined by chromatographic methods and more precisely by gas chromatography coupled with flame ionization detectors.

Furfural was measured by the same method.

Isopropyl myristate was determined by the same method.

Palmitic acid and oleic acid were determined by the same method.

Triglycerides were separated by ultracentrifugation.

Example 2: the extract of Moringa oleifera according to the invention acts as an antioxidant

The aim of this study was to evaluate the modulation of antioxidant activity of the extract of pepperylene in a decellularized in vitro colorimetric model using DPPH (2, 2-diphenyl-1-picrylhydrazyl) (2, 2-dipheny-1-picrylhydrazyl) group and also a reference antioxidant ascorbic acid. The method used is called inhibition. It is based on degradation of the purple coloured oxidative group DPPH absorbed at 540nm with the reference antioxidant ascorbic acid. This reaction served as a positive control and resulted in the formation of colorless or even pale yellow DPPH compounds. The extract of Moringa oleifera according to the invention and the reference product "ascorbic acid" were placed in contact with DPPH solution at 40 c for 30 minutes. The antioxidant activity was then evaluated by measuring the absorbance at 540 nm. The modulation of this activity is expressed as the percentage of stimulation (t+) of the maximum antioxidant activity obtained in the presence of ascorbic acid for the antioxidant activity of the test active agent versus the reference.

The scheme is as follows: the DPPH solution was incubated at 40 ℃ for 30 minutes in the absence (control) or in the presence of the arabinogalactan extract (t+) according to the present invention and in a reduced concentration of the test sample. At the end of the incubation period, after 30 minutes at 40 ℃, the antioxidant activity in the presence of the reference product and in the presence or absence of the extract of moringa oleifera was shown by staining. Thus, it was evaluated by measuring the absorbance of the reaction medium at 540 nm. For each test concentration, the modulation of the antioxidant activity of the test product was calculated according to the following formula.

[ mathematics 1]

Percent modulation of antioxidant activity = 100x [ (OD 540 control-OD 540 test product)/OD 540 reference product ].

If the result is negative, the test product is regarded as oxidized; if the result is positive, the percentage is expressed as stimulation of free radical scavenging activity. The results obtained are given below.

TABLE 2

Conclusion: the extract of moringa oleifera according to the invention is able to protect against free radicals: it has remarkable antioxidant properties at concentrations of 1% and above 1%.

Example 3: the action of the extract of Moringa oleifera according to the invention as metalloproteinase inhibitor

The aim of this study was to evaluate the modulation of metalloprotease inhibitory activity of the arabinogalactan extract according to the present invention, using collagenase type I and hyaluronidase, substrate complex and chromophore ninhydrin in an in vitro decellularization model. A buffer solution of type I collagenase and hyaluronidase reacts with a specific substrate complex and converts it to form a compound capable of activating the chromophore by incubation at 80 ℃ for 15 minutes. Collagenase and hyaluronidase activity can thus be assessed by measuring absorbance at 565 nm. The sample was placed in contact with collagenase and hyaluronidase solutions together with the enzyme substrate complex at 37 ℃ for 5 minutes. The substrate converted with the enzyme is able to activate the chromophore by incubation at 80 ℃ for 15 minutes. Collagenase and hyaluronidase activity in the presence/absence of the sample was then evaluated by measuring absorbance at 565 nm. Modulation of this activity in the absence of an active agent, i.e., in the presence of only an enzyme substrate, is expressed as a percentage of inhibition or activation of collagenase and hyaluronidase activity.

The scheme is as follows: in the absence or presence of the test pepperylene extract according to the invention, a solution of type I collagenase and hyaluronidase was incubated in its substrate for 5 minutes. The solution was then placed in contact with the chromaninhydrin followed by incubation at 80℃for 15 minutes. At the end of the incubation period, collagenase and hyaluronidase activity with and without the test or reference products was evaluated by measuring the absorbance of the reaction medium at 565 nm. For each test concentration, the modulation of collagenase and hyaluronidase enzymatic activity by the test product was calculated according to the following formula.

[ math figure 2]

Percentage modulation of collagenase/hyaluronidase enzymatic activity = 100x [ (OD test or reference product-OD collagenase/hyaluronidase only)/OD collagenase/hyaluronidase only ].

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 metalloproteases with the extract of moringa oleifera according to the present invention are given below.

TABLE 3

Conclusion: the extract of moringa oleifera according to the invention produced 62% inhibition of the strong metalloprotease (collagenase/hyaluronidase) at very low levels of 0.01%. The extract of Moringa oleifera according to the present invention is capable of strongly inhibiting these metalloproteinases and has a good potential to efficiently protect the extracellular matrix of the skin, and through such inhibition, it shows an anti-aging effect.

Example 4: the Moringa oleifera extract pair according to the present invention inhibits Histone Deacetylase (HDAC) and sirtuin I action

The purpose of this study was to demonstrate the inhibitory activity of the arabinogalactan extract according to the present invention on the enzymes HDAC and sirtuin I. The buffer solution of HDAC and sirtuin I was reacted with the substrate at 37 ℃ for 20 minutes and converted to form a compound, which was stained in the presence of a chromogenic agent (developer) after incubation at 37 ℃ for 10 minutes. Thus, the maximum deacetylation activity of sirtuin can be evaluated by measuring the absorbance at 405 nm. The arabinogalactan extract or reference product "trichostatin a (STA) inhibitor 1 μm" according to the present invention was placed in contact with a sirtuin solution together with an enzyme substrate at 37 ℃ for 20 minutes and the substrate transformed with the enzyme was stained by adding a chromogenic agent. Deacetylation activity of HDAC and sirtuin I in the presence of the active agent was then assessed by measuring absorbance at 405 nm. Modulation of this activity in the absence of an active agent, i.e., in the presence of HDAC and sirtuin I enzyme substrates alone, is expressed as a percent inhibition or percent activation of the maximum activity of HDAC and sirtuin I.

The scheme is as follows: the sirtuin enzyme solution was incubated in its substrate for 20 minutes in the absence (control) or in the presence of a reference product or an increased concentration of a test product. At the following concentrations: 2%;1%; the extract of Moringa oleifera according to the invention was tested at 0.1% (V/V). At the end of the incubation period, the activity of the sirtuin enzyme with and without the test or reference product was shown by staining with a developer solution (10 minutes at 37 ℃) and evaluated by measuring the absorbance of the reaction medium at 405 nm. For each test concentration, the modulation of the deacetylation activity of the histone deacetylase and sirtuin I enzymes by the test products was calculated according to the following formula.

[ math 3]

Percentage modulation of sirtuin enzymatic activity = 100x [ (OD) ₄₀₅ Test or reference product) - (OD ₄₀₅ HDAC and sirtuin I only)]/OD ₄₀₅ Only sirtuins.

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 inhibition of Histone Deacetylase (HDAC) enzymes are given below.

TABLE 4

Conclusion: at 2%, the arabinogalactan extract according to the present invention showed significant HDAC inhibition; this inhibition reflects the ability of skin cells to promote self-protection from genetic drift, particularly that associated with the aging process. Thus, the extract appears to be useful against one of the most common genetic drift on the skin surface, namely fibrosis, which is manifested by the appearance of "skin tags" (fibrotic protuberances). The extract may advantageously interfere with fibrosis on the skin surface and thus prevent skin aging.

Example 5: the extract of Moringa oleifera according to the invention is useful for modulating the anti-inflammatory activity of phospholipase-A2 enzymes Is used.

The aim of this study was to evaluate the modulation of the anti-inflammatory activity of the enzyme phospholipase A2 by one or more samples in an in vitro decellularization model with the aid of a "SPLA2 (type V) inhibitor screening assay kit". Phospholipase A2 is a key enzyme upstream of the inflammatory process, triggered by the arachidonic acid cascade. A buffer solution of phospholipase A2 was reacted with a specific substrate diheptylthio-PC (diheptanoyl thio-PC) and converted to a compound which was bound to chromogen DTNB with stirring at room temperature. Phospholipase A2 activity can thus be evaluated by measuring absorbance at 413 nm. The arabinogalactan extract or reference inhibition product "thioether amide-PC" according to the present invention is placed in contact with a phospholipase A2 solution simultaneously with an enzyme substrate. Substrates converted by the enzyme were stained by means of chromogen DTNB by stirring at room temperature. The activity of the extract of moringa oleifera or the reference product according to the invention was then evaluated by measuring the absorbance at 413 nm. Modulation of this activity in the absence of active agent, i.e. in the presence of only the enzyme substrate (diheptanoylthio-PC), is expressed as percent inhibition or percent activation of phospholipase A2 activity.

The scheme is as follows: incubating a solution of the enzyme phospholipase A2 in its substrate diheptyl thio-PC in the absence or presence of a reference inhibitor and an arabinogalactan extract according to the present invention under the following conditions: 2%;1%; the test was performed at 0.1% (V/V) and then the chromogen DTNB was incorporated, followed by incubation at 25 ℃ for 15 minutes. At the end of the incubation period, the activity of the enzyme phospholipase A2 with and without the test product or the reference product was evaluated by measuring the absorbance of the reaction medium at 413 nm. For each test concentration, the modulation of phospholipase A2 enzymatic activity by the test product was calculated according to the following formula.

[ mathematics 4]

Percent modulation of phospholipase A2 enzymatic activity = 100x [ (OD) ₄₀₅ Test or reference product-OD ₄₀₅ sPLA 2)/OD only ₄₀₅ sPLA2 only]。

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 modulating the anti-inflammatory activity of the enzyme phospholipase-A2 are given below.

TABLE 5

Conclusion: the arabinogalactan extract according to the present invention produces a slight, stable PLA2 inhibition at a dose of 0.1% and above 0.1%, but more preferably at 1% or 2%. This means that the extract of Moringa oleifera according to the present invention has the ability to reduce the arachidonic acid cascade/inflammatory cascade very early; therefore, the extract has good soothing or relaxing potential on the skin.

Example 6: effect of the arabinogalactan extract according to the present invention on inhibiting endothelin-1 action

Endothelin is the most effective known vasoconstrictor in humans. In addition, endothelin consumption is also known to produce vasodilatory effects [ Hirata, Y.et al.,1988,Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells,Biochemical and Biophysical Research Communications,154:3,pages868-875] [ Sharinkumar P.et al.,2018,H2S Mediates the Vasodilator Effect of Endothelin-1in the Cerebral Circulation.American Journal of Physiology.Heart Circulatory Physiology,315,pages 1759-1764].

The objective was to determine type 1 endothelin in human microvascular endothelial cells 24 hours after exposure to the extract of moringa oleifera according to the present invention.

The scheme is as follows: human microvascular endothelial cells were supplied by pelofotech corporation and cultured in 96-well plates according to the commercial production procedure. The extracts were applied to endothelial cells at 80% confluence for 24 hours at varying concentrations and then endothelin-1in the cell supernatant was quantified using the PicoKine ELISA kit (EDN 1). Viability tests were performed in advance to prescribe non-toxic doses for endothelin-1 assays. Negative controls were performed using untreated cells in the medium. The positive control in the viability test was 0.5% sds. All conditions were prepared in medium and the cells were then incubated at 36.5 ℃/5% CO ₂ Incubate for 24 hours.

a) The test solution is applied to endothelial cells:

the test product was placed in contact with endothelial cells in a 96-well plate at sub-confluence. For each concentration, the test was performed in three wells. Plate is put intoAt 36.5 ℃/5% CO ₂ Incubate for 24 hours.+ -. 1 hour.

b) Viability test:

after incubation with the product, the cells were evaluated for cell viability using the MTT method. After 24 hours of incubation, the supernatant was recovered and stored at-20 ℃ for measurement. The wells were then rinsed once with 200 μl PBS. To each well 50. Mu.L of 0.5mg/ml MTT solution was added: at 36.5 ℃/5% CO ₂ Incubate for 3 hours. To each well was added 100 μl of isopropanol. After homogenization, the absorbance was read at 550 nm. For each condition, the ratio of the average optical density value of the cells to the average optical density value of the negative control determines the viability ratio.

c) Endothelin-1 assay:

the assay was performed using an ELISA kit.

TABLE 6

Conclusion: viability tests performed at the end of the treatment did not show any toxic effect on the tested concentrations. Endothelin-1 assays were performed in cell supernatants at non-toxic concentrations. The amount of endothelin-1 under each condition was determined using ELISA kit. For negative control cells, this value was approximately 134.94pg/ml. For cells treated with different concentrations of extract, these values ranged from 63.14pg/ml (using 5% of extract according to the invention) to 101.06pg/ml (using 0.1% of extract according to the invention), which showed a very significant inhibition at 0.1% and above 0.1% of extract according to the invention, with about 25% inhibition of endothelin type 1 production and up to 53% inhibition of endothelin type 1 production with 5% of extract according to the invention.

Example 7: effect of the arabinogalactan extract according to the present invention on stimulation of telomerase activity

Telomeres are complexes of protective DNA, located at the ends of linear chromosomes, promoting chromosomal stability. Human telomere shortening is developing into the risk and progression of disease and prognostic markers of premature death in many types of cancer, especially 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,pages 1048-1057]. Telomere shortening is counteracted by the cellular enzyme telomerase.

The aim of this study was to evaluate the effect of a compound called "extract of moringa oleifera according to the invention" on telomerase activity in a model consisting of adult keratinocytes cultivated in a monolayer at low passage levels.

The scheme is as follows: human keratinocytes were obtained from a 49 year old donor. For the experiments, keratinocytes were used at low passage level (i.e. cell separation passage number 2). These cells were grown as monolayers until they reached about 75% confluence, after which they were used in experiments.

Reference product: 100ng/ml FK228 was used as a reference inducer of telomerase 1 activity.

Incubation protocol: at 0.5 in the absence (control) or in the presence of a reference product or an increased concentration of a test compound such as an extract of moringa oleifera according to the invention; cells were incubated for 24 hours at 1% and 5% (v/v).

The extract of moringa oleifera according to the invention is diluted directly in the incubation medium to achieve the various concentrations described above.

Evaluation of action:

protein measurement

At the end of the incubation period, the total cellular protein is extracted from the cells and measured by means of the spectrocolorimetry method (Bradford method). This measurement was used to determine the exact volume of extract used in the telomerase activity measurement to maintain the same amount of protein (including telomerase) for all conditions tested in the PCR step.

Measurement of telomerase Activity

At the end of the incubation period, telomerase is extracted from the cells and its activity is determined by means of a specific, sensitive kit. The principle of the telomerase kit is to measure telomerase activity by coupling a PCR step (in which the function of telomerase is related to its extension activity) to an ELISA step to semi-quantitatively determine the amount of telomerase extension product.

-statistics

Results are expressed in arbitrary units of telomerase activity level (mean ± s.d). Evaluation of the level of significance between "vehicle" and "reference product" by means of student t-test ^* :p<0.05). Evaluation of the significance level between "control" and "test compound" for each product independently by one-way analysis of variance (one-way ANOVA), followed by Holm-Sidak test ^* :p<0.05)。

The extract of moringa oleifera according to the invention tested at 0.5% and 1% (v/v) did not significantly modulate telomerase activity relative to the "control". The extract of Moringa oleifera according to the invention significantly increased telomerase activity by 18.9% (p < 0.001) when tested at 5% (v/v) compared to the "control".

A reference product named "FK228" was tested at 100ng/ml and the telomerase activity was significantly increased by 28.0% (p < 0.01). This result was expected and validated the experiment. The results of stimulation of telomerase activity are given below.

TABLE 7

Conclusion: the extract of moringa oleifera according to the invention tested at 0.5% and 1% (v/v) did not significantly modulate telomerase activity relative to the "control". The extract of Moringa oleifera according to the invention significantly increased telomerase activity by 18.9% (p < 0.001) when tested at 5% (v/v) compared to the "control". The extracts according to the invention act directly on the enzymatic pathway which establishes protective telomeres at the chromosome ends and slows down the natural senescence of genetic material. Thus, the extract according to the present invention may exert an anti-aging effect on chromosomes.

Examples 2 to 7 demonstrate that the extract of moringa oleifera according to the invention has anti-aging, anti-stress and relaxation properties, which demonstrate the properties of good skin protectants.

Example 8: the effect of the extract of Moringa oleifera in stimulating tyrosinase activity according to the present invention

The aim of this study was to evaluate the activity of the arabinogalactan extract according to the invention on enzymatic tyrosinase in an in vitro decellularization model using tyrosinase of fungal origin (Sigma-Aldrich ref.t 3824), its substrate L-tyrosine (Sigma-Aldrich ref.t 3754) and a reference inhibitor hydroquinone (Sigma-Aldrich ref.h17902, inhibitor = hydroquinone 2.5 mM). A buffered solution of tyrosinase was reacted with the substrate L-tyrosine 2.5mM at 23℃for 60 minutes and converted to form a colored compound. The maximum tyrosinase activity can thus be evaluated by measuring the absorbance at 475 nm. The arabinogalactan extract or reference product "hydroquinone" according to the present invention is placed in contact with tyrosinase solution together with enzyme substrate at 23 ℃ for 60 minutes, the substrate converted with enzyme being naturally coloured. Tyrosinase activity in the presence of the active agent was then evaluated by measuring absorbance at 475 nm. Modulation of this activity in the absence of an active agent, i.e., in the presence of only the enzyme substrate (L-tyrosine), is expressed as a percent inhibition or percent activation of the maximum tyrosinase activity.

The scheme is as follows: 2% of the extract of peppery wood according to the invention in the absence (control) or in the presence of a reference product or in an increased concentration; 1%; the tyrosinase solution was incubated in its substrate L-tyrosine for 60 minutes at 0.1% (V/V). At the end of the incubation period, tyrosinase activity with and without test or reference products was evaluated by measuring absorbance of the reaction medium at 475 nm. For each test concentration, the modulation of tyrosinase enzymatic activity by the test product was calculated according to the following formula.

[ math 5]

Percentage of modulation of tyrosinase enzymatic activity = 100x [ (OD 475 test product or reference product) - (OD 475 tyrosinase only) ]/OD475 tyrosinase only.

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 stimulation of tyrosinase activity are given below.

TABLE 8

Conclusion: the ability of the extract of moringa oleifera according to the invention to reduce basal tyrosinase activity makes it possible to demonstrate the ability of the extract to have one of the natural forms of increasing skin protection: protecting against ultraviolet rays.

Example 9: the effect of the arabinogalactan extract on inhibiting melanin production according to the present invention

The purpose of this study on human cell culture was to collate all data used and also the results obtained, in order to conduct a melanin regulating test on human melanocytes 5 days after exposure to the extract of moringa oleifera according to the invention.

The scheme is as follows: human melanocytes were cultured in 96-well plates and 24-well plates.

The arabinogalactan extract according to the present invention was allowed to act on confluent melanocytes at concentrations of 5%, 2%, 1% and 0.1% for 5 days. The viability pre-test with MTT after 24 hours allows the cytotoxicity to be assessed and the concentration of the melanin regulating test to be selected. This modulation was assessed by measuring melanin in the cell lysate 5 days after exposure to the extract. Negative controls were performed using untreated cells in the medium. The positive control for viability test was 0.5% sds. For the melanin regulating test, medium with and without α -MSH was used as negative control.

All conditions were prepared in medium and the cells were then incubated at 36.5 ℃/5% CO ₂ Incubate for 24 hours for cytotoxicity test and incubate for 5 days for melanin assay.

a) The test solution was applied to melanocytes: the test concentrations were placed in confluent black with 96-well plates (cytotoxicity test) and 24-well plates (melanogenesis assay) And (5) contacting the plain cells. For each concentration, the test was performed in three wells. The plates were incubated at 36.5deg.C/5% CO ₂ Incubate for 24 hours.+ -.1 hour and 5 days.

b) Viability test: after 24 hours incubation with the product, the cells were evaluated for cell viability using the MTT method. After 24 hours of incubation, the wells were rinsed once with 200 μl PBS. Mu.l of 0.5mg/ml MTT solution was added to each well and at 36.5 ℃/5% CO ₂ Incubation was performed for 3 hours. To each well was added 150 μl of isopropanol. After homogenization, the absorbance was read at 550 nm. For each condition, the ratio of the average optical density value of the cells to the average optical density value of the negative control determines the viability ratio.

A viability cut-off value of 70% relative to the negative control value was used to classify the test substance as cytotoxic or non-cytotoxic. For in vitro results with viability >70%, a "non-cytotoxic" classification is given, and for viability ∈70%, a "cytotoxic" classification is given.

The extract according to the invention at a concentration of 5% proved to be cytotoxic at 5% under the test conditions. Thus, concentrations of 2%, 1% and 0.1% were used for melanin regulating tests. After cell lysis, the amount of melanin present in the cells is determined. The results of inhibiting melanin production are given below.

TABLE 9

Conclusion: the extract of moringa oleifera according to the present invention inhibits melanin production in cells, which gives it skin protective properties. This inhibition also shows a relaxing effect on melanocytes, since the basal rate is lower than that of the control (no extract according to the invention in the medium); it is recalled that melanin production is a response to cellular stress. Thus, the extract of Moringa oleifera according to the present invention proves that it can prevent senile plaques.

Example 10: the comparison of the extract of the moringa oleifera according to the inventionMoringa oleifera extract with the same extraction method Analytical characterization

The extraction method according to the invention described in example 1 was applied on the basis of the arabinogalactan cake and the moringa cake. The comparative compositions of the extracted components are given below on a dry matter basis.

TABLE 10

It is seen that the two extracts have very different molecular profiles. The moringa extract contains less than 1% DFF, while the arabinoga extract contains greater than 50% DFF.

Example 11: comparison test with Moringa oleifera in-tube (in tub) collagenase test

[ math figure 6]

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 metalloprotease inhibition are given below.

TABLE 11

Conclusion: the extract of Moringa oleifera according to the present invention produces a strong metalloprotease (collagenase/hyaluronidase) inhibition. The extract of Moringa oleifera according to the present invention can exert 88% inhibition of these metalloproteinases at a concentration of 0.1% and above 0.1%, and has a good potential to efficiently protect the extracellular matrix of the skin, and through such inhibition, it shows an anti-aging effect.

This was compared with the extract according to Pierre Fabre patent FR2 946 879, and the results of the comparison are given below according to the same test.

TABLE 12

Conclusion: the extracts according to the Pierre Fabre patent showed slightly opposite dose dependent inhibition of collagenase activity with a peak inhibition of 42% for all combinations concentrations, whereas the peak inhibition of 100% for the arabinogalactan extract according to the present invention.

The anti-aging activity with respect to this parameter appears to be different and novel compared to the effect observed with the extract according to the Pierre Fabre patent.

Example 12: comparison test of anti-stress activation (by inhibition of PLA 2).

For sedative/anti-stress orientation by inhibiting PLA2 on skin, which provides anti-aging effects, two additional in-tube PLA2 tests were performed: carried out on moringa cakes via the process according to the invention (extract prepared by the same process as that of the arabinoga according to the process of the invention), one with an extract corresponding to Pierre Fabre patent FR2 946 879 and the other with a product corresponding to BASF Beauty Care Solutions patent FR 3 076 460Finally the last Chuun&The Thurot patent FR2825267./ >

The aim of this study was to evaluate the modulation of the anti-inflammatory activity of the enzyme phospholipase A2 by one or more samples in an in vitro decellularization model with the aid of a "SPLA2 (type V) inhibitor screening assay kit".

A buffer solution of phospholipase A2 was reacted with a specific substrate diheptylthio-PC (diheptanoyl thio-PC) and converted to a compound which was bound to chromogen DTNB with stirring at room temperature. Phospholipase A2 activity can thus be evaluated by measuring absorbance at 413 nm.

The product "extract of peppery ara according to the invention" or the reference inhibition product "thioether amide-PC" is placed in contact with the phospholipase A2 solution simultaneously with the enzyme substrate. Substrates converted by the enzyme were stained by means of chromogen DTNB by stirring at room temperature. The activity of phospholipase A2 in the presence/absence of the product "arabinogalactan extract according to the present invention" or the reference product was then evaluated by measuring the absorbance at 413 nm.

Modulation of this activity in the absence of active agent, i.e. in the presence of only the enzyme substrate (diheptanoylthio-PC), is expressed as percent inhibition or percent activation of phospholipase A2 activity.

The inhibitor "thioether amide-PC" at a concentration of 1mg in 100 μl was the reference product in this study (active control); the product inhibited PLA2 activity by 93%, validating the test.

A solution of the enzyme phospholipase A2 was incubated in its substrate diheptylthio-PC in the absence or presence of a reference inhibitor and test product "arabinogalactan extract according to the present invention" and then incorporated with chromogen DTNB followed by incubation at 25 ℃ for 15 minutes.

At the end of the incubation period, the activity of the enzyme phospholipase A2 with and without the test product or the reference product was shown by measuring the absorbance of the reaction medium at 413 nm. For each test concentration, the modulation of phospholipase A2 enzymatic activity by the test product was calculated according to the following formula.

[ math 7]

Percent modulation of phospholipase A2 enzymatic activity = 100x [ (OD 405 test product or reference product-OD 405 sPLA2 only)/OD 405 sPLA2 only ].

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

TABLE 13

The consistent and substantially dose-dependent activity of the arabinogalactan extract in accordance with the present invention against PLA2 inhibition shows sedative activity by decreasing the intensity upstream of the arachidonic acid cascade. This sedation well upstream of the arachidonic acid cascade reduces the impact of basal physiological stress. Accordingly, the extract of Moringa oleifera according to the present invention is an anti-stress agent.

The following results were obtained using the protocol extract from Pierre Fabre patent FR 2 946 879.

TABLE 14

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At 1%, which corresponds to its lowest working dose, the extract does not show any activity. After dilution, the activity appears, but the absence of dose-dependent effects makes it impossible to verify the specific and reliable effects of the extract on the inhibition of this enzyme.

Using products from FR 3 076 460 of BASF Beauty Care SolutionsScheme extract of LS9726, the following results were obtained.

TABLE 15

The extract shows a slight inhibition which does not reach the maximum observed with the extract of moringa oleifera according to the invention at 1%, i.e. 19% inhibition for the extract according to the invention and 10% inhibition at 1% for the extract.

Using the protocol extract from Chuun & thulot patent FR 2 825 267, the following results were observed.

TABLE 16

The extract showed no inhibition of this enzyme.

The following results were observed with the protocol extract from the method according to the invention, but applied to moringa cake instead of arabinoga cake.

TABLE 17

The extract did not show any significant or stable inhibition of this enzyme.

Conclusion: only the extract of moringa oleifera according to the invention demonstrated a remarkable inhibitory activity on the enzyme PLA 2.

Example 13: cosmetic product formulation

TABLE 18

Example 14: detergent product formulations

TABLE 19

Example 15: nursing product preparation (anti-stress anti-aging cream)

TABLE 20

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Example 16: nutritional cosmetic oral route preparation

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

Example 17: toxicology testing of Moringa oleifera extracts according to the invention

An arabinogalactan extract was prepared according to example 1:

when the fruit ripens, the harvested seeds of the peppery tree are dried to obtain an internal moisture content of about 6%, and then pressed with a mechanical headless screw press to separate the oil from the rest of the seeds, so as to obtain, on the one hand, a freshly extracted oil and, on the other hand, a cake. The cake was then separated into the form of pre-cut rolls in 1 to 2cm pieces. The cake was macerated and extracted with 96 ° ethanol pre-heated at 55 ℃ for 10 minutes at a rate of 25%/75% (m/m). The mixture was sheared with a blender for 15 minutes and then stirred by an impeller at 20 ℃ for 2 hours. The product was then filtered under vacuum through a buchner funnel to obtain a pale yellow filtrate containing 1.15% dry matter. The filtrate was used in the following test.

Determination of the mutagenic Activity of the bacterial Strain Salmonella typhimurium (Salmonella typhimurium) (TA 100) Bacterial back mutation test

The test is performed in three main phases:

performing a preliminary experiment to evaluate the cytotoxicity of the element to be tested and selecting a dosage range for the subsequent experiment,

a first genotoxicity test (test 1), with and without metabolic activation, incorporating the test system and the test (or control) directly on minimal agar, within the dose range prescribed by the preliminary study,

a second experiment (test 2), pre-incubation of the test system and test element (or control), with and without metabolic activation, the dose level being specified by the study responsible after analysis of the results of the first experiment. This second experiment was performed in order to confirm or complete the results of the first experiment, especially when ambiguous or negative results were obtained.

Dilutions of the test elements were prepared in analytical grade water.

Cytotoxicity tests were performed on the strain Salmonella typhimurium TA100 with and without S9-mixture (S9-Mix) at concentrations of 5000, 1600, 500, 160 and 50. Mu.g/plate.

Reagents for preparing the S9-mixture were prepared according to the following description:

TABLE 21

The bacteria were exposed to test extracts with and without metabolic activation system. The metabolic system used is the postmitochondrial component that supplements the cofactor (S9). The S9 fraction, the microsomal fraction of Sprague-Dawley rat liver homogenate treated with enzyme inducer, was prepared according to Maron, D.M. and Ames, B.N. (1983) and was supplied by Moltox. TM. It is stored at a temperature below-70 ℃. The S9 microsomal fraction was used at a concentration of 10% in the S9-mixture. The application protocol was as follows:

The following were introduced into three lysovessels:

o assay without metabolic activation:

test elements of various concentrations of 0.1ml,

0.5ml of sterile 0.2M, pH 7.4 phosphate buffer,

2ml of top agar for Salmonella typhimurium,

0.1ml of bacterial inoculum (TA 100).

o assay with metabolic activation:

test elements of various concentrations of 0.1ml,

2ml of top agar for Salmonella typhimurium,

0.1ml of bacterial inoculum (TA 100),

0.5ml of S9-mixture.

Mix and pour onto the bottom agar surface previously spread in the petri dish.

Incubation at 37 ℃ + -2 ℃ for 48 to 72 hours.

These assays were performed for each test: preliminary cytotoxicity test, test 1 and test 2. Untreated control, negative control and positive control generated during the pre-incubation method were incubated at 37 ℃ ± 2 ℃ for 20 to 30 minutes before pouring into the top agar.

The application protocol was as follows:

the following were introduced into four 2-ml fractions of the top agar for salmonella typhimurium:

o 0.1ml of 0.2M phosphate buffer, pH 7.4,

o 0.1ml of a solvent,

o 0.1ml of S9-mixture,

o 0.1ml of the highest concentration test element formulation,

2ml of the fraction of the top agar for Salmonella typhimurium was used to check its sterility.

Mix and pour onto the bottom agar surface previously spread in the petri dish.

Incubation at 37 ℃ + -2 ℃ for 48 to 72 hours.

This test was performed in triplicate.

Bacterial growth should not be observed.

For at least five concentrations of the test extracts, tests without and with metabolic activation were performed.

Expression and interpretation of results

Many criteria allow to determine if the result is positive, in particular an increase in the number of revertants associated with the dose of the test item, or a reproducible increase in the number of revertants at one or more concentrations, with or without metabolic activation.

-treating the test element as mutagenic if in the conclusion of the validation step a dose-effect relationship is reproducibly obtained on one or more of the five strains with and/or without metabolic activation. A given concentration is considered mutagenic when the number of revertants is at least equal to two times the spontaneous recovery (R.gtoreq.2) for strains TA98, TA100 and TA102 and at least equal to three times the spontaneous recovery (R.gtoreq.3) for strains TA1535 and TA 1537.

If in conclusion of test 1 and test 2 the frequency of revertants remains less than twice the spontaneous recovery (R < 2) for strains TA98, TA100 and TA102 and less than three times the spontaneous recovery (R < 3) for strains TA1535 and TA1537 for all concentrations of test element with and without metabolic activation, and provided that the absence of mutagenic effect is checked independent of the toxicity of the test concentration, the test element is considered non-mutagenic.

Preliminary studies showed that the test elements were not cytotoxic; thus, this concentration range was used for genotoxicity test 1.

Based on the results obtained for test 1, it was decided to use the same dilution range for test 2. Analysis of the revertants showed:

no cytotoxic effect is observed and,

with and without metabolic activation, the concentration of the test extract shows that the ratio R is greater than or equal to at least two times the spontaneous recovery for TA98, TA100 and TA102, or greater than or equal to three times the spontaneous recovery for TA1535 and TA1537,

no dose response was observed regardless of the test system or test conditions.

Based on the results obtained in this study, the extract of moringa oleifera according to example 1 can be regarded as having no mutagenic or mutagenic (promutagenic) activity.

In vitro phototoxicity test 3T3 NRU

The principle of the test is based on a comparison of cytotoxicity of the extract of moringa oleifera according to example 1 on cells in culture in the presence and absence of non-cytotoxic doses of UVA. 24 hours after treatment with reference elements and the extract of moringa oleifera, with or without UVA irradiation, by using a vital dye: neutral red determines cell viability to assess cytotoxicity. The cells used were mouse embryonic fibroblasts of Balb/c 3T3 clone 31 (ATCC-CCL 163). The positive control was chlorpromazine solution (CAS No.: 69-09-0). The negative control was a dilution (buffered saline solution ± 1% solvent) for the test extract and the reference. The extract of Moringa oleifera was tested in the presence or absence of UVA at eight concentrations in at least four culture wells/study concentrations. The fibroblasts were pancreatin digested and inoculated in two 96-well plates containing 2X 10 in complete medium ⁵ Individual cells/ml (i.e. 2X 10 ⁶ Individual cells/well) of 100 μl of cell suspension.

The plates were incubated at 37℃with 5% CO ₂ Incubate for 24 hours. At the end of the incubation, the cell lawn was examined for half-confluency. The dilutions were prepared just prior to deposition on the cells. Measuring the pH of the highest concentration; it is between 6.5 and 8. The medium was removed, each well carefully pre-washed with 150 μl of PBS maintained at room temperature, and then treated with 100 μl of each extract or reference dilution. The plates were incubated in the dark at 37℃and 5% CO ₂ Incubate for 1 h.+ -. 5 min. Irradiation was performed using a Bio Sun solar irradiator (Vilber Lourmat RMX W). The Bio Sun machine is a system for controlling UV irradiation by means of a programmable microprocessor. The system continuously tracks UV light emissions. Irradiation is automatically stopped when the delivered energy is equal to the programmed energy. The spectral irradiance of the test device was measured using a calibrated spectral radiometer over a wavelength range of 250 to 700 nanometers. One of the two plates was irradiated with its cover (cover) at room temperature, and the other plate was protected from UVA and kept at room temperature during irradiation. After irradiation, the treatment medium is extracted and the cells are rinsed. Mu.l of complete medium was then carefully added and the plates were incubated at 37℃with 5% CO ₂ Incubate for 18 to 22h. The next day, cell viability (growth, morphology, monolayer integrity) was assessed by observation using a phase contrast microscope. The medium was removed and each well was pre-rinsed and kept at room temperature prior to treatment with 100 μl of staining solution. The plates were returned to the incubator for 3 hours under the same conditions. The staining solution was removed and the cells were washed, then the washing solution was removed, and 150 μl of desorption solution was added to each well. The plate was shaken until the crystals were completely dissolved. Absorbance values were measured at 450 nm.

And (3) testing and verifying:

after the cells were exposed to increased irradiation doses, the cells were examined for UVA sensitivity approximately every 10 passages by assessing the viability of the cells. Cells were cultured at the density used in the test. The next day at a rate of 2.5 to 9J/cm ² They were irradiated and cell viability was determined by means of NRU test after one day. If the cells are at 5J/cm ² Viability after UVA irradiation is greater thanOr equal to 80% of the viability of the control maintained in the dark, the cells meet the quality criterion; at the highest dose of 9J/cm ² The viability must be at least equal to 50% of the viability of the control maintained in the dark under UVA.

Results:

the negative control has an absorbance greater than or equal to 0.4. Positive control chlorpromazine has IC in the presence of UVA ₅₀ Values of 0.1 to 2. Mu.g/ml, and 7 to 90. Mu.g/ml in the absence of UVA. These results allow verification of the test. The concentration of the extract of the moringa oleifera cake that resulted in 50% cell death in the presence or absence of UVA could not be estimated. Mortality never reaches 50%. The concentration of the arabinogalactan cake extract that produced 50% cell viability in the presence or absence of UVA could not be estimated. Viability was always greater than 50%.

Conclusion: under the experimental conditions employed, the moringa oleifera cake extract can be considered to be non-toxic.

Evaluation of eye irritation potential by studying SIRC cell line cytotoxicity in vitro using neutral Red Release method

The in vitro study was based on the determination of the concentration (IC) that resulted in 50% cell death on the cell monolayer by means of the neutral red release technique ₅₀ ) To evaluate cytotoxicity of the arabinogalactan cake extract. The cells used were mycoplasma-free SIRC rabbit corneal fibroblasts (ATCC-CCL 60).

The Moringa oleifera extract was diluted to 25% and 50% in physiological saline. The fibroblasts were pancreatin digested and in two 24-well plates to contain 2X 10 in complete medium ⁵ Ratio of individual cells/ml 1ml cell suspension. The plates were incubated at 37℃with 5% CO ₂ Incubate overnight. At the end of the incubation, confluence of cell lawn was examined. Staining solutions were prepared at 0.5mg/ml in complete medium. The medium was removed and 1ml of staining solution was placed in each well. The plate was returned to 37℃and 5% CO ₂ The incubator below was 3 hours ± 15 minutes. After this contact time, the staining solution was removed and replaced with 1ml of complete medium per well. At and extract The plates were kept at room temperature for at least 30 minutes to stabilize the system prior to object or reference contact. Each well was rinsed with 2ml of PBS, kept at room temperature, and then 500 μl of each dilution of the moringa extract or reference was placed in contact with the cell lawn. The contact time was 60 seconds (positive control was 30 seconds). At the moment of the placement of the arabinogalactan extract or reference, a stopwatch is started and the treatment is performed hole by hole. The plate was manually shaken throughout the process. After 55 seconds (or 25 seconds for positive control), the dilutions were withdrawn. At exactly 60 or 30 seconds, five sequential washes (5 x 2ml PBS, kept at room temperature) were performed. The supernatant was withdrawn after each wash and after the last wash, the wells were kept free of medium while waiting for the display phase. After the plates were completely treated, 1ml of desorption solution was placed in each well. The plate was shaken for about 15 minutes until a uniform staining was obtained. The solution obtained for each culture well was removed and split into two wells of a 96-well plate, i.e., 150. Mu.l/well.

Results:

the concentration of the moringa extract that resulted in 50% cell death was rated as >50%. The percentage of cell death of the arabinogalactan extract at 50% was rated as 17%.

Conclusion: under the experimental conditions employed, the cytotoxicity of the extract of the moringa oleifera cake can be regarded as negligible cytotoxicity.

Under dermatological control, the skin of the extract of Moringa oleifera was evaluated after a single administration for 48 hours under bandaging therapy Skin compatibility

The purpose of this study was to evaluate the degree of skin compatibility of the moringa extract by performing an epicutaneous (epikutaneous) test for 48 hours on the front and outer sides of the arm; and the ability of the extract of moringa oleifera to maintain skin in good condition is generally evaluated. 10 healthy female or male volunteers from 18 to 65 years old, with no dry skin nor sensitive skin and no dermatological lesions in the treated area, were included in the study. After 30 to 40 minutes of initial application after removal of the dressing, the skin compatibility of the arabinogalactan extract prepared in the form of a lotion comprising 5% of the arabinogalactan extract according to example 1 and 95% of the propylene glycol/sorbitol mixture was evaluated for 48 hours. Skin reactions (erythema and edema) were scored from 0 to 3 according to the following scale:

TABLE 22

Any other skin reactions (bullae, pimples, blisters, dryness, desquamation, roughness, soap effects, etc.) were evaluated and reported descriptively according to the following scale:

-0: no reaction

-0.5: very mild

-1: mild and mild

-2: medium and medium

-3: is remarkable in

At the end of the study, the average stimulation score (m.i.s.) was calculated according to the following formula:

[ math figure 8]

M.i.s. =sum of skin reactions (er+oe+bulla+pimple+bleb)/number of volunteers analyzed

The m.i.s. Obtained allows to classify the test extracts according to the grades presented in the following table:

M.I.S. less than or equal to 0.20 non-irritating

0.20< M.I.S. < 0.50 light stimulus

0.50< M.I.S. 2 or less moderate stimulation

2< M.I.S. less than or equal to 3 high stimulation

Results:the average irritation fraction (m.i.s.) of the arabinogalactan cake extract is equal to: 0.

conclusion: after 12 volunteers had been administered for 48 hours in succession, the arabinogalactan cake extract could be regarded as non-irritating.

General conclusion of the test:

the results of the tests performed above were conclusive for the arabinogalactan extract according to example 1 and demonstrated:

1. eye and skin irritation test negative

2. Phototoxicity test negative

3. The mutagenicity test was negative.

The safety of the extract of moringa oleifera according to the present invention has been demonstrated and is ideal for large-scale topical cosmetic use, without being limited by the target population.

Example 18: by measuring the percutaneous moisture loss (TEWL) on the skin barrier and for a period of 21 days in use Post acceptability evaluation

The product studied in this study was a care product in the form of a cream of example 15. The product was applied to the clean face by gentle massaging in the morning and evening, avoiding the area around the eyes. Measurements were made on the cheeks.

The evaluation criteria are:

evaluation of skin barrier effect: comparison of TEWL values before any application of the product (D1) and then after 21 days of application (D21).

Feedback on uncomfortable D21.

Cosmetic acceptability: questionnaires filled in with respect to D21 volunteers.

22 female volunteers with an average age of 50 years (age 20 to 70 years) were tested for all skin types. The following results are given for the skin barrier evaluation product ^* Change in D21 relative to D1, ^** wilcoxon (Wilcoxon) test of paired data, where s=significance (p.ltoreq.0.05 and ns=no significance (p>0.05)：

TABLE 23

Analysis of the results shows that TEWL remains stable at D21 compared to D1: the product showed a "skin protection" effect 21 days after application. Whereas there is no significant decrease in TEWL value at D21 compared to D1, the instrumental measurements fail to show the "nourishing" effect of the test product. 81% of volunteers responded positively to the "skin nourished" question in the acceptability questionnaire at D21.

Conclusion: under the conditions of this study, the cream showed a "skin protection" effect, shown by TEWL measurement, and good cosmetic acceptability, with a favorable evaluation of 86%.

Claims

1. An arabinogalactan seed extract, characterized in that the arabinogalactan seed extract is obtained by: extracting the non-dehulled seed cake solid-liquid in a solvent of predominantly alcohol in a proportion of 20 to 30% by weight of solid matter relative to the total weight used, the alcohol being selected from ethanol or methanol, optionally together with a polyhydric alcohol or subcritical water co-solvent, for a period of time of plus or minus 10% to 20% for 2 hours at a temperature of 16 to 30 ℃, the proportion being 70% to 100% by weight of alcohol relative to the total weight of the solvent of predominantly alcohol, and separating the liquid and solid phases to remove the solid phase and recover a liquid extract of moringa seed, the extract comprising more than 50% of the compound 2, 5-diformylfuran relative to the dry matter of the total extract.

2. The extract according to claim 1, characterized in that the obtained liquid extract is dried to obtain a dry extract of the seed cake of moringa oleifera comprising more than 50% by weight of 2, 5-diformylfuran with respect to the total weight of dry matter.

3. A process for obtaining an extract of seeds of moringa oleifera according to any one of claims 1 and 2, characterized in that it comprises the following steps, wherein:

c) Milling the cake obtained in step b),

d) Dispersing the milled material obtained in step c) in a predominantly alcoholic solvent selected from ethanol or methanol, optionally together with a polyhydric alcohol or subcritical water co-solvent, in a proportion of 20 to 30% by weight of solid material relative to the total weight used, in a proportion of 70 to 100% by weight of alcohol relative to the total weight of the predominantly alcoholic solvent;

e) Extracting solid and liquid under stirring at 16-30deg.C for a period of time of 10-20% or more and 2 hr,

4. A method according to claim 3, wherein the predominantly alcohol solvent is 96 ° pure ethanol.

5. A method according to claim 3, characterized in that the liquid moringa extract is purified by distillation, microfiltration, ultrafiltration and/or nanofiltration to concentrate the 2, 5-diformylfuran of the extract relative to the organic material that is also extracted.

6. Cosmetic composition, characterized in that it comprises, as active agent, an effective amount of an extract of seeds of moringa oleifera according to any one of claims 1 and 2, together with physiologically acceptable excipients.

7. The composition of claim 6, wherein the composition is a cosmetic composition formulated for topical application to skin, and wherein the moringa 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.

8. Use of a composition according to one of claims 6 to 7 in cosmetics for improving the appearance of skin, mucous membranes or integuments, for relaxing, soothing and pressing the skin, and for preventing and/or combating the signs of aging and/or photoaging of the skin, and for preventing senile plaques.