MX2008008739A - Use of an unsaponifiable extract of plant pulp in the treatment of skin ageing - Google Patents

Abstract

The field of the present invention relates to the use of an unsaponifiable extract of plant pulp for the preparation of a cosmetic, pharmaceutical or nutraceutical product intended to treat and/or prevent skin disorders related to ageing.

Description

USE OF AN INSAPONIFIABLE VEGETABLE PULP EXTRACT IN THE TREATMENT OF SKIN AGING FIELD OF THE INVENTION The domain of the present invention relates to the use of an unsaponifiable extract of vegetable pulp for the preparation of a cosmetic, pharmaceutical or nutraceutical product intended to treat and / or prevent the skin disorders associated with aging. BACKGROUND OF THE INVENTION Aging is an inevitable, slowly evolving and irreversible problem that includes anatomical and histological modifications responsible for functional abnormalities of the organs. The first signs are manifested at the level of the cutaneous tissue due to alterations in texture, color, transparency and the appearance of wrinkles. These manifestations can be potentiated by extrinsic factors such as sun, tobacco, etc. The importance of oxygenated free radicals (ROS) in the processes involved in aging is considered one of the main theories. At the skin level, the RLOs described as the early mediators of inflammatory pathologies and aging (Kress M, et al., Pain 1995; 62: 87-94). In the course of aging, all structures of the skin are modified. But the fundamental alterations predominantly in the dermis and these are the fibroblasts and the extracellular matrix that are the main objectives and the main actors. The fibroblasts are capable of entering senescence. As a result, their number decreases, their function slows down and their phenotype modifies. They then participate actively in the degradation of the extracellular dermal matrix. Furthermore, after senescence, the fibroblasts lose their reactivity and their regulation is modulated. Indeed, it is admitted that aging is associated with a reduction of a part of the response to environmental stress, and thus, an appearance of infectious diseases, autoimmune diseases and cancers (Gardner ID Rev. Infect. Dis. 1980; 2: 801-10). The appearance of wrinkles is one of the earliest signs of aging. It constitutes, for certain people, a real problem in its relations with the outside world. Thus, in the present, numerous cosmetic products aimed at the treatment of skin aging are made available to the public. Mainly, their specialties are based on plant extracts. The arganier, known in the international botanical nomenclature under the name of Argania spinosa (L.) S ells, it has been marked out especially by the cosmetic industry, and more particularly the kernel almond. The arganier is a stunted tree 6 to 10 meters high, where the aspect reminds that of the olive tree. The appearance of the leaf crown is not very variable, it can be straightened or it can be weeping. The very thorny branches have small lanceolate leaves, alternate, narrow, short (about 2 cm), often grouped into fascicles. The foliage of the arganier is generally persistent, but it happens that in a period of great dryness, it becomes obsolete. The flowers, of greenish yellow color, hermaphrodites (stamens and pistils in the same flower) and pentamers (5 petals, 5 sepals ...), regroup in inflorescences of the glomerulus type. They open from May to June. The arganier fructifies at the age of 5 years. The fruit is a sessile and oval yellow berry of 4 to 5 cm in length. It is formed of a fleshy pericarp (also called pulp), enclosing a sort of very hard "brown bone". This element is actually made of 2 to 3 crushed grains welded together and each enclosing an oilseed kernel. The most valuable applications originate from the grain kernel. This supplies an oil later, in a second time a solid residue.

The oil extracted from the grains is the subject of several invention patents: obtaining the oil by solvent (FR 2 553 788), enriched and unsaponifiable argan oil (FR 2 724 663). Substances other than oil have also been patented. This is the case of the peptides issued from the borujo of the grains obtained after the extraction of the oil: association of the oil and the peptides of borujos or solid residues for the treatment of the problems linked to skin aging (FR 2 756 183). The arganier sheet, the proteins and the saponins of the solid waste are also the subject of the patents of invention: EP 1 213 025 concerning the leaf extracts, reduced EP 1 213 024 of the solid waste proteins and EP 1 430 900 of solid waste saponins. The pulps of arganier fruits are the most recent object of patent application O2005 / 039610. The fruit of the arganier is a false drupe. It is constituted then of a fleshy pericarp called pulp (55 to 75% of fruit) and of a bone provided with a very hard shell that contains one to three almonds. The oil is extracted from the latter. The pulp of the fruit is the object of chemical studies. It consists of carbohydrates where cellulose, glucose, fructose and sucrose (Charrouf Z. Guillaume D., Ethnoeconomical, ethnomedical and phytochemical study of Argania spinosa (L.) Skeels., Journal of Ethnopharmacology, 1999, 67, 1, 7-14 - Sandret FG, Etudes p'reliminaires des glucides et du latex of the pulp of the fruit of Argen (Argania spinosa): variation au cours de la maturation, Bulletin of the Societé de Chimie Biologique, 1957, 39, 5-6, 619-631). The lipids are also present. Its content is 6%. In the unsaponifiable fraction of these lipids, 5 triterpene alcohols = erythrodiol, lupeol a and ß-amirin, betulinaldehyde and 2 sterols = spinosterol and schottenol (Charrouf Z., Fkih-Tetouani S., Charrouf M. Mouchel B., Triterpenos et al. stérols extraits from the pulp d'Argania spinosa, Plantes Medicinales et Phytothérapie, 1991, 25, 2-3, 112-117). The patent application WO2005 / 039610 deals, in general, with the use of the composition based on arganier fruit pulps for the preparation of cosmetic products. The fruit pulp extract has been more or less purified. Thus, it is preferably the use of an extract of fruit pulps contained next to a hexane extraction described (page 15). Followed by a classical saponification step known to the person skilled in the art, the authors have tested the fraction unsaponifiable thus received. Finally, the authors have visualized a step of fractionating the unsaponifiable by chromatography taking care to divert the triterpene compound of erythrodiol. This reasoning has been truly guided by the results obtained especially by the fact that erythrodiol is only present as toxic (example 1) at a lower dose than the triterpene fraction as defined in the document: fraction A devoid of erythrodiol (page 38). In addition, erythrodiol alone has only a mediocre benefit against UVA and UVB (examples 3 and 4), with respect to said triterpene fraction. The general teaching of this document starts from the use of the triterpene fraction of an arganier fruit pulp extract for the preparation of cosmetic products and preferably in the treatment of skins damaged by UVA and UVB via the stimulation of the metabolism of the fibroblasts. More specifically, this document teaches that said triterpenic fraction as disclosed in O2005 / 039610 will therefore be more active that the amount of erythrodiol will be weak. Surprisingly unexpected, the authors of the present invention have shown an effect of inhibition of senescence of the skin fibroblasts mature with an unsaponifiable extract of alganier fruit pulps rich in erythrodiol; said extract is capable of being obtained by extraction with acetone followed by a classical saponification step. However, it can be reasonably considered that the benefits of the present invention can be understood with any unsaponifiable extract of vegetable pulp having a triterpene fraction where the composition in its main compounds is close to that exit of the arganier fruit pulps. The present invention relates to the use of an unsaponifiable extract of vegetable pulp comprising a triterpene fraction, characterized in that said triterpene fraction comprises erythrodiol, -amyrin and lupeol, for the preparation of a cosmetic, pharmaceutical or nutraceutical product intended to prevent and / or treat skin disorders associated with skin aging. Preferably, said extract is obtained by an acetonic extraction followed by a classical saponification step. This unsaponifiable extract, still called initial unsaponifiable, can be solubilized in an excipient to facilitate its formulation.

Preferably said extract is obtained from a vegetable chosen from the Sapotacae or Sapotaceae family; and even more preferably said extract is obtained from arganier fruit pulps. The advantage of acetonic extraction lies in the fact that it can be released from latex, which represents the largest majority of the lipid fraction, and thus is more concentrated of unsaponifiable substance in the lipid fraction. The composition of the unsaponifiable according to the present invention differs both qualitatively and quantitatively from that described preferably in the patent application O2005 / 039610. The extracts of the present invention are characterized by their content of triterpene substances. The latter can be analyzed by gas chromatography according to an appropriate classical method that allows the identification of β-amyrin, erythrodiol. In contrast, a-amyrin and lupeol are not separated by this method, these molecules can then be commonly dosed. Advantageously, the triterpene fraction of said extract is composed of erythrodiol where the mass fraction is between about 7% and about 40% of the initial unsaponifiable, the β-amyrin where the mass fraction is between about 5% and about % of the initial unsaponifiable, the a-amirin and the lupeol where the sum of these two mass fractions is comprised between about 10% and about 50% of the initial unsaponifiable. Advantageously, said mass fraction of erythrodiol is comprised between about 10% and about 20% of the initial unsaponifiable; and even more advantageously it is equal to approximately 15% of the initial unsaponifiable. Advantageously, said mass fraction of β-amyrin is comprised between about 7% and about 20% of the unsaponifiable inciial; and even more advantageously it is equal to approximately 10% of the initial unsaponifiable. Advantageously, the sum of said a-amyrin and lupeol mass fractions is comprised between about 15% and about 30% of the initial unsaponifiable; and even more advantageously it is equal to approximately 20% of the initial unsaponifiable. The contents of these different molecules depend on the extraction conditions. These values will be less important in the cosmetic, pharmaceutical or nutraceutical product depending on the excipient (s) that will be added to the initial unsaponifiable. A remarkable point of the present invention is the important contribution of erythrodiol in the properties anti-aging of the unsaponifiable extract according to the present invention. The RLO plays an important role in the process of skin aging, the antiradical effect (antho-RLO) of the erythrodiol has been evaluated in comparison with the unsaponifiable of the arganier fruit pulps according to the present invention. The domains created by the RLO within the cells are translated by the alteration of the lipid compounds of the plasma membrane (lipoperoxidation), the proteins (denaturation and degradation) and the genetic material or DNA (mutations). Tests carried out in vitro have led to the determination: - of the protective efficacy by erythrodiol and by the unsaponifiable extract against the oxidation of membrane lipids (example 3), - - and of the protective power of erythrodiol and other triterpene molecules (-) lupeol a and β-amyrin) against the alteration of genomic DNA (example 4). These trials have made it possible to demonstrate that erythrodiol is a molecule that has an important anti-oxidant potential. In a particular embodiment of the present invention, the extraction can be carried out as follows: the pulps of the dried arganier fruit are then ground extract with acetone. It is also possible to use a mixture of acetone-water. The extraction is carried out under agitation or statically, in a plant / solvent ratio that can vary from 1/2 to 1/20, at temperatures that vary from the ambient temperature to the boiling temperature of the solvent and over a duration that It can go from 30 minutes to 24 hours. Once extracted, the solid residue of the plant is separated from the extractive solution by filtration or centrifugation. The solution can be more or less concentrated until obtaining a dry extract. In the latter case, the dry matter can be solubilized in an alcohol to allow saponification. To the solution, a metal hydroxide, in particular soda or potash, is added at concentrations ranging from 0.1 to 10 N. The saponification is conducted at temperatures ranging from room temperature to boiling temperature, with stirring and for a varying duration. from 15 minutes to 48 hours depending on the temperature. The purification is conducted by a liquid / liquid extraction. A non-miscible solvent, which can be water saturated or not with salts [NaCl, (NH4) 2S04] and added at a pH ranging from 3 to 9, is then added to the hydrolysis medium. This solvent can be an ether oxide, an ester , an alkane, a halogenated hydrocarbon, or a mixture of these solvents. One, two or three successive liquid / liquid extractions are performed. The organic phases are combined then washed with water saturated or not with salts and at a variant pH of 3 to 9. This washing phase can be repeated several times. After the purification, the organic phases are treated so as to eliminate the solvent. This treatment can be carried out by evaporation controlling the pressure. The evaporation step can be conducted to a product with a more or less waxy lipid consistency, the initial unsaponifiable. You can add an excipient that can be an animal wax (bee for example) or vegetable wax (carnauba, candelilla or jojoba wax) a vegetable oil (corn, safflower, sesame, argan ..) glycerin, the products of origin synthetic, such as petroleum jelly, polyols (such as propylene glycol, butylene glycol, glycerol ...), esterified triglycerides (such as migliol 812, miritol 318, neobee MJ), oxypropylene polymers of formula H (OCH2- CHCH3) nOH or the oxyethylenated polymers of the formula H (OCH2-CH2) nOH, the fatty alcohol diesters of variable length from Ci to C40. The proportions of the initial unsaponifiables of the arganier fruit pulp and the excipient may vary from 1/99 to 99/1.

Advantageously, the present invention allows an appreciation of the fruit pulps is anti-aging treatment, at a reasonable return cost. The unsaponifiable extract is used without a complementary purification step, which is very expensive. The composition according to the present invention can then be obtained with the aid of a process involving the conventional extraction and saponification steps known to the person skilled in the art. The use of an unsaponifiable extract of vegetable pulp according to the present invention makes it possible to obtain and / or treat skin disorders that are manifested by alterations in texture, color, transparency of the skin and the appearance of wrinkles. In a particular embodiment of the invention, the skin disorders are consecutive to a reduction or loss of response to environmental stress, especially caused by sun or tobacco. In another particular embodiment of the invention, the skin disorders are consecutive to a reduction or a loss of inducibility of HSP72 proteins. HSP proteins for "heat shock protein" are expressed constitutively in numerous cells and possess the essential functions in maintaining proteins, hence their name of "company" proteins. In effect, they inhibit the aggregation of denatured proteins, avoid inappropriate associations of proteins and are not involved in intracellular transport and maintenance in the inactive form of certain proteins (Morris SD Clin. Exp. Dermatol., 2002; 27 : 220-224). HSPs also play an essential role in the response to stress or stress and especially in the cellular protection processes that bring into play the adaptive response (Maytin EV J. "Invest. Dermatol., 1995; 104: 448-55). unexpectedly, the use of an extract according to the present invention allows restoring the induction of the HSP72 proteins in the senescent fibroblasts In the framework of the present invention, the cosmetic, pharmaceutical or nutraceutical product, enclosing an extract according to the invention, is administered orally or topically, preferably topically, for topical administration, the galenic form is selected from the group comprising creams, gels, ointments and aerosols Advantageously, the oral form is selected from the group consisting of: group comprising tablets, capsules or powders for drinkable suspensions.

Advantageously, the amount of said extract in the final cosmetic product is between about 0.001% and about 50%, preferably between about 0.01% and about 10%, and even more preferably between about 0.1% and about 2% by weight respect to the total weight of the preparation. The preparation can also contain other active ingredients well known to the person skilled in the art, for the treatment and / or prevention of skin disorders associated with cutaneous aging. Advantageously, said preparation contains other substances than arganier salts known for their "anti-age" action such as the oil obtained from the almond of the grain and the peptides of the borujos for example. The example here below of a composition of the invention is given by way of indication and not limitation. The percentages are given by weight with respect to the total weight of the composition. Example 1: Anti-loosening care of the face - Unsaponifiable extract of arganier fruit pulp 0.1 to 2% - Argan oil enriched 1 to 5% - Argan peptides 0.1 to 1% - Vitamin E derivatives 0.1 to 0.5% - Vitamin E glyceric ester 0.1 to 0.5% - Vitamin A palmitate 0.1 to 1% - Methyl glucose stearate 1 to 5% - Caprylic capric triglycerides 2 to 8% - Liquid paraffin 5 to 12% - Perfume is - Purified water csp lOOg The following examples illustrate the present invention without limiting the scope in any way. Example 2: Method of obtaining an unsaponifiable extract of arganier fruit pulps. One ton of dried arganier fruit pulps are crushed after being removed from a 5 tonne acetone reactor. The extraction is conducted under stirring for one hour at reflux. Once re-cooled, the solution is recovered by filtration, then it is concentrated under vacuum until obtaining an oily solvent extract. This residue is taken up in 500 1 of ethanol at 95% v / v. 100 l of sodium hydroxide solution is added and brought to reflux under hard stirring for one hour. After cooling, the hydrolyzed solution is placed in a decanter, 500 1 of heptane and 300 1 of water are added. The liquid / liquid extraction is conducted with caution. After decanting, the organic phase is recovered. Repeat 2 new extractions with 500 1 of heptane. The 3 heptanic phases are regrouped and washed 3 times with 500 1 of water each time. The solvent is removed from the washed organic phases. A waxy paste is thus recovered. This extract, which corresponds to the initial unsaponifiable, is dosed for its content in triterpene substances. It contains 10% of ß amirin, 15% of erythendiol and 20% of lupeol-amyrin mixture. Example 3: Analysis of the anti-radical effect of erythrodiol - analysis of lipid peroxidation. 1) Introduction The plasma membrane is the main and the first target of the RLO and, being enriched in liquid, is the site of increased peroxidation (Girotti A.W.J. Free Radie, Biol. Med 1985; 1: 87-95). The peroxides generated in the course of this lipid oxidation are also very reactive and capable of degrading the genomic protein material. To evaluate the alteration of the membrane, the inventors have measured lipid peroxidation by in vitro dosing of complexes between lipid oxidation products and thiobarbituric acid. These complexes are called TBARS (by thiobarbituric acid reactive substances) and the name is given to the test: TBARS. After imitating a chemical oxidative stress, the L929 fibroblast line has been treated by a complex composed of hydrogen peroxide (H202) and iron (Fe2 + / Fe3 +) thus reconstituting the reaction of Fenton, source of RLO and more particularly of the radical hydroxyl (0H °) (Vessey DA et al J. Invest. Dermatol. 1992; 99: 859-63): H2? 2 + Fe "+? 0H ° + OH" + Fe, 3 +. 2) Methodology Products tested: The products have been evaluated on the line of murine fibroblasts L929. The cells are pretreated with the different product concentrations (Table I) for 16 hours and then stimulated with the H202-Fe2 + / Fe3 + complex for 1 hour. Lot LK0304 of unsaponifiable extract of arganier fruit pulp has been prepared according to example 2. Table 1: Presentation of the tested solutions (* Anti-radical reference molecule) Peroxidation of membranous lipids is analyzed by measuring TBARS (according to Morlière P. et al., Biochim, Biophys, Acta 1991, 1084: 261-268). Principle of the test: In acidic medium, at 95 ° C complexes are formed, noted TBARS for reactive substance of thiobarbituric acid, between lipid oxidation products (malondialdehyde or MDA) and thiobarbituric acid (TBA) that can be dosed in fluorescence with respect to a standard range with MDA. Dosage of TBARS is indicated in pmol / μg of proteins. The proteins and TBARS are dosed in the intracellular medium. Calculation of the protection percentage of cell membranes: From the calculation of TBARS in pmol / μg proteins, the protective efficiency of different products against the oxidation of membrane lipids is calculated as follows: [TBARS control] - [TBARS (+ products) ] % protection = [TABRS control] 3) Results - discussion After a 16-hour treatment with the various products to be tested, the radical tension model used in this experiment (Fenton reaction) induces a important lipid peroxidation in fibroblasts L929.

This massive discharge of hydroxyl radical OH ° then generates an oxidative stress at the cellular level and especially at the level of the membranes. However, in this type of oxidative reaction, the products generated from the lipid peroxidation are internalized in the cells and the TBARS are then dosed in the intracellular medium. The results obtained are presented in Table II below Table II: Analysis of lipid peroxidation Vitamin E, which constitutes the anti-radical reference molecule, decreases lipid peroxidation induced by the H20-Fe2 + / Fe3 + complex, and very efficiently protects cell membranes (approximately 56%). The unsaponifiable extract of arganier fruit pulps prepared according to example 2 presents an anti-radical activity at concentrations of 1 and 3 μg / ml (30 and 37% protection of lipid membranes, respectively). Erythrodiol, a molecule contained in the triterpene fraction of the unsaponifiable extract, has a good anti-oxidant activity with a dose-dependent effect. Erythrodiol is active from 0.3 μg / ml (33% protection). The anti-radical effect of erythrodiol s 3μg / ml is very important and comparable to vitamin E. 4) Conclusion The in vitro model presented in this study reflects the consequences due to a greater oxidative stress on the main cellular target that is the membrane plasmic Thus, the dose of lipid peroxidation is a good marker of oxidative stress and allows the evaluation of the antioxidant action against the hydroxyl radical of the active principles at the level of the cell membrane. Vitamin E, an anti-oxidant molecule, allows the validation of this model. Under the experimental conditions, it is observed that the extract according to the present invention contains the erythrodiol as well as erythrodiol itself that has an important antioxidant potential. Example 4: Analysis of the anti-radical effect of erythrodiol - analysis of genomic damage 1) Introduction DNA is an objective of the RLO that induces the modification of the base (oxidation, nitration, deamination: Guetens G. et al Cli. Lab. Sci 2002; 39: 331-457), the formation of strand breaks (non-basic sites or β-elimination) and DNA-protein or DNA-hydroperoxide bridges. The alteration of the genomic material induces a cascade of cellular reactions (bifurcation blockade of replication, activation of key proteins, arrest in the cell cycle) that ends in the induction of repair mechanisms. The bases modified by stress or oxidative stress are thus mostly taken over by the base repair system or SEE by base excision repair (Friedberg E.C. et al DNA repair and mutagenesis, ASMPress, Washington DC 1995). This system acts quickly and efficiently according to three key stages: 1. Recognition of the altered base; 2. Incision and excision of the lesion 3. Re-synthesis of the gap The RLO can be produced in such a quantity that the defense systems and the cell repair can be saturated. If the effectors of apoptosis are activated, the damaged cells die. But in the case where DNA lesions are poorly repaired, there may be generation of deleterious mutations that are then involved in the initiation stage of carcenogenesis. That is why the biological incidence of stress or oxidative stress (mortality or mutagenesis) conditions longer-term events such as aging and cancer. Numerous studies have shown the strong correlation between aging and the progressive and irreversible accumulation of oxidative domains at the level of cellular macromolecules. Many research groups have shown in the rodent, that the rates of 8-0xoGuanina, measured in different tissues such as skin, increase with age (Tahara S. et al Mech, Ageiging Dev. 2001; 122: 145-426) . The works of Meccocci P. et al (Free Radie, Biol. Med. 1999; 26: 303-8) on skeletal muscle in man, show that oxidative lesions in DNA or in lipids accumulate with age. The same team has also shown that in subjects afflicted with Alzheimer's disease, the rate of oxidized bases in lymphocyte DNA and the rate of antioxidants in plasma are significantly higher and lower respectively, than in healthy subjects (Meccocci P. et al Arch Neurol, 2002; 59: 794-8). 2) Objective Continuing with example 3 and in order to control the anti-radical activity of erythrodiol on another model, the authors of the invention have analyzed its protective power against the alteration of genomic DNA induced by an oxidative stress, in comparison with the unsaponifiable extract of the arganier fruit pulps and other triterpene molecules also contained in said extract. We have chosen to generate the DNA lesions by a tension of H202 and to indirectly analyze the damage thus formed by analyzing the repair reaction. For this, a team called the 3D DNA Damage Detection (Salles B. et al Anal, Biochem, 1995; 232: 37-42 and Salles B. et al Biochemi, 1999; 81: 53-58) has been used. . The 3D test is based on the repair of DNA lesions in the medium of purified human cell extracts. In the course of the repair stage, a marker is incorporated into the DNA and this incorporation, quantitatively reflects the number of repaired lesions, is immediately revealed by chemiluminescence. 3) Methodology Products tested: The products have been evaluated on the line of murine fibroblasts L929. The cells are pre-treated with the products (Table III) for 16 hours and then stimulated with H202 (3 & amp; hydrogen peroxide - Ref. GIFRER - Laboratorire Gifrer Barbezat) at 100 μM for 30 minutes. Table III: Presentation of proven solutions 3D test: The principle is the following: after the damage of the genomic DNA (oxidative treatment), the cells are lysed. The used is deposited on a microplate coated with polylysine: 1. Adsorption of DNA 2. Incubation of DNA with a protein extract enriched with repair enzymes) and a set of nucleotides where a nucleotide was labeled with biotin (dUTP-Biotin) - Repair of lesions and incorporation into the DNA of nucleotides marked "dUTP-Biotin" 3. Incubation with an enzymatic complex "avidin-peroxidase" - avidin recognition of the dUTP-Biotin incorporated. 4. Addition of a luminescent peroxidase substrate and quantification of the emitted signal proportional to the name of repaired lesions. The protocol for carrying out the test is followed according to the instructions of the equipment or kit provider (3D Test Solycel - Ref: SFRIDN013 - AES Laboratoire). At the end of the reaction, the plate is read in a luminometer (MITHRAS LB940 - BERTHOLD). Calculation of the protection percentage of the DNA The yield immediately allows to calculate, for each concentration of the tested product, the% protection against the induction of lesions on the DNA by an oxidative stress (the luminescence intensity - or IL - explains the amount of DNA lesions) IL (H202) - IL (product)% protection of DNA = x 100 IL (H202) - IL (control) 4) Results and conclusion The results obtained in example 3 have shown that erythrodiol has the strongest anti-radical activity at 3μg / ml (6.78μM). This is because the authors have chosen to test all triterpenes (lupeol, α-amyrin, β-amyrin and erythrodiol) and the unsaponifiable extract of arganier fruit pulps at 3 μg / ml in the 3D test "Damage Detection DNA. " Said unsaponifiable extract has been obtained following the procedure of example 2. The results obtained are presented in table IV down. The values indicated in this table are the percentages of inhibition (or% protection) of the lesions in the DNA as a consequence of an exogenous oxidative stress, with respect to the cells "base control" (100%) and the cells "stressed by H202"(0%).

Table IV: Protection of DNA by erythrodiol The treatment of H202 induces a strong oxidation rate at the level of guanine with formation in particular of d-oxo-7,8-dihydro-deoxyguanosine (8-OxoGuanin) (Dizdaroglu M. et al Arch. Biochem Biophys., 1991; 285: 388-390). The 3D test shows a strong luminescence increase after treatment with H202, reflecting a strong repair activity and consequently a significant base rate damaged in the DNA. The unsaponifiable extract of arganier fruit pulps effectively protects the ADNB against oxidative stress.

Erythrodiol, the molecule contained in said unsaponifiable extract, at 3μg / ml has a very good anti-oxidant activity with 99% protection of the DNA against the formation of oxidative lesions. Comparing triterpenic molecules with equivalent molar concentration (approximately 7μM), it is the erythrodiol that is most active. Example 5: In vitro study model of the effect of unsaponifiable extract according to the invention on the induction of HSP72 proteins. 1) Bibliography Different works have shown the loss of inductibility of HSP72 proteins after aging. In mature patients, the induction of the HSP72 protein by heat is significantly reduced at the cutaneous level (Muramatsu T. et al., Br.J., Dermatol., 1996; 134: 1035-1038.) On the other hand, Gustmann -Conrad A. et al. (Exp. Cell Res. 1998; 241: 404-413) have shown that the induction of the HSP72 protein by thermal stress, significantly decreases in the fribroblasts that leave the "skin of mature subjects. with respect to those that come out of young subjects. In this same study, it has been shown that the level of induction of HSP72 is reduced equally in fibroblasts (which leave the skin young) or in the lines of fibroblasts (IMP-90) become senescent in the course of cell divisions. A moderate first strain undergoes an in vitro induction of the HSPs proteins in order to protect the cell against new stresses (Morris SD et al., J., Clin. Invest., 1996; 97: 706-12). Main protein of the HSP72 family, expressed in keratinocytes and cutaneous fibroblasts and inducible by numerous tensioning agents (heat, UV ...) (Trauntinger F. et al., J. Dermatol, 1993; 101: 334-38; Charveron M. et al., Cell Biol Toxicol, 1995; 11: 161-65) 2) Experimental protocol The authors of the present invention have chosen to analyze the level of induction, by thermal stress, of the HSP72 proteins in the IMR-90 fibroblasts (fibroblastic line) after senescence, and this in order to evaluate the "anti-age" properties of an extract of arganier fruit pulps prepared according to example 2, either containing 10% of β-amyrin , 15% of erythrodiol and 20% of the lupeol-amyrin mixture. The authors have put in place and validated a cell aging model that induces the senescence of fibroblasts by an oxidative stress. - Induced senescence model: Fibroblasts are divided into a critical state called replicative senescence and assimilated to cellular aging. But senescence can also be induced especially by an oxidative stress, if it is "Premature Senescence Induced by tension or SIPS" (Dumont et al Free Radie, Biol. Med 2000; 28: 361-373). Model used: The induction of senescence in the line of young IMR-90 fibroblasts has been revealed by treating 2h cells with H202. 72 hours after this tension, the IMR-90 cells are senescent. In a second time, they have shown the decrease of a level of induction of HSP72 followed by a thermal stress, in the senescent fibroblasts compared with the young fibroblasts. Finally, the properties of an arganier fruit pulp extract prepared according to example 2 have been evaluated, either containing 10% of ß amyrin, 15% of erythrodiol and 20% of lupeol-amyrin mixture on this model of senescence. 3) Results The invention will be better understood and the objectives, advantages and characteristics thereof will appear more clearly from the description that follows and that is made with reference to the attached drawings in which: - Figure 1 presents the analysis of the rate of Induction of HSP72 in IMR-90 fibroblasts at the transcriptional and translational level. - Figure 2 shows the Western blot analysis of the HSP72 protein rate in the IMR-90 cells treated with different concentrations of the arganier fruit pulp extract according to the invention. - Figure 3 presents the semi-quantitative analysis of the induction rate of HSP72 proteins (normalized by the level of β-actin expression) in pre-treated senescent IMR-90 fibroblasts with different concentrations of pulp extract from arganier fruit according to the invention. Analysis of the induction of HSP72 by a tension or thermal stress in the course of the senescence of IMR-90 fibroblasts: The cells cultured at 37 ° C are incubated 1 hour at 45 ° C and then incubated at 37 ° C during 2 hours (mRNA analysis) or during 4h (protein analysis): - Expression of the protein (Western Spotting) The intracellular proteins, extracted from the fibroblasts, were analyzed by the Western spotting technique, using an anti-HSP72 antibody (monoclonal antibody, CHEMICON and an indirect revelation system in luminescence.) The membrane is analyzed and the intensity of the Bands are quantified by densitometry (Logiciel ImageMaster TotalLab AMERCHAM). The expression level of HSP72 is normalized by that of a constitutively expressed protein, β-actin. Figure IA presents the semi-quantitative Western blot analysis of protein induction rates HSP72 in the young IMR-90 (B) and senescent IMR-90 (U) (senescence induced by H202). Thus, Figure IA clearly shows that the HSP72 protein rate is induced by a thermal stress in young IMR-90 fibroblasts. This induction of HSP72 decreases in senescent IMR-90 fibroblasts. • Expression of mRNA (Real-time PCR) The authors analyzed the HSP72 expression at the transcriptional level quantifying the mRNAs by the real-time PCR technique. The level of expression of the gene of interest HSP72 is calculated in the samples treated by a thermal stress and the control samples. The level of expression of the HSP72 gene is then normalized using three reference genes [β-actin, GAPDH (human glyceraldehyde-3-phosphate dehydrogenase) and YWHAZ (zeta polypeptide of the tyrosine-3-monooxygenase activation protein, tryptophan- 5-monooxygenase)], where the expression is constitutive. Finally, by setting the expression level in the control samples in 1, then it is possible to determine the induction factor of the HSP72 gene. Figure IB presents the real-time PCR analysis of the induction rate of HSP72 mRNA in young IMR-90 (B) and senescent IMR-90 (B) (senescence induced by H202). Figure IB clearly shows that the induction of HSP72 mRNA is similarly greatly diminished after the induced senescence of IMR-90 fibroblasts. - Analysis of the effectiveness of arganier fruit pulp extract: The authors of the invention have used the induced senescence model or SIPS (premature senescence induced by stress or stress) with the fibroblastic line IMR-90 to evaluate the extract of Arganier fruit pulps prepared according to example 2. The cells were incubated with the extract of arganier fruit pulps at the concentrations of 1 and 3 μg / ml for 24 hours. Then the oxidative stress rises inducing senescence. All treatments have been compared with a batch of "young" IMR90 cells and a batch of "senescent" cells (induced senescence) not pretreated by the extract of Arganier fruit pulps. Three days (72h) after stress or oxidative stress, HSP72 have been induced by heat. Finally, RNA and HSP72 proteins have been analyzed by real-time PCR and Western spotting, respectively. Figure 2 shows the Western blot analysis of the HSP72 protein rates in the IMR-90 cells treated with the different concentrations of the unsaponifiable extract prepared according to example 2. The mentions "T" and "ST" mean respectively "Control" and "Thermal stress or thermal stress". The analyzes A, B, C and D refer respectively to the young IMR-90 fibroblasts, the senescent IMR-90 fibroblasts (senescence induced by H202), the senescent IMR-90 fibroblasts incubated with the unsaponifiable extract at 1 μg / ml and finally the senescent IMR-90 fibroblasts incubated with the unsaponifiable extract at 3 μg / ml. Figure 3 presents the semi-quantitative analysis of the induction rate of HSP72 proteins (normalized by the level of β-actin expression) in senescent IMR-90 fibroblasts pre-treated with the unsaponifiable extract at lμg / ml ( C) and at 3μg / ml (D). The induction rates of HSP72 proteins in IMR-90 fibroblasts are also represented young (A) and in senescent IMR-90 fibroblasts (B) (senescence induced by H202). Figures 2 and 3 show that there is no further induction of HSP72 proteins in IMR-90 fibroblasts that become senescent, but that the extract of arganier fruit pulps, at concentrations of 1 and 3 μg / ml, restores the Induction of HSP72 by thermal stress. Finally, Table V below gives the induction factor (after normalization) of HSP72 mRNA in pre-treated senescent fibroblasts with different concentrations of arganier fruit pulp extract.

Table V: Induction factor This table confirms the results obtained at the transcriptional level and shows the almost-total restoration of the HSP72 mRNA induction by the extract of arganier fruit pulps. This is the concentration of 3μg / ml that this extract is the most active. 4) Conclusion HSP72 proteins are proteins that are inducible by numerous stresses (heat ...) and are strongly involved in adaptive response processes. It is recognized that the inducibility of HSP72 proteins, at the cutaneous level and in other tissues, decreases with age and especially after cellular senescence. In addition, it is admitted that aging is associated with a reduction in the response in the environmental stress generated by pathologies linked to age. From a model of induced senescence in cultured fibroblasts, the authors evaluated the ability of arganier fruit pulp extract to modulate the decrease in induction of HSP72 by heat. The set of results confirms on the one hand that there is a strong decrease in the induction of HSP72 (by a thermal stress) in senescent fibroblasts compared to young fibroblasts. On the other hand, these works show that the extract of arganier fruit pulp restores the induction of HSP72 proteins in senescent fibroblasts. In this model of in vitro study, arganier fruit pulp extract limits the biological consequences of cellular senescence and then presents the anti-aging properties.

Claims (13)

1. CLAIMS 1. The use of an unsaponifiable extract of vegetable pulp comprising a triterpene fraction, characterized in that said triterpene fraction comprises erythrodiol, a-amyrin, β-amyrin and lupeol, for the preparation of a cosmetic product, pharmaceutical or nutraceutical intended to prevent and / or treat skin disorders associated with skin aging, the amount of erythrodiol is comprised between 7 and 40% by weight of the unsaponifiable extract.
2. 2. The use according to claim 1, characterized in that the mass fraction of β-amyrin is comprised between 5% and 30% of the unsaponifiable extract.
3. 3. The use according to claim 1, characterized in that the sum of mass fractions of a-amirin and lupeol is comprised between 10% and 50% of the unsaponifiable extract.
4. 4. The use according to claim 1, characterized in that the amount of said extract in the final cosmetic product is between 0.001% and 50%, preferably between 0.01% and 10%, and even more preferably between 0.1 and 2% by weight of the total weight of the preparation.
5. The use according to claim 1, characterized in that said extract is obtained from a vegetable chosen from the botanical family of Sapotaceae or Sapotaceae.
6. 6. The use according to claim 1, characterized in that said extract is obtained from arganier fruit pulps.
7. The use according to claim 1, characterized in that the skin disorders are manifested by the alterations of texture, color, transparency of the skin and by the appearance of wrinkles.
8. 8. The use according to claim 1, characterized in that the skin disorders are consecutive to a reduction or a part of response to stress or environmental stress.
9. The use according to claim 8, characterized in that the environmental stress is caused by the sun, the tobacco.
10. 10. The use according to claim 1, characterized in that the skin disorders are consecutive to a reduction or a part of inductibility of HSP72 proteins.
11. The use according to claim 1, characterized in that the cosmetic, pharmaceutical or nutraceutical product is under the oral or topical form, preferably under the topical form.
12. 12. The use according to claim 11, characterized in that the topical form is chosen from the group comprising creams, gels, ointments and aerosols.
13. 13. The use according to claim 11, characterized in that the oral form is chosen from the group comprising the tablets, capsules and powders for drinkable suspensions.