HK1124779B - Use of an unsaponifiable extract of plant pulp in the treatment of skin ageing - Google Patents

Description

Use of unsaponifiable extract of plant pulp for treating skin aging

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 nutritional product for the treatment and/or prevention of skin disorders associated with aging.

Aging is an inevitable, slowly progressing and irreversible phenomenon that causes anatomical and histological changes leading to organ dysfunction. The first visible signs appear at the skin tissue level as changes in texture, color and transparency, and the appearance of wrinkles. These manifestations can be enhanced by extrinsic factors such as sunlight, tobacco, etc.

The importance of oxygen radicals (OFR) in the process of participating in aging is considered to be one of the main theories.

At the skin level, oxygen radicals are described as early mediators of inflammatory pathology as well as aging (Kress M. et al, Pain 1995; 62: 87-94).

During aging, all the structures of the skin change. However, the fundamental changes are primarily in the dermis, and fibroblasts and extracellular matrix are the primary targets and major participants of these changes. Fibroblasts are able to enter a state of senescence, as a result of which their number is reduced, function is diminished, and phenotype is altered. In this case, they are actively involved in the degradation of the skin extracellular matrix. Furthermore, during senescence, fibroblasts lose their reactivity and undergo changes in their regulation. This is because it is recognized that aging is associated with a reduction or even loss of response to environmental stress and thus also with infectious, autoimmune and cancer-like diseases (Gardner I.D. Rev. Infect.Dis.1980; 2: 801-10).

The appearance of wrinkles is one of the earliest signs of aging. For some people, this is a real problem in their relationship to the outside world. Thus, today, many cosmetic products aimed at treating skin aging are available to the public. These special products are based mainly on plant extracts.

The argania tree (arganier), known in international plant nomenclature as arganiaspepinosa (L.) shells, is multiplied by its fitness, especially its kernel, especially by the cosmetic industry.

The Aronia melanocarpa is a low tree with a height of 6-10 m, and the tree shape of the Aronia melanocarpa is reminiscent of an olive tree.

The crown shape may vary somewhat, either upright or sagged. Branches are very thorny, are covered with intercropping, narrow, short (about 2cm) aciculiform lobules, and are often gathered into bundles.

The leaves of argan are usually evergreen, but also fall off during periods of extreme dryness.

Yellow-green, hermaphrodite (stamen and pistil on the same flower), five-base (five petals, five sepals), conglomerated into globular inflorescences. The flowering period is from May to June.

The Argan trees are grown from five years old. The fruit is a yellow, oval, stemless berry, 4 to 5 cm in length. It consists of a fleshy peel (also called pulp) that harbors a very hard brown "pseudostone". The "pseudonut kernel" consists in fact of 2 to 3 flat seeds, each comprising an oily kernel, stuck to each other.

The most valuable application is in kernels, which provide oil, and consequently oil cake.

Oils of seed origin have been the object of several patents: obtaining an oil with a solvent (FR 2553788); artocarpus oil enriched in unsaponifiable fraction (FR 2724663).

Substances other than oil have also been patented. An example is a polypeptide from a seed oil cake obtained after oil extraction: the combination of oil and polypeptide in oil cake is used for treating disorders related to skin aging (FR 2756183). The protein and saponin in the leaves and oil cake of the argan tree are also the objects of the invention patent: EP 1213025 relates to leaf extract, EP 1213024 relates to protein in oil cake, and EP 1430900 relates to saponin in oil cake.

Recently, the pulp of the fruit of Argania spinosa has been the object of patent application WO 2005/039610.

The fruit of Argania spinosa is a pseudodrupe fruit. It therefore consists of a fleshy peel (55% to 75% of the fruit) called flesh and a kernel with an extremely hard outer shell, containing 1 to 3 kernels. The oil is extracted from the kernel.

Chemical studies have been performed on the flesh of the fruit. It is composed of sugars including cellulose, glucose, fructose and sucrose. (Charrouf Z. Guillame D., Ethnoeconomic, ethnomedical, and phytochemical study of Argania spinosa (L.), Skoels, Journal of Ethnohormalogy, 1999, 67, 1, 7-14; Sandret F. G., Etuder pr preferably terms glucides et du latex del de du de pu de du freit d' Argan (Argania spinosa) variation of urea de la mapping, Bulletin de la Soci de Chimie biologicals, 1957, 619, 5-6, 631). Lipids are also present. Their content was 6%. 5 triterpene alcohols have been identified in the unsaponifiable fraction of these lipids: higher glycols, lupeol, alpha-and beta-liposantalin, betulinal, and two sterols: alpha-spinasterol (alpha-spinosrol) and cactus sterol (schott nol) (Charrouf Z., Fkih-Tetouani S., Charrouf M., Mouchel B., Triterp. et sterol extrics de lapulpe d' Argania spinosa, Plates M bicinales et Phytothrapi, 1991, 25, 2-3, 112-.

Patent application WO2005/039610 generally refers to the use of compositions based on the pulp of the fruit of argan for the preparation of cosmetic products. The pulp extract has been somewhat purified. This is because the inventors have tested the extract at various stages of the process. This therefore preferentially describes the use of the pulp extract obtained after hexane extraction (page 15). Subsequently, the authors tested the unsaponifiable fraction thus collected after a conventional saponification step known to the person skilled in the art. Finally, the authors have set up a step of separating the unsaponifiable fraction by chromatography, taking care to remove the triterpenoid homodiol.

The reason for this may be based on the results obtained, in particular on the fact that higher diols alone exhibit toxicity at doses lower than the triterpene fraction defined in this document (example 1): fraction a has no high-radical diols (page 38). Furthermore, the higher diol alone showed only general advantages for UVA and UVB compared to the triterpene fraction (examples 3 and 4). The general teaching of this document therefore relates to the use of the triterpene fraction of the pulp extract of the fruit of argan tree for the preparation of cosmetic products, preferably for the treatment of skin affected by UVA and UVB by stimulation of the metabolism of fibroblasts. More specifically, this document teaches that the triterpene fractions disclosed in WO2005/039610 are more active than low levels of high-root diols.

Surprisingly and unexpectedly, the authors of the present invention demonstrated the inhibitory effect of the unsaponifiable extract of the pulp of the fruit of argan tree, rich in high radiciol, on the ageing of mature skin fibroblasts; the extract may be obtained by acetone extraction followed by a conventional saponification step. However, it is reasonable to expect that the benefits of the present invention can be extended to any unsaponifiable extract of plant pulp having a triterpene fraction whose composition approaches in terms of major compounds the triterpene fraction derived from argania pulp.

The present invention relates to the use of an unsaponifiable extract of plant pulp comprising a triterpene fraction, characterized in that said triterpene fraction comprises high-radical diols, alpha-liposantalin, beta-liposantalin and lupeol, for the preparation of a cosmetic, pharmaceutical or nutritional product for the prevention and/or treatment of skin disorders associated with skin ageing. Preferably, the extract is obtained by acetone extraction followed by a conventional saponification step.

This unsaponifiable extract, also called the initial unsaponifiable fraction, may be dissolved in excipients to facilitate formulation.

Preferably, the extract is obtained from a plant selected from the family Sapotaceae (Sapotaceae). More preferably, the extract is obtained from the pulp of the fruit of the argan tree.

One advantage of acetone extraction is that: latex can be removed, which accounts for the majority of the lipid fraction and is therefore more concentrated in the unsaponifiable matter of the lipid fraction.

The composition of the unsaponifiable matter according to the invention differs in quality and quantity from that described preferentially in patent application WO 2005/039610.

The extract according to the present invention is characterized by containing a triterpene substance. The triterpene species may be analyzed by gas chromatography according to conventional suitable methods which allow the identification of β -liposantonin and homoradiciol. In contrast, α -santalin and lupeol cannot be separated in this way, and thus, the content measurement can be generally performed for these molecules.

Advantageously, the triterpene fraction of the extract consists of: a high-root diol in a mass fraction of about 7% to about 40% of the starting unsaponifiable matter; beta-santalin in a mass fraction of about 5% to about 30% of the starting unsaponifiable matter; alpha-pterocarpin and lupeol, the sum of the two mass fractions being from about 10% to about 50% of the starting unsaponifiable matter.

Advantageously, the mass fraction of high-radical diols is from about 10% to about 20% of the starting unsaponifiable matter; more advantageously, equal to about 15% of the starting unsaponifiable matter.

Advantageously, the mass fraction of β -santalin is from about 7% to about 20% of the starting unsaponifiable matter; more advantageously, equal to about 10% of the starting unsaponifiable matter.

Advantageously, the sum of the mass fractions of α -santalin and lupeol is from about 15% to about 30% of the starting unsaponifiable matter; more advantageously, equal to about 20% of the starting unsaponifiable matter.

The content of these different molecules depends on the extraction conditions. Depending on the excipients added to the starting unsaponifiable matter, these values will be lower in the cosmetic, pharmaceutical or nutraceutical product.

One salient point of the present invention is that high-root diols contribute significantly to the anti-aging properties of the unsaponifiable extract according to the invention. Since oxygen radicals play an important role in the aging process of the skin, the anti-radical (anti-oxygen radical) effect of high-root diols was evaluated by comparison with the unsaponifiable matter of the pulp of argan according to the invention.

The damage caused by oxygen radicals in cells is manifested by changes in the lipid composition of the plasma membrane (lipid peroxidation), changes in proteins (denaturation and degradation), and changes in genetic material or DNA (mutations). In vitro tests were performed to determine:

protective efficacy of high-rooted diols and unsaponifiable extracts against membrane lipid oxidation (example 3); and

protective capacity of homoradiciol and other triterpene molecules (lupeol, α -and β -santonin) for genomic DNA alterations (example 4).

These tests have demonstrated that high root diols are molecules possessing significant antioxidant potential.

In one embodiment of the invention, the extraction may be performed as follows: the dried pulp of the fruit of the argan tree is ground and then extracted with acetone. Mixtures of acetone/water may also be employed. The extraction is carried out under stirring or in a static manner, the plant/solvent ratio being variable between 1: 2 and 1: 20, the temperature being variable between ambient temperature and the boiling point of the solvent, and the time being from 30 minutes to 24 hours.

Once extracted, the solid residue of the plant is separated from the extraction solution by filtration or centrifugation. The solution may be more or less concentrated until a dry extract is obtained. In the latter case, the dry matter may be dissolved in an alcohol to allow saponification.

To this solution is added a metal hydroxide, in particular sodium hydroxide or potassium hydroxide, in a concentration of from 0.1N to 10N. The saponification is carried out at a temperature from ambient temperature to the boiling point under stirring, the duration varying from 15 minutes to 48 hours depending on the temperature.

Purification is carried out by liquid/liquid extraction. Then, an immiscible solvent, which may be a salt adjusted to a pH of 3 to 9 [ NaCl, (NH), is added to the hydrolysis medium₄)₂SO₄]Saturated or unsaturated water. The solvent may be an oxyether, 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 and subsequently washed with water, saturated or unsaturated with salts, at a pH of 3 to 9. The washing process can be repeated several times.

After purification, the organic phase is treated to remove the solvent. This process can be done by evaporation with controlled pressure. The evaporation step may result in a product with a somewhat waxy, lipid consistency, i.e. a starting unsaponifiable material.

Excipients may be added which may be animal waxes (e.g. beeswax) or vegetable waxes (e.g. carnauba, candelilla or jojoba wax), vegetableOils (corn, safflower, sesame, argania, etc.), glycerol, products of synthetic origin such as vaseline oil, polyols (e.g. propylene glycol, butylene glycol, glycerol, etc.), esterified triglycerides (e.g. miglyol 812, myritol318, neobee MJ), molecular formula H (OCH)₂-CHCH₃)_nPropylene oxide polymers of OH or of the formula H (OCH)₂-CH₂)_nEthylene oxide polymers of OH, different lengths (C)₁To C₄₀) Diesters of fatty alcohols of (a). The initial unsaponifiable fraction of the argan pulp may be present in a ratio of 1/99 to 99/1 to the excipient.

Advantageously, the present invention enables the flesh of the fruit to be used for anti-aging treatment at a reasonable cost. The unsaponifiable extract can be used without additional purification steps, which are very expensive. The composition according to the invention can therefore be obtained by means of a process incorporating conventional extraction and saponification steps known to those skilled in the art.

The use of the unsaponifiable extract of plant pulp according to the invention makes it possible to prevent and/or treat skin disorders which are manifested by changes in the texture, color and transparency of the skin and the appearance of wrinkles.

In a particular embodiment of the invention, the skin condition is caused by a reduced or absent response to environmental stress, in particular environmental stress caused by sunlight or tobacco.

In another embodiment of the invention, the skin condition is caused by a reduction or loss of inducibility of HSP72 protein. HSP (labeled "heat shock protein") proteins are constitutively expressed in many cells and have an essential function in maintaining proteins, from which they are named "chaperone" proteins. This is because they inhibit the aggregation of denatured proteins, prevent inappropriate binding of proteins, and they are involved in intracellular trafficking and in maintaining certain proteins in an inactive form (Morris S.D.Clin. exp. Dermatol. 2002; 27: 220-). HSP's also play a fundamental role in stress responses, especially in cytoprotection that mediates adaptive responses (Maytin E.V.J.invest.Dermatol.1995; 104: 448-55).

Surprisingly, the use of the extract according to the invention makes it possible to restore the induction of HSP72 protein in senescent fibroblasts.

Within the scope of the present invention, the cosmetic, pharmaceutical or nutraceutical product comprising the extract according to the invention is administered by oral or topical route, preferably by topical route.

For administration by topical route, the galenic form is selected from creams, gels, ointments and sprays.

Advantageously, the oral form is selected from tablets, capsules and powders for drinkable suspensions.

Advantageously, the amount of the extract in the final cosmetic product is from about 0.001% to about 50%, preferably from about 0.01% to about 10%, more preferably from about 0.1% to about 2% by weight of the total weight of the formulation.

Furthermore, the formulations may also comprise other active substances known to the person skilled in the art for the treatment and/or prevention of skin disorders associated with skin ageing. Advantageously, the formulation comprises other substances from the argan tree known for their "anti-ageing" effect, such as oil obtained from the kernel and peptides in the oil cake.

The following examples of compositions according to the invention are given by way of illustration and not by way of limitation. The percentages are given in proportions by weight relative to the total weight of the composition.

Example 1: anti-facial laxity care

-unsaponifiable extract of Argania pulp 0.1-2%

-enriched argania oil 1 to 5%

0.1 to 1% of Polypeptides from Argania spinosa

-derivatives of vitamin E0.1 to 0.5%

-glycerides of vitamin F0.1 to 0.5%

-vitamin a palmitate 0.1 to 1%

-methyl glucose stearate 1 to 5%

-caprylic/capric triglyceride 2 to 8%

-liquid paraffin 5 to 12%

-a perfume q.s.

Q.s.p.100g of purified water

The following examples illustrate the invention without limiting its scope.

Example 2: method for obtaining unsaponifiable extract of Argania spinosa pulp

1 ton of dried pulp of the fruit of argan tree was ground and then extracted with 5 tons of acetone in a reactor. The extraction was carried out under reflux with stirring for 1 hour. Once cooled, the solution was recovered by filtration and then concentrated in vacuo until an oily extract was obtained which was desolventized. The residue was taken up in 500 liters of 95% (v/v) ethanol. Then, 100 liters of 10N sodium hydroxide (lessive de soude.

After cooling, the hydrolysed solution was placed in a decanter, to which 500 l heptane and 300 l water were added. The liquid/liquid extraction is carefully performed. After decanting, the organic phase was collected. An additional 2 extractions with 500 l heptane were carried out. The 3 heptane phases were combined and washed 3 times with 500 l of water each time. The washed organic phase is freed from the solvent. This gives a waxy paste. The content of triterpene species in this extract (corresponding to the starting unsaponifiable matter) was determined. It contains 10% beta-santalin, 15% homoradicol and 20% lupeol/alpha-santalin mixture.

Example 3: analysis of anti-radical Effect of high radical diol-analysis of lipid peroxidation

1) Introduction to the word

Plasma membrane is the main and primary target of oxygen radicals and, due to the lipid enrichment, is the site of increased peroxidation (Girotti A. W.J. free Radic.biol.Med.1985; 1: 87-95). Peroxides produced during lipid oxidation are also highly reactive and are capable of degrading proteins and genomic material.

To evaluate the membrane changes, the authors of the present invention measured lipid peroxidation by in vitro quantification of the complex between lipid oxidation product and thiobarbituric acid. These complexes are referred to as TBARS (for "thiobarbituric acid Reactive substrates") and the test is named this way: TBARS test.

To simulate chemical oxidative stress, a mixture of hydrogen peroxide (H)₂O₂) And iron (Fe)²⁺/Fe³⁺) The fibroblast line L929 was treated with the composed complexes, thus reproducing the Fenton (Fenton) reaction, which is the source of oxygen radicals, especially hydroxyl radicals (OH °) (vessey d.a. et al j. invest.dermatol.1992; 99: 859-63): h₂O₂+Fe²⁺→OH°+OH^-+Fe³⁺。

2) Method of producing a composite material

Test products:

the product was evaluated on the murine fibroblast line L929. Cells were pretreated with different concentrations of product (Table I) 16H then by₂O₂-Fe²⁺/Fe³⁺The complexes were stimulated for 1 hour. An unsaponifiable extract of argan pulp of batch LK0304 was prepared according to example 2.

Table I: overview of test solutions

Reference product	Mother liquor	Test solutions
			Unsaponifiable batches of argan pulp: LK0304	10mg/ml(DMEM/TWEEN20)-20℃	0.3μg/ml1μg/ml3μg/ml
High root diol/extrasynthiese batches: 05040605	10mg/ml DMSO-20℃	0.3μg/ml-0.68μM1μg/ml-2.26μM3μg/ml-6.78μM
			Vitamin E SIGMA T-1539	400mg/ml-20℃	400μg/ml-928.7μM

(a reference anti-radical molecule)

Membrane lipid peroxidation was analyzed by measuring TBARS. (according to Morri re P. et al, Biochim. Biophys. acta.1991; 1084: 261-268).

Principle of testing:

in an acidic medium, at 95 ℃, a complex is formed between the lipid oxidation product (malondialdehyde or MDA) and thiobarbituric acid (TBA), called TBARS (denoted "thiobarbituric acid reactive species"), the content of which can be determined by fluorescence with respect to the MDA standard series. The assay value for TBARS is expressed in pmol/. mu.g protein. Both protein and TBARS were assayed for their content in the intracellular environment.

Calculate the percentage of cell membrane protection:

starting from the calculation of TBARS in pmol/μ g protein, the protective efficacy of the different products against membrane lipid oxidation was calculated according to the following formula:

3) results-discussion

The free radical stress model used in this experiment (fenton reaction) induced significant lipid peroxidation in L929 fibroblasts 16 hours after treatment of various products to be tested. Thus, the release of substantial amounts of the hydroxyl radical OH ° generates oxidative stress at the cellular level, in particular at the cell membrane level. However, in this oxidation reaction, the products derived from lipid peroxidation are internalized into the cell and the TBARS content is measured in the intracellular environment.

The results obtained are listed in table II below.

Table II: analysis of lipid peroxidation

Vitamin E as a reference anti-radical molecule reduced by H₂O₂-Fe²⁺/Fe³⁺Lipid peroxidation induced by the complex, and very effective protection of cell membranes (about 56%).

The unsaponifiable extract of the pulp of Argania spinosa prepared according to example 2 showed anti-radical activity (protection of the lipid membrane by 30% and 37%, respectively) at concentrations of 1 and 3. mu.g/ml.

The high-radical diols contained in the triterpene fraction of the unsaponifiable extract exhibit good antioxidant activity and are dose-dependent. Higher diol was active from 0.3. mu.g/ml (33% protection). The anti-radical protective effect of homodiol at 3. mu.g/ml is very large, relative to vitamin E.

4) Conclusion

The in vitro model presented in this study reflects the consequences due to more oxidative stress on the major cellular target, the plasma membrane. Thus, the lipid peroxidation assay is a good marker of oxidative stress and enables the evaluation of the antioxidant effect of active ingredients against hydroxyl radicals at the level of the cell membrane.

The antioxidant molecule vitamin E enables validation of the model.

Under these experimental conditions, it was observed that both the extract of the invention comprising high root diol and high root diol itself have significant antioxidant potential.

Example 4: analysis of anti-radical Effect of high-radical diol-analysis of genome Damage

1) Introduction to the word

DNA is the target of oxygen radicals which lead to base modification (oxidation, nitration, deamination: Guetens G. et al, Glin. Lab. Sci.2002; 39: 331-. Changes in genomic material trigger a series of cellular responses (blockade of replication forks, activation of key proteins, cell cycle arrest) that ultimately lead to the induction of repair mechanisms. Thus, bases modified by oxidative stress are mainly responsible for Base Repair or the BER (meaning "Base Excision Repair") system (Friedberg E.C. et al, DNA Repair and mutagenesis, ASMPress; Washington DC 1995). This system functions quickly and efficiently through the following three key steps:

1-recognition of an altered base;

2-incising and excising the damaged part;

3-resynthesis of the cleft.

Oxygen radicals may be produced in such an amount that the cellular defense and repair systems are saturated. Damaged cells die if the effector of apoptosis is activated. However, in the case of poorly repaired DNA damage, harmful mutations may be generated, which thereby participate in the initiation step of carcinogenesis. This is why the biological effects of oxidative stress (death or mutagenesis) can restrict longer-term events such as aging and cancer.

Many studies have demonstrated that aging is closely related to the progressive and irreversible accumulation of oxidative damage at the cellular macromolecule level. Several groups showed that the levels of 8-oxoguanine measured in different tissues, such as the skin, in rodents increased with age (TaharaS et al, Mech. Ageing Dev.2001; 122: 415-426). Work by Mecocci P et al on human skeletal muscle (Free radic. biol. med. 1999; 26: 303-8) suggests that oxidative damage to DNA or lipids increases with age. This group also found that in subjects with Alzheimer's disease, the levels of oxidized bases in the DNA of lymphocytes and antioxidants in the plasma membrane were significantly higher and lower, respectively, than in healthy subjects (Mecocci P. et al, Arch. neurol.2002; 59: 794-8).

2) Purpose(s) to

Example 3 follows and in order to examine the anti-radical activity of homoradiciol in another model, the authors of the present invention analyzed its protective capacity against the alterations of genomic DNA induced by oxidative stress, in comparison with the unsaponifiable extract of the pulp of argan tree and with other triterpene molecules also contained in said extract.

Select them for passage through H₂O₂Stress produces damage, and the damage so formed is analyzed indirectly by analyzing the repair response. For this purpose, a so-called 3D (denoted "DNA damage detection: (A))DNA Damage DChoice) "). This biochemical assay mimics the repair response by excision in vitro (Salles B. et al, anal. biochem. 1995; 232: 37-42; and Salles B. et al, Biochimie 1999; 81: 53-58). The 3D test is based on the use of purified human cell extracts to repair DNA damage. During the repair phase, a marker is incorporated into the DNA, which incorporation is subsequently revealed by chemiluminescence, which is a quantitative reflection of the number of lesions repaired.

3) Method of producing a composite material

Products tested

The product was evaluated on the murine fibroblast line L929. Cells were pretreated with the product (Table III) for 16 hours and then with 100. mu.MH₂O₂(3% Hydrogen peroxide-reference GIFRER-Laboratoire Gifrer Barbezat) for 30 minutes.

Table III: overview of test solutions

Reference product	Mother liquor	Test solutions
			Unsaponifiable batches of argan pulp: LK0304	10mg/ml(DMEM/TWEEN20)-20℃	3μg/ml
High root diol/extrasynthiese batches: 05040605	10mg/ml DMSO-20℃	3μg/ml-6.78μM
			Luck alcohol (triterpene)	50mM DMSO-20℃	3μg/ml-7μM
Alpha-santalin (triterpene)	50mM DMSO-20℃	3μg/ml-7μM
			Beta-santalin (triterpene)	50mM DMSO-20℃	3μg/ml-7μM

O 3D test:

the principle is as follows: after genomic DAN damage (oxidative treatment), cells were lysed. The lysate was placed in a polylysine coated microplate:

1-adsorption of DNA;

2-incubating the DNA with a protein extract (rich in repair enzymes) and a pool of nucleotides, one of which is labeled with biotin (dUTP-biotin) -repairing the damage and incorporating the dUTP-biotin labeled nucleotide into the DNA;

3-incubation with an enzyme complex "avidin-peroxidase" -recognition of the incorporated dUTP-biotin by avidin;

4-addition of a peroxidase substrate capable of luminescence, and quantification of the signal emitted in proportion to the number of lesions repaired.

The protocol for this Test was followed according to the instructions of the kit supplier (Solyscel 3D Test-Ref: SFRIDN013-AES laboratory). At the end of the reaction, the plates were placed in a luminometer (MITHRAS LB940-BERTHOLD) for reading.

Calculation of percent DNA protection:

the following relationship enables the calculation of the protection% for each tested product concentration against the induction of DNA damage (the luminescence intensity or IL represents the amount of DNA damage) caused by oxidative stress.

4) Results and conclusions

The results obtained in example 3 show that higher diols show the strongest anti-radical activity at 3. mu.g/ml (6.78. mu.M). This is why the authors chose to test all triterpenes (lupeol, alpha-santalin, beta-santalin and homoradiciol) and unsaponifiable extracts of the pulp of the Argan tree in 3D tests (DNA damage detection). The unsaponifiable extract is obtained according to the method of example 2.

The results obtained are listed belowIn Table IV below. The values listed in the table are relative to "basal control" cells (100%) and "Via H₂O₂Stressed "cells (0%), percent inhibition (or protection%) of DNA damage caused by external oxidative stress.

Table IV: protection of DNA by high-radical diols

	Luminous intensity	DNA Damage (%)	Protection of DNA (%)
				Control	5460	0
H2O2100 mu M to 30 minutes	11576.7	100	0

High root diol 3 mug/ml-6.78 mug M	5520	1	99
				Unsaponifiable matter of Argania spinosa pulp 3 μ g/ml	6193.3	12	88
Lupeol 3 μ g/ml-7 μ M	9020	58.2	41.8
				Alpha-pterocarpin 3 mu g/ml-7 mu M	8663.3	52.4	47.6
Beta-santalin 3 mu g/ml-7 mu M	13133.3	125.4	-25.4

H₂O₂Treatment induced a significant proportion of oxidation at the guanine level, in particular the formation of 8-oxo-7, 8-dihydro-2' -deoxyguanosine (8-oxoguanine) (Dizdagroglu M. et al, Arch. biochem. Biophys.1991; 285: 388-. 3D testing showed that in H₂O₂The luminescence was significantly enhanced after the treatment, which reflects a clear increaseAnd thus reflects significant levels of damaged bases on DNA.

Unsaponifiable extracts of Argania pulp effectively protect DNA against oxidative stress.

High-rooted diols (molecules contained in the unsaponifiable extract) show very good antioxidant activity at 3. mu.g/ml, with 99% DNA protection against the formation of oxidative damage.

Higher diol is most active compared to the triterpene molecules at the same molar concentration (about 7. mu.M).

Example 5: in vitro research model of the influence of unsaponifiable extracts on the induction of HSP72 protein

1) Feed summary

Various studies have shown that HSP72 is rendered non-inducible during aging. In older patients, the heat induction of HSP72 protein was greatly reduced at the skin level (Muramatsu T. et al, Br. J. Dermatol. 1996; 134: 1035- > 1038). Gustmann-Conrad A. et al (Exp. cell. Res. 1998; 241: 404-413), on the other hand, show a significant reduction in the induction of HSP72 protein by heat stress in fibroblasts from the skin of an aged subject compared to fibroblasts from a young subject. In the same study, it was shown that the level of induction of HSP72 is also reduced in fibroblasts (from young skin) or in fibroblast lines (IMR-90) that become senescent during cell division.

Moderate first stresses are sufficient to induce HSP proteins in vitro, so that they protect cells against new stresses (Morris s.d. et al, j.clin.invest.1996; 97: 706-12). HSP72 is an important protein of the HSP70 family, which is expressed in keratinocytes and fibroblasts of the skin and can be induced by a number of stress factors (heat, UV, etc.) (traunger F. et al, J. invest. 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 chose to analyze the level of induction of HSP72 protein by thermal stress in IMR-90 fibroblasts (fibroblast cell line) during senescence in order to evaluate the "anti-aging" properties of the pulp extract of argan (containing 10% β -santalin, 5% homoradiciol and 20% lupeol/α -santalin mixture) prepared according to example 2.

First, the authors established and validated a model of cellular aging in which fibroblast senescence was induced by oxidative stress.

-model of induced senescence:

fibroblasts divide until a critical stage, called replicative senescence, similar to cellular aging. However, senescence can also be induced in particular by oxidative stress, which is referred to as "stress-induced premature senescence or SIPS" (Dumont et al, Free Radic.biol.Med.2000; 28: 361-.

The model used was: by using H₂O₂Cells were treated for 2 hours and an induction of senescence had been shown in the young fibroblast cell line IMR-90. IMR-90 cells became senescent 72 hours after this stress.

Second, they showed that the level of induction of HSP72 was reduced after heat stress in senescent fibroblasts compared to young fibroblasts. Finally, they evaluated the characteristics of the pulp extract of the argan (containing 10% β -santalin, 15% homoradiciol and 20% lupeol/α -santalin mixture) prepared according to example 2 in this aging model.

3) Results

The invention will be more fully understood and its objects, advantages and features will become more apparent from the following description taken in conjunction with the accompanying drawings in which:

FIG. 1 shows the analysis of the level of induction of HSP72 in IMR-90 fibroblasts at the transcriptional and translational level.

FIG. 2 shows Western blot analysis of HSP72 protein levels in IMR-90 cells treated with different concentrations of the extract of the pulp of Aronia melanocarpa according to the invention.

FIG. 3 shows a semi-quantitative analysis of the induction level of HSP72 protein (normalized by the expression level of β -actin) in senescent IMR-90 fibroblasts pretreated with different concentrations of the pulp extract of the Aronia melanocarpa of the present invention.

Analysis of heat stress-induced HSP72 during IMR-90 fibroblast senescence:

cells cultured at 37 ℃ were incubated at 45 ℃ for 1 hour, then at 37 ℃ for 2 hours (mRNA assay) or 4 hours (protein assay):

expression of protein (Western blot)

Intracellular proteins extracted from fibroblasts were analyzed by Western blotting technique using an anti-HSP 72 antibody (monoclonal antibody, chemoicon) and an indirect revealing system using luminescence. The film was analyzed and the intensity of the band quantified by densitometer (ImageMasterTotalLab software, AMERSHAM). The expression level of HSP72 was normalized by the expression level of β -actin, which was expressed constitutively.

FIG. 1A shows Western blotting of IMR-90(■) in young and senescent IMR-90(■) (from H)₂O₂Induced senescence) of HSP72 protein. Thus, figure 1A clearly shows that heat stress induced HSP72 protein levels in young IMR-90 fibroblasts. This induction of HSP72 is reduced in senescent IMR-90 fibroblasts.

Expression of mRNA (real-time PCR)

The authors analyzed HSP72 expression at the transcriptional level by quantifying mRNA using real-time PCR techniques.

Expression of the gene of interest HSP72 was calculated in the heat-stressed and control samples. The expression level of HSP72 gene was then normalized by using three constitutively expressed reference genes [ β -actin, GAPDH (human glyceraldehyde-3-phosphate dehydrogenase) and yhlaz (tyrosine-3-monooxygenase/tryptophan-5-monooxygenase activating protein ζ -polypeptide) ].

Finally, the expression level in the control sample was set to 1, whereby the fold induction of HSP72 gene could be determined.

FIG. 1B shows real-time PCR for IMR-90 at young age (■) and IMR-90 at old age (■) (from H)₂O₂Induced senescence) of HSP72 mRNA induction levels. FIG. 1B clearly shows that the induction of HSP72 mRNA was also greatly reduced during induced senescence of IMR-90 fibroblasts.

Analysis of the efficacy of the extract of fruit pulp of argan tree:

in order to evaluate the extract of pulp of the fruit of the Argania tree prepared according to example 2, the authors of the present invention used the induced Senescence or SIPS (stress induced Premature Senescence) model using the IMR-90 fibroblast cell line.

The cells were incubated with the extract of pulp of dried fruit of Argania spinosa at a concentration of 1. mu.g/ml to 3. mu.g/ml for 24 hours. They are then subjected to oxidative stress which induces senescence.

All treatments were compared for a batch of "young" IMR-90 cells and a batch of "senescent" (senescence-induced) cells that had not been pretreated with an extract of pulp from the argan tree.

HSP72 was induced with heat 3 days (72 hours) after oxidative stress. Finally, HSP72 RNA and HSP72 proteins were analyzed by real-time PCR and Western blot, respectively.

FIG. 2 shows Western blot analysis of HSP72 protein levels in IMR-90 cells treated with different concentrations of unsaponifiable extracts prepared according to example 2. Legends "T" and "STRespectively, the "control" and "heat stress". A. B, C and D analysis corresponded to young IMR-90 fibroblasts, senescent IMR-90 fibroblasts, respectively (from H)₂O₂Induced senescence), senescent IMR-90 fibroblasts incubated with 1. mu.g/ml of unsaponifiable extract, and senescent IMR-90 fibroblasts incubated with 3. mu.g/ml of unsaponifiable extract.

FIG. 3 shows a semi-quantitative analysis of the level of induction of HSP72 protein (normalized by the expression level of β -actin) in senescent IMR-90 fibroblasts pre-treated with 1 μ g/ml (C) and 3 μ g/ml (D) unsaponifiable extracts. It is also shown in young IMR-90 fibroblasts (A) and senescent IMR-90 fibroblasts (consisting of H)₂O₂Induced senescence) (induction level of HSP72 protein in B).

FIGS. 2 and 3 show that HSP72 protein was no longer induced in senescing IMR-90 fibroblasts, but that extracts of dried fruit pulp from the Argania spinosa at concentrations of 1. mu.g/ml and 3. mu.g/ml restored the induction of HSP72 by heat stress.

Finally, table V below shows the fold induction (after normalization) of HSP72 mRNA in senescent fibroblasts pretreated with different concentrations of the extract of fruit pulp of argan tree.

Table V: fold induction

This table confirms the results obtained at the transcriptional level and shows that the extract of pulp of argan almost completely restored induction of HSP72 mRNA. The activity of the extract was maximal at a concentration of 3. mu.g/ml.

4) Conclusion

HSP72 protein is a protein that can be induced by various stresses (heat, etc.) and is strongly involved in the process of adaptive response. It is recognized that the inducibility of HSP72 protein at the skin and other tissue levels decreases with age, particularly during cellular aging. Furthermore, it is recognized that aging is associated with a decreased response to environmental stress that causes age-related pathologies.

Starting from an induced senescence model in cultured fibroblasts, the authors evaluated the ability of the extract of fruit pulp of argan tree to modulate the heat-induced reduction of HSP 72.

Taken together, these results confirm, on the one hand, a strong reduction in the induction of HSP72 protein (by heat stress) in senescent fibroblasts compared to young fibroblasts. On the other hand, these works indicate that the extract of pulp of argan tree restores induction of HSP72 protein in senescent fibroblasts. In this in vitro study model, the extract of pulp of Argania spinosa limited the biological consequences of cellular senescence and thus exhibited anti-aging properties.

Claims (12)

1. Use of an unsaponifiable extract of plant pulp comprising a triterpene fraction, characterized in that said triterpene fraction comprises high-radiciol, alpha-santalin, beta-santalin and lupeol, wherein the mass fraction of high-radiciol is between 7% and 40% of said unsaponifiable extract, the mass fraction of beta-santalin is between 5% and 30% of said unsaponifiable extract and the sum of the mass fractions of alpha-santalin and lupeol is between 10% and 50% of said unsaponifiable extract, for the preparation of a cosmetic, pharmaceutical or nutritional product for the prevention and/or treatment of skin disorders associated with cutaneous ageing; and the extract is obtained from the pulp of the fruit of the argan tree.

2. Use according to claim 1, characterized in that the amount of said extract in the final cosmetic product is comprised between 0.001% and 50% by weight of the total weight of the formulation of said product.

3. Use according to claim 2, characterized in that the amount of said extract in the final cosmetic product is between 0.01% and 10% by weight relative to the total weight of the formulation of said product.

4. Use according to claim 3, characterized in that the amount of said extract in the final cosmetic product is comprised between 0.1% and 2% by weight of the total weight of the formulation of said product.

5. Use according to claim 1, characterized in that the skin disorders are manifested by changes in skin texture, color and transparency and the appearance of wrinkles.

6. Use according to claim 1, characterized in that the skin condition is caused by a reduced or lost response to environmental stress.

7. Use according to claim 6, characterized in that the environmental stress is caused by sunlight or tobacco.

8. Use according to claim 1, characterized in that the skin disorder is caused by a reduction or loss of inducibility of HSP72 protein.

9. Use according to claim 1, characterized in that the cosmetic, pharmaceutical or nutritional product is in oral or topical form.

10. Use according to claim 9, characterized in that the cosmetic, pharmaceutical or nutritional product is in a form for topical use.

11. Use according to claim 9 or 10, characterized in that the form for topical use is selected from creams, gels, ointments and sprays.

12. Use according to claim 9, characterized in that the oral form is selected from tablets, capsules and powders for drinkable suspensions.