METHOD FOR OBTAINING A ROSEWOOD EXTRACT, COMPOSITIONS COMPRISING IT AND ITS COSMETIC USES

Abstract

The invention relates to a rosewood extract obtained from co-products of the distillation of rosewood (Aniba rosaeodora) essential oil by supercritical CO2 extraction in the presence of a co-solvent. The invention also relates to the process for obtaining such an extract. Furthermore, the invention relates to cosmetic compositions comprising such an extract and, finally, to the cosmetic uses of such compositions for firming the skin, reducing sagging, redefining facial contours, and minimizing the appearance of wrinkles and fine lines.

Description

Title of the invention: METHOD FOR OBTAINING A ROSEWOOD EXTRACT, COMPOSITIONS COMPRISING IT AND ITS COSMETIC USES technical field

[0001] The present invention relates to the field of skincare ingredients. The invention relates to a rosewood (Aniba rosaeodora) extract composed, among other things, of nicotinic acid, cotoin, sesquiterpene derivatives, and depleted in volatile compounds and anibine, obtained by a supercritical CO2 extraction process from the co-products of rosewood distillation. The invention also relates to cosmetic compositions comprising such an extract, and finally to the uses of such compositions to prevent or limit the signs of skin aging. Technical background of the invention

[0002] Rosewood (Aniba rosaeodora Ducke or “rosewood” in English), is a tree found in the tropical and subtropical rainforests of the Amazon basin (Venezuela, Colombia, Ecuador, French Guiana, Guyana, Peru, Suriname and Brazil).

[0003] The trunk, branches or leaves of rosewood are used in perfumery and cosmetics in the form of essential oil whose distinctive olfactory characteristic is conferred by linalool, a volatile terpene alcohol present in large quantities in the essential oil (70 to 90%).

[0004] According to the literature, the non-volatile part of rosewood contains anibine, methoxy-4 paracotoine, cotoine and pinocembrine, but also (-)-rubranine, a chalcone present in the branches of this species (Mors, WB et al. J. Am. Chem. Soc., 1957, 79, 4507-4511; Gottlieb, OR and Mors, WB, J. Am. Chem. Soc., 1958, 80, 2263-2265; Galaverna, RS et al., Anal. Methods, 2015, 7, 1984-1990; De Alléluia, IB et al., Phytochem., 1978, 17, 517-521).

[0005] Several chemical classes of compounds present in rosewood, including aryl phenyl ketones, nicotinic acid and phytosterols, have shown biological activities such as antioxidant, antibacterial, antimicrobial, antifungal, anti-inflammatory, anti-cancer, vasodilatory and hypolipidemic properties (Wu, S.-B. et al., Nat. Prod. Rep., 2014, 31, 1158-1174).

[0006] The rosewood extracts described in the prior art are mostly obtained by hydrodistillation. This results in extracts enriched in volatile compounds and primarily in linalool (70 to 90%). However, a significant amount of these volatile compounds, including linalool and benzyl benzoate, are considered allergenic.

[0007] The inventors wished to develop an extract that did not exhibit these characteristics in terms of chemical composition and olfactory note. To this end, they chose to use a raw material of rosewood (trunk, branches, and leaves) that had undergone a first distillation and was therefore depleted of volatile aromatic compounds. This depleted raw material is extracted again using a supercritical fluid to obtain an extract enriched in semi-volatile to non-volatile compounds and possessing a high degree of chemical diversity.

[0008] To date, to the inventors' knowledge, no document describes the parameters for extracting rosewood using a supercritical fluid. For example, documents FR1000801B1, CN105708740, and CN105640822 describe cosmetic compositions and a massage oil comprising a rosewood essential oil obtained by supercritical CO2 extraction without specifying the parameters applied for the extraction.

[0009] Rosewood extract according to the present invention has proven useful in preventing or limiting the signs of skin aging and in particular in firming the skin, limiting the appearance of facial ptosis, redefining the contour of the face or limiting the appearance of wrinkles and fine lines. Summary of the invention

[0010] The invention has as its first object the obtaining of an extract obtained from leaves or chips of trunks and branches of rosewood (Aniba rosaeodora) previously exhausted, according to the process comprising the following steps:

[0011] a) between 5 and 25% water is added to exhausted and dried rosewood;

[0012] b) an extraction is carried out using a fluid in a supercritical state such as carbon dioxide (CO2) in the presence of a polar co-solvent, chosen from primary or secondary alcohols, or any mixture thereof;

[0013] c) a support solvent is added, chosen from polyol-type solvents, saturated or unsaturated fatty alcohols, linear or branched, comprising 8 to 30 carbons, glyceride-type solvents or long-chain carbon ester-type solvents, or any mixture thereof;

[0014] d) the extract solubilized in step c) is evaporated to eliminate all of the co-solvent,

[0015] e) The extract is filtered to remove precipitated compounds that do not solubilize in the supporting solvent and collect the filtrate,

[0016] f) the filtered extract obtained in e) is diluted in the same supporting solvent to a concentration of between 0.005 and 50% of crude extract by weight of the total weight of the final extract.

[0017] The invention has as its second object a purified extract of exhausted rosewood capable of being obtained by the process according to the invention characterized in that it is diluted to a concentration of between 0.1 and 1% in the supporting solvent and comprises from 0.3 to 0.5%, advantageously from 0.35 to 0.5% and preferably between 0.4 and 0.5% of semi- to non-volatile compounds.

[0018] The invention has as its third object a composition comprising as an active agent, an effective quantity of a purified rosewood extract, obtained according to the process of the invention, and a physiologically acceptable medium.

[0019] The invention has as its fourth object the cosmetic use of a composition comprising a purified extract of rosewood to prevent or limit the signs of skin aging, to firm the skin, limit the appearance of facial ptosis, redefine the contour of the face or limit the appearance of wrinkles and fine lines.

[0020] The invention further relates to a composition comprising a purified extract of rosewood for use in obtaining an effect similar to that of retinoids, without presenting undesirable inflammatory effects. Brief description of the figures

[0021] The following figures illustrate the advantages arising from the invention and the non-limiting embodiments presented in the description:

[0022] [Fig-1] Analytical comparison by HS-GC / MS of the volatile compound composition of exhausted rosewood distillers' grains and rosewood essential oil as described in Example 3.

[0023] [Fig.2] Analytical comparison by HPLC / UV (300 nm) of the composition in semi to non-volatile compounds of an essential oil of rosewood as described in example 3 and of the purified extract of rosewood according to example 1.

[0024] [Fig.3] Analytical comparison by HS-GC / MS of the purified rosewood extract according to example 1 and of a rosewood essential oil as described in example 3.

[0025] [Fig.4] Analytical comparison by HPLC / DEDL of the crude rosewood extract and the purified rosewood extract according to example 1 describing the difference in anibine content.

[0026] [Fig.5] Evaluation of the purified 1% rosewood extract on the expression of type I collagen, type III and hyaluronic acid on human skin biopsies.

[0027] [Fig.6] Evaluation of the purified 1% rosewood extract on the expression of CRABP2 on human skin biopsies compared with Retinoic acid.

[0028] [Fig.7] Evaluation of the 1% purified rosewood extract on the expression of the alpha 3 chain of type V collagen on human skin biopsies in com- Comparison with Retinoic Acid.

[0029] [Fig-8] Evaluation of the effect of a 1% purified rosewood extract on the volume of the oval of the face (a) and the V angle of the oval of the face (b).

[0030] [Fig.9] Evaluation of the effect of a 1% purified rosewood extract on the distance facial ptosis (a) and the volume of ptosis (b).

[0031] [Fig.10] Evaluation of the effect of a 1% purified rosewood extract on the volume of the corner of the lips (a) and on the profile roughness of the corner of the lips Ra (b) and Rz (c).

[0032] [Fig. 11] Evaluation of the effect of a 1% purified rosewood extract on roughness profile Ra (a) and Rz (b) of crow's feet wrinkles.

[0033] [Fig. 12] Evaluation of the purified 1% rosewood extract on the expression of the interleukin 1 receptor type 1 (IL1R1) on human skin biopsies compared with Retinoic acid. Detailed description of the invention Definitions

[0034] All terms used in this description have their most widely understood meanings unless otherwise stated. For the purposes of the invention, the following terms are defined as follows:

[0035] In this description, the terms "rosewood," "rosewood (Aniba rosaeodora)," "exhausted rosewood," and "co-products or by-products of rosewood essential oil distillation" all refer to the spent grains of rosewood (Aniba rosaeodora) trunk, branch, or leaf chips recovered after essential oil extraction. The essential oil content in the fresh plant varies between 0.4% and 3.5%, with an average linalool content of between 0.32% and 2.8%. The essential oil is traditionally extracted by hydrodistillation or steam distillation. The term "exhausted" therefore refers to plant material that has undergone a first extraction treatment, such as steam distillation or hydrodistillation, or any solid / liquid extraction processes, aimed at extracting between 80 and 95% of the volatile compounds present in this raw material.

[0036] The term “rosewood trunk and branches” means the parts of the rosewood corresponding to the trunk and branches with bark.

[0037] The term "polar co-solvent" means a solvent with a polarity greater than that of CO2 in the supercritical state, such as primary or secondary alcohols, or any mixture thereof.

[0038] The term "polyol type solvent" means organic compounds having at least 2 hydroxyl groups, such as diols or triols, or mixtures of these compounds.

[0039] The term “fatty alcohol type solvent” means organic compounds, saturated or unsaturated, linear or branched, possessing a hydroxyl group, or a mixture of these compounds.

[0040] The term "glyceride-type solvent" means esters of fatty acids and glycerol, or a mixture of these compounds.

[0041] The term "long carbon-chain ester type solvent" means organic compounds having an ester function substituted by long carbon chains.

[0042] The term “support solvent” means a solvent selected from polyol-type solvents, saturated or unsaturated fatty alcohols, linear or branched, comprising 8 to 30 carbons, glyceride-type solvents or long-chain carbon ester-type solvents, or any mixture thereof.

[0043] The term “volatile compounds” means organic compounds that can easily pass into the gaseous phase at atmospheric pressure and ambient temperature.

[0044] The term "semi-volatile to non-volatile compounds" refers to compounds that do not readily transition to the gaseous phase at atmospheric pressure and ambient temperature due to a higher boiling point than volatile molecules. In the context of this invention, these are moderately polar molecules, belonging primarily to the following chemical families: vitamins, aryl phenyl ketones, sesquiterpenes, sesquiterpene derivatives, and phytosterols.

[0045] The term "purified rosewood extract" means the extract in liquid form purified by partial precipitation of anibine, after dilution of the crude extract in a polyol-type solvent, saturated or unsaturated fatty alcohol, linear or branched, comprising 8 to 30 carbons, glyceride-type solvents or long-chain carbon ester solvents, or any mixture thereof, and filtered to remove the precipitate.

[0046] The term "crude extract" of rosewood means the extract in paste form obtained by steps a), b) and c') of the process by complete evaporation of the co-solvent without the addition of a supporting solvent.

[0047] When a range of values ​​is described, the bounds of that range must be understood as explicitly including the upper and lower bounds of said range, as well as all intermediate values ​​of the range. For example, a range of values ​​between 1% and 10% must be understood as including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, as well as all decimal values ​​between 1% and 10%.

[0048] The numerical values ​​in percentage are percentages by weight, that is to say the weight of a compound in relation to the total weight of the mixture envisaged, unless otherwise specified.

[0049] The compositions described in this application may "include", "consist of" or "consist essentially of", the essential compounds or the in- optional ingredients.

[0050] “Consist essentially of” means that the composition or component may include additional ingredients, but only if the additional ingredients do not alter the basic characteristics or new characteristics of the composition or use described in this application.

[0051] The term “physiologically acceptable medium” means a solvent suitable for contact with the external layers of the skin, scalp or hair, without toxicity, irritation, undue allergic response and similar or intolerance reaction, and proportionate to a reasonable benefit / risk ratio, at the concentrations used.

[0052] By "effective quantity" is meant the minimum quantity of extract according to the invention which is necessary to obtain at least one of the desired biological activities.

[0053] Facial ptosis refers to the sagging associated with the sliding and atrophy of fatty tissue and the loss of facial bone support with age. Extraction methods

[0054] Rosewood has been overexploited for its essential oil, making it fall into the category of species protected by the IUCN (International Union for Conservation of Nature) whose trade is regulated by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).

[0055] The inventors have therefore chosen to use co-products of rosewood distillation, in order to reduce the impact on the environment and on biodiversity and to allow better management of plant resources.

[0056] The leaves and the chips of trunk or branches of rosewood (Aniba ro-saeodora) recovered after extraction of the essential oil by distillation are dried in such a way that the residual moisture content in the plant material is between 1 and 10%.

[0057] The objective of the process of the invention is to concentrate and promote the chemical diversity of the extract, the content of semi- to non-volatile compounds while maintaining the highest possible extraction yield.

[0058] The extraction is carried out using a supercritical fluid such as supercritical CO2, which is an alternative process to distillation that complies with the principles of green chemistry. The addition of a polar co-solvent to supercritical CO2 increases the polarity of the CO2 and thus allows the extraction of chemical families of metabolites over a wider range of polarities, such as carbohydrates, glycosylated or non-glycosylated flavonoids, phenolic acids, triterpenes, lipids, and phytosterols.

[0059] A screening experimental design made it possible to identify the parameters most influential on the following criteria: extraction yield, chemical diversity of the extract, and content of semi-volatile to non-volatile compounds. The parameters studied were grinding, the moisture content of the plant material, and the addition or not of inert compound such as cellulose powder, co-solvent flow rate, as well as extraction temperature and pressure.

[0060] The screening experimental design revealed that, surprisingly, the co-solvent flow rate and the plant material wettability rate were the two parameters that influenced the extraction, unlike the other parameters which did not show a significant impact.

[0061] These two influential parameters were then more finely analyzed by an optimization experimental design by applying the response surface method.

[0062] Finally, the support solvent added before the evaporation of the co-solvent was selected following numerous tests carried out to determine the best solvent allowing both the concentration of the compounds of interest and also the maximum elimination of anibine by precipitation.

[0063] Anibine is a substance classified as an irritant, and belonging to a chemical class known to possess marked biological activity often associated with some toxicity.

[0064] The invention thus relates first to a process for obtaining a rosewood extract from leaves or chips of exhausted rosewood trunks and branches (Aniba rosaeodora), comprising the following steps:

[0065] a) between 5 and 25% water is added to exhausted and dried rosewood;

[0066] b) an extraction is carried out using a fluid in a supercritical state such as carbon dioxide (CO2) in the presence of a polar co-solvent, chosen from primary or secondary alcohols, or any mixture thereof;

[0067] c) a support solvent is added, selected from polyol-type solvents, saturated or unsaturated fatty alcohols, linear or branched, comprising 8 to 30 carbons, glyceride-type solvents or long-chain carbon ester-type solvents, or any mixture thereof;

[0068] d) the extract solubilized in step c) is evaporated to remove all of the co-solvent,

[0069] e) The extract is filtered to remove precipitated compounds that do not solubilize in the supporting solvent and collect the filtrate,

[0070] f) the filtered extract obtained in e) is diluted in the same supporting solvent at a concentration of between 0.1 and 1% of crude extract by weight of the total weight of the final extract.

[0071] In step a) advantageously, dried spent grains from the distillation of trunk and branch chips are used, to which between 5 and 25% water is added to exhausted and dried rosewood to obtain an optimum humidification rate.

[0072] Advantageously, the exhausted chips can come from trunks and branches of rosewood grown in Latin America.

[0073] Preferably, prior to step a), the raw material is ground in the presence or not of liquid nitrogen allowing to obtain a powder of coarse particle size.

[0074] In step b) preferably the co-solvent is ethanol at a concentration between 80 and 100% (volume / volume in water), preferably between 90 and 100% and even more preferably at a concentration of 96% in water (volume / volume).

[0075] Preferably, the mass ratio of carbon dioxide (CO2) in the supercritical state, relative to the quantity of moistened raw material involved is between 10 and 50, advantageously between 20 and 40 and preferably between 25 and 35.

[0076] Preferably, the mass ratio of the co-solvent to the supercritical solvent (carbon dioxide) is between 0.050 and 0.080, advantageously between 0.055 and 0.075 and preferably between 0.060 and 0.070.

[0077] Preferably, the co-solvent flow rate, when this is ethanol, is between 10 and 20 ml / min. Even more preferably, the co-solvent flow rate is set at 20 ml / min.

[0078] Preferably, the extraction temperature is between 35 and 85°C, advantageously between 45 and 75°C and preferably between 55 and 65°C.

[0079] Preferably, the pressure within the extractor is between 90 and 1000 bar, preferably between 150 and 700 bar and even more preferably between 250 and 350 bar.

[0080] To carry out step b) advantageously, the mixture obtained in step a) is placed in a stainless steel cartridge, this cartridge is introduced into a supercritical fluid extractor.

[0081] The presence of a polar co-solvent under the extraction conditions described above above, allows obtaining an extract possessing a notable phytochemical diversity in that it contains both a residual part of volatile compounds not extracted during distillation and semi to non-volatile compounds of variable polarity belonging to the following chemical families: vitamin and derivative, aryl phenyl ketones, sesquiterpene derivatives, sesquiterpenes, phytosterols and free fatty acids.

[0082] In step c), preferably the quantity of solvent called "support" necessary to obtain a crude extract content of between 5 and 80% by weight of the total weight of the final extract is added to the extract obtained in step b), advantageously between 10 and 50% and preferably between 15 and 25%.

[0083] Preferably the supporting solvent is chosen from octyldodecanol, 2-hexyl decanol, oleyl alcohol, glycerol tricaprylate / caprate or an octyldodecyl stearoyl stearate ester, or any mixture of these.

[0084] Even more preferably, the supporting solvent is glycerol tricaprylate / caprate.

[0085] In the presence of the supporting solvent, precipitation is observed. Analysis shows that the precipitate is mainly composed of anibine, as illustrated by the chromatographic profiles of [Fig.4].

[0086] At step d) the optimum conditions for evaporation of the co-solvent are an evaporation temperature of a maximum of 60°C and a pressure of less than 60 mbar.

[0087] In step e) the liquid extract obtained is filtered to remove the precipitated anibine. The filtrate is collected.

[0088] At step f) the filtrate is preferably diluted to a concentration of 0.5% by weight of crude extract / weight of final extract.

[0089] Advantageously, the process can be followed by at least one optional step g) of purification, according to any technique known to the person skilled in the art and in particular by chromatography or by molecular distillation.

[0090] Alternatively, following step b), another extraction process can be implemented by carrying out a step c') of total evaporation of the co-solvent, without the addition of a supporting solvent. This yields a crude extract in paste form. This extract can be used, among other things, for analytical chemistry studies that might be affected by the presence of a supporting solvent.

[0091] At the end of step c') the extraction yield is between 0.5 and 5%. Extract

[0092] The second object of the invention is a purified extract of exhausted rosewood capable of being obtained by the process according to the invention characterized in that it is diluted to a concentration of between 0.1 and 1% in the supporting solvent and comprises from 0.3 to 0.5%, advantageously from 0.35 to 0.5% and preferably between 0.4 and 0.5% of semi- to non-volatile compounds.

[0093] The purified rosewood extract is advantageously solubilized in glycerol tricaprylate / caprate.

[0094] In another embodiment, the purified rosewood extract comprises 0.5% crude extract, by weight of the total weight of the final extract.

[0095] In this particular embodiment, the semi- to non-volatile compounds comprise 0.01 to 0.4% sesquiterpene derivatives as cotoin equivalents, 0.001 to 0.1% aryl phenyl ketones of which 0.001 to 0.05% cotoin, 0.001 to 0.01% beta-sitosterol and 0.001 to 0.1% anibine.

[0096] By way of example, one can cite purified rosewood extract diluted to 0.5% in glycerol tricaprylate / caprate by weight of crude extract relative to the weight of the final extract and comprising 0.08% sesquiterpene derivatives as cotoin equivalent, 0.006% aryl phenyl ketones of which 0.005% cotoin and 0.009% anibine.

[0097] The extract of the invention differs from the rosewood extracts described in the prior art, and in particular from essential oils, in that it has a much less intense olfactory note and a significantly lower content of volatile and allergenic compounds. decreased, and semi- to non-volatile compounds of varying polarity belonging to several chemical families.

[0098] In the purified rosewood extract of the invention, volatile compounds are present at concentrations below the detection limit and therefore cannot be quantified. The linalool concentration is less than 0.01%.

[0099] The phytochemical study to characterize the compounds was carried out on the crude extract of exhausted rosewood, obtained by a process comprising steps a), b) and c').

[0100] The raw rosewood extract comprises:

[0101] Between 5 and 20% of volatile compounds, mainly sesquiterpenes and sesquiterpene alcohols, preferably between 8 and 18%, even more preferably 10 and 15% of volatile compounds and at most 1% of linalool.

[0102] Between 80 and 95% of semi- to non-volatile compounds, preferably between 82 and 92%, and even more preferably between 85 and 90%. This fraction is mainly composed of sesquiterpene derivatives (about 30% of the extract), aryl phenyl ketones (about 10% of the extract, of which about 5% is cotoin), a nicotinic acid derivative (about 20% of the extract), eudesmane and guaiane type sesquiterpenes, nicotinic acid, organic acids, as well as more nonpolar molecules belonging to the phytosterol and fatty acid type chemical families (about 25% of the extract for all these other compounds).

[0103] In a very advantageous embodiment, the crude rosewood extract comprises 13% volatile compounds and 87% semi- to non-volatile compounds of which 32% sesquiterpene derivatives, 12% aryl phenyl ketones of which 6% cotoin, 20% anibine and 23% sesquiterpene compounds, free fatty acids and phytosterols.

[0104] A non-exhaustive list of the compounds present in these two fractions analyzed by HPLC coupled with a diode array detector or an evaporative light scattering detector with external calibration is given by way of example in the following Table 1:

[0105] [Tables 1] Fraction Chemical Family Identification Volatile Fraction Sesquiterpene Alcohols, Sesquiterpenes Gamma-selinene Beta-selinene Alpha-selinene Alpha-muurolene Delta-cadinene Spathulenol Tau-cadinol Alpha-cadinol Semi- to Non-volatile Fraction Aryl phenyl ketones Vitamin and Nicotinic Acid Derivative Sesquiterpene Derivatives Sesquiterpenes Sesquiterpene Alcohols Nicotinic Acid (2,6-dihydroxy-4-methoxyphenyl)-(4-hydroxyphenyl)methanone Cotoin Teucladiol Incisumdiol 1,6-dihydroxy-4(14)-Eudesmene 1,6-dihydroxy-3-eudesmene Eudesma-4,11-dien-1-ol Alpha-dictyopterol Beta-dictyopterol Anibine

[0106] Several markers were quantified in the crude rosewood extract. This notably contains:

[0107] Between 10 and 50% sesquiterpene derivatives, advantageously between 20 and 40% and preferably between 25 and 35%;

[0108] Between 1 and 25% of aryl phenyl ketones, advantageously between 5 and 20% and preferably between 10 and 15% of which between 5 and 10% of cotoin;

[0109] Between 0.01 and 2% beta-sitosterol, advantageously between 0.1 and 1.5% and preferably between 0.5 and 1%;

[0110] Between 1 and 40% anibine, advantageously between 5 and 30% and preferably between 10 and 20%. Compositions

[0111] A third object of the invention is a composition comprising, as an active agent, an effective amount of a purified rosewood extract, obtained according to the process described above, and a physiologically acceptable medium.

[0112] Advantageously, the purified rosewood extract is added to a physiologically acceptable medium at a concentration of between 0.01% and 10% of purified rosewood extract by weight relative to the total weight of the composition, preferably at a concentration of between 0.1% and 5% by weight relative to the total weight of the composition, and even more preferably at a concentration of between 0.5% and 2% by weight relative to the total weight of the composition.

[0113] The composition of this application is formulated to be administered by any suitable route, including oral or external topical application, and the formulation of the compositions will be adapted by a person skilled in the art.

[0114] Preferably, the composition of the present application is in a form suitable for topical application. This composition must therefore contain a physiologically acceptable medium, i.e., one compatible with the skin and hair, without risk of discomfort during application.

[0115] The composition may in particular be in the form of an aqueous, hydroalcoholic or oily solution or gel, an oil-in-water, water-in-oil emulsion or multiple emulsions; they may also be in the form of suspensions, or even powders, suitable for application to the skin, mucous membranes, lips and / or hair.

[0116] The composition may be more or less viscous and may also have the appearance of a cream, lotion, fluid, milk, serum, ointment, gel, paste, balm or mousse. It may also be in solid form, such as a stick, or be applied to the skin as an aerosol.

[0117] Examples of physiologically acceptable media commonly used in the intended field of application include formulation aids such as solvents, thickeners, gelling agents, diluents, emulsifiers, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, esters, vegetable oils or butters, etc.

[0118] In all cases, a person skilled in the art will ensure that these adjuvants and their proportions are chosen in such a way as not to impair the advantageous properties sought in the composition according to the invention.

[0119] According to another advantageous embodiment, the purified rosewood extract can be encapsulated or included in a cosmetic vector such as liposomes or any other nano capsule or microcapsule used in the field of cosmetics or adsorbed onto powdered organic polymers, mineral supports such as talcs and bentonites.

[0120] Advantageously, the composition may comprise, in addition to the active agent, i.e. a purified extract of rosewood, at least one other active agent having cosmetic effects similar and / or complementary to those of the invention.

[0121] Such additional active agents may also be selected according to their chemical composition, from the group comprising: amino sugars, glucosamine, D-glucosamine, N-acetylglucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, vitamin B3 and its derivatives, niacinamide, sodium dehydroacetate, dehydroacetic acid and its salts, phytosterols, salicylic acid compounds, hexamidines, dialkanoyl dihydroxyproline compounds, soy extracts and derivatives, equol, isoflavones, flavonoids, phytantriol, farnesol, geraniol, bisabolol, peptides and their derivatives, di-, tri-, tetra-, penta-, and hexapeptides and their derivatives, lys-thr-thr-lys-ser, palmitoyl-lys-thr-thr-lys-ser, camosine, N-acyl amino acid compounds, retinoids, retinyl propionate, retinol, retinyl palmitate, retinyl acetate, retinal, retinoic acid, water-soluble vitamins, ascorbates, vitamin C, ascorbyl glucoside, ascorbyl palmitate,magnesium ascorbyl phosphate, sodium ascorbyl phosphate, vitamins and their salts and derivatives, provitamins and their salts and derivatives, ethyl panthenol, vitamin A and its derivatives, vitamin B and its derivatives, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin F, vitamin K and its derivatives, pantothenic acid and its derivatives, pan-tothenyl ethyl ether, panthenol and its derivatives, ethyl panthenol, dexpanthenol, biotin, amino acids and their salts and derivatives, water-soluble amino acids, asparagine, alanine, indole, glutamic acid, water-insoluble vitamins, beta-ionol, cedrol, and their derivatives, water-insoluble amino acids, tyrosine, tryptamine, particulate materials, butylated hydroxytoluene, butylated hydroxyanisole, allantoin, tocopherol nicotinate, tocopherol, esters of tocopherol, palmitoyl-glycerol, phytosterol, hydroxy acids, glycolic acid, lactic acid, lactobionic acid, keto acids, pyruvic acid, phytic acid,lysophosphatidic acid, stilbenes, cinnamates, resveratrol, kinetin, zeatin, dimethylaminoethanol, natural peptides, soy peptides, acidic sugar salts, manganese gluconate, zinc gluconate, piroctone olamine, 3,4,4'-trichlorocarbanilide, triclocarban, zinc pyrithione, hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucoside, pyridoxine, aloe vera, terpene alcohols, allantoin, bisabolol, dipotassium glycyrrhizate, glycerol acid, sorbitol, pentaerythritol, pyrrolidone and its salts, dihydroxyacetone, erythrulose, glyceraldehyde, tartaraldehyde, clove oil, menthol, the camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate, eicosene and vinyl pyrrolidone copolymer, iodopropyl butylcarbamate, a polysaccharide, an essential fatty acid, a salicylate, glycyrhetinic acid, carotenoids, ceramides and pseudo-ceramides,a complex lipid, oils in general of natural origin such as shea butter, oil, apricot, evening primrose oil, prune oil, palm oil, monoi oil, kahai oil, hydroquinone, HEPES, procysteine, O-octanoyl-6-D-maltose, disodium salt of methylglycine diacetate, steroids such as diosgenin and DHEA derivatives, DHEA dehydroepiandrosterone and / or a chemical or biological precursor or derivative, N-ethylcarbonyl-4-para-aminophenol, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolytic enzymes, plant extracts, phytohormones, yeast extracts, a metalloproteinase inhibitor, enzymes, enzyme inhibitors, enzyme inducers, coenzymes, chelating agents, plant extracts and derivatives of plants, essential oils, marine extracts, agents from a biofermentation and / or biotechnology process, mineral salts, cell extracts.

[0122] By way of example, we can also cite:

[0123] peptides commercially known as MATRIXYL®, ARGIRELINE®, CHRONOGEN™, LAMINIXYL IS™, PEPTIDE Q10™, COLLAXYL™ (patent FR2827170, ASHLAND®), PEPTIDE VINCI 01™ (patent FR2837098, ASHLAND®), PEPTIDE VINCI 02™ (patent FR2841781, ASHLAND®), ATPeptide™ (patent FR2846883, ASHLAND®) or the synthetic peptide of sequence Arg-Gly-Ser-NH2, marketed under the name ATPeptide™ by ASHLAND®;

[0124] Artemia salina extract, marketed under the name GP4G™ (FR2817748, ASHLAND®);

[0125] plant peptide extracts such as flax extracts (Lipigénine™, patent FR2956818, ASHLAND®), soy extracts, spelt extract, vine extract, rapeseed extract, flax extract, rice extract, corn extract, pea extract, cocoa extract;

[0126] Yeast extracts, for example Dynagen™ (patent FR2951946, ASHLAND®) or Actopontine™ (patent FR2944526, ASHLAND®). Uses

[0127] The invention has as its fourth object the cosmetic use of a composition described above comprising a purified extract of rosewood to prevent or limit the signs of skin aging and in particular to firm the skin, limit the appearance of facial ptosis, redefine the contour of the face, remodel and restructure the face and limit the appearance of wrinkles and fine lines.

[0128] .

[0129] The term “cosmetic use” means that the use is intended for individuals with healthy skin, scalp or hair.

[0130] By "signs of skin aging" is meant any change in the external appearance of the skin due to aging such as, for example, sagging of the face with loss of oval contour of the face, the appearance of ptosis or jowls, a loss of volume of the cheeks and cheekbones, the appearance of wrinkles and fine lines at the corners of the lips and crow's feet, sagging, loss of elasticity, firmness and / or tone of the skin, but also all internal changes in the skin which do not systematically result in a changed external appearance such as, for example, thinning of the skin, or all internal degradation of the skin resulting from external aggressions producing free radicals, such as pollution and solar radiation including UV.

[0131] The shape of the face is genetically determined and it is known that a large number of genetic loci influence the shape and contours of the face.

[0132] The extracellular matrix is ​​one of the first determinants of facial morphology, so rosewood extract was tested on the expression of collagens I and III, and hyaluronic acid and showed a positive effect on the expression of these proteins.

[0133] The inventors have now identified, through bioinformatic analysis of genome-wide association study data, certain genetic determinants involved in facial shape and contours. These genetic determinants include, among others, collagen XVII, fibulin-7, and harmonin.

[0134] Fibulin-7, in addition to its involvement in the shape and contours of the face, interacts with the extracellular matrix and studies have suggested that it plays a crucial role in maintaining epidermal stem cells during skin aging.

[0135] Collagen XVII is a transmembrane protein present in hemidesmosomes (HD). Collagen XVII participates in the interactions of stem cells with surrounding cells and the matrix and may also be involved in the maintenance of stem cells and their niches. Certain mutations responsible for a deficiency in collagen XVII lead to premature hair aging phenotypes.

[0136] Harmonin is a scaffolding protein, a family of proteins known to play a central role in macromolecular assemblies.

[0137] Rosewood extract was tested on the expression of fibulin-7 and collagen XVII, and harmonin and showed a positive effect on the expression of these proteins, in connection with the anti-wrinkle and facial shape and contour restoration effect observed in a clinical test.

[0138] Genetic variations in the alpha 3 chain of type V collagen have been described in a panel of Asian volunteers as being associated with genetic predispositions linked to skin sagging and wrinkles at the corner of the eye (Okuno R. et al., Exp Dermatol. 2022;31(9): 1411-1420). In addition, a deficiency in the alpha 3 chain of type V collagen induced by the action of matrix metalloproteinase 9 (MMP-9) has been significantly associated with eczema (Martin MJ et al. Genoa (Basel). 2020;11(4):442).

[0139] The rosewood extract of the invention has been tested on the expression of the alpha 3 chain of type V collagen, and has shown a positive effect on the expression of this protein, which suggests an anti-aging effect that is particularly interesting for Asian and Caucasian skin and especially for Asian skin.

[0140] The invention further relates to the cosmetic use of the composition of the invention to increase the expression of the following proteins: collagen XVII, fibulin-7, harmonin, alpha 3 chain of type V collagen and CRABP2.

[0141] The set of genes that code for these proteins can be considered as a "Face Architecture Genomic Complex" or "Face Architecture Genomic Complex".

[0142] Vitamin A and its derivatives, particularly retinol, are among the most effective substances for delaying the skin aging process. Similarly, retinoids, which are natural or synthetic derivatives of vitamin A, are widely used in dermatology for anti-aging treatments (Mukherjee S, et al. Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety. Clin Interv Aging. 2006;1(4):327-348), and their efficacy on photoaging and the restructuring of the dermis and epidermis is well documented. However, it is also known that these molecules have drying and irritating side effects.

[0143] Rosewood extract was tested on the expression of an inflammation marker: the interleukin-1 receptor (IL1R1), and showed no significant modulation of the expression of this protein. In comparison, retinoic acid caused an increase in the expression of this protein.

[0144] CRABP1 and CRABP2, the specific retinoic acid-binding proteins, bind to retinoic acid and promote its transport within the cell. It is known that a deficiency in CRABP2 expression accelerates skin aging (Bielli A. et al., Aging (Albany NY). 2019; 11(6): 1619-1632).

[0145] Rosewood extract has been tested on CRABP2 expression and has shown a positive effect on the expression of this protein and does not increase the expression of ILlRL. Purified rosewood extract shows effects comparable to retinoic acid on CRABP2 expression, and has the advantage of not causing inflammatory side effects, which most sensitive skin cannot tolerate.

[0146] The composition of the invention exhibits good tolerance, which allows its use on sensitive skin or skin exhibiting characteristics of atopic dermatitis.

[0147] The invention thus relates to a composition described in the present invention for its use to obtain an effect similar to that of retinoids, without presenting undesirable inflammatory effects (retinol-like effect).

[0148] The invention further relates to a composition comprising a purified rosewood extract to combat external aggressions that produce pro-inflammatory free radicals, such as stress, pollution, or external aggressions. Examples

[0149] The present invention will now be illustrated by means of the following non-limiting examples:

[0150] Example 1: Preparation of a crude extract and a purified extract of exhausted rosewood

[0151] For the production of the examples, trees of the species Aniba rosaeodora were cultivated in Peru.

[0152] The plant parts used are the spent grains from the trunk and branches of the Aniba rosaeodora tree, depleted of essential oil (volatile compounds) by a first steam distillation process. During the development of the process of the invention, the extraction parameters were optimized to extract the maximum amount of semi-volatile to non-volatile compounds with maximum yield. The first stage of development consisted of identifying the factors that had the greatest influence on the extraction. Thus, several parameters were studied, namely the benefits of cryo-milling, the moisture content of the plant material, the percentage of cellulose added, the co-solvent flow rate, as well as the temperature and pressure in the extractor. Only the factors that showed a real impact on the extraction were optimized within the framework of an optimization experimental design applied according to the response surface method.

[0153] Table 2 shows some simulated results obtained from the experimental design carried out to optimize the extraction parameters:

[0154] [Tables2] Co-solvent flow rate (ml / min) Plant material humidification (%) Extraction yield (%) Peak area DEDL compound 1 Peak area DEDL compound 2 10 1 1.31 1.94 18.25 20 15 2.18 47.79 24.88 15 29 1.99 17.16 17.52 20 1 1.72 7.01 13.78 10 29 1.87 8.01 23.71 20 29 2.26 31.17 16.18 10 15 1.70 4.36 16.91 15 1 1.63 4.24 12.16 15 15 2.01 13.85 19.38 15 15 2.02 13.90 18.53 15 15 2.05 14.12 20.31 13 12 1.91 7.48 18.87 17 12 1.86 12.30 25.97 15 22 1.98 10.82 18.80 15 15 2.01 11.53 20.40

[0155] The peaks selected (compound 1 and compound 2) for reprocessing the experimental design are cotoin and a cotoin derivative. Each area has been corrected here by the concentration of the solution injected by HPLC in order to eliminate variations related to sample preparation.

[0156] The extraction conditions were selected to obtain a satisfactory extraction yield while ensuring that the extract has a maximum content and chemical diversity of semi-volatile to non-volatile compounds. The extraction parameters preferably chosen are as follows:

[0157] Exhausted rosewood chips are cryogenically ground using a 4 mm sieve. The resulting plant powder is moistened with 15% water (wt.%). The moistened plant material is introduced into a stainless steel cartridge. This cartridge is placed in a supercritical fluid extractor such as the Separex SFE 5 extractor. The extraction solvent used is supercritical carbon dioxide at a flow rate of 15 kg / h with the addition of a polar co-solvent, corresponding to 96% ethanol in water (volume / volume) at a flow rate of 20 ml / min. The mass ratio of carbon dioxide to moistened plant material is 30, and the ratio of co-solvent to carbon dioxide is 0.065. The pressure and temperature within the extractor are 300 bar and 60°C, respectively. The temperature within the separator is 35°C. A dry extract measurement is performed on the ethanolic extract obtained for the preparation of the purified extract.

[0158] In the case of the crude extract, the exhausted rosewood ethanolic extract solution is evaporated under vacuum until the ethanol has completely evaporated (pressure below 90 mbar and water bath temperature at a maximum of 60°C). This yields an extract in the form of a relatively solid paste.

[0159] To prepare the purified extract, prior to evaporation under vacuum, the supporting solvent, namely glycerol tricaprylate / caprate, is added to the ethanolic extract solution to obtain a solution diluted to 20% by weight of the crude extract. The mixture is evaporated under vacuum until the ethanol has completely evaporated (pressure below 90 mbar and water bath temperature not exceeding 60°C). In the presence of glycerol tricaprylate / caprate, anibine partially precipitates.

[0160] The extract is filtered and the precipitate removed.

[0161] The tricaprylate / glycerol caprate content in the filtrate obtained is quantified by HPLC. From this result, the extract is diluted in the same solvent (tricaprylate / glycerol caprate) to obtain a clear, fluid extract containing 0.5% crude rosewood extract.

[0162] The purified extract obtained is in the form of a liquid extract.

[0163] Several markers of the extract were quantified. Thus, the purified rosewood extract contains 0.5% glycerol tricaprylate / caprate (by weight of crude extract relative to weight of final extract) and comprises 0.08% sesquiterpene derivatives as cotoin equivalents, 0.006% aryl phenyl ketones of which 0.005% is cotoin, 0.004% beta-sitosterol, and 0.009% anibine. The linalool content is below the limit of detection, i.e., less than 0.01%.

[0164] Example 2: Characterization of the crude rosewood extract obtained according to example 1

[0165] The phytochemical study was carried out on the crude extract of exhausted rosewood. This extract is obtained under the conditions described in Example 1, where the co-solvent (ethanol) is completely evaporated under vacuum (90 mbar, water bath temperature at a maximum of 60°C) without the addition of a supporting solvent. The crude extract is then obtained in a relatively solid, paste-like form.

[0166] Volatile compounds are analyzed by gas chromatography (GC) coupled with mass spectrometry (MS) and / or ionization detection of flame (FID). Confirmation of identifications is made possible by comparing linear retention indices and mass spectra contained in libraries. Quantification by GC / FID is performed by internal calibration using predicted and / or calculated response factors.

[0167] Semi-volatile to non-volatile compounds are monitored by high-performance liquid chromatography (HPLC) coupled with a diode array UV detector (DAD) and an evaporative light scattering detector (DEDL). Structural identifications are confirmed by nuclear magnetic resonance (NMR) experiments and high-resolution mass spectrometry (HRMS) analyses. Identification is also confirmed by standard injection if commercially available.

[0168] Sesquiterpene derivatives, aryl phenyl ketone compounds, and beta-sitosterol were quantified by HPLC. Cotoin was used as an external standard for the determination of sesquiterpene derivatives and aryl phenyl ketones. The content is then expressed as a cotoin equivalent. The analytical method developed for this determination has undergone method validation according to the guidelines indicated by the ICH (International Conference on Harmonisation).

[0169] A non-exhaustive list of compounds present in the volatile fraction and the semi-volatile to non-volatile fraction is detailed in Table 3 below:

[0170] [Tables3] Chemical Family Identification Volatile Fraction (sesquiterpene alcohols, sesquiterpenes) Benzyl alcohol 2-phenyl ethanol Linalool Alpha-copaene Gamma-selinene Beta-selinene Alpha-selinene Delta-cadinene Spathulenol Benzyl benzoate Semi- to Non-volatile Fraction (aryl phenyl ketones, sesquiterpene derivatives, sesquiterpenes, vitamin and nicotinic acid derivative, organic acid, fatty acid, phytosterol) (2,6-dihydroxy-4-methoxyphenyl)-(4-hydroxyoxyphenylmethanone) Cotoin Anibine Nicotinic acid Citric acid Teucladiol Incisumdiol 1,6-dihydroxy-4(14)-eudesmene 1,6-dihydroxy-3-eudesmene eudesma-4,11-dien-1-ol Alpha-dictyopterol Beta-dictyopterol Sitosterol Linoleic acid

[0171] The volatile fraction and the semi-volatile to non-volatile fraction have been quantified for this extract. It thus contains:

[0172] 13% volatile fraction; of which a maximum of 1% is linalool

[0173] 87% semi-volatile to non-volatile fraction, of which 32% are sesquiterpene derivatives, 12% of aryl phenyl ketones including 5% cotoin, 20% anibin and 23% of various compounds such as sesquiterpenes, free fatty acids, phytosterols and other lipid compounds.

[0174] Example 3: Preparation of a rosewood extract of the essential oil type

[0175] With the aim of carrying out a comparative analysis, an essential oil extraction The extraction of rosewood (not exhausted) was carried out conventionally, by steam distillation. The trunk and branches of Aniba rosaeodora are dried and then coarsely ground into chips, which are placed in separate stills and then subjected to a stream of steam. This steam releases the volatile molecules, or essential oil, which is carried along by the steam and condenses in the condenser. Since the essential oil is less dense than water and is not, or only slightly, water-soluble, it is collected at the outlet in a decanter called a solubles tank. The water, which still contains traces of essential oil, is called a hydrosol.

[0176] The essential oil is in the form of a colorless to pale yellow liquid (density 0.8789, refractive index 1.4644, optical rotation +1.15).

[0177] Example 4: Demonstration of the depletion of the raw material by steam distillation

[0178] The rosewood raw material used in the context of the invention is said to be "exhausted". This means that it has undergone a first extraction treatment, in this case steam distillation, which has made it possible to extract the volatile fraction of the rosewood, i.e. the essential oil.

[0179] Spent rosewood spent grains were analyzed by gas chromatography-mass spectrometry (GC / MS) and flame ionization detector (FID). Injection was performed in headspace. This sample preparation facilitates the analysis of the most volatile compounds. Separation was achieved on a 30 x 0.25 m, 0.25 pm nonpolar column, with elution via a temperature gradient (50°C isotherm, 5 min; then 3°C / min to 120°C; then 5°C / min to 250°C; 250°C isotherm, 5 min). Helium was the carrier gas used.

[0180] Figure 1 shows the GC / MS chromatographic profile of exhausted rosewood spent grains compared to the GC / MS chromatographic profile of rosewood essential oil. It can be seen that the essential oil is much richer in volatile compounds. These compounds are eluted before 40 minutes under the conditions of the method described above. The abundance of peaks is also much more intense than in the case of rosewood spent grains.

[0181] The chromatographic profile of the spent grains shows, on the contrary, low-intensity peaks compared to the essential oil, which elute mainly between 30 and 35 min.

[0182] Example 5: Demonstration of the major phytochemical differences between an essential oil and the purified extract obtained according to Example 1

[0183] The purified rosewood extract obtained according to Example 1 was compared to an essential oil of Aniba rosaeodora as prepared in Example 3 by analysis using high-performance liquid chromatography (HPLC) coupled to a strip detector diodes and an evaporative light scattering detector (ELSD) of solutions of the same concentration of these two extracts. Chromatographic analysis was carried out on a Core-Shell Cl8 type column according to an elution gradient using formic acid (HCO2H) acidified mobile phases consisting of a water / acetonitrile (ACN) / propane-2-ol (IPA) mixture for channel A and ACN / IPA / methanol (MeOH) for channel B.

[0184] Figure 2 shows the 300 nm UV chromatographic profile of rosewood essential oil as described in Example 3 and of the purified exhausted rosewood extract according to Example 1, analyzed by HPLC / UV-DEDL under the following elution gradient conditions: 0–5 min 100% A, 5–22 min from 100% A to 100% B, then 22–32 min 100% B, with A: H₂O / ACN / IPA / HCO₂H 95 / 2.5 / 2.5 / 0.1 (v / v / v / v) and B: IPA / ACN / MeOH / HCO₂H 40 / 40 / 20 / 0.1 (v / v / v / v). The ordinate axis represents the detector response in mAU. The abscissa represents the analysis time in minutes.

[0185] As shown in [Fig.2], the major semi-volatile to non-volatile compounds corresponding to sesquiterpene derivatives and aryl phenyl ketones are not or only weakly detected in rosewood essential oil

[0186] In parallel, the two extracts obtained according to Examples 1 and 3 were also compared by GC / MS-FID analysis. The samples were analyzed at the same headspace (HS) concentration. Separation was achieved on a 30 x 0.25 m, 0.25 pm nonpolar column, with elution via a temperature gradient (50°C isotherm, 5 min; then 3°C / min up to 120°C; then 5°C / min up to 250°C; 250°C isotherm, 5 min). Helium was the carrier gas used.

[0187] Figure 3 shows the GC / MS chromatographic profiles of rosewood essential oil as described in Example 3 and of the purified rosewood extract obtained according to Example 1, analyzed by GC / MS-FID under the conditions described above. The detector response is shown on the y-axis. The x-axis represents the analysis time in minutes.

[0188] Figure 3 illustrates the absence or very low presence of volatile compounds in the solubilized extract of exhausted rosewood compared to the essential oil diluted to 0.5% in glycerol tricaprylate / caprate, which, as described in the prior art, contains a significant amount of linalool (major peak between 18 and 19 min), with an estimated content between 70 and 90%. The chromatographic profile of the purified rosewood extract shows much lower peak abundances than the rosewood essential oil, with an estimated linalool content between 0.05 x 10⁻³ and 0.005%. This marked decrease in volatile compounds, particularly linalool, is a consequence of the steam distillation process previously applied to the rosewood, which depleted the raw material of volatile compounds.

[0189] Figure 4 shows the HPLC / DEDL chromatographic profiles of the crude rosewood extract at 1% in ethanol compared to the purified rosewood extract at 1% in glycerol tricaprylate / caprate, obtained according to Example 1. The chromatographic analysis was carried out on a Core-Shell Cl8 type column using a gradient elution with formic acid (HCO2H) acidified mobile phases consisting of a water / acetonitrile (ACN) / propane-2-ol (IPA) mixture for route A and ACN / IPA / methanol (MeOH) mixture for route B under the following gradient elution conditions: 0-5 min 100% A, 5-22 min from 100% A to 100% B, then 22-32 min 100% B with A: H2O / ACN / IPA / HCO2H 95 / 2.5 / 2.5 / 0.1 (v / v / v / v) and B: IPA / ACN / MeOH / HCO2H 40 / 40 / 20 / 0.1 (v / v / v / v). The y-axis represents the detector response in mAU. The x-axis represents the analysis time in minutes.

[0190] A marked decrease in the intensity of the anibine peak (retention time at 9 min) is observed for the purified extract, unlike the crude extract, for which anibine is among the major compounds. These profiles are illustrated in [Fig. 4] and demonstrate the depletion of anibine in the purified extract, due to precipitation of this compound following dilution in the supporting solvent.

[0191] Example 6: Evaluation of 1% purified rosewood extract on the expression of type I collagen, type III collagen and hyaluronic acid on human skin biopsies

[0192] The aim of this experiment is to demonstrate an effect of purified rosewood extract on the synthesis of collagen I, collagen III, and hyaluronic acid in ex vivo human skin biopsies.

[0193] Protocol:

[0194] The expression of collagen I, collagen III, and hyaluronic acid was evaluated by indirect immunofluorescence on skin biopsies pretreated by topical application of purified rosewood extract obtained according to Example 1 and diluted to 1% in glycerol tricaprylate / caprate for 48 hours (twice daily). Control biopsies incubated in parallel under the same conditions received placebo (Phosphate Buffer Saline, PBS). At the end of incubation, the biopsies were fixed and paraffin-embedded for histological sectioning. Detection of collagen I, collagen III, and hyaluronic acid was performed by incubation with anti-Collagen I antibody (Abcam); anti-Collagen III antibody (Proteintech); and a biotinylated HABP (hyaluronan-binding protein) probe (millipore).After an hour and a half of incubation followed by rinsing, the sections are incubated in the presence of a fluorophore-coupled anti-rabbit secondary antibody (Alexa Fluor® 488, Invitrogen) to detect collagens I and III. A biotin / streptavidin system coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) is used to detect hyaluronic acid. The sections are then examined under a microscope at [temperature missing]. Epi-fluorescence (Zeiss Axiovert 200M microscope). The expression of collagen I, collagen III, and hyaluronic acid are then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0195] Results: As shown in [Fig.5], when biopsies were treated with 1% purified rosewood extract, collagen I expression increased by 20%, collagen III expression increased by 32%, and hyaluronic acid expression increased by 36% compared to the negative control.

[0196] Conclusion: The purified rosewood extract showed a positive effect on the expression of collagen I, collagen III, and hyaluronic acid.

[0197] Example 7: Evaluation of the purified 1% rosewood extract on the expression of Fibulin-7 on human skin biopsies

[0198] The purified rosewood extract was tested on the expression of

[0199] Principle: The aim of this experiment is to demonstrate the effect of purified rosewood extract on fibulin-7 synthesis in ex vivo human skin biopsies. Fibulin-7 is a keratinocyte-stem-preserving protein identified as being involved in facial morphology.

[0200] Protocol: Fibulin-7 expression is assessed by indirect immunofluorescence on skin biopsies, processed as in Example 6. Fibulin-7 detection is performed by incubation with anti-fibulin-7 antibody (Thermo Scientific). After 1.5 hours of incubation followed by rinsing, the sections are incubated in the presence of anti-rabbit secondary antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined under an epifluorescence microscope (Zeiss Axiovert 200M microscope). Fibulin-7 expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0201] Results: when biopsies were treated with purified rosewood extract at 1%, fibulin-7 expression increased by 39% compared to the negative control (mean + / - ESM, n=6 result highly significant with Student's t-test).

[0202] Conclusion: The purified rosewood extract showed a positive effect on fibulin-7 expression.

[0203] Example 8: Evaluation of the purified 1% rosewood extract on collagen XVII expression in human skin biopsies

[0204] Purified rosewood extract has been tested on collagen XVII expression, a protein whose expression decreases with age in human skin (Xiang Y, et al., J Cell Commun Signal. 2022; 16(3):421-432.), and identified as involved in the facial morphology of Europeans (Liu F. et al., PLoS Genet. 2012;8(9):el002932).

[0205] Protocol: Collagen XVII expression is evaluated by indirect immunofluorescence on skin biopsies, treated as in Example 6. Detection of collagen XVII is carried out by incubation with anti-Coll7Al antibody (Abcam). After an incubation period of 1.5 hours followed by rinsing, the sections are incubated with a fluorophore-coupled anti-rabbit secondary antibody (Alexa Fluor® 488, Invitrogen). The sections are then examined under an epifluorescence microscope (Zeiss Axiovert 200M microscope). Collagen XVII expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0206] Results: when biopsies were treated with purified rosewood extract according to example 1 at 1%, collagen XVII expression increased by 94% compared to the negative control (mean + / - ESM, n=6 result highly significant with Student's t-test).

[0207] Conclusion: The purified rosewood extract showed a positive effect on the expression of collagen XVII.

[0208] Example 9: Evaluation of the 1% purified rosewood extract on harmonin expression in human skin biopsies

[0209] The purified rosewood extract was tested on the expression of harmonin, a protein which has scaffolding and molecular hub properties in signaling pathways (Bugge K, et al., J Biol Chem. 2021;296:100226; Friis Theisen F., et al., J Biol Chem. 2022;298(6): 101963).

[0210] Protocol: Harmonin expression is evaluated by indirect immunofluorescence on skin biopsies treated as in Example 6. Harmonin detection is performed by incubation with anti-harmonin antibody (Proteintech). After 1.5 hours of incubation followed by rinsing, the sections are incubated in the presence of anti-rabbit secondary antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined under an epifluorescence microscope (Zeiss Axiovert 200M microscope). Collagen XVII expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0211] Results: when biopsies were treated with 1% purified rosewood extract, harmonine expression increased by 44% compared to the negative control (mean + :- ESM, n=6 highly significant result with Student's t-test).

[0212] Conclusion: The purified rosewood extract showed a positive effect on the expression of harmonin.

[0213] Example 10: Evaluation of the purified 1% rosewood extract on CRABP2 expression in human skin biopsies

[0214] The purified rosewood extract was tested on the expression of CRABP2, a protein that decreases in human skin with age, and which is involved in the retinoic acid signaling pathway.

[0215] Protocol: CRABP2 expression is assessed by indirect immunofluorescence on skin biopsies, processed as in Example 6. Biopsies serving as Positive controls are incubated in parallel under the same conditions and receive 1 pg / ml and 10 pg / ml of retinoic acid (Sigma). CRABP2 detection is performed by incubation with anti-CRABP2 antibody (Abcam). After 1.5 hours of incubation followed by rinsing, the sections are incubated with a fluorophore-coupled anti-rabbit secondary antibody (Alexa Fluor® 488, Invitrogen). The sections are then examined by epifluorescence microscopy (Zeiss Axiovert 200M microscope). CRABP2 expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0216] Results: As shown in [Fig. 6], when biopsies were treated with purified rosewood extract according to Example 1 at 1%, CRABP2 expression increased by 80%. When biopsies were treated with 1 pg / ml and 10 pg / ml retinoic acid, CRABP2 expression increased by 106% and 132%, respectively.

[0217] Conclusion: The purified rosewood extract showed a positive effect on the expression of CRABP2.

[0218] Example 11: Evaluation of 1% purified rosewood extract on the expression of type V alpha 3 chain collagen on human skin biopsies

[0219] Purified rosewood extract has been tested on the expression of the alpha 3 chain of type V collagen. Genetic variations of this gene have been associated with wrinkles located in the corner of the eye.

[0220] Protocol: Alpha chain V collagen expression is assessed by indirect immunofluorescence on skin biopsies, processed as in Example 6. Biopsies serving as positive controls are incubated in parallel under the same conditions and receive 1 pg / ml of retinoic acid (Sigma). Detection of the alpha chain V collagen is performed by incubation with the alpha chain V collagen antibody (Sigma). After 1.5 hours of incubation followed by rinsing, the sections are incubated in the presence of the anti-rabbit secondary antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined by epifluorescence microscopy (Zeiss Axiovert 200M microscope). Alpha chain V collagen expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0221] Results: As shown in [Fig. 7], when biopsies were treated with 1% purified rosewood extract, alpha 3 chain expression of type V collagen increased by 23%. When biopsies were treated with 1 pg / ml retinoic acid, alpha 3 chain expression of type V collagen increased by 28%.

[0222] Conclusion: Purified rosewood extract has shown a positive effect on the expression of the alpha 3 chain of type V collagen. This anti-aging effect is particularly interesting for Asian skin.

[0223] Example 12: In vivo (clinical) evaluation of purified rosewood extract on facial sagging and signs of aging such as volume changes and the appearance of wrinkles

[0224] Principle: To study the in vivo efficacy of the purified rosewood extract according to example 1, formulated at 1%, and to link it with the in vitro results obtained on key markers of facial morphology.

[0225] Protocol: comparative double-blind study against a placebo, conducted on the face of 33 volunteers (aged 35 to 67 years) randomly divided into two groups: 16 volunteers for the placebo group and 17 volunteers for the treated group, homogeneous according to age and sex.

[0226] [Table 4]: Care formula containing the rosewood extract of Example 1 at 1% used in the clinical test on the treated group. ingrédients (trade name 1INCI) % w / w Fournisseur phase A Eau purifiée Water / Aqua Qs. 100 Local Sel EDTA tetrasodium Tetrasodium EDTA 0,05 Fisher Lubrajel* MS Free hydrogel Glycerin (and) Glyceryl Acrylate / Acrylic Acid Copolymer (and) Phe-noxyethanol 3,00 Ashland LiquaPar™ / Rokonsal™ MEP conservateur Phenoxyethanol (and) Methylparaben (and) Ethylparaben (and) Pro-pylparaben 1,00 Ashland phase B UltraThix™ P-100 polymer Acrylic Acid / VP Crosspolymer 0,60 Ashland phase C Hydroxide de sodium Sodium Hydroxide 0,02 Fisher Eau purifiée Water / Aqua 0,50 Local phase D Belsil* W3230 Bis-Stearoxydimethylsila ne (and) Stearyl Alcohol (and) Dimethicone 2,00 Wacker Shnulsol* 165 PEG-100 Stearate (and) Glyceryl Stearate 2.00 Seppic Refined Shea Butter Butyrospermum Parkii (Shea) Butter 2.00 Ashland Ceraphyl™ 28 ester Cetyl Lactate 1.50 Ashland Ceraphyl 791 ester Isocetyl Stearoyl Stearate 2.00 Ashland Ceraphyl ODS ester Octyldodecyl Stearate 3.00 Ashland Ceraphyl 368 ester Ethylhexyl Palmitate 4.00 Ashland Purified Rosewood Extract as per Example 1 Caprylic / Capric Triglyceride (and) Aniba Rosaeodora (Rosewood) Wood Extract 1.00 Ashland Phase E Sodium Hydroxide 0.03 Fisher Purified Water / Aqua 0.50 Local Total 100.00%

[0227] The placebo group received the same treatment formula but without the extract.

[0228] Study duration: 56 days.

[0229] Follow-up visits at JO (first day of the study), J28 and J56.

[0230] -Representative measurements of facial sagging: the angle of the V shape of the face, the distance between ptoses, the volume of ptoses, the volume of the oval of the face as well as the volume of the cheekbones by the AEAVA-HE® system (Eotech®).

[0231] - Measurement of lip corner volume and roughness by the system AEAVA-HE® (Eotech®).

[0232] - Topography of crow's feet skin by silicone replica, analyzed by Fringe projection and stereometry associated with the AEAVA-HE® system (Eotech®). The profile roughness of crow's feet wrinkles was measured and 2D and 3D representations of the skin topography were performed.

[0233] - Colour photos of the face with the HeadScan* V03 bench (Orion Concept*).

[0234] - Evaluation on a scale of 0 to 100 by an expert and by state volunteers of the facial skin to the touch and according to the visual appearance of the following parameters:

[0235] - Skin tone: 0 corresponding to very dull skin and 100 to a luminous and a perfect complexion.

[0236] - Crow's feet: 0 corresponding to smooth skin without wrinkles and 100 to a skin with marked micro-relief and visible fine lines.

[0237] - nasolabial fold: 0 corresponding to a very deep nasolabial fold and 100 corresponds to a nasolabial fold that is not marked and not visible

[0238] - Skin tone: 0 corresponding to flaccid skin with no tone and 100 cor suitable for very firm skin with very good tone

[0239] - Oval contour of the face: 0 corresponding to a poorly defined facial contour with pronounced ptosis and 100 corresponding to a perfectly oval face contour without ptosis

[0240] - Facial harmony: 0, corresponding to a face with poor harmony and a dull complexion. Deep wrinkles and a present nasolabial fold, sagging skin and a poorly defined facial contour with ptosis. 100 corresponds to a harmonious face with a radiant complexion, smooth, wrinkle-free skin, toned skin, and a perfectly defined facial contour.

[0241] The measurements were carried out on the subject after 15 minutes of rest in a room at 21°C + / -1 and with a relative humidity of 50% + / - 5.

[0242] The results of the facial sagging and volume change measurements show that after one and two months of application, volunteers in the treated group have a more V-shaped facial angle and a reduced facial oval volume, unlike volunteers in the placebo group. These results are illustrated in [Fig. 8]. Furthermore, as illustrated in [Fig. 9], the distance between the ptoses as well as their volumes are reduced compared to the placebo group.

[0243] These results are consistent with the results obtained in vitro on collagen XVII but also on harmonin and fibulin-7, key markers in facial morphology and which decrease with age.

[0244] Results of measurements of lip corner volume and roughness: as illustrated in [Fig. 10], after 2 months of application, a decrease in the volume and roughness of lip corner wrinkles is observed for the treated group compared to the placebo group.

[0245] These results are in agreement with the results obtained in vitro on Fibulin-7 (a key marker of corner lip wrinkles and which decreases with age).

[0246] Results of skin topography measurements at crow's feet: as illustrated in [Fig. 1 1] a decrease in the roughness of crow's feet wrinkles is observed at J28 and J56 for the treated group compared to the placebo group.

[0247] These results support the results obtained in vitro on collagen I and III.

[0248] These results are confirmed by observation of color photographs of the face and the evaluation of the expert and the volunteers.

[0249] Test conclusion: The above results demonstrate that the purified rosewood extract according to Example 1, formulated at 1%, has a firming effect and helps to reduce it addresses facial sagging and redefines the oval of the face and has an anti-wrinkle effect allowing the reduction of wrinkles at the corners of the mouth and crow's feet.

[0250] Similar results were obtained in a clinical study conducted on Asian skin, in which a reduction in wrinkles was observed after one month of application with the same cream containing the purified rosewood extract formulated at 1%. This result is consistent with the in vitro results obtained at the alpha 3 chain of type V collagen, a marker associated with wrinkles at the corners of the eyes, primarily in Asian skin.

[0251] Example 13: Evaluation of the purified 1% rosewood extract on the expression of the interleukin-1 receptor type 1 (IL1R1) on human skin biopsies

[0252] The purified rosewood extract was tested on the expression of IL1R1, a protein that increases in human skin with inflammation.

[0253] Protocol: IL1R1 expression is assessed by indirect immunofluorescence on skin biopsies, processed as in Example 6. Biopsies serving as positive controls are incubated in parallel under the same conditions with 1 pg / ml and 10 pg / ml of retinoic acid (Sigma). IL1R1 detection is performed by incubation with anti-IL1R1 antibody (Tebu bio). After 1.5 hours of incubation followed by rinsing, the sections are incubated with a fluorophore-coupled anti-rabbit secondary antibody (Alexa Fluor® 488, Invitrogen). The sections are then examined by epifluorescence microscopy (Zeiss Axiovert 200M microscope). IL1R1 expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0254] Results: As shown in [Fig. 12], when biopsies were treated with purified rosewood extract according to Example 1 at 1%, IL1R1 expression decreased by 3%. When biopsies were treated with 1 pg / ml and 10 pg / ml retinoic acid, IL1R1 expression increased by 19% and 56%, respectively.

[0255] Conclusion: The purified rosewood extract showed no significant modulation of interleukin-1 receptor type 1 (IL1R1) expression. Application of retinoic acid showed an increase in IL1R1 expression.

Claims

Demands

1. A process for obtaining a rosewood extract from exhausted rosewood (Aniba rosaeodora) leaves or chips, comprising the following steps: a. Between 5 and 25% water is added to the exhausted and dried rosewood b. An extraction is carried out using carbon dioxide (CO2) in the supercritical state in the presence of a polar co-solvent chosen from primary or secondary alcohols, or any mixture thereof; c. a support solvent is added, chosen from polyol-type solvents, saturated or unsaturated fatty alcohols, linear or branched, comprising 8 to 30 carbons, glyceride-type solvents or long-chain carbon ester-type solvents, or any mixture thereof; d. The extract solubilized in step c) is evaporated to remove all of the co-solvent, e. The extract is filtered to remove precipitated compounds that do not dissolve in the supporting solvent, and the filtrate is collected. f. the extract obtained in e) is diluted in the same supporting solvent to a concentration of between 0.005 and 50% of crude extract by weight of the total weight of the final extract.

2. A process according to claim 1 wherein in step b) the co-solvent is ethanol, advantageously at a concentration between 80 and 100% (volume percentage / volume water), preferably between 90 and 100% and more preferably at a concentration of 96%.

3. A method of any one of the preceding claims wherein in step b), the mass ratio of the co-solvent to carbon dioxide (CO2) in the supercritical state is between 0.050 and 0.080, advantageously between 0.055 and 0.075 and preferably between 0.060 and 0.

070.

4. A method of any one of claims 2 or 3 wherein in step b) the co-solvent flow rate is between 10 and 20 ml / min.

5. A method of any one of the preceding claims wherein in step b) the extraction temperature is between 35 and 85°C, advantageously between 45 and 75°C and preferably between 55 and 65°C and the pressure within the extractor is between 90 and 1000 bar, preferably between 150 and 700 bar and even more preferably between 250 and 350 bar.

6. A method of any one of the preceding claims wherein in step c) the supporting solvent is selected from polyol-type solvents, saturated or unsaturated fatty alcohols, linear or branched, comprising 8 to 30 carbons or glyceride-type solvents or long-chain carbon ester-type solvents, or any mixture thereof, preferably the supporting solvent is glycerol tricaprylate / caprate.

7. Purified rosewood extract obtainable by the process of any one of claims 1 to 6, characterized in that it is diluted to a concentration of between 0.1 and 1% in the supporting solvent and comprises from 0.3 to 0.5%, advantageously from 0.35 to 0.5% and preferably from 0.4 to 0.5% of semi-volatile to non-volatile compounds.

8. Purified rosewood extract of claim 7, characterized in that the semi- to non-volatile compounds comprise 0.01 to 0.4% sesquiterpene derivatives as cotoin equivalents, 0.001 to 0.1% aryl phenyl ketones of which 0.001 to 0.05% cotoin, 0.001 to 0.01% beta-sitosterol and 0.001 to 0.1% anibine.

9. Composition comprising an effective amount of purified rosewood extract of one of claims 7 or 8 as an active agent and a physiologically acceptable medium.

10. Composition of claim 9, characterized in that it comprises between 0.01% and 10%, preferably between 0.1% and 5% and even more preferably between 0.5% and 2% of purified rosewood extract by weight relative to the total weight of the composition.

11. Cosmetic use of the composition of claim 9 to prevent or limit the signs of skin aging, firm the skin, limit the appearance of facial ptosis, redefine the contour of the face and also to limit the appearance of wrinkles and fine lines.

12. Cosmetic use of the composition of claim 9 to increase the expression of collagen I, collagen III, collagen XVII, hyaluronic acid, fibulin-7, harmonin and CRABP2.

13. Cosmetic use of the composition of claim 9 to increase the expression of the alpha 3 chain of type V collagen and to limit the appearance of wrinkles and fine lines.

14. Cosmetic use of the composition of claim 9 to obtain an effect similar to that of retinoids on the signs of skin aging, without presenting undesirable inflammatory effects.

15. Composition according to claim 9 for its use in combating external aggressions producing pro-inflammatory free radicals, such as stress or pollution.

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