Extract of chlamydomonas acidophila, method for preparing the same, and cosmetic compositions and dermatological compositions comprising the same

Abstract

To provide a peptide extract of the microalga Chlamydomonas acidophila, as a cosmetic or dermatological composition, and a method for the preparation thereof.SOLUTION: The present invention provides a peptide extract of the microalga Chlamydomonas acidophila, and a method for the preparation thereof, wherein the extract is obtainable by the preparation method comprising at least one enzymatic hydrolysis step comprising at least 20 wt.% of peptides as the percentages expressed with respect to the total weight of the extract.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a peptide extract of the microalga Chlamydomonas acidophila and to a cosmetic, dermatological or pharmaceutical composition comprising such an extract. The present invention also relates to a method for extracting a peptide extract of Chlamydomonas acidophila and to an extract obtainable by said method. The present invention also relates to a composition or such an extract for use in the prevention or treatment of disorders or pathologies of the skin, mucous membranes or skin appendages, for use in the prevention or treatment of vascular disorders or for use in the prevention or treatment of adipose tissue degeneration. Finally, the present invention also relates to a method for the cosmetic care of the skin, mucous membranes or skin appendages, aiming at improving their condition or appearance, which method consists of administering such a composition or such an extract. [Background technology]

[0002] microalgae Microalgae are single-celled eukaryotic organisms that can perform photosynthesis and thus, like higher plants, can use CO2 from the air in their metabolism, in addition to other nutrients such as phosphorus, nitrates, etc. Microalgae were among the first species to colonize the Earth. About 30,000 species have been described, but there are likely many more. Microalgae are found in their natural state in fresh, brackish and salt waters throughout the world.

[0003] Microalgae can be cultivated in light, pH and nutrient controlled environments, such as photoreactors, according to methods known to those skilled in the art, and there are many products. Microalgae, like higher organisms, can synthesize proteins, carbohydrates and lipids. Some lipids are unique, such as complex fatty acids or pigments (xanthophylls) with specific biological properties. These lipids have become the focus of attention for the possibility of producing biofuels, and their production in bioreactors is expanding. Other products are diverse, including fish feed (aquariums and aquaculture), food and human health (spirulina protein, astaxanthin extracted from Haematococcus pluvialis) and some products in the cosmetics industry.

[0004] Prior Art -Chlamydomonas acidophila Chlamydomonas acidophila, a member of the Chlorophyceae (family Chlamydomonadaceae), is a green freshwater microalgae that grows in highly acidic waters (pH 2.3-3.4) and has adapted to environments containing heavy metals. Notably, Chlamydomonas acidophila was first identified and collected from a volcanic lake in Argentina. Chlamydomonas acidophila is said to be rich in phytochelatins, specific structures capable of chelating metals, and carotenoids (β-carotene, lutein). Little is known about its composition and uses, except for publications about its culture and development conditions (its tolerance to extreme pH and heavy metals). Its "close relative" Chlamydomonas reinhardtii is used as a model organism in various chemical disciplines, such as genetics. DISCLOSURE OF THEINVENTION

[0005] The applicant has discovered that peptide extracts of the microalga Chlamydomonas acidophila exhibit cosmetic, pharmacological and dermatological properties that have not been described before, in particular, this is the first time that such Chlamydomonas acidophila extracts have been used per se for their specific properties.

[0006] The present invention relates to a peptide extract of the microalga Chlamydomonas acidophila.

[0007] In the context of the present invention, "peptide extract" means an extract that comprises primarily peptides.

[0008] In the context of the present invention, a "peptide" means a polymer of amino acids linked together by peptide bonds. Peptides are in particular characterized by a molecular weight of between 200 and 10,000 Daltons (Da).

[0009] Advantageously, the extract of Chlamydomonas acidophila according to the invention comprises at least 20% by weight of peptides, expressed as a percentage relative to the total weight of the extract, in particular, the extract according to the invention comprises 20% to 90% by weight of peptides, advantageously 20% to 75% by weight, more advantageously 30% to 70% by weight, typically 65% ​​by weight, expressed as a percentage relative to the total weight of the extract.

[0010] Advantageously, the Chlamydomonas acidophila extract according to the invention is substantially free of proteins, in particular residual natural proteins, which, among other things, avoids allergic reactions and improves the solubility and bioavailability of the extract according to the invention.

[0011] In the context of the present invention, a "protein" refers to a biological polymer formed from one or more polypeptide chains. Each of these chains consists of a sequence of amino acid residues interconnected by peptide bonds. Proteins are characterized in particular by a molecular weight greater than 10,000 Daltons (Da).

[0012] Advantageously, the Chlamydomonas acidophila extract according to the invention is substantially free of free amino acids, the free amino acids having a molecular weight of less than 200 Da.

[0013] In the peptide extract of Chlamydomonas acidophila according to the invention, the peptides advantageously have a molecular weight of less than 3500 Da. Advantageously, these peptides include all of the compounds based on amino acids originally present in the extract.

[0014] Advantageously, in the extract according to the invention, at least 80%, more advantageously at least 90%, of the peptides have a molecular weight of less than 1000 Da.

[0015] Advantageously, in the extract according to the invention, at least 30% of the peptides have a molecular weight of less than 500 Da, more advantageously at least 35%, more advantageously at least 40% of the peptides.

[0016] The molecular weight distribution of peptides is expressed as a percentage of the total peptide concentration.

[0017] In the context of the present invention, the peptide extract of Chlamydomonas acidophila is advantageously obtained by enzymatic hydrolysis, more advantageously in the presence of at least one protease. The extract according to the invention is more advantageously obtainable by the method described herein below.

[0018] The present invention also relates to a method for preparing a peptide extract of the microalga Chlamydomonas acidophila, comprising at least one enzymatic hydrolysis step, advantageously carried out under optimal pH and temperature conditions known to the skilled artisan, in particular optimal pH and temperature conditions for the enzyme used.

[0019] Advantageously, said enzymatic hydrolysis step is carried out in the presence of at least one protease, which may advantageously be an alkaline protease or an acid protease, advantageously an alkaline protease.

[0020] Advantageously, according to the invention, the method for preparing a peptide extract of Chlamydomonas acidophila comprises at least the following steps: a) Aqueous phase dispersion of the microalga Chlamydomonas acidophila; b) enzymatic hydrolysis of the aqueous phase dispersion obtained in step a), c) heat treatment of the mixture obtained in step b), and d) Recovery of the peptide extract at the end of step c).

[0021] In step a), the aqueous phase is preferably water. Furthermore, the content of the microalgae Chlamydomonas acidophila in the aqueous dispersion preferably comprises 0.1% to 20%, more preferably 1% to 10%, of the equivalent of the dry extract of the microalgae.

[0022] The enzymatic treatment (step b) is advantageously carried out with at least one protease, advantageously under optimal pH and temperature conditions known to the person skilled in the art, for example at a pH comprised between 3.0 and 9.0, and at a temperature generally comprised between 20° C. and 90° C. In particular, the enzymatic treatment comprises the addition of an alkaline or acidic protease, advantageously an alkaline protease.

[0023] The enzymatic hydrolysis step of the method according to the invention is crucial since it "transforms" or "cuts" the native proteins present in Chlamydomonas acidophila to obtain peptides.

[0024] In the context of the present invention, the enzymatic hydrolysis step is advantageously followed by a heat treatment step to denature the enzymes, which is advantageously carried out at a temperature above 40°C, generally between 80°C and 100°C.

[0025] In step d), the peptide extract is advantageously recovered at the end of step c), by extraction of the dispersion, advantageously under stirring, advantageously at a pH comprised between 3.0 and 9.0 and at a temperature comprised between 20°C and 90°C.

[0026] Advantageously, the method comprises an additional filtration or centrifugation step located between steps c) and d), optionally followed by ultrafiltration, diafiltration or nanofiltration.

[0027] A filtration or centrifugation step, optionally followed by membrane ultrafiltration or diafiltration, is used to remove residual proteins. A nanofiltration step is used, for example, to remove inorganic salts or free amino acids.

[0028] The process according to the invention advantageously comprises an ultrafiltration step at 15 kDa, advantageously between 10 and 15 kDa, carried out between steps c) and d), which is used to remove potentially allergenic residual proteins.

[0029] Advantageously, the method according to the invention further comprises a nanofiltration step, for example with a cut-off threshold comprised between 100 and 300 Daltons, advantageously between 130 and 300 Daltons, generally between 200 and 300 Daltons, to remove part of the amino acids or inorganic salts after the ultrafiltration step. Advantageously, said nanofiltration step is carried out with a membrane of 200 Da.

[0030] The resulting aqueous hydrolysate, i.e. the peptide extract according to the invention, can be physically and microbiologically stabilized by adding a solvent, such as glycerol or a glycol, such as 1,3-propanediol, in various proportions suitable for stabilization. Advantageously, glycerol is present alone or in combination with water or glycol, advantageously in a proportion comprised between 40% and 95% by weight, preferentially between 50% and 90% by weight, relative to the total weight of the peptide extract and the solvent. Similarly, glycol, preferentially 1,3-propanediol, is advantageously present alone or in combination with water or glycerol, advantageously in a proportion comprised between 40% and 95% by weight, preferentially between 50% and 90% by weight, relative to the total weight of the peptide extract and the solvent. Thus, the invention further relates to a composition comprising a peptide extract of Chlamydomonas acidophila according to the invention, a solvent selected from glycerol, glycol and mixtures thereof, in an amount effective for a physical and microbiological stabilizing effect, and optionally water. The amounts effective for a physical and microbiological stabilizing effect are as described above.

[0031] Alternatively, the peptide extract can be stabilized in the presence or absence of a carrier, such as, for example, maltodextrin or acacia fiber (Fibergum® from CNI), by methods known to those skilled in the art. The carrier content generally varies according to the carrier ratio, ranging from 0% to 80% with respect to the percentage of dry matter obtained in the liquid extract. The extract can be dried by spraying, freeze-drying or any method known to those skilled in the art, preferentially by freeze-drying to obtain a fine powder. The fine powder advantageously comprises 30% to 70% by weight of the extract dry matter, the remainder being the freeze-dried carrier to make 100%. More advantageously, the fine powder comprises 50% of dry matter from the extract and 50% of the freeze-dried carrier, said freeze-dried carrier being preferably of the maltodextrin or acacia fiber type.

[0032] Preferentially, by way of example, the peptide extract can be obtained according to the following method: a) dissolution of the microalgae Chlamydomonas acidophila in water at a microalgae content equivalent to approximately 10% dry extract; b) enzymatic hydrolysis with alkaline protease (Alcalase from Lyven); c) heat treatment to denature the enzyme; c') centrifugation, ultrafiltration and diafiltration through a 15 kDa membrane to remove potentially allergenic residual proteins; c"), for example, 200 Da membrane nanofiltration to remove inorganic salts or free amino acids; and d) Recovery of the peptide extract obtained at the end of step c”).

[0033] For the method according to the invention, the Chlamydomonas acidophila microalgae used as raw material can be obtained from cultivation in an open environment, for example in a "waterway" (oval track tanks used for aquaculture), or from cultivation in a closed environment photobioreactor. Advantageously, the microalgae used as raw material are obtained from cultivation in a photobioreactor, in particular a stirred tank photobioreactor. More advantageously, the microalgae used as raw material are obtained from cultivation in a horizontal tubular wave-ventilated stirred tank photobioreactor, such as, for example, those developed by the company Microphyt and described in particular in patent application FR2943685 and in international application WO2011 / 058267.

[0034] The present invention also relates to a Chlamydomonas acidophila extract obtainable by the process described above, such extract meeting the specifications defined above.

[0035] The present invention also relates to cosmetic, dermatological or pharmaceutical compositions comprising as active ingredient a peptide extract of Chlamydomonas acidophila and, if necessary, suitable excipients.

[0036] Advantageously, in the composition according to the invention, the peptide extract of Chlamydomonas acidophila is as defined above or is obtainable by the method described above, said extract therefore being advantageously defined in the paragraphs above relating to the extract according to the invention per se or to the extract obtainable by the method according to the invention.

[0037] The compositions are advantageously formulated for external topical, vaginal or buccal administration.

[0038] Advantageously, the composition according to the invention comprises from 0.001% to 10% by weight, advantageously from 0.01% to 5% by weight, of said Chlamydomonas acidophila peptide extract, expressed as dry extract based on the total weight of the composition.

[0039] The compositions according to the invention may further comprise one or more other active ingredients.

[0040] According to the first option, the various preparations are suitable for topical administration and include in particular creams, emulsions, milks, ointments, lotions, oils, aqueous or hydroalcoholic or glycolic solutions, powders, patches, sprays, shampoos, polishes or any other external product, and according to the following option, the various preparations include in particular intimate hygiene care, oral care, such as toothpastes, oral solutions, gum gels.

[0041] Depending on its nature (cosmetic, pharmaceutical or dermatological), the composition according to the invention may further comprise at least one cosmetically, pharma- ceutical or dermatologically acceptable excipient. In particular, the composition according to the invention may further comprise at least one cosmetically, pharma- ceutical or dermatologically acceptable adjuvant known to those skilled in the art, selected from surfactants, thickeners, preservatives, fragrances, dyes, chemical or inorganic filters, moisturizers, spring water, etc. Those skilled in the art know how to adapt the formulation of the composition according to the invention using their general knowledge.

[0042] The optimal dose and galenical form of the composition according to the invention can be determined according to criteria taking into account the overall establishment of a pharmacological, dermatological or cosmetic treatment adapted to the patient or animal, such as, for example, the age or weight of the patient or animal, the severity of its health condition, tolerance to the treatment, observed side effects and skin type.

[0043] The present invention also provides Disorders or conditions of the skin and / or mucous membranes (e.g., gums, periodontal tissues, genital mucosa) and / or skin appendages (e.g., hair and nails); Vascular disorders; and Degeneration of adipose tissue The present invention also relates to an extract according to the invention, or an extract obtainable by the method according to the invention, or a composition according to the invention, for use in the prevention and / or treatment of

[0044] The present invention also provides Disorders or conditions of the skin and / or mucous membranes (e.g., gums, periodontal tissues, genital mucosa) and / or skin appendages (e.g., hair and nails); Vascular disorders; and Degeneration of adipose tissue The present invention also relates to the use of an extract according to the invention, or an extract obtainable by the process according to the invention, or a composition according to the invention, for the manufacture of a cosmetic, pharmaceutical or dermatological composition for use in the prevention and / or treatment of

[0045] The present invention further comprises: Disorders or conditions of the skin and / or mucous membranes (e.g., gums, periodontal tissues, genital mucosa) and / or skin appendages (e.g., hair and nails); Vascular disorders; and Degeneration of adipose tissue A method for preventing and / or treating It also relates to a method comprising administering, in particular topically administering, an effective amount of the extract according to the invention, or the extract obtainable by the method according to the invention, or the composition according to the invention to a subject in need thereof.

[0046] In particular, the extract according to the invention, or the extract obtainable by the method according to the invention, or the composition according to the invention, is intended for the prevention and / or treatment of allergic, inflammatory, irritant reactions or pathologies or disorders of the barrier or homeostasis of the skin, immature, normal or mature / aged skin appendages (hair and nails) and / or mucous membranes (gingiva, periodontium, genital mucosa).

[0047] Advantageously, the composition or extract according to the invention comprises: Acne, rosacea or erythrocouperosis, psoriasis, vascular disorders, diaper rash, atopic dermatitis, eczema, contact dermatitis, irritant dermatitis, allergic dermatitis, seborrheic dermatitis (cradle cap), psoriasis, sensitive skin, reactive skin, dry skin (xerostomia), dehydrated skin, skin with redness, skin erythema, aging or photoaging skin, photosensitized skin, pigmented skin (liver spots, post-inflammatory pigmentation, etc.), depigmented skin (vitiligo), skin with cellulite, of the skin, such as loose skin, skin with stretch marks, crusts, rough skin, puncture wounds, cracks, especially on the breasts, burns, inflammation due to any kind of light, irritation due to chemical, physical (e.g. stress in pregnant women), bacterial, fungal or viral, parasitic (lice, scabies, ringworm, mites, dermatophytes) or radiation agents, or due to a lack of innate (antimicrobial peptides) or acquired (cellular, humoral, cytokine) immunity, and / or the gums and periodontal tissues, which may exhibit gingivitis (due to sensitive gums in newborns, hygiene problems, smoking or otherwise), periodontal disease, or mucous membranes, such as the genital mucosa, which may exhibit irritation of the external or internal male or female genital areas; and / or In particular it can be used for the prevention and / or treatment of reactions, disorders or conditions of the skin appendages such as immature, normal or mature nails (brittle weak nails) and hair (alopecia, dandruff, hirsutism, seborrheic dermatitis, folliculitis) which present with disorders of the scalp such as androgenic, acute, localized, cicatricial, congenital or infantile occipital alopecia (or alopecia areata), alopecia areata, chemotherapy / radiotherapy associated alopecia or telogen effluvium, anagen effluvium, pilar dystrophy, trichotillomania, ringworm or oily or dry dandruff.

[0048] The present invention also relates to a method for the cosmetic care of the skin and / or the skin appendages and / or mucous membranes, aiming to improve their condition or appearance, which method comprises administering an extract according to the invention, or an extract obtainable by a method according to the invention, or a composition according to the invention.

[0049] In particular, this cosmetic care method firmes the skin and advantageously reduces the "orange peel" effect by topical route on the skin and / or skin appendages and / or mucous membranes.

[0050] In particular, the present invention relates to a method for the cosmetic care of the skin and / or skin appendages, in order to affect the elasticity or firmness of the skin, in particular to act as an anti-wrinkle or anti-wrinkle agent, to affect sensitive skin or to act against pollution, which method comprises applying to the skin and / or skin appendages a composition or an extract according to the invention.

[0051] In particular, the present invention relates to a method for the cosmetic care of the skin and / or skin appendages, their barrier and dehydration, which method consists in applying to the skin and / or skin appendages a composition or an extract according to the invention.

[0052] The present invention relates to a cosmetic skin care method for anti-aging purposes, which consists in applying to the skin a composition or an extract according to the invention.

[0053] The composition or extract according to the invention may also be advantageously used in the prevention and / or treatment of vascular disorders, in particular redness and cuperose.

[0054] The composition or extract according to the invention can also be advantageously used in the prevention and / or treatment of adipose tissue degeneration, in particular cellulite or the "orange peel" effect. The composition according to the invention firmes the skin.

[0055] The invention can be illustrated, but not limited to, by the following examples. [Brief description of the drawings]

[0056] [Figure 1] Figure 1 represents the results of erythema intensity measurements: active / placebo / untreated area comparison. NS: non-significant difference. *: p<0.05 (Example 3B). [Diagram 2] FIG. 2 shows the time course of changes in blood flow (Example 3B). [Diagram 3] FIG. 3 represents a graph of the TEWL results obtained at D0 and D28 (Example 3C). [Figure 4] FIG. 4 represents a graph of the water retention results obtained at D0 and D28 (Example 3C). [Diagram 5] FIG. 5 represents a graph of the amount of NMF quantified at D0 and D28 (Example 3C). [Figure 6] FIG. 6 represents a graph of the amount of ceramide quantified at D0 and D28 (Example 3C). [Figure 7] FIG. 7 represents a graph of the amount of IL1RA quantified at DO and D28 (Example 3C). [Figure 8] FIG. 8 represents a graph of the Nile Red / Involucrin ratio at D0 and D28 (Example 3C). EXAMPLES

[0057] Example 1: Extract according to the invention The peptide extract is obtained according to the following method: a) Dissolution of the microalga Chlamydomonas acidophila in water at approximately 10% dry matter; b) hydrolysis with alkaline protease (Alcalase from Lyven); c) heat treatment at temperatures comprised between 80°C and 100°C to denature the enzyme; c') centrifugation, ultrafiltration and diafiltration through a 15 kDa membrane to remove potentially allergenic residual proteins; c”) 200Da membrane nanofiltration to remove inorganic salts or free amino acids or monosaccharides; d) recovery of peptide extracts; e) Stabilization in glycerol / 1,3-propanediol mixtures.

[0058] The liquid peptide extract thus obtained has the following characteristics: 1-Physicochemical analysis (% / total dry matter) Dry extract (2 hours, 105°C, ventilated oven): 1.2% pH: 5.1 Alpha-amino nitrogen (OPA, leucine equivalent): 29% Peptide (Kjeldahl, N×6.25): 70% Total Ash: 4% 2-Peptide molecular weight distribution profile Less than 500 Da: 40% 500-1000 Da: 55% 1000-3500 Da: 4% Greater than 3500Da: 1%

[0059] Example 2: Testing the biological activity of the extract according to the invention (in vitro) The biological activity of the Chlamydomonas acidophila (CAP) extract obtained in Example 1 was demonstrated in vitro as described below.

[0060] These in vitro studies Strengthening of the skin barrier (especially the lipid barrier); Skin hydration via hyaluronan synthesis, NMF production and osmolyte transport pathways; antioxidant and anti-inflammatory defenses against non-specific stress or those associated with air pollution; Anti-aging effect, especially through the protection of the homeostasis of the skin matrix; Allergic mechanism This demonstrated the potential of CAP extract against

[0061] I. Preliminary screening of activity on skin fibroblasts and melanized reconstructed epidermis The potential biological activity of Chlamydomonas acidophila extract was investigated by gene expression modulation studies on skin fibroblasts and melanized reconstructed epidermis. Thus, the expression of 96 genes of major interest in skin and cosmetic physiology was examined by PCR array on fibroblasts and melanized reconstructed epidermis.

[0062] Materials and Methods: Chlamydomonas acidophila (CAP) extract, 0.05% dry matter, was added to the culture medium of normal human dermal fibroblasts (NHDFs) or melanized reconstructed human epidermis.

[0063] After 6 or 24 h of incubation, expression of selected markers was assessed by quantitative RT-PCR (TaqMan microfluidic cards). Changes in expression of investigated markers compared to the control were expressed as relative quantities (RQ, RQ>1: increase, RQ<1: decrease).

[0064] b.Result: The most significant results showing the effect of CAP extract on gene expression in the reconstructed epidermis are shown in Table 1 below.

[0065] [Table 1]

[0066] These results tend to indicate that Chlamydomonas acidophila extracts may be particularly noteworthy in the following activities by altering the gene expression of specific markers:

[0067] Remodeling of lipid synthesis and epidermal barrier function: The GBA / GBAP1 gene, which encodes the enzyme glucosylceramidase or β-glucocerebrosidase, is overexpressed 6 hours after CAP treatment. RAB11A encodes a GTPase (Ras-related protein Rab-11A) involved in the biogenesis of lamellar bodies in keratinocytes, highlighting the importance of RAB11A in epidermal barrier homeostasis.

[0068] Hyaluronic acid biosynthesis and epidermal hydration: Hyaluronan synthase-2 and -3 (HAS2 and HAS3) are enzymes responsible for the synthesis of hyaluronic acid (HA). The gradual expression of SLC6A6 in the spinous and granular layers of the epidermis maintains the necessary moisture retention in the epidermis in a dry environment. The PADI1, BLMH and CASP14 genes encode enzymes involved in the production of natural moisturising factors (NMFs).

[0069] Table 2 below shows the most significant results of the CAP extract on gene expression in fibroblasts.

[0070] [Table 2]

[0071] These results indicate potential activity of the CAP extract in the following areas:

[0072] Skin Anti-Aging: Three hyaluronan synthases, HAS1, HAS2 (5.76 0.008) and HAS3, produced by dermal fibroblasts, are responsible for the biosynthesis of hyaluronic acid (HA). Furthermore, it has been shown that skin aging is associated with a decrease in fibroblast proliferation. A decrease in the expression of the growth factor Ki-67 is seen in association with age [Ma et al. 2011 Br. J. Dermatol, 164(3), pp. 479-482]. CAP activators enhance the expression of MKI67 after 24 hours of treatment, demonstrating a potential increase in cell proliferation that reinforces its anti-aging effect. Expression of lamin B1 (LMNB1) in the skin has been shown to decrease with age.

[0073] Antioxidant Defense: NAD(P)H dehydrogenase, quinone 1 (NQO1) is a cytosolic flavoprotein under the control of the transcription factor Nrf-2. NQO1, upon reduction, promotes the formation of hydroquinone from quinones and prevents the production of radical species. The HSP70 protein (encoded by the HSPA1A gene) is a chaperone molecule (heat shock protein 70) that inhibits the aggregation of denatured proteins and promotes their renaturation (refolding) and controls essential mediators of the apoptotic machinery. The thioredoxin system is one of the major antioxidant defense systems. Among the three isoforms of thioredoxin reductase, isoform 1, encoded by the TXNRD1 gene and regulated by CAP extract, is the best studied. Besides the thioredoxin system, there is also the peroxiredoxin / sulfiredoxin system. Among the peroxiredoxins, peroxiredoxin-6 (PRDX6) is overexpressed by CAP extracts after 24 hours of treatment, indicating the detoxification effect of the active agent against the presence of peroxides. Finally, the CAP extract stimulates the GLO1 gene, which is part of the GLO system that detects and neutralizes certain carbonyls, thus preventing them from attacking cells and cellular components.

[0074] Skin matrix homeostasis: CAP extract stimulates the expression of PSMB1, a gene that encodes the β1 subunit of the proteasome, which plays a key role in maintaining protein homeostasis by removing damaged or malformed proteins that may alter cellular function. Gene expression of LOXL2 was also increased; this gene is part of the lysyl oxidase family of enzymes involved in the assembly of elastin and collagen fibers.

[0075] II. Anti-inflammatory effect A. Anti-inflammatory activity against PMA chemical stress a.Introduction : The inflammatory response is the body's normal, immediate and transient response to an environmental insult.

[0076] However, in certain pathological or physiological conditions, this inflammatory response can be exacerbated and, if not properly controlled, can lead to tissue damage.

[0077] In the skin, keratinocytes are among the first cells involved in eliciting an inflammatory response in response to an environmental insult.

[0078] The "attacked" keratinocytes then Primary cytokines (IL1α, IL1β or TNFα) or secondary cytokines (IL8) that trigger a cascade of reactions involving the immune system; Prostaglandin (PGE), a member of the prostanoid family. The prostaglandin synthesis pathway leading to the synthesis of PGE2 and other PGEs is induced by inflammatory stimuli.

[0079] The anti-inflammatory activity of the Chlamydomonas acidophila extract according to the invention was evaluated in an inflammation model induced in keratinocytes by treatment with PMA (phorbol 12-myristate 13-acetate). The release of cytokines TNFα and prostaglandin E2 (PGE2) was analyzed.

[0080] b. Materials and Methods : Normal human epidermal keratinocytes were pretreated for 24 h with Chlamydomonas acidophila (CAP) extract at concentrations ranging from 0.0001% to 0.05% dry matter according to Example 1, or with the anti-inflammatory reference molecules dexamethasone 0.1 μM or indomethacin 0.1 μM (these two references are used as anti-inflammatory references for cytokines and prostaglandins, respectively).

[0081] Inflammation was then induced by adding 10 μg / mL PMA overnight.

[0082] TNFα and PGE2 assays were then performed on the cell culture supernatants.

[0083] The significance of the results was confirmed by one-way ANOVA followed by Tukey's test (GraphPad Prism software version 5.02, GraphPad Software, San Diego, CA, USA).

[0084] c.Results : PMA at 10 μg / ml significantly increased TNFα release in keratinocyte supernatants and therefore did not induce inflammation. Dexamethasone at 0.1 μM and indomethacin at 0.1 μM as 24-h pretreatments reduced TNFα release, indicating their anti-inflammatory effect and validating the validity of this study.

[0085] Chlamydomonas acidophila extracts at various concentrations as a 24-h pretreatment significantly reduced TNFα release, thus demonstrating an anti-inflammatory effect against PMA.

[0086] [Table 3]

[0087] PMA at 10 μg / ml significantly increased the release of PGE2 in keratinocyte supernatant, thus inducing inflammation. Indomethacin and dexamethasone at 0.1 μM as 24-h pretreatment significantly decreased the release of PGE2. Thus, the anti-inflammatory effects of these two controls were fully validated.

[0088] Chlamydomonas acidophila extract as a 24-h pretreatment at both concentrations significantly reduced the release of PGE2, thus demonstrating an anti-inflammatory effect against PMA.

[0089] [Table 4]

[0090] d. Conclusion : The anti-inflammatory effect of Chlamydomonas acidophila extract was demonstrated by its action on the release of TNFα and prostaglandin E2 under inflammatory conditions.

[0091] B. Anti-inflammatory activity against nickel stress a.Introduction Nickel is a major cause of allergic contact dermatitis in the population with a worldwide prevalence of approximately 8.6%. The aim of the study described below was to evaluate the effect of CAP extract on the release of IL8 by nickel-stimulated keratinocytes.

[0092] b. Materials and Methods Normal human epidermal keratinocytes were pretreated for 24 hours with CAP extract at 0.01% and 0.05% dry matter or with the anti-inflammatory reference molecule dexamethasone at 1 μM. The keratinocytes were then treated with 10 μM nickel:NiSO4 for 24 hours. At the end of the incubation, the amount of IL8 produced by these cells was assessed by ELISA in the supernatant.

[0093] The assayed concentrations of IL8 were normalized to the amount of total intracellular protein assessed by BC assay.

[0094] The significance of the results was statistically analyzed by Student's t test.

[0095] c.Results CAP extract induces a significant reduction in nickel stress-induced IL8 release in keratinocytes.

[0096] [Table 5]

[0097] d. Conclusion Chlamydomonas acidophila (CAP) extract inhibits the release of IL-8, a key cytokine in nickel-induced inflammatory stress, making it of interest in nickel-related contact allergy or skin hypersensitivity.

[0098] C. Anti-inflammatory activity against cadmium stress a.Introduction The aim of this study was to evaluate the anti-inflammatory activity of Chlamydomonas acidophila (CAP) extract against heavy metal stress, represented by cadmium, in human keratinocytes.

[0099] b. Materials and Methods Normal human epidermal keratinocytes were pretreated for 24 hours with CAP extracts at 0.001% and 0.01% dry matter or with the anti-inflammatory reference molecule indomethacin at 0.1 μM. Keratinocytes were then treated with 100 μM cadmium:CdCl2 for 48 hours. At the end of the incubation, the amount of PGE2 produced by these cells was assessed by ELISA in the supernatant.

[0100] The assayed concentrations of PGE2 were normalized to the amount of total intracellular protein assessed by the BC assay.

[0101] c.Results CAP extract induces a decrease in cadmium stress-induced PGE2 release in keratinocytes.

[0102] [Table 6]

[0103] d. Conclusion Chlamydomonas acidophila (CAP) extract inhibits the production of prostaglandin E2 (PGE2) induced by cadmium stress, thus providing protection to the skin against heavy metal stress, e.g., in relation to environmental pollution.

[0104] D. Anti-inflammatory activity against SDS stress a.Introduction The anti-inflammatory activity of Chlamydomonas acidophila (CAP) extract was evaluated in an inflammation model induced by sodium dodecyl sulfate (SDS) treatment on reconstructed epidermis.

[0105] b. Materials and Methods Reconstructed human epidermis (RHE) was preincubated for 24 hours in the presence of 0.01% and 0.05% dry matter of CAP, then 0.025% SDS was applied to the surface of the epidermis and incubated again for 24 hours in the presence of CAP extract.

[0106] At the end of the incubation, the cytokine tumor necrosis factor alpha (TNFα) was assayed by ELISA in the supernatants.

[0107] Gene expression of inflammatory and barrier markers was assessed by qRT-PCR.

[0108] The significance of the results was statistically analyzed by one-way ANOVA followed by Tukey's test.

[0109] c.Results SDS treatment of reconstructed epidermis induces enhanced TNFα expression at the gene level (qRT-PCR, Table 8) and protein level (ELISA, Table 7). This proinflammatory effect is also accompanied by a decrease in the expression of keratin 1 (KRT1) (Table 8), demonstrating an impairment of the epidermal barrier function.

[0110] Under these conditions, the CAP extract significantly inhibited the overproduction of TNFα and enhanced the expression of keratin-1.

[0111] [Table 7]

[0112] [Table 8]

[0113] d. Conclusion These results confirm the anti-inflammatory potential of Chlamydomonas acidophila extract and demonstrate its ability to protect the barrier against external stresses.

[0114] III. Antioxidant effect a.Introduction Gene expression screening was performed with the Chlamydomonas acidophila extract of Example 1 which showed potential in stimulating antioxidant defense; its ability to protect cells from oxidative stress was assessed by measuring the production of reactive oxygen species (ROS) in keratinocytes exposed to oxidative stress induced by H2O2.

[0115] The antioxidant activity of the active agents is evaluated by incorporating DCFH-DA (2',7'-dichlorofluorescin diacetate) into cultured keratinocytes. This molecule is a non-fluorescent marker in non-oxidative conditions. Under oxidative conditions (here, H2O2 stress), DCFH-DA is degraded to DCF, a fluorescent molecule. The measured fluorescence is proportional to the amount of reactive oxygen species produced by the cells in the presence of H2O2 and / or the extract.

[0116] b. Materials and Methods Normal human epidermal keratinocytes were preincubated for 24 hours in the presence of 0.0001% dry matter of CAP extract, 10 μM quercetin or 500 μM vitamin C (these two molecules are used as antioxidant references).

[0117] These cells were then treated in the presence of 0.5 mM DCFH-DA for 1 hour.

[0118] Oxidation is induced by adding 100 μM H2O2 for 20 min. A second treatment with the test article is performed simultaneously with the H2O2 stress (same concentration as in the pretreatment).

[0119] Finally, the fluorescence density (DFU), which corresponds to the amount of ROS, is measured using a microplate reader.

[0120] The significance of the results was confirmed by one-way ANOVA followed by Tukey's test (GraphPad Prism software version 5.02, GraphPad Software, San Diego, CA, USA).

[0121] c.Results Increased ROS production was observed after H2O2 treatment, which validates the model. Quercetin and vitamin C significantly reduced ROS production after H2O2 treatment. The antioxidant effects of these two references were well validated in the model.

[0122] Chlamydomonas acidophila extract significantly reduced the production of ROS induced by H2O2 stress.

[0123] [Table 9]

[0124] d. Conclusion: Chlamydomonas acidophila extract exhibited antioxidant effects against H2O2-induced stress.

[0125] IV. Barrier and Moisture Retention Activity a.Introduction The gene expression screen performed on Chlamydomonas acidophila extract and presented above showed a potential effect on stimulating the expression of gene markers involved in barrier and hydration. We sought to confirm this effect on keratinocytes.

[0126] b. Materials and Methods Normal human epidermal keratinocytes were incubated for 48 hours in the presence of 0.001% dry matter of CAP extract.

[0127] Gene expression of barrier function and water retention markers was assessed by qRT-PCR.

[0128] The results were statistically analyzed by one-way ANOVA followed by Dunnett's test.

[0129] c.Results Chlamydomonas acidophila extract stimulated gene expression of GBA (β-glucocerebrosidase) and HAS3 (hyaluronan synthase-3), markers involved in the synthesis of epidermal lipids and hyaluronic acid, respectively. These results confirm the trends obtained with the genomic expression screen, supporting an effect on enhancing the epidermal permeability barrier and water retention.

[0130] Furthermore, Chlamydomonas acidophila extract also stimulated the expression of markers SLC6A6 and SLC5A3, which encode TAUT (taurine membrane transporter channel) and SMIT (myo-inositol transporter channel), respectively.

[0131] These two genes code for osmolyte transporters and are therefore involved in maintaining skin hydration and protecting cells from external stress.

[0132] Finally, the extract induced an increase in the gene expression of filaggrin (FLG) and PADI1 (peptidylarginine deiminase), proteins and enzymes involved in the synthesis of natural moisturizing factor (NMF) components.

[0133] [Table 10]

[0134] d. Conclusion These results indicate that extracts of Chlamydomonas acidophila have the ability to enhance the skin barrier and maintain skin hydration.

[0135] V. Evaluation of the Effects of Chlamydomonas acidophila Extract on Allergic Mechanisms A. Introduction The potential anti-allergic effect of Chlamydomonas acidophila extract was Proinflammatory chemokine gene expression in normal human epidermal keratinocytes (NHEKs) stimulated with a mixture of Th2 cytokines (IL-4+IL-13+IL-22+TNF-α) mimicking a late-stage "atopic dermatitis" phenotype (chronic inflammation) Activation of human basophils induced by fMLP. This activation was measured by quantification of a specific marker of activated basophil cells (CD63) in the total population of basophils identified by expression of the CCR3 marker using a specific kit and flow cytometry analysis. In parallel, activation with anti-FCεRI antibody was performed as a positive control.

[0136] B. Inhibition of chemoattractants following TH2 stress A. Materials and Methods Normal human epidermal keratinocytes were preincubated for 24 h in the presence of 0.01% dry matter (DM) CAP extract or 10 μM reference JAK inhibitor I. After preincubation, cells were re-treated with CAP extract or reference product, and then cells were stimulated with Th2 cytokine cocktail (10 ng / ml IL4+IL13+IL22+TNFα) for 24 h.

[0137] At the end of the incubation, gene expression of markers of interest was assessed by qRT-PCR.

[0138] b.Results In keratinocytes exposed to Th2 stress, Chlamydomonas acidophila extract inhibited gene expression of CCL5 (C-C motif chemokine ligand 5 or RANTES) and CCL27 (C-C motif chemokine ligand 27), which encode chemokines involved in amplifying inflammatory and allergic responses in the skin.

[0139] [Table 11]

[0140] c. Conclusion Chlamydomonas acidophila extract modulates Th2 stress-induced inflammation in keratinocytes by inhibiting gene expression of the chemoattractants CCL5 and CCL27.

[0141] C. Inhibition of basophil activation A. Materials and Methods The basophil activation test (BAT) was performed using a Flow CAST® kit (BUHLMANN, item code FKCCR).

[0142] Whole blood was preincubated for 15 min in the presence of 0.033% and 0.1% dry matter (DM) CAP extract or reference products (30 μM SB202190; or 10 mM cromoglycate).

[0143] The stimulator 1 μM fMLP was then added and the blood was incubated for a further 15 min in the presence of labelling buffer containing a mixture of monoclonal antibodies (anti-CD63-FITC and anti-CCR3-PE).

[0144] Flow cytometry analysis was then performed to enumerate the total population of basophils (CCR3+) and activated basophils (CCR3+ / CD63+).

[0145] b.Results Stimulation with fMLP peptide resulted in a very clear activation of basophils (40.1% activated cells, or 4527% stimulation).

[0146] In this study, two potential control compounds were tested in the presence of fMLP: SB202190, an inhibitor of p38 MAP kinase; activation of this kinase is essential in the activation mechanism of basophils that leads to degranulation; Cromolyn, a known antiallergic agent whose mechanism of action involves stabilization of the plasma membrane, ++ It inhibits the intracellular penetration of , and this ion is essential for mast cell degranulation.

[0147] Both SB202190, tested at 30 μM, and cromoglycate, tested at 10 mM, showed significant inhibitory effects on fMLP-induced basophil activation (26% and 46% inhibition, respectively).

[0148] Under the experimental conditions of this study, the CAP extract tested at 0.033% and 0.1% showed a clear concentration-dependent inhibitory effect on fMLP-induced basophil activation (22% and 39% inhibition, respectively).

[0149] [Table 12]

[0150] c. Conclusion Chlamydomonas acidophila extract inhibits basophil activation.

[0151] D. Conclusion By inhibiting, on the one hand, the gene expression of chemotactic cytokines in keratinocytes exposed to Th2 stress and, on the other hand, the activation of basophils, Chlamydomonas acidophila extract may contribute to the regulation of processes involved in the induction of allergic responses.

[0152] Example 3: Clinical trials of the extract according to the invention The biological activity of the Chlamydomonas acidophila (CAP) extract obtained in Example 1 was demonstrated by clinical trials as described below.

[0153] All these results show a significant effect of the "CAP" activator: Protective effect; Barrier effect; Moisturizing effect; and Anti-inflammatory / anti-redness effects.

[0154] These clinical trials are Sensitive skin; Redness; Inflammation; and allergy They highlighted the ability of CAP extract for the prevention or treatment of

[0155] A. Summary of clinical findings Chemical erythema test CAP activator (3% active ingredient) showed significant efficacy against the following parameters: Maximum erythema intensity 20 minutes after application of 0.1% methyl nicotinate solution

[0156] Blood flow intensity as measured by TiVi is significantly lower in areas treated with the active agent compared to untreated areas.

[0157] Redness, as measured by spectrocolorimetry, is significantly less in areas treated with the active agent compared to untreated areas.

[0158] Change in erythema 30 minutes after maximum intensity erythema The reduction in blood flow intensity as measured by TiVi is significantly greater in areas treated with the active agent compared to untreated areas.

[0159] The reduction in redness, as measured by spectrocolorimetry, is significantly greater in areas treated with the active agent compared to untreated areas.

[0160] Sensitive Skin Test CAP activator (3% active ingredient) showed significant efficacy against the following parameters: equipment measurement

[0161] The activator is: Transepidermal water loss was significantly reduced 28 days after application. Redness was significantly reduced 28 days after application. Water retention was significantly increased 28 days after application.

[0162] Biochemical evaluation The activator is The amount of NMF was significantly increased 28 days after application. After 28 days of application, it significantly compensates for the decrease in ceramides seen in the placebo area. The amount of IL1RA was significantly reduced 28 days after application. 28 days after application, the amount of IL1α was significantly reduced. The IL1RA / IL1α ratio was significantly reduced 28 days after application. IL8 levels were significantly reduced 28 days after application.

[0163] B. Evaluation of protective / anti-redness / anti-inflammatory efficacy of active versus placebo by assessment of blood flow with TiVi and color by spectrocolorimetry. Study design: Double-blind study. Randomized, active agent versus placebo controlled trial.

[0164] Group : Nineteen subjects were analyzed in this study. The 19 subjects received active agent and placebo in two defined areas. An untreated area was also defined.

[0165] The individuals recruited for this study were: Healthy women aged 18-60 years, mean age 38±3 years, minimum 18 years, maximum 60 years Caucasian skin with phototype I to III was used.

[0166] How to use and administer the test Subjects self-apply the product to each defined area of ​​the forearm twice daily (morning and evening) at home for 14 days, D-14 through D0t0. After 14 days, subjects return to the clinical site. A baseline D0t0 measurement is then taken. A final application of the product is made. Each area is then induced to undergo chemical erythema with a 0.1% methyl nicotinate solution. Measurements of each area are then taken 20 minutes (maximum intensity) and 50 minutes (30 minutes after maximum erythema) after erythema induction, designated D0t20, and D0t50, respectively.

[0167] For each parameter evaluated, two parameters are analyzed: Variation from D0t20 to D0t0 reflecting the preventive effect of each product on the appearance of erythema. The variation between D0t50 and D0t20 reflects the preventive effect of each product on changes in erythema.

[0168] Assessment of blood flow with TIVI The method used by the TiVi 700 is based on the fact that green light is strongly absorbed by blood vessel cells, while red light is absorbed less. Using a polarized source, the method does not take into account specular reflectance alone, but rather the light reflected by skin tissue. The device creates an intensity map where each pixel represents the density of blood cells in the skin.

[0169] → A significant decrease in the density of blood cells in the skin indicates an anti-inflammatory effect.

[0170] Results: Preventive effect on the appearance of erythema The graphs in Figures 1A, 1B, and 1C represent pairwise comparisons between active, placebo, and untreated areas for erythema intensity 20 minutes after methyl nicotinate application as measured by TiVi. The parameter on the abscissa represents the blood flow intensity (red blood cell density). The exact values ​​are given in Tables 13A and 13B below (Tables 13A and B: Mean and standard deviation of the measurements. % of subjects showing a positive effect. % difference and p-value (exact value of significance).

[0171] The graph in FIG. 2 shows the progression of changes in blood flow values.

[0172] → The results show that the active agent significantly reduces the intensity of the erythema formed compared to the untreated areas. The intensity of the erythema for the placebo areas was not different from the treated areas. The intensity of the erythema for the active areas was significantly lower than for the placebo.

[0173] [Table 13]

[0174] [Table 14]

[0175] C. Efficacy of active agent versus placebo using transepidermal water loss assessed by Tevameter, hydration assessed by corneometry, and biochemical characterization of swabs Study design: Double-blind study Active agent vs. placebo, randomized, half-face comparative study

[0176] Group : Thirty-six subjects were included in the analysis for measurements of hydration, transepidermal water loss, color and questionnaires.

[0177] Regarding biochemical analysis, 10 subjects were included in the analysis.

[0178] The subjects enrolled in this study were as follows: Healthy women aged 18 years or older, mean age 51±3 years, minimum 21 years, maximum 68 years Caucasian skin type I-IV Have sensitive facial skin (subject's skin must be reactive to at least two of the following three stresses: environmental, chemical or mechanical)

[0179] Usage and testing methods : D0: · Checking inclusion and exclusion criteria Acclimatization for 30 minutes -Define the measurement area for each half of the face Instrumental measurements and biological samples D0~D28: - Apply active agent and placebo to half of the face twice daily (morning and evening) D28: Acclimatization for 30 minutes Instrumental measurements and biological samples

[0180] Transepidermal Water Loss Assessment The skin barrier regulates water loss through evaporation. When this barrier is impaired, transepidermal water loss increases. Conversely, a strengthened barrier corresponds to a decrease in transepidermal water loss.

[0181] Transepidermal water loss was measured by a Tevameter™ 300. The principle is to apply one of its openings to the skin and measure the temperature and relative humidity inside the tube with two sensors placed at two different heights. Fick's law is then used to determine the transepidermal water loss.

[0182] The results obtained for the TEWL measurements at D0 and D28 are shown in Table 14. A graph of the changes is shown in Figure 3.

[0183] [Table 15]

[0184] The results obtained show that TEWL decreases significantly between D0 and D28 in the active agent and in the placebo. The comparison of the changes between D0 and D28 observed in the active agent with those obtained in the placebo is statistically significant in favor of the active agent.

[0185] Water Retention Rating The measurement of skin hydration was carried out with a CM 825 Corneometer. This device is based on the capacitance measurement principle, which allows the measurement of hydration in the superficial layers of the skin (depth 10-20 μM).

[0186] The results obtained for water retention at D0 and D28 are given in Table 15. A graph of the changes is shown in FIG.

[0187] The results obtained show that there is a significant increase in hydration between D0 and D28 with the active agent, but not with the placebo. The comparison of the changes between D0 and D28 observed with the active agent with those observed with the placebo is statistically significant in favor of the active agent.

[0188] [Table 16]

[0189] Biochemical evaluation The following biochemical assessments were performed: From the swab sample: Natural Moisturizing Factors (NMF) by liquid chromatography coupled with UV detection (LC / UV). The amount of NMF includes urocanic acid (UCA), pyrrolidone carboxylic acid (PCA) and serine. The NMF content provides information about the hydration status of the skin. · Ceramides by liquid chromatography coupled with mass spectrometry detection (LC / MS). The amount of ceramide includes ceramides with [S], [DS] and [P] bases. The ceramide content provides information about the condition of the skin barrier. - Inflammatory state by cytokine quantification (by ELISA) IL1RA IL1α IL8 From the D-Squames sample: Nile red / involucrin staining

[0190] NMF The results obtained for NMF on D0 and D28 are shown in Table 16. A graph of the changes is shown in FIG.

[0191] The results obtained show that the active agent significantly increases the amount of NMF between D0 and D28, whereas the placebo significantly reduces this amount. The comparison of the changes between D0 and D28 observed with the active agent with those observed with the placebo is statistically significant in favor of the active agent.

[0192] [Table 17]

[0193] Ceramide The results obtained for ceramide at D0 and D28 are shown in Table 17. A graph of the changes is shown in FIG.

[0194] The results obtained show that the amount of ceramide decreases when using placebo and that the active agent compensates for this decrease (the changes between D0 and D28 for placebo and active agent are not significant). The changes between D0 and D28 seen when using the active agent are compared with those seen when using placebo and are statistically significant in favor of the active agent.

[0195] [Table 18]

[0196] IL1RA The results obtained for IL1RA at D0 and D28 are shown in Table 18. A graph of the changes is shown in FIG.

[0197] The results obtained show that the amount of IL1RA decreases significantly between D0 and D28 with the active agent and with placebo. The changes between D0 and D28 observed with the active agent compared with those observed with placebo are statistically significant in favor of the active agent.

[0198] [Table 19]

[0199] Nile Red / Involucrin Ratio The results obtained for the Nile Red / Involucrin ratio at D0 and D28 are shown in Table 19. A graph of the changes is shown in FIG.

[0200] The results obtained show that the Nile Red / Involucrin ratio does not change significantly between D0 and D28 with the active agent and with the placebo. The changes between D0 and D28 seen with the active agent compared with those seen with the placebo are statistically significant in favor of the active agent.

[0201] [Table 20]

Claims

1. A peptide extract of the microalga Chlamydomonas acidophila obtained by a preparation process comprising at least one enzymatic hydrolysis step comprising at least 20% by weight of peptides, as a percentage expressed relative to the total weight of the extract.

2. 2. The extract according to claim 1, comprising, as a percentage expressed relative to the total weight of the extract, between 20% and 90% by weight of peptides, advantageously between 20% and 75% by weight, generally between 30% and 70% by weight.

3. 3. The extract according to claim 1 or 2, wherein the peptides have a molecular weight of less than 3,500 Daltons (Da).

4. The extract according to any one of claims 1 to 3, wherein at least 80% by weight of the peptides have a molecular weight of less than 1000 Da.

5. The extract according to any one of claims 1 to 4, wherein at least 30% by weight, advantageously at least 35% by weight, of the peptides have a molecular weight of less than 500 Da.

6. A method for preparing a peptide extract of Chlamydomonas acidophila comprising at least one enzymatic hydrolysis step.

7. 7. The process according to claim 6, wherein the enzymatic hydrolysis is carried out in the presence of at least one protease, advantageously at least one alkaline protease.

8. At least the following steps: a) an aqueous phase dispersion of the microalga Chlamydomonas acidophila; b) enzymatic hydrolysis of the aqueous phase dispersion obtained in step a), preferably in the presence of at least one protease; c) heat treatment of the mixture obtained in step b), and d) Recovery of the peptide extract at the end of step c) The method according to claim 6 or 7, comprising:

9. 9. The method according to any one of claims 6 to 8, comprising an additional filtration or centrifugation step located between steps c) and d), optionally followed by ultrafiltration, diafiltration or nanofiltration.

10. The method according to any one of claims 6 to 9, further comprising a step of 15 kDa ultrafiltration carried out between steps c) and d) or after an additional filtration or centrifugation step.

11. 11. The method of claim 10, further comprising a nanofiltration step having a cutoff of 100 to 300 Daltons, performed after the 15 kDa ultrafiltration step.

12. A composition comprising as active ingredient a peptide extract of the microalga Chlamydomonas acidophila obtained by a preparation process comprising at least one enzymatic hydrolysis step and suitable excipients.

13. The composition according to claim 12, wherein said extract is defined as described in any one of claims 1 to 5 or is obtainable by the method according to any one of claims 6 to 11.

14. 14. The composition according to claim 12 or 13, comprising from 0.001% to 10% by weight, advantageously from 0.01% to 5% by weight, of said peptide extract of Chlamydomonas acidophila, expressed as dry extract based on the total weight of the composition.

15. Disorders or conditions of the skin and / or mucous membranes and / or skin appendages, preferably allergic, inflammatory, irritant reactions or conditions or disorders of the barrier or homeostasis of the skin, immature, normal or mature / aged skin appendages and / or mucous membranes, and / or Vascular disorders, in particular redness or cuperosis, and / or Degeneration of adipose tissue 15. The extract according to any one of claims 1 to 5 or the extract obtainable by the method according to any one of claims 6 to 11 or the composition according to any one of claims 12 to 14 for use in the prevention and / or treatment of

16. An extract according to any one of claims 1 to 5 or an extract obtainable by a method according to any one of claims 6 to 11 or a composition according to any one of claims 12 to 14 for use in improving the condition and / or appearance of the skin and / or the skin appendages and / or mucous membranes.

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