Cosmetic and / or pharmaceutical composition comprising synthetic or plant extracts for increasing the expression of mitochondrial derived peptides

Abstract

The invention relates to a cosmetic or pharmaceutical composition comprising a physiologically acceptable medium and an active principle capable to increase the expression of mitochondrial derived peptide, and more particularly humanin, wherein the active principle can be a peptide, a plant extract such as an aqueous extract of date (Phoenix dactylifera) fruit pulp, an aqueous extract of germinated red clover (Trifolium pratense) seeds, an aqueous extract of quinoa (Chenopodium quinoa) seeds, or an aqueous extract of Iris pallida roots. The invention also concerns a method of cosmetic treatment to protect the skin and the appendages from external aggressions and to combat cutaneous aging.

Description

DescriptionCOSMETIC AND / OR PHARMACEUTICAL COMPOSITION COMPRISING SYNTHETIC OR PLANT EXTRACTS FOR INCREASING THE EXPRESSION OF MITOCHONDRIAL DERIVED PEPTIDESTechnical field[1] The invention belongs to the field of cosmetics and pharmaceutical domain, and more particularly to plant extracts and peptides able to increase mitochondrial derived peptide expression and their uses in cosmetic applications for mitochondrial biogenesis and skin longevity.Technical background of the invention[2] Mitochondria have been widely studied in the field of aging because of their roles in cellular energy production, calcium homeostasis, apoptosis, and cell signaling. Mitochondria possess DNA repair mechanisms less efficient than those found in the nucleus. Their inefficient DNA repair mechanism combined with the proximity with the electron transport chain generate a large amount of ROS, promotes a high number of mtDNA mutations over a lifetime.[3] Small open reading frames (ORF) in circular mt DNA can encode multiple microproteins, called mitochondria-derived peptides (MDPs). Some of these peptides act intracellularly, while others are found in the systemic circulation and target various tissues. Currently, 8 MDPs have been identified: humanin, MOTS- c, and SHLPs (small humanin-like peptides) 1-6 (Miller et al. Mitochondria-derived peptides in aging and healthspan. J Clin Invest, 2022, 132(9)).[4] Humanin (HN) is a 24 amino acid peptide encoded in the mitochondrial 16S rRNA region by the MT-RNR2 gene.[5] HN mediates mitochondrial status and cell survival by acting via an intracellular mechanism, or as a secreted factor via extracellular signals. Intracellularly, it binds Bcl2-associated X protein (BAX), Bim and tBid, and IGFBP3 to inhibit caspase activity and cell apoptosis.[6] Circulating humanin levels decreased with age in both mice, monkey and human plasma. Intriguingly, not in the long-lived naked mole rat, which is a model of negligible senescence and healthy aging. Human offspring of centenarians, who have a greater chance of living to be 100 years old, even displayed elevated levels of circulating humanin compared with age-matched controls without family history of exceptional longevity. Using a C. elegans model of HN overexpression, HN was reported to increase lifespan via the regulation of Foxo (Yen et al. The mitochondrial derived peptide humanin is a regulator of lifespan and healthspan. Aging 2020, Vol 12, N°12).[7] HN has senolytic activity since it has been shown to exacerbates the Senescence Associated Secretory Phenotype (SASP) in senescent cells by stimulating the secretion of IL-6, IL-1 (3, IL-8, IL-10 and tumor necrosis factor a. Humanin protects cells from oxidative stress, serum starvation, hypoxia, and other insults in vitro (Mendelsohn and Larrick. Mitochondrial-Derived Peptides Exacerbate Senescence. Rejuvenation Res, Aug;21 (4):369-373. 2018)[8] MOTS-c is a 16 amino acid mitochondrial derived peptide encoded from the 12S rRNA region of the mitochondrial genome. Under stress conditions, MOTS-c translocates to the nucleus where it regulates a wide range of genes in response to metabolic dysfunction. It is colocalized to mitochondria in various issues and is found in plasma, but levels decline with age. Since MOTS-c has important cellular functions as well as a possible hormonal role, it has been shown to have beneficial effects on age-related diseases and it may contribute to longevity by promoting cellular homeostasis.[9] Small humanin-like peptides 1 to 6 (SHLPs 1-6), are encoded from the 16S rRNA region and share some biological features with humanin. For example, SHLP2 protects cells from amyloid [3-induced toxicity and age-related macular degeneration and administration of SHLP2 and SHLP3 promotes mitochondrial biogenesis, reduces ROS, and decreases mtDNA oxidation. SHLP2 could also affect lifespan and health span similarly to other MDPs.

[0010] The presence of mitochondrial derived peptides including humanin was identified in primary dermal fibroblasts (Kim et al. Mitochondrial peptides modulate mitochondrial function during cellular senescence, Aging, 2018, Vol.10, No. 6)

[0011] Compounds stimulating mitochondrial derived peptides with associated activity on mitochondrial metabolism and cellular energy level are essential to improve skin longevity, and to prevent or to treat signs of cutaneous aging. They represent an important concern of medical research and cosmetics by their capacity to mitigate the skin and hair damage caused by external aggressions, such as UV rays, radiation, or exposure to toxins or to pollutants. To the knowledge of the inventors, no cosmetic including plant extracts or peptides capable of activating mitochondrial derived peptides has yet been described.Brief description of the drawings

[0012] The invention and its advantages will be better understood from the following description and non-limiting embodiments, illustrated with reference to the attached figures, in which:

[0013] [Fig. 1] Quantification of humanin expression in normal human skin biopsies treated with 5 mg / Kg of peptide SEQ ID No. 1 and SEQ ID No. 2 by immunofluorescence detection.

[0014] [Fig. 2] Quantification of collagen I expression in normal human skin biopsies treated with 5 mg / Kg of SEQ ID No. 1 and SEQ ID No. 2 by immunofluorescence detection.

[0015] [Fig. 3] Quantification of humanin expression in normal human skin biopsies treated with 1 % Phoenix dactylifera aqueous extract by immunofluorescence detection.

[0016] [Fig. 4] Quantification of humanin expression in human skin biopsies treated with 1 % Trifolium pratense aqueous extract by immunofluorescence detection.

[0017] [Fig. 5] Quantification of humanin expression in human skin biopsies treated with 1 % Chenopodium quinoa aqueous extract by immunofluorescence detection.

[0018] [Fig. 6] Quantification of humanin expression in human skin biopsies treated with 1 % Iris pallida aqueous extract by immunofluorescence detection.Summary of the invention

[0019] In one embodiment the invention relates to a peptide capable to increase mitochondrial derived peptide expression, of the following general formula (I):R1 -(AA)n- Cys-Leu-Leu-Leu-Ser-Thr-X1 -(AA)p-R2 whereinAA represents any amino acid, and n and p are whole numbers between 0 and 2,X1 represents a Glycine or no amino acid,R1 represents the primary amine function of the N-terminal amino acid, free or substituted with a protector group which may be chosen from an acetyl group, a benzoyl group, a tosyl group, or a benzyloxycarbonyl group. R1 can also represent a palmitoylation and more particularly S-palmitoylation,R2 represents the hydroxyl function of the carboxylic acid of the C-terminal amino acid, free or substituted with a protector group which can be chosen from an alkyl chain of C1 to C20, or an NH2, NHY, or NYY group, with Y representing an alkyl chain of C1 to C4.

[0020] In another embodiment, the invention relates to a cosmetic or pharmaceutical composition comprising in a physiologically acceptable medium an effective amount of an active principle able to increase mitochondrial derived peptide expression, selected among: the peptide of SEQ ID No 1 , the peptide of SEQ ID No 2, an aqueous extract of date (Phoenix dactylifera) fruit pulp, an aqueous extract of germinated red clover (Trifolium pratense) seeds, an aqueous extract of quinoa (Chenopodium quinoa) seeds, and an aqueous extract of Iris pallida roots, or any mixture thereof, or any mixture thereof.

[0021] In another embodiment the invention relates to a method of cosmetic treatment intended to protect the skin and the appendages from external aggressions and to combat cutaneous aging, comprising applying to the skin a composition according to the invention.Detailed description of the inventionDefinitions

[0022] All terms used in this description have the most widely known meaning unless otherwise stated. For the purposes of the invention the following terms are defined as follows:

[0023] When a range of values is described, the limits of that range should be understood as explicitly including all intermediate values in the range. For example, a range of values between 1 % and 10% should be understood to include 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, and also all decimal values between 1 % and 10%.

[0024] Numerical percentage values are percentages by weight, i.e. , the weight of a compound in relation to the total weight of the intended mixture, unless otherwise specified.

[0025] The compositions described in the present application may "comprise", "consist of' or "consist substantially of" the essential compounds or optional ingredients.

[0026] "Consist substantially of" means that the composition or component may include additional ingredients, but only if the additional ingredients do not alter the basic or novel characteristics of the composition or use described in the present application.

[0027] A "physiologically acceptable medium" means a vehicle that is suitable for coming into contact with the outer layers of the skin or mucous membranes, without toxicity, irritation, undue allergic response and the like or intolerance reaction, and proportionate to a reasonable benefit / risk ratio.

[0028] "Topical application" means applying or spreading peptides or plant extract obtained from plants or fungi families of interest, according to the invention, or a composition containing it, on the surface of the skin or a mucous membrane.

[0029] "Skin" refers to the skin including skin appendages (hair and body hair, eyebrow, eyelashes, nails).

[0030] It is understood that the invention relates to mammals and more particularly to human beings.

[0031] The inventors have identified several peptides and plant extracts comprising those peptides as capable of modulating mitochondrial derived peptides. Theyparticularly evidenced that some plant extracts or peptides, when applied to the skin, where able to stimulate the mitochondrial functions in a significant way. This has been demonstrated by an increase in the expression of mitochondrial derived peptides, including humanin, associated with skin benefits such as an increase in the expression of collagen I.

[0032] “Active principle” as used herein means any peptides or plant extract capable of activating mitochondrial derived peptides when applied to skin of a subject in needs.

[0033] “combat cutaneous or skin Aging” include, but are not limited to, one or more of: (a) treatment, reduction, and / or prevention of fine lines or wrinkles, (b) improvement in skin thickness, plumpness, and / or tautness; (c) improvement in skin suppleness and / or softness; (d) improvement in procollagen and / or collagen production; (e) improvement in skin texture and / or promotion of densification; (f) improvement in skin barrier repair and / or function; (g) treatment and / or prevention of skin sagging or atrophy; (h) improvement in appearance of skin contours; (i) restoration of skin luster and / or brightness; (j) improvement of skin appearance decreased by menopause; (k) improvement in skin moisturization and / or hydration; and (I) improvement of skin elasticity and / or resiliency.

[0034] An "active principle capable of activating mitochondrial derived peptide" is understood to be any substance capable of increasing the expression of mitochondrial derived peptides, either by activating proteinic synthesis (by means of direct or indirect modulation of the gene expression), or by increasing the biological activity of mitochondrial derived peptides, or by other biological processes such as the stabilization of the mitochondrial derived peptides or even the stabilization of the messenger RNA transcripts.

[0035] Preferentially, the substances capable of activating mitochondrial derived peptides according to the invention are plant extracts or pure peptides.

[0036] The present disclosure encompasses peptides that were designed by the inventors through the selection of short amino acid sequences of humanin, including sequences involved in the neuroprotection activity of humanin and in the binding to protein Bax and IGFBP-3. Several peptide sequences were designedas potentially acting on humanin mitochondrial derived peptides but only peptides showing homology with botanical sequences were selected.

[0037] Thus, the first object of the invention is a peptide capable of activating mitochondrial derived peptides, of general formula (I)R1 -(AA)n- Cys-Leu-Leu-Leu-Ser-Thr-X1 -(AA)p-R2 whereinAA represents any amino acid, and n and p are whole numbers between 0 and 2.

[0038] In a particular embodiment,X1 a represents glycine or no amino acid,

[0039] R1 represents the primary amine function of the N-terminal amino acid, free or substituted with a protector group which may be chosen from an acetyl group, a benzoyl group, a tosyl group, or a benzyloxycarbonyl group. R1 can also represent a palmitoylation and more particularly S-palmitoylation.

[0040] R2 represents the hydroxyl function of the carboxylic acid of the C-terminal amino acid, free or substituted with a protector group which can be chosen from an alkyl chain of C1 to C20, or an NH2, NHY, or NYY group, with Y representing an alkyl chain of C1 to C4.

[0041] According to a particular embodiment of the invention, the biologically active peptide consists of the sequence:(SEQ ID No. 1 ) Ser-Cys-Leu-Leu-Leu-Ser-Thr,

[0042] According to another particular embodiment of the invention, the biologically active peptide consists of the sequence:

[0043] (SEQ ID No. 2) Ser-Cys-Leu-Leu-Leu-Ser-Thr-Gly

[0044] The invention also concerns homologous forms of these sequences. The term "homologous" designates, according to the invention, any peptide sequence identical to at least 50%, or preferably to at least 80%, and even more preferentially to at least 90% of said peptide sequence, chosen from among the sequences SEQ ID No. 1 to SEQ ID No. 2. A "peptide sequence identical to at least X%" is understood to designate an identity percentage between the residues of amino acids of the two sequences to be compared, obtained after the optimalalignment of the two sequences. The optimal alignment is obtained using algorithms for local homologies such as those used by the computer software BLAST P or T BLAST N, available on the National Center for Biotechnology Information (NCBI) website.

[0045] The term "homologous" can also designate a peptide that differs from the sequence of a peptide with sequence SEQ ID No. 1 to SEQ ID No. 2 by the substitution of chemically equivalent amino acids, that is, by the substitution of one residue by another possessing the same characteristics. Thus, the classic substitutions are made between Ala, Vai, Leu, and lie; between Ser and Thr; between the acid residues of Asp and Glu; between Asn and Gin; and between the basic residues of Lys and Arg, or between the aromatic residues of Phe and Tyr.

[0046] The term "peptide" designates a chain of two or several amino acids linked to one another by peptide bonds or by modified peptide bonds.

[0047] "Peptide" must be understood as a synthetic peptide or as at least one fragment of protein, which was either obtained by proteolysis from of larger proteins or polypeptides.

[0048] The peptide with general formula (I) according to the invention can be obtained either by classical chemical synthesis (in solid phase or homogeneous liquid phase) or by enzymatic synthesis (Kullman et al. (1980), J. Biol. Chem., 225, 8234), starting from the constituent amino acids or from their derivatives.

[0049] The peptide with general formula (I) can also be obtained by fermentation of a strain of bacteria, modified or not by genetic engineering, or even by extraction of proteins of animal or vegetable origin, preferentially of vegetable origin, followed by a controlled hydrolysis which releases the peptide fragments which correspond in whole or in part to the peptides with general formula (I).

[0050] Other procedures, simpler or more complex, may be envisioned by a person skilled in the field of synthesis, extraction, and purification of proteins and peptides. Thus, the peptide according to the invention can be of natural or synthetic origin. Preferentially according to the invention, the peptide of general Formula (I) is obtained by chemical synthesis.

[0051] Many proteins found in plants are likely to contain peptide of general formula (I) within their structure. Controlled hydrolysis enables these peptide fragments to be released. It is possible, but not necessary to achieve the invention, either to extract the proteins of interest first and then to hydrolyze them, or to perform the hydrolysis first on a raw extract and to subsequently purify the peptide fragments. It is also possible to use certain hydrolyzed extracts without purifying the peptide fragments which correspond to the general formula (I) according to the invention, but at the same time ensuring the presence of the said fragments by appropriate analytical means.

[0052] In a second step, the inventors selected plants comprising sequence homology with the peptides of general formula (I), in order to provide plant extracts having the same biological activity than the peptides of general formula (I).

[0053] Sequence similarity searches to identify homologous sequences were performed using comprehensive protein sequence databases like BLAST following standard procedures (William Pearson, “An Introduction to Sequence Similarity (“Homology”) searching”, Curr Protoc Bioinformatics. June 2013).

[0054] This search allowed the inventors to identify several candidates in the following taxonomic groups: o Arecaceae family, Phoenix genus and more particularly Phoenix dactylifera species. o Fabaceae family, Trifolium genus and more particularly Trifolium pratense species. o Iridaceae family, Iris genus and more particularly Iris pallida species. o Amaranthaceae family, Chenopodium genus and more particularly Chenopodium quinoa species. o Anacardiaceae family, Pistacia genus and more particularly Pistacia vera and Pistacia atlantica species. o Sapindaceae family, Xanthoceras genus and more particularly Xanthoceras sorbifolia species. o Teratosphaeriaceae family, Friedmanniomyces genus, phylum Ascomycota and more particularly endolithic fungal species such as Friedmanniomyces endolithicus o Oceanospirillaceae family, Marinomonas genus and more particularly marinomonas species such as marinomonas mediterranea.

[0055] A series of peptides, different from those having a general formula (I), were identified as potentially acting on humanin. However, since these peptides did not show homology to botanical sequences, they were not selected.

[0056] In a particular embodiment the invention relates to an aqueous extract of date (Phoenix dactylifera) fruit pulp, obtained by crushing the pulp in water and submitting the plant material to microwave, having a dry matter content comprised between 20 and 50 g / kg.

[0057] In another particular embodiment the invention relates to an aqueous extract of germinated red clover (Trifolium pratense) seeds having a dry matter content comprised between 15 and 30 g / kg and obtained using a method described in patent US11 ,021 ,505 B2 (incorporated herein by reference).

[0058] In another particular embodiment the invention relates to an aqueous extract of quinoa (Chenopodium quinoa) seeds obtained by crushing the seeds in water and submitting the plant material to microwave, having a dry matter content comprised between 4 and 10 g / kg.

[0059] In another particular embodiment the invention relates to an aqueous extract of Iris pallida roots having a dry matter content comprised between 8 and 14 g / kg and obtained using a method described in patent US11 ,021 ,505 B2 (incorporated herein by reference).

[0060] According to the invention, the active principle can be a pure peptide, a mixture of several peptides or at least one aqueous plant extract comprising peptides. The effective amount of active principle corresponds to the minimal quantity necessary to obtain the desired result, that is to say, to increase the expression of the mitochondrial derived peptide humanin in skin cells.

[0061] A second object of the present disclosure is a cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, as an active principle, an effective amount of at least one peptide or one aqueous extract described above or any mixture thereof, capable of activating mitochondrial derived peptides.

[0062] In an advantageous embodiment the composition is a cosmetic composition.

[0063] In a particular embodiment, the cosmetic composition comprises, in a physiologically acceptable medium, an effective amount of an active principle able to increase mitochondrial derived peptide expression selected among: the peptide of SEQ ID No 1 , the peptide of SEQ ID No 2, an aqueous extract of date (Phoenix dactylifera) fruit pulp, an aqueous extract of germinated red clover (Trifolium pratense) seeds, an aqueous extract of quinoa (Chenopodium quinoa) seeds, and an aqueous extract of Iris pallida roots, or any mixture thereof.

[0064] Preferentially, the mitochondrial derived peptide is humanin.

[0065] According to an advantageous embodiment, when the active principle is a peptide of SEQ ID No 1 or SEQ ID No 1 , it is in a concentration comprised between approximately 0.0005 and 500 mg / kg (parts per million), and preferentially in a concentration between approximately 0.01 and 10 mg / kg, relative to the total weight of the final composition.

[0066] According to another embodiment, when the active principle is an aqueous extract, it is in a concentration comprised between approximately 0.01 % and 5 % preferentially in a concentration between approximately 0.1 % and 2 %, relative to the total weight of the final composition.

[0067] According to another embodiment, the active principle according to the invention is diluted in advance in one or several physiologically acceptable solvents, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, Vaseline, a vegetable oil, or any mixture of these solvents.

[0068] According to a particular embodiment, when the active principle is an aqueous extract of Iris pallida roots it can be diluted in 20% propanediol (propane-1 ,3-diol) and 10% of glycerine.

[0069] It is of course understood that the active principle according to the invention can be used alone or in association with at least one other active principle, in a cosmetic composition or for the preparation of a pharmaceutical and / or dermatological composition.

[0070] The composition according to the invention can be applied in any appropriate way, particularly oral, parenteral, or externally topical, and their formulation will beadapted by the professional, in particular for cosmetic or dermatological compositions.

[0071] Advantageously, the composition according to the invention is in a form adapted to application by topical means.

[0072] The composition usable according to the invention may be in particular, in the form of creams, oil-in-water emulsions, or water-in-oil or multiple emulsions, solutions, suspensions, gels, milks, lotions, sticks, or even powders, adapted to an application onto the skin, the lips, and / or the appendages.

[0073] The composition may include the excipients necessary for their formulation, such as solvents, thickeners, diluents, surfactants, antioxidants, colorants, preservatives, or perfumes.

[0074] Advantageously, the usable composition contains at least one other active agent promoting the action of the peptides according to the invention. Thus, the composition according to the invention may associate, with the active principle according to the invention, active agents having an antioxidant action, or else stimulating the synthesis of dermal macromolecules, or else stimulating energy metabolism. For example, as active agents having an anti-radical or antioxidant action, vitamin C, vitamin E, coenzyme Q10, and the polyphenolic extracts of plants may be cited.

[0075] The composition could be particularly in the form of an aqueous, hydroalcoholic, or oily solution, an oil-in-water or water-in-oil emulsion, or multiple emulsions. They can also be in the form of creams, suspensions, or even powders, adapted to application onto the skin, the mucous membranes, the lips, and / or the appendages. These compositions can be more or less fluid and have the appearance of a cream, a lotion, a milk, a butter, an ointment, a gel, a paste, or a mousse. They can also be in solid form like a stick or be applied on the skin in the form of an aerosol. They can be used as a care product and / or as a makeup product for the skin.

[0076] The composition may include, in addition, any additive commonly used in the application domain envisioned, as well as the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, colorants, solar filters, self-bronzing agents, pigments, vehicles, preservatives, perfumes, odorabsorbents, active cosmetic or pharmaceutical agents, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

[0077] In any case, the professional will take care that these adjuvants, as well as their proportions, are chosen in such a way as not to harm the advantageous properties studied in the composition according to the invention. These adjuvants can, for instance, correspond to 0.01 to 20% of the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase may represent 5 to 80% by weight and preferably 5 to 50% by weight relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from among those traditionally used in the domain considered. For example, they may be used in a proportion from 0.3 to 30% by weight relative to the total weight of the composition.

[0078] In another embodiment, the invention relates to a method of cosmetic treatment intended to protect the skin and the appendages from external aggressions and to combat cutaneous aging, comprising applying to the skin of a subject in needs the composition according to the invention.

[0079] In another embodiment, the invention relates to a method of cosmetic treatment intended to increase the expression of mitochondrial derived peptides and collagen I in skin cells.

[0080] Advantageously, the mitochondrial derived peptide is humanin.Examples

[0081] By way of illustration, exemplary embodiments of the process according to the invention are described below.

[0082] Example 1 : Effect of peptides of SEQ ID No. 1 and SEQ ID No. 2 on the expression of humanin in ex vivo human skin biopsies

[0083] The aim of this study was to determine the effect of the peptides of SEQ ID No. 1 and SEQ ID No. 2 on the expression of humanin mitochondrial derived peptide.

[0084] Protocol: Normal human skin biopsies were treated twice a day for 2 days with a solution of 500 mg / kg SEQ ID No. 1 or SEQ ID No. 2 diluted at 1 % in PBS. Negative controls were carried out using PBS. For immunolabelling by anti-humanin antibody, tissues were fixed and embedded in paraffin. Embedded skin biopsies were then cut, and sections were deparaffinized and rehydrated. Then, an unmasking protocol was performed before applying a specific anti-humanin antibody (Sigma, ref. H2414, rabbit polyclonal), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides were observed by epifluorescence microscope (Nikon Eclipse 80i microscope).

[0085] Results: Microscopic observation of humanin immunofluorescent staining on skin sections showed an increase of humanin expression after the treatment with the SEQ ID No. 1 and SEQ ID No. 2 peptide at 5 mg / Kg, compared to the PBS control biopsies (Fig. 1 ).

[0086] Example 2: Effect of peptides of SEQ ID No. 1 and SEQ ID No. 2 on the expression of collagen I in ex vivo human skin biopsies

[0087] The aim of this study was to determine the effect of the peptides of SEQ ID No. 1 and SEQ ID No. 2 on the expression of collagen I.

[0088] Protocol: Normal human skin biopsies were treated twice a day for 2 days with a solution of 500 mg / kg SEQ ID No. 1 or SEQ ID No. 2 diluted at 1 % in PBS. Negative controls were carried out using PBS. For immunolabelling by anticollagen I antibody, tissues were fixed and embedded in paraffin. Embedded skin biopsies were then cut, and sections were deparaffinized and rehydrated. Then, an unmasking protocol was performed before applying a specific anti-collagen I antibody (Proteintech, 14695-1 -AP, rabbit polyclonal), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides were observed by epifluorescence microscope (Nikon Eclipse 80i microscope).

[0089] Results: Microscopic observation of collagen I immunofluorescent staining on skin sections showed an increase of collagen I expression after the treatment with the SEQ ID No. 1 and SEQ ID No. 2 peptides, at 5 mg / Kg, compared to the PBS control biopsies (Fig. 2).

[0090] Example 3: Effect of a Phoenix dactylifera extract on the expression of humanin in ex vivo human skin biopsies

[0091] The aim of this study was to determine the effect of an aqueous extract of Phoenix dactylifera on the expression of humanin.

[0092] Protocol: Normal human skin biopsies were treated twice a day for 2 days with the extract of Phoenix dactylifera of Example 7, diluted at 1% in PBS. Negative controls were carried out using PBS. For immunolabelling by anti-humanin antibody, tissues were fixed and embedded in paraffin. Embedded skin biopsies were then cut, and sections were deparaffinized and rehydrated. Then, an unmasking protocol was performed before applying a specific anti-humanin antibody (ProteinTech, ref. 25885-1 -AP, rabbit polyclonal), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides were observed by epifluorescence microscope (Nikon Eclipse 80i microscope).

[0093] Results: Microscopic observation of humanin immunofluorescent staining on skin sections showed an increase of humanin expression after the treatment with 1 % Phoenix dactylifera extract, compared to the PBS control biopsies (Fig. 3).

[0094] Example 4: Effect of a Trifolium pratense extract on the expression of humanin in ex vivo human skin biopsies

[0095] The aim of this study was to determine the effect of an aqueous extract of Trifolium pratense on the expression of humanin.

[0096] Protocol: Normal human skin biopsies were treated twice a day for 2 days with the extract of Trifolium pratense of Example 7, diluted at 1 % in PBS. Negative controls were carried out using PBS. For immunolabelling by anti-humanin antibody, tissues were fixed and embedded in paraffin. Embedded skin biopsies were then cut, and sections were deparaffinized and rehydrated. Then, an unmasking protocol was performed before applying a specific anti-humanin antibody (Sigma, ref. H2414, rabbit polyclonal), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides were observed by epifluorescence microscope (Nikon Eclipse 80i microscope).

[0097] Results: Microscopic observation of humanin immunofluorescent staining on skin sections showed an increase of humanin expression after the treatment with 1 % Trifolium Pratense extract, compared to the PBS control biopsies (Fig. 4).

[0098] Example 5: Effect of a Chenopodium quinoa extract on the expression of humanin in ex vivo human skin biopsies

[0099] The aim of this study was to determine the effect of the aqueous extract of Chenopodium quinoa of Example 7 on the expression of humanin.

[0100] Protocol: Normal human skin biopsies were treated twice a day for 2 days with the extract of Chenopodium quinoa of Example 7 diluted at 1 % in PBS. Negative controls were carried out using PBS. For immunolabelling by anti-humanin antibody, tissues were fixed and embedded in paraffin. Embedded skin biopsies were then cut, and sections were deparaffinized and rehydrated. Then, an unmasking protocol was performed before applying a specific anti-humanin antibody (ProteinTech, ref. 25885-1 -AP, rabbit polyclonal), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium, the slides were observed by epifluorescence microscope (Nikon Eclipse 80i microscope).

[0101] Results: Microscopic observation of humanin immunofluorescent staining on skin sections showed an increase of humanin expression after the treatment with 1 % Chenopodium quinoa extract, compared to the PBS control biopsies (Fig. 5).

[0102] Example 6: Effect of an Iris pallida extract on the expression of humanin in ex vivo human skin biopsies

[0103] The aim of this study was to determine the effect of an aqueous extract of Iris pallida roots on the expression of humanin mitochondrial derived peptide.

[0104] Protocol: Normal human skin biopsies were treated twice a day for 2 days with the Iris pallida root extract of example 7, diluted at 1 % in PBS. Negative controls were carried out using PBS. For immunolabelling by anti-humanin antibody, tissues were fixed and embedded in paraffin. Embedded skin biopsies were then cut, and sections were deparaffinized and rehydrated. Then, an unmasking protocol was performed before applying a specific anti-humanin antibody (ProteinTech, ref. 25885-1 -AP, rabbit polyclonal), and then a secondary suitable antibody, coupled with a fluorescent dye. After mounting in a particular medium,the slides were observed by epifluorescence microscope (Nikon Eclipse 80i microscope).

[0105] Results: Microscopic observation of humanin immunofluorescent staining on skin sections showed an increase of humanin expression after the treatment with 1 % Iris pallida root extract, compared to the PBS control biopsies (Fig. 6).

[0106] Example 7: Preparation of aqueous plant extracts

[0107] Phoenix dactylifera: Phoenix dactylifera fruits pulps were crushed, then 5 % of crushed plant in water were extracted using high performance microwaves (maximal powerful magnetron 850 watts, maximal pressure 30 bars) for 1 to 10 minutes at a temperature between 60 - 80°C. The solid matter was removed by centrifugation and the supernatant was collected and clarified by sequential filtrations until a sterilizing filtration. The obtained extract of phoenix dactylifera fruits had a dry matter comprised between 20 - 50 g / kg and was used as is in biological efficacy testing.

[0108] Trifolium pratense: Trifolium pratense seeds were germinated by being immersed in water. 10% of germinated seeds were crushed and submitted to an aqueous extraction using a proprietary technology disclosed in patent US 11 ,021 ,505 B2. The solid matter was then removed by centrifugation and the supernatant was collected and clarified by sequential filtrations until sterilizing filtration. The obtained extract of trifolium pratense had a dry matter comprised between 15 - 30 g / kg and was used as is in biological efficacy testing.

[0109] Chenopodium quinoa: Chenopodium quinoa seeds were crushed then, 5% of crushed plant in water were extracted using high performance microwaves (maximal powerful magnetron 850 watts, maximal pressure 30 bars) for 1 to 10 minutes at high temperatures from 60 to 80°C. The solid matter was then removed by centrifugation and the supernatant was collected and clarified by sequential filtration until a filtration at a sterilizing porosity. The obtained extract of chenopodium quinoa seeds had a dry matter content comprised between 4-10 g / kg and was used as is in biological efficacy testing.

[0110] Iris pallida: iris pallida crushed dried roots were extracted using a proprietary technology disclosed in patent US 11 ,021 ,505 B2 by a process comprising the following steps:a) crushed dried roots of Iris pallida are extracted in water, at a concentration of 2.5 %, in the presence of 10 mM of phytic acid, at a pH comprised between 8 and 11 , preferably between 10.5 and 11 , at a temperature comprised between 40 and 80°C, for a duration comprised between 30 min and 4 hours, preferably 2 hours, b) the solid matter is removed by centrifugation and the supernatant is collected and c) the supernatant is clarified by sequential filtrations until sterilizing filtration, d) the filtered extract was then diluted in water to obtain an extract of iris pallida roots with a dry matter content comprised between 8 and 14 g / kg. This diluted extract was used as is in biological efficacy testing.In step d) the Iris pallida root extract can be alternatively diluted in solvents such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, Vaseline, a vegetable oil, or any mixture of these solvents. Preferentially, the Iris pallida root extract is diluted in 20% propanediol (propane-1 ,3-diol) and 10% glycerine.1 / 20 / 25, &:& / HIVI Sequence ListingSequence Listingfile: / / / C: / Users / a764125 / AppData / Roaming / ST26_authoring / Project_4 / generated.html 1 / 21 / 20 / 25, &:& / HIVI Sequence Listingfile: / / / C: / Users / a764125 / AppData / Roaming / ST26_authoring / Project_4 / generated.html 2 / 2

Claims

Claims1 . A peptide capable to increase the expression of mitochondrial derived peptides, of the following general formula (I):R1 -(AA)n- Cys-Leu-Leu-Leu-Ser-Thr-X1 -(AA)p-R2Wherein,AA represents any amino acid, and n and p are whole numbers between 0 and 2,X1 represents a Glycine or no amino acid,R1 represents the primary amine function of the N-terminal amino acid, free or substituted with a protector group which may be chosen from an acetyl group, a benzoyl group, a tosyl group, or a benzyloxycarbonyl group. R1 can also represent a palmitoylation and more particularly S-palmitoylation,R2 represents the hydroxyl function of the carboxylic acid of the C-terminal amino acid, free or substituted with a protector group which can be chosen from an alkyl chain of C1 to C20, or an NH2, NHY, or NYY group, with Y representing an alkyl chain of C1 to C4.

2. The peptide according to claim 1 consisting of the amino acid sequence SEQ ID No 1 : Ser-Cys-Leu-Leu-Leu-Ser-Thr.

3. The peptide according to claim 1 consisting of the amino acid sequence SEQ ID No 2: Ser-Cys-Leu-Leu-Leu-Ser-Thr-Gly4. A Cosmetic or pharmaceutical composition comprising, in a physiologically acceptable medium, an effective amount of an active principle capable to increase the expression of mitochondrial derived peptides, selected among: the peptide of SEQ ID No 1 , the peptide of SEQ ID No 2, an aqueous extract of date (Phoenix dactylifera) fruit pulp, an aqueous extract of germinated red clover (Trifolium pratense) seeds, an aqueous extract of quinoa (Chenopodium quinoa) seeds, and an aqueous extract of Iris pallida roots, or any mixture thereof, or any mixture thereof.

5. The composition according to claim 4, wherein the mitochondrial derived peptide is humanin.

6. The composition according to claim 4 wherein when the active principle is a peptide of SEQ ID No 1 or SEQ ID No 2, it is in a concentration comprised between approximately 0.0005 and 500 mg / kg, and preferentially in a concentration between approximately 0.01 and 10 mg / kg, relative to the total weight of the final composition.

7. The composition according to claim 4 wherein when the active principle is an aqueous extract, it is in a concentration comprised between approximately 0.01 % and 5 % preferentially in a concentration between approximately 0.1 % and 2 %, relative to the total weight of the final composition.

8. The composition according to claim 4 wherein the aqueous extract of Iris pallida roots is obtained by a method comprising the following steps: a) crushed dried roots of Iris pallida are extracted in water, at a concentration of 2.5 %, in the presence of 10 mM of phytic acid, at a pH comprised between 8 and 11 , preferably between 10.5 and 11 , at a temperature comprised between 40 and 80°C, for a duration comprised between 30 min and 4 hours, preferably 2 hours, b) the solid matter is removed by centrifugation and the supernatant is collected, c) the supernatant is clarified by sequential filtrations until sterilizing filtration and d) the filtered supernatant is diluted into water to obtain an extract having a dry matter content comprised between 8 and 14 g / kg.

9. The composition according to claim 8 wherein in the step d) the aqueous extract of Iris pallida roots is diluted in solvents selected among: water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, Vaseline, a vegetable oil, and any mixture of these solvents.

10. The composition according to claim 8 wherein in the step d) the aqueous extract of Iris pallida roots is diluted in 20% propanediol (propane-1 ,3-diol) and 10% of glycerine.11 . The composition according to claim 4, wherein the composition is in the form of a cream, oil-in-water emulsion or water-in-oil or multiple emulsion, solution, suspension, gel, milk, lotion, stick, or even powder.

12. The composition according to claim 4, wherein the composition is adapted to topical application onto the skin, the lips, and / or the appendages.

13. A method of cosmetic treatment intended to protect the skin and the appendages from external aggressions and to combat cutaneous aging, comprising applying to the skin the composition according to claim 4.

14. The method of cosmetic treatment according to claim 13, wherein the applied composition increases the expression of humanin in skin cells.

15. The method of cosmetic treatment according to claim 13, wherein the applied composition increases the expression of collagen I in skin cells.

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