Active complex for increasing cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis

Abstract

The present invention relates to a non-therapeutic cosmetic composition for increasing the cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis, comprising an active complex: extracts of Melaleuca alternifolia, extracts of Lavandula hybrida, and melatonin. The invention also relates to the use of this non-therapeutic cosmetic composition for increasing the cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis.

Description

Description Title of the invention: Active complex to increase cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis

[0001] The present invention relates to a non-therapeutic cosmetic composition for increasing mitochondrial cellular respiration of keratinocytes underlying the stratum corneum of the epidermis, comprising a complex of active ingredients: extracts of Melaleuca Altemifolia, extracts of Lavandula Hybrida, and melatonin. The invention also relates to the use of this non-therapeutic cosmetic composition to increase mitochondrial cellular respiration of keratinocytes underlying the stratum corneum of the epidermis. Previous Art

[0002] Previous research in the field of skin physiology has extensively explored the metabolic and energy processes of skin cells (Hourigan R. Cellular energy metabolism and oxidative stress. In: MA Farage, KW Miller, HI Maibach, eds. Textbook of Aging Skin. Heidelberg; 2010: 313-320 - Cibrian D, et al. Metabolic pathways that control skin homeostasis and inflammation. Trends Mol Med. 2020; 26: 975-986 - Mangez C, et al. An integrative multi-omic analysis reveals a major metabolic rewiring between baby foreskin keratinocytes and adult female abdominal keratinocytes. Exp Dermatol. 2022; 31:622-627).

[0003] Keratinocytes are keratin-rich cells that lose their nucleus and organelles as they migrate towards the surface. Keratin gives the stratum corneum its strength and impermeability. This outermost layer of the epidermis is composed of keratinocytes that have differentiated into keratinocytes. This protective barrier shields the skin against external aggressors such as infections, chemicals, and dehydration.

[0004] Mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis plays a crucial role in producing the energy necessary for their survival and function. Keratinocytes undergo several stages of differentiation before becoming keratinocytes and require energy for lipid synthesis, cell proliferation, and stress response, among other processes (Roe DF, et al. Topical Dissolved Oxygen Penetrates Skin: Model and Method. Journal of Surgical Research 159, e29-e36 (2010). - Stücker, M. et al. The cutaneous uptake of atmospheric oxygen contributes significantly to the oxygen supply of human dermis and epidermis. J Physiol 538, 985-994 (2002). - Baumgartl H., et al. Initial Results of Intracutaneous Measurements of PO2 Profiles, in Clinical Oxygen Pressure Measurement (eds. Ehrly, A. M. Hauss, J. & Huch, R.) 121-128 (Springer, 1987). doi: 10.1007 / 978-3-642-71226-5J 5).

[0005] The mitochondria of keratinocytes produce ATP through oxidative phosphorylation, which uses oxygen to convert energy substrates into usable energy. This energy production is crucial for keratinocytes migrating to the superficial layers of the epidermis.

[0006] In addition to their role in energy production, the mitochondria of keratinocytes participate in the regulation of apoptosis, essential for the elimination of damaged or stressed cells. Thus, mitochondrial respiration is fundamental for maintaining homeostasis and the protective function of the epidermis, which depends on the proper functioning of the keratinocytes underlying the stratum corneum of the epidermis.

[0007] There is a continuing need for effective and specific agents to enhance mitochondrial respiration in keratinocytes, thereby providing cosmetic benefits for the skin. Description of the invention

[0008] According to a first aspect, the invention relates to a non-therapeutic cosmetic composition for increasing cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis in a healthy subject, comprising melatonin.

[0009] According to one embodiment, said cosmetic composition comprises an extract of Melaleuca Alternifolia, preferably oil from the leaves of Melaleuca Alternifolia.

[0010] According to one embodiment, said cosmetic composition comprises an extract of Lavandula Hybrida, preferably oil from the flowers of Lavandula Hybrida.

[0011] According to one embodiment, the invention relates to a non-therapeutic cosmetic composition for increasing cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis in a healthy subject, comprising: - An extract of Melaleuca Alternifolia, preferably oil from the leaves of Melaleuca Alternifolia; - An extract of Lavandula Hybrida, preferably Lavandula Hybrida flower oil; - Melatonin.

[0012] According to another aspect, the invention relates to the non-therapeutic cosmetic use of increasing cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis in a healthy subject of a composition including: - An extract of Melaleuca Alternifolia, preferably oil from the leaves of Melaleuca Alternifolia; - An extract of Lavandula Hybrida, preferably Lavandula Hybrida flower oil; - Melatonin.

[0013] According to another aspect, the invention relates to a method for preparing a non-therapeutic cosmetic composition, comprising at least one step of mixing said extracts of Melaleuca Alternifolia and Lavandula Hybrida, and said melatonin.

[0014] According to another aspect, the invention relates to a kit for preparing a non-therapeutic cosmetic composition to increase cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis, comprising: - An extract of Melaleuca Alternifolia, preferably oil from the leaves of Melaleuca Alternifolia; - An extract of Lavandula Hybrida, preferably Lavandula Hybrida flower oil; - Melatonin.

[0015] According to another aspect, the invention relates to a non-therapeutic cosmetic skin care method for a healthy subject, comprising a step of applying a cosmetic composition according to the invention to the skin.

[0016] The embodiments that follow apply equally to the non-therapeutic cosmetic composition according to the invention, and to the uses, processes and kits according to the invention.

[0017] According to one embodiment, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 2%, by weight of the composition of said extract of Melaleuca Alternifolia, preferably of the oil of the leaves of Melaleuca Alternifolia.

[0018] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 1.5%, by weight of the composition of said extract of Melaleuca Alternifolia, preferably of the oil of the leaves of Melaleuca Alternifolia.

[0019] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 1% by weight of the composition of said extract of Melaleuca Alternifolia, preferably of the oil of the leaves of Melaleuca Alternifolia.

[0020] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.01% and 1% by weight of the composition of said extract of Melaleuca Alternifolia, preferably of the oil of the leaves of Melaleuca Alternifolia.

[0021] According to one embodiment, said non-therapeutic cosmetic composition comprises 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2% by weight of the composition of said extract of Melaleuca Alternifolia, preferably of the oil of the leaves of Melaleuca Alternifolia.

[0022] According to one embodiment, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 2%, by weight of the composition of said extract of Lavandula Hybrida, preferably of the oil of flowers of Lavandula Hybrida.

[0023] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 1.5%, by weight of the composition of said Lavandula Hybrida extract, preferably of Lavandula Hybrida flower oil.

[0024] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 1% by weight of the composition of said Lavandula Hybrida extract, preferably from Lavandula Hybrida flower oil.

[0025] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.01% and 1% by weight of the composition of said Lavandula Hybrida extract, preferably of the oil of Lavandula Hybrida leaves.

[0026] According to one embodiment, said non-therapeutic cosmetic composition comprises 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.05%, 1.1%, 1.15%, 1.2%, 1.25%, 1.3%, 1.35%, 1.4%, 1.45%, 1.5%, 1.55%, 1.6%, 1.65%, 1.7%, 1.75%, 1.8%, 1.85%, 1.9%, 1.95%, 2% by weight of the composition of said Lavandula Hybrida extract, preferably of Lavandula flower oil Hybrida. According to one embodiment, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 10% by weight of the melatonin composition.

[0027] Preferably, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 1% by weight of the melatonin composition.

[0028] According to one embodiment, said non-therapeutic cosmetic composition comprises 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.0015%, 0.002%, 0.0025%, 0.003%, 0.0035%, 0.004%, 0.0045%, 0.005%, 0.0055%, 0.006%, 0.0065%, 0.007%, 0.0075%, 0.008%, 0.0085%, 0.009%, 0.0095%, 0.005%, 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, or 1%, or 1.25%, or 1.5%, or 1.75%, or 2%, or 2.25%, or 2.5%, or 2.75%, or 3%, or 3.25%, or 3.5%, or 3.75%, or 4%, or 4.25%, or 4.5%, or 4.75%, or 5%, or 5.25%, or 5.5%, or 5.75%, or 6%, or 6.25%, or 6.5%, or 6.75%, or 7%, or 7.25%, or 7.5%, or 7.75%, or 8%, or 8.25%, or 8.5%, or 8.75%, or 9%, or 9.25%, or 9.5%, or 9.75%, or 10% by weight of the melatonin composition.

[0029] According to one embodiment, said melatonin is vectorized.

[0030] Preferably, said melatonin is delivered via a liposome delivery system. Even more preferably, said liposome delivery system comprises water, phospholipids, melatonin, and Lactobacillus cultures.

[0031] According to one embodiment, said liposome delivery system comprises: - 89% water by weight of said vectorization system; - 5% phospholipids by weight in said vectorization system; - 4% melatonin by weight of said delivery system; - 2% of Lactobacillus ferments by weight of said vectorization system.

[0032] According to one embodiment, said non-therapeutic cosmetic composition comprises: - Between 0.00001% and 2%, preferably between 0.00001% and 1.5%, even more preferably between 0.01% and 1%, by weight of the composition of said extract of Melaleuca Alternifolia; - Between 0.00001% and 2%, preferably between 0.00001% and 1.5%, even more preferably between 0.01% and 1%, by weight of the composition of said Lavandula Hybrida extract; - Between 0.00001% and 10% by weight of the melatonin composition.

[0033] More specifically, the composition according to the invention increases the respiration and energy production of the epidermis. This results in skin with a more beautiful appearance for the user, giving a visual impression of healthier skin.

[0034] The composition according to the invention comprises a cosmetically acceptable medium, that is to say, one compatible with the skin of the face and / or body. In other words, the medium used has a pleasant color, odor, and feel and does not generate unacceptable discomfort (such as, in particular, tingling, tightness, or redness) that might deter the consumer from using this composition.

[0035] According to one embodiment, the cosmetic composition according to the invention further comprises at least one cosmetically acceptable agent.

[0036] According to one embodiment, said non-therapeutic cosmetic composition further comprises at least one cosmetically acceptable agent selected from among soothing agents, restructuring agents, regenerating agents, revitalizing agents, sunscreens, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, smoothing agents, agents that enhance the firmness, elasticity and / or barrier effect of the skin, antioxidants, opacifiers, thermal waters, mattifying agents, chemical or mineral filters, trace elements, stabilizing agents, foaming agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, perfumes, filters, essential oils, coloring materials, pigments, hydrophilic or lipophilic actives,Lipid vesicles encapsulating one or more active ingredients and / or preservatives.

[0037] According to one embodiment, the cosmetic composition according to the invention is administered by topical application to an area of ​​the skin.

[0038] According to one embodiment, said cosmetic composition according to the invention is formulated in the form of a cream, an ointment, an ointment, a balm, a mask, a milk, a lotion, a serum, a spray, a paste, a foam, an aerosol, a stick, a shampoo, a conditioner, patches, an aqueous hydroalcoholic or oily solution, an oil-in-water or water-in-oil or multiple emulsion, an aqueous or oily gel, an anhydrous liquid, paste or solid product, and / or an oil dispersion in an aqueous phase using spherules, these spherules being able to be polymeric nanoparticles such as nanospheres and nanocapsules or ionic and / or non-ionic lipid vesicles.

[0039] According to one embodiment, an amount of approximately 0.1 to 50 mg / cm³ 2 preferably about 0.25 to 25 mg / cm³ 2 , preferably in an even higher concentration of approximately 2 to 10 mg / cm² 2, of cosmetic composition according to the invention is applied to the area of ​​skin.

[0040] According to one embodiment, the cosmetic composition according to the invention is applied once or twice a day, preferably twice a day, for at least 7 days, preferably at least 15 days, even more preferably for at least one month, and particularly preferably for at least 2 months.

[0041] According to one embodiment, the cosmetic composition according to the invention is applied in the process according to the invention twice a day for at least 1 month, preferably 2 months.

[0042] Preferably, according to the invention, said area of ​​skin is the face. Figures

[0043] [Fig 1] is a graph showing the oxygen consumption profiles of NHEK cultured for 48 hours + 30 minutes under hypoxia

[0044] [Fig 2] A, B and C are graphs showing the mitochondrial respiration parameters of NHEK cultured for 48 hours + 30 minutes under hypoxia

[0045] [Fig 3] is a graph showing the oxygen consumption profiles of NHEK cultured for 48 hours + 2 hours under hypoxia

[0046] [Fig 4] A, B and C are graphs showing the mitochondrial respiration parameters of NHEK cultured for 48 hours + 2 hours in hypoxia

[0047] [Fig 5] is a graph showing the oxygen consumption profiles of NHEK cultured over 48 hours + 24 hours.

[0048] [Fig 6] A, B and C are graphs showing the analysis of mitochondrial respiration parameters of NHEK cultured for 48 hours + 24 hours.

[0049] [Fig 7] is a graph showing the illustration of the ECAR of NHEK cultured for 48 hours + 30 minutes in hypoxia.

[0050] [Fig 8] is a graph showing the illustration of the ECAR of NHEK cultured for 48 hours + 2 hours in hypoxia.

[0051] [Fig 9] is a graph showing the illustration of the ECAR of NHEK cultured for 48 hours + 24 hours in hypoxia.

[0052] [Fig 10] is a graph showing the effect of the products on citrate synthase activity, compared to the negative control TJ2.

[0053] [Fig 11] is a graph showing the effect of the products on the activity of complex IV, compared to the negative control TJ2.

[0054] [Fig 12] is a graph showing the ATP dosage, compared to the negative control TJ2.

[0055] [Fig 13] is a graph showing the comparison of the average reference spectra of the products and the control skin, in Example 3.

[0056] [Fig 14] is a photograph showing the correlation between the visible images and the fit images showing the distribution of the "Melatonin" products in the layers of the epidermis and dermis down to a depth of 135 pm after a sampling time of 8 hours, in example 3. CORRECTED SHEET (RULE 91) ISA / EP

[0057] [Fig 15] is a graph representing the semi-quantitative percentages of skin penetration of melatonin-based products in example 4.

[0058] Figure 16 represents the activity levels of complex 4. (A) The kinetics of complex 4 activity levels from skin explant extracts as a function of treatment are presented as mean ± standard deviation (OD at 550 nm). Curve 1 corresponds to the background, curve 2 to the control, curve 3 to melatonin alone, and curve 4 to the combination of melatonin and 1% Melaleuca Alternifolia leaf oil extract and 1% Lavandula Hybrida flower oil extract. (B) Complex 4 activity obtained from the linear reaction phase according to the manufacturer's instructions. Data are reported for each group as mean (% vs. control) ± SD***, p < 0.01 - Unpaired t-test with Welch's correction for binary analyses vs. control group. Definitions

[0059] The epidermis has five distinct layers. From deepest to most superficial, there is the basal (or germinative) layer, where keratinocytes divide and renew themselves. Above this is the spinous layer, composed of keratinocytes connected by desmosomes. Next, the granular layer contains keratinocytes that accumulate keratohyalin granules. The stratum corneum, visible only in thicker skin, is a thin band of cells whose nuclei are disintegrating, giving them a translucent appearance. Finally, the outermost layer is the stratum corneum.

[0060] The stratum corneum is composed of differentiated keratinocytes, called comeocytes. These keratin-rich cells lack a nucleus and organelles, providing a protective barrier against external aggressors such as infections, chemicals, and dehydration. This impermeable and robust layer is essential for skin protection. Comeocytes originate from keratinocytes in the deeper layers that migrate and transform over time. The stratum corneum is constantly renewed through desquamation, where dead cells are shed and replaced by new ones.

[0061] Keratinocytes are specialized epithelial cells found in all layers of the epidermis, but their density varies with depth. As keratinocytes move toward the upper layers of the epidermis, they undergo a differentiation process, producing increasing amounts of keratin and eventually differentiating into keratinocytes within the stratum corneum. Keratinocytes perform several important functions, including protecting the skin from damage, regulating skin hydration, repairing wounds, and responding to pathogens. They play a key role in protecting the body against external aggressions such as infections, UV rays and water loss.

[0062] Mitochondrial respiration in epidermal keratinocytes refers to the biochemical process by which keratinocytes in the epidermis, prior to their differentiation into comeocytes and migration into the stratum corneum, use oxygen to metabolize energy substrates, such as fatty acids and sugars, through a series of enzymatic reactions in the mitochondria. This process generates adenosine triphosphate (ATP), the primary source of cellular energy used to fuel essential cellular functions, such as cell division, differentiation, migration, and the synthesis of biomolecules. Maintaining or enhancing mitochondrial respiration in keratinocytes is crucial for maintaining skin homeostasis, promoting epidermal regeneration, and responding to the skin's changing metabolic demands, while also contributing to protection against environmental stressors and external aggressions.By "increase" is understood that the composition according to the invention makes it possible to obtain a greater mitochondrial cellular respiration of the keratinocytes of the epidermis, underlying the stratum corneum, than without the application of said composition, in a significant but non-therapeutic way, in the healthy subject.

[0063] The term "underlying the stratum corneum" refers to keratinocytes present in the epidermis, "below" or "before" their migration into the stratum corneum and their differentiation into keratinocytes. These keratinocytes can therefore be found in the stratum granulosum, the stratum spinosum, or the stratum basale of the epidermis.

[0064] The term "non-therapeutic cosmetic" refers to a composition that is the subject of the invention and has a non-therapeutic technical effect, thereby improving the superficial visual appearance of the skin. Such a composition does not act as a drug for the treatment of dermatological diseases and is administered to a healthy individual.

[0065] By "healthy subject" is meant a human individual not exhibiting dermatological diseases that would be treated by the administration of the composition according to the invention, which only allows to improve the superficial visual appearance of the skin, preferably of the face.

[0066] Melaleuca alternifolia is a tree species native to Australia, also known as the tea tree. It belongs to the Myrtaceae family and is widely recognized for the medicinal properties of its oil, extracted from the tree's leaves. This oil can be obtained after harvesting and distilling the leaves. The leaves are typically harvested when mature and then crushed or shredded. Next, these leaves are placed in a still or other distillation system where steam is passed through them. This steam heats the leaves, releasing the volatile compounds contained in the leaf glands. These volatile compounds include terpenes and The phenols are then condensed and collected in liquid form. The Melaleuca Alternifolia oil obtained by this process is then filtered to remove impurities and obtain a pure oil, ready for use in the composition according to the invention. An example of such an extract is marketed under the name Regenight™. This extract comprises a fat-soluble fraction upcycled from waste products of Australian tea tree distillation and standardized for sesquiterpenes.

[0067] In detail, Melaleuca Alternifolia leaf extract can be obtained after harvesting the tree's leaves, followed by an extraction process using a plant-based solvent, such as ethanol, or an aqueous solvent. The leaves are generally harvested when mature, then crushed or shredded to increase their surface area. They are then placed in a suitable extractor, such as a stirred reactor or an infusion tank, where they are brought into contact with the extraction solvent.

[0068] The extraction process can be carried out at a controlled temperature, generally between 30°C and 80°C, preferably between 40°C and 60°C, for a duration typically ranging from 1 to 24 hours. It can be conducted at atmospheric pressure or under reduced pressure (e.g., between 200 and 800 mbar) to preserve heat-sensitive compounds. Under the influence of the solvent and temperature, the compounds of interest, including terpenes, phenols, and other aromatic substances present in the leaf glands, are extracted into the liquid phase.

[0069] The resulting mixture is then filtered to remove plant residues. The solvent is then removed, for example by vacuum evaporation, to obtain a concentrated extract. This extract can be further purified, for example by decantation, fine filtration, or chromatography, to remove impurities or concentrate the active fractions. The resulting Melaleuca alternifolia extract is in liquid or paste form and can be used as is or formulated into a composition according to the invention.

[0070] Lavandula hybrida, also known as lavandin, is a hybrid plant resulting from a cross between Lavandula angustifolia (true lavender) and Lavandula latifolia (spike lavender). This plant belongs to the Lamiaceae family and is widely cultivated for its aromatic flowers and oils. Obtaining these extracts involves harvesting the flowering tops of the plant to extract the aromatic compounds. The flowering tops are generally harvested when mature and then subjected to various extraction processes, primarily steam distillation. During distillation, steam is passed through the flowers, heating the plant's oil glands and releasing the volatile aromatic compounds. These compounds are then carried away by the steam and condensed to form a mixture. of essential oil and water, which is then separated. The lavandin oil thus obtained is then filtered to remove impurities and obtain a pure oil, ready for use in the composition according to the invention. An example of such an extract is marketed under the name Immunight™. This is a fat-soluble extract of organic lavandin, standardized in monoterpenes, obtained by a proprietary green process using plant-based solvents.

[0071] In detail, Lavandula hybrida flower oil extract can also be obtained through an extraction process using a plant-based solvent, such as ethanol, or water, alone or mixed with a co-solvent. Obtaining this extract involves collecting the flowering tops of the plant, generally harvested when fully mature, when their aromatic compound content is optimal. These flowering tops are then ground or lightly crushed to promote the release of volatile compounds.

[0072] The flowers are then placed in an extraction device, such as a stirred extractor or an infusion tank, where they are brought into contact with the chosen solvent. Extraction is carried out at a temperature between 30°C and 80°C, preferably between 40°C and 60°C, for a duration ranging from 1 to 24 hours, typically between 4 and 8 hours. The operation can be performed at atmospheric pressure or under reduced pressure (e.g., between 200 and 800 mbar), which helps to preserve the integrity of the heat-sensitive compounds.

[0073] The volatile and aromatic compounds naturally present in the oil glands of the flowers, including esters, terpenes, and aromatic alcohols, are thus extracted into the liquid phase. After extraction, the mixture is filtered to remove plant residues. The solvent is then removed, for example by vacuum evaporation, to obtain a concentrated extract. A further purification step, such as decantation, fine filtration, or chromatography, may be carried out to remove residual impurities. The resulting Lavandula hybrida extract is generally in liquid form, concentrated in aromatic compounds, and can be used as is in the composition according to the invention.

[0074] Melatonin is a hormone naturally produced by the pineal gland in the brain. It plays a crucial role in regulating circadian rhythms, which are the natural sleep-wake cycles over a period of approximately 24 hours. In addition to its role in controlling sleep-wake cycles, melatonin also plays a role in other biological processes, such as the regulation of blood pressure, the immune system, ovarian function, and thyroid function. An example of a melatonin product usable according to the invention is marketed under the name AC Melatonin Liposome™. In one embodiment, said melatonin can be delivered via a delivery system. "Delivered via a delivery system" means the inclusion of the melatonin in a liposomal system.

[0075] For the purposes of this invention, "topical application" means application to the skin (including the scalp) and mucous membranes.

[0076] For the purposes of this invention, "cosmetically acceptable" means something that is useful in the preparation of a cosmetic composition, that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and that is acceptable for cosmetic use, in particular by topical application to the skin.

[0077] For the purposes of this invention, "epidermal barrier function" means the protective function of the epidermis, particularly against external aggressions, and the regulation of transient loss of water and ions.

[0078] The ingredients of said cosmetic composition according to the invention are combined in effective quantities. For the purposes of this invention, "effective quantity" means a quantity sufficient to obtain the desired effect, as determined by a person skilled in the art.

[0079] For the purposes of this invention, "topical application" means application to the skin (including the scalp).

[0080] In the description and the following examples, unless otherwise stated, percentages are percentages by weight of the composition, and value ranges expressed as "between ... and ..." include the specified lower and upper bounds. The examples below are provided for illustrative purposes only and are not intended to limit the scope of the invention. Examples

[0081] Example 1: Effect of the composition according to the invention (MEST22M01) on mitochondrial respiration in normal human epidermal keratinocytes under hypoxic conditions

[0082] INTRODUCTION

[0083] In the present study, the effect of the composition according to the invention (MEST22M01) was evaluated on mitochondrial respiration and glycolysis in normal human epidermal keratinocytes (NHEK) cultured under hypoxic conditions using Seahorse technology.

[0084] Previously, a cytotoxicity test was performed under hypoxic conditions using a standard WST-8 reduction test in order to determine the concentrations to be tested.

[0085] MATERIALS AND METHODS

[0086] 1. Biological model - Cell type: Normal human epidermal keratinocytes (NHEK), - Synelvia BH032 reference, used on the 2nd pass - Culture conditions: 37°C, 5% CO2, under hypoxia - Culture medium: DermaLife - Test medium: DermaLife optimized for testing

[0087] 2. Tested compound

[0088] Composition: Melatonin in powder form at a concentration of 0.0015% by weight of the total composition.

[0089] Powder form

[0090] Intermediate solution: 10% in DMSO

[0091] Concentration tested: 0.0015% (0.015% DMSO)

[0092] 3. Preliminary cytotoxicity test - Cell type: NHEK in test medium - Culture conditions: 37°C, 5% CO2, under hypoxia - Cell pre-incubation time: 24 hours - Incubation time of the active ingredient: 24 hours - Evaluation parameters: WST-8 reduction. At the end of treatment, cells were incubated in the presence of WST-8 (a highly water-soluble tetrazolium salt) which was reduced to a water-soluble, orange-colored product (formazan) by succinate dehydrogenase (a mitochondrial enzyme). This transformation is proportional to the number of viable cells and their metabolic activity. Optical density (OD) was measured using a spectrophotometer at 450 nm (SPARK, TECAN).

[0093] 4. Cultivation and processing

[0094] Keratinocytes were seeded in 24-well plates and cultured in normoxic culture medium for 24 hours. The medium was then replaced with culture medium containing or not the compound and the solvent control (DMSO). After 48 hours of incubation under hypoxic conditions, a second treatment was performed. The cells were then incubated under hypoxia for 30 minutes, 2 hours, or 24 hours before assessment of mitochondrial respiration and glycolysis.

[0095] 5. Evaluation of mitochondrial activity

[0096] Seahorse XF technology (Agilent) allows simultaneous and real-time measurement of mitochondrial respiration and glycolysis within a microchamber.

[0097] Mitochondrial respiration is measured by the real-time oxygen consumption rate (OCR) of cells. The different measurement cycles are performed following sequential injections: - Oligomycin, which inhibits ATP synthase in order to determine ATP production, - of FCCP (Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone) which stimulates the OCR and allows the determination of the respiratory reserve capacity defined as the difference between the maximum breathing value and the basal breathing, - of Rotenone and Antimycin A which allow the cessation of mitochondrial respiration in order to determine non-mitochondrial respiration.

[0098] The results are evaluated against several criteria: - Basal respiration: corresponds to the oxygen consumption of cells for normal energy activity. The higher its value, the more active the cell. - The production of ATP (Adenosine triphosphate) is directly proportional to the energy needs of cells. - The "leak" of protons (H+ Leak) is often associated with low ATP production, which indicates mitochondrial damage. - Maximum respiration is directly proportional to the metabolic agility of the cell. The higher its value, the more reactive the cell.

[0099] In parallel with OCR, glycolysis is measured using the extracellular acidification rate (ECAR). ECAR is closely linked to the decrease in pH caused by glycolytic activity. The unit of ECAR is therefore mpH / min (mpH = millipH units).

[0100] Extracellular acidification is the sum of two components: - respiratory acidification, in the form of CO2, - acidification from glycolysis, in the form of lactate - + H+.

[0101] The contribution of CO2 to total extracellular acidification was considered negligible by the measurement platform used here, the Seahorse XF analyzer.

[0102] 6. Data Processing

[0103] The raw data was transferred and processed using Microsoft Excel® and GraphPad PRISM® software.

[0104] Intergroup comparisons were performed using the unpaired two-tailed Student's t-test. Statistical analyses can be interpreted if n > 5; however, for n < 5, the calculated data are provided for illustrative purposes only.

[0105] Formulas used in this example: - Standard error of the mean: esm = standard deviation (Sd) / - / ” n The standard error of the mean (SEM) represents the deviation of the sample mean from the true population mean. The SEM is calculated by dividing the Sd by the square root of the sample size. - Percentage of viability: viability (%) = (compound OD / control OD) x 100 - Percentage relative to the solvent control: (%) = (compound value / control average) x 100

[0106] RESULTS

[0107] 1. Preliminary cytotoxicity test

[0108] Table 1: Cytotoxicity results of the active ingredient MEST22M01 [Table 1] (1): Statistical significance threshold ns: > 0.05, Not significant * : 0.01 to 0.05, Significant ** : 0.001 to 0.01, Very significant *** : < 0.001, Extremely significant

[0109] In the remainder of the study, the concentration "0.0015%" will be used.

[0110] 2. Effect on mitochondrial respiration [YES] 2.2. Treatments 48 hours + 30 minutes

[0112] The analysis of mitochondrial respiration after a 48-hour treatment followed by a further 30-minute treatment, under hypoxia, is described in Figure 1 and in the following tables:

[0113] Tables 2, 3, and 4: Results of mitochondrial respiration of NHEK cultured for 48 hours + 30 minutes under hypoxia [Table 2] [Table 3] [Table 4]

[0114] For tables 2, 3 and 4: (1): Statistical significance threshold ne: not calculable ns: > 0.05, Not significant (2): Invalidated data * : 0.01 to 0.05, Significant ** : 0.001 to 0.01, Very significant *** : < 0.001, Extremely significant

[0115] In the presence of the active ingredient MEST22M01, basal and maximal respiration, as well as ATP production, increased compared to the DMSO solvent control, under hypoxic conditions (Figure 2)

[0116] 3. Treatments: 48 hours + 2 hours

[0117] The analysis of mitochondrial respiration after a 48-hour treatment followed by a further 2-hour treatment, in hypoxia, is described in Figure 3.

[0118] Tables 5, 6 and 7: Results of mitochondrial respiration of NHEK cultured for 48 hours + 2 hours in hypoxia [Table 5] [Table 6] [Table 7] Maximum Breathing Treatment

[0119] For tables 5, 6 and 7: (1): Statistical significance threshold ne: not calculable ns: > 0.05, Not significant (2): Invalidated data * : 0.01 to 0.05, Significant ** : 0.001 to 0.01, Very significant *** : < 0.001, Extremely significant

[0120] In the presence of the active ingredient MEST22M01, basal and maximal respiration, as well as ATP production, increased compared to the solvent control (Figure 4).

[0121] 2.4. 48-hour + 24-hour treatments

[0122] The analysis of mitochondrial respiration after a 48-hour treatment followed by a further 24-hour treatment is described in Figure 5.

[0123] Tables 8, 9 and 10: Results of mitochondrial respiration of NHEK cultured for 48 hours + 24 hours in hypoxia - Calculations vs solvent control (DMSO) hypoxia [Table 8] [Table 9] [Table 10]

[0124] For tables 8, 9 and 10: (1): Statistical significance threshold: not calculable ns: > 0.05, Not significant (2): Invalidated data * : 0.01 to 0.05, Significant ** : 0.001 to 0.01, Very significant *** : < 0.001, Extremely significant

[0125] After 72h, in the presence of the active ingredient MEST22M01, basal and maximum respiration, as well as ATP production, increased compared to the solvent control (Figure 6).

[0126] 3. Effect on glycolysis

[0127] 3.1. Glycolysis analysis after treatment with the active ingredient MEST22M01

[0128] Regardless of the treatment time of the cells in hypoxia, we do not observe any variation in the ECAR in the culture medium in the presence or absence of the active ingredient (Figures 7-8-9).

[0129] CONCLUSION

[0130] Under our experimental conditions in hypoxia, and compared to its control (DMSO solvent), the treatment of keratinocytes with the active ingredient MEST22M01 increased: - 97% basal respiration, 65% ATP production and 47% maximum respiration after 48h+30 min of incubation - 30% basal respiration and 29% ATP production after 48h + 2h incubation.

[0131] At 48h + 24h of treatment, the active ingredient MES T22M01 increased basal respiration by 44%, ATP production by 30% and maximum respiration by 39%.

[0132] Treatment of keratinocytes, 48 ​​hours + 30 minutes or 48 hours + 2 hours or 48 hours + 24 hours, under hypoxia, with the active ingredient MEST22M01 increased oxygen consumption at the level of basal respiration and ATP production, indicating that energy production occurs mainly via the respiratory pathway rather than the glycolytic pathway.

[0133] Example 2: Effects of the composition according to the invention (MEST22M01), in hypoxia, on the production of ROS in keratinocytes stimulated by H2O2 and on the synthesis of melanin in weakly pigmented melanocytes

[0134] INTRODUCTION

[0135] In this example, the effects of the composition according to the invention MEST22M01 were evaluated on different parameters under hypoxic conditions.

[0136] More specifically, the effects of this compound were evaluated on: - The production of reactive oxygen species (ROS) induced by H2O2 stimulation in normal human epidermal keratinocytes (NHEK). ROS production was quantified using a photo-oxidation-resistant fluorescent probe: DCFH-DA. - Melanin synthesis in normal low-pigmented human epidermal melanocytes (NHEM-LP). Previously, a cytotoxicity test was performed on NHEM-LP using a standard reduction assay. WST-8 in order to determine the concentrations to be tested.

[0137] MATERIALS AND METHODS

[0138] 1. Biological Models

[0139] Normal human epidermal keratinocytes (NHEK) - Cell type: NHEK, Bioaltematives reference K341, used in the 3rd pass - Growing conditions: 37°C, 5% CO2 - Culture medium: Keratinocyte-SFM optimized for the test, supplemented with Epidermal Growth Factor and Pituitary Extract - Test medium: Keratinocyte-SFM optimized for the test

[0140] Normal human epidermal melanocytes - low pigmentation (NHEM-LP) - Cell type: NHEM-LP, Bioaltematives reference NHEM-2 used in the 10th pass (preliminary cytotoxicity test) and in the 12th pass (melanogenesis test) - Growing conditions: 37°C, 5% CO2 - Culture medium: Medium 254 optimized for the test (HMGS-2 without PMA) - Test medium: Medium 254 optimized for testing

[0141] 2. Tested compound

[0142] Composition: Melatonin in powder form at a concentration of 0.0015%.

[0143] Powder form

[0144] Intermediate solution: 10% in DMSO

[0145] Concentration tested: 0.0015% (0.015% DMSO)

[0146] 3. Production of ROS by H2O2-stimulated NHEKs

[0147] 3.1. Culture and treatments

[0148] Keratinocytes were seeded in 96-well plates and cultured in culture medium for 24 hours. The medium was then replaced with test medium containing or not (control) the compound or solvent control (DMSO - 0.015%), and the cells were pre-incubated under hypoxia for 48 hours.

[0149] After pre-incubation, the cells were placed under normoxia and the medium was replaced with the photo-oxidation-resistant fluorescent probe DCFH-DA. The cells were then incubated for 30 minutes at 37°C. After rinsing with PBS, the treatment with the compound or solvent control was repeated, and H₂O₂ (tested at 250 pM) was added. The cells were incubated for another 30 minutes. After stimulation, the medium was replaced with PBS containing or not the compound or solvent control, and the cells were incubated for 20 minutes before ROS measurement.

[0150] All experimental conditions were carried out in n=5.

[0151] In order to determine the background noise signal, a control in the absence of a probe was performed for the stimulated control condition in hypoxia condition in n=5. For the calculation of effects, background noise values ​​were subtracted from the raw measured values.

[0152] 2. Measurement of SWR

[0153] The intensity of the emitted fluorescence (Zex = 485 nm, Xem = 538 nm) was measured using a Synergy Hl microplate reader (BioTek).

[0154] The fluorescence intensity of the metabolized probe is proportional to the amount of ROS. ROS production is expressed in fluorescence units.

[0155] 4. Tests on NHEM-LP

[0156] 4.1. Preliminary cytotoxicity test - Cell type: NHEM-LP in test medium - Incubation time: 24 hours in hypoxia followed by 24 hours in hypoxia with the compound - Evaluation parameters: WST-8 reduction and morphological observations under the microscope. At the end of the treatment, the cells were incubated in the presence of WST-8 (a highly water-soluble tetrazolium salt) which was reduced to a water-soluble, orange-colored product (formazan) by succinate dehydrogenase (a mitochondrial enzyme). This transformation is proportional to the number of live cells and their metabolic activity. Optical density (OD) was measured with a spectrophotometer at 450 nm (VERSAmax, Molecular Devices).

[0157] 4.2. Melanogenesis

[0158] 4.2.1. Culture and treatments

[0159] Melanocytes were seeded in 24-well plates and cultured in normoxic medium for 24 hours. The culture medium was then renewed, and the cells were cultured under hypoxia for an additional 24 hours. The medium was then replaced with culture medium containing or not (control) the compound or the solvent control (DMSO - 0.015%), and the cells were incubated under hypoxia for 10 days, with repeat treatments after 3 and 7 days of incubation.

[0160] All experimental conditions were carried out in n=5, except for the reference in n=3.

[0161] 4.2.2. Melanin assay

[0162] At the end of the incubation, melanin was extracted by cell lysis with a 0.5 N NaOH solution.

[0163] The optical density (OD) of the samples was measured at 405 nm, and then the amount of melanin was determined by comparison with a range of exogenous melanin (melanin curve including standards from 0.39 to 100 pg / ml). The results are expressed as pg / ml of melanin, as a percentage of the control.

[0164] 5. Data Processing

[0165] The raw data was transferred and processed using Microsoft Excel® software.

[0166] Intergroup comparisons were performed using the unpaired two-tailed Student's t-test. Statistical analyses can be interpreted if n > 5; however, for n < 5, the calculated data are provided for illustrative purposes only.

[0167] Formulas used in this report:

[0168] Standard error of the mean: esm = standard deviation (Sd) / - / ” n The standard error of the mean (SEM) represents the deviation of the sample mean from the true population mean. The SEM is calculated by dividing the Sd by the square root of the sample size.

[0169] Percentage of viability: viability (%) = (compound OD / control OD) x 100

[0170] RESULTS

[0171] 1. Production of ROS by keratinocytes stimulated with H2O2 under hypoxic conditions

[0172] Table 11: Effect of compound MEST22M01 on ROS production by keratinocytes stimulated with H2O2 under hypoxic conditions [Table 11] (1): Statistical significance threshold ns: > 0.05, Not significant * : 0.01 to 0.05, Significant ** : 0.001 to 0.01, Very significant ***: < 0.001, Extremely significant RFU: Relative Fluorescence units

[0174] Under the experimental conditions of this test, when the keratinocytes were cultivated in hypoxia, their stimulation by H2O 2 allowed us to observe a high level of reactive oxygen species (12972 RFU).

[0175] Treatment of cultured cells under hypoxia with the solvent control (DMSO tested at 0.015%) had no significant effect on the production of reactive oxygen species. In comparison with the solvent control, the compound MEST22M01, tested at 0.0015% under hypoxia, had no effect on the production of reactive oxygen species in H2O2-stimulated keratinocytes.

[0176] 2. Melanocytes under hypoxic conditions

[0177] 2.1. Preliminary cytotoxicity test

[0178] Table 12: Effect of compound MEST22M01 on the viability of lightly pigmented melanocytes after 48 hours of incubation in hypoxia, the last 24 hours of which were in the presence of the compound [Table 12] Legends + : normal population; : growth reduction; - : toxicity; 0 : cell death; g: compound granules; op: opacity due to the compound; * : morphological changes; ag: agglutinated cells

[0179] The results of the WST-8 viability test and the observation of the cell mats led to the selection of the concentration to be tested in the following example: Concentration tested: 0.0015% (0.015% DMSO).

[0180] 2.2. Effect on melanogenesis

[0181] Table 13: Effect of compound MEST22M01 on melanin synthesis in lightly pigmented melanocytes under hypoxic conditions [Table 13] (1): Statistical significance threshold ns: > 0.05, Not significant * : 0.01 to 0.05, Significant ** : 0.001 to 0.01, Very significant *** : < 0.001, Extremely significant

[0182] Compared to the control, treatment of cultured melanocytes under hypoxia with the DMSO solvent control, tested at 0.015% for 10 days, did not modulate the amount of melanin in the melanocytes. In comparison with this solvent control, the compound MEST22M01, tested at 0.0015% under hypoxia, had no significant effect on melanin synthesis by melanocytes.

[0183] CONCLUSION

[0184] The results obtained indicate that, under the experimental conditions of this study, the compound MEST22M01, at the tested concentration, has no effect on the parameters evaluated (production of reactive oxygen species by keratinocytes stimulated by H2O2 in hypoxia and synthesis of melanin by melanocytes cultured in hypoxia).

[0185] By linking this conclusion with the conclusion of Example 1, it is observed that the compound MEST22M01 increased cellular respiration, without inducing an increase in the production of reactive oxygen species; thus playing its role as an antioxidant: since without antioxidants, an increase in cellular respiration mechanically leads to an increase in the production of reactive oxygen species (Gardès-Albert M., et al. “Reactive oxygen species: How can oxygen become toxic?” (2003); Cadenas E., et al. Mitochondrial free radical generation, oxidative stress, and aging. Free Radie Biol Med 29, 222-230 (2000); Migdal C., et al. Reactive oxygen species and oxidative stress. Med Sci (Paris) 27, 405-412 (2011); Rinnerthaler M., et al. Oxidative Stress in Aging Human Skin. Biomolecules 5, 545-589 (2015)).

[0186] Example 3: Evaluation of the composition according to the invention, where melatonin is in 2 different forms, on cellular oxygenation on ex vivo human skin expiants

[0187] INTRODUCTION

[0188] This study aims to evaluate the action of an active ingredient in two forms on cellular metabolism (oxygenation) on ex vivo human skin expiants.

[0189] The ex vivo phase will allow for the reproduction of product application on the skin. The histological phase will allow for the evaluation of changes in biological parameters through staining and / or immunostaining.

[0190] The analytical phase will allow us to evaluate mitochondrial density, as well as the activity of 2 complexes of the mitochondrial respiratory chain.

[0191] The activity will be evaluated by:

[0192] A measurement of citrate synthase activity;

[0193] A measurement of cytochrome c oxidase activity (Complex IV);

[0194] An ATP assay (product of Complex V activity).

[0195] MATERIALS AND METHODS

[0196] Study outline: - The expiators were put in survival mode at Jl. - On day J0, the products are applied to the expiants. - On day 1, the products are applied to the expiants. - On day 2, the products are applied to the expiants, then the expiants are stopped after 8 hours of incubation.

[0197] Products tested - Product A = Lavandula Hybrida extract - Product B = Melaleuca Alternifolia extract - Product C = melatonin in powder form - Product D = melatonin in liposomal form

[0198] Preparation of the expiatory

[0199] Thirteen expiants of 11±1 mm in diameter were prepared from an abdominal plasty of a 37-year-old woman (reference P2933-AB37) with a phototype II according to the Fitzpatrick classification.

[0200] At Jl, the expiants were placed in survival mode in BEM (BIO-EC's Expiants Medium) at 37°C in a humid atmosphere, enriched with 5% CO2.

[0201] The study is conducted on skin tissue obtained from surgical waste (cosmetic surgery) from a donor, in accordance with the Declaration of Helsinki and Article L.1243-4 of the French Public Health Code. This study does not require prior authorization from an ethics committee for the collection and use of surgical waste.

[0202] Distribution of expiators

[0203] The expiators were divided into 3 groups as follows: - Lot T: DPBS IX - 3 expiants - Downtime on J2 - Batch PI: Product PI = C (melatonin powder) at 0.0015% + A (Lavandula Hybrida) at 1% + B (Melaleuca Alternifolia) at 1% + DMSO 0.015% in DPBS IX - 5 expiants - Stop time on Day 2 - Batch P2: Product P2 = D (liposomal melatonin) at 0.0015% + A (Lavandula Hybrida) at 1% + B (Melaleuca Alternifolia) at 1% in DPBS IX - 5 expiants - Stop time on Day 2

[0204] Product preparation: The products were prepared on day 0 and then stored at 4°C throughout the ex vivo phase.

[0205] Product application

[0206] On days 0, 1 and 2, for the expiry of batch T, DPBS IX was applied topically, at a rate of 5 liters per 1 cm² expiry area (~2 mg / cm²) 2 ) and spread using a spatula.

[0207] On days JO, J1, and J2, for the expiants of the batches concerned, products Pl and P2 were applied topically at a rate of 5 pl per 1 cm² expiant (~5 mg / cm²). 2) and spread using a spatula.

[0208] Samples

[0209] On day 2 (8 hours after the last application), 3-5 expiants from each batch were collected.

[0210] Histological treatments

[0211] After 24 hours in buffered formalin, the samples were dehydrated and impregnated with paraffin using a Leica PEARL automated dehydration system. They were then block-formed using a Leica EG 1160 embedding station.

[0212] 5 µm sections were made using a Minot type microtome, Leica RM 2125 and mounted on histological glass slides.

[0213] The frozen samples were cut into sections of 7 µm or 20 µm thickness using a Leica CM 3050 cryostat. The sections were then mounted on different supports depending on the type of shipment.

[0214] Microscopic observations were carried out using optical microscopy, with the aid of a Leica DMLB type microscope, Olympus BX43 or Olympus BX63.

[0215] The shots were taken with an Olympus DP72 or DP74 camera and the cellSens software (Olympus).

[0216] Control of cell viability

[0217] Cell viability was observed on paraffin sections after staining with Masson's trichrome Goldner variant.

[0218] It was evaluated by microscopic examination.

[0219] Biochemical and enzymatic assays

[0220] The assays were performed using excipients frozen at -80°C. The frozen skin excipients were ground in phosphate buffer pH=7.8 using a turbine homogenizer (Turax), then subjected to ultrasonic cell disruption (Ultrasonic disruptor, Bransonic). After centrifugation at 4000 rpm at 18°C, the supernatant was collected for the assays.

[0221] Total protein assay: Total proteins were detected by an automated Bradford technique, using the "Bradford reagent" from Sigma, ref B6916. The total protein concentration of the expiants (3-5 expiants per batch) is expressed in g / L (see annexes).

[0222] Citrate synthase assay: Citrate synthase activity was measured using the colorimetric technique of Srere (1969), based on measuring the appearance of coenzyme A, which carries a sulfhydryl (SH) group. This group reacts with dinitrothiobenzoic acid (DTNB) added to the reaction medium. The reaction is monitored by measuring the optical density at a wavelength of 415 nm. Citrate synthase activity (3-5 excipients per batch) is expressed in U / g of protein.

[0223] Complex IV - Cytochrome C Oxidase Assay: Complex IV activity was measured by spectrophotometric assay at 546 nm. Complex IV activity (3-5 expiants per batch) is expressed in U / g of protein.

[0224] ATP assay: ATP was measured by chemiluminescence using the CellTiter-Glo™ reagent (Promega, ref. G755A, lot 494567) according to the method described by Kimmich et al. Analytical Biochem, 1975, 69: 187-206. The ATP concentration of the expiants (3-5 expiants per lot) is expressed in pmol / g of protein.

[0225] Statistical analysis: Wilcoxon-Mann-Whitney test

[0226] The Wilcoxon-Mann Whitney test allows for the comparison of two independent and small samples, namely each product versus the control on the same day. The difference between two batches is significant if p < 0.1 (*), i.e., a 90% probability that two batches are significantly different, or p < 0.05 (**), i.e., a 95% probability that two batches are significantly different, or p < 0.01 (***), i.e., a 99% probability that two batches are significantly different.

[0227] * : significant with p<0.1 (90%)

[0228] ** : significant with p<0.05 (95%)

[0229] *** : significant with p<0.01 (99%)

[0230] Ns: not significant

[0231] RESULTS

[0232] Citrate synthase activity

[0233] Table 14: Citrate synthase activity [Table 14]

[0234] The results are presented in Figure 10.

[0235] Product 1 (P1J2) induces a significant increase of 71%*

[0236] Product 2 (P2J2) induces a significant increase of 83%*

[0237] Wilcoxon-Mann Whitney Test

[0238] Non-significant = ns; Significant *: p<0.1 (90%) **: p<0.05 (95%) ***: p<0.01 (99%)

[0239] Activity of complex IV

[0240] Table 15: Activity of Complex IV [Table 15]

[0241] The results are presented in Figure 11.

[0242] Product 1 (P1J2) induces a non-significant increase of 70% ns

[0243] Product 2 (P2J2) induces a significant increase of 83%*

[0244] Wilcoxon-Mann Whitney Test

[0245] Non-significant = ns; Significant *: p<0.1 (90%) **: p<0.05 (95%) ***: p<0.01 (99%)

[0246] ATP assay

[0247] Table 16: ATP Assay [Table 16]

[0248] The results are presented in Figure 12.

[0249] Product 1 (P1J2) induces a non-significant increase of 28% ns

[0250] Product 2 (P2J2) induces a non-significant increase of 143% ns

[0251] Wilcoxon-Mann Whitney Test

[0252] Non-significant = ns; Significant *: p<0.1 (90%) **: p<0.05 (95%) ***: p<0.01 (99%)

[0253] DISCUSSION

[0254] Products PI and P2 induce a statistically significant increase in mitochondrial density, evident in the rise in citrate synthase activity. Product P2 stimulates mitochondrial respiratory chain activity, via its complex IV, in a statistically significant manner; and via its complex V, in a non-significant but substantial manner, evident in the increased production of ATP (cellular energy).

[0255] Example 3: Evaluation of the transcutaneous penetration of "melatonin" based products by Raman micro-imaging on sections of human skin

[0256] Products tested:

[0257] Number of products: 3 (liposomal melatonin, vectorized melatonin and free melatonin)

[0258] - M Lip: Liposomal melatonin

[0259] - M Vect: Vectorized melatonin Batch: 230922001

[0260] - M Free: Free Melatonin

[0261] These products were tested at a concentration of 1% melatonin.

[0262] - For the preparation of liposomal melatonin: the initial solution with a a 4% concentration was diluted in PBS.

[0263] - Vectorized melatonin Batch: 230922 001 (1% melatonin concentration)

[0264] For the preparation of free melatonin: Feasibility tests were carried out for the solubilization of pure melatonin in ethanol, PBS, and DMSO. (See feasibility test results.)

[0265] Melatonin (1%) is not soluble in PBS, but it is soluble in DMSO and ethanol.

[0266] To minimize the potential effects of DSMO and ethanol on the skin, 1% melatonin was solubilized in a PBS solution with 10% DMSO. Dissolving the melatonin required heating to 37°C and stirring for 30 minutes.

[0267] The three 1% melatonin products were prepared

[0268] Skin samples and processing:

[0269] Skin sample:

[0270] A skin expungement was provided by BIOPREDIC INTERNATIONAL, (RENENS, France). Reference product: PRE006 (BIOPREDIC) / Batch: SKIN0S2K030.

[0271] The fresh skin samples used in this study came from a donor with phototype (II):

[0272] Donor information:

[0273] - Skin type: Caucasian phototype (II), fresh skin (full thickness).

[0274] - Sex: female

[0275] - Anatomical site: Abdomen

[0276] Four expiants of fresh abdominal skin (1.5 to 2 cm2) from the same donor are cut and distributed as follows (1 expiant per condition):

[0277] - 1 expiatory for the negative control (without treatment)

[0278] - 1 expiatory for treatment with M Lip: Liposomal melatonin

[0279] - 1 expiant for treatment with M Free: Free Melatonin

[0280] - 1 expiant for treatment with M Vect: Vectorized melatonin [Table 17]

[0281] Treatment of expiatory persons:

[0282] The products were applied topically to the surface of the expiry samples (5 mg / cm²) and then incubated at 37°C. After the incubation period (T8H), the surface of the expiry samples was cleaned to remove any excess product. An untreated expiry sample was used as a negative control.

[0283] Analysis by Raman micro-imaging:

[0284] - After application of the products, the expiants are frozen at -80 °C and then cut longitudinally using a Cryotome with a thickness of 16 pm.

[0285] - For each expat, 3 sections of skin tissue were selected and deposited on a specific CaF2 support for Raman imaging analysis.

[0286] Skin sections on a specific support for Raman (CaF2) analysis for each product

[0287] In total, 12 Raman images were recorded (3 sections per expiant (n = 3) and one image per section).

[0288] - 4 other adjacent sections of 7 µm thickness were made for HE staining control.

[0289] - The Raman images have a size of Y: 10pm / X: 135pm with a step of 5pm in X and 5pm in Y.

[0290] - Each Raman image contains 3 Y spectra and 29 X spectra (87 spectra per image).

[0291] Raman image acquisition parameters:

[0292] - Laser wavelength: 660 nm,

[0293] - Objective: 100X, long focal length with a numerical aperture of 0.75

[0294] - Acquisition time: 30 seconds,

[0295] - Accumulation: IX - Spectral range: 400 to 3100 cm-1

[0296] - Scraping: 1200T

[0297] - Confocal hole: 500 pm,

[0298] - Slit width: 100 pm (spectral resolution 6.5 cm-1)

[0299] - Not in X: 5pm, Not in Y: 5pm

[0300] To ensure the reproducibility of measurements: Before each use, the Raman spectrometer is calibrated with silicon, which gives a Raman peak of 520.7 cm-1. Continuous monitoring of the laser power at the sample level is carried out.

[0301] Raman image preprocessing:

[0302] - Elimination of aberrant spectra (fluorescence, burn-in, saturation, offset....)

[0303] - Baseline correction

[0304] - Spectral smoothing and thinning.

[0305] - Spectral normalization

[0306] Processing of spectral image analysis:

[0307] The processing of the corrected data maps was performed using in-house software based on the least-squares fitting method, which runs within the Matlab environment. This method involves mathematically modeling the reference spectra within the overall spectral image to determine the contribution and distribution of these spectra. In our case, we used the mean spectra of the "Melatonin" product and the control skin as reference spectra.

[0308] Results :

[0309] Determination of the reference spectra of "1% Melatonin" products:

[0310] The first step in this work was to determine the reference Raman spectra of the products and the skin control. To do this, several Raman measurements were repeated on drops of each product, and the average reference spectrum was calculated to account for the heterogeneity of the products.

[0311] To assess the spectral difference between the products and the control skin, the reference spectra were compared (Figure 13).

[0312] The products give Raman spectra with characteristic peaks and do not generate fluorescence background with 660 nm laser excitation in the spectral region of interest from 400 to 3100 cm-1.

[0313] The Raman spectra of the "Melatonin" products (Pure Melatonon, M Lip, M Free and M Vect) show a different spectral signature.

[0314] Specific markers of pure melatonin (highlighted peaks) can be detected in M ​​Lip, M Free and M Vect products at 1% concentration.

[0315] The spectral difference between the reference spectra of melatonin-based products (M Free, M Lip, M Vect) and the skin control is observed. (Highlighted peaks)

[0316] Comparing the spectra of melatonin-based products with skin control helps to determine the most relevant spectral characteristics that can be used as markers to track product penetration and distribution in skin sections.

[0317] Raman image analysis:

[0318] Determination of the level of penetration and distribution of "Melatonin" products in skin sections:

[0319] After image preprocessing, the spectral images were reconstructed using a fitting procedure that takes into account the reference spectra of the products and the skin control. The spectrum of each pixel is represented by a linear combination of the corresponding reference spectra.

[0320] The adjustment coefficients were averaged over 3 measurements (n=3) taken on different but adjacent skin sections. The reconstructed spectral images allow visualization of the permeation level and spatial distribution of each product in the epidermal and dermal layers down to 135 pm. after 8 hours of sampling time. (Essendoubi et al. Skin Research and Technology 2016; 22: 55-62 (Human skin penetration of hyaluronic acid of different molecular weights as probed by Raman spectroscopy)

[0321] Conclusions:

[0322] The results are presented in Figure 14.

[0323] A weak background signal was detected in untreated skin (negative control).

[0324] No penetration was observed with the free melatonin product.

[0325] Both products, liposomal melatonin and vectorized melatonin, penetrate the skin at the level of the epidermal layer.

[0326] Liposomal melatonin and vectorized melatonin products have different distribution profiles.

[0327] The Raman signal of the liposomal melatonin product (M Lip) is detected mainly at the SC level at approximately 0-15pm;

[0328] The Raman signal of vectored melatonin (M Vect) is detected mainly at the level of the epidermis by forming a reservoir in the SC at approximately 20-30 pm and the signal of the product is detected up to 55 pm deep in the epidermis.

[0329] The Raman signal of the liposomal melatonin product (M Lip) is less intense than that of the vectored melatonin product (M Vect).

[0330] An improvement in product penetration is observed with the vectorized melatonin product (M Vect).

[0331] Example 4: Semi-quantitative representation of the penetration of skin products following example 3

[0332] Semi-quantitative analysis procedures for product penetration:

[0333] After processing and normalizing the Raman spectral images, the distribution of skin products was reconstructed using a fitting procedure, and the correlation of the visible image of the skin section and the fitting mean image of the product distribution is presented as a flow.

[0334] The adjustment data can be presented in the form of tables and graphs:

[0335] The adjustment coefficients were averaged over 3 measurements taken on different skin sections and exported as a table of values ​​to determine the semi-quantitative percentage of product penetration.

[0336] These semi-quantitative percentages were calculated on the average image adjustment coefficients for each product after normalization.

[0337] The tables of adjustment coefficients can be presented as overall percentages calculated on the adjustment coefficients of the average image of each product after normalization according to the flow rate.

[0338] Results (shown in Figure 15):

[0339] No penetration was observed with the "Free Melatonin" product.

[0340] Skin penetration was observed with both products "Liposomal Melatonin" and "Vectorized Melatonin".

[0341] The product "Liposomal Melatonin" has a lower skin penetration than the product "Vectorized Melatonin".

[0342] The "Vectorized Melatonin" product exhibits better skin penetration efficacy than the "Liposomal Melatonin" and "Free Melatonin" products.

[0343] Example 5: Evaluation of the efficacy of liposomal melatonin at a concentration of 0.0015% alone and in combination with 1% extract of the oil of Melaleuca Altemifolia leaves, and 1% extract of the oil of Lavandula Hybrida flowers, on ex vivo human skin expiants at basal level.

[0344] Study objective:

[0345] This study evaluated the efficacy of liposomal melatonin at a concentration of 0.0015%, alone and in combination with Melaleuca Alternifolia leaf oil extract and 1% Lavandula Hybrida flower oil extract, on ex vivo human skin expiants at baseline. The evaluation focused on the following key markers:

[0346] - ATP: Vital indicator of cellular energy status, strongly linked to mitochondrial activity, tissue oxygenation and regeneration.

[0347] - Complex 4 activity: Cytochrome C oxidase, an essential enzyme of the mitochondrial respiratory chain, plays a key role in metabolism and oxidative phosphorylation (OXPHOS), processes that generate ATP and promote oxygenation and tissue regeneration.

[0348] Experimental procedure

[0349] A. Culture of human organotypic skin explants

[0350] The excipients were obtained with the informed consent of a 35-year-old Caucasian woman (phototype II / III, Ref SKIN0S4L124) during abdominal surgery. After surgery, they were kept alive by culture on metal grids in standard 12-well contact plates in OxiProteomics® medium at 37°C in 5% CO2 humidified air.

[0351] B. Product application, stress testing, and sampling

[0352] Upon receipt, the skin excipients were divided into 3 experimental groups (control - untreated / / 0.0015% liposomal melatonin = 1 application at D0, D1, and D2 / / liposomal melatonin at a concentration of 0.0015%, in combination with 1% Melaleuca Alternifolia leaf oil extract and 1% Lavandula Hybrida flower oil extract, 1 application at D0, D1, and D2 / / with sampling 8 hours after the last treatment at D2). The culture medium was renewed every 24 hours. The active ingredients were prepared according to the information provided by the sponsor (see appendix, page 9).

[0353] The expiry were treated topically (30 pL / cm2) with the ingredients active on day 0, day 1 and day 2. Eight hours (8h) after the last application (day 2), skin expiants were sampled, transferred into OCTs for cryopreservation, flash-frozen in liquid nitrogen and stored at -80°C until analysis.

[0354] Protein extraction and ATP quantification

[0355] Skin samples were subjected to protein extraction in an aqueous buffer using the buffer optimized for each ELISA assay. Accurate measurement of protein concentration was performed using Bradford Protein Assay Dye Reagent (Bio-Rad) according to the manufacturer's instructions. Accurate measurement of ATP levels (Abeam, ab83355) was performed using an ELISA kit according to the manufacturer's instructions. Absorbance was acquired using the Varioskan system (ThermoFisher).

[0356] Protein extraction and complex 4 activity

[0357] The activity of complex 4 is determined by colorimetry (Abeam, ab!09909) by monitoring the oxidation of reduced cytochrome c, indicated by a decrease in absorbance at 550 nm. The overall reaction is as follows:

[0358] 4 cytochrome c- + 4 H+ + 4 H+ (in) + 02 4 cytochrome c + + 2 H2O +4 H+ (out)

[0359] Extraction was performed according to the manufacturer's instructions. Specific absorbance values ​​(OD=550 nm) were collected over time, and linear kinetic ranges were used for analysis with the Varioskan system (ThermoFisher). White background was assessed using a solution containing only the extraction buffer. The activity of complex 4 was calculated using the slope of the kinetic curve for each replicate.

[0360] Illustration of histograms and statistical analysis

[0361] Five (5) replicates of the slope values ​​of ATP activity or complex 4 were collected per batch and normalized against the mean of the control (considered to be 100%), ultimately obtaining a relative mean value and a standard deviation for each group.

[0362] Statistical analyses were performed using the "GraphPad" software (La Jolla, California, USA) using the unpaired t-test with Welch's correction for binary analyses vs control group (95% confidence interval).

[0363] The raw data and their integrations are included in the Annex- D02SB2024.pptx file.

[0364] An induction value (%) was obtained for the experimental groups using the following formula: Biomarker level (group X) Induction % (group X) = - 1 ' 100 Biomarker level (Control)

[0365]

[0366] Results - ATP Level

[0367] ATP detection from skin explants was performed by a test ELISA. The quantification of ATP concentrations was measured in each sample and reported in Table 18.

[0368] The presence of 0.0015% liposomal melatonin alone or combined with A 1% extract of Melaleuca Alternifolia leaf oil and a 1% extract of Lavandula Hybrida flower oil significantly increase ATP levels. The results are presented in Table 18.

[0369] [Table 18] Table 18: ATP Levels

[0370] Results - Activity levels of complex 4

[0371] The activity of complex 4 was determined colorimetrically by monitoring the oxidation of reduced cytochrome C, indicated by a decrease in absorbance at 550 nm. The activity value of complex 4 was calculated using the slope of the kinetic curve for each group. The data are presented in Table 19 and Figure 16. The overall reaction is as follows:

[0372] 4 cytochrome c- + 4 H+ + 4 H+ (in) + 02 4 cytochrome c + + 2 H2O +4 H+ (out)

[0373] The presence of 0.0015% liposomal melatonin alone or combined with 1% extract of Melaleuca Alternifolia leaf oil, and 1% extract of Lavandula Hybrida flower oil significantly increases the activity levels of complex 4.

[0374] [Table 19] Table 19: Activity levels of complex 4

[0375] Figure 16 shows the activity levels of complex 4.

[0376] Conclusion

[0377] Compared to the control group, topical application of liposomal melatonin at a concentration of 0.0015% resulted in a significant 37% increase in ATP levels. This increase reached 321% when combined with 1% Melaleuca Alternifolia leaf oil extract and 1% Lavandula Hybrida flower oil extract. Compared to the control group, topical application of liposomal melatonin at a concentration of 0.0015% resulted in a significant 104% increase in complex 4 activity levels. This increase reaches over 650% when combined with 1% Melaleuca Alternifolia leaf oil extract and 1% Lavandula Hybrida flower oil extract.

[0378] Taken together, these results show that liposomal melatonin stimulates mitochondrial activity and function. The combination of liposomal melatonin with extracts of Melaleuca Alternifolia and Lavandula Hybrida had a greater stimulating effect than liposomal melatonin alone.

Claims

Demands

1. A non-therapeutic cosmetic composition to increase cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis in a healthy subject, comprising: - An extract of Melaleuca Alternifolia, preferably oil from the leaves of Melaleuca Alternifolia; - An extract of Lavandula Hybrida, preferably Lavandula Hybrida flower oil; - Melatonin.

2. A non-therapeutic cosmetic composition according to the preceding claim, comprising: - Between 0.00001% and 2%, preferably between 0.00001% and 1.5%, even more preferably between 0.01% and 1%, by weight of the composition of said extract of Melaleuca Alternifolia.

3. A non-therapeutic cosmetic composition according to any one of the preceding claims, comprising: - Between 0.00001% and 2%, preferably between 0.00001% and 1.5%, even more preferably between 0.01% and 1%, by weight of the composition of said Lavandula Hybrida extract.

4. A non-therapeutic cosmetic composition according to any one of the preceding claims, comprising: - Between 0.00001% and 10%, preferably between 0.00001% and 1%, by weight of the melatonin composition.

5. A non-therapeutic cosmetic composition according to any one of the preceding claims, comprising: - Between 0.00001% and 2%, preferably between 0.00001% and 1.5%, even more preferably between 0.01% and 1%, by weight of the composition of said extract of Melaleuca Alternifolia; - Between 0.00001% and 2%, preferably between 0.00001% and 1.5%, even more preferably between 0.01% and 1%, by weight of the composition of said Lavandula Hybrida extract; - Between 0.00001% and 10%, preferably between 0.00001% and 1%, by weight of the melatonin composition.

6. A non-therapeutic cosmetic composition according to any one of the preceding claims, said composition comprising in in addition to at least one cosmetically acceptable agent chosen from among soothing agents, restructuring agents, regenerating agents, revitalizing agents, sunscreens, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, smoothing agents, agents strengthening the firmness, elasticity and / or barrier effect of the skin, antioxidants, opacifiers, thermal waters, mattifying agents, chemical or mineral filters, trace elements, stabilizing agents, foaming agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestrants and chelators, perfumes, filters, essential oils, coloring materials, pigments, hydrophilic or lipophilic actives, lipid vesicles encapsulating one or more actives and / or preservatives.

7. A non-therapeutic cosmetic composition according to any one of the preceding claims, said composition being formulated as a cream, ointment, balm, mask, milk, lotion, serum, spray, paste, foam, aerosol, stick, shampoo, conditioner, patches, aqueous hydroalcoholic or oily solution, oil-in-water or water-in-oil or multiple oil-in-water emulsion, aqueous or oily gel, anhydrous liquid, paste or solid product, and / or an oil dispersion in an aqueous phase using spherules, such spherules being polymeric nanoparticles such as nanospheres and nanocapsules or ionic lipid vesicles and / or non-ionic.

8. A non-therapeutic cosmetic skincare method for a healthy subject, comprising a step of applying a cosmetic composition according to any one of claims 1 to 7 to the skin of said subject.

9. Non-therapeutic cosmetic use of a composition according to any one of claims 1 to 7, to increase cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis in a healthy subject.

10. Kit for the preparation of a non-therapeutic cosmetic composition to increase cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis, comprising: - An extract of Melaleuca Alternifolia, preferably oil from the leaves of Melaleuca Alternifolia; - An extract of Lavandula Hybrida, preferably Lavandula Hybrida flower oil; - Melatonin.

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