Active complex to increase 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 mitochondrial cellular respiration of keratinocytes underlying the stratum corneum of the epidermis, comprising a complex of active ingredients: extracts of Melaleuca Alternifolia, 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.

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 for increasing 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 move 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 protects the skin against external aggressions such as infections, chemicals, and dehydration.

[0004] Mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis plays an essential role in the production of energy necessary for their survival and function. Keratinocytes go through several stages of differentiation before becoming corneocytes and require energy for lipid synthesis, cell proliferation, and the stress response, among other things (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). - Baumgärtl 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 / 978-3-642-71 226-5_15).

[0005] The mitochondria of keratinocytes produce ATP by 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, keratinocyte mitochondria participate in the regulation of apoptosis, which is 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 of 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, preferably of the oil from the leaves, of Melaleuca Altemifolia.

[0010] According to one embodiment, said cosmetic composition comprises an extract, preferably of the flower oil, 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, preferably of the oil from the leaves, of Melaleuca Altemifolia; • An extract, preferably of flower oil, of Lavandula Hybrida; • Melatonin.

[0012] According to another aspect, the invention relates to the non-therapeutic cosmetic use of a composition comprising: to increase cellular mitochondrial respiration of keratinocytes underlying the stratum corneum of the epidermis in a healthy subject • An extract, preferably of the oil from the leaves, of Melaleuca Altemifolia; • An extract, preferably of flower oil, of Lavandula Hybrida; • 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 Altemifolia and Lavandula Hybrida, and said melatonin.

[0014] According to another aspect, the invention relates to a 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, preferably of the oil from the leaves, of Melaleuca Altemifolia; • An extract, preferably of flower oil, of Lavandula Hybrida; • 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 following embodiments 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 1% by weight of the composition of said extract, preferably of the oil of the leaves, of Melaleuca Altemifolia.

[0018] Preferably, said non-therapeutic cosmetic composition comprises: • Between 0.01% and 1% by weight of the composition of said extract, preferably of the oil of the leaves, of Melaleuca Altemifolia.

[0019] 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% by weight of the composition of said extract, preferably of the oil of the leaves, of Melaleuca Altemifolia.

[0020] According to one embodiment, said non-therapeutic cosmetic composition comprises: • Between 0.00001% and 1% by weight of the composition of said extract, preferably of flower oil, of Lavandula Hybrida.

[0021] Preferably, said non-therapeutic cosmetic composition comprises: • Between 0.01% and 1% by weight of the composition of said extract, preferably of the oil of the leaves, of Lavandula Hybrida.

[0022] 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% by weight of the composition of said extract, preferably of flower oil, of Lavandula Hybrida.

[0023] According to one embodiment, said non-therapeutic cosmetic composition comprises: • Between 0.00001% and 10% by weight of the melatonin composition.

[0024] Preferably, said non-therapeutic cosmetic composition comprises: • Between 0.00001% and 1% by weight of the melatonin composition.

[0025] 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.

[0026] According to one embodiment, said melatonin is vectorized.

[0027] Preferably, said melatonin is delivered via a liposome delivery system. Even more preferably, said liposome delivery system comprises water, phospholipids, melatonin, and Lactobacillus cultures.

[0028] 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.

[0029] According to one embodiment, said non-therapeutic cosmetic composition comprises: • Between 0.00001% and 1% by weight of the composition of said extract, preferably of the oil of the leaves, of Melaleuca Altemifolia. • Between 0.00001% and 1% by weight of the composition of said extract, preferably of flower oil, of Lavandula Hybrida. • Between 0.00001% and 10% by weight of the melatonin composition.

[0030] More specifically, the composition according to the invention increases the respiration and energy production of the epidermis of a subject. This results in skin with a more beautiful appearance for the user, giving a visual impression of healthier skin.

[0031] 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, redness) that might deter the consumer from using this composition.

[0032] According to one embodiment, the cosmetic composition according to the invention further comprises at least one cosmetically acceptable agent.

[0033] 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 agents, pigments, hydrophilic actives or lipophilic,Lipid vesicles encapsulating one or more active ingredients and / or preservatives.

[0034] According to one embodiment, the cosmetic composition according to the invention is administered by topical application to an area of ​​the skin.

[0035] 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.

[0036] According to one embodiment, an amount of about 0.1 to 50 mg / cm2, preferably about 0.25 to 25 mg / cm2, even more preferably about 2 to 10 mg / cm2, of cosmetic composition according to the invention is applied to the area of ​​skin.

[0037] 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.

[0038] 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.

[0039] Preferably, according to the invention, said area of ​​skin is the face. Figures

[0040] [Fig-1] is a graph showing the oxygen consumption profiles of NHEK cultured for 48 hours + 30 minutes under hypoxia

[0041] [Fig 2] A, B and C are graphs showing the mitochondrial respiration parameters of NHEK cultured for 48 hours + 30 minutes under hypoxia

[0042] [Fig.3] is a graph showing the oxygen consumption profiles of NHEK cultured for 48 hours + 2 hours under hypoxia

[0043] [Fig 4] A, B and C are graphs showing the mitochondrial respiration parameters of NHEK cultured for 48 hours + 2 hours under hypoxia

[0044] [Fig.5] B is a graph showing the oxygen consumption profiles of NHEK cultured over 48 hours + 24 hours.

[0045] [Fig 6] A, B and C are graphs showing the analysis of mitochondrial respiration parameters of NHEK cultured for 48 hours + 24 hours.

[0046] [Fig.7] is a graph showing the illustration of the ECAR of cultivated NHEK for 48 hours + 30 minutes in hypoxia.

[0047] [Fig.8] is a graph showing the illustration of the ECAR of NHEK cultured for 48 hours + 2 hours in hypoxia.

[0048] [Fig.9] is a graph showing the illustration of the ECAR of cultivated NHEK for 48 hours + 24 hours in hypoxia.

[0049] [Fig. 10] is a graph showing the effect of the products on citrate synthase activity, compared to the negative control TJ2.

[0050] [Fig. 11] is a graph showing the effect of the products on the activity of complex IV, compared to the negative control TJ2.

[0051] [Fig. 12] is a graph showing the ATP dosage, compared to the negative control TJ2. Definitions

[0052] The epidermis comprises 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 accumulating keratohyalin granules. The clear layer, visible only in thick skin, is a thin band of cells whose nuclei disintegrate, giving them a translucent appearance. Finally, the outermost layer is the stratum corneum.

[0053] The stratum corneum is composed of differentiated keratinocytes, called corneocytes. These keratin-rich cells lack a nucleus and organelles, providing a protective barrier against external aggressions such as infections, chemicals, and dehydration. This impermeable and robust layer is essential for skin protection. Corneocytes originate from keratinocytes in the deeper layers that migrate and transform over time. The stratum corneum is constantly renewed by desquamation, where dead cells are shed and replaced by new ones.

[0054] Keratinocytes are specialized epithelial cells found in all layers of the epidermis, but their density varies with layer 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 corneocytes 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 radiation, and water loss.

[0055] Mitochondrial cellular respiration of epidermal keratinocytes refers to the biochemical process by which keratinocytes located in the epidermis, prior to their differentiation into comeocytes and their 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 main source of cellular energy used to power essential cellular functions, such as cell division, differentiation, migration, and the synthesis of biomolecules.Maintaining or increasing 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" means that the composition according to the invention allows for 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 healthy subjects.

[0056] By "underlying the stratum corneum" is meant the keratinocytes present in the epidermis, "below" or "before" their migration into the stratum corneum and their differentiation into keratinocytes. These keratinocytes may therefore be present in the granulocytic layer, the spinous layer, or the basal layer of the epidermis.

[0057] By "non-therapeutic cosmetic" it is understood that the composition of the invention has a non-therapeutic technical effect that improves 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 subject.

[0058] 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 the improvement of the superficial visual appearance of the skin, preferably of the face.

[0059] "Melaleuca Altemifolia" 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, including terpenes and phenols, are then condensed and collected in liquid form. The Melaleuca Altemifolia oil obtained through this process is then filtered to remove impurities and obtain a pure oil, ready for use in the composition according to the invention. One example of such an extract is marketed under the name Regenight™.The latter comprises a fat-soluble fraction upcycled from waste from Australian tea tree distillation, and standardized in sesquiterpenes.

[0060] “Lavandula Hybrida”, also known as lavandin, is a plant A hybrid 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 distillation. Steam. During distillation, steam is passed through the flowers, heating the plants' oil glands and releasing volatile aromatic compounds. These compounds are then carried away with the steam and condensed to form a mixture of essential oil and water, which is then separated. The resulting lavandin oil 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 through a proprietary green process using plant-based solvents.

[0061] 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.

[0062] For the purposes of this invention, "topical application" means application to the skin (including the scalp) and mucous membranes.

[0063] 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.

[0064] By "epidermal barrier function", we mean, in the context of the present invention, the protective function of the epidermis, in particular against external aggressions, and the regulation of insensible water loss and ions.

[0065] The ingredients of said cosmetic composition according to the invention are combined in effective quantities. By "effective quantity" is meant, for the purposes of the present invention, a quantity sufficient to obtain the desired effect, which can be determined by a person skilled in the art.

[0066] For the purposes of this invention, "topical application" means application to the skin (including the scalp).

[0067] 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 lower and upper bounds specified. The examples below are presented for illustrative purposes only and are not exhaustive of the scope of the invention. Examples

[0068] Example 1: Effect of the composition according to the invention _ _( MEST22M01 ) on mitochondrial respiration in normal human epidermal keratinocytes under hypoxic conditions

[0069] INTRODUCTION

[0070] 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.

[0071] Previously, a cytotoxicity test was carried out under hypoxic conditions using a standard WST-8 reduction test in order to determine the concentrations to be tested.

[0072] MATERIALS AND METHODS

[0073] 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

[0074] 2. Tested compound

[0075] Composition: Melatonin in powder form at a concentration of 0.0015% by weight of the total composition.

[0076] Powder form

[0077] Intermediate solution: 10% in DMSO

[0078] Concentration tested: 0.0015% (0.015% DMSO)

[0079] 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 live cells and their metabolic activity. Optical density (OD) was measured using a spectrophotometer at 450 nm (SPARK, TECAN).

[0080] 4. Culture and processing

[0081] Keratinocytes were seeded in 24-well plates and cultured in culture medium for 24 hours under normoxia. 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 evaluation of mitochondrial respiration and glycolysis.

[0082] 5. Evaluation of mitochondrial activity

[0083] Seahorse XF technology (Agilent) allows simultaneous and real-time measurement of mitochondrial respiration and glycolysis within a micro-chamber.

[0084] Mitochondrial respiration is measured from the real-time oxygen consumption rate (OCR) of the 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.

[0085] The results are evaluated against several criteria: - Basal respiration: this corresponds to the oxygen consumption of cells for normal energy activity. The higher its value, the more active the cell. - ATP (Adenosine triphosphate) production 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.

[0086] In parallel with OCR, glycolysis is measured using the extracellular acidification rate (ECAR). ECAR is closely related to the decrease in pH caused by glycolytic activity. The unit of TECAR is therefore mpH / min (mpH = millipH units).

[0087] Extracellular acidification is the sum of two components: - respiratory acidification, in the form of CO2, - acidification from glycolysis, in the form of lactate - + H+.

[0088] The contribution of CO2 to total extracellular acidification was considered negligible by the measurement platform used here, the Seahorse XF analyzer.

[0089] 6. Data processing

[0090] The raw data were transferred and processed using Microsoft Excel® software and GraphPad PRISM® software.

[0091] 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.

[0092] Formulas used in this example: - Standard error of the mean: esm = standard deviation (Sd) / ^n

[0093] 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

[0094] RESULTS

[0095] 1. Preliminary cytotoxicity test

[0096] Table 1: Cytotoxicity results of the active ingredient MEST22M01 [Tables 1] Control So Ivant (DMS O) MEST22M01 Hypo xia Hypoxia Hypoxia / 0.15% 0.00015 % 0.0005 % 0.001 % 0.0015 % 0.0075 % 0.015 % Viability 98 98 85 92 93 90 89 76 (% control 99 102 96 96 93 83 97 60 in Hypoxia) 102 97 104 74 72 95 82 72 Mean e 100 99 95 87 86 89 89 69 Esm 1 2 5 7 7 4 4 5 p(i) - ns ns ns ns * ns ** (1): Statistical significance threshold

[0097] ns: > 0.05, Not significant

[0098] *: 0.01 to 0.05, Significant

[0099] ** : 0.001 to 0.01, Very significant

[0100] *** : < 0.001, Extremely significant

[0101] In the remainder of the study, the concentration "0.0015%" will be used.

[0102] 2. Effect on mitochondrial respiration

[0103] 2.2. Treatments 48 hours + 30 minutes

[0104] The analysis of mitochondrial respiration after a 48-hour treatment followed by a further 30-minute treatment, under hypoxia, is described in [Fig. 1] and in the following tables:

[0105] Tables 2, 3 and 4: Results of mitochondrial respiration of NHEK cultured for 48 hours + 30 minutes under hypoxia [Tables 2] Treatment Basal Breathing Condition Tested Compounds Basal Breathing Concentration (pmol / m in) Mean (pmol / min) Esm (pm ol / min) % Solvent Control (DMS O) Esm (%) p(i) Hypoxia Control - 4.8(2) 20.2 1.1 165 9 ne 21.2 19.1 Solvent control (DMSO) 0.015% 11.8 12.2 1.0 100 8 - 14.1 10.8 MEST22M01 0.0015% 1.3(2) 24.1 1.3 197 11 ne 22.8 25.4 [Tables 3] Treatment ATP Production Cond Compounds Concentration Average Production Esm (pm % Esm p(1) ition of eu Iture tés ation on d'ATP P (pmol / min) (pmol / min) ol / min) control solvent (%) (DMS O) Hypoxia Control - 6.5® 10.5 0.7 123 8 ne 11.1 9.8 Solvent control (DMSO) 0.015% 7.5 8.5 1 100 12 - 10.6 7.6 MEST22M01 0.0015% 3.2® 14.1 0.4 165 5 ne 13.7 14.5 [Tables 4] Treatment Maximum Respiration Condition of Water Tested Compounds Concentration Maximum Respiration (pmol / min) Mean (pmol / min) Esm (pm ol / min) % Solvent Control (DMSO) Esm (%) p(i) Hypoxia Control - 33.3® 43.3 6.3 129 19 ne 49.6 37 Solvent Control (DMSO) 0.015% 31.4 33.5 1.1 100 3 - 33.8 35.2 MEST22M01 0.0015% 36.4® 49.1 2.4 147 7 ne 46.7 51.5

[0106] 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

[0107] 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 ([Fig 2])

[0108] 3. Treatments 48 hours + 2 hours

[0109] The analysis of mitochondrial respiration after a 48-hour treatment followed by a further 2-hour treatment, in hypoxia, is described in [Fig.3].

[0110] Tables 5, 6 and 7: Results of mitochondrial respiration of NHEK cultured for 48 hours + 2 hours under hypoxia [Tables 5] Treatment Basal Breathing Eu Iture Cond ition Tested Compounds Basal Breathing Concentration (pmol / m in) Mean (pmol / min) Esm (pm ol / min) % solvent control (DMS O) Esm (%) p(i) Hypo xia Control - 16.5 21.6 3.1 104 15 ne 21.3 27.1 Control solvent (DMSO) 0.015% 24.7 20.8 3.9 100 19 - 16.8 8.5(2) MEST22M01 0.0015% 24.5 26.9 2.2 130 11 ne 24.8 31.4 [Tableauxô] Treatment ATP Production Cond Compounds Concentration Average Production Esm (pm % Esm p(1) ition of eu Iture tés ation on d'ATP P (pmol / min) (pmol / min) ol / min) control solvent (%) (DMS O) Hypoxia Control - 14.2 17.7 1.8 104 11 ne 20.2 18.7 Solvent control (DMSO) 0.015% 17.4 17.0 0.4 100 2 - 16.5 7 (2) MEST22M01 0.0015% 20.3 21.8 1.1 129 7 ne 21.1 24.1 [Paintings?] Treatment Maximum Respiration Condition of Water Tested Compounds Concentration Maximum Respiration (pmol / min) Mean (pmol / min) Esm (pm ol / min) % Solvent Control (DMSO) Esm (%) p(i) Hypoxia Control - 35 36.5 2.1 103 6 ne 33.8 40.7 Solvent Control (DMSO) 0.015% 34.9 35.4 0.6 100 2 - 36 7.1(2) MEST22M01 0.0015% 34.8 41.4 6.7 117 19 ne 34.7 54.8

[0111] 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

[0112] In the presence of the active ingredient MEST22M01, basal and maximal respiration, as well as ATP production, increased compared to the solvent control ([Fig 4]).

[0113] 2.4. Treatments 48 hours + 24 hours

[0114] The analysis of mitochondrial respiration after a 48-hour treatment followed by a further 24-hour treatment is described in [Fig.5].

[0115] 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 [Tables 8] Treatment Basal Breathing Eu Iture Condition Tested Compounds Basal Respirati on Concentration (pmol / m in) Mean (pmol / mi n) Esm (pm ol / min) % Solvent Control (DM SO) Esm (%) p(i) Hypoxia Control - 20.9 22 1.2 179 10 ne 20.6 24.3 Solvent Control nt (DMSO) 0.015% 5.5 12.3 3.7 100 30 - 13 18.4 MEST22M01 0.0015% 10.6 17.7 3.8 144 31 ne 19.1 23.5 [Tables 9] Treatment ATP Production Eu Condition Tested Compounds Concentration ATP Production P (pmol / min) Average (pmol / min) Esm (pm ol / min) % Solvent Control (DM SO) Esm (%) p(i) Hypo xia Control - 14.2 15.8 1.4 173 15 ne 14.5 18.6 Solvent control (DMSO) 0.015% 5.6 9.1 1.9 100 20 - 9.7 12 MEST22M01 0.0015% 7 11.8 2.5 130 27 ne 15 13.5 [Tables 10] Treatment Maximum Respiration Condition of Water Tested Compounds Concentration Maximum Respiration (pmol / min) Mean (pmol / min) Esm (pm ol / min) % Solvent Control (DMSO) Esm (%) p(i) Hypoxia Control - 30.7 34.1 2.8 143 12 ne 32.0 39.6 Solvent Control (DMSO) 0.015% 22.5 23.8 1.2 100 5 - 22.7 26.2 MEST22M01 0.0015% 22.1 33.1 5.6 139 23 ne 39.7 37.6

[0116] 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

[0117] 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 ([Fig 6]).

[0118] 3. Effect on glycolysis

[0119] 3.1. Glycolysis analysis after treatment with the active ingredient MEST22M01

[0120] 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).

[0121] CONCLUSION

[0122] Under our experimental conditions in hypoxia, and compared to its control (DMSO solvent), treatment of keratinocytes with the active ingredient MEST22M01 increased: - Basal respiration by 97%, ATP production by 65% ​​and maximum respiration by 47% after 48h+30 min of incubation - 30% basal respiration and 29% ATP production after 48h + 2h incubation.

[0123] At 48h + 24h of treatment, the active ingredient MEST22M01 increased basal respiration by 44%, ATP production by 30% and maximum respiration by 39%.

[0124] 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 energy production occurring mainly via the respiratory rather than glycolytic pathway.

[0125] Example 2: _ Effects _ of the composition according to the invention _ (MEST22M01), in hypoxia, on the production of ROS in keratinocytes stimulated by TH2O2 and on the synthesis of melanin in weakly pigmented melanocytes

[0126] INTRODUCTION

[0127] In the present example, the effects of the composition according to the invention MEST22M01 were evaluated on different parameters under hypoxic conditions.

[0128] 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). Prior to this, a cytotoxicity test was performed on NHEM-LP using a standard WST-8 reduction assay to determine the concentrations to be tested.

[0129] MATERIALS AND METHODS

[0130] 1. Biological models

[0131] Normal human epidermal keratinocytes (NHEK) - Cell type: NHEK, Bioalternatives 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

[0132] Normal human epidermal melanocytes - low pigmentation (NHEM-LP) - Cell type: NHEM-LP, reference Bioalternatives NHEM-2 used in the 10th passage (preliminary cytotoxicity test) and the 12th passage (melanogenesis test) - Culture conditions: 37°C, 5% CO2 - Culture medium: Medium 254 optimized for the test (HMGS-2 without PMA) - Test medium: Medium 254 optimized for testing

[0133] 2. Tested compound

[0134] Composition: Melatonin in powder form at a concentration of 0.0015%.

[0135] Powder form

[0136] Intermediate solution: 10% in DMSO

[0137] Concentration tested: 0.0015% (0.015% DMSO)

[0138] 3. Production of ROS by NHEKs stimulated by H 2O2

[0139] 3.1. Culture and treatments

[0140] 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.

[0141] 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 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.

[0142] All experimental conditions were carried out in n=5.

[0143] In order to determine the background noise signal, a control in the absence of a probe was carried out for the stimulated control condition in hypoxia in n=5. For the calculation of the effects, the background noise values ​​were subtracted from the raw measured values.

[0144] 2. Measurement of SWR

[0145] The intensity of the emitted fluorescence (Xex = 485 nm, Xem = 538 nm) was measured using a Synergy H1 microplate reader (BioTek).

[0146] The fluorescence intensity of the metabolized probe is proportional to the amount of ROS. ROS production is expressed in fluorescence units.

[0147] 4. Tests on NHEM-LP

[0148] 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).

[0149] 4.2. Melanogenesis

[0150] 4.2.1. Culture and treatments

[0151] Melanocytes were seeded in 24-well plates and cultured in culture medium for 24 hours under normoxia. The culture medium was 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.

[0152] All experimental conditions were carried out in n=5, except for the reference in n=3.

[0153] 4.2.2. Melanin assay

[0154] At the end of the incubation, melanin was extracted by lysis of the cells with a 0.5 N NaOH solution.

[0155] 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 qg / ml). The results were expressed as qg / ml of melanin, as a percentage of the control.

[0156] 5. Data processing

[0157] The raw data were transferred and processed using Microsoft Excel® software.

[0158] Intergroup comparisons were performed using the two-sided Student's t-test Unpaired. Statistical analyses can be interpreted if n>5; however, for n<5, the calculated data are provided for information purposes only.

[0159] Formulas used in this report:

[0160] 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.

[0161] Percentage of viability: viability (%) = (compound OD / control OD) x 100

[0162] RESULTS

[0163] 1. Production of ROS by keratinocytes stimulated with H2O2 under hypoxia

[0164] Table 11: Effect of compound MEST22M01 on ROS production by keratinocytes stimulated with H2O2 under hypoxic conditions [Tableauxll] Compounds tested Concentration ROS ROS -background noise (RFU ) Mean (RFU ) Esm (RFU ) % Control stim uli (hypoxia) Es m (%) p(i) % C ontr ole s olva nt Es m(%) p(i) 1. Hy ( pox1 ie IZ ( ZZ 1 IberiMbions stimulated: 1 ) .50 lM 1313 4 1076 8 1169 7 1423 1 1502 9 1297 2 785 100 6 89 5 ns Solvent control (DMSO) 0.015% 1079 0 1633 0 1475 5 1525 9 1611 8 1465 0 1006 113 8 ns 100 7 MEST22 ME 0.0015% (DMSO 1074 0 1432 9 1449 110 11 ns 98 10 ns -0.015 1692%) 5 1757 0 1536 5 1104 7 (1): Threshold of statistical significance

[0165] ns: > 0.05, Not significant

[0166] *: 0.01 to 0.05, Significant

[0167] ** : 0.001 to 0.01, Very significant

[0168] *** : < 0.001, Extremely significant

[0169] RFU: Relative Fluorescence units

[0170] Under the experimental conditions of this test, when keratinocytes were cultured in hypoxia, their stimulation by H2O2 allowed a high level of reactive oxygen species (12972 RFU) to be observed.

[0171] 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. Compared with the solvent control, compound MEST22M01, tested at 0.0015% under hypoxia, had no effect on the production of reactive oxygen species in H2O2-stimulated keratinocytes.

[0172] 2. Melanocytes under hypoxic conditions

[0173] 2.1. Preliminary cytotoxicity test

[0174] Table 12: Effect of compound MEST22M01 on the viability of lightly pigmented melanocytes after 48 hours of hypoxic incubation, the last 24 hours of which were in the presence of the compound [Tables 12] Tested compounds Absorbance Concentration (D O450n m) Mean (DO4 50nm) Esm (D O450n m) % minus (hypoxia) Es m( %) p(i) Morphological Observations Hyp Témo - 0.816 0.8020. 2 - + oxie in 0.946 0.873 0.863 0.880 MES T22M 01 7.5 x 10-5 % 0.752 0.788 0.895 0.787 0.881 0.821 0.028 94 3 ns + 1 x 1 0-4% 0.798 0.870 0.897 0.857 0.867 0.869. 0.016 98 2 ns + 7.5 x 10-4 % 0.809 0.803 0.876 0.846 0.850 0.837 0.014 96 2 ns + 1.5 x 10-3 % 0.765 0.814 0.852 0.852 0.850. 0.820 0.018 94 2 ns + 7.5 x 10-3 % 0.704 0.732 0.801 0.674 0.763 0.735 0.022 84 3 ** + 0.01 % 0.641 0.712 0.6970 0.711 0.702 0.018 80 2 *** + 0.075 % 0.244 0.241 0.258 0.243 0.261 0.249 0.004 28 0 *** + / -, * 0.1% 0.174 **0.013 + / −, * 0.181 0.184 0.188 0.189 Legends

[0175] + : normal population ; + / - : growth reduction; - : toxicity ; 0 : mortality cellular

[0176] g: compound grains; op: opacity due to the compound; *: morphological changes; ag: agglutinated cells

[0177] 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).

[0178] 2.2. Effect on melanogenesis

[0179] Table 13: Effect of compound MEST22M01 on melanin synthesis in weakly pigmented melanocytes under hypoxic conditions [Tables 13] Com Conc Mêla Average Esm ( % té Es p(i) % Esp p(i) placed s tes tees entrât ion nine (qg / m 1) nne ( qg / m 1) qg / m 1) less (hyp oxia) m(%) Control so Iva nt m(%) Hyp oxia Witness 10.4 9.9 11.4 12.2 10.8 10.9 0.4 100 4 95 4 ns Solvent control (DMSO) 0.015 % 12.0 12.1 11.2 11.3 11.0 11.5 0.2 105 2 ns 100 2 MEST22 MOI 0.001 5% (D MSO 12.4 12.0 12.4 11.9 12.0 0.2 110 1 * 105 1 ns -0.01 5%) 11.5 (1): Threshold of statistical significance

[0180] ns: > 0.05, Not significant

[0181] *: 0.01 to 0.05, Significant

[0182] ** : 0.001 to 0.01, Very significant

[0183] *** : < 0.001, Extremely significant

[0184] 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. Compared with this solvent control, the compound MEST22M01, tested at 0.0015% under hypoxia, had no significant effect on melanin synthesis by melanocytes.

[0185] CONCLUSION

[0186] 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 FH2O2 in hypoxia and synthesis of melanin by melanocytes cultured in hypoxia).

[0187] 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)).

[0188] Example 3 j. _ Evaluation _ of the composition according to the invention where melatonin is in 2 different forms, on cellular oxygenation on ex vivo human skin expiants

[0189] INTRODUCTION

[0190] This study aims to evaluate the action of an active ingredient in two forms on cellular metabolism (oxygenation) on ex vivo human skin expiants.

[0191] 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 by staining and / or immunostaining.

[0192] The analytical phase will allow evaluation of mitochondrial density, as well as the activity of 2 complexes of the mitochondrial respiratory chain.

[0193] The activity will be evaluated by:

[0194] A assay of citrate synthase activity;

[0195] An assay of cytochrome c oxidase activity (Complex IV);

[0196] An ATP assay (product of Complex V activity).

[0197] MATERIALS AND METHODS

[0198] Study outline: • The expiators were placed 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 arrested after 8 hours of incubation.

[0199] Products tested • Product A = Lavandula Hybrida extract • Product B = Melaleuca Altemifolia extract • Product C = melatonin in powder form • Product D = melatonin in liposomal form

[0200] Preparation of expiations

[0201] 13 expiants of 11+1 mm in diameter were prepared from a plasty abdominal of a 37-year-old woman (reference P2933-AB37) with a phototype II according to the Fitzpatrick classification.

[0202] 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.

[0203] 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. The latter does not require prior authorization from an ethics committee for the collection and use of surgical waste.

[0204] Distribution of Expireds

[0205] The expiators were divided into 3 lots 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 Altemifolia) 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

[0206] Product preparation: The products were prepared on day 0 and then stored at 4°C throughout the ex vivo phase.

[0207] Product application

[0208] On J0, J1 and J2, for the expiants of lot T, DPBS IX was applied topically, at a rate of 5 pl per expiant of 1 cm2 (“2 mg / cm2”) and spread using a spatula.

[0209] On J0, J1 and J2, for the expiants of the lots concerned, the products PI, P2 were applied topically, at a rate of 5 pl per expiant of 1 cm2 (“5 mg / cm2”) and spread using a spatula.

[0210] Samples

[0211] At J2 (8h after the last application), 3-5 expiants from each batch were collected.

[0212] Histological treatments

[0213] After 24 hours in buffered formalin, the samples were dehydrated and impregnated with paraffin using a Leica PEARL dehydration system. They were then block-formed using a Leica EG 1160 embedding station.

[0214] 5 pm sections were made using a Minot type microtome, Leica RM 2125 and mounted on histological glass slides.

[0215] The frozen samples were cut into sections of 7 pm or 20 pm thickness using a Leica CM 3050 cryostat. The sections were then mounted on different supports depending on the type of shipment.

[0216] Microscopic observations were carried out using optical microscopy, with the aid of a Leica DMLB type microscope, Olympus BX43 or Olympus BX63.

[0217] The shots were taken with an Olympus DP72 or DP74 camera and cellSens software (Olympus).

[0218] Control of cell viability

[0219] Cell viability was observed on paraffin sections after Masson trichrome Goldner variant staining.

[0220] It was evaluated by microscopic examination.

[0221] Biochemical and enzymatic assays

[0222] 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.

[0223] 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 lot) is expressed in g / L (see annexes).

[0224] Citrate synthase assay: Citrate synthase activity was measured using the colorimetric technique of Srere (1969), based on measuring the appearance of coenzyme A, which bears 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 expiants per batch) is expressed in U / g of protein.

[0225] 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.

[0226] ATP assay: ATP was assayed 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.

[0227] Statistical analysis: Wilcoxon-Mann-Whitney test

[0228] 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 lots is significant if p<0.1 (*), i.e., a 90% probability that two lots are significantly different, or p<0.05 (**), i.e., a 95% probability that two lots are significantly different, or p<0.01 (***), i.e., a 99% probability that two lots are significantly different.

[0229] * : significant with p<0.1 (90%)

[0230] ** : significant with p<0.05 (95%)

[0231] *** : significant with p<0.01 (99%)

[0232] Ns: not significant

[0233] RESULTS

[0234] Citrate synthase activity

[0235] Table 14: Citrate synthase activity [Tables 14] Citrate synthase (U / g protein) T-J2 P1-J2 P2-J2 Mean 43.7 74.5 79.9 Standard deviation 0.9 9.8 15.3

[0236] The results are presented in [Fig. 10].

[0237] Product 1 (P1J2) induces a significant increase of 71%*

[0238] Product 2 (P2J2) induces a significant increase of 83%*

[0239] Wilcoxon test - Mann Whitney

[0240] Non-significant = ns; Significant *: p<0.1 (90%) **: p<0.05 (95%) ***: p<0.01 (99%)

[0241] Complex IV Activity

[0242] Table 15: Activity of complex IV [Tables 15] Complex IV (U / g protein) T-J2 P1-J2 P2-J2 Mean 6.4 6.6 11.0 Standard deviation 2.2 2.0 2.3

[0243] The results are presented in [Fig.1 1].

[0244] Product 1 (P1J2) induces a non-significant increase of 70% ns

[0245] Product 2 (P2J2) induces a significant increase of 83%*

[0246] Wilcoxon test - Mann Whitney

[0247] Non-significant = ns; Significant *: p<0.1 (90%) **: p<0.05 (95%) ***: p<0.01 (99%)

[0248] ATP assay

[0249] Table 16: ATP Assay [Tables 16] ATP (pmol / g protein) T-J2 P1-J2 P2-J2 Mean 7.1 9.1 17.3 Standard deviation 6.3 3.5 9.9

[0250] The results are presented in [Fig. 12].

[0251] Product 1 (P1J2) induces a non-significant increase of 28% ns

[0252] Product 2 (P2J2) induces a non-significant increase of 143% ns

[0253] Wilcoxon test - Mann Whitney

[0254] Non-significant = ns; Significant *: p<0.1 (90%) **: p<0.05 (95%) ***: p<0.01 (99%)

[0255] DISCUSSION

[0256] Products PI and P2 induce a statistically significant increase in mitochondrial density, as evidenced by the rise in citrate synthase activity. Product P2 stimulates mitochondrial respiratory chain activity, via its complex IV, in a statistically significant manner; as well as through its complex V, in a non-significant but substantial way, visible through the increase in ATP production (cellular energy).

Claims

Demands

1. 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, preferably of the oil of the leaves, of Melaleuca Altemifolia; • An extract, preferably of the oil of the flowers, of Lavandula Hybrida; • Melatonin.

2. Non-therapeutic cosmetic composition according to the preceding claim, comprising: • Between 0.00001% and 1%, preferably between 0.01% and 1%, by weight of the composition of said extract, preferably of the oil of the leaves, of Melaleuca Altemifolia.

3. Non-therapeutic cosmetic composition according to any one of the preceding claims, comprising: • Between 0.00001% and 1%, preferably between 0.01% and 1%, by weight of the composition of said extract, preferably of the flower oil, of Lavandula Hybrida.

4. 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 1%, preferably between 0.01% and 1%, by weight of the composition of said extract, preferably of the leaf oil, of Melaleuca Altemifolia; • Between 0.00001% and 1%, preferably between 0.01% and 1%, by weight of the composition of said extract, preferably of the flower oil, of Lavandula Hybrida; • Between 0.00001% and 10%, preferably between 0.00001% and 1%, by weight of the composition of melatonin.

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 and / or non-ionic lipid vesicles.

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 keratinocytes underlying the stratum corneum of the epidermis, including: • An extract, preferably of the oil from the leaves, of Melaleuca Altemifolia; • An extract, preferably of flower oil, of Lavandula Hybrida; • Melatonin.

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