Cosmetic treatment method for improving nighttime renewal process of skin and for protecting skin from free radicals associated with blue light exposure

Abstract

The present invention relates to a method for improving the nighttime renewal process of the skin, for protecting the skin from the effects of free radicals associated with blue light exposure, and for increasing the expression of the proteins Angel2 and Timeless, comprising topical application of a cosmetic composition to the skin, the present invention relates to a cosmetic composition comprising, as an active ingredient, an aqueous extract of an overground part of Lavandula angustifolia (Lavandula angustifolia).

Description

Technical Field

[0001] The present invention is in the field of cosmetics and more particularly relates to cosmetic treatments for improving the appearance of skin.

[0002] The present invention relates to a cosmetic treatment method for improving the skin's nightly renewal process, for ensuring correct DNA replication and DNA damage response and more particularly for increasing the expression of the proteins Angel2 and Timeless, and for protecting the skin from the effects of free radicals associated with blue light exposure. Background Art

[0003] Many consumers are concerned about the appearance of their skin and the signs of skin aging, such as wrinkles and loss of firmness or freckles and age spots.

[0004] The skin is a living organ composed of several layers (dermis, epidermis and stratum corneum) that covers the entire surface of the body and provides protective, sensory, immune, metabolic or thermoregulatory functions. Like other organs, the skin ages.

[0005] The skin undergoes a complex physiological aging process. Intrinsic aging or chronological aging is the result of genetically programmed aging and biochemical changes caused by endogenous factors. In the skin, the aging process is characterized by a slowdown in the regeneration of cells and extracellular matrix, a gradual loss of dermal collagen and elastic fibers (leading to dermal and epidermal atrophy, dryness, reduced skin elasticity and firmness, the appearance of fine lines and wrinkles), hyperpigmentation or hypopigmentation blemishes, age spots, etc.

[0006] On the other hand, extrinsic aging is due to environmental insults such as pollution, solar radiation (including UV radiation), diseases, lifestyle, etc. In areas chronically exposed to these insults, extrinsic aging is superimposed on intrinsic aging; this is then called photoaging.

[0007] Angel2 is a member of the CCR4 family of deadenylases and catalyzes the removal of poly(A) tails from mRNA during RNA quality control (Godwin et al. Kiss your tail goodbye: the role of PARN, Nocturnin, and Angel deadenylases in mRNA biology. Biochim Biophys Acta. 2013; 1829(6-7): 571-579). Angel2 not only prevents the accumulation of non-functional RNA, but also regulates normal mRNA.

[0008] Angel2 is a rhythmic protein and shows peak expression in the early evening. Angel2 helps regulate diurnal gene expression patterns (Zhang, Ray, "Building and Mining an Atlas of the Mammalian Circadian Transcriptome" (2015). Publicly Accessible Penn Dissertations. 1171. http: / / repository.upenn.edu / edissertations / 1171). Hundreds of genes were found to exhibit rhythmicity in poly(A) tail length, and therefore deadenylase plays a key role in regulating poly(A) tail rhythms and circadian gene expression (Yao et al. Critical role of deadenylation in regulating poly(A) rhythms and circadian gene expression. PLoS Comput Biol. 2020; 16(4): e1007842; Yao et al. Correction: Critical role of deadenylation in regulating poly(A) rhythms and circadian gene expression. PLoS Comput Biol. 2021 17(5): e1009065). The importance of endogenous rhythmic transcriptional and translational feedback loops of genes in skin physiology has been reviewed (Matsui et al. Biological Rhythms in the Skin. Int J Mol Sci. 2016; 17(6):801; Lyons et al. Circadian Rhythm and the Skin: A Review of the literature. J Clin Aesthet Dermatol. 2019; 12(9):42-45).

[0009] Angel2 has 2',3'-cyclic phosphatase activity and can play a key role in regulating the response to cellular stress (Pinto et al. ANGEL2 is a member of the CCR4 family of deadenylases with 2',3'-cyclic phosphatase activity. Science. 2020; 369(6503): 524-530). RNA molecules are often modified with terminal 2',3'-cyclic phosphate groups, and Angel2 plays a key role in a surveillance mechanism called ribosome-associated quality control (RQC) (Pinto et al. ANGEL2 is a member of the CCR4 family of deadenylases with 2',3'-cyclic phosphatase activity. Science. 2020; 369(6503): 524-530). Ribosomes that are inappropriately stalled during protein synthesis contain truncated / misfolded polypeptides (Yip et al. Mechanism for recycling tRNAs on stalled ribosomes. Nat Struct Mol Biol. 2019; 26(5): 343-349). Angel2 protects cells from producing proteotoxic polypeptides that may have harmful effects (Pinto et al. ANGEL2 is a member of the CCR4 family of deadenylases with 2',3'-cyclic phosphatase activity. Science. 2020; 369(6503): 524-530).

[0010] Timeless is another essential protein for circadian rhythms, and it interacts with the circadian clock protein cryptochrome 2 (Unsal-Kacmaz et al., 2005. Coupling of Human Circadian and Cell Cycles by the Timeless Protein. Molecular and Cellular Biology. 3109-3116).

[0011] Timeless is a protein that belongs to the "replication protection complex" that ensures replication checkpoints and correct DNA replication.

[0012] DNA replication genes are upregulated in the afternoon and evening (E Paatela et al., Circadian Regulation in Tissue Regeneration. Int. J. Mol. Sci. 2019, 20, 2263). It requires coordination between the replication fork advance and the metabolic pathway that produces deoxynucleotide triphosphates. Timeless and another protein Tipin of the "replication protection complex" play a role in replication fork stability and sister chromatid cohesion. It has been shown that the depletion of Timeless leads to a robust replication fork slowdown (Leman AR et al., 2009. Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion. Journal of Cell Science 123: 660-670).

[0013] Timeless protein is also a core component of the cell cycle checkpoint system. It interacts with the cell cycle checkpoint protein Chk1 and the ATR-ATRIP complex and plays an important role in the DNA damage checkpoint response. Downregulation of Timeless in human cells severely impairs replication and S phase checkpoints, indicating that there is a tight connection between the circadian cycle and the DNA damage checkpoint, which is partly mediated by Timeless protein (Unsal-Kacmaz et al., 2005).

[0014] Blue light represents the portion of visible light between 400 and 495 nm. It is the main high-energy portion of the visible spectrum. Most of the blue light received comes from the sun. In our indoor environments, we are increasingly exposed to artificial blue light from LEDs (light emitting diodes) present in lighting equipment, smartphones, tablets, computers and TV screens.

[0015] Blue light has an effect on the skin. It has been demonstrated on keratinocytes and endothelial cells of human skin that blue light exerts an anti-proliferative effect and promotes the differentiation of keratinocytes.

[0016] Considering the harmful effects that blue light can have on the skin, and more particularly the DNA damage associated with free radical exposure, it seems important to be able to develop cosmetic compositions that can be used to protect human skin from the effects of blue light.

[0017] Peschel et al. (Current Biology 19, 241-247, February 10, 2009) showed that in Drosophila, the clock factor Timeless is degraded after blue light, causing the circadian clock to reset daily to its environment and adapt to its environment.

[0018] Lavender, and in particular true lavender (Lavandula angustifolia), is a species native to Provence (southeast France) that is widely used in Europe, North Africa, the Middle East and Asia for the production of essential oils, particularly for the perfume industry.

[0019] The composition used in the present disclosure comprises an aqueous extract of the aerial parts of lavender as an active ingredient, the aqueous extract being rich in small RNAs of up to 150 nucleotides in length, sugars, phenolic compounds and organic acids, and being free of DNA, the aqueous extract being disclosed in patent application WO 2021156104 (also disclosed as U.S. patent application No. US17 / 795389) (incorporated herein by reference).

[0020] The aqueous extract of lavender described above does not have the disadvantages of currently known extracts, namely strong odor, instability (such as essential oils in formulations for topical application), irritation or sensitization due to the presence of terpene molecules (such as linalool), and does not require the use of detergents and solvents that are potentially toxic in cosmetics.

[0021] The aforementioned aqueous extracts of lavender are designed to protect the skin from external aggressions and oxidation, to fight signs of skin aging, to increase photoprotection, to brighten the skin, to improve skin hydration, to enhance barrier function or to soothe the skin, as well as to improve biological mechanisms associated with nighttime skin repair.

[0022] Surprisingly, it was found that the composition comprising the aqueous extract of lavender described above has beneficial effects in its ability to ensure proper DNA replication and DNA damage checkpoint response, ensure RNA quality control checks, rhythmic control of RNA poly (A) tail length and participate in ribosome-related quality control pathways.

[0023] The present disclosure provides a method for improving the skin's nightly renewal process, for protecting the skin from the effects of blue light exposure, and more particularly for increasing the expression of the proteins Angel2 and Timeless. Summary of the invention

[0024] The present invention relates to a method for improving the skin's nightly renewal process and for protecting the skin from the effects of free radicals associated with blue light exposure, the method comprising topically applying to the skin a cosmetic composition comprising i) as an active ingredient an aqueous extract of aerial parts of lavender (Lavandula angustifolia), the aqueous extract comprising 4 to 20 g / kg of dry weight, based on the weight of the total weight of the extract, containing 0.5 to 7 g / kg of sugars, 200 to 1000 mg / kg of organic acids, 600 to 1200 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA of up to 150 nucleotides in length, and ii) a physiologically acceptable medium.

[0025] The present invention also relates to a method for improving the skin's nightly renewal process, comprising ensuring correct DNA replication and DNA damage response, ensuring RNA quality control checks, rhythmic control of RNA poly (A) tail length, engaging ribosome-related quality control pathways, and increasing the expression of proteins Angel2 and Timeless.

[0026] The present invention also relates to a method of protecting the skin from free radicals associated with blue light exposure, the method comprising reducing the level of free radicals and increasing the expression of the proteins Angel2 and Timeless in skin cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The invention and its advantages will be better understood from the following description and non-limiting embodiments illustrated with reference to the accompanying drawings, in which:

[0028] [ Figure 1 ] shows microscopic observation of Angel2 staining on keratinocytes.

[0029] [ Figure 2 ] shows microscopic observation of Timeless staining on keratinocytes.

[0030] [ Figure 3 ] Normalized ROS production in keratinocytes after 30 min of blue light irradiation.

[0031] [ Figure 4 ] Normalized ROS production in keratinocytes after 1, 2, and 4 h of blue light irradiation.

[0032] [ Figure 5 ] Normalized ROS production in keratinocytes after 1, 2, or 4 h of blue light irradiation in the presence and absence of 1% lavender extract.

[0033] [ Figure 6] shows microscopic observation of Angel2 staining on keratinocytes after blue light irradiation in the presence and absence of 0.5% lavender extract.

[0034] [ Figure 7 ] shows microscopic observation of Timeless staining on keratinocytes after blue light irradiation in the presence and absence of 0.5% lavender extract. DETAILED DESCRIPTION definition

[0035] Unless otherwise stated, all terms used in this specification have the broadest known meanings. For the purposes of the present invention, the following terms are defined as follows:

[0036] "Lavender" refers to the aerial parts of the species Lavandula angustifolia, namely the lavender flowers and the small stems that carry the flowers.

[0037] The term "small RNA" or "low molecular weight RNA" or "small RNA of up to 150 nucleotides in length" means a small molecular weight non-coding RNA (ribonucleic acid) of up to 150 nucleotides in length, such as all types of small non-messenger RNAs, such as microRNA, interfering RNA, intron, small nuclear RNA or any RNA fragment, which are single-stranded and / or double-stranded. Electrophoresis analysis shows that the small RNA present in the lavender extract of the present invention has a variety of molecular weights ranging from about 30 to 150 nucleotides.

[0038] "Organic acids" means alpha-hydroxy acids (or AHAs), carboxylic acids derived from fruit or plant sugars, such as glycolic acid, malic acid, citric acid, tartaric acid, succinic acid, and uronic acid.

[0039] "Phenolic compounds" means molecules of plant origin having an aromatic ring with one or more hydroxyl groups, such as phenolic acids, flavonoids or derivatives thereof. Phenolic compounds are known to be powerful antioxidant molecules.

[0040] "Sugar" means monosaccharides, especially glucose and fructose, as well as oligosaccharides and polysaccharides.

[0041] When describing a range of values, the limits of the range should be understood to explicitly include all intermediate values ​​in the range. For example, a range of values ​​between 1% and 10% should be understood to include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%, and all decimal values ​​between 1% and 10%.

[0042] Unless otherwise specified, numerical percentage values ​​are percentages by weight, ie, the weight of the compound to the total weight of the intended mixture.

[0043] The compositions described herein may "comprise," "consist of," or "consist essentially of," essential compounds or optional ingredients.

[0044] "Consisting essentially of" means that the composition or component may include additional ingredients, but only if the additional ingredients do not alter the basic or novel characteristics of the composition or use described in the present application.

[0045] "Physiologically acceptable medium" means a vehicle that is suitable for contact with the outer layer of the skin or mucous membranes, is not toxic, irritating, excessively allergic, or the like, or intolerable, and is commensurate with a reasonable benefit / risk ratio.

[0046] "Topical application" means applying or spreading the aqueous extract rich in small RNA with a maximum length of 150 nucleotides, sugar, phenolic compound and organic acid according to the present invention or a composition containing the aqueous extract on the skin surface or mucous membrane.

[0047] "Skin" refers to the skin including the skin appendages (hair and body hair, eyebrows, eyelashes, nails).

[0048] The “nighttime skin renewal process” has the meaning of multiple biological mechanisms related to nighttime skin repair, including at least: ensuring correct DNA replication and DNA damage response, ensuring RNA quality control checks, rhythmic control of RNA poly(A) tail length, engaging ribosome-related quality control pathways, and increasing the expression of the proteins Angel2 and Timeless.

[0049] It will be understood that the present invention relates to mammals, and more particularly to humans.

[0050] The present disclosure relates to a method for improving the skin's nightly renewal process, for increasing the expression of proteins Angel2 and Timeless, and for protecting the skin from the effects of free radicals associated with blue light exposure, the method comprising topically applying to the skin a cosmetic composition comprising i) as an active ingredient an aqueous extract of aerial parts of lavender (Lavandula angustifolia), the aqueous extract comprising 4 to 20 g / kg of dry weight, based on the weight of the total weight of the extract, containing 0.5 to 7 g / kg of sugars, 200 to 1000 mg / kg of organic acids, 600 to 1200 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA of up to 150 nucleotides in length, and no DNA, and ii) a physiologically acceptable medium. Extraction of lavender The extraction method of the aerial part of lavender (Lavandula angustifolia) is disclosed in patent application US17 / 795389, which is incorporated herein by reference. In short, the method comprises the following steps:

[0051] a) contacting the above-ground part of the lavender with water;

[0052] b) adding phytic acid at a concentration between 1 and 10 mM, and preferably at a concentration of 3 mM, to the mixture obtained in a) at a pH between 10 and 11 and maintaining it under stirring at a temperature between 20° C. and 80° C. for at least 1 hour;

[0053] c) purifying the mixture obtained in b) by eliminating the remaining solid aerial parts of lavender to recover the soluble part constituting the crude aqueous extract;

[0054] d) clarifying the plant extract by filtering the aqueous extract on filters of decreasing porosity in succession until filtering at 1 μm to remove the remaining solid fraction and collecting the filtrate;

[0055] e) adjusting the pH of the filtrate obtained in d) to a value between 6 and 8;

[0056] f) diluting the extract obtained in e) with a physiologically acceptable solvent to increase the stability and conservation of the extract over time.

[0057] g) checking the pH and, if necessary, re-adjusting the pH to a value between 6 and 7;

[0058] h) The extract is sterilized at low temperature to avoid any contamination.

[0059] The diluted extract may be further preserved using any physiologically acceptable preservative.

[0060] This method produces an extract comprising 4 to 20 g / kg of dry weight, based on the weight of the total weight of the extract, containing 0.5 to 7 g / kg of sugars, 200 to 1000 mg / kg of organic acids, 600 to 1200 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA of up to 150 nucleotides in length, and ii) a physiologically acceptable medium.

[0061] The extracts obtained by this method do not contain DNA.

[0062] Examples of the above disclosed methods include:

[0063] In step a), 35 g of dried lavender ground into a powder having a particle size ranging from 500 μm to 1 mm was mixed in 963 g of distilled water;

[0064] In step b), 2 g / L or 3 mM phytic acid is added. The pH is adjusted to 10.8 to optimally enrich the extract with low molecular weight RNA, and then the mixture is heated at 80°C for 1 hour with stirring;

[0065] In step c), after one hour of extraction, the mixture obtained in b) is filtered through a filter with a pore size of 30 μm to remove solid matter;

[0066] In step d), filtering of the extract is performed by lowering the filtration threshold from 50 μm to 20 μm and then from 1 μm to 0.1 μm;

[0067] In step e), the pH is checked and then adjusted to between 6 and 6.5 with HCl solution to preserve the small RNA in the extract, and preferably the pH is adjusted to 6.3;

[0068] In step f), dilution is performed with plant-derived glycerol to obtain a final concentration of 30% glycerol and 70% lavender extract, and 1.5% phenoxyethanol is added for preservation;

[0069] In step g), the pH is controlled and adjusted to a value between 8 and 6.3 using hydrochloric acid (HCl);

[0070] In step h), the diluted extract is low temperature sterilized at 72°C for 8 hours.

[0071] The diluted extract obtained had a dry weight of 9.5 g / kg and had the following concentrations of substances: 3.7 g / kg total sugars, 698 mg / kg total organic acids, 1040 mg / kg total phenolics and 64 mg / kg low molecular weight RNA with a length of up to 150 nucleotides.

[0072] Thus, the lavender extracts disclosed herein contain a wide range of plant molecules that can have beneficial effects on the skin without presenting the risk of skin irritation or other health damage. Composition

[0073] The cosmetic composition used in the present disclosure comprises i) as an active ingredient an aqueous extract of aerial parts of lavender (Lavandula angustifolia), the aqueous extract comprising 4 to 20 g / kg of dry weight based on the weight of the total weight of the extract, containing 0.5 to 7 g / kg of sugar, 200 to 1000 mg / kg of organic acids, 600 to 1200 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA of up to 150 nucleotides in length, and ii) a physiologically acceptable medium.

[0074] Advantageously, the extract of aerial parts of lavender obtained by the above-described method is added to the composition in a concentration ranging from 0.5% to 2% by weight relative to the total weight of the composition.

[0075] The compositions used in the present disclosure may be applied by any suitable route, and the formulation of the compositions will be adjusted by those skilled in the art.

[0076] Preferably, the compositions used in the present disclosure are in a form suitable for topical application. These compositions must therefore contain a physiologically acceptable medium, ie compatible with the skin and skin appendages, without any risk of discomfort during their application, and must cover all suitable cosmetic forms.

[0077] The compositions used to implement the invention may in particular be in the form of aqueous, hydroalcoholic or oily solutions, oil-in-water emulsions, water-in-oil emulsions or multiple emulsions; they may also be in the form of suspensions or powders.

[0078] The compositions used to practice the invention may in particular be suitable for application to the skin, mucous membranes, lips and / or hair.

[0079] These compositions may be more or less fluid, and may also have the appearance of a cream, lotion, milk, serum, ointment, gel, paste or foam. They may also be in solid form, such as a stick, or may be applied to the skin in the form of an aerosol.

[0080] Examples of physiologically acceptable media customarily used in the envisaged field of application are adjuvants required for formulations, such as solvents, thickeners, diluents, antioxidants, colorants, filters, tanning agents, pigments, fillers, preservatives, fragrances, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

[0081] Advantageously, the composition according to the invention may comprise, in addition to the active ingredient according to the invention, at least one other active agent having cosmetic effects similar and / or complementary to those of the invention.

[0082] For example, the one or more additional active agents can be selected from: anti-aging agents, firming agents, brightening agents, moisturizing agents, drainage agents, microcirculation promoters, exfoliating agents, desquamation agents, extracellular matrix stimulators, energy metabolism activators, antibacterial agents, antifungal agents, soothing agents, anti-free radical agents, anti-ultraviolet agents, anti-acne agents, anti-inflammatory agents, anesthetics, warm sensation inducing agents, cool sensation inducing agents and slimming agents. Cosmetic treatment methods

[0083] The present disclosure relates to a cosmetic treatment method for improving the skin's nightly renewal process and for protecting the skin from the effects of free radicals associated with blue light exposure, the method comprising topically applying to the skin a cosmetic composition comprising i) as an active ingredient an aqueous extract of aerial parts of lavender (Lavandula angustifolia), the aqueous extract comprising 4 to 20 g / kg of dry weight, based on the weight of the total weight of the extract, containing 0.5 to 7 g / kg of sugars, 200 to 1000 mg / kg of organic acids, 600 to 1200 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA of up to 150 nucleotides in length, and ii) a physiologically acceptable medium.

[0084] In a specific embodiment, the above method comprises applying a cosmetic composition comprising an aqueous extract of aerial parts of lavender (Lavandula angustifolia) obtained by the above method of the present disclosure.

[0085] In a particular embodiment, the above method comprises applying a cosmetic composition comprising 0.5% to 2% by weight of an aqueous extract of aerial parts of lavender (Lavandula angustifolia) relative to the total weight of the composition.

[0086] In a specific embodiment, the present disclosure also relates to a method for improving the skin's nighttime renewal process, wherein the skin's nighttime renewal process includes ensuring correct DNA replication and DNA damage response, ensuring RNA quality control checks, rhythmic control of RNA poly (A) tail length, participating in ribosome-related quality control pathways, and increasing the expression of proteins Angel2 and Timeless, the method comprising topically applying a cosmetic composition to the skin, wherein the cosmetic composition comprises an aqueous extract of lavender of the present disclosure.

[0087] In another specific embodiment, the present disclosure also relates to a method for protecting the skin from the effects of free radicals associated with blue light exposure, wherein the levels of free radicals in skin cells are reduced and the levels of proteins Angel2 and Timeless are increased, the method comprising topically applying to the skin a cosmetic composition comprising an aqueous extract of lavender of the present disclosure.

[0088] The extract of aerial parts of lavender of the present invention has been tested for key biomarkers related to nighttime skin repair mechanisms, proper DNA replication and DNA damage response.

[0089] More particularly, the extract of aerial parts of lavender of the present invention has been tested for the expression of the proteins Angel2 and Timeless, and for the protection against free radicals associated with blue light exposure.

[0090] The extract of aerial parts of lavender of the present invention has advantageous effects in its ability to ensure correct DNA replication and DNA damage checkpoint response, ensure RNA quality control checks, rhythmic control of RNA poly (A) tail length, and participate in ribosome-related quality control pathways. Example

[0091] By way of illustration, exemplary embodiments of methods according to the present invention are described below.

[0092] Example 1: Evaluation of the effect of lavender water extract on Angel2 expression in human keratinocytes

[0093] principle: The aim of this study was to show the effect of the application of lavender water extract versus conventional extract of lavender on the expression of Angel2 in epidermal keratinocytes.

[0094] plan: Normal human keratinocytes were treated twice daily with 0.5% vol / vol lavender water extract solution prepared according to the method described in the present disclosure or with 0.5% vol / vol lavender conventional extract solution for 48 hours. In order to be immunolabeled by anti-Angel2 antibody, the cells were washed and fixed with 3.7% paraformaldehyde at room temperature for 15 minutes. The cell membrane was permeabilized with 0.2% Triton X-100 (Sigma) solution for 10 minutes. Nonspecific binding sites were blocked with 1% BSA (Sigma) solution for 30 minutes. The cells were then incubated in the presence of specific anti-Angel2 antibody (Sigma, reference HPA030796, rabbit polyclonal), and then incubated in the presence of a suitable secondary antibody coupled to a fluorescent dye. After sealing in a specific medium, the slides were observed by epifluorescence microscopy (Nikon Eclipse 80i microscope). Fluorescence intensity was quantified by using Volocity 6.3. software to analyze the image.

[0095] For comparison purposes, a conventional extract was prepared as follows: Lavender aerial parts were placed in water at the same ratio as the extract of the present disclosure, under gentle stirring at room temperature for one hour. The mixture obtained was filtered sequentially to clarify the extract from 30 μm until sterile filtration at 0.2 μm. The pH was adjusted to between 6.0 and 6.5.

[0096] result: Microscopic observations showed that Angel2 staining on keratinocytes was significantly increased after treatment with lavender water extract (Student's t-test). Conventional extracts showed significantly lower activity than lavender water extract. The results are presented in Figure 1 middle.

[0097] Table 1 - Quantification of microscopic observations of Angel2 expression on keratinocytes *: significant; ***: highly significant, using Student's t test

[0098] in conclusion: In vitro studies have shown that the application of the 0.5% lavender water extract of the present invention for 48 hours is associated with an increase in the expression of Angel2 protein in the cell nucleus and has better efficacy than the use of conventional lavender extracts.

[0099] Example 2: Evaluation of the effect of lavender water extract on Timeless expression in human keratinocytes

[0100] principle: The aim of this study was to show the effect of application of lavender water extract versus conventional extract of lavender on Timeless expression in epidermal keratinocytes.

[0101] plan: Normal human keratinocytes were treated twice daily with 0.5% vol / vol lavender water extract solution prepared according to the method described in the present disclosure or with 0.5% vol / vol lavender conventional extract solution for 48 hours. The preparation of lavender conventional extract is described in Example 1. In order to be immunolabeled by anti-timeless antibody, cells were washed and fixed with cold methanol for 4 minutes at 4°C. Nonspecific binding sites were blocked with 1% BSA (Sigma) solution for 30 minutes. The cells were then incubated in the presence of specific anti-Timeless antibody (Abcam, reference ab72458, rabbit polyclonal), and then incubated in the presence of a suitable secondary antibody coupled to a fluorescent dye. After sealing in a specific medium, the slides were observed by epifluorescence microscopy (Nikon Eclipse 80i microscope). Fluorescence intensity was quantified by using Volocity 6.3. software to analyze the image.

[0102] result: Microscopic observations showed that Timeless staining on keratinocytes was significantly increased after treatment with lavender water extract (Student's t-test). Conventional extracts showed significantly lower activity than lavender water extract. The results are presented in Figure 2 middle.

[0103] Table 2 - Quantification of microscopic observations of Timeless expression on keratinocytes *: significant; ***: highly significant, using Student's t test

[0104] in conclusion: In vitro studies have shown that application of 0.5% lavender water extract for 48 hours was associated with an increase in Timeless protein expression in the cell nucleus and had better efficacy than conventional extracts.

[0105] Example 3: Normalized ROS production in keratinocytes after 30 minutes of blue light irradiation

[0106] Protocol - 30 Minutes Experiment Normal human epidermal keratinocytes (NHEK, 45 years old, Life Technologies; Catalog No. A13401; Lot No. 2068397) were seeded at 40,000 cells / well into 2-4 well chamber slides with complete supplemented medium (Epilife; Life Technologies; Catalog No. MEPI500CA and HKGS; Life Technologies; Catalog No. S0015). The plated cells were incubated overnight under standard conditions (37°C and 5% CO2). After overnight incubation, cells were treated with a 1% (vol / vol) solution of lavender (aqueous lavender extract prepared according to the methods described in this disclosure and similar to the lavender extract used in Examples 1 and 2) in fully supplemented medium. After 24 hours, cells were washed twice with Dulbecco's phosphate buffered saline (DPBS; Corning; catalog number 21-031-CV) and then supplemented with 250 μl of DPBS containing glucose supplement (Life Technologies; catalog number 14287-080). Afterwards, one chamber slide was kept in complete darkness, while the other chamber slide was placed in a blue light chamber where it was exposed to 100 J / cm 2 The chamber slide was irradiated with blue light (a light source emitting at 410 nm) (410 nm) (duration 30 minutes). After 30 minutes of irradiation, cells were immediately stained for ROS and nuclei using CellROX Oxidative Stress Green Reagent (488) and HCS NuclearMask Blue Stain (Life Technologies; Catalog No. H10325). 250 μl of CellROX and 250 μl of HCS NuclearMask were added to each well and placed in a 37°C incubator for 30 minutes. For CellROX, a final concentration of 5 μM was administered to the cells. For HCS Nuclear Mask, 5 μl of the final dilution of stain in 10 ml of complete medium was administered to the cells. Once the time had elapsed, the medium was removed, washed once with DPBS, and imaged and analyzed on a Keyence BX-7 (ROS acquired at 488 nm filter and DAPI acquired at 405 nm filter). After all images were taken, they were analyzed for ROS production and cell counts on a Nikon Elements. Obtaining cell count values ​​using the Object Count function in Nikon Elements The ROS generation value was obtained by setting a binary threshold based on the intensity of the signal and then taking the total intensity of the binary threshold, which was automatically given by Nikon Elements.

[0107] The results are Figure 3 and showed a 93% reduction in free radicals in the case of treatment with 1% lavender water extract.

[0108] Example 4: Normalized ROS production in keratinocytes after 1, 2 and 4 hours of blue light irradiation

[0109] Protocol: Multiple blue light exposures Normal human epidermal keratinocytes (NHEK, 45 years old, Life Technologies; Catalog No. A13401; Lot No. 2068397) were seeded at 40,000 cells / well into 6-4 well chamber slides with complete supplemented medium (Epilife; Life Technologies; Catalog No. MEPI500CA and HKGS; Life Technologies; Catalog No. S0015). The plated cells were incubated overnight under standard conditions (37°C and 5% CO2). After overnight incubation, cells were washed twice with Dulbecco's Phosphate Buffered Saline (DPBS; Corning; Catalog No. 21-031-CV) and then supplemented with 250 μl of DPBS containing glucose supplement (Life Technologies; Catalog No. 14287-080). Afterwards, three chamber slides were kept in complete darkness, while the other chamber slides were placed in a blue light chamber, where they were irradiated with blue light (a light source emitting 410 nm) (410 nm) for durations of 1, 2, and 4 hours. After each blue light irradiation time point, cells were immediately stained for ROS and nuclei using CellROX oxidative stress green reagent (488 nm) and HCS NuclearMask blue stain (Life Technologies; catalog number H10325). 250 μl of CellROX and 250 μl of HCS NuclearMask were added to each well and placed in an incubator at 37°C for 30 minutes. a. For CellROX, a final concentration of 5 μM was administered to cells. b. For HCS Nuclear Mask, 5 μl of the final dilution of stain in 10 ml of complete medium was administered to the cells. Once the time had elapsed, the medium was removed, the cells were washed once with DPBS, and imaged and analyzed on a Keyence BX-7 (ROS was acquired at a 488 nm filter, and DAPI was acquired at a 405 nm filter). After all images were taken, they were analyzed for ROS production and cell counts on a Nikon Elements. c. Obtain cell count values ​​using the object counting function in Nikon Elements. d. The ROS generation value was obtained by setting a binary threshold based on the intensity of the signal and then obtaining the total intensity of the binary threshold, which was automatically given by Nikon Elements.

[0110] The results are Figure 4 and showed that blue light exposure caused very high levels of free radical production.

[0111] Example 5: Normalized ROS production in keratinocytes after 1, 2 or 4 hours of blue light irradiation with and without lavender extract

[0112] Protocol: Multiple blue light exposure and treatment Normal human epidermal keratinocytes (NHEK, 45 years old, Life Technologies; Catalog No. A13401; Lot No. 2068397) were seeded at 40,000 cells / well into 9-4 well chamber slides with complete supplemented medium (Epilife; Life Technologies; Catalog No. MEPI500CA and HKGS; Life Technologies; Catalog No. S0015). The plated cells were incubated overnight under standard conditions (37°C and 5% CO2). After overnight incubation, three slides were treated with a 1% (vol / vol) solution of lavender (aqueous lavender extract prepared according to the methods described in this disclosure and similar to the lavender extract used in Examples 1 and 2) in fully supplemented medium. After overnight incubation, cells were washed twice with Dulbecco's Phosphate Buffered Saline (DPBS; Corning; Catalog No. 21-031-CV) and then supplemented with 250 μl of DPBS containing glucose supplement (Life Technologies; Catalog No. 14287-080). Afterwards, three chamber slides were kept in complete darkness, while the other chamber slides were placed in a blue light chamber, where they were irradiated with blue light (a light source emitting 410 nm) (410 nm) for durations of 1, 2, and 4 hours. After each blue light irradiation time point, cells were immediately stained for ROS and nuclei using CellROX oxidative stress green reagent (488 nm) and HCS NuclearMask blue stain (Life Technologies; catalog number H10325). 250 μl of CellROX and 250 μl of HCS NuclearMask were added to each well and placed in an incubator at 37°C for 30 minutes. a. For CellROX, a final concentration of 5 μM was administered to cells. b. For HCS Nuclear Mask, 5 μl of the final dilution of stain in 10 ml of complete medium was administered to the cells. Once the time had elapsed, the medium was removed, the cells were washed once with DPBS, and imaged and analyzed on a Keyence BX-7 (ROS was acquired at a 488 nm filter, and DAPI was acquired at a 405 nm filter). After all images were taken, they were analyzed for ROS production and cell counts on a Nikon Elements. c. Obtain cell count values ​​using the object counting function in Nikon Elements. d. The ROS generation value was obtained by setting a binary threshold based on the intensity of the signal and then obtaining the total intensity of the binary threshold, which was automatically given by Nikon Elements. e. Statistics were performed using one-way ANOVA with Dunnett's Post Test. *If P < 0.05.

[0113] The results are Figure 5 and showed that pretreatment with 1% lavender extract produced an anti-free radical effect.

[0114] Example 6: Evaluation of the effect of lavender water extract on Angel2 expression in human keratinocytes after blue light irradiation

[0115] principle: The aim of this study was to show the effect of application of lavender water extract on Angel2 expression in epidermal keratinocytes.

[0116] plan: Normal human keratinocytes were treated twice with a 0.5% vol / vol lavender water extract solution prepared according to the method described in the present disclosure for 24 hours. The cells were then irradiated with blue light (415nm) for 30 minutes and treated twice again for 24 hours. In order to be immunolabeled by anti-Angel2 antibodies, the cells were washed and fixed with 3.7% paraformaldehyde at room temperature for 15 minutes. The cell membrane was permeabilized with 0.2% Triton X-100 (Sigma) solution for 10 minutes. Nonspecific binding sites were blocked with 1% BSA (Sigma) solution for 30 minutes. The cells were then incubated in the presence of specific anti-Angel2 antibodies (Sigma, reference HPA030796, rabbit polyclonal), and then incubated in the presence of a suitable secondary antibody coupled to a fluorescent dye. After sealing in a specific medium, the slides were observed by an epifluorescence microscope (NikonEclipse 80i microscope). Fluorescence intensity was quantified by analyzing images using Volocity 6.3. software.

[0117] result: Microscopic observations showed that Angel2 staining on keratinocytes was significantly reduced (Student's t-test) after blue light irradiation. Treatment with lavender water extract allowed to increase Angel2 in stressed cells. The results are presented in Figure 6 middle.

[0118] Table 3 - Quantification of microscopic observations of Angel2 expression on keratinocytes after blue light irradiation *: significant; **: very significant, using Student's t test

[0119] Example 7: Evaluation of the effect of lavender water extract on Timeless expression in human keratinocytes after blue light irradiation

[0120] principle: The aim of this study was to show the effect of the application of lavender water extract on the expression of Timeless in epidermal keratinocytes after blue light irradiation.

[0121] plan: Normal human keratinocytes were treated twice with a 0.5% vol / vol lavender water extract solution prepared according to the method described in the present disclosure for 24 hours. The cells were then irradiated with blue light (415nm) for 30 minutes and treated twice again for 24 hours. In order to be immunolabeled by anti-timeless antibodies, the cells were washed and fixed with cold methanol for 4 minutes at 4°C. Nonspecific binding sites were blocked with 1% BSA (Sigma) solution for 30 minutes. The cells were then incubated in the presence of specific anti-Timeless antibodies (Abcam, reference ab72458, rabbit polyclonal), and then incubated in the presence of a suitable secondary antibody coupled to a fluorescent dye. After sealing in a specific medium, the slides were observed by an epifluorescence microscope (Nikon Eclipse 80i microscope). Fluorescence intensity was quantified by using Volocity 6.3. software to analyze the image.

[0122] result: Microscopic observations showed that Timeless staining on keratinocytes was significantly reduced (Student's t-test) after blue light irradiation. Treatment with lavender water extract allowed to increase Timeless in stressed cells. The results are presented in Figure 7 middle.

[0123] Table 4 - Quantification of microscopic observations of Timeless expression on keratinocytes following blue light irradiation. *: significant; ***: highly significant, using Student's t test

Claims

1. A method for improving the skin's nightly renewal process and for protecting the skin from the effects of free radicals associated with blue light exposure, the method comprising topically applying to the skin a cosmetic composition comprising i) as an active ingredient an aqueous extract of aerial parts of lavender (Lavandula angustifolia), the aqueous extract comprising 4 to 20 g / kg of dry weight, based on the weight of the total weight of the extract, containing 0.5 to 7 g / kg of sugars, 200 to 1000 mg / kg of organic acids, 600 to 1200 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA of up to 150 nucleotides in length, and ii) a physiologically acceptable medium.

2. The method according to claim 1, wherein the aqueous extract of lavender (Lavandula angustifolia) is obtained by a method comprising the following steps: a) contacting the above-ground part of the lavender with water; b) adding phytic acid at a concentration between 1 and 10 mM to the mixture obtained in a) at a pH between 10 and 11 and maintaining it under stirring at a temperature between 20° C. and 80° C. for at least 1 hour; c) purifying the mixture obtained in b) by eliminating residual solid lavender to recover the soluble portion constituting the crude aqueous extract; d) filtering the aqueous extract obtained in step d) by continuous sequential filtration on filters of decreasing porosity until filtration at 1 μm; e) adjusting the pH of the filtrate obtained in d) to a value between 6 and 8; f) diluting the extract obtained in e) with a physiologically acceptable solvent; g) checking the pH and, if necessary, re-adjusting the pH to a value between 6 and 7; h) sterilizing the extract at low temperature.

3. The method according to claim 2, wherein in step b), the concentration of phytic acid is 3 mM.

4. The method of claim 1, wherein the skin night renewal process includes ensuring correct DNA replication and DNA damage response, ensuring RNA quality control checks, ensuring rhythmic control of RNA poly (A) tail length, engaging ribosome-related quality control pathways, and increasing the expression of proteins Angel2 and Timeless.

5. The method of claim 1, wherein protecting the skin from free radicals associated with blue light exposure comprises reducing the level of free radicals associated with blue light exposure and increasing the levels of the proteins Angel2 and Timeless in skin cells.

6. The method of claim 1, wherein the cosmetic composition comprises 0.5% to 2% by weight of the aqueous extract of aerial parts of lavender (Lavandula angustifolia), based on the total weight of the composition.

7. The method of claim 1, wherein the cosmetic composition is in a form selected from the group consisting of an aqueous solution, a hydroalcoholic solution, an oily solution, an oil-in-water emulsion, a water-in-oil emulsion, a multiple emulsion, a suspension, or a powder.

8. The method of claim 1, wherein the cosmetic composition is suitable for application to the skin, mucous membranes, and lips.

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