CITRUS EXTRACT AND ITS USE AS A COSMETIC AGENT

Abstract

This application relates to a citrus extract, preferably obtained from Citrus aurantium fruit, by hydroalcoholic extraction and hydrolysis, and its use as a cosmetic agent to protect the skin against bacterial stress. This application also relates to a composition comprising such an extract and a process for manufacturing said extract.

Description

Title of the invention: CITRUS EXTRACT and ITS USE AS A COSMETIC AGENT Technical field

[0001] The present invention relates to the field of cosmetic active ingredients and their uses, in particular for combating the effects of bacterial stress on the skin.

[0002] CONTEXT OF THE INVENTION

[0003] The skin is the first barrier protecting the body from external aggressions. This organ is composed of several layers of tissue. We distinguish (i) the epidermis which is the outermost part of the skin, (ii) the dermis, a connective tissue made up of fibroblasts and an extracellular matrix which ensures the functions of cohesion and nutrition of the skin, and (iii) the hypodermis made up of adipocytes.

[0004] The epidermis is made up of several cellular strata of keratinocytes. We distinguish, among others, the germinative layer of the epidermis, called the basal layer, containing, in particular, the cutaneous stem cells, the spinous layer, Stratum spinosum, made up of several layers of polygonal cells, the granular layer, Stratum granulosum, comprising one to three layers of flattened cells containing cytoplasmic inclusions, the keratohyaline grains, and finally, the horny layer, Stratum corneum which is composed of anucleated and keratin-rich cells called corneocytes which correspond to the terminal stage of differentiation of keratinocytes.

[0005] The outermost cells of the stratum corneum are continually shed and replaced by cells from a lower layer, in a process called desquamation. Cell regeneration of the stratum corneum is based on a process of cell maturation in which cells from the basal layer of the epidermis differentiate and gradually migrate through the different strata of the epidermis until they reach the stratum corneum in the form of corneocytes.

[0006] Furthermore, the cells of the stratum corneum are linked together by epidermal lipids. These lipids create a protective barrier and have a water-retaining power. In the event of a lipid deficiency, the skin can become dry, tight and rough. The epidermis is also covered with an emulsion of water and lipids (fats), called the hydrolipidic film. This film, renewed by the secretions of the sebaceous and sweat glands, helps keep the skin supple and acts as an additional barrier against pathogens and irritants.

[0007] Much more than an external envelope, the skin is a real organ ensuring a barrier function that protects our body against multiple external aggressions, whether microbiological, chemical or mechanical. The skin is supported in this protective function by the skin microbiota (also known as skin flora) made up of various types of microorganisms including bacteria such as Staphylococcus epidermidis. When a pathogen attempts to penetrate the body via the epidermis, the skin and the skin microbiota can act together to counteract this aggression: the skin microbiota prevents the pathogen from attaching and can secrete bacteriocins and other toxins while the skin's innate immune system is activated, involving the release of pro-inflammatory cytokines, the recruitment of immune cells such as neutrophils, NK cells and macrophages and the secretion of antibacterial peptides.

[0008] The balance between the skin and the skin microbiota is fragile. Due to repeated aggressions of all kinds, the skin can become more reactive, more sensitive and respond disproportionately to bacterial stress resulting from bacteria present in the environment, but also generated, in certain circumstances, by commensal bacteria. This disproportionate inflammatory reaction can further impair its barrier function and increase its reactivity to external aggressions. Indeed, a reduction in the skin's barrier function leads to an increase in its permeability to irritants and an alteration in its capacity to retain water. The skin suffers from cutaneous dryness and irritants can penetrate more deeply into the epidermis, leading in particular to inflammatory reactions.Eventually, the skin's tolerance threshold decreases, the skin becomes drier, rougher and more red, and skin discomfort sets in. This is called sensitive or reactive skin.

[0009] The care of sensitive skin is essentially based on the use of cosmetic products capable of intensely nourishing the skin and relieving feelings of discomfort. It is based on the combined use of moisturizing, emollient and lipid-replenishing agents to nourish and soften the skin.

[0010] However, such treatments do not aim to correct the hyperreactivity of the skin to toxins secreted by bacteria present on the surface of the skin.

[0011] There is therefore a need for new cosmetic agents capable of protecting the skin against the effects of bacterial stress. Summary of the invention

[0012] The invention relates to the cosmetic use of a Citrus fruit extract as a cosmetic agent protecting healthy skin against bacterial stress. The Citrus fruit extract is preferably a hydroalcoholic and hydrolyzed extract. A preferred species is Citrus aurantium.

[0013] In some embodiments, the bacterial stress results from exposure of the skin to bacterial secretome, for example produced by commensal bacteria, preferably S. aureus, S. epidermidis and C. acnes and / or to endotoxin present in the environment.

[0014] The extract according to the invention can be used to normalize the skin's response to bacterial stress and / or to increase the skin's tolerance to bacterial stress. In certain embodiments, the extract according to the invention is used to prevent, alleviate and / or treat one or more non-pathological alterations of the skin induced by bacterial stress, for example a decrease in the barrier function of the skin, a decrease in skin hydration, an increase in skin roughness, the appearance of redness, a loss of evenness of skin tone, a loss of suppleness, a loss of skin radiance and combinations thereof. In other embodiments, the extract is further used as a cosmetic agent to prevent, alleviate, soothe or treat skin discomfort resulting from exposure of the skin to bacterial stress.

[0015] The extract according to the invention can in particular act as an activator of the TAS2R14 signaling pathway, preferably as an agonist of the TAS2R14 receptor.

[0016] In certain embodiments, the extract is applied to sensitive skin, acne-prone skin or atopic-prone skin.

[0017] According to a particular aspect, the invention relates to an extract of Citrus fruit, preferably Citrus aurantium, obtained by a process comprising the following steps:

[0018] (a) extracting a Citrus fruit, preferably a Citrus aurantium fruit from using a hydroalcoholic solvent, and

[0019] (b) hydrolysis of the extract obtained in step (a)

[0020] A further subject matter according to the invention is a cosmetic ingredient comprising the extract as defined herein in a cosmetically acceptable vehicle selected from the group consisting of triethyl citrate, glycerol, alkanediols such as pro-panediol, propylene glycol, butylene glycol, pentylene glycol, ketal esters e.g. ketal levulinates such as methyl or ethyl levulinate or glycerol ketal levulinate, water, and mixtures thereof.

[0021] The invention also relates to a cosmetic composition intended to protect the skin against bacterial stress comprising the extract or cosmetic ingredient as defined herein and one or more cosmetically acceptable excipients.

[0022] The cosmetic composition according to the invention may be in the form of a cosmetic treatment, a makeup product or a body hygiene product preferably chosen from the group consisting of a lotion, a milk, a serum, an aqueous or oily gel, an emulsion, a cream, a gel-cream, a treatment water, an ointment, a balm, a foundation, a spray, a mascara, a stick, a lipstick, a gloss, a mousse, a deodorant, a nourishing mask, a shower gel, a cleansing oil, a soap, a shampoo or conditioner, a mask or a hair oil.

[0023] Finally, the invention also relates to a method for manufacturing a Citrus extract, preferably Citrus aurantium, comprising the following steps:

[0024] - the extraction of Citrus fruits, preferably in crushed and dried form, using of a hydroalcoholic solvent, and

[0025] - a hydrolysis step, preferably using an acidic aqueous solution. FIGURES

[0026] [Fig.l] shows the results of Example 2. Exposure of skin explants to the bacterial secretome (bacterial secretome) leads to a significant increase in the expression of the IL1a and IL-8 genes and a significant decrease in the expression of the TAS2R14 gene compared to skin explants not exposed to the secretome (Untreated). The extract according to the Invention makes it possible to counteract the effects of the bacterial secretome on the expression of the genes coding for IL1a, IL-8 and TAS2R14 (bacterial secretome + Extract according to the Invention).

[0027] [Fig.2] shows the results of Example 2. Exposure of skin fibroblasts to IL1-a stimulates the production of IL-8. Incubation of fibroblasts with the extract according to the invention significantly reduces the production of IL-8. This effect is blocked in the presence of SBA, a TAS2R14 antagonist.

[0028] [Fig.3] shows the results of Example 3. Exposure of skin fibroblasts to LPS causes an increase in the production of IL-8 by cells (IL-8 group). The extract according to the invention used at a dose of 0.01 mg / ml or 0.05 mg / ml significantly decreases the production of IL-8, and this in a similar manner to dexamethasone tested at 10 pM. Hesperidin has no notable effect on the production of IL-8. A non-hydrolyzed extract (bitter orange extract) and hesperetin decrease the production of IL-8 but much less effectively than the extract according to the invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] After extensive research, the Applicant has developed a new extract of Citrus aurantium prepared by hydroalcoholic extraction followed by a hydrolysis step. Surprisingly, the Applicant has shown that this extract was capable of modulating the immune response induced in skin explants by exposure to a bacterial secretome obtained from the culture medium of bacteria present in the human skin microbiota, namely Staphylococcus epidermidis, Staphylococcus aureus and Cutibacterium acnes. The extract according to the Invention thus made it possible to significantly reduce the expression of several pro genes inflammatory. The extract according to the invention also made it possible to prevent the decrease in the expression of a bitter taste receptor, namely TAS2R14: the level of expression of TAS2R14 in skin explants exposed to the bacterial secretome but treated with the extract according to the invention is similar to that observed for control explants, not exposed to the bacterial secretome.

[0030] The Applicant has also shown that the extract according to the invention was capable of reducing IL-8 production induced by IL-1a in skin fibroblasts and that this effect was inhibited in the presence of SBA, a TAS2R14 antagonist. These results strongly suggest that the extract according to the invention acts, at least in part, by activating the TAS2R14 signaling pathway.

[0031] The Applicant has also shown that the extract according to the Invention was capable of protecting skin fibroblasts against the pro-inflammatory effect induced by bacterial lipopolysaccharides, which are endotoxins, major constituents of the wall of Gram-negative bacteria. Notably, the extract according to the Invention proved to be significantly more effective than an equivalent non-hydrolyzed extract or than hesperetin tested at a dose equivalent to its content in the extract. Such a result underlines that the effect of the extract results specifically from its composition, in particular in aglycone compounds.

[0032] Finally, the Applicant has demonstrated that repeated application of the extract according to the Invention to the skin was well tolerated and did not generate any sensitization or irritation effect.

[0033] Thus, according to a first aspect, the invention relates to a hydroalcoholic and hydrolyzed extract of a species of Cil rus, in particular of the fruit of Citrus aurantium (also known as bitter orange) and its use as a cosmetic agent.

[0034] The extract according to the invention can be used as a protective agent for the skin against bacterial stress.

[0035] More specifically, the extract according to the invention can be used to protect the skin against non-pathological alterations of the skin resulting from its exposure to bacterial stress, in particular to a bacterial secretome. According to a particular aspect, the invention relates to the cosmetic use of an extract according to the invention as a cosmetic agent for balancing or normalizing the innate immune response of the skin against bacterial stress.

[0036] According to an additional aspect, the extract according to the invention is used in the cosmetic field to strengthen the skin barrier function and / or maintain or increase the resistance or tolerance of the skin against external aggression or stress, in particular of microbial origin.

[0037] The present invention also relates to a cosmetic ingredient or a com cosmetic position comprising an extract according to the Invention, as well as a cosmetic process comprising the application of an extract according to the Invention, eg formulated in the form of a cosmetic composition, to the skin.

[0038] The extract according to the Invention:

[0039] According to a first aspect, the invention relates to a hydroalcoholic and hydrolyzed extract of Citrus.

[0040] The extract is obtained from an aerial part of the plant, for example the leaves, stems, flowers, fruits and mixtures thereof. In a preferred embodiment, the extract is obtained from Citrus fruits.

[0041] Citrus means any plant species, including plant varieties, belonging to the genus Citrus. Citrus species include, but are not limited to, Citrus sinensis, Citrus aurantium, Citrus bergamia Risso, and Citrus reticulata.

[0042] In a most preferred embodiment, the extract according to the invention is obtained from fruits of Citrus aurantium, also called bitter orange.

[0043] Advantageously, the extract according to the invention can be prepared from immature fruits, that is to say, fruits having a level of maturity unsuitable for use in the agri-food sector. In other words, the extract according to the invention can be prepared from fruits discarded during harvesting, and therefore from by-products of agricultural activity.

[0044] The extract according to the invention can be prepared from the whole fruit or from any part of the fruit, in particular the skin and / or the bark, in particular from the bark or from any part thereof such as the epicarp, the mesocarp, the endocarp and their combinations.

[0045] Preferably, the extract according to the invention is prepared from the whole fruit of Citrus, in particular from whole fruits of Citrus aurantium

[0046] The extract according to the invention is called “hydroalcoholic”. For the purposes of the invention, a hydroalcoholic extract refers to an extract whose manufacturing process comprises a hydroalcoholic extraction step, i.e. an extraction using a water / alcohol mixture.

[0047] The extract according to the invention is said to be “hydrolyzed”. For the purposes of the invention, a hydrolyzed extract refers to an extract whose manufacturing process comprises a hydrolysis step, that is to say a step in which certain covalent bonds present in the extracted molecules are broken by reaction with a water molecule. The hydrolysis can be of any type, in particular acid hydrolysis (i.e. hydrolysis catalyzed by the presence of an acid), basic hydrolysis (i.e. hydrolysis catalyzed by the presence of a base) or enzymatic hydrolysis.

[0048] In certain embodiments, the extract according to the invention is prepared by a process comprising the following steps:

[0049] (a) Extracting a Citrus fruit, preferably a Citrus aurantium fruit from using a hydroalcoholic solvent,

[0050] (b) Hydrolysis of the extract obtained in step (a)

[0051] Before carrying out step (a), the Citrus fruit, preferably a Citrus aurantium fruit, may undergo one or more treatments such as washing, drying, grinding, cryogenic grinding, freezing, drying and combinations thereof. The Citrus fruit is preferably dried and ground before carrying out step (a). Step (a) is a solid-liquid extraction step.

[0052] The hydroalcoholic solvent used in step (a) is typically a mixture of water with an alcohol. For the purposes of the invention, the term “alcohol” includes lower alcohols, in particular C2-C5, glycerin, C2-C5 alkanediols and mixtures thereof.

[0053] For example, the alcohol may be selected from glycerin, ethanol, propanol, butanol, isomers thereof such as isopropanol, tert-butanol, pentanol, ethylene glycol, 1,3-propanediol, and mixtures thereof. Preferred solvents are ethanol, isopropanol, and glycerol.

[0054] The “alcohol / water” volume ratio in the hydroalcoholic mixture is typically in a range from 0.01 to 20.0, for example from about 1 to about 15.0, for example from about 5.0 to about 15.0, for example from about 7.0 to about 8.0.

[0055] A preferred “alcohol / water” mixture is an “EtOH / water” mixture, for example in a volume ratio of 5.0 to 10.0, for example 7.0 to 8.0.

[0056] The volume of hydroalcoholic solvent used in step (a) is preferably sufficient to cover the Citrus fruits. In general, the volume of hydroalcoholic solvent is generally between 1 L and 30 L, preferably from 5 L to 15 L per kg of Citrus fruits. Step (a) can be carried out with stirring. Step (a) can be carried out at room temperature or at a higher temperature. Preferably, step (a) is carried out under reflux for a sufficient time to allow efficient extraction of the molecules of interest. Typically, the extraction step (a) can last between a few minutes to a few hours, for example between 10 min and 6 h, preferably from 1 h to 3 h.

[0057] At the end of the extraction, the liquid phase and the solid phase of the reaction medium are separated. Any method allowing such separation can be used, such as filtration or centrifugation.

[0058] The liquid phase is recovered and is condensed, at least partially, preferably under reduced pressure. The residue obtained can be directly used in step (b).

[0059] Step (b) is a hydrolysis step. This step aims in particular to cleave the flavonoid heterosides present in the extract so as to release the parts aglycones of these compounds. For example, step (b) allows the conversion of hesperidin into hesperetin and naringin into naringenin. Preferably, step (b) allows an almost total conversion (of at least 90 mol%) of the hesperidin and naringin present in the extract obtained in step (a).

[0060] This step can be carried out by contacting the residue obtained in step (a) with an acidic aqueous solution, a basic aqueous solution or in the presence of an enzyme such as Rhamnosidases, hesperedinase, [3-glucosidase, [3-glucosidase and their combinations. In particular, commercial enzyme cocktails such as the Snailase mixture marketed by Abbexa can be used. In a preferred embodiment, step (b) is carried out by acid hydrolysis, for example using a concentrated aqueous solution of hydrochloric acid, for example an aqueous HCl solution at 1N.

[0061] Step (b) may be carried out at room temperature or at a higher temperature. Typically, the reaction medium is refluxed, for example in the case of acid hydrolysis. The reaction may last from a few minutes to a few hours, for example from 10 min to 5 h. At the end of the reflux step, the reaction medium may be cooled and then filtered. The filtrate may be brought back to a pH of approximately 5.0 to 6.0 in order to precipitate the extract. The precipitated extract may be recovered by any known method, for example by filtration. The precipitate thus recovered may be washed, dried and / or ground.

[0062] The extract according to the invention can therefore be obtained in the form of a powder. This extract can be directly implemented in the uses and methods described in the present application.

[0063] Where appropriate, the preparation process may comprise one or more additional steps, in particular a step of formulating the extract in a suitable vehicle.

[0064] For example, the Citrus extract according to the invention may be diluted in a solvent, for example chosen from triethyl citrate, glycerol (also called glycerin), alkanediols such as propanediol, butylene glycol, pentylene glycol and their isomers, ketal esters, e.g. ketal levulinates such as methyl or ethyl levulinate or glycerol ketal levulinate, water, and mixtures thereof. The extract according to the invention may also be encapsulated in a vectorization system, for example liposomes, or dissolved in a suitable solvent and then absorbed onto a solid support such as maltodextrin.

[0065] By way of example, the extract according to the invention may be formulated in a mixture of glycerol / water or alkanediol / water. Preferably, it is a C2-C5 alkanediol / water mixture. Said alkanediol is preferably a propanediol such as 1,3-propanediol and 1,2-propanediol (propylene glycol). The extract present gene- generally in a content ranging from 0.1% to 10%, e / g. approximately 1% by weight of the total weight of the formulation.

[0066] Antioxidant and preservative-type compounds (namely compounds having certain antibacterial or bacteriostatic activities) such as caprylyl glycol or glycerol caprylate or agents for adjusting the pH (eg buffering agent) can also be added to the formulation.

[0067] It goes without saying that the present invention also relates to the process for preparing the extract according to the invention described above, as such.

[0068] In a particular embodiment, the extract according to the invention comprises from 18% to 35% of hesperetin and from 8% to 20% of naringenin.

[0069] Preferably, the extract according to the invention comprises less than 3.0%, preferably less than 1.0% or less than 0.5% by weight of naringin and hesperetin,

[0070] The above percentages are expressed by weight relative to the total dry point of the extract. The quantitative composition of the extract can be determined in particular by HPLC analysis, as illustrated in the examples.

[0071] Uses, methods and cosmetic compositions according to the Invention:

[0072] According to another aspect, the invention also relates to the cosmetic, non-therapeutic use of a hydroalcoholic, hydrolyzed extract of Citrus, preferably obtained from the fruit of Citrus aurantium as described herein as a cosmetic agent. The extract according to the invention is preferably intended for application to healthy skin including a healthy scalp.

[0073] The term “cosmetic effect” means any non-therapeutic effect aimed at modifying and / or improving the appearance of the skin or mucous membranes such as the lips, at protecting them from external aggressions, in particular bacterial stress, at preventing, treating and / or correcting non-pathological alterations of the skin or its appendages and / or at preventing or treating sensations of discomfort of the skin or one of its appendages (in other words cutaneous discomfort).

[0074] For the purposes of the invention, the term "skin" means any part of the skin of the human body, in particular the skin of the face, including the lips and eyelids, the scalp, the neck, the skin of the hands and the skin of the feet. The appendages of the skin include nails, hair, eyebrows, eyelashes and hair, preferably hair.

[0075] For the purposes of the Invention, the term “healthy skin” means skin which does not present any lesion or pathology such as a skin infection.

[0076] The cosmetic use according to the invention does not include the treatment of pathologies, in particular skin pathologies such as skin infections.

[0077] According to a particular aspect, the invention relates to the cosmetic, non-therapeutic use of a hydroalcoholic, hydrolyzed extract of Citrus, preferably obtained from the fruit of Citrus aurantium as described herein, as a skin protective agent against bacterial stress.

[0078] For the purposes of the invention, the term "bacterial stress" means the exposure of the skin to certain bacterial strains, and / or to biomolecules derived from bacteria. Bacterial stress may be caused in particular by biomolecules derived from bacteria present in the environment or on the skin, for example environmental bacteria. Bacterial biomolecules include in particular bacterial secretomes and endotoxins, for example present in the environment. Exposure of the skin to certain bacterial strains and / or to the bacterial secretome and / or to endotoxins, in particular environmental ones, without there being a bacterial infection, may cause a disproportionate response in the skin, in particular an inflammatory response, leading to non-pathological alterations of the skin in the long term.

[0079] For the purposes of the invention, the term "secretome" refers to all the biomolecules secreted by bacteria. It is mainly a part of the proteome located at the level of the bacterial envelope or secreted into the extracellular environment. The bacterial secretome includes in particular exotoxins and enzymes. The secretome may be derived from bacteria belonging to the commensal flora, or to the transient flora of the skin, or even from bacteria present in the environment. In certain embodiments, the bacterial secretome is a secretome of commensal bacteria. Commensal bacteria of the skin include, but are not limited to, bacteria of the genus Staphylococcus (eg S. epidermidis or S. aureus), Corynebacterium, Cubacterium (eg C. acnes), Lactobacillus and Streptococcus.

[0080] In certain embodiments, the extract according to the invention is used as an agent for protecting the skin against stress resulting from exposure of the skin to a bacterial secretome, in particular S. epidermidis, S. aureus, and / or C. acnes.

[0081] For the purposes of the invention, the term "endotoxin" means complex molecules present in the wall of Gram-negative bacteria. These molecules are released during the multiplication or lysis of the bacteria, and persist for a long time in the environment and are resistant to numerous chemical or physical agents. For example, LPS are endotoxins. For the purposes of the invention, the term "exotoxin" means a toxin secreted by

[0082] a bacterium when it is still alive, unlike an endotoxin which is released during the growth phase or during cell lysis. The exotoxin is generally of a protein nature. In a particular embodiment, the extract according to the invention is used as a skin protective agent against stress resulting from exposure of the skin to a bacterial toxin, for example an endotoxin or an exotoxin, preferably originating from a commensal bacterium.

[0083] In another particular embodiment, the extract according to the invention is used as a protective agent for the skin against bacterial stress resulting from exposure of the skin to an environmental exotoxin.

[0084] The commensal flora includes, in addition to bacteria, other microbial agents such as fungi like Malassezia furfur or Candida albicans or mites like mites of the genus Demodex.

[0085] More generally, the extract according to the invention is used as a protective agent for the skin against stress resulting from exposure of the skin to microbial stress. Preferably, the microbial stress results from microorganisms present in the skin microbiota or from biomolecules originating from microorganisms present in the environment.

[0086] The term "protective agent against bacterial stress" or "protective agent against microbial stress" means a cosmetic agent capable of preventing, treating and / or alleviating one or more non-pathological alterations of the skin or its appendages and / or of preventing or treating sensations of discomfort of the skin or one of its appendages, resulting from the exposure of the skin to bacterial (or microbial) stress, in particular by normalizing the response of the skin to bacterial (or microbial) stress and / or by increasing the resistance or tolerance of the skin to bacterial (or microbial) stress.

[0087] Indeed, exposure of the skin to bacterial (or microbial) stress, and / or a disproportionate response of the skin to this stress can cause an alteration of the barrier, visual and / or mechanical properties of the skin.

[0088] In certain embodiments, the extract according to the invention is used as a cosmetic agent for preventing, alleviating and / or treating one or more non-pathological alterations of the skin caused by exposure to bacterial stress.

[0089] Such alterations include, but are not limited to: decreased skin barrier function, decreased skin hydration, increased skin roughness, the appearance of redness, loss of evenness of skin tone, loss of suppleness, loss of skin radiance and combinations thereof.

[0090] In other embodiments, the extract according to the invention is used as a cosmetic agent to prevent, alleviate, soothe or treat skin discomfort resulting from exposure of the skin to bacterial (or microbial) stress.

[0091] “Skin discomfort” means a set of unpleasant sensations, not linked to a pathology, such as sensations of tightness, tingling, itching, or heating, sensations of dry or dehydrated skin, transient redness and combinations thereof.

[0092] By way of example, the extract according to the invention, in particular the extract of the fruit of Citrus aurantium as described herein, can be used for:

[0093] - reduce, relieve or prevent sensations of tightness, tingling and / or itching, and / or

[0094] - reduce, relieve or prevent feelings of dryness or dehydration cutaneous, and / or

[0095] - treat or prevent transient redness, and / or

[0096] - reduce, relieve or prevent discomfort of the skin or scalp, and / or

[0097] - increase skin tolerance, and / or

[0098] - improve or maintain the barrier effect of the skin, and / or

[0099] - make the skin less rough and / or more supple.

[0100] In certain embodiments, the Extract according to the Invention is used on sensitive skin or on skin with atopic tendency.

[0101] “Sensitive, reactive or intolerant skin” means skin characterized by disproportionate reactivity to external factors (such as bacterial stress) which causes skin discomfort. This increased reactivity may result from a non-pathological alteration of the skin's barrier function which reduces the skin's tolerance threshold to external stimuli.

[0102] The term "atopic-prone skin" means sensitive skin in an individual who has suffered from atopic dermatitis, and / or has an allergic background, i.e. has a history of allergies and / or has a family history of allergies.

[0103] The phenomenon of "sensitive skin" can also be caused or accentuated by dysbiosis of the skin microbiota, that is to say by an imbalance of the skin microbiota (also called skin flora), as observed in certain skins with atopic tendency.

[0104] In certain embodiments, the extract according to the invention is used to normalize the skin's response to bacterial stress, and / or increase the skin's tolerance to bacterial stress. In this case, the extract according to the invention can be used in particular in sensitive or atopic-prone skin.

[0105] Without wishing to be bound by any theory, the Applicant is of the opinion that the protective action of the Extract according to the Invention against bacterial stress is linked to the activation of the TAS2R14 signaling pathway.

[0106] Thus, in certain embodiments, the extract according to the invention is used as an activating agent of the TAS2R14 signaling pathway in the skin. In particular, the extract according to the invention can be used as an agonist of the TAS2R14 receptor.

[0107] In certain embodiments, the extract according to the invention can be used to prevent an imbalance (or dysbiosis) of the skin microbiota and / or to promote the rebalancing of the skin microbiota. Indeed, without wishing to be bound by any theory, the Applicant is of the opinion that an exaggerated immune response of the skin against bacterial stress can be accompanied by an increase in the expression of receptors to microorganisms (TLR), or an increase in numerous inflammatory factors disrupting the balance between the skin and the cutaneous microbiota. Disruption of this balance can affect the composition of the cutaneous microbiota by promoting the expansion of certain bacterial strains to the detriment of others, leading to dysbiosis.

[0108] The term "acne-prone skin" or "skin with a tendency to blemishes" refers to a type of healthy skin with a tendency to develop comedones and pimples. The appearance of these skin imperfections may be associated with an imbalance in the microbiota, and in particular with overexposure of the skin to so-called acne-prone strains of C. acnes, such as strains of phylotype 1A1.

[0109] In certain embodiments, the extract according to the invention can be used on acne-prone skin or blemish-prone skin. The extract according to the invention is administered topically, that is to say applied to an area of ​​the skin or one of its appendages, including the scalp.

[0110] This is preferably a healthy area of ​​the skin or scalp, i.e. one that does not have wounds or skin pathologies.

[0111] The extract according to the invention can be used in any type of subject, in particular in subjects with sensitive, reactive or atopic-prone skin or in a subject with skin with imperfections.

[0112] The subject may be of any age and / or gender. For example, the subject may be an infant, a child, an elderly person (over 65 years old) or a woman of any age with sensitive or reactive skin. Preferably, the subject is at least 3 years old, for example at least 12 years old.

[0113] In certain embodiments, the extract according to the invention, preferably the hydroalcoholic, hydrolyzed extract of Citrus aurantium fruit, is applied to the skin in the form of a cosmetic composition.

[0114] Thus, the extract according to the invention is typically integrated into a cosmetic composition as an active cosmetic agent.

[0115] The extract according to the invention represents at least 0.0001%, for example at least 0.001% by weight of the composition. The extract is generally present in a content ranging from 0.0001% to 10% by weight, preferably from 0.0005% to 5% by weight, more preferably from 0.005% to 1% by weight relative to the total weight of said composition.

[0116] Said composition, which is also an object of the present invention, may comprise one or more cosmetically acceptable excipients.

[0117] Cosmetically acceptable excipients generally represent at least ù, for example at least 60%, 70% or 90% by weight relative to the total weight of the composition.

[0118] They may represent from 50% to 99.9999% by weight, preferably from 60% to 99.9995%, for example from 70% to 99.995% by weight relative to the total weight of the composition.

[0119] The composition may further comprise one or more additional active ingredients with a cosmetic effect.

[0120] Typically, said cosmetic composition comprises:

[0121] - from 0.0001% to 10% of extract according to the Invention,

[0122] - from 0% to 20% of one or more additional active agents with a cosmetic effect, and

[0123] - from 70% to 99.9999% of one or more excipients acceptable in terms of cosmetic,

[0124] the percentages being expressed by weight relative to the total weight of the cosmetic composition.

[0125] In certain embodiments, said composition comprises

[0126] - from 0.001% to 5% by weight of an extract according to the Invention,

[0127] - from 0.001% to 10% of one or more additional active agents with a cosmetic effect, and

[0128] - from 85% to 99.998% of one or more cosmetically acceptable excipients.

[0129] The term "active ingredient with a cosmetic effect, active agent with a cosmetic effect, cosmetic agent or active ingredient with a cosmetic effect" means a compound capable of exerting at least one cosmetic effect on the skin or its appendages. The additional active agent may exert any non-therapeutic effect aimed at modifying and / or improving the appearance of the skin or mucous membranes such as the lips, protecting them from external aggressions, or preventing and / or correcting phenomena linked to their aging.

[0130] The additional active agent(s) with a cosmetic effect may be chosen from the group consisting of vitamins, sunscreens and filters, anti-aging agents, lipid-replenishing agents, anti-wrinkle agents, tightening agents, anti-redness agents, emollient agents, antioxidants, firming agents, moisturizing agents, soothing agents, scrubbing or exfoliating agents, mattifying agents, sebum-regulating agents, lightening active ingredients, anti-blemish active ingredients, moisturizing agents and combinations thereof.

[0131] By way of example, the composition may comprise an active agent chosen from an anti-wrinkle agent, an anti-redness agent, a moisturizing active agent, an emollient agent, a re-lipidizing agent, a soothing agent, an anti-blemish agent, a filter or a sunscreen, and combinations thereof.

[0132] As examples of moisturizing agents, mention may be made of urea, pidolic acid (PCA) and its derivatives, in particular its salts such as arginine PCA, chitosan PCA, its copper salts (Copper PCA), magnesium salts (magnesium PCA), sodium salts (sodium PCA) or zinc salts, ethylhexyl PCA, calcium gluconate, hyaluronic acid and its salts and other glycosaminoglycans, hyaluronic acid, fructose, glucose, isomaltose, lactose, trehalose, polydextrose, sucrose, maltitol, mannitol, sorbitol, xylitol and other carbohydrates and derivatives, polyethylene glycols such as PEG-7, PEG-8, PEG-10, PEG-12 or PEG-14, glycerin, propylene glycol, pentylene glycol, butylene glycol, butanediol, betaine, citrulline, collagen and its derivatives, rhistidine, silk, keratin or soy hydrolysates, plant extracts rich in polysaccharides and / or polyphenols, for example extracts of Aloe, cornflower (Centaurea cyanus), and combinations thereof.

[0133] As lipid-replenishing or nourishing agents, mention may be made of vegetable oils such as olive oil, sweet almond oil, evening primrose oil, borage oil, shea butter, fatty acids, triglycerides, and ceramides.

[0134] Examples of emollient agents include coconut oil, cetearyl alcohol, petrolatum, liquid paraffin, lanolin, polyglycerides of fatty acids and combinations thereof.

[0135] Examples of soothing agents include allantoin, panthenol, bisabolol, extracts of aloe, marigold (Calendula officinalis), birch (e.g. Betula albd), oat seedlings, licorice, or willow herb (Epilobium angustifolium).

[0136] Examples of antioxidant agents include HMR (hydroxy methyl re-sorcinol), ascorbic acid and its derivatives, BHT (Butyl Hydroxy Toluene), vitamin B9, histidine hydrochloride, vitamin E and its derivatives, or an extract of willow herb (Epilobium augustifolium).

[0137] As anti-redness agents, mention may be made of saponins, flavonoids, rus-cogenins, esculosides, and extracts containing them, for example extracts of Ruscus, as well as certain essential oils, for example rosemary.

[0138] Examples of anti-stain agents include extracts such as licorice (Glycyrrhyza glabra), jackfruit extract (Artocarpus heterophyllus), Rumex extract (R.occidentalis), plant extracts belonging to the citrus genus, re-sveratrol, peptides such as oligopeptide-68, nonapeptide-1, kojic acid, magnesium ascorbyl phosphate and combinations thereof.

[0139] In certain embodiments, the cosmetic composition according to the invention comprises at least one additional plant extract, in addition to the extract according to the invention. The plant extract may be of any type. It may in particular be an essential oil, an extract obtained by decoction, extraction or maceration using an organic solvent such as an alkane, an ester, an ether, or a glycolic solution or by supercritical fluid extraction.

[0140] These extracts may have antioxidant, soothing, anti-aging or lightening (anti-blemish) properties or be present to provide a fragrant note to the com- position.

[0141] The cosmetically acceptable excipient(s) present in the cosmetic composition may be chosen from diluting agents, dispersing agents, gelling agents, emollients, vectorizing agents such as polycationic polymers or phospholipids, gums, resins, solvents in particular lower alcohols including ethanol, isopropanol, dipropylene glycol, butylene glycol, propanediol, glycerin, sorbitol, and propylene glycol, fillers such as modified and polymerized starches, titanium dioxide, or a metal stearate, preservatives, essential oils, pearlescent agents, colorants, odor absorbers, pH regulating agents or neutralizing agents, lubricating agents, thickening agents, surfactants including anionic, cationic, amphoteric or non-amphoteric surfactants. ionic, humectants such as glycerin or sorbitol,wetting agents, dispersing agents, perfumes, organic or mineral pigments such as iron oxides, oily agents such as oils or fats of vegetable origin, fats of animal origin, synthetic oils such as petroleum jelly, silicone oils, fatty alcohol esters, fluorinated oils, waxes, modified clays, bentonites, metallic salts of fatty acids, silica, mica, preservatives, vehicles such as mineral, thermal or floral water, and / or other substances commonly used in formulation in the cosmetic or pharmaceutical field.

[0142] The extract according to the invention can be incorporated into any type of composition. Preferably, it is a composition having a form suitable for topical administration, in particular suitable for application to the skin or scalp. Said cosmetic composition can be in the form of aqueous, hydroalcoholic solutions, oil-in-water (O / W) or water-in-oil (W / O) emulsions or multiple emulsions (triple: W / O / W or O / W / O), nanoemulsions, in particular O / W nanoemulsions, aqueous gels, dispersions or even a powder.

[0143] The composition according to the invention may be in the form of a lotion, a milk, a cream, an ointment, a balm including a lip balm, a gel, a mousse, a solution, a serum, or an aerosol,

[0144] More generally, the composition according to the invention may also be in the form of a cosmetic or dermocosmetic product of any type. It may be a cosmetic treatment, or a makeup or body hygiene product, for example a lotion, a milk, a serum, an aqueous or oily gel, an emulsion, a cream, a gel-cream, a treatment water, an ointment, a balm, a foundation, a spray, a mascara, a stick, a lipstick, a gloss, a mousse, a deodorant, a nourishing mask, a shower gel, a cleansing oil, a soap, a shampoo or conditioner, hair mask or oil.

[0145] By way of illustration, the extract may be present in a facial care cream.

[0146] The extract according to the invention may be incorporated as such in the cosmetic composition according to the invention, or in the form of a cosmetic ingredient.

[0147] According to a particular aspect, the invention relates to a cosmetic ingredient comprising the extract according to the invention, preferably a hydroalcoholic and hydrolyzed extract of C. aurantium in a cosmetically acceptable vehicle. The vehicle is typically chosen from triethyl citrate, glycerol (also called glycerin), alkanediols such as propylene glycol, butylene glycol, pentylene glycol, ketal esters e.g. ketal levulinates such as methyl or ethyl levulinate or glycerol ketal levulinate, water, and mixtures thereof. For example, the vehicle is a mixture of glycerol / water or alkanediol / water in a volume ratio of 0.2 to 10. Antioxidant and preservative-type compounds such as caprylyl glycol or glycerol caprylate or a buffering agent can also be added to the cosmetic ingredient.The extract represents at least 0.05% by weight and generally at most 10%, preferably at most 5% by weight or even at most 3% by weight of the cosmetic ingredient. The extract according to the invention is generally present in a content of 0.1% to 10%, for example from 0.0% to 5.0% by weight in the cosmetic ingredient according to the invention.

[0148] An additional subject matter according to the invention is a non-therapeutic, cosmetic method of a subject comprising administering a cosmetically effective amount of a Citrus extract as described herein, according to the invention, preferably a hydroalcoholic and hydrolyzed extract of C. aurantium topically, to said subject. The extract is typically applied to the area to be treated in the form of a cosmetic composition or product described above.

[0149] The cosmetic process according to the invention is implemented to obtain one or more cosmetic effects as described above. It is notably implemented to protect the skin against the effects of bacterial stress. For example, the cosmetic process according to the invention can be implemented to relieve or prevent skin discomfort, notably sensations of irritation, tingling, tightness, itching, dry skin, or dehydrated skin or skin redness or to maintain or improve the barrier function in skin exposed to bacterial stress. As an additional example, the process according to the invention can be implemented to prevent, attenuate and / or treat one or more non-pathological alterations of the skin caused by exposure to bacterial stress.

[0150] The subject may have sensitive, reactive or atopic-prone skin.

[0151] The extract according to the invention can be used in the method as an agent for modulating or normalizing the response of skin cells to bacterial stress, for increase the skin's tolerance threshold to bacterial stress and / or to maintain the barrier function of the skin exposed to bacterial stress. The extract according to the invention can be used as an activating agent of the TAS2R14 signaling pathway.

[0152] In the cosmetic methods and uses according to the invention, a Citrus extract obtained from C. aurantium fruits, previously dried and crushed, is preferably used, by a process comprising a hydroalcoholic extraction step followed by a hydrolysis step.

[0153] In the cosmetic methods and uses according to the Invention, the dose to be administered and the frequency of administration of the extract according to the Invention vary according to the desired cosmetic effect, the area of ​​application (skin or scalp), the characteristics of the individual, in particular their sex, age and skin type.

[0154] Typically, the extract according to the Invention can be applied at least once a week, or even 2 to 3 times a week, or daily when the area to be treated is the scalp.

[0155] As a further example, the extract according to the invention can be applied once or twice a day, typically in the morning and / or evening, when the area to be treated is an area of ​​skin on the body, neck, face, or hands.

[0156] By way of example, the subject can apply a dose of 0.5 g to 2 g of cosmetic composition comprising from 0.001% to 0.5% by weight of extract according to the Invention to his face, morning and evening.

[0157] The treatments described above can last several weeks, or even several months.

[0158] Other aspects and advantages of the present invention will appear on reading the following examples, which must be considered as illustrative and in no case as limiting. EXAMPLES

[0159] Example 1: Preparation of the extract according to the invention and chemical characterization

[0160] Immature bitter oranges (Citrus Aurantium) were dried and crushed. They were then placed in a hydroalcoholic solvent (EtOH / H2O in a volume ratio of 6.5 to 8.5) and heated for 2°C at a temperature ranging from 45°C to 55°C.

[0161] This mixture was then centrifuged to remove the orange residues. The liquid phase was recovered and then filtered. The solvent was then evaporated under reduced pressure. The residue obtained was added to an aqueous solution of hydrochloric acid HCl (0.5N-1N) at a ratio of 1 kg of residue to 20 L of aqueous solution (W / V) and refluxed for between 2 and 4 hours to carry out a complete hydrolysis step. The hydrolysis step serves in particular to cleave the flavonoid heterosides present in the extract in order to release the aglycone parts of these compounds. The extract precipitates during the hydrolysis reaction.

[0162] The reaction mixture is then cooled and the precipitated extract is drained and then washed with a NaOH solution in an amount of 1 / 20 (W / V) at a pH of 5-6. The powder obtained is then dried and ground to give the extract according to the invention.

[0163] The obtained extract was characterized by HPLC analysis with an Agilent Poroshel 120 SB C18 column (150 mm x 4.6 mm, 2.7 pm). The mobile phase consists of a mixture of water and acetonitrile with 0.1% (V / V) trifluoroacetic acid in each phase. The injected quantity is 3 pL, at a flow rate of 1 mL / min. Detection is carried out at 272 nm.

[0164] The extract according to the Invention mainly comprises:

[0165] - Hesperetin between 22% - 35%

[0166] - Naringenin between 10% - 20%

[0167] - Hesperidin < 1.0%

[0168] -Naringin <1.0%

[0169] The percentages are expressed in mass relative to the total mass of the composition.

[0170] Example 2: The extract according to the Invention protects against attacks caused by bacterial secretomes.

[0171] Objective: The objective of this study was to show that the extract according to the Invention exerts a protective action against attacks caused by toxins released by bacteria naturally present on the skin.

[0172] Protocol:

[0173] Preparation of bacterial secretomes

[0174] Bacterial strains Staphylococcus epidermidis (ATCC 14990), Staphylococcus aureus (ATCC 29213) and Cutibacterium acnes (ATCC 6919) were used for this study. Staphylococci were grown on Mueller-Hinton agar medium (MH, 0X0ID) and C. acnes on MH agar supplemented with 5% defibrinated horse blood (Thermo Scientific). Bacterial cultures were incubated at 37 °C under aerobic conditions for staphylococci and anaerobic conditions for C. acnes. For secretome production, strains were grown in Epilife medium (Gibco) for 20 h at 37 °C with orbital shaking at 200 rpm. Bacterial cultures were then centrifuged at 9,000 g for 15 min and supernatants were collected and filtered through 0.22 μm pore membranes (Sarstedt) before being stored at -80 °C until use. The mixture of the three secretomes (S. epidermidis, S. aureus and C.acnes) was carried out in equivalent proportion (vol / vol).

[0175] Ex vivo explant culture and stimulation conditions

[0176] Skin samples were obtained from mammoplasties or abdominal surgeries. After mechanical dissociation of the hypodermis, the skin pieces containing the dermis and epidermis were washed 3 times in PBS. 8 mm diameter biopsies were then taken and cultured on 500 μm diameter nylon filters (Sefar) in 12-well plates. The explants were cultured in DMEM (“Dulbecco's Modified Eagle Medium, GlutaMAX™ Supplement”, Gibco) supplemented with 10% fetal calf serum (FCS, Gibco) decomplemented FCS and 1% penicillin-streptomycin (Gibco) and incubated at 37 °C in a humid atmosphere with 5% CO2. The explants were treated with 10 pL of the extract according to the invention at 0.1 mg / mL for 1 h then stimulated for 24 h with 10 pL of the mixture of secretomes deposited on a cellulose disc (diameter 6 mm) on the surface of the explant (topical stimulation on the stratum corneum).

[0177] At the end of the incubation period, the explants were lysed in 1 mL of RAI buffer from the NucleoSpin® RNA extraction kit (Macherey & Nagel) supplemented with 10 μL of [3-mercaptoethanol (Sigma-Aldrich) containing 300 mg of glass beads (diameter less than 106 μm, Sigma) and cryogenically ground at 0°C using the Cryolys tissue homogenizer (Bertin technologies) at 8800 rpm (4 cycles of 20 seconds interspersed with 30-second pauses) then centrifuged for 10 min at 11,000 g. The cell lysates were stored at -80°C until extraction.

[0178] Nucleic acid extraction, reverse transcription

[0179] Total RNA was extracted using the NucleoSpin® RNA Extraction Kit (Macherey & Nagel) recommendations. RNA concentrations and sample purity were assessed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Then, reverse transcription was performed from 1 pg of the RNA extracts using the SuperScript II kit (Invitrogen). The resulting cDNAs were diluted 1 / 10 in ultrapure water and were kept at 4°C before use or stored at -20°C.

[0180] Real-time quantitative PCR

[0181] From the cDNAs generated by reverse transcription, a transcriptional analysis by quantitative PCR was carried out in a 96-well plate and read by the LightCyler 480 (Roche) where each well (sample) contains 5 μl of SYBR Green Master Mix (AceQ qPCR, Vazyme) including SYBR Green, Taq polymerase, MgC12 and dNTPs, as well as 1 μM of sense and antisense oligonucleotide primers, and 2 μl of cDNA diluted to 1 / 10. Gene expression was normalized with the housekeeping genes GAPDH (Glyceraldehyde-3-phosphate Dehydrogenase) and 28S (28S ribosomal protein), allowing the relative expression to be carried out in relation to the housekeeping genes calculated as follows: 2-ACT where ACT = CT gene of interest - average CT of the genes of household, or the expression of the transcripts was reported to the control condition in the experiments according to the AACT:2-AACT method with AACT = ACT (control) - ACT (sample).

[0182] Results:

[0183] The results are illustrated in [Fig.l]. During this study, it was found that the secretome released by the bacterial mixture composed of C.Acnes, S.Aureus and S.Epidermidis induced an increase in skin inflammation. Indeed, a significant increase in pro-inflammatory markers (IL-1a and IL-8) was shown for explants treated with the bacterial secretome compared to control explants. In addition, a significant decrease in the expression level of the TAS2R14 receptor was also observed in explants treated with the bacterial secretome compared to control explants.

[0184] The treatment of skin explants with the extract according to the invention makes it possible to very significantly reduce the inflammatory immune response induced by the bacterial secretome at the level of the skin explant. Indeed, the extract according to the invention made it possible to significantly prevent the increase in the expression of several pro-inflammatory genes. The extract also made it possible to prevent the decrease in the expression of the bitter taste receptor (TAS2R14): the level of expression of TAS2R14 in skin explants exposed to the bacterial secretome but treated with the extract according to the invention is similar to that observed for the control explants. This demonstrates that the extract according to the invention makes it possible to protect the skin against toxins produced by bacteria naturally present in the skin microbiota.

[0185] [Tables 1] Fold change Bacterial secretome vs. Control explant P-value Fold change Bacterial secretome with extract according to the invention vs. Control explant P-value vs. Bacterial secretome IL-1a 3.20 0.008 1.15 0.04 IL-8 4.92 0.027 1.14 0.006 TAS2R14 0.47 0.004 0.88 NS

[0186] Table 1: modulation of the expression of the IL-1a, IL-8 and TAS2R14 genes in skin explants exposed to the bacterial secretome + / - with the extract according to the Invention versus control explants (not exposed to the bacterial secretome and not treated with the extract according to the Invention).

[0187] Example 3: Involvement of the TAS2R14 pathway (extract + / -SBA, U-73122)

[0188] Objective: The objective of this study was to show that the protective effect of the extract according to the invention against bacterial toxins is mediated by the activation of the TAS2R14 receptor. To verify this, a TAS2R14 receptor antagonist was used to block the activation of this receptor in human dermal fibroblasts (NHDF).

[0189] Protocol: NHDFs were isolated from human dermis (women aged 30, 39 and 53) and maintained in a specific medium composed of DMEM (Dutscher, L0060-500) containing 10% serum (Sigma, F7524), 1% antibiotics (penicillin / streptomycin / Sigma, P0781) at 37°C under 5% CO2 and 95% humidity.

[0190] NHDFs were seeded at a concentration of 2.104 cells / well in microplates in complete DMEM. After 1 day, the medium with serum was removed and the cells were treated with the TAS2R14 antagonist, sulfamoylbenzoic acid (SBA at 500pM) for 1 hour. Then, IL1-a, an inducer of IL-8 production, was added in the presence or absence of the extract according to the invention. After 24h, the supernatants were collected and the IL-8 released by the fibroblasts was determined by ELISA (DY208, R&D System). Cell viability was also measured (MTS, Promega, G1111). ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed to detect and quantify peptides, proteins, or hormones in cell supernatants. Following the supplier's instructions for antibody dilutions, the absorbance of each well is then measured by a plate reader at 450 nm, with a correction at 540 nm.Results are reported as percentage of IL-8 synthesis relative to cell viability. The obtained data and percentage changes were subjected to the two-way Student's t-test for paired data. The statistical significance value is p<0.05 (*p<0.05, **p<0.01, ***p<0.001).

[0191] Statistical analyses

[0192] The obtained data and percentage changes were subjected to the Iway ANOVA (Kruskal-Wallis) test. The statistical significance value is p<0.05 (#p<0.1, *p<0.05, **p<0.01, ***p<0.001).

[0193] Results: The results are illustrated in [Fig.2]. As expected, IL-1a induced the production of IL8 by NHDFs. When the addition of IL-1a is carried out in the presence of the extract according to the invention, the induction of IL8 production is decreased by approximately 30% at the concentration of 0.01 mg / mL.

[0194] When the cells were treated simultaneously with IL-1a and the extract according to the invention in the presence of SBA, a TAS2R14 antagonist, the level of IL-8 production is similar to that of the cells treated with IL-1a only: in other words, the protective effect of the extract according to the invention is no longer visible. Such a result confirms that the protective effect of the extract according to the invention involves an action on the TAS2R14 receiver

[0195] Example 4: The extract according to the invention has a protective effect against bacterial toxins which is superior to that of hesperetin, hesperidin and a non-hydrolyzed bitter orange extract.

[0196] Objective: The objective of this study was to evaluate the effects of the extract according to the Invention on the inflammatory response of human dermal fibroblasts (NHDF) induced by lipopolysaccharides (LPS), bacterial endotoxins.

[0197] Protocol: NHDFs are fibroblasts isolated from human dermis and maintained in a specific medium composed of DMEM (Dutscher, L0060-500) containing 10% serum (Sigma, F7524), 1% antibiotics (penicillin / streptomycin / Sigma #P0781) at 37°C under 5% CO2 and 95% humidity.

[0198] NHDFs were seeded at a concentration of 2.104 cells / well in microplates in complete DMEM. After 1 day, the medium with serum was removed and the cells were treated with LPS at 100 ng / mL, as an inducer of IL-8 production, in the presence / absence of the extract according to the invention, a classic bitter orange extract (i.e., the precursor extract before hydrolysis), hesperetin, or positive control (dexamethasone at 10pM). Hesperetin was used at a concentration of 8 and 4 pM, which corresponds to the concentration present in the extract according to the invention at 0.01 mg / mL and 0.005 mg / mL, respectively. At the end of the incubation period, supernatants were collected and IL-8 released by fibroblasts was determined by ELISA (DY208, R&D System) and cell viability by a viability assay (MTS, Promega, G1111).ELIS A (enzyme-linked im-munosorbent assay) is a plate-based assay technique designed to detect and quantify peptides, proteins, or hormones in cell supernatants. Following the supplier's instructions for antibody dilutions, the absorbance of each well is then measured by a plate reader at 450 nm, with a correction at 540 nm. Results are reported as a percentage of IL-8 synthesis relative to cell viability.

[0199] Statistical analyses

[0200] The obtained data and percentage changes were subjected to the ANOVA-one way Fisher LSD test. The statistical significance value is p<0.05 (*p<0.05, **p<0.01, ***p<0.001).

[0201] Results:

[0202] The results are illustrated in [Fig.3]. As expected, LPS activates IL-8 production at NHDFs. Dexamethasone significantly decreased IL-8 production. After 24 hours of treatment, the extract according to the invention significantly reduced IL-8 production by 49% and 41% at 0.01 and 0.005 mg / ml, respectively. It is noted that the decrease in IL-8 production obtained with the extract according to the Invention is similar to that obtained with dexamethasone.

[0203] It is noted that the extract according to the invention has a protective effect against LPS-induced IL-8 production that is greater than a bitter orange extract without hydrolysis (-21% and -23% at 0.01 mg / mL and 0.005 mg / mL, respectively). Notably, it is noted that at a dose equivalent to that contained in the extract according to the invention, hesperetin alone is much less effective in counteracting the effect of LPS than the extract according to the invention: such a result demonstrates that the protective action of the extract according to the invention results from its complex composition, said composition making it possible to potentiate the effect of hesperetin.

[0204] Example 5: Effect of the extract on irritation and sensitivity (HRIPT test)

[0205] Objective: The objective of this study was to determine the potential of the extract according to the Invention to cause skin irritation and / or to induce sensitization after repeated patch applications.

[0206] Protocol: A total of 120 male and female subjects, aged 18 to 70 years, were selected to participate in the study. This clinical study includes an induction phase, a challenge phase, and a new challenge test. During the induction phase, the extract according to the Invention is applied to the same site on the back of each subject three times a week for a total of nine applications, with 24-hour rest periods, except for the Friday applications, which allows for a 48-hour rest period. The extract according to the Invention is tested at Img / mL. The test sites are examined at each visit to verify the absence of skin irritation before proceeding with a new application of the extract according to the Invention. Approximately 10 to 21 days after the last visit of the induction phase, the subjects return to the laboratory for the challenge phase.The test product is applied to a blank site on the back under a suitable patch and is removed by clinic staff approximately 24 hours later. The test sites are examined for signs of skin irritation or sensitization. A subject may be asked to return to the laboratory for further challenge testing if reactions indicating sensitization are observed during the challenge phase. During this phase, the test product is applied to a blank site under the same conditions as during the challenge phase, and the subject is examined for skin reactions. Any visible irritation is recorded.

[0207] The following dermal scoring system was used:

[0208] 0: No visible skin reaction

[0209] +: Barely perceptible erythema

[0210] 1+: Mild erythema

[0211] 2+: Well-defined erythema

[0212] 3+: Severe erythema and edema

[0213] 4+: Erythema and edema with vesiculation

[0214] Results:

[0215] Based on the tested population, 116 subjects completed the study.

[0216] The study showed that the extract according to the Invention is very well tolerated: the Extract did not induce erythema and / or induce sensitization. The Extract according to the invention does not present a significant risk of skin irritation or sensitization. The 116 volunteers who completed the study all presented a score of "0"

[0217] Example 6: Example of a cosmetic product incorporating the extract according to the Invention

[0218] Day cream comprising 1% of a cosmetic ingredient comprising 1% by weight of the extract according to the Invention prepared according to Example 1 in 1,3-propanediol (final percentage of the extract according to the Invention in the cream: 0.01% by weight).

[0219] [Tables2] INCI % by weight AQUA (Water) 79.30 XANTHAN GUM 0.50 GLYCERYL STEARATE CITRATE 2.00 POLYGLYCERYL-3 STEARATE 2.00 PRUNUS AMYGDALUS DULCIS (SWEET ALMOND) OIL 3.00 CETYL ALCOHOL 2.00 BUTYROSPERMUM PARKII (SHEA) BUTTER 3.00 COCO-CAPRYLATE 3.00 TOCOPHEROL (AND) HELIANTHUS ANNUUS (SUNFLOWER) SEED OIL 0.20 GLYCERIN (AND) SODIUM ANISATE (AND) SODIUM LE-VULINATE (AND) AQUA (WATER) 4.00 PROPANEDIOL (AND) CITRUS AURANTIUM AMARA (BITTER ORANGE) FRUIT EXTRACT according to the Invention 1.00 100.00

Claims

Claims

1. Cosmetic use of a Citrus fruit extract as a cosmetic agent protecting healthy skin against bacterial stress.

2. Use according to claim 1, wherein the Citrus fruit extract is a hydroalcoholic and hydrolyzed extract.

3. Use according to claim 1 or 2, wherein the Citrus fruit extract is a Citrus aurantium fruit extract.

4. Use according to any one of claims 1 to 3 wherein the bacterial stress results from exposure of the skin to a bacterial secretome, for example produced by commensal bacteria, preferably S. aureus, S. epidermidis and C. acnes and / or to an endotoxin present in the environment.

5. Use according to any one of claims 1 to 4 wherein the extract is used to normalize the skin's response to bacterial stress and / or to increase the skin's tolerance to bacterial stress.

6. Use according to any one of claims 1 to 5 wherein the extract is used to prevent, alleviate and / or treat one or more non-pathological alterations of the skin induced by bacterial stress, for example a decrease in the barrier function of the skin, a decrease in the hydration of the skin, an increase in the roughness of the skin, the appearance of redness, a loss of homogeneity of the complexion, a loss of suppleness, a loss of radiance of the skin and combinations thereof.

7. Use according to any one of claims 1 to 6 wherein the extract is further used as a cosmetic agent for preventing, alleviating, soothing or treating skin discomfort resulting from exposure of the skin to bacterial stress.

8. Use according to any one of claims 1 to 7 wherein the extract acts as an activator of the TAS2R14 signaling pathway, preferably as an agonist of the TAS2R14 receptor.

9. Use according to any one of claims 1 to 8 in which the extract is applied to sensitive skin, acne-prone skin or atopic-prone skin.

10. 0 Citrus fruit extract, preferably Citrus aurantium, obtained by a process comprising the following steps: (a) extracting a Citrus fruit, preferably a Citrus fruit aurantium using a hydroalcoholic solvent, and (b) hydrolysis of the extract obtained in step (a).

11. 11. A cosmetic ingredient comprising the extract of claim 10 in a cosmetically acceptable vehicle selected from the group consisting of triethyl citrate, glycerol, alkanediols such as propanediol, propylene glycol, butylene glycol, pentylene glycol, ketal esters e.g. ketal levulinates such as methyl or ethyl levulinate or glycerol ketal levulinate, water, and mixtures thereof.

12. 2 Cosmetic composition for protecting the skin against bacterial stress comprising the extract according to claim 10 or the cosmetic ingredient according to claim 11 and one or more cosmetically acceptable excipients.

13. 3 Cosmetic composition according to claim 12 characterized in that it is in the form of a cosmetic treatment, a makeup product or a body hygiene product preferably chosen from the group consisting of a lotion, a milk, a serum, an aqueous or oily gel, an emulsion, a cream, a gel-cream, a treatment water, an ointment, a balm, a foundation, a spray, a mascara, a stick, a lipstick, a gloss, a mousse, a deodorant, a nourishing mask, a shower gel, a washing oil, a soap, a shampoo or a conditioner, a mask or a hair oil.

14. 4 A method of manufacturing an extract of Citrus, preferably Citrus aurantium, comprising the following steps: - extraction of Citrus fruits, preferably in crushed and dried form, using a hydroalcoholic solvent, and - a hydrolysis step, preferably using an acidic aqueous solution.

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