Production process of an extract of the microalga Emiliania huxleyi essentially free of coccoliths, extract thus obtained, composition including this extract, and associated uses.

Abstract

The invention relates to a method for producing an extract of the microalga Emiliania luxleyi essentially without the coccoliths usually produced by this microalga. The invention also relates to such an extract of Emiliania luxleyi obtained by this method. The invention further relates to compositions comprising this extract. The invention also relates to the cosmetic uses of such an extract or such compositions to prepare the skin to combat oxidative stress from the external environment, and / or to prepare the skin to maintain its homeostasis.

Description

Title of the invention: Process for producing an extract of the microalga Emiliania huxleyi essentially free of coccoliths, extract thus obtained, composition comprising this extract, and associated uses. technical field

[0001] The field of the invention is that of cosmetology. More specifically, the invention relates to a process for producing an extract of the microalga Emiliania huxleyi (recently renamed Gephyrocapsa huxleyi), essentially without the coccoliths usually produced by this microalga, such an extract of Emiliania huxleyi obtained by this process, compositions comprising this extract, as well as the cosmetic uses of such an extract or such compositions to prepare the skin to fight against oxidative stresses from the external environment, and / or to prepare the skin to maintain its homeostasis.

[0002] Such an extract makes it possible, in particular, to stimulate the expression of genes that have an impact on preparing the skin to react to aggressions from the external environment. Prior Art

[0003] Age and chronic exposure to external stressors, particularly environmental stressors including ultraviolet (UV) radiation or oxidizing compounds such as reactive oxygen species (ROS), can have deleterious effects on the skin. Such effects can be illustrated by the alteration of certain proteins, or by the degradation of extracellular matrix (ECM) components, for example collagen or elastin.

[0004] Cosmetic solutions are known to combat these harmful effects, such as compositions including anti-aging active ingredients.

[0005] It is also known to use biomineral compounds, such as coccoliths secreted by algae, or compositions comprising them, in particular to protect the skin against UV radiation while mitigating unsightly skin features, such as dark circles or wrinkles.

[0006] Coccoliths are formed of calcite (CaCO3) and a polysaccharide matrix, and synthesized by coccolithophores.

[0007] Coccolithophores are unicellular marine microalgae characterized by the presence on their outer surface of an assembly of calcareous plates protecting them, which are these coccoliths.

[0008] One objective of the present invention is therefore to propose cos compositions metics including active ingredients designed to prepare the skin to better react to environmental aggressions.

[0009] There is also a strong and constant need for effective cosmetic compositions comprising natural active ingredients instead of synthetic chemical compounds. Summary of the invention

[0010] This objective is achieved through the invention, which relates to a process for producing an extract of the microalga Emiliania huxleyi essentially without coccoliths.

[0011] The inventors have demonstrated, in a surprising manner, that the use of an extract of the microalga Emiliania huxleyi that has produced few, if any, coccoliths yields interesting effects on skin protection and maintaining its hydration. In particular, such use prepares the skin to better react to environmental aggressors by stimulating the skin's cell detoxification systems and defenses. Topical application of this particular Emiliania huxleyi microalga therefore protects the skin against aggressive external elements, counteracting oxidative effects from external sources such as ultraviolet radiation, pollution, and heavy metals.

[0012] The microalga Emiliania huxleyi, now known as Gephyrocapsa huxleyi, is known to have a very significant impact on the climate. It is considered to be responsible for the homeostasis of the Earth.

[0013] Emiliania huxleyi plays a very important role in the carbon cycle. In particular, it captures carbon dioxide (CO2). Its ability to produce oxygen also makes it a very important element in the oxygenation of the atmosphere, the formation of the ozone layer, and therefore life on Earth.

[0014] Emiliania huxleyi also produces dimethylsulfoniopropionate (DMSP), which is active in thermal regulation by reflecting solar radiation, thus contributing to the reduction of the greenhouse effect. The DMSP produced by Emiliania huxleyi is transformed into dimethyl sulfide (DMS), an organosulfur gas known to have a significant impact on the climate and to be involved in cloud formation. This compound is an antioxidant and has a structure similar to glycine betaines produced by higher plants. Emiliania huxleyi can convert DMSP into DMS, just as marine bacteria can also do, thanks to a DMSP lyase. During the degradation of DMSP into DMS, Emiliania huxleyi also releases acrylates, which are antimicrobial.

[0015] Emiliania huxleyi also captures calcium and produces calcite, via the production of coccoliths. Emiliania huxleyi adapts to its environment, as the size The number of cells and coccoliths is increased when the CO2 level increases.

[0016] Emiliania huxleyi also adapts very well to changes in its environment thanks to a very high degree of genetic flexibility. It captures selenium from seawater and produces selenoproteins which are similar in structure and function to the antioxidant proteins of skin cells, in particular thioredoxin reductase 1 (TRI).

[0017] Thus, the invention is based on an original approach which consisted of studying the effect of topical application of extracts of Emiliania huxleyi which essentially do not contain coccoliths on the expression of genes involved in the fight against the oxidative effects / oxidative stresses of the environment / external.

[0018] Thus, a first object relates to a process for producing an extract of the microalga Emiliania huxleyi comprising: - a photobioreactor culture step of Emiliania huxleyi, using a culture medium comprising seawater, one or more microelements, such as selenium, and preferably between 10 and 100 pM (micromoles.L') of phosphate, more preferably between 20 and 60 pM of phosphate. This step allows the culture of Emiliania huxleyi essentially without the production of coccoliths, that is to say that only a few percent of the cells produced of Emiliania huxleyi possess a coccolith, preferably less than 1% of the cells produced, and even more preferably less than 0.1% of the cells produced.

[0019] The following steps of this process are: -a stage of harvesting the crop; -a step of freezing the culture; -a step of thawing the frozen culture and adding propane-1,3-diol; and -at least one filtration step.

[0020] Thus, it has been demonstrated that the absence, or near absence, of coccoliths in the extract obtained from Emiliania huxleyi plays a major role in preparing the skin to better react to aggressions from the external environment. Furthermore, this process is simple to implement and inexpensive.

[0021] According to an advantageous embodiment, said at least one final filtration step of this process comprises: -a tangential filtration stage through a filter with a cutoff threshold of 0.2 µm; and -a sterilizing filtration step.

[0022] Thus, the extract obtained by this method is suitable for use in cosmetic formulation.

[0023] A second object of the invention consists of an extract of the microalga Emiliania huxleyi obtained by the process described above, this extract not comprising essential virtually no coccoliths, that is to say that only a few percent of the cells produced of Emiliania huxleyi possess a coccolith, preferentially less than 1% of the cells produced, and even more preferentially less than 0.1% of the cells produced.

[0024] According to one aspect of the invention, this extract stimulates the expression of genes encoding proteosome proteins, and / or metallothioneins, and / or stress proteins (“Heat Shock Proteins”), and / or extracellular matrix proteins, and / or the production of HSP proteins and ubiquitin.

[0025] Stimulation of these genes and increased production of these proteins have a positive impact on the skin's antioxidant defenses, in particular to better prepare the skin to fight against the imbalance caused by stress and to maintain its homeostasis.

[0026] A third object relates to a cosmetic composition for topical application comprising an extract of the microalga Emiliania huxleyi as previously described, as well as a physiologically acceptable medium.

[0027] According to one embodiment, this composition according to the invention comprises between 0.01% and 10% by weight of extract of Emiliania huxleyi, relative to the total weight of the composition, preferably between 0.1% and 1% by weight, relative to the total weight of the composition.

[0028] At these concentrations of extract in cosmetic compositions, an appropriate cosmetic formulation can be achieved, allowing the generation of the desired cosmetic effects on the skin, without side effects.

[0029] A fourth object relates to the use of an extract of Emiliania huxleyi as previously described as a cosmetic active ingredient.

[0030] A fifth object relates to the use of a composition according to the invention as previously introduced as a cosmetic composition intended to be applied to healthy skin.

[0031] According to one embodiment, this composition is used in particular to prepare the skin to fight against external oxidative stresses, and / or to prepare the skin to maintain its homeostasis. Brief description of the figures

[0032] [Fig. 1] is a histogram representing the amount of carbonylated proteins, illustrating oxidative stress, on areas of skin that have been exposed to UV rays, compared with areas of skin that have not been exposed to such radiation.

[0033] [Fig.2] is a histogram illustrating the value of the antioxidant protection index during topical application of the Emiliania huxleyi extract according to the invention, in comparison with the application of a placebo.

[0034] [Fig.3] is a graph showing the evolution of transepidermal water loss in the time in g / m2 / h, in the case of topical application of the extract of Emiliania huxleyi according to the invention, compared with the application of a placebo.

[0035] [Fig.4] is a histogram representing the variations in transepidermal water loss dermal over time in percentage, in the case of topical application of the extract of Emiliania huxleyi according to the invention, in the case of application of a placebo and in the comparative case extract according to the invention / placebo. Detailed description of the invention

[0036] Method for producing an extract of Emiliania huxleyi according to the invention

[0037] According to a first aspect of the invention, an extract of Emiliania huxleyi is produced according to the following general process: -a first stage of culture of Emiliania huxleyi is implemented in a photobioreactor, in the presence of seawater, at least one microelement, such as selenium, and preferably with a phosphate content between 10 and 100 pM, more preferably between 20 and 60 pM; -this is followed by a stage of harvesting the crop; -the harvested crop is frozen; -a subsequent step involves thawing this frozen medium and adding propane-1,3-diol; and -a final filtration step is implemented, allowing the obtaining of an extract of Emiliania huxleyi which does not contain, or at least very few, coccoliths.

[0038] The culture stage of Emiliania huxleyi is carried out in a photobioreactor with a capacity of 30 L to 750 L, with seawater, illuminated by artificial light.

[0039] The microalga Emiliania huxleyi is used whole, in the form of individual cells.

[0040] The addition of a small amount of selenium to the culture medium, from 1 to 100 nM (nanomoles.L1), more preferably from 2 to 10 nM, is necessary for the production of Emiliania huxleyi, as this microalga is selenium-dependent for its growth. Indeed, the selenium taken up by Emiliania huxleyi is then incorporated into selenoproteins that are essential for cell function, allowing them to multiply rapidly.

[0041] Upstream, the photobioreactor is chemically sterilized using sodium hypochlorite NaClO, and neutralized with a sodium sulfite solution Na2SO3.

[0042] The inoculation rate is approximately 4%.

[0043] A preliminary pre-culture is carried out in a 10 L carboy, under the same medium conditions as those applied to the culture stage of the mid- croalgue.

[0044] During the culture, the parameters monitored are optical density, in particular at 750 nm, cell counting, using a Malassez slide (allowing the number of cells suspended in a solution to be counted), and pH.

[0045] The culture lasts between 10 and 15 days. It is harvested when the number of cells exceeds 2 x 10⁶ cells / mL*. This concentration ensures the presence of a minimum quantity of cells necessary for the production of the Emiliania huxleyi extract. For example, the inventors succeeded in obtaining 4 billion cells per liter of culture in three weeks.

[0046] The distinctive feature of this Emiliania huxleyi production process is that the culture conditions used do not promote the production of Emiliania huxleyi coccoliths, unlike existing processes for producing coccolithophore extracts, particularly those intended for cosmetic use. Specifically, in the process according to the invention, the microalgal strain used loses its ability to produce coccoliths, notably through repeated subculturing in the culture medium, which is slightly richer in phosphate than the media typically used to promote algal coccolith production. Thus, this process according to the invention drastically limits coccolith production, so that, for example, only a few percent, advantageously less than 1%, and even more advantageously less than 0.1%, of the Emiliania huxleyi cells produced actually produce coccoliths.

[0047] The final extract obtained is a hydro-glycolic extract, also comprising seawater and propanediol. Extract of Emiliania huxleyi according to the invention

[0048] According to a second aspect of the invention, an extract of Emiliania huxleyi which has not, or at least very little, produced coccoliths is obtained.

[0049] It has been demonstrated by the inventors that such an extract of Emiliania huxleyi without coccoliths, or with very few coccoliths, stimulates the expression of genes encoding stress proteins (“Heat Shock Proteins”, HSP).

[0050] When the first HSPs were discovered, they were initially observed when cells were exposed to heat stress (Ritossa, 1962). It is now known that HSPs are of different sizes and play diverse roles in cells, including repairing damaged proteins, assisting in the synthesis of new proteins, and assembling newly produced proteins. They enable cell survival under various types of stress.

[0051] The main function of HSPs is to allow newly formed or incorrectly formed proteins to acquire the correct shape in which they will be functional. In the absence of this conformational change, proteins can be inactive and non-functional.

[0052] The inventors have also demonstrated that the extract of Emiliania huxleyi according to the invention stimulates the expression of genes encoding proteasome proteins.

[0053] The proteasome helps to detoxify cells by eliminating altered proteins that accumulate with age.

[0054] It has also been demonstrated that the extract of Emiliania huxleyi according to the invention stimulates the expression of genes encoding metallothioneins (MT).

[0055] Metallothioneins also help to detoxify cells against heavy metals.

[0056] It has finally been demonstrated that the extract of Emiliania huxleyi according to the invention stimulates the expression of genes encoding extracellular matrix proteins. Composition according to the invention

[0057] A third object of the invention relates to a cosmetic composition comprising an extract of the microalga Emiliania huxleyi according to the invention, therefore containing no, or very little, coccoliths of this microalga. Such a composition further comprises a physiologically acceptable medium.

[0058] A physiologically acceptable medium is defined as any medium suitable for cosmetic use, particularly for application to the skin. For example, a physiologically acceptable medium may be selected from or include water or a water-soluble organic solvent, texturizing agents, including a gelling agent, perfumes, preservatives, colorants, essential oils of plants, plant extracts, or mixtures thereof. Water suitable for the invention may be thermal and / or mineral water, in particular Vittel water, water from the Vichy basin, and La Roche-Posay water.A gelling agent suitable for the invention may be selected from pectin, guar gum, cellulose, dextrin, maltodextrin, starch, tara gum, locust bean gum, inulin, acacia gum, gum arabic, fucose-rich polymers, carrageenans, konjac gum, xanthan gum, dextran, chitosan, tragacanth gum, ghatti gum, karaya gum, tamarind gum, agar-agar, alginate, gellan gum and mixtures thereof.

[0059] These additives are well known for their use in cosmetic compositions, and make it possible to improve the organoleptic characteristics of the composition and the quality of the user experience of the composition.

[0060] The compositions according to the invention may also include one or more other active ingredients, provided that these are compatible with the Emiliania huxleyi extract as described above. These active ingredients may, in particular, be chosen for their moisturizing, pigmenting, depigmenting, soothing, oxygenating, regenerating, anti-aging, anti-wrinkle, anti-redness activity, and combinations thereof.

[0061] A composition according to the invention can be in all the pharmaceutical forms normally used for topical application to the skin. It can then take the form of an aqueous or oily solution, a lotion, a serum, an emulsion, a milk, a cream, a gel, an ointment, a butter, an oil-in-water emulsion, a water-in-oil emulsion, a triple emulsion, microcapsules, or microparticles. These compositions can be prepared according to methods known in the art.

[0062] These formulations and textures are simple to produce and are compatible with application to the skin. They will be chosen according to the desired application and skin type. For example, it is more advantageous to formulate a lotion rather than a cream for application to the entire body, while a butter proves to be better suited for application to dry skin.

[0063] Emiliania huxleyi extract can also be incorporated into two-in-one or three-in-one makeup removers for deep cleansing and to prepare the skin to better combat the effects of stress. This extract can also be used as a complement to surface anti-pollution active ingredients in such cosmetic compositions.

[0064] Preferably, the extract of Emiliania huxleyi of the invention is present in the composition at a concentration of between 0.01 and 10% by weight, relative to the total weight of the composition, preferably between 0.1 and 1% by weight, relative to the total weight of the composition.

[0065] It has been shown that at these concentrations, a cosmetic composition comprising such an extract of Emiliania huxleyi and applied to the skin effectively and satisfactorily stimulates the expression of genes encoding HSPs, and / or proteosome proteins, and / or metallothioneins, and / or extracellular matrix proteins. These proteins are involved in cellular detoxification and the protection of the skin against aggressive external elements.

[0066] Use of the extract of Emiliuniu huxleyi and / or a composition thereof comprising, according to the invention

[0067] According to fourth and fifth aspects of the invention, an extract of Emiliania huxleyi and / or compositions comprising it is / are used topically to prepare the skin to fight against the imbalance caused by oxidative stress, and / or to maintain its homeostasis.

[0068] Such compositions according to the invention can be used on the face or body.

[0069] The use of the composition according to the invention may be daily or twice daily. It may be less frequent, such as once a week, twice a week, three times a week, four times a week, five times a week or six times a week.

[0070] Thus, the frequency of use of the composition according to the invention can be adapted according to the improvement of the preparation of the skin to better react to environmental aggressions.

[0071] The invention will now be illustrated by the following non-limiting examples. Examples

[0072] Example 1: Obtaining the extract of Emiliania huxleyi according to the invention

[0073] This example illustrates the obtaining of an extract of Emiliania huxleyi according to the invention, according to a process implemented according to the following steps: -a first stage of Emiliania huxleyi culture was implemented in a photobioreactor, including seawater, a selenium content of between 1 and 100 nM, and a phosphate content of between 10 and 100 pM, and being illuminated by artificial light.

[0074] The particularity of this culture step is based on the fact that it allows the culture of Emiliania huxleyi essentially without the production of microalgal coccoliths, that is to say that less than 1% of the Emiliania huxleyi cells have a coccolith, and more preferably less than 0.1%; -a harvesting step of this culture was then carried out, in particular when the number of Emiliania huxleyi cells produced was greater than 2xl06 cells.mL 1; -in a subsequent step, the harvested crop was frozen; -then, the frozen culture underwent a thawing step, and propane-1,3-diol was added to the medium; -a tangential flow filtration step was then implemented, using a filter with a cutoff threshold of 0.2 µm; and -the medium underwent a final sterilizing filtration stage.

[0075] The implementation of all the steps of this process has therefore made it possible to obtain an extract of Emiliania huxleyi according to the invention, essentially not comprising coccoliths of this microalga.

[0076] Example 2: Effects of the Emiliania huxleyi extract obtained on the expression of genes involved in the preparation and protection of the skin against environmental stress - in vitro experimentation

[0077] Example 2 demonstrates the effects of the Emiliania huxleyi extract obtained in Example 1 on the protection of the skin against environmental oxidative stress.

[0078] A first in vitro experiment was conducted to measure the effects of the extract of Emiliania huxleyi according to the invention on the expression of genes encoding stress proteins, proteasome proteins, metallothioneins and extracellular matrix proteins.

[0079] Test conditions

[0080] The Emiliania huxleyi extract produced was tested at 0.1% by weight in cell culture medium. It was incubated for 24 hours with normal human dermal fibroblasts (NHF), isolated from a fragment of human skin, and used after either a low number of subculturings (simulating young fibroblasts) or a high number of subculturings (simulating aged fibroblasts). A comparative test was performed with untreated cells.

[0081] After 24 hours, the mRNA from cells that had been in contact with the extract and that from cells that had not undergone such contact were extracted, and a comparative analysis was carried out in order to highlight the effects of the extract of Emiliania huxleyi on the expression of genes encoding stress proteins, proteasome proteins, metallothioneins and extracellular matrix proteins.

[0082] Expression of genes encoding stress proteins (HSPs)

[0083] Analysis of the topical application of the Emiliania huxleyi extract obtained demonstrated that the expression of the following genes, encoding HSPs, chaperone molecules, was increased:

[0084] - the HFS4 gene, encoding a transcription factor that stimulates the expression of HSP. This gene is therefore a regulator of HSP expression.

[0085] - the HSPA5 gene, encoding subunits of the HSP70 protein. This protein The chaperone is known for its protective role against temperature changes, thus enabling thermotolerance in cells exposed to temperature stress. It is also an important chaperone during protein transport into mitochondria, and is therefore involved in mitochondrial function. The HSP70 protein binds to numerous proteins and ensures their correct conformation, increases cell survival under stress (Nonaka et al., Inhibitory effect of heat shock protein 70 on apoptosis induced by photodynamic therapy in vitro. Photochem Photobiol, 2004, 79(1):94-98), and helps determine whether a damaged protein will be selected for repair or sent to the proteasome recycling system (Wagstaff et al., 2007).

[0086] - the 7 genes HSPB6, HSPB8, HSPB1, HSPB3, DNAJC24, DNAJC16, and DNAJB9, encoding subunits of HSPs, which tend to decrease with the age of fibroblasts.

[0087] - the 3 genes DNAJC24, DNAJB9, DNAJC16, encoding subunits of the HSP40 protein. The HSP40 protein is a chaperone known to bind to HSP70 proteins and confer specificity of action and localization (Fan et al., Mechanisms for regulation of Hsp70 function by Hsp40, Cell Stress Chaperones. 2003 Oct; 8(4): 309-316). HSP40 is therefore a regulator of HSP70.

[0088] -the 4 genes HSPB6, HSPB8, HSPB1, HSPB3, encoding subunits of Small HSPs (HSP20, HSP22, and HSP27). Notably, the HSP27 protein is a chaperone molecule with a function in cell survival. HSP27 also plays an important role in the architecture and repair of newly formed tissues, resulting in a tensing and firming effect (Hirano et al., HSP27 regulates fibroblast adhesion, motility, and matrix contraction. Cell Stress Chaperones. 2004;9(l):29-37). HSP27 is phosphorylated in the presence of high levels of TGF-beta, which stimulates the production of type 1 collagen. HSP27 allows for better adhesion of fibroblasts to collagen fibers. In addition, HSP27 has intrinsic chaperone activity, and stimulates the degradation by the 26S proteasome of altered and ubiquitin-tagged proteins, thus limiting the accumulation of these altered proteins in the skin, and in particular lipofuscin (Arrigo AP, Heat Shock Proteins as molecular chaperones).Med Sci (Paris) 2005; 21:619-625). .

[0089] Therefore, by stimulating HSP subunits involved in particular in the formation of HSP27, HSP40 and HSP70, the Emiliania huxleyi extract obtained, which has not, or very little, formed coccoliths, is able to promote the response to environmental stresses, the repair of proteins or their degradation by the proteasome system, and to have an effect on the collagen matrix, in particular on its synthesis, structure and contraction.

[0090] These results are summarized in Table 1 below.

[0091] [Tables 1] Gene Variation in gene expression (in %) with application of 0.1% Emiliania huxleyi extract HFS4 +46 HSPA5 +27 DNAJC24 +21 DNAJC16 +38 DNAJB9 +25 HSPB6 +22 HSPB1 +33 HSPB8 +24 HSPB3 +31

[0092] Effect of Emiliania huxleyi extract on HSP gene expression (results expressed as % increase, compared to the control not including the extract).

[0093] Expression of genes encoding proteasome proteins

[0094] Analysis of the application of the extract of Emiliania huxleyi also demonstrated that the expression of the 8 genes PSMA3, PSMA4, PSMA6, PSMB8, PSMB9, PSMB10, PSME1 and PSME2, ​​encoding proteasome subunits or proteasome activators, was increased, in both young and old fibroblasts.

[0095] Stimulation of these genes therefore demonstrates the ability of the extract according to the invention to eliminate altered proteins.

[0096] These results are summarized in Table 2 below.

[0097] [Tables2] Gene Variation in gene expression (in %) with application of 0.1% Emiliania huxleyi extract PSMA3 +22 PSMA4 +33 PSMA6 +37 PSMB8 +175 PSMB9 +392 PSMB10 +147 PSME1 +131 PSME2 +265

[0098] Effect of Emiliania huxleyi extract on proteasome gene expression (results expressed as % increase, compared to the control not including the extract).

[0099] Expression of genes encoding metallothioneins (MT)

[0100] Analysis of the application of the Emiliania huxleyi extract also demonstrated that the expression of 8 genes (MTF1, MT1A, MT1B, MT1E, MT1F, MT1M, MT1X, MT2A) was increased in young fibroblasts. These genes code for different MTs.

[0101] These results are summarized in Table 3 below.

[0102] [Tables3] Gene Variation in gene expression (in %) with application of 0.1% Emiliania huxleyi extract MTF1 +39 MT1A +175 MT1B +268 MT1E +89 MT1F +157 MT1M +144 MT IX +208 MT2A +287

[0103] Effect of Emiliania huxleyi extract on the expression of metallothionein genes (results expressed as % increase, compared to the control not including the extract).

[0104] The extract obtained also stimulates the expression of genes encoding selenoproteins, including the DIO2 gene, which decreases with age; the GPX6 gene encoding glutathione peroxidase GPx-6; the SELENOM gene encoding selenoprotein M and the TXN gene encoding thioredoxin 1 (+24%).

[0105] Finally, the extract stimulates the expression of the SOD2 gene encoding Superoxide Dismutase type 2 (mitochondrial and manganese dependent SOD) on aged fibroblasts (+546%).

[0106] Stimulation of these genes also demonstrates the ability of the extract according to the invention to detoxify cells against heavy metals.

[0107] Expression of genes encoding extracellular matrix (ECM) proteins

[0108] Analysis of the application of the extract of Emiliania huxleyi also demonstrated that this extract has an effect on proteins involved in the interactions of cells with the extracellular matrix.

[0109] In particular, it increases the expression of the four genes ITGA3, ITGA5, FN1, and COL8A1, which are involved in the interactions of dermal cells with the ECM. These are receptors or matrix proteins that decrease with cell age. Their increase in aged cells is therefore of interest.

[0110] Furthermore, it reduces the expression of the MMP3 gene, encoding type 3 metalloproteinase (MMP3), which is involved in collagen degradation in aged fibroblasts, by 22%. Reducing this protein is particularly important because MMP3 expression increases with cell age (2.2-fold).

[0111] Stimulation of these genes also demonstrates the action of applying the extract according to the invention at the level of the ECM.

[0112] Thus, by stimulating antioxidant defenses, through the stimulation of HSPs and MTs, and by promoting the elimination of altered proteins through proteasome activation, topical application of the Emiliania huxleyi extract obtained according to the invention makes it possible to prepare the skin to fight against the imbalance caused by stress and to maintain its homeostasis.

[0113] A second in vitro experiment was also conducted on human dermal fibroblasts to evaluate the effect of the Emiliania huxleyi extract according to the invention on the stimulation of genes encoding HSPs at the protein level, and also encoding ubiquitin.

[0114] Test conditions

[0115] The cells used in this experiment were normal human dermal fibroblasts (NHF) from a 51-year-old donor, used in the 7th passage. The cells were seeded at 1.1 x 10⁶ cells in a monolayer in 75 cm² culture flasks for 7 days in a suitable culture medium (Zen Bio). On day 7 and at 80% confluence, the cells were treated, or not (control), with 1% (v / v) Emiliania huxleyi extract in the culture medium, in triplicate, for 24 hours. After 24 hours, the cells were treated with trypsin to detach them, then lysed with 350 pL of a buffer provided by the supplier of the Human Heat Shock Protein Antibody Array kit, to which a cocktail of protease inhibitors had been added. Total protein content was measured using a BCA assay kit (Pierce). HSP and ubiquitin were measured in the cell lysate using the RayBio® Heat Shock Protein Antibody Array kit.Each membrane in the kit was printed with antibodies capable of specifically recognizing nine different HSPs. Cell lysates of cells treated and untreated with 1% (v / v) Emiliania huxleyi extract were deposited onto each membrane. After thorough washing, the membranes were incubated with a cocktail of biotin-labeled anti-HSP antibodies. Following incubation with a Steptavidin-HRP complex, the signal was visualized by chemiluminescence. This analysis aimed to evaluate the effects of the Emiliania huxleyi extract according to the invention on the stimulation of gene expression encoding HSPs at the protein level, and also encoding ubiquitin.

[0116] Analysis of the systemic application (in the culture medium) of the extract of Emiliania huxleyi according to the invention at 1% (v / v) thus made it possible to demonstrate a significant increase (p<0.01) in the synthesis of HSP proteins, compared with the control not including the extract: HSP10 (+87%), HSP27 (+76%), HSP32 (+145%), HSP40 (+213%), HSP70 (+94%), and also of ubiquitin (+74%).

[0117] This therefore confirms the stimulation of HSP production at the protein level, as well as ubiquitin, by the extract according to the invention.

[0118] These results are summarized in Table 4 below.

[0119] [Tables4] Gene Variation in gene expression (in %) with application of 1% Emiliania huxleyi extract HSP10 +87 HSP27 +76 HSP32 +145 HSP40 +213 HSP70 +94 UBIQUITIN +74

[0120] Effect of Emiliania huxleyi extract on HSP and ubiquitin synthesis (results expressed as % increase in gene expression, compared to the control not including the extract).

[0121] Example 3: Effects of the Emiliania huxleyi extract obtained on the expression of genes involved in the preparation and protection of the skin against environmental stress - in vivo experimentation

[0122] Example 3a: Effects of applying the extract according to the invention on the protection of the skin against the harmful effects of UV rays

[0123] Example 3a demonstrates the effects of the extract of Emiliania huxleyi obtained in Example 1 on the skin, in particular its properties of protecting the skin against external aggressions, in particular with regard to UV radiation.

[0124] Test conditions

[0125] Extract of Emiliania huxleyi in the form of an emulsion stored at room temperature was applied to the back area of ​​21 female and male volunteers aged 23 to 53 years. 14% of these volunteers had oily skin, and 86% had skin considered normal.

[0126] For this experiment, four zones were delimited on the backs of the volunteers: -an area that has been exposed to UV light and on which the extract according to the invention has been applied (area 1); -an area that has been exposed to UV light and on which a placebo has been applied (area 2); -an area that has been exposed only to UV light (zone 3); and -an area that has not been exposed to UV light, and has not had any product applied (zone 4).

[0127] A sample was taken from each of these four areas of the back using an adhesive strip, for analysis.

[0128] i) Initially, the oxidative stress actually induced in the skin by UV exposure, i.e., in zones 3 and 4, was analyzed. Carbonyl groups of the stratum corneum collected by adhesive strips were labeled with fluorescein-5-thiosemicarbazide (FTZ). Indeed, carbonyl proteins are induced after UV stress and are therefore a good marker of oxidative stress. The staining images of these carbonyl groups were observed by fluorescence microscopy, and the average fluorescence intensity of the stratum corneum extracted from the images was calculated as the 'stratum corneum carbonylated protein level' (SCCP).

[0129] The results of this analysis are presented in Table 5 below.

[0130] [Tables5] Oxidative Stress Marker Zone Oxidative Stress Marker Concentration Statistical Analyses Mean and Standard Deviation (in IU* / mg of protein content) A% [zone 3 - zone 4] (absolute value) P Significance Level of SCCP Zone 4 (unexposed, and no topical application) 15.13+ / - 8.82 65% <0.000 1 yes Zone 3 (exposed, and no topical application) 24.97 + / - 10.09

[0131] Validation of oxidative stress induced by UV exposure (untreated and exposed areas VS untreated and neither exposed areas)

[0132] * IU: International Unit

[0133] Figure 1 also illustrates these results, which represents the quantity of carbonylated proteins in IU per mg of total protein, in the case of a skin area not exposed to UV radiation (zone 4) and in the case of a skin area that has been exposed to UV radiation. such an application (zone 3), none of these zones having received the product application. It is therefore clearly visible that UV exposure induces oxidative stress, as a 65% increase in the amount of carboxylated proteins is observed in the exposed zone 3.

[0134] It is therefore clear from these results that exposure of the skin to UV rays induces oxidative stress.

[0135] ii) In a second step, the protective effect of the extract according to the invention against oxidative stress induced by UV exposure was analyzed. The results of this analysis are presented in Table 6 below.

[0136] [Tableauxô] Oxidative Stress Marker Zone Variation in Oxidative Stress Marker Concentration Antioxidant Protection Index Statistical Analyses A (zone 1 or 2 - zone 3) (mean and standard deviation) A% (zone 1 or 2 - zone 4) P Significance SCCP Level Extract according to the invention (zone 1) -9.03 + / - 7.20 92% 0.0003 yes Placebo (zone 2) -6.96 + / - 6.67 71% 0.0068 yes

[0137] Protective effect of the extract according to the invention against oxidative stress induced by UV exposure.

[0138] Figure 2 also illustrates these results, showing the value of the antioxidant protection index calculated from the analyzed quantities of carbonylated proteins in areas of skin to which the Emiliania huxleyi extract according to the invention and the placebo were applied, namely areas 1 and 2 defined above. It is also clearly visible here that the application of the extract according to the invention protects skin exposed to UV radiation (protection index of 92%), and does so more effectively than a placebo (protection index of 71%).

[0139] It has therefore been demonstrated that topical application of the tested products (Emiliania huxleyi extract according to the invention and placebo) significantly reduced the effect of UV-induced stress on the skin. The decrease in SCCP levels of an area exposed and having received the application of these products compared to an exposed area having received no application is, respectively, 36% on average for the extract of Emiliania huxleyi, and 28% on average for the placebo.

[0140] In view of these results, an antioxidant protection index could be determined for each product, which is 92% for the Emiliania huxleyi extract, and only 71% for the placebo.

[0141] It may be noted that the effect of the extract of Emiliania huxleyi is significantly better than that of the placebo.

[0142] Thus, from all the results of this example 3a, and in particular the reduction of oxidative stress markers in the skin exposed to UV and having received a topical application of the extract of Emiliania huxleyi according to the invention, i.e. not containing, or only very few, coccoliths, it has been demonstrated that the reduction of protein carbonylation in the skin by this extract has been demonstrated, which clearly illustrates the protective effect on the skin of the extract according to the invention, in particular with regard to external aggressions by UV radiation.

[0143] Example 3b: Effects of applying the extract according to the invention on skin repair against external aggression such as a chemical product

[0144] Example 3b demonstrates the effects on the skin of the extract of Emiliania huxleyi obtained in Example 1, in particular its skin repair properties, especially after aggression by a detergent.

[0145] Test conditions

[0146] Extract of Emiliania huxleyi in the form of an emulsion stored at room temperature was applied to the forearm area of ​​23 female volunteers, aged 18 to 56 years.

[0147] For this experiment, four areas were marked on the forearms of the volunteers. Patches containing 300 qL of 0.5% detergent, which is sodium lauryl sulfate, were applied to some of these areas. The areas are as follows: -a patched area on which extract according to the invention has been applied (area 1); -a patched area on which a placebo has been applied (area 2); -a patched area that has not received any product application (zone 3); -an area that has not been patched and has not received any product application (area 4).

[0148] The application of the extract according to the invention and of the placebo was done twice daily, for 6 days.

[0149] Transepidermal water loss (TEWL) was measured in the four areas using a Tewameter TM 300® (Courage & Khazaka). The skin barrier acts as a regulator of the skin's water balance. When it is damaged, the system regulating water exchange is When the skin barrier is destabilized, water migrates more easily to the external environment, increasing the water-loss threshold (WLT). Conversely, if the skin barrier improves, water loss decreases because the mechanism regulating water exchange regains its balance. Measuring water loss therefore allows us to assess the barrier effect of the stratum corneum and the hydrolipidic film.

[0150] The results of this analysis are presented in Tables 7 to 9 below:

[0151] [Tables?] Kinetic Product Variations of the TEWL factor (in gm².h') (mean and standard deviation) A% (mean) P Significance % of volunteers with restructuring and repairing effect Extract according to the invention (zone 1) VS zone 3 AJ**3 -2.5 + / - 1.2 -10% 0.0478 yes 70% AJ4 -5.9 + / - 1.5 -20% <0.0001 yes 78% AJ7 -5.2 + / - 1.8 -11% 0.0014 yes 70% AJ8 -4.0 + / - 2.0 -6% 0.0100 yes 70%

[0152] Comparison of the repairing and restructuring effect of the extract according to the invention compared to an area that has not received product application

[0153] **D: day

[0154] [Tables8] Kinetic Product Variations in TEWL factor (in gm 2.h') (mean and standard deviation) A% (mean) P Sign. % of volunteers with restructuring and repairing effect Placebo (zone 2) VS zone 3 AJ3 -0.5+ / - 1.1 -1% 0.7153 no 48% AJ4 -1.1 + / - 1.4 -3% 0.3868 no 56% AJ7 -2.0+ / - 1.5 -4% 0.1224 no 52% AJ8 -1.7+ / - 1.6 -2% 0.1943 no 52%

[0155] Comparison of the repairing and restructuring effect of placebo versus an area that had not received product application

[0156] [Tables9] Kinetic Product Variations in TEWL factor (in gm².h') (mean and standard deviation) A% (mean) P Sign. % of volunteers with restructuring and repairing effect Extract according to the invention (zone 1) VS Placebo (zone 2) AJ3 -2.4 + / - 0.7 -9% 0.0492 yes 78% AJ4 -5.7 + / - 0.9 -17% <0.0001 yes 78% AJ7 -3.2 + / - 1.4 -7% 0.0208 yes 57% AJ8 -2.5 + / - 1.5 -4% 0.0740 no 57%

[0157] Comparison of the repairing and restructuring effect of the extract according to the invention versus placebo

[0158] Figures [Fig. 3] and 4 also illustrate these results.

[0159] Figure 3 represents transepidermal water loss over time in g / m² / h, in zones 1 (application of the extract according to the invention) and 2 (application of a placebo), when the skin has been exposed to a chemical, in particular a detergent. The lower curve corresponds to zone 1 and the upper curve corresponds to zone 2. The position of the lower curve thus demonstrates that the application of the extract according to the invention reduces skin water loss more effectively than the application of a placebo.

[0160] Figure 4 also represents transepidermal water loss, as a percentage. This parameter is compared over time in zones 1 and 2, as well as in the case of the difference between these two zones, always when the skin has been exposed to detergent. It is therefore clearly illustrated in each case that topical application of the Emiliania huxleyi extract according to the invention reduces water loss, and that this reduction is always greater than in the case of topical application of the placebo.

[0161] Therefore, from all the results of this example 3b, it has been demonstrated that, compared to an area of ​​skin damaged with a detergent and on which no product application (extract according to the invention and placebo) was carried out, and after twice-daily topical application from day 2, the extract of Emiliania huxleyi according to the invention presented a restructuring / repairing effect from day 3 to day 8. This effect is in particular illustrated by a significant decrease in transepidermal water loss (TEWL factor) of 20% between days 2 and 4, observed in 78% of the volunteers.

[0162] Conversely, it has been shown that the placebo did not exhibit a restructuring / re-repairing effect.

[0163] Thus, the results of these two examples, 2 and 3, demonstrate that the Emiliania huxleyi extract according to the invention, which essentially does not contain coccoliths, prepares the skin to react optimally to environmental aggressions and protects it, particularly against oxidative stress that can be induced by multiple external factors, such as UV radiation or contact with chemicals. This extract also helps repair the skin in the event of such aggressions.

Claims

Claims

1. A method for producing an extract of the microalga Emiliania huxleyi comprising: - a step of culturing Emiliania huxleyi in a photobioreactor, using a culture medium comprising seawater, one or more microelements, such as selenium, and between 10 and 100 pM (micromoles.L1) of phosphate, said step allowing the culture of Emiliania huxleyi essentially without production of coccoliths, that is to say that only a few percent of the produced cells of Emiliania huxleyi possess a coccolith, preferably less than 1% of the produced cells, and even more preferably less than 0.1% of the produced cells; - a step of harvesting said culture; - a step of freezing said culture; - a step of thawing said frozen culture and adding propane-1,3-diol; and - at least one filtration step.

2. Method according to claim 1, wherein said at least one filtration step comprises: - a tangential filtration step through a filter having a cut-off threshold of 0.2 pm; and - a sterilizing filtration step.

3. Extract of the microalga Emiliania huxleyi obtained by the method according to claim 1 or 2, characterized in that it comprises essentially and practically no coccoliths, that is to say that only a few percent of the cells produced & Emiliania huxleyi possess a coccolith, preferably less than 1% of the cells produced, and even more preferably less than 0.1% of the cells produced.

4. Extract according to claim 3 stimulating the expression of genes coding for proteasome proteins, and / or metallothioneins, and / or stress proteins (“Heat Shock Proteins”), and / or extracellular matrix proteins.

5. Extract according to claim 3 or 4 stimulating the expression of genes coding for HSP proteins and for ubiquitin.

6. Cosmetic composition for topical application comprising an extract of the microalga Emiliania huxleyi according to any one of claims 3 to 5, as well as a physiologically acceptable medium.

7. Composition according to claim 6, comprising between 0.01% and 10% by weight of Emiliania huxleyi extract, relative to the total weight of said composition, preferably between 0.1% and 1% by weight, relative to the total weight of said composition.

8. Use of an extract according to any one of claims 3 to 5 as a cosmetic active ingredient.

9. Composition according to claim 6 or 7 as a cosmetic composition intended to be applied to healthy skin.

10. Composition according to claim 9 for its use in the preparation of the skin to combat external oxidative stress, and / or in the preparation of the skin to maintain its homeostasis.

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