FR3064481A1 - USE OF A CHLORELLA SOROKINIANA EXTRACT IN COSMETICS AND PARTICULAR EXTRACTS OF CHLORELLA SOROKINIANA - Google Patents

Abstract

The subject of the invention is the cosmetic use of an extract of Chlorella sorokiniana in a cosmetic composition as a cosmetic agent, as well as a particular extract of Chlorella sorokiniana, compositions comprising it and a cosmetic process of skin care.

Description

Holder (s): SOCIETE INDUSTRIELLE LIMOUSINE BIOLOGICAL APPLICATION Société anonyme.

Extension request (s)

Agent (s): AQUINOV.

104 / USE OF AN EXTRACT OF CHLORELLA SOROKINIANA IN COSMETICS AND SPECIAL EXTRACTS OF CHLORELLA SOROKINIANA.

_ The object of the invention is the cosmetic use of an extract of Chlorella sorokiniana in a cosmetic composition as a cosmetic agent, as well as a particular extract of Chlorella sorokiniana, compositions including it and a cosmetic process of skin care.

FR 3 064 481 - A1

USE OF A CHLORELLA SOROKINIANA EXTRACT IN COSMETICS

AND PARTICULARS OF CHLORELLA SOROKINIANA

The present invention relates to the cosmetic use of extracts of Chlorella sorokiniana as a cosmetic agent, in particular for reviving the radiance of the complexion of the skin of the face, as well as to specific extracts of Chlorella sorokiniana. A subject of the invention is also cosmetic compositions comprising such extracts, and a cosmetic method for caring for the skin.

The cosmetic active agents act on different ways to provide their effectiveness and improve the aesthetic appearance of the skin. Among the different pathways, the action on the mitochondria of skin cells has recently been studied.

In fact, skin cells are equipped with mitochondria, organelles capable of producing energy in the form of adenosine triphosphate (ATP) and which have their own DNA, mitochondrial DNA (mtDNA). The mitochondria are capable of changing morphology depending on the conditions to which they are subjected. These modifications are made possible thanks to a finely regulated balance between two processes: fission and mitochondrial fusion.

Depending on their state of health, the mitochondria condition the fate of cells and tissues. They are notably known to influence proliferation and epidermal differentiation (B Hamanaka R., Navdeep S. C. Mitochondrial metabolism as a regulator of keratinocyte differentiation. Cellular Logistics, 3,1-5 (2013)).

When healthy, the mitochondrion acts as an energy factory by producing ΙΆΤΡ. However, being particularly sensitive to stress, particularly oxidative stress, its functioning can be modified in the event of damage. The production of energy is then compromised and the production of toxic molecules such as free radicals is abnormally increased. These free radicals damage the mitochondrial DNA which, unlike its nuclear counterpart, has limited means of protection, thus causing an aggravation of organelle dysfunction. In addition, the mitochondria being the seat of proteins involved in cell death, in acute stress, proteins such as cytochrome C can be released and cause cell death. In the long term, chronic mitochondrial dysfunction leads to accelerated skin aging.

Depending on the level of stress and the mitochondrial damage induced, three defense systems will come into play (Andreux P., Riekelt H. Houtkooper R., Auwerx J. Pharmacological approaches to restore mitochondrial function. Nature reviews, 12, 465-483 (2013)).

In the event of low stress, a first line of defense is mobilized. Its mission is to prevent or even repair minor mitochondrial damage. It involves chaperone proteins which act on damaged proteins and antioxidant systems which will prevent the oxidative damage induced on proteins and mtDNA. Three proteins actively participate in this first level of defense: sirtuin 3 (SIRT3), aconitase and HSP27.

In moderate stress, the level of damage is greater and compromises the functionality of the mitochondria. A second line of defense is then put in place. The dynamic properties of the mitochondria are called upon. A mitochondrial fusion mechanism is initiated so that the contents of healthy and slightly damaged mitochondria can be mixed. This results in obtaining a pool of homogeneous and again functional mitochondria.

In acute stress, the mitochondria suffer irreversible damage and become potentially dangerous for the cell. They must then be eliminated. Faced with this problem, the third line of defense is put into action through the mitophagy process, a degradation by autophagy specifically dedicated to mitochondria.

Due to chronological aging or environmental damage (ultraviolet radiation, pollution, etc.), the skin is constantly in a “state of stress”. At the cellular level, the mitochondria are the first to be damaged. Their influence on the tissue being very important, there is an important need to provide a natural cosmetic solution aimed at effectively preserving the functioning of these organelles to act on the aesthetic appearance of the skin and in particular for an energizing effect making it possible to revive the complexion.

The objective of the present invention is to meet this need. To this end, the invention proposes to use an extract of a specific microalga Chlorella sorokiniana.

Microalgae are the basis of life. Among the first organisms to appear on earth, they play a crucial role in the biological cycles and terrestrial biodiversity. They arouse great interest because they have many advantages:

- extraordinary biodiversity: there are 30,000 species described and between 200,000 to 1 million estimated. Due to their great adaptability, these organisms are able to modify their metabolism and develop defense systems that allow them to survive independently of their environment;

- an inexhaustible richness in very varied active molecules;

- industrially cultivable, their production has no impact on the environment. All microalgae species are different and have distinct molecular compositions which give them their properties. Some of them, or certain extracts of these microalgae, have cosmetic properties but the cosmetic properties of an extract of a particular microalga cannot be applied to the same extract of another microalga. Thus, with an identical extraction process, the use of another species of Chlorella does not make it possible to obtain a cosmetic ingredient having the desired cosmetic efficacy.

Chlorella algae are single-celled algae that live in isolation or in small groups of cells. The cell is globular or ellipsoidal. The cell wall is smooth and contains a glucosamine (chitosan). It has only one nucleus and one chloroplast, usually located on the edges of the cell. The chloroplast contains only one pyrenoid, covered with a layer of starch. There are many different species of the genus Chlorella. The best known, Chlorella vulgoris, is used for many applications, in particular as a food supplement, but also in cosmetics.

The Chlorella extracts currently known and used in cosmetics are extracts essentially comprising proteins, as described in particular in patent application FR3031985.

Chlorella sorokiniana has a special composition that is different from that of Chlorella vulgoris. Surprisingly and advantageously, an extract of Chlorella sorokiniana, when applied to the skin, is capable of acting on the mitochondria of the cells of the epidermis, ensuring their protection, whatever the level of stress. It allows the mitochondria to adapt to their environment and promotes its metabolism. In good health, the mitochondria then participate in the effective renewal of the barrier to obtain glowing skin.

The subject of the invention is therefore the cosmetic use of an extract of Chlorella sorokiniana in a cosmetic composition as a cosmetic agent, as well as a cosmetic treatment method for improving the aesthetic appearance of the skin, comprising: application to the skin of at least one extract of Chlorella sorokiniana or a composition containing it.

The invention also relates to a specific extract of Chlorella sorokiniana comprising at least carbohydrates and proteins, the carbohydrates representing at least 36% by weight of dry matter of the extract, as well as cosmetic compositions containing such an extract.

Other characteristics and advantages of the invention will emerge from the detailed description of the invention which will follow, with regard to examples, efficiency tests and the appended figures in which:

- Figure 1 shows a microscope photograph of the biomass of Chlorella sorokiniana;

- Figure 2 shows a microscope photograph, taken under the same conditions as Figurel, after solubilization and hydrolysis of the biomass and before separation of the soluble and insoluble phases;

- Figure 3 shows a microscope photograph, taken under the same conditions as the Figurel, of the extract according to the invention.

Definitions

By “cosmetic agent” within the meaning of the invention, is also meant a cosmetic active principle or a cosmetic active agent, that is to say at least one molecule, preferably a set of several molecules having an effect on the skin cells. .

By "containing an" X "" or "including an" X ". ”, Unless otherwise indicated, within the meaning of the invention means which contains or comprises at least one“ X ”.

By “extract” of Chlorella sorokiniana within the meaning of the invention means any molecule or mixture of molecules derived from the structure and / or from the intracellular medium of the microalga Chlorella sorokiniana. They can be native molecules of the microalga or molecules obtained by any type of extraction and transformation of the native molecules of the microalga, for example by hydrolysis. The term "extract" excludes the biomass of Chlorella sorokiniana, as is.

By “hydrolyzate” is meant any extract of Chlorella sorokiniana, obtained with a process comprising at least one step of enzymatic or chemical hydrolysis of Chlorella sorokiniana, preferably at least one step of enzymatic hydrolysis.

Detailed description of the invention

The subject of the invention is therefore the use of an extract of Chlorella sorokiniana in cosmetics, not therapeutically.

In fact, according to the invention, an extract of Chlorella sorokiniana when applied to the skin, is capable of improving the aesthetic appearance and the texture of the skin.

It is in fact capable of acting on the three mechanical pathways which condition the balance and the proper functioning of the mitochondria of epidermal cells, namely:

- control and regulation of mitochondrial quality,

- mitochondrial dynamics, and

- mitophagy.

In order to keep cells and tissues in good working order, the mitochondria use different proteins which will ensure mitochondrial quality control. These proteins have different missions: to protect the organelle from radical stresses, to promote its metabolism and to prevent it from emitting a death signal.

Among these proteins, there are three major players:

- Sirtuin 3 (SIRT3) is a member of the sirtuin family, a family known for its positive effects on cell metabolism, the maintenance of genome integrity and resistance to stress. Located at the mitochondrial level, it plays a vital protective role. On the one hand, it makes it possible to fight against oxidative stress by de-acetylating or by activating the production of anti-radical enzymes (superoxide dismutase or catalase) (HHTseng A., Shieh S.-S., LingWang D. SIRT3 deacetylates FOXO3 to protect mitochondria against oxidative damage. Free Radical Biology and Medicine, 63, 222-234 (2013)). On the other hand, it plays a favorable role on metabolism by boosting the proteins of the respiratory chain (Kincaid B., Bossy-Wetzel E. Forever young: SIRT3 a shield against mitochondrial meltdown, aging, and neurodegeneration. Frontiers in Aging Neuroscience , 5.1-13 (2013));

- Aconitase is described as the bodyguard of the mitochondria. The mitochondrial form of this enzyme is known to have a dual function. On the one hand, it actively participates in metabolism since it is one of the enzymes of the respiratory chain. On the other hand, it preserves mtDNA by binding to it and by regulating the expression of mitochondrial genes (S. Shadel G. Mitochondrial DNA, aconitase 'wraps' it up. Trends in Biochemical Sciences, 30, 294- 296 (2005));

The chaperone protein HSP27 is localized at the cytoplasmic level and involved in numerous cellular processes. Among the functions assigned to it, it maintains the functionality of the mitochondrial proteome and promotes cell survival. In fact, the mitochondria contain various proteins potentially dangerous for the cell. By preventing the release of these proteins, the HSP27 protein will protect the mitochondria and the cell from a stress-induced death process (Bakthisaran R., Ramakrishna T., Mohan Ch. R. Small heat shock proteins: Role in cellular functions and pathology. Journal of Investigative Dermatology, 291-319 (2015)).

These 3 proteins involved in the good state of mitochondrial functioning are deregulated during skin aging and when the skin is subjected to low stress. This has been demonstrated in particular on two models mimicking aging (see point I). According to the invention, the use of an extract of Chlorella sorokianina on the skin, in particular on aged skin, makes it possible to increase the synthesis of SIRT3 and aconitase, and to normalize the expression of the chaperone protein HSP27 . Advantageously, by boosting these three major mitochondrial actors, the application of an extract of Chlorella sorokianina on the skin makes it possible to correct the loss of quality of the mitochondria and to control the aesthetic defects which appear during aging or when the skin is subjected. to low stress.

Furthermore, we know that mitochondria are extremely dynamic organelles, capable of moving along microtubules, frequently changing direction and interacting with each other. The mitochondrial network constantly adopts different morphologies through cycles (Westermann B. Mitochondrial fusion and fission in cell life and death. Molecular Cell Biology, 11, 872-884 (2010)):

- fusion, characterized by the creation of a tubular mitochondrial network with large elongated mitochondria;

- fission, characterized by the production of small punctate and spherical mitochondria.

These shape changes influence not only the integrity of the mitochondria but also many cellular functions of the mitochondria, namely the production of energy in the form of ATP or the release of free radicals (Yu T., Robotham LJ, Yoon Y. Increased production of reactive oxygen species in hyperglycemia conditions requires dynamic change of mitochondrial morphology. PNAS, 103, 2653-2658 (2006)).

The number and morphology of mitochondria mainly depend on the balance between these mitochondrial fission and fusion mechanisms and can vary depending on physiological conditions or the cellular environment (Jugé R., Breugnot J., Da Silva C., Bordes S., Closs B., Aouacheria A. Quantification and Characterization of UVB-Induced Mitochondrial Fragmentation in Normal Primary Human Kératinocytes. Scientific Reports, 6, 1-9 (2016)).

Mitochondrial fusion allows certain moderately damaged mitochondria to mix their material with healthy mitochondria, in particular matrix proteins and their mtDNA. This process ensures homogeneity within the mitochondrial population of the cell. In case of greater damage, mitochondrial fission allows the mitochondria to divide asymmetrically to form a healthy mitochondria and a damaged mitochondria which will be eliminated by a process of selective autophagy called mitophagia (Twig G., Elorza A., Molina JA. A., Mohamed H., Wikstrom DJ, Walzer G., Stiles L., Haigh ES, Katz S., Las G., Alroy J., Wu M., Py FB, Yuan J., Deeney TJ, Barbara E Corkey EB, Orian S Shirihai SO Fission and selective fusion govern mitochondrial segregation and elimination by autophagy. The EMBO Journal, 27, 433-446 (2008)).

Aging of the skin is associated with an increase in the mitochondrial fission process. This has been demonstrated in particular in tests carried out on keratinocytes from young and old donors, as presented in the section relating to the evaluation of the cosmetic efficacy of an extract of Chlorella sorokianina in point I.

According to the invention, the use of an extract of Chlorella sorokianina on the skin, in particular on aged skin, makes it possible to restore the mitochondrial fusion. Advantageously, the application of an extract of Chlorella sorokianina to the skin thus makes it possible to obtain and maintain a homogeneous and functional mitochondrial population, during skin aging or in the event of moderate stress.

In addition, when the damage becomes critical and generalizes to many mitochondria, the latter must be managed to be degraded. This elimination is made possible thanks to a mechanism of self-digestion of cellular compounds called autophagy and more particularly via one of its specific forms: mitophagia.

Autophagy is characterized by the formation of a double lipid membrane, called autophagosome which will surround a part of the cytoplasm. This autophagosome then fuses with a lysosome inside which the cytoplasmic content will be degraded. This process allows the cell to get rid of the various damaged elements present in its cytoplasm such as proteins and oxidized lipids (P Codogno, AJ Meijer. Autophagy and signaling: their role in cell survival and cell death. Cell Death and Differentiation, 12, 1509-1518 (2005)). By ensuring basal and non-discriminating quality control, autophagy is a first line of detoxification.

Mitophagy is a specific form of autophagy targeting damaged mitochondria. Different proteins are involved in this process: PINK1, Parkin, NIX or BNIP3 (Youle J.R., Narendra P.D. Mechanisms of mitophagy. Molecular Cell Biology, 12, 914 (2011)).

They allow the specific recruitment of mitochondria damaged by autophagososms and the formation of mitophagosomes. By eliminating defective free radical-producing mitochondria, mitophagy prevents oxidative damage to proteins. This is how, unlike autophagy which will eliminate damaged biomolecules, mitophagy attacks the source of cellular waste, free radicals (ROS), by targeting dysfunctional mitochondria that overproduce ROS. This specialized autophagic "subsystem" detoxifies in a discriminating manner. It constitutes a second, more powerful detoxification line.

Acute mitochondrial stress is associated with the initiation of a mitophagy process. This has been demonstrated in particular in tests carried out on keratinocytes, as presented in the section relating to the evaluation of the cosmetic efficacy of an extract of Chlorella sorokianina in point I.

According to the invention, the use of an extract of Chlorella sorokianina on the skin, in particular on skin which has undergone acute mitochondrial stress, makes it possible to help the degradation of damaged mitochondria by promoting mitophagy. Advantageously, the application of an extract of Chlorella sorokianina to the skin thus makes it possible to promote homeostasis and the renewal of the mitochondrial pool when the skin is subjected to aggressions.

On the other hand, it is known that there are many anti-radical or anti-oxidant products which make it possible to limit the presence of free radicals in the skin, but this cosmetic efficacy is different from that of the invention. Indeed, an anti-radical or anti-oxidant product will limit the presence of ROS in the skin, but will have no effectiveness on the state and quality of the mitochondria.

By reverse effect, a product acting on the state and quality of the mitochondria will not necessarily be an anti-radical or anti-oxidant product, these two efficiencies being very distinct. The extract of Chlorella sorokiniana according to the invention is moreover not an anti-radical product.

An extract of Chlorella sorokianina on the skin therefore allows:

- to control and regulate the mitochondrial quality,

- stimulate mitochondrial dynamics, and

- to increase mitophagy.

Thus, it can be used to improve the aesthetic appearance and texture of the skin, in particular to combat skin aging, in particular to combat the unsightly manifestations of aging of the skin, the dysfunction of the mitochondria being at origin of accelerated aging.

It can also be used to strengthen the barrier function and promote the radiance of the complexion. Indeed, mitochondrial dysfunction also has an influence on keratinocyte differentiation (G. Feichtinger R., Sperl W., W. Bauer J., Kofler B. Mitochondrial dysfunction: a neglected component of skin diseases. Experimental Dermotology, 23 , 607-614 (2014)). The absence of expression of certain mitochondrial genes leads to a modification of the functioning of the organelle which then exhibits a different production of ATP and free radicals. At the skin scale, repercussions have been highlighted since the epidermal differentiation is modified (Hamanaka et a!., 2013). Another study attributes the defects of cornification to an alteration of the mitochondrial dynamics in the stem cells of the epidermis. These data show that mitochondrial dynamics play an important role and that the fusion and establishment of a good distribution of mitochondria is essential for differentiation (R. Baris O., Klose A., E. Kloepper J., Weiland D ., Neuhaus J., Schauen M., Wille A., Müller A., Merkwirth C., Langert.,

Larsson N.-G., Krieg T., J. Tobin D., Ralf Paus R., J. Wiesner R. The Mitochondrial Electron Transport Chain Is Dispensable for Proliferation and Differentiation of Epidermal Progenitor Cells. Stem Cells, 29,1459-1468 (2011)).

Beyond epidermal differentiation, mitochondrial dysfunction also changes the quality of the skin. The massive production of free radicals by the mitochondria will:

damage the mitochondria themselves, thereby aggravating the stressful situation;

alter the various cellular components including proteins. This has important consequences on the skin since the oxidation of proteins leads to an alteration of the keratin fibers and of the optical properties of the stratum corneum (Iwai I., Ikuta K., Murayama K., Hirao T. Change in optical properties of stratum corneum induced by protein carbonylation in vitro. Internotionol Journal of Cosmetic science, 30, 41-46 (2008)) exert deleterious effects on the activity of skin cells. The skin thus loses its ability to regenerate.

Establishing a pool of homogeneous and healthy mitochondria is therefore an essential parameter for obtaining quality skin. It seems essential to stimulate the production of cellular energy while limiting the excessive production of free radicals in order to obtain an effective barrier function, constant cell renewal and a radiant complexion.

Advantageously, the application of an extract of Chlorella sorokianina on the skin makes it possible to increase the production of ATP by the keratinocytes of aged skin or under stress conditions. This is correlated with an increase in epidermal regeneration and epidermal differentiation. The skin is clearer, brighter and more even.

The invention therefore specifically relates to the cosmetic use of an extract of Chlorella sorokiniana in a cosmetic composition as a cosmetic agent, in particular as a cosmetic agent intended to improve the aesthetic appearance and / or the skin texture, especially for:

- improve the functioning of the mitochondria of the cells of the epidermis,

- improve the radiance of the complexion,

- stimulate the renewal of epidermal cells and / or strengthen the skin barrier.

The invention also relates to a cosmetic, non-therapeutic treatment method for improving the aesthetic appearance and / or the texture of the skin, comprising the application to the skin of at least one Chlorella sorokiniana extract or of a composition containing it, in particular to improve the radiance of the complexion.

The extracts of Chlorella sorokiniana useful according to the invention can be of any type. Preferably, it is a hydrolyzate of Chlorella sorokiniana. It can be a chemical hydrolyzate (basic or acid) and / or an enzymatic hydrolyzate. Preferably it is an enzymatic hydrolyzate.

The biomass of Chlorella sorokiniana used as raw material at the origin of the extracts of Chlorella sorokiniana useful according to the invention, consists of microalgae cultivated according to any process known by the skilled person. Currently, there are several methods of producing microalgae:

- in the presence of light and carbon dioxide, the culture is said to be photoautotrophic. It involves the chloroplasts, seat of photosynthesis;

- in the absence of light, we speak of heterotrophic culture. This second type of culture is done independently of photosynthesis. The microalga then develops by consuming the carbon sources which are brought directly to it in its culture medium.

Due to their adaptability properties, microalgae are able to pass from one metabolism to another in order to ensure their survival and growth regardless of the conditions imposed on them.

The biomass of Chlorella sorokiniana used as raw material at the origin of the extracts of Chlorella sorokiniana useful according to the invention, consists of microalgae which can be cultivated in heterotrophy or in autotrophy.

Preferably, the biomass of Chlorella sorokiniana used as raw material at the origin of the extracts of Chlorella sorokiniana useful according to the invention, consists of microalgae cultivated in heterotrophy.

Advantageously, the culture in heterotrophy ensures:

- control of the bioreactor culture which is done independently of climatic conditions. These methods give the process for obtaining the extract according to the invention robustness, reproducibility and repeatability;

- the production in large quantity of biomass, an essential element for the industrial production of extracts;

- sustainability because it requires a single sample of the strain of interest and therefore has no impact on biodiversity;

- traceability of supply since the strain comes from a collection.

In addition, from a biological point of view, in this heterotrophic culture mode the microalga develops an atypical metabolism. Indeed, this culture being done in the dark, the photosynthetic machinery is inactivated and the microalga uses its resources to produce metabolites other than pigments which is an advantage in the white cosmetic industry. This metabolism uses only the mitochondria. It is thanks to an increase in the number of mitochondria and a 100% mitochondrial metabolism that the microalga is able to survive and proliferate. The heterotrophic culture method is therefore preferred for the Chlorella sorokiniana extracts which are useful according to the invention.

The biomass of Chlorella sorokiniana cultivated in heterotrophy, can be characterized using the same methods of analysis as the extracts. Thus the biomass of Chlorella sorokiniana cultivated in heterotrophy includes:

Proteins: between 30 and 40%

Carbohydrates: between 30 and 45%

Fat: between 5 and 10%

Mineral ash: <5%

Unidentified: <20% the percentages being expressed by weight (m / m).

The biomass of Chlorella sorokiniana has whole microalgae cells. Figure 1 shows a microscope photograph of a solution containing 5% biomass of Chlorella sorokiniana. The presence of numerous whole cells is observed. The extracts of Chlorella sorokiniana which are useful according to the invention are preferably the extracts as described below.

Indeed, the subject of the invention is also specific extracts of Chlorella sorokiniono, namely extracts of Chlorella sorokiniono, comprising at least carbohydrates and proteins, characterized in that the carbohydrates represent at least 36% by weight of dry matter extract. Chlorella sorokiniana extracts can also contain minerals.

The sugar content in the extract can be determined by the DUBOIS method (Dubois M. et al., Analytical chemistry, 28, 3, 350-356,1956).

These extracts therefore include a significant amount of carbohydrates. Preferably, the sugar content of the extract according to the invention is between 36% and 94%, by weight of dry matter of the extract.

Preferably, the carbohydrates of the extract have the following characteristics:

- the carbohydrates contained in the extract are sugars with a molar mass of less than 8400 Da, and / or

- at least 70% of the carbohydrates are oligosaccharides and glucose polysaccharides with molar masses between 345 and 8400Da (degree of polymerization between 2 and 47), the percentage being given by weight relative to the total weight of the carbohydrates of extract,

- Less than 30% of the carbohydrates are monosaccharides, the percentage being given by weight relative to the total weight of the carbohydrates of the extract.

The simple sugars constituting the carbohydrates of the Chlorella sorokiniana extract according to the invention are preferably glucose and galactose.

With regard to proteins, the extracts of Chlorella sorokiniana according to the invention preferably have a protein content of between 6% and 40% by weight relative to the total weight of dry matter of the extract.

The protein content is preferably determined by the LOWRY method (Lowry et al., Protein measurement with the folin reagent, J. Biol. Chem., 193, 265,1951). Preferably, at least 90% of the proteins contained in the extract according to the invention are proteins with a molar mass of less than 3500 Da, the percentage being given by weight relative to the total weight of the proteins of the extract.

Regarding the minerals, the extracts of Chlorella sorokiniana according to the invention preferably have a mineral ash content of less than 20% by weight relative to the total weight of dry matter of the extract.

The crude ash content can be determined by weighing the residues from the incineration of the hydrolyzate samples at 550 ° C in an electric muffle furnace. The extract according to the invention does not include a biomass of Chlorella sorokiniono, it does not contain whole cells, nor cell fragments. Figure 3 shows a microscopic photograph of an extract of Chlorella sorokiniana at 5% in water. The photography was carried out under the same conditions as FIG. 1. A single cell is not observed, and no cell fragment is present in the extract according to the invention. Preferably, the extract according to the invention is a hydrolyzate of Chlorella sorokiniana. It can be a chemical hydrolyzate (acid or basic) and / or an enzymatic hydrolyzate of Chlorella sorokiniana. Preferably, the extract according to the invention is an enzymatic hydrolyzate, that is to say obtained by a process comprising at least one enzymatic hydrolysis. The enzyme (s) used can be any enzyme (s) known by those skilled in the art to allow an extract having the characteristics of the extract according to the invention to be obtained . The enzyme (s) may be in particular proteases, which are capable of hydrolyzing the proteins and glycoproteins constituting Chlorella sorokiniana or carbohydrases, which are capable of hydrolyzing the carbohydrates and glycoproteins constituting Chlorella sorokiniana.

Preferably, the biomass used to obtain the extract according to the invention is a biomass cultivated in heterotrophy.

The extract according to the invention can be in solid form or in liquid form, preferably in liquid form.

When it is in liquid form, it is preferably in the form of a clear or opalescent liquid, with a yellow color.

The extract according to the invention can be obtained by any process making it possible to obtain an extract of Chlorella sorokiniana having the characteristics claimed. Preferably, it is a process comprising at least one step of hydrolysis of Chlorella sorokiniono, in particular at least one step of hydrolysis of the proteins and glycoproteins constituting the microalga. Even more preferably, it is obtained by a process comprising at least one step of enzymatic hydrolysis, in particular of enzymatic hydrolysis of proteins and glycoproteins, that is to say hydrolysis carried out enzymatically by means of proteolytic enzymes. They may, for example, be proteases, endoproteases and exopeptidases of plant origin (papain, bromelain, ficin) and of bacterial origin (Aspergillus, Rhizopus, Bacillus, etc.). Preferably, it is a process comprising at least one step of hydrolysis of Chlorella sorokiniono, in particular at least one step of hydrolysis of the carbohydrates and glycoproteins constituting the microalga. Even more preferably, it is obtained by a process comprising at least one step of enzymatic hydrolysis, in particular of enzymatic hydrolysis of carbohydrates and glycoproteins, that is to say a hydrolysis carried out enzymatically by means of enzymes such as carbohydrases. The extract according to the invention can be obtained by a process combining at least one chemical hydrolysis and at least one enzymatic hydrolysis.

Before the process for obtaining the hydrolyzate as such, it is advisable to produce the biomass of Chlorella sorokiniana. This step is carried out according to the mode of culture of the yeasts in a medium suitable for their development, in a conventional manner for the skilled person. Preferably, the culture is carried out in heterotrophy.

Once the biomass has been obtained, the extraction process should be carried out. According to a particularly suitable embodiment, the active principle is obtained by the implementation of the following steps:

- solubilization of the biomass of Chlorella sorokiniana, preferably between 20 and

200g / L in water (Figure 1 was carried out at this stage),

- chemical hydrolysis,

- enzymatic hydrolysis (s):

o the hydrolysis conditions are chosen to hydrolyze proteins and glycoproteins; preferably the enzymatic hydrolysis is carried out by means of a protease, and / or o the hydrolysis conditions are chosen to hydrolyze the carbohydrates and the glycoproteins; preferably the enzymatic hydrolysis is carried out by means of a carbohydrase,

- inactivation of the enzyme (s) preferably by heat treatment: this inactivation is carried out according to the technical recommendation of the supplier (s) of the enzyme (s) (Figure 2 has been carried out at this stage; we observe the presence of a few whole microalgae cells, but especially the presence of cell fragments),

- Separation of the soluble and insoluble phases preferably by centrifugation, and recovery of the soluble phase containing inter alia sugars, proteins and possibly soluble minerals;

- filtration to remove particles still in suspension, and recovery of the filtrate;

- purification by filtration in order to eliminate the molecules of high molecular weight, preferably the molecules of molecular weight higher than lOkDa; this step is preferably carried out by membrane nanofiltration;

- Obtaining a filtrate, which constitutes the extract according to the invention, which is in liquid form (Figure 3 was carried out at this stage).

The hydrolyzate obtained at this stage can be further concentrated and / or purified in order to select the low molecular weight fractions, preferably less than 5kDa, by successive stages of filtration and sterilizing filtration through filters of different porosity, retaining the filtrates at each stage and / or by a chromatographic type method, in order, for example, to specifically enrich the hydrolyzate in these molecules.

The process can also include a bleaching and / or deodorizing step.

The steps of the processes described above, taken individually, are usual in the field of extraction of active ingredients from natural raw materials and the person skilled in the art is able to adjust the reaction parameters based on his general knowledge. .

The extract according to the invention is preferably used as a cosmetic agent in compositions, these compositions comprising a cosmetically acceptable medium. These are compositions in different dosage forms, suitable for application to the skin, in particular the skin of the face.

These compositions may be in particular in the form of oil-in-water emulsions, water-in-oil emulsions, multiple emulsions (Water / Oil / Water or Oil / Water / Oil) which may optionally be microemulsions or nanoemulsions, or in the form of solutions, suspensions, hydrodispersions, aqueous gels or powders. They can be more or less fluid and have the appearance of a lotion, shampoo, cream or foam.

They may be compositions comprising at least 0.5% of an extract of Chlorella sorokiniana according to the invention, preferably between 0.5 and 5%, the percentages being given by weight relative to the weight of the composition.

These compositions comprise, in addition to the extract of Chlorella sorokiniana, a physiologically acceptable and preferably cosmetically acceptable medium, that is to say one which does not cause feelings of discomfort unacceptable to the user such as redness, tightness or tingling.

The compositions according to the invention may contain as adjuvant at least one compound chosen from:

- oils, which can be chosen in particular from silicone oils, linear or cyclic, volatile or non-volatile;

- waxes, such as ozokerite, polyethylene wax, beeswax or carnauba wax,

- silicone elastomers,

- surfactants, preferably emulsifiers, whether they are nonionic, anionic, cationic or amphoteric,

- co-surfactants, such as linear fatty alcohols,

- thickeners and / or gelling agents,

- humectants, such as polyols such as glycerin,

- dyes, preservatives, fillers,

- tensors,

- sequestrants,

- the perfumes,

- and their mixtures, without this list being exhaustive.

Examples of such adjuvants are cited in particular in the CTFA Dictionary (International Cosmetic Ingredient Dictionary and Handbook published by the Personal Core Product Council).

Of course, a person skilled in the art will take care to choose any additional compounds, active or non-active, and their quantity, so that the advantageous properties of the mixture are not, or not substantially, altered by the addition envisaged.

These compositions are in particular intended to be used on the skin to improve the aesthetic appearance of the skin, in particular to hydrate the skin and / or improve the radiance of the complexion.

In order to illustrate the cosmetic effects of an extract of Chlorella sorokiniono, the following examples with their test results are presented.

EXAMPLES

Examples of extracts

Several extracts according to the invention were produced by varying the steps of hydrolysis, concentration, discoloration / deodorization.

The general steps are as follows:

-solubilization in water of the biomass of Chlorella sorokiniana,

- chemical hydrolysis (s) and / or enzymatic hydrolysis (s). The conditions for optimal use of enzymes are those recommended by suppliers.

-Inactivation by heat treatment, according to the recommendations of enzyme suppliers

-Separation of the soluble and insoluble phases by centrifugation and recovery of the soluble phase

- Filtration on OkDa and recovery of the filtrate

- Filtration and sterilizing filtration on 0.22pm filter.

The variable process conditions for the different extracts from the examples are described in Tables A and B:

Conditions Solubilization AT 20g / l B 50g / l VS 100g / l D 150g / l E 200g / l

Conditions Hydrolysis (s) at Basic and protease b Basic and carbohydrase vs Basic and protease and carbohydrase d Acid and protease e Acid and carbohydrase f Acid and protease and carbohydrase

Table IB

Table IA

Table IA and IB: Process conditions for obtaining the various examples of extracts according to the invention

The extracts according to the invention obtained are characterized as follows:

Proteins are determined by weight of dry matter, by the LOWRY method,

- Carbohydrates are determined by weight of dry matter, by the DUBOIS method,

- The ashes are determined by weight of dry matter, by weighing the residues from the incineration of the hydrolyzate samples at 550 ° C in an electric muffle furnace.

The characteristics obtained for several examples are presented in Table 2 below:

Examples Dry matter Protein content Carbohydrate content Content ashes mineral Example 1 31.4g / l 24% 60% 11% Example 2 48.7g / l 23% 57% 20% Example 3 48.3g / l 34% 57% 9% Example 4 36.3g / l 25% 63% 12% Example 5 33.7g / l 29.4% 52% 18.6% Example 6 71.1g / l 27% 53% 20%

Table 2: Characterization of different examples of extracts according to the invention

The extraction method of Example 1 was applied to the biomass of Chlorella vulgaris. 10 The analytical characterization of the extract obtained is presented in Table 3:

Examples Dry matter Protein content Carbohydrate content Content ashes mineral Example 7 On Chlorella vulgaris 49.1g / l 48% 28% 10%

Table 3: Characterization of an extract of Chlorella vulgaris obtained by the same process as that used to obtain an extract of Chlorella sorokiniana according to Example 1

The peptides were characterized so as to determine their molecular weights and to quantify the various protein fractions by steric exclusion FPLC chromatography.

The extract is analyzed under the following conditions:

- Pump: FPLC AKTA (Pharmacia)

- Column: Superdex Peptides TRICORN 10/300 GL (Pharmacia)

- Mobile phase: 20 mM potassium phosphate buffer, pH 7.2 with 0.25 M NaCl.

- Detector: UV at 280 nm

- Flow rate: 0.5 mL / min

- Injection volume: 200 pL

A calibration curve for the retention times of the markers as a function of their molar mass is constructed (logarithmic fit). Table 4 summarizes the retention times obtained for the different markers.

Molecules Molecular weight (Da) Retention time (min) Cytochrome C 12,500 20.0 Aprotinin 6,512 23.5 Vitamin B12 1,355 33.4 Cytidine 243 41.4

Table 4. Retention times of the various markers.

Thanks to the calibration curve, the following retention times could be estimated: protein of 10,000 Da: tr = 21.4 min protein of 3,500 Da: tr = 25.2 min protein of 2,000 Da: tr = 30 , 3 mins

Then, a fractionation program is constructed, as a function of the retention times, in order to collect the following fractions (MM = molecular weight):

o fraction 1: : 10,000 Da <MIVI o fraction 2: : 3,500 Da <MM < 10,000 Da o fraction 3: : 2,000Da <MM < 3,500 Da o fraction 4: 243 Da <MM < 2,000 Da o fraction 5: MM < 243 Da

The different fractions collected from examples of extracts according to the invention are quantified by spectrophotometric assay according to the LOWRY method (Lowry et al., Protein measurement with the folin reagent, J. Biol. Chem., 193, 265,1951).

The chromatographic profile and the fractionation of the peptides of the extract and the distribution of each fraction determined by spectrophotometric method is presented in Table 5.

Molecular weight (Da) Division (%) Fraction 1: 10,000 Da <MM 0.0 Fraction 2: 3,500 Da <MIVI <10,000 Da 6.3 Fraction 3: 2,000 Da <MM <3,500 Da 17.7 Fraction 4: 243 Da <MM <2,000 Da 75.8 Fraction 5: MM <243 Da 0; 3

Table 5. Distribution and quantification of the proteins of the extracts according to the invention (average of the examples)

More than 93% of peptides of the extracts according to the invention have molar masses of between 243 and 3500 Da.

Likewise, the carbohydrate fraction of the extracts was characterized.

The molecular weights are determined by HPLC with Rl detection according to the following conditions. The samples of the extracts according to the invention were diluted to% in the mobile phase before injection on the HPLC / RI system.

The molar masses of the carbohydrates are evaluated by comparison of the retention times of the peaks detected in the samples with the retention times of standards injected beforehand. The extract is analyzed under the following conditions:

Column: Polymer Laboratories (Varian) PL aquagel-OH 60, aquagel-OH 40 and aquagel-OH 30 in size 300 x 7.5 mm (in series) with pre-column with the same characteristics

Flow rate: 1 mL / min

Solvent: 0.3 M NaNÜ3 buffer and 0.01 M NaH2PO4-2H2O, pH = 7 in isocratic mode

Detector: Rl (Refractive Index)

- Injection volume: 10 pL

The identification and quantification of simple sugars are carried out by ionic liquid chromatography. The samples were hydrolyzed with 2 M trifluoroacetic acid (TFA) for 2 hours at 105 ° C. They were then cooled and neutralized with 4 M sodium hydroxide (NaOH), then diluted to the appropriate concentration in order to correspond to the calibration range.

A calibration range is prepared for each neutral dare from standards between 10 mg / L and 50 mg / L.

Table 6 gives the distribution of the molar masses of the carbohydrates present in the extracts according to the invention obtained from the areas of the chromatogram obtained.

Molar mass (Da) and degree of polymerization corresponding Carbohydrate level (%) Monosaccharides MM <180 (DPI) 15% Oligosaccharides Polysaccharides and 180 <MM <8,400 DPI <DP <DP47 85%

Table 6: Distribution of the molar masses of the carbohydrates present in the extracts of the examples (average)

The extract according to the invention consists mainly of oligosaccharides and polysaccharides with a molar mass of less than 8400Da. The carbohydrates of the extracts of the examples have an average molar mass of 697 Da.

The composition of simple sugars of the extracts of the examples is determined by ionic liquid chromatography, is presented in Table 7.

Type of sugar Proportion (%) Glucose 95 Galactose 5

Table 7: Composition of simple sugars from the extracts of the examples (average)

This analysis therefore makes it possible to demonstrate that the extracts of the examples mainly consist of glucose and galactose.

Examples of compositions

Example 8 Night care comprising an extract of Chlorella sorokiniana:

An example of a composition according to the invention in the form of a night care gel consists of:

A. Water qs100%

Glycerin 6%

Preservative 1%

Chlorella sorokiniana extract 2.5%

DUBCG7 (Stéarinerie Dubois) 1% Phytowax Olive 8L25 (Sophim) 1.5% Biophytosebum (Sophim) 2.5% Vegeline 65 (Sophim) 0.5% DUB Zenoat (Stéarinerie Dubois) 3% DUB 5545 (Stéarinerie Dubois) 6% Simulsoll65 (Seppic) 1.5% DUB PTCC (Stéarinerie Dubois) 1% Almond butter (Sophim) 2.5% Montanov L (Seppic) 1.5% Lanol 1688 (Seppic) 3.0% Sepinov EMT10 (Seppic) 2% H121 Sunspheres (AGC Chemicals) 0.5% Sepiplus 400 (Seppic) 0.3%

This emulsified gel has a pH of 5.1. It is white, thick, shiny, odorless, with a supple texture.

Its other characteristics are easy grip, fresh and slippery spreading, rapid penetration, a soft and slightly film-forming finish.

The composition can be obtained by implementing the following steps:

- Mix and heat separately A and B, in a water bath at 80 ° C

- Emulsify B in A, under stator rotor at 2500 rpm

- At 30 ° C, add C

- Leave to agitate (at 2000 rpm) for complete homogenization.

Example 9: Blur formula comprising an extract of Chlorella sorokiniana:

An example of a composition according to the invention in the form of the Blur formula consists of:

A. Water qs100%

Glycerin 4%

Conservative

1%

Chlorella sorokiniana extract 2.5% DUB Lilirose (Stéarinerie Dubois) 2% Biophytosebum (Sophim) 1% Vegeline65 (Sophim) 1% DUB Zenoat (Stéarinerie Dubois) 4% Sepimax Zen (Seppic) 1% Easynov (Seppic) 3% Timiron Splendid Violet (Merck) 0.05% Timiron Glam Sylver (Merck) 0.02%

This white gel, shiny, flexible and odorless, has a pH of 6.

Its other characteristics are: a creamy grip, a slippery and soft spreading, and a very fast penetration, as well as a silky finish.

This composition can be obtained by implementing the following steps:

- Mix A

- Heat in a water bath at 80 ° C with stirring

- Mix B

- Heat in a water bath at 80 ° C with stirring

- Emulsify B in A, under stator rotor at 1500 rpm

- At 50 ° C, add C and allow to cool always under stator rotor at 1500 rpm.

Example 10: Day Care Formula comprising an extract of Chlorella sorokiniana:

An example of composition according to the invention in the form of day care consists of:

Water qs 100% Glycerin 4% Conservative 1% Chlorella sorokiniana extract 2.5% Sepimax Zen (Seppic) 0.5% DUB 5545 (Stéarinerie Dubois) 3%

EuthanolG (BASF) 2% Tegosoft tn2 (BASF) 1% Glycerol oleate (Stéarinerie Dubois) 2% DUB Vinyl (Stéarinerie Dubois) 0.6% DUB Cocorose (Stéarinerie Dubois) 0.8% Easynov (Seppic) 4% H121 Sunspheres (AGC Chemicals) 1% DC71300 (Dow Corning) 1%

This white, shiny, fluid and odorless emulsion has a pH of 5.6.

Its other characteristics are: a liquid grip, a slippery spread, a fairly rapid penetration and a soft, dry finish.

This composition can be obtained by implementing the following steps:

- Mix A

- Heat in a water bath at 80 ° C with stirring

- Mix B

- Heat in a water bath at 80 ° C with stirring

- Emulsify B in A, under stator rotor at 2000 rpm

- At 50 ° C, add C and allow to homogenize under rotor stator at 1500 rpm, until complete cooling.

EVALUATION OF COSMETIC EFFICIENCY ACCORDING TO THE INVENTION

/. EFFECT OF CHLORELLA SOROKINIANA EXTRACT ACCORDING TO THE INVENTION ON DEFENSE LEVERS

MITOCHONDRIALE

The tests were carried out with several extracts according to the invention, as presented above. The results given in the tests described below represent the average of the results of each of these extracts.

o 1.1. Effect of an extract according to the invention on mitochondrial quality control The objective of this study is to assess the effect of an extract of Chlorella sorokiniana on mitochondrial quality control.

For this, three markers involved in controlling the quality of the good vitality of the mitochondria were studied.

- the expression of HSP27 (Heat Shock protein 27), chaperone protein involved in cell survival;

- the synthesis of SIRT3 (Sirtuin 3), the predominant mitochondrial enzyme involved in metabolism and in defense processes in the event of stress;

- the synthesis of aconitase, a true bodyguard of the mitochondria which acts both on metabolism and on the preservation of mtDNA.

The expression of HSP27 was studied on young and old human keratinocytes by quantitative PCR. The syntheses of SIRT3 and aconitase were evaluated after immunohistological markings on reconstructed epidermis (SILABSKIN ”RE) made from young or aged keratinocytes by successive replications.

The operating protocols are described as follows:

- A. Test on keratinocytes

The keratinocytes are seeded in a suitable culture medium and incubated at 37 ° C for several days. They are then treated with the active principle according to the invention at 0.40% and 0.70%. after 48 hours of treatment, the RNAs are collected and the complementary DNAs are analyzed by the quantitative PCR technique.

B. Study on reconstructed epidermis:

At D0, normal human keratinocytes or cells aged by replication are seeded on the insert.

The culture medium is changed every 2 days with or without the extract according to the invention at 0.20% and 0.40% (V / V).

At D15, the reconstructed epidermis are fixed, dehydrated and included in paraffin. Sections (4 µm) are made using a microtome.

Immunohistological labeling of SIRT3 and aconitase:

- Xylene dewaxing

- Unmasking of antigenic sites

- Saturation

- Primary monoclonal anti-SIRT3 or anti-aconitase antibodies:

- Coupled anti-IgG secondary antibodies for SIRT3 or aconitase:

Reading of the immunostaining: the visualization is carried out on a confocal microscope coupled with an image analysis system. The syntheses of SIRT3 and aconitase are proportional to the intensity of the fluorescence (green color) present on the reconstructed epidermis.

A quantitative analysis of the images was carried out using software.

The results are given in Tables 8 to 10.

Expression of HSP27 (%) Ability to restore expression of HSP27 (%) Young human keratinocytes Witness 100 Extract according to the invention 0.70% 98 Aged human keratinocytes Witness 69 ** Extract according to the invention 0.40% 79 32 Extract according to the invention 0.70% 95 * 84

Table 8 Effect of the extract according to the invention on the expression of HSP27 by young and old human keratinocytes.

: significant result according to Student's t test / auxSILABSKIN * RE normal control (p <0.01).

*: significant results according to Student's t test / at SILABSKIN * RE aged control (p <0.05).

Synthesis of SIRT3 (xlO ² AU) Ability to restore SIRT3 synthesis (%) SILABSKIN RE normal Witness 1016 Extract according to the invention 0.40% 1025 SILABSKIN RE aged Witness 695 ^ψψ Extract according to the invention 0.20% 907 * 66 Extract according to the invention 0.40% 1115 * 131

Table 9 Effect of the extract according to the invention on the synthesis of SIRT3 in normal and aged SILABSKIN RE.

: significant result according to Student's t test / at normal control SILABSKIN * RE (p <0.01).

* significant results according to Student's t test / auxSILABSKIN * RE aged control (p <0.05).

Aconitase synthesis (x10 ⁴ AU) Ability to restore aconitase synthesis (%) SILABSKIN RE normal Witness 207 Extract according to the invention 0.40% 210 SILABSKIN RE aged Witness 146 ^ Extract according to the invention 0.20% 188 ** 69 Extract according to the invention 0.40% 208 ** 102

Table 10. Effect of the extract according to the invention on the synthesis of aconitase in normal and aged SILABSKIN * RE.

: significant result according to Student's t test / at normal control SILABSKIN * RE (p <0.01).

** significant results according to Student's t test / auxSILABSKIN * RE aged control (p <0.01).

These results show that the expression of HSP27 and the syntheses of SIRT3 and aconitase are reduced with aging.

There is also an extract of Chlorella sorokiniana used to increase 3 main parameters of mitochondrial quality control.

In particular, tested at 0.70% on aged keratinocytes, the extract according to the invention significantly stimulates the expression of HSP27 by 84%. In addition, tested at 0.40% on aged SILABSKIN® RE, it significantly activates the syntheses of SIRT3 by 131% and aconitase by 102%. Thus, the use of an extract of Chlorella sorokiniana makes it possible to restore the defects of mitochondrial quality control during aging.

ο 1.2. Effect of an extract according to the invention on mitochondrial dynamics

The objective of this study is to assess the effect of an extract of Chlorella sorokiniana on mitochondrial dynamics.

For this, the morphology of the mitochondrial network, dictated by the balance between the mechanisms of fusion (elongated mitochondria) and of fission (punctate and spherical mitochondria) was studied.

Fragmentation of the mitochondrial network is often associated with loss of mitochondrial integrity.

Therefore, the morphological study of mitochondria provides information on the state of cellular stress. Two shape parameters of the mitochondria were studied: circularity and aspect ratio.

These studies were carried out by labeling with fluorescent probes on normal young and old human keratinocytes.

The operating protocol is described below.

At OJ, the keratinocytes are seeded in culture medium and incubated at 37 ° C.

After several days of culture, the keratinocytes are treated with the extract according to the invention at 0.70% (V / V final) for 24 hours.

The culture medium is removed and replaced and the keratinocytes are incubated at 37 ° C.

After 6 hours, two markings are made with the MitoTracker ”Green FM probe and the Hoechst 33342. with the CellMask ™ Orange plasma membrane stain probe.

The visualization is carried out on a confocal microscope coupled with an Mlitoshape® image analysis system.

The mitochondria appear colored green.

Staining of the plasma membrane and the nucleus is used for automatic detection of cells during quantification.

Quantification of the parameters of circularity and aspect ratio of the mitochondrial network for each cell is carried out numerically:

o The circularity corresponds to the degree of branching and the length of the mitochondria.

o The aspect ratio corresponds to the ratio: length of the mitochondria / width of the mitochondria.

The 2 parameters used are inversely correlated. The more the mitochondrial network is fragmented, the higher the circularity and the lower aspect ratio.

The results are given in Tables 11 and 12.

Circularity (%) Ability to restore circularity (%) Young human keratinocytes Witness 0.636 Extract according to the invention 0.70% 0.649 Aged human keratinocytes Witness 0.663 * Extract according to the invention 0.70% 0.650 42

Table 11 Effect of the extract according to the invention on the circularity of the mitochondrial network in young and old human keratinocytes.

: significant result according to Student's t test / to young control human keratinocytes (p <0.05).

Aspect Ratio (%) Ability to restore aspect ratio (%) Young human keratinocytes Witness 1,258 Extract according to the invention 0.70% 1,219 Aged human keratinocytes Witness 1,117 ** Extract according to the invention 0.70% 1,196 * 48

Table 12 Effect of an extract of Chlorella sorokiniana on the aspect ratio of the mitochondrial network in young and old human keratinocytes.

: significant result according to Student's t test / to young control human keratinocytes (p <0.01).

*: significant result according to Student's t test / to control aged human keratinocytes (p <0.05).

We note that with aging, the morphology of the mitochondrial network is modified. Increased circularity and a decreased aspect ratio are observed in older human keratinocytes compared to younger human keratinocytes.

These results also show that the use of an extract of Chlorella sorokiniana allows an improvement in the integrity of the mitochondria in aged human keratinocytes, by restoring the morphological parameters of young human keratinocytes.

In particular, under the conditions of the study, tested at 0.70% on aged keratinocytes, it significantly restores the two shape parameters of the mitochondria up to:

- 42% for circularity

- 48% for the aspect ratio ο 1.3 Effect of an extract according to the invention on mitophagia and comparison to the effect of an extract of Chlorella vulgoris

The objective of this study is to evaluate the effect of an extract according to the invention and of an extract of Chlorella vulgoris (extract of Chlorella vulgoris from Example 7) on mitophagia.

For this, the collocation between the markers GRP75 (mitochondria) and LC3 (autophagosomes), direct proof of the management of mitochondria by mitophagy, was studied.

These studies were carried out by immunofluorescence labeling on young normal human keratinocytes subjected to stress CCCP (carbonyl cyanide mchlorophenylhydrazone) known to induce mitophagy. The operating protocol is described below.

At OJ, the keratinocytes are seeded in culture medium and incubated at 37 ° C.

After a few days, the keratinocytes are treated with medium containing CCCP at 1 μΜ with different products.

On D3, an immunostaining is carried out under the following conditions:

- Primary anti-GRP75 or anti-LC3B antibodies:

- Coupled anti-IgG secondary antibodies for GRP75 or LC3B:

The marking is visualized on a confocal microscope coupled to an image analysis system.

The mitochondria appear colored green.

The autophagosomes appear colored red.

The percentage of mitophagosomes corresponds to the number of autophagosomes containing a mitochondria compared to the total number of autophagosomes present in the cells.

The quantification of the percentage of mitophagosomes is carried out using an analysis program implemented in image analysis software.

Mitophagy is characterized by an increase in the percentage of mitophagosomes. The results are presented in Table 13.

Mitophagosomes (%) Ability to increase mitophagosomes (%) Young human keratinocytes Witness 7.88 Witness + 18.73 CCCP + extract according to the invention 0.70% 23.85 27 CCCP + Example 7 (excluding invention) 0.70% 19.10 2

Table 13 Effect of different products on the production of mitophagosomes in young human keratinocytes treated with CCCP.

These results show that the CCCP treatment of keratinocytes leads to activation of mitophagy as shown by the colocalization between the markers GRP75 (mitochondria) and LC3 (autophagosomes): direct proof of the management of mitochondria.

It is found that the extract according to the invention makes it possible to increase the mitophagosomes. Under the conditions of the study, this increase is 27%. Thus an extract of Chlorella sorokiniana makes it possible to potentiate the activation of mitophagy in human keratinocytes.

It is also found that an extract of Chlorella vulgoris, another species of microalgae of the same genus, obtained by the same process, does not exhibit such an effect.

//. LACK OF ANTI-RADICAL EFFECT OF THE EXTRACT OF CHLORELLA SOROKINIANA ACCORDING TO THE INVENTION

The objective of this study is to evaluate the anti-radical activity of an extract according to the invention, according to the DPPH method.

Di Phenyl Picrylhydrazyl Hydrate (DPPH) is a free radical, absorbing in violet at 15 517nm. An anti-radical product causes a disappearance of the purple coloration.

The DPPH solution used for this study is a solution prepared from 4.8 mg of DPPH dissolved in methanol,

The experimental study protocol is described below.

DPPH solution is added to hemolysis tubes and the diluted solution of the test product (extract according to the invention at 10% and 20%) or distilled water (for the blank) is added.

Stir and wait 20 minutes before reading the optical densities (OD) at 517nm against the air.

The results obtained are presented in percentage of anti-radical activity, in Table 14.

Tested dose of active ingredient according to the invention DPPH activity PM 17.023.1 35g / l ° 10% 7% 1% 5% PM 17.025.1 70g / l 10% 7% 1% 4%

Table 14 Effect of different products on the production of mitophagosomes in young human keratinocytes treated with CCCP.

The extract according to the invention does not have anti-radical activity according to the DPPH method, because the person skilled in the art considers that an active agent has anti-radical / anti-oxidant efficacy from 25% activity for a concentration maximum 10%.

III. EFFECT OF CHLORELLA SOROKINIANA EXTRACT ACCORDING TO THE INVENTION ON CELLULAR METABOLISM AND

TISSUE o 1111 Effect of an extract according to the invention on the production of ATP

Two studies have been conducted to show the effect on ATP production, this is an in vitro study and an in vivo study.

In vitro study

The objective of this study is to assess the effect of an extract of Chlorella sorokiniana on the production of ATP.

The tests were carried out with several extracts according to the invention, as presented above. As for the other studies, the results given in this test represent the average of the individual results of these extracts.

ATP is a source of energy directly usable by the cell and is essential for the proper functioning of cells.

The intracellular ATP assay was carried out by bioluminescence on normal young and old human keratinocytes treated or not treated with CCCP.

The operating protocol is described below.

On D0, the keratinocytes are seeded in culture medium and incubated at 37 ° C.

After several days of culture, the keratinocytes are treated with medium containing or not containing CCCP at 1 μΜ with an extract according to the invention at 0.40% and 0.70% (final V / V).

After 24 hours, the intracellular ATP is quantified using a dedicated kit.

The results are given in Tables 15 and 16.

Concentration ATP (μΜ) Ability to increase ATP (%) Young human keratinocytes Witness 1.92 Extract according to the invention 0.40% 2.21 ** 15 Extract according to the invention 0.70% 2.58 ** 34 Aged human keratinocytes Witness 1.50 ** Extract according to the invention 0.40% 1.60 7 Extract according to the invention 0.70% 1.84 ** 23

Table 15 Effect of an extract of Chlorella sorokiniana on the production of ATP by young and old human keratinocytes.

: significant results according to Student's t test / to young control human keratinocytes (p <0.01).

**: significant result according to Student's t test / to control aged human keratinocytes (p <0.01).

Concentration ATP (μΜ) Ability to increase ATP (%) Young human keratinocytes CCCP Witness 0.93 CCCP + Extract according to the invention 0.40% 1.61 73 CCCP + Extract according to the invention 0.70% 1.76 89 Aged human keratinocytes CCCP Witness 0.89 CCCP + Extract according to the invention 0.40% 1.29 45 CCCP + Extract according to the invention 0.70% 1.47 65

Table 16 Effect of an extract of Chlorella sorokiniana on the production of ATP by young and old human keratinocytes subjected to CCCP aggression.

: significant result according to Student's t test / with young human keratinocytes, CCCP control (p <0.01).

* significant result according to Student's t test / to aged human keratinocytes, CCCP control (p <0.05).

It is found that aged human keratinocytes subjected or not to CCCP stress produce significantly less ATP than young keratinocytes under the same conditions.

These results also show that the use of an extract of Chlorella sorokiniana makes it possible to activate the production of the energy necessary for good cellular functioning.

In particular, under the conditions of the study, tested at 0.70%, it makes it possible to significantly increase the production of ATP by:

-34% on young keratinocytes,

-23% on aged keratinocytes,

-89% on young keratinocytes treated with CCCP,

-65% on aged keratinocytes treated with CCCP.

In vivo study

The objective of this study was to evaluate, in vivo, the effect of an extract of Chlorella sorokiniana formulated at 2% in emulsion on the production of ATP, in comparison with a placebo.

The effect of the extract was studied on 20 volunteers, healthy, Caucasian, female, average age 42 + 5 years.

The ATP concentration is determined by luminometry. The samples were taken, at the cheeks on asymmetric zones using D-squames', before and 2 hours after a single application as well as after 7 days of twice-daily use of the formulas.

A summary of the results corresponding to the effect of extracts of Chlorella sorokiniana formulated at 2% and compared with placebo, on ATP production is presented in Table 17.

Change / Placebo (%) T2H +51.1 (p = 0.0083) D7 +54.2 (p = 0.0156)

Table 17 Effect of an extract of Chlorella sorokiniana formulated at 2% on the production of ATP compared to placebo.

These results also show that the use of an extract according to the invention makes it possible to increase the production of ATP.

In particular, we note that under the conditions of this study, and in comparison with placebo, from 2 hours after a single application, the use of an extract of Chlorella sorokiniana allows to significantly boost the production of ATP by 51%. It continues to activate ATP production after 7 days of twice-daily applications.

///.2 Effect of an extract according to the invention on cell renewal and barrier function

Two studies have been conducted to show the effect on ATP production, this is an in vitro study and an in vivo study.

In vitro study

The objective of this study is to assess the capacity of an extract according to the invention to promote the construction of a stratified and functional epidermis.

The tests were carried out with several extracts according to the invention, as presented above. As for the other tests, the results given in this test represent the average of the individual results of these extracts.

For this, a proliferation marker (Ki-67) which validates the functionality of the reconstructed epidermis has been studied.

This study was carried out by immunohistological markings on reconstructed epidermis (SILABSKIN® RE) from human keratinocytes aged by successive replications.

At OJ, normal or aged human keratinocytes are seeded on the insert.

The culture medium is changed every 2 days with or without the presence of an extract of Chlorella sorokiniana at 0.20% and 0.40% (V / V).

After several days of culture, the reconstructed epidermis are fixed, dehydrated and included in paraffin. Sections (4 µm) are made using a microtome.

Immunohistological labeling is carried out under the following conditions:

-Primary anti-Ki67 antibody:

- Coupled anti-IgG secondary antibody for Ki67

The visualization is carried out on a microscope coupled with an image analysis system.

The synthesis of Ki-67 is proportional to the intensity of the fluorescence (yellow color) present on the sections of SILABSKIN® RE.

The nuclei of the cells appear colored blue.

The results are given in Table 18.

Number of cells Ki-67 positive (%) Ability to restore the synthesis of Ki-67 (%) SILABSKIN RE normal Witness 58 Extract according to the invention 0.40% 57 SILABSKIN RE aged Witness 53 Extract according to the invention 0.20% 61 160 Extract according to the invention 0.40% 63 200

Table 18. Effect of an extract of Chlorella sorokiniana on the synthesis of Ki-67 in normal and aged SILABSKIN * RE.

r significant result according to Student's t test / to normal control SILABSKIN * RE (p <0.05).

**: significant results according to Student's t test / at SILABSKIN * RE aged control (p <0.01).

These results show that the synthesis of Ki-67 is reduced in aged SILABSKIN RE compared to normal SILABSKIN® RE.

It is noted that the use of an extract of Chlorella sorokiniana makes it possible to restore the renewal of aged SILABSKIN® RE.

In particular, under the conditions of the study, tested at 0.40% on aged SILABSKIN® RE, it makes it possible to significantly increase the synthesis of Ki-67, by 200%.

In vivo study

The objective of this study was to evaluate in vivo the effect of an extract of Chlorella sorokiniono, formulated at 2% as an emulsion on cell renewal compared to a placebo. The study was carried out on 18 healthy, Caucasian, female, middle-aged volunteers

39 ± 5 years.

The measurements of the skin color were carried out on areas located at the level of the belly, after coloring with dihydroxyacetone (DHA), using a Chromameter®. The measurements were taken before, 72 hours after coloring, and then 5 times during the 14 days of twice-daily applications of the composition containing the extract of Chlorella sorokiniana and the placebo.

Two parameters were studied:

parameter b * (characteristic of melanin yellow skin pigmentation). The more the parameter b * decreases, the more the skin becomes lighter.

the parameter ΔΕ (characteristic of the variation in color). The more this parameter increases, the greater the discoloration of the area stained with DHA

A summary of the results corresponding to the effect of an extract of Chlorella sorokiniana 5 formulated at 2.5% and compared with placebo on the evolution of the parameters b * and ΔΕ, characteristic of cell renewal is presented in Tables 19 and 20 .

Evolution of the parameter b * D2-D0 D4-D0 D7-D0 Jll-JO D14-D0 Untreated area -0.9 -1.3 -2.0 -4.5 -5.3 Placebo -0.8 -1.3 -2.7 -4.6 -5.0 Chlorella sorokiniana extract 2.5% -1.1 -2.4 -3.6 -5.2 -5.3

p-value / placebo 0.2040 0.0012 0.0035 0.0490 0.3475 Table 19 Effect of an extract of Chlorella sorokiniana formulated with 2% emulsion on the parameter b * after 14 days of twice-daily applications. Evolution of the parameter ΔΕ D2 D4 17 Jll D14 Untreated area 2.6 3.1 4.5 6.9 8.8 Placebo 2.8 4.0 5.4 7.9 9.0 Chlorella extract 2.8 4.4 6.3 8.6 9.2 sorokiniana 2.5% p-value / placebo 0.5346 0.0958 0.0490 0.0154 0.3289

Table 20 Effect of an extract of Chlorella sorokiniana formulated at 2% as an emulsion on the ΔΕ parameter after 14 days of twice-daily applications.

It is noted that the use of an extract of Chlorella sorokiniana makes it possible to accelerate cell renewal. In particular, under the conditions of this study, after 14 days of twice-daily applications, in comparison with placebo, an extract of Chlorella sorokiniana formulated at 2% more rapidly decreases the parameter b * representative of the melanic yellow color and increases the parameter ΔΕ characteristic of skin lightening.

III.3 Effect of an extract according to the invention on the barrier function Two studies have been carried out to show the effect of the extract of Chlorella sorokiniana according to the invention on the skin barrier function. A first in vitro study made it possible to assess the effect on epidermal differentiation and a second in vivo study completed by the study of insensitive water losses under condition or not of stress.

In vitro study

The objective of this study is to assess the capacity of an extract according to the invention to promote the construction of a stratified and functional epidermis.

The tests were carried out with several extracts according to the invention, as presented above. As for the other tests, the results given in this test represent the average of the individual results of these extracts.

For this, differentiation markers (cytokeratin 10 and loricrin) which allow the functionality of the reconstructed epidermis to be validated have been studied.

These studies were carried out by immunohistological markings on reconstructed epidermis (SILABSKIN® RE) from human keratinocytes aged by successive replications.

At OJ, normal or aged human keratinocytes are seeded on the insert.

The culture medium is changed every 2 days with or without the presence of an extract of Chlorella sorokiniana at 0.20% and 0.40% (V / V).

After several days of culture, the reconstructed epidermis are fixed, dehydrated and included in paraffin. Sections (4 µm) are made using a microtome.

Immunohistological labeling is carried out under the following conditions:

- Primary anti-CKlO or anti-loricrin antibodies:

- Secondary anti-IgG antibodies coupled for CK10 or loricrin

The visualization is carried out on a microscope coupled with an image analysis system.

The syntheses of cytokeratin 10 and loricrin are proportional to the intensity of the green fluorescence present on the sections of SILABSKIN ’'RE.

The nuclei of the cells appear colored blue.

The results are given in Tables 21 and 22.

Synthesis of CK10 (xlO ⁴ AU) Ability to restore CK10 synthesis (%) SILABSKIN RE normal Witness 1990 Extract according to the invention 0.40% 1991 SILABSKIN RE aged Witness 1731 * Extract according to the invention 0.20% 1856 * 48 Extract according to the invention 0.40% 1878 ** 57

Table 21 Effect of an extract of Chlorella sorokiniana on the synthesis of CK10 in normal and aged SILABSKIN * RE.

significant result according to Student's t test / at normal control SILABSKIN * RE (p <0.05) *: significant result according to Student's t test / at control aged SILABSKIN * RE (p <0.05) **: result significant according to Student's t test / with aged control SILABSKIN * RE (p <0.01)

Loricrin synthesis (x10 ⁴ AU) Ability to restore the synthesis of loricrin (%) SILABSKIN RE normal Witness four hundred ninety seven Extract according to the invention 0.40% 491 SILABSKIN RE aged Witness 330 ** Extract according to the invention 0.20% 398 * 41 Extract according to the invention 0.40% 445 * 69

Table 22 Effect of an extract of Chlorella sorokiniana on the synthesis of loricrin in normal and aged SILABSKIN * RE.

: significant result according to Student's t test / at normal control SILABSKIN * RE (p <0.01).

* significant results according to Student's t test / at SILABSKIN * RE aged control (p <0.05).

These results show that the syntheses of cytokeratin 10 and loricrin are reduced in aged SILABSKIN ⁸ RE compared to normal SILABSKIN ⁸ RE.

It is noted that the use of an extract of Chlorella sorokiniana makes it possible to restore the renewal and the differentiation of aged SILABSKIN ⁸ RE.

In particular, under the conditions of the study, tested at 0.40% on aged SILABSKIN® RE, it significantly increases cytokeratin 10 and loricrin synthesis by 57% and 69% respectively.

In vivo study

The objective of this study was to evaluate, in vivo, the effect of an extract of Chlorella sorokiniana formulated at 2% on the quality of the barrier function of the skin with and without stress.

In a first study, the capacity of an extract of Chlorella sorokiniana to improve the quality of the skin barrier was studied on 20 healthy, female, Caucasian volunteers, average age 45 ± 4 years.

The measurements were taken on the face using a Tewameter ’before and after 14 and 28 days of twice-daily placebo applications and of a composition comprising an extract of Chlorella sorokiniana in the hemi-face.

In a second study, the extract of Chlorella sorokiniana was evaluated on its ability to regulate excessive water losses caused by repeated aggression using a detergent, Lauryl Sodium Sulfate (SLS) 10% for 14 days.

Indeed, the skin barrier plays a regulatory role in the water balance of the skin. When the latter is damaged, there are disturbances in the regulation of water exchanges. Water then migrates more easily to the outside environment, which increases the insensible loss of water. On the other hand, if the condition of the skin barrier improves, the water loss values will decrease because the regulation of water exchanges will be ensured correctly.

This effect was observed on 22 healthy, female, Caucasian volunteers, mean age 44 ± 4 years.

The water loss measurements of the skin were carried out with a Tewameter ”at the level of the external anterolateral face of the arm before and after 7 and 14 days of twice-daily applications of the placebo and of the composition comprising an extract of Chlorella sorokiniana .

Regarding the effect on insensitive water loss without aggression, a summary of the results corresponding to the effect of an extract of Chlorella sorokiniana formulated at 2% on the insensitive water loss of the skin measured using a Tewameter® is presented in Table 23.

Change / Placebo (%) D14 -11.2 D28 -18.1

Table 23 Effect of an extract of Chlorella sorokiniana formulated at 2% on the insensitive water loss

It is found that the use of an extract of Chlorella sorokiniana on the skin makes it possible to reduce the insensitive loss of water and consequently to improve the quality of the barrier function of the skin.

In particular, under the conditions of this study, from 14 days of twice-daily applications and in comparison with placebo, an extract of Chlorella sorokiniana formulated at 2% in emulsion, significantly reduces the insensible water loss of the skin by 11.2% . This effect continues after 28 days of twice-daily applications to achieve a significant reduction in water loss, on healthy skin, of 18.1%.

Regarding the effect on insensible water loss with stress (or aggression)

A summary of the results corresponding to the effect of an extract of Chlorella sorokiniana formulated at 2% compared to placebo on the PIE measured using a Tewameter ’after aggression at SLS 10% is presented in Table 24.

Change / Placebo (%) D7 -9.3 D14 -24.2 ²⁰

Table 24 Effect of an extract of Chlorella sorokiniana formulated at 2% on the insensible water loss after repeated attacks with SLS

It is noted that the use of an extract of Chlorella sorokiniana on the skin, makes it possible to regulate the excessive water losses engendered by a repeated aggression with SLS.

In particular, under the conditions of this study, from 7 days of twice-daily applications, an extract of Chlorella sorokiniana formulated at 2% in emulsified gel significantly reduces the insensible water loss after repeated aggression by SLS by 9.3% in comparison placebo. This effect continues after 14 days of application, where the reduction in water loss observed reaches 24.2%. This effect was validated in 73% of the volunteers.

IV. Effect of chlorella sorokiniana extract on skin parameters

For the following studies, the compositions used have the following formulas:

- For Caucasian volunteers

Isononyl isononanoate (Lanol 99, Seppic) 5.0%

Behenyl alcohol / arachidyl glucoside / arachidyl alcohol (Montanov 202,

Seppic) ³ ' ^{0 /} °

Chlorella sorokiniana extract 2.0%

Cetearyl alcohol / cetearyl glucoside (Montanov 68, Seppic) 2.0%

Preservatives 0.7%

Polyacrylamide / C13-14 isoparaffin / laureth 7 (Sepigel 305, Seppic) 0.3%

Water qs 100%

For Asian volunteers

Glycerin 6.2%

Cetearyl Ethylhexanoate (Lanol 1688, Seppic) 3.0%

Coco-Caprylate / Caprate (Lanol 2681, Seppic) 3.0%

Chlorella sorokiniana extract 2.0%

Isononyl isononanoate (Lanol 99, Seppic) 2.0%

Preservatives 1.0%

Polyacrylate Crosspolymer-6 (Sepimax Zen, Seppic) 0.5%

Hydroxyethyl Acrylate / Sodium Acryloyldimethyl Taurate Copolymer θ (Sepinov EMT 10, Seppic) '°

Water qs 100% ο IV. 1. Effect of an extract according to the invention on the lightening of the skin The objective of this study was to evaluate, in vivo, the effect of an extract of Chlorella sorokiniana formulated at 2% in emulsion on skin lightening versus placebo.

The effect of a Chlorella sorokiniana extract on skin lightening has been studied:

on a Caucasian panel, composed of 20 healthy, female volunteers, average age 45 + 4 years.

on an Asian panel made up of 32 healthy, female volunteers, average age 40 + 6 years.

The Caucasian panel study was carried out using chromametric measurements on the cheeks.

Two parameters were studied:

the L * parameter (characteristic of skin clarity). The higher the L * parameter, the lighter the skin becomes.

the ITA (Individual Typological Angle) parameter is used to assess the development of the radiant appearance of the skin. An increase in the ITA parameter is characteristic of clearer and luminous skin.

As for the Asian panel study, a colorimetric analysis on digital photographs was carried out.

Three parameters were chosen:

the parameter L * represents the clarity (from dark to pale) the parameter ITA °, makes it possible to evaluate the evolution of the radiant appearance of the skin.

homogeneity is obtained from a texture analysis which aims to look at the variation of colors in all directions of the surface of the skin.

In both cases, the measurements were taken before and after 14 and 28 days of twice-daily applications of the placebo and of the composition comprising the Chlorella sorokiniana extract in the hemi-face.

Caucasian panel study

A summary of the results corresponding to the effect of a composition comprising an extract of Chlorella sorokiniana formulated at 2.5% compared to placebo, on the parameters characteristic of the lightening of the skin of Caucasian type people, is presented in the Tables 25 and 26.

ITA ° parameter OJ D14 D28 Placebo 29.81 29.92 31.33 Chlorella sorokiniana extract 27.65 28.96 32.47

Table 25. Effect of an extract of Chlorella sorokiniana formulated at 2% on the ITA ° parameter representative of the lightening of the skin.

Parameter L * OJ D14 D28 Placebo 59.59 59.83 60.23 Chlorella sorokiniana extract 58.77 59.45 60.46

Table 26 Effect of an extract of Chlorella sorokiniana formulated at 2%. On the L * parameter representative of the lightening of the skin.

It is found that under the conditions of this study, after 28 days of twice-daily applications and in comparison with placebo, an extract of Chlorella sorokiniana formulated at 2% in emulsion lightens the skin of people of Caucasian type.

Indeed, a significant increase in the L * and ITA ° parameters, characteristic of clearer and brighter skin, is observed after 28 days of twice-daily applications of a composition comprising an extract of Chlorella sorokiniana.

Asian panel study

A summary of the results corresponding to the effect of a composition comprising an extract of Chlorella sorokiniana formulated at 2% compared to placebo, on the parameters characteristic of the lightening of the skin of people of Asian type, is presented in Tables 27 to 29.

ITA ° parameter OJ D14 D28 Placebo 72.55 72.55 72.83 Chlorella sorokiniana extract 72.63 72.85 73.43

Table 27 Effect of an extract of Chlorella sorokiniana formulated at 2% on the ITA ° parameter representative of the lightening of the skin

Parameter L * OJ D14 D28 Placebo 59.01 59.22 58.93 Chlorella sorokiniana extract 59.02 59.38 59.61

Table 28 Effect of an extract of Chlorella sorokiniana formulated at 2% on the L * parameter representative of the skin lightening.

Homogeneity parameter D0 D14 D28 Placebo 0.025 0.025 0.026 Chlorella sorokiniana extract 0.025 0.026 0.027

Table 29 Effect of an extract of Chlorella sorokiniana formulated at 2% on the homogeneity parameter of the skin.

It is found that under the conditions of this study, after 28 days of twice-daily applications and in comparison with placebo, an extract of Chlorella sorokiniana formulated at 2% in emulsion lightens the skin of people of Asian origin.

Indeed, a significant increase in the L * and ITA ° parameters, characteristic of clearer and more luminous skin, is observed after 28 days of twice-daily applications of an extract of Chlorella sorokiniana as well as a significant improvement in the homogeneity of the skin.

o IV.2. Coexistence of an extract according to the invention to improve the complexion complexion The objective of this study was to evaluate, in vivo, the effect of an extract of Chlorella sorokiniana formulated at 2% in emulsion on the radiance compared to a placebo.

This study was carried out on 20 healthy, female, Caucasian volunteers, average age 45 ± 4 years.

The evaluation of the radiance of the complexion of the face was carried out blind, by two trained experts, before and after 14 and 28 days of twice-daily applications of the placebo or of a composition comprising an extract of Chlorella sorokiniana in hemi-face. .

A summary of the results corresponding to the effect of an extract of Chlorella sorokiniana formulated at 2% compared to placebo, on the main parameters characteristic of the radiance of the complexion evaluated by experts is presented in Table 30.

Change / Placebo (%) D14 D28 Radiation +5.3 +8.8 Pink color +7.7 +16.3 Olive color -6.1 -11.0 Eye fatigue -7.1 -9.2

Table 30 Effect of an extract of Chlorella sorokiniana formulated at 2% on the radiance of the complexion.

We note that under the conditions of this study, from 14 days of twice-daily applications and in comparison with placebo, an extract of Chlorella sorokiniana formulated at 2% in emulsion:

- significantly improves skin radiance by 5.3% as well as the pink color, characteristic of a fresh complexion by 7.7%,

- significantly decreases the olive color by 6.1% and the tiredness of the eyes by 7.1%.

This effect continues after 28 days of application. It is thus found that the application of an extract of Chlorella sorokiniana:

- significantly increases the skin's radiation by 8.8% as well as the pink color, by 16.3% (effect validated in 85% of volunteers),

- allows a significant decrease in the olive color of 11.0% and in the tiredness of the eyes by 9.2% (effect validated in 75% of the volunteers).

Claims (18)

1. Cosmetic use of an extract of Chlorella sorokiniana in a cosmetic composition as a cosmetic agent. 2. Cosmetic use of an extract of Chlorella sorokiniana according to claim 1, as a cosmetic agent improving the aesthetic appearance and / or the texture of the skin. 3. Cosmetic use of an extract of Chlorella sorokiniana according to claim 1 or 2, as a cosmetic agent improving the radiance of the complexion. 4. Cosmetic use of an extract of Chlorella sorokiniana according to one of the preceding claims, as a cosmetic agent stimulating the renewal of epidermal cells and / or strengthening the skin barrier. 5. Cosmetic use of an extract of Chlorella sorokiniana according to one of the preceding claims, as a cosmetic agent improving the functioning of the mitochondria of the cells of the epidermis. 6. Cosmetic use of an extract of Chlorella sorokiniana according to one of the preceding claims, characterized in that said extract is a hydrolyzate of Chlorella sorokiniana. 7. Chlorella sorokiniono extract, comprising at least carbohydrates, proteins and minerals, characterized in that the carbohydrates represent at least 36% by weight of dry matter of the extract. 8. Chlorella sorokiniana extract according to claim 7, characterized in that it is characterized by: a carbohydrate content of between 36 and 94% a protein content of between 6 and 40%, the percentages being given by weight relative to the total dry matter weight of the extract. 9. Chlorella sorokiniana extract according to claim 7 or 8, characterized in that at least 70% of the carbohydrates are oligosaccharides and polysaccharides of glucose of molar masses between 345 and 8400Da, the percentage being given by weight relative to the total weight of the carbohydrates of the extract. 10. Chlorella sorokiniana extract according to one of claims 7 to 9, characterized in that at least 90% of the proteins have a molar mass of less than 3500 Da, the percentage being given by weight relative to the total weight of the proteins the extract. 11. Chlorella sorokiniana extract according to one of claims 7 to 10, characterized in 5 that it is a hydrolyzate of Chlorella sorokiniana. 12. Chlorella sorokiniana extract according to one of claims 7 to 11, characterized in that it is an enzymatic hydrolyzate of Chlorella sorokiniana. 13. Cosmetic use of an extract of Chlorella sorokiniana according to one of claims 1 to 6, characterized in that said extract is an extract according to one of 10 claims 7 to 12. 14. Cosmetic composition comprising at least 0.5% by weight of an extract of Chlorella sorokiniana. 15. Cosmetic composition according to claim 14, characterized in that it is in the form of a cream, lotion, milk, serum, ointment, gel, paste, foam, or under 15 solid form. 16. Cosmetic treatment method for improving the aesthetic appearance of the skin, characterized in that it comprises the application to the skin of at least one extract of Chlorella sorokiniana or of a composition containing it. 17. Cosmetic treatment method according to claim 16, for improving the radiance of the complexion of the skin. 18. Cosmetic treatment method according to claim 16 or 17, characterized in that the extract of Chlorella sorokiniana is an extract according to one of claims 7 to 12. 1/2