WO2015071477A1

WO2015071477A1 - Method for obtaining extracts of marine algae

Info

Publication number: WO2015071477A1
Application number: PCT/EP2014/074816
Authority: WO
Inventors: André PRIGENT
Original assignee: Agrimer
Priority date: 2013-11-18
Filing date: 2014-11-18
Publication date: 2015-05-21
Also published as: FR3013219A1; FR3013219B1

Abstract

The invention relates to a method for the fractionation of marina alga, comprising a step of treating at least one pre-cut alga, by the action of a protease, in an aqueous medium with a basic pH.

Description

Process for obtaining seaweed extracts

The present invention relates to a method for fractionating seaweed in its various biochemical compartments.

Marine algae contain active compounds such as sugars, amino acids, reserve and parietal polysaccharides, secondary metabolites such as polyphenols, lipids. These compounds can be used as active ingredients for the manufacture of cosmetic preparations. For this, it is necessary to develop efficient methods that make it possible to extract the active ingredient selectively under optimized conditions, that is to say without loss of the effectiveness of the active molecule and with acceptable yields for an exploitation. on an industrial scale.

Extraction processes are known, such as that described in document FR 2 946 878, making it possible to obtain an extract of algae by a process using an aqueous or hydro-glycerinated extraction with heating maintained for a period of time. less than two hours. It is possible to obtain an extract by this method, however it is not mentioned the valuation of extractive co-products that may contain active ingredients and that it would be interesting to value for the purpose of preparation of the cosmetic compositions, even therapeutic. In addition, such an aqueous extraction process makes it possible to have access only to a cytoplasmic extract containing only the water-soluble compounds of low molecular weight and does not give access to other types of extracts for the purpose of promoting all active substances of the algae. In order to overcome the deficiencies of the state of the art and to make it possible to obtain extracts containing a greater diversity of active principles from algae under good conditions, the subject of the present invention is a process for fractionating a aqueous suspension of seaweed, comprising the successive steps of:

a) cutting said algae after harvesting and drying; b) add 30 to 98% by weight of water;

c) add a base to adjust the pH of the solution between 7 and 10;

bringing the mixture obtained in step c) to a temperature of 60 ° C to 80 ° C;

e) adding a protease and allowing to stir for at least 2 hours by regulating the pH between 7 and 10;

f) lowering the temperature of the mixture to below 45 ° C; g) acidifying such that the mixture has a pH of 1.5 to 2.5; and h) recovering the slurry obtained by decantation and filtration, and collecting the filtrate.

The percentages by weight are expressed relative to the total mass of the mixture obtained comprising the dry algae. Alternatively, a powdered alga can be used.

Advantageously, the pH is 7.5 and 10, or it is maintained between 7.5 and 8.5, ideally 8, during steps c) to f). In step f), the temperature is advantageously lowered below

40 ° C.

The inventors have demonstrated that the action of a protease on an aqueous suspension in which a material composed of at least one marine alga was previously dispersed made it possible to obtain not only an enzymatic extract (polypeptide) incorporating substances active but also increased the solubilization of other constituents such as polyphenols for the preparation of cosmetic compositions. The species of algae used can also come from three branches: red algae (Rhodophyta), brown algae (Class Phaeophyceae in the Ochrophyta branch) and green algae (Chlorophyta). The process according to the invention has made it possible to obtain extracts useful in the cosmetic field of the brown macroalga Pylaiella littoralis belonging to the order Ectocarpales of which no species has yet been valued for its cosmetic properties. The text of the description that follows gives other examples of valued algae illustrating the three categories referred to above (brown, red and green).

The paste obtained is then taken up in water and treated with the protease as indicated previously in the context of the present invention.

Preferably, the alga which is treated in the process according to the invention is chosen from Pylaiella spp (genus), Calliblepharis Jubata (species), Bifurcaria spp (genus), Polysiphonia spp (genus) and / or Cladophora spp (genus) . That is to say, the source of algae used for the implementation of the method according to the invention may be a mixture of at least one of the species of algae mentioned above or belonging to the genera listed above. The alga treated according to the process according to the invention is advantageously selected from at least one of the following species: Pylaiella littoralis, Calliblepharis Jubata, Bifurcaria bifurcata, Polysiphonia elongata, and Cladophora rupestris.

The proteases of choice for carrying out the enzymatic treatment in the context of the process according to the invention are advantageously serine proteases, preferably subtilisin is used.

Preferably, the method according to the invention further comprises, after the action of the protease in the aqueous medium, a treatment step with at least one acid. The enzymatic extract may be contained in a filtrate. The filtrate is brought into contact with the solid residues (rinsing) at least once to extract the maximum of active substances trapped in the solid residues which are generally in the form of a paste. The acidification of the medium makes it possible to denature the enzyme. A nylon fabric can be used to separate the filtrate from the solid residues. The pH of the filtrate is preferably adjusted to 4.5 by adding 10% sodium hydroxide and then stored, for example, with sodium benzoate. The pH is then adjusted to between 4 and 5 (inclusive) by adding the necessary amount of an organic acid, such as citric acid. The filtrate is advantageously taken up on a press filter equipped with plates with pores (porosity) of 0.5 to 0.8 μηι, or even 0.6 to 0.7 μηι, diameter.

Between step b) and step c) of the process according to the invention as described above, 30 to 70% by weight of glycerin are advantageously added.

The present invention also relates to a method for treating solid residues recovered in the last stage of the process described above in the context of the invention, by taking up in water the solid residues obtained, for example in the form of a paste, and treating them by adding an acid to a pH of 1.5 to 2.5, then applying heating for at least 3 hours to at least 70 ° C, and finally adding a base to obtain a pH of 6-8 to obtain a polysaccharide extract after filtration. The paste and filtrate are separated and the paste is rinsed at least once with the filtrate. A nylon fabric can be used to effect this separation. The filtrate obtained comprises the polysaccharide extract. The filtrate is preserved for example with sodium benzoate. The pH is then adjusted to between 4 and 5 (inclusive) by adding the necessary amount of an organic acid, such as citric acid. The filtrate is advantageously taken up on a filter press fitted with plates having pores (porosity) of 0.5 to 0.8 μηι, or even 0.6 to 0.7 μηι, in diameter, to recover the expected polysaccharide extract at the outlet.

Polysaccharides are extracted under mild conditions without excess acid. Catalysis of the hydrolysis reaction occurs through the internal protons of the anionic polysaccharides. This reaction causes the size reduction of the polymers and facilitates extraction during neutralization.

Preferably, the aqueous suspension prepared in step b) is stirred at room temperature for at least 30 minutes, then a separation is carried out by filtration to obtain a paste and a cytoplasmic extract, only the paste is then treated according to the steps c ) to h). Thus, before the step using the protease, an extraction of the raw material in dehydrated form is carried out by an aqueous solution. Fresh seaweed after harvest or frozen can be used. The algae are optionally milled, but the use of algae that have undergone a simple cutting is preferred, the algae are advantageously pre-cut into small pieces before treatment. Extraction involves stirring the aqueous suspension comprising the algae at room temperature for at least 30 minutes. It is an osmotic shock to break up the cells of the pieces of algae suspended in the liquid. From the suspension, a filtrate and a paste which decants after stopping the stirring are obtained. Filtration on nylon cloth separates the pulp from the filtrate. The filtrate is brought into contact at least once on the paste by rinsing in order to recover the maximum of active substance. The filtrate is passed through a filter press fitted with plates enabling clarifying and then sterilizing filtration with pores 0.2 μm in diameter to obtain the cytoplasmic extract. The filtrate obtained is a cytoplasmic extract useful for the preparation of cosmetic compositions.

The present invention also relates to a process for extracting the insoluble fraction obtained at the end of the solid residue treatment process as described above, which is taken up, dehydrated and extracted with supercritical CO ₂ . Supercritical C0 ₂ extraction can be carried out with a mixture of capric and caprylic acid triglycerides as co-solvent and at high pressure. An oily extract is obtained which is advantageously promoted as an active ingredient of a cosmetic composition.

The present invention also relates to a composition obtained from at least one of the algae extracts used in or obtained according to the method as described above, comprising a molecule selected from: a carbohydrate or a derivative thereof, such as mannitol and a fructose oligosaccharide such as oligo-fructose; an amino acid or derivative, such as proline and taurine; a fatty acid or derivatives; a carotenoid; a diterpene; and a polyphenol, such as phlorotannin or vanillic acid or a derivative thereof.

In the context of the invention, the term carbohydrate includes oses (simple sugars or monosaccharides and their derivatives) and osides (hydrolysable sugars). Ooses include polyols such as mannitol, sorbitol, dulcitol and volemitol. Osseids include oligosides or oligosaccharides, polysaccharides or polysaccharides and glycosaccharides whose hydrolysis releases oses and non-osidic compounds.

Advantageously, the composition comprises a carbohydrate selected from starch floridéen preferably hydrolyzed, mannitol, floridoside (2-0- glycerol-D-galactopyranoside as described in the article by WE ^'IWER M. et al, Journal of Carbohydrate Chemistry, 2008, 27, pages 420-127), digeneaside (α-D-mannopyranosyl- (1 → 2) -D-glycerate, as described in the article by Ascencio

5. D. et al., Carbohydrate Research, 2006, Volume 341, Issue 5, Pages 677-682), an oligo-fructose, preferably hydrolysed laminarin (in English).

"Laminarin"), a galactan, preferably a hydrolysed sulfate galactan sulfate, or even a hydrolysed arabino-galactan sulfates, a preferably hydrolyzed fucan and an alginate preferably hydrolysed. Advantageously, the composition comprises an amino acid or derivative selected from proline, leucine, valine, taurine and glutamic acid,

Advantageously, the composition comprises a polyunsaturated fatty acid selected from an omega 3 fatty acid, preferably eicosapentaenoic acid (EPA), or selected from omega-type fatty acids.

6, preferably arachidonic acid.

Floridated starch, arabino-galactan sulphates, sulphate galactans, laminarines, alginates and fucans are advantageously hydrolysed. The hydrolysis of the polysaccharides makes it possible to reduce their size. The inventors have found that such hydrolysed polysaccharides have beneficial effects on the synthesis of collagen by the cells.

The present invention also relates to at least one cosmetic use of the composition as described above in the context of the invention, selected from: protection of cellular enzyme systems, stimulation of collagen synthesis and cytostimulation of cells. The present invention will be better understood on reading the experimental part which will follow, examples and figures of which are given for illustrative purposes only and can not be considered as limiting. EXPERIMENTAL PART

I. PREPARATION OF EXTRACTS

1.1) Preparation of a cytoplasmic extract from Pylaiella littoralis:

At the phylogenetic level, Pylaiella littoralis belongs to the phylum Ochrophytes, Phaeophyceae, Ectocarpales, and the family Acinetosporaceae.

The dehydrated algae (4% by weight), whole, are extracted with a mixture of water (38.4%) and glycerin (57.6%) (40/60) at ambient temperature for 30 minutes. The insoluble fraction is then separated from the filtrate on a nylon cloth. The filtrate is preserved for example with potassium sorbate. The pH is then adjusted to a value between 4 and 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.2 μηι to obtain the cytoplasmic extract.

Phlorotannins and mannitol were assayed in the cytoplasmic extract. Phlorotannins are polyphenols composed of a complex mixture of oligomers and phloroglucinol polymers.

The determination of the total polyphenols was carried out by the Folin method associated with an assay using the DMBA method. This makes it possible to calculate a ratio (Phlorotannin (Folin method) / Phlorotannin (DMBA method)). The higher this ratio, the more phlorotannin is polymerized (see Table 1):

The average phlorotannin content is 0.28 g / L (or even 0.05 to 0.35 g / L) of the extract. The extract also contains mannitol at an average content of 0.81 g / L (or even 0.5 to 1.5 g / L). 1.2) Preparation of an enzymatic extract from Pylaiella littoralis: The insoluble fraction obtained during the treatment described in part 1.1) is resuspended in water in order to obtain an aqueous solution of weight equivalent to that of the mixture starting point described in 1.1). A 10% sodium hydroxide solution is added and the pH is controlled to obtain a pH = 8. The mixture is heated to 60 ° C., then a protease, subtilisin (Alcalase® 2.4 L FG marketed by the company Novozyme®) is added to carry out the proteolysis and the mixture is stirred at this temperature and pH for 2 hours. The temperature is lowered to 40 ° C and the pH is adjusted to 2 with 10% hydrochloric acid. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is adjusted to a pH of 4.5 by adding 10% sodium hydroxide and then stored, for example with sodium benzoate. The pH is then adjusted to a value between 4 and 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the enzymatic extract.

The phlorotannins content is 0.1 1 g / L (or even 0.05 to 0.15 g / L) of the extract. Phlorotannins are specific to brown algae and are particularly involved in the defense mechanisms of algae against the deleterious effects of UV radiation by neutralizing the oxidizing molecules (activated species of oxygen, free radicals). The phlorotannins present in the algopeptides of Pylaiella littoralis are small in size. The determination of the total polyphenols by the Folin method associated with an assay using the DMBA method makes it possible to calculate a ratio (Phlorotannin (Folin method) / Phlorotannin (DMBA method)). The higher this ratio, the more phlorotannin is polymerized (see Table 4).

Table 4: Phlorotannin characteristics of algopeptides of Pylaiella littoralis

Analysis of the enzymatic extract showed the presence of glutamic acid at a content of 0.13 g / L (or even 0.1 to 0.2 g / L), measured by HPLC. 1.3) Preparation of a polysaccharide extract from Pylaiella littoralis

The insoluble fraction obtained at the end of part 1.2) is resuspended in water to obtain an aqueous solution of equivalent weight to that of the starting mixture described in 1.2). The pH is adjusted to 2 with 10% hydrochloric acid solution and stirred for 4 hours at 80 ° C. The suspension obtained is then neutralized to pH = 7 by adding a 10% sodium hydroxide solution. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is preserved, for example, with sodium benzoate. The pH is then adjusted to a value between 4 and 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the polysaccharide extract.

The insoluble fraction is dehydrated by lyophilization.

The majority polysaccharides of the extract were characterized and assayed after purification by ultrafiltration with a cut-off of 1000 Da. Their content in the extract is 2.6 g / L (or even a content of 2 to 3 g / L). Polysaccharides are mainly composed of neutral dares. Very few alginates (uronic acids) are present in the extract.

The major neutral ose is glucose from laminarine. Laminarin is a 1,3-linked beta-glucans with 1, 6 branches.

The other neutral oses come from fucans consisting mainly of xylose and fucose.

Fucans are sulfated polysaccharides. The molar ratio is 0.88 sulfate unit per fucose unit. The counterions of the anionic groups are mainly sodium and calcium and are in a smaller proportion of magnesium and potassium.

Self-hydrolysis reduced the weight average weight of the polysaccharide mixture to 33,000 g / mole, which makes an average polysaccharide of 133 units (sodium sulphate fucose unit).

Fucose was measured by colorimetry. The average fucose concentration is 0.22 g / L (the fucose content is between 0.1 to 0.35 g / L). Glucose was assayed by enzymatic assay. The average glucose concentration is 0.53 g / L (the glucose content is between 0.1 to 1 g / L).

I.4) Preparation of an oily extract from Pylaiella littoralis: The insoluble fraction obtained at the end of the treatment of part 1.3) is extracted with supercritical CO ₂ with a mixture of capric and caprylic acid triglycerides as co-solvent and operating at high pressure in a specific extractor. The oily extract obtained is diluted with this same oil to obtain a yield of 30% with respect to the raw material.

1.5) Material balance The following table summarizes the mass percentages obtained for the different aqueous extracts compared to the mass of treated dry algae:

Table 2: Distribution of matter

It is found that more than 50% of the algal dry matter was recovered in the 3 extracts.

1.6) Preparation of a cytoplasmic extract from Calliblepharis Jubata:

Calliblepharis jubata is a red alga belonging to the order Gigartinales and the family Cystocloniaceae (Classification: Rhodophyta / Floridaophyceae / Gigartinales / Cystocloniaceae). It consists of a foliose thallus, thick, red-brown color 10 to 30 cm long fixed by a set of branched crampons. The blade has spiny proliferations laterally. An original aspect of its reproduction methods is its ability to cut down thanks to its thorny proliferations, which detach in summer, drift in the form of balls between two waters and settle in autumn. It is a perennial seaweed that starts from its bases. It is located in the sub-littoral zone: that is to say at the bottom of the tidal sway zone.

The dehydrated algae (4% by weight), whole, are extracted with water (96%) at room temperature for 30 minutes. The insoluble fraction is then separated from the filtrate on a nylon cloth of mesh size of 500 μηι at 1 mm internal diameter. The filtrate is preserved with 0.5% sodium benzoate and its pH adjusted to a value of between 4 and 5 by addition of citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.2 μηι to obtain the cytoplasmic extract.

The cytoplasmic extract contains a floridoside content of 0.66 g / L (or even 0.40 to 1 g / L) determined by an enzymatic assay after hydrolysis. The floridoside which consists of galactose and glycerol has been characterized by 1 H NMR.

The extract also contains taurine in a content of 0.34 g / L (or even 0.25 to 0.45 g / L) determined by an HPLC assay.

I.7) Preparation of an enzymatic extract from Calliblepharis Jubata:

The insoluble fraction obtained during the treatment described in part I.6) is resuspended in water in order to obtain an aqueous solution of equivalent weight to that of the starting mixture described in 1.6). A 10% sodium hydroxide solution is added and the pH is adjusted to a value of 8. The mixture is heated to 60 ° C. and then a protease, subtilisin (Alcalase® 2.4 L FG marketed by the company, is added. Novozyme®) to carry out the proteolysis and is kept stirring at this temperature and this pH for 2 hours. The temperature is lowered to 40 ° C and the pH is adjusted to 2 with 10% hydrochloric acid. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is adjusted to a pH of 4.5 by addition of 10% sodium hydroxide and then preserved, for example with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the enzymatic extract.

1.8) Preparation of a polysaccharide extract from Calliblepharis Jubata:

The insoluble fraction obtained at the end of part 1.7) is resuspended in water in order to obtain an aqueous solution of equivalent weight to that of the starting mixture described in 1.7). The pH is adjusted to 2 with 10% hydrochloric acid solution and stirred for 3 hours at 80 ° C. The suspension obtained is then neutralized to pH = 7 by adding a 10% sodium hydroxide solution. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is preserved for example with sodium benzoate. The pH is then adjusted to a value of from 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the polysaccharide extract.

The insoluble fraction is dehydrated by lyophilization.

The extract comprises a mixture of sulphated galactans and floridated starch which are hydrolysed by the self-hydrolysis process.

Floridized starch is the so-called polysaccharide reserve of red algae. It is a 1,4-linked D-glucose polymer with alpha 1, 6 branches, whose structure is between that of amylopectin and that of glycogen. Floridized starch was determined after hydrolysis of the polysaccharides, reduction and methylation, and chromatographic analysis of the methylated derivatives. It represents 3% of the polysaccharide mixture. The polysaccharides represent 65% of the dry mass of the extract (average material of the extract = 1 .3%). They consist mainly of neutral oste, anhydro-galactose and sulphate.

Sulphated galactans are highly depolymerized. The molecular weight is 7600 g / mol, which corresponds to an average degree of polymerization of 26 units of sulfated galactose and anhydro-galactose type. The number of sulphate is important compared to the two main constituents, galactose and anhydro-galactose. The polysaccharide is strongly anionically charged and contains a significant proportion of counter ions, especially in calcium and magnesium form.

The enzymatic analysis reveals a content of 1 .7 g / L in galactose (or even 1 to 2.3 g / L), and a content of 0.4 g / L in glucose (or even 0.5 to 1 g / L).

1.9) Preparation of a cytoplasmic extract from Polysiphonia elongata:

Polysiphonia elongata is a red alga belonging to the Order Ceramiales (Rhodophyta / Floridaophyceae / Ceramiales / Rhodomelaceae). It forms tufts of bright red filaments, 20 to 30 cm high.

The dehydrated algae (4% by weight), whole, are extracted with water (96%) at room temperature for 30 minutes. The insoluble fraction is then separated from the filtrate on a nylon cloth of mesh size of 500 μηι at 1 mm internal diameter. The filtrate is preserved, for example with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.2 μηι to obtain the cytoplasmic extract.

Digeneas was assayed by enzymatic assay of mannose after hydrolysis. The digenase content is 0.81 g / L - even 0.5 to 1 .3 g / L. Beforehand, digenesis was identified in the extract by 1 H NMR with respect to the purified digenase spectrum.

Prolines were also measured at a level of 0.19 g / L - even 0.1 to 0.4 g / L - (HPLC) and 50 mg / L or even 25 to 70 mg / L - of mycosporin-like amino acids ( UV spectrometry).

1.10) Preparation of a polysaccharide extract from Polysiphonia elongata:

The insoluble fraction obtained after treatment in I.9) followed by a treatment to collect an enzymatic extract (not described) is resuspended in water to obtain an aqueous solution of weight equivalent to that of the mixture departure. The pH is adjusted to 2 with 10% hydrochloric acid solution and stirred for 3 hours at 80 ° C. The suspension obtained is then neutralized to pH = 7 by adding a 10% sodium hydroxide solution. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is preserved for example with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press fitted with plates with 0.65 μηι pores to obtain the polysaccharide extract.

The extraction process is a mild hydrolysis called "Auto-hydrolysis". Protons, catalyzing the hydrolysis of glycosidic bonds, come from the polysaccharide itself. This process promotes the solubilization and stability of polysaccharides by reducing their size.

The polysaccharide isolated in the extract is a sulfate galactan. It contains mainly neutral oses, sulphates and anhydro-galactose. The polysaccharides were purified and then characterized by ¹ H NMR. The neutral ose composition was determined after total hydrolysis, reduction, methylation and gas chromatography of the methyl derivatives. Sulfur and cations determined by elemental analysis and anhydro-galactose were assayed by colorimetry.

The major neutral ose is galactose. The glucose comes from floridin starch extracted together with sulfate galactans.

The major cation is calcium. Magnesium is also bound to the polysaccharide.

The contents determined in the extract for anhydro-galactose are 0.9 g / l (or even 0.6 to 1.1 g / l), and for galactose 2.0 g / l (or even 1.4 to 2.6 g). / L).

1.11) Preparation of a cytoplasmic extract from Cladophora rupestris:

Cladophora rupestris is a green alga of the family Cladophoraceae, Cladophorales, Ulvophyceae and Chlorophyta phylum.

This species forms very branched tufts up to 30 cm in length. The cell walls can become very thick which gives the thallus stiffness. It is abundant at mid-tide on sandy rocks.

The dehydrated algae (4% by weight), whole, are extracted with water (96%) at room temperature for 30 minutes. The insoluble fraction is then separated from the filtrate on a nylon cloth of mesh size of 500 μηι at 1 mm internal diameter. The filtrate is preserved, for example with sodium benzoate. The pH is then adjusted from 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press fitted with plates having pores 0.2 μm in diameter to obtain the cytoplasmic extract.

The extract comprises low molecular weight sugars, for example sucrose and oligosaccharides of fructose (oligo-fructose). Polyphenols were determined by the FOLIN method with reference to calibration with vanillic acid.

1.12) Preparation of an Enzymatic Extract from Cladophora rupestris: The insoluble fraction obtained during the treatment described in part 1.1 1) is resuspended in water in order to obtain an aqueous solution of equivalent weight to that of the starting mixture described in 1.1 (1). A 10% sodium hydroxide solution is added and the pH is controlled to obtain a pH = 8. The mixture is heated to 60 ° C., then a protease, subtilisin (Alcalase® 2.4 L FG marketed by the company Novozyme®) is added to carry out the proteolysis and the mixture is stirred at this temperature and pH for 2 hours. Allowed to warm to 40 ° C and adjust the pH to 2 with 10% hydrochloric acid. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is adjusted to a pH of 4.5 by adding 10% sodium hydroxide and then stored, for example with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the enzymatic extract.

1.13) Preparation of a polysaccharide extract from Cladophora rupestris:

The insoluble fraction obtained at the end of part 1.12) is resuspended in water to obtain an aqueous solution of weight equivalent to that of the starting mixture described in 1.12). The pH is adjusted to 2 with 10% hydrochloric acid solution and stirred for 4 hours at 80 ° C. The suspension obtained is then neutralized to pH = 7 by adding a 10% sodium hydroxide solution. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is preserved for example with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the polysaccharide extract.

The extract contains a mixture of sulfated arabino-galactans and starch. They are hydrolysed by an original process of self-hydrolysis.

Auto-hydrolysis reduced the weight average weight of the polysaccharide mixture to 28,000 g / mole, which makes an average polysaccharide of 173 units (galactose equivalent unit).

1.14) Preparation of a cytoplasmic extract from Bifurcaria bifurcata:

Bifurcaria bifurcata is a brown alga belonging to the family Sargassaceae, order Fucales, class Pheophyceae and branch Ochrophyta. It is a perennial seaweed. It can measure up to 40 cm long and is in the form of dichotomous cylindrical cords with a diameter of about 3 to 5 mm. It is located in the upper part of the sub-littoral stage and is very rarely emerged, except at the time of the large tidal coefficients. It is present higher on the foreshore in basins on rock faces (beaten mode) and characterizes a puddle in Bifurcaria (specific ecological enclave) located generally below the fucaceae. The presence of Bifurcaria bifurcata in cuvettes at higher levels on the foreshore exposes it to significant amplitudes of physicochemical conditions such as salt concentration and temperature. Bifurcaria bifurcata is therefore able to regulate intra-cellular osmotic pressure. Faced with strong oxidative stress, Bifurcaria bifurcata defends itself by synthesizing phlorotannins which are antioxidants.

The dehydrated algae (5% by weight), whole, are extracted with a mixture of water (57%) and glycerol (38%) at room temperature for 30 minutes. The insoluble fraction is then separated from the filtrate on a nylon cloth of mesh size of 500 μηι at 1 mm internal diameter. The filtrate is preserved, for example with potassium sorbate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.2 μηι to obtain the cytoplasmic extract.

The presence of phlorotannins, which are antioxidants, in a content of 0.23 g / L (or even 0.20 to 0.27 g / L) of the extract, has been identified. The extract also contains mannitol at a level of 1.3 g / L (or even 1.0 to 1.7 g / L).

1.15) Preparation of an enzymatic extract from Bifurcaria bifurcata:

The insoluble fraction obtained during the treatment described in part 1.14) is resuspended in water to obtain an aqueous solution of equivalent weight to that of the starting mixture described in 1.14). A 10% sodium hydroxide solution is added and the pH is controlled to obtain a pH = 8. The mixture is heated to 60 ° C., then a protease, subtilisin (Alcalase® 2.4 L FG marketed by the company Novozyme®) is added to carry out the proteolysis and the mixture is stirred at this temperature and pH for 2 hours. Allowed to warm to 40 ° C and adjust the pH to 2 with 10% hydrochloric acid. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is adjusted to a pH of 4.5 by adding 10% sodium hydroxide and then stored, for example, with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the enzymatic extract.

1.16) Preparation of a pegasaccharide extract from Bifurcaria bifurcata:

The insoluble fraction obtained at the end of part 1.15) is resuspended in water in order to obtain an aqueous solution of equivalent weight to that of the starting mixture described in 1.15). The pH is adjusted to 2 with 10% hydrochloric acid solution and stirred for 4 hours at 80 ° C. The suspension obtained is then neutralized to pH = 7 by adding a 10% sodium hydroxide solution. The insoluble fraction is separated from the filtrate on a nylon cloth. Then the insoluble fraction (paste) is rinsed with the filtrate. The filtrate is preserved for example with sodium benzoate. The pH is then adjusted to a value of 4 to 5 by adding the necessary amount of an organic acid, such as citric acid. The filtrate obtained is then filtered on a filter press equipped with plates having pores of 0.65 μηι to obtain the polysaccharide extract. The major hydrolysed polysaccharides of the extract were characterized and assayed after ultrafiltration purification with a cut-off of 1000 Da. Their content in the extract is 13.1 g / L (or even 10 to 17 g / L). These polysaccharides consist mainly of uronic acids and a minor fraction of neutral osteos. Uronic acids come from alginates and consist of mannuronic and guluronic acids.

The majority neutral oses are xylose and fucose that come from fucans. Fucans are sulfated polysaccharides. The molar ratio is 1.55 sulfate units per unit fucose.

The counter-ions of the anionic groups (sulphate of fucans and carboxylate of uronic acids salified) are mainly sodium and potassium and are in a smaller proportion of calcium and magnesium. These are very negatively charged polysaccharides with a cation exchange capacity of 367 meq / 100 g.

II. COSMETIC EFFECTIVENESS TESTS ON EXTRACTS

PROTOCOLS:

BCA method:

Evaluation of the concentration of proteins using the so-called "Bicinchoninic acid Protein Assay" kit (sold under the reference BCA1, by Sigma ™, France). Preparation of the BCA reagent by working by combining reagents A and B (50: 1). Mix the BCA reagent for work until the solution is uniform, and light green in color. Preparation of the BSA protein standards in order to obtain the following concentrations: 25 to 800 mg / ml (or 200 to 1000 mg / ml), addition of 25 μl of each sample. Add 200 μl of working reagent - BCA. Rotate the plate gently and incubate for 30 min at 37 ° C. Measure the absorbance at 570 nm with a Multiskan ® EX (Thermo Labsystems, France).

Test at WST1:

Plate culture of 32,000 cells / well / 80 μl HEK001 cells in their 96-well plate culture medium. Incubation at 37 ° C and 5% C0 ₂ . Discard the culture medium. Addition of test concentrations of test substances, positive controls and solvents (8 μΙ). Incubation at 37 ° C and 5% CO ₂ for 24 h. Heating at 45 ° C for 90 min in a hybridization oven. Incubation at 37 ° C and 5% C0 ₂ for 22h30. Addition of WST-1 reagent for cell proliferation in each well (10 μl / well). Incubation of the cells for a period of 30 minutes to 4 hours, at 37 ° C. and with 5% of C0 ₂ . Agitation of 96-well plates for 1 min with Multiskan® EX devices (Thermo Labsystems ™, France).

11.1) Test of the cosmetic efficacy of the cvtoplasmic extract of Pylaiella littoralis: Protection of keratinocytes against heat shock:

Stressed by heat, keratinocytes produce heat-protein protein (or HSP) proteins that combine with proteins to protect them from denaturation. Osmolytes are compatible solutes; that is to say, they accumulate in the cells without intoxicating them and while contributing to the maintenance of protein conformations. The following experiment demonstrates the protective activity of the cytoplasmic extract.

HEK001 keratinocytes are cultured on plate until confluent and then incubated for 24 hours with 0.05% and 0.1% by weight (non-cytotoxic doses demonstrated by the determination of cell viability by the WST1 test) of the cytoplasmic extract. in the culture medium before undergoing thermal shock at 45 ° C for 1 h 30 (or 2 h 30). After 22 h of incubation (thermal post-treatment), the proteins are extracted, assayed by the BCA method and the HSP70 assayed by an ELISA method.

Quantities of HSP70 normalized to proteins are determined. The tests were carried out in triplicate, three plates per concentration. The statistical treatment applied is a comparison of the means between the tests in the presence of the cytoplasmic extract and the test carried out with the nutrient medium. The test applied is Student's t-test (standardized statistical test to eliminate outliers and check if the results are significant). The averages are considered significantly different for a risk p <0.05.

At the two concentrations tested (0.05% and 0.1%), the amounts of HSP70 were increased by 34% and 55% relative to the stressed control in the presence of the culture medium at 45 ° C. The results are plotted in Diagram 1, whose numbers on the y-axis quantify HSP70 in ng per μg of protein (the two sticks on the right represent the quantities measured for the culture medium at 37 ° C. and 45 ° C.).

Conclusion: It has therefore been shown that the cytoplasmic extract of Pylaiella littoralis significantly increases the amount of HSP 70 produced by heat-stressed keratinocytes, thus contributing to the protection of cellular enzyme systems. The molecules identified in the cytoplasmic extract of which mannitol stimulate the defenses of skin cells. These molecules involved in the osmotic adjustment of cells stimulate the synthesis of chaperone proteins involved in the protection of biological macromolecules.

Inhibition of melanin synthesis:

B16-F10 murine melanocytes were implanted in their culture medium in 6-well plates. They are grown for 24 hours before adding the different solutions:

- The cytoplasmic extract of Pylaiella littoralis at 0.1% and 0.5%;

Kojic acid at 200 μg / ml as a positive control;

- The culture medium as a witness. The cells are incubated for 48 hours then the proteins and melanin are extracted and assayed. Melanin is determined spectrophotometrically at 405 nm and the melanin concentration is determined relative to a calibration curve of a standard melanin solution. The proteins, after reaction with the BCA, are assayed by colorimetry at 570 nm. The concentrations are determined with respect to a calibration line of a solution of bovine serum albumin. The results are expressed in μg melanin / μg protein and compared to the medium and kojic acid. The tests are carried out in triplicate. The test applied is Student's t-test. Averages are considered significantly different for p <0.05.

The amounts of melanin (normalized with respect to the proteins) are respectively decreased by 18% and 25% relative to the culture medium for 0.10% and 0.50% incorporation. At 0.5%, the decrease is equal to that of the positive control known for its inhibitory properties of melanin synthesis. The results are shown in Diagram 2, whose numbers on the y-axis quantify melanin in ng per μg of protein (the two bars on the right represent the quantities measured for kojic acid at 200 μg / ml and the culture medium (= Ref. )).

Conclusion: The cytoplasmic extract of Pylaiella littoralis inhibits melanin synthesis at both concentrations tested. The phlorotannins contained in the extract are at the origin of this property.

Antioxidant properties:

The reactive species of oxygen (superoxide radical, hydrogen peroxide and hydroxyl radical) are overproduced in the event of oxidative stress. These compounds are responsible for numerous oxidative damage to DNA, proteins and lipids. The lipid oxidation products are peroxyl radicals that propagate the lipid peroxidation chains. Molecules capable of capturing its peroxyl radicals thus stop these chain reactions. The most common test for measuring this property is the ORAC test.

The antioxidant capacity of the cytoplasmic extract of Pylaiella littoralis was determined by the ORAC test compared to Trolox which is a soluble derivative of vitamin E (diagram 3). Vitamin E is also the main sensor of peroxyl radicals at the cellular level.

The test measures the protection of the oxidation of fluorescein by a peroxyl radical (APPH). Fluorescein is assayed by fluorescence. It is therefore possible to follow in time for 1 hour, the oxidation of fluorescein with (curve with diamond points and with cross points) and without antioxidant molecules (curve with points in triangle).

The measurement of the area between the two curves correlated with the Trolox concentration makes it possible to calculate the antioxidant capacity of the extract tested.

The antioxidant capacity is expressed in Trolox equivalent in

(ORAC value), it is reported in Table 3. Table 3: ORAC value of the cvtoplasmic extract of Pylaiella littoralis

Safety tests have demonstrated, moreover, that the extract is entirely compatible with a cosmetic use.

II.2) Test of the cosmetic efficacy of the enzymatic extract of Pylaiella littoralis:

Algopeptides are produced by the biotechnological process of proteolysis. The enzyme solubilizes peptides by cleaving the peptide bonds between two amino acids within the protein, and the inventors have demonstrated that it promotes the extraction of other compounds such as phlorotannins. The active mixture stimulates lipolysis.

Stimulation of lipolysis by the enzymatic extract of Pylaiella littoralis:

Pre-adipocytes 3T3-L1 are cultured for 5 days. Their differentiation into adipocytes is initiated 3 days after the start of the culture by the addition of a lipogenic cocktail. The culture media are changed on day 5, that is to say on the fifth day, (post induction of differentiation) and replaced by fresh media containing the polypeptides at different concentrations, caffeine (control of lipolysis), the differentiation cocktail (control of lipogenesis) and the culture medium are added. This change of environment is renewed at J7. At day 10, the cells are stained with red oil and the lipids extracted with isopropanol and then assayed by spectrophotometry at 500 nm. The optical densities obtained are compared with the control (culture medium, indicated as Ref. In Diagram 4), with a positive control of lipolysis (caffeine at 0.1 mM) and with a positive control of lipogenesis (differentiation cocktail). . As a positive control of lipogenesis, the mixture solution of insulin, 3 'isobutyl 1 methylxanthine and dexamethasone was chosen, which induced up to 150-200% lipogenesis compared to an untreated control. As a positive control of lipolysis, caffeine was chosen which induces up to 40% of lipolysis compared to untreated control.

Two independent experiments were performed and each concentration was tested in duplicate. The statistical treatment applied is a comparison of the means between the tests in the presence of the algopeptides and the test carried out with the nutrient medium. The test applied is Student's t-test. Averages are considered significantly different for p <0.05.

As shown in diagram 4, at a concentration of 0.075%, the enzymatic extract of Pylaiella littoralis reduces the triglyceride level of the adipocytes of 29% relative to the culture medium. This effect is equivalent to a caffeine solution at 0.1 mM (-33%). In parallel, the antioxidant capacity of the enzymatic extract of Pylaiella littoralis was determined by the ORAC test, as explained previously for the cytoplasmic extract. The ORAC value of the enzymatic extract of Pylaillela is 2074 μηιοΐβ of trolox equivalent per L.

In addition, safety tests have shown that the extract is fully compatible with a cosmetic use.

11.3) Test of the cosmetic efficacy of the polysaccharide extract of Pylaiella littoralis:

Stimulation of the growth of keratinocytes in culture:

Human keratinocytes HEK001 were cultured for 96 h and then incubated for 48 h in the presence of 0.05% and 0.1% of the polysaccharide extract of Pylaiella littoralis prepared in I.3). Cell viability was determined by measurement of mitochondrial activity by the WST1 assay and compared to cells treated with the culture medium. WST1 is reduced in the mitochondria of viable soluble formazan derivative cells that absorb at 450 nm. The tests are carried out in triplicate. The test applied is Student's t-test. Averages are considered significantly different for p <0.05.

Diagram 5 shows the percentage of cytostimulation relative to the ordinate medium for different media compared to the reference medium (Ref.). The growth of keratinocytes is respectively increased by 20% and 41% relative to the culture medium for 0.10% and 0.05% incorporation, relative to the culture medium.

II.4) Test of the cosmetic efficacy of the cytoplasmic extract of Calliblepharis Jubata:

Protection of keratinocytes following heat stress:

Keratinocytes are cultured to confluence and then incubated for 24 hours with 0.1% and 0.25% (non-cytotoxic doses demonstrated by the determination of cell viability by the WST1 test) of the cytoplasmic extract in the culture medium. before undergoing a thermal shock at 45 ° C for 2 h 30. After 22 h 30 incubation (post-heat treatment), the proteins are extracted, assayed by the BCA method and the HSP70 assayed by an ELISA method.

Quantities of HSP70 normalized to proteins are determined. The tests were carried out in triplicate. The statistical treatment applied is a comparison of the means between the tests in the presence of cytoplasmic extract and the test carried out with the nutrient medium. The applied test is Student's t test. Averages are considered significantly different for p <0.05. The results are shown in the diagram 6 whose numbers on the y-axis quantify the HSP70 in ng per μg of protein (the two right-hand bars represent the quantities measured for the culture medium at 37 ° C. and 45 ° C.).

At the 2 concentrations tested, the amounts of HSP70 were increased by 47% and 70% relative to the stressed control in the presence of the culture medium. The cytoplasmic extract of Calliblepharis jubata significantly increases the amount of HSP70 produced by heat-stressed keratinocytes, thus contributing to the protection of cellular enzymatic systems.

11.5) Test of the cosmetic efficacy of the polysaccharide extract of

Calliblepharis Jubata:

Reduction of inflammation by the polysaccharides of Calliblepharis jubata:

Human keratinocytes (HEK001 line) are grown up to 70% confluency for 4 days. The cells are incubated with doses 0.1%, 0.25% and 1% of the polysaccharide extract prepared in I.8) for 24 hours before being treated with UV B radiation (32 mJ / cm 2 at 312 nm ). After irradiation, the cells are incubated 24h. After these 24 hours of culture, the cells are separated from the culture medium by centrifugation. IL-6 is assayed in the supernatant by an ELISA method. Proteins are extracted from the cell pellet and assayed by the BCA method against a BSA calibration curve. The results are expressed in μg of proteins and compared to those obtained on the culture medium. A positive Vitamin D3 control is also tested to validate the study. The tests are carried out in triplicate. The statistical average comparison test applied to the results is Student's t-test. Averages are considered significantly different for p <0.05.

The levels of IL6 expressed in ordinate and given in pg on the diagram 7, (cytokine involved in the inflammatory process) are related to the protein concentrations expressed in μg, it is found that they decrease proportionally to the content of polysaccharides. For a content of 1% of the extract, there is a decrease of 74% compared to the reference (Ref., Which is the medium with UV, shown by the right stick shown in black). For a content of 0.25% of the extract, there is a decrease of 61% compared to the reference, and for a content of 0.1% of the extract, there is a decrease of 48% compared to the reference. A control with vitamin D3 is shown for comparison, which only allows a decrease of 37%.

Conclusion: The levels of IL6, (cytokine involved in the inflammatory process) relative to the protein concentrations decrease proportionally to the content of polysaccharide extract Calliblepharis jubata (dose dependent effect). The polysaccharide extract of Calliblepharis jubata therefore has a soothing effect. Stimulation of collagen synthesis by the peivsaccharides of Calliblepharis jubata:

Human fibroblasts (line MRC-5) are cultured for 96 h (confluence) and then incubated for 48 h in the presence of 0.4% (non-cytotoxic dose) of the extract prepared in 1.8). The proteins are extracted and assayed by the BCA method. Their contents are expressed in μg / ml of BSA and compared with that of the control medium (duplicate). The total collagen is extracted and assayed by the sirius red method (colorimetry at 500 nm). The optical density is compared with that of the control medium (triplicate).

The ratio of the results of the two dosages expressed with respect to the nutrient medium is calculated. A positive control containing two growth factors (TGF3 and EGF) is tested in parallel to validate the study.

Diagram 8 shows the results obtained, it is measured on the ordinate that the amount of total collagen (normalized with respect to the proteins, that is to say that the ordinate expresses the ratio (collagen / proteins) / medium) is increased by 72% compared to the cells treated with the culture medium. This stimulation is equivalent to that caused by a mixture of two growth factors, TGF and EGF (Transformation Growth Factor and Epidermal Growth Factor).

Conclusion: The polysaccharide extract of Calliblepharis jubata stimulates the production of collagen by skin cells.

Safety tests have demonstrated, moreover, that the extract is entirely compatible with a cosmetic use. 11.6) Test of the cosmetic efficacy of the cytoplasmic extract of Polysiphonia Elongata:

Inversion of keratinocytes:

Human keratinocytes (line HEK001) were grown 24h and then incubated for 96h in the presence of 0.1% of the cytoplasmic extract of Polysiphonia elongata prepared in I.9). Cell viability was determined by measurement of mitochondrial activity by the WST1 assay and compared to cells treated with the culture medium. WST1 is reduced in the mitochondria of viable soluble formazan derivative cells that absorb at 450 nm. The tests are done in quadricat. The test applied is Student's t-test. Averages are considered significantly different for p <0.05.

Diagram 9 shows that the extract prepared in I.9) significantly stimulates the growth of keratinocytes since, in fact, a 40% increase in the number of cells relative to the control (Ref.) Was measured after 96 hours of culture. (the ordinate measuring% cytostimulation / reference medium).

Conclusion: There is therefore cytostimulation of skin cells in the presence of the cytoplasmic extract of Polysiphonia elongata. In parallel, tests have shown a stimulation of collagen synthesis and that the extract is entirely compatible with a cosmetic use. 11.7) Test of the cosmetic efficacy of polysaccharide extract of Polysiphonia Elongata:

Cytostimulation of keratinocytes by Polysiphonia elongata polysaccharides:

Human keratinocytes HEK001 were cultured for 96 h and then incubated for 48 h in the presence of 0.1% of the Polysiphonia Elongata extract prepared in 1.1 (1). Cell viability was determined by measurement of mitochondrial activity by the WST1 assay and compared to cells treated with the culture medium. WST1 is reduced in the mitochondria of viable cells in soluble formazan drift which absorbs at 450 nm. The tests are carried out in triplicate. The applied test is Student's t test. Averages are considered significantly different for p <0.05.

The results are shown in the diagram 10 (the ordinate measuring% cytostimulation / reference medium), there is an increase of 18% compared to the control (Ref.).

Conclusion: it is shown that the extract prepared in 1.1 (1) of Polysiphonia elongata stimulates the growth of keratinocytes.

Anti-inflammatory effect of Polysiphonia elongata polysaccharides:

Human keratinocytes (HEK001 line) are grown up to 70% confluency for 4 days. The cells are incubated with 0.1% of the extract prepared in 1.1 1) of Polysiphonia elongata for 24 hours, before being treated with UV B radiation (32 mJ / cm ² at 312 nm). After irradiation, the cells are incubated 24h. After these 24 hours of culture, the cells are separated from the culture medium by centrifugation. IL-6 is assayed in the supernatant by an ELISA method. Proteins are extracted from the cell pellet and assayed by the BCA method against a BSA calibration curve. The results are expressed in μg / μg of proteins and compared to those obtained on the culture medium. A positive Vitamin D3 control is also tested to validate the study. The tests are carried out in triplicate. The statistical average comparison test applied to the results is Student's t-test. Averages are considered significantly different for p <0.05.

The results are shown on the chart 1 1 (the ordinate measures NL6 in pg / mL / proteins in μg / mL), the polysaccharide extract of Polysiphonia elongata reduces the ratio ll-6 / protein 39%, compared to the control ( Ref.) Demonstrating an anti-inflammatory effect. As an indication, we see in the diagram the hatched hatch center corresponding to a treatment with vitamin D3, with a decrease of 37%. Conclusion: Polysiphonia elongata polysaccharide extract has an anti-inflammatory effect. Stimulation of the secretion of collagen by Polysiphonia elongata polysaccharides:

Human fibroblasts (line MRC-5) are cultured for 96h (confluence) then 48h incubates in the presence of 0.1% (non-cytotoxic dose) of the Polysiphonia elongata extract prepared in 1.1 (1). The proteins are extracted and assayed by the BCA method. Their contents are expressed in μg / ml of BSA and compared to that of the control medium (Ref - duplicate). The total collagen is extracted and assayed by the sirius red method (colorimetry at 500 nm). The optical density is compared with that of the control medium (Ref - triplicate). The ratio of the results of the two dosages expressed with respect to the nutrient medium is calculated. A positive control containing two growth factors (TGF b and EGF) is tested in parallel in order to validate the study.

Diagram 12 shows the results obtained, it shows on the ordinate the amount of total collagen (normalized with respect to the proteins, that is to say that the ordinate expresses the ratio (collagen / proteins) in percentage), and Polysiphonia elongata polysaccharide extract was found to stimulate collagen excretion by 41% compared with the control. By way of comparison, the positive control containing two growth factors (TGF β and EGF) stimulates it by 65%.

Conclusion: Polysiphonia elongata polysaccharide extract stimulates the excretion of collagen by the cells of the skin.

In parallel, tests have shown that the extract is entirely compatible with a cosmetic use.

II.8) Test of the cosmetic efficacy of the cytoplasmic extract of Cladophora rupestris:

Protection against thermal shock:

Stressed by heat, keratinocytes produce "Heat Shock Proteins" (HSP) or chaperone proteins that associate with proteins to protect them from denaturation. Osmolytes are compatible solutes; that is to say, they accumulate in the cells without intoxicating them and while contributing to the maintenance of protein conformations. The protective properties of the cytoplasmic extract of this alga were therefore tested.

Keratinocytes are cultured to confluence and then incubated for 24 hours with 0.05% and 0.1% and 0.25% (non-cytotoxic doses demonstrated by the determination of cell viability by the WST1 test) of the cytoplasmic extract in the culture medium before undergoing thermal shock at 45 ° C for 2 h 30 min. After 22 h of incubation (thermal post-treatment), the proteins are extracted, assayed by the BCA method and the HSP70 assayed by an ELISA method.

Quantities of HSP70 normalized to proteins are determined. The tests were carried out in triplicate. The statistical treatment applied is a comparison of the means between the tests in the presence of the cytoplasmic extract and the test carried out with the nutrient medium. The test applied is Student's t-test. Averages are considered significantly different for p <0.05. The results are shown in the diagram 13, whose numbers on the y-axis quantify the HSP70 in ng per μg of protein (the two sticks on the right represent the quantities measured for the culture medium at 37 ° C. and 45 ° C.).

At the 3 tested concentrations of 0.05%, 0.10 and 0.25%, the amounts of HSP70 were increased by 49%, 56% and 58%, respectively, relative to the stressed control in the presence of the culture medium.

Conclusion: The cytoplasmic extract of Cladophora rupestris significantly increases the amount of HSP70 produced by heat-stressed keratinocytes, thus contributing to the protection of cellular enzymatic systems.

Stimulation of melanin synthesis:

B16-F10 murine melanocytes are implanted in their culture medium in 6-well plates. They are grown for 24 hours before adding the different solutions:

the cytoplasmic extract of Cladophora rupestris at 0.25%;

the culture medium as a control.

The cells are incubated for 48 hours then the proteins and melanin are extracted and assayed. Melanin is determined spectrophotometrically at 405 nm and the melanin concentration is determined relative to a calibration curve of a standard melanin solution. The proteins, after reaction with the BCA, are assayed by colorimetry at 570 nm. The concentrations are determined with respect to a calibration line of a solution of bovine serum albumin. The results are expressed in μg melanin / g protein (ordered) and compared to the medium. The tests are carried out in triplicate. The test applied is Student's t-test. Averages are considered significantly different for p <0.05. The results are shown in Diagram 14.

Conclusion: The synthesis of melanin is stimulated by the addition of the cytoplasmic extract of Cladophora rupestris in the culture medium. The cytoplasmic extract of Cladophora rupestris, by increasing the synthesis of melanin contributes to the sun protection of the skin.

In parallel, tests have shown that the extract is entirely compatible with a cosmetic use. 11.9) Test of the cosmetic efficacy of the polysaccharide extract of Cladophora rupestris:

Reduction of inflammation by the polvsaccharides of Cladophora rupestris:

Human keratinocytes (HEK001 line) are grown up to 70% confluency for 4 days. The cells are incubated with doses of 0.10%, 0.25% and 1% of the polysaccharide extract prepared in 1.14) for 24 hours before being treated with UV B radiation (32 mJ / cm ² at 312 nm). .

After irradiation, the cells are incubated for 24 hours. After these 24 h of culture, the cells are separated from the culture medium by centrifugation. IL-6 is assayed in the supernatant by an ELISA method. Proteins are extracted from the cell pellet and assayed by the BCA method against a BSA calibration curve. The results are expressed in μg / μg of proteins and compared to those obtained on the culture medium. A positive Vitamin D3 control is also tested to validate the study. The tests are carried out in triplicate. The statistical average comparison test applied to the results is Student's t-test. Averages are considered significantly different for p <0.05.

The results are shown in the diagram (the ordinate measures NL6 in μg / g protein), the polysaccharide extract of Cladophora rupestris reduces the IL-6 / protein ratio to 39%, compared to the control (Ref.) demonstrating a soothing effect.

Conclusion: The polysaccharide extract of Cladophora rupestris shows a soothing effect on the cells of the skin.

11.10) Test of the cosmetic efficacy of the cytoplasmic extract of Bifurcaria bifurcata:

Protection against thermal shock:

Stressed by heat, keratinocytes produce "Heat Shock Proteins" (HSP) or chaperone proteins that associate with proteins to protect them from denaturation. Osmolytes are compatible solutes; that is to say, they accumulate in the cells without intoxicating them and while contributing to the maintenance of protein conformations.

Keratinocytes are cultured to confluence and then incubated for 24 hours with 0.1% and 0.25% (non-cytotoxic doses demonstrated by the determination of cell viability by the WST1 test) of the cytoplasmic extract in the culture medium. before undergoing a thermal shock at 45 ° C for 2 h 30. After 22 h 30 incubation (post-heat treatment), the proteins are extracted, assayed by the BCA method and the HSP70 assayed by an ELISA method. Quantities of HSP70 normalized to proteins are determined. The tests were carried out in triplicate. The statistical treatment applied is a comparison of the means between the tests in the presence of the cytoplasmic extract and the test carried out with the nutrient medium. The test applied is Student's t-test. Averages are considered significantly different for p <0.05. The results are shown in diagram 16 whose numbers on the y-axis quantify HSP70 in ng per μg of protein (the two sticks on the right represent the quantities measured for the culture medium at 37 ° C. and 45 ° C.).

At the 2 concentrations tested, the amounts of HSP70 were increased by 21% and 58% relative to the stressed control in the presence of the culture medium.

Conclusion: The cytoplasmic extract of Bifurcaria bifurcata significantly increases the amount of HSP70 produced by heat-stressed keratinocytes, thus contributing to the protection of cellular enzymatic systems.

Inhibition of melanin synthesis:

the cytoplasmic extract of Bifurcaria bifurcata at 0.25%;

kojic acid at 200 μg / ml as a positive control; the culture medium as a control.

The cells are incubated for 48 hours then the proteins and melanin are extracted and assayed. Melanin is determined spectrophotometrically at 405 nm and the melanin concentration is determined relative to a calibration curve of a standard melanin solution. The proteins, after reaction with the BCA, are assayed by colorimetry at 570 nm. The concentrations are determined with respect to a calibration line of a solution of bovine serum albumin. The results are expressed in μg of melanin and protein and compared to medium and kojic acid. The tests are carried out in triplicate. The test applied is Student's t-test. Averages are considered significantly different for p <0.05. The results are shown in Diagram 17.

The quantities of melanin (normalized with respect to the proteins: expressed in μg mg of proteins) are respectively decreased by 17% relative to the culture medium for a rate of 0.25% incorporation. The decrease is equal to that of the positive control (kojic acid) known for its inhibitory properties of melanin synthesis.

Conclusion: The cytoplasmic extract of Bifurcaria bifurcata inhibits melanin synthesis. This effect is attributed to the presence of phlorotannins (0.2 to 0.27 g / L) which are antioxidants. The extract also contains mannitol at a level of 1 to 1.7 g / L. Antioxidant properties

In addition, the antioxidant capacity of the cytoplasmic extract of Bifurcaria bifurcata was determined by the ORAC test compared to Trolox which is a soluble derivative of vitamin E. Vitamin E is also the main sensor of pyroxylated radicals at the cellular level.

The test measures the protection of the oxidation of fluorescein by a pyroxylated radical (APPH). Fluorescein is assayed by fluorescence. It is therefore possible to follow in time for 1 hour the oxidation of fluorescein with (curve with diamond points and with cross points) and without antioxidant molecules (curve with points in triangle).

The measurement of the area between the two curves correlated with the Trolox concentration makes it possible to calculate the antioxidant capacity of the extract tested (see diagram 18).

(value

Table 5 ORAC value of the cytoplasmic extract of Bifurcaria bifurcata

11.11) Test of the cosmetic efficacy of the polysaccharide extract of Bifurcaria bifurcata: Anti-inflammatory activity of the polysaccharide extract of Bifurcaria bifurcata:

Human keratinocytes (HEK001 line) are grown up to 70% confluency for 4 days. The cells are incubated with 0.01% and 0.05% polysaccharide extract of bifurcaria bifurcata for 24 h before being treated with UV B radiation (32 mJ / cm ² at 312 nm). After irradiation, the cells are incubated for 24 hours. After these 24 h of culture, the cells are separated from the culture medium by centrifugation.

IL-6 is assayed in the supernatant by an ELISA method. Proteins are extracted from the cell pellet and assayed by the BCA method against a BSA calibration curve. The results are expressed in μg of proteins and compared to those obtained on the culture medium. A positive Vitamin D3 control is also tested to validate the study. The tests are carried out in triplicate. The statistical average comparison test applied to the results is Student's t-test. Averages are considered significantly different for p <0.05. The results are shown in diagram 19.

The levels of IL6, (cytokine involved in the inflammatory process) relative to the protein concentrations decrease for the tests with enriched media polysaccharide extract of Bifurcaria bifurcata and vitamin D3 compared with those with the nutrient medium alone.

Conclusion: The polysaccharide extract of Bifurcaria bifurcata therefore has a soothing effect. This beneficial effect is explained by the fact that the polysaccharides identified in the extract have a protective effect by limiting the inflammation.

In addition enrichment of the nutrient medium with a polysaccharide extract of Bifurcaria bifurcata or vitamin D3 protects the keratinocytes against UV-B radiation (see diagram 20). Cell viability (WST1 test) is superior for tests with polysaccharide extract of Bifurcaria bifurcata or vitamin D3 compared to nutrient medium alone.

Claims

A method of fractionating an aqueous suspension of seaweed, comprising the successive steps of:

a) cutting said algae after harvesting and drying;

b) add 30 to 98% by weight of water;

c) add a base to adjust the pH of the solution between 7 and 10;

d) bringing the mixture obtained in step c) to a temperature of 60 ° C to 80 ° C;

A process for fractionating marine algae according to claim 1, characterized in that said alga is selected from at least one of the following categories: Pylaiella spp, Calliblepharis Jubata, Bifurcaria spp, Polysiphonia spp and Cladophora spp.

Process for fractionating marine algae according to one of Claims 1 to 2, characterized in that the protease is a serine protease.

A method of fractioning marine algae according to one of claims 1 to 3, further comprising, after the action of the protease in the aqueous medium, a treatment step with at least one acid.

Fractionation process according to one of claims 1 to 4, characterized in that between step b) and step c) is added 30 to 70% by weight of glycerin.

Fractionation process according to one of Claims 1 to 5, characterized in that the solid residues obtained as a result of the action of the protease are further taken up in water and treated with additives. an acid to reach a pH of 1.5 to 2.5, followed by heating for at least 3 hours at least 70 ° C, then adding a base to obtain a pH of 6 to 8, to obtain a polysaccharide extract after filtration. 7. The fractionation method according to one of claims 1 to 6, characterized in that the aqueous suspension prepared in step b) is stirred at room temperature for at least 30 minutes, and then a separation is carried out by filtration to obtain a paste and a cytoplasmic extract, only the paste is then treated according to steps c) to h).

8. Fractionation process according to claim 7, characterized in that the insoluble fraction obtained at the end of treatment is taken up, dehydrated and extracted with supercritical CO ₂ , to obtain an oily extract.

9. Composition obtained from at least one algae extract used in or obtained according to the method according to one of claims 1 to 8, comprising a molecule selected from: a carbohydrate or a derivative thereof, an amino acid or a derivative thereof, a fatty acid or a derivative thereof, a carotenoid, a diterpene and a polyphenol.

The composition of claim 9 wherein the composition comprises a carbohydrate selected from a florid starch, mannitol, floridoside, digeneaster, oligo-fructose, laminarin, galactan, fucan and alginate.

1 1. Composition according to one of claims 9 and 10, wherein the composition comprises an amino acid or derivative selected from proline, leucine, valine, taurine and glutamic acid

12. Composition according to one of claims 9 to 11, wherein the composition comprises a polyunsaturated fatty acid selected from an omega 3 and an omega 6.

13. Cosmetic use of one of the compositions according to claims 9 to 12 for the protection of cellular enzyme systems. Cosmetic use of one of the compositions according to claims 9 to 12 for stimulation of collagen synthesis.

Cosmetic use of one of the compositions according to claims 9 to 12 for the cytostimulation of cells.