FR3003762A1 - PREPARATION BASED ON ALGAE BIOMASS, ITS OBTAINING METHOD AND TOPICAL APPLICATIONS IN THE FIELDS OF THERMALISM AND COSMETOLOGY - Google Patents

Abstract

The present invention relates to a preparation based on at least one algae characterized in that it comprises, in a water-based medium, a mixture of: (a) at least one total algal biomass; and (b) at least one soluble copper salt in said medium.

Description

The present invention relates to a preparation based on at least one algae, to a method for obtaining it and to its applications. topical in the fields of hydrotherapy and cosmetology, applications related to the biological and / or pharmacological properties of this preparation.

Numerous works show the interest of cyanophyte algae or cyanobacteria naturally present in the sources and emergence of natural mineral waters. They are used in very different forms of extracts, purified fractions, alone or in combination with other compounds. They constitute an active element of thermal mud used for therapeutic purposes in the original station of mineral water. These sludges are based on a clay or loam substrate, soaked with mineral water and include algal and bacterial biocenosis. Examples of thermal mud include the thermal mud "TERDAX, the peloid of Dax" which established the reputation of the thermal establishments of the station of Dax which exploit it for rheumatology care within the framework of a Spa treatment.

French Patent No. 2,710,921 describes the process for preparing thermal muds, the sludges obtained and their applications, implemented at Dax. The algae used in the preparation of this preparation correspond to the algal and bacterial flora that develops naturally in the thermal waters of Dax, rich in therapeutically active ions, as soon as the water arrives the emergence in the presence of light. This specific flora is adapted to the high temperature of the water, its characteristic mineralization and the local conditions of natural lighting. French Patent No. 2,710,921 describes the species used in the composition of thermal mud combining cyanophycea (or cyanobacteria), chlorophyceae and diatoms. This patent also very precisely describes the culture process used to obtain these large-scale algae, their harvest and their mode of preservation. Thus, this process comprises the continuous streaming under natural and / or artificial light of the mineral water of Dax, constituting an algae culture nutrient medium, at a temperature suitable for the development thereof, on a culture support in inclined plane. The algae strains are seeded on this support or are provided by the water used, which is advantageously hyperthermal water whose temperature is greater than 45 ° C. The algae are harvested by scraping the surface of the culture, drained and then wrung out by centrifugation. For conservation purposes, these algae are frozen at a low temperature and then used for the preparation of the thermal mud "TERDAX, the peloid of Dax". This peloid, used in thermal establishments of the agglomeration dacquoise, is obtained after a particular maturation carried out, preferably, in anaerobiosis, in the presence of selected bacteria. This operation aims in particular to induce the formation of reduced sulfur compounds, which give the peloid a blackish color and a characteristic odor.

The Applicant has sought a new active for various topical applications, having pleasant organoleptic characteristics, especially on the olfactory plane, without losing the effectiveness of using the total thermal flora. The objective was therefore to use, not an algae extract, but the totum of the algae or total algal biomass comprising all the flora without eliminating by fractionation or separation of the membranes or insoluble cellular organelles and presenting all the properties of the algal flora whose effects are recognized.

The Applicant's research led her to discover unexpected properties of the combination of the totum of at least one algae with at least one soluble salt of copper, advantageously at a concentration chosen in order to increase the activity of each of the two components, the properties of this combination of total algal biomass with a soluble copper salt can then be used in conventional applications, namely in the pharmaceutical field, in particular in hydrotherapy for care in rheumatology, and in the paramedical and dermo-cosmetological fields. The tests carried out by the Applicant have shown, on the one hand, a perfect acceptability of the product and a very clear improvement of the approval of the user, and, on the other hand, a very advantageous efficiency of the new association in the field. all the applications mentioned above. French Patent Application No. 2,849,596 discloses a composition containing, in a physiologically acceptable medium, at least one u-hydroxy acid, at least one copper salt, at least one zinc salt, at least one alga and at least one haloalkynyl carbamate. This mandatory combination of four assets with complementary effects is intended for the treatment of oily skin and acne by keratolytic and antiseptic actions. The algae extract used is a cytosolic extract. The preparation according to the invention differs, inter alia, from this known composition by the combination of only two active constituents and by the use of a total algal biomass and not of an extract. The present invention therefore relates to a preparation based on at least one algae characterized in that it comprises, in a water-based medium, a mixture of: (a) at least one total algal biomass; and (b) at least one soluble copper salt in said medium. The algae (s) of the total algal biomass (s) are advantageously thermophilic algae 15 selected from marine algae, freshwater algae or algae selected by the specific temperature of the mineral waters. In particular, the algae of the total algal biomass (s) (a) belong to the following families: Cyanophyceae or Cyanobacteria, in particular of the order Chroococcales, such as Chroococcus minutus and Synechococcus elongatus, or of the order Oscillatoriales, such as Oscillatoria formosa, Oscillatoria germinata, Oscillatoria granulata, Phormidium ambigum, Phormidium autumnale, Phormidium corium, Phormidium luridum, Phormidium held, Phormidium uncinatum, Phormidium valderianum, Anabaena sp., Anabaena constricta, Anabaena variabilis, Nostoc sp., Cylindrospermum stagnale Mastigocladus laminosus, Mastigocladus laminosus forma anabaenoides, Stigonema sp., Calothrix thermalis; Chlorophytae, especially of the order Volvocales, of the Ulothrical order, such as Hormidium crassum, or of the order Chloroccales, such as Scenedesmus quadricauda, Scenedesmus acutiformis, Scenedesmus obliquus, Ankistrodesmus falcatus or Micractinium pusillum; Diatoms, in particular of the Central Order, such as Cyclotella sp. or Nalosira varians or order Pennales, such as Nitzschia palea, Nitzschia thermalis, Pinnularia interrupta forma minutissima, Hantzchia amphioxys, Amphora hosatica.

In a very particularly preferred manner, the algae of the total algal biomass (s) (a) can be chosen from the algae of the algal flora that naturally grows in the thermal waters of Dax when the waters arrive at emergence. in the presence of light, and in particular the algae of the mineral water of Dax, obtained by continuous streaming under natural and / or artificial light of the mineral water of Dax, at the temperature of development of said algae, on a culture support in inclined plane; culturing strains of said algae provided by said mineral water of Dax and / or seeded on said support; harvesting said algae by scraping the surface of the culture, dewatering and spinning by centrifugation. In the preparation according to the invention, the total algal biomass or biomasses (a) may be in a form which is free of a natural interfering bacterial flora and which is micro-milled in particular at an average particle size of not more than 50 μm, in particular not exceeding 5 pm, being in particular 1 to 5 pm.

The total algal biomass (s) may be in the form of a suspension in a vehicle stabilizing the biomass or biomasses, forming part of the said aqueous-based medium, and advantageously at least partially solubilising the salt or salts thereof. copper (b), said vehicle being in particular glycerol. The copper salt (s) (b) may be selected from non-toxic salts of copper and organic acids, such as copper acetate, copper butyrate, copper citrate, copper tartrate, copper salicylate, copper glycinate, copper lactate or copper gluconate, especially copper acetate and copper gluconate, and mineral acids, such as copper chloride, copper bromide , copper nitrate or copper sulphate. Among the copper salts, copper acetate and copper gluconate can be mentioned in particular because of their local tolerance and the absence of topical irritant action. The copper salt (s) (b) may also be associated with at least one acid, such as lactic acid, in an amount to adjust the pH of the composition to less than 7 and prevent the formation of insoluble copper derivatives in an alkaline medium. Also, the total algal biomass (s) may be suspended in the aqueous medium at 5 to 50% by weight based on the mixture (a) + (b) + aqueous medium, and the copper salt (s) (b) may be present in an amount of 0.05 to 10 g per 100 g of the mixture (a) + (b) + aqueous medium. The maximum concentration of the copper salt (s) is conditioned by the limiting solubility of this salt or salts in the aqueous-based medium. It is specified here that, by means of aqueous medium, is meant the medium in which the total algal mass or masses are in suspension and which is constituted by water or which comprises water, said one or more copper salts being dissolved in said medium, in particular in the water constituting or included in said medium.

According to a particular embodiment of the preparation according to the invention, the aqueous-based medium contains water and glycerol, and the glycerol represents 30 to 85% by weight, in particular 60 to 80% by weight, of said water-based medium.

The preparation according to the invention may especially be in the form of a stable suspension, of a deep green color, of a density of 1.08 to 1.23, in particular of 1.16 to 1.22, and of a pH of 4.5 to 6.5, especially 5 to 6.

The present invention also relates to a process for obtaining the preparation as defined below, characterized in that at least one soluble copper salt in a water-based medium is added to at least one total algal mass. in suspension in said aqueous-based medium. The total algal biomass (s) can be prepared by conducting the following steps: (1) continuous streaming under natural and / or artificial light of the mineral water of Dax, at the temperature of development of said algae, on a culture support in inclined plane ; (2) cultivation of strains of said algae provided by said mineral water of Dax and / or seeded on said support; (3) harvesting said algae by scraping the culture surface, draining and spinning by centrifugation; (4) decontaminating and deodorizing said algae under conditions maintaining the activity of the total biomass; (5) micromilling and micronization of the total algal biomass or biomasses thus obtained up to a particle size of not more than 5 μm. The decontaminations and deodorization (point 4 above) can be carried out by contacting the harvested, drained and dewatered algae with a decontaminating agent consisting of hydrogen peroxide. The micromilling (point 5 above) of the total algal biomass (s) may be carried out in the presence of a vehicle stabilizing said total algal biomass (s) and used in proportions of between 15% and 85% by weight of said one or more total algal biomasses, said vehicle entering the composition of said aqueous-based medium, and consisting in particular of glycerol. The copper salt (s) (b) can be added to the total algal biomass (s) (a) before or after micronization, at a rate of 0.05 to 10 g of copper salt (s) (b) per 100 grams of the mixture (a) + (b) + water-based medium, preferably with pH adjustment to less than 7 by addition of an acid such as lactic acid. The present invention also relates to the topical applications of the preparation as defined above or prepared by the method as defined above in hydrotherapy and cosmetology. The present invention also relates to a composition for topical use comprising the preparation as defined above or prepared by the method as defined above.

According to a first embodiment, the composition for topical use according to the invention consists of a mud bath or a mud / wrap treatment, comprising, apart from said preparation, a vehicle facilitating the application of the latter on the surface. skin, for example composed of at least one clay substrate such as kaolin and the peloid of Dax and / or at least one of paraffin, lanovaseline, methylcellulose, hydroxymethylcellulose, carbomethylcellulose and or wheat starch or corn starch and / or water, especially thermal water of Dax. According to a second embodiment, the topical composition according to the invention consists of a cosmetological composition, such as a mask for the face, a base for a mask for the face, a care cream, in particular a revitalizing care cream, and a body milk, comprising , apart from said preparation, a suitable vehicle for applications in cosmetology. A mask may comprise, as a carrier, at least one clay substrate such as kaolin and Dax peloid; and, where appropriate, sorbitol and / or wheat starch and / or corn starch. A care cream, particularly a conditioning skincare cream, or a body milk may be in the form of an emulsion which comprises an oil phase and an aqueous phase, for example 22 to 30 parts by weight of oily phase and 78 to 70 parts by weight of aqueous phase per 100 parts by weight of oily phase + aqueous phase, and wherein said preparation has been incorporated, after formation of the emulsion, a concentration of between 0.05 and 5% by weight preferably between 0.1 and 2% by weight of said emulsion.

The invention also relates to the applications of the preparation according to the invention obtained by the process according to the invention as an active ingredient in pharmaceutical preparations for humans and animals, in particular against skin and / or joint aging, to treat or prevent rheumatism, or para-medical or dermo-cosmetological preparations, as an anti-aging skin agent. The preparation according to the invention is a natural product obtained by a preparation process using mechanical treatments and respecting the environment; it does not contain synthetic antiseptics. It brings together all the constitutive elements of the algal biomass in the native form in the proportions defined by nature and having the synergy that exists between the recognized pharmacological properties, in particular: membrane polysaccharides and mucilages (alginates, carrageenans, for example) as well as other related substances used for their anti-inflammatory, anti-thrombin or anticancer action: - water-soluble fraction rich in active constituents: antiradicals, cytostimulants. Note the presence of cyanobacteria-specific phycocyanins which are present in their entirety; liposoluble fraction, consisting mainly of chlorophyll pigments, phaeophytins, carotenoids, tocotrienols whose antioxidant, anti-inflammatory and antiradical properties are well known. All these properties therefore justify the use of this asset particularly in the field of rheumatology and dermo-cosmetology, as H treatment of age-related changes and in the fight against the effects of aging. Recall also that copper and its salts have antiseptic properties, algicides, fungicides and spermicides. They are active constituents of intrauterine devices. The copper salts have an astringent action on the mucous membranes. Copper sulphate is traditionally used, combined with zinc sulphate, in "Dalibour waters" as skin protector in the treatment of irritations. Oral copper is used in oligotherapy. Copper gluconate Oligosol is prescribed in oral (or even sublingual) ampoules as a field modifier, particularly during infectious and viral conditions, influenza conditions and inflammatory rheumatic diseases (Vidal). The following Examples illustrate the present invention without however limiting its scope.

EXAMPLE 1 General Procedure for Obtaining Total Algal Biomass This example describes the general conditions for obtaining a total algal biomass based on thermal algae from Dax in the form of a suspension in glycerol. also called "Dax Thermal Algae Glycerin". (1) Runoff of the mineral water of Dax The natural mineral water of Dax constituting the nutrient medium of culture is continuously trickled off onto a culture support in inclined plane, under natural and / or artificial light. temperature above 42 ° C, between 42 and 53 ° C. (2) Cultivation of strains The strains of the algae brought by said mineral water of Dax (spontaneous development) are cultivated. (3) Harvesting strains The algae are harvested by scraping the surface of the culture, drained and wrung by centrifugation.

For storage purposes, these algae are frozen and stored at a low temperature. (4) Contaminated decontamination and deodorization of algae The use of such an open culture system, as described above, has a disadvantage: the presence of an interfering flora accompanying the expected biomass. This natural bacterial flora is most often eliminated during the successive phases of known algae treatments resulting in a purified extract. These known treatments, usually consisting of sterilizing filtration or ultrafiltration, make it possible to eliminate the germs of contamination and to obtain a sterile fraction. In return, these diverse and varied extracts, which are described in the literature or exploited, can not claim the activity of the total biomass, since they do not contain all the insoluble fractions of the total algae, in particular the cellular organelles, the membranes to which the active biological compounds are bound: chlorophyll, phaeophytins, phycocyanines, phycoerythrins and mucilages, in natural combination with each other. According to the invention, it was necessary to preserve the totum of the algal biomass, but to reduce or eliminate the unwanted accompanying flora of the algae obtained, it was however necessary to carry out a decontamination phase, which should be a decontamination. For this purpose, various contact decontamination agents may be added: ethyl alcohol and / or isopropyl alcohol, active chlorine, antiseptics, sulfites. The preferred choice was, however, an agent which makes it possible to obtain, after treatment, an almost total sterile and odorless total algal biomass without any alteration of the membranes, nor any discolouration of the pigments (chlorophyll in particular) and without antiseptic residue. capable of reducing the "natural, chemical preservative-free" nature of the new active ingredient according to the invention. This agent is hydrogen peroxide, which has been used at a selected concentration and for a selected time in order to obtain optimum efficiency without alteration or degradation of the biomass. The decontamination solution is a dilution of hydrogen peroxide such that its activity expressed in volumes of oxygen is between 1.5 and 6 volumes. The duration of contact is generally between 1 and 24 hours. Any residual traces of active oxygen are eliminated by simple rinsing with Dax mineral water, sterilized by filtration on a membrane of 0.2 μm pore size. (5) Micromilling and micronization The decontaminated biomass is diluted in a vehicle chosen for its good tolerance in topical use and for its solvent and stabilizing effect vis-à-vis the constituents of the algal biomass. This diluent is glycerol, a natural substance of vegetable origin, used in the proportions between 30 and 85% of the weight of the biomass. It is added to the decontaminated biomass so as to obtain a fluid, homogeneous and stable mass. The suspension of total algae is ground using a mixer and then micronized. Among the various micronization processes conventionally used, the ultrasonic treatment has been retained which makes it possible both to break the membranes of the cell walls and to homogenize the suspension without causing denaturation or degradation of the biological constituents. However, this process requires operating in a refrigerated circuit in order to avoid the possible deleterious effects of an excessive rise in temperature above 40-45 ° C.

Thus, the suspension obtained consists of particles whose diameter does not exceed 3 to 5 pm. The addition of copper salt is carried out at the end of this micronization operation.

Example 2: Obtaining 10 kg of Dax Thermal Algae Glyceride 10 kg of a total glycerin of thermal algae from Dax were produced by proceeding as follows: Streaming continuously under natural light and / or or artificial 5 Dax natural mineral water at a temperature between 42 and 53 ° C on a sloping culture support. The mineral water of Dax constituted the nutritive medium of the strains of algae sown on this support or brought by the water used. Algae were harvested by scraping the culture surface. 2.2 kg of thermal algae biomass from Dax were recovered and placed in a sieve with a mesh size of 0.5 mm and washed extensively with natural Dax mineral water (about 20 to 25 liters). . The biomass was drained and squeezed by gentle pressure. This washed biomass was placed in a tank equipped with a lid and an agitator. A decontamination solution consisting of a solution of hydrogen peroxide titrating between 3 and 6 volumes was added to the vessel and the contact was maintained at room temperature, with gentle stirring, for a time sufficient to obtain decontamination. gentle deodorization, avoiding any alteration, including discoloration, of the total algal biomass. This duration was between 1 and 24 hours. The presence of a foam on the surface of the algal mass indicated the release of active oxygen in the form of gaseous bubbles and showed the activity of this treatment. At the end of this operation, the total algae biomass was collected on a sterilized sieve, with a mesh size of 0.2 mm, rinsed rapidly with Dax mineral water filtered through a sterilizing membrane (approx. 4 to 5 liters of mineral water) then drained and wrung out. At this stage, the algal biomass could be stored after freezing at low temperature or subjected to micromilling. All of these operations and the final microbrush have been carried out in compliance with the rules of hygiene and asepsis of common use for the skilled person. The micromilling was carried out in the following manner: 2 kg of total algae biomass obtained at the end of this decontamination-deodorizing operation 15 made and therefore almost sterile, were diluted in 8 kg of glycerol of vegetable origin. This suspension was cold-milled using a mixer and then subjected to the action of ultrasound under conditions making it possible finally to obtain particles whose diameter did not exceed 3 to 5 μm, avoiding any heating of the mass, ie keeping the temperature below 40-45 ° C. It should be noted that these results can be obtained at the laboratory level with a Helscher 25 UP 400S apparatus which makes it possible to prepare batches of 100 g of product, and at the pilot and industrial level, with a device combining the action of ultrasound and pressure. The UP 1000 type equipment, of the same brand, makes it possible to recirculate quantities of the order of 2 to 4 kg of product. The cooling of the circuit makes it possible to limit the heating of the mass.

EXAMPLE 3 Association of Copper Gluconate with Total Glycerate of Dax Thermal Algae to Form a Preparation of the Invention After micronization, copper gluconate was combined with the total glycerin of algae obtained in Example 2. It may be noted that this association could equally well have been achieved during micronization. Copper gluconate has been favored among the various copper salts that can be envisaged, because of the good results of the biological activity tests described below, and has been used at the concentration of 2 grams of copper gluconate in 100 grams of glycerol. As a precaution, the copper salt has been associated with lactic acid in order to adjust the pH of the combination to a value of less than 7 so as to stabilize the suspension and to avoid the formation of insoluble copper derivatives. alkaline medium. The preparation of the invention thus obtained had the following characteristics: appearance: stable suspension of a deep green color particle size: their diameter does not exceed 5 μm density: close to 1.2 pH: diluted In distilled water, in the vicinity of 4.5-5.5. Example 4 Action of the Preparation According to the Invention in the Prevention and Treatment of Tissue and Biological Changes The present example demonstrates the action of the preparation according to the invention in the prevention and treatment of tissue and biological modifications, especially at the level of the connective tissue tissue matrix. The aging of the body leads to inevitable changes, particularly affecting the connective tissue, a ubiquitous structure of tissue support. It affects especially the joints and the skin. This aging is manifested by pain, a decrease in motor function, and, at the level of the skin, by a decrease in the tone of the dermis, a loss of skin elasticity, the formation of wrinkles and the impasto of the face. A: The preparation according to the invention exhibits an inhibitory effect on the activity of the metalloprotease MMP-9. It appears that one of the common parameters determining all these manifestations is the decrease in collagen synthesis and the reduction biological activity of chondrocytes, located in cartilage, and fibroblasts, located in the dermis. The Applicant has been able to demonstrate that the preparation according to the invention makes it possible to reinforce the protection of the tissue matrix constituted by collagen, by inhibiting the collagenase activity. The biochemical test used consisted of analyzing the MMP-9 activity, a metalloprotease of human origin, according to the protocol below. Al-Biochemical Analysis of the MMP-9 Activity The compounds and their mixtures as well as the 0-phenanthroline reference were pre-incubated in the presence of MMP-9 for 10 minutes on ice, and then a fluorescent substrate (Mca-Pro- Leu-Gly-Leu-Dpa-Ala-Arg-NH2) was added. The plates were then incubated for 2 hours at 37 ° C. A control of the compound in the presence of the enzyme-free reaction medium (measurement of the autofluorescence) as well as a control of the possible absorption of the fluorescence by the compound ("quenching") were carried out in parallel. All the conditions were realized in n = 3 except for the controls which were carried out in n = 2. At the end of the incubation, the fluorescence emitted, as a result of substrate degradation, was measured by fluorimetry at 328 nm and 393 nm on SpectroMax Gemini, Molecular Devices. The fluorescence values presented in Table 1 below correspond to the measured and corrected value of the fluorescence of the compound alone. The results are expressed as percent inhibition. It should be noted that in case of quenching, this effect is taken into account in the interpretation of the results. 20 A2 - Data Processing The raw data has been transferred and processed by Microsoft Excel. Intergroup comparisons were performed using the unpaired bilateral Student T test. Statistical analyzes can be interpreted if n is greater than or equal to 5. However, for n less than 5, the calculated data are provided for information purposes only. The formulas used are as follows: (Control Medium + UV - Value) x 100 Percentage of Protection - Average Control + UV - Control Medium - UV 100 - (Value x 100) Percentage Inhibition - Mean of Control Standard Error of Mean : esm = standard deviation (Sd) / n 1/2 The standard error of the mean (esm) represents the deviation of the sample mean from the average of the true population. The esm is calculated by dividing the Sd by the square root of the sample size. Percent viability: viability% = (compound OD / control OD) x 100 A3 - Results of the effect on the enzymatic activity of MMP -9 The O-phenanthroline reference, tested at 0.1 and 1 mM, significantly inhibited the enzymatic activity of MMP-9. This result was expected and validate the test. The compounds - total algal biomass and copper gluconate - tested at 0.4 and 2% (w / w) significantly inhibited the activity of MMP-9. A clear inhibition of the activity of the enzyme MMP-9 is also observed with the combination of the compounds - total algal biomass + copper gluconate. In this case, the inhibitory effect is greater than that observed with the compounds tested alone, suggesting a potential additive effect between these two compounds. The results are shown in Table 1 below. 21 Table 1: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE ACTIVITY OF MMP-9 Treatment Basic Data Standardized Data Quenching Compound-tested Noise Concentration MMP-9 background MMP-Average esm % esm Pn) Inhibition esm pn) Equivalent MMP-9 #% (Equivalent 9 # _9 Indicator Indicator MMP-9) (UA) (UA) (UA) (UA) (AU) (%) (%) (%) ( UA) Negative Control 705 02 (Enzyme 731 752 02 1 1 0 0 *** 100 0 *** Inactive) 756 4 12867 12115 Indicator 12552 752 11800 12052 131 100 1 - 0 1 12052 0 12992 12240 8645 7805 0.1 mM 7801 840 6961 7473 259 62 2 *** 38 2 *** 12734 0 0-phen-8492 7652 thrombin 1399 12 1 mM 1373 1387 02 15 10 0 0 *** 100 0 *** 9889 18 1420 34 22 Table 1 : EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE ACTIVITY OF MMP-9 (continued 1) Processing Basic Data Standardized Data Quenching Compound Test Noise Concentration MMP-9 Background MMP- Medium m4p_9 esm% esm len) Inhibition esm pn) Equivalent MMP-9 #% (Equivalent 9 # Indicator Lamp MMP-9) (UA) (UA) (UA) (UA) (AU) (%) (%) (%) (AU) 13236 12000 0.08 % 13080 1236 11844 11931 46 99 0 ns 1 0 ns 12469 0 13185 11949 12744 10559 BIOMASS ALGALE 0.4% 12484 2185 10299 10432 75 87 1 *** 13 1 *** 11652 3 TOTAL 12625 10439 10911 6732 2% 10813 4179 6634 6726 52 56 0 *** 44 0 *** 8717 28 10992 6813 23 Table 1: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE ACTIVITY OF MMP-9 (continued 2 ) Processing Master Data Standard Data Quenching Compound Test Noise Concentration MMP-9 Background MMP-Average Esm% Esm len) Inhibition esm pn) Equivalent MMP-9 #% (Equivalent 9 # _9 Control Indicator MMP-9) (UA) ( UA) (AU) (AU) (AU) (%) (%) (%) (AU) 13290 12408 0.08% 12687 882 11805 12016 196 100 2 ns 0 2 ns 12863 0 12719 11837 10690 9827 GLUCONATE *** *** COPPER 0.4% 10507 863 9644 9782 70 81 1 19 1 13218 0 10737 9874 6278 5414 2% 5824 864 4960 5074 173 42 1 *** 58 1 *** 12606 0 5710 4846 24 Table 1: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE ACTIVITY OF MMP-9 (continued 3) Treatment Basic Data Standardized Data Quenching Tested Compound Concentration MMP-9 Noise of MMP- Average esm% esm n) Inhibition esm pn) Equivalent MMP-9 #% (UA) background 9 # _9 (UA) Control (%) (%) (%) (UA) Control (Equivalent (UA) (UA) ) MMP-9) (UA) MIXTURE OF TWO CONSTITUENTS (50/50) 0.08% / 0.08% 12729 1216 11513 11168 176 93 1 7 1 12204 0 12151 10934 12273 11057 0'4% / 0.4% 10660 2001 8659 8424 140 70 1 *** 30 1 *** 11327 6 10176 8175 10440 8439 2% / 2% 6064 2553 3510 3299 106 27 1 *** 73 1 *** 9847 18 5726 3173 5769 3215 # the noise background of products alone (interference control) has been deducted from raw values (1): Threshold of statistical significance ns:> 0.05, Not significant *: 0.01 to 0.05, significant **: 0.001 to 0.01, Very significant ***: <0.001, Extremely significant 2: Extrapolated value B - The preparation according to the invention has a significant anti-radical effect. Another common parameter determining the whole of the manifestations related to age is the accumulation of free radicals, consecutive to the formation of reactive oxygen species that induce "oxidative stress". The Applicant has been able to demonstrate that the combination according to the invention of the total algal biomass with a soluble salt of copper has a significant antiradical activity. The cell test used consisted of studying the production of hydrogen peroxide in Jurkat cells irradiated with ultraviolet light (UV A and UV B). B1 - Production of H202 flow cytometry Treatment and analysis by Jurkat cells were incubated for 45 minutes in the presence or absence (control) of test or reference compounds (BHA, 100 μM) and in the presence of the probe specific for the formation of H202 (DHR, 10-6 M) whose fluorescence intensity is proportional to the formation of H2O2. Probeless control was also performed in parallel and all conditions were realized at n = 3. At the end of the incubation, the cells were irradiated or not (unirradiated control) by UV A (dose of 2.6 J / cm 2) and UV B (dose of 180 mJ / cm 2). At the end of the irradiation, the cells were incubated for 30 minutes at 37 ° C. and 5% CO 2, before being analyzed by flow cytometry.

B2 - Data Processing The raw data was transferred under Microsoft Excel software and processed according to the method 5 described in Study A above of the MMP-9 activity. B3 - Results of the effect on drH202 production The irradiation of Jurkat cells by UV A + B induced a clear increase in the fluorescence intensity of the DHR probe, reflecting an increase in the production of drH2O2. Under these conditions, the reference, BHA tested at 100M, clearly and significantly protects the Jurkat cells from irradiation: 83% protection. The induction of radical stress by the ultra-violet and the protective effect of BHA were expected and validate the test. The copper gluconate compound, tested at 0.5%, significantly inhibits the production of hydrogen peroxide by irradiated Jurkat cells: 27% protection. The total algal biomass compound tested between 0.0001 and 0.05% did not have a significant effect on the hydrogen peroxide production by Jurkat cells. The results of the test are shown in Table 2. Table 2: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE PRODUCTION OF HYDROGEN PEROXIDE IN UVA AND UVB-IRONIZED JURKAT CELLS Treatment Basic data Standardized data Compounds tested Concentration Fluorescence intensity Average esm% Control. (%) Protection (%) Condition No Control without probe 92 88 4 - irradiated 81 106 82 83 83 Control 1053 1184 1425 1332 1394 1342 1288 58 6 0 *** 100 0 * * * 28 Table 2: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE PRODUCTION OF HYDROGEN PEROXIDE IN UVA AND UVB-IRONIZED JURKAT CELLS (continued 1) Treatment Basic Data Standardized Data Compounds tested Concentration Fluorescence intensity Medium (UA) esm% esm p (1)% esm p (1) (AU) (AU) Control (%) Protection (%) irradiated Irradiated conditions Control - 24153 22521 816 100 4 - 0 4 UVA (2.6 J / cm2) + UVB (180 mJ / cm2) 21657 21754 BHA 100 1_1M 4931 4859 4784 22334 22787 14257 15298 4858 42 22 0 *** 83 0 *** 29 Table 2: EFFECTS OF TOTAL ALGAL BIOMASS AND OF COPPER SALT AND THEIR ASSOCIATION ON THE PRODUCTION OF HYDROGEN PEROXIDE IN JURKAT CELLS IRRADIATED BY UVA AND UVB (continued 2) Treatment Data Basic data Standardized data Compounds tested Concentration Fluorescence intensity Average esm% esm pn)% esm P (I) (UA) (UA) (AU) Control (%) Protection (%) irradiated Irradiated conditions BIOMASS ALGALE TOTAL 0.0001% 22301 23880 899 106 4 ns -6 4/1 S UVA (2.6 23924 J / cm2) + 25415 UVB (180 mJ / cm2) 0.0005% 13430 (2) 27314 3783 121 17 ns -23 18/15 31096 23531 0.001% 21860 22284 252 99 1 ns 1 1 liS 22260 22731 0.05% 19627 21583 987 96 4 ns 4 5 ns 22334 22787 30 Table 2: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON PEROXIDE PRODUCTION OF HYDROGEN IN JURKAT CELLS IRRADIATED BY UVA AND UVB (continued 3) Processing Basic Data Standardized Data Compound (s) Tested Concentration Fluorescence Intensity Mean Esm% esm pn)% esm P (I) (UA) (UA) (UA) Control (%) Irradiated protection (%) Irradiated conditions COPPER GLUCONATE 0.01% 21917 22046 82 98 0 ns 2 0 ns UVA (2.6 22197 J / cm2) + 22025 UVB (180 mJ / cm2 0.5% 16707 16828 349 75 2 ** 27 2 ** 16293 17484 31 Table 2: EFFECTS OF TOTAL ALGAL BIOMASS AND COPPER SALT AND THEIR ASSOCIATION ON THE PRODUCTION OF HYDROGEN PEROXIDE IN UVA AND UVB-IRONIZED JURKAT CELLS Treatment Basic Data Standardized Data% Tested Compounds Concentration Fluorescence Intensity Mean esm Exposure Control (AU) (AU) (%) (%)% 25259 0.01% / 0.1% 21808 21104 453 94 2 ns 7 2 ns MIXTURE 21246 OF TWO CONSTITUENTS 13591 0.05% / 0.5% 15586 14342 626 64 3 ** 39 3 ** Irradiated conditions 13849 UVA (2.6 J / cm2) + UVB (180 21881 mJ / cm2 0.0001% / 0.1% 20879 21850 552 97 2 ns 3 3 ns MIXTURE 22791 OF TWO CONSTITUENTS 13694 0.0005% / 0.5% 14257 14416 470 64 2 ** 38 2 ** 15298 (1 ): Threshold of statistical significance ***: <0.001, Extremely significant ns:> 0.05, Not significant (2): Invalid data: 0.01 to 0.05, Significant nc: not calculable **: 0, 001 at 0, 01, Very significant x x: Standard deviation The combination of the two compounds: total algal biomass and copper gluconate significantly inhibits the production of hydrogen peroxide at the highest concentrations tested (approximately 39% protection). Under these conditions, the protective effect observed is greater than that of copper gluconate compound tested alone, thus suggesting a possible synergistic effect between the two compounds. The two experimental studies A and B carried out make it possible to establish the exceptional interest of the preparation according to the invention (combination of total algal biomass and copper salt) in the prevention and treatment of tissue and biological modifications, especially at the level of the tissue matrix that constitutes the connective tissue. The individual compounds: total algal biomass and copper salt have an inhibitory effect on the activity of MMP-9. An anti-radical effect is also demonstrated for the copper salt compound, via an inhibition of the production of H202 by UV irradiated Jurkat cells. The combination of these two compounds also makes it possible to effectively inhibit the enzymatic activity of MMP-9 and the production of H202 by irradiated Jurkat cells. It should also be noted that in these two cases, the effect of the combination is greater than that of the compounds tested individually. The effects of this combination on the inhibition of a protease (MMP-9) coupled with an anti-radical effect are in favor of anti-aging activity.

Examples 5 and 6: Sludge baths employing the preparation according to the invention Sludge baths having the following composition were prepared: Constituents Example 5 Example 6 Hydroxyethylcellulose 0.3 g 0.3 g Wheat starch 2 g 2 g Kaolin 2 g - Terdax, the peloid of - 2.5 g Dax Preparation according to 0.5 g 0.5 g Example 3 (active) Thermal water of Dax qs 100g qs 100 g Hydroxyethylcellulose, starch was mixed of wheat, kaolin or Terdax and the preparation of Example 10 3 above in Dax thermal water at emergence temperature (> 50 ° C) to form a fluid milky suspension, constituting the mud bath . These mud baths were used at a skin-friendly temperature of 37-45 ° C under the conditions well known in hydrotherapy. Examples 7 and 8: Sludge Care / Wraps Using the Preparation of the Invention Sludge / wrap treatments having the following composition were prepared: Component Example 7 Example 8 Carboxymethylcellulose 1.8 g 1.8 g Corn starch 3 g 3 g Kaolin 2.5 g - Terdax, the peloid of - 3 g Dax Preparation according to 1 g 1 g Example 3 (active) Thermal water 30 g 30 g of pasting Thermal water of Dax qs 100 g qs 100 g Carboxymethylcellulose, corn starch, kaolin or terdax and the preparation of Example 3 above were mixed in Dax thermal spring water (> 50 ° C) to form a dough. The latter was diluted in this water to form after swelling a smooth, unctuous paste that could be applied to the part of the body to be treated. The operator has made the application of the preparations at a skin-friendly temperature (37-45 ° C) under the conditions well known in hydrotherapy. Examples 9 and 10: Face masks employing the preparation according to the invention Face masks having the following composition were prepared: Constituents Example 9 Example 10 Sorbitol 1.7 g 1.7 g Corn starch 1, 7 g 1.7 g Kaolin 6.5 g - Terdax, the peloid - 8 g of Dax Preparation according to 0.1 g 0.1 g Example 3 (active) Thermal water of qs 20 g qs 20 g Dax mixed with sorbitol, corn starch, kaolin or Terdax and the preparation of Example 3, then extemporaneously, this preparation was diluted with its weight of mineral water of Dax to form a creamy pasty gel, that we applied on the face. These masks could be held in place for 10 -15 minutes and then rinsed off with a wipe impregnated with mineral water.

EXAMPLE 11 Body Milk An oil phase and an aqueous phase were prepared having the following formulations: Oily phase: vegetable oil - squalane - glycerol stearate - cetostearyl alcohol Aqueous phase: xanthan gum - purified water - preservatives, perfumes The two above formulations were heated separately at 65-70 ° C, the oily phase being 22 parts by weight and the aqueous phase 78 parts by weight per 100 parts by weight of the oily phase + aqueous phase. While stirring, the oily phase was added to the aqueous phase to form an emulsion. It was cooled with stirring to 30 ° C. The preparation of Example 3 (active) was added at 1 g per 100 g of the emulsion and homogenized. Example 12: Revitalizing Care Cream An oil phase and an aqueous phase were prepared having the following formulations: Oily phase: - PEG 1500 monostearate - PEG 300 monostearate 20 - Paraffin oil Aqueous phase: - Purified water - Preservatives, Perfumes The procedure was as in Example 11 except that the oily and aqueous phases respectively represented 30 and 70 parts by weight per 100 parts by weight of the oily phase + the aqueous phase and that the preparation of Example 3 (active ) was incorporated at a rate of 2 g per 100 g of the emulsion. 30

Claims (4)

CLAIMS1 - Preparation based on at least one alga characterized in that it comprises, in a water-based medium, a mixture of: (a) at least one total algal biomass; and (b) at least one soluble copper salt in said medium. 2 - Preparation according to claim 1, characterized in that the algae or the total algal biomasses (a) are selected from the algae of the algal flora that grows naturally in the thermal waters of Dax when the waters arrive at emergence in the presence of light. 3 - Preparation according to one of claims 1 and 2, characterized in that the algae or the total algal biomasses (a) are the algae of the mineral water of Dax, obtained by continuous streaming under light natural and / or artificial mineral water of Dax, at the temperature of development of said algae, on a culture support inclined plane; culturing strains of said algae provided by said mineral water of Dax and / or seeded on said support; harvesting said algae by scraping the surface of the culture, dewatering and spinning by centrifugation. 4 - Preparation according to one of claims 1 to 3, characterized in that the or the total algal biomasses (a) are in a form which is free of a natural bacterial flora interfering and which is microbrilled especially to a size mean particle not exceeding 50 μm, in particular not exceeding 5 μm, being in particular from 1 to 5 μm. - Preparation according to one of claims 1 5 to 4, characterized in that the or the total algal biomasses (a) are in the form of a suspension in a vehicle stabilizing the biomass or biomasses, forming part of the constitution of said water-based medium, and advantageously at least partially solubilizing or copper (b) salts, said vehicle being in particular glycerol. 6 - Preparation according to one of claims 1 to 5, characterized in that the copper salt or salts (b) are chosen from non-toxic salts of copper and organic acids, such as copper acetate copper butyrate, copper citrate, copper tartrate, copper salicylate, copper glycinate, copper lactate or copper gluconate, especially copper acetate and copper gluconate, and mineral acids, such as copper chloride, copper bromide, copper nitrate or copper sulfate. 7 - Preparation according to one of claims 1 to 6, characterized in that the or copper salts (b) are associated with at least one acid, such as lactic acid, in an amount to adjust the pH of the composition is below 7 and to prevent the formation of insoluble copper derivatives in an alkaline medium. 8 - Preparation according to one of claims 1 7, characterized in that the or the total biomassesalgales (a) are suspended in the aqueous medium, at a rate of 5 to 50% by weight of the algal biomass or biomasses total relative to the mixture (a) + (b) + aqueous-based medium, and that the copper salt (s) (b) are present at a rate of 0.05 to 10 g per 100 g of mixture (a) + ( b) + water-based medium. 9 - Preparation according to claim 8, characterized in that the aqueous-based medium contains water and glycerol, and the glycerol represents 30 to 85% by weight, in particular 60 to 80% by weight, of said medium water-based. 10 - Preparation according to one of claims 1 to 9, characterized in that it is in the form of a stable suspension of green color, a density of 1.08 to 1.23, in in particular from 1.16 to 1.22, and from pH 4.5 to 6.5, in particular from 5 to 6. 11 - Process for the preparation of the preparation as defined in one of claims 1 to 10, characterized in that at least one soluble copper salt in a water-based medium is added to at least one total algal mass suspended in said aqueous-based medium. 12. Process according to claim 11, characterized in that the total algal biomass (s) is prepared by carrying out the following steps: (1) continuous streaming under natural and / or artificial light of the mineral water of Dax, at the temperature of development of said algae, on a culture support inclined plane; (2) cultivation of strains of said algae provided by said mineral water of Dax and / or seeded on said support; harvesting said algae by scraping the culture surface, draining and spinning by centrifugation; decontaminating and deodorizing said algae under conditions maintaining the activity of the total biomass; microbrilling and micronization of the total algal biomass or biomasses thus obtained up to a particle size of not more than 5 μm. (5) 13 Process according to Claim 12, characterized in that the decontamination and deodorization are carried out by contacting the algae harvested, drained and dewatered with a decontamination agent consisting of hydrogen peroxide. 14 - Method according to one of claims 12 and 13, characterized in that one carries out the micromilling of the total algal biomass or biomasses in the presence of a vehicle stabilizing the said total algal biomass or these and used in proportions included between 30 and 85% by weight of said total algal biomass or said biomasses, said vehicle entering into the composition of said aqueous-based medium, and consisting in particular of glycerol. 15 - Method according to one of claims 12 to 14, characterized in that the one or more salts of copper (b) in the total algal biomass (s) (a) before or after micronization, at the rate of 0.05 to 10 g of copper salt (s) (b) per 100 grams of the mixture (a) + (b) + aqueous medium, preferably with pH adjustment to less than 7 by addition of an acid such as lactic acid. 16 - topical applications of the preparation as defined in one of claims 1 to 10 or obtained by the process as defined in one of claims 11 to 15 in hydrotherapy and cosmetology. 17 - Composition for topical use comprising the preparation as defined in one of claims 1 to 10 or prepared by the process as defined in one of claims 11 to 15. 18 - composition for topical use according to claim 17 characterized in that it consists of a mud bath or mud / wrap treatment, comprising, besides said preparation, a vehicle facilitating the application of the latter to the skin. 19 - Composition for topical use according to claim 17, characterized in that it consists of a cosmetological composition, such as mask for the face, base for mask for the face, care cream, in particular revitalizing care cream, body milk, comprising, apart from said preparation, a suitable vehicle for cosmetology applications.