RAW EXCERPTS OF THE BLUE ALGA, MANUFACTURING METHOD AND USE IN THE KOSMETOLOGIE AND DERMATOLOGIE

The present invention relates to crude extracts of blue-green algae (blue), processes for their preparation and their use in cosmetology and dermatology. The skin is a frangible barrier, subject to numerous attacks. Many products have been provided in order to enhance its protection or for processing; this in particular include hyaluronic acid, elastin, collagen or DNA. However, these products have the major drawback of having a surface effect and fugitive, especially because of the absence of their entry to the dermis. The applicant has found that crude extracts of blue algae have, surprisingly, barrier properties and resurfacing in cosmetology and dermatology. The present invention relates to crude extracts of blue-green algae, of the type comprising at least one active substance selected from the derivatives porphin ring following: chlorophylls and carotene, characterized in that they further contain at least one of the following substances: [...], chlorophyllins, [...] and [...], and in that it is obtainable from a blue-green algae, algae or by decoction or maceration, freeze dried or fresh, to a temperature between 60 °c 20° and, in a solvent selected from the group consisting of mineral oils, vegetable oils, synthetic substitutes for natural oils, organic solvents of low polarity and water. The present invention also relates to crude extracts of blue-green algae, the kills comprising at least one active substance selected from the derivatives porphin ring following: chlorophylls and carotene, characterized in that it further contains at least one of the following substances: chlorophylls, [...], chlorophyllins, [...] and [...] and in that it is obtainable from the genus of blue [...], by decoction or maceration of said algae, lyophilized or fresh, to a temperature between 60 °c 20° and, in a solvent selected from the group consisting of mineral oils, vegetable oils, synthetic substitutes for natural oils, organic solvents of low polarity, water and water miscible solvents. Advantageously, it is crude extracts of blue [...]. The strain [...] of [...] (removing any bacterial contaminant) is in the form of gelatinous mass emerald green colored. Generally, this algae comprises:

• carbohydrates, in free form and in condensed form, mainly mannose and glucose.
• lipids: the total lipids represent 2 to 12% of the dry weight of the algae, including in particular the triterpene alcohol, vitamin a and fatty acids whose main participating in the composition of the algae are as follows:

  Palmitic acid

  about 50% of the total fatty acids

  Oleic acid

  about 15% of the total fatty acids

  C. the omega&18:1; 7.

  about 10% of the total fatty acids

  C.&18:2 omega-; 6.

  about 4% of the total fatty acids

  Stearic acid

  about 3% of the total fatty acids

  C. the omega&16:1; 7.

  about 1% of the total fatty acids.

• proteinaceous materials, which occupy a place very important chemical composition of the seaweed (of 20 to 70% of the dry weight).
• free amino acids, which comprise from 1 to 7% of the dry weight of the alga, and essentially: of alanine, phenylalanine, glutamic acid, valine and
• pigments:
  • structural porphine: chlorophylls, [...], [...], [...],
  • type [...]: beta-&; - carotene, zeaxanthin, unidentified and other carotenoids
  • the phycocyanin: unstable blue pigment of proteinaceous nature and characteristic of blue stain.

• decoction or maceration of blue alga, freeze-dried or fresh, to a temperature between 60 °c 20° and, in a solvent selected from the group consisting of mineral oils, vegetable oils, synthetic substitutes for natural oils, organic solvents of low polarity, water and water miscible solvents (when the blue alga is of the genus [...]) and, if necessary
• filtering the resulting extract.

It is prepared exclusively from algae lyophilized, because the presence of water is detrimental to good removal.

Extracting a phase fat-soluble by maceration of lyophilized alga, therefore dry in a mineral or vegetable oil, in a synthetic natural oil such as isopropyl myristate, decyl oleate or dibutyl adipate. This maceration can be performed at a temperature between 20 °c and 60 °c.

a kilogram of algae lyophilized is placed in 20 liters of oil cores and this is maintained 40 °c during 48 hours. The oily solution obtained is sieved on a sieve of 100 µm stainless steel to eliminate the greater amount of seaweed, and then filtered at 40 °c onto a forming wire 10 µm porosity. 19.7 liters obtained a clear solution is greenish yellow color usable in cosmetic formulations. This extract is referred as the fat-soluble extract no. 1.

The alga is extracted by decoction or lyophilized maceration in an organic solvent of low polarity such as chloroform, methylene chloride, petroleum ether or hexane. The solvent is then evaporated to leave a metallic coloring and extract composition depends on the solvent employed. Chloroform will, for example, extracting the pigments like chlorophyll while hexane extracts exclusively pigments [...].

A kilogram of freeze dried algae are subjected to three successive decoctions at reflux for one hour in chloroform. The solutions chloroform on paper, joined, and then evaporated to dry under reduced pressure. Obtained 124 g of a dark green paste to characteristic smell of seaweed. This extract, subjected to analysis contains pigments [...] utterances highest following: chlorophylls, [...], [...], [...]. It also contains all pigments [...] present in the seaweed.

The water-soluble extracts are prepared by steeping cold or heat-treated algae fresh or freeze dried, by distilled water. Added a small proportion of chloroform in the water to prevent the fermentations being extracted. Algae can also be extracted first by a water-miscible solvent such as ethanol, methanol or acetone, and then evaporating the solvents under reduced pressure and resume the extract thus obtained by distilled water.

Five kilograms fresh seaweeds are placed in 20 liters of distilled water which has been previously added 10 ml of chloroform. Is subjected to agitation during 24 hours permanent. Aqueous solution is sifted through a sieve of 100 µm stainless steel is then filtered and the solution on a fabric 10 µm porosity. The resulting aqueous solution is then lyophilized. 67 g of lyophilizate obtained. This extract contains polysaccharides, free sugars, minerals, amino acids and proteins but very little pigment.

A kilogram of algae are lyophilized [...] three times in succession by 50% ethanol at reflux for one hour. The [...] are filtered hot on paper, then combined and evaporated to dry under reduced pressure. Obtained 164 g of dry extract. The dry extract thus obtained is taken up by a liter of distilled water to 60 °c under constant agitation for one hour. Is filtered on paper to 60^DEGREES C., and then the aqueous solution is lyophilized. This then 27 g of lyophilisate. This extract contains little proteins, polysaccharides, free sugars, mineral salts and pigments [...]. The extracts may be particularly obtained from the algae [...], from the griffin source [...] Vichy and especially from the strain [...], termed [...], deposited 29 May 1991, under the n° I-a 1101. These different extracts, which contain the different aforementioned active substances at varying concentrations, can be used alone or in admixture as may be desired.

Is distributed in four petri dishes of 8 cm diameter, 20 ml of a solution at 1% agarose in tris buffer at pH 8.8 suspensions which contain 20 mg of insoluble fibrous collagen and 0, 0.1 0.3 and 0.5 ml of a 5% solution of product to be tested (crude extract) as the boxes, are concentrations of 0, 0,025%, 0,075%, 0,125%. As most of the products to be tested are not soluble in the buffer, it starts with a stock solution in dimethyl sulfoxide (DMSO in). This distribution occurs at 45^DEGREES C.. After solidification of agarose, is perforated 5 3 mm diameter wells in the gel through a die. In these five well, placed 50 µl of pancreatic in collagenase solution at pH 8.8 Tris concentration: 10 mg/ml. (the U/ml to 3800). The petri dishes are incubated 15 hours at 37 °c, then the gel is stained with a solution to 0.25% picric acid to 0.25% during 3 minutes, then a solution of 0.1% to 5 during the Sirius red minutes. The excess dye is removed by washing with water.

Is distributed in four petri dishes of 8 cm diameter, 20 ml of a solution at 1% agarose in tris buffer at pH 8.8, containing suspended 20 mg insoluble fibrous collagen. This distribution occurs at 45 °c. After solidification of agarose, is perforated 5 3 mm diameter wells in the gel through a die. In these five wells, is placed 25 µl of a pancreatic collagenase at pH 8.8 Tris in concentrations of: 10 mg/ml. (the U/ml to 3800) and 25 µl of solution of the substance to be assayed in the same buffer at a concentration of 4%, 3%, 2%, 1% and 0%. As most of the products to be tested are not soluble in the buffer, it starts with a stock solution in dimethyl sulfoxide. The petri dishes are incubated 15 hours at 37 °c, then the gel is stained with a solution to 0.25% picric acid to 0.25% during 3 minutes, then a solution of 0.1% to 5 during the Sirius red minutes. The excess dye is removed by washing with water.

Pancreatic elastase activity is determined on a synthetic substrate. It is found that the products described above have an effect on this enzyme while the substrate peptide derivative is a low molecular weight: n n-succinyl-Ala-Ala-P-[...]. Substrate solution: solution from the substrate above to 1% in phosphate buffer pH 7.6. Enzyme solution: U-pancreatic elastase to 1000/ml in the buffer pH 7.6. Solvent: DMSO to be 50% in distilled water. Test solution: crude extract dissolved in solvent to 0.1%. In a first test tube (white), is placed:

2.2 ml

of buffer pH 7.6

0.3 ml

enzyme solution

0.5 ml

solvent.

1 ml

of substrate solution

1.2 ml

of buffer pH 7.6

0.3 ml

enzyme solution

0.5 ml

solution to be tested.

1 ml

of substrate solution

1.2 ml

of buffer pH 7.6

0.3 ml

enzyme solution

0.5 ml

solvent.

A study of cytotoxicity is previously carried out for determining the concentrations of various extracts maximum test. Cells used: human fibroblasts [...], at 28ème passageway. The cells are seeded on a 96 well plate, a proportion of 2.10⁵ cells per well about, and set to grow in medium minimum Eagle medium supplemented with 1% (MEMs) 200 mm glutamine, 1% solution of vitamins, 100 U-/ ml penicillin, 100 U-/ ml streptomycin and 2% fetal calf serum. The test solution (crude extract) is added in a proportion of 100 µl per well at the concentration of 50 ug/ml.. The cells are incubated at 37 °c under 5% CO2 atmosphere₂ . Is taken from the culture medium after 48, 72, 96 and 120 hours of cultivation. The total protein synthesized are determined after staining of the supernatants by coomassie blue and measuring the O.D. to 595 nm. The increase of protein synthesis is measured relative to the control (solvent of the test substance).

Two techniques can be used to highlight the effect antiradical the in vitro crude extracts according to the invention.

Substrate:

The EDTA: 0.05 grams Xanthin: 0.0125 grams A cytochrome c: 0.02 grams Phosphate buffer pH 7.5: 200 ml.

The enzyme:

xanthine oxidase to 1% in the buffer.

Solvent:

DMSO to be 50% in distilled water.

White solution is:

2 ml substrate buffer pH 7.5: 0.5 ml solvent: 0.5 ml

Control solution:

substrate: 2 ml enzyme solution: 0.5 ml solvent: 0.5 ml.

The solution test:

substrate: 2 ml enzyme solution: 0.5 ml the composition antiradical: 0.5 ml.

Reagent: 1.1-diphenyl 2 a-picrylhydrazyl: 2.5 mg in 100 ml of methanol. Solvent crude extracts to antiradical activity: methanol. The composition antiradical: 2% in methanol. This reagent is a stable free radical which can be degraded by substances eliminating free radicals. Are made a white for control and a white for the test:

White cookie

: phosphate buffer pH 7: 1.5 ml methanol: 3 ml

Witness:

the solution reagent: 3 ml phosphate buffer pH 7: 1.5 ml

White test:

phosphate buffer pH 7: 1.35 ml methanol: 3.15 ml

Test:

the solution reagent: 3 ml phosphate buffer pH 7: 1.35 ml solution of antiradical: 0.15 ml.

For each formula, the overall concentration in crude extract is between 0.1 and 3%.