Ribosomal fraction and composition containing it

 

(57) Abstract:

Ribosomal fraction obtained from at least one non-photosynthetic filamentous bacteria detachment Beggiatoales, of the genus Beggiatoa, Vitreoscilla, Flexithrix or Leucothrix. Cosmetic or pharmaceutical composition contains the specified ribosomal fraction in the amount of from 0.0001 to 20% by weight of the total composition and has an immunostimulating action. Method of cosmetic treatment is applied to the skin, hair and/or mucous membranes cosmetic compositions containing the specified ribosomal fraction. Ribosomal fraction, obtained on the basis of new microorganisms, provides restoration of immune functions, particularly cells of the epidermis, enhancing the natural protection of the skin. 3 S. and 4 C.p. f-crystals, 2 tab.

The present invention relates to ribosomal fractions of at least one non-photosynthetic filamentous bacteria.

Ribosomes are corpuscular education ranging in size from 140 to 230 angstroms, consisting of ribonucleic acids associated with proteins that are present in cells of all organisms, in particular bacteria. Ribosomes are the site of protein synthesis, due to the multienzyme complex: international unit, measuring the speed of sedimentation of substances subject to a standard centrifugation).

Morphologically ribosomes consist of 2 subparticles of different sizes. These 2 subunit are respectively constant sedimentation order 50S and 30S.

The 50S subunit contains two RNA, respectively, with 23S and 5S and about thirty proteins, while the 30S subunit contains one RNA with 16S and about twenty proteins.

Thus, the expression of ribosomal fraction corresponds to the fraction of the environment, enriched in ribosomes, which may be obtained, for example, after centrifugation, separating the various organelles that make up the cells of the body.

Ribosomal fraction may, therefore, contain, regardless of the degree of its purity, or at least one whole ribosome, or at least one cutting element of the ribosome, or an element of one of the subparticles of the ribosome.

Currently ribosomal fractions are used, for example, in cosmetics, for the preparation of compositions intended to slow down skin aging by stimulating cell growth and modulation of maturation of the connective tissue (European the STI, patients who received severe burns and sensitive to the result of infectious diseases caused by conditionally pathogenic microorganisms, bacteria, viruses or fungi (international patent 91/11174) or, even, with regard to diseases otorhinolaryngologic sphere (French patents 2253499, 2360314, 2388563, 2674755, patent South Africa 8801071), allergies (U.S. patent 4946945). Ribosomal fractions used in obtaining additives to vaccines (French patent 2374911, patent Germany 1617809).

Usually ribosomal fractions used in these inventions are derived from gram-negative bacteria, such as bacteria of the family Enterobacteriacees (for example, Klebsiella pneumoniae, Escherichia coli, Serratia marces - sens) or family Pasteurellacees (e.g., Haemophilus influenzae), or gram-positive bacteria, such as bacteria of the Bacillus family (e.g., Bacillus subtilis), family Streptococcacees (for example, Streptococcus pneumoniae, Streptococcus pyogenes Gr. A), or family Lactobacillacees (for example, Acidophilus, Bifidum), or on the basis of yeast, such as Candida albicans or Candida tropicalis.

Although known ribosomal fractions have been applied, there is a need to search for new ribosomal fractions derived from organisms that up to the present vremenena fraction, which can be used, at least in the areas specified above and, where appropriate, in new areas.

Object of the invention is a new product that is a ribosomal fraction derived from at least one non-photosynthetic filamentous bacteria.

Ribosomal fraction, as it was pointed out before that can contain, whatever its degree of purity, or at least one whole ribosome, or at least one cutting element of the ribosome, or an element of one of the subparticles of the ribosome.

The immune system contains a set of specialized cells subjected to numerous control mechanisms, enabling them to update their activation and differentiation, required for normal levels of immunocompetence. The role of the immune system is to recognize "their" and "them" to eliminate pathogenic agents and spontaneous tumors. Any depletion of the population of cells, any rosregulirovanie immune system or any functional failure can contribute to the unexpected appearance of pathological manifestations, characterized by disorders of recognition mechanisms "see and processes neoplasms.

The skin is an important organ of the body and is considered one of the main active elements of the system of immune protection. Three types of epidermal cells are involved in this system: keratinocytes, melanocytes and cells of Langerhans. These cells, which are found only in the skin, play a major role in the immune response and, in particular, in view of the antigen.

Healthy skin is able to protect against external influences due to the funds available. However, it is subject to continuous exposure to environmental chemicals, radiation. In particular, cells of Langerhans are a preferential target for ultraviolet radiation.

These aggression expressed in the effect of suppression of the immune defenses, leading to a reduction of resistance to pathogenic agents and to increase the frequency of some cancers.

Thus, of great interest are the products that stimulate the skin's immune system.

On the other hand, it is known that the immune system and, in particular, the immune system of the skin is weakened during chronobiological aging.

This weakening also appears in fremontii, in particular, cells of the epidermis, enhancing the natural protection of the skin.

Another aim of the invention is a new adjuvant, more specifically, for the immune defense of the skin.

Another object of the invention is a cosmetic or pharmaceutical composition containing ribosomal fraction, at least one non-photosynthetic filamentous bacteria. Ribosomal fraction, at least one photosite - shirouma filamentous bacteria possesses a remarkable ability to stimulate the immune system.

Thus, the task of the invention is a cosmetic or pharmaceutical composition containing as an immunostimulating beginning at least one ribosomal fraction, at least one non-photosynthetic filamentous bacteria.

Preferably, the ribosomal fraction is designed to stimulate the immune system of the skin. This stimulation of the immune system of the skin is most needed during chronobiological aging and/or photoaging.

Ribosomal fraction according to the invention comes from a bacteria selected among non-photosynthetic filamentous bacteria, such as those described in the tutorial systemswith bacteria, belonging to the order Beggiatoales, and, more specifically, bacteria belonging to the genera Beggiatoa, Vitreoscilla, Flexithrix or Leucothrix.

The above bacteria are usually aquatic habitat and can be found, especially in marine waters or in thermal waters. Among such bacteria include, for example:

Vitreoscilla filiformis (ATCC 15551)

Vitreoscilla beggiatoides (ATCC 43181)

Beggiatoa alba (ATCC 33555)

Flexithrix dorotheae (ATCC 23163)

Leucothrix mucor (ATCC 25107)

Sphaerotilus natans (ATCC 13338)

It is preferable to use a strain of Vitreoscilla filiformis.

To obtain ribosomal fractions of the present invention may be used any method known to the expert.

In particular, it is possible to call the methods described by Norris and Ribbons in the book "Methods in Microbiology", 1973 (Academic Press).

In the compositions according to the invention ribosomal fraction of non-photosynthetic filamentous bacteria from 0.0001 to 20% by weight of the total composition and preferably from 0.01 to 10% by weight of the total composition.

Depending on the method of introduction of the composition, orally, by injection, or applied to the skin (any skin area of the body), the hair, the nails or the mucous membranes (mouth, cheek, gingival, genital, anal, conjunctiva), Yesenia on the skin, the composition may be in the form of aqueous or oily solution or of the dispersion solution, the type of lotion or serum, of emulsions of liquid or viscous consistency, type of milk, obtained by dispersion of a fatty phase in an aqueous phase (M/W) or conversely (W/M), or suspensions or emulsions of soft consistency, type of cream or gel, aqueous or anhydrous, or, even, microcapsules or microparticles, or of vesicular dispersions, type ionic and/or nonionic. These compositions have the usual ways.

In the case of application to the hair or scalp they can be used in the form of aqueous, alcoholic or hydroalcoholic solutions or in the form of creams, gels, emulsions, foams or, even, in the form of aerosol compositions containing a stimulating agent, under pressure.

For injection, the composition may be in the form of an aqueous lotion, oily lotion or serum. To the eye it may be in the form of drops for oral administration, it can be in the form of capsules, granules, syrups or tablets.

The amounts of the various components of the compositions according to the invention, are the same as commonly used in the fields.

For external grease is Yes for the skin of the face, for the hands, for the feet, for the large anatomical folds or for the body (for example day creams, night creams, creams for removing make-up, creams, sunscreens), liquid, powder, lotion, makeup remover, cleansing milk for the protection of the body or for body care, sunscreen lotion, lotions, gels or foams for caring for the skin, such as cleansing lotions, sun lotions, lotions, artificial tanning compositions for baths, deodorants containing a bactericidal agent, gels or lotions after shaving, creams for hair removal, compositions against insect bites, anesthetics compositions, compositions for treating certain skin diseases such as eczema, red acne, psoriasis, herpes, strong Sudy, dermatitis.

The composition can also be made in the form of an aerosol composition containing a stimulating agent under pressure.

Ribosomal fraction of the present invention, at least one non-photosynthetic filamentous bacteria can also be introduced into various compositions for hair care products, especially shampoos, lotions, hair styling, treatment lotions, creams or gels for hair styling, coloring compositions (especially oxidizing krassi for Curling hair (especially compositions for primary perms), lotions or gels from alopecia, disinfectant shampoos, etc.

The composition can also be used for the care of mouth, teeth, for example, toothpaste. In this case, the composition may contain auxiliary AIDS and additives for compositions for oral administration, particularly, surfactants, thickeners, moisturizing agents, abrasive agents, such as silica, various active ingredients such as fluorides, in particular sodium fluoride, and, if necessary, sweetening agents as sodium saccharinate.

When the composition is an emulsion, the fatty phase can range from 5 to 80 wt.%, preferably, from 5 to 50 wt.% in relation to the total weight of the composition. Oils, waxes, emulsifiers and simulatory used in the composition in the form of an emulsion, choose among those that are commonly used in the cosmetic area. Emulsifiers and simulatory present in the composition in an amount of from 0.3 to 30 wt.%, preferably from 0.5 to 20 wt. % relative to the total weight of the composition. In addition, the emulsion may contain lipid vesicles.

When the composition is a solution or oily Geka composition may, similarly, to contain auxiliary means customary in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, fragrances, fillers, filters, odor absorbers and colorants. The amounts of these various additives such as that usually used in the cosmetic field, for example, from 0.01 to 10% by weight of the total composition. These auxiliary means in accordance with their nature can be introduced into the fatty phase, into the aqueous phase and/or into the lipid balls.

As oils and waxes suitable for use in the invention, can be called mineral oil (liquid paraffin), vegetable oils (liquid fraction of the oil, Shea butter, sunflower oil), animal oils (perhydrosqualene), synthetic oils (porcelanowe oil), silicone oils or waxes (cyclomethicone) and fluorinated oils (parfocality), beeswax, Carnauba wax or paraffin wax. These oils can be added fatty alcohols and fatty acids (stearic acid).

As emulsifiers suitable for use in the invention include, for example, glycerites.

As solvents suitable for use in the invention, can be called the lower alcohols, especially ethanol and isopropanol, propylene glycol.

As hydrophilic gelling agents suitable for use in the invention, can be called carboxyvinyl polymers (carbomer), acrylic copolymers such as copolymers of acrylates/alkylacrylate, polyacrylamides, polysaccharides such as hydroxypropylcellulose, natural resins and clays, and lipophilic gelling agents can be called, modified clays, such as bentonites, metal salts of fatty acids such as aluminum stearates, and hydrophobic silica, ethylcellulose, polyethylene.

The composition may contain other hydrophilic active substances, such as proteins or products of hydrolysis of proteins, amino acids, polyols, urea, allantoin, sugars and sugar derivatives, water-soluble vitamins, plant extracts and hydroxy acids.

As lipophilic active substances can be used retinol (vitamin A) and its derivatives, tocopherol (vitamin E) and its derivatives, essential fatty acids, salicylic acid and its PR is, the one non-photosynthetic filamentous bacteria with other active agents intended in particular for the prophylaxis and/or treatment of skin diseases. Among these active agents as examples:

agents, modulating the differentiation and/or proliferation, and/or skin pigmentation, such as retinoic acid and its isomers, retinol and its esters, vitamin D and its derivatives, oestrogens such as oestradiol, kojic acid or hydroquinone;

- antibacterial, such as clindamycine, erythromycin or antibiotics of the class tetracycline;

- anti-parasitic, in particular metronidazole, crotamiton or pyrethrinoid;

- antifungal, in particular compounds belonging to the class of imidazoles, such as econazole, ketoconazole or miconazole or their salts, polyene compounds such as amphotericin b, compounds of the family of allylamines, such as terbinafine or more octopirox;

- antiviral agents such as acyclovir;

- steroid anti-inflammatory agents such as hydrocortisone, betamethasone or globalatlanta, or nonsteroidal anti-inflammatory agents such as ibuprofen and its salts, diclofenac and its salts, azorher lidocaine and its derivatives;

- antipruritic agents, such as analgin, trimeprazine or cyproheptadine at;

- keratolytic agents such as alpha - and beta - hydroxycarbonate acids or beta-ketocarboxylic acids, their salts, amides or esters and, more particularly hydroxy acids such as glycolic acid, lactic acid, salicylic acid, citric acid and fruit acids and n-5-octylsilane acid;

agents that suppress free radicals, such as alpha-tocopherol or its esters, peroxide-dismutase, some agents of the formation of chelate complexes with metals or ascorbic acid and its esters;

- antisabotage, such as progesterone;

- anti-dandruff, such as octopirox or zinc pyrithione;

- anti-acne, such as retinoic acid or benzoyl peroxide.

Thus, the invention relates to compositions containing at least one ribosomal fraction of non-photosynthetic filamentous bacteria and at least one agent chosen among antibacterial, antiparasitic, antifungal, antiviral, anti-inflammatory, antipruritic, anaesthetic, keratolytic agents that suppress the situation, and/or skin pigmentation.

In addition, the subject of the present invention is a method of cosmetic treatment to stimulate the immune system and, in particular, the immune system of the skin in which the skin, hair and/or mucous membranes cause the composition described above.

Method of cosmetic treatment according to the invention can be carried out, especially, the application of hygienic or cosmetic compositions as described above, in accordance with the usual methods of using these compositions. For example: application of creams, gels, serums, lotions, milk makeup remover or sunscreen compositions to the skin or to dry hair, application of lotion for hair soaked hair washing shampoo or more, application of toothpaste to the gums.

The following examples and compositions illustrate the invention but in no way limit its scope. In the compositions of the contents of the components are indicated in mass percent, if not marked otherwise.

Example 1

Getting ribosomal fractions Vitreoscilla fliiformis:

The culture of Vitreoscilla filiformis get in the way described by the applicant in French patent application published under number 2700172.

/P> The supernatant layer is removed. Add ammonium sulfate to pH 7.5 calculation of 210 mg/ml.

Subsequent centrifugation is carried out in the same conditions as before.

The supernatant layer is subjected to dialysis (membrane spectral dialysis, porosity 1000 MWCO (approximately 1000 daltons)) against a buffer solution of Tris-HCl with a pH of 7.6.

Get 70 ml of medium enriched in ribosomes.

Example 2

The influence of the ribosomal fraction of Vitreoscilla filiformis on the growth and differentiation of mouse splenocytes Balb/c mice aged 7 weeks

Ribosomal fraction of Vitreoscilla filiformis example 1 was tested at different concentrations in a representative system for determining the activity of the effects of the product on the immune system: growth and differentiation of splenocytes from mouse Balb/c mice aged 7 weeks.

Assess the potential activity of ribosomal fractions:

(a) by measuring the implementation of thymidine into deoxyribonucleic acid (coefficient of proliferation).

The negative control is represented by the influence of culture medium on splenocytes; positive control - effect of lipopolysaccharides (LPS) that serve as a standard of comparison, at concentrations of 0.001% and 0.0001%. Used LPS of t is:

The mouse splenocytes distributed in 96-well plates to cultivation NUNC round bottom, at the rate of 250,000 cells per well in a final volume of 200 ml. of the Test products are placed in the wells at various concentrations. Cell proliferation is assessed according to the introduction tritylodontidae thymidine (1 µci/well). For 18 h before the end of the cultivation 10 ál tritylodontidae thymidine with an activity of 100 µci/ml added to each well with the culture. After 18 h cells remove the filter using the tool for the selection of cell type Tomtec. The filter is then dried and coated with liquid scintillator. The radioactivity deposited on the filter is determined by the count in the counter-type. The proliferation rely on stimulation coefficient, calculated according to the following formula:

[Measured radioactivity Control radioactivity (cells+medium)] /Control radioactivity

The results are presented in table. 1 (the coefficients of stimulation, calculated according to the formula above).

Ribosomal fraction significantly increase the rate of proliferation. The maximum effect is obtained at a concentration of 2.5% and after 48 h of cultivation.

The effect is comparable to the effect obtained by the all splenocytes cells (lymphocytes).

The negative control is represented by the influence of culture medium on splenocytes; positive control - effect of lipopolysaccharides (LPS) that serve as a standard of comparison, at concentrations of 0.001% and 0.0001%. Used LPS are FSC Esherichia coil, sold by the company Sigma.

Analysis of immunoglobulins:

Cells are placed in culture medium in the same conditions that were described before. After 2 days of cultivation the supernatant layers were removed and determine the amount present of immunoglobulins (Ig).

This quantification is carried out at a set Pharmingen according to the instructions of the supplier.

Immunoglobulins are proteins present in the blood that attach to antigens, so they can be recognized and identified as a foreign body lymphocytes Century

Immunoglobulins are divided into different types and subtypes depending on the structure of the constant part of their heavy chains. In mice there are 5 classes (Ig M, G, A, D and E). Immunoglobulin G is an immunoglobulin, the most common somatic fluids, particularly, extravascular, in which he fights against microorganisms and toxins. You can kassinove immunoglobulin in sereznosti secretions, where it protects the outer surface of the body.

Immunoglobulin M is a very effective agglutinating agent, very quickly produced in an immune response. It forms the first line of defense against bacteraemia.

In this test separately determine the number of Ig Gl, G2a, G2b, G3, M and A.

The results are presented in table. 2

Ribosomal fraction significantly increases the production of immunoglobulins of all studied classes and subclasses. Optimal active concentration equal to 1.25%.

These results are comparable with the results obtained with the positive control.

Example 3

Example compositions according to the invention. These compositions were obtained by classical methods known to the expert.

Cream cleanser

Ribosomal fraction of example 1 and 0.5

Cetyl alcohol - 2,00

Literallayout - 2,00

Stearic acid - 2,0

Polyglyceryl-3-gidrometeorology ether - 5,0

Mineral oil Codex to 12.0

Carbomer - 0,35

Sodium hydroxide - 0,15

Fragrant substance, a Sufficient amount of

Methylparaben - 0,20

Sterile demineralized water Sufficient to 100.00

Cleansing milk
Literallayout - 1,00

Cetyl alcohol - 0,50

PEG-100 stearate - 0,80

Methylparaben - 0,20

Fragrant substance, a Sufficient amount of

Sterile demineralized water Sufficient to 100.00

Lotion to care

Ribosomal fraction of example 1 - 1

Glycerin - 2,00

Methylparaben - 0,15

Fragrant substance, a Sufficient amount of

Sterile demineralized water Sufficient to 100.00

Care cream

Ribosomal fraction of example 1 or 2

Literallayout - 1,00

PEG-100 stearate - 1,00

Stearic acid - 1,00

Cetyl alcohol - 2,00

Soybean oil - 3,00

Palm oil is 2.00

Cyclomethicone - 2,00

Dimethicone - 1,00

Polyacrylamide - 0,20

Glycerin - 3,00

Methylparaben - 0,20

Fragrant substance, a Sufficient amount of

Sterile demineralized water Sufficient to 100.00 t

1. Ribosomal fraction derived from at least one non-photosynthetic filamentous bacteria detachment Beggiatoales, of the genus Beggiatoa, Vitreoscilla, Flexithrix or Leucothrix.

2. Ribosomal fraction under item 1, characterized in that the above-mentioned bacterium selected from strains of Vitreoscilla filiformis.

3. Insha least one ribosomal fraction described in paragraph 1 or 2, in the amount of from 0.0001% to 20% by weight of the total composition.

4. The composition according to p. 3, characterized in that the ribosomal fraction is an immunostimulating agent.

5. The composition according to p. 4, characterized in that the ribosomal fraction is an immunostimulating agent to the immune system of the skin.

6. Composition according to any one of paragraphs.3 to 5, characterized in that the ribosomal fraction ranges from 0.01 to 10% by weight of the total composition.

7. Method of cosmetic treatment, characterized in that on the skin, hair and/or mucous membranes causing at least one cosmetic composition according to any one of paragraphs.3 to 6.

 

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