Arthrospira-based compositions and their application

FIELD: medicine.

SUBSTANCE: invention relates to composition, which contains Arthrospira maxima subjected to physiological stress, for application as biocide and/or therapeutic medication. Invention also relates to method of prevention or treatment of infection or contamination of subject with organism, where method includes stage of introduction of efficient amount of composition, containing Arthrospira maxima subjected to physiological stress, to subject.

EFFECT: increase of efficiency of biocidal compositions and therapeutic preparations.

30 cl, 34 dwg, 23 tbl, 13 ex

 

The technical FIELD TO WHICH the INVENTION RELATES

The present invention relates to compositions containingArthrospiraand the use of a composition as a biocide and/or a therapeutic agent.

The LEVEL of TECHNOLOGY

Arthrospira(previously known asSpirulina) is a cyanobacterium, which is grown primarily for use as food and/or protein source. It also describes therapeutic useArthrospiraincluding use as antiviral agents, anticancer means, means lowering cholesterol, antidiabetic agents, antihypertensive agents and immunomodulator. See, for example, links placed on the website of Spirulina Source.com, http://www.spirulinasource.com/library.html.

The INVENTION

The aim of the present invention is a compositionArthrospirafor use as a biocide and/or a therapeutic agent.

Subjected to physiological stressArthrospirahas biocidal activity

The authors of the present invention have found that under physiological stressArthrospiraproduces at least one type of bioactive substances which are active against fungi, bacteria and possibly viruses.Arthrospiracan be subjected to physiological stress voltage is emer, depriving the body of nutrients or light, or by dehydration/drying of the body. Indication that the filamentArthrospira(trichomes) is in a state of "physiological stress"is its occurrence in a state of suspended animation, in this state, the filament is twisted, and formed a thick mucous membrane. The filament and/or thick mucous membrane, as suggested by the authors of the present invention contains one or more bioactive substances that protect the body from predators. One or more bioactive substances can destroy the cell wall or outer skeleton of the body of the predator. One or more bioactive substances may constitute, for example, splitting and modifying substances, such as chitinase, chitosanase and chitin-deacetylase that break down chitin, a derivative of chitin (chitosan) or other polymers of the cell wall that contains, for example, N-acetylglucosamine and D-glucosamine as the subunits of the polymer. One such polymer is peptidoglycan of gram-positive bacteria, consisting of N-acetylglucosamine and N-acetylmuramic acid.

According to the first aspect, the present invention relates to compositions for degradation or modification of chitin, a derivative of chitin or polymer containing N-acetyl-D-glucosamine as when jedinica polymer, where the specified composition comprises subjected to physiological stressArthrospira.

According to the second aspect, the present invention relates to a method for cleavage or modification of chitin, a derivative of chitin or polymer containing N-acetyl-D-glucosamine as a subunit of the polymer, where the method involves the step of contacting chitin, a derivative of chitin or polymer containing N-acetyl-D-glucosamine as a subunit of a polymer composition containing subjected to physiological stressArthrospira.

Chitin, a derivative of chitin or the polymer can be in any suitable form. Chitin, a derivative of chitin or the polymer may be in a substantially purified form, or can be parts of the body, such as the cell wall of fungi or gram-positive bacteria, or a part of the external skeleton of an insect.

According to a third aspect, the present invention relates to a method for the identification of bioactive substances fromArthrospirawhere this method involves the following stages:

(I) combining the composition containing subjected to physiological stressArthrospirawith at least one test substrate, modified bioactive substance from theArthrospira; and

(II) analysis of the modification of the test substrate.

Can be used any suitable t the p test substrate. Analysis of the modification of the test substrate can be held in any suitable way. However, the preferred large-scale screening.

Preferably, the bioactive substance is a chitinase, chitosanase or chitin-deacetylase, and the test substrate is chitin, a derivative of chitin or a polymer containing N-acetyl-D-glucosamine as a subunit of the polymer. Preferably, for the analysis of the modification (i.e. degradation) of chitin, a derivative of chitin or other polymer used respirometry.

The method may additionally include the stage of purification of bioactive substances. Cleaning can be carried out in any suitable way. The method may also include the stage of cloning and expression of a gene/genes bioactive substances. These stages can be implemented using any suitable way. For example, the stage can be a stage that is described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, shown here as a cross-reference in full.

Application subjected to physiological stressArthrospiraas a biocide and/or a therapeutic agent

Chitin, a derivative of chitin and polymers containing glucosamine, found everywhere in nature, usually in the form of a component of the cell wall or outer skeleton, for example, algae, fungus is in, protozoa, ciliates, coelenterates, bryozoans, molluscs, annelids, arthropods, garanovich, phoronid, pogonophora and bacteria (gram-positive bacteria andRhizobia).

Therefore subjected to physiological stressArthrospirawith splitting or modifying activity towards chitin, a derivative of chitin and polymers containing glucosamine, could potentially be used as a biocide against a wide variety of organisms. Used herein, the term "biocide" refers to either the destruction of the body, or to inhibition of the growth or replication of the organism (that is, in fact, the body can not be killed). You should take into account that some applications of the biocide will have a therapeutic purpose.

According to a fourth aspect, the present invention relates to compositions for use as a biocide and/or a therapeutic agent, where this composition contains an effective amount subjected to physiological stressArthrospira.

According to the fifth aspect, the present invention relates to a method for prevention or treatment of infection or contamination of the subject body, where the method includes the stage of introduction to the subject an effective amount of a composition containing subjected to physiological stressArthospira .

Preferably, the method includes a step of identifying a subject in need of such treatment or in need of such prevention.

Provided also use subjected to physiological stressArthrospirain the manufacture of a medicine for the prevention or treatment of infection or contamination of the subject organism.

Unexpectedly, the authors of the present invention, it was found that removal ofArthrospirafrom its growth environment may be sufficient for the onset of physiological stress and induction of expression of one or more bioactive substances that have biocidal activity. That is, to haveArthrospirabiocidal activity is not needed in carrying out the stages of summoning her physiological stress, as indicated in the descriptions of patents in New Zealand No. 336620 and No. 336619, no further treatment (destruction, explosive decompression, potenzirovaniem, grinding, re-soaking in sodium bicarbonate or freeze drying).

Moreover, it was found that commercially available preparations (tablets, capsules and powders)Arthrospirathat still were tested by the authors of the present invention had activity against the substrate, and that such activity, according to the authors, indicates the biocidal activity of These drugs is made, as a rule, by collectingArthrospirawashing and dryingArthrospira, bypassing the stage-specific stress effects on the body. In fact, commercial manufacturersArthrospiratry to reduce stress on the body, as thick mucosa tends to clog the equipment for processing of an organism.

For any one or more of the described in this paper compositionsArthrospiramay be of any suitable type, or a mixture of species or variants ofArthrospiraincluding, but without limitation,A.maxima. It should be noted thatArthrospiraandSpirulinaare two different genera. The differences between these two genera has been proved on the basis of many characteristics, such as the helicity and the size of trichomes, the structure of the cell wall and the structure of the pores, the contents of the vacuoles, the structure of the thylakoids, mobility and fragmentation of trichomes, GC-composition, oligonucleotide directory 16S rRNA and mutations in the locus cpcB-cpcA. See, for example, J.F. Manen and J. Falquet (2002) "The cpcB-cpcA locus as a tool for the genetic characterization of the genusArthrospira(Cyanobacteria): evidence for horizontal transfer. International Journal of Systematic and Evolutionary Microbiology, Vol. 52, 861-867; and Chapter 1: Morphology, Ultrastructure and Taxonomy ofArthrospira (Spirulina) MaximaandArthrospira (Spirulina) Platensisby Luisa Tomaselli, ofSpirulina platensis (Arthrospira): Discrimination, Cell-biology and Biotechnology. Taylor and Francis. Avigad Vonshak (Ed) 1997. To the position, presented in these descriptions do not include the species of the genusSpirulina.

The composition may contain intact filamentsArthrospirasegments of the filaments is broken/destroyed segments or their extracts/fractions. The composition may include both living and non-livingArthrospira. Preferably, the composition is comprised destroyed filaments and segmentsArthrospira.

The composition is based onArthrospiracan be made in any suitable way, while maintaining biocidal activity. OK, it could include stages: (I) cultivation ofArthrospiraany appropriate way; (II) collecting grownArthrospiraany suitable method; and (III) dryingArthrospiraany suitable means. Additional stages include exposureArthrospiraphysiological stress in whatever way is appropriate, the destruction of the filaments and segments in whatever way is appropriate, the sterilisation of the body in any suitable way, discolorationArthrospiraany suitable means, removal or destruction of chlorophyll by any suitable means and the grinding driedArthrospira.

Some of the above stages are described, for example, in the descriptions of the patents of New Zealand No. 336620 and 336619, the full content of which is given here as a cross-reference. For example, stress may be caused during sat the RA biomass or depriving Arthrospiraessential nutrients (reducing the amount of nutrients or light for a time sufficient to stress, but not death, or creating the conditions for growth. To cause stress can also at partial drying or gatheringArthrospiraand keeping it in wet conditions live up to the evocation of stress, but not death. For example, for formation of a fine powder of damaged cellsArthrospiracan be spray dried. The spray drying can be carried out, for example, when 50-190°C within a few seconds. For example, drying can be performed in any suitable way, including vortex drying, drying by means of a heat pump drying in heated tubes, refractory drying or drying at a temperature of about 67°C for a short period of time. For example, the filaments may be destroyed by shredding or explosive decompression, as described in the patents of New Zealand No. 328013 and No. 328740, the full content of which is shown here as a cross-reference.

The concentration ofArthrospirain the composition may vary depending on the task, i.e. to be any in the range from 0.01% to 100%. The composition may include any suitable solvent, carrier, base molding, filler, binder, plasticizer, emulsifier, one hundred is ilitator, lubricant, buffer agent, a softener, a substance that enhances the solubility, suspendisse agent, thickener, perfume, dye and preservative.

Depending on the purpose, the composition may be supplemented by one or several active substances not derived fromArthrospirasuch as antimicrobial agents (e.g., a bactericide or fungicide), therapeutic agent (e.g., tools for wound healing, steroids) or nutrients (e.g. amino acids, vitamins). Such active substances is well known in this field.

The composition may be in any suitable form. The composition can be administered or applied in any suitable way. The composition may be liquid, gel or solid, or a mixture thereof. The composition may be a liquid cultureArthrospiraused in aerosol form. Alternatively, the composition may be a powder, which is evenly distributed over the surface of the area being treated using the devices for shaking or spraying. Infected or infected subject may be placed in a bath containing a liquid form of the composition. The composition and method of use or administration may be as set forth in the descriptions of the patents of New Zealand No. 336620 and No. 336619.

For therapeutic purposes compositeimage be imposed or applied, for example, oral, parenteral, spray inhalation, topically, rectally, through the nose, transbukkalno or vaginally. The composition may be in the form of tablets, solution, aerosol, spray, cream, ointment, lotion, emulsion, gel or powder. The composition may be in the form of filledArthrospirabandage clothing, adhesive plaster, suppository, pessary, or lotions.

Preferably, the biocide active against fungi, gram-positive bacteria, protozoa, viruses, crustaceans, mites and insects, some of these cases are examined in more detail below.

According to the sixth aspect, the present invention relates to a method for producing a composition based onArthrospirafor use as a biocide and/or a therapeutic agent, where the method involves the following stages:

(1) exposureArthrospiraphysiological stress; and

(2) the Association under stressArthrospiraunder item(1) with a suitable carrier, solvent, base or filler.

According to the seventh aspect, the present invention relates to a method for producing a composition based onArthrospirafor use as a biocide and/or medicinal product, where the method includes the steps:

(1) inducing physiological stressArthrospiraby removing approximately 80% of the fluid, W is Arthrospiragrown;

(2) leaching under stressArthrospirain order to remove contaminants;

(3) drying the washedArthrospira; and

(4) combining the washedArthrospirawith suitable carrier, solvent, base or filler.

Any of the above methods can be used to prepare one or more of the compositions described herein.

Preferably, the above-described methods exclude specific stage of explosive decompression and/or drying by freezing subjected to physiological stressArthrospirabefore adding the carrier, mortar, base or filler.

Application subjected to physiological stressArthrospiraas antifungal agents

According to the eighth aspect, the present invention relates to compositions for use as antifungal agents, where this composition contains an effective amount subjected to physiological stressArthrospira.

According to the ninth aspect, the present invention relates to a method for prevention or treatment of fungal infection or contamination of the subject, where the method includes a step of introducing to a subject an effective amount of a composition containing subjected to physiological stressArthrospira.

Preferably, the ESP includes a step of identifying a subject, in need of such treatment or in need of such prevention.

Provided also use subjected to physiological stressArthrospirain the manufacture of a medicine for the prevention or treatment of fungal infection or contamination of the subject.

Subjected to physiological stressArthrospiracan be used to handle at least the following types of mushrooms:Zygomycotina;Ascomycotina;Fungi Deuteromycota,Malassezia;Microsporum;TrichophytonandEpidermophyton.

In the first embodiment, the subject is a human or an animal such as a mammal or vertebrate. When the subject is a human, the composition may be used to treat:

- tinea corporis (for example, athletes foot, inguinal athlete's foot, ringworm, deep infection of the nail bed), which is mainly generaMicrosporum,Trichophyton,CandidaandEpidermophyton;

is thrush caused byCandida;

- dandruff caused byMalassezia;

- tropical mycetoma - granular spreading education in the skin, muscle tissue and lymph nodes caused byMadurella;

- sporotrichosis - granular spreading education in skin and lymph nodes, calledSporothrix schenkii; and

- histoplasmosis - chronic pneumonia with the soap is common systemic infection, calledHistoplasma capsulatum.

When the subject is an animal, the composition can be used to treat, for example, ringworm, infections of the hoof (Candida,Malassezia), dermatitis and tinea (Microsporum,Trichophyton,Alternaria,Fusarium). Local fungal disease in cats is usually caused by the same fungi that and people.

In the second embodiment, the subject is agricultural or horticultural product, such as, for example, plant, flower, fruit, vegetable, cereal, grain, bean, mushroom micelli, pasture or lawn. The composition may be used for processing, for example, rot panicle and root collar (Fusarium), eczema surface (Pithomyces Chartarum), grey mould (Botrytiscineria), spots on leaves (Septoria,Alteria,Bipolaris), powdery mildew (Sphaerotheca macularis,Erysiphe,Sphaerotheca pannosa), brown rot (Monilinia fruiticolaand rust leaves and stems (Puccinia).

In the third embodiment, the subject is soil, timber, building material or building. The composition can be applied, for example, to handle mold inside or outside of buildings and for treatment of fungal stains on wood building materials and power lines.

Unexpectedly, the authors of the present invention found that subjected physiologists is a mini-stress Arthrospirasynergistically interacts with known antifungal agents (fungistatic substances), which provides a broader spectrum of antifungal activity. The authors found that under stressArthrospiracan inhibit the growth of fungi, to destroy the fungi and prevent re-growth of fungi.

According to the tenth aspect, the present invention relates to fungicidal compositions containing a synergistic combination subjected to physiological stressArthrospirawith at least one fungistatic substance.

According to the eleventh aspect, the present invention relates to a method for prevention or treatment of fungal infection or contamination of the subject, where the method includes a step of introducing to a subject an effective amount of a composition containing a synergistic combination subjected to physiological stressArthrospirawith at least one fungistatic substance.

Preferably, the method includes a step of identifying a subject in need of such treatment or in need of such prevention.

Provided also use subjected to physiological stressArthrospirain combination with at least one fungistatic substance in the manufacture of a medicine for the prevention and treatment of fungal infection or contamination of the subject.

Can be used any suitable fungistatic substance (known substance), such as terbinafine, bifonazole, clotrimazole, miconazole, econazole, ketoconazole or tolnaftate. Depending on the intended application of the composition can be used in any suitable concentration of a known substance.

Application subjected to physiological stressArthrospiraas an antibacterial agent

The authors of the present invention, it was found that subjected to physiological stressArthrospirais an effective antibacterial agent against gram-positive bacteria. The nature of the antibacterial action are not well understood. Perhaps the cell wall of gram-positive bacteria are destroyed or otherwise modified by the influence of one or more bioactive substancesArthrospira. Cell walls contain peptidoglycan (consisting of N-acetylglucosamine and N-acetylmuramic acid and peptidoglycan may split one or more bioactive substances.

According to the twelfth aspect, the present invention relates to compositions for use as an antibacterial agent, where this composition contains an effective amount subjected to physiological stressArthrospira.

According Proc. of the twelfth aspect, the present invention relates to a method for prevention or treatment of a bacterial infection or contamination of the subject, where the method includes a step of introducing to a subject an effective amount of a composition containing subjected to physiological stressArthrospira.

Preferably, the method includes a step of identifying a subject in need of such treatment or in need of such prevention.

Provided also use subjected to physiological stressArthrospirain the manufacture of a medicinal product for preventing or treating a bacterial infection or contamination of the subject.

Preferably, bacteria belong to the gram-positive bacteria and may constitute, for example,Bacilli,Clostridia,SusceptibleorPneumococci. Bacteria, preferably, arePropionibacterium acnethat cause acne.

In the first embodiment, the subject is a human or an animal such as a mammal or vertebrate. When the subject is a human, the composition may be applied for treatment of acne, dermatitis, ulcers or wounds caused by or infected with gram-positive bacteria. The composition may be applied to processing internal infections, such as respiratory tract, oral cavity, digestive and urogenital tract (in the example, streptococcal infection of the pharynx, middle ear).

In the second embodiment, the subject is agricultural or horticultural product, such as a plant, flower, fruit, vegetable, cereal, grain, bean or mushroom micelli.

In the third embodiment, the subject is, for example, contaminated artificial structure or photoproton, which can grow bacteria.

Application subjected to physiological stressArthrospiraas pesticide

According to the fourteenth aspect, the present invention relates to compositions for use as a pesticide, where this composition contains an effective amount subjected to physiological stressArthrospira.

According to the fifteenth aspect, the present invention relates to a method of suppressing or eliminating pests, where the method includes the introduction or use of an effective amount of a composition containing subjected to physiological stressArthrospira.

The pesticide may be applied against pests, such as crustaceans and mites. The pesticide may be applied against insects such as flies (e.g.Simulidaeblack flies, wasps, mosquitoes and termites. The pesticide can be applied to stop the spread of parasites such as those causing malaria and drugmetabolizing, portable insects.

In the first embodiment, the composition may be defined as a repellent, or bait.

In the second embodiment, the composition can be used for agricultural or horticultural product, such as a plant, flower, fruit, vegetable, cereal, grain, bean or mushroom micelli.

In the third embodiment, the composition may be applied, for example, contaminated artificial structures, buildings, construction material, river, lake or wetland.

The use ofArthrospiraas a therapeutic agent

The authors of the present invention discovered that by using subjected to physiological stressArthrospiraas a biocide and/or medicines for skin treatment one or more other substitutesArthrospirasynergistically interact with one or more bioactive substances, which helps to cure skin. Data other substitutes, as suggested, include beta-carotene, which provides skin nutrition; phycocyanin, which has anti-inflammatory effect; other proteins, and other nutrients, including vitamins, minerals, trace elements, antioxidants, essential oils and hydrocarbons.

At first subjected to the stress is the Arthrospirainhibits or destroys the germs whether fungi, bacteria or viruses, and then other substitutesArthrospirarestore damage caused by a microbe.

The authors of the present invention found that even not subjected to physiological stressArthrospirahas therapeutic properties, for example, in the treatment of skin diseases and skin restoration.

According to the sixteenth aspect, the present invention relates to compositions for the repair or prevention of damage to the skin of a mammal, where the specified composition contains an effective amountArthrospira.

Preferably, the composition comprises subjected to physiological stressArthrospira.

According to the seventeenth aspect, the present invention relates to a method for healing or prevention of damage to the skin of a mammal, where the method involves administering an effective amount of a composition containingArthrospira.

Preferably, the composition comprises subjected to physiological stressArthrospira.

Preferably, the method includes a step of identifying a mammal in need of such treatment or in need of such prevention.

Also envisaged is the use ofArthrospirapreferably subjected to physiological stress, in the manufacture of a medicament is built tools for healing or prevention of skin defect in a mammal.

According to the sixteenth and seventeenth aspects of the invention, the composition may have one or more component parts or properties, as described for the compositions according to other aspects of the invention that have been defined.

According to the sixteenth and seventeenth aspects of the invention, the composition is preferably obtained by any of the methods described above.

The skin defect can be, for example, pock, prevue damage, red acne, red stain, crack, burn, blister, psoriasis, eczema, peeling, wrinkle, pimple, stomatitis, abrasion, pustules, wound, seborrheic dermatitis, diaper rash, ulcers, herpes, skin rash after shaving, chickenpox, eczema, cracked heels and elbows. The composition can be used to treat burns, insect bites and animals and to eliminate skin irritation. The composition can be used to alleviate itching on the skin. The composition can be used for healing scar tissue, sunburn and dry and flaky skin that has lost its elasticity.

Other aspects and embodiments of the invention will become apparent from the subsequent detailed description.

BRIEF DESCRIPTION of DRAWINGS

In Fig. 1 shows the activity subjected to physiological stressArthrospira(AMYCOT®and raw powder) in relation to the chitin on the basis of the data is espirometria;

In Fig. 2 shows antifungal activity AMYCOT®in relation toC.albicans;

In Fig. 3 shows antibacterial activity AMYCOT®in relation toP.acnebased on the data of respirometry;

In Fig. 4 shows the antifungal activity of different commercial preparationsArthrospira(Spirulina) with respect toT.rubrum;

In Fig. 5 shows the antifungal activity of commercial product (Life Stream)Arthrospirain relation toA.niger;

In Fig. 6 shows antifungal activity AMYCOT®in relation toM.canis;

In Fig. 7 shows the antifungal activity of AMYCOT®in relation toF.dhamek;

In Fig. 8 shows antifungal activity AMYCOT®in relation toM.furfur;

In Fig. 9a shows the antifungal activity of shampoo AMYCOT®in relation toT.rubrum;

In Fig. 9b shows the antifungal activity of shampoo AMYCOT®in relation toC.albicans;

In Fig. 10 shows antifungal activity AMYCOT®in relation toP.chartarum;

In Fig. 11 shows antifungal activity AMYCOT®in relation toB.cinerea;

In Fig. 12 shows results of inhibition ofT.rubrumusing AMYCOT®and various well-known antifungal substances;

In Fig. 13 PR is dstanley data of inhibition of T.mentagrophytesusing AMYCOT®and various known substances;

In Fig. 14 shows results of inhibition ofC.albicansusing AMYCOT®and various known substances;

In Fig. 15 shows results of inhibition ofC.albicansusing various known substances;

In Fig. 16 shows results of inhibition ofT.mentagrophytesusing AMYCOT®in combination with the known substance and various known substances;

In Fig. 17 shows results of inhibition ofC.albicansusing AMYCOT®in combination with the known substance and various known substances;

In Fig. 18 shows results of inhibition ofT.mentagrophytesusing AMYCOT®in combination with the known substances;

In Fig. 19 shows results of inhibition ofT.mentagrophytesusing AMYCOT®in combination with the known substances;

In Fig. 20 shows results of inhibition ofT.mentagrophytesusing AMYCOT®in combination with the known substance and various known substances;

In Fig. 21 shows results of inhibition ofC.albicansusing AMYCOT®in combination with the known substance and various known substances;

In Fig. 22 presents data fungicidal action againstT.mentagrophytesusing AMYCOT®combined the AI with known substances;

In Fig. 23 presents data fungicide AMYCOT®in combination with the known substances in relation toT.rubrumusing;

In Fig. 24 presents data fungicide AMYCOT®in combination with the known substances in relation toT.mentagrophytes;

In Fig. 25 presents data fungicide AMYCOT®in combination with the known substances in relation toT.mentagrophytes;

In Fig. 26 presents data fungicide AMYCOT®in combination with the known substances in relation toT.mentagrophytes;

In Fig. 27 presents data fungicide AMYCOT®in combination with the known substance and various known substances in relation toC.albicansusing;

In Fig. 28 presents data fungicidal action againstT.rubrumusing AMYCOT®and known substances;

In Fig. 29 shows the effect subjected to physiological stressArthrospira(AMYCOT®against chitin at different temperatures based on the data of respirometry;

In Fig. 30 shows the effect subjected to physiological stressArthrospira(AMYCOT®against chitin at different pH values based on the data of respirometry;

In Fig. 31 shows the antifungal activity ofArthrospiraon attributed the h to T.mentagrophytes;

In Fig. 32 shows the antifungal activity ofArthrospirain relation toT.rubrum;

In Fig. 33 shows the antifungal activity ofArthrospirain relation toM.fructicola;

In Fig. 34 shows the antifungal activity of "enhanced AMYCOT®and the original AMYCOT®" toAlternaria sp.

The BEST OPTION, AND OTHER OPTIONS of carrying out the INVENTION

Preferred embodiments of the present invention are described in detail with reference to the following series of examples only as an illustration.

Example 1 - Getting subjected to physiological stressArthrospira

CultureArthrospira maxima(obtained from Biovite Australia Pty Ltd) was subjected to stress by reducing the amount of nutrients or partial drying.Arthrospirawas dehydrated and dried, the result was obtained raw powder. DriedArthrospirathen was subjected to explosive decompression (potenzirovania) using the method described in the patents of New Zealand No. 328013 and No. 328740.Arthrospirawas re-wetted with water, bleached, dried and crushed, the result was received "advanced powder mixture. Preliminary powder mixture was then dissolved in a suitable medium (for example, in commercially available is m the cream is water-based production British Pharmacopoeia or in the water). Preliminary powder mixture is called "pre-powder mixture AMYCOT®".

Example 2 - determination of the activity under stressArthrospira

These respirometry suggest that subjected to physiological stressArthrospirait has the ability to modify or destroy the substrates containing chitin, chitosan and/or N-acetyl-D-glucosamine.

Chitin as substrate

The standard number of pre-mixed powder AMYCOT®and the raw powder separately used in the reaction with the polymer chitin BHD of the four links (Sigma, No. in catalogue C7170) Warburg respirometer using the standard method. Each composition was mixed with chitin, and was measured quantity of gas evolved. The reaction was carried out for one hour, then within 30 minutes was trim. The same composition, but pre-boiled for 5 minutes in a water bath, do not lead to the formation of any gas, which suggests that activity may be enzymatic and may be due to one or more types susceptible to denaturation of proteins.

The results in the graphs shown in Fig. 1. Specific activity (presumably, enzymatic activity) of the raw powder of the example is as 694,9 ml/g cell mass/g substrate/hour. The specific activity of pre-mixed powder AMYCOT®was 729,65 ml/g cell mass/g substrate/hour.

By using respirometry was also determined temperature optimum for the activity. Preliminary powder mixture AMYCOT®(0.02 g and 0.06 g) was incubated with chitin (0.02 g) at 20°C, 30°C, 33°C and 40°C. Prior powder mixture AMYCOT®the basis for the cream (0.05 g) was also incubated with chitin (0.02 g) at the same temperature. The results are graphically presented in Fig. 29. The optimum temperature for activity in both cases was 33°C.

By using respirometry was also optimal for the activity of the pH value. Preliminary powder mixture AMYCOT®(0.02 g) was suspended in buffers with different pH values (60-70% ethanol, 30-40% water, 0-0,05% phenolphthalein, 0-0,03% bronchiology blue, 0-0,02% methyl red, sodium salt, 0-0,01% sodium hydroxide and 0-0,01% methyl orange, sodium salt, pH 5, 7, 9 and 11) and was incubated with chitin (0.02 g) at 33°C. the Results are graphically presented in Fig. 30. It was determined that for activity the optimum pH value is 6.

These results suggest that the local application on the skin of the bioactive substance or substances must be active, because the skin has a similar temperature and pH.

<> N-acetyl-D-glucosamine and chitosan as substrates

Pre-mixed powder AMYCOT®(0.02 g) was separately incubated with N-acetyl-D-glucosamine (0.02 g, No. A8625-5G in the directory Sigma) and chitosan (0.02 g, No. C3646 in the directory Sigma) as substrates in a Warburg respirometer at 33°C. Each reaction was carried out for one hour, then within 30 minutes was trim.

Results for AMYCOT®plus N-acetyl-D-glucosamine presented in the table below.

Table 1
AMYCOT®+ N-acetyl-D-glucosamine
Time (min)mm gasmm gasmm gas
0000
5967
1012911
30121011
6012 1011

The table below shows the results in case AMYCOT®plus chitosan.

Table 2
AMYCOT®+ chitosan
Time (min)mm gasmm gasmm gas
0000
5121010
10141214
30171315
60171416

For comparison, the table below shows the results in case AMYCOT®plus chitin (0.02 g), incubated at 33°C.

td align="center"> mm gas
Table 3
AMYCOT®+ chitin
Time (min)mm gasmm gas
0000
5121015
10181820
30191821
60191921

These results suggest that subjected to physiological stressArthrospiracontains one or more bioactive substances that destroy or modify chitin, chitosan and/or N-acetyl-D-glucosamine, or perhaps another type of polymer containing N-acetyl-D-glucosamine as a subunit of the polymer. The bioactive substance may be, for example, chitinase, chitosanase or chitin-deacetylase.

Example 3 - Antifungal and antibacterial activity under stressArthrospira

Biocidal activity of the powder mixture prior AMYCOT®was tested byin vitrolive fungi and gram-positive bacter the s.

Live cultures of selected pathogens were obtained from American Type Culture Collection (ATCC), Australian Collection of Microorganisms (ACM) and Sullivan Niccolaides. Tested culture are presented in Table 4.

Table 4
PathogenNo. in ATCCNo. in ACMIsolates
Trichophyton mentagrophytes4808, 95335068
Candida albicans7534574
Trichophyton rubrumSN01 (Sullivan Niccolaides)
Epidermophyton floccosumSN02 (Sullivan Niccolaides)
Propionibacterium acne257465109

Uniform lawn of each pathogen was grown on plates containing medium potato dextrose agar (PDA). After 3-4 days of incubation in the PDA environment using sterile punch the Torah for tubes with a diameter of 8 mm were cut 4 holes. To each well was placed a certain amount of preliminary powder mixture AMYCOT®and control creams/powders. The Cup was cultivated in an environment intended for the respective pathogen, and viewed on a daily basis.

In 2-4 days around the holes were shown two concentric zones.

1. Less transparent circular area "enlightenment" around the hole. This area, in which the mushrooms were killed, leaving only residual material in the cytoplasm (which could be seen against the light and electron microscope).

2. Large translucent zone "damage"extending from the perimeter of enlightenment. The damage zone is less thick lawn, his lesser density and greater transparency in comparison with the pristine lawn. This is the area in which the cell wall of fungi were badly damaged, but some was still present, mainly in the form of rounded cytoplasmic areas (which could be seen against the light and electron microscope).

In each case, the measurement zones was carried out from the outer perimeter of the hole to the outer perimeter of the zone. This value is marked as the size of the zone (RZ). The radius of the zone of non-linear way reflects the efficiency of biocidal substances/substances in relation to the pathogenin vitro.

The results of the checkin vitropresented in Tab is itzá 5.

Table 5
No. in ACMPathogenResultThe size of enlightenment (RP) (mm)Size range (mm)
5068Trichophyton mentagrophytesEnlightenment610
4574Enlightenment6,5-
5109Propionibacterium acne(was cultivated under anaerobic conditions)There is no enlightenment--
No. in ATCC
4808Trichophyton mentagrophytesEnlightenment58
9533Trichophyton mentagrophytes Enlightenment610
753Candida albicansEnlightenment6,5-
25746Propionibacterium acne(was cultivated under anaerobic conditions)There is no enlightenment--
No. of isolates
SN01Trichophyton rubrumEnlightenment6-
SN02Epidermophyton floccosumEnlightenment58
301IsolateTinea pedisEnlightenment610
307The isolate causing the itch in the jockeysEnlightenment 10

In Fig. 2 shows an enlightenment forCandida albicans(ACM 4574) using AMYCOT®that testifies to the effectiveness of the actions of one or more biocidal substances in relation to disease thrush. Preliminary powder mixture AMYCOT®is in the 6 o'clock position, and "cream for testing on humans, containing 12,5% (mass/mass) AMYCOT®the cream is water-based - at 12 o'clock (RA = 9 mm).

In the case of anaerobic bacteriaPropionibacterium acnecausing acne, when tested in anaerobic conditions, the enlightenment was absent. The explanation for this is given in Example 4.

Example 4 is an Antibacterial activity under stressArthrospirain relation toPropionibacterium acne

According to respirometry, AMYCOT®has antibacterial activity againstP.acne.

Preliminary powder mixture AMYCOT®(0.02 g) and 5g "cream for testing on humans, containing 12,5% (mass/mass) AMYCOT®were separately added to the liquid medium in an anaerobic organismP.acnecausing acne (ACM No. 5109), Warburg respirometer in the presence or absence of oxygen. The results were compared with those for a control containing liquid medium withP.acnewithout AMYCOT®. In the absence of oxygen gas is not visualaids is. In the presence of oxygen gas were released, and each song had antibacterial activity.

Results for AMYCOT®and cream for tests on humans in the presence of oxygen are graphically presented in Fig. 3. (Presumably enzymatic) specific activity prior powder mixture AMYCOT®was 626,00 ml/g cell mass/g substrate/hour. The specific activity for cream for testing on humans was at 12.00 ml/g cell mass/g substrate/hour.

The results showed that the composition could have antibacterial actionin vivoin relation toP.acnerequires the presence of oxygen. Therefore, at least one biocidal substances can be oxidase.

The authors of the present invention is believed that one or more biocidal against fungi substances can destroy or modify the cell wall of fungi. Such substances can be chitinase, chitosanase or chitin-deacetylase.

AlthoughP.acneand other gram-positive bacteria are believed to not contain chitin or chitosan in their cell walls, it is assumed that one or more biocidal substances destroy or modify N-acetyl-D-glucosamine/composition of the bacterial cell wall.

Example 5 is Different is KOMMERCHESKIY available drugs Arthrospirapossess antifungal activity

Commercial preparations of driedArthrospira(powders, tablets and capsules) were obtained from various manufacturers in different departmental subordination, and were tested for the presence of antifungal activity. Typically, the powders are made by collectingArthrospira, washing the collectedArthrospiraand dryingArthrospira. Some vendors crushedArthrospirain the wet state. In the manufacture of sinks stage exposureArthrospiraspecific stress after collecting biomass, whichArthrospiraenters a state of suspended animation. Indeed, as noted above, commercial manufacturersArthrospiratry to reduce stress on the body, as thick mucosa tends to clog the equipment for processing of an organism.

PowdersArthrospira(Spirulina) were received from the following manufacturers:

1. China Spirulina - Jiangsu Cibainian Nutrition Food Co., Ltd. China;

2. Febico - Far East Biotech Co. Ltd. Taiwan;

3. Life Stream/Earthrise - DIC. California, USA;

4. Pacifica - Cyanotech. Hawaii, USA;

5. Siam Algae Co. - DIC. Thailand;

6. Spirin - Yunnan Spirin Co. Ltd. China;

7. Synergy - DIC. China.

Each powder was mixed with cream water-based concentration of 12.5% mass/mass. Then each cream was obession is. Uniform lawnT.rubrumwas grown on plates with the PDA environment. After 3-4 days of incubation in the PDA environment using sterile punch for tubes were made wells. Certain amount of each of the cream were placed in the appropriate hole, and the cups were then incubated for 2-4 days.

In Fig. 4 shows the antifungal activity of 12.5% (mass/mass) Siam Algae Co. (position 3 h)12,5% (mass/mass) Life Stream (6 o'clock) and 12.5% (mass/mass) China Spirulina (9 o'clock).

The results show that all tested commercial powders have some antifungal activity. These respirometry confirmed that all tested commercial powders by incubation with chitin possessed activity (presumably enzymatic) (results not shown).

One of the powders (Life Stream) was randomly selected for mixing with the nonprescription cream water-based concentration of 12.5% (mass/mass) without further processing and used for testing antifungal activity againstAspergillus niger. Cream led to the formation of enlightenment 4 mm, as seen in Fig. 5 in positions 12, 3, 6 and 9 hours. It showed that perhaps all commercial preparations of driedArthrospiraapparently possessed some level of antifungal activity, which removes neobhodimosti additional stage for the induction of stress or stage further processing, for example, the destruction of the filaments.

Example 6 Activity under stressArthrospiraagainst fungal diseases of mammals

Antifungal activity AMYCOT®was tested byin vitroagainst various fungal diseases in mammals. The following pathogens were obtained from the Department of Primary Industries, Department of Agriculture, ACM and kindergartens:

- Candida albicans;

Microsporum canis;

Trichophyton mentagrophytes;

Trichophyton rubrum;

- Epidermophyton floccosum;

- Fusarium frost;

- Alternaria sp.;

- Malassezia furfur.

Uniform lawn of each pathogen was grown on PDA environment. After 1-2 days of incubation in the medium PDA were made wells using sterile punch for tubes with diameter of 8 mm In each well was placed a certain amount of AMYCOT®. The cups were incubated at a suitable temperature, depending on the pathogen. Cups with holes were reviewed daily. Transparency was measured after 2-3 days. Area measurement was carried out from the perimeter of the hole to the outer perimeter of the zone.

This area was free from living cells of fungi that talked about the effectiveness of AMYCOT®against the selected pathogens.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment in 8 mm pathogenT.rubrum.T.rubrumcauses infection of the feet and ringworm is Yishai.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment in 15 mm pathogenT.mentagrophytes.T.mentagrophytescauses infection of the feet and ringworm.

In Fig. 6 shows the hole from 12.5% (mass/mass) cream AMYCOT®that led to the formation of enlightenment 3 mm pathogenM.canis.M.caniscauses infection of the feet and ringworm.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment 3 mm pathogenE.floccosum.E.floccosumcauses infection of the feet and ringworm.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment 4.5 mm pathogenC.albicans.C.albicanscauses ringworm and dermatitis.

In Fig. 7 shows the hole from 12.5% (mass/mass) cream AMYCOT®that led to the formation of enlightenment in 12 mmF.frost.F.frostcauses infection of the hoof.

In Fig. 8 shows the hole from 12.5% (mass/mass) cream AMYCOT®that led to the formation of enlightenmentM.furfur.M.furfurcauses dandruff.

In Fig. 9a shows a hole with 5% (mass/mass) shampoo AMYCOT®that led to the formation of enlightenment 3 mm pathogenT.rubrum.

In Fig. 9b shows the hole with 5% (mass/mass) shampoo AMYCOT®that led to the formation of enlightenment 4 mm pathogenC.albicans.

Example 7 Akti is the ability under stress Arthrospiraagainst fungal diseases of plants

Antifungal activity AMYCOT®was tested byin vitroagainst various fungal diseases of plants. The following pathogens were obtained from the Department of Primary Industries, Department of Agriculture, ACM and kindergartens:

- Fusarium frost;

- Pithomyces Chartarum;

-Botrytis cinerea;

- Alternaria sp.

Uniform lawn of each pathogen was grown on PDA environment. After 1-2 days of incubation in the medium PDA were made wells using sterile punch for tubes with diameter of 8 mm In each well were made a certain number of AMYCOT®. The cups were incubated at a suitable temperature depending on the pathogen. Cups with holes viewed daily.

Transparency was measured after 2-3 days. Area measurement was performed from the perimeter of the hole to the outer perimeter of the zone.

The area was free from living cells of fungi that talked about the effectiveness of AMYCOT®against the selected pathogens.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment in 13 mm pathogenF.frost.F.frostcauses the ears wilting and rotting of the root collar for small grains.

In Fig. 10 shows the hole from 12.5% (mass/mass) cream AMYCOT®that led to the formation of enlightenment in 10 mmP.chartarum.P.chartarumyou who indicates eczema muzzle in sheep.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment in 8 mm pathogenB.cinerea.B.cinereacauses grey mould on fruits.

12,5% (mass/mass) cream AMYCOT®resulted in the formation of enlightenment in 9 mm pathogenAlternaria sp., which causes leaf spot fruit plants.

Example 8 Testing AMYCOT®in the form of a spray against fungal plant pathogens

AMYCOT®in the form of antifungal aerosol was tested byin vitroagainst widespread among plant pathogenBotrytis cinerea. 5% (mass/mass) powder AMYCOT®was moistened with water, but turned out to be poorly soluble.

Uniform lawnB.cinereawas grown on the Cup with the PDA environment. Then the right side of the Cup was treated with aerosol and incubated for 24 hours at room temperature. The left side of the Cup aerosol has not been previously processed. In Fig. 11 shows that the treated spray the side of the Cup contains dead mushrooms. The spray was effective for the destruction of gray rot and to suppress further sporulation.

Example 9 - Antimicrobial testsin vitrowith subjected to stressArthrospiraand known drugs

In this and other examples were tested for antifungal and antibacterial activity of various nonprescription against Gribkova and antibacterial creams (including Lamisil TM, Dakta GoldTM, CanestenTM, TinadermTM, DaktarinTM, Tripod LabsTM, Resolve TineaTM, Resolve BalmTM, Resolve PlusTM, ClearasilTM, Benzac WTMand AMYCOT®.

In this example, antifungal and antibacterial activity of various nonprescription creams and AMYCOT®been tested in an independent laboratory (ConMac Laboratory Services) against selected pathogensTrichophyton mentagrophytes,Epidermophyton floccosum,Trichophyton rubrumandPropionibacterium acne.

The results are presented in Table 6.

Table 6
Selected pathogenTest creamEnlightenment Day 1Enlightenment Day 2Enlightenment Day 3
Trichophyton mentagrophytes1)Controlnot foundnot foundnot found
2)Amycot®0.5-1 mm opaque, round4mm opaque, round1-2 mm transparent, round 3-4 mm opaque, round
3)Lamisilnot foundnot foundnot found
4)Dakta Goldnot foundnot foundnot found
Epidermophyton floccosum1)Controlnot foundnot foundnot found
2)Amycot®1 mm, transparent, round2.5 mm transparent, round2.5 mm transparent, round
5 mm opaque, round
3)Canestennot foundnot foundnot found
4)Lamisilnot foundnot foundnot found
Trichophyton rubrum1)Controlnot foundnot found2)Amycot®1 mm, transparent, round5.5 mm transparent, round5.5 mm transparent, round
4mm opaque, round
3)Tinadermnot foundnot foundnot found
4)Lamisilnot found1 mm transparent, rugged1.5 mm transparent, rugged
Propionibacterium acne1)Controlnot foundnot foundnot found
2)Amycot®not foundnot foundnot found
3)Clearasilnot foundnot foundnot found
4)Benzac Wnot foundnot the detection is but not found

The results show that only AMYCOT®was active against each pathogen. The only non-AMYCOT®cream with activity, was cream LamisilTMwith activity againstT.rubrum.

Example 10 -Testsin vitroc known substances and combinations AMYCOT®

Antifungal agents such as miconazole, tolnaftate, bifonazole and clotrimazole, act by suppressing the formation of specific proteins, interfering with normal functions such as reproduction. The antifungal agent terbinafine inhibits the formation of vital steroids.

The first aim of these tests isin vitroit was the determination of the efficiencies of various over-the-counter creams or make up their composition active components as inhibitors of fungi and comparison with the efficiency of AMYCOT®. The second objective was to determine the effectiveness of actions AMYCOT®in combination with one or more nonprescription creams or their parts with the active compounds as inhibitors of fungi.

Liquid Sabouraud's medium was planted-selected pathogen. Then the culture was incubated for one week at 28°C. In a medium containing the pathogen, immersed sterile the tampon, and cups with medium PDA planted strokes at right angles, providing a smooth lawn in cups.

Known substances that are not included in the basis for cream, mixed with an appropriate basis for the cream. Selected the cream was placed on a disk of filter paper with a diameter of 10 mm, after which the disc was placed on a newly seeded Cup. One Cup was located up to four different drives. The Cup was then incubated for 24-48 hours at a suitable temperature, depending on the pathogen. After 24-48 hours took pictures.

The results of the tests on inhibition shown in Fig. 12-21 and in tables 7-16 below.

Table 7
Test inhibition (Fig. 12)
Pathogen:Trichophyton rubrum
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream8
3:00Tinaderm (tolnaftate 2%)0
6:00Placebo0
9:00Daktarin (miconazole 2%) 15

Table 8
Test inhibition (Fig. 13)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream4
3:00Tinaderm (tolnaftate 2%)1
6:00Placebo0
9:00Daktarin (miconazole 2%)11

Table 9
Test inhibition (Fig. 14)
Pathogen:Candida albicans
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream2
3:00Tinaderm (tolnaftate 2%)3
6:00 Placebo0
9:00Daktarin (miconazole 2%)15

Table 10
Test inhibition (Fig. 15)
Pathogen:Candida albicans
SubstanceThe size of the enlightenment (mm)
Tripod Labs (clotrimazole 1% + tea tree oil)10

Table 11
Test inhibition (Fig. 16)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:0050% AMYCOT®12.5% cream + 50% Daktarin (miconazole 2%)10
3:00Resolve Tinea (miconazole 2%)10
6:00Resolve Balm (miconazole 2%)7
9:00Resolve Plus (miconazole 2%)11

Table 12
Test inhibition (Fig. 17)
Pathogen:Candida albicans
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®+ Daktarin10
3:00Resolve Tinea (miconazole 2%)6
6:00Resolve Balm (miconazole 2%)7
9:00Resolve Plus (miconazole 2%)11

Table 13
Test inhibition (Fig. 18)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream1
3:00AMYCOT®12% + clotrimazole 10%13
6:00AMYCOT®12% + terbinafine 1% 1
9:00AMYCOT®12% + tolnaftate 10%1

Table 14
Test inhibition (Fig. 19)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream1
3:00AMYCOT®12% + clotrimazole 10%11
6:00AMYCOT®12% + terbinafine 1%2
9:00AMYCOT®12% + tolnaftate 10%4

Table 15
Test inhibition (Fig. 20)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream + miconazole 2%11
3:00Placebo0
6:00Resolve Balm (miconazole 2%)7
9:00Resolve Plus (miconazole 2%)7

Table 16
Test inhibition (Fig. 21)
Pathogen:Candida albicans
PositionSubstanceThe size of the enlightenment (mm)
12:0050% AMYCOT®12.5% cream + 50% Daktarin (miconazole 2%)14
3:001% clotrimazole4
6:0010% tolnaftate0
9:002% miconazole9

In the case of Fig. 21 of a known substance contained in the cream is water-based, unlike the commonly used alcohol based.

In testsin vitroit turned out that miconazole and clotrimazole are the most effective inhibitors pathologically the fungi (see Fig. 12-21). Fig. 16 and 17 and tables 11 and 12 prove that miconazole is an effective inhibitor of fungi. AMYCOT®also acts as an inhibitor, but its efficiency is about 40% efficiency clotrimazole and miconazole (see Fig. 12-14). Fig. 18-21 and tables 13-16 suggest that combination AMYCOT®plus miconazole and AMYCOT®plus clotrimazole are the most effective fungal inhibitorsin vitrocompared with terbinafine and tolnaftate.

The third objective of these testsin vitroit was the determination of the efficiencies of various over-the-counter creams or part of them active compounds as fungicides and comparison with the efficiency of AMYCOT®. The fourth objective was to determine the effectiveness of actions AMYCOT®in combination with known substances.

Fungicidal test involved the cultivation of the lawn with the pathogen on cups with medium PDA within 2-3 days and then exposed grown pathogen with antifungal agents. This was done by cutting holes in covered lawn agar, remove agar and fill holes with cream.

The results fungicidal test shown in Fig. 22-28 and tables 17-23 below.

Table 17 Fungicidal test (Fig. 22)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream6
3:00AMYCOT®12.5% cream 1% terbinafine6
6:00AMYCOT®12.5% cream + 10% tolnaftate6
9:00AMYCOT®12.5% cream + 10% clotrimazole5

Table 18
Fungicidal test (Fig. 23)
Pathogen:Trichophyton rubrum
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream9
3:00AMYCOT®12.5% cream + 10% clotrimazole7
6:00AMYCOT®12.5% cream + 1% miconazole 8
9:00Placebo0

Table 19
Fungicidal test (Fig. 24)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream6
3:00AMYCOT®12.5% cream 1% terbinafine5
6:00AMYCOT®12.5% cream + 10% tolnaftate5
9:00AMYCOT®12.5% cream + 10% clotrimazole4

Table 20
Fungicidal test (Fig. 25)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream8
3:00AMYCOT®12.5% cream 1% terbinafine7
6:00AMYCOT®12.5% cream + 10% tolnaftate5
9:00AMYCOT®12.5% cream + 10% clotrimazole4

Table 21
Fungicidal test (Fig. 26)
Pathogen:Trichophyton mentagrophytes
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream9
3:00AMYCOT®12.5% cream 1% terbinafine4
6:00AMYCOT®12.5% cream + 10% tolnaftate5
9:00AMYCOT®12.5% cream + 10% clotrimazole5

Table 22
Fungicidal test (Fig. 27)
Pathogen:Candida albicans
ulozhenie SubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream8
3:00Daktarin (miconazole 1%)0
6:00Canesten (clotrimazole 1%)0
9:00AMYCOT®125% + clotrimazole 1%5

Table 23
Test inhibition (Fig. 28)
Pathogen:Trichophyton rubrum
PositionSubstanceThe size of the enlightenment (mm)
12:00AMYCOT®12.5% cream6
3:0010% clotrimazole0
6:0010% tolnaftate0
9:002% miconazole0

In the case of Fig. 28 known substances are contained in to the Birmingham water based unlike alcohol based, commonly used.

The results confirm that the nonprescription antifungal substances and known substances are fungistatic substances and fungicides are not. Fig. 22-28 and tables 17-23 show that AMYCOT®is an effective fungicide in conjunction with known substances.

The combination AMYCOT®with miconazole or clotrimazole did not lead to a significant reduction in the effectiveness of each. The combination of increased inhibitory effect (see Fig. 17-21). This is talking about creating a very effective product with a broad spectrum of action.

The combination AMYCOT®with clotrimazolum weakly inhibited antifungal activity AMYCOT®(see Fig. 22-28), but the combination AMYCOT®with miconazole had no significant adverse effects on inhibitory or antifungal activity combined cream (see Fig. 17-26).

Combined cream with AMYCOT®and miconazole was more effective as a product of broad-spectrum compared to the other due to the inhibitory properties of miconazole and how inhibitory and fungicidal activity AMYCOT®.

Cream containing such a combination, acted as an inhibitor and then did not take a position", whereas enlightenment is obtained only from the ne by miconazole, violated in the re-growth, as in the case of pathogenT.mentagrophytes(see Fig. 20).

It was also found that the combination of cream AMYCOT®with 2% miconazole inhibits the re-growth of the pathogen after enlightenment, formed around the hole.

Example 11 - TestArthrospirawhen various skin diseases

The subject had a large athletic foot deep lesions between the toes. The subject reported that is caused by the fungus itching stopped after 15 minutes after the first application of 12.5% (mass/mass) of cream AMYCOT®. Irritation decreased and in some cases completely disappeared within 12 hours. The damage began to take 24 hours and fully healed in 4-5 days.

Similar picture was observed in the case of acne in subjects who noted that concomitant irritation disappeared and that their skin got normal color and become soft and pliable like a new skin. Pustules were also dried up.

According to the authors of the present invention, one or more biocidal substances (such as chitinase, chitosanase, chitin-deacetylase) interact synergistically with other components of theArthrospirathat enhances therapeutic effect. Fungal infections itching is an indicator of the release of mushrooms digestive enzymes or metal the litas. Itching stops as soon as interferes with the normal metabolism of fungi that occurs when the destruction of the cell wall.

Similar results were obtained when using the cream for diseases that are not caused by fungus, in the case of dry and cracked heels and elbows, rosacea, eczema, sunburn and psoriasis. Subjects reported almost immediate cessation of itching with dermatitis.

Example 12 - Antifungal activity under stressArthrospirawhen using raw powder

Example 5 confirmed that commercial preparations of driedArthrospirahad, apparently, a certain level of antifungal activity that does not require additional stages of the stressful effects or stage of processing of the collected biomass, such as the destruction of the filaments. Example 12 further confirms this observation.

The dried drugArthrospira maximawas obtained from a commercial manufacturer. The drug was mixed with nonprescription cream water-based concentration of 12.5% (mass/mass) without further processing, and its antifungal activity was tested againstT.mentagrophytes,T.rubrumandM.fructicola(as described in Example 5).

As shown in Fig. 31, cream resulted in the formation of enlightenmentT.mentagrophytesin positions 2, 3, 6 and 9 o'clock.

As can be seen in Fig. 32, cream resulted in the formation of enlightenmentT.rubrumat 6 o'clock.

As can be seen in Fig. 33, cream resulted in the formation of enlightenmentM.fructicolaat 6 o'clock.

These observations confirm that to receive theArthrospirapossessing fungicidal activity, there is no need for the stages following the collection of biomass, such as explosive decompression, freeze drying, discoloration and grinding.

Example 13 - Antifungal activity ofArthrospiranot podvergnutoi explosive decompression

This example further confirms that to receive theArthrospirapossessing antifungal activity, which does not require a stage of explosive decompression (potentiation), which follows the stage of biomass harvesting.

The dried drugArthrospira maximawas obtained from a commercial manufacturer. In one case, driedArthrospirawas destroyed with the help of explosive decompression (potenzirovania) using the method described in the patents of New Zealand No. 328013 and No. 328740.Arthrospirawas re-soaked, bleached, dried and premalatha, the result was obtained dry "pre-powder mixture. Preliminary powder mixture was then mixed with a suitable carrier (12.5%) and got called by the W "reinforced AMYCOT ®".

In another case, when manufacturing was excluded method explosive decompression described in the patents of New Zealand No. 328013 and No. 328740, and such a drug called "original AMYCOT®".

As can be seen in Fig. 34 as "original AMYCOT®and reinforced AMYCOT®" had resulted in almost the same enlightenmentAlternaria sp. in positions 12 and 7 hours, respectively. Thus, to obtainArthrospirapossessing fungicidal activity, the stage of explosive decompression following the collection of biomass is not significant.

You should additionally take into account that can be made many changes in the composition, methods of application and receipt of, illustrated in the above examples, without going beyond certain limits and scope of the invention.

The term "contain" and variations of that term "contains" and "contain" is used here to denote the inclusion of a stated number or the stated numbers, but does not exclude any other number or other number, unless the context or use does not require special interpretation of the term.

Any references cited here are not published, as in the Australian open are public knowledge.

1. A method of obtaining a composition based on Arthrospira for local use in individas as a biocide or as a means to restore or prevent defects in the skin of a mammal, which includes stages
(1) maintain physiological stress Arthrospira, but not drying freezing Arthrospira; and
(2) Association number, effective as a biocide or as a means for recovery or prevention of skin defects, Arthrospira under stress, (1) with a carrier, solvent, base or excipient suitable for topical application in the individual, and effective as a biocide amount is effective against fungi, gram-positive bacteria or viruses.

2. The method according to claim 1, in which Arthrospira is a A.maxima.

3. Composition based Arthrospira for local use at the individual as the biocide or the means to restore or prevent defects in the skin of a mammal, obtained by the method according to claim 1.

4. A method of obtaining a composition based on Arthrospira for local use at the individual as a biocide against fungi, gram-positive bacteria or viruses, or the means to restore or prevention of skin defects in a mammal, which includes stages
(1) maintain physiological stress Arthrospira by removing approximately 80% of the liquid in which the Arthrospira is cultivated;
(2) washing stressed Arthrospira to remove contaminants;
(3) drying the washed Arthrospira; and
(4) Association number, effective as of the biocide or the means to restore or prevention of skin defects, Arthrospira obtained at stage (3), with a carrier, solvent, base or excipient suitable for topical application in person or mammal, and Arthrospira not dried by the freeze.

5. Composition based Arthrospira for local use at the individual as the biocide or the means to restore or prevent defects in the skin of a mammal, obtained by the method according to claim 4.

6. Fungicidal composition for prevention or treatment of local fungal infection or infection at the individual, containing the number, effective as fungicide subjected to physiological stress Arthrospira and a carrier, solvent, base or excipient suitable for topical application in the individual, and Arthrospira not dried by the freeze.

7. The method of prevention or treatment of local fungal infection or infection in the individual, which includes a step of local administration of the individual composition according to claim 6.

8. The method according to claim 7, further comprising the initial stage of identification of an individual in need of such treatment or in need of such prevention.

9. The method according to claim 7, in which the individual is a person, animal, agricultural or horticultural product, soil or an artificial structure.

10. A method of obtaining a fungicidal composition for topical application according to claim 6 d is I the prevention or treatment of local fungal infection in the individual, which includes stages
(1) maintain physiological stress Arthrospira;
(2) Association number, effective as a fungicide, stressed Arthrospira (1) with a carrier, solvent, base or excipient suitable for topical application in the specified individual, and Arthrospira not dried by the freeze.

11. Fungicidal composition for topical use for the prevention or treatment of local fungal infection or infection of an individual, contain a synergistic combination of number, effective as fungicide subjected to physiological stress Arthrospira and at least one mould tools and media, solvent, base or excipient suitable for topical application in the specified individual, and Arthrospira not dried by the freeze.

12. Fungicidal composition for topical application according to claim 11, in which fungistatic agent selected from the group consisting of terbinafine, bifonazole, clotrimazole, miconazole, econazole, ketoconazole and tolnaftate.

13. The method of prevention or treatment of local fungal infection or infection in the individual, which includes a step of local administration of the individual the composition according to item 11.

14. The method according to item 13, in which fungistatic agent selected from the group consisting of terbinafine, bifonazole, LOTR is masala, miconazole, econazole, ketoconazole and tolnaftate.

15. A method of obtaining a fungicidal composition for topical application according to claim 11 for the prevention or treatment of local fungal infection or contamination of the individual, which includes stages
(1) maintain physiological stress Arthrospira;
(2) combining the composition effective as a fungicide, stressed Arthrospira (1) at least one fungistatic agent and a carrier, solvent, base or excipient suitable for topical application in the individual, and Arthrospira not dried by the freeze.

16. Bactericidal composition for topical application to prevent or treat local infections caused by gram-positive bacteria, the individual that contains the number, effective as bactericidal subjected to physiological stress Arthrospira and a carrier, solvent, base or excipient suitable for topical application in the individual, and Arthrospira not dried by the freeze.

17. The method of prevention or treatment of local infections caused by gram-positive bacteria or infection in the individual, which includes a step of local administration of the individual composition according to item 16.

18. The method according to 17, further comprising the initial stage of identification of an individual in need of such treatment is whether in need of such prevention.

19. The method according to 17, in which the bacterial infection is caused by gram-positive bacteria Propionibacterium acne.

20. The method according to 17, in which the individual is the man.

21. The method of obtaining the antibacterial composition against gram-positive bacteria for local use in article 16, for the prevention or treatment of local infections caused by gram-positive bacteria, which includes stages
(1) maintain physiological stress Arthrospira; and
(2) Association number, effective as bactericidal against gram-positive bacteria, stressed Arthrospira (1) with a carrier, solvent, base or excipient suitable for topical application in the specified individual, and Arthrospira not dried by the freeze.

22. Antiviral composition for topical application to prevent or treat local viral infection in the individual, containing the number, effective as antiviral, stressed Arthrospira and a carrier, solvent, base or excipient suitable for topical application in the specified individual, and Arthrospira not dried by the freeze.

23. The method of prevention or treatment of local viral infection in the individual, which includes a step of local administration of the individual an effective amount of the composition according to item 22.

24. The way pop, additionally includes the stage of initial detection of an individual in need of such treatment or in need of such prevention.

25. The method according to paragraph 24, in which the individual is the man.

26. The method of obtaining antiviral composition for local application on p.22 for the prevention or treatment of local viral infection in the individual, which includes stages
(1) maintain physiological stress Arthrospira; and
(2) Association number, effective as antiviral, stressed Arthrospira (1) with a carrier, solvent, base or excipient suitable for topical application in the specified individual, and Arthrospira not dried by the freeze.

27. Composition for healing or prevention of skin defect for topical application in a mammal, where the specified composition comprises an amount, effective as a biocide subjected to physiological stress Arthrospira and a carrier, solvent, base or excipient suitable for topical application in a mammal, the amount, effective as a biocide that is effective against fungi, gram-positive bacteria or viruses, it Arthrospira not dried by the freeze.

28. Method for healing or prevention of skin defect in a mammal, which includes the local stage is about the introduction to the mammal a composition according to item 27.

29. The method according to p, further comprising the initial stage of identifying a mammal in need of such treatment or in need of such prevention.

30. The method according to p, in which the skin defect selected from the group consisting of pockmarks, prevage damage, pink eel, red spots, cracks, burns, blisters, psoriasis, eczema, peeling, wrinkles, pimples, sores, lesions, blisters, wounds, seborrheic dermatitis, diaper rash, sores, herpes, rashes after shaving, chicken pox, dermatitis and cracks on the heels and elbows.



 

Same patents:

FIELD: chemistry; biochemistry.

SUBSTANCE: present invention pertains to biochemistry. To obtain lysozyme enzyme and avidin protein from albumen, chromatography of albumen homogenisate is carried out on silochrome C-80 at neutral pH (7.0-7.5). Ballast proteins are removed in a 0.2 M glycine - NaOH buffer in the presence of 0.3 M NaCl at pH 9.3-9.5. Target proteins are eluted together with carbonate buffer with 0.5-0.8 M NaCl, with pH 10.8-11.0, and subsequent separation of proteins through gel filtration on a column with sephadex G-75.

EFFECT: simplification of the method of obtaining electrophoretically pure proteins and increasing the efficiency of the method.

1 dwg, 4 ex

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to the microorganism strain Klebsiella pneumoniae GISK № 278 isolated from a patent feces suffering from intestine dysbacteriosis. The strain is used for preparing an agent for producing the lysozyme inhibitor. The level of activity of lysozyme inhibitor produced by this strain is 1.64-2.16 mcg/ml of *OD value.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

The invention relates to a process for the production of lysozyme from egg white

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The invention relates to the Enzymology and biotechnology and can be used in microbiological industry to obtain the complex enzyme preparation containing-1,3-glucanase and chitinase, and to obtain each of these enzymes separately

FIELD: medicine.

SUBSTANCE: antagonistic activity of lactobacterium and/or bifidobacterium is determined simultaneously by method of double-layer agar and method of inverted agar, inoculation being performed by Gold. Power of antagonistic impact of probiotic strains of opportunistic microorganisms (OM) in comparison with control is estimated by degree of strain inhibition intensity. Antagonistic activity of probiotics is detected by one or two of said methods. In case of individual detection of antagonistic activity of probiotic medications, containing lactobacteria, in case of absence of antagonistic activity or detection of low or medium degree of intensity of OM inhibition, antagonistic activity is additionally detected by drop method.

EFFECT: increase of method accuracy and extension of spectrum of optimally selected probiotic medications for particular patient, simplification of inoculation and account of obtained result.

2 cl,3 dwg, 10 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and methods of cleaning soil and the surface of solid objects from petroleum and petroleum products. The method involves treatment of the contaminated object with a microbial preparation consisting of a mixture of natural hydrocarbon-oxidising cultures of microorganisms isolated through selection from a natural biocenosis of microorganisms. The preparation used is based on a biocenosis of microorganisms inhabiting sea brown algae of at least one genus, and the treated object is further treated with water and a biogeneous additive.

EFFECT: invention increases the rate and efficiency of cleaning solid objects from petroleum and petroleum products even with high salt concentration and relatively low temperature, including in regions with a short warm period.

4 cl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and methods of cleaning salty and fresh water from petroleum and petroleum products. The method involves addition to contaminated water of a microbial preparation which consists of a mixture of natural hydrocarbon-oxidising cultures of microorganisms, which are isolated through selection from a natural biocenosis of microorganisms, where the preparation used is based on a biocenosis of microorganisms inhabiting sea brown algae of at least one genus.

EFFECT: invention increases the rate and efficiency of cleaning water from petroleum and petroleum products even with high salt concentration and relatively low temperature, including in regions with a short warm period.

2 cl, 10 ex

FIELD: medicine.

SUBSTANCE: method related to field of medicine, namely to transfusiology. Method includes collecting umbilical cord blood (UCB) into container, weighing it with calculation of its volume, determining type of human leukocyte antigen for comparing hypocompatibility, presence or absence of infection, introduction into packet with UCB, anticoagulant and hydroxyethyl starch, mechanical mixing of packet content in various planes, incubation after mixing at room temperature with separation of erythrocyte mass (EM) by sedimentation, placement of centrifuge chamber in separator, connecting to it on symmetry axis main pipeline, the latter being connected via distribution unit via pipelines to sacks for collection of blood components and to packet with mixture of UCB, anticoagulant and hydroxyethyl starch. After that, chamber is filled with packet content, and mixture is subjected to separation, with its separation into EM, plasma and leukocyte concentrate with their distribution into corresponding sacks and into empty packet. After separation, viability and amount of SC are determined, they are checked on biological inoculation of sample of plasma and EM mixture, dose of plasma is tested for detection of antibodies, hemotransmissive infections, dose of EM is used to determine blood group and Rhesus factor.

EFFECT: method application makes it possible to increase quality and reliability of determining SM from various biological contaminations.

1 dwg

FIELD: food industry.

SUBSTANCE: invention is intended for use in production of fodders. The method envisages production of liquid cultures of Bacillus subtilis VKPM V-8130, Bacillus subtilis VKPM V-2984, Bacillus subtilis VKPM V-4099 and Bacillus licheniformis VKPM V-4162 by way of their separate aerobic in-depth cultivation in nutrient media of a specified composition. The produced liquid cultures of Bacillus subtilis are mixed at a 6:6:1 ratio accordingly, applied onto a sterile mixture of beet cake and wheat bran preliminarily treated with a cellulolytic enzyme dissolved in a nutrient medium. PH and batch moisture content are brought to 7.5-8.0 and 43-48% accordingly; the mixture is thoroughly stirred and one proceeds with solid phase fermentation under restricted oxygen access conditions during 48 h at a temperature of 45-50°C and drying for moisture content to be 8-10%. Bacillus licheniformis VKPM V-4162 liquid culture is immediately mixed with beet cake and dried till moisture content is 8-9%. The dried products are mixed and crushed for a homogenous product production.

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3 cl, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: by means of expression vector into plant cell introduced are nucleotide sequences, coding light and heavy chains of antibody, binding human VEGF. Antibody against human VEGF can be used, in particular, for reduction of microvascular permeability of human tumours and treatment of diabetic and age-related neovascular retinopathy.

EFFECT: antibody production in plant cells provides possibility of its obtaining in industrial scale, at a significantly lower cost than in obtaining in expression system on mammalian cell culture base, presence in obtained preparation of antibody of causative agents of prion, mycoplasmal and viral diseases of mammals is excluded.

39 cl, 11 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention describes modified genetically constructed fluorescence-possessing fluorescent protein. Protein possesses higher speed of ripening at temperature 20°C and higher. Also described are nucleic acids, coding it, vectors, expression cassettes and host-cells, containing said nucleic acids.

EFFECT: group of inventions can be used in many various applications and methods, in particular for labelling of biomolecules, cells or cell organelles.

15 cl, 8 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention describes modified genetically constructed fluorescence-possessing fluorescent protein. Protein possesses higher speed of ripening at temperature 20°C and higher. Also described are nucleic acids, coding it, vectors, expression cassettes and host-cells, containing said nucleic acids.

EFFECT: group of inventions can be used in many various applications and methods, in particular for labelling of biomolecules, cells or cell organelles.

15 cl, 8 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: invention relates to method of display with application of filamentous phage, as well as to phage or phagmid vectors and libraries of such vectors, applied in the method. Method lies in constituting vector in form of filamentous phage or phagmid, containing expression cassette for fused polypeptide, which has terminal signal sequence, selected from group of signal sequences TorT, SfmC, TolB and DsbA. After that, combinatorial library of phage or phagmid vectors is constructed by cloning library of DNA, coding analysed polypeptides in expression cassette of obtained at previous stage vector, corresponding Gram-negative bacteria are transformed by such library and selection cycle is realised by method of phage display.

EFFECT: method makes it possible to display polypeptides, about which it is known that they can be badly displayed on phages, as well as for proteins of cDNA libraries and other combinatorial libraries, especially those, which are formed as a result of very fast packing, stable protein support.

5 cl, 6 dwg, 3 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: antagonistic activity of lactobacterium and/or bifidobacterium is determined simultaneously by method of double-layer agar and method of inverted agar, inoculation being performed by Gold. Power of antagonistic impact of probiotic strains of opportunistic microorganisms (OM) in comparison with control is estimated by degree of strain inhibition intensity. Antagonistic activity of probiotics is detected by one or two of said methods. In case of individual detection of antagonistic activity of probiotic medications, containing lactobacteria, in case of absence of antagonistic activity or detection of low or medium degree of intensity of OM inhibition, antagonistic activity is additionally detected by drop method.

EFFECT: increase of method accuracy and extension of spectrum of optimally selected probiotic medications for particular patient, simplification of inoculation and account of obtained result.

2 cl,3 dwg, 10 tbl

FIELD: medicine.

SUBSTANCE: invention relates to medicine, in particular to cosmetology. Anti-eczema medication contains propylene glycol extract of medicinal herbs of common tormentil, Achillea millefolium, Aloe arborescens, German chamomile, St John's wort, three-lobe beggarticks taken in definite ratio, polypropylene extract of pig placenta and, cyclomethicone DC 345, elastomer DC 9045, avocado oil, emulsifier DC 5329, acrylate emulsion of copolymer Salcare SC80, triethanolamine, fragrance, preservative Kathon CG and slightly mineralised spring water, with definite component content.

EFFECT: medication is efficient in case of eczema, possesses anti-inflammatory and bactericidal action, regenerates, enhances skin tone and elasticity.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: method related to field of medicine, namely to transfusiology. Method includes collecting umbilical cord blood (UCB) into container, weighing it with calculation of its volume, determining type of human leukocyte antigen for comparing hypocompatibility, presence or absence of infection, introduction into packet with UCB, anticoagulant and hydroxyethyl starch, mechanical mixing of packet content in various planes, incubation after mixing at room temperature with separation of erythrocyte mass (EM) by sedimentation, placement of centrifuge chamber in separator, connecting to it on symmetry axis main pipeline, the latter being connected via distribution unit via pipelines to sacks for collection of blood components and to packet with mixture of UCB, anticoagulant and hydroxyethyl starch. After that, chamber is filled with packet content, and mixture is subjected to separation, with its separation into EM, plasma and leukocyte concentrate with their distribution into corresponding sacks and into empty packet. After separation, viability and amount of SC are determined, they are checked on biological inoculation of sample of plasma and EM mixture, dose of plasma is tested for detection of antibodies, hemotransmissive infections, dose of EM is used to determine blood group and Rhesus factor.

EFFECT: method application makes it possible to increase quality and reliability of determining SM from various biological contaminations.

1 dwg

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