Method of obtaining melanin from chaga

FIELD: chemistry.

SUBSTANCE: method of obtaining melanin from chaga, includes addition of 25% solution of hydrochloric acid to a water extract of chaga, separation of the obtained melanin sediment, addition to the sediment of petroleum-ether, mixing until a mixture of a homogeneous consistency is obtained, the obtained mixture is frozen, after the mixture defrosting a layer of an organic extract is removed, and a sediment of melanin is separated from the water layer and dried under the specified conditions.

EFFECT: method makes it possible to reduce a quantity of sol substances in melanin without changing its biological activity.

1 tbl, 4 ex

 

The invention relates to chemical-pharmaceutical industry, namely to obtain melanin from chaga low ash content and high biological activity.

The main active component of the fungus, providing its therapeutic activity is melanin, previously called Chromogens or polyphenols, or polifenoloksikarbonovy complex. When the water extraction of the fungus the fungus forms a colloidal system in which the melanin forms a dispersed phase. Adding to aqueous extract of chaga some electrolytes from among neutral salts and mineral acids leads to the deposition of melanin (Yakimov P.A. Comprehensive study of physiologically active substances of lower plants. - M.-L. Science. - 1961. - S. 113-119). The method of obtaining the extract, and the nature of the precipitating agent affect the structural organization and biological activity of secreted melanin, in particular on the ash content of the substances involved in the formation of melanin particles.

Known methods for producing melanin by adding a 20%aqueous solution of hydrochloric acid to the extracts of the fungus, obtained using aqueous solutions of certain concentrations of dimethyl sulfoxide (patent RU №2392952, A61K 36/06, A61P 39/06, 2010), sodium hydroxide or potassium (patent RU №2465911, A61K 36/06, B01D 11/02, 2012). The precipitate melanin finish Aut and dried.

Know the use of 9-20%-aqueous solution of calcium chloride for the deposition of melanin from aqueous extracts of the fungus (patent RU 2442596, A61K 36/06, B01D 11/02, 2012).

The disadvantage of these methods is the high content of ash matter in the obtained melanin, which is partly due to the participation of ash elements contained in the extractants and precipitating agents in the formation of melanin particles.

High content medicines ash elements, particularly potassium and sodium, disrupts the osmotic pressure of the cells and electrolyte balance of the gastrointestinal tract, irritates the mucous membranes, prevents rapid healing of erosions and ulcers (Bandages F.T. Some aspects of the toxicity of metal ions. - M.: Mir, 1993.).

The closest technical solution, taken as a prototype, is a method for melanin, in which water extract of chaga add 25% hydrochloric acid solution to pH 1.0 to 2.0, the resulting mixture is stirred and left for 30 minutes, precipitated dark brown melanin precipitate is separated and dried (the State Pharmacopoeia of the USSR XI. V.2. General methods of analysis. - M.: Medicine; 1990. - S. 342-343.).

The disadvantage of this method is the high content of ash matter in the obtained melanin.

The technical object of the present invention is to reduce the content of C is selected substances in the melanin fungus.

This object is achieved by a method in which water extract of chaga add 25% hydrochloric acid solution to pH 1.0 to 2.0, separating the precipitate of melanin, to the precipitate add petroleum ether at a mass ratio of melanin:petroleum ether 1:5, stirred to obtain a mixture of uniform consistency, the mixture is then freeze after defrosting remove the organic layer of the extract and the aqueous layer is separated precipitate of melanin and dried.

The technical result consists in obtaining melanin low content of ash substances.

The method consists in the following.

The proposed method can be used an aqueous extract of the fungus obtained by any known method. After adding the extract of chaga 25% hydrochloric acid is the deposition of melanin. The precipitate was separated and wet melanin add petroleum ether at a mass ratio of melanin:ether 1:5, stirred to obtain a mixture of uniform consistency. The resulting mixture consists of petroleum ether, water and melanin. The choice of solvent due to its selectivity for hydrophobic components involved in the formation of melanin particles. The mass ratio of the melanin:ether 1:5 is optimal for homogeneous stable under normal the conditions of the mixture. The mixture is then frozen. After thawing the mixture is divided into two layers: organic and water. The top layer of the organic extract containing hydrophobic components of melanin, remove. The melanin contained in the water layer in the form of sediment, has a structural organization that is different from the original (obtained by precipitation with hydrochloric acid) melanin. This leads to the fact that part of the ash substances involved in the formation of particles of the source of melanin, goes into the dispersion medium of the aqueous layer, and reduces the ash content of melanin obtained by the present method.

The invention is illustrated by the following examples of specific performance.

Used raw chaga LLC "KFH "centaur", party 011209. The technical result of the invention is achieved regardless of the method of obtaining an aqueous extract of the fungus. To obtain melanin were used the following aqueous extracts of the fungus.

Aqueous extract of chaga 1, obtained by the Pharmacopoeia requirements (the State Pharmacopoeia of the USSR: Vol. 2. - M.: Medicine, 1990. - 400 C.). 50 g of the fungus was filled with 1500 ml of water, insisted 1 h at room temperature, then boiled for 2 hours, separating the extract and brought the volume of the extract to 2500 ml;

Aqueous extract of chaga 2 obtained by the method of remuneratio (patent RU №2343930, A61K 36/06, B01D 11/02, 2007). 50 g of the fungus was filled with 300 ml of water, insisted 5 h at is the temperature 70°C, separating the extract from the first stage, the remaining meal fungus was added 200 ml of water, insisted another 5 h at 70°C, separating the extract from the second stage and combined with the extract from the first stage.

Example 1 (method prototype).

Water extract of chaga 1 was added a 25% solution of hydrochloric acid to pH 1.0 to 2.0, the precipitation of melanin (50±2 g) was separated by filtration under vacuum and dried.

Example 2.

Similar to example 1, but after the separation to the precipitate melanin was added 250 ml of petroleum ether and stirred to obtain a mixture of uniform consistency, then the mixture was frozen. After thawing of the upper layer of the organic extract was removed from the aqueous layer melanin precipitate was separated by filtration under vacuum and dried.

Example 3 (method prototype).

Used an aqueous extract of the fungus 2 (390±10 ml)by addition of 25% hydrochloric acid to pH 1.0 to 2.0 besieged 50±3 g of melanin, the precipitate was separated by filtration under vacuum and dried.

Example 4. Similar to example 3, but after the separation to melanin was added 250 ml of petroleum ether and stirred to obtain a mixture of uniform consistency, then the mixture was frozen. After thawing of the upper layer of the organic extract was removed from the aqueous layer melanin was separated by filtration under vacuum and isusually.

For all the obtained melanin was determined amount of mineral substances and solubility (the State Pharmacopoeia of the USSR: Vol. 2. General methods of analysis. - 1990.).

Antioxidant activity of melanin was determined by coulometric method (EN 2253114, G01N 33/48).

The results repeatedly conducted experiments processed using Statistics 6" (at P=0.95, n=5) and given in the table.

Table
Physico-chemical characteristics of the obtained melanin
IndexExample
aqueous extract of chaga 1aqueous extract of chaga 2
1 (prototype)23 (prototype)4
Ash, g/100 g1,80±0,101,50±0,103,30±0,200,80±0,20
Solubility, %98,80±0,6099,10±0,8092,20±0,20 91,20±0,30
Antioxidant activity, kcal/100 g51±250±237±238±2

As shown by table data, the ash content of substances obtained melanin fungus depend on the method of obtaining water extracts of the fungus, from which they were selected. The ash content of substances in the melanin of the aqueous extract obtained by the method of remuneratio (example 3), 1.8 times higher compared to the melanin of the aqueous extract obtained by boiling the fungus (example 1).

Getting melanin in the proposed method, regardless of the method of obtaining an aqueous extract of the fungus leads to a decrease in the ash content, but the amount of ash substances varies. In particular, in the melanin of the aqueous extract obtained by the method of remuneratio (example 4), the amount of mineral substances in 4 times less than in the melanin of the aqueous extract obtained by boiling the fungus (example 2).

Was determined the solubility of melanin in phosphate buffer at pH=7,17, i.e. in conditions close to the physiological conditions in the gastrointestinal tract of man. Reducing the ash content of substances in the melanin obtained by the proposed method does not affect the solubility of melanin, which makes it possible R is the this on the basis of their dietary supplements and medicines.

One of the indicators of biological activity of melanin fungus is its antioxidant activity. As shown by these tables, the antioxidant activity of melanin obtained by the method prototype (examples 1,3), varies depending on the method of obtaining an aqueous extract of the fungus. While the proposed method does not alter the antioxidant activity of melanin (examples 2, 4).

Thus, the inventive method allows to obtain melanin with a low content of ash substances without changing their antioxidant properties and solubility. The obtained melanin will be different structural organization and physico-chemical properties depending on the method of obtaining an aqueous extract of the fungus. Physiology of the fungus, obtained by the proposed method is a promising target for development on their basis of biologically active additives and drugs for the treatment of, for example, erosive conditions of the gastrointestinal tract.

The method of obtaining the melanin of the fungus, comprising adding to the water extract of chaga 25% hydrochloric acid to pH 1.0 to 2.0, the Department received melanin precipitate and drying, characterized in that after separation to the precipitate add petroleum ether at a mass ratio of melanin:petroleum ether 1:5, mix zapolucheniya mixture of uniform consistency, the resulting mixture is frozen, after thawing of the mixture layer of the organic extract was removed and the aqueous layer is separated precipitate of melanin.



 

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