RU2523414C1 - Method of obtaining melanin from chaga - Google Patents

Abstract

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.

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Description

The invention relates to the pharmaceutical industry, in particular to the production of melanin from chaga with a low ash content and high biological activity.

The main active component of the chaga, providing its therapeutic activity, is melanin, previously called chromogens or polyphenols, or the polyphenoloxycarboxylic complex. During aqueous extraction of the fungus fungus, a colloidal system is formed in which melanin forms a dispersed phase. Addition of some electrolytes from the number of neutral salts and mineral acids to the chaga water extract leads to precipitation of melanin (PA Yakimov. Comprehensive study of physiologically active substances of lower plants. - M.-L. Nauka. - 1961. - P. 113-119) . The method of obtaining the extract and the nature of the precipitating agent affect the structural organization and biological activity of the secreted melanins, in particular, the content of ash substances involved in the formation of melanin particles.

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

It is known to use a 9-20% solution of calcium chloride to precipitate melanin from aqueous extracts of chaga (patent RU 2442596, A61K 36/06, B01D 11/02, 2012).

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

The high content of ash elements in drugs, in particular salts of potassium and sodium, violates the osmotic pressure of cells and the electrolyte balance of the gastrointestinal tract, irritates the mucous membranes, and prevents the rapid healing of erosion and ulcers (F. Bintam, Some questions about the toxicity of metal ions. - M .: Mir, 1993.).

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

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

The technical task of the invention is to reduce the ash content of chaga melanin.

The problem is achieved by the method in which a 25% solution of hydrochloric acid is added to the aqueous extract of chaga to a pH of 1.0-2.0, the resulting melanin precipitate is separated, petroleum ether is added to the precipitate with a mass ratio of melanin: petroleum ether 1: 5, and the mixture is stirred until to obtain a mixture of uniform consistency, then the mixture is frozen, after thawing, the organic extract layer is removed, and the melanin precipitate is separated from the aqueous layer and dried.

The technical result is to obtain melanin with a low ash content.

The essence of the method is as follows.

In the proposed method can be used an aqueous extract of chaga obtained by any known method. After adding a 25% hydrochloric acid solution to the chaga extract, melanin precipitates. The precipitate is separated and petroleum ether is added to the wet melanin in a mass ratio of melanin: ether of 1: 5, stirred until a mixture of uniform consistency is obtained. The resulting mixture consists of petroleum ether, water and melanin. The choice of solvent is due to its selectivity with respect to the hydrophobic components involved in the formation of melanin particles. The mass ratio of melanin: ether 1: 5 is optimal to obtain a homogeneous mixture, stable under normal conditions. Then the mixture is frozen. After thawing, the mixture is divided into two layers: organic and water. The top layer of the organic extract containing the hydrophobic components of melanin is removed. Melanin, which is in the form of a precipitate in the aqueous layer, has a structural organization that is different from the initial (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 original melanin passes into the dispersion medium of the aqueous layer, due to which the ash content of melanin obtained by the present method is reduced.

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

Used the raw material of chaga LLC “KFH“ Centaur ”, lot 011209. The technical result of the invention is achieved regardless of the method of obtaining an aqueous extract of chaga. The following aqueous extracts of chaga were used to produce melanin.

An aqueous extract of chaga 1 obtained according to the pharmacopeia article (State Pharmacopoeia of the USSR: Issue 2. - M .: Medicine, 1990. - 400 p.). 50 g of chaga were poured into 1500 ml of water, insisted for 1 h at room temperature, then boiled for 2 h, the extract was separated and the extract volume was adjusted to 2500 ml;

An aqueous extract of chaga 2 obtained by the method of remaceration (patent RU No. 2343930, A61K 36/06, B01D 11/02, 2007). 50 g of chaga were poured into 300 ml of water, insisted for 5 hours at 70 ° C, the extract was separated from the first stage, 200 ml of water was added to the remaining chaga meal, insisted for another 5 hours at 70 ° C, the extract was separated from the second stage and combined with extract from the first stage.

Example 1 (by the prototype method).

A 25% hydrochloric acid solution was added to the aqueous extract of chaga 1 to a pH of 1.0-2.0, the precipitated melanin precipitate (50 ± 2 g) was separated by suction filtration and dried.

Example 2

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

Example 3 (by the prototype method).

An aqueous extract of chaga 2 (390 ± 10 ml) was used, from which 50 ± 3 g of melanin was precipitated by adding 25% hydrochloric acid to a pH of 1.0–2.0, and the precipitate was suction filtered and dried.

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

For all melanins obtained, the amount of ash substances and solubility were determined (State Pharmacopoeia of the USSR: Issue 2. General methods of analysis. - 1990.).

The antioxidant activity of melanins was determined by coulometric method (RU 2253114, G01N 33/48).

The results of repeated experiments were processed using the "Statistics 6" program (at P = 0.95, n = 5) and are shown in the table.

Table Physico-chemical characteristics of the resulting melanins Indicator Example chaga water extract 1 chaga water extract 2 1 (prototype) 2 3 (prototype) four Ash content, g / 100 g 1.80 ± 0.10 1.50 ± 0.10 3.30 ± 0.20 0.80 ± 0.20 Solubility% 98.80 ± 0.60 99.10 ± 0.80 92.20 ± 0.20 91.20 ± 0.30 Antioxidant activity, ccl / 100 g 51 ± 2 50 ± 2 37 ± 2 38 ± 2

As the tabular data shows, the ash content of the obtained chaga melanins depends on the method of obtaining aqueous chaga extracts from which they were isolated. The ash content in the melanin from the aqueous extract obtained by the remaceration method (Example 3) is 1.8 times higher than the melanin from the aqueous extract obtained by boiling the chaga (Example 1).

Obtaining melanin according to the proposed method, regardless of the method of obtaining an aqueous extract of chaga, leads to a decrease in its ash content, but the amount of ash substances removed varies. In particular, in the melanin from the aqueous extract obtained by the remaceration method (Example 4), the amount of ash substances is 4 times less than in melanin from the aqueous extract obtained by boiling the chaga (Example 2).

The solubility of melanins in phosphate buffer was determined at pH = 7.17, i.e. in conditions close to physiological conditions in the human gastrointestinal tract. The reduction in the content of ash substances in melanins obtained by the proposed method does not affect the solubility of melanins, which makes it possible to develop biologically active additives and drugs on their basis.

One of the indicators of the biological activity of chaga melanin is its antioxidant activity. As the data in the table show, the antioxidant activity of melanins obtained by the prototype method (examples 1,3) varies depending on the method of obtaining an aqueous extract of chaga. Moreover, the proposed method does not change the antioxidant activity of melanins (examples 2, 4).

Thus, the claimed method allows to obtain melanins with a low ash content without changing their antioxidant properties and solubility. In this case, the resulting melanins will differ in structural organization and physico-chemical properties, depending on the method of obtaining an aqueous extract of chaga. Chaga melanin obtained by the proposed method is a promising object for the development on their basis of biologically active additives and drugs for the treatment, for example, of erosive conditions of the gastrointestinal tract.

Claims (1)

A method for producing chaga melanin, including adding 25% hydrochloric acid solution to the chaga water extract to a pH of 1.0-2.0, separating the resulting melanin precipitate and drying it, characterized in that after separation, petroleum ether is added to the precipitate in a mass ratio of melanin : petroleum ether 1: 5, stirred until a mixture of uniform consistency is obtained, the resulting mixture is frozen, after thawing the mixture, the organic extract layer is removed and the melanin precipitate is separated from the aqueous layer.