RU2708034C1 - Method of producing agaricic acid from a mycelium of a fomes fomentarius - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention represents a method for producing agaricic acid from deep cultivated mycelium mushroom of Fomes fomentarius (Fomitopsis of ficinalis Will. Bond. Et Sing.), by extraction with isopropyl alcohol at ratio of raw material and extractant equal to 1 g of raw material per 50–60 ml of extractant, during boiling and stirring for at least 6 hours, obtained mixture is cooled, filtered and residue is repeatedly extracted at ratio of raw material and extraction agent 1 g of raw material to 25–30 ml of extractant, during boiling and stirring for at least 6 hours, obtained mixture is cooled, filtered, obtained extracts are evaporated at temperature not higher than +50 °C to constant weight.EFFECT: disclosed method simplifies the process, increases output of agaricic acid, enables to obtain the desired product with high degree of purity.1 cl, 4 dwg, 2 ex

Description

The invention relates to the pharmaceutical industry, and in particular to methods of isolating biologically active substances from plant materials, in particular to a technology for producing agaricin acid.

Agaricic acid is used to analyze and obtain drugs [Garcia, N. Agaric acid induces mitochondrial permeability transition through its interaction with the adenine nucleotide translocase. Its dependence on membrane fluidity. / Garcia N., Zazueta C., Pavon N., Chavez E. // Mitochondrion. - 2005. - V.5. - Is. 4. - P.272-281; Chavez, E. The effect of agaric on citrate transport in rat liver mitochondria. / Chavez E., Chavez R., Carrasco N. // Life Sciences. - 1978. - V.23. - Is. 14. - P.1423-1429].

There is a method of complex processing of fungus tinder fungus (Fomitopsis officinalis Will., Family tinder fungi - Polyporaceae, class of basidiomycetes - Basidiomycetes), which is characterized by the fact that the feedstock is ground, then subjected to three-stage extraction in a Soxhlet apparatus with a 25-fold excess of organic solvent during 5-6 hours, followed by distillation of the solvent, in the first stage extraction is carried out with hexane, in the second stage with ethyl acetate, in the third stage with methyl alcohol, then the residue obtained by after extraction with methyl alcohol, it is extracted with water at a 4-8-fold excess of extractant for 4 hours, filtering and repeating the extraction three more times, then a 6% sodium hydroxide solution is added to the insoluble residue in the weight ratio of residue: sodium hydroxide, equal to 1:10, the mixture is heated for 5-6 hours, cooled, filtered and washed with water, a 2% aqueous solution of hydrogen chloride is added to the resulting precipitate, with a ratio of residue: solution of hydrogen chloride equal to 1:10, the mixture is heated in within 5-6 hours, cooling t, filtered and the precipitate washed with water. (Patent RU No. 2619551, IPC A61K 36/06, B01D 11/00, published on 05.16.2017).

The disadvantages of the method are: the complexity and multi-stage extraction, the use of a large set of reagents, the use of natural raw materials (fruit bodies of the fungus), a small yield of agaricin acid (20.0-24.0% with impurities of carotenoids, bioflavonoids, vitamin K).

A known method of producing agaricic acid from the fungus of the medicinal tinder fungus (Fomitopsis of ficinalis Will. Bond. Et Sing.). The method is based on the fact that the raw material is first extracted with liquefied carbon dioxide with the release of a carbon dioxide extract containing essential oil, fatty unsaturated acids, carotenoids, vitamins E, A and sterols, then the residue is extracted with diethyl ether at a temperature of 34-36 ° C for 2- 3 hours with the release of an extract containing agaricic acid and a lipid-carotenoid complex (Patent RU No. 2257222, IPC A61K 35/84, publ. July 27, 2005).

The disadvantages of the method include: the use of extraction with carbon dioxide, which requires complex sealed equipment, the use of expensive diethyl ether as a solvent, the preparation of the finished product with an admixture of lipid-carotenoid complex.

A known method of obtaining extracts from the mycelium of medicinal mushrooms (including from Fomitopsis of ficinalis Will. Bond. Et Sing.) Using different alcohols. The method is based on the fact that the mycelium is grown on grain (rice) or wood for 30-180 days, then extracted with a mixture of ethanol-water (50%) or other alcohols for 14 days. (Patent US20060171958A1, IPC A61K 36/09, published 03.08.2006).

However, this method is not suitable for producing agaricic acid, since gas-liquid analysis showed the absence of agaricic acid in the extract.

Closest to the claimed technical solution is a method for producing agaricic acid from the fungus of the medicinal tinder fungus (Fomitopsis officinalis Will., The tinder family - Polyporaceae, the class of basidiomycetes - Basidiomycetes). The method includes grinding tinder fungus to a size of not more than 3 mm, extraction with 95% ethanol with a total ratio of raw materials and extractant 1:25 when heated in a water bath at a temperature of + 78 ° C until the raw materials are exhausted, evaporation of the obtained extract to a total ratio of raw materials and extract 1 parts by weight raw materials: 6 vol. extract, followed by exposure at a temperature of -4 ° C to -5 ° C, for 12 hours until a precipitate forms. The separation of the precipitate and washing it on the filter with 95% ethanol with a total ratio of raw materials: ethanol 95% 1 wt.h. draft: 0.5 vol.h. ethanol (ethanol temperature no more than + 5 ° С). As a result, technical agaricic acid is obtained, which requires further purification. The completeness of the extraction of agaricic acid is controlled by chromatographic method.

In order to obtain pure agaricic acid, an additional purification is carried out. Why technical agaricic acid is dissolved by heating, at +50 - + 55 ° C, in 95% ethanol, taken in the ratio agaricic acid: solvent 1:10, cooled, kept at -4 ° C - -5 ° C for 2 hours, the precipitate is separated by filtration through a glass filter. Recrystallization is repeated four more times to obtain purified agaricinic acid. The resulting product is dried in an oven to constant weight at + 50 ° C to + 55 ° C (patent RU No. 2330676, IPC A61K 36/074, publ. 10.08.2008).

The disadvantages of the method are: the complexity and duration of the process (about 5 days), a low yield of agaricic acid (9.5 - 14.2%), the need for additional purification from impurities of the target product, use to control the completeness of the extraction of the chromatographic method that does not allow to identify agaricin acid quickly and with a high degree of reliability.

The technical problem solved by the invention is to simplify the process, increase the yield of agaricinic acid, obtain a pure target product, without impurities of other substances, reduce the cost of the process of obtaining the target product, as well as the conservation of biological resources.

The problem is solved in that in the known method for producing agaricinic acid from the mycelium of the fungus of the medicinal fungus (Fomitopsis of ficinalis Will. Bond. Et Sing.), Including the extraction of raw materials according to the invention, the deeply cultivated mycelium of the fungus of the medicinal fungus is taken as raw material, the extraction is carried out isopropyl alcohol with a ratio of raw material and extractant equal to 1 g of raw material per 50-60 ml of extractant, with boiling and stirring for at least 6 hours, the resulting mixture is cooled, filtered and the residue is re-extracted when the ratio of raw material and extractant is 1 g of raw material per 25-30 ml of extractant, when boiling and stirring for at least 6 hours, the resulting mixture is cooled, filtered, the obtained extracts are evaporated at a temperature not exceeding + 50 ° С to constant weight.

An important distinguishing feature of our invention is the raw material - mycelium of the fungus grown in a deep way. The applicant has not found any source of information describing a method for producing agaricinic acid from the mycelium of the fungus of the medicinal tinder fungus (Fomitopsis officinalis Will., Family of tinder fungi - Polyporaceae, class of basidiomycetes - Basidiomycetes), cultivated by the deep method. The use of mycelium makes it possible to avoid the use of natural resources of this species, since it is listed in many regional Red Books as a rare endangered species. In the Far Eastern Federal District, as an endangered species, larch sponge is listed in the Red Books of Primorsky Krai, Sakhalin Oblast, Jewish Autonomous Oblast and Kamchatka. This is one of the types of fungus fungi, which is planned to be included in the next edition of the Red Book of Russia. Therefore, this work is aimed at solving not only one of the priority tasks facing domestic science - “Chemical and biological synthesis of medicines and food products” (Decree of the Government of the Russian Federation N 2727 / p-P8 dated July 21, 1996), but also to address fundamental issues of biodiversity conservation through the development of resource-saving technologies.

Moreover, the use of medicinal fungus mushroom mycelium as a raw material can also significantly simplify and reduce the duration of the process of obtaining the target product, and also provides the target product of a high degree of purity without the use of an additional cleaning operation.

The use of isopropyl alcohol as an extractant not only provides a simplification, reduction in the duration of the process of obtaining the target product with a high degree of purity, but also significantly reduces its cost.

Carrying out the extraction at a ratio of raw materials and extractant of 1 g of raw material per 50-60 ml of extractant by boiling and stirring for at least 6 hours allows you to quickly extract the bulk of agaricic acid from raw materials with a high degree of purity. Extraction with isopropyl alcohol in an amount of less than 50 ml per 1 g of raw material does not provide a high yield of the target product, and an increase in the amount of isopropyl alcohol more than 60 ml per 1 g of raw material no longer affects the yield of the target product.

Carrying out the extraction with stirring and boiling for less than 6 hours does not lead to the extraction of the target product in good yield.

In order to increase the degree of extraction of agaricic acid, re-extraction of the residue after the first extraction is carried out. Extraction, in this case, is carried out at a ratio of raw material and extractant of 1 g of raw material per 25-30 ml of extractant with boiling and stirring for at least 6 hours. It was experimentally established that the use of an extractant in an amount of less than 25 ml per 1 g of raw material does not lead to additional extraction of agaricic acid, while extracting the residue with an extractant in an amount of more than 30 ml per 1 g of raw material practically does not increase the yield of agaricic acid, as a result, it is impractical.

Thus, a deviation from the parameters indicated in the claims leads either to incomplete isolation or to an overspending of raw materials and extractant.

To obtain agaricic acid, preliminarily, the initial raw material of the mycelium of the fungus tinder fungus was obtained by a known method.

The fungal mycelium is obtained by deep cultivation in a fermenter (Applikon Biotechnology, Netherlands) on a nutrient medium of the following composition: glucose - 20.0 g, NH ₄ NO ₃ - 3.5 g, KCl - 0.5 g, K ₂ PO ₄ - 1.0 g, MgSO ₄ - 0.5 g , beer mash 15 ° Balling - 115.0 ml, water - up to 1 liter. subject to the following conditions: temperature 25–28 ° С, stirring speed 180 rpm, for 14 days. The method of obtaining mycelium is described in more detail in Sidorenko ML, Semal VA Production of submerged mycelium of Laricifomes officinalis (Vill) Kotl.et Pouzar // World applied sciences journal. - 2013 .-- Vol.23. - pp. 685-689.

The invention is illustrated by the following figures:

- in FIG. 1 shows the MALDI TOF (MALDI VP MS) mass spectrum of agaricinic acid in the mode of registration of negative ions using 2,5-dihydroxybenzoic acid as the matrix. The deprotonated agaricin acid molecule [MH] ^{- is} represented by a peak with m / z 415.3;

- in FIG. 2. presents the MALDI TOF / TOF (MALDI VP MS / MS) mass spectrum of the deprotonated agaricinic acid molecule in the mode of registration of negative ions using 2,5-dihydroxybenzoic acid as the matrix. Typical fragment ions are represented by peaks with m / z 397, 379, 353, 335,309, 281;

- in FIG. Figure 3 shows the ESI-MS (IER MS) mass spectrum of agaricinic acid in the mode of registration of negative ions. The deprotonated agaricin acid molecule [MH] ^{- is} represented by a peak with m / z 415.3;

- in FIG. Figure 4 shows the ESI-MS / MS (ESI MS / MS) mass spectrum of a deprotonated agaricinic acid molecule in a negative ion registration mode. Typical fragment ions are represented by peaks with m / z 397, 379, 353, 335,309, 281.

The method is as follows:

Example 1

Take 1 g of mycelium of the fungus of the medicinal fungus (Fomitopsis of ficinalis Will. Bond. Et Sing.) Obtained by deep cultivation, and placed in a flat-bottomed flask with reflux condenser, add 50 ml of isopropyl alcohol. Boil for 7 hours with stirring on a magnetic stirrer 200 rpm (Magnetic stirrer with heating Agimatic-ED, Selecta, Spain). The mixture is cooled, filtered through a glass filter, extract 1 is obtained. The residue is re-extracted with 25 ml of isopropyl alcohol under the same conditions, extract 2 is obtained. The obtained extracts 1 and 2 are combined and evaporated on a rotary evaporator (RV 8 Flex, IKA, Germany). The insoluble residue is dried in an oven to constant weight at + 50 ° C. Get a crystalline powder of white color with a slight yellowish tint, odorless, practically insoluble in water, soluble in ethanol 95%. Weigh. The total yield of the extractable fraction is 30.0% in terms of absolutely dry raw materials. To identify agaricic acid, a mass spectrometric analysis method using matrix-activated laser desorption / ionization (MALDI) using an Ultraflex III MALDI-TOF / TOF mass spectrometer (Bruker, Germany) was used. A sample of agaricic acid Sigma Aldrich, USA (Sigma Catalog No. 666-99-9 FC No. 211-566-5 was used as a standard. The purity of agaricic acid was determined by electrospray mass spectrometry on an Agilent 6510 Q-TOF instrument and was 97 ,8%

Example 2

Take 1 g of mycelium of the fungus tinder fungus (Fomitopsis of ficinalis Will. Bond. Et Sing.), Placed in a flat-bottomed flask with reflux condenser, add 60 ml of isopropyl alcohol. Boil for 6 hours with stirring on a magnetic stirrer 200 rpm (Magnetic stirrer with heating Agimatic-ED, Selecta, Spain). The mixture is cooled, filtered through a glass filter, extract 1 is obtained. The residue is re-extracted with 30 ml of isopropyl alcohol under the same conditions, extract 2 is obtained. The obtained extracts 1 and 2 are combined and evaporated on a rotary evaporator (RV 8 Flex, IKA, Germany). The insoluble residue is dried in an oven to constant weight at + 50 ° C. Get a crystalline powder of white color with a slight yellowish tint, odorless, practically insoluble in water, soluble in ethanol 95%. Weigh. The total yield of the extracted fraction is 30.3% in terms of absolutely dry raw materials. To identify agaricic acid, a mass spectrometric analysis method using matrix-activated laser desorption / ionization (MALDI) using an Ultraflex III MALDI-TOF / TOF mass spectrometer (Bruker, Germany) was used. A sample of agaricic acid Sigma Aldrich, USA (Sigma Catalog No. 666-99-9 FC No. 211-566-5) was used as a standard. The purity of agaricic acid was determined by electrospray mass spectrometry on an Agilent 6510 Q-TOF instrument and amounted to 99.0%.

Identification of the obtained substance as agaricic acid was performed by mass spectrometric analysis using matrix-activated laser desorption / ionization (MALDI) using an Ultraflex III MALDI-TOF / TOF mass spectrometer (Bruker, Germany). For this, the obtained samples were dissolved in ethanol and centrifuged for 10 min at 10,000 rpm, passed through a filter (pore size 5 μm). 1 μl of the extract was mixed with 1 μl of the matrix, applied to the target and dried in air. As the matrices, 2,5-dihydroxybenzoic acid (10 mg / ml in 33% aqueous acetonitrile), alpha-cyanocinnamic acid (saturated solution in 33% aqueous acetonitrile), 3-hydroxypicolinic acid (10 mg / ml in 33% aqueous) were used acetonitrile), and the method of applying extracts to the target in the absence of a matrix was also used. The best sensitivity results were achieved when using 2,5-dihydroxybenzoic acid as a matrix in the mode of registration of negative ions. Agaricinic acid was identified by the presence of a peak of the deprotonated molecule [MH] ^- at m / z 415.2701 ± 10 ppm (see Fig. 1, Fig. 3) and the peaks of fragment ions at m / z 397, 379, 353, 335, 309 and 281 in the MS / MS mode (high energy collisional dissociation mode, high energy CID) (see FIG. 2).

To compare and determine the effectiveness of our method for determining agaricic acid, we used the method of electrospray mass spectrometry on an Agilent 6510 LC Q-TOF mass spectrometer with ionization in the mode of negative ions. Analysis conditions: direct injection, flow rate 5 μl / min.

Laricic acid was identified in the mass spectra obtained by electrospray ESI MS (ESI MS) by the presence of the high resolution deprotonated molecule peak mode [MH] ^- at m / z 415.2701 ± 2 ppm (calculated m / z 415.2701 for [MH] ^- ) and characteristic values of m / z fragment ions: at m / z 397, 379, 353, 335, 309 and 281 in the MS / MS mode (low energy collisional dissociation mode, low energy CID) (Fig. 1-4).

As can be seen from the presented results, the inventive method greatly simplifies the process of producing agaricic acid in comparison with the prototype, does not require additional purification, since it immediately provides a pure target product having a purity of 97.8 - 99.0%. In addition, the inventive method allows to increase the yield of agaricinic acid by 2.1 times.

Claims (1)

A method of producing agaricic acid from the fungus of the medicinal fungus (Fomitopsis of ficinalis Will. Bond. Et Sing.), Including the extraction of raw materials, characterized in that the deeply cultured mycelium of the fungus is taken as raw material, extraction is carried out with isopropyl alcohol in the ratio of raw material and extractant 1 g of raw material 50-60 ml of extractant, while boiling and stirring for at least 6 hours, the resulting mixture is cooled, filtered and the residue is re-extracted with a ratio of raw material and extractant 1 g of raw material per 25-30 ml of extractant, while boiling and stirring not less its 6 hours the mixture was cooled, filtered, the extracts are combined and evaporated at 50 ° C.

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