Method for producing of preparation possessing anxiolytic activity. RU patent 2460534.

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutics, particularly a method for producing a preparation possessing anxiolytic activity. A method for producing the preparation possessing anxiolytic activity wherein ground hop cones are sequentially extracted in 60-70% ethanol under certain conditions; thereafter aqueous-alcoholic extracts are concentrated under vacuum; stillage residue are combined with the aqueous extract, filtered, boiled out, purified by separation, boiled out additionally, dried in a vacuum drier and ground.

EFFECT: preparation produced by the method described above is characterised by higher active substance content and exhibits high anxiolytic activity.

2 dwg, 13 tbl, 2 ex

The invention relates to a pharmacy and relates to a method of receiving means having anxiolytic activity.

Due to the high prevalence of neuropsychiatric diseases relevant is the development of drugs aimed at the correction of functional disorders of the nervous system. It is widely used at the present time, the anxiety, have a negative impact on the body: gipersedation, myorelaxation and others (Mashkovsky, 2002).

Promising pharmacological correction of violations arising from functional disorders of the nervous system, are agents of vegetable origin. The use of herbal remedies is considered a safer therapy as a complex of biologically active substances contributing to therapeutic effect, has low toxicity and better tolerability. Herbal preparations that have significantly fewer side effects, can be considered as an alternative therapy of anxiety and depressive disorders or could be used to increase the efficiency of basic drugs (Vorobyov, Akarachkova, 2007; Ushkalov, Illarionov, 2008).

Great interest in this direction are of hop cones ordinary as a source of new drugs with anxiolytic activity.

Know the use of hop cones ordinary members: "Collection reassuring": peppermint leaves - 33,3%, leaf watch three leaf - 33,3%, rhizomes with roots Valerian - 16,7%, hop cones by 16.7% (FS 42-1026-91). "Soothing collection №2. Powder - filter packages": motherwort herb - 40%, hop stems - 20%, peppermint leaves - 15%, Valerian rhizomes with roots - 15%, licorice roots - 10% (mm 42-8796-07). A method of obtaining "Hops extract liquid 1:1 95%alcohol, which is used for production of medicines (the Fund 42-8271-06). A method of obtaining "Hop dry extract". Substance (15:1, the extractant is water)used for manufacture of sterile medicinal products (FSE-42-8459-07).

The disadvantages of the known sources are low yield of extractives, manufacturing time and less pronounced pharmacological action of these funds compared to the means obtained by the claimed method.

The objective of this invention to provide means on the basis of plants with enhanced pharmacological activity by increasing the output of extractive and active substances by successive extraction of raw materials by various extractants.

To achieve the objectives of the crushed plant material (raw material) hop cones ordinary - Humulus lupulus L. family Konopleva - Cannabaceae extracted with alcohol 60-70% in the ratio of raw materials-extractant 1:(20-25) taking into account the coefficient of water absorption of 1:5 at a temperature (60-70°C with constant stirring for 2.5 hours. Removing the filter in the collection. Next meal sequentially extracted with alcohol 40-50% and purified water, feeding every once in extractor extractant in an amount equal to the amount drained retrieval. Water-alcohol extract concentrated under vacuum and distillation residues combine.

Extraction with alcohol 60-70% and purified water is conducted once, and alcohol 40-50% - twice. Water extraction combined with kubovy balance (alcohol extracts) and evaporated to 1/15 of the original volume. The concentrated extract was purified by separation, then darpariaeth up to 1/3 of this volume, is dried in a vacuum drier and pulverized in the mill propeller type. Yield is 21-24% by weight of the feedstock.

Identified features that distinguish the proposed technical solution to the prototype, are significant and allow to draw a conclusion on the conformity of the proposed method the criterion of "novelty".

The proposed method allows to obtain the finished product, representing an amorphous hygroscopic powder from greenish-brown to brown color with a specific smell and bitter taste. Weight loss at drying is 3.1 to 3.7%.

The claimed method of obtaining funds allows you to get dry polistrant hop ordinary, contains a complex of biologically active substances with pronounced anxiolytic activity.

The definition of authenticity and quantitative content of extractive and active ingredients of the dry preextracted hop ordinary set of modern physico-chemical methods.

On the basis of phytochemical analysis in dry preextracted hops are established:

1). Phenolic compounds.

0.5 g of dry preextracted dissolved in 50 ml of water by heating on a magnetic stirrer", cooled, filtered through a paper filter.

To 1 ml of the filtrate add 1 ml of 3% solution of iron oxide chloride; appears black and green staining (polyphenolic compounds).

To 2 ml of the filtrate add 2-3 drops of 2% sodium hydroxide solution; the solution becomes yellow-orange color (flavonoids).

To 3 ml of the filtrate add 1 ml of 2% solution of aluminum chloride, at a low heat for 10-15 minutes; the solution turns greenish-yellow color (flavonoids).

To 1 ml of the filtrate add 5 ml of 5% hydrochloric acid solution in butanol and heated on water bath for 15 min; the solution becomes pink (leucoanthocyanin).

From an ethyl acetate extracts by chromatography in thin layer of sorbent (TLC) plates "Kieselgel G60 F 254 in the solvent system ethyl acetate-glacial acetic acid-water (5:1:1), viewing under UV light before and after processing of chromatograms 3% alcohol solution of aluminum chloride; identified by bright yellow and greenish-yellow fluorescence standards: rutin - R f ~0,24; hesperidin - R f ~0,31; hyperoside - R f ~0,38; myricetin - R f ~0,86; quercetin - R f ~0,90; kaempferol - R f ~0,94.

Chromatography in thin layer of sorbent on the plates "Sorbfil" PTSH P-a-UV-254 or plates "Kieselgel G60 F 254 in the solvent system benzene-ethyl acetate-formic acid (4:4:2), after developing a 10% alcoholic solution of sodium hydroxide detected phenol carbonic acids: chlorogenic - R f ~0,22; Gallic - R f ~0,63; coffee - R f ~0,74; cinnamon - R f ~0,85.

2). Carbohydrate.

To 1 ml of the filtrate add 3 ml of 96% alcohol; a white precipitate is formed (polysaccharides).

3). By TLC hexane extraction from dry preextracted chromatographic on the plates "Sorbfil" PTSH P-a-UV-254 or plates "Kieselgel G60 F 254 " in the solvent system hexane-diethyl ether (80:20). When the manifestation of chromatograms 10% alcohol solution phosphomolybdenum acid detected: stain yellowish-green color with R f ~0,85 (β-carotene), stain blue with R f ~0,50 (tocopherol) at the level of the witnesses.

4). Of amino acids. To 2 ml of the filtrate add 3 ml of 0.1% aqueous solution of ninhydrin; when heated forms a blue-violet staining solution (amino acids).

By TLC in aqueous extract of dry preextracted, on the plates "Kieselgel G60 F 254 " in the system of solvents propanol-water (7:3), after processing the chromatograms of 0.2% alcoholic solution of ninhydrin, when heated to visualize spots pinkish-purple, found 10 amino acids: histidine - R f ~0,04; glycine - R f ~0,41; glutamic acid - R f ~0,51; γ-aminomalonate acid R f ~0,36; asparagine - R f ~0,29; alanine - R f ~0,50; serine - R f ~0,28; Proline - R f ~0,42; threonine R f ~0,48; tyrosine - R f ~0,73. In a dual system PDR (4:1:5) and (8:3:1) even found 5 amino acids: lysine - R f ~0,08; arginine - R f ~0,10; aspartic acid - R f ~0,21; cysteine - R f ~0,43; tryptophan - R f ~0,66.

By HPLC. 0.6 g of dry preextracted dissolved in 25 ml of ethanol 70%. 20 μl of the resulting solution was injected into high performance liquid chromatograph "Gilston (model 305, France) (column of 4.6×250 mm, Kromasil 18, particle size 5 μm); mobile phase: methanol-water-phosphoric acid concentrated (400:600:5). When detecting with UV-detector "Gilston" UV/VIS model 151, at a wavelength of 254 nm identified tannin, Gallic, chlorogenic, ferulic acid, rutin, hyperoside, quercetin, luteolin-7-glucoside, luteolin, kaempferol, coumarin (Fig.1).

Identification of amino acids.

Free amino acids. 1 g (accurately weighed) of preextracted was dissolved in 50 ml of purified water and heated on water bath for 10-15 min, the Extract was cooled and filtered. Next, the sample preparation of the sample for the study included treatment with chloroform, to remove lipids and hydrophobic pigments, precipitation of polysaccharides double the number of alcohol 96%, removing the precipitate by centrifugation and the concentration of the aqueous extract in a vacuum. The solids (accurately weighed) was dissolved in a citrate ion buffer (pH 2.2) was in the amount determined depending on the concentration of amino acids and was applied on the column amino acid analyzer type AAA-339 (Czech Republic).

Related amino acids. Was determined after acid hydrolysis of the sample 6 N solution of hydrochloric acid at 110°C for 24 hours in the ampoule. After the ampoule was opened, the acid was evaporated on a rotary evaporator, the residue was dissolved in sodium citrate buffer solution (pH 2.2) was in the amount determined depending on the concentration aminocyclopentane about 20 amino acids, 10 of which are irreplaceable (table 1, 2).

5). The essential oils. Essential oils were investigated by capillary GC-MS. 30 µl of essential oil was dissolved in 1.5 ml n-hexane. The volume of the sample 1 μl of the solution with separation of stream 20:1, was introduced into the injector of the gas chromatograph.

Identification of substances on the chromatogram was carried out by taking into account three factors: the retention time of the substance, the spectrum of electron impact and ion mass chromatogram in the database Chromatography-Mass Spectrometer NBS 54 (figure 2).

Quantitative determination. The method of quantitative determination of the amount of flavonoids rutin standard using the method of differential spectrophotometry.

Method. Approximately 0.4 g (accurately weighed) of preextracted dry hops are placed in a glass, poured 30 ml of ethanol 70%, dissolved in low heat, quantitatively transferred into a volumetric flask with a capacity of 50 ml, the volume was adjusted solution with alcohol 70% up to the mark. The contents of the flask was filtered through a paper filter into a dry flask, discarding the first 10 ml of the filtrate (solution A).

3 ml of the solution And placed in a volumetric flask with a capacity of 25 ml, add 3 ml of 2% alcohol solution of aluminum chloride solution to bring the volume of solution with alcohol 70% up to the mark and mix (solution B).

After 30 minutes, measure the optical density of a solution in a spectrophotometer at a wavelength of 412 nm in a cell with a layer thickness of 10 mm as the reference solution using solution consisting of 3 ml And 1-2 drops of 1% acetic acid was placed in a volumetric flask with a capacity of 25 ml and increased alcohol 70% up to the mark.

In parallel, under the same conditions, measure the optical density of the solution B GSO routine prepared similarly subject to the solution.

The content of total flavonoids rutin and absolutely dry substance (X) in percent is calculated by the formula:

where D is the optical density of the test solution,

D O is the optical density of the solution of the state security service routine,

m is the mass of preextracted in grams,

m O is the mass of the state security service routine in grams,

W is the loss in weight on drying of preextracted in percent.

The total content of flavonoids should be not less than 0.8% in preextracted rutin.

The proposed method of producing quite simple, does not require complicated purification, allows to obtain a product of constant composition with the maximum number of active substances through the use of the three extractants.

The technology can be implemented at the enterprises producing drugs and prophylactics.

To prove the materiality of the proposed features of the process, to identify the optimal technological regimes we have studied the main factors influencing the process of extraction of plant materials: the composition and nature of the extractant, his relationship with the raw material, the crushing of the raw material, temperature, duration and the ratio of the number of extraction.

Quantitative evaluation was carried out according to the output of extractives (GF XI, issue 1. S) and total flavonoids rutin standard.

Metrological characteristics of the methodology are presented in table 3. As can be seen from the table, the relative error of a single determination with 95% probability is ±2,77%.

In the selection of the extractant were used purified water and ethyl alcohol in various concentrations. From table 4 it follows that the extraction at concentrations above 70% alcohol, the yield of flavonoids is not increased, the optimum is 50%, 60%, 70% alcohol and hot water, which gives the maximum amount of extractives and flavonoid compounds.

As is known, the particle size of the raw material determines the completeness and speed of transition in removing extractables. To study the effect of fineness on the yield of extractive substances and flavonoids, raw materials were subjected to grinding to particle diameter 0,5; 1,0; 2,0; 3,0 mm, table 5 presents the results of the research. It is established that the optimal degree of grinding of the raw material is a particle size of not more than 1 mm

The optimal correlation of phases raw material extractant, the corresponding 1:(20-25) for a single Bay of raw materials (table 6). Further increase in the volume of the extractant is impractical, because the content of active substances is reduced. For intensification of the process of extraction of raw materials need to take into account the temperature factor. Considering the obtained data (table 7), the extraction of raw materials should be at a temperature (60-70)°C.

To determine the duration and the ratio of the number of extraction set time to reach the equilibrium concentration in the system "raw-extractant for alcohol and water extraction by the following method. The portions 1 g of the crushed material (1 mm) was added (70% alcohol, 50% alcohol) in a ratio of 1:25 with regard To/in 1:5 and extracted at room temperature on a vibrating shaker". After a certain period of time removing the merge and analyze the content of extractives (1 contact phase). The extraction was repeated three times, pouring raw new portion of the extractant in the amount equal to the slit extraction (2 and 3 contacts phases). Plum performed every 30 min, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours. Similarly, in the same conditions, the hot water extraction performed on "water bath".

Experimental data are shown in table 8. The obtained results indicate that the equilibrium state when the first, second, and third contact phase is reached after 2.5 hours. The first contact extraction with alcohol 70% and hot water extraction goes 65-75% extractives. By extraction with alcohol 50% is removed within the first two contacts of the phases up to 90% extractives. Therefore, in order to save extractants and extraction time are excluded from the application of 2 and 3 contacts phases during extraction with alcohol 70% and purified water and 3 contact phase by extraction with alcohol 50%.

The method is illustrated below by the following examples.

Example 1

1 kg of hop cones ordinary crushed to particle size diameter not exceeding 1 mm (sieve No. 10 GOST 214-83). The crushed raw material is loaded into the extractor with a stirrer and external steam heating. Pour 25 l of ethyl alcohol 60% in the ratio of raw materials-extractant equal to 1:20 with regard To/in 1:5. Extracted at 60°C with constant stirring for 2.5 hours. Next meal is extracted with alcohol 40% - two times, and purified water once, giving every once in extractor extractant in an amount equal to the amount drained retrieval. 1st drain - 19,8 l; 2 plums - 19,5 l; 3 drain - 19,3 l; 4-th drain - 18,8 L. hydroalcoholic extract concentrated under vacuum. Distillation residues combined with the aqueous extract, filtered and evaporated to 1/15 of the original volume. The concentrated extract was purified by separation, darpariaeth up to 1/3 of this volume, is dried in a vacuum dryer for 5 hours and pulverized in the mill. Get 237 g of the finished product, representing 23.7% of the weight of raw material.

Polistrant dry hops is an amorphous brown powder with a characteristic odor. Hygroscopic. The moisture content of 3.1%.

Example 2

1 kg of hop cones ordinary crushed to particle size diameter not exceeding 1 mm (sieve No. 10 GOST 214-83). The crushed raw material is loaded into the extractor with a stirrer and external steam heating. Pour 30 l of ethyl alcohol 70% in the ratio of raw materials-extractant equal to 1:25 with regard To/in 1:5. Extracted at 70°C with constant stirring for 2.5 hours. Next meal is extracted with alcohol 50% twice and purified water once, giving every once in extractor extractant in an amount equal to the amount drained retrieval. 1st drain - 25,4 l; 2nd drain - 24,9 l; 3 drain - 24,8 l; 4-th drain - 24,2 L. hydroalcoholic extract concentrated under vacuum. Distillation residues combined with the aqueous extract, filtered and evaporated to 1/15 of the original volume. The concentrated extract was purified by separation, darpariaeth up to 1/3 of this volume, is dried in a vacuum dryer for 4.5 hours and pulverized in the mill. Obtain 210 g of the finished product, which is 21% by weight of the feedstock.

Polistrant hop is a dry amorphous powder greenish-brown color with a specific smell. Hygroscopic. The moisture content of 3.7%.

Thus, the proposed method of production as compared with the known (table) allows to obtain polistrant dry hops, containing biologically active substances, which cause its pronounced anxiolytic activity through the use of rational technology of production.

Pharmacological study of preextracted hop ordinary dry

Experimental work done on white Wistar rats of both sexes, with an initial weight of 160-180, the Animals were housed under standard conditions and feeding in vivarium (MOH Order No. 1179 from 10.10.83 year).

Experiments on animals were performed in accordance with the rules of the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes.

Animals were divided into 5 groups. Animals I-III experimental groups during the 5 days before the experiment was administered intragastrically polistrant dry hops in doses of 100, 200 and 300 mg/kg, respectively, diluted in purified water. Rats of the fourth experimental group received the drug comparisons pills Valerian extract at a dose of 120 mg/kg, the animals of the control group - treated water in the equivalent volume on a similar scheme introduction.

Animal testing in the experimental setups were performed within 1 hour after the last injection of the investigational tools.

Anxiolytic effects of preextracted hops dry investigated according to methodical instructions for the study of anxiolytic (anxiolytic) actions of pharmacological substances (Voronin, Seredenin, 2005).

The significance of the differences between these parameters among experimental and control groups of animals were evaluated using the nonparametric criterion of Mann-Whitney. The differences were considered significant at P≤0,05 (Sergienko, Bondareva, 2006).

The influence of preextracted hop ordinary dry animal behavior in the elevated cross maze

Method the elevated cross maze (PCL) - based skill preferences rodents dark holes and a natural fear of being in open areas and falls from a height. Immediately before the experiment, animals were kept for 3 minutes in dark cells.

Then the animals were placed in a PC to the Central site, head to the open sleeve, and within 10 minutes were recorded during the stay of animals in open and closed arms, as well as on the Central platform, the number of entries in open and closed sleeves, the number of sesivany and vertical columns, and the number of defecations.

The results of the test animals in a crosswise maze showed (table 10)that control animals prefer most of its time in the closed arms than open. Course introduction animals preextracted dry hops in doses of 200 and 300 mg/kg and Valerian extract suppressed in animals fear of open spaces, resulting in the number of entries into the open sleeve installation in animals data group increased by 1.6; 1.4 and 1.9 times. The time spent in the open sleeve of animals in experimental groups was higher than that observed for animals in the control group on average 1.5 times. The greatest time in the open sleeve was in the animals treated with the study polistrant at a dose of 300 mg/kg

The time spent on the Central platform, allowing to estimate the rate of decision-making, animal experimental and control groups were not significantly different (p>0,05).

The analysis of the results showed that in animals of the experimental groups in PCL was observed and higher overall locomotor activity compared to control animals (table 10). The so-treated rats polistrant dry hops in the dose of 200 and 300 mg/kg and Valerian extract increased the number of entries into closed arms of the maze and the number of vertical pillars, and in animals treated with the study polistrant at a dose of 100 mg/kg, the number of sesivany.

The increase in research activity and decrease feelings of fear and anxiety in animals of the experimental group can be attributed to a decrease in their level of emotionality (table 10). Thus, in animals of the experimental groups, the number of boles was lower than that observed for control animals on average 1.6 times.

The influence of preextracted hop ordinary dry animal behavior in open field

The influence of preextracted dry hops and tablets of Valerian extract on spontaneous locomotor activity, orienting-exploratory behavior and emotionality in animals studied with the help of test "open field". The behavior of rats in the experimental setup was observed within 3 minutes (Voronin, 2005). Recorded vertical activity (number of crossed Central and peripheral squares), vertical activity (number of rises on its hind legs), the number of zaglyadyvanie in holes (mink reflex), the number of animals visiting the Central squares, and the latent period of time in the Central square. The emotional state of the animals was determined by the number defecation balls and acts of grooming. About General motor activity was assessed by the sum of the vertical and horizontal components and mink reflex (Voronin, 2005).

The results showed that the placement of white rats receiving polistrant dry hops in doses of 200 and 300 mg/kg and Valerian extract at a dose of 120 mg/kg, in unfamiliar conditions have led to an increase in total motor activity 1.6; 2.8 and 2.1 times, respectively (table) compared with those in animals of the control group. However, the imposition of rats studied preextracted in these doses and Valerian extract significantly increased the number of visits are not only peripheral but also Central squares. The latent period of time in the Central squares in rats treated with dry polistrant hops in the doses of 200 and 300 mg/kg and Valerian extract was lower than that seen in animals of the control group of 22, 35 and 26%, respectively.

50% of the animals treated with polistrant dry hops in a dose of 300 mg/kg and Valerian extract, visited the Central squares, whereas the animals of the control group, this figure was 30%. Also in animals of the experimental groups to the increase in horizontal activity was observed a significant increase in vertical activity and mink reflex (table). The increase of approximately research activity in albino rats experimental groups can be attributed to a decrease in their level of emotionality (table). Thus, animals treated with polistrant in doses of 200 and 300 have noted a decrease in the number defecation balls on average by 38% and the number of acts of grooming 1.8 and 3.1 times, respectively, compared to those values in control animals. On the background of the introduction of the reference preparation of Valerian extract in rats observed a decrease in the number defecation balls 2.2 times in comparison with the same indicators in animals of the control group. Therefore, the introduction of animals studied preextracted hops in the doses of 200 and 300 mg/kg and Valerian extract results in rats acceleration of the processes of adaptation and reduction of anxiety-defensive reaction.

The influence of preextracted hop ordinary dry animal behavior in the light/dark chamber"

The method of "light/dark" camera is based on the natural desire of rodents to avoid brightly lit places. In this experiment, animals were placed in a brightly lit compartment, double chamber "light/dark" installation and within 5 minutes we registered the number of transitions between light and dark compartments, as well as the duration of stay in the light and dark compartments.

The results of the test animals in the "light/dark chamber" showed that of the control animals prefer most of the time spend in a dark chamber (tabl). All 100% of the animals of the control group went into the dark compartment and performed the entire testing period. Course introduction animals preextracted dry hops in doses of 100, 200 and 300 mg/kg and Valerian extract at a dose of 120 mg/kg caused depression feelings of fear and anxiety, as evidenced by the increase in the number of transitions between chambers 2.6; 3,3; 3.5 and 2.5 times increase in the animals stay in the bright compartment installation 2.9; 3,5; 2.5 and 2.7 times in comparison with the same indicators in animals of the control group (table). The study polistrant in doses of 200 and 300 mg/kg in this experiment had a more pronounced anxiolytic effects compared to Valerian extract.

The influence of preextracted hop ordinary dry on the behavior of white rats in a conflict situation in the variant Vogel

The conflict situation in the variant Vogel was created, pushing the drinking and defensive motivation, by suppressing painful electric stimulus drinking reflex consumption by rats of water from a Cup. For this animals within 2 days deprived of water, without limiting feed intake, on the 3rd day had developed the skill of taking water from the trough, placing the rat in the chamber, where she found the drinking water and began to drink. On the 4th day the rat was placed in a cell for 3 minutes and 10 seconds after the start of drinking every taking of water were punished electrovalves irritation (1 mA). In order to meet drinking motivation, the rat had to overcome the feeling of fear of punishment. About the anxiolytic effect of preextracted dry hops and Valerian extract was judged by the number of punishable takes water animals within 3 minutes of being in the camera. The results showed (table 13)that the course introduction of the investigated preextracted in doses of 100, 200 and 300 mg/kg eliminates the feeling of anxiety and fear, increasing the number of punishable takes water 5.3; 5.8 and 7.0% respectively compared with that indicator in animals of the control group. However, the imposition of preextracted at a dose of 300 mg/kg exerted a more pronounced anxiolytic effects compared to the reference preparation of Valerian extract.

Thus, the course introduction to the animals of preextracted hop ordinary dry in doses of 200 and 300 mg/kg has a pronounced anxiolytic effects in terms of punishable behavior, increasing the number of visits and time spent in open arms elevated cross maze, horizontal, vertical activity, and mink reflex in the open field, and the number of transitions and time spent in light compartment "dark/light"camera.

This effect is comparable with that of the reference preparation of Valerian extract.

Course introduction animals preextracted hop ordinary dry in doses of 100, 200 and 300 mg/kg has a pronounced anxiolytic effects in terms of punishable behavior, significantly increasing the number of punishable takes water in the methodology conflict Vogel. While the introduction of the investigated preextracted at a dose of 300 mg/kg has more pronounced effect compared to the reference preparation of Valerian extract.

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The method of obtaining funds with anxiolytic activity by sequential extraction of plant material with ethyl alcohol, characterized in that crushed to particle size of 1 mm hop cones ordinary sequentially extracted with 60-70% ethyl alcohol in the ratio of raw material:solvent 1:(20-25) taking into account the coefficient of water absorption of 1:5 - once, 40-50% ethyl alcohol twice and purified water - once, feeding every once in extractor extractant in an amount equal to the amount drained extract, 2.5 hours at a temperature of 60-70°C with constant stirring, then a water-alcohol extract concentrated under vacuum distillation residues combined with the aqueous extract, filtered, evaporated, and purified by separation, darpariaeth, dried in a vacuum dryer, followed by grinding.