RU2613312C2 - Method of producing agent with sedative properties, anticonvulsant and neuromodulating anti-alcohol activity - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to method of producing agent with sedative properties, anticonvulsant and neuromodulating anti-alcohol activity. Above method involves extraction of vegetal raw material – cow wheat with 50 % ethanol, by repercolation with equal load of raw material, with complete cycle of in batch of four unequally batched percolators in ratio of raw material:extractant – 1:2 at infusing at each stage of extraction for six hours, then combined extraction are settled, filtered, decanted, evaporated to dry residue in rotary evaporator.

EFFECT: invention provides preparation of agent with evident sedative properties, anticonvulsant and neuromodulating anti-alcohol activity.

1 cl, 5 dwg, 8 tbl, 1 ex

Description

FIELD OF THE INVENTION

The invention relates to the field of pharmacy and consists in a method for producing a medicinal product from plant materials with sedative, anticonvulsant activity and reducing the neurotoxic effect of alcohol.

State of the art

An urgent problem of modern pharmaceutical science is the search for new effective drugs for the treatment of diseases of the nervous system that have the ability to eliminate anxiety, fear, anxiety, tension, increased irritability, insomnia and other manifestations of neurotic, neurosis, psychopathic conditions, autonomic dysfunctions, and various convulsive conditions, which are severe and widespread diseases. Convulsive conditions can occur with various diseases and intoxications. These include encephalitis, meningitis, injuries to the skull and brain, tumors, cerebrovascular accident, impaired function of the endocrine glands and related metabolic disorders.

Almost all modern drugs cause side effects and complications, in addition, with prolonged use, drug dependence may occur [7].

Of interest are herbal preparations containing a complex of biologically active substances, which have a greater therapeutic breadth in comparison with existing ones, which have a unique combination of properties. The spectrum of pharmacological activity of new drugs for the treatment of diseases of the nervous system should include such actions as anticonvulsant, sedative, neuromodulatory. The search is conducted to reduce side effects and toxicity, as well as expand the scope of drugs.

Herbal medicines have several advantages over synthetic ones - low toxicity, mild action, therefore, the study of plants with the above types of biological activity is relevant.

Among the wild plants that affect the nervous system, the species of the genus Melampyrum L. are distinguished. Forest Mariannik is widespread in Russia and has a sufficient raw material base [1]. The aerial part of the plant has long been used in folk medicine. Water extracts (infusions, decoctions) are prepared from it, which in small doses are prescribed as effective agents for heart diseases [6], but they contain a low amount of biologically active substances (Table 1). Currently, a number of scientific studies are being carried out on the possibility of using bovine and preparations from it in scientific medicine [4, 5].

The phytochemical analysis of the aerial part with the roots of forest bryophytes made it possible to establish the presence of such biologically active substances as flavonoids (cinaroside, hyperoside, quercetin glycosides), iridoids (aucubin), phenol carboxylic acids (chlorogenic acid), alkaloids, amino acids [2] that take participation in complex pharmacological action.

Of the methods for producing complex natural products that have a sedative effect, there is a known method for producing valerian extract (RF patent No. 2098115). The production method includes extraction of the aerial part of the plant (65-75)% with ethanol, settling in a refrigerator, separation of the phases by decantation and filtration, evaporation and drying.

This method is advisable to use as a prototype due to the presence of the general stages of obtaining and the similarity of the pharmacological action of the final product.

The prototype has several disadvantages:

- the complexity and duration of the production method;

- use as extractant (65-75)% ethanol;

- high concentrations of ethanol extract a large amount of lipophilic substances (chlorophylls, carotenoids, resins) from plant materials.

As a result, a stage of separation of the hydrophilic and lipophilic phases formed is required - settling for 5 hours at a temperature of 10 ° C and purification of the obtained extract by decantation and subsequent filtration.

In turn, this leads to the loss of part of the biologically active components and a decrease in the quality of the biological properties of the target product.

Another disadvantage is the lack of neuromodulatory anti-alcohol activity in the drug.

Disclosure of invention

The purpose of the invention is to obtain a drug containing a complex of natural compounds with sedative and anticonvulsant activity, as well as expanding the arsenal of drugs used to treat diseases of the nervous system. The method has simplicity, accessibility and the ability of the extractant to extract a certain complex of biologically active substances (BAS), the absence of stages using toxic substances, and also a sufficient raw material base.

Extractant Effect

A significant effect on the concentration of active substances and the pharmacological activity of the final product is exerted by the extractant, as well as the ratio of raw materials and extractant. When selecting the optimal extractant, the quantitative content of the main groups of active substances in the water extract from the herb m. Forest, in infusion, alcohol extract was investigated. The maximum amount of extractives was observed in dry alcohol extract. The selection of the optimal ratio was carried out by determining the content of the main groups of biologically active substances with the following ratios of raw materials and extractant in the dry alcohol extract 1: 1 and 1: 2. The maximum concentration of compounds was observed at a ratio of 1: 2.

The source of the preparation is grass (the aerial part with roots) of forest meadowsweet (Herba melampyri), harvested during the flowering phase.

SUMMARY OF THE INVENTION

The grass of the browning is crushed to a particle size of (1-3) mm, extracted by repercolation with an equal load of raw materials with a complete cycle in a battery of four percolators in the ratio of raw material: extractant - 1: 2. As extractant use 50% ethanol. The infusion time is 6 hours. The combined extracts are settled, filtered, decanted and evaporated in a rotary vacuum evaporator at a temperature of 50 ° C to obtain a dry residue.

Example: 500 g of an air-dry aerial part with roots of forest bryophytes are ground to a particle size of 2 mm in diameter, loaded into four percolators in an equal amount. On the first day, plant material in percolator No. 1 is poured with extractant - 50% ethanol to a “mirror” and insisted for 6 hours. After 6 hours, it is filled with percolator No. 2 by extraction obtained in percolator No. 1, and fresh extractant is poured into the first percolator until "Mirrors". After 6 hours, load percolator No. 3 with an extract from percolator No. 2, and percolator No. 2 with an extract from percolator No. 1. After 6 hours, fill percolator No. 4 with extract from percolator No. 3, percolator No. 3 with extraction from percolator No. 2, and percolator No. 2 with an extract from percolator No. 1. After 6 hours, from the percolator No. 4, the first discharge of the finished product is obtained, extraction from percolator No. 3 is poured into percolator No. 4, from percolator No. 2 to percolator No. 3, and from percolator No. 1 to percolator No. 2; the raw material from percolator No. 1 is squeezed, the percolator itself is disconnected from the battery. After the next 6 hours, the second part of the finished product is obtained from percolator No. 4. The extract from percolator No. 3 is poured into percolator No. 4, and from percolator No. 2 to percolator No. 3, percolator No. 2 is disconnected from the battery, the raw materials are squeezed out. After 6 hours, the third portion of the extract from the fourth percolator is drained, the liquid from the third percolator is poured into percolator No. 4, and percolator No. 3 is disconnected from the battery, the raw materials from the third percolator are squeezed. After 6 hours, the last portion of the extract is obtained, the raw material from the fourth percolator is squeezed. Four servings of the finished product are combined. The volume of extract obtained exceeds the mass of raw materials twice. Next, the combined extracts are evaporated in vacuo at a temperature of the reaction mass (50 ± 2) ° C and a pressure of 0.01 kgf / cm ² (9.81 hPa) to obtain a dry residue. The yield of the extract is 12% by weight of the raw material.

The dry extract is a dark brown hygroscopic mass with a characteristic specific odor, bitter taste, soluble in water. The content of flavonoids (11.74 ± 0.14)%, moisture (6.67 ± 0.012)%. The shelf life of the extract is 3 years.

Final Product Standardization Options

The moisture content was determined by the method of GF XI [3].

The authenticity of the drug is confirmed by qualitative reactions, chromatographic tests and UV spectral characteristics.

1. Reactions of authenticity to the content of biologically active substances of forest meadowsweet:

a) Flavonoids

10 mg of crystalline magnesium and 5 drops of concentrated hydrochloric acid are added to 1 ml of a 10% aqueous solution of the extract, heated in a boiling water bath for 3 minutes, a red-brown color appears (Synod sample);

b) Iridoids

To 1 ml of a 10% aqueous solution of the extract, add 0.5 ml of Trimm Hill reagent (2 ml of conc. HCl, 10 ml of 0.2% aqueous solution of CuSO ₄ , glacial acetic acid to 100 ml), 2.5 ml of 50% acetic acid, heated in boiling water bath for 10 minutes, blue staining appears;

c) Phenolcarboxylic acids

5 drops of iron-ammonium alum are added to 1 ml of a 10% aqueous solution of the extract, heated in a boiling water bath for 5 minutes, a black precipitate appears;

d) Alkaloids

2 drops of hydrochloric acid and 2 drops of Bushard's Reagent (a solution of 1.27 g of iodine and 2 g of potassium iodide in 100 ml of water) are added to 1 drop of a 10% aqueous solution of the extract; a brown, slightly soluble precipitate appears in water;

e) Coumarins

5-10 drops of a 10% alcohol solution of NaOH are added to 1 ml of a 10% aqueous solution of the extract and heated in a water bath for several minutes. In the presence of coumarins, the solution turns yellow. (5-10) ml of distilled water is added to the contents of the tube and mixed. After the addition of 5-10 drops of 10% hydrochloric acid, turbidity or, less commonly, precipitation (lactone test) is observed.

The results of the qualitative reactions carried out with extract of woody biloba are shown in table 2.

2. Chromatographic characterization of flavonoids

On a sheet of chromatographic paper of the Leningradskaya-S brand (30 × 40) cm in size, 0.1 ml of the extract is applied and chromatographed in a glass chamber using the ascending method in a solvent system of isopropanol - formic acid - water (2: 5: 6) for a period of 4 hours. The chromatogram is removed from the chamber, dried, rotated 90 ° so that the flavonoids distributed in the first direction are at the bottom, placed in the solvent system butanol - acetic acid - water (4: 1: 2), where they are kept for 8 hours ( second direction). After a time, the chromatogram is removed from the chamber, dried and viewed in UV light before and after treatment with ammonia vapors, and it is also treated with a chromogenic reagent - 1% alcohol solution of aluminum chloride. The results of chromatographic analysis are presented in table 3.

On the basis of luminescent chromatographic properties and reactions with chromogenic reagents, it was found that 5 substances of flavonoid nature are contained in the extract of forest bryophyta. The value of the distribution coefficients (R _f ) of these substances is an indicator of the authenticity of the drug.

3. UV spectral response

The UV spectra of the extract at a 1:50 dilution are presented in Table 4. As follows from the obtained data, one absorption maximum at 335 nm is clearly expressed in the UV spectrum of the extract of woody biloba. When a chromogenic reagent (AlCl ₃ ) is added, in addition to the indicated one (335 nm), two peaks appear in the short-wavelength (275 nm) and long-wavelength (396 nm) spectral regions, and the absorption intensity at the maximum at 335 nm slightly decreases with the addition of aluminum chloride. Changes in the UV spectrum confirm the presence of flavonoids in the preparation and their participation in complexation reactions.

The differential spectrum also showed the presence of a maximum absorption in the long-wavelength region of the spectrum (396 nm), free from absorption of simple phenolic compounds (simple phenols, hydroxycinnamic acids, coumarins, etc.). This maximum can be used both for authentication and for developing a method for the quantitative determination of flavonoids in an extract.

4. Quantification of flavonoids

In a volumetric flask with a capacity of 25 ml is placed 0.5 g of the drug (accurately weighed), adjusted to the mark with purified water (solution A). 1 ml of solution A is placed in a 100 ml volumetric flask, 3 ml of 2% aluminum chloride solution is added, the volume of the solution is brought to the mark with purified water and stirred (solution B). Stand for 30 minutes and measure the optical density of solution B on an SF-2000 spectrophotometer in a cuvette with a layer thickness of 10 mm at a wavelength of 396 nm. The following solution is used as a comparison solution: 1 ml of solution A is placed in a 100 ml volumetric flask and the volume of the solution is adjusted to the mark with purified water. The mass fraction of the sum of flavonoids in the extract of biloba in terms of cinaroside and absolutely dry raw materials X,%, is calculated by the formula:

where D is the optical density of the test solution;

- удельный показатель поглощения комплекса цинарозида с хлоридом алюминия при λ₃₉₆=387;

- specific absorption rate of the complex of cinaroside with aluminum chloride at λ ₃₉₆ = 387;

a is the exact portion of the extract, g;

W is the moisture content of the extract,%.

The flavonoid content in the extract is 11.74%.

5. Quantification of Iridoids

0.1 g (accurately weighed) of the extract is placed in a 25 ml volumetric flask, adjusted to the mark with purified water, and mixed (solution A). To 1 ml of solution A add 2.5 ml of 50% acetic acid, 0.5 ml of Trim-Hill reagent, heated for 15 minutes (solution B). The absorbance is measured at KFK-2M at a wavelength of 590 nm (yellow filter).

Comparison solution: 2.5 ml of 50% acetic acid, 0.5 ml of Trim Hill reagent are added to 1 ml of purified water, heated for 15 minutes. Cool.

The content of the sum of iridoids in the extract of M. forest in terms of aucubin and absolutely dry raw materials X,%, is calculated by the formula:

where C is the content of the sum of iridoids in terms of aucubin, found according to the schedule (Fig. 1), μg / ml;

25 - the volume of solution A, ml;

a - an exact sample of the extract, g;

W is the loss in mass upon drying of the extract,%.

The calibration graph (Fig. 1) shows the dependence of the optical density of the solution on the concentration of aucubin. The concentration of aucubin according to the schedule is given in μg / ml, the translation in grams is taken into account in the formula.

Pharmacological tests

The acute toxicity of the extract was determined in accordance with the basic document “Methodological recommendations for studying the general toxic effect of drugs” [8], when administered orally on nonlinear white mice weighing (17-25) g. M. sylvaticum extract was administered at a dose of 5000 mg / kg. Mortality in the experimental group of animals was absent. LD ₅₀ could not be detected. A further increase in the dose of the extract is impractical due to the lack of toxicity at a dose of 5000 mg / kg and the difficulty of administering a larger amount of the extract.

Sedation test

The experiments were carried out on 40 non-linear mice of both sexes weighing (20-25) g. The animals were kept on a standard diet of vivarium in accordance with the "Rules for the work using experimental animals" and the requirements of the Pharmaceutical Committee of the Russian Federation for preclinical trials. Animal experiments were performed in accordance with the European Convention for the Protection and Use of Vertebrate Animals for Experimental and Other Purposes EST No. 123 (1986), Art. 37 and Art. 40 of the Federal Law “On Medicines” (1998), “Rules of Laboratory Practice in the Russian Federation” (2003). The animals were kept under controlled environmental conditions at an air temperature of (18-26) ° C, humidity not more than 50%, and the volume of air exchange (exhaust: supply) as 8:10. A 12-hour lighting cycle was maintained in the room. The animals were in standard plastic cages, cut autoclaved paper was used as a bedding. A routine check was routinely carried out for microbiological contamination. Animals adapted in the laboratory within 10 days before the introduction of the test substances.

Five days before the start of the experiment, the initial characteristics of the behavior of mice were recorded, after which the animals were divided into 4 groups. Animals were randomly assigned to groups. As a criterion, body weight was taken so that its individual value did not deviate from the average value within the same sex by more than 10%. Measurement of behavioral indicators in each of the four groups of animals was carried out before the start of the experiment (control) and then 10, 20 and 30 days after drug administration.

Sedative activity was evaluated in the Open Field test. The study was carried out in a special chamber with a diameter of 34 cm and a height of 17 cm, the bottom of which is divided into circles and sectors with an area of 9 cm ² . In FIG. 2 shows the installation diagram.

The extract was administered orally in the form of an aqueous solution at a dose of 200 mg / kg. As a control, the animals were treated with purified sterile water at a dose of 1 ml / kg. As a comparison drug, we used a tincture of rhizomes with the roots of Valerian officinalis (FS 42-3865-99) at a dose of 0.5 ml / kg. Before the introduction, the tincture was evaporated to 1/3 of the volume to remove alcohol and adjusted to the original volume with water. The selected comparison drug and the method of its preparation described above avoids the formation of a water-insoluble precipitate in the experiment, which ensures maximum bioavailability of the biologically active substances complex. Testing of experimental animals was carried out before the start of the experiment, then every ten days for a month.

The effect of the extract on horizontal activity (the number of animal exits to the center of the site and transitions by sectors), vertical activity (by the number of mice rising on the hind legs with emphasis on the field wall and weight), grooming (short and long) were evaluated. Statistical processing of pharmacological experiment data was carried out according to Student t-test, the effect was considered significant at P≤0.05.

Already with a single use of extract of woody biloba 30 minutes after oral administration, a change in motor activity was observed, comparable to the effect of valerian (Table 5.). With prolonged use of the extract of browning there is a significant decrease in vertical activity (Fig. 3, Table 5). A decrease in the number of “uprights” indicates the presence of a sedative effect in the extract of browning. The horizontal activity along the periphery, as well as the number of animal exits to the center of the site, is significantly reduced, which indicates the inhibition of tentative research behavior. After a ten-day intake of the extract, short and long grooming increases, which is an indicator of comfortable behavior for this group of animals (Fig. 3, Table 5).

Hippocampal Neural Activity Test

A study of the effect of the extract on the spontaneous activity of neurons was carried out in a dissociated hippocampal culture that reached 3 weeks of age in vitro. In the manufacture, incubation and maintenance of the culture followed the protocol described in detail in [9]. At the age of 3 weeks, a fairly dense functional network of neurons forms on the glass, forming excitatory and inhibitory synapses, which form the entire complex of receptors and neuromeriators. The functional and morphological parameters of the hippocampus synapses in culture are close to those in the intact animal brain [13].

For the study, the method of dynamic visualization of intracellular calcium was used, which allows one to evaluate the spontaneous activity in the neural networks of the studied culture and to identify any variations and deviations in its pattern. The study was based on the method of laser confocal microscopy, described in detail in [9].

At first, the background spontaneous activity was recorded, its frequency and stability were checked. After the establishment of these parameters, the neural culture was washed with a solution containing various concentrations of alcoholic extract of biloba from a concentrated mother liquor. Used dilutions of the alcoholic extract of browning per 1 ml of washing solution, shown in table 6.

In 6 experiments, on 35 cells, it was found that with the addition of 0.3, 0.6, and 1.3 μl of an aqueous solution of the extract, there was no statistically significant difference in spike activity for 100 s from the control level.

At 2, 3.3, and especially 6.6 μl of the extract, a very significant, more than threefold, decrease in the number of spikes per 100 sec was detected (P <0.0001) (Fig. 4). The experimental data indicate a significant decrease in the spike activity of hippocampal neurons already at 0.07 mg / ml of dry extract.

Anticonvulsant Activity Test

Determination of anticonvulsant activity was carried out on nonlinear white mice of both sexes, weighing (17-25) g [7]. Seizures were caused by intraperitoneal administration of corazole at a dose of 120 mg / kg in the form of a 1% aqueous solution. From this dose, all animals experience tonic-clonic convulsions with a latent time (0.80 ± 0.02) min and the death of 100% of the animals in the tonic extension phase. The extract was administered orally in the form of an aqueous solution 30 minutes before the injection of corazole. The anticonvulsant effect was judged by the prevention of the tonic phase of seizures. Statistical processing of data from a pharmacological experiment was carried out according to student criterion, the effect was considered significant at P≤0.05 [3]. The results are shown in table 7.

The data in the table show that the extract at a dose of 100 mg / kg does not affect the latent period of seizures, but significantly increases the life expectancy. At a dose of 200 mg / kg, the latent period and life expectancy are significantly increased, and the survival rate of animals is 20%.

Neuromodulatory Antialcohol Test

As a result of a study of the effect of alcohol on neuronal culture [9], it was found that physiological doses of alcohol increase the spontaneous activity of hippocampal cells, thereby disrupting its normal functioning. These disorders affect the cognitive functions of the brain, depending on the hippocampus, such as: RAM, spatial orientation, spatial learning, and others [10, 11]. By virtue of this study, on the cruciform labyrinth, during the passage of which the normal functioning of the hippocampus plays a special role [12], they are an adequate test for studying the anti-alcoholic effect of alcohol extract of browning.

The determination of anti-alcohol activity was carried out on non-linear white mice of both sexes, weighing (17-25) g, in a cruciform labyrinth test.

A study of the effect of physiological doses of alcohol in vivo (corresponding blood concentrations in the range of 0.01% –0.50%) was carried out with intraperitoneal administration of 20% ethyl alcohol [7]. The control group of animals was injected intraperitoneally with 0.9% sodium chloride solution.

In alcoholized animals, a decrease in the latent period, the total time in the maze, and the time of complete crawl in comparison with the control was noted in the cruciform labyrinth test, which confirms the increase in activity established in experiments in culture [9]. In addition, there was a pronounced stereotypic behavior and asymmetry of locomotion in comparison with the control group of animals (Table 8).

The effect of the indicated doses of alcohol was studied against the background of the action of the extract of M. forest. The extract was administered orally in the form of an aqueous solution 30 minutes before the injection of ethyl alcohol. In animals, there was an increase in the total time in the labyrinth compared to alcoholized animals, the time spent by the animals to complete a round, and also a decrease in the stereotypic behavior and asymmetry of locomotion, which indicates the prevention of the occurrence of spontaneous activity of nerve cells caused by the action of alcohol.

Alcohol Activity Test in Neural Culture

Physiological alcohol concentrations (0.1-0.5)% cause a significant increase in the level of spontaneous activity in the culture of hippocampal neurons [9]. Functionally, these changes correlate with such in vivo disorders as coordination of movements, hyperreactivity, euphoria, decreased concentration, and a general decrease in inhibitory functions of the brain [14, 15]. In experiments, the activity of the extract of biloba was studied against the background of exposure to 0.5%, i.e. physiological concentration of alcohol.

Against the background of alcohol exposure, the level of recorded spontaneous activity of cultured hippocampal neurons increased, as judged on the basis of dynamic fluorescence imaging of intracellular calcium. Against the background of increased activity of neural networks, various concentrations of extract of biloba were applied by perfusion (see Table 8). Already at concentrations of 0.3 and 0.6 μl (which corresponds to 0.01-0.02 mg of dry extract), a significant decrease in neuronal activity was observed, shown in the histogram of FIG. 5 (P <0.01). A further increase in concentration led to an even greater suppression of spontaneous activity in neural networks. It should be noted that when washing, the initial level of spontaneous activity was completely restored (4 experiments, 36 cells).

The recorded effect indicates the presence of a pronounced anti-alcoholic effect of low concentrations of browning on the spontaneous activity of cultured mouse hippocampal neurons, even before the onset of the inhibitory (sedative) effect. A further increase in the concentration of bismatic leads to a significant decrease in spontaneous activity (Fig. 5, right columns), as was shown in Fig. four.

It has been experimentally proved that extract of grass of biloba has pronounced sedative, anticonvulsant and neuromodulatory anti-alcoholic activity.

The proposed method allows to obtain a drug with a high content of active substances, low toxicity and pronounced biological activity, as well as control the stages of the process and standardize the finished product, convenient for storage and transportation. These advantages confirm the promise of introducing this invention into the pharmaceutical industry, which allows us to expand the range of herbal medicines for the treatment of diseases of the nervous system.

Literature

1. Galishevskaya E.E., "Resource characteristics of two species of plants of the genus Mariannik (Melampyrum L.)", Galishevskaya E.E., Scriabin E.N., Levinova V.F. Materialy VII Międzynarodowej naukowi-praktycznej konferencji “Perspektywiczne opracowania są nauką i technikami - 2011” Volume 42. Nauk biologicznych .: Przemyśl. Nauka i studia. - Str. 14-18

2. Galishevskaya E.E. "Phenolic compounds of plants of the genus Mariannik" Galishevskaya E.E., Petrichenko V.M., Scriabin E.N. // Pharmacy: Scientific. - prakt. journal - 2011. - No. 2. - S. 25-29

3. State Pharmacopoeia of the USSR: Issue. 1. General methods of analysis / Ministry of Health of the USSR. XI - ed., Ext. - M.: Medicine. - 1987. - 336 p.

4. Otinova E.V. "The effect of aqueous and alcoholic extracts of various species of Mariannik on blood coagulability." Otinova E.V., Petrichenko V.M., Syropyatov B.Ya. and others // Ukrainian scientific and medical youth magazine. - 2011. - No. 1. - S. 29-31.

5. Petrichenko, V. M., “Biological and chemical properties of plants of the Norichnikov family”. Petrichenko V.M., Sukhinina T.V., Syropyatov B.Ya., Galishevskaya, E.E. Shestakova T.S., Mischenkova, K.A. Regional competition RFBR-Ural. The results of scientific research obtained in 2007. Digest of articles. Part II Permian. Yekaterinburg. 2008. - S. 120-123.

6. Plant resources of the USSR. Flowering plants, their chem. Composition, Spanish, family Caprifoliaceae-Plantaginaceae. - M .: Nauka, 1990 .-- 328 p.

7. Guidelines for preclinical research of medicines ”, M., 2012

8. Guidelines for preclinical studies of drugs. Part one. - M .: Grif and K, 2012 .-- 944 p.

9. E. Korkotian, T. Bombela, T. Odegova, P. Zubov, M. Segal "Ethanol Affects Network Activity in Cultured Rat Hippocampus: Mediation by Potassium Channels" // PLOS one - 2013 - DOI: 10.1371 / journal.pone .0075988.

10. J.A. King, I. Trinkler, T. Hartley, F. Vargha-Khadem, N. Burgess "The Hippocampal Role in Spatial Memory and the Familiarity-Recollection Distinction: A Case Study" // Neuropsychology - 2004 - Vol. 18, No. 3 - pp. 405-417

11. L.E. Jarrard "On the Role of the Hippocampus in Learning and Memory in the Rat" // Behavioral and neural biology - 1993 - No. 60 - pp. 9-26.

12. D.M. Bannerman, R. Sprengel, D.J. Sanderson, S.B. McHugh, J.N.P. Rawlins, H. Monyer, P.H. Seeburg "Hippocampal synaptic plasticity, spatial memory and anxiety" // Nature Reviews Neuroscience - 2014 - No. 15, pp. 181-192.

13. C. Boyer, T. Schikorski, C.F. Stevens "Comparison of Hippocampal Dendritic Spines in Culture and in Brain" // The Journal of Neuroscience, - 1998 - No. 18 (14) - pp. 5294-5300.

14. B. Smith, B. Molina, W. Pelham "The Clinically Meaningful Link Between Alcohol Use and Attention Deficit Hyperactivity Disorder" // Alcohol research and Health - 2002 - No. 26 (2) -pp. 122-129.

15. C.F. Valenzuela "Alcohol and Neurotransmitter Interactions" // Alcohol health and research World - 1997 - No. 21 (2) -pp. 144-148.

Claims (1)

The method of obtaining funds with sedative, anticonvulsant and neuromodulatory anti-alcoholic activity, including extraction of plant materials - forest grass with 50% ethanol, repercolation with equal loading of raw materials, with a completed cycle in a battery of four percolators in the ratio of raw material: extractant - 1: 2, when insisting on each extraction step for six hours, the combined extracts are left to stand, filtered, decanted, evaporated to a dry residue in a rotary vacuum evaporator.

Images