RU2064300C1 - Method of agent preparing showing antiinflammatory activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: knot-grass herb is extracted with 35-45% ethanol at ratio raw : extractant = 1:(12-16) for 1 hr, twice. The third extraction is carried out for 30 min. Combined extracts were filtered and evaporated. Evaporated extract is separated, subjected for additional evaporation and dried in vacuum-drying apparatus. EFFECT: enhanced antiinflammatory activity.

Description

 The invention relates to medicine and relates to a method for producing biologically active substances from plant materials.

 A known method of obtaining the infusion of the mountaineer bird, used as an anti-inflammatory agent. To prepare the infusion, the grass of the mountaineer bird is crushed to particles no larger than 5 mm in size. The crushed medicinal plant material is placed in a porcelain, enameled or stainless steel infundirka, doused with water at room temperature, taking into account the coefficient of water absorption, closed with a lid and heated in a boiling water bath with frequent stirring for 15 minutes. Cool for 45 minutes, filter (squeezing the remainder of the plant material) and add water to the prescribed extract volume (GF X, Art. 349, FS-42-2570-87; GF-XI, p.147-148).

 The disadvantage of this method is the relatively low anti-inflammatory activity compared to the amount obtained by the claimed method.

The aim of the invention is to obtain the sum of extractive substances to increase anti-inflammatory activity. To achieve this goal, the plant material is crushed to particles no larger than 7 mm, extracted with 35 45% ethanol in the ratio of raw material extractant 1: (12-16) (taking into account the coefficient of water absorption and false bottom of the extractor) for 60 minutes at a temperature of 60 ± 2.5 ^o C and constant stirring. The process is repeated twice. In a similar manner, a third extraction is carried out for 30 minutes. The combined extracts are filtered through a cloth, evaporated to 1/15 of the original volume. One stripped off extract is separated, concentrated by an additional park to 1/5 of the initial volume, then dried in a vacuum dryer and crushed in a propeller-type mill. The yield of the finished product is 15.6% by weight of the plant material. According to the applicant, the features that distinguish the proposed technical solution from the prototype are significant, since no patent studies were found.

 The search and development of new effective medicines intended for the prevention and treatment of kidney and urinary tract diseases is due to the wide distribution and special severity of their course, leading to disability of the population. The relevance of the development of medicinal products from plant materials is due to low toxicity and the many-sided mild effects of a wide range of biologically active substances of plants on the main pathogenesis factors. Given the needs of practical health care in herbal medicines for the treatment of diseases of the kidneys and urinary tract, a method has been developed to obtain the amount of biologically active substances from the mountaineer bird.

The proposed method allows to obtain the final product, which is a fine brown powder, loss in mass upon drying of 2.3%
1. Based on a qualitative phytochemical analysis of the amount of extractive substances, the presence of:
1) Flavonoids:
two-dimensional ascending chromatography on paper brand "Filtrak" FN 12 in solvent systems:
I isopropanol acetic acid water (2: 5: 5)
II H butanol acetic acid water (40:12:28).

When viewing the chromatogram in UV light at λ = 365 nm before and after developing with a 5% aluminum chloride solution, the following were found: quercetin Rf 0.22 (I), R _f 0.73 (II); myricetin R _f 0.60 (I), R _f 0.40 (II); hyperoside R _f 0.66 (I), R _f 0.55 (II); avicularin R _f 0.60 (I), Rf 0.65 (II). Identification was performed with authentic samples.

2) coumarins:
thin-layer chromatography on Silufol plates in the toluene-ethyl acetate acetic acid system (5: 4: 1), spraying the chromatograms with 2 N alcoholic solution of potassium hydroxide. When viewing the chromatogram in UV light at λ = 365 nm, umbelliferon (R _f = 0.60) and scopoletin (R _f 0.42) were detected.

 Identification was performed with authentic samples.

3) tannins:
when a 1% solution of gelatin is added dropwise to aqueous extraction, a haze forms, disappearing with an excess of gelatin; when 2 3 drops of a solution of iron-ammonium alum are added to aqueous extraction, a black-and-blue staining and precipitate appear.

4) Phenolcarboxylic acids:
one-dimensional ascending chromatography on paper brand FN 17 in a solvent system: 1 n butanol acetic acid water (4: 1: 2); 11 15% acetic acid.

 After processing the chromatograms in ammonia vapors and viewing in UV light, they were identified with reliable samples.

Caffeic acid R _f 0.80 (I), R _f 0.42 (II).

Chlorogenic acid R _f 0.67 (I), R _f 0.70 (II).

 2. Quantitative determination.

Sum of flavonoids
Spectrophotometric method (GF XI ed.) Found 4.72 ± 0.1527 total flavonoids in terms of avicularin. The proposed method of obtaining is quite simple, does not require a complex purification scheme, allows to obtain a product of constant composition. The technology can be introduced at the enterprises of the Ministry of Medical Industry.

 We have studied various factors affecting the extraction process, the composition and nature of the extractant, its ratio with raw materials, the degree of grinding of raw materials, temperature, time to reach equilibrium concentration in the raw material-extractant system. A quantitative assessment was carried out according to the yield of extractives and the content of flavonoids in them (see Table 1).

 The selection of the optimal extractant is one of the main points in obtaining extracts. The extractant, acting as an active component of the system, affects the speed, completeness and quality of the extraction of biologically active substances from plant material.

As extractants were used hot water (80 90 ^o C) and ethyl alcohol of the following concentrations: 20, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 96.

 Based on experimental data, it was found that the optimal extractant is 35 45% ethyl alcohol (Table 2).

 We have studied the dependence of the yield of the sum of extractive substances on the ratio of raw materials to extractant. The results of the study are presented in table 3.

 Based on the data obtained, it can be concluded that the optimal ratio of raw material to extractant is 1: (12 16), a further increase in the amount of solvent is impractical, since extraction efficiency is not achieved.

 It is well known that the degree of comminution of plant material is affected, which leads to an increase in the surface of the extracted material due to cell rupture, and due to an increase in its contact with the solvent, to accelerate the extraction process.

 To study the effect of the degree of grinding on the output of the sum of extractive substances, the raw material was subjected to grinding to a particle diameter of 0.5; 1.0; 3.0; 5.0; 7.0; 10.0 mm. The results are shown in table 4.

 It was found that the optimum degree of grinding for raw materials is 7 mm.

In order to select the optimal extraction temperature, the yield of the sum of extractive substances and the sum of flavonoids was studied in the temperature range from 20 ^o C to 100 ^o C.

 The results are presented in table 5.

Based on the analysis data, the optimum extraction temperature was established + 60 ± 2.5 ^o C. A further increase in temperature is impractical, destruction of thermolabile substances is possible.

 As can be seen from the above data, the optimal extraction time is 150 minutes, while during the first phase contact (60 minutes) 65 70% of the extractives and the sum of flavonoids are extracted. II phase contact (60 min) provides a yield of 15 20% and III 5 10% i.e. triple extraction is sufficient for complete depletion of raw materials (see table 6).

To prove the drying temperature, we studied drying methods using a vacuum dryer and spray method. The finished product that meets the requirements of preliminary regulatory and technical documentation, obtained by 1 method in a vacuum drying machine. To determine the mechanism of decomposition of substances in the finished product, thermographic studies were carried out (derivatogram was taken). The derivatogram confirms that the decomposition of substances begins at a temperature of 112 122 ^o C, the main removal of moisture occurs at a temperature of 47 72 ^o C. In this regard, the rate of loss in mass during drying was determined at a temperature of +80 - 85 ^o C.

Example N 1. 5 kg of grass of the mountaineer bird is crushed in a mill to a particle size of 7 mm in diameter (sieve N 70 GOST 21 70). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 65l of 35% ethanol (taken in a ratio of raw material extractant 1:12 taking into account the coefficient of water absorption). Extracted at a temperature of 55 ^o C and constant stirring for 60 minutes. The extract is filtered through a tin cloth in the collection. Pour 35% ethyl alcohol in an amount equal to the drained, and again extracted 60 minutes. The third extraction is carried out under similar conditions for 30 minutes. I discharge 46 l, II discharge 42 l, III discharge 42 l. The combined extracts are evaporated to 1/15 of the original volume. The concentrated extract is separated, doubled to 1/5 of the original volume, then dried in a vacuum dryer. Get 755 g of the finished product, which is 15.1% of extractive substances by weight of the feedstock.

Brown amorphous powder, crumple, 2.1% loss in mass upon drying
The anti-inflammatory activity of the finished product was evaluated according to the classical Menkin method (Oyvin, Shetel, 1961). The experiments were performed on white outbred male rats with an initial weight of 160-180 g. The inflammatory reaction was reproduced by subcutaneous injection of white rats with a 9% solution of acetic acid in a volume of 0.5 ml in the back, with a simultaneous intraperitoneal injection of a solution of dextran B at a dose of 300 mg / kg. The sum of extracts of the mountaineer was introduced intragastrically in the form of an aqueous solution in the optimal conditionally-therapeutic dose of 200 mg / kg (the optimal conditionally-therapeutic dose of the sum of the extractives was determined in preliminary experiments) in a volume of 1 ml / 100 g. funds were carried out 1 hour before the introduction of acetic acid, and then daily 1 time per day for 25 days. Animals of the control group received distilled water in a volume of 1 ml / 100 g according to a similar scheme. As an example of comparison, we used an infusion of grass of the mountaineer bird, prepared according to GF XI, which was injected according to a similar scheme in a volume of 1 ml / 100 g. On the 2nd, 7th, and 25th days of the study, the area of necrotic tissue was estimated by applying a necrosis contour onto a transparent film. It was established that the course administration of the amount of extractives in a dose of 200 mg / kg has an anti-inflammatory effect, reducing the degree of tissue alteration when exposed to a phlogogenic agent (Table 7). Moreover, the activity of the agent used on the 2nd day of the study was similar to that of the comparison drug.

 Example 2. 5 kg of grass of the mountaineer bird is crushed in a mill to a particle size of 7 mm in diameter (Sieve N 70 GOST 214-70).

The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 65 liters of 40% ethyl alcohol (taken in the ratio of raw material-extractant 1: 12, taking into account the coefficient of water absorption). Extracted at a temperature of +60 ^o C and constant stirring for 60 minutes The extract is filtered through a tin cloth in the collection. Pour 40% ethyl alcohol in an amount equal to the drained, and again extracted. The third extraction is carried out under similar conditions for 30 minutes I discharge 47.0 l; II discharge 46.0 l; III discharge 45.0 liters

 The combined extracts are evaporated to 1/15 of the original volume. The concentrated extract is separated, doubled to 1/5 of the original volume, then dried in a vacuum dryer. Get 780 g of the finished product, which is 15.6% of extractive substances from the weight of the feedstock.

Brown amorphous powder, crumbles, loss in mass upon drying 2.3%
The acute toxicity of the amount of extractive substances was investigated by the conventional Kerber method (Requirements 1984). The experiments were carried out on 36 outbred white rats of both sexes with an initial weight of 160 180 g. The test substance in the form of an aqueous solution was administered intraperitoneally at doses from 1000 to 2250 mg / kg. Observation of animals was carried out for 14 days.

Studies have shown that the LD _{50 of the} amount of extractive substances when administered intraperitoneally to white rats is 1895.8 mg / kg (1489 ^° C2301). The results obtained allow us to classify the test drug as a low-toxic substance according to the classification of K. Sidorov. (1973). The anti-inflammatory activity of the sum of extractive substances was evaluated using the model of inflammation according to the method of Menkin (Oyvin, Shetel, 1961) in 18 white rats. For a description of the model and experimental design, see the previous section. The test product in the form of an aqueous solution was administered at a dose of 200 mg / kg.

 The results are shown in table. 8.

 As follows from the table, the sum of extractives in a dose of 200 mg / kg has an anti-inflammatory effect, as evidenced by a significant decrease in the area of necrotic tissue in rats treated with the test substance compared to the control. At the same time, the anti-inflammatory activity of the proposed agent was higher than that of the infusion of the mountaineer.

 In the next series of experiments, the anti-inflammatory activity of the sum of extractive substances was studied in a model of acute peritonitis (Alexandrov et al. 1986). The experiments were performed on 24 white outbred male rats weighing 180,200 g. Acute peritonitis was caused by intraperitoneal administration of 1 ml of a 0.2% aqueous solution of silver nitrate. The amount of extractives in the form of an aqueous solution was administered intragastrically at a dose of 200 mg / kg in a volume of 1 ml / 100 g for 30 minutes and 1 hour after silver nitrate injection. The animals in the control group were injected with the same volume of distilled water. Rats of the following group were injected with an infusion of mountaineer 1 ml / 100 g, which served as a comparison drug. 3 hours after administration of silver nitrate, the animals were decapitated. The mesentery was fixed with a Carnoy mixture and stained with a 0.05% toluidine blue solution. In order to evaluate anti-inflammatory activity, the volume of exudate in the abdominal cavity of animals was measured and the number of degranulated mast cells in the mesentery was counted for every 100 cells in the field of view. (See tab. 9).

 The data presented in table 9 indicate that the amount of extractives introduced against the background of acute peritonitis contributes to a significant decrease in the volume of exudate in the abdominal cavity, however, the tested agent does not significantly affect the degree of degranulation of mast cells in the mesentery. Moreover, the activity of the sum of extractive substances was similar to that of the infusion of the mountaineer.

 In an additional series of experiments, the effect of the sum of extractive substances on the course of acute renal failure (ARF) was investigated, since, as is known, many kidney diseases occur against the background of inflammatory processes (Tareev, 1973).

 The experiments were carried out on 108 outbred white male rats weighing 160 180 g. The model of acute renal failure was reproduced by intraperitoneal administration of 1 ml of 2% mercuric chloride solution to animals (Sokolova et al. 1975). The amount of extractives in the form of an aqueous solution was administered intragastrically at a dose of 200 mg / kg in a volume of 1 ml / 100 g, preliminarily 1 hour before the introduction of mercuric chloride, and then 1 time per day throughout the experiment. Rats in the control group received an equivolume amount of distilled water at the same time. As a comparison drug, we used an infusion of highlander grass in a volume of 1 ml / 100 g. The pharmacotherapeutic efficacy of the drugs was evaluated by the level of hyperazotemia developing against the background of acute renal failure. For this, on the 1st, 3rd and 7th day of the experiment, the animals were killed by decapitation and the urea content in the blood serum was determined by the diacetyl monoxime method and creatinine by the Popper method using the BIO LA Test reagent kit (Czechoslovakia).

 The results are presented in table. 10.

 As follows from the table, the preventive therapeutic introduction of the amount of extractives at a dose of 200 mg / kg against the background of acute renal failure has a pronounced hypoazotemic effect, contributing to a significant decrease in the concentration of urea and creatinine in the blood serum of animals treated with the test drug at all times of the study. Moreover, the pharmacotherapeutic efficacy of the sum of extractive substances exceeded that of the comparison drug, as evidenced by the lower level of nitrogen metabolites in the blood of animals treated with the test substance.

Example 3. 5 kg of grass of the mountaineer bird is crushed in a mill to a particle size of 7 mm in diameter (Sieve N 70 GOST 214 70). The crushed raw materials are loaded into an extraction apparatus with a stirrer and external steam heating. Pour 65 liters of 45% ethyl alcohol (taken in the ratio of raw material-extractant 1:12 taking into account the coefficient of water absorption). It is extracted at a temperature of 65 ^° C. with constant stirring for 60 minutes. The extracts are filtered through a gray-tinted cloth in the collection. Pour 45% ethyl alcohol in an amount equal to the drained, and again extracted for 60 minutes The third extraction is carried out under similar conditions for 30 minutes I drain 47.5 l; II discharge - 45.0 l; III discharge 44.0 liters

The combined extracts are evaporated to 1/15 of the original volume. The concentrated extract is separated, doubled to 1/5 of the original volume, then dried in a vacuum dryer. Get 770 g of the finished product, which is 15.2% of extractive substances from the weight of the feedstock. Brown amorphous powder, crumbles, loss in mass upon drying 2.5%
We studied the anti-inflammatory activity of the sum of extractive substances using the inflammation model according to the Menkin method (Oyvin, Shetel, 1961) (for a description of the model and experimental design, see "Example 1"). The test agent in the form of an aqueous solution was administered intragastrically at a dose of 200 mg / kg according to the above scheme.

It was established that the course administration of the sum of extractive substances in the specified dose has an anti-inflammatory effect, reducing the degree of tissue damage when exposed to a phlogogenic agent. (See table 11.)
Comparative characteristics of the proposed method with the selected prototype are presented in table. 12.

Information sources:
1. Alexandrov P.N. Speranskaya T.V. Bobkov Yu.G. et al. The effect of rutin and esculamine on some models of aseptic inflammation // Farmakol. and toxicol. 1986. N 1, p. 84-86.

 2. Gerashchenko G.L. Bandyukova V.A. Samokhin I.I. Anti-inflammatory activity of bioflavonoids. Materials of the 3rd congress of pharmacists of the BSSR. 1977. p. 187-188.

 3. State Pharmacopia X edition. M. Medicine, 1968.

 4. State Pharmacopia of the XIth edition. M. Medicine, 1987.

 5. Medicines approved for use in the USSR. Ed. A.A. Klyuyev and E.A. Babayana. M. 1979.

 6. Mashkovsky M. D. Drugs. Part I. M.Meditsina, 1988.S. 624.

 7. Oyvin I. A. Shetel S. L. Methodology for studying local disorders of capillary permeability // Materials on the pathogenesis of inflammation and pathology of blood proteins. Dushanbe. 1961. V. 49, N 5. s. 167-173.

8. Ponomarev VD Extraction of medicinal raw materials. M. Medicine, 1976. p. 202
9. Sidorov K. K. On the classification of toxicity of poisons with parenteral routes of administration // Toxicology of new industrial chemicals. M. 1973. S. 47-51.

 10. Sokolova V.E. Vasilchenko E.A. Lyubartseva L.A. The effect of drugs from lespedeci species on the nitrogen metabolism of rabbits // Growing. resources. 1975.V.2, N I. p. 90-94.

 11. Tareev E.M. Acute renal failure // Sov. medicine. 1973. N 10. S. 39-46.

 12. Requirements for the preclinical study of the general toxic effects of new pharmacological substances: Temporary guidelines. M. 1984.

 13. Brushwood P. P. Komissarenko N.F. Flavonoids Polugonum aviculare L. // Chemistry nat. connection 1980. N 6. c. 840.

 14. Yakovlev A.I. Churilov T.I. Skokova I.G. Promakhova S.S. Chemical research of the mountaineer bird // Thesis. doc. III All-Union. Congress of pharmacists. Chisinau: Timpul, 1980. c. 210.

 15. Swiatek, Lucyan; Dombrowcz, Elzbieta. Phenolic acides in medicinal Plant drugs of Polygonum species. Farm. Pol. 1987, 43 (7-8), 420-423.

 16. Schimmer, Oskar. Flavonoids. Their role as biologically active natural product. Dtsch. Apoth. Ztg. 1986. 126 (35). 1811-16. TTT12

Claims (1)

 The method of obtaining funds with anti-inflammatory activity by extracting the grass of the mountaineer bird, characterized in that the plant material changed to particles no larger than 7 mm is extracted with 35-45% ethyl alcohol in the ratio of raw material extractant 1: (12-16) 2 times 60 min, the third extraction is carried out for 30 min, the combined extracts are evaporated, purified by separation, concentrated with a double park and dried in a vacuum dryer.

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