RU2491951C1 - Method of obtaining vegetable preparation, which has diuretic and anticoagulation activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and deals with creation of medication of vegetable origin based on biologically active substances of Linaria vulgaris, which has diuretic and anticoagulation activity. Method of obtaining medication, which has diuretic and anticoagulation activity, consists in the following: extraction of milled grass of Linaria vulgaris with ethyl alcohol is carried out with successive drawing with 3 parts of extractant by method of remaceration with heating and connecting vacuum at the stage of discharge, under specified conditions.

EFFECT: method makes it possible to obtain medication, which has high diuretic and anticoagulation activity.

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Description

The invention relates to the field of medicine and the pharmaceutical industry and relates to the creation of a herbal product based on the biologically active substances of common flax Linaria vulgaris Mill (family Scrophulariaceae), which has diuretic activity.

Diuretic agents are widely used in medical practice. They are actively prescribed for diseases of the cardiovascular system: for edema associated with chronic heart failure, in the treatment of hypertensive patients. Very often, these diseases are accompanied by the formation of vascular blood clots, which is due to the use of anticoagulant agents in complex therapy. At present, the effectiveness of the use of herbal medicines for the treatment of diseases of the cardiovascular system has been proven, while herbal preparations do not have side effects and are harmless with prolonged use.

A number of herbal remedies that have a diuretic effect are known. Diuretic activity was found in extracts from the underground organs of the marsh cinquefoil [Patent RU 2318531 C1], in pear leaves and grass of the mountaineers unnoticed and variegated [Denisenko ON Pharmacognostic study of plants of the diuretic effect of pear and mountaineer species in the south-east of Ukraine, author. diss. for a job. Candidate degree farm. Sciences, 1991, Lviv], in bearberry, lingonberry [Turova A.D., Sapozhnikova E.N. Medicinal plants of the USSR and their use. M., 1982.]. The use of horsetail leaves as a diuretic is known. The broth is prepared from cut grass, briquettes and granules from the grass of horsetail [Mashkovsky M.D. "Medicinal plants", Kharkov, Torsing, 1997, Ed. 13, v. 1, p. 498]. Anticoagulant activity was found in extracts from plants of dandelion officinalis [Patent RU 2085205 C1], tree pion [Patent RU 93033687 A].

The closest in complexity of action and method of obtaining is a tool that includes dry extracts of the mountaineer, bird leaves, orthosyphon leaves, bearberry leaves and possessing diuretic and hypoazotemic activities [Patent RU 2008142861 A], taken by us for the prototype.

The main disadvantages of the prototype

1. The technology for obtaining funds is multistage: each raw material is extracted separately.

2. The extraction temperature can reach 80 ° C, which leads to additional costs.

3. The product does not have anticoagulant activity.

The aim of the invention is to obtain a preparation based on grass flax flax and the identification of its diuretic and anticoagulant effects.

To achieve this goal, raw flax flaxseed, crushed to the size of particles passing through a sieve with holes with a diameter of 2 mm and screened from dust, was extracted by remaceration by dividing the extractant into 3 parts with heating and connecting a vacuum at the stage of discharge. The raw materials were sequentially insisted with the first part of the extractant (5 parts), then with the second and third (2.5 parts each), each time draining the extract. As extractant use 60% ethanol. The infusion time with each part of the extractant is 40 minutes at a temperature of 42 ° C. The resulting extract was concentrated in a rotary evaporator at a temperature of 60-70 ° C, then dried in an oven at a temperature of 60 ° C. The yield of the finished product is 24% by weight of plant materials. In the future, the goal is achieved by conducting experiments characterizing the strength of the diuretic effect of common flax.

The final product has the following qualitative and quantitative characteristics.

Flavonoid analysis:

Dry extract (0.1 g) was dissolved in 10 ml of 60% ethyl alcohol

1. To 3 ml of solution add 1 ml of a 2% solution of aluminum chloride, a yellow color appears.

2. 10 mg of crystalline magnesium and 5 drops of concentrated hydrochloric acid are added to 3 ml of the solution, heated in a boiling water bath for 3 minutes, and an orange-red color appears.

The quantitative content of flavonoids:

An analytical sample of air-dry raw materials is crushed to the size of particles passing through a sieve according to GOST 214-83 with holes with a diameter of 1 mm. About 0.5 g (accurately weighed) of the raw material is placed in a conical flask with a capacity of 200 ml with thin section, 100 ml of 60% ethanol are added. The flask is connected to a reflux condenser and heated in a boiling water bath for 60 minutes, counting from the moment the extractant is boiled. The flask was cooled to room temperature, the extract was filtered through a cotton swab into a flat-bottomed volumetric flask with a capacity of 100 ml, and through the same filter the solution volume was brought up to 60% with ethanol to the mark. The resulting extract was filtered into a flask through a paper filter (solution A), the first 10 ml of the filtrate was discarded; 2 ml of solution A was placed in a volumetric flask with a capacity of 50 ml, the volume of the solution was adjusted with 60% ethanol to the mark and stirred (solution B). The optical density of solution B was measured on an SF-2000 spectrophotometer at a wavelength of 332 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution using 60% ethyl alcohol. The content of the sum of flavonoids in the grass of common flax in terms of acetylpectolinarine is calculated by the formula:

$$x\left(\%\right)=\frac{A\cdot \mathrm{one\; hundred}\cdot \mathrm{fifty}\cdot \mathrm{one\; hundred}\%}{379\cdot m\cdot V\cdot \left(\mathrm{one\; hundred}-w\right)}$$

where A is the optical density of the test solution; m is a sample of raw materials, g; V is the volume of solution A taken for dilution; 100 - the volume of solution A, ml; 50 - the volume of solution B, ml; W is the loss in mass upon drying of the raw material,%; 379 is the specific absorbance of acetylpectolinarine at a wavelength of 332 nm.

Iridoid Content Analysis:

The dry extract (0.1 g) was dissolved in 10 ml of 60% ethyl alcohol.

1. 5 drops of Trim Hill reagent (a mixture of 5 ml of concentrated hydrochloric acid, 10 ml of a 0.2% copper sulfate solution and 100 ml of glacial acetic acid) are added to 5 ml of the resulting solution, heated in a boiling water bath for 10 min, blue staining appears.

2. 5 drops of Stahl's reagent (1 g of n-diethylaminobenzaldehyde, 5 g of phosphoric acid and 50 g of acetic acid, diluted with water to 100 ml) are added to 5 ml of the resulting solution, brought to a boil in a water bath, a blue-blue color appears.

The quantitative content of iridoids:

An analytical sample of the raw material is crushed to the size of the particles passing through a sieve with holes with a diameter of 1 mm. About 2.0 g (accurately weighed) of the crushed and dust-screened raw materials are placed in a conical flask with a thin section with a capacity of 100 ml, 0.05 calcium carbonate and 50 ml of 20% ethyl alcohol are added. The flask was attached to a reflux condenser and heated in a boiling water bath for 40 minutes. The hot extract is filtered through cotton wool into a 50 ml volumetric flask. After cooling, the extraction volume was adjusted to the mark with ethyl alcohol 20% and stirred. The resulting solution was transferred to a flat-bottomed flask with a capacity of 100 ml with 1.0 g of neutral alumina, stirred on a shaker for 20 minutes and filtered through a paper filter.

For photocolorimetric determination, take 1 ml of the resulting solution into a test tube, add 0.5 ml of Trim-Hill reagent (HCl conc .; 0.2% CuSO ₄ ; CH ₃ COOH ice) and 2.5 ml of 50% acetic acid. The resulting volume is heated in a water bath for 15 minutes, cooled and the absorbance is determined at a wavelength of 590 nm (yellow filter) in a cuvette with a layer thickness of 0.5 cm. As a comparison solution, a mixture of 1 ml of ethyl alcohol 20%, 0.2 ml Trim Hill reagent and 2.5 ml 50% acetic acid. The content of iridoids is determined by the calibration graph, where the optical density of the solution (D) is indicated on the ordinate axis, and the concentration of aucubin (μg / ml) on the abscissa axis.

The content of the amount of iridoids in terms of aucubin as a percentage of absolutely dry raw materials is calculated by the formula:

X = C * 50 * 100 * 100 / m * (100-W) * 1000 * 1000, where

C is the content of the sum of iridoids in terms of aucubin, found according to the schedule, μg / ml; m is a sample of raw materials, g; W - loss in mass during drying of raw materials,%.

The method of obtaining funds is illustrated by the following example:

0.1 kg of common flax grass (only the aerial part) is crushed to a particle size <0.25≤2.0. The crushed raw material is loaded into the extractor, pour 500 ml of 60% ethanol and extracted for 40 min at a temperature of 42 ° C. The resulting extract is drained by connecting a vacuum. Partially depleted raw materials are again poured with 250 ml of 60% ethanol and insist similarly to the first stage of extraction. In total, the infusion procedure is repeated 3 times. The total consumption of the extractant is 1000 ml, which corresponds to the ratio of raw materials - extractant 1:10. The resulting hood is concentrated in a rotary evaporator at a temperature of 60-70 ° C, then dried in an oven at a temperature of 60 ° C. Get 0,024 kg of the finished product, which is 24% of the dry extract by weight of the feedstock. Dry alcoholic extract is a mass of black-brown color. The content of flavonoids in the extract is 23.7 ± 0.20%, iridoids 0.28 ± 0.001%, humidity 7.9 ± 0.10%.

The proposed method of obtaining allows to obtain a product of constant composition, prevents gelation of the resulting extraction and loss of biologically active substances. The method of obtaining can be implemented in enterprises producing drugs.

Test

Conducting experimental studies of the toxicity and specific activity of common flax grass.

1. The study of acute toxicity.

The study of acute toxicity of the extracts was carried out on white mice according to Prozorovsky [Prozorovsky VB, Prozorovskaya MP, Demchenko VM Express method for determining the average dose efficiency and its errors. // Farmakol. and toxicol. - 1978 - No. 4 - S.497-503.]. The dry extract of flax flax, diluted in distilled water, was studied with a single oral administration at doses of 1000 mg / kg, 2000 mg / kg, 5000 mg / kg.

Studies conducted on white mice with a wide range of doses, aimed at the possibility of identifying toxic effects of varying severity and even deaths. Common flax flax does not have a toxic effect on experimental animals even with the introduction of high concentrations. The results are presented in table 1.

Table 1 The results of determination of acute toxicity of dry extract of common flax (Linaria vulgaris) Extract (extractant) The number of animals in the experiment The number of dead animals Surviving animals,% LD ₅₀ , mg / kg Horsetail grass (water) 6 0 one hundred > 5000 Common flax grass (60% ethanol) 6 0 one hundred > 5000

As can be seen from the table, the oral administration of extracts of the studied plants in the indicated doses did not cause the death of animals. In five experimental groups of animals treated with extracts, there were no visual changes compared with the control group of animals. Determination of LD ₅₀ for mice is not possible. A further increase in the dose of extracts is impractical due to the lack of toxicity at a dose of 5000 mg / kg and the difficulty of administering more extract. Based on the results obtained, it can be concluded that the flax flax extracts are practically harmless and belong to hazard class 4 (low-hazard substances) according to GOST 12.1.007-76, (average lethal dose when injected into the stomach more than 5000 mg / kg).

2. The study of diuretic (diuretic) activity.

The diuretic activity of the flax flax herb extract was evaluated in accordance with the Methodological Recommendations (1989). The experiments were performed on male white rats of the Wistar strain weighing 190–200 g. The extract of the common flax grass without the use of a water load was administered once orally at a dose of 50 mg / kg dissolved in 5 ml of distilled water. Animals of the control group received an equivolume amount of distilled water according to a similar scheme. Field horsetail prepared by HF XI followed by distillation of the extractant (distilled water) to a dry state was used as a comparison drug. The resulting extract was administered at a dose of 50 mg / kg according to the above scheme. Urine collection was carried out in metabolic cells within 6 hours after administration of the extract. The volume of urine excreted was measured. The results are presented in table 2.

table 2 Study of the diuretic activity of dry extract of common flax grass (Linaria vulgaris) Extract (extractant) Urine volume First 3 hours Total for 6 hours Control ml Experience ml Change in urine volume,% Control ml Experience ml Change in urine volume,% Horsetail grass (water) 1.22 ± 0.44 2.0 ± 0.046 64 1.30 ± 0.45 2.61 ± 0.16 one hundred Common flax grass (60% ethanol) 2.3 ± 0.87 86 3.14 ± 0.38 141

As follows from the data given in table 2, a single administration of a dry extract in the specified dose had a pronounced stimulating effect on the excretion of water. At the same time, the diuretic activity of the tested extract exceeded that in 41 hours by 41% in the comparison drug.

3. The study of anticoagulant activity.

Evaluation of anticoagulant activity was carried out in vitro using a Minilab-701 coagulometer at a concentration of 1 mg / ml. For research, we used dog blood stabilized with a 3.8% sodium citrate solution in a 9: 1 ratio. A 0.28% calcium chloride solution was used as an inducer of blood coagulation. The standard of anticoagulant activity was heparin, which was tested at a concentration of 1 U / ml under similar conditions. The effects of all pharmacological tests were considered significant at P≤0.05 [Belenky M.L. Elements of a quantitative assessment of the pharmacological effect, 2nd ed., L .: Medical literature, 1963]. The data obtained are shown in table 3.

Table 3 Anticoagulant activity of dry extract of common flax No. Extract, comparison drug Coagulation time The change in coagulability,% R the control experience one Common flaxseed extract 21.9 ± 0.91 25.2 ± 0.65 -15.1 <0.01 2 heparin 29.9 ± 0.48 36.6 ± 1.82 -22.4 <0.01

As follows from the data in table 3, the extract of flax flax has anticoagulant activity. It significantly slows down the coagulation time by 15%.

It has been established that the common flax grass has a high diuretic and anticoagulant activity. The simultaneous combination of anticoagulant and diuretic properties allows the use of the drug for the prevention and treatment of diseases of the cardiovascular system, often complicated by thrombosis and accompanied by an increase in the volume of circulating blood.

The essence of the invention lies in the fact that the proposed method of obtaining a drug from a plant with a diuretic and anticoagulant effect, not previously used in scientific medicine.

Literature

1. Belenky M.L. Elements of a quantitative assessment of the pharmacological effect, 2nd ed., L .: Medical literature, 1963.

2. Denisenko ON, Pharmacognostic study of plants of the diuretic effect of species of pear and mountaineer in the conditions of southeast Ukraine, author. diss. for a job. Candidate degree farm. Sciences, 1991, Lviv.

3. Mashkovsky M.D. "Medicinal plants", Kharkov, Torsing, 1997, Ed. 13, v. 1, p. 498.

4. Prozorovsky V. B., Prozorovskaya M. P., Demchenko V. M. Express method for determining the average dose efficiency and its errors. // Farmakol. and toxicol., 1978, No. 4, C.497-503.

5. A method of obtaining a complex having fibrinolytic, thrombolytic and anticoagulant activity Patent RU No. 93033687 A from 11/20/1995.

6. The method of obtaining funds with diuretic and anti-inflammatory activity Patent No. 2318531 C1 of 03/10/2008.

7. A tool with anticoagulant action Patent RU 2085205 C1 of 07.27.1997.

8. A tool with diuretic and hypoazotemic activity (options), and a method for its preparation Patent No. 2008142861 A of 05/10/2010.

9. Turova AD, Sapozhnikova EN, medicinal plants of the USSR and their use. M., 1982.

Claims (1)

The method of obtaining funds with a diuretic and anticoagulant effect, consisting in the fact that the extraction of the crushed grass of common flax is carried out with 60% ethanol in the ratio of raw materials: extractant 1:10 when sequentially infused with 5, 2.5 and 2.5 parts of the extractant by remaceration dividing the extractant into 3 parts with heating and connecting a vacuum at the stage of discharge, and the infusion time with each part of the extractant is 40 minutes at a temperature of 42 ° C, and the resulting extract is concentrated in a rotary evaporator at a temperature ur 60-70 ° C, and then dried in an oven at a temperature of 60 ° C.