RU2504391C2 - Method for preparing herbal agent possessing anticoagulant, hypotensive and diuretic action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely a method for preparing an agent possessing anticoagulant, hypotensive and diuretic action. The method for preparing the agent possessing anticoagulant, hypotensive and diuretic action, consisting in the fact that milled dark mullein is extracted in 60% ethanol by re-percolation in a cluster of 3 unequally batched percolators; the prepared extract is settled, filtered, boiled down and dried in a vacuum drier in the certain environment.

EFFECT: agent prepared by the above method possess pronounced anticoagulant, hypotensive and diuretic action.

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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 black mullein (Verbascum nigrum L.) fam. Scrophulariaceae, which has both anticoagulant, hypotensive and diuretic activities.

The search and development of new drugs intended for the treatment and prevention of diseases of the cardiovascular system are due to the wide distribution and complexity of the pathogenesis of these diseases. The most common diseases from this nosological group are various types of hypertension, atherosclerosis, coronary heart disease, thromboembolism and others. Very often, these diseases are interdependent in nature and are accompanied by high blood pressure, swelling, and the formation of vascular blood clots. In the complex therapy of diseases of the cardiovascular system, hypotensive, anticoagulant and diuretic agents are used, which are prescribed simultaneously. Currently, 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 have a selective anticoagulant effect. So, anticoagulant activity was found in extracts from plants of dandelion officinalis [Patent RU 2085205 C1], tree pion [Patent RU 93033687A], lungwort softest [Chiryatiev EA, Rusakova OA Types of Siberian flora as sources of direct-acting anticoagulants. Rast. resources 1994. T 30. No. 4, C.21-28], extracts from the products of microbial destruction of plant substances of sapropel [Patent RU 2101023 C1]. Extracts of medicinal eyebright from grass [Patent RU 2220735 C1], meadow grass [Patent RU 2222339 C2], collection of five types of medicinal plants (marshmallow cherries, hawthorn berries, Scutellaria baicalensis, bird highlander, rose hips) have hypotensive activity [Patent RU 2182487 C2]. Diuretic activity was found in extracts from underground organs of the marsh cinquefoil [Patent RU 2318531 C1], orthosyphon stamen grass [Patent RU 1737797 C1] and wild strawberry [Patent RU 2342945 C1], as well as in extracts from collections of medicinal plants containing lingonberry leaves, bearberry, highlander grass and other species [Patent RU 2008142861 A; Patent RU 2237488 C1].

The closest in the direction and complexity of the action, as well as in the method of preparation, is a tool that is a purified extract from the roots of middle patrinia, having a sedative, hypotensive, anticoagulant, antiplatelet and hypolipidemic action [Patent RU 2189241 C1], taken by us as a prototype.

The main disadvantages of the prototype are that:

1. The plant source of the remedy is middle patrinia {Patrinia in-tercetia Roem et Sehult) fam. Valerianaceae has a limited distribution area and does not form dense thickets, which limits the raw material base of production.

2. The source of the preparation is the underground organs of middle patrinia. The process of harvesting the roots is time-consuming, and bringing the raw materials to the standard state is energetically and economically costly (washing the roots from the ground, drying, grinding).

3. The crushed plant material is extracted with high concentration alcohol (80%), which leads to a rise in the cost of the drug.

4. The technology for producing the agent is multi-stage; there is an additional stage of purification of the recovery from ballast substances.

5. In the process of obtaining funds highly toxic organic solvents are used - chloroform and butanol.

6. The product does not have a diuretic effect.

The objective of the present invention is to increase the biological value, improve healing properties and expand the spectrum of pharmacological activity due to the manifestation of diuretic activity. A drug is proposed that simultaneously combines anticoagulant, antihypertensive and diuretic properties, allowing it to be used for the prevention and treatment of diseases of the cardiovascular system (hypertension, coronary heart disease, etc.).

To achieve this goal, the dried aerial part (grass) of Black Mullein (Verbascum nigrum L. sem. Scrophulariaceae) is crushed to a particle size of not more than 2 mm, extracted with 60% ethyl alcohol in the ratio of raw material - extractant 1: 6, by repercolation in a battery of three percolators with unequal loading of raw materials (20:30:50), the infusion time in each percolator is 8 hours. The obtained extract was asserted at a temperature not exceeding + 10 ° C for 2 days, filtered off, concentrated, dried under vacuum and a dry extract was obtained. The yield of the finished product is 38% by weight of plant materials.

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.

3. 0.01 ml of the solution is applied to a 40 × 40 cm Filtrak FN-1 chromatographic paper sheet and chromatographed by the ascending method, first in a solvent system butanol — acetic acid — water (4: 1: 2) for 17 hours, and after drying, a sheet of paper is rotated 90 ° and chromatographed in 15% acetic acid for 8 hours. The chromatogram is dried and viewed in UV light before and after treatment with ammonia vapors. In this case, 6 spots appear on the chromatogram. The value R _{f of} these spots should correspond to the data in table No. 1.

Quantification of flavonoids:

An analytical sample of a dry extract of about 0.2 g (accurately weighed) is placed in a 100 ml volumetric flask, dissolved in dioxane-water (1: 1) and adjusted to the mark (solution A), 10 ml of solution A is placed in a volumetric flask with a capacity 50 ml, add 1 ml of a 1% solution of aluminum chloride, make up to the mark with a mixture of dioxane - water (1: 1) and mix (solution B). After 10 minutes, the optical density of solution B was measured on a Spectr-2000 spectrophotometer at a wavelength of 383 nm in a cuvette with an absorbing layer thickness of 10 mm. As a comparison solution, use 10 ml of solution A, brought with a mixture of dioxane - water (1: 1) to the mark in a volumetric flask with a capacity of 50 ml. The content of the sum of flavonoids in the dry extract in terms of hesperidin is calculated by the formula:

,

,

where D is the optical density of the test solution; m is the mass of the extract, g; V is the volume of solution A taken for dilution, ml; 50 - the volume of solution B; 100 is the volume of solution A; W is the moisture content of the extract,%; 120 - specific absorption rate of a complex of 100% of the substance hesperidin with a reagent at a wavelength of 383 nm.

Iridoid Content Analysis:

The dry extract (0.1 g) was dissolved in 10 ml of 60% ethyl alcohol. 5 drops of Stahl's reagent (1 g of p-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.

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

Quantitative determination of iridoids:

Conducted by the reaction with the Trim Hill reagent according to the method of Gregor and Simhen. A sample of the substance for quantification is 0.2 g.

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

0.1 kg of black mullein grass (only the aerial part) is ground to a particle size <0.25≤2.0. The crushed raw materials are loaded into three percolators, with 20 g placed in the first percolator, 30 g in the second and 50 g in the second. The raw materials in the first percolator are poured to a “mirror” and insisted for 8 hours. The obtained extracts are poured and used for extraction of raw materials in the second percolator, and then each last portion of the raw material is extracted with the extract obtained from the previous percolator. The volume of discharge (ml) from each percolator is equal to the weight of the loaded raw materials (g). The infusion time in each percolator is 8 hours, the total consumption of the extractant is 600 ml, which corresponds to the ratio of raw material - extractant 1: 6. The extracts obtained from the last percolator are combined, defended at a temperature of + 10 ° C for two days, filtered, evaporated and dried in vacuum. Get 0,038 kg of the finished product, which is 38% of the dry extract by weight of the feedstock.

The dry extract is a dark brown absorbent mass with a characteristic specific odor. The content of flavonoids in the extract is 8.2 ± 0.4%, iridoids 0.83 ± 0.04%, humidity 6.5 ± 0.5%.

The proposed method of obtaining is quite simple, does not require a complex cleaning system, allows to obtain a product of constant composition. The method of obtaining can be implemented in enterprises producing drugs.

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. Platelet-free and platelet-free plasma was obtained according to the standard method [Guide for the experimental (preclinical) study of new pharmacological substances. M., 2000].

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 2.

As follows from the data in table 2, the extract of black mullein has an anticoagulant effect. It significantly slows down the coagulation time by 17-18%. " The biologically active substances of the extract do not affect the erythrocyte component of thrombus formation. Experiments with platelet-rich plasma and platelet-free plasma indicate that black mullein extract does not block platelet function, but has an effect on plasma coagulation factors similar to heparin.

The test for antihypertensive activity was carried out in vivo on cats [Berezina MP, Vasilevskaya N.E., Averbakh M.S. and others. A large workshop on the physiology of humans and animals. / Edited by L. Vasiliev and Vetyukova I.A. - M .: Higher school, 1961. p.675], as a reference standard used a solution of papaverine hydrochloride at a dose of 3 mg / kg (0.1 LD ₅₀ ). It causes a significant decrease in blood pressure by 34.4% for a duration of 5 minutes. The results in table 3.

As follows from table 3, the dry extract of Mullein black causes a significant decrease in blood pressure by 12% for 10 minutes.

Diuretic activity was determined on 30 white outbred rats males weighing 250-300 g. Dry extracts were administered orally at a dose of 50 mg / kg, previously dissolved in 5 ml of water. Urine collection was performed 3 and 6 hours after drug administration. The control group of animals was injected with 5 ml of water. Comparison preparations (dry aqueous extracts of horsetail and leaves of common lingonberry) were tested under similar conditions. The results are presented in table 4. From the presented data it is seen that the extracts of black mullein have diuretic activity. It should be noted that the extracts exhibit maximum activity during the first three hours after administration of the drug. The diuretic effect of black mullein extract obtained with 60% ethanol is twice as high in the first three hours, and three times as active as the extract obtained in 40% ethanol in 6 hours. The diuretic effect of the claimed agent for 6 hours is reliable and comparable with the activity of the extract from lingonberry leaves and is twice as high as that of the traditional diuretic - horsetail grass.

LITERATURE

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

Berezina M.P., Vasilevskaya N.E., Averbakh M.S. and others. A large workshop on the physiology of man and animals. / edited by Vasiliev L.L. and Vetyukova I.A. - M.: Higher School, 1961, p.675.

Guidelines for the experimental (preclinical) study of new pharmacological substances. M., 2000.

A method of obtaining biologically active substances with anti-aggregation and antihypertensive effect Patent No. 2222339 from 01/27/2004.

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

The method of obtaining funds with anticoagulant activity Patent RU No. 2101023 C1 from 01/10/1998.

The method of obtaining funds with antihypertensive activity Patent No. 2182487 C2 05/20/2002.

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

The method of obtaining funds with diuretic and anti-inflammatory activity Patent No. 2342945 C1 from 10.01.2009.

The method of obtaining funds with anti-inflammatory and antihypertensive activity Patent No. 2220735 from 01/10/2004.

The method of obtaining the sum of extractive substances with diuretic activity Patent No. 1737797 C dated 11/30/1994.

The tool with anticoagulant effect Patent RU No. 2085205 C1 from 07/27/1997.

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

Means "Patrimin" with sedative, antihypertensive, anticoagulant, antiplatelet and antiplatelet effect. Patent No. 2189241 C1 dated 06/26/2001.

Means "Baikal Tea - 6" with diuretic activity and the method for its preparation Patent No. 2237488 C1 of 10.10.2004.

Chiryatiev E.A., Rusakova O.A. Types of Siberian flora as sources of direct-acting anticoagulants. Rast. resources. 1994. T 30, No. 4, p.21-28.

Claims (1)

The method of obtaining funds with anticoagulant, hypotensive and diuretic effects, which consists in the fact that the extraction of black mullein grass, ground to no more than 2 mm, is carried out with 60% ethyl alcohol in the ratio of raw material-extractant 1: 6, by repercolation in a battery of 3 percolators with unequal loading of raw materials in the ratio of 20:30:50 and the infusion time in each percolator 8 hours, the obtained extract is assured at a temperature not exceeding 10 ° C, filtered, evaporated and dried in a vacuum drying apparatus.