RU2240814C1 - Method for preparing agent eliciting cardioprotective activity - Google Patents

Abstract

FIELD: medicine, cardiology, pharmacy.

SUBSTANCE: Cacalia hastate leaves as milled vegetable material are extracted with 70% ethanol twice in the ratio raw : extractant = 1:16 for 16 h and 10 h and concentrated by evaporation under vacuum. After alcoholic extraction raw is extracted with boiling water additionally twice for 60 min in the ratio raw : extractant = 1:15. Aqueous extracts are evaporated, combined with alcoholic extraction an aqueous residue and dried in vacuum-drying device. The yield of ready product is 40.3% of vegetable material mass. Invention provides realization of the indicated designation.

EFFECT: improved preparing method.

7 tbl, 3 ex

Description

The invention relates to the field of pharmacy and relates to a method for producing funds from plant materials with cardioprotective activity.

A known method of obtaining tincture of hawthorn, which is based on a countercurrent 3-stage extraction of raw materials with 70% ethanol in a battery of three percolators [Bredneva ND, Muravyov I.A. Development of a technology for the production of tinctures from freshly picked hawthorn fruits // Pharmacy. - 1987.- T.XXXVI. - No. 4. - C.33-37]. The disadvantages of this method are the duration of production (6 days) and low yield of extractive substances (2.5%).

The technical result of the invention is the obtaining of funds from the leaves of cacalia spear-shaped, with pronounced cardioprotective activity, which is achieved by increasing the yield of active substances.

To achieve the technical result, the crushed plant material (cacalya leaves) is extracted with 70% ethanol in the ratio of raw materials: extractant 1: (16-18), taking into account the coefficient of water absorption at room temperature and constant stirring. Extract filtered into the collection. The extraction is repeated twice. The extraction time at the 1st phase contact is 16 hours, the 2nd phase contact is 10 hours. Alcohol extracts are combined and evaporated to 1/4 of the original volume. Meal after alcohol extraction is additionally extracted with water at 100 ° C in the ratio of raw materials: extractant 1: (15-20), taking into account the coefficient of water absorption and constant stirring. Extract filtered into the collection. The extraction is repeated twice. The extraction time at the 1st and 2nd phase contacts is 60 min. Aqueous extracts are combined with alcohol and evaporated to 1/3 of the original volume. The resulting product is purified by separation, dried in a vacuum dryer and ground in a propeller-type mill. The yield of the finished product is 40.3%.

Identified distinguishing features allow us to conclude that the proposed technological solution meets the criterion of "novelty."

The proposed method allows to obtain a substance, which is a crumbly amorphous non-hygroscopic powder of brown color with a bitter-acid astringent taste and a peculiar smell. The mass loss upon drying is 2.14%.

The method is illustrated by the following examples.

Example 1

1 kg of speckled cacalya leaves is ground in a mill to a particle size in diameter (sieve No. 50 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and an external steam heater and pour 16 l of 70% ethanol. It is extracted at room temperature and batchwise stirred for 16 hours. The extract is filtered into a collection vessel. Another extraction is carried out under the same conditions for 10 hours, feeding 70% ethanol to the extractor in an amount equal to the volume drained. The first discharge is 12.4 liters, the second is 11.8 liters. The combined alcoholic extracts are evaporated to about 1/4 of the original volume. Raw materials after alcohol extraction pour 15 liters of water. Extracted at 100 ° C and stirring for 60 minutes. Extract filtered into the collection. Another extraction is carried out under the same conditions, supplying water to the extractor in an amount equal to the volume of the drained extract. The first discharge is 14.2 liters, the second discharge is 14.0 liters. Alcohol and water extracts are combined, evaporated to about 1/3 of the original volume. The bottom residue from the four extracts is subjected to purification by separation and dried on stainless baking sheets in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 0.403 kg of the finished product, which is 40.3% of extractive substances by weight of the feedstock. The dry extract is a crumbly amorphous non-hygroscopic powder of brown color with a bitter-acid astringent taste and a pleasant aromatic odor. The mass loss upon drying is 2.14%.

Example 2

1 kg of speckled cacalya leaves is ground in a mill to a particle size in diameter (sieve No. 50 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and an external steam heater, and 14 l of 70% ethanol are poured. It is extracted at room temperature and batchwise stirred for 10 hours. The extract is filtered into a collector. Another extraction is carried out under the same conditions for 8 hours, feeding 70% ethanol to the extractor in an amount equal to the volume drained. The first drain is 10.7 liters, the second is 10.2 liters. The combined alcoholic extracts are evaporated to about 1/4 of the original volume. The raw material after alcohol extraction is poured into 20 liters of water. Extracted at 100 ° C and stirring for 60 minutes. Extract filtered into the collection. Another extraction is carried out under the same conditions, supplying water to the extractor in an amount equal to the volume of the drained extract. The first discharge is 18.5 liters, the second discharge is 18.2 liters. Alcohol and water extracts are combined, evaporated to about 1/3 of the original volume. The bottom residue from the four extracts is subjected to purification by separation and dried on stainless baking sheets in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 0.396 kg of the finished product, which is 39.6% of extractive substances by weight of the feedstock. The dry extract is a crumbly amorphous non-hygroscopic powder of brown color with a bitter-acid astringent taste and a pleasant aromatic odor. The loss in weight on drying is 2.00%.

Example 3

1 kg of speckled cacalya leaves is ground in a mill to a particle size in diameter (sieve No. 50 GOST 214-83). The crushed raw materials are loaded into an extraction apparatus with a stirrer and an external steam heater, and 14 l of 60% ethanol are poured. It is extracted at room temperature and batchwise stirred for 12 hours. The extract is filtered into a collection vessel. Another extraction is carried out under the same conditions for 10 hours, feeding 60% ethanol to the extractor in an amount equal to the volume drained. The first discharge is 10.0 liters, the second is 9.8 liters. The combined alcoholic extracts are evaporated to about 1/4 of the original volume. The raw material after alcohol extraction is poured into 18 liters of water. Extracted at 100 ° C and stirring for 75 minutes. Extract filtered into the collection. Another extraction is carried out for 30 minutes under the same conditions, supplying water to the extractor in an amount equal to the volume of the drained extract. The first discharge is 16.5 liters, the second discharge is 15.8 liters. Alcohol and water extracts are combined, evaporated to about 1/3 of the original volume. The bottom residue from the four extracts is subjected to purification by separation and dried on stainless baking sheets in a vacuum dryer at a temperature of 60 ° C for 8 hours and ground in a propeller-type mill. Get 0.392 kg of the finished product, which is 39.2% of extractive substances by weight of the feedstock. The dry extract is a crumbly amorphous non-hygroscopic powder of brown color with a bitter-acid astringent taste and a pleasant aromatic odor. The mass loss upon drying is 2.16%.

1. We conducted a phytochemical analysis of the dry extract. Based on the qualitative phytochemical analysis of the dry extract, the presence of:

1) organic acids

Chromatography on paper (FN-16, 10 ° С, rising solvent current) in solvent systems butanol - formic acid - water (18: 2: 5) (I direction), ethyl ether - formic acid - water (18: 5: 9) (II direction) after treatment with a 0.5% solution of bromophenol blue (pH 9.2), tartaric acid was detected - R _f = 0.32 (I), R _f = 0.45 (II); citric acid - R _f = 0.48 (I), R _f = 0.56 (II); oxalic acid - R _f = 0.53 (I), R _f = 0.60 (II); malic acid - R _f = 0.68 (I), R _f = 0.63 (II); fumaric acid - r _f = 0.82 (I), R _f = 0.96 (II); succinic acid - R _f = 0.85 (I), R _f = 0.90 (II); pyruvic acid - R _f = 0.91 (I), R _f = 0.98 (II). Identification was carried out in comparison with authentic samples.

2) β-carotene

0.05 g of the obtained extract is dissolved in 10 ml of hexane, pour 2 ml of a saturated solution of antimony (III) chloride in chloroform to 2 ml of the solution, shake vigorously, a blue color develops (β-carotene).

Chromatography in a thin layer of a sorbent (Sorbfil PTSX-P-A-UV plate) in a chloroform-ethyl alcohol system (19: 1) during the development of chromatograms with a 10% solution of phosphoromolybdenum acid in ethyl alcohol and incubation in an oven at a temperature of 80 ° C for 5 minutes, 2 blue zones were detected on a yellow-green background with R _f = 0.87 (β-carotene) and with R _f = 0.92 (unidentified).

3) phenolic acids

Chromatography on paper (FN-6, solvent upflow) in solvent systems butanol - acetic acid - water (4: 1: 2) (I direction), 2% acetic acid (II direction) when viewing the chromatogram in UV light up to and after treatment with a 10% solution of potassium hydroxide in 96% ethanol, caffeic acid was detected - R _f = 0.80 (II), R _f = 0.30 (II); chlorogenic acid - R _f = 0.63 (I), R _f = 0.56 (II). Identification was carried out in comparison with authentic samples.

4) tannins

0.05 g of the obtained extract is dissolved in 5 ml of purified water, 10 ml of a 10% solution of acetic acid and 5 ml of a 10% solution of lead acetic acid are added to 2 ml of a solution, a precipitate forms (hydrolyzable tannins). The reaction mixture is filtered, 2 drops of a 1% solution of iron-ammonium alum and 0.1 g of lead acetic acid are added to the filtrate, a black-green color develops (condensed tannins).

5) coumarins

Chromatography in a thin layer of sorbent (Silufol plate) in a chloroform-ethyl alcohol system (9: 1) after processing the chromatograms with Pauli's reagent and keeping in an oven at a temperature of 105 ° C for 10 minutes revealed three red zones with R _f = 0.50 (esculletin), R _f = 0.76 (umbelliferone), R _f = 0.88 (scopoletin). Identification was carried out in comparison with authentic samples.

2. A method has been developed for the quantitative determination of the sum of organic acids (in terms of malic acid) using the potentiotitrimetric method.

About 0.200 g (accurately weighed) of the dry extract was placed in a conical flat-bottomed flask with a capacity of 250 ml with a ground stopper, 100 ml of distilled water was added, the contents of the flask in a water bath were heated to 70 ° C and this temperature was maintained for one hour. After cooling, the extract is filtered into a 200 ml volumetric flask through a paper filter, which is washed with 10 ml of distilled water and the solution volume is adjusted to the mark with distilled water (solution A).

10 ml of solution A is passed through an ion exchange column, which is washed with 50 ml of water. The eluate and washings are transferred to a beaker for potentiometric titration. Titration is carried out with 0.1 N NaOH solution to a pH value of 7.0. The titer of a NaOH solution is determined by potassium hydrophthalate (State Pharmacopoeia Vol. 11 - Issue 1. - 1990).

The content of the sum of organic acids X (%) in the dry extract in terms of malic acid is calculated by the formula

where V _NaOH is the volume of 0.1 N NaOH, which went to bring the pH of the test solution to a value of 7.0, ml;

V 'is the volume of the dry extract solution, ml (200 ml);

V '' is the volume of the sample passed through an ion-exchange column, ml (10 ml);

K - correction for titer 0.01 N NaOH;

0.0067 - the amount of malic acid in g, corresponding exactly 1 ml of 0.1 N NaOH;

m is the mass of a sample of dry extract, g;

W - loss in mass during drying of raw materials,%.

Found 25.5 ± 0.23%

3. A method has been developed for the quantitative determination of the sum of carotenoids (in terms of β-carotene) using the spectrophotometric method.

About 0.200 g (accurately weighed) of the dry extract was placed in a conical flat-bottomed flask with a capacity of 250 ml with a ground stopper, 50 ml of hexane were added, and thoroughly mixed on the shaker for one hour. After that, the extract is filtered into a 100 ml volumetric flask through a paper filter, which is washed with 10 ml of hexane and the volume of the solution is adjusted to the mark with distilled water (solution A).

The optical density of the solution is measured on a spectrophotometer at a wavelength of 450 nm in a cell with a layer thickness of 10 mm

Hexane is used as a comparison solution.

The content of the sum of carotenoids X (mg%) in terms of β-carotene in the dry extract is calculated by the formula

where D is the optical density of the test solution;

V is the volume of the dry extract solution, ml (100 ml);

m is the mass of a sample of dry extract, g;

W is the loss in mass upon drying of the raw material,%;

β-каротина при длине волны 450 нм в гексане.2770 - specific absorption rate

β-carotene at a wavelength of 450 nm in hexane.

Found the amount of carotenoids in terms of β-carotene 560 mg%.

The proposed method of obtaining does not require a complex purification scheme, allows you to extract the maximum amount of active substances through the use of two extractants, allows you to get a product of constant composition. The technology can be introduced at the enterprises for the production of pharmaceuticals and wellness products.

To establish the optimal technological regimes, the influence of the main factors was studied: the composition and nature of the extractant, its ratio with raw materials, temperature conditions, the degree of grinding of raw materials, the duration and frequency of extraction on the extraction processes of plant material.

Quantification was carried out by the sum of organic acids (in terms of malic acid) and the sum of carotenoids (in terms of β-carotene).

The metrological characteristic of the determination of the sum of organic acids and the sum of carotenoids was carried out on spear-shaped cacalya leaves collected in the Mukhorshibir district of the Republic of Buryatia in 2002 (table 1).

One of the main factors determining the completeness of extraction is the type and concentration of the extractant, which acts as an active component of the system, significantly affecting the quality, completeness and speed of extraction of biologically active substances from plant material. Hot water and ethyl alcohol of the following concentrations were used as extractants: 20, 30, 40, 50, 60, 70, 80, 90, 95%. The results are presented in table 2.

It was found that when extraction with ethyl alcohol at a concentration above 70%, the yield of carotenoids does not significantly increase; hot water extracts the maximum amount of organic acids. Based on the results obtained, 70% ethanol was selected for the extraction of carotenoids, and hot water was selected for the extraction of organic acids.

Grinding a solid product in order to accelerate the process of diffusion extraction of biologically active substances is one of the important stages of the preparation of raw materials, which ensures the completeness of the extraction of extractives. To study the effect of the degree of grinding of plant material on the yield of the sum of organic acids and the sum of carotenoids, the raw material was crushed to particles passing through a sieve with a hole diameter of 0.5; 1.0; 2.0; 3.0; 5.0 mm. It was found that the optimal particle size for this type of raw material is a particle size of 0.5-1.0 mm (table 3).

The dependence of the yield of biologically active substances on the ratio of raw material and extractant was studied, as a result of which the optimal ratios were selected for extraction with alcohol 1: (16:18) and for extraction with water 1: (15-20) (table 4).

To intensify the processes of transition of biologically active substances from plant materials, it is necessary to take into account the temperature factor. With increasing temperature of extraction, the solubility of the components increases. Based on the analysis data, it was found that boiling water is the best extractant for the extraction of organic acids, while room temperature was chosen for alcohol extraction to avoid the formation of artifacts and the destruction of thermolabile carotenoids (table 5).

To determine the duration and multiplicity of the number of extractions, we studied the dependence of the time to reach equilibrium concentration in the raw material system: extractant for alcohol (A) and water (B) extraction.

A. Spent the extraction of crushed raw materials with 70% ethyl alcohol in a ratio of 1:16 at room temperature and periodic stirring. At predetermined time intervals (1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24 h), the extraction was filtered (1 phase contact), evaporated to an aqueous residue, hexane was added and the amount of carotenoids was determined in in terms of β-carotene. Two subsequent extraction of the squeezed raw material was carried out in a similar manner, adding 70% ethanol each time in a volume equal to the merged (2nd and 3rd phase contacts), while the content of the sum of carotenoids was also analyzed.

B. Spent the extraction of crushed raw materials with boiling water in a ratio of 1:15 in a water bath under reflux at 100 ° C with constant stirring. At predetermined time intervals (30, 45, 60, 75, 90, 120 min), the extraction was filtered (1 phase contact) and the amount of organic acids in terms of malic acid was determined. Two subsequent extraction of the squeezed raw material was carried out in a similar manner, each time adding water in a volume equal to the drained (2nd and 3rd phase contacts), while the content of the sum of organic acids was also analyzed.

The experimental data indicate that the equilibrium state occurs at the first contact of the phases after 16 hours and the second contact of the phases after 10 hours in the case of alcohol extraction (table 6); at the first and second contact of the phases after 60 min in the case of aqueous extraction (table 7).

In the case of alcohol and water extraction during the first phase contact, about 75 and 60% of the sum of carotenoids and organic acids (respectively) are extracted, the second - up to 25 and 35%, the third - up to 5%, i.e. up to 95% of carotenoids and organic acids are extracted during the first two phase contacts; therefore, in order to save, the use of the third phase contact was considered inappropriate for both extractants.

The proposed method in comparison with the known allows you to get a drug of constant composition with pronounced cardioprotective activity, which is caused by a significantly higher content of active substances.

It is currently not possible to calculate the economic feasibility of the proposed method, however, the above advantages, combined with a simple production scheme, contribute to the rational use of medicinal plant materials and determine the prospects of introducing this method into the pharmaceutical industry.

PHARMACOLOGICAL STUDY OF EXTRACT FROM LEAVES OF KAKALIA SPEED-LIKE.

Cardioprotective effect of lanceolate extract

The experiments were carried out on 50 white Wistar rats of both sexes weighing 180-200 g. The object of the study was a dry extract isolated from spear-shaped cakalia leaves. To assess its pharmacological activity, acute myocardial damage with catecholamines was reproduced [Bartikov, 1999]. The model of adrenaline myocardial necrosis was induced by a single intramuscular injection of 0.1% adrenaline hydrochloride solution at a dose of 2 mg / kg rat mass into the thigh area of animals. For pharmacotherapeutic purposes, the experimental group of rats was intragastrally administered 1 time per day with extract from the leaves of cacalia spear-shaped in a dose of 5 mg / kg of rat mass, in the form of an aqueous solution in a volume of 1 ml, 5 days before the administration of adrenaline and then for 3 days before the end of the observation . Amiodarone at a dose of 9 mg / kg was used as a comparison drug [Mashkovsky, 1993]. The control group of animals received distilled water in an equivalent volume according to a similar scheme. Studies of the functional state of the animal myocardium were performed 2, 8, 24, and 72 hours after the administration of adrenaline.

The following methods were used to assess the functional state of the myocardium and the pharmacotherapeutic effect of spear-shaped leaf extract: c) determination of serum biochemical parameters: activity of aspartate aminotransferase (AsAT) and alanine aminotransferase (AlAT), lactate dehydrogenase and its LDH fraction ₁ , and de Ritis coefficient was calculated generally accepted methods [Menshikov et al., 1987]; b) a study of the level of malondialdehyde (MDA) in the homogenate of the heart muscle [Steel and others, 1977], diene conjugates (DC) [Gavrilov et al., 1983] and MDA in serum [Temirbulatov et al., 1981]; c) a study of the blood serum cholesterol and total lipids by generally accepted methods [Menshikov et al., 1987]; d) registration of electrocardiographic indicators on an electrocardiograph EK1K - 1 in six standard leads. In this case, the frequency and rhythm of heart contractions, changes in the QRS complex, ST segment and T wave were evaluated [Zyuzenkov, 1998; Murashko et al., 1998]; e) determination of body weight coefficients in the study of the antistress activity of the test drug [Avtandilov, 1976; Selye, 1950]; f) the study of the mechanism of blood coagulation using the anticoagulant test of Bercard modified by Barkagan [Methods of hemostasis, 1993]; g) the study of the development and severity of the stress response. In this case, the criteria were taken into account: an increase in adrenal mass, thymus involution, the appearance of ulcerative lesions in the gastric mucosa.

The data obtained during the experiments were processed by the method of variation statistics using Student t-criterion [Sergienko V.I., Bondareva IB Mathematical statistics in clinical studies. - M.: GEOTAR-MED. 2001. - 256 p.].

During the experiment it was found that already 2 hours after the administration of dry extract of this plant to white rats at the indicated dose, a decrease in the toxic effect of adrenaline on the myocardium was observed. A decrease in the intensity of LPO processes was noted: a decrease in MDA in the homogenate of the heart muscle, MDA and DC in the blood serum of animals by 6, 47, 46%, respectively, than in rats in the control. Along with this, a tendency toward a decrease in the activity of AcAT was observed, and the de Ritis coefficient decreased by 23% compared with the control. In addition, LDH activity decreased by 16% and its cardiac fraction - by 19% in rats receiving dry extract. Cholesterol- and lipid-lowering effects of the extract by 21 and 14%, respectively, are also noted against this background, compared with the control. The observed beneficial effect of the studied extract on the myocardial function in rats is observed 8-24 hours after administration of adrenaline. After 72 hours of observation, the studied herbal remedy contributes to a decrease in the activity of AcAT and AlAT by 36 and 23%, a decrease in the content of MDA and DC in the blood serum by 5 and 34%, respectively. In addition, animals showed a decrease in the level of activity of LDH and LDH ₁ by 22 and 43%, a decrease in the amount of cholesterol and total lipids by 36 and 67%, respectively, with the introduction of dry spear-shaped cactia extract. Comparison drug amiodarone was significantly inferior in the degree of influence on similar indicators of myocardial function in rats with myocardial damage.

During an electrocardiographic study, it was observed that in the control group of animals in 1 case out of 5 in the first 2 hours after administration of adrenaline, signs of transmural myocardial infarction were noted (an increase in the S wave and the formation of a negative QS wave). In all other cases, pronounced myocardial ischemia was noted - an ST segment elevation by 2.5-4 mm above the isoline and T wave inversion, reflecting diffuse metabolic disturbances in the white rat myocardium. Rhythm disturbance was recorded in the form of single and group extrasystoles, up to ventricular fibrillation. In the experimental groups, against the background of the use of the extract and amiodarone, the number of extrasystoles decreased by 3 and 4 times compared with the control. QS wave formation was not observed in groups of animals treated with amiodarone and the studied herbal remedy. Signs of myocardial ischemia were less pronounced with the use of these agents, the ST elevation was 1-3 mm, and the T wave inversion was recorded with less constancy.

When studying the mechanism of blood coagulation, it was found that for 2 hours of experiment in animals of the control group, the rate of thromboplastin and thrombin formation decreases by 16%. Against the background of the introduction of a dry extract and amiodarone, blood plasma coagulation is accelerated. After 8 hours from the start of adrenaline administration, in the control group of rats, activation of fibrinolysis was observed against the background of moderate activation of plasma coagulation. With the introduction of these funds (extract and reference drug), the coagulation of coagulation and fibrinolytic systems is ensured. At 24 hours from the start of adrenaline administration, a high coagulation activity of plasma was observed in the control group of rats. With the introduction of a dry extract of cacalium spear, the liquid state of the blood was provided with a higher hemostatic potential. With the introduction of the extract, inhibition of the formation of thromboplastin and thrombin by 76% is noted in comparison with the control. Comparison drug amiodarone inferior to the dry extract by a similar effect.

Claims (1)

The method of obtaining funds with cardioprotective activity, by extraction with ethanol and water, evaporation and drying, characterized in that the pre-crushed to a particle size of 0.5-1.0 mm dry vegetable raw materials - speckled cacalya leaves - extracted twice at room temperature 70% ethanol at a ratio of raw materials: extractant (1:16) for 16 hours and 10 hours, respectively, followed by combining the extracts, distillation of ethanol, and twice 60 minutes with boiling water at a ratio of raw materials: extractant (1:15), followed by union of the extracts, filtration and combining with the aqueous residue alcoholic extraction, evaporation and drying in a vacuum drying apparatus.