RU2601407C1 - Agent with antiplatelet and anticoagulant activity - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry, namely to agent with antiplatelet and anticoagulant activity. Agent with antiplatelet and anticoagulant activity, based on dry ethanol extract of Maackia amurensis, which is root bark extract of Maackia amurensis Rupr et., Maxim, containing complex of isoflavonoids, consisting of daidzein 7-O-gentiobiosides, genistein, afromosine, pseudobaptigenine, formononetin and 5-O-metildaidzein, daidzein 7-O-β-D-glucopyranoside and genistein, pseudobaptigenine 7-O-primverosides and formononetin, maackiaine 3-O-gentiobiosides and medikarpine, produced by triple extraction of Maackia amurensis dry roots bark with 90 % ethanol under certain conditions.

EFFECT: agent has high antiplatelet and anticoagulant activity and is capable of inhibiting vascular-thrombocyte and coagulation hemostasis processes.

1 cl, 2 dwg, 6 tbl, 9 ex

Description

The invention relates to medicine, specifically to pharmacology, and relates to agents having antiplatelet and anticoagulant properties.

Known drugs that have antiaggregant properties - acetylsalicylic acid, ticlopidine, clopidogrel, dipyridamole [1-5], as well as drugs derived from plant materials that have the ability to inhibit platelet aggregation [6-10].

Known drug "PATRIMIN" with sedative, hypotensive, anticoagulant, antiplatelet and hypolipidemic action. The tool is characterized by the fact that it is a dry extract of triterpene glycosides-patrinosides D, B and C, obtained from the roots of middle patrinia [11].

Known drugs with anticoagulant properties - heparin, warfarin, dabigatran, rivaroxaban [12-17], as well as funds based on extracts from plant materials that can inhibit the blood coagulation process [18-21].

Known anticoagulant agent having inhibitory activity against thrombin and representing a solution of a biologically active complex from birch bark, obtained by extraction of crushed birch bark with water during boiling [22].

The prototype of the claimed drug is the preparation "Maakii Amur dry extract" with hemorheological, antiplatelet and antithrombogenic activity [23, 24]. This preparation is a vacuum-dried ethanol extract prepared from crushed sound wood of the Amurian maakia plant (Maackia amurensis Rupr et. Maxim, fam. Fabaceae) [25]. It contains the sum of natural polyphenols, consisting of flavonoids, isoflavonoids, pterocarpans, monomeric stilbenes, dimeric stilbenes, stilbenolignans, isoflavonostilbenes and chalcon [26, 27]. The preparation "Maakii Amur dry extract" is registered in the Russian Federation as a substance (P N003309 / 01 dated 04/12/2004) for the preparation of the drug "Maksar® coated tablets, film coating, 60 mg" (P N003294 / 01 dated 12.04 .20011), intended for the treatment of chronic hepatitis [28-30].

However, the level of antiplatelet and antiplatelet activity of the well-known means "Maakii Amur dry extract" is not high enough.

The objective of the invention is to expand the arsenal of herbal products with antiplatelet and anticoagulant activity.

The problem is solved by creating a new tool with antiplatelet and anticoagulant activity, based on the dry ethanol extract of the root bark of Maackia amurensis Rupr et. Maxim containing a complex of isoflavonoids. This complex contains over 95% glycosidic forms of isoflavones and pterocarpans and consists of 7-O-gentiobiosios daidzein (1), genistein (3), afromosin (4), pseudobaptigenin (6), formononetin (7) and 5-O-methyldaidine ( 12), 7-O-β-D-glucopyranosides of daidzein (2) and genistein (5), 7-O-primeroosides of pseudobaptigenin (8) and formononetin (9), 3-O-gentiobioside maakiain (10) and medicarpine (11) )

: - 19,0° (с 1,0 С₂Н₅ОН); ИК-спектр (KBr): v мах 3406, 1620, 1560, 1441 см^-1. Его структура установлена методами масс-спектрометрии и спектроскопии ЯМР как 7-O-[β-D-ксилопиранозил-(1→6)]-β-D-глюкопиранозид псевдобаптигенина. Данные ¹H и ¹³С ЯМР спектров соединения (8) представлены в таблице 1.All compounds are known, with the exception of 7-O-primerosyl pseudobaptigenin (8), which was isolated by the authors from alcohol extract of the root bark of M. amurensis on a column filled with Toyopearl HW-50F sorbent (2 × 30 cm) in a water-alcohol solvent system (9: 1) containing 0.02% formic acid. Compound (8) is obtained as a white amorphous powder;

: - 19.0 ° (s 1.0 C ₂ H ₅ OH); IR Spectrum (KBr): v max 3406, 1620, 1560, 1441 cm ^-1 . Its structure was established by mass spectrometry and NMR spectroscopy as 7-O- [β-D-xylopyranosyl- (1 → 6)] - β-D-glucopyranoside pseudobaptigenin. Data ¹ H and ¹³ C NMR spectra of the compound (8) are presented in table 1.

In FIG. 1 shows the structures of the above isoflavonoids.

In FIG. Figure 2 shows the HPLC of the isoflavonoid complex from the root bark of Maackia amurensis. Dihydroquercetin (DHA) is used as an internal standard.

In the patent and other scientific and technical literature available, an agent based on an extract from the bark of Maakia Amur roots, possessing antiplatelet and anticoagulant activity, was not found. Previously, the authors patented an agent that is an extract of Amurian maakia roots, containing up to 70% of 7-O- [β-D-glucopyranosyl- (1 → 6)] - β-D-glucopyranosides of genistein, pseudobaptigenin, formononetin, 5-methoxididine and maakiain with hepatoprotective activity [31].

Since the composition of the polyphenolic complexes of sound wood and the bark of the roots of Amurian maakia is different, it was impossible to clearly assume that the latter had antiplatelet and anticoagulant properties.

The technical result provided by the invention is the manifestation of the claimed means of higher antiplatelet and anticoagulant activity in comparison with the drug "Maakia Amur dry extract" with the simultaneous ability to inhibit the processes of vascular-platelet and coagulation hemostasis.

The method of obtaining the claimed funds is as follows.

Dry root bark of Maackia amurensis is extracted three times with 90% ethanol at 45 ° C in the ratio of raw materials: ethanol 1: 5. The resulting total alcoholic extract was evaporated in vacuo to a syrupy state, then dissolved in 60% ethanol and extracted four times with hexane to remove ballast compounds. The aqueous-alcoholic extract of the root bark was evaporated in vacuo at 40 ° С with the addition of 96% ethanol in the ratio of the aqueous-alcoholic extract: ethanol 1: 5 to remove residual solvent, then dried by spraying until a dry powder of light yellow color was formed.

The yield of the final product is 15-16% in terms of dry bark of the roots of Amur maakia.

The root bark of Amur maakia roots contains a consistently high amount of isoflavones and pterocarpans glycosides, as can be seen from table 2.

The chemical composition of isoflavonoids and their relative content in the dry extract obtained from the bark of the roots of Amuria maakia was determined using NMR spectroscopy and chromatomass spectrometry and are presented in table 3.

The proposed method allows to obtain a highly purified complex of isoflavonoids, previously not described in the accessible literature, free of vegetable waxes, terpenoids, sterols and other ballast compounds.

Antiplatelet and anticoagulant properties of the claimed funds were found experimentally.

Experiments on the study of vascular-platelet hemostasis using a purified complex of isoflavonoids containing isoflavones and pterocarpans glycosides from the bark of the roots of Amurian maakia (MAKK) were carried out on 39 male Wistar rats weighing 240-290 g.

The animals were divided into 3 groups of 13 rats: the first (control) group received 1 ml of starch mucus intragastrically every day for 10 days; the second group - the preparation "Maakii Amur dry extract" (25 mg / kg daily for 10 days); the third group is MAcc in the same dose over the same period of time. 2 hours after the last injection of substances or starchy mucus under mild ether anesthesia, blood was taken from the rat abdominal aorta and platelet count (cells / μl) was determined using an automatic hematological analyzer.

The normal range of fluctuations in the number of blood platelets in rats is from 430 thousand to 1 million in 1 mm ³ [32, 33], which corresponded to the results obtained in experiments.

After determining the platelet count for the study of vascular platelet hemostasis, the method of induced ADP platelet aggregation was used using an optical aggregometer that records aggregation in platelet-rich plasma by changing its optical density (light transmission,%).

Coagulation hemostasis experiments were carried out on 40 male Wistar rats weighing 260-280 g. Animals were divided into 3 groups: the first group (14 rats) received 1 ml of starch mucus intragastrically every day (control); animals of the second group (13 rats) were administered enterally for 10 days with the preparation “Maakii Amur dry extract” at a dose of 25 mg / kg daily; the third group - MAcc (13 rats) in the same dose over the same period of time.

2 hours after the last injection of substances or starchy mucus under mild ether anesthesia, blood was taken from the rat abdominal aorta and coagulation hemostasis was examined. For this purpose, a number of coagulogram parameters were determined: activated partial (partial) thromboplastin time (APTT, sec), prothrombin time (sec), thrombin time (sec), fibrinogen concentration (g / l), plasma fibrin-monomeric complexes (ortho-phenanthroline test, RFMC, mg / 100 ml); the activity of the physiological anticoagulant antithrombin III (%), as well as fibrinolysis (lysis time, min).

The principle of the method for determining APTT is based on measuring the coagulation time of blood plasma under standardized contact (kaolin) and phospholipid (cephalin) activation of the process in the presence of Ca ^{2+ ions} .

The basis of measuring prothrombin time is the ability of thromboplastin (factor III, thrombinase) to convert blood prothrombin in the presence of Ca ²⁺ into active thrombin, which, in turn, transforms blood fibrin into insoluble fibrin. PV is the time of fibrin formation in the presence of Ca ²⁺ and tissue thromboplastin.

The measurement of thrombin time consists in determining the clotting time of blood plasma under the influence of thrombin of standard activity.

The principle for determining fibrinogen is to measure the clotting time of diluted citrate plasma with excess thrombin. The clotting time is proportional to the concentration of fibrinogen, which is determined according to the calibration graph.

The principle of the method for determining fibrin-monomer complexes in blood plasma is to fix the time of appearance in the plasma containing these complexes of grains (paracoagulates) of fibrin after adding a solution of phenanthroline to it.

The essence of the determination of antithrombin III is that AIII of the diluted test plasma in the presence of heparin rapidly inactivates α-thrombin. This leads to a prolongation of the clotting time by which the activity of the AIII sample is evaluated. Activity AIII, expressed as a percentage of normal, is found from a specially constructed calibration curve.

Fibrinolysis was evaluated by the time of spontaneous lysis of the clot (min) obtained from the euglobulin fraction of plasma when a solution of calcium chloride was added to it.

For the purpose of graphical registration of blood coagulation and fibrinolysis, a thromboelastography technique was used. The following indicators were recorded on the elastogram: CT - time of coagulation onset, sec; CFT — clot formation time, sec; MCF — maximum clot hardness, mm; ML - maximum clot lysis,%; angle α — kinetics of clot formation, °.

The results of the study are presented in examples 1-9.

Example 1. Under the conditions of 10-day enteral administration of starch mucus, the platelet count in the control group of rats was 477 ± 23.1 · 10 ³ cells / μl, the amplitude of ADP-induced platelet aggregation in standardized plasma was 34 ± 3.5%.

Example 2. A 10-day enteral administration of the drug "Maakii Amur dry extract" at a dose of 25 mg / kg caused a significant increase in platelet count: 564 ± 13.7 · 10 ³ cells / μl; an increase of 18.2%. In this case, significant changes in the amplitude of ADP-platelet aggregation when using the drug in this dose were not recorded.

Example 3. Enteral administration of the MAcc compound for 10 days at a dose of 25 mg / kg daily resulted in a slight increase in platelet count by 9.6% (up to 523 ± 15.6 · 10 ³ cells / μl), without reaching significant differences in comparison with indicators of intact rats. In this case, the amplitude indicator of ADP-induced platelet aggregation decreased sharply, by 40.4% yielding to the numbers of control animals.

The results obtained in examples 1-3 are presented in table 4.

Thus, the total dry ethanol extract of the bark of Maakia Amur roots during 10-day enteral administration to rats at a dose of 25 mg / kg has a pronounced antiplatelet effect, significantly exceeding the effect of the preparation “Maakii Amur dry extract” taken in the same dose for the same duration of administration .

Example 4. Under the conditions of a 10-day enteric use of starch mucus, figures were obtained characterizing the indicators of coagulation hemostasis in this rat population.

Example 5. Intragastric administration of the drug "Maakii Amur extract dry" for 10 days at a dose of 25 mg / kg led to a significant increase in prothrombin and thrombin time (by 24% and 11.6%, respectively). In addition, an increase in antithrombin III of 8.8% was recorded. Noteworthy is a slight increase in the activity of antithrombin III (by 8.8%), which indicates the possibility of direct anticoagulant activity.

Example 6. Enteral administration of the MAcc compound for 10 days at a dose of 25 mg / kg resulted in a significant lengthening of the prothrombin and thrombin times (by 36.3% and 64.2%, respectively), significantly superior to the effect of the preparation "Maakii Amur dry extract." In addition, a slight increase in antithrombin III activity (by 5.9%) was found, comparable with the effect of the comparison drug. Noteworthy is the sharp decrease in fibrinolysis time recorded under the influence of MAcc. Under the conditions of using MAcc, the lysis of the formed clot occurred 40-45% faster than in intact rats and animals treated with the preparation “Maakii Amur dry extract”.

The results obtained in examples 4-6 are presented in table 5.

Thus, it was found that under conditions of 10-day administration at a dose of 25 mg / kg, MAKC has a pronounced effect on blood coagulation, slowing down this process to a greater extent than the preparation "Maakii Amur dry extract."

Fixing changes in the blood using a thromboelastograph basically confirmed the ability of isoflavonoids from the root cortex of Amurian maakia (MAKK) to interfere with the process of hemocoagulation, slowing down the blood coagulation process.

Example 7. Under the conditions of a 10-day use of starch mucus, thromboelastogram parameters characteristic of this rat population were established.

Example 8. The enteral use of the preparation "Maakii Amur dry extract" for 10 days at a dose of 25 mg / kg did not significantly change the indicators of thromboelastogram except for a 36.9% increase in the maximum hardness of the formed clot.

Example 9. 10-day enteral administration of MAcc led to a significant change in the time of the beginning of coagulation, which increased by 37.7%.

The results obtained in examples 7-9 are presented in table 6.

The indicators shown in examples 7-9 and table 6, confirm that the total dry extract of the root bark of Maakia Amur roots significantly inhibits the process of blood coagulation.

Thus, the claimed tool has a pronounced antiplatelet and anticoagulant effect, while inhibiting the processes of vascular-platelet and coagulation hemostasis.

The present invention expands the arsenal of antiplatelet and anticoagulant drugs of plant origin, used for disorders of microcirculation, for the treatment and prevention of thrombophlebitis, in the treatment of myocardial infarction, ischemic stroke, for the prevention of thromboembolism.

Information sources

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Claims (1)

An agent with antiplatelet and anticoagulant activity based on dry ethanol extract of Maackia amurensis, characterized in that the agent is an extract of the root bark of Maackia amurensis Rupr et. Maxim containing a complex of isoflavonoids consisting of 7-O-gentiobiozides of daidzein, genistein, afromosin, pseudobaptigenin, formononetin and 5-O-methyldaidzein, 7-O-β-D-glucopyranosides of daidzein and genistein, 7-O-primeronetenides , 3-O-gentiobiosides of maakiain and medicarpin, obtained by triple extraction of dry root bark of Maackia amurensis with 90% ethanol at 45 ° C in the ratio of raw materials: ethanol 1: 5, evaporation of the resulting extract in vacuum to a syrupy state, followed by its dissolution in 60 % ethanol and quadruple extraction with hexane to remove ballast compounds; followed by evaporation of the obtained aqueous-alcoholic extract of root bark in vacuum at 40 ° C with the addition of 96% ethanol in the ratio of aqueous-alcoholic extract: ethanol 1: 5 to remove the residual solvent and drying by spraying until a dry powder of light yellow color is formed.

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