JPH0717507B2 - Antithrombotic agent - Google Patents

Description

TECHNICAL FIELD The present invention relates to an antithrombotic agent.

[Prior Art and Problems] The adult diseases (encephalomalacia, myocardial infarction, etc.), which have been increasing in number and are of great interest lately, include thrombosis as a cause thereof. It is important to improve the fluidity of the blood itself that flows in blood vessels such as thrombosis, that is, to reduce the blood viscosity and to suppress the aggregation of platelets in the blood, and to have such medicinal effects. Development of a drug was desired.

[Means for Solving Problems] The inventors of the present invention have conducted extensive studies to provide an antithrombotic agent effective for treating thrombosis, and as a result of conducting a drug efficacy test on a methanol extract of Enchoron, It was found that the methanol extract has such an effect.

Corydalis Tuber is a herbal medicine that has been used since ancient times for the purpose of sedation and analgesia.
ceae) Corydalis plant tubers.

Since many alkaloids are contained in this cord of cord, the present inventors further screened the component of cord cord for inhibiting platelet aggregation and reducing blood viscosity, and as a result, dehydrocoridaline has a strong platelet aggregation inhibitory action. It was also found that it has a blood viscosity lowering effect.

The present invention is based on this finding, and provides an antithrombotic agent containing dehydrocoridaline as an active ingredient.

The structural formula of dehydrocoridarin is represented as follows.

Dehydrocoridaline can be obtained from the cord of chordae, for example, as follows.

An extraction liquid is obtained by using at least one mixed solvent selected from water, methanol, ethanol, acetone, ethyl acetate, ether, methylene chloride, benzene, n-hexane, and petroleum ether. This extract as it is,
Alternatively, it is concentrated and dried, and after removing the fat-soluble components with petroleum ether, etc., it is distributed with an aqueous diethyl ether-acetic acid solution, chloroform is added to the acetic acid layer for extraction, and the extract is concentrated and dried to give a crude fraction. To get

This crude fraction is used as it is, or concentrated and dried to obtain at least one selected from water, acetonitrile, tetrahydrofuran, methanol, ethanol, acetone, ethyl acetate, ether, chloroform, methylene chloride, benzene, n-hexane and petroleum ether. Column chromatography is carried out using a mixed solvent to confirm the eluted components of the fraction, the fraction containing dehydrocoridaline is merged, and the solvent is distilled off to obtain dehydrocoridaline.

Further, if necessary, purification by recrystallization may be carried out using a suitable solvent that is usually used, and at this time, hydrochloric acid, iodic acid or the like may be added to form a pharmaceutically acceptable salt. .

Example 1 20 kg of Chinese lacquer cords are treated with 45 liters of methanol for 4 hours each.
The mixture was extracted under reflux, and the methanol extract was concentrated under reduced pressure. 670 g of the obtained methanol extract was dissolved in 2 l of 10% acetic acid and degreased with petroleum ether 1. The insoluble matter in the 10% acetic acid layer was removed by filtration, and the mixture was partitioned and extracted 5 times with diethyl ether 1.

The 10% acetic acid layer was partitioned and extracted with chloroform 5 times, dehydrated with sodium sulfate, and the solvent was distilled off. Chloroform extract 24.6
I got 4g.

Chloroform was further added to this chloroform extract to obtain 22.03 g of a chloroform-soluble portion, which was subjected to silica gel column chromatography, and the polarities were sequentially increased with a mixed solvent of methanol-chloroform to elute. Among the fractions eluted with chloroform-methanol (20: 1, 10: 1, 5: 1), the fractions containing the components showing Rf value 0.46 in thin layer chromatography (developing solvent chloroform: methanol = 4: 1) were extracted. Residue after merger and evaporation of solvent
Dissolve 8.59 g in methanol and add a few drops of concentrated hydrochloric acid to pH 2-3
After adjusting to 1, and leaving it for 24 hours, the yellow needle-shaped substance precipitated 7.65.
g was collected by filtration.

The physicochemical properties of this yellow needle-like substance are described in the literature [Heihachiro Taguchi, Izumi Imekize, Pharmaceutical Journal, vol 82,1214 (1962) / vol8.
3,578 (1963) / vol 84,773 (1964)], which is consistent with the physicochemical properties of dehydrocoridaline chloride.

Mass spectrum (FD-MS): m / z 366 (M ⁺ ) Proton nuclear magnetic resonance spectrum (δ ppm in CDCl ₃ ): 3.03 (3H, s), 3,30 (2H, m), 3.92 (3H, s), 3,96 (3H, s), 4.11 (3H, s) ), 4,21 (3H, s), 7.13 (1H, s), 7,39 (1H, s), 8.15 (2H, s), 9,78 (1H, s), ¹³ C-nuclear magnetic resonance spectrum δ ppm in CDCl ₃ ): 18.3 (q), 28.8 (t), 58.9 (t), 56.8 (q), 57.3 (q), 57.7 (q), 62.7 (q), 112.2 (d), 116.1 (d ), 120.8 (s), 122.0 (d), 122.6 (s), 127.4 (d), 131.6 (s), 133.3 (s), 135.3 (s), 138.1 (s), 144.9 (d), 146.2 (s ), 149.4 (s), 151.7 (s), 153.0 (s) [Effects of the Invention] Next, the fact that the drug of the present invention has an antithrombotic effect will be described with reference to experimental examples.

Experimental Example 1 Wistar male rats (10 to 12 weeks old) preliminarily bred for 1 week were blood-collected from the abdominal aorta under ether anesthesia, and 40% ethylenediaminetetraacetic acid-2 potassium [(EDTA. 2K) physiological saline] was added at a rate of 3 μl per 1 ml. Blood is centrifuged (3000 rpm,
After 4 minutes at 4 ° C, the supernatant and the red blood cell layer were separated. The supernatant obtained by further centrifugation (3000 rpm, 4 ° C., 15 min) was used as plasma. Several erythrocyte layers and plasma were combined, the hematocrit value (hereinafter referred to as HT value) of the erythrocyte layer was measured, the HT value was adjusted to 45%, and the blood viscosity was measured.

Final concentration of dehydrocoridarin in 1 ml of blood for measurement 4.8 × 10
^{-Dissolve in 4} % ethanol in 50% ethanol,
50 μl of this solution was added to 1 ml of blood for blood viscosity measurement, and 37
Incubated for 60 minutes at ℃. Next, 0.5 ml of the incubated blood was sampled and the shear rate was adjusted to 7.5 using a viscometer.
The viscosity was measured at S ^-1 and the degree of decrease in blood viscosity was calculated from the following formula.

However, A: blood viscosity without dehydrocoridarin B: blood viscosity with dehydrocoridarin As a result, the blood viscosity decrease was 12.7%.

Experimental Example 2 Citric acid (using 1/10 volume of 3.8% sodium citrate) was collected from a femoral artery of a lower extremity of a male rabbit having a body weight of 3 to 3.5 kg, and then centrifuged at 800 rpm for 10 minutes, and the supernatant was platelet-rich plasma. (Hereinafter referred to as PRP). Further sediment 15 at 3000 rpm
After centrifugation for 5 minutes, the supernatant was used as platelet plasma (hereinafter referred to as PPP). Next, using a collector counter, PRP
The number of platelets was measured and diluted with PPP to 230,000 to 320,000 cells / μl, and this was used as a regulated PRP for the measurement of the platelet aggregation inhibitory effect.

Dehydrocoridarine chloride was dissolved in physiological saline, and physiological saline was used as a control. Adenosine diphosphate physiological saline solution (hereinafter referred to as ADP) having a final concentration of 10 μM was used as a platelet aggregation inducer. Adjustment PRP 400 μl
50 μl of the sample was added to the cuvette containing the mixture and incubated at 37 ° C. for 3 minutes at a low temperature, and then 50 μl of ADP was added to induce aggregation.

For the aggregation inhibition rate, the aggregation inhibition rate was calculated by obtaining the aggregation rate from the light transmittance at the time of maximum aggregation and comparing with the control.

As a result, the inhibition rate of dehydrocoridarin chloride aggregation was 62% at the final concentration of 50 μg / ml.

From the above results, it was confirmed that dehydrocoridaline has a remarkable antithrombotic effect.

Next, an acute toxicity test of dehydrocoridarin was carried out using male ddY mice.
The safety was confirmed by the intraperitoneal administration of 00 mg / kg without death.

Dehydrocoridaline can also be used as a pharmaceutically acceptable salt such as hydrochloride, iodate or bromate.

Next, the dose and formulation of dehydrocoridarin will be described.

Dehydrocoridarin can be administered to animals and humans either neat or in combination with conventional pharmaceutical carriers. The dosage form is not particularly limited and may be appropriately selected and used as needed. Oral preparations such as tablets, capsules, granules, fine granules and powders, parenteral preparations such as injections and suppositories may be used. Can be mentioned.

In order to exert the intended effect as an oral agent, it depends on the patient's age, body weight, and degree of disease, but usually 30 to 400 mg of dehydrocoridarin should be taken in divided doses daily in adults. Seems appropriate.

In the present invention, oral preparations such as tablets, capsules, granules and the like are produced by a conventional method using, for example, starch, lactose, sucrose, mannitol, carboxymethyl cellulose, corn starch, inorganic salts and the like.

In addition to the above-mentioned excipients, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a fragrance and the like can be appropriately used in this type of formulation. Specific examples of each are as follows.

[Binder] Starch, dextrin, gum arabic powder, gelatin,
Hydroxypropyl starch, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol.

[Disintegrant] Starch, hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80.

[Lubricant] Talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol.

[Fluidity promoter] Light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

The agent of the present invention can also be administered as a suspension, emulsion, syrup or elixir, and these various dosage forms may contain a flavoring agent and a coloring agent.

In order to exert the intended effect as a parenteral agent, it depends on the patient's age, body weight, and degree of disease, but in normal adults, the dehydrocoridarin weight of 5 to 45 mg / day IV or IV Injection, subcutaneous injection, and intramuscular injection seem appropriate.

This parenteral preparation is produced according to a conventional method, and generally uses distilled water for injection, physiological saline, glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. as a diluent. be able to. Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. Further, from the viewpoint of stability, this parenteral preparation may be filled in a vial or the like, frozen, and then water may be removed by an ordinary freeze-drying technique, and a liquid preparation may be re-prepared from the freeze-dried product immediately before use. Further, if necessary, a tonicity agent, a stabilizer, a preservative, a soothing agent and the like may be added appropriately.

Other parenteral agents include external preparations, coating agents such as ointments, suppositories for rectal administration, etc., and they are manufactured by a conventional method.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Corn starch 23.5 g Crystalline cellulose 15 g Carboxymethyl cellulose calcium 5 g Light anhydrous silicic acid 0.5 g Magnesium stearate 1 g Dehydrocoridarine 5 g Total 50 g According to the above formulation, 1 to are uniformly mixed and compressed into a tablet using a tablet machine. 200 mg tablets were obtained.

This tablet contains 20 mg of dehydrocoridaline, and 2 to 20 tablets for adults are to be taken in divided doses.

Example 2 Crystalline cellulose 39.5 g Magnesium stearate 0.5 g Carboxymethyl cellulose calcium 5 g Dehydrocoridarine 5 g Total 50 g According to the above formulation, a part of and was uniformly mixed, compression molded, crushed, and the remaining amount of was added. The resulting mixture was mixed and compression-molded with a tableting machine to obtain 200 mg tablets.

This tablet contains 20 mg of dehydrocoridaline, and 2 to 20 tablets for adults are to be taken in divided doses.

Example 3 Corn starch 43 g Magnesium stearate 0.5 g Carboxymethyl cellulose calcium 5 g Light anhydrous silicic acid 0.5 g Dehydrocoridarine 1 g Total 50 g According to the above prescription, were uniformly mixed with, and after compression molding with a compression molding machine, crushed with a crusher Then, it was sieved to obtain granules.

1 g of this granule contains 20 mg of dehydrocoridarine, and 2 to 20 g of an adult is taken in several divided doses a day.

Example 4 Crystalline cellulose 29g 10% Hydroxypropyl cellulose ethanol solution 20g Dehydrocoridarine 1g Total 50g According to the above-mentioned prescription, were uniformly mixed and kneaded. After granulating with an extrusion granulator, it was dried and sieved to obtain granules.

1 g of this granule contains 20 mg of dehydrocoridarine, and 2 to 20 g of an adult is taken in several divided doses a day.

Example 5 Cornstarch 44.5g Light anhydrous silicic acid 0.5g Dehydrocoridarine 5g Total 50g
No. capsules were filled.

One capsule of this capsule contains 20 dehydrocoridarine.
Contains 2 to 20 capsules daily for adults in several divided doses.

Example 6 Distilled water for injection Appropriate amount Glucose 200 mg Dehydrocoridaline 20 mg Total amount 5 ml After dissolving and in distilled water for injection, the mixture was poured into an ampoule of 5 ml and autoclaved at 121 ° C for 15 minutes to obtain an injection. .

Claims (1)

[Claims] 1. An antithrombotic agent containing dehydrocoridaline as an active ingredient.