JPH0761951B2 - Platelet aggregation inhibitor and thrombosis remedy containing the ingredient of Chinese cabbage as an active ingredient - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for inhibiting platelet aggregation and treating thrombosis, which contains as an active ingredient the ingredients contained in Chinese cabbage.

[0002]

2. Description of the Related Art Thromboembolis
As for m), it is currently receiving much attention as one of the ten major causes of death in society. Thrombosis is a disease in which blood in a blood vessel is in a coagulated state, and not only general heart disease and diseases of the circulatory system, but also many diseases such as kidney disease, diabetes and cancer cause damage to the vascular endothelium, Blood clots may form. Myocardial infarction and cerebral thrombosis have become a large proportion of adult diseases in recent years, and the emergence of antithrombotic agents that effectively prevent and treat them is desired. It is known that when the arterial thrombus has damage to the endothelium of the artery, platelets adhere to that part, and based on this, thrombosis is formed by the two actions of platelet aggregation and release. That is, metabolism of phosphoinositide, thromboxane and platelet ac
There are complicated biochemical changes such as the formation of activating factor (PAF) or the release of adenosine diphosphate (ADP) and fibrinogen substances. Until now, only a small number of hemagglutination inhibitors such as aspirin and dipyridamole have been used as the drugs mainly suppressing the physiological process of hemagglutination. Regarding this, many drugs have been recently researched and developed for suppressing platelet aggregation, but no significant progress has been reported.

On the other hand, abnormal blood vessels and vasospasm are also factors that form thrombi. A blood vessel abnormality is an abnormality of the inner wall of a blood vessel that causes cholesterol, triglyceride-deposited arteriosclerosis on the blood vessel wall of an artery, or other causes. Since vasospasm lasts for a long period of time, it is different from mere functional vasoconstriction, and is accompanied by organic changes, edema or necrosis of endothelial cells and smooth muscle cells, and ischemic symptoms due to platelet adhesion and aggregation to cell injury parts. And is involved in the development of infarction.
Therefore, vasospasm is a vasodilation, and is called prostacyclin (PGI ₂ ) or EDRF.
(Endothelium delivered rela
xing factor) is released into blood vessels, and blood flow increases. To date, nifedipine or verapamil can be used to suppress the extracellular Ca ^{++ from going} into the cell and relieve the symptoms of vasospasm. Other than this Ca ⁺⁺ channel blocker, there are not many drugs that prevent abnormalities in the inner wall of blood vessels or drugs that can promote the release of prostacyclin and EDRF.

An extract obtained by extracting the water from spicy sauce with a solvent such as water or alcohol has been shown to suppress blood pressure in rats by intramuscular injection and subcutaneous injection, but the components suppressing blood pressure have not yet been announced. .

[0005]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention The inventors of the present invention have conducted extensive research on treatments for various thrombosis, such as platelet aggregation inhibitors and antiarrhythmic drugs. When the effects on thrombosis, vasospasm, and arrhythmia were examined by pharmacological experiments in rats and rabbits, they found that they exhibited a markedly improving effect, and completed the present invention.

[0006] Therefore, an object of the present invention is to provide a therapeutic agent for platelet aggregation inhibitory or thrombosis, which contains a pungent ingredient as an active ingredient.

[0007]

[Means for Solving the Problem] The spicy sauce is Magnolia
nolia kobus DC. ) And homologous plants M. f
argesii , M .; acuminata , M .; sal
A dried flower bud of icifolia , which is used for the treatment of rhinitis, pyometra, headache, heavy headache, etc. for the purpose of sedation and analgesia in Chinese medicine. Braquet et al. Have reported that a component of Veraguensin was isolated from the buds of Chinese cabbage [Pharmacol. Rev. 39 , 97-145
(1987)], Kadsurenone, Ver.
The effects of compounds of aguensin on platelets in animal experiments were examined, and the results were examined.
reported by Uet et al. [Trends in
Pharmacol. Sci. Oct. 397-403
(1986)].

[0008] The ingredient of the Chinese cabbage used in the present invention can be produced by the following method. In other words, dried Chinese herbs, or dried and crushed or powdered, are used as alcohols (eg methanol, ethanol, etc.), ketones (eg acetone, methyl ethyl ketone, etc.), chloroform, etc., alone or as a mixture thereof. Partitioning and extraction with a solvent, distilling off the organic solvent from the resulting organic layer under reduced pressure, subjecting the residue to silica gel chromatography, and concentrating the fractions eluted alone or with a mixed solvent thereof, the effect is obtained. The ingredients can be obtained. The active ingredients of the present invention were separately examined in an activity test for inhibiting platelet aggregation in rabbits, and a therapeutic effect on the thrombosis of acne was found and the present invention was completed.

The active ingredient in the present invention is a compound represented by [Formula I] or [Formula II].

[0010]

[Chemical 1]

(In the formula, A is a phenyl group having a lower alkyl group or a lower alkoxy group as a substituent, or [Formula II
I], R ₁ is a lower alkoxy group, R ₂
And R ₃ represents a hydrogen atom or an allyl group,
R ₄ represents a hydrogen atom or a lower alkoxy group)

[0012]

[Chemical 2]

[0013]

[Chemical 3] (In the formula, A is a phenyl group having a lower alkoxy group as a substituent) In the present invention, the lower alkyl group is a methyl group, an ethyl group, a propyl group, an n-butyl group, a sec-butyl group, a tert-butyl group. And the lower alkoxy group includes methoxy group, ethoxy group, propoxy group and the like. Moreover, the following groups can be illustrated as a phenyl group substituted by the lower alkyl group or the lower alkoxy group.

[0014]

[Chemical 4]

[0015]

[Chemical 5]

[0016]

[Chemical 6] Examples of the compound represented by [Formula I] or [Formula II] include Denudatin B (Denud) as shown below.
atin B), Veraguens
in) or Fargesone. These can use the component extracted from the Chinese pepper, but you may use the thing by synthetic | combination as needed.

[0017]

[Chemical 7]

That is, the present invention is directed to arachidonic acid (arachidonic ac), which is an inducer of platelet aggregation in rabbits.
id), collagen, thrombin, Platelet-
An activating factor (PAF) was added, and the platelet aggregation rate (%) was measured by a turbidimetric method. [Formula I]
Alternatively, the component of [Formula II] exhibits a remarkable effect of inhibiting the aggregation of platelets, and the component of [Formula I] or [Formula II] is added thereto at a concentration of 10 to 50 μg / ml, or 100 μM arachidonic acid, 10 μg / collagen. When ml and thrombin 0.1 U / ml are separately cultured in rabbit platelets, thromboxane B ₂ having a strong platelet aggregating action is released from the platelets, but the above components were previously added for culturing. Then it can be blocked. This inhibitory action has different strengths depending on the ingredients.

As shown in the experiment, rabbit platelets contained 0.1 U / ml of thrombin, 10 μg / ml of collagen,
And PAF 2 ng / ml were added separately and the release amount of inositol phosphate (IP) was observed, and the degree of inhibition of either release amount of the ingredient (Formula I) or [Formula II] A significant reduction is seen with the addition of the ingredients. However, it was also revealed that this effect had no effect on the reaction between platelets and fibrinogen. As shown in FIG. 1 and FIG. 2, denudatin B and veraguencin suppress the platelet aggregation action of rabbits depending on their doses. This action is a competitive inhibitory action since it shifts the PAF curve to the right. The IC ₅₀ of denudatin B is about 10 μg / ml and that of veraguencin is weaker than this.

The rat thoracic artery ring specimen is immersed in a high-concentration potassium solution, and if a calcium solution is added, the thoracic artery blood vessels show the phenomenon of spasm, which is represented by the formula [I] or [II] In the thoracic artery ring pretreated with the ingredients and the potassium solution, the spasm phenomenon does not appear even when the calcium solution is added. When a similar specimen is dipped in a calcium solution and norepinephrine is present, the arterial ring exhibits a transient spastic (tonic) and tonic (tonic) reaction. This reaction is carried out before adding the norepinephrine to the component [formula I] or [formula I].
It is shown that it does not appear when the component I] is added. As shown in FIG. 3 to FIG. 6, all four ingredients of Chinese radish suppress the vasoconstrictor action caused by different calcium ion concentration depending on the dose. IC _{50 for} denudatin B, veraguencin, fargeson A and fargeson B
Were 10, 25, 28 and 12 μg / ml, respectively.

Examining the cardiac direction, the components represented by [Formula I] and [Formula II] have no significant effect on the heart rate of rabbits, but in the atrioventricular conduction of rabbits, conduction by electrocardiogram is conducted. Seen to be delayed. On the other hand, in the rat ventricle, the components represented by [Formula I] and [Formula II] exert a strong inhibitory effect on the contraction of the ventricular muscle, but the Ca ⁺⁺ inflow action in the ventricular cells also has a significant inhibitory effect. Have. Moreover, it does not reduce the heart rate.

The present invention comprises a compound of formula [I] and [formula II] or a salt thereof and a pharmaceutically acceptable pharmaceutical additive (eg, carrier, excipient, diluent, etc.), which is usually an effective amount of radish. It can be safely administered to patients in the form of tablets, granules, powders, capsules, injections, suppositories, patches and the like. The dose may vary depending on the patient's symptoms, weight, age, etc., but is usually 10 to 500 m per day for an adult.
It is preferable to administer the drug in the range of g once or several times a day. [Acute toxicity test] Using a group of ICR male mice having a body weight of 18 to 25 g, denudatin B, veraguencin and fargeson A and fargeson B were separately separated from Litchfield & Wilco.
Result of xon method (acute toxicity test) LD ₅₀ >
200 mg / kg (IP injection 200 mg / kg, 2
After 4 hours, no 8 mice / group of all types of drug administered die. )

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited thereto. Example 1 Magnolia Kobus
D. C. ) And M.M. fargesii , M .; salic
ifolia , M .; dried flower buds of acuminata, was left to stir or at room temperature by adding them to the 5-fold amount of chloroform and filtered. This process is repeated 3 times. The obtained chloroform extract is concentrated under reduced pressure.
The obtained residue was subjected to silica gel chromatography, fraction I eluted with n-hexane: ethyl acetate (10: 1), fraction II subsequently eluted with ethyl acetate, and finally a fraction eluted with methanol. III are obtained separately. Fraction II
Was also subjected to silica gel chromatography, and the fraction eluted with n-hexane: ethyl acetate (13: 1) was purified. Each of the obtained fractions was denudatin B (denudat).
in B), fargesone A,
B, C), and Veraguensin (Veraguensi)
n) Consistent with the standard of the compound in the physical constants such as NMR, fusion test and MS.

Example 2 [Platelet Aggregation Inhibitory Action] Preparation of Rabbit Platelet—Ethylenediaminetetraacetic acid was adjusted to a concentration of 6 mM in rabbit blood collected from the ear vein of a rabbit, and 90 Xg at room temperature,
After centrifugation for 10 minutes, the upper layer is platelet rich plasma (Plate).
let rich Plasma, PRP). Furthermore, the lower layer was centrifuged at 500 xg for 10 minutes, and Tyr was added.
By washing with ode's solution and repeating it, the upper layer was made platelet poor (PPP). The above platelet-rich plasma was measured with a hemocytometer (ZM type) and the concentration was 4.5 x 1
0 ⁸ platelets / ml prepared in ethylenediaminetetraacetic acid. Platelet aggregation test-method of O'Brien et al. (J. C.
lin. Path. 15,452-456,1962)
Adenosine diphosphate (AD
P), collagen, arachidonic acid, thrombin, ionophore A23187 and PAF were added, and changes in absorbance of platelet-rich plasma caused by platelet aggregation were recorded. The aggregation rate is calculated by the following formula, assuming that the absorbance of platelets present in Tyrode's solution is 100%.

[0025]

[Equation 1]

With respect to the inhibitory effect of the analyte on platelet aggregation, the inhibitory rate of the analyte is calculated from the ratio of the maximum aggregation of the analyte added to the maximum aggregation when the control solvent of 0.5% DMSO is added. The results of the pharmacological experiments conducted on denudatin B are shown in FIGS. 1 and 2.

Example 3 Denudatin B (denuda) obtained by the method of Example 1
tin) 10 mg, salt 0.9 mg, distilled water 100 ml
Was dissolved in the mixture, filtered, filled in an ampoule for 2 ml, and sterilized by heating by a conventional method to prepare an intramuscular injection solution.

Example 4 Denudatin B (denuda) obtained by the method of Example 1
tin) 500 g, sodium bicarbonate 250 g, D-mannite 4000 g, hydroxypropyl cellulose 4
5 g and 5 g of talc were used to prepare a fine granule according to a conventional method.

[Brief description of drawings]

FIG. 1 shows the inhibitory effect of veraguensin on collagen platelet aggregation.

FIG. 2 shows the inhibitory effect of denudatin B (Denudatin) on the platelet aggregation effect of collagen.

FIG. 3 shows the effect of denudatin B on rat thoracic artery ring.

FIG. 4 shows the effect of veraguencin on rat thoracic artery ring.

FIG. 5 shows the effect of fergueson A on rat thoracic artery rings.

FIG. 6 shows the effect of fargeson B on the rat thoracic artery ring.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication location C07D 407/04 (72) Inventor Fandef 16 Ungokai, Taipei City, Republic of China 7 Gou 14 Gui ( 72) Inventor Yushme, San Jung City, Republic of China, San-Holo, 4 Dan, 181 Gou, 7 Gou, 2 Guy (72) Inventor, Fan Yu Lin, Shinden City, Republic of, China, 391, Gou, 2 Go, Dan, 2 Goan, (72) Inventor, Chen Jeff, Chinese Republic of Shinden, Republic of Korea 2 Dan 391 Goi 2 Gou

Claims (4)

[Claims] 1. A compound having an inhibitory effect on platelet aggregation and a therapeutic effect on thrombosis, which is obtained by extracting spicy sauce with any of alcohols, ketones, chloroform or a mixed solvent thereof and subjecting it to silica gel chromatography as an active ingredient. Platelet aggregation inhibitor and thrombosis therapeutic agent. 2. The active ingredient is denudatin B (Denuda).
The agent according to claim 1, which is tin B). 3. The active ingredient is fargeson A (Fargeson).
Sone A) or Fargeson B (Fargeso)
The agent according to claim 1, which is ne B). 4. An active ingredient, veragensin (Verag).
The agent according to claim 1, which is uensin).