JPS629567B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a thrombolytic agent containing herbal medicines that enhances the thrombolytic action of urokinase using herbal medicine extracts, and more specifically, ginseng (carrot), water leech (suitetsu), horsetail (bochiyu), peach kernel (tōnin), and apricot kernel (kiyounin). The present invention relates to a thrombolytic agent containing crude drugs in which the thrombolytic action of urokinase is enhanced by using extracts of one or more crude drugs selected from the following in combination with urokinase. In Japan, the incidence of thrombosis has increased to the same level as in Europe and the United States with changes in dietary habits, and it has become an important problem as it is associated with various diseases including cerebrovascular and coronary artery diseases. Platelet function inhibitors, anticoagulants, thrombolytic agents, and the like are used to prevent and treat such thrombosis, and among these, urokinase is mainly used as the thrombolytic agent. However, since urokinase is obtained from fresh human urine, it usually contains other components, such as clotting substances and inhibitors, and requires high-purity purification.
In addition, there are problems with stability, and the cost is extremely high. Therefore, it is highly active,
In order to obtain products at low cost, various separation and purification methods and methods for maintaining and activating the activity have been researched, but at present no satisfactory method has been found yet. Under these circumstances, the present inventors have conducted extensive research on various substances in order to obtain a composition that promotes the activity of urokinase and exhibits a sufficiently high thrombolytic effect with a small amount of urokinase. As a result, the inventors discovered that certain crude drug extracts have an excellent effect of promoting urokinase activity, and completed the present invention. That is, according to the research of the present inventors, ginseng,
It has been found that the thrombolytic action of urokinase can be significantly enhanced by using extracts of herbal medicines selected from water leech, phlegm, peach kernel, and apricot kernel alone or in combination with urokinase. Next, we will show the experimental results of the urokinase activity promoting effect of various herbal medicine extracts using the fibrin plate method. Preparation of sample solution: 100 g of dried shredded crude drug powder was heated under reflux for 3 hours with 300 ml of 50% aqueous alcohol, then filtered.
The crude drug extract obtained by concentrating the liquid to dryness is 1
Sample solutions were prepared by dissolving them in 0.01M phosphate buffer with 0.15M sodium chloride to give concentrations of mg/ml, 10 mg/ml, and 10 mg/ml. Plate preparation: 100 mg according to the method of Wallen et al.
Dissolve agarose by heating in 10 ml of 0.15 M salt-added 0.01 M phosphate buffer (PH7.8), cool to 40°C, add and dissolve 16.6 mg of plasminogen-containing fibrinogen (manufactured by Hanui Chemical Co., Ltd.), and dissolve this solution. 0.1 ml of thrombin solution (100 units/ml, manufactured by Mochida Pharmaceutical Co., Ltd.) was added dropwise to the solution, and then poured into a shear dish (diameter 9 cm). After cooling at room temperature, a hole with a diameter of 6 mm was made. Activity measurement and results: To 0.1 ml of the above sample solution, add urokinase solution (10
Unit/ml, 100 units/ml, manufactured by Midori + Ajisha) 0.1ml
and injected 20 μ each into the holes of the plate, and heated at 37℃.
After being left for 16 hours, the area of the fibrin dissolution window was measured, and the rate of increase was determined from the ratio to the area of the fibrin dissolution window when no sample was added. The results are shown in Table 1.

【table】

[Table] As is clear from the above experimental results, the extracts of ginseng, water leech, horsetail, peach kernel, apricot kernel, etc. have an excellent effect on promoting urokinase activity. In terms of urokinase units, it exhibits a thrombolytic effect that is twice or more than ten times stronger than that of urokinase. The herbal medicine extract used in the present invention is obtained by extracting with water or aqueous alcohols and then concentrating to dryness using a conventional method. If desired, this may be further fractionated with another organic solvent or dialysis. It may be purified by subjecting it to various types of chromatography. The herbal medicine used in the present invention has been widely used since ancient times, is highly safe, has a mild action, and its dosage is easy to control, making it extremely preferable for use as a thrombolytic agent. . The herbal medicine combination thrombolytic agent of the present invention enhances the thrombolytic activity of urokinase by using a herbal medicine extract and urokinase together, and may be in a single dosage form in which urokinase and herbal medicine extract are formulated together. Alternatively, a two-dosage form consisting of a urokinase agent and a preparation containing a crude drug extract may be administered separately before and after the administration of the urokinase agent. The administration method may be oral or parenteral, but an injection form is preferred from the viewpoint of the administration method of urokinase.
When administered separately from the urokinase drug, it may be administered in the form of ordinary oral preparations such as extracts, powders, granules, or tablets, or in the form of other parenteral preparations such as suppositories. Good too. The dosage varies depending on the patient's age, weight, degree of disease, and degree of purification of the herbal medicine extract, but usually, the amount of crude drug extract per 1000 units of urokinase is
About 0.002 to 2 g is used. Next, the present invention will be explained with reference to Examples. Example 1 Urokinase 1200 extracted and purified from fresh urine
Add 120 mg of sterilized dry 50% ethanol extract of ginseng to the freeze-dried product equivalent to one unit to prepare a thrombolytic preparation, and place in a sterilized 10 ml glass vial. Example 2 Instead of the ginseng extract in Example 1, 120 mg of dried extracts extracted with 50% aqueous alcohol from water leeches, horsetails, peach kernels, and apricot kernels were used, and urokinase was added to each in the same manner as in Example 1. Four types of thrombolytic preparations are available. Example 3 In the same manner as in Example 1, urokinase-sterilized ginseng and water leech sterilized dry extracts, respectively.
Add 60 mg each to make a thrombolytic preparation. Example 4 Extracts of ginseng and peach kernel are sterilized and dried, and 60 mg of each of the obtained dried products are mixed together, used as a promoter of urokinase activity, and used simultaneously with a urokinase preparation. When used, the accelerator and urokinase are mixed together in a physiological saline solution. Example 5 A mixture of equal amounts of water leeches and peach kernels is extracted with 50% aqueous alcohol, concentrated to dryness, and the resulting dry product (120 mg) is used as a promoter of urokinase activity and used simultaneously when urokinase is used. The artificial thrombolytic effect of the composition obtained in the above example was investigated by the following method. Thrombolytic rate was measured using the Chandler loop method, which allows thrombus formation to occur in a manner similar to that of a living body. Pour 1 ml of fresh blood from a healthy adult male to which 1/9 volume 3.8% sodium citrate has been added into a plastic tube (TOALON-G, Toa, inner diameter 3 mm, length 272 mm), and add a silicone tube (diameter 5 mm, length 15 mm).
mm) to create a loop. in this
Add 0.1ml of 0.25M calcium chloride and
It was rotated at an angle of 80° and a rotational speed of 12 rpm while maintaining the temperature at 37°C. After 30 minutes, thrombus formation was confirmed, and the composition of each example (urokinase 500U + crude drug extract 50U) was confirmed.
0.1 ml (all prepared to mg/ml) was added and rotated for an additional 4 hours. After spinning, pour the contents into a shear dish to remove the thrombus, add water to make 10 ml, homogenize, centrifuge (2000 rpm, 10 minutes), and calculate the amount of hemoglobin in the blood cells in the thrombus that comes out in the supernatant.
The amount of thrombus was determined by absorbance at 540 nm. The thrombolysis rate was determined from the absorbance when only physiological saline was added instead of the drug. The results are shown in Table 2.

[Table] As is clear from the above results, when the crude drug extract of the present invention is used in combination with urokinase, it exhibits a significantly improved thrombolytic effect compared to when urokinase is used alone.

Claims (1)

[Scope of Claims] 1. Thrombolytic agent containing crude drug, which is characterized in that the urokinase-containing thrombolytic agent contains extracts of one or more crude drugs selected from ginseng, water leech, horsetail, peach kernel, and apricot kernel. agent. 2 Crude drug extract 0.002 per 1000 units of urokinase
The herbal medicine combination thrombolytic agent according to item 1 above, which contains ~2g.