JPS6213927B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a thrombosis prevention and treatment agent containing ginsenoside-Ro as an active ingredient. Ginsenoside-Ro is a compound represented by the following formula, and is derived from Panax ginseng (Panax ginseng C.
It is known to be contained in the roots of A.Mey). Panax ginseng, also known as Panax ginseng, has been used as a tonic or tonic since ancient times. Among its components, saponins include, in addition to the above-mentioned ginsenoside-Ro, ginsenosides- _{Rb 1} , Rb ₂ , Rb ₃ , Rc, Re, Rg ₁ , Rg ₂ , Rh, etc. Among them, ginsenoside −
Only Ro has the structure of a pentacyclic tritenpene derivative as described above, and the others are tetracyclic triterpene derivatives and are compounds with completely different basic skeletons. It has not been previously known that these saponins exhibit pharmacological effects such as thrombosis preventive or therapeutic agents. As a result of research on these saponins, the inventor found that only ginsenoside-Ro exhibits a strong antithrombin effect, while the others do not exhibit a strong antithrombin effect. Since thrombin is an enzyme that causes blood coagulation, we learned that ginsenoside-Ro has the effect of inhibiting blood coagulation and can be used as a thrombosis preventive agent. The inventor also found that ginsenoside-Ro promotes the plasminogen activation effect of urokinase at low concentrations. They also learned that ginsenoside-Ro has the effect of promoting the dissolution of coagulated blood, and can be used as a therapeutic agent for thrombosis, since activation of plasminogen produces plasmin, which dissolves coagulated blood. Furthermore, there is concern about the hemolytic effect that saponins tend to have as a side effect of ginzenoside-Ro, but the inventor found that no hemolytic effect was observed in doses used as a thrombosis preventive or therapeutic agent, and no significant hemolytic effect was observed. I learned that there is no toxicity. This invention was made based on such knowledge. Ginsenoside-Ro used in this invention is the whole plant of Panax ginseng or its roots (water ginseng, white ginseng,
Manufactured by extraction from red ginseng, white ginseng) or synthesis. Extraction methods include, for example, the following methods. That is, the whole plant of Panax ginseng or its root is mixed with a water-containing lower alcohol (e.g. 70%
% methanol) and concentrate the extract to dryness to obtain an extract. This was extracted with a mixture of ethyl acetate and water, the aqueous layer was extracted with water-saturated n-butanol, the organic layer was subjected to column chromatography using silica gel, and the fraction containing ginsenoside-Ro was concentrated to dryness. Ginzenocide
Ro is obtained. As a synthetic method, for example, oleanolic acid (formula below) contained in sugar beet, olive leaves, apple peel, etc. It is produced by combining a sugar component with a known method. At this time, if a derivative of glucose or glucuronic acid (for example, one in which the hydroxyl group is protected with a protective group) is used as the sugar component,
A derivative of ginsenoside-Ro is obtained, which can also be used as a prophylactic and therapeutic agent for thrombosis according to the present invention. The above-mentioned protecting group is preferably one that is easily removed by chemical treatment or in vivo. Furthermore, when other sugars or sugar acids are used in place of glucose or glucuronic acid, analogs of ginsenoside-Ro are obtained, which can also be used as prophylactic and therapeutic agents for thrombosis. The thrombosis prevention and treatment agent of this invention includes:
A preparation containing ginsenoside-Ro as a main component and a pharmaceutically acceptable carrier, such as an organic or inorganic, solid or liquid excipient for oral or parenteral use, is used. Specifically, oral preparations include powders, tablets, granules, capsules, solutions, tinctures, liquid extracts, sugar syrups, suspensions, lemonades, syrups, and the like. In addition, parenteral preparations include injections, drips, suppositories, and the like. The excipients used here are:
Water, lactose, starch, dextrin, calcium phosphate, calcium carbonate, aluminum silicate, magnesium oxide, magnesium stearate, dry aluminum hydroxide, etc. are used. Further, the above-mentioned formulation may contain stabilizers, emulsifiers, buffers, etc. The dosage of the thrombosis prevention and treatment agent of this invention varies depending on the patient's age, medical condition, etc., but for example, in the case of oral preparations, 10 to 1000 mg of ginzenoside-Ro is used per day for adults, and about 50 to 500 mg. is preferred. Furthermore, when used as a therapeutic agent for thrombosis, it is desirable to administer it simultaneously with urokinase or before and after the administration of urokinase, in which case 10 to 1000 mg of ginsenoside-Ro can be used per 1000 units of urokinase. Next, Examples of the present invention will be shown, and the effects of the present invention will be clarified through test examples and comparative examples. Example 1 Ginzenoside-Ro 50mg in a sterilized vial
was added aseptically and the water was removed to obtain a drug in a vial for injection. When using this drug, add lidocaine 0.5% injection solution to make an injection solution. Example 2 Lactose was added to ginsenoside-Ro, mixed uniformly, and tableted using a tablet machine to obtain tablets containing 20 mg of ginsenoside-Ro in each tablet. Example 3 1200 units of commercially available urokinase (lyophilized product) was added to 120 mg of ginsenoside-Ro, and the mixture was aseptically placed in a sterilized vial and water was removed to obtain a drug in a vial for injection. Before use, this drug is diluted 100 times with physiological saline and made into an injection solution. Experimental Example 1 (Antithrombin effect) To a 0.5% bovine fibrinogen solution dissolved in 0.15M NaCl-0.05M Tris-acetate buffer (PH7.4), add 0.1 ginzenoside-Ro solution at the following concentration or a solvent (DMSO) as a control. ml, and 1 minute later, a thrombin solution was added so that the final concentration was 0.2 units/ml, and the clotting time was measured at room temperature (20 to 30°C). The results are as follows.

[Table] From the above results, it was found that ginzenoside-Ro exhibits a strong antithrombin effect and can be used as a thrombosis preventive agent. Comparative Example 1 (Antithrombin effect) In Test Example 1, instead of ginzenoside-Ro, ginzenoside- _{Rb 1} , Rb ₂ , Rc, Re,
The same procedure was carried out using Rg ₁ , Rg ₂ and Rh. The results are as follows.

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[Preparation of fibrin plate]

A 1% agarose solution was heated and dissolved according to the method of Nolen (Haemostasis Vol. 4, p. 100).
The mixture was cooled to 45°C and fibrinogen (containing 200 mg/100 ml of plasminogen) was added. Add this solution to 10
After dispensing 0.1 ml of thrombin solution (100 NIHU/ml) into test tubes, the mixture was poured into a shear plate (9 cm in diameter) to prepare a fibrin plate containing plasminogen. [Test method] According to the method of Astrap (Arch.Biochem.Biophys. Vol. 40, p. 346), the final concentration was added to 0.1 ml of Ginsenoside-Ro solution at the following concentration or the solvent (Nolen's phosphate buffer) as a control. Add 0.1 ml of urokinase solution so that the concentration is 5 units/ml, and after 30 minutes add 20μ
The fibrin dissolution area was measured after injecting the cells at 30° C. for 15 hours, and the activation rate was determined thereby. The results are as follows.

[Table] From the above results, it was found that ginzenoside-Ro exhibits a strong urokinase activation effect at low concentrations. Comparative Example 2 In Test Example 2, instead of ginzenoside-Ro, ginzenoside-Rb ₁ , Rb ₂ , Rc, Re,
The same procedure was carried out using Rg ₁ and Rg ₂ . Result is,
It is as follows.

[Acute toxicity value of Ginsenoside-Ro]

[Test Method] Ginsenoside-Ro was administered orally and intraperitoneally to groups of five male or female mice and rats to examine acute toxicity values. For oral administration,
Ginsenoside-Ro dissolved in distilled water was used as a test solution, and for intraperitoneal administration, a solution prepared by dissolving this in physiological saline for injection was used as a test solution. Also,
For oral administration, administer the test solution per 100 g of body weight to animals that have been fasted for 18 hours, with 2 ml for mice and 1 ml for rats.
was administered intragastrically using an oral probe.
In the case of intraperitoneal administration, the test solution should be administered per 100 g of body weight to non-fasted animals, 4 ml for mice, 2 ml for rats.
ml. In addition, the same volume of distilled water or physiological saline for injection was administered to the control group. After administering the test solution, observe changes in the symptoms of each animal,
After that, the body weight was measured every day for 7 days, and after the final weight measurement, an autopsy was performed to observe the presence or absence of abnormalities in major organs.The heart, lungs, liver, kidneys, and spleen were removed, and the weights of each organ were measured and abnormalities were detected. I looked into it. [Test Results] (In the case of oral administration) The test results showed that in all administration groups up to the highest dose, only mild sedative symptoms lasting approximately 30 minutes were observed immediately after administration, and no other symptoms were observed. No noteworthy symptoms of toxicity were observed. In addition, their weight increased steadily, the same as in the control group, and autopsy results showed no abnormalities in major organs, and there were no cases of death. (In the case of intraperitoneal administration) The test results showed that in all dose groups up to the highest dose, there was only mild sedation symptoms that lasted for about 90 minutes immediately after administration, and other than that, there were no noteworthy symptoms of toxicity. I didn't approve of it. In addition, the body weight was 1 after administration.
It only decreased on the first day, but increased from the second day onward, showing a steady increase similar to that of the control group.
Further, autopsy results showed only slight abnormalities in the liver at the highest dose, but no abnormalities in other organs. Moreover, the abnormalities in the liver were only a slight white spot on its surface and mild adhesions with surrounding organs, but there was no significant difference compared to the control group, and there were no fatal cases. There wasn't any. As a result, the 50% lethal dose (LD ₅₀ ) of Ginsenoside-Ro was as shown below, and its acute toxic effect was recognized to be extremely weak.

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Claims (1)

[Claims] 1. A thrombosis prevention and treatment agent containing Ginsenoside-Ro as an active ingredient.