JPS6129326B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a pharmaceutical composition containing as an active ingredient a propionic acid derivative, which is a hemostatic and platelet increasing active ingredient obtained from Tashichi. Panax noto−
The main production areas of ginseng Chen (Araliaceae) are Yunnan, Guangdong, and Jiangxi in China, and the powdered root (stem) has been used since ancient times to stop bleeding in cuts and bruises, relieve swelling, and relieve pain, and is used mainly in the surgical field. Used as an important drug. It is also said to be effective against bleeding disorders such as gastric and duodenal ulcers. However, the hemostatic substance contained in Tashichi has not yet been elucidated. Therefore, the purpose of the present invention is to extract and isolate a hemostatic active ingredient from Tashichi, elucidate its structure and action, and provide a useful pharmaceutical composition. The present inventor discovered that the active ingredient of Tashichi is poorly soluble in methanol and soluble in water, and succeeded in extracting it. The extraction method of the present invention is characterized by extracting Tashichi cutlets or powder with methanol, separating the precipitated part and the methanol-soluble part by centrifugation, and further extracting the precipitated part with water. Extraction can be performed at room temperature. It is also advantageous to carry out the extraction operation two to three times in order to completely extract the active substance. According to the present invention, since most of the organic substances are first removed by methanol extraction, the subsequent separation and purification of the active components contained in the residue becomes easy.
That is, the active substance can be obtained by extracting the residue after methanol extraction with water and evaporating the water extract to dryness. The active substance can be further purified in conventional manner, for example by chromatography or recrystallization. The purified active substance has a melting point of 208°C (decomposition)
[α] It is a columnar crystal of ²⁴ _D -17° (C = 2, 4NHCl), and from various equipment data and hydrolysis reaction products, the formula (); HOOC・CONH・CH ₂ CH (NH ₂ )COOH・H ₂ O
A propionic acid derivative represented by (), that is, L-β-
N-oxalo-α/β-diaminopropionic acid.
Estimated to be H ₂ O. Its structure was confirmed because it completely matched that of a standard product synthesized according to Journal of Organic Chemistry, Vol. 41, No. 1, p. 159 (1976). The propionic acid derivative represented by the formula () is described in the above literature and Current Science (India) Volume 32.
153 pages (1963), Biochemistry Vol. 3, No. 3
432 pages (1964), Biochemistry Vol. 11, No. 22
It is a known substance disclosed on page 4053 (1972). However, these documents do not mention that it is an active ingredient of Tashichi at all.
It is only described as a substance contained in the seeds of Lathyrus Sativus and has neurotoxic effects, and no other pharmacological effects are described. The inventor's research has shown that when the compound is ingested in large amounts, it exhibits neurotoxic effects, but when ingested in small amounts, it exhibits no neurotoxic effects, and exhibits a strong hemostatic effect as well as a platelet increasing effect. There was found. That is, the present invention provides L-β-N-oxalo-α・
The present invention relates to a pharmaceutical composition containing β-diaminopropionic acid/H ₂ O as an active ingredient. The active ingredient is not only used as a hemostatic agent, but also has a thrombocytopenic effect, so it can be used as a therapeutic agent for thrombocytosis, thrombocytopenia, thrombocytopenic hemorrhagic purpura, and the like. For example, in an experiment on hemostatic effect on mice, 100 mg of the known transamine took 30 minutes to stop bleeding.
In contrast, the propionic acid derivative of the present invention shortens the hemostasis time by 5 minutes at 1 mg and about 3 minutes at 0.5 mg. In addition, in experiments using mice to increase platelet activity, propionic acid derivatives showed an approximately 30% increase in the number of platelets compared to the control. Acute toxicity tests were also conducted on the active ingredients of the present invention. The results were as follows. Acute Toxicity Value [Test Method] Four-week-old ddY male mice were preliminarily bred for one week, and 10 mice per group were used for the experiment. The test drug was dissolved in physiological saline (10 mg/ml) and administered intravenously at the maximum dose of 200 mg/Kg (0.2 ml/10 g). The LD ₅₀ value is calculated using the up amp down method. [Test Results] No noteworthy toxicity symptoms were observed. L-β-N-oxalo-α・β-diaminopropionic acid LD ₅₀ >200mg/Kg (iv) The compound of formula () can be formulated into tablets, powders, capsules, external preparations, injections, etc. in a conventional manner. It can be administered orally or parenterally. The dosage depends on the symptoms to be treated and the method of administration;
When administered orally to adults, the effect is usually seen with a single dose of 1 to 2 mg. Next, the present invention will be described in detail based on Examples, and the transfer of active ingredients due to extraction was monitored by the following hemostasis test. Hemostasis test method: Inject 0.5 ml of Lock solution into the abdominal cavity of a male mouse weighing 15-20 g. After 10 minutes, place the mouse in a mouse fixator and inject the left tail vein at the middle of the tail (3-4 cm from the base of the tail). Injure it with a scalpel and make it bleed. Gently place a piece of filter paper on the wound at 30 second intervals and observe whether blood adheres to the filter paper. The time until blood no longer adheres to the filter paper piece is measured, and this is taken as the hemostasis time for the control. Subsequently, the test drug was added to the same mouse in a locking solution.
Dissolve in 0.5 ml and administer intraperitoneally to mice. After 10 minutes, place the animal in a fixator and fix it. Injure the right tail vein in the middle of the tail with a scalpel and measure the time for hemostasis in the same manner as the control. The difference from the control hemostasis time is defined as the hemostasis shortening time. Example 1 Add 200 ml of methanol to 100 g of Tashichi powder and extract with stirring at room temperature for 1 hour. Next, centrifugation is performed to separate the precipitated portion and the methanol-soluble portion. Add 200 ml of methanol to the precipitate and repeat the extraction three times in the same manner as above. Next, add 300 ml of water to this precipitate, extract while stirring at room temperature for 1 hour, and centrifuge. Add 300ml of water to the sedimentation part,
Repeat the extraction three times as above. Combine each water-soluble part. A hemostasis test was performed by adding 5 mg each of the methanol-soluble portion and the water-soluble portion to 0.5 ml of the lock solution. The results are shown in the table below.

[Table] From this result, it is clear that there is no hemostatic active substance in the methanol-soluble part, but only in the water-soluble part. Next, this aqueous part was mixed with butanol and water in a ratio of 1:1.
The aqueous portion is dissolved by shaking with a mixture of , and the insoluble matter is removed by centrifugation, and the upper butanol phase is further extracted with water 10 times. The upper and lower layers are separately concentrated to dryness at 40°C under reduced pressure. The results of the hemostasis test are as follows.

[Table] As a result, it was found that hemostatic active substances were concentrated in the lower layer, so this fraction was
Applied to LH-20 and eluted with water. When a hemostatic test was performed on each elution fraction, it was found that the hemostatic active substances were concentrated in the elution volume range of 120 ml to 150 ml, and the range that showed a reduction in hemostasis time of 3 minutes and 30 seconds or more was collected. And it became 625 mg. When this 625 mg was subjected to column chromatography on CM-Sephadex C-25 and eluted with water, crystals precipitated in the elution volume range of 200 ml to 220 ml, so these fractions were collected (50 mg) and reconstituted with water. Upon crystallization, 16 mg of thin columnar crystals were obtained.
This substance has a melting point of 208°C and [α] ²⁴ _D -17° (C=
2,4NHCl), and from the results of instrumental analysis etc., it was found to have the following structural formula. Elemental analysis values (as C ₅ H ₈ N ₂ O ₅・H ₂ O) C H N Calculated value 30.93 5.19 14.43 Actual value 31.02 5.21 14.13 The results of the hemostasis test for this substance were as follows.

[Table] Example 2 Test method for platelet increase effect A group of 10 DDY female mice weighing 15 to 20 g were intraperitoneally administered with 1 mg/0.5 ml/body of the compound of the present invention dissolved in Ringer-Locke nutrient solution. did. Ten
Minutes later, one eyeball was removed to cause bleeding, blood was collected, and the platelet count was measured by the direct method (Fonio Ujihara method). The control group received 0.5ml of Ringer-Locke nutrient solution.
was administered intraperitoneally.

【table】

Claims (1)

[Claims] 1 Formula: HOOC・CO NHCH ₂ CH (NH ₂ )
L-β-N-oxalo- represented by COOH・H ₂ O
A hemostatic and platelet increasing agent characterized by containing α/β-diaminopropionic acid as an active ingredient.