JPS60193996A - Extraction of hemostatic substance from "sokuhakuyo" and hemostat containing the same - Google Patents

Abstract

PURPOSE:An aqueous extract of leaves of SOKUHAKU (Thujae orientalis cacumen) is extracted with an organ ic solvent, then subjected to gel-filtration column chromatography and chromatography to effect siolation and purification to obtain simply quercitrin which is used as a hemostatic. CONSTITUTION:Leaves of SOKUHAKU is extracted with hot water and the extract is fractionated with an ethyl acetate-water solvent mix system and the ethyl acetate phase is separated. The resultant lipid-soluble substances are separated by means of gel-filtration column chromatography using methanol as a eluent to collect the hemostatic fractions. Further, the fractions are purified by subjecting them to silica-gel chromatography and gel-filtration column chromatography to obtain the objective quercitrin.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for extracting a hemostatically active substance from a sycamore leaf, and a pharmaceutical composition containing the same. The sycamore leaf, which is the raw material of the present invention, is a species of Cypress cypress +7 in the Cupressaceae family.
(Biota orientalis Endl,
) In Chinese medicine, it is used as Shishomaru, Aiba-to, Saiso-san, Dankogetsei N-to, etc. to treat nosebleeds, blood in the stool, and hematemesis.

The sexual substances used as hemostatic agents for internal bleeding, antitussive expectorants, and burn treatment agents have not yet been elucidated.

As a result of intensive research, the present inventors discovered that the hemostasis-active substance contained in Quercus leaves is quercitrin, and that highly purified quercitrin can be obtained by a simple procedure, thereby completing the present invention. reached.

That is, the gist of the present invention is to provide a method for extracting quercitrin represented by formula (1) from Cypress leaves;
and hemostatic agents containing quercitrin as an active ingredient.

The extraction method of the present invention involves water-extracting the oak leaves, extracting the resulting water-extracted extract with an organic solvent to remove water-soluble substances, and then separating the resulting extract using gel filtration column chromatography. It is characterized in that it is further purified by chromatography.

To describe the method of the present invention in more detail, the leaves of the oak are extracted with hot water, and the obtained water extract is partitioned and separated in a solvent system of ethyl acetate and water to obtain an ethyl acetate layer. Excluding substances. Next, the obtained fat-soluble substance is separated by gel filtration column chromatography using methanol as an elution solvent to obtain a fraction having hemostatic activity.The obtained fraction having hemostatic activity is then separated by silica gel column chromatography. The hemostatic active substance quercitrin (1) can be obtained by purification by gel filtration column chromatography.

Quercitrin ('I), which is the active ingredient of the present invention, is described in Pharmaceutical Journal, 56, 441 (1936), Eie, 40.
(1949), Foot, 1173 (1951), 97, 1
09 (1977), Mu 1, 1288 (1978),
It is a known substance already disclosed in Shionogi Research Institute Report, 2°162 (1952), etc.

According to these documents, quercitrin is found in various plants,
For example, it has been isolated from Hokutami, fisheye, white peach, episcus, etc., but all isolation methods involve repeated fractional precipitation and recrystallization, and the operations are complicated and the process to obtain pure elcitrine is difficult. It was long.

The extraction method of the present invention is characterized in that the operation method is simpler than in conventional methods, and highly pure quercitrin can be obtained.

Quercitrin, which is the active ingredient of the present invention, is also known as a compound having various pharmacological activities such as diuretic action, antibacterial action, and capillary reinforcing action. However, it was not known at all that quercitrin had a hemostatic effect.

It is known that IJ has a capillary reinforcing effect as a pharmacological activity similar to the hemostatic effect of IJ. The hemostatic action of quercitrin, which was invented by the present inventors, is a hemostatic action that acts on blood coagulation factors, and has the same mechanism of action as vitamin 1.

Therefore, the conventional clinical use of quertitrin was effective only for prophylactic use to strengthen blood vessels or to prevent bleeding, but based on the knowledge obtained by the present invention,
Since it has become clear that it is very effective as a hemostatic agent for bleeding from wounds, it can be said that its clinical use has been expanded. That is, quercitrin has a strong hemostatic effect and is extremely useful as a therapeutic agent for pulmonary hemorrhage, cerebral hemorrhage, nasal hemorrhage, retinal hemorrhage, purpura, bleeding from wounds, aplastic anemia, and the like.

In addition, when we conducted a hemostatic activity test to be described later on rutin, myricetin, hesveridin, and quercitrin of the present invention, which are known substances that have a capillary reinforcing effect, it was found that a dose of 5 mg/Quercitin at the mountain pass was 1.
Whereas l-rucin-myricetin and hesveridin showed no activity at all, fc.

Quercitrin (1) obtained according to the present invention can be prepared into tablets by a conventional method! ? ! It can be made into preparations such as i.p., capsules, injections, or external preparations, and can be administered orally or parenterally, systemically or locally for clinical use. administration ff
l depends on the symptom to be treated and the method of administration, but
Usually, when administered orally to adults, 0.5 to 619
It is.

The hemostatic activity test conducted in the Examples was measured and calculated by the method described in F.

[Hemostatic activity test method]

Weight 20! A 1% methylcellulose/physiological saline solution (0.5%) was administered intraperitoneally to a ddY strain male mouse, and 10 minutes later, the mouse was fixed in a fixation box and the left leg vein near the middle of the tail was injured with a scalpel to cause bleeding. Thereafter, a piece of P paper is lightly applied to the wound at 60 second intervals, and the time until blood no longer adheres to the paper is measured. This time is used as the control bleeding time q4
After a period of time, a test sample suspension prepared by suspending the test sample in 0.5 ml of a 1% methylcellulose/physiological saline solution is intraperitoneally administered to the same mouse. After 10 minutes, the mouse is immobilized, the right leg vein near the middle of the tail is incised with a scalpel, and the bleeding time is measured in the same manner as in the control case.The test is performed twice for the same sample, and the average value is used.

Calculation method of hemostatic activity: Here, bleeding reduction time after administration of test sample (bleeding time of control) - (bleeding time after administration of test sample).

Hereinafter, the present invention will be explained by examples.

Example Side Phase #400g (dry weight) was ground, 4t of water was added, and the mixture was heated and refluxed for 1 hour. After cooling to zero, the resulting extract was centrifuged and freeze-dried to obtain 35.3g of water extract. The obtained water-extracted extract is dissolved in 750 g of water, the same amount of ethyl acetate is added and shaken, and the ethyl acetate layer is separated. The same operation was repeated 5 times and the ethyl acetate layers were combined. The solvent was distilled off to obtain an extract of 3.73, li'.

Hemostatic activity of ethyl acetate extract; 60% (dose 135
x9/kq) Ethyl acetate extract 3.73.9 was dissolved in a small amount of methanol and fractionated by gel p percolumn chromatography (Cef7 Denox LH-20, 159 g) using methanol as the /lo elution solvent, Each fraction of the eluate (7Il1
1 to 4) were obtained. The hemostatic activity of each fraction is measured, and 3,123.5 mg of the fraction with the strongest hemostatic activity is obtained.

Hemostatic activity of fraction j≦3: 50% (administration m50 this fraction (
A3) 123.51 g is dissolved in a small amount of a mixed solution of tetrahydrofuran and chloroform (1:1), and purified by silica gel column chromatography. Elution is performed with a mixture (11) of tetrahydro7ran and chloroform, and then with tetrahydrofuran. Concentrate the tetrahydro 7 run eluate to obtain a 73.5 my residue.

The residue was dissolved in a small amount of methanol and subjected to gel p percolumn chromatography (Sephadex LH-20, 14
F, elution solvent: methanol) to obtain 67iIg of quercitrin.

Recrystallized from a mixture of methanol and water, melting point 175~
44.5 mg of pale yellow crystals of 176° are obtained.

This product is from the Merck Index (the MerckIndex).
dex) 10th edition, and matched with quercitrin according to various equipment data.

The hemostatic activity of quercitrin and commercially available hemostatic agents ethansylate and tranexamic acid was compared, and the results are shown in the table.

Table Hemostatic activity

Claims (1)

[Scope of Claims] 1. Extract the Quercitrin leaf with water, extract the water extract with an organic solvent, and then separate by gel p percolumn chromatography. 1. Further purify by chromatography to obtain quercitrin represented by the formula How to extract. 2. A hemostatic agent containing quercitrin represented by the formula % as an active ingredient.