JPH0741430A - Fibrinolysis-resistant substance - Google Patents

Abstract

PURPOSE:To provide a novel fibrinolysis-resistant substance and a method for obtaining the same, and to apply the fibrinolysis-resistant substance as an agent for treating or preventing hemorrhagic diseases. CONSTITUTION:A method for producing the saccharide-originated fibrinolysis- resistant substance from the saccharide or a plant containing the saccharide, and its property, and the production of a preparation for preventing hemorrhage.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an antifibrinolytic substance produced by heating or oxidizing a saccharide and a method for obtaining the same. Today, this food-derived substance is considered to have high safety and high utility value for the treatment or prevention of various bleeding caused by many diseases.

[0002]

2. Description of the Related Art Epsilone aminocaproic acid (ε-), which is a synthetic substance, has a relatively high specificity as an antifibrinolytic agent that suppresses bleeding caused by abnormal blood coagulation system or acceleration of fibrinolytic enzyme system. Only ACA) or tranexamic acid (t-AMCHA). On the other hand, as a substance that acts on the blood coagulation / fibrinolysis system in natural products, heparin in animal organs or sugar components such as alginic acid of seaweed have anticoagulant ability, and these are also used as antiulcer agents. However, there are very few reports on substances that act on antifibrinolysis (edited by Shibata: New Bioactive Natural Substances, Ito Denshi Shuppan, p. 305, 1988). Furthermore, there is nothing related to substances derived from sugar. Although there are some reports about the traditional effects of Japanese medicine "Kuroyaki" (Koizumi, "Kuroyaki", Miyazawa Shoten, 1921). There have been few studies, let alone reports of sugar-derived substances.

[0003]

Problems such as ε-ACA and t-AMCHA, which are currently used clinically as antifibrinolytic agents, are synthetic products and are therefore inexpensive, but their history is short and excessive. Side effects such as increased coagulation and thrombus formation due to administration have been pointed out.

[0004]

[Means for Solving the Problems] We have made diligent efforts to search for various fibrinolysis-related substances in natural materials centering on foods, and have discovered and reported nattokinase and the like (H. Sumi et al., Experientia
43: III0, 1987). The reason why the food was selected as a target in this way is that it is known that there is no problem in safety even if it is taken for many years. The present invention is a carbohydrate-derived antifibrinolytic substance that has been discovered in the course of research on the active ingredient in various "Kuroyaki" that have been used for a long time in Japan. The various sugars are not limited to monosaccharides, disaccharides, and oligosaccharides, and polysaccharides such as dextran, starch, and celluloses, or plants containing them may be used. In any case, no anti-fibrinolytic substance derived from sugar, which is produced by the heating of saccharides or its oxidation reaction, has been reported so far. It is expected to be applied and developed as a medicine for treating and preventing ulcers, hemorrhoids and various other inflammation-related diseases.

[0005]

Next, the present invention will be described in detail with reference to Examples.

First Example Various sugars were put in a crucible and heated by an open flame, and five-stage samples having different browning degrees were taken, and the effect of addition of the water-soluble fraction on various fibrinolytic enzyme activities was examined. Plasmin converts Glu-plasminogen into Robbins & S
ummaria method (Methods in Enzym)
ol. 19: 184, 1970) activated about 25C
U / mg protein, urokinase (Midori Juyusha),
Tissue plasminogen activator (Boehringer) was purchased as a commercial product. The fibrinolytic activity of urokinase and tissue plasminogen activator was determined by the standard filin plate method using bovine fibrinogen (Armer). It measured by the plasminogen free plate method using the Lys-Sepharose processing fibrinogen. As a result, it was found that a strong fibrinolysis-inhibiting activity was generated at a time when the degree of browning was relatively low, regardless of the degree of browning due to heating. The strength of the antifibrinolytic activity examined using urokinase was the fructose heating product, maltose heating product,
The order was arabinose heat product, glucose heat product, lactose heat product, and starch heat product. In addition to urokinase, they also inhibited tissue plasminogen activator and plasmin. The urokinase inhibitory activity of the heated fructose product is 25 international units (IU) / mg after pre-incubation at 37 ° C for 2.5 hours.
The above was very strong.

Second Example In addition, the figure shows the antifibrinolytic substance produced by heating fructose as S
As a result of gel filtration using ephadex G-25, it was found that the activity peak was divided into three or more kinds. In particular, the major activity peak, fraction 9-1
No. 1 was a colorless and transparent substance having a higher molecular weight than fructose. On the other hand, vanillin, coumarin, and 3-hydroxy-2-, which are known to be produced by heating fructose,
Methyl-4-pyrone, ethyl maltol, etc. did not have such strong antifibrinolytic activity.

Third Example 6N HCl was added to fructose and incubated for 12 hours, and the browning substance produced was separated by the same method as in the second example.
After gel filtration on hadex G-25, fractions 9-11 were collected and sent to Centrifugal conce
It was concentrated with an intrator (TOMY CC-100). This substance was also found to have a urokinase inhibitory ability of 7 IU / mg or more.

Fourth Example Eggplants were placed in a unglazed pan and heated to a brown color with an open flame. After crushing this product, it was further made into a fine powder in a mortar, and 20 times the weight of distilled water was added thereto, and extraction was carried out at room temperature for 2 hours. The freeze-dried product of this extract was found to have an antifibrinolytic activity of about 7 IU / mg by the fibrin plate method. The elution position of this product by gel filtration coincided with the position of the antifibrinolytic substance generated by heating the fructose in the second example.

Fifth Example Fructose was put in a crucible, heated by an open flame, caramelized and then dissolved in distilled water. This solution has an absorption at 280 nm (0.1 m) not found in the original aqueous solution of fructose.
There was ΔOD ₂₈₀ = 1.8) in g / ml aqueous solution.
After 2.5 ml of this 20% aqueous solution was drunk to 7 Wistar rats (male, 420 to 470 g), exercise load was applied to induce blood fibrinolysis. As an object, the same amount of water or fructose solution was given to 7 rats in the same group and exercise load was applied. As for exercise load, Tamura's method was applied to each animal (Jpn Pharmacol. 59:78, 196).
In 3), he was forced to swim for 25 minutes. Blood was collected 1 hour after administration, and the lysis area of standard fibrin plates by plasma euglobin fractionation (H. Sumi et al, Com.
p. Biochem. Biophysiol. 102
B: 159-162, 1992),
It was found that the caramel-administered group had a diameter of 53 ± 14 mm ² and the caramel-administered group had a diameter of 29 ± 11 mm ² , and that the heat-generated component of this sugar suppressed fibrinolytic activity.

Example 6 A toothpaste was prepared by mixing 9 parts by weight of salt with 1 of the antifibrinolytic substance obtained by heat treatment of fructose, and bleeding was observed during toothpaste. When we used 3 g of a patient with alveolar pyorrhea at a rate of 3 times a day for brushing teeth for 1 month, all patients had no bleeding, and the inflammation of mycorrhiza was improved. Obtained.

[0012]

INDUSTRIAL APPLICABILITY As described above, the novel antifibrinolytic substance of the present invention is the first product derived from carbohydrates and has a very strong inhibition against plasmin, plasminogen activator and the like. In addition to its activity, it is safe from oral administration because it is derived from food. Therefore, it is useful as an oral therapeutic agent or preventive agent for hemorrhagic diseases such as alveolar pyorrhea and gastric ulcer.

[Brief description of drawings]

Gel filtration pattern of heated fructose. For equilibration, 0.05M borate buffer containing 0.15M sodium chloride, p
H8.5 was used. The vertical axis represents the fibrinolysis inhibition rate calculated from the dissolution area of the fibrin plate using 30 μl after addition of urokinase (5 IU / ml) to a fixed amount of each fraction and preincubation at 37 ° C. for 2 hours. It can be seen that as the antifibrinolytic substance, three or more kinds of substances having a larger molecular weight than the original fructose are contained.

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[Procedure amendment]

[Submission date] January 26, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1 is a diagram showing a gel filtration pattern of a heated fructose product.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Figure 1

[Correction method] Change

[Correction content]

[Figure 1]

Claims (2)

[Claims] 1. An antifibrinolytic substance derived from a sugar, which is produced by heating a sugar or an oxidation reaction thereof. 2. The obtained product as it is, or after being subjected to a suitable polar organic solvent, salting-out and ultrafiltration treatment,
The method according to claim 1, wherein one or more operations of adsorption, ion exchange chromatography, gel filtration, affinity chromatography or isoelectric focusing are combined for purification.