JPS61204130A - Thrombus solubilizer - Google Patents

Abstract

PURPOSE:A thrombus solubilizer comprising an aqueous extract of animal belonging to the family GOKAI (Nereis japonica) or ISOME (Eunice viridis) as an active ingredient. CONSTITUTION:Groud material of animal belonging to the family GOKAI (Nereis japonica) (e.g., perinereis brevicirris, Tylorrhynchus heterochaetus, etc.) or ISOME (e.g., Eunice viridis) (e.g., Eunice aphroditois, marphysa sanguinea, Diopatra sugokai Izuka, AKAMUSHI, etc.) is blended with water, allowed to stand, its supernatant liquid is collected, an aqueous extract of animal belonging to the family GOKAIKA (Nereis japonica) or ISOME (Eunice viridis) is isolated by adding a salt such as ammonium sulfate, etc., to the supernatant liquid, by adding an organic solvent such as an alcohol, etc., to precipitate it, by concentrating the supernatant liquid by the use of an ultrafiltration, and lopphilizing it, and fractionated and purified by using ion exchange chromatography, etc. The extract shows improved thrombus solubilizing ability by oral administration, low toxicity and no side effects. The raw material can be fed stably.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a thrombolytic agent, and more particularly to a thrombolytic agent containing an aqueous extract of a member of the family Rugraceae or the family Isomidae as an active ingredient.

[Conventional technology]

Thrombi are associated with various diseases including peripheral arteriovenous thrombosis, pulmonary embolism, myocardial infarction, coronary artery occlusion, cerebrovascular occlusion, and retinal arteriovenous thrombosis, and have become a major problem as a pathogenic factor. .

Currently, fibrinolytic therapy using urokinase is mainly used to treat thrombosis.

[Problem that the invention seeks to solve]

However, urokinase is ineffective when administered orally, and its administration method is limited to intravenous drip injection; it has a short half-life and is easily deactivated; and because it is obtained from the human urine of healthy male adults, it is difficult to use as a raw material. There are various problems such as restrictions, and even if administered, it has a short half-life in the blood and low affinity for ficillin, so large doses must be administered, and there is an increased tendency for bleeding with large doses. To have a point,
The development of a new thrombolytic 41 to replace urokinase has been eagerly awaited.

Means for solving the problem C] In view of the above circumstances, the present inventors have proposed a method that can be administered orally,
We have been diligently researching the development of thrombolytic agents with no restrictions in terms of raw materials, and have discovered that aqueous extracts from Annelida, Polychaete, Caricinidae, or Isomidae have thrombolytic effects, and have completed the present invention. .

That is, the present invention provides a thrombolytic agent containing as an active ingredient an aqueous extract of an animal belonging to the family Rugraceae or the family Isomidae.

Annelidae and polychaetes are the largest group in the Annelida phylum, with over 10,000 species.Among these, lugworms and isomidae are prized as bait for fishing, and both wild and farmed species are prized. A considerable amount is produced, and a stable supply of raw materials is possible.

The lugworms used in the present invention include 1.
As for the isomidae, it is possible to use any of the snails, the snails, the snails, the red worms, and the like, and although the species is not limited to any particular type, the snails and the snails are preferable.

An aqueous extract of a lugworm or a lugworm is obtained by mixing a lugworm or a lugworm or a pulverized product thereof with water.

The aqueous extract of this family, the family Lugacidae or the family Isomidae, is rich in protease activity, and the protease isolated from the aqueous extract by a conventional enzyme separation/n production method showed an even stronger thrombolytic ability.

The method for separating and purifying protease from an aqueous extract of a Lugraceae or Isomidae is not particularly limited, and may be carried out by a known method. For example, a ground product of a lugworm or a seaweed is mixed with water, left to stand, and then the supernatant is collected;
A method of salting out by adding a salt such as ammonium sulfate to the supernatant; a method of adding an organic solvent such as alcohol or acetone to precipitate the supernatant; an ultrafiltration method (for example, Diaflow Membrane YC manufactured by Amicon) After concentration, it is isolated by freeze-drying, etc.
It is fractionated and purified using ion exchange chromatography using DKAE)-cellulose or DEAE-Sephadex, and gluchromatography such as Sephadex G-500, either alone or in combination. When isolated by salting out, it can be powdered by filtration or centrifugation, desalting, and freeze-drying. Examples of the desalting method include dialysis and a gel filtration method using Sephadex G-25 and the like.

The method of administering the aqueous extract of the lugworm or isomidae of the present invention is not particularly limited, but when administered orally, it exhibits an extremely excellent ability to dissolve the face.

In the case of oral administration, the dosage of the aqueous krill extract is preferably 10 to 5001 q/day as the amount of protease.
It is also possible to further increase the dosage depending on the severity of the symptoms.

In addition, the toxicity of protease, which is an aqueous extract of a lugworm or isomidae, to rats is LD5° of 9 or more at 5f/ when administered intraperitoneally, and 2°C/M/ when administered orally.
It was rare to get more than Nigu.

[Effect]

Although the mechanism of the thrombolytic action of the aqueous extract of the Lugulidae or Isomidae of the present invention has not yet been elucidated, it has been found that the protease contained in the aqueous extract of the Lugulidae or Isomidae has high activity. It is presumed that the protease lyses the surface frame.

〔Effect of the invention〕

As mentioned above, the aqueous extract of the Lugminidae or Isomidae of the present invention, in particular the protease derived from the Lugminidae or Isomidae, exhibits excellent thrombolytic ability upon oral administration, has low toxicity, and has no side effects. It is an excellent thrombolytic agent.

〔Example〕

EXAMPLES The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples. In addition,
The protease activity in Example 1 was measured by the following method.

Measurement of protease activity> Milk casein 12 was dissolved in 0.1M phosphate buffer (pH 7,
6) Suspend in 100 rnl and boil for 15 minutes to completely dissolve, which was used as a substrate solution. The same buffer solution 4dK substrate solution l- was added, the sample was added O, l-, and the mixture was reacted at 30°C for 60 minutes. 11nl of 12% trichloroacetic acid! After stopping the reaction, the mixture was left at room temperature for 30 minutes or more. 10”C1
Centrifuge at +4000xr for 20 min and remove the supernatant at 280 n.
The absorption of m was measured. Activity unit is 280 n in 10 minutes
The activity that increases the absorbance at m by 1 was defined as 1 unit.

Example 1 Fresh sea lugworm (Perinerelm brevl)
ei-rrla) is finely crushed, and this crushed material is 100
P in 0.1M phosphate buffer (pH 7,0) 1.5 A
and ground for 2 minutes at 0°C using a homogenizer. The homogenate was then incubated at 6800 x g at 0°C for 30
The mixture was centrifuged for a minute to obtain a supernatant.

Ammonium sulfate was added to the supernatant to achieve 65% saturation;
After being left at 0°C for 1 hour, it was centrifuged and a salted-out precipitate fraction was collected. Then, both portions were added to 0.1M phosphate buffer p) 17.5
After desalting by dialysis against distilled water using a cellophane tube at 4°C, the mixture was lyophilized to obtain protease 1.929. The protease activity of this product is 1
It happened at 670U/f.

You can also buy fresh sardines (Vlarphysa sang).
When the same operation as above was carried out using uln@a) 100t, 1.689 protease was obtained. The protease activity of this product was negligible at 850 U/9.

The physiological properties of the so-obtained rockworm grotease and rotifer protease are as follows.

■ Optimum pH Pi of Japanese rockfish grotease and Coryx grotease
(-An activity curve was determined using milk casein as a substrate. The results are shown in FIG. 1.

Note that the pH buffer solution is 100 mM acetic acid (
pi (4,0-6.0), phosphoric acid (ptts, s-8.
0) and Tris-HCl (pH 7.5 to 9.5) buffer solutions were used. Ingo shellfish grotease and Corylus protease exhibit strong activity in a fairly wide range of pH from 6.0 to 9.0, and have a wide range of pH % isomerism. Optimum p■ is pH 8
It happened nearby.

■ Using milk casein as a substrate at the optimum temperature pfN7.6 at each temperature (0
The reaction was carried out at 60° C. for 60 minutes to examine the effect of temperature on the activities of Japanese lugworm grotease and Corylus protease. The results are shown in Figure 2. Optimum temperature is 40' for both
It happened near C.

C3) pH-stable Japanese rockweed grotease, Corylus protease
Breincubation was performed at 3-10.30°C and its pH stability was 14. The results are shown in Figures 3 and 4, respectively.
As shown in the figure.

Both the Japanese barley shellfish grotease and the coriander protease are stable in weakly acidic to weakly alkaline conditions, but they are somewhat unstable in acidic and alkaline conditions, which is rare.

■ Temperature-stable rockfish grotease, coriander protease with O,
After being left at a predetermined temperature for 30 minutes in 1M phosphate buffer (pH 7, 6), the activity was measured using milk casein as a substrate, and the temperature stability was examined. The results are shown in Figure 5. It was observed that both the Japanese rockfish grotease and the Corylus protease were inactivated at temperatures above 60°C.

Example 2 Fibrinolytic activity of Japanese lugworm grotease and Corylus protease: (1) Fibrin plate method The fibrinolytic activity of the protease obtained in Example 1 (65% saturated ammonium sulfate fraction) was measured by fibrin plate method.

For the fibrin plate, bovine fibrinodan (manufactured by Sigma) was added to a 1/15M phosphate buffer solution pi 7.5 at a concentration of 0.
1 dissolved to 2% in a petri dish (diameter 90
mm) K8m/pour, and add thrombin solution (50U/
rrIl.

Made by Mochida Muyaku) o, i1n! It was prepared by dropping the mixture and immediately stirring it while rotating from side to side. Next, on this fibrin plate, an enzyme bath of $30 μt (the above protease 1
00 p9) was added and kept at 37°C for 30 minutes, and fibrin dissolution was clearly observed. The results of measuring the fibrin dissolution window area at this time are shown in Table 1. As a control, physiological saline was used instead of the enzyme solution.

Below is Table 1 in the margin (il Example 1 of fibrinolysis promotion effect by oral administration to rats)
If the protease obtained (65% saturated ammonium sulfate fraction)
10 rats (male wimtar) per group at a ratio of /Kf
(7 weeks old) Orally administered using a K sonde. Ninety minutes after oral administration, blood was collected from the abdominal aorta under ether anesthesia.

In addition, a group in which physiological saline was administered instead of protease,
Blood was collected in the same manner from the group to which urokinase and urokinase (2000 IU/[9) were administered.

The fibrinolysis promoting effect was evaluated based on the knee globulin dissolution test. Table 2 shows the results of measuring the weight of the fibrin solution in each administration group.

Table 2 *Ammonium sulfate fraction obtained in Example 1.

As shown in Table 2, more than 30% of the fibrinolytic promoting effect was observed in the protease administration group compared to the control group, and unlike urokinase, the fibrinolytic promoting effect of proteases from lugworms or grasshoppers was observed even when administered orally. It was found that it exhibited.

[BRIEF DESCRIPTION OF THE DRAWINGS] Fig. 1 is a drawing showing the pfl dependence of the Lothica 1f rotiase or Corvus protease activity obtained in Example 1, Fig. 2 is a drawing showing the same temperature dependence, and Fig. 3 is a drawing showing the pfl dependence of the activity of the Japanese locust 1f rotiase or the Corylus protease obtained in Example 1. Example 1
Figure 4 is a diagram showing the pH stability of the Corylus protease obtained in Example 1. Figure 5 is a diagram showing the pH stability of the Corylus protease obtained in Example 1. A diagram showing the temperature stability of protease is shown. that's all

Claims (1)

[Scope of Claims] 1. A thrombolytic agent containing an aqueous extract of a Lugraceae or Isomidae as an active ingredient. 2. The thrombolytic agent according to claim 1, wherein the aqueous extract is a protease.