JPH09309839A - Platelet coagulation inhibiting material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an extremely strong platelet coagulation inhibiting material, having good thermal stability by compounding an extract of a food widely available in Japan as an active component. SOLUTION: NATTOU (fermented soybean) or a cultured liquid of Bacillus natto is extracted singly with water or with an extracting liquid prepared by combining water with an alcohol or several kinds of salts. The obtained material can be used as an active component as it is. Further, the obtained material is optionally purified by adequately carrying out a polar organic solvent treatment, salting-out and ultrafiltration and subsequently further performing purification operation of one or more kinds selected from absorption, ion-exchange chromatography, gel filtration, affinity chromatography, etc. The obtained platelet coagulation-inhibiting material is effective for treating or prophylaxis of circulatory failure attributable to thrombus formation in a blood vessel. Further, the raw material is derived from a food, and accordingly the platelet coagulation-inhibiting material is excellent in safety.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a heat-stable platelet aggregation inhibitor extracted from natto or a natto culture solution with water alone or an extract containing water and alcohol or several salts in combination. . Today, for the treatment or prevention of circulatory failure due to thrombus formation in blood vessels related to the development of many adult diseases, this food-derived substance is highly safe, has good stability, and has high utility value. Conceivable.

[0002]

2. Description of the Related Art Up to now, various synthetic drugs such as aspirin, ticlopidine, and trapidil have been known to inhibit the platelet aggregation reaction that triggers various thrombotic diseases. Since such side effects are strong, caution was required when using it in clinical settings. It has been clarified that the extract of crude drugs such as (u) contains substances that have inhibitory activity against platelet aggregation (Kouhei Miyao: Research and development on functional food materials and bioactive substances derived from food). , P.61, Japan Society for Industrial Technology, 198.
9; Ryozo Yamaguchi et al .: These vegetables prevent blood clots, Kodansha, 1
991). In particular, vegetables derived from vegetables are ingested in fairly large amounts on a daily basis, and thus their physiological effects were of interest.

[0003]

[Problems to be Solved by the Invention] Aspirin, ticlopidine, which are platelet aggregation inhibitors currently in clinical use,
All of them, such as trapidil, were cheap because they were synthetic products, but side effects such as gastric ulcer, gastrointestinal bleeding, and liver damage were also pointed out.

[0004]

[Means for Solving the Problems] We have made diligent efforts to search for various blood circulation-related substances in natural materials centered on foods, and used a thrombolytic enzyme in natto, which is a traditional fermented food in Japan. A certain nattokinase has been discovered, and its acquisition method and enzymatic properties have been reported (Sumi et al., Research and development on functional food materials, food-derived physiologically active substances, p.88, Industrial Technology Association, 1989). The reason why the food was selected as a target is that there are few safety problems even if it is taken for many years. The present invention relates to natto or a platelet aggregation inhibitor derived from a natto culture solution, which was discovered in the course of the research. Its action is extremely strong, and it is mildly orally administered for a long period of time. Therefore, it is expected to be applied and developed as a preventive agent for various adult diseases caused by blood circulation failure that is effective when eaten. In addition,
So far, regarding the components that act on the platelets of natto, Kosug
e, a pyrazine compound (K
osage T.S. et al. , Agr. Biol. C
hem. , 35: 693, 1971; edited by Japan Society of Technology:
Research and development on functional food materials, bioactive substances derived from foods, Industrial Technology Association, p. 61, 1989) and nothing else was known. The present invention searches for substances that act on platelet aggregation from among many natto products in Japan, or natto bacterium culture broth, and finds an extremely potent platelet aggregation inhibitor that is different from pyrazine compounds and has high thermal stability. Is.

[0005]

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

Example 1 A commercially available natto (Takano Foods Co., Ltd.) was mixed with a double volume of physiological saline, treated with a mixer, and allowed to stand at room temperature for 1 hour. Then, the effect of natto extract obtained by centrifugation at 3,000 rpm for 10 minutes on human platelet aggregation was examined. That is, blood samples of citrate (3.8% sodium citrate) were collected from 5 healthy adult male volunteers, and platelet-rich fractions (PRP-1 to 5) separated by centrifugation (1,000 rpm, 10 minutes) were collected. 200 μl and 22 μl of Sigma ADP (adenosine 5′-2 phosphate) were added to induce aggregation, and the aggregation pattern was centrifuged more strongly than each PRP (2,500 rpm).
m platelet fraction (P for 10 minutes)
200 μl of PP-1 to 5) was used as a control and observed with an aggregometer (NBS HEMATORACER 601). Two of the volunteers had a final concentration of 2 μM in this reaction system, and three had 10 μM of ADP.
The maximum agglutination rate (Max agg) was not less than%, but in each case, addition of the natto extract (control using physiological saline) prior to the addition of ADP almost completely inhibited each agglutination reaction. I found out. Figure 1 shows a final concentration of 2 μM
An example showing a platelet aggregation pattern when 1/20 amount of natto extract (a physiological saline was used as a control) was added to the aggregation reaction of certain human PRP (PRP-2) by the addition of ADP Is. The platelet aggregation inhibitor contained in this natto was fairly thermostable, and 90% or more of the activity remained even after boiling at 100 ° C for 1 hour. In addition, the activity of this product was not easily lost even with water dialysis (stirring for one day) using a cellophane membrane, unlike the pyrazine compound.

Second Example Commercially available natto (Eishoku Co., Ltd.) was divided into three equal parts, each of which was added with twice the volume of methanol, and the mixture was slowly stirred for 1 hour for extraction, and the resulting supernatant was used with an evaporator. Physiological saline was added to the dried natto to the original natto capacity, and the platelet aggregation inhibitory activity of the solubilized natto (methanol extract) was measured by the same method as in Example 1. as a result,
The control group was 63 ± 11% (n =
In contrast to the maximum aggregation rate (Max agg) of 5), the reaction system to which 1/20 amount of the methanol extract was added did not cause any aggregation under the same conditions, and the natto extract It was found that strong aggregation inhibition was observed in some of them.

Third Example Nutrient break (Difco La
b. ) To which 5% soy protein was added (pH
The culture solution obtained by culturing Bacillus subtilis natto (Naruse fungus) at 40 ° C. for 24 hours using 7.0) as a basic medium is freeze-dried, and 0.01 M phosphate buffer of 4 times the volume of the wet weight , PH
7.0 was added, and the supernatant obtained by homogenization was used as an extract to determine the platelet aggregation inhibitory activity by the same method as in Example 1.
As a result of an examination using P (final concentration 10 μM) and collagen as aggregation inducers, it was found that this extract strongly inhibited maximum aggregation (Max agg) by 65% or more in all cases. In addition, platelet aggregation by collagen is H
Orm's bovine Achilles tendon derived final concentration 0.3m
Used at g / ml.

Fourth Example 2 for 1 kg of commercially available natto (Takano Foods Co., Ltd.)
To the extract obtained by adding 0 L of water and stirring at room temperature for 2 hours, ethanol was added so that the amount of the extract was 80%, and the resulting fibrous viscous substance was removed with a metallic filter. 25φ × 10cm Cel washed with distilled water
ite 545 column (manufactured by Wako Pure Chemical Industries; Sumi H.e
t al. , Comp. Biochem. Physio
l. , 102B: 163, 1992). The non-adsorbed fraction, thus obtained, was filtered and dried at 50 ° C. under vacuum, and the dried powder thus obtained was analyzed by the same method as in Examples 1 and 3 for aggregation of human platelets by ADP and collagen. An extremely strong inhibitory activity was observed (all inhibit Max agg by 80% or more). In addition, the method of Kosuge et al. (Agr. Biol. Che.
m. , 35: 693, 1971) and the addition of tetramethylpyrazine (provided by Takano Foods Co., Ltd.) at a high concentration of about 5 times (0.2 μg / ml) the pyrazine content in natto confirmed by quantification. Platelet aggregation (Max a
The inhibitory activity against gg) was 5% or less, which was extremely low with any of the aggregation inducers.

Fifth Example 100% ethanol, 80% ethanol, or distilled water was added to 25 g of commercially available natto (Malkin Co., Ltd.) to make 50 ml, and the mixture was stirred overnight, then at 3,000 rpm, 10 rpm.
The supernatant obtained by centrifugation for 30 minutes is vacuum dried at 35 ° C.,
Using a solution prepared by adding physiological saline to 200 ml and dissolving it, AD of human platelets was prepared in the same manner as in Example 1.
The effect on P aggregation was investigated. The results are shown in FIG. 2. Unlike the results with various pyrazine compounds, the strongest inhibitory activity was observed in the distilled water extract.

Sixth Example Since the dry powder prepared from natto according to the fourth example is delicious to eat and safe because it is naturally derived from foods, 7 healthy adults (volunteers aged 18 to 48 years old as a platelet aggregation inhibitor preparation were used. ) Was orally administered at a dose of 500 mg / kg, and blood was collected from citrate over time, and the platelet aggregating ability and blood fibrinolytic activity were measured. The aggregating ability of platelets was measured by the same method as in Example 1 using ADP (final concentration 2 μM) as the inducer and the maximum agglutination rate (Max agg,%). Further, the blood fibrinolytic activity was measured by the lysis area of plasma euglobulin fraction on a fibrin plate (EFA; Sumi H. et al., A).
cta Haematol. , 84: 139, 199
0). As a result, as shown in Table 1, it was found that the platelet aggregation ability in blood was significantly decreased by oral administration, and the fibrinolytic activity in blood also tended to increase.

[Table 1]

[0012]

INDUSTRIAL APPLICABILITY According to the present invention, a platelet aggregation inhibitor which is easily available and safe to eat and drink is provided.

[Brief description of drawings]

FIG. 1 shows the inhibitory activity of natto extract on human platelet (PRP-2) aggregation. In addition, 10 μl of the natto extract was added to the reaction system, and 2 μM of ADP was used to induce platelet aggregation. In addition, A shows a control and B shows the aggregation pattern of adding the natto extract.

FIG. 2 shows the inhibitory activity of natto extract on human platelet (PRP-5) aggregation. 10 μl of each physiological saline solution was added to the reaction system, and ADP at a final concentration of 10 μM was used to induce platelet aggregation. C is a control, D is 100% ethanol extract added, E is
Shows the aggregation pattern of addition of 80% ethanol extract and F shows the addition of distilled water extract.

Claims (2)

[Claims] 1. A heat-stable platelet aggregation inhibitor which is extracted from natto or a natto culture solution with water alone or with an extract obtained by combining water and alcohol or several salts. 2. The obtained substance as it is, or after treatment with a suitable polar organic solvent, salting-out and ultrafiltration treatment,
The method according to claim 1, wherein one or more operations such as adsorption, ion exchange chromatography, gel filtration and affinity chromatography are combined for purification.