JPH0881385A - Platelet agglutination-resistant substance originated from mushroom - Google Patents

Abstract

PURPOSE: To obtain a new substance high in safety, excellent in stability, and useful for treating or preventing circulatory disorders caused by the formation of thrombus in blood vessels relating to the crisis of degenerative diseases from the extract of a mushroom. CONSTITUTION: The platelet agglutination-resistant substance comprises the extract of a mushroom, especially the fruit body or mycelium of Pleurotus ostreatus, with water alone, or water and an alcohol, or the water or alcohol containing several kinds of salts. The substance is used as such or preferably after subjected to a polar organic solvent treatment, a salting-out treatment, an ultrafiltration treatment, an adsorption treatment, a gel filtration treatment, etc., to purify the substance. For example, Pleurotus ostreatus can be mixed with physiological saline in an amount of 10 times that the wet weight of the Pleurotus ostreatus, treated with a mixer, left at room temperature for a day, and subsequently filtered with a filter comprising four superposed gauze sheets to obtain the substance as a pale yellow solution having a pH of 6-7. The extract of the Pleurotus ostreatus has an especially strong agglutination-inhibiting activity, and maintains the activity of >=90% also after boiled at 100 deg.C.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a heat-stable antibacterial agent extracted from mushrooms, especially fruiting bodies or mycelium of Pleurotus ostreatus by water alone or an extract obtained by combining water with alcohol or several salts. It relates to platelet aggregating substances. 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 as substances that suppress the platelet aggregation phenomenon that triggers various thrombotic diseases. The side effects of were strong and required clinical attention. It has been reported that the exudates contain substances that suppressively act on platelet aggregation (Kouhei Miyao, Research and Development on Functional Food Materials, Food-derived Bioactive Substances, p.
61, Institute of Industrial Technology, 1989), especially because vegetables are eaten in large quantities on a daily basis, their effects were of interest.

[0003]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention Aspirin, ticlopidine, which are platelet aggregation inhibitors currently in clinical use,
Although trapidil and the like are all synthetic products and therefore inexpensive, side effects such as gastric ulcer, gastrointestinal bleeding, and liver disorder have been pointed out.

[0004]

[Means for Solving the Problems] We made diligent efforts to search for various blood circulation-related substances in natural materials, mainly foods, discovered nattokinase, a thrombolytic enzyme in natto, and obtained it. Laws and properties have been reported (Sumi et al., Research and development on functional food materials, food-derived physiologically active substances, p. 88, Industrial Branch Society, 198).
9). The reason why the food was selected as a target in this way is that there are few safety problems even if it is taken for many years. The present invention
Among them, the mushrooms, especially the oyster mushroom-derived antiplatelet aggregating substance, have been discovered in the course of research on the active ingredients in various mushrooms that have been eaten since ancient times in Japan. 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. Up to now, reports on the components that act on platelets regarding mushrooms have been reported by Shiori, Mori and Hayakawa et al. (Taku Mizuno, Masamitsu Kawai: Mushroom Chemistry and Biochemistry, Academic Publishing Center, p. 2).
33, 1992), or recently Ganoderma lucidum on the riverbank (Journal of the Japanese Society of Agricultural Chemistry, 68: 769, 1994), and the properties of these active ingredients have not been well understood. The present invention has searched for substances that act on platelet aggregation in many mushrooms, and found the strongest antiplatelet aggregation substance not only in the fruit body of Pleurotus ostreatus but also in the mycelium. It should be noted that there have been no reports up to now on platelet aggregation derived from oyster mushrooms.

[0005]

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

First Example: Wet weight of shiitake mushrooms or oyster mushrooms (generally known as shimeji mushrooms) cultivated in a fungus bed at the Asano Industrial and Bio Research Institute (Tamano City), and five other mushrooms commercially available in Okayama City Ten times as much physiological saline or ion-exchanged water was added to each, treated with a mixer, and then allowed to stand at room temperature for 1 day. The pH of these mushroom extracts obtained by filtering gauze (4 layers)
6 to 7 light yellow solutions, for example, those extracted from oyster mushrooms with ion-exchanged water showed the absorbance characteristics as shown in FIG. Next, the influence of the extract on human platelet aggregation was examined. Blood was taken from 5 healthy adult male porantia citrate (3.8% sodium citrate), and 200 μl of platelet rich fraction (PRP) obtained by centrifugation (1,000 rpm, 5 minutes), and Sigma 20, 100 and 200 μMADP (adenosine 5 '
-2 phosphate) was added in an amount of 22 μl to induce aggregation, and the aggregation pattern was measured using an aggregometer (NBS HEMATORAC
It was observed by ER 601). Two of the volunteers showed agglutination with a final concentration of 2 μM and three with 10 μM of ADP. In all cases, addition of mushroom extract (control using physiological saline) prior to ADP addition Inhibition of agglutination is observed, especially in the case of oyster mushroom
When 40 to 1/20 amount was added, platelet aggregation (primary and secondary aggregation) was almost completely suppressed. Figure 2 shows a human P
1/40 volume of shiitake extract (2) or oyster mushroom extract (3) was added to the agglutination reaction (1; physiological saline was used as a control) with the final concentration of 2 μMADP added to RP (PRP-5). 3 is an example showing a platelet aggregation pattern when added, and FIG. 3 shows another human PRP (PRP-
7), using a final concentration of 2 μMADP added to the agglutination reaction (1; physiological saline is used as a control), a 1/40 volume of the shiitake extract (2) or the oyster mushroom extract (3)
This is an example showing the platelet aggregation pattern of the case of addition of the above. It was found that the extract from oyster mushrooms has an aggregation inhibitory activity that is twice as strong as that of the extract from shiitake mushrooms on average. The anti-platelet aggregating substance contained in this oyster mushroom is quite thermostable, and remains active even after boiling for 1 hour at 100 ° C
Left over 0%. Further, even when this was dialyzed with water using a cellophane membrane (stirring overnight), the activity was not completely lost.

Second Example As in the first example, an edible part which is a general commercial product of oyster mushrooms cultivated in a fungus bed, a root part which is not commonly eaten among fruiting bodies, and a fungal bed containing a large amount of mycelium after culturing For the part,
As in the first example, 10 times the amount of physiological saline or ion-exchanged water was added, and the mixture was boiled and extracted for 1 hour (using Toyo filter paper No. 50 for filtration).
As a result of comparing the platelet aggregation inhibitory activity of each of the extracts, it was found that each extract has a strong inhibitory activity against human platelet aggregation reaction by ADP and collagen. The platelet aggregation by collagen was induced by using a bovine Achilles tendon-derived product from Horm, which was used at a final concentration of 0.3 mg / ml.

Third Example To 1 kg (wet weight) of Pleurotus ostreatus purchased in Okayama city, 20 l of 20% ethanol was added, and the mixture was stirred at room temperature for 2 days, and the obtained pale yellow extract was activated with 4N hydrochloric acid. 5φ × 15cm Celite washed with distilled water after
545 column (manufactured by Wako Pure Chemical Industries; Sumi H. eta)
l. , Comp. Biochem. Physiol. ，
102B: 163, 1992). After washing this column with distilled water, the fraction eluted with 1% ammonia was added to 5
The dried powder obtained by vacuum drying at 0 ° C. also showed a strong inhibitory activity against ADP aggregation of human platelets.

Fourth Example To oyster mushrooms cultivated in a fungus bed at the Asano Sangyo Bio Research Institute (Tamano City), 10 times the wet weight of distilled water was added, treated with a mixer for 20 minutes, and then treated with 1N hydrochloric acid to a pH of 4. In accordance with 5, the polysaccharide degrading enzyme [SMIZYM AC and MC (Shin Nippon Kagaku Kogyo,
Aichi) was mixed at a ratio of 1: 1], and the reaction was carried out at 45 ° C. for 24 hours to stop the enzymatic reaction. After spontaneous cooling, the mixture was filtered through gauze (4 pieces stacked) to obtain ASK-1L. This product is delicious for humans to eat and, of course, is safe because it is derived from food. Therefore, 500 mg / kg body weight is orally administered to 7 healthy adults (volunteers) aged 18 to 48 years as an antiplatelet aggregation preparation.
Blood was collected from citrate over time, and platelet aggregation ability and fibrinolytic activity in plasma were measured. Platelet aggregation is the same as in Example 1
It measured using DP (final concentration 2 and 10 micromol). The fibrinolytic activity in blood is determined by the standard fibrin plate lysis method (EFA; Sumi H. et al.,
Acta Haematol. , 84: 139, 199
0), tissue plasminogen activator activity (t-
PA activity; Verheijen J .; H. et a
l. , Thromb. Haemost. , 51:39
2, 1984) and zymography pattern (Tis
sot J, D. et al. J. Clin. Inv
est. , 70: 1320, 1982). The results are shown in Table 1, and the platelet aggregation ability of blood obtained by oral administration for a considerably long time, particularly at 5 hours after administration, was significantly decreased, and meanwhile, the blood fibrinolytic activity was increased. I understood. In the zymography pattern, it was confirmed that t-PA-like plasminogen activator having a molecular weight of 70,000 to 100,000 was increased in plasma one hour after oral administration.

[Table 1]

Fifth Example ASK-1L prepared by the method shown in the fourth example was freeze-dried to obtain a pale yellow powder (ASK-1P). This is 10
The solution was dissolved in physiological saline so as to be mg / ml, and was intraperitoneally administered to Wistar rats (male, 200 to 250 g). The citrate blood was collected over time, and the aggregation ability was examined using the platelet rich fraction (PRP) obtained by centrifugation. Table 2 shows the agglutination ability measured using ADP at a final concentration of 10 μM and the fibrinolytic activity (dissolved area EFA of a standard fibrin plate) investigated using a euglobulin fraction separated from platelet-free plasma (PPP) with respect to the reaction system. As shown, it was found that the platelet aggregation ability was clearly reduced 1 hour after the administration, and the blood fibrinolytic activity by EFA was weakly increased.

[Table 2]

[0011]

EFFECTS OF THE INVENTION According to the present invention, an antiplatelet aggregation substance that is easily available and safe to eat and drink is provided.

[Brief description of drawings]

FIG. 1 is a diagram showing the absorbance characteristics of a mushroom extract.

FIG. 2 Human platelets (PRP-5) possessed by mushroom extract
It is a figure which shows the inhibitory activity with respect to aggregation.

FIG. 3 Human platelets (PRP-7) possessed by mushroom extract
It is a figure which shows the inhibitory activity with respect to aggregation.

Claims (2)

[Claims] 1. A heat-stable antiplatelet aggregating substance extracted from mushrooms, in particular oyster mushroom fruiting bodies or mycelium, with water alone or with an extract obtained by combining water with 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.