JP4383822B2 - Cancer cell distant metastasis inhibitor - Google Patents

Description

  The present invention relates to a cancer cell distant metastasis inhibitor containing nattokinase as an active ingredient. More specifically, the present invention relates to a cancer cell distant metastasis inhibitor containing, as an active ingredient, nattokinase that prevents distant metastasis of cancer cells caused by a decrease in oxygen partial pressure in blood due to the formation of microthrombus.

Natto is one of the traditional Japanese foods and has been used since ancient times as a folk medicine for heart and blood vessel diseases, or as a treatment for fatigue recovery and beriberi.
In recent years, natto has been reviewed, research on natto has become active, and it has been announced that fibrinolytic enzymes produced by natto have a strong thrombolytic action (for example, Patent Document 1 and Patent Document 2).
Furthermore, it has also been revealed that nattokinase-containing processed food (crude product) produced and isolated by Bacillus natto exerts a thrombolytic action and a thrombus formation inhibitory action (see, for example, Patent Document 3). .
Therefore, since nattokinase has been known to have thrombolytic and thrombus formation-inhibiting effects, nattokinase-containing foods (coarse and refined) are used as foods for preventing ischemic heart disease (myocardial infarction) and cerebral infarction thrombosis. Products) are now commercially available (see, for example, Non-patent Document 1), and ingestion of this nattokinase-containing food (crude product) for the prevention of ischemic heart disease (myocardial infarction) and thrombosis of cerebral infarction The recommended amount is 250-500 mg per adult.

On the other hand, it has also been proposed that a culture of Bacillus subtilis such as Bacillus natto contains a water-soluble vitamin K derivative, and that this culture is useful as an agent for preventing and treating osteoporosis (see, for example, Patent Document 4). .
However, there is no report on the literature that nattokinase has an action of suppressing cancer cells per se, and the present inventors actually ingested about 250 to 500 mg of nattokinase-containing food (crude product) per adult. It was not possible to confirm the effect of suppressing cancer cells per se.

However, in recent years, the probability of death due to cancer has increased, and the main cause of such a high probability of death due to cancer is the distant metastasis to many other parts of the human body during the process of cancer cell growth. As a method for preventing such distant metastasis of cancer cells, there is currently no particularly effective means.
However, since the local control effect in radiotherapy changes greatly due to the difference in oxygen partial pressure, the so-called oxygen effect, the application of hyperbaric oxygen therapy and the hypoxic cell sensitizer (see Non-Patent Document 2, for example) ) And other oxygen therapy studies have been conducted.
However, when these oxygen therapies become clinical treatments, good results have not been obtained regarding the relationship between oxygen effects and radiosensitivity.

Therefore, the present inventors have advanced research, and the cause of the embolization of vascular endothelial cells and the remote metastasis of cancer cells in hypoxia is first caused by the proliferation of cancer cells and the oxygen and proliferation supporting liquid properties via blood flow. Lactic acid is produced by necrosis of cancer cells in the tumor due to the occurrence of chronic hypoxia, which is an imbalance in supply with factors, resulting in a low pH, resulting in damage to vascular endothelial cells, etc. Platelets tend to adhere to the damaged part, become fibrin, and thrombus begins to form.
Next, when the blood vessel is embolized by this fibrin, stimulation of injury, expansion, contraction, etc. is given to the vascular wall cells, and tissue plasminogen activator (tPA) is released from the vascular wall cells. Then, a fibrinolysis phenomenon occurs in which fibrin forming the thrombus dissolves, and when the vascular endothelial cell embolus disappears, the damaged portion of the vascular endothelial cell is exposed.
Therefore, ICAM-1 (cell-cell adhesion) after reperfusion in the process of repeated reperfusion injury of vascular endothelial cells caused by repeated embolization and disappearance of vascular endothelial cells every tens of minutes. Molecules), IL-6 (interleukin-6), IL-1 (interleukin-1) and other inflammatory cytokines flow, and IL-6 and IL-1 are captured via ICAM-1, The hypothesis of invasion from the site of reperfusion injury of vascular endothelial cells was announced, and it was estimated that metastasis of cancer cells would occur (for example, see Non-Patent Document 3).

Japanese Patent Laid-Open No. 1-180834 (page 1-5) JP-A-8-208512 (page 1-5) JP 2001-352929 A (second page) JP 2001-136959 A (page 1-19) "Dried natto culture filtrate NKCP nattokinase compound New food material born from Japanese traditional food" Natto "" Catalog made by Daiwa Pharmaceutical Co., Ltd. Journal of Cancer and Radiation Therapy 2002, -III. Fundamentals of Radiation Biology-, Shigekazu Nakatsugawa, Hideaki Nakamura, Kazuyuki Koyama, "Fundamentals and Applications of Radiosensitizers-History and Prospects", 166 ~ 186 pages, 2002, published by Shinohara Publishing New Company, Magazine "Radiobiology Research" Vol. 4, 37, 393-405, 2002, Shigekazu Nakatsugawa, and 4 other authors "Based on hypothesis in hypoxia and classical radiobiology traps"

  Therefore, in order to reduce the probability of death due to cancer in radiotherapy, even if the oxygen partial pressure in the blood is reduced due to the formation of microthrombus, the vascular endothelial cells are not destroyed and the cancer cells It is intended to suppress distant metastasis through.

As a result of further intensive studies on the relationship between embolization of vascular endothelial cells and distant metastasis of cancer cells in hypoxia, the present inventor promoted embolization of cancer cell endothelial blood vessels in the process of distant metastasis of the cancer cells. When the intercellular binding molecule (ICAM-1) to be reduced can be reduced or disabled by nattokinase, the discovery of cancer cell distant metastasis and malignant progression can be obtained, and the present invention has been completed. It is a thing.
That is, the cancer cell distant metastasis inhibitor of the present invention is characterized by containing nattokinase as an active ingredient.

Such a cancer cell distant metastasis inhibitor of the present invention can reduce blood glucose concentration or blood total cholesterol (T-CHO) or triglyceride (TG) by oral administration. Therefore, it can be used as a drug for inhibiting distant metastasis of cancer cells.
Moreover, since it is a food extracted from natto, it has no side effects and is extremely safe.

[I] Cancer cell distant metastasis inhibitor
(1) Active ingredient
(A) Nattokinase As the nattokinase used as the cancer cell distant metastasis inhibitor of the present invention, nattokinase produced by various methods can be used, and in particular, natto obtained by culturing natto bacteria using soybean as a medium. After separating the solid content of the fungal culture using an ultrafiltration membrane, etc., it is concentrated and dried, and then the natto-kinase cultured nattokinase content (crude product: nattokinase compound) consisting of solid components obtained by drying It is preferable to use purified nattokinase (nattokinase fraction) adsorbed on an ion exchange resin and separated and purified.

(a) Nattokinase cultivated nattokinase (crude product: nattokinase compound)
Nattokinase as a main component per 100 g of nattokinase-containing nattokinase-containing product (crude product: nattokinase compound) separated from the natto culture obtained by cultivating natto using the soybean as a medium About 12 mg is contained, and in addition to the nattokinase, enzymes such as amylase, protease and ribose, glutamic acid polypeptide, fructose polymer fructan and the like are contained.
Specifically, the amount of nattokinase accumulated in the culture of natto bacteria varies depending on the microbial cells used, the type of culture medium, culture conditions, and the like, but usually 10 to 20 mg of nattokinase is contained in the culture. It is a vacuum-dried powder contained at a ratio of / 100 g.

(b) Purified nattokinase (nattokinase fraction)
Since the natto-kinase cultured nattokinase-containing product (crude product: nattokinase compound) contains only about 12 mg of nattokinase per 100 g, it has been purified to a high purity, for example, 100 times or more, preferably 200 to 1, A purified nattokinase (nattokinase fraction) purified to 000 times, specifically purified to a concentration of 20 to 100% by weight can also be used.
Purification method The method of purifying the above highly purified purified nattokinase (nattokinase fraction) can be carried out in the same manner as the usual method for purifying enzymes, and in particular, a natto-cultured nattokinase-containing product (crude product: The nattokinase compound) is preferably purified by dissolving it in a Tris-HCl buffer solution, adsorbing it onto an ion exchange resin, separating impurities, and then eluting it again in a Tris-HCl buffer solution.

(c) Properties As physicochemical properties of the nattokinase, the molecular weight is 24,000 to 40,000, preferably 30,000 to 36,000 (257 amino acid residues), and the isoelectric point is preferably 8.6. It is stable to heating up to 60 ° C. in a basic aqueous solution of ± 0.3 (svensson column method) and pH 6-12.

(2) Intake The dose as a cancer cell distant metastasis inhibitor in the present invention varies depending on the degree of symptoms, patient age, weight, treatment period, etc., and the exact amount is determined by a doctor, When this drug is administered as a cancer cell distant metastasis inhibitor, it is usually 0.3-3.0 mg per day, preferably 0.4-2.0 mg, particularly preferably in terms of the dose of the pure nattokinase. 0.5-1.0 mg is administered in one or several divided doses.
When the natto-kinase cultured nattokinase-containing product (crude product: nattokinase compound) is used, 2.5 to 25 g, preferably 2.8 to 20 g, per day per day for adults in terms of the dose of the culture. Particularly preferably, 3.0 to 18 g is administered once or divided into several times.
When the purified nattokinase (cultured purified product: nattokinase fraction) is used, it is 12.5 to 125 mg, preferably 14 to 100 mg, particularly preferably 14 to 100 mg per day per day for adults in terms of the dose of the cultured purified product. Administer 15-80 mg in one or several divided doses.
If the amount of intake is too small, the cancer cell distant metastasis inhibiting effect of the present invention is reduced. On the other hand, if the amount is too large, side effects such as excessive reduction in blood coagulation ability tend to occur.

(3) Drug Form The cancer cell distant metastasis inhibitor of the present invention is orally administered, and no effect can be found even if this drug is administered into blood.
Therefore, the form of the cancer cell distant metastasis inhibitor of the present invention may be the above culture alone or as a tablet, pill, powder, powder, granule, syrup, solution, suspension, emulsion, capsule. It is common to administer to patients orally as such.
The form of these cancer cell distant metastasis inhibitors is appropriately selected by a doctor according to the age, sex, constitution, symptom, treatment time, etc. of the patient, but may be formulated in foods described later.

(4) Additive When the cancer cell distant metastasis inhibitor in the present invention is a solid preparation such as a tablet, pill, powder, powder, granule, etc., the culture is treated with an appropriate additive according to a conventional method, for example, , Lactose, sucrose, mannitol, corn starch, synthetic or natural gum, excipients such as crystalline cellulose, binders such as starch, cellulose derivatives, gum arabic, gelatin, polyvinyl pyrrolidone, carboxymethylcellulose cellulose, carbo Disintegrants such as sodium xymethylcellulose, starch, corn starch, sodium alginate, lubricants such as talc, magnesium stearate, sodium stearate, fillers or diluents such as calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate Etc., and can be mixed as appropriate.
In addition, tablets and the like may be coated with sugar coating, gelatin, enteric coating, film coating, etc. using an appropriate coating base as necessary.

(5) Formulation in food As described above, the nattokinase-containing material may be mixed as it is or dissolved in water, and may be contained in foods and beverages that can be eaten normally. .

[II] Production of nattokinase
(1) Culturing of Bacillus natto The cancer cell distant metastasis inhibitor of the present invention is effective in separating nattokinase obtained by separating solid components from a culture obtained by cultivating Bacillus natto using soybean as a medium and concentrating and drying. It is contained as a component.

(A) Bacteria The Bacillus subtilis (Bacillus subtilis) is considered as a Bacillus natto used to obtain a cancer cell distant metastasis inhibitor containing the nattokinase of the present invention as an active ingredient in consideration of safety, nattokinase production and the like. The known Bacillus subtilis natto belonging to) is used.
Although it does not restrict | limit especially as Bacillus natto, Takahashi bacillus (Yuzo Takahashi laboratory, Yamagata), Fungus Naruse (Naruse fermentation chemical laboratory, Tokyo), Miyagino bacillus (Miyagino natto factory, Sendai) Commercially available natto bacteria such as Asahi bacteria (Asahi Kogyo Co., Ltd., Tokyo), Nitto bacteria (Nitto Yakuhin Kogyo Co., Ltd., Kyoto), Meguro (Meguro Laboratories, Osaka); and Yunnan SL-001 Examples include bacteria.
In addition, Yunnan SL-001 bacteria was internationally deposited under the accession number FERM BP-6713 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry on May 7, 1999.

(B) Preparation of Medium As a medium used for culturing Bacillus natto, a medium similar to a known medium generally used for culturing Bacillus natto is used.
Specifically, for example, okara, soybeans, soybean soup produced as a by-product during production of miso or natto, tofu cake produced as a by-product during production of tofu or fried soybeans, soybean meal produced as a by-product during oil production using soybean as a raw material, Materials that can be fermented by Bacillus natto, such as soybean seed coat, which is a by-product, may be used.
Therefore, it can be prepared by mixing various culture components as appropriate, or a commercially available medium can be used as it is, or the following carbon source, nitrogen source, inorganic salt and other nutrients can be added to the commercially available medium. A medium appropriately added as an auxiliary component may be used.
At this time, the medium may be either a solid or liquid medium, but is preferably a liquid medium from the viewpoint of productivity, and is appropriately selected depending on the purpose of use, and contains nutrients that can be assimilated by the microorganism to be used. As long as it is a medium, either a synthetic medium or a natural medium may be used.

(a) Carbon source The carbon source that can be used for cultivation of Bacillus natto in the present invention is not particularly limited as long as it varies depending on the species used and the strain used grows well and can efficiently produce nattokinase.
Specifically, for example, starch or a composition fraction thereof, roasted dextrin, modified starch, starch derivative, physically treated starch, α-starch, soluble starch, amylose, amylopectin, malto-oligosaccharide, cyclodextrin, pullulan, corn starch, Mention may be made of potato starch, sweet potato starch and carbohydrates such as dextrin, glycerin, sorbitol, wort and glucose.
Among these carbon sources, glucose and starch are preferably used from the viewpoint of production of nattokinase. These carbon sources can be used alone or in the form of a mixture of two or more.

(b) Nitrogen source The nitrogen source that can be used in the culture of Bacillus natto according to the present invention is also not particularly limited as long as it varies depending on the species used and the strain used grows well and can efficiently produce nattokinase.
Specifically, for example, meat extract, malt extract, peptone, soybean-derived polypeptone (for example, polypeptone-S), yeast extract, taste liquid (soy protein acid hydrolyzate), soybean powder, milk casein, casamino acid, Examples thereof include organic nitrogen compounds such as various amino acids and corn steep liquor, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate and ammonium chloride, nitrates such as sodium nitrate, and inorganic nitrogen compounds such as urea.
Among these nitrogen sources, soybean-derived polypeptone (for example, polypeptone-S) and soybean powder can be preferably used from the viewpoint of production of nattokinase. These nitrogen sources can also be used alone or in the form of a mixture of two or more.

(c) Inorganic salt The inorganic salt that can be used for cultivation of Bacillus natto according to the present invention is also not particularly limited as long as it varies depending on the species used and the strain used can grow well and produce nattokinase well.
Specifically, for example, one or more selected from phosphates, hydrochlorides, sulfates, acetates and the like such as magnesium, manganese, calcium, sodium, potassium, copper, iron and zinc are used. You can also

(C) Culture conditions In the present invention, culture of Bacillus natto is performed in the same manner as a conventionally known method, and the culture conditions at that time are appropriately selected and used according to the strain used, the composition of the medium and the culture method. The strain is not particularly limited as long as it can grow and efficiently produce nattokinase.
The culture temperature is usually 20 to 45 ° C., preferably 37 to 42 ° C., and the pH of the medium suitable for the culture is usually 6.0 to 9.5, preferably 7.0 to 8.5. It is.

(2) Separation of Bacillus natto The solid components of the Bacillus natto culture cultivated by the method of the present invention are collected by a known method such as filtration, ultrafiltration or centrifugation, and the filtrate is concentrated.

(3) Drying It can be obtained as a powder by drying by a known method such as freeze drying, air drying, vacuum heat drying and the like.
The obtained powder can be ingested as a therapeutic agent for diabetes as it is as a natto-cultured nattokinase-containing product (crude product: nattokinase compound).

(4) Purification The powdered natto-bacterial nattokinase-containing product (crude product: nattokinase compound) can be further purified to obtain a highly purified nattokinase (nattokinase fraction).
Specifically, powdered natto bacteria cultivated nattokinase-containing material (crude product: nattokinase compound) is dissolved in Tris-HCl buffer, fractionated with 25 wt% ammonium sulfate, centrifuged, After removing the precipitate, the liquid is adsorbed on an ion exchange resin, the supernatant is removed, and then eluted with a Tris-HCl buffer to obtain a purified nattokinase having a purity of about 200 to 1,000 times. (Nattokinase fraction) can be purified.
Further, it can be further purified to obtain ultrapurified nattokinase with a further ultrahigh purity.

(5) Constituents of dried filtrate of Bacillus natto culture
(A) Nattokinase The nattokinase culture filtrate obtained by cultivating Bacillus natto using the soybeans as a medium contains nattokinase as a main active ingredient.
Specifically, the amount of nattokinase accumulated in the filtrate dried product of the culture of Bacillus natto varies depending on the cells used, the type of culture medium, the culture conditions, and the like, but usually 10 to 200 mg / 100 g. It is a vacuum heat-dried microbial cell.

(B) Other components In addition to the above nattokinase, 0.002 to 0.005% by weight of an enzyme such as amylase, protease, or ribose, or 0.5 to 1.5% by weight of a glutamic acid polypeptide Or 0.5 to 1.5% by weight of fructan of a fructose polymer.

[III] Medicinal effect The cancer cell distant metastasis inhibitor of the present invention containing nattokinase produced as described above as an active ingredient has a medicinal effect as a patient who has developed cancer, or cancer has occurred but cancer has occurred. If a person who has not yet noticed it continues to take it, it can exert an effect of suppressing distant metastasis from a site where cancer cells are generated to another organ, particularly a different organ.

  The present invention will be described more specifically with reference to the following examples and comparative examples.

Example 1
[I] Preparation of nattokinase
(1) Culturing of Bacillus natto colonies After adding Bacillus natto (Takahashi) to pure water and stirring it, it was put into a plate medium prepared in a petri dish using a standard agar medium (Nissui). A small amount is added (inoculated) and cultured at 37 ° C. for 24 hours to obtain Bacillus natto colonies on the plate medium.

(2) Preparation of culture solution On the other hand, 200 ml of a liquid medium consisting of 1 g of glucose as a raw material and 4 g of powdered soy protein (Nisshin Cosmo Foods Co., Ltd., trade name: Solpy NY) is placed in an Erlenmeyer flask having an internal volume of 500 ml. Prepare and sterilize the Erlenmeyer flask using an autoclave at 120 ° C. for 30 minutes.
The powdery soy protein is purified from soybean. As a raw material, soybean powder obtained by pulverizing soybean may be used.
Then, from the Bacillus natto colonies cultivated on the plate medium, Bacillus natto is collected once with a platinum loop having a diameter of a platinum wire ring of 2 mm, and the collected Bacillus natto is inoculated into the liquid medium in the Erlenmeyer flask.
Next, the Erlenmeyer flask in which the liquid medium inoculated with Bacillus natto is housed is rotated and shaken for two days without being sealed while being kept at 40 ° C., and the liquid medium in the Erlenmeyer flask is cultured.

(3) Cultivation Further, in a jar fermenter with an internal volume of 30 liters, 20 liters of a liquid medium comprising 100 g of glucose, 400 g of powdered soy protein and soluble starch (SF-400 manufactured by Shikishima Starch Co., Ltd.) was prepared. Prepare a sterilized product.
In addition, 200 ml of the Bacillus natto culture solution cultured in the Erlenmeyer flask is added to the liquid medium in the jar fermenter, and cultured while being kept stirring for 2 days while bubbling in a state heated to 42 ° C.
Three similar ones are prepared to obtain about 50 liters of soybean protein culture solution.

(4) Separation of Bacillus natto Next, Bacillus natto and impurities are removed from the obtained soybean protein culture solution by centrifugation, and 1 mol of ammonium sulfate is added. By the addition of ammonium sulfate, proteins containing proteinaceous polymer compounds such as various enzymes dispersed in the fermentation broth are made hydrophobic and easily aggregated.
Further, insoluble matters are newly formed by addition of ammonium sulfate, and these insoluble matters are filtered through glass fiber filter paper.

(5) Aggregation Next, a resin for hydrophobic chromatography equilibrated with 1 molar ammonium sulfate solution (trade name: Butyrute Yopal 650M) equilibrated with 1 molar ammonium sulfate solution was immersed in the filtrate. The hydrophobic polymer compound is aggregated (attached) on top.

(6) Concentration Next, the above-mentioned resin is immersed in a 0.1 molar ammonium hydrogen carbonate aqueous solution to elute the polymer compound adhering to the resin, thereby concentrating the soy protein culture solution to a concentrated culture solution 40. Get a liter.

(7) Ultrafiltration Next, the preb scale M.I. W. The low molecular weight material is separated by ultrafiltration using a 10,000 ultrafiltration membrane, and 40 liters of the concentrated culture solution is concentrated to 10 liters.
By ultrafiltration, 10 liters of pure water is added to the concentrate concentrated by the ultrafiltration membrane to make 20 liters, and again by the ultrafiltration described above, low-molecular substances are separated and again 10 liters. The soy protein fermented product solution from which most low molecular weight substances have been removed is obtained from the concentrated culture solution obtained by concentrating soy protein culture solution three times.

Ultrafiltration The above ultrafiltration is generally performed to separate fine particles such as colloidal particles from a dispersion medium. Therefore, the ultrafiltration membrane used in the ultrafiltration is a collodion attached to a cloth or an unglazed porous plate. A membrane, a gelatin film hardened with formalin, a silicate film, a cellophane film or the like is used, and a colloidal solution is filtered through these films under pressure.
In order to adjust the size of the membrane, in the case of collodion, the concentration of alcohol and ether in the mixed solvent, adjustment of drying conditions, etc., in the case of cellophane, adjustment of the degree of swelling when immersed in an aqueous salt solution, or filtration of the collodion solution through the membrane However, it can be performed by depositing collodion on the membrane.

What is separated by filtration As what is filtered by ultrafiltration, the main components of odor unique to natto are dimethylpyrazine (molecular weight 108.14), trimethylpyrazine (molecular weight 122.17), tetramethylpyrazine (molecular weight) 126.20), 2-methylbutyric acid (molecular weight 102.13), isovaleric acid (molecular weight 102.13), and ammonia (molecular weight 17.03).
Vitamin K ₂ contained in natto has a molecular weight of hydrocarbon compounds 649. On the other hand, proteins such as nattokinase are high molecular compounds having a molecular weight of tens of thousands or more.
Therefore, the removal of low molecular weight substances by ultrafiltration described above results in a state in which the low-molecular weight substances such as natto-specific odor components and vitamin K ₂ are substantially removed from the soybean protein culture solution.
Vitamin K ₂ contained in natto, there is a problem of suppressing the effect of the antagonist to be used for heart disease, such as myocardial infarction "Warfarin" (pharmaceutical).
However, according to this embodiment, since vitamin K ₂ is removed, it is possible to solve this problem.

(8) Drying Finally, 2 kg of lactose as an excipient is added to the soy protein fermented product solution from which low molecular weight substances have been removed by the ultrafiltration and freeze-dried, soy protein fermented product powder (processed food) ) About 2.1 kg is obtained.

(9) Odor test When two subjects who were not good at natto were unable to eat and confirmed the presence of odor in the fermented soy protein powder according to this embodiment, both subjects felt natto odor. It never happened.

(10) Purification 10 g of the above powdered nattokinase-containing product (crude product: nattokinase compound) is dissolved in 500 ml of 30 mM Tris-HCl buffer (pH 9.0), and 166 ml of 25 wt% saturated ammonium sulfate is added. The mixture was stirred for 15 minutes, allowed to stand for 15 minutes, centrifuged at 11,000 rpm for 30 minutes, and fractionated to remove precipitates.
Thereafter, 50 ml of ion exchange resin (“Butyl Toyopearl” manufactured by Tosoh Corporation) (30 mmol of Tris-HCl buffer, 25 wt% saturated ammonium sulfate, pH 9.0) was added to the liquid, and the mixture was stirred for 2 hours for ionization. It was made to adsorb | suck to exchange resin, and rotation speed 3000rpm was performed again for 5 minutes, and the supernatant liquid was removed, Then, it wash | cleaned by 30 mmol Tris-HCl buffer solution, 25 weight% saturated ammonium sulfate, pH 9.0.
After centrifuging again at 3000 rpm for 5 minutes and removing the supernatant, the nattokinase fraction (200-fold purified product) purified by elution with 30 mM Tris-HCl buffer, pH 9.0 was obtained. Prepared.

[II] Evaluation Using the above nattokinase, the effect of inhibiting distant metastasis of cancer cells by experimental animals was measured and evaluated.
(1) Experimental animals and rearing conditions Ten 7-week-old male mice were purchased from Japan SLC Co., Ltd. and reared in an SPF animal experimental facility with a room temperature of 22 ± 2 ° C. and a 12-hour light-dark cycle. The bait (manufactured by Claire Japan, trade name: CE-2) and water were used as free intake and subjected to an experiment after preliminary breeding for 1 week.

(2) Administration The human lung cancer-derived AO1 cells were transplanted into the abdomen of the nude mice BALB / nu10.
The size of the abdominal tumor 1 day after the transplantation was measured.
As shown in Table 1, in 5 mice of Examples 1-1 to 1-5, nattokinase compound (crude product) was dissolved in water after 2 weeks while feeding, and 100 mg / kg · day was reduced to 60 mg. Oral administration was continued for consecutive days.
Moreover, in the five mice of Comparative Examples 1-1 to 1-5, only water was orally administered except that food was given.
After 60 days from the date of transplantation, the lungs of the 10 mice were dissected, the size of the abdominal tumor was measured, and the number of colonies of the metastasized tumor was measured.
The results are shown in Table 1.

Comparative Example 1
The same procedure as described in Example 1 was performed except that natto kinase was not administered in Example 1.
The results are shown in Table 1.

From these results, it can be understood that in the mice to which nattokinase compound (crude product) was administered, the tumor of the transplanted part was growing, but the dissected lung colony number was 0, and it did not metastasize to the lung. .
However, in the mice to which nattokinase compound (crude product) was not administered, the transplanted tumor had grown, but the average number of dissected lung colonies was 10.4, and the abdominal tumor metastasized to the lung. I can understand that.

  As described above, the cancer cell distant metastasis inhibitor containing the nattokinase of the present invention as an active ingredient can be used as a cancer if it continues to be taken even by patients who have developed cancer or those who have developed cancer but have not yet noticed the occurrence of cancer. Since it has an effect of suppressing distant metastasis from a site where cells are generated to another organ, in particular, a different organ, it is extremely useful as a pharmaceutical or health food.

Claims (5)

  A cancer cell distant metastasis inhibitor comprising nattokinase as an active ingredient.   2. A natto-kinase cultured nattokinase-containing product obtained by culturing natto bacteria or a purified nattokinase obtained by adsorbing and purifying natto-bacterium-cultivated nattokinase-containing product obtained by culturing natto bacteria on an ion exchange resin. The cancer cell distant metastasis inhibitor described in 1.   The cancer cell distant metastasis inhibitor according to claim 2, wherein the intake of the natto-cultured nattokinase-containing material is 2,500 mg to 25,000 mg per day per adult.   The cancer cell distant metastasis inhibitor according to claim 2, wherein the intake of purified nattokinase is 12.5 mg to 125 mg per adult per day.   Purified nattokinase is purified by dissolving the natto-cultured nattokinase-containing material with Tris-HCl buffer, adsorbing it to an ion exchange resin, separating impurities, and then eluting with Tris-HCl buffer again. The cancer cell distant metastasis suppressor according to claim 4.