US20040043014A1 - Platelet aggregation inhibitor and supplement food effective for inhibiting platelet aggregation - Google Patents
Platelet aggregation inhibitor and supplement food effective for inhibiting platelet aggregation Download PDFInfo
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- US20040043014A1 US20040043014A1 US10/353,300 US35330003A US2004043014A1 US 20040043014 A1 US20040043014 A1 US 20040043014A1 US 35330003 A US35330003 A US 35330003A US 2004043014 A1 US2004043014 A1 US 2004043014A1
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- nattokinase
- platelet aggregation
- platelet
- collagen
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- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 16
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- 101000783577 Dendroaspis angusticeps Thrombostatin Proteins 0.000 title claims abstract description 11
- 101000783578 Dendroaspis jamesoni kaimosae Dendroaspin Proteins 0.000 title claims abstract description 11
- 239000000106 platelet aggregation inhibitor Substances 0.000 title claims abstract description 11
- 239000013589 supplement Substances 0.000 title claims abstract description 10
- 229940086319 nattokinase Drugs 0.000 claims abstract description 52
- 108010073682 nattokinase Proteins 0.000 claims abstract description 52
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- DKHGMERMDICWDU-GHDNBGIDSA-N menaquinone-4 Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 DKHGMERMDICWDU-GHDNBGIDSA-N 0.000 claims abstract description 13
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- PHWBOXQYWZNQIN-UHFFFAOYSA-N ticlopidine Chemical compound ClC1=CC=CC=C1CN1CC(C=CS2)=C2CC1 PHWBOXQYWZNQIN-UHFFFAOYSA-N 0.000 description 2
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- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 206010061172 Gastrointestinal injury Diseases 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- 229920002494 Zein Polymers 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 238000011047 acute toxicity test Methods 0.000 description 1
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- 208000010125 myocardial infarction Diseases 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
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- 229940046010 vitamin k Drugs 0.000 description 1
- 239000005019 zein Substances 0.000 description 1
- 229940093612 zein Drugs 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/45—Transferases (2)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates to a platelet aggregation inhibitor and, more particularly, to a platelet aggregation inhibitor whose active ingredient is nattokinase and a supplement food using the same.
- nattokinase has been known as a thrombolytic enzyme, and a thrombolytic activity of the nattokinase has been known to be excellent not only for preventing thrombosis but also for treating the thrombosis (Shinsaku Takaoka: Japan Food Science, 39 (9), 55-60, 2000).
- the nattokinase is not contained in soybeans, but produced in the course of fermentation when Bacillus natto acts on the soybeans, and further, the nattokinase also contains vitamin K 2 as a blood coagulation factor. Therefore, ingestion of natto or Bacillus natto culture extract which contains /the nattokinase as a thrombolytic enzyme, for the purpose of preventing thrombosis, means that the vitamin K 2 is also ingested simultaneously. Thus, there has been a problem that an effect of the vitamin K-dependent coagulation factor synthesis inhibitor is counteracted. Consequently, a technique for producing Bacillus natto culture extract whose vitamin K 2 content is 1 mg/g or less on a dry weight basis has been developed (see Japanese Patent Laid-Open No. 2001-299277).
- nattokinase has inhibitory action on platelet aggregation.
- aspirin or ticlopidine for example is orally administered.
- these agents produce side effects. For example, side effects such as gastrointestinal injury and aspirin-induced asthma are produced if the aspirin is administered, whereas side effects such as thrombocytopenic purpura (TTP), agranulocytosis, and serious hepatopathy are produced if the ticlopidine is administered.
- an object of the present invention is to provide a platelet aggregation inhibitor without side effects and a supplement food effective for inhibiting platelet aggregation.
- a platelet aggregation inhibitor according to the present invention is intended to have nattokinase as an active ingredient and to have Bacillus natto culture extract, containing a high proportion of nattokinase containing 1 mg/g or less of vitamin K 2 on a dry weight basis, as an active ingredient.
- a supplement food effective for inhibiting platelet aggregation is intended to have nattokinase as a principal ingredient and to have Bacillus natto culture extract, containing a high proportion of nattokinase containing 1 mg/g or less of vitamin K 2 on a dry weight basis, as a principal ingredient.
- nattokinase has been ingested by eating natto since hundreds years before, so that its safety has empirically been proven. Also, in the case of nattokinase contained in the Bacillus natto culture extract (of powder type), it has been observed that the nattokinase does not affect general conditions of mouse at all in an acute toxicity test. In this case, an LD 50 value was considered to be 2,000 mg/kg or more. Further, it has also been demonstrated that results from a reverse mutation test are negative (Shinsaku Takaoka: Japan Food Science, 39 (9), 55-60, 2000).
- nattokinase When a vitamin K 2 content in the nattokinase is 1 mg/g or less on a dry weight basis, the above described disorders caused by the vitamin K 2 are not produced.
- the nattokinase which has been proved to be safe as described above, is available not only for agents but also for foods (supplement foods).
- the present invention it is possible to provide a platelet aggregation inhibitor which does not cause problems such as side effects and to provide a supplement food which is safe and effective for inhibiting platelet aggregation. According to the present invention, it is also possible to treat and prevent myocardial infarction or brain infarction, because blood clots are hardly formed inside a blood vessel and growth of the blood clots is also inhibited.
- FIG. 1 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers;
- FIG. 2 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers whose concentrations are different from those shown in FIG. 1;
- FIG. 3 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers whose concentrations are different from those shown in FIG. 1 and FIG. 2; and
- FIG. 4 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers whose concentrations are different from those shown in FIGS. 1, 2, and 3 .
- Bacillus natto culture extract containing a high proportion of nattokinase whose vitamin K 2 content is 1 mg/g or less on a dry weight basis is as follows.
- Bacillus natto is taken into a liquid medium which contains soybeans as principal materials and is cultured to produce a culture solution containing a high proportion of nattokinase. Then the Bacillus natto and the vitamin K 2 are removed by coagulation sedimentation filtration using a chitosan solution. The obtained culture solution, which has been subjected to sterile filtration through a membrane filter, is dried and processed into powder.
- Bacillus natto culture extract can be processed into a certain form such as a capsule, a tablet, a drinkable preparation, a granule, or paste. Examples of processing the culture extract will be described bellow.
- an enteric capsule including an acid-resistant coat
- 10 mg of soybean lecithin, 133.3 mg of soybean oil, 15 mg of beewax, and 15 mg of glycerin fatty acid ester are mixed and emulsified to obtain 210 mg in total of an emulsified mixture, and then the emulsified mixture is filled into a film cell made of 130 mg in total of a material composed 100 mg of gelatin and 30 mg of glycerin. Consequently, a soft capsule having a total weight of 340 mg is formed.
- This capsule thus formed is coated with 30 mg of zein to form an enteric capsule having a total weight of 370 mg.
- Administration of 3 to 6 capsules thus formed per day is equivalent to ingestion of nattokinase contained in 1 or 2 packs (50 to 100 g) of natto on the market.
- nattokinase corresponding to 2 packs of natto, 100 g
- a platelet aggregation activity was measured by collecting his blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration.
- Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate.
- An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total.
- collagen As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, final concentrations of the collagen were 1 ⁇ g/ml and 2 ⁇ g/ml, and a final concentration of the ADP was 2 ⁇ M.
- a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used.
- the apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof.
- the absorbance was lowered only after the aggregates comprising thousands of platelets were produced.
- the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity.
- the platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV ⁇ a small aggregate (particle size, 9 to 25 ⁇ m) ⁇ 200 mV, 200 mV ⁇ a medium aggregate (particle size, 25 to 50 ⁇ m) ⁇ 600 mV, and 600 mV ⁇ a large aggregate (particle size, 50 to 70 ⁇ m) ⁇ 2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin.- Pharmacol. Ther., 32, 223-230 (2001)].
- X scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y scattering intensity or OD obtained by adding ADP or collagen before ingesting nattokinase.
- nattokinase corresponding to 2 packs of natto, 100 g
- a platelet aggregation activity was measured by collecting his blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration.
- Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate.
- An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total.
- collagen As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, a final concentration of the collagen was 2 ⁇ g/ml, and final concentrations of the ADP were 2 ⁇ M and 5 ⁇ M.
- a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used.
- the apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof.
- the absorbance was lowered only after the aggregates comprising thousands of platelets were produced.
- the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity.
- the platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV ⁇ a small aggregate (particle size, 9 to 25 ⁇ m) ⁇ 200 mV, 200 mv ⁇ a medium aggregate (particle size, 25 to 50 ⁇ m) ⁇ 600 mV, and 600 mV ⁇ a large aggregate (particle size, 50-to 70 ⁇ m) ⁇ 2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
- X scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y scattering intensity or OD obtained by adding ADP or collagen before ingesting nattokinase.
- nattokinase For a normal healthy subject, female: 1, to whom 6 soft capsules according to the above described Processing Example (potency of nattokinase corresponding to 2 packs of natto, 100 g) were administered, a platelet aggregation activity was measured by collecting her blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration. Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate. An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total.
- collagen As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, final concentrations of the collagen were 1 ⁇ g/ml and 2 ⁇ g/ml, and final concentrations of the ADP were 2 ⁇ M and 5 ⁇ M.
- a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used.
- the apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof.
- the absorbance was lowered only after the aggregates comprising thousands of platelets were produced.
- the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity.
- the platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV ⁇ a small aggregate (particle size, 9 to 25 tm) ⁇ 200 mV, 200 mV ⁇ a medium aggregate (particle size, 25 to 50 ⁇ m) ⁇ 600 mV, and 600 mV ⁇ a large aggregate (particle size, 50 to 70 ⁇ m) ⁇ 2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
- X scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y scattering intensity or OD.obtained by adding ADP or collagen before ingesting nattokinase.
- nattokinase enteric capsules For a normal healthy subject, male: 1, to whom 6 nattokinase enteric capsules according to the above described Processing Example (potency of nattokinase corresponding to 2 packs of natto, 100 g) were administered, a platelet aggregation activity was measured by collecting his blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration. Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate. An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total.
- collagen As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, final concentrations of the collagen were 0.5 ⁇ g/ml, 1 ⁇ g/ml, and 2 ⁇ g/ml, and a final concentration of the ADP was 2 ⁇ M.
- a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used.
- the apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof.
- the absorbance was lowered only after the aggregates comprising thousands of platelets were produced.
- the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity.
- the platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV ⁇ a small aggregate (particle size, 9 to 25 ⁇ m) ⁇ 200 mV, 200 mV ⁇ a medium aggregate (particle size, 25 to 50 ⁇ m) ⁇ 600 mv, and 600 mV ⁇ a large aggregate (particle size, 50 to 70 ⁇ m) ⁇ 2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
- X scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y scattering intensity or OD obtained by adding ADP or collagen before ingesting nattokinase.
Abstract
An object of the present invention is to provide a platelet aggregation inhibitor without side effects and a supplement food effective for inhibiting platelet aggregation. The platelet aggregation inhibitor has nattokinase as an active ingredient and has Bacillus natto culture extract, containing a high proportion of nattokinase containing 1 mg/g or less of vitamin K2 on a dry weight basis, as an active ingredient. The supplement food effective for inhibiting platelet aggregation has nattokinase as an active ingredient and has Bacillus natto culture extract, containing a high proportion of nattokinase containing 1 mg/g or less of vitamin K2 on a dry weight basis, as an active ingredient.
Description
- 1. Field of the Invention
- The present invention relates to a platelet aggregation inhibitor and, more particularly, to a platelet aggregation inhibitor whose active ingredient is nattokinase and a supplement food using the same.
- 2. Description of the Related Art
- Previously, nattokinase has been known as a thrombolytic enzyme, and a thrombolytic activity of the nattokinase has been known to be excellent not only for preventing thrombosis but also for treating the thrombosis (Shinsaku Takaoka: Japan Food Science, 39 (9), 55-60, 2000).
- The nattokinase is not contained in soybeans, but produced in the course of fermentation when Bacillus natto acts on the soybeans, and further, the nattokinase also contains vitamin K2 as a blood coagulation factor. Therefore, ingestion of natto or Bacillus natto culture extract which contains /the nattokinase as a thrombolytic enzyme, for the purpose of preventing thrombosis, means that the vitamin K2 is also ingested simultaneously. Thus, there has been a problem that an effect of the vitamin K-dependent coagulation factor synthesis inhibitor is counteracted. Consequently, a technique for producing Bacillus natto culture extract whose vitamin K2 content is 1 mg/g or less on a dry weight basis has been developed (see Japanese Patent Laid-Open No. 2001-299277).
- As a result of making an intensive study on effective actions of nattokinase on human bodies in addition to thrombolytic action thereof, the inventors have now found that the nattokinase has inhibitory action on platelet aggregation. As an example of a platelet aggregation inhibitor which has practically been used as an anti-platelet agent clinically, aspirin or ticlopidine for example is orally administered. However, these agents produce side effects. For example, side effects such as gastrointestinal injury and aspirin-induced asthma are produced if the aspirin is administered, whereas side effects such as thrombocytopenic purpura (TTP), agranulocytosis, and serious hepatopathy are produced if the ticlopidine is administered.
- Therefore, an object of the present invention is to provide a platelet aggregation inhibitor without side effects and a supplement food effective for inhibiting platelet aggregation.
- To achieve the above described object, a platelet aggregation inhibitor according to the present invention is intended to have nattokinase as an active ingredient and to have Bacillus natto culture extract, containing a high proportion of nattokinase containing 1 mg/g or less of vitamin K2 on a dry weight basis, as an active ingredient.
- In addition, a supplement food effective for inhibiting platelet aggregation according to the present invention is intended to have nattokinase as a principal ingredient and to have Bacillus natto culture extract, containing a high proportion of nattokinase containing 1 mg/g or less of vitamin K2 on a dry weight basis, as a principal ingredient.
- The nattokinase has been ingested by eating natto since hundreds years before, so that its safety has empirically been proven. Also, in the case of nattokinase contained in the Bacillus natto culture extract (of powder type), it has been observed that the nattokinase does not affect general conditions of mouse at all in an acute toxicity test. In this case, an LD50 value was considered to be 2,000 mg/kg or more. Further, it has also been demonstrated that results from a reverse mutation test are negative (Shinsaku Takaoka: Japan Food Science, 39 (9), 55-60, 2000).
- When a vitamin K2 content in the nattokinase is 1 mg/g or less on a dry weight basis, the above described disorders caused by the vitamin K2 are not produced. The nattokinase, which has been proved to be safe as described above, is available not only for agents but also for foods (supplement foods).
- According to the present invention, it is possible to provide a platelet aggregation inhibitor which does not cause problems such as side effects and to provide a supplement food which is safe and effective for inhibiting platelet aggregation. According to the present invention, it is also possible to treat and prevent myocardial infarction or brain infarction, because blood clots are hardly formed inside a blood vessel and growth of the blood clots is also inhibited.
- FIG. 1 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers;
- FIG. 2 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers whose concentrations are different from those shown in FIG. 1;
- FIG. 3 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers whose concentrations are different from those shown in FIG. 1 and FIG. 2; and
- FIG. 4 is a diagram showing a change of a platelet inhibition rate with respect to time after the ingestion of nattokinase, when using collagen and ADP as inducers whose concentrations are different from those shown in FIGS. 1, 2, and3.
- Examples of producing an extract and processing the extract and examples of the present invention will be descried below.
- An example of producing Bacillus natto culture extract containing a high proportion of nattokinase whose vitamin K2 content is 1 mg/g or less on a dry weight basis is as follows.
- Bacillus natto is taken into a liquid medium which contains soybeans as principal materials and is cultured to produce a culture solution containing a high proportion of nattokinase. Then the Bacillus natto and the vitamin K2 are removed by coagulation sedimentation filtration using a chitosan solution. The obtained culture solution, which has been subjected to sterile filtration through a membrane filter, is dried and processed into powder.
- The above described Bacillus natto culture extract can be processed into a certain form such as a capsule, a tablet, a drinkable preparation, a granule, or paste. Examples of processing the culture extract will be described bellow.
- When the above described culture extract is processed into a soft capsule form for example, 36.7 mg of Bacillus natto culture extract powder (20000 FU/g), 10 mg of soybean lecithin, 133.3 mg of soybean oil, 15 mg of beewax, and 15 mg of glycerin fatty acid ester are mixed and emulsified to obtain 210 mg in total of an emulsified mixture, and then the emulsified mixture is filled into a film cell made of 130 mg in total of a material composed of 100 mg of gelatin and 30 mg of glycerin. Consequently, a soft capsule having a total weight of 340 mg is formed. Administration of 3 to 6 capsules thus formed per day is equivalent to ingestion of nattokinase contained in 1 or 2 packs (50 to 100 g) of natto on the market.
- Similarly, when a hard capsule is formed, 36.7 mg of Bacillus natto culture extract powder (20000 FU/g), 209.8 mg of dextrin, and 13.5 mg of sucrose fatty acid ester are mixed to obtain 260 mg in total of a mixture, and then the mixture is filled into a gelatin hard capsule (70 mg) to make a No. 2 gelatin hard capsule having a total weight of 330 mg. Administration of 3 to 6 capsules thus formed per day is equivalent to ingestion of nattokinase contained in 1 or 2 packs (50 to 100 g) of natto on the market.
- When an enteric capsule (including an acid-resistant coat) is formed for example, 36.7 mg of Bacillus natto culture extract powder (20000 FU/g), 10 mg of soybean lecithin, 133.3 mg of soybean oil, 15 mg of beewax, and 15 mg of glycerin fatty acid ester are mixed and emulsified to obtain 210 mg in total of an emulsified mixture, and then the emulsified mixture is filled into a film cell made of 130 mg in total of a material composed 100 mg of gelatin and 30 mg of glycerin. Consequently, a soft capsule having a total weight of 340 mg is formed. This capsule thus formed is coated with 30 mg of zein to form an enteric capsule having a total weight of 370 mg. Administration of 3 to 6 capsules thus formed per day is equivalent to ingestion of nattokinase contained in 1 or 2 packs (50 to 100 g) of natto on the market.
- The above described methods can also be applied to tablets, drinkable preparations, granules, pastes or the like.
- Experimental Method:
- For a normal healthy subject, male: 1, to whom 6 soft capsules according to the above described Processing Example (potency of nattokinase corresponding to 2 packs of natto, 100 g) were administered, a platelet aggregation activity was measured by collecting his blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration. Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate. An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total. Although this male subject did not need to be fasted, a lapse of 2 or more hours after the breakfast was required because of the nature of this examination, for the purpose of getting knowledge of the platelet aggregation activity in a certain condition similar to an actual administration condition. Each blood specimen thus obtained was subjected to centrifugation for 10 minutes at 180×g, and the obtained supernatant was used as platelet rich plasma (PRP). The remaining specimen was subjected to centrifugation for 15 minutes at 1600×g and used as platelet poor plasma (PPP). The PRP was diluted with the PPP to prepare a specimen which contains platelet at a concentration of 25±3×104/l. As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, final concentrations of the collagen were 1 μg/ml and 2 μg/ml, and a final concentration of the ADP was 2 μM.
- For measurement of the platelet aggregation activity, a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used. The apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof. In the conventional extinction method, the absorbance was lowered only after the aggregates comprising thousands of platelets were produced. However, the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity. The platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV<a small aggregate (particle size, 9 to 25 μm)<200 mV, 200 mV<a medium aggregate (particle size, 25 to 50 μm)<600 mV, and 600 mV<a large aggregate (particle size, 50 to 70 μm)<2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin.- Pharmacol. Ther., 32, 223-230 (2001)].
- Platelet inhibition rates were calculated from an equation as follows:
- Platelet inhibition rate (%)=(1−X/Y)×100
- X: scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y: scattering intensity or OD obtained by adding ADP or collagen before ingesting nattokinase.
- Experimental Result:
- When 1.0 μg/ml of collagen was added as a coagulant, a strong inhibitory action was observed after a lapse of 4 hours from the ingestion (FIG. 1). The platelet aggregates were reduced to their minimum sizes after 8 hours, and the inhibitory action of nattokinase on the platelets were observed (Table1A, 1B, and 1C described below).
TABLE 1A platelet Collagen 1.0 μg/ml aggregate 0 h 2 h 4 h 6 h 8 h Large 12 6 1 1 0 (50-70 ìm) Medium 25 20 12 4 3 (25-50 ìm) Small 63 75 88 95 97 (9-25 ìm) -
TABLE 1B Platelet Collagen 2.0 μg/ml aggregate 0 h 2 h 4 h 6 h 8 h Large 31 35 31 27 25 (50-70 ìm) Medium 30 25 26 28 27 (25-50 ìm) Small 39 40 43 45 28 (9-25 ìm) -
TABLE 1C Platelet ADP 2.0 μM aggregate 0 h 2 h 4 h 6 h 8 h Large 41 33 42 35 20 (50-70 ìm) Medium 23 30 26 27 31 (25-50 ìm) Small 36 37 33 37 49 (9-25 ìm) - Experimental Method:
- For a normal healthy subject, male: 1, to whom 6 soft capsules according to the above described Processing Example (potency of nattokinase corresponding to 2 packs of natto, 100 g) were administered, a platelet aggregation activity was measured by collecting his blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration. Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate. An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total. Although this male subject did not need to be fasted, a lapse of 2 or more hours after the breakfast was required because of the nature of this examination, for the purpose of getting knowledge of the platelet aggregation activity in a certain condition similar to an actual administration condition. Each blood specimen thus obtained was subjected to centrifugation for 10 minutes at 180×g, and the obtained supernatant was used as platelet rich plasma (PRP). The remaining specimen was subjected to centrifugation for 15 minutes at 1600×g and used as platelet poor plasma (PPP). The PRP was diluted with the PPP to prepare a specimen which contains platelet at a concentration of 25+3×104 μl. As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, a final concentration of the collagen was 2 μg/ml, and final concentrations of the ADP were 2 μM and 5 μM.
- For measurement of the platelet aggregation activity, a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used. The apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof. In the conventional extinction method, the absorbance was lowered only after the aggregates comprising thousands of platelets were produced. However, the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity. The platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV<a small aggregate (particle size, 9 to 25 μm)<200 mV, 200 mv<a medium aggregate (particle size, 25 to 50 μm)<600 mV, and 600 mV<a large aggregate (particle size, 50-to 70 μm)<2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
- Platelet inhibition rates were calculated from an equation as follows:
- Platelet inhibition rate (%)=(1−X/Y)×100
- X: scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y: scattering intensity or OD obtained by adding ADP or collagen before ingesting nattokinase.
- Experimental Result:
- When 1.0 μg/ml of collagen was added as a coagulant, a strong inhibitory action was observed after a lapse of 4 hours from the ingestion (FIG. 2).
- Experimental Method:
- For a normal healthy subject, female: 1, to whom 6 soft capsules according to the above described Processing Example (potency of nattokinase corresponding to 2 packs of natto, 100 g) were administered, a platelet aggregation activity was measured by collecting her blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration. Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate. An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total. Although this female subject did not need to be fasted, a lapse of 2 or more hours after the breakfast was required because of the nature of this examination, for the purpose of getting knowledge of the platelet aggregation activity in a certain condition similar to an actual administration condition. Each blood specimen thus obtained was subjected to centrifugation for 10 minutes at 180×g, and the obtained supernatant was used as platelet rich plasma (PRP). The remaining specimen was subjected to centrifugation for 15 minutes at 1600×g and used as platelet poor plasma (PPP). The PRP was diluted with the PPP to prepare a specimen which contains platelet at a concentration of 25+3×104 μl. As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, final concentrations of the collagen were 1 μg/ml and 2 μg/ml, and final concentrations of the ADP were 2 μM and 5 μM.
- For measurement of the platelet aggregation activity, a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used. The apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof. In the conventional extinction method, the absorbance was lowered only after the aggregates comprising thousands of platelets were produced. However, the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity. The platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV<a small aggregate (particle size, 9 to 25 tm)<200 mV, 200 mV<a medium aggregate (particle size, 25 to 50 μm)<600 mV, and 600 mV<a large aggregate (particle size, 50 to 70 μm)<2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
- Platelet inhibition rates were calculated from an equation as follows:
- Platelet inhibition rate (%)=(1−X/Y)×100
- X: scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y: scattering intensity or OD.obtained by adding ADP or collagen before ingesting nattokinase.
- Experimental Result:
- When 1.0 μg/ml of collagen was added as a coagulant, a strong inhibitory action was observed after a lapse of 4 hours from the ingestion (FIG. 3). The platelet aggregates were reduced to their minimum sizes after 8 hours, and the inhibitory action of nattokinase on the platelets were observed (Table 2A and 2B described below).
TABLE 2A Platelet Collagen 1.0 μg/ml aggregate 0 h 2 h 4 h 6 h 8 h Large 15 9 15 17 7 (50-70 ìm) Medium 9 10 6 10 8 (25-50 ìm) Small 76 81 79 73 86 (9-25 ìm) -
TABLE 2B Platelet Collagen 2.0 μg/ml aggregate 0 h 2 h 4 h 6 h 8 h Large 42 48 18 2 8 (50-70 ìm) Medium 27 26 28 2 4 (25-50 ìm) Small 32 26 55 96 88 (9-25 ìm) - Experimental Method:
- For a normal healthy subject, male: 1, to whom 6 nattokinase enteric capsules according to the above described Processing Example (potency of nattokinase corresponding to 2 packs of natto, 100 g) were administered, a platelet aggregation activity was measured by collecting his blood before the administration, and 2 hours, 4 hours, 6 hours, and 8 hours after the administration. Each blood sampling was performed from a brachial median vein via a 21G needle, using a tube containing 3.8% sodium citrate. An amount of the blood collected by single sampling was 11 ml, and thus 55 ml of blood was collected in total. Although this male subject did not need to be fasted, a lapse of 2 or more hours after the breakfast was required because of the nature of this examination, for the purpose of getting knowledge of the platelet aggregation activity in a certain condition similar to an actual administration condition. Each blood specimen thus obtained was subjected to centrifugation for 10 minutes at 180×g, and the obtained supernatant was used as platelet rich plasma (PRP). The remaining specimen was subjected to centrifugation for 15 minutes at 1600×g and used as platelet poor plasma (PPP). The PRP was diluted with the PPP to prepare a specimen which contains platelet at a concentration of 25±3×104/μl. As an aggregation inducer, collagen [MC Medical Inc.] and ADP [MC Medical Inc.] were used. In this case, final concentrations of the collagen were 0.5 μg/ml, 1 μg/ml, and 2 μg/ml, and a final concentration of the ADP was 2 μM.
- For measurement of the platelet aggregation activity, a particle measurement type of platelet aggregation activity measuring apparatus [PA-20: Kowa Co, Ltd.] employing laser scattered light was used. The apparatus PA-20 was developed on the principle that an intensity of scattered light which is generated by a beam of light impinging on a fine particle increases in proportion to a square of the particle size, and further, this apparatus can compute a platelet aggregation rate as well as a size of the produced platelet aggregate and the number thereof. In the conventional extinction method, the absorbance was lowered only after the aggregates comprising thousands of platelets were produced. However, the apparatus of the present invention can measure even small aggregates composed of tens of platelets, that is, this apparatus is excellent in its detection sensitivity. The platelet aggregate sizes were classified into three groups as follows, depending on the scattered light intensities: 25 mV<a small aggregate (particle size, 9 to 25 μm)<200 mV, 200 mV<a medium aggregate (particle size, 25 to 50 μm)<600 mv, and 600 mV<a large aggregate (particle size, 50 to 70 μm)<2,047 mV [Hoshi K., Zhou X., Terazono M., Satou Y., Yamazaki M., Miyake F., Jpn. J. Clin. Pharmacol. Ther., 32, 223-230 (2001)].
- Platelet inhibition rates were calculated from an equation as follows:
- Platelet inhibition rate (%)=(1−X/Y)×100
- X: scattering intensity or OD obtained by adding ADP or collagen after ingesting nattokinase
- Y: scattering intensity or OD obtained by adding ADP or collagen before ingesting nattokinase.
- Experimental Result:
- When 1.0 μg/ml of collagen was added as a coagulant, a strong inhibitory action was observed after a lapse of 4 hours from the ingestion (FIG. 4). The platelet aggregates were reduced to their minimum sizes after 8 hours, and the inhibitory action of nattokinase on the platelets were observed (Table 3A and 3B described below).
TABLE 3A Platelet Collagen 0.5 μg/ml aggregate 0 h 2 h 4 h 6 h 8 h Large 31 8 6 6 1 (50-70 ìm) Medium 23 6 2 3 1 (25-50 ìm) Small 47 85 93 91 99 (9-25 ìm) -
TABLE 3B Platelet Collagen 1.0 μg/ml aggregate 0 h 2 h 4 h 6 h 8 h Large 37 11 2 3 5 (50-70 ìm) Medium 22 21 14 2 3 (25-50 ìm) Small 41 68 84 95 93 (9-25 ìm)
Claims (4)
1. A platelet aggregation inhibitor whose active ingredient is nattokinase.
2. A platelet aggregation inhibitor whose active ingredient is Bacillus natto culture extract containing a high proportion of nattokinase, the nattokinase containing 1 mg/g or less of vitamin K2 on a dry weight basis.
3. A supplement food effective for inhibiting platelet aggregation whose principal ingredient is nattokinase.
4. A supplement food effective for inhibiting platelet aggregation whose principal ingredient is Bacillus natto culture extract containing a high proportion of nattokinase, the nattokinase containing 1 mg/g or less of vitamin K2 on a dry weight basis.
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US10/353,300 Abandoned US20040043014A1 (en) | 2002-08-30 | 2003-01-29 | Platelet aggregation inhibitor and supplement food effective for inhibiting platelet aggregation |
US10/465,193 Abandoned US20040043015A1 (en) | 2002-08-30 | 2003-06-19 | Platelet aggregation inhibitor and supplement food effective for inhibiting platelet aggregation |
US10/932,806 Expired - Lifetime US7014851B2 (en) | 2002-08-30 | 2004-09-02 | Process for inhibiting platelet aggregation |
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US10/932,806 Expired - Lifetime US7014851B2 (en) | 2002-08-30 | 2004-09-02 | Process for inhibiting platelet aggregation |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US20040223962A1 (en) * | 2003-05-07 | 2004-11-11 | Riordan Neil H. | Method and composition for preventing or reducing edema, deep vein thrombosis and/or pulmonary embolism |
EP1657304A1 (en) * | 2004-11-16 | 2006-05-17 | Daiwa Pharmaceutical Co., Ltd | Blood-viscosity reducing agent |
US20060141095A1 (en) * | 2004-12-28 | 2006-06-29 | Japan Bio Science Laboratory Co., Ltd. | Method for producing foods from culture of Bacillus natto |
US20070116699A1 (en) * | 2005-06-24 | 2007-05-24 | N-Zymeceuticals, Inc. | Nattokinase for reducing whole blood viscosity |
US20090136648A1 (en) * | 2005-10-17 | 2009-05-28 | Hiroyoshi Moriyama | Platelet aggregation inhibitor and health food effective in inhibiting platelet aggregation |
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JP2006151950A (en) * | 2004-11-05 | 2006-06-15 | Oto Corporation:Kk | Bacillus natto culture extract-containing preparation and method for preserving bacillus natto culture extract |
CN1857722A (en) * | 2005-04-30 | 2006-11-08 | 成都地奥九泓制药厂 | Use of cereour bacillus in preparing thrombus treating medicine |
US20080044398A1 (en) * | 2006-07-13 | 2008-02-21 | Vesta Ingredients, Inc. | Soluble composition having enzymatic activity and improved stability |
US20080073351A1 (en) * | 2006-08-25 | 2008-03-27 | Jrw Properties, Inc. | Container for housing documents outdoors |
KR100986880B1 (en) | 2008-06-11 | 2010-10-08 | 조아제약주식회사 | Composition for improving male regenerative function |
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Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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DK167878B1 (en) * | 1985-04-19 | 1993-12-27 | Sankyo Co | PIGEOLIC ACID DERIVATIVES |
US5139871A (en) * | 1988-01-13 | 1992-08-18 | The United States Of America As Represented By The Secretary Of The Navy | Thermochemically treated oligomeric and/or polymeric derived silicon carbide fibers |
US5977160A (en) * | 1992-04-10 | 1999-11-02 | Brigham And Women's Hospital, Inc. | Methods for reducing risk of repeat myocardial infarction and increasing survival in heart attack victims |
JP3299100B2 (en) * | 1995-12-05 | 2002-07-08 | 株式会社資生堂 | Phenylenediamine derivative and radical scavenger, cerebral infarction inhibitor, cerebral edema inhibitor |
AU4753697A (en) * | 1996-10-01 | 1998-04-24 | South Alabama Medical Science Foundation | Method for diminishing myocardial infarction using protein phosphatase inhibitors |
JP3881494B2 (en) * | 2000-04-21 | 2007-02-14 | 株式会社日本生物科学研究所 | Natto bacteria culture extract |
US20040223962A1 (en) * | 2003-05-07 | 2004-11-11 | Riordan Neil H. | Method and composition for preventing or reducing edema, deep vein thrombosis and/or pulmonary embolism |
-
2003
- 2003-01-29 US US10/353,300 patent/US20040043014A1/en not_active Abandoned
- 2003-04-28 CA CA2427080A patent/CA2427080C/en not_active Expired - Lifetime
- 2003-06-19 US US10/465,193 patent/US20040043015A1/en not_active Abandoned
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2004
- 2004-09-02 US US10/932,806 patent/US7014851B2/en not_active Expired - Lifetime
Cited By (12)
Publication number | Priority date | Publication date | Assignee | Title |
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US20040223962A1 (en) * | 2003-05-07 | 2004-11-11 | Riordan Neil H. | Method and composition for preventing or reducing edema, deep vein thrombosis and/or pulmonary embolism |
US20060222641A1 (en) * | 2003-05-07 | 2006-10-05 | Riordan Neil H | Method and composition for preventing or reducing edema, deep vein thrombosis and/or pulmonary embolism |
EP1657304A1 (en) * | 2004-11-16 | 2006-05-17 | Daiwa Pharmaceutical Co., Ltd | Blood-viscosity reducing agent |
KR100709300B1 (en) * | 2004-11-16 | 2007-04-20 | 다이와 야꾸힝 가부시키가이샤 | Blood-Viscosity Reducing Agent |
US20090202982A1 (en) * | 2004-11-16 | 2009-08-13 | Masahito Hitosugi | Blood-viscosity reducing agent |
US7972835B2 (en) | 2004-11-16 | 2011-07-05 | Daiwa Pharmaceutical Co., Ltd. | Blood-viscosity reducing agent |
US20060141095A1 (en) * | 2004-12-28 | 2006-06-29 | Japan Bio Science Laboratory Co., Ltd. | Method for producing foods from culture of Bacillus natto |
US20070254347A1 (en) * | 2004-12-28 | 2007-11-01 | Japan Bio Science Laboratory Co, Ltd. | Method for producing vitamin K2 from culture of Bacillus natto |
US8114642B2 (en) | 2004-12-28 | 2012-02-14 | Japan Bio Science Laboratory Co., Ltd. | Method for producing vitamin K2 from culture of Bacillus natto |
US8603552B2 (en) | 2004-12-28 | 2013-12-10 | Japan Bio Science Laboratory Co., Ltd. | Method for producing foods from culture of Bacillus natto |
US20070116699A1 (en) * | 2005-06-24 | 2007-05-24 | N-Zymeceuticals, Inc. | Nattokinase for reducing whole blood viscosity |
US20090136648A1 (en) * | 2005-10-17 | 2009-05-28 | Hiroyoshi Moriyama | Platelet aggregation inhibitor and health food effective in inhibiting platelet aggregation |
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US20050025759A1 (en) | 2005-02-03 |
US20040043015A1 (en) | 2004-03-04 |
CA2427080A1 (en) | 2004-02-29 |
CA2427080C (en) | 2012-02-21 |
US7014851B2 (en) | 2006-03-21 |
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