JP6059578B2 - Hemostatic promoter containing fucoidan - Google Patents

Description

  The present invention relates to a hemostasis promoter and the like. Specifically, the present invention relates to a hemostasis promoter, a platelet aggregation promoter and the like containing fucoidan as an active ingredient.

  In humans and animals, when bleeding occurs in daily life, such as trauma or surgery, or in bleeding disorders such as hemophilia patients, the risk of blood loss increases when hemostasis is not successful. Therefore, in order to prevent bleeding, physical methods such as compression method and tourniquet method, hemostatic agents such as tranexamic acid, ε-aminocaproic acid, thrombin, factor VIII, factor IX or recombinant factor VII, etc. Various measures have been taken, such as administration of blood coagulation factor preparation (see Non-Patent Document 1, etc.), infusion of concentrated platelets, covering wounds with adhesive polymers, and using surgical procedures such as using a laser knife.

However, these drugs are administered by doctors or veterinarians in principle. Blood clotting factor preparations such as factor VIII generate antibodies (inhibitors) by repeated infusions and lose their effects. Moreover, blood coagulation factor preparations are expensive. Moreover, physical methods such as tourniquets and surgical treatment methods are merely symptomatic treatments. That is, it is no exaggeration to say that there is no radical treatment that can be routinely and easily performed by a human or animal owner in preparation for the need for hemostasis.

Hidehiko Saito: History and prospects of anticoagulants, thrombosis and hemostasis 19: 284-291, 2008

  The solution of the present invention was to find a radical treatment method for promoting hemostasis that is safe, simple, inexpensive, and free from the occurrence of inhibitors, and that can be routinely performed by humans or animal owners. .

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that fucoidan, particularly fucoidan derived from Okinawa Mozuku, has a hemostatic promoting action, and that the hemostatic action is caused by platelet aggregation ability, an inhibitor against fucoidan. The inventors have found that there is no occurrence of the problem and have completed the present invention.

That is, the present invention provides the following:
(1) A hemostasis promoter comprising fucoidan as an active ingredient.
(2) The hemostasis promoter according to (1), wherein fucoidan is derived from mozuku.
(3) The hemostasis promoter according to (1) or (2), which is for dogs.
(4) The hemostasis promoter according to any one of (1) to (3), wherein the molecular weight of fucoidan is 300,000 or more.
(5) A platelet aggregation promoter comprising fucoidan as an active ingredient.
(6) The platelet aggregation promoter according to (5), wherein the fucoidan is derived from mozuku.
(7) The platelet aggregation promoter according to (5) or (6), which is for dogs.
(8) The platelet aggregation promoter according to any one of (5) to (7), wherein the molecular weight of fucoidan is 110,000 or more.
(9) Mammal feed containing fucoidan.
(10) The feed according to (7), wherein the fucoidan is derived from mozuku.
(11) The feed according to (9) or (10), which is for dogs.
(12) The feed according to any one of (9) to (11), wherein the molecular weight of fucoidan is 110,000 or more.
(13) A method for producing a food or drink or a mammal feed having a hemostasis promoting action and / or a platelet aggregation promoting action, comprising adding fucoidan.
(14) The method according to (13), wherein the fucoidan is derived from Mozuku.
(15) The method according to (13) or (14), wherein the molecular weight of fucoidan is 110,000 or more.
(16) A method for promoting hemostasis of a mammal, comprising administering fucoidan to a mammal (excluding a human).
(17) The method according to (16), wherein the fucoidan is derived from Mozuku.
(18) The method according to (16) or (17), wherein the mammal is a dog.
(19) A method for promoting platelet aggregation in mammals, comprising administering fucoidan to mammals (excluding humans).
(20) The method according to (19), wherein the fucoidan is derived from mozuku.
(21) The method according to (19) or (20), wherein the mammal is a dog.

According to the present invention, safety, convenience, cheaper, and there is no fear of occurrence of an inhibitor, yet human or animal owners are provided curative therapy for hemostatic capable of performing on a daily basis. Fucoidan, which is an active ingredient of the hemostasis promoter of the present invention, is safe because it is naturally derived, is contained in a lot of seaweeds such as mozuku, and is easy to extract, so it is inexpensive. Moreover, no inhibitor is generated by fucoidan. By taking the hemostasis promoter of the present invention in daily life, it is possible to prepare for unexpected bleeding and improve the patient's QOL.

The upper row shows the results of examining the hemostatic function of whole blood of dogs with hemophilia before administration of fucoidan by thromboelastography (TEG). (A) shows TEG of a sample obtained by adding genetically modified human tissue factor (rhTF) and calcium chloride to citrated whole blood. (B) shows TEG of a sample obtained by adding calcium chloride to citrated whole blood. The lower panel shows the results of examining the hemostatic function of whole blood of dogs suffering from hemophilia after administration for 30 days with fucoidan by TEG. (C) shows TEG of a sample obtained by adding genetically modified human tissue factor (rhTF) and calcium chloride to whole blood citrate of a dog with hemophilia. (D) shows TEG of a sample obtained by adding calcium chloride to whole blood of citrate from a dog with hemophilia. In the figure, the vertical axis indicates clot firmness (clot firmness, unit: mm), and the horizontal axis indicates reaction time (min). In the figure, EXTEM is a hemostasis response test item that starts with tissue factor and calcium chloride, NATEM is a hemostasis response test item that starts only with calcium chloride, and CT (clotting time) is from measurement until fibrin formation starts (with an amplitude of 2 mm) CFT (coagulation formation time) is the time until the amplitude (vertical) reaches 20 mm from 2 mm, and α (α angle) is the tangent of the crotting curve through the point where the amplitude is 2 mm, MCF ( (Maximum coagulation stiffness) is the maximum amplitude, A10 is the clot stiffness (amplitude) 10 minutes after the start of measurement, and A15 is the clot stiffness (amplitude) 15 minutes after the start of measurement. (A) shows the addition of fucoidan to a whole blood citrate of a dog suffering from hemophilia before administration of fucoidan so that the final concentration is 5 μg / ml, and after allowing to stand for 10 minutes, a sample TEG added with calcium chloride is added. (B) shows a sample in which fucoidan was added to whole blood citrate of a dog suffering from hemophilia before administration of fucoidan so that the final concentration was 10 μg / ml and allowed to stand for 10 minutes, and then calcium chloride was added. (C) shows TEG, fucoidan was added to whole blood citrate of dogs suffering from hemophilia after 30 days administration of fucoidan to a final concentration of 5 μg / ml and allowed to stand for 10 minutes, and then calcium chloride was added. The TEG of the added sample is shown, and (d) shows that after adding fucoidan at a final concentration of 10 μg / ml to whole blood citrate of dogs with hemophilia after administration for 30 days after fucoidan, the mixture was allowed to stand for 10 minutes. ,chloride Shows a TEG of the sample was added calcium. The vertical and horizontal axes in the figure and the abbreviations in the figure are the same as those in FIG. (E) shows a sample in which fucoidan was added to whole blood citrate of dogs with hemophilia after administration for 30 days after fucoidan was administered to a final concentration of 5 μg / ml, allowed to stand for 10 minutes, and then calcium chloride was added. TEG is shown. (F) shows a sample in which fucoidan was added to whole blood citrate of a dog suffering from hemophilia after administration for 30 days after fucoidan was added to a final concentration of 10 μg / ml, allowed to stand for 10 minutes, and then calcium chloride was added. TEG is shown. (G) shows a sample in which fucoidan was added to whole blood citrate of dogs with hemophilia after administration for 30 days after fucoidan was administered to a final concentration of 15 μg / ml, allowed to stand for 10 minutes, and then calcium chloride was added. TEG is shown. (H) shows TEG of a sample to which fucoidan was added to citric acid plasma of a dog with hemophilia after administration for 10 days so that the final concentration was 15 μg / ml, and allowed to stand for 10 minutes, and then calcium chloride was added. Indicates. The vertical and horizontal axes in the figure and the abbreviations in the figure are the same as those in FIG. FIG. 3 is a diagram showing the results of examining the influence of the molecular weight of fucoidan on the hemostatic function. Fucoidan of each molecular weight was added to whole blood citrate from dogs with hemophilia so that the final concentration was 15 μg / ml. Fucoidan molecular weights are 300,000, 110,000 and 0.540,000 from the top. The hemostatic reaction test item is NATEM. FIG. 4 is a diagram showing the result of confirming that platelet aggregation is caused in a concentration-dependent manner by adding fucoidan to thrombin-inhibited whole blood of a healthy dog using an impedance method platelet aggregometer. Final concentrations of fucoidan in thrombin-inhibited whole blood collected from healthy dogs are 100 μg / ml (a), 50 μg / ml (b), 10 μg / ml (c), 5 μg / ml (d), 1 μg / ml (e). The platelet aggregation ability of the sample added as such is shown. (F) shows the platelet aggregation ability of a sample obtained by adding ADP (adenosine diphosphate, final concentration 10 μM) to thrombin-inhibited whole blood collected from the same healthy dog (positive control). (G) shows the platelet aggregation ability of a sample obtained by adding collagen (final concentration 5 μg / ml) to thrombin-inhibited whole blood collected from the same healthy dog (positive control). (H) shows the platelet aggregation ability of a sample obtained by adding physiological saline (20 μl) to thrombin-inhibited whole blood collected from the same healthy dog (negative control). The horizontal axis of the figure represents time (min). Measurement was performed for 12 minutes (one scale is 80 seconds). The vertical axis in the figure represents arbitrary aggregation units (AU) indicating the degree of platelet aggregation by this apparatus. The two curves in the figure show the results of duplicate measurements with one measurement cuvette. FIG. 5 is a diagram showing the results of examining the influence of fucoidan administration concentration on platelet aggregation ability using an impedance method platelet aggregometer. Fucoidan was administered for 90 days to dogs with hemophilia. The upper row shows platelets of a sample obtained by adding fucoidan to thrombin-inhibited whole blood collected from a dog suffering from hemophilia so that the final concentration is 100 μg / ml (a), 50 μg / ml (b), or 10 μg / ml (c). Shows aggregating ability. The middle row shows the platelet aggregation ability of a sample in which fucoidan was added to thrombin-inhibited whole blood collected from hemophilia-affected dogs to a final concentration of 5 μg / ml (d) and 1 μg / ml (e). Indicates. The lower row shows thrombin-inhibited whole blood collected from the same hemophilia dog with hemophilia, ADP (adenosine diphosphate, final concentration 10 μM) (f-positive control), collagen (final concentration 5 μg / ml) ( (g-positive control) and platelet aggregation ability of a sample added with physiological saline (20 μl) (h-negative control). The horizontal axis, vertical axis, and two curves in the figure are the same as those in FIG. FIG. 6 is a graph showing the results of examining the influence of fucoidan molecular weight on platelet aggregation ability using an impedance method platelet aggregometer. Fucoidan of each molecular weight was added to thrombin-inhibited whole blood of healthy dogs to a final concentration of 50 μg / ml. (A) has a molecular weight of 300,000, (b) has a molecular weight of 170,000, (c) has a molecular weight of 110,000, (d) has a molecular weight of 40,000, and (e) has a molecular weight of 0.540,000. The measurement conditions and the vertical and horizontal axes in the figure are the same as in FIG. FIG. 7 is a graph showing the results of examining whether or not platelet aggregation is enhanced after 14 and 30 days after administration of fucoidan having a molecular weight of 300,000 to a healthy dog at 40 mg / kg body weight / day. It is. In the upper, middle and lower stages, the left figure shows the results before fucoidan administration, the middle figure shows the results 14 days after the start of fucoidan administration, and the right figure shows the results 30 days after the start of fucoidan administration. Collagen was added to a final concentration of 5 μg / ml (upper), 2.5 μg / ml (middle), and 1.25 μg / ml (lower). The measurement conditions and the vertical and horizontal axes in the figure are the same as in FIG. FIG. 8 is a graph showing the results of examining the effect of fucoidan on enhancing the platelet aggregation ability by adding ADP in the same test system as in FIG. In the upper, middle and lower stages, the left figure shows the results before fucoidan administration, the middle figure shows the results 14 days after the start of fucoidan administration, and the right figure shows the results 30 days after the start of fucoidan administration. In the upper row, ADP was added to a final concentration of 10 μM, in the middle row, ADP was added to a final concentration of 5 μM, and in the lower row, ADP was added to a final concentration of 2.5 μM. The measurement conditions and the vertical and horizontal axes in the figure are the same as in FIG.

  In the first aspect, the present invention provides a hemostasis promoter containing fucoidan as an active ingredient. In addition, “hemostasis” means a reaction that occurs physiologically when bleeding occurs medically, and preventing a bleeding event that may occur in the future refers to “preventing bleeding by enhancing hemostasis” However, as used herein, “hemostasis” includes not only suppressing bleeding that actually occurs from the subject, but also preventing or suppressing bleeding events that may occur in the subject in the future.

  Fucoidan used as an active ingredient of the hemostatic promoter of the present invention is a sulfur-containing polysaccharide found in nature and is abundant in seaweeds such as mozuku and seaweed. In recent years, it has been discovered that fucoidan is useful for promoting tissue regeneration, adjusting immune balance, suicide of cancer cells, suppressing hypertension and the like. However, it has not been demonstrated that fucoidan has an excellent hemostasis promoting action and platelet aggregation promoting action. The present invention is based on the finding that fucoidan has an excellent hemostasis promoting action and platelet aggregation promoting action.

  The fucoidan, which is an active ingredient of the hemostasis promoter of the present invention, may be a purified product or a crude product, for example, an extract from seaweed such as mozuku. Fucoidan is a natural polysaccharide that is abundant in seaweeds, especially brown algae. Examples of brown algae containing fucoidan include mozuku (Okinawa mozuku, itomozuk, ftmozuku, etc.), wakame, arame, gagome, macomb, kurome, kajime, mitsuishimikobu, yoremoku, hijiki, hondawala, yamatamamok, akamoku, hibamata However, it is not limited to these. A fucoidan that is preferable as an active ingredient of the hemostasis promoter of the present invention is fucoidan derived from Okinawa mozuku.

  In the present invention, an extract or purified product of fucoidan can be used. Moreover, the processed material of an extract, for example, a concentrate, a paste, a dried product, an emulsion, a liquid product, a diluted product, etc., can be used.

  Methods for extracting and purifying fucoidan from seaweed are known. Typically, purified fucoidan can be obtained by hot water extraction of seaweed and subjecting the extract to processes such as centrifugation, filtration, and desalting. The obtained purified fucoidan may be further subjected to steps such as alcohol precipitation, freeze drying, and spray drying.

Fucoidan, which is an active ingredient of the hemostasis promoter of the present invention, is more preferable as it is a polymer. The molecular weight of fucoidan is preferably about 120,000 or more, for example, about 150,000 or more, about 170,000 or more, and more preferably about 200,000 or more, for example, about 250,000 or more, about 300,000 or more. In the present specification, the molecular weight of fucoidan is a weight average molecular weight, and its unit is dalton. In general, the molecular weight of fucoidan is at most several hundred thousand to several million.

  The administration target of the hemostasis promoter of the present invention is a mammal, preferably a human, a dog, a cat, a monkey, a horse, a cow, a sheep, a goat and the like. Particularly preferred administration subjects of the hemostasis promoter of the present invention are humans, dogs and cats. The administration route may be any route, but oral administration is preferred. In the case of oral administration, it is convenient because the subject can ingest it by a method such as mixing fucoidan in food, drinking water or food.

  The dosage form of the hemostatic promoter of the present invention may be any dosage form. For example, for oral administration, it can be formulated into various oral dosage forms such as concentrates, powders, granules, tablets, capsules, drinks and the like. Methods for producing these dosage forms are known, and processes such as mixing, dissolution, pulverization, tableting, and drying can be used as appropriate, and carriers and excipients according to the purpose can be used. A fragrance, a sweetener, a colorant and the like may be appropriately added to the oral administration agent of the present invention. Although the oral administration agent of the present invention can be administered as it is, the fucoidan which is an active ingredient is tasteless and odorless so that it can be easily taken orally, for example, miso soup, tea, other foods or beverages, or Oral administration agents of the present invention can optionally be added to animal feed. Thus, since the hemostasis promoter of the present invention is easy to be taken by the subject, the burden on the administration subject is extremely small.

The hemostasis promoter of the present invention can be used prophylactically and therapeutically. For example, the hemostasis promoter of the present invention may be continuously administered in preparation for bleeding due to unexpected trauma or the like. The hemostasis promoter of the present invention may be continuously administered to subjects who are constitutionally bleeding. By such administration, the QOL of humans and animals can be improved. Alternatively, the hemostasis promoter of the present invention may be administered in preparation for bleeding events such as trauma or surgery.

When the hemostasis promoter of the present invention is orally administered, the dose of fucoidan as an active ingredient may generally be about 10 mg to about 10,000 mg per day for an adult. The dose can be appropriately increased or decreased in consideration of age, weight, other treatments the subject is receiving, and the like. Oral doses to animals other than humans can also be determined by a veterinarian according to the above oral doses to adults. The frequency of administration of the hemostasis promoter of the present invention may be once to several times a day, or may be once to several times a few days.

  The hemostatic promoter of the present invention may be used in combination with other hemostatic agents, blood coagulants and the like. In addition, other components related to blood coagulation and hemostasis such as blood coagulation factor IX may be contained in the hemostasis promoter of the present invention.

  The present inventors have elucidated for the first time that the action of the hemostasis promoter of the present invention is expressed by promoting platelet aggregation. Therefore, in the second aspect, the present invention provides a platelet aggregation promoter containing fucoidan as an active ingredient. Regarding the platelet aggregation promoter of the present invention, the description of fucoidan as an active ingredient, the administration route, the dose and other explanations are the same as the description of the hemostasis promoter of the present invention.

  Fucoidan, which is an active ingredient of the platelet aggregation promoter of the present invention, is preferably a polymer. The molecular weight of fucoidan is preferably about 20,000 or more, such as about 40,000 or more, about 70,000 or more, about 110,000 or more, more preferably about 170,000 or more, such as about 200,000 or more, about 250,000 or more, About 300,000 or more.

In a further aspect, the present invention provides the following.
A method for promoting hemostasis of a mammal, comprising administering fucoidan to a mammal in need of hemostasis promotion,
A method of promoting platelet aggregation in mammals, comprising administering fucoidan to a mammal in need of platelet aggregation promotion;
The use of fucoidan for the production of hemostatic agents in mammals,
The use of fucoidan for the production of platelet aggregation promoters in mammals,
-A feed for mammals containing fucoidan and having a hemostasis-promoting action or platelet aggregation-promoting action, and a food and drink for humans, and a food and drink having a hemostasis-promoting action or a platelet aggregation-promoting action characterized by adding fucoidan Production method.
Preferred mammals in these inventions include dogs, cats, monkeys, horses, cows, sheep, goats and the like, with dogs and cats being particularly preferred. Humans may or may not be included as subjects in these methods and uses. A preferred fucoidan for use in these methods and uses is fucoidan from Okinawa mozuku. Regarding the fucoidan in these methods and uses, the administration route, the dosage and the like, the above explanation concerning the hemostasis promoter of the present invention can be referred to.

  As described above, fucoidan is tasteless and odorless and can be easily taken orally. Therefore, foods and animal feeds containing fucoidan can be accepted by the subject without resistance.

  Fucoidan may be added to the raw material, may be added in the production process, or may be added to the finished product.

  The present invention will be described in more detail and specifically with reference to the following examples. However, the examples should not be construed as limiting the present invention.

Example 1 Effect of Fucoidan on Whole Blood Hemostasis Function in Dogs with Hemophilia In this experiment and the experiment described below, a Rotation thromboelastometry, ROTEM (registered trademark) system, Pentapharm GmbH, Germany was used. The measurement sample was citrate whole blood obtained by mixing 3.8% citrate solution and whole blood 1: 9, and then recombinant human tissue factor (rhTF prothrombin time measurement kit Dadeinobin Sysmex Co., Ltd.) Alternatively, calcium chloride (0.2 M CaCl ₂ star-TEM Fingallink Co., Ltd.) was added and the hemostatic function was recorded with TEG for 60 minutes.

As the fucoidan used in the experiment, a high-molecular mozuku fucoidan manufactured by Kimuraya Co., Ltd., a marine product, was prepared as follows.
Mozuku-derived fucoidan is treated with mozuku (Okinawa mozuku) for 30 minutes to 3 hours using hot water or acidic hot water of 60 ° C. or higher, preferably 80 ° C. or higher, and the resulting extract is electrodialyzed. Can be obtained by desalting. Thereafter, it may be purified by quaternary ammonium salt treatment, alcohol precipitation, ion exchange resin chromatography, gel filtration chromatography, ultrafiltration, ultrafine filtration or the like. A mozuku-derived fucoidan-containing liquid was obtained as described above.
The molecular weight of the obtained mozuku-derived fucoidan was analyzed by gel filtration column chromatography (GPC) by HPLC. The analysis conditions are as follows. Column: Shodex Asahipak GS520 + GS320 + GS220, eluent: 0.1 M NaNO3, flow rate: 1.0 ml / min, detection: RID, column temperature: 40 ° C., calibration curve standard: pullulan standard. The molecular weight, based on pullulan molecular weight, was about 300,000, and the sulfate group content was 14%.

  Prior to administration of fucoidan, blood (2 ml) was collected from the forearm cephalic vein of dogs with hemophilia (dog type: toy poodle, age: 4 years, sex: male), and citric acid was added to the whole citric acid. It was blood. FIG. 1A shows a TEG of a sample obtained by adding genetically modified human tissue factor (rhTF) and calcium chloride to the obtained citrated whole blood. The TEG of the sample obtained by adding calcium chloride to the obtained citrated whole blood is shown in FIG. In the blood of dogs with hemophilia, compared to normal blood, the rhTF addition system has increased CT (coagulation time) and CFT (coagulation time) (654 seconds and 465 seconds, respectively) and MCF (coagulation). It was confirmed that the hemostasis function was lowered (55 mm). In the calcium addition system, CT was remarkably prolonged (2089 seconds), and the blood hardly clotted. In the TEG of normal citrated whole blood, CT 103 seconds, CFT 79 seconds, MCF 62 mm in the rhTF addition system, CT 238 seconds, CFT 82 seconds, MCF 63 mm in the calcium addition system.

  When the above-mentioned dog suffering from hemophilia was ingested fucoidan for 30 days (packed with 20 mg fucoidan per kg body weight and orally administered after meal), the same test as described above was conducted. In the rhTF addition system, CT and CFT were used. Was shortened (FIG. 1 (c)). In addition, CT and CFT were significantly shortened even in the calcium addition system, and MCF was enlarged (FIG. 1 (d)). See above. From these results, it was confirmed that the hemostatic function of dogs with hemophilia was significantly improved by the addition of fucoidan.

Table 1 summarizes PT (prothrombin time), activated partial thromboplastin time (APTT), and fibrinogen concentration before and after fucoidan administration for 30 days in the above-mentioned dogs with hemophilia.

In hemophilia, the activated partial thromboplastin time (APTT) was prolonged, but this value did not change after 30 days of administration of fucoidan. From this result, it became clear that the effect of improving hemostatic function by fucoidan cannot be evaluated by APTT which is a general blood coagulation test item. Therefore, it was considered that the hemostasis promoting effect by fucoidan is based on a mechanism called APTT, which cannot be evaluated by a detection system in which blood coagulation factors are activated in a chain in a test tube to precipitate fibrin.

  In the above experiment, no spontaneous bleeding and other adverse reactions were observed after 30 days of administration of fucoidan.

Example 2 In Vitro Fucoidan Addition Test-Examination of Concentration-Dependent Effect In the same manner as in Example 1, blood was collected before and after fucoidan was given for 30 days in the above-mentioned dogs with hemophilia, and citrated whole blood was prepared. Then, fucoidan was added to the obtained whole citrated blood so that the final concentration would be 5 μg / ml, 10 μg / ml or 15 μg / ml, and allowed to stand for 10 minutes, followed by addition of calcium chloride and hemostasis with TEG. The function was evaluated. The fucoidan obtained by the method described in Example 1 was used.

  When fucoidan was added to a citrate whole blood sample before administration of fucoidan, slight improvement in hemostatic function was observed (system with 10 μg / ml addition). However, it was found that when fucoidan (5 μg / ml or 10 μg / ml) was added to a citrated whole blood sample after administration of fucoidan for 30 days, the hemostatic function was improved according to the amount of fucoidan added. The hemostatic function was significantly improved when 10 μg / ml fucoidan was added. See FIGS. 2A to 2D.

  It was found that when fucoidan (5 μg / ml, 10 μg / ml or 15 μg / ml) was added to a citrated whole blood sample after administration of fucoidan for 30 days, the hemostatic function was improved according to the amount of fucoidan added. See FIGS. 2E to 2G. However, when fucoidan was added to citrate plasma after administration of fucoidan for 30 days to 15 μg / ml, no improvement in hemostatic function was observed. See FIG. 2 (h).

  From these results, it was concluded that the hemostasis function was improved in whole blood containing platelets depending on the concentration of fucoidan added, but there was no change in the hemostasis function in plasma without platelets. Therefore, it was considered that the hemostatic function was improved by fucoidan acting on platelets.

Example 3 Examination of molecular weight of fucoidan on hemostasis function in vitro 15 μg / ml of fucoidan having a molecular weight of 0.54 million, 110,000 or 300,000 in the whole blood citrate of dogs suffering from hemophilia NATEM (the hemostasis response test item started only with calcium chloride) when added so that

Fucoidan of each molecular weight was prepared as follows.
As the fucoidan having a molecular weight of 300,000, the high molecular weight mozuku fucoidan manufactured by Kimuraya Co., Ltd., a seafood product prepared in Example 1, was used. Fucoidans having molecular weights of 0.54 million and 110,000 were prepared by hydrothermal treatment described in JP-A-2008-266299. An aqueous fucoidan solution was prepared by dissolving 800 mg of fucoidan having a molecular weight of 300,000 in 80 ml of water (pH was unadjusted, pH 5.2), and the treatment temperatures were 120 ° C, 140 ° C, 160 ° C, and 180 ° C. Hold for 20 minutes. HTP-50 / 250 (manufactured by Nisaka Seisakusho) was used as the hydrothermal treatment apparatus.

  It was found that the higher the molecular weight of fucoidan, the higher the hemostatic function. See FIG.

Example 4 Effect of Fucoidan on Platelet Aggregation Capacity—Concentration Dependence In order to prove that hemostasis is promoted by the action of fucoidan on platelets, an impedance-based platelet aggregometer (Multiplate (registered trademark), Dynabyte) GmbH, Germany) was used to evaluate the effect of fucoidan on platelet aggregation ability. The fucoidan obtained by the method described in Example 1 was used.

  The platelet aggregation ability when 1 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml, and 100 μg / ml fucoidan (final concentrations) were added to tonlongine-inhibited whole blood collected from healthy dogs was examined. Fucoidan having a molecular weight of 300,000 obtained by the method described in Example 1 was used. As a result, fucoidan aggregated platelets in a healthy dog sample in a concentration-dependent manner, and showed a slight platelet aggregation ability even at a concentration of 5 μg / ml (FIG. 4 (d)). In the healthy dog sample, in the system to which fucoidan was added at 10 μg / ml or more (FIGS. 4A to 4C), the platelet aggregation ability was equal to or higher than that of ADP and collagen.

  Fucoidan was administered to hemophilia-affected dogs (breed: Chihuahua, age: 1 year, sex: male) for 90 days (filled with 20 mg fucoidan per kg body weight in capsules, administered twice a day, orally after meals) Thereafter, the platelet aggregation ability when 1 μg / ml, 5 μg / ml, 10 μg / ml, 50 μg / ml, and 100 μg / ml fucoidan were added to thrombin-inhibited whole blood was examined. Fucoidan having a molecular weight of 300,000 obtained by the method described in Example 1 was used. As a result, fucoidan aggregated platelets in a concentration-dependent manner in samples of dogs suffering from hemophilia, and sufficient platelet aggregation ability was shown even at a concentration of 5 μg / ml (FIG. 5 (d)). In a sample of hemophilia-affected dog, platelet aggregation ability was equal to or higher than that of ADP and collagen in the system to which fucoidan was added at 10 μg / ml or more (FIGS. 5A to 5C). In addition, it is thought that the platelet aggregation by fucoidan is stronger in the sample of the hemophilia affected dog of FIG. 5 than that of the healthy dog sample shown in FIG. 4 because fucoidan was administered for 90 days.

Example 5 Effect of Fucoidan on Platelet Aggregation Capacity-Examination of Molecular Weight of Fucoidan Fucoidan having a molecular weight of 0.54, 40,000, 110,000, 170,000 and 300,000 in thrombin-inhibited whole blood of healthy dogs at a final concentration of 50 μg / ml The platelet aggregation ability when added as such was examined. The experiment was performed using an impedance platelet aggregometer (described above). Fucoidan of each molecular weight was prepared in the same manner as described in Example 3.

  It was found that the higher fucoidan polymer aggregates platelets. Mild platelet aggregation ability was obtained with fucoidan having a molecular weight of 40,000 and 110,000. Sufficient platelet aggregation ability was obtained with fucoidan having a molecular weight of 170,000 or more. See FIG.

Example 6 Effect of Fucoidan on Promoting Platelet Function A mozuku-derived fucoidan having a molecular weight of 300,000 was administered to healthy dogs (40 mg / kg body weight / day), and it was evaluated whether platelet function was enhanced after 14 and 30 days. Thrombin-inhibited whole blood collected after administration, 14 days and 30 days after administration, was evaluated for the platelet aggregation ability when collagen was added to final concentrations of 5 μg / ml, 2.5 μg / ml, and 1.25 μg / ml. The results of investigation using a platelet aggregometer (described above) are shown in FIG. It was confirmed that the platelet aggregation ability was enhanced 14 days after the administration of fucoidan and the platelet aggregation ability was enhanced 30 days later.

  A similar experiment was performed by adding ADP to the final concentrations of 10 μM, 5 μM, and 2.5 μM instead of collagen. As a result, it was confirmed that the platelet aggregation ability was enhanced 14 days after administration of fucoidan and the platelet aggregation ability was enhanced even 30 days later. See FIG.

As shown in the above examples, when healthy dogs take fucoidan, increased platelet function is observed, so fucoidan promotes not only hemophilia but also hemostasis abnormalities different from hemophilia It was suggested to be useful for.

  The present invention can be used in fields such as pharmaceuticals, foods, feeds, and research reagents.

Claims (8)

An orally administered hemostasis promoter comprising fucoidan having a molecular weight of 300,000 or more derived from mozuku as an active ingredient.   The hemostasis promoter according to claim 1, which is for dogs. An orally administered platelet aggregation promoter comprising fucoidan having a molecular weight of 300,000 or more derived from mozuku as an active ingredient.   The platelet aggregation promoter according to claim 3, which is for dogs. A mammal feed composition for promoting hemostasis and / or promoting platelet aggregation, comprising fucoidan having a molecular weight of 300,000 or more derived from mozuku. The feed composition according to claim 5, which is for dogs. A method for producing a food and drink or a mammal feed for promoting hemostasis and / or promoting platelet aggregation , comprising adding fucoidan having a molecular weight of 300,000 or more derived from mozuku. A food composition for promoting hemostasis and / or promoting platelet aggregation, comprising fucoidan having a molecular weight of 300,000 or more derived from mozuku.