JP5651834B2 - Hemagglutination inhibitor and method for inhibiting erythrocyte aggregation - Google Patents

Description

  The present invention relates to a hemagglutination inhibitor and a method for inhibiting erythrocyte aggregation.

  Red blood cells are known to aggregate in the blood of patients with various diseases (eg, allergic diseases, diabetes, collagen disease, cancer, etc.).

  Red blood cells have the function of transporting oxygen to each cell via hemoglobin, so if the red blood cells agglutinate, the red blood cells cannot flow through the capillaries and the internal tissues become oxygen deficient. Or immunity is expected to decline. Therefore, it is effective to inhibit red blood cell aggregation to prevent new diseases caused by red blood cell aggregation (for example, necrosis due to lack of oxygen in tissues). An alkyl derivative of 2-phenylbenzimidazole has been developed (see Patent Document 1). However, since the inhibitor is a chemically synthesized product and may require attention for its use, an erythrocyte inhibitor that is highly safe for the human body has been demanded.

  By the way, the Taceae eye has long been used as a raw material for dyes, and its use as a pharmaceutical is also known. For example, eye seeds are said to have antipyretic effects. In addition, the juice obtained from the raw leaves of the eye is used by being applied to stings such as poisonous insects. However, there are various reports on the active ingredient of eye, and it has not been put into practical use as a pharmaceutical product.

  The present inventor has so far discovered and filed that the water-soluble component contained in the stem and root of the eye has a physiological action such as strong antiviral properties (see Patent Document 2). . Since such an antiviral agent is mainly composed of a component derived from an eye that is a natural product, it is expected to be highly safe for the human body.

German Patent Application Publication No. 383060 JP 2008-19227 A

  This invention is made | formed in view of the above situations, and this inventor raise | lifted the subject to provide a hemagglutination inhibitor safe to a human body using the component derived from a natural product.

  As a result of intensive studies to solve the above problems, the present inventor has found that the water-soluble component of the eye (component derived from natural products) has an action of inhibiting the aggregation of erythrocytes. It came. That is, the hemagglutination inhibitor of the present invention that has solved the above problems is characterized by containing a water-soluble component of eye. In addition, the eye as used in this specification means the eye (scientific name: Polygonum tinctorium Lour.) Of the genus Tadaceae.

  The hemagglutination inhibitor of the present invention uses a water-soluble component of eye and is derived from a natural product, so that it is possible to provide a highly safe inhibitor for the human body.

  The hemagglutination inhibitor of the present invention is preferably for administering 0.002 mg or more and less than 0.2 mg of the water-soluble component to 1 μl of blood.

  Moreover, it is a preferable embodiment that the water-soluble component is obtained from the stem of the eye. As for the eye, only a leaf part is usually used as a dye material, and a stem part and a root part are discarded. Therefore, by obtaining the hemagglutination inhibitor of the present invention from the stem of the eye, the discarded stem can be effectively used as the dye material. In addition, the water-soluble component obtained from the stem is expected to exhibit a stronger hemagglutination-inhibiting effect than that derived from the leaf of the eye used as a dye raw material.

  The present invention also includes a method for inhibiting erythrocyte aggregation, which comprises administering the hemagglutination inhibitor.

  The water-soluble component of eye according to the present invention can inhibit erythrocyte aggregation. For this reason, it is expected to contribute to the treatment of various diseases caused by erythrocyte aggregation.

  The hemagglutination inhibitor of the present invention is characterized by containing a water-soluble component of eye. Hereinafter, the hemagglutination inhibitor of the present invention will be described in detail.

(Eye)
The eye used in the present invention may be an eye belonging to the genus Capaceae, and may be naturally grown or artificially cultivated. Further, the state of the eye is not particularly limited, and examples thereof include those containing moisture immediately after cutting, frozen ones, dried ones, and fermented dried ones. These may be used alone or in combination of two or more. In the present invention, it is preferable to use a dried eye from the viewpoint of easy storage and transportation.

  In the present invention, any part of the eye root, stem, and leaf may be used, but the stem is preferably used. The present inventor has found that the water-soluble component obtained from the stem of the eye among the water-soluble components of the eye exhibits higher physiological activity than the water-soluble component obtained from the root and leaf portions. In addition, by using the stem part, waste (such as the stem part) after removing the eye leaf used as a dye raw material can be used effectively, so the hemagglutination inhibitor of the present invention is supplied at a low cost. Is possible.

  In the present invention, the eye may be crushed, crushed or the like. Thereby, a water-soluble component can be efficiently extracted from the eye.

(Water-soluble ingredients)
The water-soluble component of the present invention is an extract extracted from the eye with an aqueous solvent. Although the specific details of this water-soluble component are currently being identified and are unknown, the present inventor has confirmed that various components are contained by analysis by liquid chromatography.

  The method for extracting the water-soluble component from the eye is not particularly limited. For example, after immersing the eye in an aqueous solvent, if necessary, the eye is subjected to at least one treatment selected from stirring treatment, heat treatment, pressure treatment, ultrasonic treatment, etc. The method of elution in a solvent is mentioned. When performing the heat treatment in the present invention, it is preferable to heat the solution so that the liquid temperature is 60 ° C. or higher (more preferably 70 ° C. or higher, more preferably 80 ° C. or higher). By setting the liquid temperature to 60 ° C. or higher, the water-soluble component contained in the eye can be efficiently eluted into the solvent. The upper limit of the liquid temperature is not particularly limited, and the liquid temperature may be heated to the boiling point of the solvent. The heating time is preferably 5 minutes or longer (more preferably 15 minutes or longer, more preferably 25 minutes or longer), and 24 hours or shorter (more preferably 60 minutes or shorter, more preferably 50 minutes or shorter). Is preferred. By heating within the above time, the water-soluble component contained in the eye can be effectively extracted into the solvent.

  Examples of the aqueous solvent used for extraction of the water-soluble component include water such as tap water, pure water, and ion exchange water; a mixed solvent of alcohols such as methanol and ethanol and the above water; ketones such as acetone and the above water. And a mixed solvent. The mixing ratio of alcohols and ketones to water is preferably 50% by mass or less (more preferably 40% by mass or less, and still more preferably 30% by mass or less). These solvents may be used alone or in combination of two or more. Since the hemagglutination inhibitor of the present invention is used by being administered to the human body, from the viewpoint of safety, it is preferable to use water or a mixed solvent of ethanol and water as the solvent, and more preferably only water. Further, the pH of the aqueous solvent may be adjusted appropriately. Usually, the pH is preferably in the range of 2 to 13, and more preferably in the range of 5 to 8.

  The amount of the solvent used for the extraction of the water-soluble component is not particularly limited, and considering the effect and efficiency, the amount of the solvent is 10 g or more (more preferably 20 g or more, more preferably 40 g or more) with respect to 1 g of the dried eye. Preferably, it is 1000 g or less (more preferably 100 g or less, more preferably 40 g or less).

  The water-soluble component of the present invention separates solids (such as eye residues after extraction of the water-soluble component) from the water-soluble component-containing solution obtained through the above extraction operation by filtration, centrifugation, decantation, or the like. It is preferable to obtain by this.

  In the present invention, an antioxidant, an antibacterial agent, a pH adjuster, or the like may be added in order to suppress the occurrence of discoloration, a strange odor, or the growth of microorganisms during the preparation of the water-soluble component. The addition timing and addition amount of these components are not particularly limited as long as the effects of the present invention are not hindered.

  In this invention, you may use the water-soluble component (water-soluble component containing solution) obtained by the said operation as a hemagglutination inhibitor as it is. Moreover, in order to improve safety | security or to raise active ingredient content, you may perform a refinement | purification process to a water-soluble component containing solution. Examples of the purification treatment include dialysis and liquid chromatography.

  Furthermore, in the present invention, the water-soluble component-containing solution may be subjected to a treatment such as concentration and drying, or the water-soluble component obtained by such concentration or the like may be powdered, granulated, or pasty. Thereby, handling of a water-soluble component becomes easy. Examples of the drying and concentration method include vacuum drying, freeze drying, spray drying, and ultrafiltration concentration. These methods may be used alone or in combination of two or more.

(Hemagglutination inhibitor)
As described above, the hemagglutination inhibitor of the present invention has a water-soluble component of eye as a main component and is derived from a natural plant, and thus has high safety to the human body.

(Method of inhibiting red blood cell aggregation)
In the present invention, the hemagglutination inhibitor may be used as a food, medicine, quasi-drug, or the like, for example, to inhibit erythrocyte aggregation by oral administration or parenteral administration. Oral administration can be carried out by eating the above-mentioned water-soluble component-containing solution, diluted solutions thereof, dried products, concentrates (powder, granules, pastes, etc.) as they are or added to other foods. Parenteral administration can be performed by intravenously or subcutaneously administering the extract, or by applying an ointment or cream containing the water-soluble component to the affected area. Alternatively, the water-soluble component may be ingested by spraying the water-soluble component-containing solution or a diluted solution thereof in the air, or impregnating the mask and sucking the water-soluble component. In addition, the present inventors have confirmed that the hemagglutination inhibitor of the present invention is basically safe for the human body.

(Dose)
The hemagglutination inhibitor of the present invention is preferably administered in an amount of 0.002 mg or more (more preferably 0.005 mg or more) per 1 μl of blood, less than 0.2 mg (more preferably 0.15 mg or less, more preferably 0). .10 mg or less) is preferable. When the dose is less than 0.002 mg, the aggregation of red blood cells may not be sufficiently suppressed. In addition, when the dose is 0.2 mg or more, red blood cell deposition (aggregation-like reaction) may occur.

  Therefore, it is preferable to administer the hemagglutination inhibitor of the present invention to 160 mg / kg body weight or more (more preferably 400 mg / kg body weight or more) per day for humans whose blood volume accounts for about 8% of body weight. , Less than 16 g / kg body weight (more preferably 12 g / kg body weight or less, more preferably 8 g / kg body weight or less).

  Hereinafter, the present invention will be described in detail based on examples. However, the following examples are not intended to limit the present invention, and all modifications made without departing from the spirit of the preceding and following descriptions are included in the technical scope of the present invention. In the examples, “%” means mass%.

Example 1 (Extraction of water-soluble components)
20 g of a sample obtained by pulverizing a dried eye stalk of Tokushima Prefecture to about 1 cm or less is immersed in 1000 g of an aqueous solvent (purified water) in a flask equipped with a reflux tube, and the water temperature becomes 100 ° C. The state was maintained for 30 minutes. Thereafter, the sample (solid content) was filtered off using a stainless steel sieve, and the filtrate was sterilized with a sterilizing filter to obtain an aqueous solution containing 0.208% by mass of the water-soluble component of eye according to the present invention. . In addition, the water-soluble component density | concentration in aqueous solution was calculated | required by measuring the mass of the residue obtained by heating aqueous solution at 90 degreeC and evaporating an aqueous solvent.

Production Example 1 (Preparation of test substance)
The above aqueous solution was used as test substance a, and this test substance a was diluted 1/10 times with phosphate buffered saline (PBS) to prepare test substance b. A dilution series of each test substance was prepared from the test substance a or b by a 2-fold serial dilution method.

The composition of the phosphate buffered saline (PBS) is as follows.
8.0 g / L Sodium chloride 0.2 g / L Potassium chloride 2.87 g / L Disodium hydrogen phosphate dodecahydrate 0.2 g / L Potassium dihydrogen phosphate

Production Example 2 (0.5% blood preparation)
Chicken stored blood (manufactured by Nippon Biotest Laboratories, Inc., product number 0050420, prepared by mixing the storage solution (Alsever's solution) and chicken blood 1: 1) was defined as 50% blood. 10% of this 50% blood and 90 mL of PBS were mixed to prepare 5% blood. Subsequently, 10% of this 5% blood and 90 mL of PBS were mixed to prepare 0.5% blood.

Production Example 3 (Preparation of virus dilution)
Virus dilutions 1 and 2 were prepared by diluting Influenza virus A (IFVA) (strain name PR / 8/34) 25.6 times or 160 times with PBS.

Test Example 1 (Hemagglutination Inhibition Test)
Mix 50 μL of the test substance dilution at each concentration with 50 μL of virus dilution 1 or 2 and let it incubate at room temperature (23 ° C. ± 2 ° C.) for 1 hour, then add 100 μL of 0.5% blood. The mixture was allowed to stand at room temperature for an additional hour. After standing at a constant temperature, the presence or absence of erythrocyte aggregation was visually observed. The results are shown in Table 1.

  The presence or absence of aggregation was evaluated as “+” when erythrocyte aggregation was confirmed, “±” when incomplete erythrocyte aggregation was observed, and “−” when no erythrocyte aggregation was confirmed. In addition, the case where erythrocytes were deposited unevenly was evaluated as “*”.

Test example 2 (negative control test 1)
The presence or absence of erythrocyte aggregation was observed in the same manner as in Test Example 1 except that test substance a or b (dilution ratio 1/1) was used as the test substance and PBS was used instead of the virus dilution. The results are shown in Table 1.

Test Example 3 (Negative Control Test 2)
Existence of erythrocyte aggregation was observed in the same manner as in Test Example 1, except that PBS was used instead of the test substance dilution. The results are shown in Table 1.

  According to the results, it can be seen that the water-soluble component of eye according to the present invention has an effect of inhibiting hemagglutination. In addition, in order for the hemagglutination inhibitor of the present invention to fully perform its function in the presence of a 25.6-fold diluted virus, it is preferable to administer 0.0104 mg or more of a water-soluble component to 1 μl of blood. The Furthermore, in order for the hemagglutination inhibitor of the present invention to fully exert its function in the presence of a 160-fold diluted virus, it can be seen that 0.0026 mg or more of a water-soluble component is preferably administered to 1 μl of blood.

  In negative control test 1 using test substance a (dilution ratio 1/1), hemagglutination-like reaction was observed in which erythrocytes were deposited unevenly. Therefore, administration of a water-soluble component to 1 μl of blood was less than 0.208 mg. It has been found that it is preferable to

  Further, even in Test Example 1 using the test substance a, a hemagglutination-like reaction in which erythrocytes are deposited unevenly (that is, an agglutination-like reaction was exhibited regardless of the presence or absence of virus). These hemagglutination inhibitors are presumed to have some effect on erythrocytes.

Claims (2)

Hemagglutination inhibition agent characterized der Rukoto intended to contain a water-soluble component obtained from the stem portion of the eye, the water-soluble components to 1μl of blood administered less than 0.002 mg 0.2 mg. The hemagglutination inhibitor according to claim 1, which is administered at a dose of 160 mg / kg body weight or more and 16 g / kg body weight or less per day.