JP4606715B2 - Immune activity enhancer, and food and quasi drug containing the same - Google Patents

Description

    The present invention relates to an immune activity enhancer, and foods and quasi drugs containing the same.

  Senso is a herbal medicine that has been solidified from the venom gland secretions of the Chinese toad (Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider) and is a herbal medicine that has been used as a component of cardiotonic and anti-inflammatory agents since ancient times. Buphadienolides such as bufalin, cinobufagin, and resibufogenin are known as pharmacologically active components, and pharmacological actions such as cardiotonia, respiratory excitement, diuresis, antitumor action, and tumor cell apoptosis induction have been reported (Patent Document 1).

JP-A-11-279067

    However, it is difficult to explain all the physiological functions of Senso using only these components. Senso contains a relatively large amount of proteins and polysaccharides, but there has been no report on the physiological activity of these polymer components. In addition, although the skin of certain toads is known to contain peptides that exhibit antibacterial activity against bacteria, there are no reports that such substances are contained in the senso component. .

  Therefore, the present invention finds an immunopotentiator useful for the immune system, that is, a defense system against xenobiotics such as viruses, bacteria, and malignant tumor cells, and provides a food, quasi-drug or pharmaceutical containing the same. That is.

  The leading cause of death among Japanese is still malignant neoplasms or malignant tumors. Improving the immunity of a living body leads to preventing the growth of xenobiotics such as malignant tumor cells in the body. Various tumor cell growth inhibitors have been developed so far, and it is desired to develop drugs that are effective and have few side effects. In addition, from the viewpoint of self-medication, the development of foods and quasi-drugs that do not act directly on tumor cells but activate the immune function of the living body to prevent tumor growth is also desired. . Therefore, an object of the present invention is to provide a novel immune activity enhancer having an antitumor action, and a food and quasi drug containing the same.

  Furthermore, it is known that immunity decreases with stress and aging. In today's so-called stress society and aging society, the susceptibility to various foreign substances (pathogenic bacteria, viruses, etc.) due to a decline in immunity has not only reduced people's production capacity but also their quality of life (QOL). Invite. Therefore, the present invention aims to provide an immune activity enhancer capable of enhancing the immunity of a living body and indirectly suppressing a decrease in production capacity and QOL, and a food and quasi-drug containing the same. And

  In order to achieve the above object, the present inventors have conducted extensive research and found that a water-soluble extract of Senso is excellent in enhancing immune activity. That is, the present invention is an immune activity enhancer comprising a water-soluble extract of Senso.

  As described above, according to the present invention, by including a water-soluble extract of senso, an immune activity enhancer useful for the immune system, that is, a defense system from xenobiotics such as viruses, bacteria, and malignant tumor cells. , And foods and quasi drugs containing the same can be provided.

  The immune activity enhancer according to the present invention desirably has at least one of the lymphocyte blastogenesis promoting action, interleukin 2 and interleukin 12 production promoting action, and natural killer cell activity enhancing action.

  In the immune activity enhancer according to the present invention, the water-soluble extract of Senso is a protein, glycoprotein or polysaccharide having a molecular weight of 118,000 to 214,000, or a molecular weight of 6,800 to Desirably any of 20,800 proteins, glycoproteins and polysaccharides.

  The immunoreactivity enhancer according to the present invention has a use as a tissue cell agent such as an immunogenic preparation or an antineoplastic agent, and is orally administered in the form of a liquid, powder, fine granule, granule, capsule or tablet. Alternatively, it is administered parenterally (intracerebral, intramuscular, subcutaneous intravenous, intravenous infusion, etc.).

  The dose varies depending on the degree of symptoms, disease differences, patient age, weight, health status, type of concurrent treatment, the type of desired effect, frequency of treatment, etc., and is not particularly limited, but per adult day Orally, about 10 mg to 1000 mg in terms of the drug substance is administered once or more times a day.

  In the case of oral administration, liquids, powders, fine granules, granules, capsules, pills, tablets, and the like applied to them are commonly used excipients, binders, disintegrations in their compositions. You may contain additives, such as an agent, a lubricant, a coloring agent, and a flavoring agent. When used as an oral liquid preparation, it may be in the form of a suspension or syrup, or it may be in the form of a dried product that is redissolved before use. Further, such a liquid preparation may contain any commonly used additive and preservative.

  Examples of excipients include lactose, corn starch, sucrose, glucose, sorbit, mannitol, crystalline cellulose, starch, silicon dioxide, and inorganic salts. Examples of binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, methyl cellulose, and arabic. Gum, tragacanth, gelatin, shellac, hydroxypropyl cellulose, hydroxypropyl starch, polyvinylpyrrolidone, etc., disintegrating agents such as starch, agar, gelatin powder, crystalline cellulose, carboxymethylcellulose, carboxymethylcellulose calcium, carboxymethylcellulose sodium, calcium carbonate Sodium bicarbonate, calcium citrate, dextrin, pectin, sucrose fatty acid ester, lecithin, polysorbate 80, sodium lauryl sulfate, etc., as lubricants, for example, magnesium stearate, talc, polyethylene glycol, silica, hydrogenated vegetable oil, etc. As the flavoring agent, for example, cocoa powder, mint brain, aromatic acid, mint oil, dragon brain, cinnamon powder and the like are used. Of course, these tablets and granules may be appropriately coated with sugar coating, gelatin coating, etc. as required. Of course, these tablets, granules, and capsules may be used as sustained-release agents.

  For injection, the composition may contain additives such as stabilizers, buffers, preservatives, tonicity agents and the like. The composition may be in the form of a suspension, solution, or oil.

  Other parenteral agents include suppositories for rectal administration, and are produced according to a conventional method.

  In addition, the present invention is a food characterized by containing the above-mentioned immune activity enhancer, and includes, for example, soft drinks such as tea and juice, jelly, rice cake, chocolate, chewing gum, and nutritional supplements. The food according to the present invention includes health foods (functional foods, health supplements) and the like, and is manufactured in the form of liquids, powders, granules, capsules, and tablets.

  Furthermore, the present invention is a quasi-drug containing the above-described immunological activity enhancer, such as a throat refreshing agent or a healthy stomach refreshing agent. There are vitamin-containing health agents, etc., which are manufactured in the form of liquids, powders, fine granules, granules, capsules, pills, and tablets.

  Next, Examples 1 and 2 of the immune activity enhancer according to the present invention will be described, but the present invention is not limited to these Examples.

First, add 15 times the amount of methanol to Senso, shake for 10 minutes, centrifuge and remove the supernatant three times, then ventilate the precipitate with nitrogen gas to remove the methanol. Add 10 mM phosphate buffered saline (pH 7.4, hereinafter PBS) containing mM 2-mercaptoethanol and 0.1 mM phenylmethanesulfonyl fluoride, shake and mix for 11 hours, 15,000 rpm, 25 minutes, 4 ° C centrifugation After separation, the supernatant was collected. The supernatant was placed in a dialysis membrane, dialyzed against water for 3 days, and the dialysis contents were lyophilized to obtain a dry powder (fraction 1). Moreover, fraction 1 is taken, PBS containing 0.1 mM MnCl ₂ and 0.1 mM CaCl ₂ is added and dissolved, passed through concanavalin A-agarose to remove unbound components, and 0.2 M α-methyl mannose The binding component was eluted with PBS containing, and the eluate was placed in a dialysis membrane and dialyzed against water for 2 days. The dialyzed content was freeze-dried to obtain a dry powder (fraction 2). These dry powders according to fraction 1 are used as the immune activity enhancer according to Example 1, and the dry powder according to fraction 2 is used as the immune activity enhancer according to Example 2.

Experimental Example 1 Effects on mouse spleen lymphocyte blastogenesis
After aseptically removing the spleen from C3H / HeN mice, lightly on a frosted glass slide (ground glass) in a cell culture medium (RPMI 1640 medium containing 5% fetal bovine serum, 3% glutamine, and penicillin-streptomycin) Cells were removed by pressing. The cells were dispersed by pipetting and then centrifuged (500 × g, 5 minutes, 4 ° C.). After erythrocytes were removed by hemolysis, the precipitate was washed twice with a cell culture medium to obtain spleen lymphocytes. The obtained spleen lymphocytes were suspended in a cell culture medium (RPMI 1640 medium containing 5% fetal bovine serum, 3% glutamine, 100 U / ml penicillin, 0.1 mg / ml streptomycin mixed solution), and then an automatic hemocytometer ( MEK-4300, determining the number of cells using Nihon Kohden Corporation), and 2 × ¹⁰ 5/40 μl as diluted with preceding medium was splenic lymphocytes suspension. In a well of a flat bottom 96-well microplate, 0.045 ml of cell culture medium, 0.005 ml of 1 mM 2-mercaptoethanol, 0.04 ml of spleen lymphocyte suspension (2 × 10 ⁵ cells) and immunity according to Example 1 A test drug solution containing an activity enhancer or 0.01 ml of a test drug solution containing an immune activity enhancer according to Example 2 was added and cultured at 5% CO ₂ and 37 ° C. for 48 hours. Formazan produced by intracellular NADH-generating dehydrogenase as a result of addition of cell proliferation assay kit (Cell Titer 96AQueous, Promega) solution to each well and uptake of tetrazolium salt into living cells 4 hours before the end of culture The absorbance of the dye at 492 nm was measured with a microplate reader. The result is shown in FIG.

  As shown in FIG. 1, the immune activity enhancer according to Example 1 and the immune activity enhancer according to Example 2 promoted mouse spleen lymphocyte blastogenesis reaction in a concentration-dependent manner. In addition, since the action was enhanced by concancvalin A binding column purification, it was confirmed that most of the lymphocyte blastogenesis-promoting substances contained in senso bind to concanavalin A.

Experimental Example 2 Effects on lymphocyte culture supernatant cytokine concentration
Spleen lymphocytes were prepared by the method described in Experimental Example 1, and the immune activity enhancer according to Example 2 or concanavalin A as a comparison was added thereto, followed by culturing at 5% CO ₂ and 37 ° C. for 48 hours. The culture broth was collected, and the amount of cytokine (interleukin 2, interleukin 4, and interleukin 12) contained in the supernatant obtained by centrifugation (1,500 rpm, 5 minutes) was measured using an ELISA kit (BioSourse International). The measurement was performed in the same manner using a culture solution to which no immune activity enhancer or concanavalin A was added as Control. These results are shown in Table 1.

  As shown in Table 1, a significant increase in the interleukin 2 and interleukin 12 concentrations and a significant decrease in the interleukin 4 concentration in the culture medium were observed by the addition of the immune activity enhancer according to Example 2.

Experimental Example 3 Effect on mouse spleen cell natural killer activity 1 mg / kg of the immunoreactivity enhancer according to Example 2 or, as a comparative example, 10 ml / kg of sterilized physiological saline, once a day for C3H / HeN mice. It was administered intraperitoneally for days. On the fourth day after administration, the spleen was removed, and the lymphocyte fraction was purified by passing through a nylon column to obtain effector cells. Separately, mouse lymphoid-derived YAC-1 cells were subcultured at 10-fold FBS-containing RPMI 1640 times, and the culture medium was changed the day before and used as target cells. It was. That is, 0.2 ml of 2 × 10 ² / μl of target cells is added to 0.2 ml of 1 × 10 ⁴ / μl of effector cells (effector cells: target cells = 50: 1) to promote cell-cell contact. After centrifugation (1,500 rpm, 5 minutes, 20 ° C.), the cells were cultured at 37 ° C. and 5% CO _{2 for} 4 hours. After completion, the mixture is centrifuged (1,500 rpm, 5 minutes, 20 ° C.), 0.05 ml of the supernatant is taken into a well of a flat-bottomed microplate, and the activity (A) of lactate dehydrogenase leaking as a result of cell damage Measured with a kit (Cytotox 96, Promega). (B) is obtained by adding 0.2 ml of culture medium instead of 0.2 ml of YAC-1 cells, and (C) is obtained by adding 0.2 ml of culture medium instead of 0.2 ml of effector cells. (D) is the addition of 0.4 ml of culture medium, and (E) and (F) are the addition of 0.05 ml of 10% Triton X-100 to (C) and (D). Was calculated by Equation 1. The result is shown in FIG.

  As shown in FIG. 2, the spleen cell natural killer activity of the mice administered with the immune activity enhancer according to Example 2 was significantly higher than that of the mice administered with the sterile physiological saline according to the comparative example. It became.

Experimental Example 4 Analysis of Immunoreactivity Enhancer According to Example 1 by Electrophoresis and Estimation of Active Ingredient Polyacrylamide gel (vertical on a 15% separation gel of 60 mm length, 80 mm width, 1 mm thickness) 20 mm, 80 mm wide, 1 mm thick layered 4% sample concentration gel), 5 μl of molecular weight marker stained in the sample addition well prepared at the top of the concentrated gel and Example 1 200 μg of such an immune activity enhancer was added. This gel was attached to a slab gel electrophoresis apparatus, and 40 mA in a mixed solution of 0.05 M Tris (hydroxymethylaminomethane), 0.384 M glycine, 0.1% SDS, about 60 minutes (the bromophenolblue dye in the sample solution was Electricity was applied (until elution from the lower end of the gel), and electrophoresis (SDS-PAGE) was performed. The immunoreactivity enhancer according to Example 1 is a sample solution (0.03 M Tris-HCl buffer containing 1% SDS, 10% sucrose, 0.5 V / V% 2-mercaptoethanol, 0.025% bromophenolblue. The solution was dissolved in pH 6.8, heated in a boiling water bath for 3 minutes, then cooled to room temperature and added.

  The immunoreactivity enhancer according to Example 1 was separated by SDS-PAGE, the gel was cut out and extracted, and the lymphocyte rejuvenation action was examined according to the method of Example 1. The result is shown in FIG.

  As shown in FIG. 3, relatively strong lymphocyte blastogenic activity is recognized in the fractions having molecular weights of 118,000 to 214,000 and 6,800 to 20,800.

Experimental Example 5 Analysis of Immunoreactivity Enhancer According to Example 2 by Electrophoresis and Lectin Blotting and Estimation of Active Components The action of the immunoreactivity enhancer according to Example 1 is enhanced by purification of a lectin concanvalin A binding column. In order to estimate the active component, components contained in the immune activity enhancer according to Example 2 were separated by electrophoresis and reacted with concanavalin A labeled with enzyme after transcription on a PVDF membrane to detect the binding component did. That is, 5 μl of a stained molecular weight marker and 25 μg of an immunopotentiator according to Example 2 (dissolved in the same sample solution as in Experimental Example 4) were added to polyacrylamide gel prepared in the same manner as in Experimental Example 4, and Electrophoresis was performed by SDS-PAGE under the same conditions. The gel was taken out and electrically transferred to a PVDF film with a transfer device (200 mA, 4 hours, 14 ° C.). After the transfer, the PVDF membrane was washed with Tris-buffered saline (TBS) and then immersed in TBS containing 3% bovine serum albumin at 4 ° C. for 16 hours for blocking. Thereafter, the plate was washed with TBS containing 0.05% Tween 20 (TTBS), immersed in a TTBS solution of 5 μg / ml concanavalin A-alkaline phosphatase conjugate, and reacted at 4 ° C. for 1 hour. The membrane was washed 3 times with TTBS and then once with TBS, and then an alkaline phosphatase substrate solution (mixture of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate) was added and allowed to react at room temperature. The result is shown in FIG.

  As shown in FIG. 4, concanavalin A binding components having molecular weights of around 18,000 and around 13,000 are confirmed from the immunopotentiator according to Example 2.

  Concanavalin A is a lectin that has an affinity for D-glucose and D-mannose. From the results of Experimental Example 4, at least one of the immunoreactivity enhancing components of the senso extract has a molecular weight of 18,000 including these sugar chains. It is possible that there is a glycoprotein or polysaccharide of around 13,000 or around 13,000.

It is a graph which shows the effect | action with respect to the mouse | mouth spleen lymphocyte rejuvenation reaction of the immune activity enhancer which concerns on Example 1 and Example 2. FIG. It is a graph which shows the effect | action with respect to mouse | mouth spleen NK cell activity of the immune activity enhancer which concerns on Example 2. FIG. The vertical axis represents NK cell activity (cytotoxicity) with standard error. The relationship between the isolation | separation of the immune activity enhancer which concerns on Example 1 by SDS-polyacrylamide electrophoresis, and its lymphocyte rejuvenation effect is shown. The lectin blot result by the enzyme label | marker concanavalin A of the immune activity enhancer which concerns on Example 2 is shown.

Claims (5)

An immune activity enhancer comprising a water-soluble extract of Senso,
The water-soluble extract of Senso is one or more of proteins, glycoproteins and polysaccharides having a molecular weight of 118,000 to 214,000. An immune activity enhancer comprising a water-soluble extract of Senso,
The water-soluble extract of Senso is an immune activity enhancer characterized by being any one or more of proteins, glycoproteins and polysaccharides having a molecular weight of 6,800 to 20,800.   3. The immunoreactivity enhancer according to claim 1 or 2, which has an effect of promoting blastogenesis of lymphocytes.   3. The immune activity enhancer according to claim 1 or 2, which has an action of promoting production of interleukin 2 and interleukin 12.   3. The immune activity enhancer according to claim 1 or 2, which has a natural killer cell activity enhancing action.