JP2011121918A - Vegetable component neutralizing antigenicity of virus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To clarify a vegetable component effective for neutralizing antigenicity of a virus and provide the vegetable component having immunoglobulin-like activity. <P>SOLUTION: A vegetable eaten by human as a food is defatted with ethanol and extracted with a weakly alkaline water. The extracted liquid is treated with a centrifugal concentration filter to extract a component having a molecular weight of 150,000-180,000. The skin of the vegetable eaten by human is potato skin, rice bran, adzuki bean skin or squeezed cake of Panax ginseng. Viral infection through mucosa can be prevented by including the component in candy, chewing gum, troche, etc. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a plant component that neutralizes the antigenicity of a virus and has an immunoglobulin-like function.

The theory is that "immunoglobulin", a protein produced by animal immune cells, does not exist in plants.
JP 10-67669 A

  The inventor of the present application has clarified the existence of an infection-protecting component produced by a plant by previous studies. The problem to be solved by the present invention is to clarify a plant component that neutralizes the antigenicity of a virus and to provide a plant component having an immunoglobulin-like action.

  From the skin of a plant with a human history, a water-soluble and ethanol-insoluble component having a molecular weight of between 150,000 and 180,000 is extracted. Plant skin with a human history can use potato skin, rice bran, red bean skin, ginseng ginseng.

  It neutralizes the antigenicity of rotavirus, poliovirus, influenza virus, hepatitis A virus, hepatitis B virus, herpes virus, norovirus, etc. that cause mucosal infection, and neutralizes the infectivity of the virus. It is possible to specify an effective concentration that suppresses the virus by 95% or more in a concentration range having no toxicity.

FIG. 1 is an HPCL chart showing the molecular weight distribution of Phyto-IgA. FIG. 2 shows that Phyto-IgA extracted from potato skin suppresses the growth of influenza virus. An in vitro model was used in which the canine kidney-derived cell line MDCK was used as a host cell and mixed and cultured with various concentrations of Phyto-IgA under or without influenza virus H1N1 infection. FIG. 3 shows that Phyto-IgA extracted from rice bran suppresses the growth of influenza virus. An in vitro model was used in which the canine kidney-derived cell line MDCK was used as a host cell and mixed and cultured with various concentrations of Phyto-IgA under or without influenza virus H1N1 infection. FIG. 4 shows that Phyto-IgA extracted from red bean peel suppresses the growth of influenza virus. An in vitro model was used in which the canine kidney-derived cell line MDCK was used as a host cell and mixed and cultured with various concentrations of Phyto-IgA under or without influenza virus H1N1 infection. FIG. 5 shows that Phyto-IgA extracted from ginseng suppresses the growth of influenza virus. An in vitro model was used in which the canine kidney-derived cell line MDCK was used as a host cell and mixed and cultured with various concentrations of Phyto-IgA under or without influenza virus H1N1 infection. FIG. 6 is a graph showing that Phyto-IgA saves mice infected with lethal doses of influenza virus. FIG. 7 is a graph showing that Phyto-IgA neutralizes the antigenicity of the virus. FIG. 8 is a graph showing experimental results on the antiviral activity effective time of Phyto-IgA Candy.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings.

  Since the plant component of the present invention has an immunoglobulin-like action as described later, and has an effect of neutralizing the infectivity of a virus that causes mucosal infection, its function is similar to IgA. Therefore, the inventor of the present invention named the plant component according to the present invention as plant immunoglobulin A (Phytoimmunoglobulin A, Phyto-IgA). It is coined by the inventor of the present application. In addition, the molecular weight of IgA is about 160,000.

  In order to produce Phyto-IgA, a plant with a human dietary history is degreased with ethanol, extracted with weak alkaline water, and then the extract is applied to a centrifugal concentration filter to obtain a molecular weight of 150,000 to 180,000. By getting the ingredients in between. Plants with a human history can use potato skin, rice bran, red bean skin, ginseng ginseng. The human dietary history refers to what human beings have eaten since history.

[Production of Phyto-IgA] Phyto-IgA is a material with a human dietary history, defatted with ethanol, and extracted with 10 times 2.5% aqueous bicarbonate (NaHCO3) at 90 ° C for 1 hour. . The extract is filtered through a 0.45 micron membrane filter (Millipore Co., Ltd.), and a centrifugal filtration unit for protein concentration and desalting provided by Hitech Co., Ltd. (Vivapin 4, product number VS0443, molecular weight cut off 100,000 MWCO) And concentrated fractionation.

  The molecular weight distribution of the fractionated Phyto-IgA is shown in FIG. From the figure, rice bran (B), red bean peel (C), potato peel (D), ginseng (E), Phyto-IgA having a molecular weight distribution similar to that of γ immunoglobulin (A) used as a molecular weight marker You can see that it was fractionated. The molecular weight was measured by a HPLC method (solvent: water, flow rate: 1 ml / min, column temperature: 30 ° C.) using a size exclusion chromatography (SEC) column (manufactured by Showa Denko).

  Next, we show that Phyto-IgA suppresses the growth of influenza virus. In this test, the canine kidney-derived cell line MDCK is used as a host cell in vitro with various concentrations (0.15-2.5 mg / ml) of Phyto-IgA, either uninfected or infected with influenza virus (H1N1). A model was used. The results are shown in Table 1 and FIG. Here, Phyto-IgA extracted from potato skin was used.

[Test Method] MDCK cells were prepared at 2 × 10 ⁴ / ml and seeded in 100 μL each in a 96-well microculture plate in an EMEM medium supplemented with 10% FCS. The cells were cultured at 37 ° C. and 5% CO ₂ for 2 days. After removing the culture solution and washing with PBS, a medium containing influenza virus (Inf / A H1N1) solution and a sample was inoculated into MDCK cells and cultured at 34.5 ° C. for 3 days. After removing the culture solution, 100 μL of EMEM medium (no FCS added) was added. After culturing at 34.5 ° C. for 2 days, the supernatant was removed and measured by the MTT method (viable cells were calculated by comparing viable cells with blanks).

  FIG. 2 is a graph showing the results of Table 1. The horizontal axis of FIG. 2 shows the added concentration of Pyhto-IgA, and the vertical axis shows the survival rate of the host. Under influenza virus non-infection (open vertical bars in FIG. 2, virus non-infected), no host cell death was observed. This indicates that Phyto-IgA is not toxic to host cells at concentrations of 0.15 to 2.5 mg / ml.

  On the other hand, under influenza virus infection (black vertical bar in FIG. 2, virus infected), it can be seen that 100% of the host cells were killed without the addition of Phyto-IgA. However, the survival rate of the host cells increased with increasing concentration of Phyto-IgA. In other words, Phyto-IgA suppressed virus infection in a concentration-dependent manner.

  From the value of the concentration 313 and the value of the concentration 625, an effective concentration that suppresses virus infection by 95 percent and an effective concentration that suppresses 99.9 percent can be obtained by calculation of an interpolation method (interpolation method). Table 2 shows the calculation of the interpolation method.

  Table 3 and FIG. 3 show the same experiment as in Example 2 performed on Phyto-IgA extracted from rice bran. The test method is the same as in Example 2.

  As in Example 2, the effective concentration that suppresses viral infection by 90 percent and the effective concentration that suppresses 99.9 percent are calculated from the value of concentration 313 and the value of concentration 625 by the calculation of the interpolation method (interpolation method). Can be sought. Table 4 shows the calculation of the interpolation method.

  Table 5 and FIG. 4 show the same experiment as in Examples 2 and 3 performed on Phyto-IgA extracted from red bean skin. The test method is the same as in Examples 2 and 3.

  As in Examples 2 and 3, the effective concentration of suppressing virus infection by 90% and the effective of suppressing 99.9% by calculation of the interpolation method (interpolation method) from the value of concentration 313 and the value of concentration 625. The concentration can be determined. Table 6 shows the calculation of the interpolation method.

  Table 7 and FIG. 5 show the same experiment as in Examples 2 and 3 performed on Phyto-IgA extracted from ginseng. The test method is the same as in Examples 2, 3, and 4.

  As in Examples 2 and 3, the effective concentration of suppressing virus infection by 90% and the effective of suppressing 99.9% by calculation of the interpolation method (interpolation method) from the value of concentration 313 and the value of concentration 625. The concentration can be determined. Table 8 shows the calculation of the interpolation method.

  FIG. 5 is a graph showing that Phyto-IgA saves mice infected with lethal doses of influenza virus. The test used an in vivo model in which mice were infected with a lethal influenza virus (H1N1) in the brain in the absence or presence of Phyto-IgA, and the survival rate thereafter was observed daily. In FIG. 5, the horizontal axis represents the number of days elapsed after infection, and the vertical axis represents the survival rate of the host animal.

  In the absence of Phyto-IgA (open circles), the host animals died after 6 days of virus infection, and only 30 percent finally escaped infection. On the other hand, in the presence of Phyto-IgA (black circle), no host animal died after 6 days from virus infection, and all (100 percent) finally escaped from the infection. This indicates that Phyto-IgA suppressed viral infection. These results indicate that Phyto-IgA saves host animals from influenza virus infection. This can be said to prove the test tube level results of Examples 1 to 3 at the animal level. In this experiment, Phyto-IgA extracted from rice bran was used.

  In recent years, the hyperimmune reaction (cytokine storms) induced by the H5N1 virus has been a problem, and it has also been a problem with SARS, but Phyto-IgA according to the present invention provides a means to avoid the cytokine storm. We examined whether or not.

  In the test, mouse spleen cells were used as IgA antibody-producing cells, mixed with various concentrations of Phyto-IgA, and sensitized with influenza virus or not. We used an in vitro model to compare Whether cytokine storms can be avoided can be evaluated by suppressing IgA antibody production induced as a result of immune responses.

The virus inactivator is prepared by incubating the influenza H1N1 virus in host cells and then inactivating the virus solution with formalin. Spleen cells are prepared by removing the spleen from normal mice (Day 14), adjusting the spleen cells, and using RPMI1640 containing 5% fetal bovine serum (FBS) and culturing the cells in a flat-bottom 96-well plate. The specimen is a lignin PBS solution (5 mg / ml). The test schedule was as follows: Each lignin PBS solution (concentration: 100, 200, 400 μg / ml) and inactivated influenza virus were mixed, and after 1 hour incubation, spleen cells (1 × 10 ⁶ / well) were added and the temperature was 37 degrees Celsius. Incubate in the presence of 5 percent CO ₂ (n = 2). Set virus-free and lignin-free as negative controls. For IgA measurement, influenza virus-specific IgA in the culture supernatant of mouse spleen cell culture day 7 is measured by ELISA.

  FIG. 7 is a graph showing that Phyto-IgA avoids cytokine storms by neutralizing the antigenicity of the virus. In the figure, the horizontal axis represents the added concentration of Phyto-IgA, and the vertical axis represents IgA antibody production. When Phyto-IgA is not added (0.0 μg / ml), the amount of IgA antibody produced is about 930 pg / ml in the presence of virus (black circle, Influenza virus +), and about 130 pg in the absence of virus (white circle, Influenza virus −). A difference of about 800 pg / ml was observed. This difference in the amount of production can be said to refer to the amount of antibody biosynthesized due to virus stimulation. It is thought that cytokine production is always involved in this antibody production, and if the virus stimulation becomes lethal, a rapid immune reaction is induced to defend it, and a large amount of cytokine is released, that is, a cytokine storm is generated.

  Under these conditions, the production of IgA antibody induced in the presence of virus (black circle, Influenza virus +) decreases as the concentration of Phyto-IgA added increases, and at concentrations above 200 μg / ml (200 ppm) It was found that it was reduced to the level of no virus. That is, it is shown that Phyto-IgA neutralized the antigenicity of the virus and suppressed IgA antibody production induced by virus stimulation by 100 percent. This means that Phyto-IgA itself acts like IgA, so that spleen cells no longer need to produce IgA antibodies. In other words, it was clarified that Phyto-IgA can avoid the occurrence of hyperimmune reaction (cytokine storm). Phyto-IgA adsorbs non-specifically with viruses and deactivates its function, so it is expected to succeed in preventing infection regardless of the type of virus.

  Next, we examined the protective effect of Phyto-IgA against herpes virus (HSV). An in vitro model was used in which plaques formed by HSV infection were counted. As a result, it was revealed that Phyto-IgA suppresses plaque formation (proliferation) by HSV that cannot be suppressed by antitumor polysaccharides such as mushrooms (Table 9). This is considered to be a result of Phyto-IgA binding to HSV to inhibit virus adsorption / invasion / growth, that is, infection.

  Examples of adaptive viruses of Phyto-IgA include rotavirus, poliovirus, influenza virus, hepatitis A virus, hepatitis B virus, herpes virus, norovirus and the like that cause mucosal infection.

  As an adaptive dosage form of Phyto-IgA, a troche, candy, gum or the like that can be continuously released into the saliva in the oral cavity is preferable for activating mucosal immunity.

  Table 10 shows the nutritional components of Phyto-IgA Candy. Ingredients are sugar, chickenpox, Phyto-IgA extracted from rice bran, strawberry concentrated juice, acidulant, flavor, purple corn pigment. In this example, the content of Phyto-IgA in one pod was 0.5 percent.

  According to experiments, the concentration of Phyto-IgA required to inhibit viral infection by 99.9 percent is 560 ppm. Since the weight of one Phyto-IgA Candy is 3.5 grams, when the Phyto-IgA content is 0.5 percent, the amount of Phyto-IgA contained in one candy is 17.5 mg . It took about 30 minutes to hold one pod after it had melted without doing anything (without biting). If saliva is normal, about 1 ml is secreted per minute, so the amount of saliva secreted in 30 minutes is 30 ml, and the concentration of Phyto-IgA in saliva is 0.583 mg / ml (583 ppm). Therefore, the duration of inhibiting virus infection by 99.9% or more is about 30 minutes per pod.

  The adaptive intake is preferably 0.5% to 1.5%. Table 12 and FIG. 9 show the experimental results on the antiviral activity effective time. 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes after starting to lick the cocoon containing 0.5 percent Phyto-IgA extracted from rice bran and 1.5 percent The saliva sample was collected and the concentration of Phyto-IgA was examined.

  From this experimental result, the effective time for inhibiting 99.9% virus infection was calculated by interpolation (interpolation). As shown in Table 13, in the case of 0.5% wrinkle, 54 minutes, 1 In the case of 5% wrinkle, 68 minutes is obtained.

  Viral infection from the mucous membrane can be protected by including it in sputum, chewing gum, troche and the like. In addition to humans, examples of adaptive animals include livestock, poultry farming, racehorses, and fish farming.

Claims (5)

  A plant component having a molecular weight between 150,000 and 180,000 extracted from a plant with a human dietary history, which neutralizes the antigenicity of the virus.   A plant component that has a molecular weight of between 150,000 and 180,000 extracted from plants with a human dietary history and neutralizes the infectivity of viruses.   A plant component with a molecular weight of between 150,000 and 180,000 extracted from a plant with a human dietary history, which can specify an effective concentration that suppresses the virus by 95% or more in a non-toxic range.   The plant component according to any one of claims 1, 2, and 3, wherein the plant is one of the group consisting of potato skin, rice bran, red bean skin, ginseng ginseng. A plant component characterized by that.   The plant component according to any one of claims 1, 2, and 3, wherein the virus comprises rotavirus, poliovirus, influenza virus, hepatitis A virus, hepatitis B virus, herpes virus, norovirus. A plant component which is any one of the above.

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