JP4842507B2 - Anti-influenza virus activator - Google Patents

Description

  The present invention relates to an anti-influenza virus activator derived from maitake and a food or pharmaceutical product containing the anti-virus activator.

  Influenza is an acute inflammation caused by infection with influenza viruses (types A, B, and C), which often affects inflammation of the lower respiratory tract, such as the bronchi, rather than the upper respiratory tract. Systemic symptoms such as headache, muscle pain, and joint pain are prominent. Influenza A is known to cause a global pandemic compared to the other two types. In recent years, it has been reported that both types of influenza are prevalent, and influenza A and B are often associated with pneumonia and bronchitis, and encephalitis and encephalopathy occur in younger children under 5 years old Some examples have been reported. On the other hand, in Japan, with the increase in the number of elderly people, the effects of influenza A and B influenza are significant, and the risk of heart disease and pulmonary disease carriers is also increased. Regarding the effects of influenza vaccines, the antigenicity of influenza viruses is likely to change, and it is said that there may be cases where the desired effects cannot be expected due to fluctuations in the effects.

  Accordingly, it is highly desired that an anti-influenza virus active substance can be obtained from a natural product that is safe and easily available or an extract thereof.

Japanese Patent Laid-Open No. 7-69913 JP-A-9-238697 JP 2000-119650 A JP 2001-172194 A

  An object of the present invention is to obtain an anti-influenza virus activator from a natural product or an extract thereof.

  In order to solve the above-mentioned problems, the inventors of the present invention have tried to examine various natural product extracts, and have focused attention on maitake, which is a kind of mushroom that has recently become easy to obtain.

As for the maitake extract, various actions are known due to the development efforts of the present applicants. For example, regarding the improvement effect of AIDS disease, JP-A-7-69913 (Patent Document 1) to 9-238697 (Patent Document 2), to the active oxygen scavenging activity Patent 200 0 -119 650 (Patent Document 3), Japanese Unexamined Patent Publication No. 2001-172194 Patent for NO production inducing activity (Patent Document 4 ) Etc.

  As a result of intensive studies, the present inventors have found that an extract of maitake has anti-influenza virus activity and completed the present invention.

That is, the present invention
(1) an anti-influenza virus activator in the presence of macrophages comprising an extract obtained by extracting one or more of raw maitake, dried maitake and maitake dry powder with water or hot water,
(2) Anti-influenza virus activator in the presence of macrophages consisting of a precipitate formed by adding alcohol to an extract obtained by extracting one or more of raw maitake, dried maitake and dried maitake with water or hot water ,
(3) Add alcohol to the extract obtained by extracting one or more of raw maitake, dried maitake and dried maitake with water or hot water to the extent that precipitation does not occur. An anti-influenza virus activator in the presence of macrophages, consisting of a material that floats or adheres to the wall of the container and then dries the solution;
(4) Add alcohol to the extract obtained by extracting one or more of raw maitake, dried maitake and dried maitake with water or hot water to the extent that precipitation does not occur. An anti-influenza virus activator in the presence of macrophages consisting of a precipitate formed by removing floating or adhering substances on the wall of the container, and then adding more alcohol;
(5) The anti-influenza virus activator in the presence of macrophages according to (1) to (4), wherein the mechanism of activity expression is through TNF-α production induction,
(6) A food comprising the anti-influenza virus activator according to any one of (1) to (5),
(7) A pharmaceutical product comprising the anti-influenza virus activator according to any one of (1) to (5).

  According to the present invention, a maitake extract that can be obtained safely and easily can be used to prevent or prevent symptoms of the common cold such as influenza, and can be used as a food or a medicine.

  The present invention is described in detail below.

In the present invention, maitake ( Grifola frondosa ), white maitake ( Grifola albicans ), choreimaitake ( Dendropolyporus umbellatus ), tombii maitake ( Grifola gigantia ), etc. can be used. Both fruit bodies and mycelium of these maitakes can be used, but recently, fruit bodies of maitake can be artificially grown, and it is preferable to use the fruit bodies of maitake from the viewpoint of securing a stable raw material.

  Raw maitake is preferably as fresh as possible after harvesting, but can be used as long as it is ready for food. The use site is not particularly specified and can be used above the stem. As the dried maitake, any of sun, hot air dried or freeze dried can be used. Maitake dry powder can be used from coarse to fine particles.

  The extraction method is performed with water or hot water. “Water or hot water” means from water to hot water, and naturally includes hot water. Therefore, extraction can be appropriately performed according to a conventional method at room temperature, under heating or heating, under normal pressure or under pressure.

  For example, it is performed at room temperature to 120 ° C. for 5 minutes to several hours. In order to perform efficiently in a short time, it is preferable to perform extraction at about 100 ° C. or higher under pressure, for example, at about 120 ° C. under pressure for about 30 minutes to about 1 hour under pressure.

  Distilled water, ion exchange water, reverse osmosis membrane (RO) water, tap water, and natural water can be used as water. About 4 to 30 times the weight of water is used per 1 weight of dry maitake or dry maitake powder. When using raw maitake, use about 2 to 10 times the weight of water.

  As described above, the water or hot water extract can be used as a dry extract powder by further concentrating it and drying the concentrated extract or concentrated extract.

  Water or hot water extract can be purified by alcohol precipitation. For example, an alcohol is added to the extract to collect a precipitated substance. The precipitate is a fraction mainly composed of high-molecular polysaccharide β-glucan contained in maitake and can be used as it is. Further purification can be performed as necessary. For example, alcohol is added to such an extent that precipitation does not occur in water or hot water extract, and preferably at 1 to 25 ° C., the liquid surface left standing, the substance floating in the liquid or the material adhering to the wall of the container is removed. Can be purified. The removal can be appropriately performed by filtration, suction, or scrubbing with a net-like material.

The amount of alcohol added to such an extent that precipitation does not occur differs depending on the concentration and temperature of the extract, and although it is generally difficult to determine, the final volume concentration of alcohol is relatively low, that is, about 30-60% by volume. To do.
In addition, although lower alcohol can be used as alcohol, especially ethanol is preferable.

  The solution thus obtained may be dried, or if necessary, alcohol is added to a relatively high concentration, that is, the final volume concentration is 60% by volume or more, and the precipitated substance is collected. It can also be purified by taking measures. In addition, although any means, such as spray drying, concentration drying, or freeze drying, can be used for the said drying, spray drying is especially preferable.

  The dried product or precipitate obtained as described above is soluble in water and contains β-glucan which is a polysaccharide. It can be further purified by using an aqueous solution and repeating the alcohol precipitation method as described above, and the purity of β-glucan can be increased by chromatography. For example, gel filtration, ion chromatography, affinity chromatography and the like can be used.

  The specific maitake extract obtained as described above was added to MDCK cells derived from canine kidney inoculated with influenza virus at various concentrations directly in the absence of macrophages. Almost no inhibition was observed.

  However, when the culture supernatant collected after various times of culturing macrophage-derived RAW cells and maitake extract was added to MDCK cells inoculated with influenza virus, the influenza virus decreased in a concentration-dependent manner over time. It was recognized that

  Furthermore, it was revealed by RT-PCR that TNF-α mRNA expression was induced in the concentration of maitake extract after stimulating RAW cells with maitake extract. In addition, the fact that TNF-α mRNA was actually translated and TNF-α increased over time was found using an ELISA kit.

  From the above results, it was found that the maitake extract stimulated macrophages, induced TNF-α, and induced anti-influenza virus activity. Therefore, it can be applied as a food or medicine for prevention of colds such as influenza or reduction of symptoms after illness.

  The maitake extract of the present invention has been confirmed to be non-toxic and can be used as a new form of food or as a pharmaceutical product.

  The oral intake is generally 50 to 500 mg per day (concentrated dry powder) for adults, but can be increased or decreased as necessary.

  For example, when applied as food, it can be used as health foods, supplements, functional foods, but general foods such as confectionery / breads, noodles, seasonings, spices, edible powders, dairy products, meat It can be blended into products, processed marine products, processed fruits and vegetables, various beverages and juices, tea, and instant foods.

When used as health foods, supplements, functional foods or pharmaceuticals, excipients and extenders are added as appropriate, and processed into various preparations such as tablets, capsules, granules, powders, pills, liquids, suspensions, etc. Can do.
Examples are shown below, but the present invention is not limited thereto.

Manufacture of anti-influenza virus active substance (1) 2.5 kg of dried maitake fruit body powder is charged with 30 L of ion exchange water in a pressure kettle, treated under pressure at 115-120 ° C. for about 0.5 hours, and then filtered to brown About 20 L of liquid (A) was obtained. 5 L of the brown liquid (A) is taken and concentrated under reduced pressure to obtain a concentrated liquid having a Brix concentration of 20%. The concentrated liquid is spray-dried using a spray drying apparatus to obtain a brown dry powder (Mitake extract [1]) About 152 g was obtained.
(2) Take 3 L of the brown liquid (A) obtained in (1) above, concentrate to 0.5 L under reduced pressure, add the same volume of 99% ethanol at room temperature, and leave it for about 10 hours to recover the brown precipitate. It was dried under reduced pressure to obtain 5 g of a brown substance (Maitake extract [2]).
(3) Take 10 L of the brown liquid (A) obtained in (1) above, concentrate to about 1 L under reduced pressure, add the same volume of 99% ethanol at room temperature, and leave it at 10 ° C. or less for about 10 hours to leave the liquid level. A brownish substance floating in the liquid and adhering to the wall of the container was formed. These materials are removed to give a brown solution. About 1.9 L of the brown liquid was subjected to removal of ethanol under reduced pressure and further concentrated to obtain a concentrated liquid having a Brix concentration of 35%. The concentrated solution was spray-dried using a spray-drying apparatus to obtain about 230 g of a brown dry powder (Mitake extract [3]).

In the acute oral toxicity test using rats on the maitake extract [3], the LD50 value was 2 g / kg or more, indicating no toxicity.
Further, as a result of a reverse mutation test using bacteria such as Salmonella typhimurium and Escherichia coli, it was confirmed that there was no mutagenic activity.

(1) Preparation of materials The materials used in the experiment for examining the anti-influenza virus activity are as follows.
Test-subject stimulating cells: Mouse macrophage-derived RAW264.7 cells (cultured in Dulbecco's MEM with 10% inactivated fetal bovine serum) (hereinafter referred to as RAW cells) were used.
Influenza virus-infected cells: MDCK cells (cultured in 8% inactivated fetal bovine serum-added MEM) were used.

Influenza virus: Influenza virus A / Aich / 2/68 (H3N2) [inoculated into the chorioallantoic cavity of the developing chicken egg and the obtained infected chorioallantoic fluid diluted appropriately with PBS as an active viral fluid] was used.
Test substance: Maitake extract [1] to Maitake extract [3] produced in Example 1
The results for the maitake extract [3] are illustrated below.

(2) Anti-influenza virus activity
As conditioned medium, RAW cells cultured confluently in 6 centimeters were washed once with serum free DMED, and then cultured in medium containing various concentrations of maitake extract [3] (hereinafter abbreviated as maitake extract). Thereafter, the culture supernatant appropriately collected was used as a conditioned medium (hereinafter abbreviated as CM).

In the proliferation test, influenza virus solution was inoculated with MOI 5 PFU / cell on MDCK cells cultured in 24-well plates, adsorbed at room temperature for 60 minutes, and then washed 3 times with PBS to remove unadsorbed virus. . Thereafter, CM cultured with various concentrations of maitake extract was added, and the culture was started at 37 ° C.
The start of this culture was defined as 0 hours of infection, and the amount of virus after 12 hours of culture was quantified by the plaque method.

  Prior to the test, the cytotoxicity of maitake extract to RAW cells was examined by MTT method. The result is shown in FIG. As shown in the graph, when RAW cells were stimulated with 1 mg / ml of maitake extract for 12 hours, cytotoxicity was observed compared to serum free DMEM as a control. However, cytotoxicity was not observed at concentrations of 300 or less maitake extract. Although not shown in the figure, 300 μg / ml of maitake extract did not show cytotoxicity to MDCK cells. From these results, it was decided to use 300 μg / ml or less as the concentration of the maitake extract in the subsequent experiments.

The results are shown in FIGS.
As shown in the graph on the left side of FIG. 2, in the group using CM after stimulation for 12 hours, the virus amount of CM addition of 300 μg / ml of maitake extract was 47.7 × 10 ⁴ PFU / ml, CM addition of 100 μg / ml CM The amount of virus was 2.5 × 10 ⁶ PFU / ml. When the amount of virus in the unstimulated control CM was taken as 100%, the amount of virus decreased by 12% and 64%, respectively, depending on the concentration.

  On the other hand, the graph on the right shows the result of adding the maitake extract directly to the infected MDCK cells and quantifying the amount of virus after culturing by the plaque method. The maitake extract was only DMEM for both 300 μg / ml and 100 μg / ml. There was no significant difference compared to the amount of virus in the control.

  FIG. 3 shows the results of a similar proliferation experiment performed on CM collected with maitake extract 300 μg / ml and collected over time.

In this case, when CM collected after 30 minutes and 1 hour was used, no significant virus growth inhibition was observed with respect to the control control CM. However, the viral load gradually decreased as the stimulation time became longer, and when using CM for 20 hours after stimulation, the viral load was 50 × 10 ⁴ PFU / ml, decreasing to 0.6% of the control. It was.

(3) Expression of TNF-α mRNA When induction of TNF-α mRNA expression after stimulation with maitake extract 300 μg / ml and 100 μg / ml for 12 hours was observed by RT-PCR, as shown in FIG. Was found to induce TNF-α mRNA expression in a concentration-dependent manner.

Furthermore, changes in TNF-α mRNA expression level over time when RAW cells were stimulated with the concentration of the maitake extract fixed at 300 μg / ml were examined.
The result is shown in FIG. As shown in the graph, TNF-α mRNA expression level was not observed in RAW cells 30 minutes after stimulation when compared with the mRNA expression level in GAPDH using TNF-α mRNA expression level as an internal standard. It increased rapidly after 1 hour, reached a peak at 2 hours, and then gradually decreased its expression.

(4) Production of TNF-α Next, it was examined using ELISA Kit whether TNF-α mRNA was actually translated and TNF-α was produced.
The result is shown in FIG.

The amount of TNF-α slightly increased in the case of only 300 μg / ml of maitake extract, which is the control control on the rightmost side of the graph. In contrast, the amount of TNF-α in CM stimulated with RAW cells with 300 μg / ml of maitake extract increased with time, and increased to a maximum of 800 pg / ml.
This result is consistent with the change over time in the expression of TNF-α mRNA shown above.

  That is, it was confirmed that the maitake extract of the present invention stimulates RAW cells to induce the production of TNF-α and thus enhance the activity of anti-influenza virus.

The figure which shows the cytotoxicity of a maitake extract. The figure which shows the virus growth inhibitory effect of a maitake extract-CM and a maitake extract direct addition. The figure which shows the time-dependent change of the virus growth suppression in a maitake extract-CM. The figure which shows the detection of TNF- (alpha) mRNA expression in a maitake extract stimulated RAW cell. The figure which shows the time-dependent change of TNF- (alpha) mRNA expression in a maitake-stimulated RAW cell. The figure which shows the time-dependent change of TNF- (alpha) production in a RAW cell.

Claims (3)

  Alcohol is added to the extract obtained by extracting one or more of raw maitake, dried maitake, and dried maitake with water or hot water to the extent that precipitation does not occur, and then left floating or in the liquid surface or in the liquid The anti-influenza virus agent characterized by including the extract obtained by removing the substance adhering to the wall surface.   To the extract obtained by extracting any one or more of raw maitake, dried maitake and dried maitake with water or hot water, alcohol is added to the final volume concentration of 30 to 60% as a guideline so that precipitation does not occur. An anti-influenza virus agent characterized by containing a substance obtained by removing substances that float on the liquid surface or in the liquid after standing or adhere to the wall surface of the container, and then dry the solution. Claim 1 or 2 anti-influenza virus drugs made by blending an anti-influenza virus agent according.

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