JP5653034B2 - Antiviral agent - Google Patents

Description

  The present invention relates to an antiviral agent. Specifically, this invention relates to the antiviral agent which uses a green tea component as an active ingredient.

In recent years, emerging viruses have spread pandemicly, and viral infections have become a global problem.
Among them, influenza caused by influenza virus is one of the most widespread zoonotic diseases in the world, and once a new type of influenza virus appears, not only will human lives be lost in a short period of time, but it will also have a profound impact on social functions. give.
In addition, in recent years, swine flu has been reported in Japan and fatalities have been observed, and further spread of infection is expected.

Vaccination is mainly used for the prevention of influenza, and neuraminidase inhibitors and amantadine are used for the treatment of influenza.
Patent Document 1 discloses a proanthocyanidin polymer composition having a structure containing a flavonoid unit such as catechin having antiviral activity.

Japanese Patent No. 3448052

However, vaccines against viruses such as influenza vaccines need to cope with constant mutations in antigens, and the current situation is that development takes time and effort.
In addition, viruses having resistance have appeared against conventional antiviral agents.

  Therefore, it is desired to develop a drug that can prevent virus infection without depending on antigenicity and that can be used stably without causing harmful side effects on the human body.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found a novel antiviral agent containing a green tea component as an active ingredient and completed the present invention.

The present invention is as follows.
[1]
An antiviral agent containing a green tea ingredient as an active ingredient.
[2]
The antiviral agent according to [1], wherein the green tea component is one or more selected from the group consisting of ellagitannins, epigallocatechin gallate, and pharmaceutically acceptable salts thereof.
[3]
The antiviral agent according to [2], wherein the ellagitannin is strictinin.
[4]
The antiviral agent according to any one of [1] to [3], wherein the virus is an influenza virus.
[5]
The antiviral agent according to [4], wherein the influenza virus is H1N1 type.

  According to the present invention, a new antiviral agent can be provided.

The structural formula of strictinin is shown. The protocol of the measuring method of the anti-influenza virus activity of strictinin is shown. In the figure, the protocols 1 to 4 show only the part where the reaction was performed by adding strictinin, and all the parts where the strictinin was not added were reacted under the same conditions as the control. FIG. 3A shows the measurement results of anti-influenza virus activity when reacted according to Protocol 1 (Attachment) and Protocol 2 (Entry). The vertical axis represents the virus titer, and the horizontal axis represents the strictinin concentration. FIG. 3B shows the measurement results of anti-influenza virus activity when reacted according to protocol 3. The vertical axis represents the virus titer, and the horizontal axis represents the strictinin concentration. The measurement result of the anti-influenza virus activity of strictinin and epigallocatechin gallate (EGCG) at the time of reacting by the protocol 1 is shown. The vertical axis represents the inhibition rate, and the horizontal axis represents the concentration of each compound. The measurement result of anti-influenza virus activity at the time of infecting a vero cell is shown. The vertical axis represents the virus titer, and the horizontal axis represents the strictinin concentration. The measurement result of the anti-influenza activity with respect to a B type strain | stump | stock (B / Lee / 40) is shown. The vertical axis represents the infection rate, and the horizontal axis represents the concentration of strictinin.

  Hereinafter, embodiments for carrying out the present invention will be described in detail. In addition, this invention is not limited to the following embodiment, It can implement by changing variously within the range of the summary.

The antiviral agent of the present invention contains a green tea component as an active ingredient.
In the present invention, the green tea component refers to a component contained in green tea, and is a component obtained by extraction from a green tea raw material (for example, tea leaves).
The green tea component may be a compound as a component contained in green tea such as epigallocatechin gallate or strictinin, or may be a green tea extract obtained by extraction from a green tea raw material by a conventionally known method. Green tea itself can also be used as the antiviral agent of the present invention. Moreover, green tea components, such as flavonoids other than epigallocatechin gallate and strictinin, may be included as a green tea component.

In the present invention, “epigallocatechin gallate” is an ester of epigallocatechin and gallic acid, and pharmaceutically acceptable salts of epigallocatechin gallate are alkali metals such as sodium and potassium, calcium And the like, and salts of alkaline earth metals such as epigallocatechin gallate, and quaternary ammonium salts such as ammonium salt of epigallocatechin gallate.
The epigallocatechin gallate may be a prodrug that releases epigallocatechin gallate in vivo.

In the present invention, “elagitannin” means a compound having saccharide and ellagic acid, which is a dimer of gallic acid, as hydrolysis products in the molecule, and is also called hydrolyzable tannin.
Examples of ellagitannins include strictinin, geraniin, kebrinic acid, elaeocarpsin, keblagic acid, corilagin, embricanin, punygluconin, pedunculagine, cornucin A, agrimoniin, terimalangin, casarrictin, lugosin C, casulinin and the like. It is done.
Examples of pharmaceutically acceptable salts of ellagitannins include alkali metals such as sodium and potassium, salts of alkaline earth metals such as calcium and ellagitannins, and quaternary ammonium salts such as ammonium salts of ellagitannins.
The ellagitannin may be a prodrug that releases ellagitannin in vivo.

Epigallocatechin gallate and ellagitannin are components widely contained in green tea and the like, and have high safety.
Epigallocatechin gallate or ellagitannin used in the present invention may be epigallocatechin gallate or ellagitannin as an extract of green tea leaves from the viewpoint of safety. As epigallocatechin gallate and ellagitannin, commercially available products may be used, or those extracted from green tea raw materials by a conventionally known method may be used. Further, it may be used as a green tea leaf extract containing epigallocatechin gallate or ellagitannin. Furthermore, epigallocatechin gallate and ellagitannin may be obtained from plants other than green tea leaves.

  The antiviral agent of this invention can be used as food-drinks or a pharmaceutical.

Green tea ingredients such as ellagitannin and epigallocatechin gallate can be used as foods and drinks, foods for specified health use, nutritional functional foods, health foods, functional foods, health supplements, etc. It can be used as such. Moreover, the fortified food which added the green tea component to the existing food may be sufficient.
The food / beverage product may be a food / beverage product with an indication that it has an antiviral effect.
Epigallocatechin gallate and ellagitannin are abundant in green tea and can be used as highly safe antiviral agents. Among them, they are widely used as anti-influenza virus agents independent of influenza virus types and subtypes. Can be used. Epigallocatechin gallate and ellagitannin in green tea show infection-preventing action against various types of viruses including the new influenza (H1N1 type) and are effective in viral infection regardless of type. It is suggested that it is effective for prevention.

  Preferred forms as food and drink are foods and beverages such as candy, jelly, tablet confectionery, beverages, soup, noodles, rice crackers, Japanese confectionery, frozen confectionery, baked confectionery, etc., preferably fruit juice beverages, vegetable juices, fruit vegetable juices, teas Container-packed beverages such as beverages (including green tea beverages), coffee beverages, and sports drinks.

  Green tea ingredients, including epigallocatechin gallate and ellagitannin, can be ingested by oral administration in liquid or solid form as food or drink, and powdered by itself or appropriately mixed with excipients etc. for convenience of formulation , Granules, tablets, capsules and the like.

When the beverage contains the green tea component, it may be a beverage to which the green tea component is added, and a liquid extracted from green tea leaves may be used as the beverage as the green tea extract. The content of the green tea component is not particularly limited, but the active ingredient content is preferably, for example, 5 ppm to 600 ppm, preferably 5 ppm to 50000 ppm, more preferably 20 ppm to 5000 ppm. is there.
The method for extracting the green tea component from the plant is not particularly limited. For example, when the plant is tea, a method of extracting tea leaves by immersing tea leaves in hot water or the like, or a dry powder of the plant is used. A method of extraction by suspending in hot water or the like can be mentioned. The extract itself may be used as it is as a tea beverage as an antiviral agent, or a green tea component such as epigallocatechin gallate or ellagitannin may be separated and purified from the extract and used as an antiviral agent.

The green tea component can be used as a pharmaceutical preparation by adding a pharmaceutically acceptable excipient as a pharmaceutical product.
As a pharmaceutical preparation, it can be formulated into a known dosage form such as powder, granule or tablet, and can also be used as a liquid preparation such as liquid or paste.

  Pharmaceutical preparations include tablets, capsules, granules, fine granules, powders, solutions, syrups, chews, lozenges and other oral preparations, ointments, gels, creams, patches, external preparations, injections , Sublingual agents, inhalants, eye drops, suppositories and the like.

The pharmaceutical preparation is useful as an antiviral agent in animals, especially mammals, and can be administered to animals, especially mammals.
Examples of mammals include humans and livestock animals such as dogs, cats, cows, and horses, with humans being preferred.

The antiviral agent containing the green tea component of the present invention as an active ingredient is an antiviral composition appropriately mixed with other additives as a food or drink or as a medicine so that the following intake or dosage of the green tea component can be achieved. It can be.
The intake or dosage of the green tea component, including epigallocatechin gallate and ellagitannin, can be appropriately adjusted according to the use, but is preferably 1 mg to 10000 mg at a time, more preferably 5 mg to 1000 mg at a time, More preferably, it is 10 mg to 500 mg at a time.
The number of times of intake or the number of times of administration is not particularly limited, but is preferably 1 to 3 times a day, and the number of times of intake may be increased or decreased as necessary.

The antiviral agent containing a green tea component as an active ingredient can be used for prevention or treatment of viral diseases.
Examples of viruses include human parainfluenza virus, viruses with envelopes such as hepatitis B virus, influenza virus, and human immunodeficiency virus.
Especially, the antiviral agent of this invention can be used for the prevention or treatment of influenza as an anti-influenza virus agent. Since the green tea component is also a component contained in tea, it can be used as an active component of a highly safe anti-influenza virus agent having influenza preventive action. Moreover, it is effective for prevention of influenza by ingesting the food / beverage products containing the green tea component in this invention, for example, green tea etc. on a daily basis.

  EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention concretely, this invention is not limited to these Examples at all.

The influenza virus used in the following experiments was prepared as follows.
Virus was inoculated into the chorioallantoic cavity of the growing egg on day 10, cultured at 34 ° C. for 2 days, and allowed to stand at 4 ° C. overnight to obtain a chorioallantoic fluid containing virus. After removing the cell debris from this chorioallantoic fluid at 4 ° C. and 3000 rpm (Hitachi SCR20B centrifuge, RPR9 rotor), the supernatant was centrifuged at 4 ° C. and 8500 rpm (Hitachi SCR20B centrifuge, RPR9 rotor) for 3 hours. The pellet obtained by removing the supernatant contains phosphate buffer (131 mM NaCl, 14 mM Na ₂ HPO ₄ , 1.5 mM KH ₂ PO ₄ , 2.7 mM KCl at pH 7.2. Hereinafter, the phosphate buffer. The solution was sometimes suspended as “PBS”) to obtain concentrated virus. This concentrated virus was centrifuged for 2 hours at 38000 rpm (Hitachi CP65B ultracentrifuge, P40ST rotor) at 4 ° C. by sucrose density gradient centrifugation with a 50% (v / v) glycerol-PBS solution. separated. The purified virus obtained by separation was used in the following experiment.
The purified and concentrated virus was suspended in HSFM (which may optionally contain strictinin and the like) and used below.
As human influenza virus strains, A / Memphis / 1/71 (H3N2), A / Aichi / 2/68 (H3N2), A / Shizuoka / 838/2009 (H1N1), A / Puerto Rico / 8/34 (H1N1) (Hereafter, it may be described as “A / PR / 8/34”). A / duck / 313/4/78 (H5N3) was used as the avian influenza virus strain, and A / sine / Hokaido / 10/85 was used as the swine influenza virus strain.

Hemagglutination activity (HAU) was measured as follows.
PBS (50 μL) was added to a microplate (Falcon 3911 Test3 ™ Flexible Assay Plate) to dilute the purified virus twice. 0.5% (v / v) guinea pig erythrocyte-PBS suspension (50 μL) was added, shaken for 30 seconds, and allowed to stand at 4 ° C. for 2 hours to observe an aggregated image. Hemagglutination activity (HAU) was expressed as the highest dilution factor at which erythrocyte aggregation was observed.

(1-1. Anti-influenza virus activity)
The anti-influenza virus activity of strictinin against influenza A virus was reacted according to the methods of the following protocols 1 to 4, and the virus infectivity in the supernatant from the infected cells was measured by a focus-forming assay. Details of protocols 1 to 4 are shown in FIG. 2 and below.
Madin-Darby canine kidney (MDCK) cells (used from the National Institute of Genetics) were used in 5% (v / v) heat-inactivated fetal bovine serum (FBS) (Bio The cells were cultured in a minimal essential medium (MEM: Nissui Pharmaceutical) supplemented with (Logical Industry) and 50 μg / mL gentamicin (Gibco).

In protocol 1, the cultured MDCK cells were added to a 96-well microplate at about 1.5 × 10 ⁴ cells / well and cultured at 37 ° C. for 12 hours to adhere the cells to the plate. Washing was performed 3 times by adding PBS and removing the supernatant. After washing, type A H3N2 strain (A / Memphis / 1/71) was reacted with 1 FFU / cell.
A mixture of HSFM (Hybridoma-SFM complete DPM, Invitrogen) containing various concentrations (0-50 μM) of strictinin (Nagara Science) and virus (100 μL / well) was allowed to react with MDCK cells in an ice bath for 1 hour. It was. In order to remove unadsorbed virus, MDCK cells were washed three times or more with HSFM (100 μL / well). And HSFM (100 microliters / well) containing the strictinin of the same density | concentration of 0-50 micromol was added, and it was made to react at 37 degreeC for 1 hour. Thereafter, MDCK cells were similarly washed with HSFM, and then reacted with HSFM (100 μL / well) containing strictinin at the same concentration of 0-50 μM at 37 ° C. for 12 hours.

In protocol 2, MDCK cells cultured on a 96-well microplate were added at about 1.5 × 10 ⁴ cells / well, and a type A H3N2 strain (A / Memphis / 1/71) was reacted at 1 FFU / cell.
HSFM and virus mixtures containing various concentrations (0-50 μM) of strictinin were added and allowed to react with MDCK cells in an ice bath for 1 hour for virus adsorption. In order to remove unadsorbed virus, MDCK cells were washed three times or more with HSFM (100 μL / well). Then, in the same manner as in the case of 0 μM strictinin in Protocol 1 (without strictin, in HSFM), the mixture was reacted at 37 ° C. for 1 hour, then washed and then at 37 ° C. for 12 hours.

In protocol 3, MDCK cells cultured on a 96-well microplate were added at about 1.5 × 10 ⁴ cells / well, and a type A H3N2 strain (A / Memphis / 1/71) was reacted at 1 FFU / cell.
A mixed solution of HSFM and virus (100 μL) containing no strictin was reacted with MDCK cells in an ice bath for 1 hour. In order to remove unadsorbed virus, MDCK cells were washed three times or more with HSFM (100 μL / well). Then, HSFM (100 μL / well) containing strictinin at various concentrations (0-50 μM) was added and reacted at 37 ° C. for 1 hour for virus uptake. Thereafter, MDCK cells were washed in the same manner with HSFM (100 μL / well), and then reacted with HSFM (100 μL / well) containing no strictin for 12 hours at 37 ° C.

In protocol 4, MDCK cells cultured on a 12-well plate were added at about 1.5 × 10 ⁴ cells / well, and a type A H3N2 strain (A / Memphis / 1/71) was reacted at 1 FFU / cell.
A mixed solution of HSFM and virus (100 μL / well) containing no strictinin was reacted with MDCK cells in an ice bath for 1 hour. In order to remove unadsorbed virus, MDCK cells were washed three times or more with HSFM (100 μL / well). And it was made to react at 37 degreeC with HSFM (100 microliter / well) which does not contain strictin for 1 hour. MDCK cells were then washed in the same way with HSFM (100 μL / well), then HSFM (100 μL / well) containing various concentrations (0-50 μM) of strictinin was added, and 37 ° C. for virus replication. For 12 hours.

The focus-forming assay is described in Biol. Pharm. Bull. , 2009, 32, 1188-1192, as follows.
MDCK cells reacted with virus were fixed with ice-cold methanol for 30 seconds, and after removing the methanol, an anti-NP monoclonal antibody solution (MAb J. Virol., 2008, 82, 5940-5950 diluted 4 times with PBS was added). Mice were immunized with influenza virus by the method described, spleen cells were collected, hybridomas were prepared by a general method, and MAb was obtained.) And reacted for 30 minutes. Subsequently, horseradish peroxidase (HRP) -conjugated anti-rabbit IgG (Jackson Immuno Research, diluted 3000-fold with PBS) was added and incubated at room temperature for 30 minutes. Virus-infected cells were treated with DEPDA color solution (1.2 mM N, N-diethyl-p-phenylenediamine dihydrochloride, 0.003% hydrogen peroxide, 100 mM citrate buffer containing 2 mM 4-chloro-1-naphthol). (PH 6.0)) was added and detected. All images were counted for virus-infected cells using Photoshop software with OLYMPUS SZX7 at a magnification of 12x.

  The measurement result of virus titer in protocol 1-3 (n number = 3) is shown in FIG.

Culture was performed in the same manner as in Protocol 1 using EGCG (Nagara Science) and strictinin, and anti-influenza virus activity was measured.
The measurement results (n number = 3) of strictinin and EGCG in Protocol 1 are shown in FIG.

  Table 1 shows the inhibitory activity of strictinin measured using other influenza viruses in place of the type A H3N2 strain (A / Memphis / 1/71) according to protocol 1. In addition, EGCG and amantadine were also measured for similar anti-influenza virus activity instead of strictinin. The results are shown in Table 1. Each measurement was performed three times.

(1-2. Anti-influenza virus activity)
Anti-influenza virus activity of strictinin against influenza A virus was measured by focus-forming assay using vero cells.
Vero cells (using those obtained from RIKEN BioResource Center) were treated with 5% (v / v) heat-inactivated fetal bovine serum (FBS) (biological industry) and 50 μg / mL gentamicin ( The cells were cultured in MEM medium (Nissui Pharmaceutical) supplemented with Gibco).
The cultured vero cells were added to a 12-well plate at about 1.5 × 10 ⁵ cells / well, and the A-type H3N2 strain (A / Memphis / 1/71) was reacted at 1 FFU / cell.
Vero cells were reacted with HSFM containing various concentrations (0-50 μM) of strictinin at 37 ° C. for 48 hours with the virus.
Virus infected cells were measured by focus-forming assay. The measurement results are shown in FIG. Each measurement was performed twice.

(1-3. Anti-influenza virus activity)
Anti-influenza virus activity of strictinin against influenza B virus was measured by a focus-forming assay.
MDCK cells cultured on a 12-well plate were added at about 1.0 × 10 ⁵ cells / well, and a B-type strain (B / Lee / 40) was reacted at 0.001 FFU / cell.
MDCK cells were reacted with various concentrations (0-30 μM) of strictinin at 34 ° C. for 1 hour with a pre-culture mixture containing virus. MDCK cells were washed with HSFM. The cells were cultured at 34 ° C. for 48 hours in HSFM containing strictinin at the same concentration of 0-30 μM, 1.2% Avicel (FMC Corporation, USA), 2 μg / mL acetylated trypsin.
Virus infected cells were measured by focus-forming assay. The measurement results (n number = 3) are shown in FIG.

The cytotoxicity of strictinin on MDCK cells was measured by measuring the lactose dehydrogenase activity released from dead cells.
Strictinin did not show cytotoxicity against MDCK cells in the 0-200 μM dose range.

From the above experiments, it was found that epigallocatechin gallate, which is a green tea component, and strictinin, which is one of ellagitannins, have anti-influenza virus inhibitory activity. It was also found that strictinin inhibits influenza A virus replication in a dose-dependent manner when added at the time of viral infection of cells. In addition, strictinin is considered to inhibit the initial stage of infection upon attachment / invasion to influenza virus cells, and strictinin has an unprecedented strong inhibitory activity due to such inhibitory action. Moreover, it can also be used in screening for compounds having an anti-influenza virus inhibitory effect using strictinin and utilizing this action. It was also confirmed that strictinin has an inhibitory activity against influenza B virus. As shown in Table 1, green tea components such as strictinin and epigallocatechin gallate also have inhibitory activity against a wide range of subtypes of influenza viruses, so the highly pathogenic avian influenza H5N1 virus, It is also considered to have inhibitory activity against the swine influenza H1N1 virus and the like. Moreover, it has influenza inhibitory activity with respect to any of A type, B type, and C type, and is effective as an influenza preventive agent because of its action mechanism that inhibits the early stage of infection.
In addition, this example suggests that green tea is effective in preventing influenza.

  In the present invention, the green tea component has an antiviral effect. Among these, epigallocatechin gallate and ellagitannin have industrial applicability as anti-influenza preventive and therapeutic agents for pharmaceuticals and foods.

Claims (3)

An anti-influenza virus agent for pharmaceutical use, containing as an active ingredient one or more selected from the group consisting of strictinin and pharmaceutically acceptable salts thereof.   The anti-influenza virus agent according to claim 1, further comprising one or more selected from the group consisting of epigallocatechin gallate and pharmaceutically acceptable salts thereof. The anti-influenza virus agent according to claim 1 or 2 , wherein the influenza virus is H1N1 type.