JP2011046636A - Antiviral composition and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antiviral composition which can effectively deactivate a virus and its utilization. <P>SOLUTION: The antiviral composition contains a bamboo extract and this extract is obtained by the method including a step (a) of extracting a bamboo plant with an aqueous solvent to obtaine an extract, a step (b) of removing the aqueous solvent from the extract to obtain a dried substance, and a step (c) of dissolving the dried substance in water to obtain a water soluble fraction. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a composition having antiviral activity and use thereof, and specifically to an antiviral composition containing a bamboo extract and use thereof.

  Viruses have so far threatened the survival of many lives. For example, pandemic outbreaks of new influenza viruses are always a concern, and once a pandemic occurs, it is predicted that many lives will be lost. In addition, half of the causes of food poisoning are caused by norovirus, and life is often lost due to food poisoning.

  Viruses with high infectivity, such as influenza viruses and noroviruses, tend to cause mass infection, and tend to become severe when these viruses are infected. Therefore, how to prevent infection with these viruses is an important issue. Under such circumstances, various antiviral drugs are being developed.

  For example, Patent Document 1 describes a pharmaceutical composition containing a phenol derivative. More specifically, in Patent Document 1, n-octyl gallate artificially produced by chemical synthesis exhibits an antiviral effect against herpes simplex virus type 1, influenza virus A, poliovirus type 1, and the like. It is described.

  However, an antiviral drug that is artificially produced by chemical synthesis as described in Patent Document 1 exhibits an immediate effect, but is resistant to the antiviral drug when used over a long period of time. It has a problem that the virus shown is easily generated. The antiviral drugs as described above have been developed mainly for the purpose of killing viruses that have infected host cells. For this reason, when such an antiviral drug is used, not only the virus but also the host cell infected with the virus is killed, resulting in a side effect.

  Therefore, instead of using an artificially produced antiviral drug by chemical synthesis, an attempt has been made to screen a substance exhibiting an antiviral effect from a group of substances originally existing in nature.

  For example, Patent Document 2 describes that a p-benzoquinone derivative, which is an extract component of bamboo, exhibits antifungal, antibacterial and antiviral properties.

  Patent Document 3 describes that an extract component of a gramineous plant exhibits antibacterial and antiviral properties.

JP 2006-306836 A (published on November 9, 2006) JP 2003-290613 A (released on October 14, 2003) JP 2006-320310 A (published November 30, 2006)

  However, the conventional antiviral drugs screened from the natural world have a problem that the virus cannot be effectively inactivated.

  For example, Patent Document 2 describes that a p-benzoquinone derivative, which is an extract component of bamboo, has antiviral properties. However, in Examples, the growth inhibitory effect of p-benzoquinone derivative on Escherichia coli O-81 is limited. Not considered. However, since the mechanisms of growth and the like are completely different between bacteria and viruses, it is not possible to conclude that a p-benzoquinone derivative has an effect of inactivating viruses in the same way because of its growth-inhibiting effect on bacteria. Is possible.

  Patent Document 3 describes that an extract component of a gramineous plant has antiviral properties, but only an effect on protozoa has been studied in Examples. Since the protozoa and the virus are completely different, just because they are effective against the protozoa, it is not known whether the extracted components are effective against the virus as well.

  That is, it can be said that, as in Patent Documents 2 and 3, there are almost no materials that have been demonstrated to have antiviral activity for naturally-occurring components that are conventionally considered to have an antiviral effect.

  This invention is made | formed in view of said problem, The objective is to provide the antiviral composition which can inactivate a virus effectively, and its utilization.

  It has been known for a long time that the extract of Moso bamboo epidermis and bamboo extract has an antibacterial effect (for example, A. Nishina, K. Hasegawa, et al.: J. Agric. Food Chem. 1991; 39: 266- 269 or A. Nishina, T. Uchibori,: Agric. Biol. Chem. 1991; 55 (9): 2395-2398). However, as described above, it was completely unknown whether or not the bamboo plant extract shows an antiviral effect.

  As a result of intensive studies in view of the above problems, the present inventors have found that bamboo extract that is extracted with an aqueous solvent and is soluble in water has antiviral activity, and the present invention has been developed. It came to complete. Furthermore, it has also been found that the bamboo extract has a wide range of antiviral activity not only against influenza virus but also against feline calicivirus (a related species of norovirus).

  That is, the present invention includes the following inventions.

In order to solve the above problems, the antiviral composition of the present invention contains a bamboo extract, and the bamboo extract is obtained (or obtained) by a method including the following steps (a) to (c). It can be obtained or obtained). That means
(A) a step of obtaining an extract by extracting bamboo plants with an aqueous solvent;
(B) removing the aqueous solvent from the extract to obtain a dried product; and
(C) A step of dissolving the dried product in water to obtain a water-soluble fraction.

  In the antiviral composition of the present invention, in the step (c), the dried product is preferably dissolved in water having a temperature of 60 ° C. or higher.

  In the antiviral composition of the present invention, the pH is preferably 3.0 or less.

  In the antiviral composition of the present invention, the dried product is preferably contained in an amount of 1% by weight or more.

  In the antiviral composition of the present invention, the bamboo plant belongs to the genus Mushroom, Narihirada, Tochiku, Okamesa, Sasa, Azumasasa, Yadatake, Medaka, Kanchiku, Houraichiku of the family Gramineae. Bamboo or cocoon to which it belongs is preferred.

  In the antiviral composition of the present invention, the aqueous solvent is preferably ethanol, methanol, n-propanol, isopropanol, or n-butanol.

  The antiviral composition of the present invention is preferably used against influenza virus, feline calicivirus or norovirus.

  In order to solve the above-mentioned problems, the method for inactivating the virus of the present invention is characterized by including a step of administering any of the above antiviral compositions to the virus.

The kit for inactivating the virus of the present invention comprises a bamboo extract obtained by a method comprising the following steps (a) to (c) in order to solve the above-mentioned problems: Yes. That means
(A) a step of obtaining an extract by extracting bamboo plants with an aqueous solvent;
(B) removing the aqueous solvent from the extract to obtain a dried product; and
(C) A step of dissolving the dried product in water to obtain a water-soluble fraction.

  The antiviral composition of the present invention can inactivate various viruses such as influenza virus, feline calicivirus and norovirus.

  Moreover, since the antiviral composition of this invention contains a natural bamboo extract component as an active ingredient, it is very safe with respect to a test subject. That is, according to the present invention, it is possible to provide a highly safe and effective antiviral composition.

  Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited to this, and can be implemented in a mode in which various modifications are made within the described range. Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, “A to B” indicating a numerical range indicates “A or more and B or less”.

[1. Antiviral composition)
The antiviral composition of this embodiment contains the bamboo extract obtained by the method mentioned later as an active ingredient. In the present specification, the “antivirus” is intended to inactivate a virus, for example, to inhibit infection of a virus to a host by damaging the virus. Therefore, in the present specification, “having antiviral activity” is intended to have an activity of preventing and suppressing infection of a virus to a host. Whether or not it has “antiviral activity” can be confirmed using, for example, a virus infectivity titer measured by a conventionally known method such as the TCID ₅₀ method as an index.

  The virus that can be targeted by the antiviral composition of the present embodiment is not particularly limited. For example, poxvirus, herpesvirus, adenovirus, parvovirus, baculovirus, piconal virus, togavirus, retrovirus, Mention of orthomyxovirus, paramyxovirus, rhabdovirus, reovirus, coronavirus, SARS coronavirus, AIDS virus, influenza virus, tobacco mosaic virus, potyvirus, comovirus, kaurimovirus, feline calicivirus or norovirus it can. Among these, influenza virus, feline calicivirus, or norovirus is more preferable, but not limited thereto. The feline calicivirus is a related species of norovirus and is often used in experiments as a substitute virus for norovirus.

  Moreover, the antiviral composition of this embodiment may contain other components (for example, a pharmaceutically acceptable carrier) in addition to the active ingredients. Ingredients other than the active ingredient can be appropriately designed by those skilled in the art according to the application site and application form of the antiviral composition of the present embodiment. The antiviral composition of this embodiment may be used alone or in combination with other substances or compositions.

  The antiviral composition of this embodiment contains a bamboo extract. And, the bamboo extract is (a) a step of obtaining an extract by extracting a bamboo plant with an aqueous solvent, and (b) a step of removing the aqueous solvent from the extract to obtain a dried product, And (c) obtaining a water-soluble fraction by dissolving the dried product in water. Below, each process (a)-(c) is demonstrated.

(1-1. Process (a))
Step (a) is a step of obtaining an extract by extracting a bamboo plant with an aqueous solvent.

  Examples of the above “bamboo plant” include bamboo belonging to the genus Mushroom, Narihirada, Tochiku, Okamezasa, Sasa, Azamasa, Yadatake, Medaka, Kantiku, Horaichiku, etc. The present invention is not limited to these examples. Of these, Mosouchi, Madoake, or Hachiku belonging to the genus Mushroom are preferable, and Mosouchi is most preferable.

  As a raw material for the bamboo extract, it is more preferable to use the skin and / or bamboo of the bamboo plant rather than the whole bamboo plant described above. Here, “Bamboo” is in the range of about 0.1 to 0.5 mm from the epidermis and is a yellow-green color located between the green bamboo skin layer and the white or creamy flesh. Refers to the site. Many bioactive substances are contained in the skin of bamboo plants and bamboo. And if these parts are used, the antiviral composition of this embodiment can be produced more efficiently.

  In the case of a bamboo plant with a thin stem, it is not practical to separate only the epidermis or only bamboo. For this reason, the whole stem including the epidermis, bamboo, and the fleshy part, or the whole bamboo including the stem and leaf can also be used as a raw material for the bamboo extract.

  In the step (a), the above-described bamboo plant or the portion of the bamboo plant is extracted with an aqueous solvent. In the present specification, the “aqueous solvent” is intended to be a polar solvent having a high polarity such as water.

  Although it does not specifically limit as a specific structure of the said aqueous solvent, It is preferable that it is a C1-C4 alcohol or a mixed solution of C1-C4 alcohol and water. Examples of the alcohol having 1 to 4 carbon atoms include methanol, ethanol, n-propanol, isopropanol, and n-butanol. 1 type of C1-C4 alcohol may be used independently, and may use 2 or more types together. Most preferably, the alcohol having 1 to 4 carbon atoms is ethanol. If it is the said structure, while being able to extract the substance which shows an antiviral effect effectively, an aqueous medium can be easily removed from an extract.

  When using the mixed solution of C1-C4 alcohol and water as an aqueous solvent, the ratio of the alcohol in the whole aqueous solvent is not limited. For example, the proportion of alcohol contained in the aqueous solvent is preferably 60% by volume or more, and more preferably 80% by volume or more.

  When using a C1-4 alcohol or a mixed solution of C1-4 alcohol and water, compared with using a water-insoluble solvent such as chloroform or ethyl acetate or a water-soluble solvent such as acetone. Thus, the obtained bamboo extract has excellent antiviral properties. C1-C4 alcohol or a mixed solution of C1-C4 alcohol and water has better permeability into bamboo plant fiber tissue than other solvents, and is effective against antiviral components. And the solubility is high, the extraction effect can be improved.

  The method for extracting the bamboo plant with an aqueous solvent to obtain an extract is not particularly limited, and it can be appropriately extracted based on a known method. In the above extract, the bamboo plant residue may or may not be mixed, but considering the ease of administration of the antiviral composition to the virus, the residue is It can be said that it is preferable that no contamination occurs. An example of the method is shown below, but the present invention is not limited to this.

  For example, the method for extracting a bamboo plant preferably includes an immersion step of immersing the bamboo plant in an aqueous solvent, and a removal step of removing the bamboo plant residue from the extract after the immersion step. .

  The said immersion process is a process of immersing a bamboo plant in an aqueous solvent. By this step, the antiviral component contained in the bamboo plant (skin and / or bamboo) is transferred from the bamboo plant fiber tissue to the aqueous solvent and extracted.

  Although the temperature which performs the said immersion process is not specifically limited, For example, it is preferable to carry out at room temperature-70 degreeC. Although the time for performing the immersion step is not particularly limited, for example, it is preferably performed for 30 minutes or more, and more preferably for 1 to 2 hours. From the viewpoint of improving the extraction efficiency, the dipping process is preferably performed while stirring. The amount of the aqueous solvent used in the dipping process is not particularly limited as long as it is an amount that can completely immerse the bamboo plant. For example, if the powdery extract is 100 g to 200 g, about 1000 mL of the aqueous solvent is used. It can be said that it is preferable.

  The removal step is a step of removing bamboo plant residues from the extract obtained after the dipping step. The removal step is not particularly limited as long as it is a step capable of removing bamboo plant residues from the extract. For example, the bamboo plant residue (in other words, insoluble matter) can be removed from the extract by filtration through a filter or centrifugation. The extract thus obtained may be used as it is in the next step (b), but after adding, for example, activated carbon to the extract and further stirring, the activated carbon is removed by filtration or centrifugation. Thus, impurities such as coloring components and odor components peculiar to bamboo plants can be further removed from the extract.

  As described above, the method for extracting bamboo plants may include a dipping step and a removing step, but preferably further includes a steam treatment step and / or a fragmentation step before the dipping step. Below, a water vapor treatment process and a fragmentation process are demonstrated.

  The steam treatment process is a process of treating a bamboo plant with steam, and the hard fiber tissue of the bamboo plant is decomposed or expanded by the process. As a result, the extraction efficiency of the antiviral component can be increased.

  For example, when a volatile substance or protein is an antiviral component, its antiviral activity is lost by heat treatment. However, as the inventors have clarified in the examples, the antiviral composition of the present embodiment is stable even at high temperatures, and the antiviral effect is not impaired. Therefore, in the steam treatment step, the antiviral component extraction effect can be enhanced without impairing the antiviral effect.

Although the specific structure of the said water vapor treatment process is not specifically limited, For example, while putting a bamboo plant into a pressure-resistant container, sealing the said pressure-resistant container, it is preferable to carry out by blowing water vapor | steam in it. Although the processing temperature of a water vapor treatment process is not specifically limited, For example, it is preferable that it is 120 to 180 degreeC, and it is still more preferable that it is 130 to 170 degreeC. Although the pressure in the said pressure vessel is not specifically limited, For example, it is preferable that it is 3-7 kg / cm < ² >. Moreover, the treatment time of the water vapor treatment step is not particularly limited, but for example, it is preferably 30 minutes to 5 hours, and more preferably 1 to 4 hours. The bamboo plant after the steam treatment step is preferably cooled to room temperature, and then subjected to the next step.

  The fragmentation step is a step of fragmenting bamboo plants into chips or powders in order to facilitate extraction of antiviral components from bamboo plants. Note that the fragmentation step can be performed before the steam treatment or can be performed after the steam treatment.

  The specific configuration of the fragmentation step is not particularly limited. For example, when only the epidermis is used as the extraction raw material, the bamboo is contacted with the cutting blade of the cylindrical polishing machine while rotating the stem. This can be achieved by polishing the outer peripheral part. When only bamboo is used as a raw material for extraction, for example, it can be achieved by stripping the skin of bamboo, cutting the portion of bamboo with a scissors, cutting it into small pieces, drying it, and pulverizing it with a fine pulverizer. obtain.

  The extract obtained as described above is subjected to the next step (b).

(1-2. Step (b))
Step (b) is a step of removing the aqueous solvent from the extract obtained in step (a) to obtain a dried product. Here, the “dried product” is intended to mean an extract that is solidified or semi-solidified by removing the liquid aqueous solvent.

  The step (b) may be any step that can remove the aqueous solvent from the extract, and its specific configuration is not particularly limited. For example, a vacuum distillation method, a freeze drying method, a spray drying method, or the like can be used, but the method is not limited to these. Among these, a vacuum distillation method or a freeze drying method can be suitably used.

  According to the said structure, since an aqueous solvent is removed from the antiviral composition of this embodiment, an antiviral composition with high safety | security for a biological body can be produced. Moreover, according to the said structure, since an aqueous solvent can be removed reliably, in the process (c) mentioned later, the substance with high solubility to water can be dissolved in water more selectively.

(1-3. Step (c))
Step (c) is a step of obtaining a fraction dissolved in water (water-soluble fraction) after dissolving the dried product obtained in step (b) in water. By the step (c), a substance having low solubility in water is removed from the antiviral composition of this embodiment. That is, the substance contained in the antiviral composition of this embodiment can be mainly made into a water-soluble substance by the said process (c).

  Step (c) may be any step that can dissolve the dried product obtained in step (b) in water, and its specific configuration is not particularly limited. For example, the step (c) is preferably a step of adding water to the dried solid and then stirring.

  The amount of water to be added to the dried product is not particularly limited. For example, it is preferable to add water having a mass 20 times or more the mass of the dried product. If it is the said structure, the substance of the dry solid with high solubility to water can be reliably dissolved in water. The upper limit value of the added water is not particularly limited, and may be set so that the antiviral activity per unit volume does not become too low.

  The temperature of water added to the dried product is not particularly limited. For example, it is preferably 60 ° C. or higher, more preferably 70 ° C. or higher, further preferably 80 ° C. or higher, and 85 ° C. or higher. It is more preferable that the temperature is 90 ° C. or higher. In other words, the step (c) is preferably a step of dissolving the dried product in water at the above temperature. According to the said structure, the antiviral activity of an antiviral composition can be raised. Moreover, according to the said structure, the substance which is easy to volatilize by high temperature can be removed from an antiviral composition. Moreover, according to the said structure, the substance which is easy to modify | denature by high temperature can be denatured. That is, according to the said structure, the unwanted effect of the unnecessary substance contained in an antiviral composition can also be suppressed.

  As described above, it is preferable to stir the mixture of water and dry matter after adding water to the dry matter. According to the above configuration, the water-soluble substance contained in the dried product can be more easily dissolved in water. In addition, what is necessary is just to perform stirring using a well-known structure suitably.

  The step (c) further preferably includes a step of removing a water-insoluble substance (in other words, an insoluble substance) from the obtained water / dried mixture. Although it does not specifically limit as a specific structure of the said process, For example, it is preferable to remove a water-insoluble substance from the mixture of water and a dry-solid thing by filtration or centrifugation. According to the said structure, the substance contained in the antiviral composition of this embodiment can be mainly made into a water-soluble substance. In other words, since a water-insoluble substance unrelated to the antiviral activity can be removed from the antiviral composition, the antiviral effect of the antiviral composition of the present embodiment can be increased. Moreover, according to the said structure, since a water-insoluble substance is removed, it becomes easy to administer the antiviral composition of this embodiment to a biological body etc.

  The bamboo extract obtained as described above can be used as it is as the antiviral composition of the present embodiment, or can be used as the antiviral composition of the present embodiment after adding other substances. Is possible.

  The antiviral composition of this embodiment preferably has a pH of 3.0 or less, and more preferably has a pH of 2.5 or less. If it is the said structure, compared with the case where pH is not adjusted, the antiviral activity of an antiviral composition can be improved more.

  The method for adjusting the pH of the antiviral composition is not particularly limited. For example, it is preferably performed by adding a buffer solution capable of adjusting the pH to an acidic value to the bamboo extract. Although it does not specifically limit as said buffer solution, For example, it is preferable that they are a citrate buffer solution, an acetate buffer solution, a tartrate buffer solution, or a lactic acid buffer solution. According to the said structure, pH of an antiviral composition can be adjusted to a desired value easily and stably. From the viewpoint of low toxicity to a living body and easy handling, it can be said that among the above buffer solutions, a citrate buffer solution or a lactic acid buffer solution is preferable.

  The antiviral composition of the present embodiment preferably contains 1% by weight or more of the dried product, more preferably 2% by weight or more, further preferably 3% by weight or more, and more preferably 4% by weight. The above content is more preferable, and the content is more preferably 5.0% or more. If it is the said structure, a virus can be inactivated effectively. In step (c) described above, it can be said that it is preferable to add water to the dried solid so as to have such a concentration.

  As described above, the antiviral composition of the present embodiment can be produced.

  In addition, as shown also in the Example (for example, refer to Experimental Example 6), the antiviral composition of the present embodiment has a higher antiviral effect than the p-benzoquinone derivative alone. Therefore, the antiviral composition of the present embodiment is considered to contain a novel antiviral component.

  In general, when a virus is inactivated using a drug, it is considered that the sensitivity to the drug varies depending on whether or not the virus has an envelope. Specifically, since envelopes are relatively easily destroyed by drugs, viruses with envelopes such as influenza viruses tend to be inactivated, and viruses without envelopes such as noroviruses are influenza viruses. It is thought that it is hard to be inactivated compared with. However, as clearly shown in the Examples, the antiviral composition of the present invention can inactivate such various viruses.

  Since the antiviral composition of this embodiment contains a bamboo extract component as a main component, it is very safe for a subject (for example, a virus host). For this reason, the antiviral composition of this embodiment may be administered orally to a subject or may be administered parenterally. Of course, the antiviral composition of this embodiment may be administered directly to the virus.

  The antiviral composition of this embodiment is preferably an external preparation for parenteral administration to the skin or mucous membrane. Here, the “external preparation” is intended to be administered to the skin or mucous membrane, and the skin is intended to include skin of the face, neck, chest, back, arms, legs, hands, scalp, etc. As such, intranasal mucosa and oral mucosa are intended.

  Examples of the external preparation include solid, semi-solid or liquid preparations, and can be provided as, for example, an ointment (eg, oily ointment, hydrophilic ointment), an emulsion (eg, emulsion, lotion, etc.), and the like. Further, the external preparation may be in the form of an aerosol.

  The antiviral composition of this embodiment may be used in a so-called drug bath, and in this case, it is preferably in the form of a liquid preparation. Moreover, in order to apply | coat easily to the whole body, it is preferable that the antiviral composition of this embodiment is a form of an emulsion.

  As the base component of the ointment, for example, fats, polyhydric alcohols, hydrocarbons and the like can be used, and a surfactant and the like can be added. In order to utilize antibacterial activity due to hydrogen peroxide, the ointment is preferably hydrophilic, but the present invention is not limited thereto. The oily ointment may be one based on an oily base, or one based on an oil / water or water / oil type emulsion base. The oily base is not particularly limited, and examples thereof include vegetable oils, animal oils, synthetic oils, fatty acids, and natural or synthetic glycerides.

  The antiviral composition of the present embodiment can also be used as a disinfecting solution or a cleaning solution for medical instruments such as scissors, scalpels, and catheters.

[2. Method of inactivating virus
The method for inactivating the virus of this embodiment includes the step of administering the antiviral composition of the present invention described above to the virus.

  The method for administering the antiviral composition of the present invention to a virus is not particularly limited, and the antiviral composition may be directly administered to the virus, or the antiviral composition may be administered to the virus host. The host is not particularly limited, and may be an individual organism, a tissue of an organism, or a cell.

  For example, if the antiviral composition of the present invention is in the form of a liquid preparation, it may be administered to the skin or mucous membrane of a subject by application, spraying, suction, etc., and the antiviral composition of the present invention is in the form of an ointment. If so, it may be administered to the skin or mucous membrane of a subject by application or the like, but the present invention is not limited thereto.

  The method for inactivating the virus of this embodiment may include steps other than those described above. Specific examples of these steps are not particularly limited, and examples include, but are not limited to, a step of detecting the number and type of viruses that have not been inactivated. The step of detecting the number and type of viruses that have not been inactivated can be appropriately performed according to a known method.

[3. Kit for virus inactivation]
The kit for inactivating the virus of this embodiment is characterized by comprising a bamboo extract. The specific configuration of the “bamboo extract” is the same as that described in “1. Antiviral composition”, and will not be described here.

  In addition to the bamboo extract, the kit of this embodiment may include other components such as a solvent for dissolving the bamboo extract. The kit of this embodiment may be provided by mixing bamboo extract and other components in the same container, or may be provided in separate containers. It may also contain one or more containers (eg, vials, tubes, ampoules, bottles, etc.) for storing the components making up the kit. Moreover, you may provide the instruction manual for using the kit of this embodiment.

  The kit of this embodiment is provided with the bamboo extract as described above. Therefore, if the kit of this embodiment is used, a virus can be inactivated easily.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and the present invention can be obtained by appropriately combining technical means disclosed in different embodiments and examples. Such embodiments are also included in the technical scope of the present invention.

  In the examples, “%” representing the concentration of Moso bamboo extract water represents the ratio (% by weight) of the weight of the dried product to the weight of purified water to be dissolved. Each test was performed three times independently to confirm reproducibility.

[1. Preparation of water extract
(1) The surface of Mosouchiku and bamboo were polished using a cylindrical polishing machine (product name: SKC-10, Amitek Co., Ltd.) to collect polishing powder. The particle size of the abrasive powder was about 355 μm (42 mesh) or less.
(2) 30 g of Moso Chiku abrasive powder was immersed in 200 g of the extraction solvent and stirred at room temperature to 60 ° C. for 3 hours or more. Ethanol was used as the extraction solvent.
(3) The immersion liquid obtained in the above step (2) was subjected to solid-liquid separation by a centrifugal separation method to remove residues.
(4) Ethanol was distilled off from the ethanol extraction solution obtained in the above step (3) under reduced pressure to obtain a paste-like dried product (Mosouchiku extract).
(5) 100 g of purified water was added to 5.00 g of the dried product obtained in the above step (4), and the mixture was stirred for 1 hour while heating to 85 ° C. to completely dissolve the dried product. % Solution was obtained.
(6) After cooling the solution obtained in the above step (5) to room temperature, it is centrifuged (3000 rpm, 10 minutes), and the supernatant obtained by centrifugation is further treated with a 0.45 μm membrane filter (product name). : Minisart, manufactured by Sartorius Stedim biotech) to obtain Moso bamboo extract water.

  The obtained Moso bamboo extract water was a yellow transparent liquid, pH 3.8-4.2, specific gravity 1.000 (15 degreeC). In the obtained Moso bamboo extract water, the content of 2,6-dimethoxy-1,4-benzoquinone which is the main component of the Moso bamboo extract was analyzed by HPLC and found to be 30 μg / ml.

  The analysis conditions of HPLC are shown below.

Column: A stainless steel tube with a diameter of 6.0 mm and a length of 200 mm filled with cyanopropyl chemically bonded silica gel (ERC CN-1171 manufactured by Yokohama Rika Co., Ltd.)
Mobile phase: hexane / ethanol mixed solution (volume ratio 49: 1)
Detection wavelength: 286 nm.

[2. Effect test on feline calicivirus]
As a test product, the Moso bamboo extract water obtained above was used. Since the culture method of norovirus has not been established, feline calicivirus (F9 strain), which is a related species of norovirus recommended by the US Environmental Protection Agency, was used to examine the effect of moso bamboo extract water.

First, in a culture bottle, feline kidney cells (CRFK cells) were monolayer cultured using DMEM (F10). The culture solution in the bottle was removed, feline calicivirus (FCV) was inoculated, and adsorbed to feline kidney cells at 37 ° C. for 1 hour. The inoculum was removed, FCS Free DMEM was added, and cat kidney cells were cultured in a CO ₂ incubator (5% CO ₂ ) at 37 ° C. for 5 days. After confirming the cytopathic effect (CPE) under a microscope, the culture bottle was freeze-thawed twice and centrifuged (2,500 rpm, 5 min). The obtained supernatant was further centrifuged (15,000 rpm, 20 min) to obtain a crude virus solution. The obtained crude virus solution was overlaid on 30% sucrose, and then centrifuged (35,000 rpm, 4 ° C., 1.5 hr) to remove the supernatant. The sediment was resuspended in sterilized water to obtain a purified virus solution.

  Details of the test method are shown below.

  (1) Feline calicivirus was diluted 10-fold with sterile phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA).

(2) CRFK cell suspension (2.5 × 10 ⁵ cells / ml) 0.1 ml is seeded in each well of a 96-well microplate (300 μl / well) and cultured overnight in a 37 ° C. CO ₂ incubator. did. After culturing, the culture solution is removed, MEM (F0) (manufactured by Wako Pure Chemical Industries, Ltd.) is added at 0.2 ml / well, and CRFK cells are rinsed twice. Further, 0.2 ml / well of the culture solution is added to each well. In addition, a monolayer culture plate of host cells was prepared.

  (3) 0.9 ml of the test product (Mosouchi extract water) and 0.1 ml of the virus solution were mixed in a test tube and reacted at 25 ° C. for a certain time. As a negative control, the same test was performed using sterile PBS instead of the test product.

  (4) Dilute the test product-virus mixture 10-fold, 100-fold, 1000-fold, etc. 10 times using a sterile MEM (F1) medium containing antibiotics, and add 0.1 ml of each diluted solution. The host cells prepared in the above step (2) were inoculated (4 wells were inoculated for each dilution step).

(5) The plate inoculated with the virus in the above step (4) was cultured in a CO ₂ incubator at 37 ° C. for 5 days, and the denaturation of CRFK cells was observed with a microscope to confirm the presence or absence of virus growth. Then, the 50% tissue culture infectious dose (TCID ₅₀ ) in the reaction solution is changed to the method of Reed and Muench et al. (See Am. J. Hyg., 27, 493-497, 1938). Based on the calculation.

[3. (Effectiveness test against avian influenza virus)
As a test product, the Moso bamboo extract water obtained above was used. H5N3 type avian influenza virus (A / whistling swan / Shimane / 499/83) was used as the avian influenza virus. This avian influenza virus was isolated from wild swan in Shimane Prefecture in 1983.

  The virus was inoculated into the chorioallantoic cavity of a 10-day-old chicken egg, cultured at 37 ° C. for 2 days, and then aseptically recovered chorioallantoic fluid was used as a viral fluid for the test.

Details of the test method are shown below.
(1) Avian influenza virus was diluted 10-fold with sterile PBS and used as a virus solution in the next step.
(2) 0.5 ml of the test product and 0.5 ml of the virus solution were mixed in a test tube and reacted at room temperature for a certain time. A similar test was performed using sterile distilled water instead of the test product as a negative control.
(3) 10-fold, 100-fold, 1000-fold, etc., 10-fold dilution of the test product-virus mixture solution using sterile PBS containing antibiotics, of which 0.1 ml is diluted with 10-day-old chicken eggs Inoculated into the allantoic cavity (5 inoculated at each dilution stage).
(4) After the cultured chicken eggs are cultured at 37 ° C. for 2 days, erythrocyte aggregation (HA) test (see Server, JL: Application of microtechnique to viral serological investigations. J. Immunol., 88: 320-329) The presence or absence of virus growth in the membrane cavity was confirmed. In addition, the 50% egg-infectious dose (EID ₅₀ ) in the reaction solution was determined using the method of Reed and Muench et al. (See Am. J. Hyg., 27, 493-497, 1938). Calculated.

(Experimental example 1)
[1. The presence or absence of antiviral activity against feline calicivirus was confirmed using 5% Moso bamboo extract water (pH 4.0) obtained in [Preparation of Moso bamboo extract water]. The reaction time between Moso bamboo extract water and the virus solution was 24 hours, and the reaction temperature was 25 ° C. The results are shown in Table 1.

  As shown in Table 1, when the virus infection titer after mixing 5% Mosouchi extract water (pH 4.0) and the virus solution and reacting for 24 hours was examined, the virus infection titer was below the detection limit. . On the other hand, feline calicivirus infection was observed when sterile PBS was used instead of 5% Mosouchi extract water (pH 4.0) as a negative control.

(Experimental example 2)
The effect of pH of mosouchi extract water on antiviral activity against feline calicivirus was examined. Moso bamboo extract water having a concentration of 5% of the dried solid was used, and the pH of moso bamboo extract water was adjusted using a citrate buffer. In addition, the reaction time between Moso bamboo extract water and the virus solution was 30 minutes and the reaction temperature was 25 ° C. As a negative control, pH 3.0 citrate buffer or sterile PBS was used instead of Moso bamboo extract water. The results are shown in Table 2.

  As shown in Table 2, a strong antiviral effect was observed when Moso bamboo extract water having a pH of 3.0 or less was used. When a pH 3.0 citrate buffer was used as a negative control, since feline calicivirus infection was observed, it can be said that the antiviral effect is derived from Mosouchi extract water. In general, alcoholic preparations for disinfection are considered to have an antiviral effect against feline calicivirus by lowering the pH, but it was speculated that stronger antiviral activity can be obtained when used in combination with Moso bamboo extract water.

(Experimental example 3)
The optimal concentration of dry matter in Mosouchi extract water to exert antiviral activity against feline calicivirus was investigated. Moso bamboo extract water was adjusted to pH 3.0. In addition, the reaction time between Moso bamboo extract water and the virus solution was 30 minutes, and the reaction temperature was 25 ° C. The results are shown in Table 3.

  Even when the concentration of the dried solid was 1%, an antiviral effect against feline calicivirus was observed compared to the negative control. Moreover, when the concentration of the dried solid was 5%, the virus infectivity titer was below the detection limit.

(Experimental example 4)
The optimal working time of Moso bamboo extract water to exert antiviral activity against feline calicivirus was investigated. 5% Moso bamboo extract water (pH 3.0) was used. As a negative control, sterilized PBS was used instead of Moso bamboo extract water, and sterilized PBS and virus solution were reacted for 0.5 to 30 minutes. The reaction temperature between Moso bamboo extract water and the virus solution was 25 ° C. The results are shown in Table 4.

  As shown in Table 4, when Mosouchi extract water and virus solution were allowed to act for 30 minutes, the virus infection titer was below the detection limit.

(Experimental example 5)
The effect of the temperature when dissolving the dried product in purified water on the antiviral effect against feline calicivirus was investigated. The dried solid was dissolved in purified water to a concentration of 5% and pH 3.0. The reaction time between Moso bamboo extract water and the virus solution was 30 minutes, and the reaction temperature was 25 ° C. The results are shown in Table 5.

  When the dried product was dissolved in purified water at 90 ° C., the infectivity titer decreased to below the detection limit. This is because, by dissolving the dried product in high-temperature purified water, the slightly water-soluble active ingredient contained in the dried product is easily dissolved in water, and substances that are likely to volatilize at a high temperature are removed or high temperature is removed. This is considered to be because an undesired effect of an unnecessary substance contained in a dried product is suppressed by modifying a substance that is easily denatured by the above.

(Experimental example 6)
Moso bamboo extract water contains 2,6-dimethoxy-1,4-benzoquinone as a main component. The 2,6-dimethoxy-1,4-benzoquinone may be a component exhibiting an antiviral effect. Therefore, the antiviral effect against feline calicivirus was compared between compound 2,6-dimethoxy-1,4-benzoquinone (hereinafter abbreviated as “DMBQ”) and Moso bamboo extract water. As Mosouchiku extract water, one having a dry solid concentration of 5% was used. The pH of Moso bamboo extract water or DMBQ solution was 3.0, the reaction time with the virus solution was 30 minutes, and the reaction temperature was 25 ° C. The results are shown in Table 6.

  5% Mosouchi extract water contains DMBQ at a concentration of 30 μg / ml. However, as shown in Table 6, compared with the virus infectivity when the DMBQ solution having the same concentration was used, 5% Moso bamboo extract water showed stronger antiviral activity. This indicates that other substances have antiviral activity along with DMBQ contained in the extract of Moso bamboo.

(Experimental example 7)
The presence or absence of antiviral activity against avian influenza virus was confirmed using 5% Mosouchi extract water whose pH was not adjusted. The results are shown in Table 7.

  As shown in Table 7, it is possible to inactivate the avian influenza virus until the viral infectivity falls below the detection limit by reacting 5% Moso bamboo extract water and avian influenza virus that have not been adjusted for pH for 2 hours. did it.

  The present invention is very safe for the human body and has excellent antiviral activity. Therefore, the present invention can be used in the fields of pharmaceuticals and hygiene products. More specifically, it can be used as an antiviral agent, a disinfectant or the like.

Claims (9)

An antiviral composition containing a bamboo extract, wherein the bamboo extract is obtained by a method including the following steps (a) to (c):
(A) a step of obtaining an extract by extracting bamboo plants with an aqueous solvent;
(B) removing the aqueous solvent from the extract to obtain a dried product; and (c) obtaining a water-soluble fraction by dissolving the dried product in water.   The antiviral composition according to claim 1, wherein in the step (c), the dried product is dissolved in water having a temperature of 60 ° C or higher.   The antiviral composition according to claim 1 or 2, wherein the pH is 3.0 or less.   4. The antiviral composition according to claim 1, wherein the dried product is contained in an amount of 1% by weight or more.   The bamboo plant is a bamboo or camellia belonging to the genus Mushroom, Narihirada, Tochiku, Okamesa, Sasa, Azamasa, Yadatake, Medaka, Kanchiku, Horaichiku of the family Gramineae The antiviral composition according to any one of claims 1 to 4.   The antiviral composition according to any one of claims 1 to 5, wherein the aqueous solvent is ethanol, methanol, n-propanol, isopropanol, or n-butanol.   The antiviral composition according to any one of claims 1 to 6, which is used for influenza virus, feline calicivirus or norovirus. A method for inactivating a virus comprising the steps of:
A method comprising the step of administering the antiviral composition according to any one of claims 1 to 7 to a virus. A kit for inactivating a virus,
A kit comprising a bamboo extract obtained by a method comprising the following steps (a) to (c):
(A) a step of obtaining an extract by extracting bamboo plants with an aqueous solvent;
(B) removing the aqueous solvent from the extract to obtain a dried product; and (c) obtaining a water-soluble fraction by dissolving the dried product in water.