JP7175467B2 - Antiviral agents and antiviral compositions against norovirus and its alternative viruses - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law Published by: Yukie Fujisaka, Satoshi Shimamoto, Hirofumi Nariya, Mitsufumi Yoshida, Ryo Sonehara, Toru, Sei Shimamoto Publisher: Teiichi Nishikawa, President of the 39th Annual Scientific Meeting of the Japanese Society for Food Microbiology Publication: Abstracts of the 39th Annual Meeting of the Japanese Society for Food Microbiology, page 129 Publication date: August 31, 2018

The present invention relates to antiviral agents and antiviral compositions effective against norovirus or its surrogates. The present invention also relates to respective uses of extracts of fruits such as persimmon and seeds such as dates.

Norovirus is a positive, single-stranded RNA virus with a diameter of about 30 nm, which is classified in the genus Norovirus in the family Caliciviridae. Since it was discovered in the United States in 1968 as a pathogen of food poisoning and acute gastroenteritis, it has been detected in many countries around the world, and many cases of food poisoning caused by norovirus have been reported every year in Japan. Sources of infection include oysters and other bivalve-causing foods, and secondary infections from patient vomit and excrement. Norovirus is highly infectious and is said to be infected by more than 10 viruses.

In order to control the spread of norovirus infection, it is extremely important to prevent primary infection by heating causative foods, as well as prevent secondary infection by disinfecting cooking utensils and the fingers of cooks. However, norovirus is a virus (non-enveloped virus) that does not have a membranous structure called an envelope in the outer layer of the virus particle (non-enveloped virus). etc.) cannot be disinfected. Benzalkonium chloride and ethanol, which have been widely used as antibacterial agents in places where food is prepared and processed, such as restaurants, school lunch facilities, and factories, have not been found to be effective against norovirus, and chlorine bleach (hypochlorite Sodium phosphate, etc.), iodine agents (povidone iodine, etc.), aldehyde agents (glutaral, etc.), etc. have been considered effective to some extent. For example, heating at 85° C. or higher for 1 minute or treatment with sodium hypochlorite of 200 ppm or higher is officially recognized as a disinfection method for norovirus (Ministry of Health, Labor and Welfare). However, considering the safety of the human body, it is not appropriate to use these chemicals on workers' fingers or cooking utensils. is practically difficult.

From the above point of view, research and development of antiviral agents and antiviral compositions containing them, which are plant-derived extracts that are equivalent to food additives and are effective in disinfecting norovirus (or other non-enveloped viruses), are progressing. It is Active ingredients of antiviral agents against norovirus and the like include, for example, fruit extracts of plants of the genus Diospyros (Patent Documents 1 and 2), extracts of plants of the genus Chaenomeles represented by Chinese quince ( Patent Document 3), a plant extract of the genus Musa represented by banana (Patent Document 4), proanthocyanidins that are polyphenols derived from grape seeds (Patent Document 5), and chestnut astringent skin extract (Patent Document 6). proposed so far.

On the other hand, in Japan, dates (fruits of date palms) are used as a raw material for okonomiyaki sauce to bring out sweetness and richness, but the seeds are disposed of in large quantities as industrial waste. It has not been known that date seed extracts can be used as active ingredients of antiviral agents.

WO2008/153077 WO2009/123183 Japanese Unexamined Patent Application Publication No. 2013-087101 JP 2013-087102 A JP 2013-047196 A JP 2017-071585 A

The present invention provides an antiviral agent that has an excellent antiviral effect against norovirus (or its alternative virus) and is highly safe for the human body, and can be produced using raw materials that have not been effectively utilized in the past. The task is to provide

The present inventors have developed an extract of the fruits of plants belonging to the genus Diospyros, typified by Diospyros kaki (herein referred to as "persimmon extract"), and Phoenix dactylifera, typified by The extract of the seed of the plant of the genus Phoenix (referred to herein as the "date seed extract") was noted. And even if sufficient anti-norovirus action is not observed when the above two types of extracts are used alone at relatively low concentrations (for example, the infectious titer reduction value described later is less than 3), they can be used. When used in combination, unexpectedly sufficient anti-norovirus action is observed (for example, the infectious titer reduction value described later becomes 3 or more), that is, a dramatic (preferably synergistic) anti-norovirus action. It has been found that an enhancing effect is exhibited.

The present inventors further found that for the above synergistic effect, among the components contained in the date seed extract, tannins (high molecular weight tannins) with relatively high molecular weights that have the ability to aggregate with proteins such as BSA are unnecessary. , that is, tannins with a relatively low molecular weight (low-molecular-weight tannins) or other ingredients that do not have the ability to aggregate with proteins such as BSA are important. While high-molecular-weight tannins seem to enhance anti-norovirus effects by interacting with capsid proteins of norovirus, components such as low-molecular-weight tannins that do not cause such interactions have the effect of enhancing anti-norovirus effects. It is surprising to have

That is, the present invention includes the following items.
[1]
An extract of fruit of Diospyros plant containing at least condensed tannins (hereinafter referred to as "oyster extract") and an extract of seeds of Phoenix plant containing at least tannins (hereinafter referred to as "persimmon extract") (referred to as "date seed extract").
[2]
Item 2. The antiviral agent according to Item 1, wherein the persimmon extract is an extract of fruits of Diospyros kaki.
[3]
Item 3. The antiviral agent according to Item 1 or 2, wherein the persimmon extract is extracted with an aqueous ethanol solution.
[4]
Item 4. The antiviral agent according to any one of Items 1 to 3, wherein the date seed extract is a date seed extract (Phoenix dactylifera).
[5]
Item 5. The antiviral agent according to any one of Items 1 to 4, wherein the date seed extract is extracted with an aqueous ethanol solution.
[6]
Item 6. The antiviral agent according to any one of Items 1 to 5, wherein the date seed extract is enriched for low-molecular-weight tannins.
[7]
Items 1 to 6, containing the date seed extract equivalent to 10 to 100 parts by weight of the seeds of the date palm relative to the persimmon extract equivalent to 1 part by weight of the fruit of the persimmon genus plant. Antiviral agent according to any one of the paragraphs.
[8]
Item containing the persimmon extract equivalent to 0.0005 to 0.1 parts by weight of the fruits of the persimmon genus plant with respect to the date seed extract equivalent to 1 part by weight of the seeds of the date palm plant 7. The antiviral agent according to any one of 1 to 6.
[9]
Item 9. The antiviral agent according to any one of Items 1 to 8 and at least one selected from the group consisting of alcohols, organic acids and/or salts thereof, surfactants, antibacterial agents, moisturizers and cosmetic oils and fats. An antiviral composition against norovirus or its surrogate virus, comprising:
[10]
Item 10. The antiviral composition according to Item 9, containing ethanol and/or propanol as the alcohol.
[11]
Item 11. The antiviral composition according to Item 9 or 10, containing at least one selected from the group consisting of lactic acid, citric acid, ferulic acid, and salts thereof as the organic acid and/or salt thereof.
[12]
Items 9 to 11 containing at least one selected from the group consisting of glycerin fatty acid ester, sucrose fatty acid ester, lauryl glucoside, sodium lauryl sulfate, ammonium lauryl sulfate, sodium laureth sulfate and sodium olefin sulfonate as the surfactant. The antiviral composition according to any one of .
[13]
Any one of items 9 to 12, which contains the antiviral agent containing the oyster extract corresponding to 0.05 to 1% by weight of the fruit of the persimmon genus plant relative to 100% by weight of the entire composition. The antiviral composition according to item.
[14]
Items 9 to 13, wherein the antiviral agent comprises the date seed extract corresponding to 1.25 to 2.5% by weight of the date seed plant seeds relative to 100% by weight of the entire composition. Antiviral composition according to any one of clauses.
[15]
A method for synergistically enhancing the antiviral action of each of the persimmon extract and the date seed extract alone against norovirus or its substitute virus by using the persimmon extract and the date seed extract in combination.

By using oyster extract and date seed extract in combination as antiviral agents, a synergistic effect is exhibited in the antiviral action against norovirus (or its alternative virus), and excellent antiviral action (antiviral activity) is exhibited. Therefore, even at such a low concentration that each alone does not exert a sufficient antiviral effect, it becomes possible to mix and blend it in a composition, so the use of oyster extract and date seed extract is increasing. spread.

In one aspect of the present invention, antiviral agents and antiviral compositions containing oyster extracts, which are already known to have excellent anti-norovirus action (antiviral activity), are improved from the viewpoint of coloring and coloring. expected to be possible. Conventionally, persimmon extracts are slightly colored due to persimmon condensation-type tannins. Such coloring and color development were suppressed. However, in the present invention, the date seed extract from which the high molecular weight tannins have been removed, which is almost colorless and transparent but contributes to synergistically enhancing the antiviral action of the oyster extract, is used in combination with the oyster extract. can be used less. Therefore, in the production of antiviral agents and antiviral compositions, it is possible to more easily suppress the coloring and coloring of the following extracts, or to make the coloring and coloring inconspicuous.

In addition, date seeds, which have been treated as industrial waste until now, can be effectively used as a raw material for the date seed extract, which is one of the components of the antiviral agent of the present invention. It also makes a significant contribution in terms of cost for business operators.

Such an antiviral agent of the present invention and an antiviral composition containing it as an active ingredient can be used as a product for disinfecting norovirus and preventing infection in situations where food is handled, medical institutions, etc., in terms of performance and cost. It can be an excellent product with high competitiveness in both aspects.

The "antiviral effect" (also referred to herein as "anti-norovirus effect") of the antiviral agent of the present invention against norovirus (or its alternative virus) means that at least infection and proliferation of norovirus It includes the effect of incapacitating or inactivating Norovirus, and usually further includes the effect of reducing or substantially eliminating the genome of Norovirus. The antiviral effect is evaluated and confirmed, for example, by an infectious titer expressed as log PFU/mL or log _TCID50 /mL (reduced value when compared to the infectious titer of the control, Δlog PFU/mL or Δlog _TCID50 /mL). can do. In addition, among the antiviral effects, the effect of extinguishing the norovirus genome is, for example, the Ct value measured by real-time RT-PCR or the copy number of the norovirus genome gene converted from it (genomic survival rate when compared with the control ) can be evaluated and confirmed by These evaluation methods are well known and commonly used for antiviral effects against norovirus and other viruses, and can be carried out by standard techniques such as those shown in the examples of the present specification. The antiviral activity in the present invention may be evaluated by any of the reduction value of the infectious titer, the genome survival rate, or other methods. In this specification, the effect brought about by the antiviral action as described above is sometimes referred to as "disinfectant effect".

The antiviral action only needs to reach a level that achieves the intended purpose according to the application of the antiviral agent and antiviral composition of the present invention, and the strength of the action is not particularly limited. do not have. One criterion for general antiviral activity is whether the infectious titer reduction value (Δlog PFU/mL or Δlog TCID ₅₀ /mL) is 3 or more. As used herein, an antiviral agent or antiviral composition “has antiviral activity” if the “antiviral effect” results in an infectious titer reduction value of 3 or more (“anti-norovirus activity have”). In addition, considering that the reduction of the norovirus genome tends to underestimate the reduction of the infectious titer, when the infectious titer reduction value is 2 or more, it is evaluated as having "antiviral activity" (this specification ) is also possible.

In addition, in the antiviral agent of the present invention, the "synergistic effect" on the antiviral action of the combined use of oyster extract and date seed extract can be evaluated and confirmed by applying the FIC index (Fractional Inhibitory Concentration Index). can be done. The FIC index is a well-known and commonly used method for evaluating the effect of drugs on bacteria, and measures the MIC (Minimum Inhibitory Concentration) of each drug alone and the MIC when two drugs are used in combination. Then, the FIC index is calculated by a predetermined formula using it, and the value is compared with the standard to determine the presence or absence of a synergistic effect (see Examples below for the formula for calculating the FIC index and the criteria for determination). Since MIC cannot be measured for viruses, in the present invention, the concentration at which the infectious value reduction value (Δlog PFU/mL) is 3, or the concentration at which the infectious value reduction value is 2 in consideration of the above is MIC can be used as an alternative to

As disclosed in the Examples (including Reference Examples and Preparation Examples) of the present specification, the present invention provides a dramatic antiviral effect against both norovirus and alternative viruses of norovirus. A strong (synergistic) enhancing effect is observed. Since it has such an enhancing effect and antiviral activity against alternative viruses of norovirus (infectivity titer reduction value is 3 or more), the antiviral agent of the present invention also has antiviral activity against norovirus. can be presumed to have

FIG. 1 is a graph showing the tannin content of each date seed extract (DSE) with different ratios of date seed and extraction solvent, measured in Preparation Example 2. FIG. 2 is a graph showing the tannin content of each date seed extract (DSE) and each date fruit extract (DFE) with different extraction solvents, measured in Preparation Example 3 (n=3). FIG. 3 shows the antiviral action (infectivity titer) of the test solution containing each date seed extract (DSE) or each date fruit extract (DFE) alone with different extraction solvents, measured in Reference Example 2, against MS2 phage Decrease value) (n=3). FIG. 4 shows the antiviral action against norovirus of the test solution containing each date seed extract (DSE) alone with a different ratio of date seed and 50% ethanol aqueous solution as an extraction solvent, measured in Reference Example 3 (genome It is a graph showing survival rate). FIG. 5 shows the antiviral action against norovirus of the test solution containing each date seed extract (DSE) alone with a different ratio of date seed and 70% ethanol aqueous solution as an extraction solvent, measured in Reference Example 4 (genome It is a graph showing survival rate). FIG. 6 shows the antiviral effect of oyster extract (PE) alone, date seed extract (DSE) alone, or a test solution containing both PE and DSE measured in Example 1 on MS2 phage (infectivity titer decreased value) (n=3). Figure 7 shows the antiviral effect (infectious titer reduction value) on MS2 phage by combining oyster extract (PE) with date seed extract (DSE) in Example 1 (2. Verification of presence or absence of synergistic effect). It is a graph related to the evaluation of the synergistic effect in. [A] When the PE concentration was fixed at 0.05% and the DSE concentration was varied. [B] When the PE concentration was fixed at 0.1% and the DSE concentration was varied. Figure 8 shows the antiviral effect (decreased value of infectious titer) on MS2 phage by combining date seed extract (DSE) with oyster extract (PE) in Example 1 (2. Verification of presence or absence of synergistic effect). It is a graph related to the evaluation of the synergistic effect in. [A] When the concentration of DSE was fixed at 1.25% and the concentration of PE was varied. [B] When the concentration of DSE was fixed at 2.5% and the concentration of PE was varied. FIG. 9 shows the tannin content (right vertical axis) and the antiviral activity against MS2 phage (infection Figure 10 is a graph showing the results of valence reduction value, left vertical axis) (n=3). The photographs below the horizontal axis are photographs of each BSA-treated DSE, in which aggregated (precipitated) macromolecular tannins are observed. 10 is a photograph showing the color reaction by the vanillin hydrochloride method when the components of the date seed extract (DSE) and BSA-treated DSE in Preparation Example 5 were analyzed by extract thin layer chromatography (TLC). etc. FIG. 11 is a graph showing the antiviral action (infectious titer reduction value) against MS2 phage by persimmon extract (PE), BSA-treated date seed extract (DSE), and a mixture thereof, as measured in Example 2. There is (n=3). FIG. 12 is a graph showing the antiviral action (genome survival rate) against norovirus by oyster extract (PE), date seed extract (DSE), and mixtures thereof, measured in Example 3 (n = 4).

- Antiviral agent -
The antiviral agent of the present invention is against norovirus or its substitute virus and contains at least a condensed tannin-containing fruit extract (persimmon extract) of a plant belonging to the genus Diospyros, and at least a tannin. and an extract of seeds of plants of the genus Phoenix (date seed extract). Such an antiviral agent may consist of oyster extract and date seed extract alone, or in addition to these two extracts, suitable solvents and excipients, and norovirus and its surrogate viruses. It may contain a substance that can equalize (enhance) the antiviral action against.

Natural fruits of plants belonging to the genus Persimmon and seeds of plants belonging to the genus Date palm have the ability to aggregate proteins (metamorphic action, astringent action) and the ability to bind metal ions. It is rich in various compounds collectively called "tannins". Therefore, the oyster extract and the date seed extract of the present invention, which are those extracts, have tannins (for the oyster extract, Condensed tannins, high-molecular-weight tannins and low-molecular-weight tannins in the case of date seed extract can be considered to naturally contain tannins.

Generally, tannins can be broadly classified into "condensed tannins" and "hydrolyzed tannins". Condensed tannins include catechins (see the structural formula below) such as epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECg), and epigallocatechin gallate (EGCg) having a flavan skeleton. It is a compound condensed by C8 (or between C4-C6) carbon-carbon bonds. Condensed tannins are not hydrolyzed, but are also called proanthocyanidins because they produce anthocyanidins when heated under acidic conditions. On the other hand, hydrolyzable tannins have a structure in which a sugar alcohol such as glucose and a carboxylic acid (polyhydric phenol) such as gallic acid are linked via an ester bond, and are hydrolyzed. Condensed tannins and hydrolyzed tannins include polymers and oligomers with different structural unit structures and different numbers of bonds, ranging from high molecular weight to low molecular weight. Note that ECg and EGCg (respectively EC or EGC gallic acid esters whose ester bonds can be hydrolyzed) exemplified as structural units of condensed tannins are also classified as hydrolyzable tannins. Sometimes. In this specification, catechins such as EC, EGC, ECg, EGCg, and other non-polymerized compounds are also included in the broad definition of "tannin", particularly in "low-molecular-weight tannin".

Between oyster extract and date seed extract (and between extracts derived from other plant materials), the structure of compounds contained as tannin and the ratio (composition) of each compound are different, In this specification, tannins with their respective characteristic compositions are sometimes referred to as "persimmon tannins", "date seed tannins", and the like. Although the details will be described below, persimmon extracts (persimmon tannins) contain a large amount of condensed tannins, which are high-molecular-weight tannins, but only a small amount of other tannins. On the other hand, the date seed extract (date seed tannin) contains not only high-molecular-weight tannins but also relatively large amounts of low-molecular-weight tannins.

• Persimmon extract The persimmon extract used in the present invention is an aggregate of substances contained in the fruits of plants belonging to the genus Persimmon, typically persimmon, and has an anti-norovirus effect. Natural oyster extracts contain tannins and various other substances, but preferably contain at least oyster condensed tannins represented by the following estimated structural formula as substances having antiviral activity. There are various theories about the average molecular weight and the ratio of constituents of persimmon condensed tannins, but according to Li et al. EGC:EGCg:EC:ECg is reported to be 4:11:3:8.

Most of the tannins contained in persimmon extracts are usually condensed tannins, and only a small amount of hydrolyzed tannins and catechins such as EGC. The antiviral action of oyster extracts against norovirus etc. is thought to be mainly due to condensed tannins, and epigallocatechin gallate etc. alone shows almost no antiviral action against norovirus etc. Therefore, the oyster extract used in the present invention The higher the purity of the condensed tannins (the ratio of the condensed tannins to the total solid content of the oyster extract) is preferred. The condensed tannin is about 10 to 50% by weight, usually about 20 to 30% by weight, relative to the total solid content of the oyster extract obtained by the extraction method described later (as a raw material for the oyster extract, Based on the moisture-containing raw oyster fruit, it is contained at, for example, about 0.1 to 1% by weight). However, substances other than condensed tannins in persimmon extracts may enhance or assist the antiviral effects of persimmon extracts (condensed tannins, etc.) and/or date seed extracts against norovirus, etc. not excluded.

In addition, the persimmon extract of the present invention is rich in condensed tannins soluble in water and / or alcohol, conversely, condensed tannins are not insolubilized (insolubilized condensed tannins). Substantially free of tannins, or even if insoluble condensed tannins are present, the proportion thereof is preferably low). Persimmon condensed tannins, which are abundantly contained in so-called astringent persimmons, are basically soluble in water, etc., and exhibit the typical properties of tannins, such as giving an astringent taste in the oral cavity and aggregating proteins. be done. On the other hand, so-called sweet persimmons and astringent persimmons obtained by removing astringency using alcohol or carbon dioxide gas do not have an astringent taste because persimmon condensation-type tannins, which are originally soluble in water, are insoluble. The tannin properties mentioned above are less likely to appear. It is speculated that in the persimmon condensation-type insolubilization process, the formaldehyde generated by the ripening of the fruit and the astringency removal process further crosslinks the persimmon condensation-type tannins at the 8-position (see the reaction formula below, arrow represents the 8th site). Considering the antiviral activity according to the present invention, the behavior when blended in the composition, etc., the persimmon condensed tannin is preferably soluble in water and/or alcohol.

The raw material for the persimmon extract is not particularly limited, and various plants belonging to the genus Diospyros, typically the fruits of the persimmon (Diospyros kaki), can be used as raw materials. It is efficient and economical to use immature fruits of astringent persimmons (eg Hachiya and Hiratanashi varieties) rich in type tannin. Among fruits, especially the pulp portion contains abundant condensed tannins, but the whole fruit can be used as a raw material in a state where the peel and seeds are attached to the pulp.

For the persimmon extract, for example, astringent persimmons from which the husks have been removed are pulverized and squeezed to recover the squeezed juice, or cut into an appropriate size, blended to a liquid state, and centrifuged to recover the supernatant. method, or a method of extracting with an appropriate solvent and recovering the extract. Embodiments of the "persimmon extract" also include the squeezed liquid of persimmon fruit that has been subjected to the processing described later as necessary, and a mixed liquid of this with water and/or ethanol (or other alcohol). .

As the extraction solvent for the oyster extract, a common solvent used in preparing an extract from plant-derived raw materials can be used, such as water; methanol, ethanol, n-propanol, n-butanol, etc. polyhydric alcohols such as propylene glycol and glycerin; polar or dipolar solvents such as dimethylformamide and dimethylsulfoxide. Considering the antiviral action and safety to the living body, it is preferable to use a solvent containing water and/or ethanol (or other alcohol), such as an aqueous ethanol solution. The concentration of ethanol in the ethanol aqueous solution can be adjusted as appropriate, but is usually 10 vol% or more, preferably 30 vol% or more, more preferably 50 vol% or more, and usually 90 vol% or less, preferably 80 vol% or less, more preferably 70 vol%. % or less.

The ratio of the raw material of the oyster extract and the extraction solvent is determined so that the desired concentration of the extract can be obtained, preferably so that as many components (especially condensed tannins) in the raw material as possible can be extracted. It can be appropriately adjusted according to the extraction conditions.

The temperature, pressure, time, and other conditions for extracting the persimmon extract can also be adjusted as appropriate. The persimmon extract can be obtained, for example, by extraction treatment at room temperature and normal pressure for about 10 minutes to overnight, but heating, pressurization, or prolonged treatment may be used as necessary. The extraction process can be appropriately combined with a centrifugal separation process, a filtration process for the residue, and the like.

The method for preparing the persimmon extract is not particularly limited as long as it has an anti-norovirus effect, exhibits a synergistic effect when used in combination with the date seed extract, and exhibits anti-norovirus activity. However, in addition to the extraction step by the method as exemplified above, a heating/alcohol treatment step, a purification step, a freeze-drying step, a powderization step, and the like can be included as necessary. These steps are desirably carried out under conditions in which the anti-norovirus action of the oyster condensed tannins and other components contained in the oyster extract is not reduced as much as possible, and those skilled in the art can set such conditions as appropriate. can be done.

As the persimmon extract of the present invention, the extract of the fruit of the persimmon genus plant as described above can be used as it is, but it is desirable to use an extract that has been heated or treated with alcohol, particularly for the squeezed liquid. By such treatment, it is possible to suppress the rotting, coloring, generation of odor, etc. of the oyster extract (squeezed juice, etc.), and it is possible to make the oyster extract highly preservable and easy to use industrially. Since the enzyme derived from the fruit of the persimmon genus plant used as the raw material can be inactivated, it is possible to reduce the risk of interfering with the measurement for verifying the antiviral property (improper evaluation).

The temperature and time conditions for the heat treatment may be conditions that can deactivate the enzymes derived from plants of the genus Persimmon contained in the squeezed juice or the like. conditions can be adopted. That is, the temperature is usually 60 to 130° C., and the time is usually 5 seconds to 30 minutes. Do it. In addition, the aspect of the heat treatment step is not particularly limited. Heating and the like can also be considered a heat treatment step.

On the other hand, the alcohol treatment can be carried out using an alcohol such as ethanol, usually 10 to 90 vol%, preferably 30 to 80 vol%, more preferably 50 to 70 vol%. For example, it is preferable to add about the same amount of 95 vol% ethanol to the squeezed oyster juice (to make the concentration of ethanol in the whole treatment liquid 47.5 vol%), and then store it in a sealed light-shielding container. The alcohol-treated squeezed oyster juice thus obtained is usually a pale brown liquid. In addition, the aspect of the alcohol treatment step is not particularly limited. As one step of (2), adding alcohol to the oyster extract that has been heat-treated as described above and mixing the extract can be regarded as an alcohol treatment step.

When the oyster extract obtained by the above process is in the form of an extract, it is solidified by a freeze-drying process from the viewpoint of storage stability (prevention of deterioration such as coloring) and handling, and then powdered. It is preferable to pulverize the solidified product into powder. The freeze-dried powder of oyster extract prepared in this way is usually pale yellow. The lyophilized powder is also preferably stored frozen until use.

In addition, "Persimmon tannin" (product name / alias = persimmon juice, persimmon extract. Origin, manufacturing method, essence = Persimmon family persimmon (Diospyros kaki THUNB.) It is obtained by squeezing the juice from the oyster fruit, or by extracting with water or ethanol.The main ingredients are tannin and tannic acid.) is added to the persimmon extract of the present invention. may be used as

The persimmon extract in the present invention is a liquid obtained by squeezing immature astringent persimmons, fermenting and maturing for a long time (about 1 to 3 years), and containing solids and organic acids produced by fermentation. Containing "persimmon juice" (for example, containing about 10% by weight of solids relative to the total amount of liquid and about half of that, ie, about 5% by weight of condensed tannin) may be used. Such persimmon tannin has been conventionally used as a folk medicine, paint, etc., and is generally commercially available as a product. Such "persimmon tannin" may be obtained by adding a persimmon-derived yeast culture solution to astringent persimmon juice and fermenting the mixture at 20 to 25° C. for 1 to 3 months (usually a reddish brown liquid).

Furthermore, a substance that reproduces or mimics an extract of the fruit of a persimmon genus plant, for example, a component equivalent to an extract obtained from a persimmon genus fruit fruit (particularly equivalent content of condensed tannin) and an anti-norovirus effect It is possible to use an extract having the persimmon extract prepared from parts (leaves, bark, etc.) other than the fruits of plants belonging to the genus Persimmon or artificially prepared as the persimmon extract in the present invention.

- Date seed extract The date seed extract used in the present invention is an aggregate of substances contained in seeds of plants belonging to the genus Date palm, and has an anti-norovirus effect. Although natural date seed extract contains various substances, it preferably contains at least low-molecular-weight tannins (fractions obtained by removing high-molecular-weight tannins from total tannins) as substances having anti-norovirus activity. (ratio of low-molecular-weight tannins to the total solid content of the date seed extract) is preferably high. However, substances other than low-molecular-weight tannins in date seed extracts, such as high-molecular-weight tannins or other tannin-like compounds, may themselves have anti-norovirus activity, and date seed extracts (low-molecular-weight tannins) and/or oyster The possibility of enhancing or assisting the anti-norovirus action in the extract is not excluded.

As used herein, the term "tannin" contained in the date seed extract refers to a substance that can be quantified by the vanillin hydrochloride method, the Folin-Ciocalteu method, the EcCl ₂ method, or other measurement methods, preferably by the vanillin hydrochloride method. . "High-molecular-weight tannin" refers to a fraction of the above-mentioned "tannin" that has the ability to aggregate with BSA or other proteins, preferably BSA, in other words, a fraction that can be removed from a solution by reacting with BSA or the like. point to "Low-molecular-weight tannin" is a fraction of the above-mentioned "tannin" that does not have the ability to aggregate with BSA or the like, in other words, it remains dissolved in the solution (supernatant) when reacted with BSA or the like. refers to fractions. The “low-molecular-weight tannins” contained in the date seed extract specifically include at least epigallocatechin gallate and/or epicatechin. Generally, "high-molecular-weight tannins" are yellowish brown, and "low-molecular-weight tannins" are almost colorless. becomes.

As shown in the examples below, the synergistic effect in the present invention is also exhibited by the combined use of the oyster extract and the date seed extract containing the full fraction of tannins, but the oyster extract and It is also effective when combined with a fraction of the date seed extract from which "high-molecular-weight tannins" have been removed, that is, a fraction of "low-molecular-weight tannins". Therefore, the date seed extract in the present invention preferably contains at least low-molecular-weight tannins.

The date seed extract is preferably one enriched with low-molecular-weight tannins, that is, one in which the ratio (weight ratio) of high-molecular-weight tannins contained in the total tannins is reduced and the ratio of low-molecular-weight tannins is increased. In one aspect of the present invention, the date seed extract preferably contains substantially only low-molecular-weight tannins as tannins by removing high-molecular-weight tannins from the extracted total tannins by purification treatment or the like. The term "substantially" means that a trace amount (or below the detection limit) of molecular tannins that could not be completely removed by purification treatment or the like for removing high-molecular-weight tannins is allowed to remain. Such embodiments are able to reduce the high-molecular tannin coloration of date seed extracts while retaining a level of synergistic effect that is considered to have antiviral activity (infectious titer reduction value of at least 3). This is preferable because it can be done.

The means for reducing (removing) high-molecular-weight tannins in the date seed extract is not particularly limited. For example, a method can be used in which a solution of date seed extract is mixed with a solution of BSA or other proteins, aggregates of high-molecular-weight tannins and proteins such as BSA are precipitated, and the supernatant is recovered. The concentration of BSA at this time is usually 1 mg/mL or higher, preferably 2 mg/mL or higher, more preferably 3 mg/mL or higher.

In one embodiment of the present invention, the date seed extract containing the antiviral agent is 50% by weight or more, 75% by weight or more, or 80% by weight or more of the total tannin content in the date seed extract. Molecular tannins are removed, in other words enriched in low-molecular-weight tannins by a factor of 2 or more, 4 or more or 5 or more. Using these ratios, the weight of "tannin" converted from "date seed extract equivalent to 1 part by weight of seeds of the genus date palm" described herein is replaced by the weight of "low molecular weight tannin" can be converted to

As a means for reducing (removing) high-molecular-weight tannins in date seed extracts, silica gel, ion-exchange resins, and active adsorbents are used, which utilize the difference in behavior between high-molecular-weight tannins and low-molecular-weight tannins. It is also possible to use a method of recovering the low-molecular-weight tannin fraction (discarding the high-molecular-weight tannin fraction) by column chromatography, contact treatment with polyvinylpolypyrrolidone (PVPP), or the like.

The method for preparing the date seed extract is not particularly limited as long as it has an anti-norovirus effect, exhibits a synergistic effect when used in combination with the oyster extract, and exhibits anti-norovirus activity. However, in addition to the extraction step by the method as exemplified above, a concentration step, a purification step, a (freeze) drying step, a pulverization/powdering step, and the like can be included as necessary. These steps are preferably carried out under conditions in which the anti-norovirus action of the low-molecular-weight tannins and other components contained in the date seed extract is not reduced as much as possible, and a person skilled in the art can set such conditions as appropriate. can be done.

The raw material for the date seed extract is not particularly limited, and various plants of the genus Phoenix (Phoenix), typically seeds of the date palm (Phoenix dactylifera) can be used as raw materials. ) can be said to be an efficient use of industrial waste, which is economical and can reduce the burden on the environment. Since the tannin content of dates is high in the seeds and low in the pulp, it is desirable to use only the seeds as a raw material, but the seeds with the pulp attached can also be used as the raw material.

The date seed extract is recovered as an extract by, for example, immersing date seed pulverized or freeze-dried powder in an extraction solvent at an appropriate temperature and time.

As the extraction solvent for the date seed extract, common solvents used in preparing extracts from plant-derived raw materials can be used, such as water; methanol, ethanol, n-propanol, n-butanol. polyhydric alcohols such as propylene glycol and glycerin; polar solvents or dipolar solvents such as dimethylformamide and dimethylsulfoxide. Solvents containing water and/or ethanol (or other alcohols) are preferable in consideration of antiviral effects and safety to living organisms, and further considering the amount of tannin contained in the extract and antiviral effects, for example, ethanol Aqueous solutions (or other aqueous solutions of alcohols) are preferably used. The concentration of ethanol in the ethanol aqueous solution can be adjusted as appropriate, but is usually 10 vol% or more, preferably 30 vol% or more, more preferably 50 vol% or more, and usually 90 vol% or less, preferably 80 vol% or less, more preferably 70 vol%. % or less.

As shown in Examples (Reference Examples) below, the date seed extract has a higher tannin content when an aqueous ethanol solution is used as the extraction solvent than when water is used as the extraction solvent. tend to become However, the present invention is not limited to the latter embodiment using the date seed extract, and depending on the application of the antiviral agent or the antiviral composition containing it, the desired antiviral activity (disinfection effect), the former embodiment using the date seed extract may be used.

The ratio of the raw material of the date seed extract and the extraction solvent is determined so that the desired concentration of the extract can be obtained, preferably so that as many components (especially tannins) in the raw material as possible can be extracted. It can be appropriately adjusted depending on the conditions. For example, when using an ethanol aqueous solution as an extraction solvent, the larger the weight ratio of the raw material (date seed) to the ethanol aqueous solution, the greater the amount of tannin in the extract until the weight ratio reaches at least 10% by weight. There is a tendency to obtain extracts that are favorable in terms of action.

The temperature, pressure, time, and other conditions for extracting the date seed extract can also be adjusted as appropriate. The date seed extract can be obtained, for example, by an extraction treatment at about room temperature and normal pressure for about 10 minutes to overnight, but if necessary, it may be heated, pressurized, or prolonged. The extraction process can be appropriately combined with a centrifugal separation process, a filtration process for the residue, and the like.

For the date seed extract, if necessary, the heating/alcohol treatment step, freeze-drying step, pulverization step, and the like described above for the oyster extract may be performed in the same manner (with appropriate modifications as necessary).

Furthermore, a substance that reproduces or imitates the extract of the seeds of the genus date palm, for example, the same components as the extract obtained from the seeds of the genus date palm (especially the low-molecular-weight tannins with the same content) and anti-norovirus activity An extract having date seed prepared from a part other than the seed of a plant belonging to the genus Date, or prepared artificially may be used as the date seed extract in the present invention.

• Norovirus The norovirus targeted by the antiviral agent of the present invention is a general term for viruses belonging to the genus Norovirus of the family Caliciviridae, and includes human norovirus, mouse norovirus, and the like. In addition, human norovirus has two genogroups (GI and GII) so far, and furthermore, there are 9 types of GI (GI.1-GI.9) and 22 types of GII (GII.1-GII.22). Although it is known that it can be classified into genotypes, the antiviral agent of the present invention can target any genogroup and genotype of human norovirus.

The antiviral agent of the present invention can target not only norovirus but also alternative viruses such as MS2 phage and feline calicivirus. As shown in the examples below, the present invention exhibits excellent antiviral action (antiviral activity) against both norovirus and its alternative viruses.

-Preparation and use of antiviral agent The fact that the antiviral agent of the present invention is "against norovirus or its alternative virus" means that when the antiviral agent is dissolved and brought into contact with those viruses, the above-mentioned It means that it exhibits an "antiviral effect" or that it has a use capable of exhibiting an "antiviral effect." At this time, the strength of the "antiviral action" of the antiviral agent is not particularly limited, but at least it is necessary to lose (inactivate) the ability to infect and multiply norovirus, etc., or to extinguish the genes of norovirus, etc. It suffices if it can be confirmed by an objective index, measurement data, or the like. As an index for evaluating the antiviral action, for example, the infectious titer reduction value (Δlog PFU/mL or Δlog TCID ₅₀ /mL) or the genome residual rate can be used. In a preferred embodiment of the present invention, "having an antiviral effect" means "having an antiviral activity" as described above, for example, the value of reducing the infection titer against norovirus or the like is 3 or more, preferably means 4 or more, more preferably 5 or more.

The antiviral agent of the present invention can be prepared by mixing the oyster extract and the date seed extract obtained as described above in an appropriate ratio. The ratio of the oyster extract and the date seed extract in the antiviral agent can be appropriately adjusted so that the antiviral activity of the antiviral agent is at the desired level, and is not particularly limited. This ratio depends on whether the persimmon extract is enriched for persimmon condensed tannins (purity of persimmon condensed tannins in the persimmon extract) or the date seed extract is enriched for low molecular weight tannins. Further adjustment can be made while taking into account the degree of squid (purity of low-molecular-weight tannins in the date seed extract) and the like.

In one embodiment of the present invention, the antiviral agent of the present invention is 10 parts by weight or more, or 20 parts by weight or more of seeds of a plant of the genus Date persimmon extract corresponding to 1 part by weight of fruit of a plant of the genus Persimmon. , preferably contains date seed extract corresponding to 100 parts by weight or less or 50 parts by weight or less.

In one embodiment of the present invention, the antiviral agent of the present invention contains 0.0005 parts by weight or more of fruit of the genus Persimmon, or 0.0005 part by weight of the date seed extract equivalent to 1 part by weight of the seed of the genus Date palm. 0.01 parts by weight or more, preferably 0.1 parts by weight or less or 0.05 parts by weight or less equivalent of oyster extract.

In the present specification, "persimmon extract equivalent to 1 part by weight of fruits of plants belonging to the genus Persimmon" refers to the ingredients contained in 1 part by weight of fruits of plants belonging to the genus Persimmon (raw fruits containing moisture etc.) (condensed tannins, etc.), and the extraction solvent as described above, such as water and/or a solvent containing ethanol (or other alcohol), preferably an aqueous ethanol solution (e.g., 50% aqueous ethanol solution) ) is an oyster extract containing components extracted by using In one embodiment of the present invention, the "persimmon extract equivalent to 1 part by weight of the fruit of the persimmon genus plant" contains about 0.001 to 0.01 part by weight (about 0.1 to 1% by weight) of condensation Contains type tannins. Using this ratio, the weight of "persimmon extract equivalent to 1 part by weight of persimmon fruit" described herein may be converted to the weight of "condensed tannins."

As used herein, the term "date seed extract equivalent to 1 part by weight of plant seeds of the genus Date palm" refers to the components contained in 1 part by weight of plant seeds of the genus Date (raw seeds containing moisture etc.) ( tannins, etc.) and an extraction solvent as described above, such as water and/or a solvent containing ethanol (or other alcohol), preferably an aqueous ethanol solution (e.g. 50% or 70% ethanol It is a date seed extract containing components extracted by using an aqueous solution). In one embodiment of the present invention, the "date seed extract equivalent to 1 part by weight of plant seeds of the genus Date palm" contains about 0.01 to 0.03 parts by weight of tannin (preparation example below 2 and Preparation Example 3, see Figures 1 and 2). These proportions may be used to convert the weight of "date seed extract equivalent to 1 part by weight of date seed" described herein to the weight of "tannin."

The antiviral agent of the present invention is a raw material for persimmon extract (typically persimmon fruit, preferably freeze-dried powder thereof) and a raw material for date seed extract (typically date seed, preferably frozen powder thereof). Dry powder) is added to the extraction solvent, extracted at the same time, and then filtered to recover a mixture (mixture) of oyster extract and date seed extract from the beginning, or the residue is filtered. It is also possible to use it as it is.

The use of the antiviral agent of the present invention is not particularly limited, and it can be used in various embodiments where antiviral activity against norovirus or its alternative viruses is required. The antiviral agent of the present invention may be used in solid form (e.g., lyophilized powder) or in solution (non-composition form) dissolved in water, buffer solution (PBS, etc.), or other suitable solvent. may be prepared and used. A solution of an antiviral agent can also be considered a base for the antiviral composition or other product of the present invention, and the content of oyster extract and date seed extract in the antiviral composition of the present invention described below. Regulations can be applied to define the content of oyster extract and date seed extract in solutions of such antiviral agents.

As a representative use of the antiviral agent of the present invention, it is incorporated as an active ingredient against norovirus and the like in the antiviral composition of the present invention (e.g., alcohol preparation, cleaning composition, cosmetic composition) as described below. is mentioned. The antiviral agent of the present invention may be prepared in advance by mixing an oyster extract and a date seed extract prior to the production of the antiviral composition. When producing the antiviral composition, the oyster extract and the date seed extract are added separately, and the coexistence of the oyster extract and the date seed extract in the composition is confirmed by the antiviral agent of the present invention. can also be considered as the use of

In one aspect of the present invention, the antiviral agent of the present invention is used in combination with an oyster extract and a date seed extract, whereby the antiviral action of each of the oyster extract and the date seed extract against norovirus or its alternative virus can be replaced by a method of synergistic enhancement.

In one embodiment of the present invention, the antiviral agent of the present invention is used as a reagent in in vitro or ex vivo tests and studies, for example, by adding it to a medium and bringing it into contact with a norovirus or its substitute virus to It can be used as a substance for analyzing the influence of antiviral action (activity) against At that time, if necessary, it is also possible to add a candidate drug for further enhancing the antiviral action or to change the conditions affecting the antiviral action.

As a further aspect of the present invention, the antiviral agent of the present invention may be incorporated into pharmaceutical compositions or food compositions as an active ingredient against norovirus and the like. In other words, the antiviral agent of the present invention is brought into contact with norovirus or the like in vivo (in vivo in humans or other animals), for example, to treat food poisoning caused by norovirus or to prevent infection with norovirus. It can also be used as a substance for

In the antiviral composition, the persimmon extract (persimmon condensed tannin) in the antiviral agent can function as a component having anti-norovirus activity and moisturizing action (skin astringent action, etc.).

- Antiviral composition -
The antiviral composition of the present invention has an antiseptic effect against at least norovirus or its alternative virus, and comprises at least the antiviral agent of the present invention, alcohol, organic acid and/or salt thereof (referred to herein as " Organic acids, etc.”), surfactants, antibacterial agents, moisturizing agents, and at least one component selected from the group consisting of cosmetic oils and fats, and if necessary, other components. may contain.

The embodiment of the antiviral composition is not particularly limited, and it can be prepared in the form of various compositions containing the above ingredients. products, and cosmetic compositions. These antiviral compositions are used to treat (paint, spray, immerse, etc.) a desired object or location to disinfect any norovirus present there or to detect the future presence of norovirus thereon. Can be used for prophylaxis. In addition, the antiviral composition of the present invention may be prepared so as to have not only a disinfecting effect against norovirus, but also a disinfecting effect against non-enveloped viruses other than norovirus, enveloped viruses, bacteria, and other microorganisms, if desired. can also

The content of the antiviral agent in the antiviral composition, on the other hand, has a disinfecting effect against norovirus, etc., product coloring (coloring due to the participation of oyster condensed tannins), and other purposes (actions) according to the composition. and properties, while considering the composition of the antiviral agent (components and amounts of oyster extract and date seed extract), etc., so that the desired antiviral effect (antiviral activity) is achieved. can be adjusted. The antiviral composition of the present invention can have a content that satisfies, for example, the following conditions (A) and/or (B), preferably both conditions (A) and (B):
(A) 0.00002% by weight or more, 0.0001% by weight or more, 0.0002% by weight of fruits of plants of the genus Persimmon relative to 100% by weight of the entire composition (including components other than active ingredients such as solvents) % by weight or more, 0.0004% by weight or more, 0.002% by weight or more, 0.003% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.02% by weight or more, or 0.05% by weight % by weight or more and 10% by weight or less, 5% by weight or less, 2% by weight or less, 1% by weight or less, 0.5% by weight or less, 0.1% by weight or less, 0.05% by weight or less, or 0.01% by weight % or less (excluding combinations in which the lower limit value is greater than the upper limit value), containing an antiviral agent containing an oyster extract;
(B) 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight of plant seeds of the genus Date palm, based on 100% by weight of the entire composition (including components other than active ingredients such as solvents) % or more, 0.5 wt% or more, 1.0 wt% or more, 1.5 wt% or more, or 2.0 wt% or more, and 20 wt% or less, 10 wt% or less, 5.0 wt% or less, 2 .5% by weight or less, 2.0% by weight or less, 1.5% by weight or less, 1.0% by weight or less, 0.5% by weight or less, or 0.2% by weight or less (however, the numerical value of the lower limit is the upper limit Excludes combinations that are greater than the numerical value of ) contains an antiviral agent containing a date seed extract equivalent to

In addition, when the antiviral composition of the present invention is an embodiment that is assumed to be diluted at a predetermined rate at the time of use (concentrated type), the oyster extract and / or date seed extract at the time of use The concentration of oyster extract and/or date seed extract during storage should be adjusted to be higher than the above numerical range (by the dilution factor) so that the concentration satisfies the above numerical range. can be done.

In a preferred embodiment of the present invention, the antiviral composition comprises an antiviral agent that exhibits a "synergistic effect" when the oyster extract and the date seed extract are present in a predetermined range of ratios, in an amount of a predetermined range. By containing it, it "has antiviral activity" (infectious titer reduction value is 3 or more).

A "synergistic effect" is achieved by "mixing" (combining) the persimmon extract and the date seed extract. "Mixing" is usually not distinguished between "adding" the latter to the former and vice versa. For example, considering embodiments in which an antiviral agent is incorporated into the composition, by first setting the concentration of either oyster extract or date seed extract and then adding the other at an appropriate concentration, The antiviral action can be enhanced synergistically as compared with the case where it is not added.

Here, the "synergistic effect" includes both the cases where the determination by the "FIC index" (see the formula below) is the "synergistic effect" and the case where the determination is the "partial synergistic effect". For example, as shown in the examples below, for an oyster extract prepared using 0.05% by weight of the fruits of a plant belonging to the genus Persimmon (persimmon) with respect to the extraction solvent (50% ethanol aqueous solution), Oysters prepared using the fruit of the persimmon genus (persimmon) at 0.1% by weight to the extraction solvent (50% ethanol aqueous solution) determined to have a "synergistic effect" by the addition of a date seed extract For extracts, the addition of the date seed extract is determined to have a "partial synergistic effect", but both are considered to produce a "synergistic effect". Conversely, for a date seed extract prepared using 1.25% by weight of date seeds relative to the extraction solvent (50% ethanol aqueous solution), the addition of persimmon extract For a date seed extract that was determined to have a "synergistic effect" and was prepared using 2.5% by weight of date seeds relative to the extraction solvent (50% aqueous ethanol solution) , the addition of oyster extract is determined to have a "partial synergistic effect", but both are considered to produce a "synergistic effect". Other concentrations of oyster extract or date seed extract are also considered to exhibit a "synergistic effect" if they are determined to have a "synergistic effect" or "partial synergistic effect". In this way, if the antiviral composition "has antiviral activity" in addition to the "synergistic effect", it will be effective with a smaller amount of oyster extract and date seed extract than normally assumed. It can be said that it has antiviral activity.

In a more preferred embodiment of the present invention, the concentration of at least one of the oyster extract and the date seed extract, preferably both, in the antiviral composition is such that the oyster extract alone and/or the date seed extract alone is "anti- It is within the range of “no viral activity”. In other words, in a more preferred embodiment of the present invention, the antiviral composition "has antiviral activity" for the first time by containing both the oyster extract and the date seed extract, each in a predetermined concentration range. ” has become a thing.

For the persimmon extract, for example, as exemplified in Example 1 (FIG. 6), 0.1% by weight or 0.1% by weight of the persimmon plant (persimmon) fruit is added to the extraction solvent (50% ethanol). 05% by weight of the test solution alone, that is, when considering the volume of the virus (MS2 phage) solution mixed therewith, persimmon genus per 100 parts by weight of the composition (mixture) When the test solution prepared using about 0.093 parts by weight or 0.047 parts by weight of the fruit of the plant was used alone, the infectious titer reduction value was 1.12 and 0.24, respectively, indicating that it had antiviral activity. 2. As shown in the verification of the presence or absence of a synergistic effect (Figs. 7 and 8), the concomitant use of the date seed extract increased the infectious titer reduction value to at least 3, and was recognized as having "antiviral activity." will be available. Therefore, in one preferred embodiment of the present invention, the composition of the present invention contains no more than 0.1 parts by weight or no more than 0.05 parts by weight of persimmon fruit per 100 parts by weight of the total composition, e.g. Contains an antiviral agent containing oyster extract equivalent to 0.05-0.1 parts by weight.

For a date seed extract, for example, as exemplified in Example 1 (FIG. 6), 2.5% by weight or 1% by weight date seeds (dates) relative to the extraction solvent (50% ethanol) When the test solution prepared using .25% by weight is used alone, that is, when considering the volume of the virus (MS2 phage) solution mixed with it, Persimmon genus per 100 parts by weight of the composition (mixture solution) When the test solution prepared using about 2.33 parts by weight or 1.16 parts by weight of the fruit of the plant is used alone, the infectious titer reduction value is 0.81 and almost 0 (below the detection limit), respectively. 2. does not have antiviral activity. As shown in the verification of the presence or absence of a synergistic effect (Figs. 7 and 8), the concomitant use of oyster seed extract raised the infectious titer reduction value to at least 3, and was recognized as having "antiviral activity." will be available. Therefore, in a preferred embodiment of the present invention, the composition of the present invention contains no more than 2.5 parts by weight or no more than 1.25 parts by weight of date seeds, for example 1 part by weight, per 100 parts by weight of the total composition. .25 to 2.5 parts by weight of antiviral agent containing date seed extract.

- Alcoholic preparation One embodiment of the virus composition of the present invention is an alcoholic preparation containing at least the antiviral agent of the present invention and (a relatively large amount) alcohol. Preferably, the alcohol formulation further contains a surfactant, such as an organic acid, or both. Such alcohol preparations adhered to workers' fingers, food, tableware, cooking utensils, refrigerators, tableware, showcases, tables, chairs, doorknobs and handrails of facilities, toilets, equipment used to handle patient's waste, etc. , or a composition for disinfecting norovirus or the like that may adhere, and can be prepared, for example, as a spray similar to conventional ethanol formulations.

Cleaning composition One embodiment of the antiviral composition of the present invention is a cleaning composition containing at least the antiviral agent of the present invention and a surfactant (in an amount and type suitable for cleaning). be done. The cleaning composition preferably further contains at least one selected from the group consisting of surfactants and disinfectants such as alcohols, organic acids, and more preferably all of them. Such a cleaning composition is a composition for removing stains from workers' fingers, food, tableware, cooking utensils, clothes, etc., and for disinfecting norovirus, etc. that have adhered or may adhere thereto. There are, for example, liquid or solid detergents (typically hand soaps) that can be prepared.

-Cosmetic composition One embodiment of the antiviral composition of the present invention includes a cosmetic composition containing at least the antiviral agent of the present invention, a moisturizing agent and/or cosmetic oils and fats. The cosmetic composition preferably further contains at least one selected from the group consisting of alcohols, organic acids, moisturizing agents and anti-inflammatory agents, and more preferably contains all of them. Such a cosmetic composition is a composition for skin care by applying it to the hands and fingers of workers who tend to get rough due to washing, etc., and for disinfecting norovirus or the like that has adhered or may adhere to the fingers, For example, they can be prepared as lotions, creams, emulsions, and other basic cosmetics (typically hand lotions).

Alcohol Alcohol is a component that can improve the solubility of various components contained in the antiviral composition, such as tannins contained in persimmon extracts and date seed extracts, especially condensed tannins contained in persimmon extracts. is. Since alcohol also has a disinfecting effect against enveloped viruses and bacteria, alcohol preparations or other antiviral compositions containing alcohol have a disinfecting effect (antibacterial action) against not only norovirus but also enveloped viruses and bacteria. can be endowed. Alcohol can also be incorporated into cosmetic compositions as a component that imparts astringency and antiseptic properties to the skin.

Examples of alcohols that can be blended and used in the antiviral composition of the present invention include lower alcohols such as methanol, ethanol, propanol (n-propanol) and butanol (n-butanol). Ethanol and propanol are preferred, and ethanol is particularly preferred. Any one of these alcohols may be used alone, or a plurality of types may be used in combination.

The content of alcohol in the antiviral composition can be appropriately adjusted in consideration of the purpose (effect), type of alcohol, and the like. For example, alcohol can be blended in an amount of usually 10 to 80% by weight, preferably 30 to 70% by weight, of the entire antiviral composition (including components other than active ingredients such as solvents).

In addition, if the oyster extract and/or date seed extract has been subjected to a process such as extraction using an alcohol such as ethanol and is in a state of being mixed with the alcohol used for the process, the alcohol is used as an antiviral It may be incorporated as part or all of the components of the composition. If the amount of alcohol used in the alcohol treatment is less than the amount that should be contained in the antiviral composition, the antiviral composition may be prepared by adding alcohol to make up for the shortage.

・Organic acids, etc. (organic acids and/or salts thereof)
Organic acids and the like adjust the pH of various components contained in the antiviral composition, such as tannins contained in persimmon extracts and date seed extracts, and in particular, the solubility of condensed tannins contained in persimmon extracts. As a chelating agent that improves the color of tannins and prevents them from coloring when they come into contact with iron, or as an antioxidant that suppresses coloration due to oxidation of tannins (for example, the red coloration of oyster condensation-type tannins in oyster extracts), It can be used by blending in a composition. Organic acids and the like can also be used as components that have the effect of disinfecting some non-enveloped viruses, enveloped viruses and bacteria.

In addition, the organic acid and/or its salt as a component of the antiviral composition of the present invention is usually added at the time of production, in addition to what the oyster extract and/or date seed extract may contain in its natural state. It refers to what is added.

Organic acids that can be used in the present invention include, for example, lactic acid, malic acid, citric acid, tartaric acid, salicylic acid, succinic acid, fumaric acid, itaconic acid, ferulic acid, and ascorbic acid (vitamin C). Examples of salts of these organic acids include sodium salts, potassium salts and calcium salts. Any one of these organic acids and/or salts thereof may be used alone, or two or more of them may be used in combination.

In a preferred embodiment of the present invention, the alcohol preparation as the antiviral composition of the present invention is selected from the group consisting of lactic acid, citric acid, ferulic acid, lactic acid and salts thereof as organic acids and/or salts thereof. Contains at least one. For example, lactic acid and calcium lactate as "acidulants" and "strengthening agents" respectively, citric acid and sodium citrate (trisodium citrate) as "acidulants" and "flavoring agents" respectively, and ferulic acid as "oxidizing It is a substance listed in the "List of Food Additives" (Ministry of Health, Labor and Welfare) as an inhibitor", and it is a component that exhibits the above-mentioned effects in the antiviral composition of the present invention.

The content of the organic acid or the like in the antiviral composition can be appropriately adjusted in consideration of the purpose (effect) and the type of the organic acid or the like. For example, the organic acid or the like is usually 0.01 to 5.0% by weight, preferably 0.05 to 2.5% by weight, based on the total antiviral composition (including components other than active ingredients such as solvents). It can be added in an amount to give a percentage by weight. Alternatively, an organic acid or the like can be added in an amount such that the pH of the antiviral composition is usually 6.0 to 2.0, preferably 5.0 to 3.0.

-Surfactant Surfactant acts as an emulsifier for emulsifying various components contained in the antiviral composition, and can be used as a component that has the effect of disinfecting enveloped viruses and bacteria. In addition, surfactants are added to cleaning compositions as components for cleaning fingers, food, tableware, cooking utensils, etc., and are used to mix oil phases and water phases in lotions, creams, milky lotions, etc. It can also be blended into a cosmetic composition as an ingredient.

Surfactants include cationic, anionic, amphoteric and non-ionic surfactants, including oyster condensed tannins contained in persimmon extracts and low-molecular-weight tannins (so-called polyphenols) contained in date seed extracts. Also, considering the chemical properties of the optionally blended organic acid and/or its salt, it is preferable to use an anionic surfactant and/or a nonionic surfactant in the present invention.

Examples of anionic surfactants include soap (alkali salts of higher fatty acids), monoalkyl sulfates (olefin (C14-16) sodium sulfonate, sodium lauryl sulfate, ammonium lauryl sulfate, etc.), alkylpolyoxyethylene sulfates ( sodium laureth sulfate, etc.), alkylbenzene sulfonates, and monoalkyl phosphates.

Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene fatty acid esters, glycerin fatty acid esters, sugar alcohol fatty acid esters (sorbitan fatty acid esters, sucrose fatty acid esters, lauryl glucoside, etc.), and fatty acid diethanolamides. mentioned.

Among these surfactants, nonionic surfactants recognized as food additives such as glycerin fatty acid ester, sorbitan fatty acid partial ester, sucrose fatty acid ester, and lauryl glucoside can be It is particularly preferable in that there is no problem even if it adheres to the surface. Also preferred are monoalkyl sulfates such as sodium olefin (C14-16) sulfonate, sodium lauryl sulfate and ammonium lauryl sulfate, and alkyl polyoxyethylene sulfates such as sodium laureth sulfate.

The content of the surfactant in the antiviral composition can be appropriately adjusted in consideration of the purpose of addition, the type of surfactant to be used, and the like. For example, the surfactant is usually 0.05 to 5.0% by weight, preferably 0.1 to 2.5% by weight, based on the total antiviral composition (including components other than active ingredients, such as solvents). It can be blended in an amount that provides a percentage by weight.

· Antibacterial agent Antibacterial agents (also called bactericides, disinfectants, and bacteriostats.) The antiviral composition of the present invention has a disinfecting effect against bacteria in addition to norovirus, for example, during food processing It can be blended into a composition and used in order to impart an antiseptic effect against Escherichia coli, Staphylococcus aureus, MRSA, Salmonella, Vibrio parahaemolyticus, Pseudomonas aeruginosa, etc., which are problematic in infections or nosocomial infections. Antimicrobial agents can also be incorporated into cosmetic compositions as preservatives.

The antibacterial agent as a component of the antiviral composition of the present invention is listed as another component of the antiviral composition, such as alcohol (ethanol, etc.), organic acid and/or its salt (citric acid, etc.). Other than those, it refers to those added at the time of manufacture.

Examples of antibacterial agents include the following. Any one of these antibacterial agents may be used alone, or two or more of them may be used in combination.
・Natural antibacterial agents: proteins (milk protein, egg white lysozyme, etc.), peptides (polylysine, etc.), etc.;
Antibiotics: penicillin antibiotics, chloramphenicol, streptomycin, tetracycline antibiotics, cephalosporin antibiotics, etc.;
・Synthetic antibacterial agents: Chlorine compounds (such as triclosan), iodine compounds (such as povidone-iodine), zinc compounds (such as cetylpyridinium zinc), benzenecarboxylic acids (benzoic acid, salicylic acid, isopropylmethylphenol (called cymen-5-ol) ), butyl parahydroxybenzoate (= butylparaben), etc.), organic acid esters (glycerin ester, sucrose ester, etc.), aldehydes (glutaraldehyde, formaldehyde, etc.), biguanide compounds (chlorhexidine gluconate, etc.) ), quaternary ammonium salts (benzalkonium chloride, cetyl ammonium bromide, etc.), and the like.

Among these antibacterial agents, isopropylmethylphenol (cymen-5-ol), butyl p-hydroxybenzoate, triclosan, etc. have excellent antibacterial action and have good compatibility with oyster extract and date seed extract. Therefore, it is a preferred antibacterial agent in the present invention.

Moisturizing agent A moisturizing agent (sometimes called a humectant) is a component mainly used when manufacturing the antiviral composition of the present invention as a cosmetic composition, and removes moisture from the stratum corneum of the skin. It is a component that retains and maintains the flexibility and elasticity of the skin.

Moisturizers include those used in general cosmetics such as lotions, milky lotions, and creams, such as glycerin, propylene glycol, sorbitol, polyethylene glycol, hyaluronic acid, sodium chondroitin sulfate, ceramide, and aloe extract. Among these moisturizing agents, for example, glycerin and propylene glycol are preferred as those to be incorporated into the antiviral composition (cosmetic composition) of the present invention.

・Cosmetic oils and fats Cosmetic oils and fats form a film on the surface of the skin to protect the skin, provide flexibility and lubricity, and are also ingredients that give cosmetics an appropriate feeling of use. is.

As the cosmetic oils and fats, the same cosmetic oils and fats as those used in cosmetics such as general milky lotions and creams can be used, and examples thereof include the following.
・ Fats and oils (esters of higher fatty acids and glycerin): vegetable oils, animal oils, or hydrogenated products thereof (partially hydrogenated rapeseed oil, etc.), synthetic triglycerides (tri(caprylic/capric acid) glyceryl, etc.);
・Waxes (esters of higher fatty acids and higher alcohols that are solid at room temperature): vegetable waxes, animal waxes (beeswax, lanolin, etc.);
・ Hydrocarbons: mineral hydrocarbons (liquid paraffin, vaseline, paraffin, etc.), animal hydrocarbons (squalane, etc.), etc.;
- Higher fatty acids: lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, etc.;
・ Higher alcohols: cetanol, stearyl alcohol, lanolin alcohol, etc.;
・ Esters (esters of fatty acids and alcohols other than waxes): Myristyl myristate, propylene glycol dioleate, cetyl lactate, etc.

Among these cosmetic oils and fats, for example, tri(caprylic/capric acid)glyceryl is preferable as one to be blended in the antiviral composition (cosmetic composition) of the present invention.

Other components The antiviral composition of the present invention is typically prepared in the form of a solution using water (or an aqueous alcohol solution as described above) as a solvent, and the balance other than the above components is water (purified water, etc.). ) and/or other solvents.

In addition to the above components, the antiviral composition of the present invention may optionally contain general or known components that can be contained in conventional alcohol preparations, cleaning compositions, cosmetic compositions, etc. can contain. Such optional ingredients include, for example, antioxidants/preservatives (sodium sulfite, etc.), fragrances, pigments, and other various ingredients or additives that can be incorporated in pharmaceuticals, cosmetics, foods, and the like.

An example of an optional ingredient is an anti-inflammatory agent that can be incorporated into the cosmetic composition. Examples of anti-inflammatory agents include dipotassium glycyrrhizinate and allantoin.

<Method for using and manufacturing antiviral composition>
When the antiviral composition of the present invention is "against norovirus or its alternative virus", it contains an antiviral agent "against norovirus or its alternative virus", so that when it is brought into contact with the virus, the above-mentioned It means that the "antiviral effect" as described above is exhibited, or that it has a use capable of exhibiting the "antiviral effect". The strength of the "antiviral action" of the antiviral composition at this time is not particularly limited, and at least the ability to infect and proliferate norovirus, etc. is lost (inactivated), preferably further, norovirus, etc. It suffices if it can be confirmed by objective measurement data or the like that the particles or genes of the method are extinguished. In a preferred embodiment of the present invention, "having an antiviral effect" means "having an antiviral activity" as described above, for example, the infectious titer reduction value (Δlog PFU/mL or Δlog TCID ₅₀ /mL ) is 3 or more, preferably 4 or more, more preferably 5 or more.

The antiviral composition of the present invention can be used in the same manner as conventional antiviral compositions, depending on its embodiment. The site to which the antiviral composition is applied may be a site (inside or on the body surface) of a human or other animal, or may be other sites.

For example, when antiviral agents are prepared as sprays, hand soaps, etc., they can be treated in the same way as before, where non-enveloped viruses may exist (food, tableware, cooking utensils, workers' fingers, clothing, etc.). In addition, antiviral agents can be prepared as liquid agents for impregnating masks, air conditioner filters, clothing, wet wipes, etc., or as concentrated liquid agents that are diluted to an appropriate concentration at the time of use. It is possible.

When the antiviral agent of the present invention takes the form of a pharmaceutical composition, the dosage and administration method (administration route, administration schedule, etc.) will depend on the dosage form, taking into consideration the patient's age, weight, symptoms, etc. It can be determined within an appropriate range. The application (administration) subject of the antiviral agent of the present invention as a pharmaceutical composition is not limited to humans, and the application (administration) subject may be animals other than humans that can be infected with non-enveloped viruses. can.

The antiviral composition of the present invention uses an oyster extract and a date seed extract (antiviral agent of the present invention) prepared by the method described above as active ingredients against norovirus, and Other ingredients according to the requirements, that is, at least one ingredient selected from the group consisting of alcohols, organic acids and/or salts thereof, surfactants, antibacterial agents, moisturizers and cosmetic oils and fats, and other optional ingredients as necessary After using the ingredients, it can be produced by a process and a production apparatus according to the production method of conventional antiviral compositions.

[Preparation Example 1] Preparation of persimmon extract The immature persimmon fruit, which had been sufficiently sterilized (NaClO), washed, treated to prevent discoloration (vitamin C), etc., was cut into dice and crushed. . The resulting fruit/fruit juice solution is passed through a 200-mesh sieve, centrifuged, subjected to high-temperature sterilization (120 to 130 ° C., 7 to 10 seconds), and then freeze-dried to freeze-dry persimmon fruit (FD). A powder was prepared. About 5% of the obtained FD powder was oyster condensed tannin.

At a ratio of 10 mL of 50% ethanol aqueous solution to 0.01 g of this oyster fruit FD powder, both were mixed by vortex, left overnight at room temperature, centrifuged (13 k × g, 5 minutes), and filtered with an ADVANTEC filter. sterilized. The obtained oyster extract is used as a test liquid and is described as "0.1% PE/50% EtOH" (PE: Persimmon Extract).

[Preparation Example 2] Preparation of date seed extract (DSE) and quantification of tannin in each extract Date seed (DS: Date Seed) pulverized and powdered (date seed powder) 0.5 g and 50 % or 70% ethanol aqueous solution or 10 mL of distilled water were mixed by vortexing, allowed to stand overnight at room temperature, centrifuged (13,000×g, 5 minutes), and sterilized with an ADVANTEC filter. The resulting three date seed extracts were used as test liquids, which are denoted as "5% DSE/50% EtOH", "5% DSE/70% EtOH" and "5% DSE/Water", respectively.

For each of the three test solutions obtained as described above, a color reaction was performed on filter paper by the vanillin hydrochloride method, the Folin-Ciocalteu method, and the _EcCl2 method to qualitatively determine the tannin content. When evaluated systematically, "5% DSE/50% EtOH" had the deepest coloration (not shown). Also, when this test solution was subjected to an agglutination reaction with BSA, BSA aggregates were formed, confirming the presence of sufficient tannin (not shown).

For "5% DSE/50% EtOH" and "5% DSE/70% EtOH", the tannin content in the test solution was quantified by the vanillin hydrochloride method according to the following procedure. In a 96-well plate, 15 μL of each test solution was mixed with a reagent consisting of 90 μL of 4% vanillin/methanol solution and 45 μL of concentrated hydrochloric acid. After allowing to stand at room temperature for 20 minutes under light shielding, the absorbance at 500 nm was measured. The concentration of tannin in each test solution was quantified from the measured absorbance and a calibration curve prepared using a catechin solution with a known concentration as a standard solution.

Furthermore, additional test solutions were prepared by changing the amounts of raw materials used in preparing the date seed extract, and the tannin content was quantified by the vanillin hydrochloride method in the same manner as described above. Using 10 mL of 50% EtOH, the amount of date powder was increased from 0.5 g (5%) to 1 g (10%), or 0.45 g, 0.4 g, 0.3 g, 0.25 g, Prepare stepwise test solutions such as 0.1 g (4.5%, 4%, 3%, 2.5%, 1%, respectively), respectively, "10% DSE / 50% EtOH", " 4.5% DSE/50% EtOH", "4% DSE/50% EtOH", "3% DSE/50% EtOH", "2.5% DSE/50% EtOH", "1% DSE/50% EtOH". Also, the amount of date seed powder was increased from 0.5 g (5%) to 1 g (10%) or decreased to 0.1 g (1%) using 10 mL of 70% EtOH. Liquids are prepared and labeled as "10% DSE/70% EtOH" and "1% DSE/70% EtOH", respectively.

FIG. 1 shows the results of quantifying the tannin content of each of the above test solutions by the vanillin hydrochloride method. Similar to the tendency described above, the "50% EtOH" test solution generally tended to have a higher tannin content than the "70% EtOH" test solution.

[Preparation Example 3] Preparation of date seed extract (DSE) and date fruit extract (DFE), and quantification of tannin in each extract /50% EtOH", "5% DSE/70% EtOH" and "5% DSE/Water" were prepared. In addition, instead of pulverized and powdered date seeds (date seed powder), pulverized and powdered date fruit (DF) from which date seeds are removed (date fruit powder) is used as a raw material. Prepare two new test solutions “5% DFE/50% EtOH” and “5% DFE/Water” in the same procedure as “5% DSE/50% EtOH” and “5% DSE/Water” except did.

The tannin content of these test solutions was quantified by the vanillin hydrochloride method in the same procedure as in Preparation Example 2. The results are shown in FIG. Tannins are almost absent in date fruit (DF) extracts with water or 50% aqueous ethanol solution, but are present in small amounts in date seed (DS) extracts with water, 50% or 70% It was found to be abundant in a date seed (DS) extract using an aqueous ethanol solution.

[Reference Example 1] Evaluation of antiviral action against MS2 phage by date seed (DS) extract alone -Part 1-
The JIS standard method was partially modified and the following procedure was used to evaluate the antiviral activity (infectious titer reduction value) against MS2 phage (an alternative virus to norovirus) when the date seed extract was used alone.

15 μL of the MS2 phage solution whose titer had been adjusted to 10 ⁷ to 10 ⁸ PFU/mL in advance was mixed with 135 μL of each test solution prepared using 50% EtOH in Preparation Example 2, and allowed to react at room temperature for 2 minutes. rice field. 100 μL of this mixture was added to 900 μL of LB medium to stop the reaction. It was serially diluted in 10-fold dilution series using LB medium to obtain an MS2 phage reagent.

To 2 mL of soft agar LB medium (0.5%) was added 100 μL of previously prepared E. coli solution, to which 100 μL of MS2 phage reagent was further added. After mixing well, the mixture was poured onto a BL agar medium, the titer was measured from the number of plaques, and the infectious titer reduction value (Δlog PFU/mL) was calculated.

Table 1 shows the results. 10% DSE / 50% EtOH and 5% DSE / 50% under the standard that antiviral activity (disinfectant effect) is recognized when the infectious titer reduction value is 3 or more (one significant figure) EtOH is recognized to have antiviral activity. On the other hand, 4.5% DSE/50% EtOH to 0.5% DSE/50% EtOH showed a certain level of antiviral activity (decreased infectious titer), but were not found to have antiviral activity. .

Referring to the data in Preparative Example 2 (Figure 1), 5% DS/50% EtOH contains tannins at a concentration of 0.81 mg/mL, thus containing tannins at a concentration of approximately 0.80 mg/mL ( It can be said that the date seed extract alone, which also contains components other than tannins, has antiviral activity against MS2 phage.

[Reference Example 2] Evaluation of antiviral action against MS2 phage by date seed extract (DSE) alone -Part 2-
A total of 5 test solutions prepared in Preparation Example 3 and "50% EtOH" and "70% EtOH" as controls (extraction solvent itself that does not contain date seed extract) were used in the same procedure as in Reference Example 1. Antiviral activity (infectious titer reduction value) against MS2 phage was evaluated.

The results are shown in FIG. Under the standard that it has antiviral activity (disinfection effect) when the infectious titer reduction value is 3 or more (with one significant figure), "5% DSE / 50% EtOH" and "5% DSE /70% EtOH" is recognized to have antiviral activity. "5% DFE/50% EtOH" is not recognized to have antiviral activity, although a certain decrease in infectious titer is observed.

[Reference Example 3] Evaluation of antiviral action against norovirus by date seed extract (DSE) alone -Part 1-
A norovirus suspension derived from feces of a patient with norovirus was purified, stored and used. Norovirus concentrations were 10 ⁶ -10 ⁷ /mL. 1.2 μL of this norovirus suspension and 10.8 μL of the test solution “5% DSE/50% EtOH” (see Preparation Example 2) were mixed and allowed to react at room temperature for 2 minutes. Additionally, the reaction was stopped (100-fold dilution). Norovirus RNA was extracted from this reaction solution using a commercially available kit, treated with DNase, and cDNA was synthesized. Using this cDNA solution, real-time PCR was performed according to a conventional method, the amount of norovirus remaining after the above reaction was quantified, and the genome residual rate (%) relative to the control was calculated.

As controls, the following two were performed. One is a control (Total control). Another is to add 9 µL of the test solution "5% DSE/50% EtOH" to 990 µL of LB medium instead of contacting the test solution with the norovirus suspension before adding it to the medium as in the above procedure. 1 μL of the norovirus suspension is added from the above and contacted, and the amount of norovirus is quantified by real-time PCR from 140 μL of the medium after the reaction in the same procedure as described above ("5% DSE / 50% EtOH-C"). do.). The above test was repeated twice, and the genome survival rate was determined as the mean±SD.

The results are shown in FIG. The anti-norovirus effect of "5% DSE/50% EtOH" was not very strong (genome survival rate of about 65%), but "10% DSE/50% EtOH" had a certain degree of anti-norovirus effect (genome about 15% survival rate).

[Reference Example 4] Evaluation of antiviral action against norovirus by date seed extract (DSE) alone -Part 2-
As the test solution, instead of "5% DSE / 50% EtOH", "5% DSE / 70% EtOH" and "10% DSE / 70% EtOH" were used, and the controls were changed to those corresponding to each. measured the genome residual rate (%) in the same manner as in Reference Example 3.

The results are shown in FIG. "10% DSE/70% EtOH" had strong anti-norovirus activity (genome survival rate of about 5%), and "5% DSE/70% EtOH" also had a certain degree of anti-norovirus activity ( genome survival rate of about 18%).

[Example 1] Evaluation of antiviral action against MS2 phage by combined use of persimmon extract (PE) and date seed extract (DSE)

1. Measurement of infectious titer reduction value An oyster extract (PE) test solution "0.1% PE/50% EtOH" was prepared in the same manner as in Preparation Example 1, and the test solution was further diluted with 50% EtOH to give the test solution " 0.05% PE/50% EtOH" was prepared. In addition, a date seed extract (DSE) test solution "2.5% DSE/50% EtOH" similar to Preparation Example 2 was prepared, and the test solution was further diluted with 50% EtOH to obtain a test solution "1.25 % DSE/50% EtOH" was prepared.

In the procedure of Reference Example 1, the mixture of 15 μL of the MS2 phage solution and 135 μL of each test solution was changed as shown in (i) to (vi) below, and otherwise similarly, in the case of oyster extract alone, date The antiviral action (infectious titer reduction value) against MS2 phage was compared between the seed extract alone and the oyster extract and date seed extract in combination.
(i) Mix 15 μL of MS2 phage solution and 135 μL of test solution “0.1% PE/50% EtOH”.
(ii) Mix 15 μL of MS2 phage solution and 135 μL of test solution “2.5% DSE/50% EtOH”.
(iii) 15 μL of MS2 phage solution, 135 μL of test solution “0.1% PE/50% EtOH” and 135 μL of test solution “2.5% DSE/50% EtOH” were mixed.
(iv) 15 μL of MS2 phage solution and 135 μL of test solution “0.05% PE/50% EtOH” were mixed.
(v) Mix 15 μL of MS2 phage solution and 135 μL of test solution “1.25% DSE/50% EtOH”.
(vi) Mix 15 μL of the MS2 phage solution, 135 μL of the test solution “0.05% PE/50% EtOH” and 135 μL of the test solution “1.25% DSE/50% EtOH”.

The results are shown in FIG. Under the standard that it is recognized to have antiviral activity (disinfectant effect) when the infectious titer reduction value is 3 or more (with one significant figure), first comparing (i) to (iii), "0 1% PE/50% EtOH alone had an infectious titer reduction of 1.12, and 2.5% DSE/50% EtOH alone had an infectious titer reduction of 0.81, both of which are said to have antiviral activity. However, when they were used together, the infectious titer reduction value increased dramatically to 5.52, and it was recognized that they had antiviral activity. Similarly, when comparing (iv) to (vi), the infection titer reduction value of “0.05% PE / 50% EtOH” alone is 0.24, and the infection value of “2.5% DSE / 50% EtOH” alone The titer reduction value was 0 (below the limit of measurement), and both were not found to have antiviral activity, but when they were used together, the infectious titer reduction value increased dramatically to 3.27, indicating that they had antiviral activity. has become accepted.

2. Verification of Presence or Absence of Synergistic Effect Among the results of the above "1. Measurement of decrease in infectious titer", it was verified as follows whether or not a synergistic effect can be recognized in cases (iii) and (vi).

In the present invention (Example), the synergistic effect is determined by the FIC index. The FIC index calculation formula and criteria are as follows. Synergy is considered to exist when the FIC index determines "synergy" and "partial synergy".

First, the "MIC value of single drug A" in the above FIC index calculation formula was obtained by the following procedure. Dilute the test solution "0.1% PE / 50% EtOH" to prepare test solutions with various PE concentrations in the oyster extract (PE) test solution, and use these test solutions alone As a result of measuring the infectious titer reduction value, it was found that the infectious titer reduction value was 3 when the PE concentration of the test solution was 0.34% (recognized as having a disinfecting effect). Therefore, the "MIC value of single drug A" is "0.34". Similarly, using the date seed extract (DSE) test solution, the "MIC value of drug B alone" was determined to be "4.31".

Subsequently, in order to measure the infectivity titer reduction value of the mixed test solution of PE and DSE, the concentration of the PE test solution was fixed at 0.05% and the concentration of the DSE test solution was varied. It was found that the infectious titer reduction value was 3 (disinfectant effect was recognized) when the concentration of was 1.26% (see Fig. 7 [A]). Therefore, the "MIC value of drug A used in combination" is "0.05" and the "MIC value of drug B used in combination" is "1.26".

Substituting each MIC value into the above FIC index formula, the FIC index is calculated as (0.05/0.34)+(1.26/4.31)≈0.44, and the “synergistic effect” (0. 5 or less). In other words, although the PE test solution with a concentration of 0.05% does not have a disinfecting effect, it can be improved to have a disinfecting effect by adding DSE with a concentration higher than a certain level. It can be said that a “synergistic effect” is exhibited in the antiviral action.

Furthermore, in the above procedure, when the concentration of the DSE test solution was changed while the concentration of the PE test solution was fixed at 0.1%, the infectivity value decreased when the concentration of the DSE test solution was 1.22%. 3 (disinfection effect is observed) (see FIG. 7B). At this time, the "MIC value of drug A when used in combination" is "0.1", the "MIC value of drug B when used in combination" is "1.22", and the FIC index is (0.1/0.34) + ( 1.22/4.31) ≈ 0.58 and is determined to be "partially synergistic" (greater than 0.5 and less than or equal to 0.75). In other words, even for the PE test solution with a concentration of 0.1%, where no disinfection effect is observed, by adding DSE with a concentration higher than a certain level, a "partial synergistic effect" in antiviral action is exhibited (this This is also considered to have a synergistic effect in the invention), and can be improved so that the disinfecting effect is recognized.

Conversely, an evaluation was also made in the case where the concentration of the DSE test liquid was fixed and the concentration of the PE test liquid was changed. When the concentration of the DSE test solution was fixed at 1.25%, when the concentration of the PE test solution was 0.023%, the infectious titer reduction value was 3 and the disinfection effect was recognized (Figure 8 [ A]), the FIC index is calculated as (0.023/0.34)+(1.25/4.31)≈0.36, and is judged to have a “synergistic effect” (0.5 or less). When the concentration of the DSE test solution was fixed at 2.5%, when the concentration of the PE test solution was 0.0016%, the infectious titer reduction value was 3, indicating a disinfecting effect (Fig. 8 [ B]), the FIC index was calculated to be (0.0016/0.34) + (2.5/4.31) ≈ 0.56, indicating a “partial synergistic effect” (0.5 greater than 0.75 below). In other words, for DSE test solutions with concentrations of 2.5% and 1.25%, where no antiseptic effect was observed, addition of PE at concentrations higher than a certain level resulted in a “synergistic” or “partial effect” in antiviral action. A synergistic effect" is exhibited (in the present invention, both are considered to have a synergistic effect), and can be improved so that a disinfecting effect can be recognized.

[Preparation Example 4] Preparation of BSA-treated date seed extract (Part 1), quantification of tannins, and evaluation of antiviral activity against MS2 phage High-molecular-weight tannins bind to bovine serum albumin (BSA) and form aggregates It is well known to do Aqueous solutions were prepared by dissolving BSA in sterilized water so that the BSA concentration was 0 mg/mL (control), 1 mg/mL, 2 mg/mL and 3 mg/mL, and the date seed extract test solution "10% DSE/50% EtOH" in equal amounts. The mixed solution was allowed to stand at room temperature for 15 minutes, centrifuged at 10,000×g for 10 minutes, and the supernatant was collected to obtain a “BSA-treated test solution” (EtOH concentration: 25%). The test solution before BSA treatment was pale yellowish brown, but yellowish brown aggregates (precipitates) were generated by BSA, and the supernatant became almost colorless and transparent (see FIG. 9).

For the BSA-treated test solution, the same procedure as in Preparation Example 2 was used to measure the tannin content in the BSA-treated test solution by the vanillin hydrochloride method. In addition, the same procedure as in Reference Example 1 was used to evaluate the antiviral activity (infectivity titer reduction value) against MS2 phage.

The results are shown in FIG. The higher the BSA concentration used in the treatment, the lower the tannin content, and the antiviral effect tends to decrease. A certain amount of tannin was found to remain. From these results, tannins having the ability to aggregate BSA, that is, "high-molecular-weight tannins" were removed from the test solution treated with high-concentration BSA, and the antiviral activity when the test solution was used alone was Although it is almost lost, it is suggested that tannins that do not have the aggregation ability of BSA but are colored by the vanillin hydrochloride method, that is, "low-molecular-weight tannins" remain.

[Preparation Example 5] Preparation of BSA-treated date seed extract (Part 2) and analysis of components contained therein The date seed extract test solution "2.5% DSE / 50% EtOH" and the 1 mg / mL A BSA-treated test solution "2.5% DSE/50% EtOH/BSA (1 mg/mL) treatment" was prepared in the same manner as in Preparation Example 4 using the BSA solution of the concentration.

The components contained in the above two test solutions were analyzed by thin layer chromatography (TLC). A mixed solvent of toluene:acetone:acetic acid=3:3:1 was used as the developing solution. After development in a development tank and drying, the spot was sprayed with a vanillin hydrochloride method reagent (4% vanillin/methanol:hydrochloric acid=2:1) to develop color. As controls, flavan-3-ols (epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate), which are catechin monomer standard substances, were used.

The results are shown in FIG. Both '2.5% DSE/50% EtOH' and '2.5% DSE/50% EtOH/BSA (1 mg/mL) treatment' were effective for epigallocatechin gallate (EGCg) and epicatechin (EC), respectively. There are corresponding color reactions in spots II and III, presuming that these two compounds are "low-molecular-weight tannins". On the other hand, spot I seen only in "2.5% DSE/50% EtOH" is presumed to represent "high molecular weight tannin".

As shown in the figure, when the components of the persimmon extract were similarly analyzed by TLC, coloration was observed only at the position of spot I. It is presumed that almost all of the tannins contained in the persimmon extract are “high-molecular-weight tannins” (persimmon-condensed tannins) and that “low-molecular-weight tannins” do not exist.

[Example 2] Evaluation of antiviral action against MS2 phage by combined use of oyster extract and BSA-treated date seed extract i'), (vii) and (viii), and otherwise in the same manner for oyster extract alone, date seed extract alone, and oyster extract and date seed extract in combination The antiviral action (infectivity titer reduction value) against MS2 phage was compared in the case of In addition, the following (i') is the result of repeating the test under the same conditions as in Example 1 (i).
(i′) Mix 15 μL of the MS2 phage solution and 135 μL of the test solution “0.1% PE/50% EtOH”.
(vii) Mix 15 μL of the MS2 phage solution and 135 μL of the test solution “treated with 2.5% DSE/50% EtOH/BSA (1 mg/mL)”.
(viii) 15 μL of the MS2 phage solution and 135 μL of the test solution “treated with 0.1% PE/2.5% DSE/50% EtOH/BSA (1 mg/mL)” were mixed.

The results are shown in FIG. The antiviral action (infectious titer reduction value = 0.10) when "2.5% DSE/50% EtOH/BSA (1 mg/mL) treatment" was used alone was compared with the test solution "2.5% DSE/50% EtOH/BSA (1 mg/mL) treatment" alone. 5% DSE/50% EtOH” alone ((ii) of Example 1, see FIG. 6, infectivity titer reduction value=0.81). However, the antiviral effect when "2.5% DSE/50% EtOH/BSA (1 mg/mL) treatment" was used in combination with "0.1% PE/50% EtOH" (infectious titer reduction value = 3.59) is when "0.1% PE/50% EtOH" and "2.5% DSE/50% EtOH" are used in combination ((iii) of Example 1, see FIG. 6, infectivity titer reduction value=5. Although not as high as 52), it has reached a level recognized as having "antiviral activity." These results suggest that date seed extract contains both high-molecular-weight and low-molecular-weight tannins, but both are not essential to enhance the antiviral activity of oyster extract (to exert a synergistic effect). , suggesting that only low-molecular-weight tannins are sufficient.

[Example 3] Evaluation of antiviral action against norovirus by combined use of oyster extract and date seed extract .05% PE/50% EtOH" and date seed extract (DSE) in an aqueous ethanol solution with a concentration of 2.5% "2.5% DSE/50% EtOH" were prepared.

In the procedure of Reference Example 3, 1.2 μL of the norovirus suspension and 10.8 μL of the test solution "5% DS/50% EtOH" were mixed as described in (iv) to (xii) below, except that Similarly, PE alone, DSE alone, and PE and DSE in combination were compared for antiviral activity (genome survival rate) against norovirus. Controls were the cases where only PBS or only 50% ethanol aqueous solution was used as the test solution.
(iv) Mix 1.2 μL of norovirus suspension and 10.8 μL of PBS.
(x) Mix 1.2 μL of norovirus suspension and 10.8 μL of 50% ethanol aqueous solution.
(xi) Mix 1.2 µL of norovirus solution and 10.8 µL of test solution "2.5% DSE/50% EtOH".
(xii) Mix 1.2 μL of norovirus solution and 10.8 μL of test solution “0.05% PE/50% EtOH”.
(xiii) Mix 1.2 µL of norovirus solution and 10.8 µL of test solution "2.5% DSE/0.05% PE/50% EtOH".

The results are shown in FIG. The antiviral activity of "2.5% DSE/50% EtOH" and "0.05% PE/50% EtOH" (genome survival rate of about 51%) was higher than that of their solvent "50% EtOH" used as a control. There was no significant difference from the antiviral action (genome residual rate of about 60%), but when they were used in combination, the antiviral action was remarkably enhanced (genome residual rate of about 7%).

Claims (15)

An extract of fruit of Diospyros plant containing at least condensed tannins (hereinafter referred to as "oyster extract") and an extract of seeds of Phoenix plant containing at least tannins (hereinafter referred to as "persimmon extract") (referred to as "date seed extract"). 2. The antiviral agent according to claim 1, wherein the persimmon extract is a persimmon (Diospyros kaki) fruit extract. The antiviral agent according to claim 1 or 2, wherein the persimmon extract is an extract with an aqueous ethanol solution. The antiviral agent according to any one of claims 1 to 3, wherein the date seed extract is a date seed (Phoenix dactylifera) seed extract. The antiviral agent according to any one of claims 1 to 4, wherein the date seed extract is an ethanol aqueous solution extract. Antiviral agent according to any one of claims 1 to 5, wherein the date seed extract is enriched for low-molecular-weight tannins. Claims 1 to 6, containing the date seed extract corresponding to 10 to 100 parts by weight of the seeds of the date palm plant relative to the oyster extract corresponding to 1 part by weight of the fruit of the persimmon genus plant. Antiviral agent according to any one of. It contains the persimmon extract equivalent to 0.0005 to 0.1 parts by weight of the fruits of the persimmon genus plant with respect to the date seed extract equivalent to 1 part by weight of the seeds of the date palm plant. Item 7. The antiviral agent according to any one of Items 1 to 6. The antiviral agent according to any one of claims 1 to 8 and at least one selected from the group consisting of alcohols, organic acids and/or salts thereof, surfactants, antibacterial agents, moisturizers and cosmetic oils and fats. An antiviral composition against norovirus or its surrogate virus MS2 phage or feline calicivirus, comprising: 10. The antiviral composition according to claim 9, containing ethanol and/or propanol as said alcohol. 11. The antiviral composition according to claim 9 or 10, wherein said organic acid and/or salt thereof comprises at least one selected from the group consisting of lactic acid, citric acid, ferulic acid and salts thereof. Claims 9 to 9, wherein the surfactant contains at least one selected from the group consisting of glycerin fatty acid ester, sucrose fatty acid ester, lauryl glucoside, sodium lauryl sulfate, ammonium lauryl sulfate, sodium laureth sulfate and sodium olefin sulfonate. 12. The antiviral composition according to any one of 11. Any one of claims 9 to 12, which contains the antiviral agent containing the oyster extract corresponding to 0.05 to 1% by weight of the persimmon fruit per 100% by weight of the total composition. The antiviral composition according to item 1. Claims 9 to 13, wherein the antiviral agent comprises the date seed extract corresponding to 1.25 to 2.5% by weight of the plant seeds of the date palm relative to 100% by weight of the entire composition. The antiviral composition according to any one of . An extract of fruit of Diospyros plant containing at least condensed tannins (hereinafter referred to as "oyster extract") and an extract of seeds of Phoenix plant containing at least tannins (hereinafter referred to as "persimmon extract") (referred to as "date seed extract")), the antiviral effect of the oyster extract and the date seed extract alone against norovirus or its alternative virus, MS2 phage or feline calicivirus, is enhanced by a synergistic effect. methods of treating humans (except when performed inside or on the surface of the human body as a method of treating humans) .

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