WO2021070551A1

WO2021070551A1 - Virus inactivating agent

Info

Publication number: WO2021070551A1
Application number: PCT/JP2020/034360
Authority: WO
Inventors: 剛正坂口; 智弘進藤
Original assignee: フマキラー株式会社
Priority date: 2019-10-08
Filing date: 2020-09-10
Publication date: 2021-04-15
Also published as: JP2021059512A; JP7406226B2

Abstract

This virus inactivating agent is characterized by comprising a buffer in an aqueous solution of a grapefruit seed extract and having a pH adjusted to 8 or above.

Description

Virus inactivating agent

The present invention relates to a virus inactivating agent that inactivates a virus.

Conventionally, it has been known that an antiviral composition, an antibacterial agent, an antifungal agent, a disinfectant, etc. contain a grapefruit seed extract as an active ingredient (see, for example, Patent Documents 1 to 6).

Patent Document 1 discloses an antiviral agent containing a grapefruit seed extract, which contains a solvent such as water or alcohol.

Patent Document 2 discloses a sterilizing composition containing a strong alkaline electrolyzed water having a pH of 11 or higher and a grapefruit seed extract.

Patent Document 3 discloses an anti-norovirus composition containing a grapefruit seed extract and alkaline electrolyzed water and having a pH of 11.5 to 14.

Patent Document 4 discloses a root canal cleaning solution in which a grapefruit seed extract is dissolved in water and the pH is adjusted to the range of 6.5 to 8.5 with sodium hydrogen carbonate.

Patent Document 5 discloses an antibacterial / antifungal composition containing calcium oxide or calcium hydroxide and grapefruit seed extract.

Patent Document 6 discloses a treatment liquid for textile products, which comprises a grapefruit seed extract adjusted to pH 6 to 13. This treatment liquid contains alkaline substances such as sodium carbonate, potassium carbonate, sodium hydroxide and potassium hydroxide, and alkali generating substances such as sodium hydrogen carbonate and potassium hydrogen carbonate as alkaline agents.

Japanese Unexamined Patent Publication No. 2011-42579 Japanese Patent No. 4846292 Japanese Patent No. 5388325 Japanese Patent No. 4582572 Japanese Patent No. 4774072 Japanese Unexamined Patent Publication No. 2009-41169

By the way, in Patent Documents 1 to 6, since edible grapefruit seed extract is used, there is an advantage that it is highly safe.

The preparation of Patent Document 1 is an antiviral agent, and its efficacy against norovirus after standing for 10 minutes is disclosed. However, in daily use, standing for 10 minutes is not realistic, and the effect in a shorter time is required. Therefore, the effect in a short time on norovirus in the constitution of Patent Document 1 is unknown. Further, Patent Document 1 does not describe the relationship between pH and efficacy. The pH of the antiviral agent produced in Patent Document 1 is unknown, but it is considered to be neutral to weakly acidic.

Further, Patent Documents 2 and 3 describe that the grapefruit seed extract and alkaline electrolyzed water are essential constituents to exert a sterilizing effect and an anti-norovirus effect, but the alkaline electrolyzed water in a weak alkaline region having a pH of 11 or less is exhibited. The stability is low, for example, the pH when stored for about one month may be significantly lowered. In this case, it is considered that the desired sterilizing effect and anti-norovirus effect cannot be exhibited.

Further, in Patent Document 4, the pH is adjusted to the range of 6.5 to 8.5 with sodium hydrogen carbonate, but this is a root canal cleaning solution having a bactericidal action, and has an effect on the above-mentioned non-enveloped virus. Is unknown. In addition, sodium hydrogen carbonate has doubts in terms of stability, and there is a possibility that the initial efficacy cannot be maintained when stored for several months, for example.

Further, in Patent Document 5, although it contains calcium oxide or calcium hydroxide, it is an antibacterial / antifungal composition, and its efficacy against the above-mentioned non-enveloped virus is unknown. In addition, calcium oxide and calcium hydroxide have doubts in terms of stability, and there is a possibility that the initial efficacy cannot be maintained when stored for several months, for example.

Further, in Patent Document 6, although it contains an alkaline substance, an alkali generating substance, etc., it is a treatment liquid for textile products, a preparation for sterilizing Klebsiella pneumoniae, and its efficacy against the above-mentioned non-enveloped virus is unknown. .. Also, the stability of the formulation is unknown.

The present invention has been made in view of the above points, and an object of the present invention is to sufficiently enhance not only the sterilizing effect but also the efficacy against non-enveloped viruses and the like while maintaining high safety and stability. There is.

In order to achieve the above object, in the present invention, a buffer solution makes it possible to maintain a pH above a predetermined level for a long period of time.

The first invention is characterized in that an aqueous solution of grapefruit seed extract contains a buffer and is adjusted to pH 8 or higher.

According to this configuration, not only the high sterilizing effect of the grapefruit seed extract, but also the synergistic action of the grapefruit seed extract and the alkali having a pH of 8 or higher provides a high virus removing effect against non-enveloped viruses and the like. In addition, a buffer solution is generated by containing a buffer agent in the aqueous solution of the grapefruit seed extract. As a result, the initial pH is maintained for a long period of time.

The second invention is characterized in that the buffer contains sodium carbonate.

The third invention is characterized in that the buffer contains sodium hydrogen carbonate.

According to the second and third inventions, the stability during long-term storage is further enhanced.

The fourth invention is characterized in that the pH is adjusted to 8.5 or higher.

According to this configuration, the virus removal effect against non-enveloped viruses is further enhanced.

The fifth invention is characterized in that the pH is adjusted to 10.0 or higher.

According to this configuration, a high virus removing effect can be obtained even if the contact time with the non-enveloped virus is short.

The sixth invention is characterized in that the pH is adjusted to 11.0 or less.

According to this configuration, it does not show strong alkali, so it is safer to handle. Moreover, the virus inactivating agent may be adjusted to pH 10.5 or less.

According to the present invention, since the aqueous solution of the grapefruit seed extract contains a buffer and is adjusted to pH 8 or higher, it has not only a sterilizing effect but also a non-enveloped virus and the like while maintaining high safety and stability. The effect on the virus can be sufficiently enhanced.

It is a graph which shows the pH stability test result of an Example and a comparative example. It is a graph which shows the antiviral test result of an Example and a comparative example.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. It should be noted that the following description of the preferred embodiment is essentially merely an example and is not intended to limit the present invention, its application or its use.

The virus inactivating agent according to the embodiment of the present invention contains a buffer in an aqueous solution of grapefruit seed extract and is adjusted to pH 8 or higher. The aqueous solution of grapefruit seed extract is obtained by dissolving the grapefruit seed extract in ion-exchanged water. The concentration of the grapefruit seed extract in this aqueous solution can be set in the range of 0.1% by mass to 5.0% by mass. The lower limit of the concentration of the grapefruit seed extract is preferably 0.15% by mass, more preferably 0.2% by mass. The upper limit of the concentration of the grapefruit seed extract is preferably 3.0% by mass, more preferably 0.8% by mass.

Grapefruit seed extract is extracted and purified from the seeds of grapefruit fruit, and is generally accepted as a food additive. When the grapefruit seed extract is obtained from grapefruit, seeds can be taken out from the harvested grapefruit, the extracted seeds can be crushed, and the crushed one can be extracted. At this time, the grapefruit seed extract may be extracted from the uncried product in the undried state, or the grapefruit seed extract may be extracted from the crushed product in the freeze-dried state.

When extracting the grapefruit seed extract, a solution such as water or alcohol can be used. Examples of the alcohol used as the solvent for extraction include ethanol and the like. When extracting the grapefruit seed extract, the seeds may be heated to, for example, 30 ° C. or higher. Grapefruit seed extract contains fatty acids, flavonoids and the like. The grapefruit seed extract is preferably food grade, but not necessarily food grade.

The buffer contains sodium carbonate and sodium hydrogen carbonate. The pH of the virus inactivating agent can be adjusted by the amount of sodium carbonate and sodium hydrogen carbonate. In this embodiment, the contents of sodium carbonate and sodium hydrogen carbonate are set so that the pH of the virus inactivating agent is 8 or more. The contents of sodium carbonate and sodium hydrogen carbonate are preferably set so that the pH of the virus inactivating agent is 8.5 or more, and more preferably the pH of the virus inactivating agent is 10.0 or more. It is set to be. When determining the contents of sodium carbonate and sodium hydrogen carbonate, sodium carbonate and sodium hydrogen carbonate are added while measuring the pH of the virus inactivating agent, and sodium carbonate and sodium hydrogen carbonate at the time when the desired pH is reached. It suffices to know the content of sodium hydrogen carbonate.

The upper limit of the pH of the virus inactivating agent can be, for example, 11.5, and it is preferable to set the contents of sodium carbonate and sodium hydrogen carbonate so that the pH is 11.5 or less. More preferred is pH 11.0. As a result, the virus inactivating agent does not show strong alkalinity, which enhances safety during handling.

The present embodiment is characterized in that it contains sodium carbonate and sodium hydrogen carbonate as a buffer. By containing sodium carbonate and sodium hydrogen carbonate as a buffer, the aqueous solution becomes a buffer solution and stabilizes. Stable means maintaining the initial pH for a long period of time, for example, several months to half a year, or about one year.

Hereinafter, the reason why the virus inactivating agent of the present embodiment is stable for a long period of time will be described. Since the entire amount of sodium carbonate is ionized in the aqueous solution, it can be represented by the following formula 1.

Na2CO3 → CO32- + 2Na + ... 1

On the other hand, when sodium hydrogen carbonate is soluble in water, it can be expressed by the following formula 2.

NaHCO3 → HCO3- + Na + ... 2

At this time, there is an equilibrium between the hydrogen carbonate ion and the carbonate ion, and this state can be expressed by the following equation 3.

CO32- + H + ← → HCO3- ... 3

The equilibrium constant in the second dissociation reaction of this carbonate ion can be expressed by the following formula.

Since the degree of ionization of carbon dioxide is low, HCO3- and CO32-are almost equal to the concentrations of sodium carbonate and sodium hydrogen carbonate added in the preparation from formulas 1 and 2.

Therefore, the pH can be determined by modifying the equation.

At this time, Ka2 is a constant, and pKa2 in carbonate ion is about 10.33.

That is, in this system, the pH is determined by the ratio of sodium carbonate and sodium hydrogen carbonate added.

Normally, the pH changes significantly due to dilution or addition of acid, but as shown in Equation 3, when H + is supplied from the outside, carbonate ions are bound to form hydrogen carbonate ions, which suppresses the pH change. On the contrary, when H + is deprived, it becomes a carbonate ion and releases hydrogen ion into the system to suppress the pH change.

On the other hand, when adjusting the pH with sodium hydrogen carbonate alone, there is an upper limit to the pH that can be reached due to the problem of ionization, and it is difficult in principle to adjust the pH to, for example, the pH set this time. On the other hand, when adjusting the pH with sodium carbonate alone, since it is not a buffer solution, it does not have an effect of suppressing the pH change, and the pH fluctuates, for example, when carbon dioxide in the air dissolves. In particular, when the solution is adjusted to a weak alkali (for example, pH 10) as in this case, the pH of sodium carbonate alone is significantly lower than that of a buffer solution, so it is large even if a small amount of carbon dioxide is dissolved. to be influenced. Therefore, the pH tends to fluctuate greatly, and stabilization becomes difficult.

It is also possible to use alkaline electrolyzed water to make it alkaline, but in the case of alkaline electrolyzed water, the lower the pH, the smaller the absolute amount of alkali, so even a small amount of carbon dioxide, etc., has a large effect. Receive. Therefore, there is a problem in stability over time in the weak alkaline region.

It is also conceivable to use calcium oxide as an active ingredient of a pH adjuster, but in the case of calcium oxide, calcium ions may combine with carbonate ions dissolved from the air, causing precipitation. In order to avoid this, it becomes necessary to add other additives.

That is, in the present embodiment, by containing sodium carbonate and sodium hydrogen carbonate, the stability over time can be improved as compared with alkaline electrolyzed water and calcium oxide.

The virus inactivating agent can be contained in a container having a spraying lever and sprayed on various articles and the like. Examples of the container having the spray lever include various containers conventionally used, and any container may be used. The virus inactivating agent can also be sprayed by a spraying device equipped with a hand-push pump or an electric pump. The virus inactivating agent can also be used by applying it to an article or dropping it. The virus inactivating agent can also be used by directly spraying it on, for example, cooking utensils such as cutting boards and kitchen knives, countertops, tableware, towels, and towels. The virus inactivating agent can also be used by spraying on clothing, floors, walls, toilet bowls, wash basins, and automobile interiors. The virus inactivating agent may be sprayed on the hands.

Also, the virus inactivating agent does not contain alcohol. That is, the virus inactivating agent does not have a bactericidal effect or an antiviral effect due to alcohol, and exhibits a bactericidal effect and an antiviral effect depending on the pH value and the grapefruit seed extract.

(PH stability test)
In the pH stability test, the samples shown in Table 1 were prepared.

The active ingredients of Example 1 and Comparative Examples 1 and 2 are grapefruit seed extract (grapefruit seed extract). The pH adjuster of Example 1 is sodium carbonate and sodium hydrogen carbonate. The pH adjuster of Comparative Example 1 is alkaline electrolyzed water, and the pH adjuster of Comparative Example 2 is sodium carbonate. The rest is ion-exchanged water. The amount of the pH adjuster added is set so as to have an initial pH value described later. Comparative Example 3 is a commercially available antiviral agent, which is strongly alkaline due to the active ingredient calcium oxide. A chelating agent preparation is added as an additive to Comparative Example 3.

The pH stability test method is shown below.

1. Place each sample in a glass vial.

2. The glass vial containing the sample is stored in a constant temperature chamber at 60 ° C.

3. Take out the glass vial in the constant temperature chamber at regular intervals, measure the pH and check the appearance.

The reason for using a 60 ° C constant temperature chamber is to obtain so-called accelerated test results.

Table 2 shows the initial pH of Example 1 and Comparative Examples 1 to 3 and the change over time in pH.

The initial pH of Example 1 and Comparative Examples 1 and 2 was approximately 10, but the initial pH of Comparative Example 3 was approximately 12. As shown in FIG. 1, in the case of the example in which sodium carbonate and sodium hydrogen carbonate are used as the pH adjuster, the pH after 3 weeks is about 9.7, and there is almost no difference from the initial pH. It was. On the other hand, in the case of Comparative Example 1 using alkaline electrolyzed water, the pH after 1 week was about 9.3, the pH after 3 weeks was about 8.2, and the rate of decrease from the initial pH was extremely high. It was big. Further, in the case of Comparative Example 2 using sodium carbonate, the pH after 1 week was about 9.4, the pH after 3 weeks was about 8.8, and the rate of decrease from the initial pH was extremely large. It was. Further, in the case of Comparative Example 3 using calcium oxide, a precipitate was formed after 1 week, and then the pH after 2 weeks was about 10.5 and the pH after 3 weeks was 10.2. The rate of decrease from the initial pH was extremely large. From the above, it can be seen that the virus inactivating agent according to the present embodiment has high stability over time.

(Antiviral test)
Next, the virus infectious titer measurement test before and after the treatment of the virus inactivating agent will be described. Feline calicivirus is used in this test, and this feline calicivirus is used in the test as a substitute for norovirus, which belongs to the same family in terms of classification and has a similar structure. This is because norovirus is difficult to culture and an easy method for evaluating the infectious titer has not yet been established. That is, it is generally considered that any agent that exhibits sufficient antiviral properties in a test using feline calicivirus will also exhibit sufficient antiviral properties against norovirus.

When conducting a virus infectivity titer measurement test, first, cells are monolayer-cultured in a cell culture microplate (96 holes) using a cell growth medium. The cells are CRFK cells. Then, the monolayer cultured cells were inoculated with a virus suspension solution diluted with cat calicivirus (FCV) and adsorbed on the cells in a carbon dioxide incubator (CO2 concentration: 5%) at 37 ° C. ± 1 ° C. for 1 hour. After that, remove the virus inoculum, add cell maintenance medium, and incubate for 4 to 7 days. Then, the cells are stained with amide black, the life and death of the cells are confirmed, and the 50% tissue culture infectivity titer (TCID50 / ml) is calculated by the Red-Muench method. The lower the value, the lower the infectivity.

In general, non-enveloped viruses such as feline calicivirus have stronger resistance to various disinfectants and antibacterial agents than enveloped viruses such as influenza virus. Therefore, any agent that is effective against non-enveloped viruses is likely to be effective against enveloped viruses in a shorter period of time.

The test agents are as shown in Table 3. The rest is ion-exchanged water.

Figure 2 shows the results of the virus infection titer measurement test. In the graph, "30 seconds" indicates that the contact time between the test agent and the virus is 30 seconds, and "120 seconds" means that the contact time between the test agent and the virus is 120 seconds. Is shown. In the case of "30 seconds", it can be called a short-time contact test, and in the case of "120 seconds", it can be called a long-time contact test. The value in the graph is a log value (log TCID 50 / ml) having an infectious value of TCID 50 / ml. As a control, sterilized water was used. It can be said that the lower the log TCID 50 / ml of the test agent as compared with the control, the higher the antiviral property.

As shown in FIG. 2, in Example 2 of pH 8.0, the decrease in logTCID 50 / ml from the control in the case of “120 seconds” shows an extremely large value of 2.5. Further, in Example 3 of pH 8.5, Example 4 of pH 9.0, and Example 5 of pH 10.0, the decrease in log TCID 50 / ml from the control at "120 seconds" was 3.0 or more. , Had sufficient antiviral properties. Further, in Example 3 of pH 8.5, Example 4 of pH 9.0, and Example 5 of pH 10.0, the decrease in log TCID 50 / ml from the control at "30 seconds" was 1.5 or more. , It had sufficient antiviral properties even if the contact time was short. In particular, in Example 5 of pH 10.0, the log TCID of 50 / ml in the case of "30 seconds" was 3.0 or more, and even if the contact time was short, it had extremely high antiviral properties. It was. Further, although not shown, the cases of pH 10.5 and pH 11.0 also have high antiviral properties similar to those of Example 5.

On the other hand, in Comparative Example 4 of pH 7.0, the decrease in log TCID 50 / ml from the control was 0.5 or less at both “30 seconds” and “120 seconds”.

Although not shown, in the case of a preparation containing 0.18% by mass of grapefruit seed extract at pH 3, the decrease in log TCID 50 / ml from the control at "120 seconds" is about 0.3, and grapefruit at pH 3. In the case of a preparation containing 0.36% by mass of seed extract, the decrease in logTCID50 / ml from the control was about 0.5 at "120 seconds", and 0.54% by mass of grapefruit seed extract at pH3. In the case of the containing preparation, the decrease in log TCID 50 / ml from the control is about 1.0 at "120 seconds", and when the pH is low, even if the content of the grapefruit seed extract is increased. , Low antiviral effect. As described above, in the neutral to acidic region, even if the grapefruit seed extract is contained, a sufficient antiviral effect cannot be obtained with a daily and practical contact time (30 to 120 seconds or less). On the other hand, in the case of a preparation containing sodium carbonate and sodium hydrogen carbonate aqueous solution having a pH of 10 and not containing the grapefruit seed extract, the value was -0.5, and the antiviral effect was low.

Further, in the case of a test using influenza virus (A / Udorn / 72 (H3N2)), trypsin may be added to the cell maintenance medium using the cells as MDCK cells. The liquid preparations of Examples 1 to 5 exhibited antiviral properties equivalent to those of feline calicivirus against influenza virus.

As described above, the virus inactivating agent of this example exerts sufficient efficacy not only against enveloped viruses such as influenza virus but also against non-enveloped viruses such as feline calicivirus. Moreover, since it showed sufficient antiviral properties against feline calicivirus, it is highly possible that the virus inactivating agent of the present embodiment is an anti-norovirus agent having sufficient anti-norovirus properties.

(Antibacterial test)
Next, the antibacterial test will be described. When conducting an antibacterial test, first, 0.1 ml of 109 cfu / ml Escherichia coli solution is added to 10 ml of the test agent to make 107 cfu / ml. At this time, as a control, a product using 10 ml of physiological saline instead of the test agent is also prepared. After 10 seconds have passed by liquid-liquid contact, 1 ml is withdrawn and placed in 9 ml of SCDLP liquid medium to inactivate. Then, serial dilution is performed and 200 μl is seeded on SCDLP agar medium. The number of colonies is counted for the control and each test agent, and the sterilization rate is measured. The sterilization rate is calculated from the formula of the number of colonies of the test agent / the number of colonies of the control. Examples 1 to 5 and Comparative Examples 1 to 4 were prepared as test agents. The antibacterial test results were 99.99% or more in all of Examples 1 to 5 and Comparative Examples 1 to 4.

(Pollutivity)
Next, the pollutability will be described. Contaminability can be determined by, for example, spraying a test agent onto a black object, completely drying it, and then visually observing whether or not powder residue is observed. If no powder residue is found, it can be determined that there is contamination, and if no powder residue is found, it can be determined that there is no contamination. Regarding the contaminating property, all of Examples 1 to 5 and Comparative Examples 1 to 4 were non-staining.

(Action and effect of the embodiment)
As described above, since the virus inactivating agent according to this embodiment contains a buffer in the aqueous solution of the grapefruit seed extract and is adjusted to pH 8 or higher, it has high safety and stability. In addition to the bactericidal effect, the efficacy against non-enveloped viruses such as norovirus can be sufficiently enhanced. Further, it can be used as a virus inactivating agent having almost no contamination.

Moreover, since the virus inactivating agent is weakly alkaline, it can reduce the irritation to the eyes and skin.

The above embodiment is merely an example in all respects and should not be construed in a limited way. Furthermore, all modifications and modifications that fall within the equivalent scope of the claims are within the scope of the present invention.

As described above, the virus inactivating agent according to the present invention can be used, for example, by spraying it on a cooking utensil or the like.

Claims

A virus inactivating agent characterized in that a buffer is contained in an aqueous solution of grapefruit seed extract and the pH is adjusted to 8 or higher.
In the virus inactivating agent according to claim 1,
The buffer is a virus inactivating agent containing sodium carbonate.
In the virus inactivating agent according to claim 1 or 2.
The buffer is a virus inactivating agent containing sodium hydrogen carbonate.
The virus inactivating agent according to any one of claims 1 to 3.
A virus inactivating agent characterized in that the pH is adjusted to 8.5 or higher.
In the virus inactivating agent according to claim 4,
A virus inactivating agent characterized in that the pH is adjusted to 10.0 or higher.
The virus inactivating agent according to any one of claims 1 to 5.
A virus inactivating agent characterized in that the pH is adjusted to 11.0 or less.