JP2011084503A - Antiviral agent and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antiviral agent that has high antiviral effect and is safely useful for living body and a method for producing such an antiviral agent. <P>SOLUTION: The antiviral agent contains an extract of fir leaves, especially emulsified extract of fir leaves. The extract contains bornyl acetate, camphene, pinene and limonene. The method for producing an antiviral agent includes an extraction step of extracting an extract from fir leaves by steam distillation and an emulsification step of emulsifying the extract extracted by the extraction step. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an antiviral agent and a method for producing the antiviral agent. Specifically, the present invention relates to an antiviral agent using a natural material and a method for producing the antiviral agent.

Viruses are microscopic structures that can replicate themselves using cells of other organisms, and are composed of a protein shell and a nucleic acid packed inside the protein shell.
Among viruses, for example, influenza virus is a virus that infects humans and causes contagious influenza.

  In addition, there is a process in which particles are exposed so that they are released from and adsorbed by host cells during the process of virus infection. Chlorine disinfectants and iodine agents are used for such situations, but these existing drugs have problems such as environmental protection, safety to the living body, irritation, metal corrosion, off-flavor, and sustainability. In practice, there are limits to application.

Therefore, antiviral agents using natural materials have been demanded.
For example, Patent Literature 1 describes an influenza virus infection preventive agent containing tea polyphenol as an active ingredient.
Patent Document 2 describes an anti-influenza virus agent containing tea saponin as an active ingredient.
Furthermore, Patent Document 3 describes a plant virus inhibitor containing a conjugate of a tannin substance extracted from tea leaves and a monosaccharide or polysaccharide as an active ingredient.

JP-A-3-101623 JP-A-11-193242 JP-A-6-199619

  However, plant-derived natural materials that are known to have antiviral activity are limited to tea-derived natural materials. To combat a wider range of pathogenic viruses, other plant species have anti-viral activity. It has been desired to search for viral agents.

  The present invention was devised in view of the above points, and provides an antiviral agent that has a high antiviral effect and can be used safely with respect to a living body, and a method for producing such an antiviral agent. For the purpose.

As a result of diligent research to solve the above-mentioned problems, the present inventors have found that the components extracted from fir leaves are effective as medicinal components of antiviral agents, and have completed the present invention. .
That is, the antiviral agent of the present invention contains a fir leaf extract.

  Moreover, the antiviral agent of this invention contains the emulsified thing of the extract of a fir leaf.

  Here, since the extract is emulsified, the antiviral agent can be sprayed, for example, by a simple spray.

In the antiviral agent of the present invention, the extract preferably contains bornyl acetate, camphene, pinene, and limonene.
In the antiviral agent of the present invention, the total amount of bornyl acetate, camphene, pinene, and limonene is preferably 90% by mass or more based on the total amount of the extract.
In the antiviral agent of the present invention, the extract preferably contains 30% by mass or more of bornyl acetate based on the total amount of the extract.

  Moreover, in order to achieve said objective, the manufacturing method of the antiviral agent of this invention emulsifies the extract extracted by the extraction process extracted from the fir leaf by steam distillation, and the extraction process An emulsification step.

Here, the fir extract can be sufficiently extracted by performing the extraction by steam distillation.
Moreover, an emulsified liquid antiviral agent can be obtained by emulsifying the extract. For example, the antiviral agent can be sprayed by a simple spray.

The antiviral agent according to the present invention has a high antiviral effect and can be safely used for a living body.
The method for producing an antiviral agent according to the present invention can produce an antiviral agent that has a high antiviral effect and can be safely used for a living body.

It is the schematic explaining an example of the process of manufacturing the antiviral agent of this invention.

  The antiviral agent of the present invention contains an extract of fir leaves. Moreover, the antiviral agent of this invention contains the emulsified thing of the extract of a fir leaf.

Fir is an evergreen conifer widely distributed in the Russian Far East. The so-called fir essential oil can be extracted from the tufts full of needles. The bunch contains about 2 to 2.5% essential oil, but the essential oil content varies depending on the distillation time, fir growing environment, tree age, and the like. This essential oil content is 5 times that of pine and 9 times that of larch.
In addition, fir essential oil (fir leaf extract) is obtained by steam distillation from the foliage under the fir tree and the foliage of the cut fir tree. Extracting fir essential oil will cause devastation of the forest It doesn't matter. In other words, it leads to the effective use of resources, which in turn increases the income of forest workers.

Also, fir essential oil has many volatile components, so it works softly and quickly and effectively on human organs. Moreover, the component of fir essential oil contains 35-45 types of bioactive substances. When analyzed by a chromatographic method or the like, 100 or more kinds of active substances have been detected, but there are some components that have not yet been identified. Some of the identified components include bornyl acetate, α-pinene, camphene, β-pinene, limonene, myrcene and the like. The total amount of bornyl acetate, camphene, pinene, and limonene is preferably 90% by mass or more based on the total amount of fir essential oil.
In addition, fir and todomatsu are the only plants containing bornyl acetate, but only fir is a plant containing a large amount of bornyl acetate.

  The fir essential oil preferably contains 30% by mass or more of bornyl acetate based on the total amount of fir essential oil.

Moreover, it is preferable that fir essential oil contains 10 mass% or more of camphene on the basis of the total amount of fir essential oil.
Moreover, it is preferable that fir essential oil contains 25 mass% or more alpha-pinene on the basis of the total amount of fir essential oil.
Moreover, it is preferable that fir essential oil contains 15 mass% or more of β-pinene based on the total amount of fir essential oil.
Moreover, it is preferable that fir essential oil contains 5 mass% or more of limonene on the basis of the total amount of fir essential oil.

FIG. 1 is a schematic diagram illustrating an example of a process for producing the antiviral agent of the present invention.
Pruned fresh Siberian fir leaves are collected, and the fir leaves are put into a steam distillation apparatus (step S1).
And water vapor | steam is generated, fir leaf is steamed and water vapor | steam distillation is performed (step S2).
Next, the vapor component extracted by the water vapor is cooled and the liquid is collected in a container to obtain fir essential oil (fir leaf extract) accumulated in the upper layer of the container (step S3).
Furthermore, the obtained fir essential oil is emulsified in an emulsifying device to obtain an emulsified liquid of fir essential oil (step S4).

[Influenza virus inactivation test]
105 ml of fir essential oil extracted by steam distillation of fir leaves from Siberia and 3000 ml of purified water were put into a stirring tank, and these were further emulsified in an emulsifier, and the resulting emulsified fir essential oil was used as a specimen. .
Next, 0.1 ml of a virus suspension of influenza virus was added to 1 ml of the obtained specimen and mixed to obtain a working solution. Then, it was allowed to act at room temperature, and after 1 hour from the start and 24 hours after the start, the working fluid was diluted 10,000 times using a cell maintenance medium, and the virus infection titer of the working fluid was measured. The working temperature was room temperature. As a comparative control, the virus suspension was also added and mixed with purified water and allowed to act at room temperature. The virus infection titer of the working solution was measured at the beginning of the action, 1 hour after the start, and 24 hours after the start. did.
The virus infectivity titer immediately after the start of the action of purified water was defined as the virus infectivity titer at the start of the action of the working fluid. The results are shown in Table 1.
A preliminary test was conducted in advance to examine the method for measuring the virus infectivity titer. As a result, it was confirmed that the virus infectivity titer can be measured without being affected by the specimen by diluting the working fluid 10,000 times in the cell maintenance medium. .

Moreover, the virus infectivity titer was measured as follows.
That is, using a cell growth medium, the cells used were monolayer cultured in a tissue culture microplate (96 wells), and then the cell growth medium was removed and 0.1 ml of cell maintenance medium was added. Next, 0.1 ml of the diluted solution of the working solution was inoculated every 4 holes and cultured in a carbon dioxide incubator (CO ₂ concentration: 5%) at 37 ° C. ± 1 ° C. for 4 to 7 days. After culturing, the presence or absence of cell morphological change (cytopathic effect) was observed using an inverted phase contrast microscope, and the 50% tissue culture infectious dose (TCID ₅₀ ) was calculated by the Reed-Muench method. The virus infection titer and the virus infection titer per ml of purified water were converted.

Moreover, the virus suspension was prepared as follows.
That is, using a cell growth medium, the cells were monolayer cultured in a tissue culture flask.
After monolayer culture, the cell growth medium was removed from the flask and inoculated with the test virus. Next, a cell maintenance medium was added to the test virus and cultured in a carbon dioxide incubator (CO ₂ concentration: 5%) at 37 ° C. ± 1 ° C. for 1 to 5 days.
After culturing, the morphology of the cells was observed using an inverted phase contrast microscope, and it was confirmed that morphological changes (cytopathic effect) occurred in the cells. The culture solution was centrifuged (3000 r / min, 10 minutes), and the resulting supernatant was used as a virus suspension.

Moreover, the virus used for the test is influenza virus type A (H1N1).
Moreover, the cell used for the test is MDCK (NBL-2) cell ATCC CCL-34 strain (manufactured by Dainippon Pharmaceutical Co., Ltd.).
Further, the cell growth medium used in the test is obtained by adding 10% of fetal calf serum to Eagle MEM medium “Nissui” (1) (manufactured by Nissui Pharmaceutical Co., Ltd.).
Moreover, the cell maintenance medium used for the test is a medium having the following composition.
Eagle MEM medium "Nissui" (1) 1000 ml
14% 10% NaHCO ₃
L-glutamine (30 g / l) 9.8 ml
100 x MEM vitamin solution 30 ml
20% 10% albumin
0.25% trypsin 20 ml

In Table 1, “log TCID ₅₀ / ml” is a logarithmic value of TCID ₅₀ per ml.
As can be seen from Table 1, one hour after adding the influenza virus suspension to the specimen, TCID ₅₀ decreased from 7.7 to 6.7, and the infectivity to cells decreased by 10%. After 24 hours, TCID ₅₀ was 4.5 or less, and no infectivity to cells was detected. That is, it was determined that “influenza virus was inactivated”.

  As described above, the emulsified extract (fir essential oil) extracted from fir leaves by steam distillation has a 10% decrease in infectivity to cells 1 hour after the addition of the influenza virus suspension. Since it has a high antiviral effect that no infectivity to cells is detected after a period of time and it is an extract extracted from fir leaves, which are natural materials, it can be safely used for living organisms.

  Moreover, since the emulsified liquid of the extract of fir leaves is liquid, it can be sprayed with a simple spray or the like and can be used very easily.

Claims (6)

Antiviral agent containing fir leaf extract. Antiviral agent including emulsified extract of fir leaf. The extract is
With bornyl acetate,
With camphene,
With pinene,
The antiviral agent according to claim 1 or 2, comprising limonene. The antiviral agent according to claim 3, wherein the total amount of bornyl acetate, camphene, pinene, and limonene is 90% by mass or more based on the total amount of the extract. The antiviral agent according to claim 4, wherein the extract contains 30% by mass or more of bornyl acetate based on the total amount of the extract. An extraction process for extracting the extract from fir leaves by steam distillation;
And an emulsifying step for emulsifying the extract extracted by the extraction step.

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