JP5506023B2 - Influenza virus infection inhibitor - Google Patents

Description

  The present invention relates to a highly safe influenza virus infection-preventing agent suitable for use in disinfection of skin and the like.

  Every year, influenza infections are repeated throughout the world. To prevent or treat influenza, generally vaccinate or take anti-influenza virus drugs such as amantadine, oseltamivir phosphate (Tamiflu: registered trademark) or zanamivir hydrate (Relenza: registered commercial) To be done. However, the antigenicity of influenza is easily mutated, and its epidemic cannot be suppressed by vaccine alone. In addition, although anti-influenza virus agents exhibit high anti-influenza activity, there are problems such as appearance of drug-resistant viruses and side effects such as headache and dyspnea. For this reason, development of an anti-influenza virus agent that has no side effects and is safe and effective for the human body is required.

Although there is vaccination as an infection prevention against influenza infection, the type of influenza virus that prevails in that year is different every year, and there is a situation where the production of vaccines corresponding to that type is not in time. Therefore, infection prevention is the most important for influenza viruses.
From such a point of view, various agents for preventing influenza virus infection have been proposed so far. For example, tea polyphenol as an active ingredient (see, for example, Patent Document 1), and chlorogenic acid ester as an active ingredient. (For example, refer patent document 2) etc. are proposed.
However, the present condition is that the effective influenza virus infection preventive agent has not appeared so far.

On the other hand, glycerin fatty acid esters and polyglycerin fatty acid esters are approved as food additives, and they are widely used, such as food emulsifiers, foaming agents, antistatic agents for plastics, and antibacterial agents. It is.
As the pharmacological action of this glycerin fatty acid ester, for example, anti-herpesvirus activity, anti-AIDS virus activity (see, for example, Patent Document 3), polyglycerin fatty acid ester, monoglycerin fatty acid ester, amino acid and lower alcohol are blended. A method for sterilizing norovirus (for example, see Patent Document 4) has been proposed.
However, the effect of the polyglycerol fatty acid ester aimed at by the present invention on influenza virus is not known.

The inventors of the present invention have studied various pharmacological actions of polyglycerol fatty acid esters with high safety, and found that this has anti-influenza virus activity, that is, infection prevention action against influenza virus, against influenza virus. The aqueous solution or emulsion containing this product was confirmed to be extremely effective as an influenza virus infection inhibitor such as skin disinfection, and the present invention was completed.
JP-A-3-101623 JP 2004-345971 A JP-T-2-502915 JP 2008-156329 A

  Therefore, the subject of this invention is providing the influenza virus infection prevention agent which has few skin irritation, is safe for a human body and a foodstuff, and shows high inactivation performance.

The present invention for solving this problem has the following configuration. That is, the present invention
1. An influenza virus infection inhibitor comprising a polyglycerol fatty acid ester having a monoester purity of 50% by mass or more;
2. 2. The influenza virus infection preventive agent according to 1, wherein the fatty acid constituting the polyglycerin fatty acid ester is a saturated fatty acid having 8 to 14 carbon atoms or an unsaturated fatty acid having 9 to 22 carbon atoms;
3. 3. Influenza virus infection inhibitor according to the above 1 and 2, wherein the polyglycerol constituting the polyglycerol fatty acid ester has a polymerization degree of 2 to 5;
4). Influenza virus infection prevention agent of said 1-3 characterized by being in the form of aqueous solution or an emulsion liquid,
It is.

The polyglycerol fatty acid ester, which is an active ingredient in the influenza virus infection preventive agent proposed by the present invention, exhibits a high anti-influenza virus effect, is a food additive, has low skin irritation, and is in contact with the human body and food. It is a compound that can be used safely.
Therefore, it is possible to provide an influenza virus infection inhibitor having high safety, no skin irritation, and no side effects, particularly an influenza virus infection inhibitor in the form of an aqueous solution or an emulsion, and can be used easily. The medical value is tremendous.

As described above, the basic aspect of the present invention is an influenza virus infection inhibitor characterized by containing a polyglycerol fatty acid ester having a monoester purity of 50% by mass or more.
Details of the present invention will be described below.

In the present invention, the polyglycerol fatty acid ester contained as an active ingredient is an esterification reaction by a known method of polyglycerol and a fatty acid, or a transesterification reaction by a known method of polyglycerol and a fatty acid lower alkyl alcohol ester. The reaction product obtained by the method is a polyglycerol fatty acid ester obtained by increasing the monoester purity to 50% by mass or more by a known method such as molecular distillation, chromatography or solvent fractionation.
In the present invention, in order to exert anti-influenza virus activity, the monoester purity needs to be 50% by mass or more, and more preferably the monoester purity is increased to 70% by mass or more. Is preferred.
When the monoester purity is less than 50% by mass, sufficient anti-influenza virus properties are not exhibited.

On the other hand, the fatty acid constituting the polyglycerol fatty acid ester of the present invention is a saturated fatty acid having 8 to 14 carbon atoms or an unsaturated fatty acid having 9 to 22 carbon atoms.
Specific examples of such fatty acids include caprylic acid, capric acid, lauric acid, myristic acid, palmitoyl acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, arachidonic acid, ricinoleic acid, and the like. Can be mentioned.
When the number of carbon atoms is shorter than 8, the fatty acid irritation becomes stronger or the unpleasant odor becomes stronger. Moreover, when carbon number becomes longer than 22, sufficient anti-influenza virus property will not be shown. As a fatty acid to be used, 1 type may be sufficient and 2 or more types may be used together.

Examples of the polyglycerin constituting the polyglycerin fatty acid ester used in the present invention include diglycerin, triglycerin, tetraglycerin, and pentaglycerin.
These polyglycerols are prepared by adding a small amount of acid or alkali to glycerol as a catalyst, and heating at a temperature of, for example, 180 ° C. or higher under an arbitrary inert gas atmosphere such as nitrogen or carbon dioxide to cause a polycondensation reaction. It can be obtained by a method of separation and purification by molecular distillation or column chromatography, or a method of reacting epichlorohydrin and solketal using raw materials as a phase transfer catalyst.
In the case of polyglycerin, even if the average degree of polymerization obtained from the hydroxyl value is 2 to 5, if the degree of polymerization is 2, 3, 4, 5 is not the main component, sufficient anti-influenza virus properties The effect is not expressed.
The method for analyzing the degree of polymerization will be described later.

Therefore, as the polyglycerol fatty acid ester as an active ingredient contained as the anti-influenza virus agent of the present invention, specifically, diglycerol monocaprylate, diglycerol monocaprate, diglycerol monolaurate, diglycerol monomilliyl Stylate, Triglycerol monocaprylate, Triglycerol monocaprate, Triglycerol monolaurate, Triglycerol monomyristate, Tetraglycerol monocaprylate, Tetraglycerol monocaprate, Tetraglycerol monolaurate, Tetraglycerol monomyristate , Pentaglycerol monocaprylate, pentaglycerol monocaprate, pentaglycerol monolaurate, pentaglycerol monomyristate, diglycerol monooleate, Glycerol monolinoleate, diglycerin monoelcate, triglycerin monooleate, triglycerin monolinoleate, triglycerin monoelcate, tetraglycerin monooleate, tetraglycerin monolinoleate, tetraglycerin monoelcate, pentaglycerin monooleate, Examples thereof include pentaglycerin monolinoleate and pentaglycerin monoelcate.
Among them, diglycerol monolaurate, triglycerol monolaurate, tetraglycerol monolaurate, and pentaglycerol monolaurate are preferable.

Examples of the usage form of the influenza virus infection inhibitor containing the polyglycerol fatty acid ester provided by the present invention as an active ingredient include aqueous solutions and emulsions.
Influenza virus infection prevention agent as this aqueous solution or emulsified liquid is used for spraying food, tableware, etc., or impregnating in masks, wet tissues, etc., as well as hospital walls, curtains, medical instruments, It can be used for disinfection of the human body, such as spraying or application of toilet seats, spraying or application to the skin or mouth.

The content of the polyglycerol fatty acid ester which is an active ingredient in the aqueous solution or emulsion as the influenza virus infection inhibitor of the present invention cannot be generally limited, but the anti-influenza virus effect can be achieved by coating or spraying. It is sufficient to contain an effective amount that exhibits the above, specifically 0.1 to 20% by mass.
When the amount exceeds 20% by mass, problems such as stickiness and handling occur, which is not preferable. Further, if the blending amount is less than 0.1% by mass, sufficient anti-influenza virus activity cannot be exhibited.

When preparing an aqueous solution or emulsion as an influenza virus infection-preventing agent provided by the present invention, other additives may be blended within a range that does not inhibit the anti-influenza virus effect of the present invention.
Examples of such additives include preservatives, preservatives, antioxidants, light stabilizers, fragrances, and coloring agents. Moreover, you may use together with other antiviral agents, antibacterial agents, etc., such as polyphenols and quaternary ammonium salts.

  Hereinafter, the present invention will be described in more detail with reference to specific production examples, test examples, and examples, but the present invention is not limited to these test examples and examples.

Production Example 1: Synthesis of diglycerin monocaprylate After adding 40 g of CaO (manufactured by Wako Pure Chemical Industries, Ltd .; the same shall apply hereinafter) to 20 kg of glycerin, and performing a condensation reaction of glycerin at 260 ° C. for 5 hours under a nitrogen gas stream, 72 g of phosphoric acid (85% product manufactured by Wako Pure Chemical Industries, Ltd .; the same applies hereinafter) was added for neutralization. Next, glycerin was removed from the obtained composition using a centrifugal molecular distiller (manufactured by ULVAC, Inc .; hereinafter the same) under a vacuum of 40 Pa at 120 ° C. to 160 ° C., followed by 20 Pa. A diglycerin composition having a diglycerin content of 94% was obtained at 190 ° C. under vacuum. The diglycerin content was analyzed according to analysis method 1 described later.
6.4 kg of the obtained diglycerin composition and 3.6 kg of caprylic acid (NAA-82, purity 99%, manufactured by NOF Corporation) were charged into a reaction kettle and subjected to esterification reaction at 220 ° C. for 3 hours in a nitrogen atmosphere. went. The obtained reaction product was removed from unreacted diglycerin at 160 ° C. under a vacuum of 1 Pa using a centrifugal molecular distillation machine, and then monoester at 200 ° C. under a vacuum of 1 Pa. A diglycerin monocaprylate composition having a content of 85% was obtained. The monoester content was analyzed according to analysis method 2 described later.

Production Example 2: Synthesis of Diglycerin Monoerkate 4.3 kg of diglycerin composition produced in the same manner as in Production Example 1 and 5.6 kg of erucic acid (purity 90%, manufactured by NOF Corporation) were charged into a reaction kettle. The esterification reaction was performed at 240 ° C. for 3 hours in a nitrogen atmosphere. The obtained reaction product was removed from unreacted diglycerin at 160 ° C. under a vacuum of 1 Pa using a centrifugal molecular distillation machine, and then monoester at 220 ° C. under a vacuum of 1 Pa. A diglycerin monoelkeate composition having a content of 81% was obtained. The monoester content was analyzed according to analysis method 2 described later.

Production Example 3: Synthesis of triglycerin monolaurate 40 g of CaO was added to 20 kg of glycerin and subjected to a condensation reaction of glycerin at 260 ° C. for 5 hours under a nitrogen gas stream, and then neutralized by adding 72 g of phosphoric acid. Next, the diglycerin was removed from the obtained composition using a centrifugal molecular distiller at 200 ° C. under a vacuum of 2 Pa, and subsequently a triglycerin content of 90 ° C. under a vacuum of 2 Pa at 230 ° C. % Triglycerin composition was obtained. The triglycerin content was analyzed according to analysis method 1 described later.
After purifying the triglycerin by activated carbon treatment, 6.5 kg of the triglycerin composition and 3.5 kg of lauric acid (NAA-122, purity 99%, manufactured by NOF Corporation) were charged into the reaction kettle, The esterification reaction was carried out at 240 ° C. for 3 hours. Unreacted triglycerin was removed from the obtained reaction product using a centrifugal molecular distillation machine at 230 ° C. under a vacuum of 1 Pa, followed by a monoester at 250 ° C. under a vacuum of 1 Pa. A triglycerin monolaurate composition having a content of 80% was obtained. The monoester content was analyzed according to analysis method 2 described later.

Production Example 4 Synthesis of Tetraglycerin Monolaurate Epichlorohydrin (Wako Pure Chemical Industries, Ltd .: hereinafter the same) 16 g and Solketal (Tokyo Chemical Industry Co., Ltd .: the same) 50 g, tetrabutylammonium iodine (Wako Pure Chemical Industries, Ltd.) Reacted by using 3.6 g of a phase transfer catalyst and 200 mL of 30% NaOH aqueous solution and 200 mL of hexane as a solvent. The temperature was vigorously stirred at 80 ° C. for 5 hours. After completion of the reaction, the hexane layer was fractionated with a separatory funnel, neutralized with a saturated aqueous solution of ammonium chloride, dried over an appropriate amount of anhydrous sodium sulfate, the solvent was distilled off with an evaporator, and the residue was 130 ° C., 100 Pa. The product was purified by vacuum distillation under the above conditions to obtain 44 g of linear triglycerol diacetonide. Further, 32 g of the obtained linear triglycerin acetonide and 10 g of epichlorohydrin were reacted using 1.8 g of tetrabutylammonium iodine as a phase transfer catalyst, 200 mL of 30% NaOH aqueous solution, and 200 mL of hexane as a solvent. . The reaction temperature was vigorously stirred at 40 ° C. After completion of the reaction, the hexane layer was fractionated with a separatory funnel, neutralized with a saturated aqueous ammonium chloride solution, dried over an appropriate amount of anhydrous sodium sulfate, the solvent was distilled off with an evaporator, and the developing solvent was hexane / ethyl acetate. = 7/3 and purified by silica gel column chromatography to obtain 30 g of a triglycerin acetonide glycidyl ether content of 95%.
In addition, the content of glycidyl ether of triglycerin acetonide was analyzed by analysis method 1 described later.
After reacting 19 g of glycidyl ether of triglycerol acetonide and 10 g of lauric acid at 120 ° C. for 1 hour, a reaction product was obtained. This was purified by silica gel chromatography using hexane / ethyl acetate = 7/3 as a developing solvent, dissolved in a solvent of THF / H ₂ O = 8/2, added with a catalytic amount of hydrochloric acid, and heated to 40 ° C. Then, acetonide was deprotected and purified again by silica gel chromatography using hexane / ethyl acetate = 7/3 as a developing solvent to obtain 19 g of a tetraglycerin monolaurate composition having a monoester content of 96%.
The monoester content was analyzed according to analysis method 2 described later.

Production Example 5: Synthesis of Pentaglycerin Monolaurate Using 38 g of glycidyl ether of triglycerin acetonide and 13 g of solketal obtained by the same method as in Production Example 4, using 1.8 g of tetrabutylammonium iodine as a phase transfer catalyst, Using 30% aqueous NaOH solution (200 mL) and hexane (200 mL) as a solvent, the mixture was vigorously stirred and reacted at 80 ° C. for 5 hours. After completion of the reaction, the hexane layer was fractionated with a separatory funnel, neutralized with a saturated aqueous solution of ammonium chloride, dehydrated with an appropriate amount of anhydrous sodium sulfate, and the residue obtained by distilling off the solvent with an evaporator, Purification by silica gel column chromatography using hexane / ethyl acetate = 7/3 as a developing solvent gave 40 g of a composition of pentaglycerin having a triacetonide content of 95%. The content of acetonide of pentaglycerin was analyzed according to analysis method 1 described later.
25 g of the resulting triglyceride of pentaglycerin and 12 g of lauric acid chloride (purity 99%, Wako Pure Chemical Industries, Ltd.) are dissolved in 40 mL of pyridine, a catalytic amount of N, N-dimethylammonium pyridine is added, and the mixture is stirred overnight at room temperature. Reaction and esterification. The residue obtained by distilling off the solvent was purified by silica gel column chromatography using hexane / ethyl acetate = 7/3 as a developing solvent, and dissolved in a solvent of THF / H ₂ O = 8/2. Then, a catalytic amount of hydrochloric acid was added and the mixture was heated to 40 ° C. to deprotect acetonide, and again purified by silica gel chromatography using hexane / ethyl acetate = 7/3 as a developing solvent, and a pentaester having a monoester content of 97%. 23 g of glycerol monolaurate was obtained.
The monoester content was analyzed according to analysis method 2 described later.

Production Example 6: Synthesis of diglycerin laurate 3.7 kg of the diglycerin composition produced in the same manner as in Production Example 1 and 6.3 kg of lauric acid were placed in a reaction kettle and subjected to esterification at 220 ° C. for 3 hours in a nitrogen atmosphere. went. The obtained reaction product was left and separated to remove diglycerin which was liberated, thereby obtaining diglycerin laurate having a monoester content of 32%, a diester content of 42%, a triester content of 22%, and a tetraester content of 4%.
The ester distribution was analyzed according to analysis method 2 described later.

Production Example 7: Synthesis of triglycerol monolaurate (type with polyglycerol polymerization degree distribution)
While charging 200 g of lauric acid into a 1 L reaction vessel and supplying nitrogen gas at a flow rate of 100 mL / min, the pressure was reduced to 550 mmHg and heated to 130 ° C. Subsequently, 741 g of glycidol (Wako Pure Chemical Industries, Ltd.) was added dropwise over 5 hours. When 20% of glycidol was dropped, 0.38 g of phosphoric acid was added. After completion of dropwise addition of glycidol, the system temperature was set to 140 ° C., and the reaction was continued for 12 hours to obtain polyglycerin laurate.
The monoester content of the obtained composition is 99%, and the average degree of polymerization of polyglycerol determined from the hydroxyl value is 3, but the distribution of the degree of polymerization is 38% glycerol, 19% diglycerol, 10% triglycerol, Tetraglycerol 9%, pentaglycerol 8%, hexaglycerol 6%, heptaglycerol 4%, octaglycerol 3%, nonaglycerol 2%, decaglycerol 1%.
The monoester content was analyzed according to analysis method 2 described later.
The polymerization degree of polyglycerin was saponified and decomposed, and the obtained polyglycerin was analyzed according to analysis method 1 described later.

Analysis method 1: Method for measuring the degree of polymerization of polyglycerol Take 10 mg of the glycerin polymer or triglycerin acetonide glycidyl ether or pentaglycerin acetonide prepared, dissolve in 1 mL of the following mobile phase, and GPC under the following conditions: The degree of polymerization was analyzed.
Apparatus: Shimadzu HPLC
Detector: RI detector Column temperature: 40 ° C
Column: Shodex-AsahipackGS220HQ × 2
Mobile phase: methanol / water = 30% / 70%
Mobile phase flow rate: 0.4 mL / min

Analysis Method 2: Method for Measuring Monoester Content 10 mg of the produced polyglycerol ester was taken and dissolved in 1 mL of the following mobile phase, and the degree of polymerization was analyzed by GPC under the following conditions.
Apparatus: Shimadzu HPLC
Detector: RI detector Column temperature: 40 ° C
Column: Shim-packGPC-801 × 2
Mobile phase: Tetrahydrofuran Mobile phase flow rate: 1.0 mL / min

Test Example 1: Confirmation of anti-influenza effect [ test sample ]
The anti-influenza virus properties were evaluated using 300 ppm aqueous solutions of the respective samples shown in Table 1 below.

* 1: Monoester content (%): Proportion of hydroxyl group of (poly) glycerin having only one ester bond * 2: Polyglycerin content (%): Ingredient name in constituent (poly) glycerin Proportion of (poly) glycerin shown * 3: Fatty acid purity (%): Proportion of fatty acid shown in component name in constituent fatty acids

Anti-influenza virus test [Test virus]
Influenza virus type A (H1N1 type)
[Used cells]
MDCK (NBL-2) cell ATCC CCL-34 strain (Dainippon Pharmaceutical Co., Ltd.)

[Preparation of virus solution]
Using a cell growth medium, MDCK cells were monolayer cultured in a tissue culture flask. After culture, the cell growth medium was removed from the flask and inoculated with the test virus.
Next, a cell maintenance medium was added and cultured in a 37 ± 1 ° C. carbon dioxide incubator for 1 to 3 days.
After culturing, the cell morphology was observed using an inverted phase contrast microscope, and it was confirmed that a cell morphology change (cytopathic effect) occurred. After confirmation, the supernatant obtained by centrifuging the culture solution (3000 r / min, 10 minutes) was used as the virus solution.

[Medium used]
(1) Cell growth medium Use a MEM medium “Nissui” (Nissui Pharmaceutical Co., Ltd.) plus 10% fetal calf serum.
(2) Cell maintenance medium A medium having the following composition was used.
Eagle MEM medium (Nissui) 1000mL
14% 10% NaHCO ₃
L-glutamine (30 g / L) 9.8 mL
100 x MEM vitamin solution 30mL
20% 10% albumin
0.25% trypsin 20 mL

[Virus inactivation test]
0.1 mL of the virus solution was added to 1 mL of the sample, mixed, and stored at room temperature for 1 hour and 24 hours. After storage, the sample virus solution was washed out with 2 mL of cell maintenance medium to prepare a test solution.
MDCK cells were monolayer-cultured on a tissue culture microplate (96 wells) using a cell growth medium, then the cell growth medium was removed, and 0.1 mL of cell maintenance medium was added. Then, 0.1 mL of the test solution and the test dilution solution were inoculated every 4 holes and cultured in a 37 ± 1 ° C. carbon dioxide incubator for 4 to 7 days. After culturing, the presence or absence of cell morphological change (cytopathic effect) was observed using a microscope, the 50% tissue culture infectious dose (TCID ₅₀ ) was calculated by the Reed-Muench method, and the virus infection titer per mL of the test solution Converted into

[result]
The results are shown in Table 2 below.

  As can be seen from the results shown in Table 2, the polyglycerol fatty acid ester of the present invention is found to have an excellent anti-influenza virus effect.

Formulation Example 1: Preparation of aqueous solution formulation Using diglycerin monocaprylate prepared in Production Example 1 and distilled water, an aqueous solution formulation containing 1000 ppm of diglycerin monocaprylate was prepared.
The obtained aqueous solution preparation was filled in a commercially available spray container to prepare an aqueous solution preparation for spray spraying.
In addition, the prepared aqueous solution preparation was further added with benzalkonium chloride to prepare an aqueous solution preparation having an anti-influenza virus effect and a bactericidal function.

Formulation Example 2: Preparation of Emulsion Formulation 20 parts of diglycerin monolaurate (manufactured by Riken Vitamin Co., Ltd .: Poem DL-100), 10 parts of sodium dioctylsulfosuccinate, 20 parts of dimethyl sulfoxide, 20 parts of isopropanol and distilled water 30 parts were heated and mixed to obtain an emulsifier.

A solution obtained by diluting the aqueous solution of Formulation Example 1 and the emulsion of Formulation Example 2 30 times with distilled water is dipped in a cotton cloth, and squeezed so that an amount of liquid equal to the weight of the cotton cloth adheres. And dried for 10 minutes.
Next, the cotton cloth was cut into 30 mm × 30 mm, placed in an autoclave at 120 ° C. for 20 minutes and dried to obtain a test piece.
This was put in a plastic petri dish, and 0.2 mL of the virus solution described above was added dropwise and stored at room temperature for 24 hours. After storage, the virus solution of the sample was washed out with 2 mL of cell maintenance medium and used as a test solution, and anti-influenza virus properties were confirmed by the same method as in the virus inactivation test.
The results are shown in Table 3 below.

  As can be seen from the results shown in Table 3, the fiber product to which the polyglycerol fatty acid ester of the present invention has been imparted is found to have an excellent anti-influenza virus effect.

  As described above, the present invention is an influenza virus infection inhibitor characterized by containing a polyglycerol fatty acid ester having a monoester purity of 50% by mass or more, and the polyglycerol fatty acid ester contained as an active ingredient is Because it is a food additive, safe and has little skin irritation, an influenza virus infection inhibitor as an aqueous solution or emulsified solution with excellent safety is provided, impregnation into mask, disinfectant, etc. It is suitable for use as a medical device, and its medical contribution is great.

Claims (3)

The influenza virus infection prevention agent characterized by containing the polyglycerol fatty acid ester shown to following (a), (b) and (c).
(A) The monoester purity is 50% by mass or more,
(B) the fatty acid is a saturated fatty acid having 8 to 14 carbon atoms or an unsaturated fatty acid having 9 to 22 carbon atoms,
(C) The degree of polymerization of polyglycerol is 2-5. 2. The influenza virus infection inhibitor according to claim 1, wherein the polyglycerol fatty acid ester is diglycerol monolaurate, triglycerol monolaurate, tetraglycerol monolaurate or pentaglycerol monolaurate. The influenza virus infection-preventing agent according to claim 1 or 2 , wherein the mask is impregnated and used in the form of an aqueous solution or an emulsion.