JP2012229184A - Antiviral drug - Google Patents

Abstract

PROBLEM TO BE SOLVED: To elucidate antiviral activity of a monoester of a hydroxyfatty acid having one hydroxy group and glycerol, and to provide a new antiviral drug.SOLUTION: This antiviral drug includes a glycerol ricinoleic acid monoester as an effective ingredient, and suitable as a blending component in antiviral targets selected from food, a food package, dinnerwares, cosmetics, skin care preparations, skin cleansing agents, disinfectants, external lotion, hair preparations, wiping type sterilizers, medicines, quasi-drugs and sanitary materials for oral cavity.

Description

  The present invention relates to an antiviral agent, and particularly to an antiviral agent containing glycerin ricinoleic acid monoester as an active ingredient.

  As countermeasures against viral diseases, prevention by vaccine is the greatest measure, but there are many viral diseases for which vaccine development is difficult, and antiviral agents effective after onset are very limited. In recent years, there is an urgent need for prevention and treatment measures against emerging and reviving viral diseases, such as the emergence of a virus resistant to Tamiflu, a therapeutic drug for the new avian influenza virus, which has become a social problem.

  As a conventional technique, there is an antiviral preparation containing, as essential ingredients, a non-pharmacological base and an effective amount of one or more compounds selected from the group consisting of fatty acids and monoglycerides thereof ( (See Patent Document 1). In Patent Document 1, as a constituent fatty acid of a fatty acid monoglyceride, saturated or unsaturated such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, linoleic acid, linolenic acid, etc. Saturated fatty acids are disclosed.

  However, the fatty acid constituting the fatty acid monoglyceride is a saturated or unsaturated fatty acid, and an antiviral agent containing a monoester of a hydroxy fatty acid having one hydroxyl group and glycerin as an active ingredient is not disclosed.

Japanese Patent Laid-Open No. 02-502915

  This invention is made | formed in view of the said situation, It makes it a subject to clarify the antiviral activity of the monoester of the hydroxy fatty acid which has one hydroxyl group, and glycerol, and to provide a novel antiviral agent.

  As a result of intensive studies to solve the above problems, the present inventors have found that glycerin ricinoleic acid monoester exhibits an antiviral action against influenza virus, and have completed the present invention.

That is, the gist of the present invention is as follows.
[1] An antiviral agent comprising glycerin ricinoleic acid monoester as an active ingredient.
[2] A skin external preparation containing the antiviral agent according to [1].
[3] Glycerin ricinoleic acid monoester is used as food, food packaging materials, tableware, cosmetics, cosmetics, skin external preparations, skin cleansing agents, disinfectants, external lotions, hair preparations, wipe disinfectants, pharmaceuticals, pharmaceutical departments A method for enhancing the antiviral ability of an antiviral object by blending it with an antiviral object selected from external products and oral hygiene materials.

  According to the present invention, for example, an antiviral agent having excellent antiviral activity against influenza virus is provided. Antiviral agent of the present invention is food, food packaging material, tableware, cosmetics, cosmetics, skin external preparation, skin cleanser, disinfectant, external lotion, hair preparation, wipe disinfectant, pharmaceutical, quasi-drug It can be applied to products and oral hygiene materials.

It is a figure which shows the antiviral activity of this invention goods.

  As described above, the antiviral agent of the present invention is characterized in that glycerin ricinoleic acid monoester is an active ingredient. The glycerin ricinoleic acid monoester is a compound in which one molecule of ricinoleic acid and one molecule of glycerin are ester-bonded.

  The antiviral agent of the present invention exhibits excellent antiviral activity mainly against viruses having an envelope. Examples of viruses having an envelope include herpes virus, influenza virus, parainfluenza virus, coronavirus, SARS-related coronavirus, togavirus, paramyxovirus, orthomyxovirus, rhabdovirus, bunyavirus, arenavirus, retrovirus, baculo Examples include viruses.

  A glycerin ricinoleic acid monoester can be manufactured by a well-known method, for example, the method of esterifying a ricinoleic acid and glycerin using a chemical catalyst or an enzyme (lipase) can be mentioned. In the present invention, among the above methods, a method using lipase is preferable because it can be produced under mild conditions.

  The lipase used as a catalyst is not particularly limited as long as it recognizes glycerides as a substrate. Examples include monoglyceride lipase, mono and diglyceride lipase, triglyceride lipase, cutinase, esterase and the like. Among these, a lipase is preferable, and a lipase that recognizes fatty acid monoglyceride as a substrate and recognizes fatty acid triglyceride as a substrate is particularly preferable. Examples of such lipase include monoglyceride lipase.

  Examples of the lipase having the above properties include, for example, the genus Penicillium, Pseudomonas, Burkholderia, Alcaligenes, Staphylococcus, and Bacillus. Lipases derived from microorganisms such as the genus Candida, Geotrichum, Rhizopus, Rhizomucor, Mucor, Aspergillus, Pseudozyme Used. More preferred are lipases derived from the genus Penicillium and Bacillus. These lipases are generally commercially available and are readily available.

  A purified lipase (including crude and partial purification) may be used. Furthermore, it may be used in a free form or may be used by being immobilized on a carrier such as an ion exchange resin, a porous resin, ceramics or calcium carbonate.

  The amount of the lipase used in the esterification reaction may be appropriately determined depending on the reaction temperature, reaction time, pressure (degree of decompression) and the like, and is not particularly limited, but is preferably 1 unit per gram of reaction mixture ( U) -10000U. In the case of lipase, 1 U of enzyme activity refers to the amount of enzyme that liberates 1 μmol of fatty acid per minute in the hydrolysis of olive oil. In the case of monoglyceride lipase, the amount of enzyme that liberates 1 μmol of oleic acid per minute in the hydrolysis of oleic acid monoglyceride.

  The ricinoleic acid used in the esterification reaction may be in a free form, a metal salt form, or an ester form. In the present invention, the free form is preferred in that the esterification reaction easily proceeds.

  The amount of glycerin used for the esterification reaction is not particularly limited. Usually, the amount is preferably 1 to 10 times, more preferably 1.5 to 5 times the amount of 1 mol of free ricinoleic acid.

  In the present invention, the glycerin ricinoleic acid monoester can be produced with high purity by appropriately adjusting the reaction temperature, reaction time, pressure (degree of reduced pressure) and the like in the esterification reaction. The reaction temperature is preferably 30 to 60 ° C., the reaction time is preferably 30 to 60 hours, and the pressure is preferably 2 to 30 mmHg. In order to maintain the activity of lipase, it is preferable to add 0.3 to 3% by weight of water with respect to the total amount of ricinoleic acid and glycerin.

  The esterification reaction may be a stationary reaction, various stirring methods, shaking methods, ultrasonic methods, nitrogen blowing methods, circulating mixing methods using pumps, mixing methods using valves or pistons, or combinations thereof May be carried out while mixing the reaction solution.

  Any isolation / purification method can be adopted as a method for isolating / purifying glycerin ricinoleic acid monoester from the reaction mixture. Examples of the isolation / purification method include deoxidation, washing with water, distillation, solvent extraction, ion exchange chromatography, thin layer chromatography, membrane separation, and combinations of these methods.

  The antiviral agent of the present invention exhibits excellent antiviral activity against the various viruses described above. For this reason, for example, food, food packaging materials, tableware, cosmetics, cosmetics, skin external preparations, skin cleansing agents, disinfectants, external lotions, hair preparations, wipe disinfectants, pharmaceuticals, quasi drugs, oral cavity If the antiviral agent of this invention is mix | blended by using the hygiene material etc. for antiviral object, the antiviral effect of this antiviral object can be improved. As an embodiment thereof, for example, the antiviral agent of the present invention is blended with an antiviral object, and an indication that the antiviral agent is used for promoting antiviral action is attached. An aspect of selling a product can be exemplified.

  The antiviral agent of the present invention may be composed only of the above-described glycerin ricinoleic acid monoester, and additionally contains a dispersion medium such as water, ethanol or propanol; a dispersion accelerator such as starch. Can do. The content of the antiviral agent of the present invention in the antiviral target is usually 0.0001 to 50% by weight, preferably 0.001 to 10% by weight, of glycerin ricinoleic acid monoester as an active ingredient. It should be set as follows.

  The antiviral agent of the present invention can contain one or more antibacterial agents as optional components. Antibacterial agents that can be used include, for example, cetylpyridinium chloride, decalinium chloride, benzalkonium chloride, chlorohexidine, triclosan, isopropylmethylphenol, ofloxacin, iodine, sodium fluoride, benzoic acid, sorbic acid, organic halogen, Benzimidazole fungicides, metal ions such as silver and copper, lecithin, sucrose fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, ethanol, propylene glycol, polylysine, lysozyme, chitosan, thymol, eugenol, oily licorice Examples include extracts, mulberry bark extract, ashitaba extract, spice extract, and plant extract extracts such as polyphenols.

  The form of the antiviral agent of the present invention can be appropriately changed according to the above-described antiviral target, and for example, granular, pasty, solid, liquid and the like can be adopted.

  When the antiviral agent of the present invention is added to the above-described antiviral target, a known apparatus (a paddle mixer, a homomixer, a homogenizer, etc.) capable of producing the above-described form can be suitably used. Since the antiviral agent of the present invention is excellent in blending characteristics, the antiviral agent does not precipitate as crystals from various manufactured antiviral objects.

  The antiviral agent of this invention can be mix | blended also as an antiviral component of a skin external preparation, By doing in this way, the antiviral power of this skin external preparation can be improved. The content of the antiviral agent of the present invention in the external preparation for skin is such that the glycerin ricinoleic acid monoester as an active ingredient is usually contained in the range of 0.001 to 10% by weight, preferably 0.01 to 1% by weight. Should be set.

  In addition to the antiviral agent of the present invention, the skin external preparation of the present invention can be blended with various optional components used in normal skin external preparations. Examples of various optional components include purified water, alcohols, oil components, surfactants, thickeners, preservatives, moisturizers, powders, fragrances, pigments, emulsifiers, pH adjusters, ceramides, sterols, Antioxidants, singlet oxygen scavengers, UV absorbers, whitening agents, anti-inflammatory agents, other antibacterial agents, and the like.

  Specifically, oily components include liquid paraffin, petrolatum, solid paraffin, lanolin, lanolin fatty acid derivatives, dimethylpolysiloxane, higher alcohol higher fatty acid esters, fatty acids, long-chain amidoamines, animal and vegetable oils, and the like. Activators include polyoxyethylene hydrogenated castor oil, isostearyl glycerin ether, polyoxyethylene alkyl ether, glycerin fatty acid ester, polyethylene glycol, sorbitan monostearate, polyoxyethylene monostearate sorbitan, polyoxyethylene lauryl ether phosphate Salt, N-stearyloyl-N-methyl taurate, lauryl phosphate, monomyristyl phosphate, monocetyl phosphate, polyoxyethylene lauryl ether sulfate, lauryl sulfate trie Nolamine, polyoxyethylene lauryl ether sulfate triethanolamine, etc., and thickeners include water-soluble polymer compounds such as carboxyvinyl polymer, carboxymethylcellulose, polyvinyl alcohol, carrageenan, gelatin, etc. , Propylene glycol, glycerin, sorbitol, xylitol, maltitol and the like, and examples of the powder include talc, sericite, mica, kaolin, silica, bentonite, zinc white, mica and the like.

  The form of the external preparation for skin is not particularly limited, and depending on the intended use, cream, gel, emulsion, lotion, ointment, powder, haptic, powder, dripping, patch, aerosol, etc. Can be adopted.

  When the antiviral agent of the present invention is blended with the external preparation for skin, a known device (paddle mixer, homomixer, homogenizer, etc.) capable of producing the above-mentioned form can be suitably used. Since the antiviral agent of the present invention is excellent in blending characteristics, the antiviral agent does not precipitate as crystals from the manufactured skin external preparation.

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

1. Synthesis example of glycerin ricinoleic acid monoester 1-1. Immobilization of lipase A carrier (manufactured by Sumika Chemtex Co., Ltd., weakly basic anion exchange resin, trade name “Duolite A-568K”) was stirred in 1/10 N NaOH for 30 minutes, the carrier was filtered, and then ion-exchanged water Then, 200 mM phosphate buffer (pH 7) was added to equilibrate the pH. The phosphate buffer containing the carrier equilibrated with pH was subjected to ethanol substitution for 10 minutes, and then maintained for 20 minutes using a solution of ricinoleic acid / ethanol = 1/10 (weight ratio) to maintain the enzyme activity. Ricinoleic acid was adsorbed on the support. Subsequently, the carrier on which ricinoleic acid was adsorbed was filtered, and then washed with 200 mM phosphate buffer (pH 7) added to the carrier. Then, the washed carrier is recovered by filtration, and 2 ml of a 5000 U / ml lipase solution (manufactured by Amano Enzyme, derived from P. camembertii, trade name “Lipase G”) per 1 g of the carrier is collected. The lipase was immobilized on a carrier by contact for 2 hours. Finally, the carrier on which the lipase was immobilized was filtered to recover the carrier, and the carrier washed with ion-exchanged water was used as an immobilized enzyme for subsequent reactions.

1-2. Synthetic reaction In an approximately 30 ml vial, 10 g of ricinoleic acid / glycerin (1/3 (molar ratio)) mixture, 0.1 g of water, and 0.5 g of immobilization prepared in “1.1 Immobilization of Lipase” The enzyme was added, and the mixture was reacted at 50 ° C. and 15 mmHg for 48 hours while stirring with a magnetic stirrer. After completion of the reaction, a composition having an oil layer with a content of ricinoleic acid monoglyceride of 80% by weight was obtained. The obtained reaction product was repeatedly extracted with a thin layer chromatograph to obtain a purified product having a glycerin ricinoleic acid monoester content of 96%.

2. 2. Measurement of anti-influenza virus activity of glycerin ricinoleic acid monoester 2-1. Test Material / Test Virus: Influenza virus type A (hereinafter referred to as “Flu-A”) PR8 strain was used.
-Cells used: MDCK cells (canine kidney cells) (source: Dainippon Pharmaceutical Co., Ltd.).
Cell growth medium: A medium obtained by adding 10% fetal bovine serum (FBS) to Dulbecco's minimum essential medium (DMEM) was used.
Cell maintenance medium: A medium obtained by adding 0.1% bovine serum albumin and 0.25% trypsin to DMEM was used.
Test liquid: A glycerin ricinoleic acid monoester mixed and dispersed in DMEM to have a glycerin ricinoleic acid monoester concentration of 1111 ppm, 333 ppm, 111 ppm.

2-2. Preparation of virus suspension (Flu-A stock solution) MDCK cells were monolayer cultured in a tissue culture flask, the cell growth medium was removed from the flask, and the test virus was inoculated. Next, cell maintenance medium was added and cultured at 37 ° C. for 2-7 days in the presence of 5% CO ₂ . 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. Next, the culture solution was centrifuged (3,000 r / min, 10 minutes), and the obtained supernatant was used as a virus suspension (Flu-A stock solution) (2 × 10 ⁸ TCID ₅₀ / ml).

2-3. Test operation 50 μl of Flu-A stock solution (2 × 10 ⁸ TCID ₅₀ / ml) was added to 450 μl of each test solution, and reacted for 60 seconds at room temperature (25 ° C.) with stirring (hereinafter referred to as “reaction solution”). ). In addition, the density | concentration of the glycerol ricinoleic acid monoester in the said reaction liquid will be 1000 ppm, 300 ppm, and 100 ppm. After completion of the reaction, 50 μl of each of the above reaction solutions is added to 450 μl of a phosphate buffer solution (hereinafter referred to as “PBS (−)”) containing 1% bovine serum albumin and not containing Mg ²⁺ or Ca ²⁺ to give 10 ^−1. A 10-fold serial dilution of 10 ⁻² to 10 ^{−6 was} sequentially prepared (4 ° C.).
The test operation was performed in the same manner as described above using DMEM as a control instead of the test solution.

2-4. Measurement of virus infectivity titer Using a cell growth medium, MDCK cells were monolayer cultured in a 24-well microplate for tissue culture, then the cell growth medium was removed and washed once with DMEM, and then 1 ml each of cell maintenance medium was added. . Subsequently, 100 μl of each of the dilutions prepared above was inoculated into each well in 4 wells and cultured at 37 ° C. for 5 days in the presence of 5% CO ₂ .
After culturing, the cells were fixed / stained with a 2.5% crystal violet / 30% ethanol / 1% ammonium oxalate solution, washed twice with PBS (−), decolorized, and then sterilized and dried under ultraviolet light.
The presence or absence of cytopathic effect (CPE) was observed, the 50% tissue culture infectious dose (TCID ₅₀ ) was calculated by the Reed-Muench method, and converted to the virus infection titer per ml of the reaction solution. Table 1 shows the results.

As shown in Table 1, when the concentration of glycerin ricinoleic acid monoester in the reaction solution was increased from 100 ppm to 300 ppm, TCID ₅₀ decreased to 1/100. Therefore, glycerin ricinoleic acid monoester was changed to influenza virus type A. It was found to have antiviral activity.

3. Virus inactivation test of hand lotion containing glycerin ricinoleic acid monoester Test solution 1 (invention product) and test solution 2 (comparative product) shown in Table 2 were used as test solutions. Except that the reaction time with the Flu-A stock solution was 0, 10, 20, 30, 60 seconds and the reaction time between the test solution 2 and the Flu-A stock solution was 0, 60 seconds, the above “2. The test was conducted in the same manner as in “Measurement of anti-influenza virus activity of ricinoleic acid monoester”, and the virus infectivity per ml of the reaction solution was determined. The results are shown in FIG.

As shown in FIG. 1, even when the Flu-A stock solution was added to the hand lotion of the test solution 2 (comparative product), the value of TCID ₅₀ / ml did not change even after 60 seconds. On the other hand, when the Flu-A stock solution was added to the test solution 1 (product of the present invention) containing glycerin ricinoleic acid monoester, the TCID ₅₀ / ml value was 1/20 compared to the test solution 2 when reacted for 20 seconds. When the reaction was carried out for 60 seconds, the value of TCID ₅₀ / ml was reduced to 1/2000 compared to test solution 2. From this result, it was found that the test liquid 1 containing glycerin ricinoleic acid monoester has excellent antiviral activity against influenza virus type A.

  Since the antiviral agent of the present invention has excellent antiviral activity and excellent blending characteristics, foods, food packaging materials, tableware, cosmetics, cosmetics, skin external preparations, skin cleansing agents, disinfectants, and external use It is suitable as a compounding component of an antiviral target selected from lotions, hair preparations, wiping disinfectants, pharmaceuticals, quasi drugs, and oral hygiene materials.

Claims (3)

  An antiviral agent containing glycerin ricinoleic acid monoester as an active ingredient.   A skin external preparation containing the antiviral agent according to claim 1. Glycerin ricinoleic acid monoester is a food, food packaging material, tableware, cosmetics, cosmetics, skin external preparation, skin cleanser, disinfectant, external lotion, hair preparation, wipe disinfectant, pharmaceutical, quasi-drug, A method for enhancing the antiviral ability of an antiviral object by blending with an antiviral object selected from oral hygiene materials.

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