WO2018079514A1 - Composition of maitake extract for treatment and/or prevention of herpes infection - Google Patents

Composition of maitake extract for treatment and/or prevention of herpes infection Download PDF

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Publication number
WO2018079514A1
WO2018079514A1 PCT/JP2017/038262 JP2017038262W WO2018079514A1 WO 2018079514 A1 WO2018079514 A1 WO 2018079514A1 JP 2017038262 W JP2017038262 W JP 2017038262W WO 2018079514 A1 WO2018079514 A1 WO 2018079514A1
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extract
maitake
dried
composition
vacuum
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PCT/JP2017/038262
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French (fr)
Japanese (ja)
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京子 林
昭弘 田中
研太 内藤
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株式会社雪国まいたけ
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

  • the present invention relates to a composition for the treatment and / or prevention of herpes infections including a heat-dried maitake extract and a vacuum-dried maitake extract, a method for producing the composition, and the like.
  • Herpes virus is a DNA virus whose genome is double-stranded DNA, and causes various diseases to humans.
  • a herpesvirus that has entered a living body latently infects specific cells, tissues, or organs regardless of the presence or absence of onset.
  • the site of latent infection varies depending on the herpesvirus species and shows diversity.
  • Herpesviruses are latent in the DNA state, but when the host falls into a state of stress or immunosuppression, the latently infected herpesvirus reactivates and proliferates, causing pathological conditions to the host again (Regression onset). Symptoms of herpes virus infection are greatly affected by the patient's immune status, and exhibit various pathological conditions from almost asymptomatic to fatal infection. Herpesviruses persist in the host with recurrent episodes and latent infections.
  • antiviral drugs that can remove or reduce herpesviruses that are latent in the state of DNA in cells, tissues, or organs. For this reason, antiviral drugs are used for the purpose of alleviating discomfort at the time of recurrence, reducing the number of recurrences, and shortening the period of illness.
  • chemical antiviral drugs such as acyclovir (ACV) inevitably produce drug-resistant viruses when used for a long time.
  • acyclovir has significant side effects such as acute renal failure, liver dysfunction, interstitial pneumonia, pneumonia, thrombocytopenia, delirium (consciousness disorder), epilepsy, agranulocytosis, respiratory depression, toxic epidermis It is known to cause necrosis.
  • Patent Document 1 discloses an anti-herpes agent containing LPS (lipopolysaccharide).
  • LPS lipopolysaccharide
  • the applicant of the present application has studied maitake extract, which is considered to be highly safe from many years of eating experience, and so far the maitake extract is effective for the treatment and / or prevention of herpes virus infection.
  • Patent Document 2 discloses an anti-herpes agent containing LPS (lipopolysaccharide).
  • the applicant of the present application has studied maitake extract, which is considered to be highly safe from many years of eating experience, and so far the maitake extract is effective for the treatment and / or prevention of herpes virus infection.
  • An object of the present invention is to provide a natural product-derived anti-herpes agent having a high effect on herpes infection.
  • the present inventor has found that a composition comprising a heat-dried maitake extract and a vacuum-dried maitake extract has a higher effect on herpes infection than a heat-dried maitake extract or a vacuum-dried maitake extract alone. I found it.
  • the present invention is based on the above findings and includes the following aspects.
  • a step of mixing the vacuum dried maitake extract B at a weight ratio of 0.05 to 10 with respect to the heat dried maitake extract A weight ratio of 1, A method for producing a composition for treatment and / or prevention of herpes virus infection.
  • the method according to any one of (6) to (8), wherein the composition is an external preparation.
  • Extract A has the following steps: Heating and drying the maitake, Any of (6) to (10) obtained by adding a solvent to the obtained heat-dried maitake to obtain an extract fraction, and removing the solvent in the obtained extract fraction to obtain a dried product The method of crab. (12) The method according to (11), comprising a step of adding an alcohol to the extracted fraction and removing a precipitated substance before the step of obtaining a dried product. (13) Extract B has the following steps: Vacuum drying the maitake, Any of (6) to (12) obtained by adding a solvent to the obtained vacuum-dried maitake to obtain an extract fraction, and removing the solvent in the obtained extract fraction to obtain a dried product The method of crab. (14) The method according to (13), wherein the step of vacuum drying the maitake is performed by freeze drying.
  • composition containing the heat-dried maitake extract and the vacuum-dried maitake extract of the present invention has a high effect on herpes infection. Moreover, since the composition of this invention is manufactured from the maitake which has a long experience of eating, it is thought that it is highly safe and has few side effects.
  • FIG. 1 shows the results of HPLC analysis of the heat-dried maitake extract obtained in Example 1.
  • FIG. 2 shows the analysis result by HPLC of the vacuum dried maitake extract obtained in Example 2.
  • FIG. 3 shows the average value of the degree of symptom (score) up to 14 days after HSV-1 inoculation in the mice treated with each sample.
  • (Negative) PBS as a control, acyclovir (ACV) as a positive control, as a sample MD Fr prepared in Example 1, and a mixture of MD Fr and YM-6A prepared in Example 3 (MD Fr: YM- 6A ratio 1: 1 mixture).
  • FIG. 4 shows the measurement results of virus titer 3 days after inoculation with HSV-1 in mice treated with each sample.
  • FIG. 5 shows the average value of the degree of symptom (score) up to 14 days after HSV-2 inoculation in mice treated with each sample.
  • FIG. 5 shows the average value of the degree of symptom (score) up to 14 days after HSV-2 inoculation in mice treated with each sample.
  • FIG. 6 shows the measurement results of virus titer 3 days after inoculation with HSV-2 in mice treated with each sample.
  • the controls and samples used are the same as in FIG. * Indicates a significant difference (* p ⁇ 0.05, ** p ⁇ 0.01) in t-test.
  • FIG. 7 shows the average value of the degree of symptom (score) up to 14 days after inoculation with HSV-2 in mice treated with each sample. (Negative) PBS was used as a control, and acyclovir (ACV) was used as a positive control.
  • FIG. 8 shows the virus titer measurement results 3 days after HSV-2 inoculation in mice treated with each sample.
  • the controls and samples used are the same as in FIG. * Indicates a significant difference (* p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001) in t-test.
  • composition for treatment and / or prevention of herpes virus infection comprising heat dried maitake extract and vacuum dried maitake extract. Relates to the composition.
  • “maitake” means a genus Grifola mushroom.
  • the mushroom genus mushrooms include not only maitake (Grifola frondosa) in the narrow sense, but also white maitake (Grifola albicans), chorei maitake (Grifola umbellata), and tomb maitake (Grifola gigantea), etc.
  • Maitake in the book refers to “Grifola frondosa” in the narrow sense.
  • both fruit bodies and mycelia of maitake can be used, but it is preferable to use fruit bodies.
  • the fruit bodies of maitake (Grifola frondosa) and white maitake (Grifola albicans) can be artificially cultivated. Therefore, it is preferable to use the above two fruit bodies from the viewpoint of securing a stable raw material.
  • dried maitake extract refers to a product obtained by increasing the content of active ingredients by drying maitake by heat drying or vacuum drying and solvent extraction.
  • the dried maitake extract includes both an extract obtained by using dried maitake as an extraction raw material and these dried products, and preferably a dried product of the maitake extract.
  • the type of solvent for obtaining the maitake extract is not limited, and examples thereof include water or alcohols such as lower alcohols, such as methanol, ethanol, and propanol. These extraction solvents can be used alone or in combination of two or more at an appropriate mixing ratio.
  • the solvent for obtaining the maitake extract is preferably a solvent mainly containing water, for example, the weight of water in the extraction solvent is 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, It is preferably 99% or more or 100%.
  • water any of distilled water, ion-exchanged water, tap water, and natural water can be used.
  • the extraction solvent may contain an additive such as a buffer as long as the extraction efficiency is not significantly impaired.
  • the weight ratio of the vacuum-dried maitake extract B when the heat-dried maitake extract A is 1 is 0.05 or more, 0.08 or more, 0.1 or more, 0.2 or more, 0.5 or more, 0.8 or more, preferably 1 or more, 1.5 or more, 2 or more, 2.5 or more, or 3 or more, and 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, preferably 5 or less, 4.5 or less, 4 or less, 3.5 or less, Or it may be 3 or less.
  • the weight ratio of the extract B when the extract A is 1 may be, for example, 0.05 to 10, 0.1 to 5, preferably 1 to 5, 2 to 4, 2.5 to 3.5, or 3.
  • the heat-dried maitake extract can be produced by methods known to those skilled in the art.
  • the mycelium or fruiting body of maitake preferably the fruiting body, is dried by heating, and after chopping or powdering as necessary, extraction can be performed by adding a solvent.
  • the method of heat-drying the maitake there is no particular limitation on the method of heat-drying the maitake, and a known heat-drying method such as hot air drying can be used. If the heating temperature is too low, the drying efficiency is lowered. If the heating temperature is too high, there is a possibility that a chemical change may occur in the components of the maitake. Therefore, it is preferable to set the heating temperature in an appropriate range. Drying is performed at 40 ° C or higher, 50 ° C or higher, 60 ° C or higher, preferably 70 ° C or higher, or 80 ° C or higher, 150 ° C or lower, 120 ° C or lower, 100 ° C or lower, preferably 90 ° C or lower or 80 ° C or lower.
  • the heating time is not particularly limited as long as moisture in the maitake is removed, and can be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, all night, or all day.
  • An extraction process can be performed by adding the said solvent to dry maitake.
  • the addition amount of the extraction solvent is not particularly limited.
  • the extraction solvent can be used in an amount of 1 to 100 times, 2 to 50 times, or 4 to 30 times the weight of the dried maitake.
  • the extraction step may be performed at room temperature, but heating may be performed to increase extraction efficiency. Enzymes that affect the components in the maitake mushrooms lose their function at temperatures above 60 ° C., and therefore extraction is preferably performed at 60 ° C. or higher, preferably 70 ° C. or higher, or 80 ° C. or higher. Further, it is preferable to further increase the extraction efficiency by pressure heating, and the heating conditions in the case of pressure heating are 110 ° C. or higher, 115 ° C. or higher, or 120 ° C. or higher, 140 ° C. or lower, 130 ° C. or lower, or 125 ° C. It may be the following.
  • the pressurizing conditions are not limited, but can be, for example, 1.2 to 3 atm, 1.5 to 2.5 atm, 1.8 to 2.2 atm, 1.9 to 2.1 atm or 2 atm using a pressure cooker.
  • the extraction time can be appropriately set according to the extraction conditions, and may be, for example, 5 minutes to 1 day, 10 minutes to 6 hours, 15 minutes to 1 hour, or 20 minutes to 40 minutes.
  • the extraction is preferably performed at a temperature of 100 ° C. or higher under pressure for a short time, for example, at 115 ° C. to 125 ° C. for 5 minutes to 1 hour under pressure.
  • the purity of the extract can be further increased by clarification by filtration and / or removal of precipitates by alcohol as necessary.
  • Clarification by filtration can be performed by a known method.
  • the removal of the precipitate by alcohol can be performed, for example, by the following steps. That is, the alcohol is added to the extract obtained by the above extraction step or a solution obtained by concentrating the extract as necessary so that the final volume is 50% by volume, 60% by volume, or 70% by volume or more. For example, by leaving it at 1 to 25 ° C. or at room temperature, it is possible to remove substances deposited on the liquid surface or in the liquid, or on the wall of the container, thereby increasing the purity of the extract.
  • the removal of the precipitate can be performed by, for example, filtering or sieving through a net or the like.
  • the alcohol lower alcohols such as methanol, ethanol, and propanol can be used alone or in combination of two or more, and it is particularly preferable to use ethanol alone.
  • the extract obtained as described above can be dried by a conventional method, for example, heat drying, vacuum drying, vacuum drying, spray drying, or the like alone or in combination of two or more. At this time, it is preferable to completely remove the solvent.
  • the vacuum dried maitake extract can be produced by methods known to those skilled in the art.
  • the fruit body part of the maitake may be vacuum-dried and, if necessary, chopped or powdered, and then extracted with a solvent.
  • the vacuum drying method of maitake is not particularly limited, and a known vacuum drying method such as freeze drying can be used.
  • a known vacuum drying method such as freeze drying can be used.
  • the vacuum drying may be performed under a complete vacuum or under a substantial vacuum, for example, 100 Pa or less, 50 Pa or less, or 20 Pa or less.
  • the vacuum drying can be performed by a method known to those skilled in the art, for example, using a commercially available vacuum dryer, and the time for vacuum drying is not particularly limited as long as moisture is removed from the maitake, for example, 1 hour or more, 2 It can be performed for more than 4 hours, more than 4 hours, more than 6 hours, all night or all day.
  • Herpesviruses that can be treated and / or prevented by the compositions of the present invention are preferably HSV-1 and HSV-2.
  • herpes infections that can be treated and / or prevented by the compositions of the present invention include, but are not limited to, herpes dermatitis, herpes keratitis, herpes conjunctivitis, cold sores, oral herpes, pharyngeal herpes, Examples include genital herpes, herpes encephalitis, and neonatal herpes.
  • the composition of the present invention treats patients suffering from these herpes infections with herpes infections or symptoms associated therewith, such as subjective symptoms such as itching and pain, and / or skin symptoms such as blisters and erythema, It may be relaxed or reduced.
  • composition of the present invention can be used prophylactically for patients at risk of suffering from herpes infection, for example, for patients suffering from herpes infection and who are likely to have relapse. .
  • the compositions of the invention can be used for the treatment and / or prevention of recurrent episodes of herpes virus.
  • herpesvirus reactivated from the state in which a pathogen is present in the living body without showing clinically recognized symptoms (latent infection), It means causing a disease state.
  • the usage mode of the composition of the present invention is not particularly limited, and may be any of foods such as health supplements, supplements, and pharmaceutical compositions, but is preferably a pharmaceutical composition.
  • composition of the present invention can be formulated by a conventional method.
  • formulation reference can be made, for example, to the method described in Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.).
  • the dosage form is not particularly limited and is appropriately selected as necessary.
  • oral forms such as tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, and elixirs are used.
  • parenteral preparations such as injections, drops, suppositories, inhalants, external preparations such as transdermal absorption agents, transmucosal absorption agents, patches and ointments, preferably transdermal absorption agents And administered as an external preparation such as a transmucosal absorbent, a patch, and an ointment, or as a topical administration agent.
  • the administration site is not particularly limited, and examples thereof include skin, cornea, conjunctiva, lips, oral cavity, pharynx, and genitals, preferably cornea. Lips and genitals.
  • the composition of the present invention comprises other components such as excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters in addition to the above-mentioned heat-dried maitake extract and vacuum-dried maitake extract. , Flavoring agents, coloring agents, fragrances and the like may be included, and the other components are appropriately selected according to the dosage form.
  • the ratio of the heat-dried maitake extract and the vacuum-dried maitake extract in the composition is not particularly limited.
  • the total amount of the two extracts is the total weight of the composition.
  • it may be 1% to 99%, 10% to 90%, or 20% to 80%.
  • Excipients include starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.
  • Binders include, but are not limited to, crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylethylcellulose, starch, polyvinylpyrrolidone, and tragacanth Is mentioned.
  • disintegrant examples include, but are not limited to, crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, starch, and tragacanth.
  • Surfactants include, but are not limited to, soy lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, sorbitan sesquioleate, sorbitan trioleate, polysorbate, glyceryl monostearate, and A sodium lauryl sulfate is mentioned.
  • Lubricants include, but are not limited to, starch, stearic acid, stearate, hydrous silicon dioxide, light silicic anhydride, talc, waxes, hydrogenated vegetable oil, and polyethylene glycol.
  • Fluidity promoters include, but are not limited to, hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.
  • compositions of the present invention include, but are not limited to, mammals, primates such as humans and chimpanzees, laboratory animals such as rats and mice, pigs, cows, horses, sheep, And domestic animals such as goats, and pet animals such as dogs and cats, preferably humans or mice, most preferably humans.
  • the dosage, administration interval, and administration period of the composition of the present invention can be determined by those skilled in the art from various factors such as the subject's sex, weight, age (month and age), and the course and symptoms of the disease. Can be determined as appropriate.
  • the dosage of the composition may be about 0.1 mg to 10 g / kg body weight, preferably 1 mg to 1000 mg / kg body weight, or 10 to 100 mg / kg body weight.
  • the number of doses is not limited, but 3 times a day, 2 times a day, 1 time a day, 1 time every 2 days, 1 time every 3 days, once a week, 1 time every 2 weeks Once a month, etc.
  • the administration period is not limited, but may be 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
  • the present invention relates to a method for treating and / or preventing herpes virus infection comprising administering the composition to a subject. Since the subject, the herpes virus infection, the dose of the composition, the administration interval, and the administration period in the present method are as described above, description thereof is omitted here. 2.
  • Method for producing composition for treatment and / or prevention of herpes virus infection In one aspect, the present invention relates to a method for producing a composition for prevention and / or treatment of herpes virus infection as described in 1 above. .
  • the production method of the present invention includes a step of mixing the heat-dried maitake extract and the vacuum-dried maitake extract as an essential step.
  • the mixing ratio in this step is such that the weight ratio of the vacuum-dried maitake extract B when the heat-dried maitake extract A is 1 is 0.05 or more, 0.08 or more, 0.1 or more, 0.2 or more, 0.5 or more, 0.8 or more, preferably 1 or more, 1.5 or more, 2 or more, 2.5 or more, or 3 or more, and 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, preferably 5 or less, 4.5 or less, 4 or less, 3.5 or less, Or it may be 3 or less.
  • the weight ratio of the extract B when the extract A is 1 may be, for example, 0.05 to 10, 0.1 to 5, preferably 1 to 5, 2 to 4, 2.5 to 3.5, or 3.
  • the heat-dried maitake extract and the vacuum-dried maitake extract may be mixed, or other components may be added to and mixed with the heat-dried maitake extract and the vacuum-dried maitake extract.
  • the composition is finally obtained at the above mixing ratio, it is possible to divide the mixing process. For example, after mixing the heat-dried maitake extract and other components, the mixture is vacuum-dried maitake extract You can also add things.
  • the production method of the present invention may include a step of obtaining a heat-dried maitake extract before the mixing step.
  • the heat-dried maitake extract includes (A) a step of heat-drying maitake, (B) a step of adding a solvent to the obtained heat-dried maitake to obtain an extract fraction, and (C) a step in which the extract is obtained.
  • the solvent can be removed to obtain a dry product.
  • the process of heat-drying maitake is not particularly limited, and a known heat-drying method such as hot air drying can be used. If the heating temperature is too low, the drying efficiency is lowered. If the heating temperature is too high, there is a possibility that a chemical change may occur in the components of the maitake. Therefore, it is preferable to set the heating temperature in an appropriate range. Drying is performed at 40 ° C or higher, 50 ° C or higher, 60 ° C or higher, preferably 70 ° C or higher, or 80 ° C or higher, 150 ° C or lower, 120 ° C or lower, 100 ° C or lower, preferably 90 ° C or lower or 80 ° C or lower.
  • the heating time is not particularly limited as long as moisture in the maitake is removed, and can be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, all night, or all day.
  • the process of obtaining an extraction fraction can be performed by adding the said solvent to the dried maitake which was chopped or powdered as needed.
  • the addition amount of the extraction solvent is not particularly limited.
  • the extraction solvent can be used 1 to 100, 2 to 50, or 4 to 30 times by weight with respect to the dried maitake.
  • the extraction efficiency can be increased by heating. Enzymes that affect the components in maitake work up to about 60 ° C, but work declines at temperatures above 60 ° C, so extraction should be done at 60 ° C or higher, preferably 70 ° C or higher, or 80 ° C or higher. Is preferred. Further, it is preferable to further increase the extraction efficiency by pressure heating, and the heating conditions in the case of pressure heating are 110 ° C. or higher, 115 ° C. or higher, or 120 ° C. or higher, 140 ° C. or lower, 130 ° C. or lower, or 125 ° C.
  • the pressurizing condition may be as follows, but is not limited to, for example, using a pressure cooker or the like to 1.2 atm to 3 atm, 1.5 to 2.5 atm, 1.8 to 2.2 atm, 1.9 to 2.1 atm or 2 atm can do.
  • the extraction time can be appropriately set according to the extraction conditions, and may be, for example, 5 minutes to 1 day, 10 minutes to 6 hours, 15 minutes to 1 hour, or 20 minutes to 40 minutes.
  • the extraction is preferably performed at a temperature of 100 ° C. or higher under pressure for a short time, for example, at 115 ° C. to 125 ° C. for 5 minutes to 1 hour under pressure.
  • the step of removing the solvent from the obtained extract fraction to obtain a dried product can be carried out by a conventional method.
  • heat drying, reduced pressure drying, vacuum drying, spray drying and the like can be performed alone or in combination. It can carry out by using in combination above.
  • It may further comprise a step of increasing purity by clarification by filtration and / or removal of precipitates by alcohol. Clarification by filtration can be performed by a known method, and removal of the precipitate by alcohol can be performed, for example, by the following steps. That is, (B) the alcohol obtained with the extract obtained in the step of obtaining the extract fraction or a solution obtained by concentrating the extract, if necessary, is 50% by volume or more, 60% by volume or more, or 70% by volume.
  • the purity of the extract can be increased by removing substances deposited on the liquid surface, in the liquid, or on the wall of the container, for example, by leaving at 1 to 25 ° C. or normal temperature.
  • the removal of the precipitate can be performed by, for example, filtering or sieving through a net or the like.
  • the alcohol lower alcohols such as methanol, ethanol, and propanol can be used alone or in combination of two or more, and it is particularly preferable to use ethanol alone.
  • the production method of the present invention includes a step of obtaining a vacuum-dried maitake extract in addition to the step of obtaining a heat-dried maitake extract before or after the mixing step. Good.
  • the production method of the present invention includes both a step of obtaining a heat-dried maitake extract and a step of obtaining a vacuum-dried maitake extract, the order of both steps is not particularly limited.
  • the vacuum-dried maitake extract includes (D) a step of vacuum-drying maitake, (E) a step of adding a solvent to the obtained vacuum-dried maitake, and obtaining an extract fraction, and (F) in the obtained extract fraction.
  • the solvent can be removed to obtain a dry product.
  • the step of vacuum drying the maitake is not particularly limited, and a known vacuum drying method such as freeze drying can be used.
  • a known vacuum drying method such as freeze drying can be used.
  • the vacuum drying may be performed under a complete vacuum or under a substantial vacuum, for example, 100 Pa or less, 50 Pa or less, or 20 Pa or less.
  • the vacuum drying can be performed by a method known to those skilled in the art, for example, using a commercially available vacuum dryer.
  • the time for vacuum drying is not particularly limited as long as moisture is removed from the maitake, and for example, it can be performed for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, or overnight.
  • (E ') process since it is the same as having described in the (B') process of the process of obtaining the said heat-dried maitake extract, description is abbreviate
  • Example 1 Preparation of heat-dried maitake extract
  • the fruit body (stem and umbrella) obtained by artificial cultivation is placed on the shelf of the shelf-type drying room, and the temperature is increased gradually from 70 ° C to 80 ° C, and it takes about 1 day. Dried. Next, the dried maitake powder was pulverized with a mill to obtain heat-dried maitake of fine powder.
  • Example 2 Preparation of vacuum dried maitake extract
  • the fruit bodies (stalks and umbrellas) obtained by artificial cultivation were frozen at ⁇ 30 ° C. to ⁇ 40 ° C. and dried using a vacuum freeze dryer. The inside of the dryer was depressurized to 20 Pa and dried in about 1 day. Next, the dried maitake powder was pulverized with a mill to make a fine powder.
  • Example 3 Preparation 1 of a mixture of heat-dried maitake extract and vacuum-dried maitake extract
  • MD Fr obtained in Example 1 and YM-6A obtained in Example 2 were mixed using a blender at the following weight ratios to prepare a mixture of a heat-dried maitake extract and a vacuum-dried maitake extract. .
  • Example 4 Preventive and therapeutic effects on herpes virus infection
  • Acyclovir ACV
  • Mice received PBS, acyclovir (0.2 mg / 40 ⁇ L / day) or the sample prepared in Example 1 or 3 (1 mg / 40 ⁇ L / day) twice a day between 3 days before and 7 days after virus inoculation ( At 9 am and 6 pm), it was administered locally to the genitals.
  • Symptom degree (score) and deaths were recorded daily for 2 weeks after virus inoculation (no deaths after 2 weeks). The degree of symptom is as shown in Table 2.
  • Example 5 Preparation of mixture of heat-dried maitake extract and vacuum-dried maitake extract 2
  • MD Fr obtained in Example 1 and YM-6A obtained in Example 2 were mixed using a blender at a weight ratio shown in Table 4, and a mixture of a heat-dried maitake extract and a vacuum-dried maitake extract was prepared.
  • Example 6 Preventive and therapeutic effects on herpes virus infection
  • Acyclovir ACV
  • Mice received PBS, acyclovir (0.2 mg / 40 ⁇ L / day) or the samples prepared in Examples 1, 2 and 5 (1 mg / 40 ⁇ L / day) 2 days a day between 3 days before and 7 days after virus inoculation. Times (9am and 6pm), administered locally to the genitals.
  • the degree of symptom (score) and death cases were recorded every day for 2 weeks after virus inoculation (no deaths after 2 weeks).
  • the degree of symptom is as shown in Table 2.
  • the mixture of heat-dried maitake extract (MD Fr) and vacuum-dried maitake extract (YM-6A) was superior to virus infection than heat-dried maitake extract or vacuum-dried maitake extract alone. It has been shown that it has an effect, particularly when the ratio of MDAFr and YM-6A is 1: 1 to 1: 5.
  • Example 7 Preparation 3 of a mixture of heat-dried maitake extract and vacuum-dried maitake extract
  • MD Fr obtained in Example 1 and YM-6A obtained in Example 2 were mixed using a blender at the weight ratio shown in Table 6, and a mixture of the heat-dried maitake extract and the vacuum-dried maitake extract was mixed. Prepared.
  • Example 8 Prevention and treatment effect on herpes virus infection
  • the experimental method was the same as that described in Example 6, except that each mixture prepared in Example 7 was used.
  • the mortality rate and the number of death days in each group are shown in Table 7, and the average score is shown in FIG.
  • Example 9 Inhibitory effect on recurrence from herpes simplex virus latent infection
  • acyclovir was orally administered.
  • mice About half of 32 died by the 14th day of infection, and among the 28 surviving animals, there were 4 mice with no symptoms in the cornea and no virus detected in the cornea. For this reason, the remaining 24 mice were subjected to the test equally divided into 6 groups per group and 3 groups based on the degree of onset and left and right cornea virus load data shown in Table 8. .
  • the MD ⁇ Fr: YM-6A mixture was locally administered twice a day (1 mg / 40 ⁇ l / day, 9:00 and 18:00).
  • sterile water was orally administered to the control group at 0.4 ml / day
  • ACV was orally administered to the ACV administration group at 1 mg / 0.4 ml / day.
  • the left and right eyeballs were washed with PBS (0.1 ml), the washing solution was subjected to a plaque assay, and the amount of virus was measured. If it was detected, it was evaluated that the patient had a regression.
  • Table 9 shows the number of regression episodes and the regression episode rate in each group.
  • Table 10 shows the neutralizing antibody titers in each group.
  • the composition containing the heat-dried maitake extract and the vacuum-dried maitake extract of the present invention has a high effect on herpes infection. Moreover, since the composition of this invention is manufactured from the maitake which has a long experience of eating, it is thought that it is highly safe and has few side effects. Therefore, the present invention can provide a therapeutic and / or preventive agent for herpes infections (including recurrent onset) that is effective against herpes infections and has few side effects.

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Abstract

In one embodiment, the present invention addresses the problem of providing a naturally derived anti-herpetic agent having a potent effect against herpes infection. In one embodiment, the present invention relates to: a composition for the treatment and/or prevention of herpes infection, said composition including a heat-dried maitake extract and a vacuum-dried maitake extract; and a method for producing said composition.

Description

マイタケ抽出物のヘルペス感染症の治療及び／又は予防のための組成物Composition for treating and / or preventing herpes infection of Maitake extract
　本発明は、加熱乾燥マイタケ抽出物及び真空乾燥マイタケ抽出物を含むヘルペス感染症の治療及び／又は予防のための組成物、及び該組成物の製造方法等に関する。 The present invention relates to a composition for the treatment and / or prevention of herpes infections including a heat-dried maitake extract and a vacuum-dried maitake extract, a method for producing the composition, and the like.
　ヘルペスウイルスは2本鎖DNAをゲノムとするDNAウイルスであり、ヒトに対して様々な病気を引き起こす。生体内に侵入したヘルペスウイルスは、発症の有無にかかわらず特定の細胞、組織、又は臓器に潜伏感染する。潜伏感染部位はヘルペスウイルス種によって異なり、多様性を示す。ヘルペスウイルスはDNAの状態で潜伏しているが、宿主がストレスや免疫抑制といった状態に陥った時等に、潜伏感染しているヘルペスウイルスは、再活性化・増殖し、宿主に再び病態を引き起こす（回帰発症）。ヘルペスウイルス感染症状は患者の免疫状態に大きく左右され、ほぼ無症状から致死的感染まで様々な病態を呈する。ヘルペスウイルスは回帰発症と潜伏感染を繰り返して、宿主に終生存在する。 Herpes virus is a DNA virus whose genome is double-stranded DNA, and causes various diseases to humans. A herpesvirus that has entered a living body latently infects specific cells, tissues, or organs regardless of the presence or absence of onset. The site of latent infection varies depending on the herpesvirus species and shows diversity. Herpesviruses are latent in the DNA state, but when the host falls into a state of stress or immunosuppression, the latently infected herpesvirus reactivates and proliferates, causing pathological conditions to the host again (Regression onset). Symptoms of herpes virus infection are greatly affected by the patient's immune status, and exhibit various pathological conditions from almost asymptomatic to fatal infection. Herpesviruses persist in the host with recurrent episodes and latent infections.
　細胞、組織、又は臓器においてDNAの状態で潜伏しているヘルペスウイルスを、体内から除去あるいは減少させることができる抗ウイルス薬は今のところ無い。この為、抗ウイルス薬は、再発時の不快感の緩和や再発回数の減少、病気の期間の短縮を目的に使用されている。しかしながら、アシクロビル（ACV）等の化学物質の抗ウイルス薬は、長期に渡って使用すると必然的に薬剤耐性ウイルスを生じるという問題がある。加えて、アシクロビルには重大な副作用として、急性腎不全、肝機能障害、間質性肺炎、肺炎、血小板減少症、せん妄（意識障害）、てんかん発作、無顆粒球症、呼吸抑制、中毒性表皮壊死症等を生じることが知られている。 There are currently no antiviral drugs that can remove or reduce herpesviruses that are latent in the state of DNA in cells, tissues, or organs. For this reason, antiviral drugs are used for the purpose of alleviating discomfort at the time of recurrence, reducing the number of recurrences, and shortening the period of illness. However, chemical antiviral drugs such as acyclovir (ACV) inevitably produce drug-resistant viruses when used for a long time. In addition, acyclovir has significant side effects such as acute renal failure, liver dysfunction, interstitial pneumonia, pneumonia, thrombocytopenia, delirium (consciousness disorder), epilepsy, agranulocytosis, respiratory depression, toxic epidermis It is known to cause necrosis.
　化学物質だけでなく、天然物に由来する抗ヘルペス剤についても研究がなされている。例えば、特許文献１にはLPS（リポポリサッカライド）を含む抗ヘルペス剤が開示されている。また、本願出願人は、長年の食経験から安全性が高いと考えられるマイタケ抽出物について研究を行い、これまでに、マイタケ抽出物がヘルペスウイルス感染症の治療及び／又は予防に有効であることを見出している（特許文献２）。 Investigating not only chemical substances but also anti-herpes drugs derived from natural products. For example, Patent Document 1 discloses an anti-herpes agent containing LPS (lipopolysaccharide). In addition, the applicant of the present application has studied maitake extract, which is considered to be highly safe from many years of eating experience, and so far the maitake extract is effective for the treatment and / or prevention of herpes virus infection. (Patent Document 2).
　しかしながら、これらの特許文献に開示される天然物由来の抗ウイルス剤のヘルペスウイルス感染症に対する効果は、必ずしも満足できるものではなかった。 However, the effects of natural products-derived antiviral agents disclosed in these patent documents on herpes virus infections are not always satisfactory.
特開平０４－０４９２４２号公報Japanese Patent Laid-Open No. 04-049242 特開２０１４－８０３７３号公報JP 2014-80373 A
　本発明は、ヘルペス感染症に対して高い効果を有する、天然物由来の抗ヘルペス剤を提供することを課題とする。 An object of the present invention is to provide a natural product-derived anti-herpes agent having a high effect on herpes infection.
　本発明者は、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物を含む組成物が、加熱乾燥マイタケ抽出物又は真空乾燥マイタケ抽出物単独と比べて、ヘルペス感染症に対して高い効果を有することを見出した。 The present inventor has found that a composition comprising a heat-dried maitake extract and a vacuum-dried maitake extract has a higher effect on herpes infection than a heat-dried maitake extract or a vacuum-dried maitake extract alone. I found it.
　本発明は上記知見に基づくものであり、以下の態様を包含する。
（１）加熱乾燥マイタケ抽出物A及び真空乾燥マイタケ抽出物Bを含み、
　抽出物Aを1としたときの抽出物Bの重量比が0.05～10である、ヘルペスウイルス感染症の治療及び／又は予防のための組成物。
（２）抽出物Aを1としたときの抽出物Bの重量比が1～5である、（１）に記載の組成物。
（３）抽出物A及び抽出物Bがいずれも水抽出物である、（１）又は（２）に記載の組成物。
（４）外用剤である、（１）～（３）のいずれかに記載の組成物。
（５）ヘルペスウイルスの回帰発症の治療及び／又は予防のための、（１）～（４）のいずれかに記載の組成物。
（６）加熱乾燥マイタケ抽出物A重量比1に対し、真空乾燥マイタケ抽出物Bを重量比0.05～10で混合する工程を含む、
　ヘルペスウイルス感染症の治療及び／又は予防のための組成物の製造方法。
（７）抽出物A重量比1に対し、抽出物Bを重量比1～5で混合する、（６）に記載の方法。
（８）抽出物A及び抽出物Bがいずれも水抽出物である、（６）又は（７）に記載の方法。
（９）前記組成物が外用剤である、（６）～（８）のいずれかに記載の方法。
（１０）前記組成物がヘルペスウイルスの回帰発症の治療及び／又は予防のためのものである、（６）～（９）のいずれかに記載の方法。
（１１）抽出物Aが、以下の工程：
　マイタケを加熱乾燥する工程、
　得られた加熱乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び
　得られた抽出画分中の溶媒を除去し、乾燥物を得る工程
により得られる、（６）～（１０）のいずれかに記載の方法。
（１２）乾燥物を得る工程の前に、抽出画分にアルコールを添加し、析出する物質を除去する工程を含む、（１１）に記載の方法。
（１３）抽出物Bが、以下の工程：
　マイタケを真空乾燥する工程、
　得られた真空乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び
　得られた抽出画分中の溶媒を除去し、乾燥物を得る工程
により得られる、（６）～（１２）のいずれかに記載の方法。
（１４）マイタケを真空乾燥する工程が、凍結乾燥により行われる、（１３）に記載の方法。 The present invention is based on the above findings and includes the following aspects.
(1) Including heat-dried maitake extract A and vacuum-dried maitake extract B,
A composition for the treatment and / or prevention of herpes virus infection, wherein the weight ratio of the extract B when the extract A is 1 is 0.05 to 10.
(2) The composition according to (1), wherein the weight ratio of the extract B is 1 to 5 when the extract A is 1.
(3) The composition according to (1) or (2), wherein both the extract A and the extract B are water extracts.
(4) The composition according to any one of (1) to (3), which is an external preparation.
(5) The composition according to any one of (1) to (4), which is used for treatment and / or prevention of recurrent onset of herpesvirus.
(6) A step of mixing the vacuum dried maitake extract B at a weight ratio of 0.05 to 10 with respect to the heat dried maitake extract A weight ratio of 1,
A method for producing a composition for treatment and / or prevention of herpes virus infection.
(7) The method according to (6), wherein the extract B is mixed at a weight ratio of 1 to 5 with respect to the extract A weight ratio of 1.
(8) The method according to (6) or (7), wherein both the extract A and the extract B are water extracts.
(9) The method according to any one of (6) to (8), wherein the composition is an external preparation.
(10) The method according to any one of (6) to (9), wherein the composition is for the treatment and / or prevention of herpesvirus recurrent episodes.
(11) Extract A has the following steps:
Heating and drying the maitake,
Any of (6) to (10) obtained by adding a solvent to the obtained heat-dried maitake to obtain an extract fraction, and removing the solvent in the obtained extract fraction to obtain a dried product The method of crab.
(12) The method according to (11), comprising a step of adding an alcohol to the extracted fraction and removing a precipitated substance before the step of obtaining a dried product.
(13) Extract B has the following steps:
Vacuum drying the maitake,
Any of (6) to (12) obtained by adding a solvent to the obtained vacuum-dried maitake to obtain an extract fraction, and removing the solvent in the obtained extract fraction to obtain a dried product The method of crab.
(14) The method according to (13), wherein the step of vacuum drying the maitake is performed by freeze drying.
　本明細書は本願の優先権の基礎となる日本国特許出願番号2016-211243号の開示内容を包含する。 This specification includes the disclosure of Japanese Patent Application No. 2016-211243 which is the basis of the priority of the present application.
　本発明の加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物を含む組成物は、ヘルペス感染症に対して高い効果を示す。また、本発明の組成物は、長年の食経験を有するマイタケから製造される為、安全性が高く、副作用が少ないと考えられる。 The composition containing the heat-dried maitake extract and the vacuum-dried maitake extract of the present invention has a high effect on herpes infection. Moreover, since the composition of this invention is manufactured from the maitake which has a long experience of eating, it is thought that it is highly safe and has few side effects.
図1は、実施例1で得られた加熱乾燥マイタケ抽出物のHPLCによる分析結果を示す。FIG. 1 shows the results of HPLC analysis of the heat-dried maitake extract obtained in Example 1. 図2は、実施例2で得られた真空乾燥マイタケ抽出物のHPLCによる分析結果を示す。FIG. 2 shows the analysis result by HPLC of the vacuum dried maitake extract obtained in Example 2. 図3は、各サンプルで処理したマウスにおける、HSV-1の接種14日後までの症状の程度（スコア）の平均値を示す。（陰性）対照としてPBS、陽性対照としてアシクロビル（ACV）を用い、サンプルとしては実施例1で調製したMD Fr、及び実施例3で調製したMD FrとYM-6Aの混合物（MD Fr：YM-6A比＝1:1混合物）を用いた。FIG. 3 shows the average value of the degree of symptom (score) up to 14 days after HSV-1 inoculation in the mice treated with each sample. (Negative) PBS as a control, acyclovir (ACV) as a positive control, as a sample MD Fr prepared in Example 1, and a mixture of MD Fr and YM-6A prepared in Example 3 (MD Fr: YM- 6A ratio = 1: 1 mixture). 図4は、各サンプルで処理したマウスにおける、HSV-1の接種3日後のウイルス力価の測定結果を示す。用いた対照及びサンプルについては図3と同様である。*は、t検定における有意差（*p<0.05）を示す。FIG. 4 shows the measurement results of virus titer 3 days after inoculation with HSV-1 in mice treated with each sample. The controls and samples used are the same as in FIG. * Indicates a significant difference (* p <0.05) in t-test. 図5は、各サンプルで処理したマウスにおける、HSV-2の接種14日後までの症状の程度（スコア）の平均値を示す。（陰性）対照としてPBS、陽性対照としてアシクロビル（ACV）を用い、サンプルとしては実施例1で調製したMD Fr、実施例2で調製したYM-6A、及び実施例5で調製したMD FrとYM-6Aの混合物（MD Fr：YM-6A比＝1:0.1、1:0.2、1:1、1:5、及び1:10混合物）を用いた。FIG. 5 shows the average value of the degree of symptom (score) up to 14 days after HSV-2 inoculation in mice treated with each sample. (Negative) PBS as a control, acyclovir (ACV) as a positive control, as samples MD Fr prepared in Example 1, YM-6A prepared in Example 2, and MD Fr and YM prepared in Example 5 A mixture of -6A (MD Fr: YM-6A ratio = 1: 0.1, 1: 0.2, 1: 1, 1: 5, and 1:10 mixture) was used. 図6は、各サンプルで処理したマウスにおける、HSV-2の接種3日後のウイルス力価の測定結果を示す。用いた対照及びサンプルについては図5と同様である。*は、t検定における有意差（*p<0.05、**p<0.01）を示す。FIG. 6 shows the measurement results of virus titer 3 days after inoculation with HSV-2 in mice treated with each sample. The controls and samples used are the same as in FIG. * Indicates a significant difference (* p <0.05, ** p <0.01) in t-test. 図7は、各サンプルで処理したマウスにおける、HSV-2の接種14日後までの症状の程度（スコア）の平均値を示す。（陰性）対照としてPBS、陽性対照としてアシクロビル（ACV）を用い、サンプルとしては、実施例7で調製したMD FrとYM-6Aの混合物（MD Fr：YM-6A比＝1:1、1：3、及び1:5混合物）を用いた。FIG. 7 shows the average value of the degree of symptom (score) up to 14 days after inoculation with HSV-2 in mice treated with each sample. (Negative) PBS was used as a control, and acyclovir (ACV) was used as a positive control. As a sample, a mixture of MDMFr and YM-6A prepared in Example 7 (MD Fr: YM-6A ratio = 1: 1, 1: 3 and 1: 5 mixtures). 図8は、各サンプルで処理したマウスにおける、HSV-2の接種3日後のウイルス力価の測定結果を示す。用いた対照及びサンプルについては図7と同様である。*は、t検定における有意差（*p<0.05、**p<0.01、*** p<0.001）を示す。FIG. 8 shows the virus titer measurement results 3 days after HSV-2 inoculation in mice treated with each sample. The controls and samples used are the same as in FIG. * Indicates a significant difference (* p <0.05, ** p <0.01, *** p <0.001) in t-test.
１．ヘルペスウイルス感染症の治療及び／又は予防のための組成物
　一態様において、本発明は、加熱乾燥マイタケ抽出物及び真空乾燥マイタケ抽出物を含む、ヘルペスウイルス感染症の治療及び／又は予防のための組成物に関する。 1. Composition for treatment and / or prevention of herpes virus infection In one aspect, the present invention provides for the treatment and / or prevention of herpes virus infection comprising heat dried maitake extract and vacuum dried maitake extract. Relates to the composition.
　本明細書において、「マイタケ」とは、マイタケ属〔Grifola〕キノコを意味する。マイタケ属キノコには、狭義のマイタケ〔Grifola frondosa〕のみならず、シロマイタケ〔Grifola albicans〕、チョレイマイタケ〔Grifola umbellata〕、及びトンビマイタケ〔Grifola gigantea〕等が含まれるが、好ましくは、本明細書におけるマイタケは、狭義のマイタケ〔Grifola frondosa〕を指す。本発明の抽出物の原料としては、マイタケの子実体及び菌糸体のいずれも用いることができるが、子実体を用いることが好ましい。最近ではマイタケ〔Grifola frondosa〕及びシロマイタケ〔Grifola albicans〕の子実体の人工栽培が可能となっているため、安定した原料確保の点から上記二者の子実体を使用するのが好ましい。 In this specification, “maitake” means a genus Grifola mushroom. The mushroom genus mushrooms include not only maitake (Grifola frondosa) in the narrow sense, but also white maitake (Grifola albicans), chorei maitake (Grifola umbellata), and tomb maitake (Grifola gigantea), etc. Maitake in the book refers to “Grifola frondosa” in the narrow sense. As the raw material of the extract of the present invention, both fruit bodies and mycelia of maitake can be used, but it is preferable to use fruit bodies. Recently, the fruit bodies of maitake (Grifola frondosa) and white maitake (Grifola albicans) can be artificially cultivated. Therefore, it is preferable to use the above two fruit bodies from the viewpoint of securing a stable raw material.
　本明細書において、「乾燥マイタケ抽出物」とは、マイタケを加熱乾燥又は真空乾燥により乾燥させ、溶媒抽出するなどして、有効成分の含有量を高めたものを指す。具体的には、乾燥マイタケ抽出物には、乾燥マイタケを抽出原料として得られる抽出液、及びこれらの乾燥物のいずれもが含まれ、好ましくはマイタケ抽出液の乾燥物である。 In this specification, “dried maitake extract” refers to a product obtained by increasing the content of active ingredients by drying maitake by heat drying or vacuum drying and solvent extraction. Specifically, the dried maitake extract includes both an extract obtained by using dried maitake as an extraction raw material and these dried products, and preferably a dried product of the maitake extract.
　マイタケ抽出物を得るための溶媒の種類は限定しないが、例えば、水又は低級アルコール等のアルコール、例えばメタノール、エタノール、及びプロパノール等が挙げられる。これらの抽出溶媒は単独で又は2種以上を適切な混合比で組み合わせて使用することができる。マイタケ抽出物を得るための溶媒は、好ましくは水を主とする溶媒であり、例えば抽出溶媒中の水分重量が、70%以上、80%以上、90%以上、95%以上、98%以上、99%以上、又は100%であることが好ましい。水としては、蒸留水、イオン交換水、水道水、及び天然水のいずれも使用し得る。抽出溶媒は、抽出効率を著しく損なわない範囲で添加剤、例えば緩衝剤を含んでいてもよい。 The type of solvent for obtaining the maitake extract is not limited, and examples thereof include water or alcohols such as lower alcohols, such as methanol, ethanol, and propanol. These extraction solvents can be used alone or in combination of two or more at an appropriate mixing ratio. The solvent for obtaining the maitake extract is preferably a solvent mainly containing water, for example, the weight of water in the extraction solvent is 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, It is preferably 99% or more or 100%. As water, any of distilled water, ion-exchanged water, tap water, and natural water can be used. The extraction solvent may contain an additive such as a buffer as long as the extraction efficiency is not significantly impaired.
　本発明の組成物において、加熱乾燥マイタケ抽出物Aを1としたときの真空乾燥マイタケ抽出物Bの重量比は、0.05以上、0.08以上、0.1以上、0.2以上、0.5以上、0.8以上、好ましくは1以上、1.5以上、2以上、2.5以上、又は3以上であってよく、また10以下、9以下、8以下、7以下、6以下、好ましくは5以下、4.5以下、4以下、3.5以下、又は3以下であってよい。抽出物Aを1としたときの抽出物Bの重量比は、例えば、0.05～10、0.1～5、好ましくは1～5、2～4、2.5～3.5、又は3であってよい。 In the composition of the present invention, the weight ratio of the vacuum-dried maitake extract B when the heat-dried maitake extract A is 1 is 0.05 or more, 0.08 or more, 0.1 or more, 0.2 or more, 0.5 or more, 0.8 or more, preferably 1 or more, 1.5 or more, 2 or more, 2.5 or more, or 3 or more, and 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, preferably 5 or less, 4.5 or less, 4 or less, 3.5 or less, Or it may be 3 or less. The weight ratio of the extract B when the extract A is 1 may be, for example, 0.05 to 10, 0.1 to 5, preferably 1 to 5, 2 to 4, 2.5 to 3.5, or 3.
　加熱乾燥マイタケ抽出物は、当業者に公知の方法により製造することができる。例えば、マイタケの菌糸体又は子実体、好ましくは子実体を加熱乾燥し、必要に応じて細断又は粉末化した後に、溶媒を加え、抽出を行うことができる。 The heat-dried maitake extract can be produced by methods known to those skilled in the art. For example, the mycelium or fruiting body of maitake, preferably the fruiting body, is dried by heating, and after chopping or powdering as necessary, extraction can be performed by adding a solvent.
　マイタケの加熱乾燥方法は特に限定せず、公知の加熱乾燥法、例えば、熱風乾燥を用いることができる。加熱温度は、低すぎると乾燥効率が低下し、高すぎるとマイタケの成分に化学変化が生じる恐れがあるため、適切な範囲に設定することが好ましい。乾燥は、40℃以上、50℃以上、60℃以上、好ましくは70℃以上、又は80℃以上、また150℃以下、120℃以下、100℃以下、好ましくは90℃以下又は80℃以下の条件で、例えば40℃～100℃、50℃～90℃、又は60℃～80℃の条件で、例えば熱風を目的の物質に送風することによって行うことができる。加熱時間はマイタケ中の水分が除かれる限り特に限定されず、例えば1時間以上、2時間以上、4時間以上、6時間以上、終夜、又は一昼夜とすることができる。 There is no particular limitation on the method of heat-drying the maitake, and a known heat-drying method such as hot air drying can be used. If the heating temperature is too low, the drying efficiency is lowered. If the heating temperature is too high, there is a possibility that a chemical change may occur in the components of the maitake. Therefore, it is preferable to set the heating temperature in an appropriate range. Drying is performed at 40 ° C or higher, 50 ° C or higher, 60 ° C or higher, preferably 70 ° C or higher, or 80 ° C or higher, 150 ° C or lower, 120 ° C or lower, 100 ° C or lower, preferably 90 ° C or lower or 80 ° C or lower. For example, it can be carried out by blowing hot air, for example, on the target substance under the conditions of 40 ° C. to 100 ° C., 50 ° C. to 90 ° C., or 60 ° C. to 80 ° C. The heating time is not particularly limited as long as moisture in the maitake is removed, and can be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, all night, or all day.
　マイタケ抽出物を得るための溶媒については、上記の通りであるからここでは記載を省略する。抽出工程は、上記溶媒を乾燥マイタケに加えることにより行うことができる。抽出溶媒の添加量は特に限定しないが、例えば乾燥マイタケに対して抽出溶媒を1～100倍、2～50倍又は4～30倍重量使用することができる。 Since the solvent for obtaining the maitake extract is as described above, description thereof is omitted here. An extraction process can be performed by adding the said solvent to dry maitake. The addition amount of the extraction solvent is not particularly limited. For example, the extraction solvent can be used in an amount of 1 to 100 times, 2 to 50 times, or 4 to 30 times the weight of the dried maitake.
　抽出工程は、常温で行ってもよいが、加熱を行って抽出効率を高めてもよい。マイタケ中の成分に影響を与えるような酵素は60℃を超える温度では働きが衰えるので、60℃以上、好ましくは70℃以上、又は80℃以上で抽出を行うのが好ましい。また、加圧加熱によりさらに抽出効率を高めることが好ましく、加圧加熱の場合の加熱条件は、110℃以上、115℃以上、又は120℃以上、また140℃以下、130℃以下、又は125℃以下であってよい。加圧条件は、限定しないが、例えば、圧力釜等を用いて1.2気圧～3気圧、1.5気圧～2.5気圧、1.8気圧～2.2気圧、1.9気圧～2.1気圧又は2気圧にすることができる。抽出時間は抽出条件に応じて適宜設定することができるが、例えば5分～1日、10分～6時間、15分～1時間、又は20分～40分であってよい。抽出は、好ましくは加圧下100℃以上で短時間、例えば加圧下115℃～125℃で5分～1時間行う。 The extraction step may be performed at room temperature, but heating may be performed to increase extraction efficiency. Enzymes that affect the components in the maitake mushrooms lose their function at temperatures above 60 ° C., and therefore extraction is preferably performed at 60 ° C. or higher, preferably 70 ° C. or higher, or 80 ° C. or higher. Further, it is preferable to further increase the extraction efficiency by pressure heating, and the heating conditions in the case of pressure heating are 110 ° C. or higher, 115 ° C. or higher, or 120 ° C. or higher, 140 ° C. or lower, 130 ° C. or lower, or 125 ° C. It may be the following. The pressurizing conditions are not limited, but can be, for example, 1.2 to 3 atm, 1.5 to 2.5 atm, 1.8 to 2.2 atm, 1.9 to 2.1 atm or 2 atm using a pressure cooker. The extraction time can be appropriately set according to the extraction conditions, and may be, for example, 5 minutes to 1 day, 10 minutes to 6 hours, 15 minutes to 1 hour, or 20 minutes to 40 minutes. The extraction is preferably performed at a temperature of 100 ° C. or higher under pressure for a short time, for example, at 115 ° C. to 125 ° C. for 5 minutes to 1 hour under pressure.
　抽出物は、必要に応じて濾過による清澄化及び／又はアルコールによる析出物の除去によって、さらに純度を高めることができる。濾過による清澄化は公知の方法により行うことができる。アルコールによる析出物の除去は、例えば以下の工程により行うことができる。すなわち、上記抽出工程によって得られた抽出液又は必要に応じてこれを濃縮した溶液に対し、アルコールを、最終容量で50容量％以上、60容量％以上、又は70容量％以上になるように加え、例えば1～25℃又は常温で、放置することによって液面若しくは液中、又は容器の壁等に析出する物質を除去して抽出物の純度を高めることができる。析出物の除去は、例えば濾過又は網等で掬うことによって行い得る。アルコールとしては、低級アルコール、例えばメタノール、エタノール、及びプロパノール等を単独で、又は二以上組み合わせて用いることができ、エタノールを単独で用いることが特に好ましい。 The purity of the extract can be further increased by clarification by filtration and / or removal of precipitates by alcohol as necessary. Clarification by filtration can be performed by a known method. The removal of the precipitate by alcohol can be performed, for example, by the following steps. That is, the alcohol is added to the extract obtained by the above extraction step or a solution obtained by concentrating the extract as necessary so that the final volume is 50% by volume, 60% by volume, or 70% by volume or more. For example, by leaving it at 1 to 25 ° C. or at room temperature, it is possible to remove substances deposited on the liquid surface or in the liquid, or on the wall of the container, thereby increasing the purity of the extract. The removal of the precipitate can be performed by, for example, filtering or sieving through a net or the like. As the alcohol, lower alcohols such as methanol, ethanol, and propanol can be used alone or in combination of two or more, and it is particularly preferable to use ethanol alone.
　上記の様にして得られた抽出液は、常法により、例えば加熱乾燥、減圧乾燥、真空乾燥、スプレードライ等を単独で、又は二以上組み合わせて用いることによって乾燥させることができる。このとき、溶媒を完全に除去することが好ましい。 The extract obtained as described above can be dried by a conventional method, for example, heat drying, vacuum drying, vacuum drying, spray drying, or the like alone or in combination of two or more. At this time, it is preferable to completely remove the solvent.
　真空乾燥マイタケ抽出物は、当業者に公知の方法により製造することができる。例えば、マイタケの子実体部分を真空乾燥し、必要に応じて細断又は粉末化した後に、溶媒を加え、抽出を行うことができる。 The vacuum dried maitake extract can be produced by methods known to those skilled in the art. For example, the fruit body part of the maitake may be vacuum-dried and, if necessary, chopped or powdered, and then extracted with a solvent.
　マイタケの真空乾燥方法は特に限定せず、公知の真空乾燥法、例えば、凍結乾燥を用いることができる。真空乾燥は、マイタケを加熱する必要がなく、低温で乾燥を行うことから、マイタケの成分に化学変化が生じる恐れが少ない。真空乾燥は、完全な真空下で行ってもよいし、実質的な真空下、例えば100Pa以下、50Pa以下、又は20Pa以下等で行ってもよい。真空乾燥は、当業者に公知の方法により、例えば市販の真空乾燥機を用いて行うことができ、真空乾燥の時間は、マイタケから水分が除かれる限り特に限定されず、例えば1時間以上、2時間以上、4時間以上、6時間以上、終夜、又は一昼夜行うことができる。 The vacuum drying method of maitake is not particularly limited, and a known vacuum drying method such as freeze drying can be used. In vacuum drying, there is no need to heat the maitake and the drying is performed at a low temperature, so that there is little risk of chemical changes in the components of maitake. The vacuum drying may be performed under a complete vacuum or under a substantial vacuum, for example, 100 Pa or less, 50 Pa or less, or 20 Pa or less. The vacuum drying can be performed by a method known to those skilled in the art, for example, using a commercially available vacuum dryer, and the time for vacuum drying is not particularly limited as long as moisture is removed from the maitake, for example, 1 hour or more, 2 It can be performed for more than 4 hours, more than 4 hours, more than 6 hours, all night or all day.
　真空乾燥を行ったマイタケについて、マイタケ抽出物を得るための溶媒、抽出工程、清澄化及び／又はアルコールによる析出物の除去、抽出液の乾燥については、上記加熱乾燥マイタケ抽出物について記載したものと同様であるからここでは記載を省略する。 About the maitake mushrooms that have been vacuum-dried, the solvent for obtaining the maitake extract, extraction process, clarification and / or removal of precipitates by alcohol, and drying of the extract are those described for the above-mentioned heat-dried maitake extract Since it is the same, the description is omitted here.
　本発明の組成物によって治療及び／又は予防され得るヘルペスウイルスの種類は限定しないが、単純ヘルペスウイルス（HSV-1：単純ヘルペスウイルス1型、又はHSV-2：単純ヘルペスウイルス2型に分けられる）、水痘帯状疱疹ウイルス、エプステインバールウイルス、ヒトサイトメガロウイルス、ヒトヘルペスウイルス6、ヒトヘルペスウイルス7、及びヒトヘルペスウイルス8が挙げられる。本発明の組成物によって治療及び／又は予防され得るヘルペスウイルスは、好ましくはHSV-1及びHSV-2である。本発明の組成物により治療及び／又は予防され得るヘルペス感染症の例として、限定するものではないが、ヘルペス性皮膚炎、ヘルペス性角膜炎、ヘルペス性結膜炎、口唇ヘルペス、口腔ヘルペス、咽頭ヘルペス、性器ヘルペス、ヘルペス性脳炎、及び新生児ヘルペス等が挙げられる。本発明の組成物は、これらのヘルペス感染症に罹患した患者に対して、ヘルペス感染症又はそれに伴う症状、例えば痒み及び痛み等の自覚症状、並びに／又は水疱及び紅斑等の皮膚症状を治療、緩和、又は低減するものであってもよい。また、本発明の組成物は、ヘルペス感染症に罹患するリスクのある患者に対して、例えば、ヘルペス感染症に罹患し、回帰発症のおそれがある患者に対して、予防的に用いることができる。一実施形態において、本発明の組成物は、ヘルペスウイルスの回帰発症の治療及び／又は予防のために使用することができる。 The type of herpes virus that can be treated and / or prevented by the composition of the present invention is not limited, but herpes simplex virus (HSV-1: herpes simplex virus type 1 or HSV-2: herpes simplex virus type 2) , Varicella-zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpes virus 6, human herpes virus 7, and human herpes virus 8. Herpesviruses that can be treated and / or prevented by the compositions of the present invention are preferably HSV-1 and HSV-2. Examples of herpes infections that can be treated and / or prevented by the compositions of the present invention include, but are not limited to, herpes dermatitis, herpes keratitis, herpes conjunctivitis, cold sores, oral herpes, pharyngeal herpes, Examples include genital herpes, herpes encephalitis, and neonatal herpes. The composition of the present invention treats patients suffering from these herpes infections with herpes infections or symptoms associated therewith, such as subjective symptoms such as itching and pain, and / or skin symptoms such as blisters and erythema, It may be relaxed or reduced. In addition, the composition of the present invention can be used prophylactically for patients at risk of suffering from herpes infection, for example, for patients suffering from herpes infection and who are likely to have relapse. . In one embodiment, the compositions of the invention can be used for the treatment and / or prevention of recurrent episodes of herpes virus.
　本明細書において、ヘルペスウイルスの「回帰発症」とは、臨床的に認められる症状を示さずに生体内に病原体が存在する状態（潜伏感染）から、ヘルペスウイルスが再活性化して、宿主に再び病態を引き起こすことを意味する。 In this specification, “relapsed onset” of herpesvirus means that the herpesvirus is reactivated from the state in which a pathogen is present in the living body without showing clinically recognized symptoms (latent infection), It means causing a disease state.
　本発明の組成物の使用態様は特に限定されず、例えば健康補助食品等の食品、サプリメント、及び医薬組成物等のいずれであってもよいが、好ましくは医薬組成物である。 The usage mode of the composition of the present invention is not particularly limited, and may be any of foods such as health supplements, supplements, and pharmaceutical compositions, but is preferably a pharmaceutical composition.
　本発明の組成物は、常法により製剤化され得る。製剤化は、例えば、Remington’s Pharmaceutical Sciences（Merck Publishing Co．，Easton，Pa．）に記載の方法を参照することができる。 The composition of the present invention can be formulated by a conventional method. For formulation, reference can be made, for example, to the method described in Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.).
　投与形態は特に制限されず、必要に応じ適宜選択されるが、一般には錠剤、カプセル剤、顆粒剤、細粒剤、散剤、液剤、シロップ剤、懸濁剤、乳剤、及びエリキシル剤等の経口剤、又は注射剤、点滴剤、坐剤、吸入剤、外用剤、例えば経皮吸収剤、経粘膜吸収剤、貼付剤、及び軟膏剤等の非経口剤として投与され、好ましくは経皮吸収剤、経粘膜吸収剤、貼付剤、及び軟膏剤等の外用剤又は局所投与剤として投与される。 The dosage form is not particularly limited and is appropriately selected as necessary. Generally, oral forms such as tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, and elixirs are used. Or as parenteral preparations such as injections, drops, suppositories, inhalants, external preparations such as transdermal absorption agents, transmucosal absorption agents, patches and ointments, preferably transdermal absorption agents And administered as an external preparation such as a transmucosal absorbent, a patch, and an ointment, or as a topical administration agent.
　本発明の組成物が外用剤又は局所投与剤として投与される場合、その投与部位は特に限定しないが、例えば皮膚、角膜、結膜、口唇、口腔、咽頭、及び性器等が挙げられ、好ましくは角膜、口唇及び性器である。 When the composition of the present invention is administered as an external preparation or a topical preparation, the administration site is not particularly limited, and examples thereof include skin, cornea, conjunctiva, lips, oral cavity, pharynx, and genitals, preferably cornea. Lips and genitals.
　本発明の組成物は、上記加熱乾燥マイタケ抽出物及び真空乾燥マイタケ抽出物に加えて、他の成分、例えば賦形剤、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、着色剤、及び香料等を含んでもよく、他の成分は上記投与形態に応じて適宜選択される。本発明の組成物が他の成分を含む場合、組成物中の加熱乾燥マイタケ抽出物及び真空乾燥マイタケ抽出物の割合は特に限定されず、例えば二つの抽出物の総量が、組成物全体の重量に対して、例えば1%～99%、10%～90％、又は20%～80％であってよい。 The composition of the present invention comprises other components such as excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters in addition to the above-mentioned heat-dried maitake extract and vacuum-dried maitake extract. , Flavoring agents, coloring agents, fragrances and the like may be included, and the other components are appropriately selected according to the dosage form. When the composition of the present invention contains other components, the ratio of the heat-dried maitake extract and the vacuum-dried maitake extract in the composition is not particularly limited. For example, the total amount of the two extracts is the total weight of the composition. For example, it may be 1% to 99%, 10% to 90%, or 20% to 80%.
　賦形剤としては、デンプン、乳糖、白糖、マンニット、カルボキシメチルセルロース、コーンスターチ、及び無機塩類等が挙げられる。 Excipients include starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.
　結合剤としては、限定するものではないが、結晶セルロース、結晶セルロース・カルメロースナトリウム、メチルセルロース、ヒドロキシプロピルセルロース、低置換度ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルエチルセルロース、デンプン、ポリビニルピロリドン、及びトラガントが挙げられる。 Binders include, but are not limited to, crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylethylcellulose, starch, polyvinylpyrrolidone, and tragacanth Is mentioned.
　崩壊剤としては、限定するものではないが、結晶セルロース、メチルセルロース、低置換度ヒドロキシプロピルセルロース、カルメロース、カルメロースカルシウム、カルメロースナトリウム、クロスカルメロースナトリウム、デンプン、及びトラガント等が挙げられる。 Examples of the disintegrant include, but are not limited to, crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, starch, and tragacanth.
　界面活性剤としては、限定するものではないが、大豆レシチン、ショ糖脂肪酸エステル、ステアリン酸ポリオキシル、ポリオキシエチレン硬化ヒマシ油、セスキオレイン酸ソルビタン、トリオレイン酸ソルビタン、ポリソルベート、モノステアリン酸グリセリン、及びラウリル硫酸ナトリウムが挙げられる。 Surfactants include, but are not limited to, soy lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, sorbitan sesquioleate, sorbitan trioleate, polysorbate, glyceryl monostearate, and A sodium lauryl sulfate is mentioned.
　滑沢剤としては、限定するものではないが、デンプン、ステアリン酸、ステアリン酸塩、含水二酸化ケイ素、軽質無水ケイ酸、タルク、ロウ類、水素添加植物油、及びポリエチレングリコールが挙げられる。 Lubricants include, but are not limited to, starch, stearic acid, stearate, hydrous silicon dioxide, light silicic anhydride, talc, waxes, hydrogenated vegetable oil, and polyethylene glycol.
　流動性促進剤としては、限定するものではないが、含水二酸化ケイ素、軽質無水ケイ酸、乾燥水酸化アルミニウムゲル、合成ケイ酸アルミニウム、及びケイ酸マグネシウムが挙げられる。 Fluidity promoters include, but are not limited to, hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.
　本発明の組成物が投与され得る被験体としては、限定されるものではないが、哺乳動物、例えばヒト及びチンパンジー等の霊長類、ラット及びマウス等の実験動物、ブタ、ウシ、ウマ、ヒツジ、及びヤギ等の家畜動物、並びにイヌ及びネコ等の愛玩動物が挙げられ、好ましくはヒト又はマウス、最も好ましくはヒトである。 Subjects to which the composition of the present invention can be administered include, but are not limited to, mammals, primates such as humans and chimpanzees, laboratory animals such as rats and mice, pigs, cows, horses, sheep, And domestic animals such as goats, and pet animals such as dogs and cats, preferably humans or mice, most preferably humans.
　本発明の組成物の投与量、投与間隔、及び投与期間は、当業者であれば、被験体の性別、体重、年齢（月齢、週齢）、並びに疾患等の経過及び症状等の種々の要因を考慮して適宜定めることができる。例えば、組成物の投与量は、約0.1mg～10g/kg体重、好ましくは1mg～1000mg/kg体重、又は10～100mg/kg体重であってよい。また、投与回数は、限定するものではないが、1日3回、1日2回、1日1回、2日に1回、3日に1回、1週間に1回、2週間に1回、1カ月に1回等であってよい。また、投与期間は、限定するものではないが、1日、2日、3日、1週間、2週間、1カ月、半年、一年、又はそれ以上であってよい。 The dosage, administration interval, and administration period of the composition of the present invention can be determined by those skilled in the art from various factors such as the subject's sex, weight, age (month and age), and the course and symptoms of the disease. Can be determined as appropriate. For example, the dosage of the composition may be about 0.1 mg to 10 g / kg body weight, preferably 1 mg to 1000 mg / kg body weight, or 10 to 100 mg / kg body weight. The number of doses is not limited, but 3 times a day, 2 times a day, 1 time a day, 1 time every 2 days, 1 time every 3 days, once a week, 1 time every 2 weeks Once a month, etc. Further, the administration period is not limited, but may be 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.
　一態様において、本発明は、被験体に上記組成物を投与することを含む、ヘルペスウイルス感染症の治療及び／又は予防方法に関する。本方法における被験体、ヘルペスウイルス感染症、組成物の投与量、投与間隔、及び投与期間は、上記の通りであるからここでは記載を省略する。
２．ヘルペスウイルス感染症の治療及び／又は予防のための組成物の製造方法
　一態様において、本発明は、上記１に記載のヘルペスウイルス感染症の予防及び／又は治療のための組成物の製造方法に関する。 In one aspect, the present invention relates to a method for treating and / or preventing herpes virus infection comprising administering the composition to a subject. Since the subject, the herpes virus infection, the dose of the composition, the administration interval, and the administration period in the present method are as described above, description thereof is omitted here.
2. Method for producing composition for treatment and / or prevention of herpes virus infection In one aspect, the present invention relates to a method for producing a composition for prevention and / or treatment of herpes virus infection as described in 1 above. .
　本発明の製造方法は、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物を混合する工程を、必須の工程として含む。本工程における混合比は、加熱乾燥マイタケ抽出物Aを1としたときの真空乾燥マイタケ抽出物Bの重量比が、0.05以上、0.08以上、0.1以上、0.2以上、0.5以上、0.8以上、好ましくは1以上、1.5以上、2以上、2.5以上、又は3以上であってよく、また10以下、9以下、8以下、7以下、6以下、好ましくは5以下、4.5以下、4以下、3.5以下、又は3以下であってよい。例えば抽出物Aを1としたときの抽出物Bの重量比は、例えば、0.05～10、0.1～5、好ましくは1～5、2～4、2.5～3.5、又は3であってよい。 The production method of the present invention includes a step of mixing the heat-dried maitake extract and the vacuum-dried maitake extract as an essential step. The mixing ratio in this step is such that the weight ratio of the vacuum-dried maitake extract B when the heat-dried maitake extract A is 1 is 0.05 or more, 0.08 or more, 0.1 or more, 0.2 or more, 0.5 or more, 0.8 or more, preferably 1 or more, 1.5 or more, 2 or more, 2.5 or more, or 3 or more, and 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, preferably 5 or less, 4.5 or less, 4 or less, 3.5 or less, Or it may be 3 or less. For example, the weight ratio of the extract B when the extract A is 1 may be, for example, 0.05 to 10, 0.1 to 5, preferably 1 to 5, 2 to 4, 2.5 to 3.5, or 3.
　本混合工程では、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物のみを混合してもよいし、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物に、他の成分を加えて混合してもよい。また、最終的に上記混合比で組成物が得られる限り、混合の工程を分けることも可能であり、例えば加熱乾燥マイタケ抽出物と他の成分を混合した後に、混合物に対して真空乾燥マイタケ抽出物を加えることもできる。 In this mixing step, only the heat-dried maitake extract and the vacuum-dried maitake extract may be mixed, or other components may be added to and mixed with the heat-dried maitake extract and the vacuum-dried maitake extract. In addition, as long as the composition is finally obtained at the above mixing ratio, it is possible to divide the mixing process. For example, after mixing the heat-dried maitake extract and other components, the mixture is vacuum-dried maitake extract You can also add things.
　本発明の製造方法は、上記混合工程の前に、加熱乾燥マイタケ抽出物を得る工程を含んでよい。 The production method of the present invention may include a step of obtaining a heat-dried maitake extract before the mixing step.
　加熱乾燥マイタケ抽出物は、（A）マイタケを加熱乾燥する工程、（B）得られた加熱乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び（C）得られた抽出画分中の溶媒を除去し、乾燥物を得る工程により得ることができる。 The heat-dried maitake extract includes (A) a step of heat-drying maitake, (B) a step of adding a solvent to the obtained heat-dried maitake to obtain an extract fraction, and (C) a step in which the extract is obtained. The solvent can be removed to obtain a dry product.
　（A）マイタケを加熱乾燥する工程は特に限定せず、公知の加熱乾燥法、例えば、熱風乾燥を用いることができる。加熱温度は、低すぎると乾燥効率が低下し、高すぎるとマイタケの成分に化学変化が生じる恐れがあるため、適切な範囲に設定することが好ましい。乾燥は、40℃以上、50℃以上、60℃以上、好ましくは70℃以上、又は80℃以上、また150℃以下、120℃以下、100℃以下、好ましくは90℃以下又は80℃以下の条件で、例えば40℃～100℃、50℃～90℃、又は60℃～80℃の条件で、例えば前記温度の熱風を目的の物質に送風することによって行うことができる。加熱時間はマイタケ中の水分が除かれる限り特に限定されず、例えば1時間以上、2時間以上、4時間以上、6時間以上、終夜、又は一昼夜とすることができる。 (A) The process of heat-drying maitake is not particularly limited, and a known heat-drying method such as hot air drying can be used. If the heating temperature is too low, the drying efficiency is lowered. If the heating temperature is too high, there is a possibility that a chemical change may occur in the components of the maitake. Therefore, it is preferable to set the heating temperature in an appropriate range. Drying is performed at 40 ° C or higher, 50 ° C or higher, 60 ° C or higher, preferably 70 ° C or higher, or 80 ° C or higher, 150 ° C or lower, 120 ° C or lower, 100 ° C or lower, preferably 90 ° C or lower or 80 ° C or lower. For example, it can be carried out by blowing hot air at the above temperature to the target substance under the conditions of 40 ° C. to 100 ° C., 50 ° C. to 90 ° C., or 60 ° C. to 80 ° C., for example. The heating time is not particularly limited as long as moisture in the maitake is removed, and can be, for example, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, all night, or all day.
　マイタケ抽出物を得るための溶媒については、上記１において記載した通りであるからここでは記載を省略する。（B）抽出画分を得る工程は、上記溶媒を必要に応じて細断又は粉末化した乾燥マイタケに加えることにより行うことができる。抽出溶媒の添加量は特に限定しないが、例えば乾燥マイタケに対して抽出溶媒を1～100、2～50又は4～30重量倍使用することができる。 Since the solvent for obtaining the maitake extract is as described in 1 above, the description is omitted here. (B) The process of obtaining an extraction fraction can be performed by adding the said solvent to the dried maitake which was chopped or powdered as needed. The addition amount of the extraction solvent is not particularly limited. For example, the extraction solvent can be used 1 to 100, 2 to 50, or 4 to 30 times by weight with respect to the dried maitake.
　（B）抽出画分を得る工程においては、加熱を行って抽出効率を高めることができる。マイタケ中の成分に影響を与えるような酵素は約60℃までは働くが、60℃を超える温度では働きが衰えるので、60℃以上、好ましくは70℃以上、又は80℃以上で抽出を行うのが好ましい。また、加圧加熱によりさらに抽出効率を高めることが好ましく、加圧加熱の場合の加熱条件は、110℃以上、115℃以上、又は120℃以上、また140℃以下、130℃以下、又は125℃以下であってよく、加圧条件は、限定しないが、例えば、圧力釜等を用いて1.2気圧～3気圧、1.5気圧～2.5気圧、1.8気圧～2.2気圧、1.9気圧～2.1気圧又は2気圧にすることができる。抽出時間は抽出条件に応じて適宜設定することができるが、例えば5分～1日、10分～6時間、15分～1時間、又は20分～40分であってよい。抽出は、好ましくは加圧下100℃以上で短時間、例えば加圧下115℃～125℃で5分～1時間行う。 (B) In the step of obtaining the extracted fraction, the extraction efficiency can be increased by heating. Enzymes that affect the components in maitake work up to about 60 ° C, but work declines at temperatures above 60 ° C, so extraction should be done at 60 ° C or higher, preferably 70 ° C or higher, or 80 ° C or higher. Is preferred. Further, it is preferable to further increase the extraction efficiency by pressure heating, and the heating conditions in the case of pressure heating are 110 ° C. or higher, 115 ° C. or higher, or 120 ° C. or higher, 140 ° C. or lower, 130 ° C. or lower, or 125 ° C. The pressurizing condition may be as follows, but is not limited to, for example, using a pressure cooker or the like to 1.2 atm to 3 atm, 1.5 to 2.5 atm, 1.8 to 2.2 atm, 1.9 to 2.1 atm or 2 atm can do. The extraction time can be appropriately set according to the extraction conditions, and may be, for example, 5 minutes to 1 day, 10 minutes to 6 hours, 15 minutes to 1 hour, or 20 minutes to 40 minutes. The extraction is preferably performed at a temperature of 100 ° C. or higher under pressure for a short time, for example, at 115 ° C. to 125 ° C. for 5 minutes to 1 hour under pressure.
　（C）得られた抽出画分中の溶媒を除去し、乾燥物を得る工程は、常法により行うことができ、例えば加熱乾燥、減圧乾燥、真空乾燥、スプレードライ等を単独で、又は二以上組み合わせて用いることによって行うことができる。 (C) The step of removing the solvent from the obtained extract fraction to obtain a dried product can be carried out by a conventional method. For example, heat drying, reduced pressure drying, vacuum drying, spray drying and the like can be performed alone or in combination. It can carry out by using in combination above.
　本発明の製造方法は、任意に、（B）抽出画分を得る工程の後、（C）得られた抽出画分中の溶媒を除去し、乾燥物を得る工程の前に、（B'）濾過による清澄化及び／又はアルコールによる析出物の除去によって、さらに純度を高める工程を含んでよい。濾過による清澄化は公知の方法により行うことができ、アルコールによる析出物の除去は、例えば以下の工程により行うことができる。すなわち、（B）抽出画分を得る工程によって得られた抽出液又は必要に応じてこれを濃縮した溶液に対し、アルコールを、最終容量で50容量％以上、60容量％以上、又は70容量％以上になるように加え、例えば1～25℃又は常温で、放置することによって液面若しくは液中、又は容器の壁等に析出する物質を除去して抽出物の純度を高めることができる。析出物の除去は、例えば濾過又は網等で掬うことによって行い得る。アルコールとしては、低級アルコール、例えばメタノール、エタノール、及びプロパノール等を単独で、又は二以上組み合わせて用いることができ、エタノールを単独で用いることが特に好ましい。 In the production method of the present invention, optionally, (B) after the step of obtaining the extract fraction, (C) before the step of removing the solvent in the obtained extract fraction and obtaining the dried product (B ′ ) It may further comprise a step of increasing purity by clarification by filtration and / or removal of precipitates by alcohol. Clarification by filtration can be performed by a known method, and removal of the precipitate by alcohol can be performed, for example, by the following steps. That is, (B) the alcohol obtained with the extract obtained in the step of obtaining the extract fraction or a solution obtained by concentrating the extract, if necessary, is 50% by volume or more, 60% by volume or more, or 70% by volume. In addition, the purity of the extract can be increased by removing substances deposited on the liquid surface, in the liquid, or on the wall of the container, for example, by leaving at 1 to 25 ° C. or normal temperature. The removal of the precipitate can be performed by, for example, filtering or sieving through a net or the like. As the alcohol, lower alcohols such as methanol, ethanol, and propanol can be used alone or in combination of two or more, and it is particularly preferable to use ethanol alone.
　本発明の製造方法は、上記混合工程の前に、加熱乾燥マイタケ抽出物を得る工程に加えて、又は加熱乾燥マイタケ抽出物を得る工程とは別に、真空乾燥マイタケ抽出物を得る工程を含んでよい。本発明の製造方法が、加熱乾燥マイタケ抽出物を得る工程と真空乾燥マイタケ抽出物を得る工程の両方を含む場合、両工程の順序は特に限定しない。 The production method of the present invention includes a step of obtaining a vacuum-dried maitake extract in addition to the step of obtaining a heat-dried maitake extract before or after the mixing step. Good. When the production method of the present invention includes both a step of obtaining a heat-dried maitake extract and a step of obtaining a vacuum-dried maitake extract, the order of both steps is not particularly limited.
　真空乾燥マイタケ抽出物は、（D）マイタケを真空乾燥する工程、（E）得られた真空乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び（F）得られた抽出画分中の溶媒を除去し、乾燥物を得る工程により得ることができる。 The vacuum-dried maitake extract includes (D) a step of vacuum-drying maitake, (E) a step of adding a solvent to the obtained vacuum-dried maitake, and obtaining an extract fraction, and (F) in the obtained extract fraction. The solvent can be removed to obtain a dry product.
　（D）マイタケを真空乾燥する工程は特に限定せず、公知の真空乾燥法、例えば、凍結乾燥を用いることができる。真空乾燥は、マイタケを加熱する必要がなく、低温で乾燥を行うことから、マイタケの成分に化学変化が生じる恐れが少ない。真空乾燥は、完全な真空下で行ってもよいし、実質的な真空下、例えば100Pa以下、50Pa以下、又は20Pa以下等で行ってもよい。真空乾燥は、当業者に公知の方法により、例えば市販の真空乾燥機を用いて行うことができる。真空乾燥の時間は、マイタケから水分が除かれる限り特に限定されず、例えば1時間以上、2時間以上、4時間以上、6時間以上、又は終夜行うことができる。 (D) The step of vacuum drying the maitake is not particularly limited, and a known vacuum drying method such as freeze drying can be used. In vacuum drying, there is no need to heat the maitake and the drying is performed at a low temperature, so that there is little risk of chemical changes in the components of maitake. The vacuum drying may be performed under a complete vacuum or under a substantial vacuum, for example, 100 Pa or less, 50 Pa or less, or 20 Pa or less. The vacuum drying can be performed by a method known to those skilled in the art, for example, using a commercially available vacuum dryer. The time for vacuum drying is not particularly limited as long as moisture is removed from the maitake, and for example, it can be performed for 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, or overnight.
　（E）得られた真空乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び（F）得られた抽出画分中の溶媒を除去し、乾燥物を得る工程については、上記加熱乾燥マイタケ抽出物を得る工程の（B）工程及び（C）工程において記載した通りであるから記載を省略する。 (E) For the step of adding a solvent to the obtained vacuum-dried maitake to obtain an extract fraction, and (F) removing the solvent in the obtained extract fraction to obtain a dried product, the above-mentioned heat-dried maitake Since it is as having described in the (B) process and the (C) process of obtaining an extract, description is abbreviate | omitted.
　本発明の製造方法は、任意に、（E）抽出画分を得る工程の後、（F）得られた抽出画分中の溶媒を除去し、乾燥物を得る工程の前に、（E'）濾過による清澄化及び／又はアルコールによる析出物の除去によって、さらに純度を高める工程を含んでよい。（E'）工程については、上記加熱乾燥マイタケ抽出物を得る工程の（B'）工程において記載したのと同様であるからここでは記載を省略する。 In the production method of the present invention, optionally, (E) after the step of obtaining an extract fraction, (F) before the step of removing the solvent in the obtained extract fraction and obtaining a dry product (E ′ ) It may further comprise a step of increasing purity by clarification by filtration and / or removal of precipitates by alcohol. About (E ') process, since it is the same as having described in the (B') process of the process of obtaining the said heat-dried maitake extract, description is abbreviate | omitted here.
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.
　以下、実施例を参照して本発明をさらに詳細に説明するが、本発明はこれらの実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
［実施例1：加熱乾燥マイタケ抽出物の調製］
　人工栽培により得られたマイタケの子実体（茎部及び傘部）を棚型乾燥室の棚にならべ、最初は70℃から段階的に温度を上げ最終的には80℃でほぼ1日かけて乾燥した。ついで乾燥マイタケ粉末を製粉機で粉砕し、微粉末の加熱乾燥マイタケを得た。 [Example 1: Preparation of heat-dried maitake extract]
The fruit body (stem and umbrella) obtained by artificial cultivation is placed on the shelf of the shelf-type drying room, and the temperature is increased gradually from 70 ° C to 80 ° C, and it takes about 1 day. Dried. Next, the dried maitake powder was pulverized with a mill to obtain heat-dried maitake of fine powder.
　加熱乾燥マイタケ粉末13.5kgを、イオン交換水310Lに投入し、2気圧加圧下、121℃で30分間の熱水抽出を行った。抽出液は、フィルタープレスを用いて濾過し、黒褐色の抽出液約260Ｌを得た。該溶液を減圧下で60L程度まで濃縮して、室温でエタノール66Lを加え10℃以下で約数時間放置すると、液面、液中に浮遊又は壁面に付着する茶褐色の物質が生成した。これらの物質を金網で掬って除去し、褐色の溶液を得た。該溶液を減圧下でエタノールを除き、さらに減圧下で濃縮後、スプレードライ装置を用いて噴霧乾燥し、微細な褐色粉末5.2kgを得た。得られた物質をMD Frとした。 13.5 kg of heat-dried maitake powder was added to 310 L of ion-exchanged water, and hot water extraction was performed at 121 ° C. for 30 minutes under 2 atm pressure. The extract was filtered using a filter press to obtain about 260 L of a blackish brown extract. The solution was concentrated to about 60 L under reduced pressure, 66 L of ethanol was added at room temperature, and the mixture was allowed to stand at 10 ° C. or lower for about several hours. As a result, a brown substance was formed that floated in the liquid or adhered to the wall. These materials were removed with a wire mesh to give a brown solution. The solution was freed from ethanol under reduced pressure, further concentrated under reduced pressure, and then spray-dried using a spray dryer to obtain 5.2 kg of a fine brown powder. The obtained substance was designated as MD Fr.
　得られたMD Frにつき、以下の条件でHPLCを行ったところ、図1に示すピークが得られた。
溶離液　　：0.1M NaNO3(0.02% NaN3)水溶液
検出器　　：示差屈折計
分析カラム：SB-806M HQ
温度　　　：40℃
流速　　　：1.0mL/min When the obtained MD Fr was subjected to HPLC under the following conditions, the peak shown in FIG. 1 was obtained.
Eluent: 0.1M NaNO 3 (0.02% NaN 3 ) aqueous solution detector: Differential refractometer analytical column: SB-806M HQ
Temperature: 40 ° C
Flow rate: 1.0mL / min
［実施例2：真空乾燥マイタケ抽出物の調製］
　人工栽培により得られたマイタケの子実体（茎部及び傘部）を－30℃～－40℃で凍結させ、真空凍結乾燥機を用いて乾燥した。乾燥機内は20Paまで減圧され、およそ1日で乾燥した。次いで、乾燥マイタケ粉末を製粉機で粉砕し、微粉末化した。 [Example 2: Preparation of vacuum dried maitake extract]
The fruit bodies (stalks and umbrellas) obtained by artificial cultivation were frozen at −30 ° C. to −40 ° C. and dried using a vacuum freeze dryer. The inside of the dryer was depressurized to 20 Pa and dried in about 1 day. Next, the dried maitake powder was pulverized with a mill to make a fine powder.
　微粉末化した乾燥マイタケ粉末10kgを、あらかじめ80℃まで加熱しておいたイオン交換水210Lに投入し、2気圧加圧下、121℃で30分間の熱水抽出を行った。抽出液は、フィルタープレスを用いて濾過し、黒褐色の抽出液159Lを得た。該溶液を減圧下で濃縮後、スプレードライ装置を用いて噴霧乾燥し、微細な褐色粉末3.6kgを得た。得られた物質をYM-6Aとした。 10 kg of finely pulverized dry maitake powder was put into 210 L of ion-exchanged water that had been heated to 80 ° C. in advance, and hot water extraction was performed at 121 ° C. for 30 minutes under 2 atm pressure. The extract was filtered using a filter press to obtain a black-brown extract 159L. The solution was concentrated under reduced pressure and then spray-dried using a spray dryer to obtain 3.6 kg of a fine brown powder. The obtained substance was designated as YM-6A.
　得られたYM-6Aにつき、以下の条件でHPLCを行ったところ、図2に示すピークが得られた。
溶離液　　：0.1M NaNO3(0.02% NaN3)水溶液
検出器　　：示差屈折計
分析カラム：SB-806M HQ
温度　　　：40℃
流速　　　：1.0mL/min When the obtained YM-6A was subjected to HPLC under the following conditions, the peak shown in FIG. 2 was obtained.
Eluent: 0.1M NaNO 3 (0.02% NaN 3 ) aqueous solution detector: Differential refractometer analytical column: SB-806M HQ
Temperature: 40 ° C
Flow rate: 1.0mL / min
［実施例3: 加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物の混合物の調製1］
　以下の重量割合で、実施例1で得られたMD Frと実施例2で得られたYM-6Aをブレンダーを用いて混合し、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物の混合物を調製した。
Figure JPOXMLDOC01-appb-T000001
 [Example 3: Preparation 1 of a mixture of heat-dried maitake extract and vacuum-dried maitake extract]
MD Fr obtained in Example 1 and YM-6A obtained in Example 2 were mixed using a blender at the following weight ratios to prepare a mixture of a heat-dried maitake extract and a vacuum-dried maitake extract. .
Figure JPOXMLDOC01-appb-T000001
［実施例4：ヘルペスウイルス感染症に対する予防及び治療効果］
　本実施例では、BALB/cマウスを用いて、実施例1、3で調製した各試料によるヘルペスウイルス感染症に対する効果を検証した。陽性対照としてはアシクロビル（ACV）を用いた。 [Example 4: Preventive and therapeutic effects on herpes virus infection]
In this example, the effect of each sample prepared in Examples 1 and 3 on herpes virus infection was verified using BALB / c mice. Acyclovir (ACV) was used as a positive control.
＜方法＞
　BALB/cマウス（6週齢、♀、n=5）にmedroxyprogesterone 17-acetate (3 mg/mouse)を、ウイルス接種6日前及び1日前に皮下注射した。マウスには、PBS、アシクロビル（0.2mg/40μL/日）又は実施例1又は3で調製したサンプル（1mg/40μL/日）を、ウイルス接種3日前から7日後までの間、1日2回（午前9時及び午後6時）、性器に局所投与した。 <Method>
BALB / c mice (6 weeks old, sputum, n = 5) were subcutaneously injected with medroxyprogesterone 17-acetate (3 mg / mouse) 6 days before and 1 day before virus inoculation. Mice received PBS, acyclovir (0.2 mg / 40 μL / day) or the sample prepared in Example 1 or 3 (1 mg / 40 μL / day) twice a day between 3 days before and 7 days after virus inoculation ( At 9 am and 6 pm), it was administered locally to the genitals.
　ウイルス（単純ヘルペスウイルス1型：HSV-1、KOS株、富山県衛生試験所からの分与株、1x106 PFU/20μL/mouse）を性器に局所接種した3日後に、局所をPBSで洗浄し、その中のウイルス力価をプラークアッセイ法によって測定した。 Three days after the local inoculation of the virus (herpes simplex virus type 1: HSV-1, KOS strain, dispenser from Toyama Sanitation Laboratory, 1x10 6 PFU / 20μL / mouse) into the genital area, the area was washed with PBS. The virus titer therein was measured by plaque assay.
　症状の程度（スコア）及び死亡例をウイルスの接種後2週間にわたって毎日記録した（2週間以降には死亡例はない）。症状の程度は表2のスコアで示す通りである。
Figure JPOXMLDOC01-appb-T000002
 Symptom degree (score) and deaths were recorded daily for 2 weeks after virus inoculation (no deaths after 2 weeks). The degree of symptom is as shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
＜結果＞
　各群における死亡率及び死亡日数を表3に、スコアの平均値を、図3に示した。
Figure JPOXMLDOC01-appb-T000003
 <Result>
The mortality rate and the number of days of death in each group are shown in Table 3, and the average score is shown in FIG.
Figure JPOXMLDOC01-appb-T000003
　表3及び図3から明らかな様に、MD Fr:YM-6A=1:1混合物において、ACVと同等の高い発症抑制効果がみられた。 As is clear from Table 3 and FIG. 3, the MD Fr: YM-6A = 1: 1 mixture showed a high onset suppression effect equivalent to ACV.
　また、ウイルス接種の3日後にウイルス量を測定したところ、MD Fr:YM-6A=1:1混合物において、対照群に比べて有意に低値であった（図4）。 Moreover, when the viral load was measured 3 days after the virus inoculation, the MD Fr: YM-6A = 1: 1 mixture was significantly lower than the control group (FIG. 4).
　以上の通り、加熱乾燥マイタケ抽出物（MD Fr）と真空乾燥マイタケ抽出物（YM-6A）の混合物が、ウイルス感染に対して優れた効果を有することが示された。 As described above, it was shown that the mixture of the heat-dried maitake extract (MD Fr) and the vacuum-dried maitake extract (YM-6A) has an excellent effect on virus infection.
［実施例5: 加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物の混合物の調製2］
　表4で示した重量割合で、実施例1で得られたMD Frと実施例2で得られたYM-6Aをブレンダーを用いて混合し、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物の混合物を調製した。
Figure JPOXMLDOC01-appb-T000004
 [Example 5: Preparation of mixture of heat-dried maitake extract and vacuum-dried maitake extract 2]
MD Fr obtained in Example 1 and YM-6A obtained in Example 2 were mixed using a blender at a weight ratio shown in Table 4, and a mixture of a heat-dried maitake extract and a vacuum-dried maitake extract Was prepared.
Figure JPOXMLDOC01-appb-T000004
［実施例6：ヘルペスウイルス感染症に対する予防及び治療効果］
　本実施例では、BALB/cマウスを用いて、実施例1、2及び5で調製した各試料によるヘルペスウイルス感染症に対する効果を検証した。陽性対照としてはアシクロビル（ACV）を用いた。 [Example 6: Preventive and therapeutic effects on herpes virus infection]
In this example, the effect of each sample prepared in Examples 1, 2 and 5 on herpes virus infection was verified using BALB / c mice. Acyclovir (ACV) was used as a positive control.
＜方法＞
　BALB/cマウス（6週齢、♀、n=5）にmedroxyprogesterone 17-acetate (3 mg/mouse)を、ウイルス接種6日前及び1日前に皮下注射した。マウスには、PBS、アシクロビル（0.2mg/40μL/日）又は実施例1、2及び5で調製したサンプル（1mg/40μL/日）を、ウイルス接種3日前から7日後までの間、1日2回（午前9時及び午後6時）、性器に局所投与した。 <Method>
BALB / c mice (6 weeks old, sputum, n = 5) were subcutaneously injected with medroxyprogesterone 17-acetate (3 mg / mouse) 6 days before and 1 day before virus inoculation. Mice received PBS, acyclovir (0.2 mg / 40 μL / day) or the samples prepared in Examples 1, 2 and 5 (1 mg / 40 μL / day) 2 days a day between 3 days before and 7 days after virus inoculation. Times (9am and 6pm), administered locally to the genitals.
　ウイルス（単純ヘルペスウイルス2型：HSV-2、UW 268株、富山県衛生試験所からの分与株、1x103 PFU/20μL/mouse）を性器に局所接種した3日後に、局所をPBSで洗浄し、その中のウイルス力価をプラークアッセイ法によって測定した。 Three days after the local inoculation of the virus (herpes simplex virus type 2: HSV-2, UW 268, distributed strain from Toyama Sanitation Laboratory, 1x10 3 PFU / 20μL / mouse) into the genital area, the area was washed with PBS. The virus titer therein was measured by plaque assay.
　症状の程度（スコア）及び死亡例をウイルスの接種後2週間にわたって毎日記録した（2週間以降には死亡例はない）。症状の程度は表2のスコアで示す通りである。 ¡The degree of symptom (score) and death cases were recorded every day for 2 weeks after virus inoculation (no deaths after 2 weeks). The degree of symptom is as shown in Table 2.
＜結果＞
　各群における死亡率及び死亡日数を表5に、スコアの平均値を、図5に示した。
Figure JPOXMLDOC01-appb-T000005
 <Result>
The mortality and the number of days of death in each group are shown in Table 5, and the average score is shown in FIG.
Figure JPOXMLDOC01-appb-T000005
　また、ウイルス接種の3日後にウイルス量を測定したところ、全ての投与群において、有意な増殖抑制効果がみられた。特にMD Fr：YM-6A＝1：1及び1：5の混合物で顕著な効果がみられた（図6）。 Moreover, when the viral load was measured 3 days after the virus inoculation, a significant growth inhibitory effect was observed in all administration groups. In particular, a remarkable effect was observed in the mixture of MD Fr: YM-6A = 1: 1 and 1: 5 (FIG. 6).
　以上の通り、加熱乾燥マイタケ抽出物（MD Fr）と真空乾燥マイタケ抽出物（YM-6A）の混合物が、加熱乾燥マイタケ抽出物又は真空乾燥マイタケ抽出物単独よりも、ウイルス感染に対して優れた効果を有し、特にMD FrとYM-6Aの比が1:1～1:5である場合に優れた効果を有することが示された。 As described above, the mixture of heat-dried maitake extract (MD Fr) and vacuum-dried maitake extract (YM-6A) was superior to virus infection than heat-dried maitake extract or vacuum-dried maitake extract alone. It has been shown that it has an effect, particularly when the ratio of MDAFr and YM-6A is 1: 1 to 1: 5.
［実施例7: 加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物の混合物の調製3］
　表6で示した重量割合で実施例1で得られたMD Frと実施例2で得られたYM-6Aをブレンダーを用いて混合し、加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物の混合物を調製した。
Figure JPOXMLDOC01-appb-T000006
 [Example 7: Preparation 3 of a mixture of heat-dried maitake extract and vacuum-dried maitake extract]
MD Fr obtained in Example 1 and YM-6A obtained in Example 2 were mixed using a blender at the weight ratio shown in Table 6, and a mixture of the heat-dried maitake extract and the vacuum-dried maitake extract was mixed. Prepared.
Figure JPOXMLDOC01-appb-T000006
［実施例8：ヘルペスウイルス感染症に対する予防及び治療効果］
　本実施例では、MD Fr：YM-6Aの最適な混合比を検討するため、BALB/cマウスを用いて、実施例7で調製した各混合物によるヘルペスウイルス感染症に対する効果を検証した。陽性対照としてはアシクロビル（ACV）を用いた。 [Example 8: Prevention and treatment effect on herpes virus infection]
In this example, in order to examine the optimal mixing ratio of MD Fr: YM-6A, the effect of each mixture prepared in Example 7 on herpes virus infection was verified using BALB / c mice. Acyclovir (ACV) was used as a positive control.
　実験方法は、実施例7で調製した各混合物を用いる以外は、実施例6に記載の方法に従った。 The experimental method was the same as that described in Example 6, except that each mixture prepared in Example 7 was used.
　各群における死亡率及び死亡日数を表7に、スコアの平均値を図7に示す。
Figure JPOXMLDOC01-appb-T000007
 The mortality rate and the number of death days in each group are shown in Table 7, and the average score is shown in FIG.
Figure JPOXMLDOC01-appb-T000007
　表7及び図7から明らかな様に、MD Fr:YM-6A=1:3混合物において、特に優れたウイルス感染症に対する効果が認められた。 As is clear from Table 7 and FIG. 7, the MD Fr: YM-6A = 1: 3 mixture showed a particularly excellent effect on viral infections.
　また、ウイルス接種の3日後にウイルス量を測定したところ、MD Fr:YM-6A=1:3混合物において、特に優れたウイルス増殖抑制効果が認められた（図8）。 Further, when the amount of virus was measured 3 days after virus inoculation, a particularly excellent virus growth-inhibiting effect was observed in the MD Fr: YM-6A = 1: 3 mixture (FIG. 8).
　以上の結果は、MD Fr:YM-6Aの混合比率を1:3とすることで、特に優れた効果が奏されることを示している。 The above results indicate that a particularly excellent effect can be achieved by setting the mixing ratio of MD Fr: YM-6A to 1: 3.
［実施例9：単純ヘルペスウイルス潜伏感染からの回帰発症に対する阻止効果］
　本実施例では、BALB/cマウスを用いて、実施例7で調製したMD Fr：YM-6A=1:3混合物による回帰発症に対する阻止効果を検証した。陽性対象としてはアシクロビルを経口投与で用いた。 [Example 9: Inhibitory effect on recurrence from herpes simplex virus latent infection]
In this example, the inhibitory effect on regression onset by the MD Fr: YM-6A = 1: 3 mixture prepared in Example 7 was verified using BALB / c mice. As a positive subject, acyclovir was orally administered.
＜方法＞
　BALB/cマウス60匹に麻酔下で、左右の角膜を23ゲージ針束で5回ずつ交差して擦過後、HSV-1 McKrae株（北陸大学 大黒徹先生からの分与株、1x105 PFU/ 5 μl/ mouse）を接種し、その後14日間にわたって、角膜の症状(Lesion scores)を記録した。感染3日後及び5日後に、PBS 0.1 mlずつで左右の角膜を洗浄し、回収した洗浄液をプラークアッセイに供した。約半数の32匹が、感染14日までに死亡し、さらに生存した28匹中、角膜に症状が認められず、かつ角膜からウイルスが検出されなかったマウスが4匹いた。この為、生存した残りの24匹のマウスについて、表8に示した発症の程度及び左右角膜のウイルス量のデータに基づいて、均等に1群6匹ずつ、3群に分けて試験に供した。
Figure JPOXMLDOC01-appb-T000008
 <Method>
Under anesthesia in 60 BALB / c mice, the left and right corneas were crossed with a 23-gauge needle bundle 5 times each, and then scraped, and then HSV-1 McKrae strain (distributed strain from Prof. Toru Oguro, Hokuriku University, 1x10 5 PFU / 5 μl / mouse) was inoculated and the corneal symptoms (Lesion scores) were recorded for 14 days. Three and five days after infection, the left and right corneas were washed with 0.1 ml of PBS, respectively, and the collected washings were subjected to a plaque assay. About half of 32 died by the 14th day of infection, and among the 28 surviving animals, there were 4 mice with no symptoms in the cornea and no virus detected in the cornea. For this reason, the remaining 24 mice were subjected to the test equally divided into 6 groups per group and 3 groups based on the degree of onset and left and right cornea virus load data shown in Table 8. .
Figure JPOXMLDOC01-appb-T000008
　感染28日後から38日後まで、MD Fr:YM-6A混合物を、1日2回（1mg/40μl/day、9時及び18時）局所投与した。同様に、コントロール群には、滅菌水を0.4ml/dayで経口投与し、ACV投与群には、ACVを1 mg/0.4 ml/dayで経口投与した。次いで、回帰発症を誘導させる為、酪酸ナトリウム(NaBu)を、感染35日後に1200 mg/kg（＝ 27 mg/22 g/0.2 ml/mouse)、さらに感染36日後に、600 mg/kg（＝13.5 mg/22 g/0.2 ml/mouse)を腹腔内注射した。 From 28 to 38 days after infection, the MD 混合 Fr: YM-6A mixture was locally administered twice a day (1 mg / 40 μl / day, 9:00 and 18:00). Similarly, sterile water was orally administered to the control group at 0.4 ml / day, and ACV was orally administered to the ACV administration group at 1 mg / 0.4 ml / day. Next, in order to induce recurrent onset, sodium butyrate (NaBu) was added at 1200 mg / kg (= 27 mg / 22 g / 0.2 ml / mouse) 35 days after infection, and further 600 mg / kg (= 13.5 mg / 22 mg / 0.2 mg / mouse) was injected intraperitoneally.
　その後、回帰発症の有無を確認する為に、感染36日後、及び感染37日後に、左右の眼球をPBS(0.1 ml)で洗浄し、洗浄液をプラークアッセイに供し、ウイルス量を測定し、ウイルスが検出されれば回帰発症していると評価した。 Then, in order to confirm the presence or absence of recurrence, 36 days after infection and 37 days after infection, the left and right eyeballs were washed with PBS (0.1 ml), the washing solution was subjected to a plaque assay, and the amount of virus was measured. If it was detected, it was evaluated that the patient had a regression.
　感染39日後（初回のNaBu処置後4日後）に、全血、左右の眼球、左右の三叉神経節（TG）、脳を採取した。眼球、TG、脳を超音波処理し、遠心後、上清からウイルスを検定した。 Whole blood, left and right eyeballs, left and right trigeminal ganglia (TG), and brain were collected 39 days after infection (4 days after the first NaBu treatment). The eyeball, TG, and brain were sonicated and centrifuged, and the virus was assayed from the supernatant.
　血清からは、以下の方法に従って中和抗体価を測定した。
1)マウスから採血し、血清を遠心分離する（3,000 rpm、5分、4 ℃）。
2) PBSで適宜希釈する。
3)ウイルス（2000 PFU/ml）100 μlを２)の希釈液100 μlと混合する (200 PFU/200 μl)（Controlとして、血清の代わりにPBSを加えたものも用意する）。
4)37 ℃、１時間処理する。
5)35-mm dishesに単層状に培養したVero 細胞に、４）の混合液を100 μl/dish (= 約 100 PFU/dish) 加え、室温で１時間感染させる。
6)0.8%メチルセルロースを加えた培地を重層し（2 ml/dish）、37 ℃で2日間培養する。
7)培地を除去後、クリスタルバイオレット液で固定・染色する。
8)顕微鏡下でプラーク数を測定する。
9)Controlのプラーク数を100%とした時の各希釈液のプラーク数の％を計算する。プラーク形成を50%阻害する血清希釈倍数をグラフ上で求め、それを中和抗体価とする。 From the serum, the neutralizing antibody titer was measured according to the following method.
1) Blood is collected from the mouse, and the serum is centrifuged (3,000 rpm, 5 minutes, 4 ° C).
2) Dilute appropriately with PBS.
3) Mix 100 μl of virus (2000 PFU / ml) with 100 μl of 2) dilution (200 PFU / 200 μl) (also prepare PBS with PBS instead of serum).
4) Treat at 37 ° C for 1 hour.
5) Add 100 μl / dish (= about 100 PFU / dish) of the mixture of 4) to Vero cells cultured in a single layer in 35-mm dishes and infect at room temperature for 1 hour.
6) Overlay the medium supplemented with 0.8% methylcellulose (2 ml / dish) and incubate at 37 ° C for 2 days.
7) After removing the medium, fix and stain with crystal violet solution.
8) Measure the number of plaques under a microscope.
9) Calculate% of the number of plaques in each dilution when the number of plaques in Control is 100%. The serum dilution factor that inhibits plaque formation by 50% is determined on the graph and used as the neutralizing antibody titer.
＜結果＞
　各群における回帰発症数及び回帰発症率を表9に示す。
Figure JPOXMLDOC01-appb-T000009
 <Result>
Table 9 shows the number of regression episodes and the regression episode rate in each group.
Figure JPOXMLDOC01-appb-T000009
　また、各群における中和抗体価を表10に示す。
Figure JPOXMLDOC01-appb-T000010
 Table 10 shows the neutralizing antibody titers in each group.
Figure JPOXMLDOC01-appb-T000010
　以上の通り、ACV及びMD Fr：YM-6A=1：3混合物の投与によって回帰発症率が減少した。さらに、中和抗体価に関しては、ACVは、ウイルスへ直接作用するため中和抗体価が上がっていないが、MD Fr：YM-6A=1：3混合物は、コントロールと比べ中和抗体価を大きく上昇させた。この結果から、MD Fr：YM-6A=1：3混合物が中和抗体を上昇させることによりウイルスの増殖を抑え、回帰発症率が低下したと推察された。 As described above, administration of ACV and MD Fr: YM-6A = 1: 3 reduced the regression rate. Furthermore, regarding neutralizing antibody titer, ACV does not increase the neutralizing antibody titer because it acts directly on the virus, but the MD Fr: YM-6A = 1: 3 mixture has a larger neutralizing antibody titer than the control. Raised. From this result, it was speculated that the MD Fr: YM-6A = 1: 3 mixture increased the neutralizing antibody, thereby suppressing the growth of the virus and reducing the regression rate.
　本発明の加熱乾燥マイタケ抽出物と真空乾燥マイタケ抽出物を含む組成物は、ヘルペス感染症に対して高い効果を示す。また、本発明の組成物は、長年の食経験を有するマイタケから製造される為、安全性が高く、副作用が少ないと考えられる。したがって、本発明によって、ヘルペス感染症に対して有効であり、かつ副作用が少ないヘルペス感染症（回帰発症を含む）の治療及び／又は予防剤が提供され得る。 The composition containing the heat-dried maitake extract and the vacuum-dried maitake extract of the present invention has a high effect on herpes infection. Moreover, since the composition of this invention is manufactured from the maitake which has a long experience of eating, it is thought that it is highly safe and has few side effects. Therefore, the present invention can provide a therapeutic and / or preventive agent for herpes infections (including recurrent onset) that is effective against herpes infections and has few side effects.

Claims (14)

  1. 　加熱乾燥マイタケ抽出物A及び真空乾燥マイタケ抽出物Bを含み、
    　抽出物Aを1としたときの抽出物Bの重量比が0.05～10である、ヘルペスウイルス感染症の治療及び／又は予防のための組成物。     Heat-dried maitake extract A and vacuum-dried maitake extract B,
    A composition for the treatment and / or prevention of herpes virus infection, wherein the weight ratio of the extract B when the extract A is 1 is 0.05 to 10.
  2. 　抽出物Aを1としたときの抽出物Bの重量比が1～5である、請求項１に記載の組成物。 The composition according to claim 1, wherein the weight ratio of the extract B when the extract A is 1 is 1 to 5.
  3. 　抽出物A及び抽出物Bがいずれも水抽出物である、請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, wherein both the extract A and the extract B are water extracts.
  4. 　外用剤である、請求項１～３のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 3, which is an external preparation.
  5. 　ヘルペスウイルスの回帰発症の治療及び／又は予防のための、請求項１～４のいずれか一項に記載の組成物。 The composition according to any one of claims 1 to 4, which is used for treatment and / or prevention of recurrent onset of herpesvirus.
  6. 　加熱乾燥マイタケ抽出物A重量比1に対し、真空乾燥マイタケ抽出物Bを重量比0.05～10で混合する工程を含む、
    　ヘルペスウイルス感染症の治療及び／又は予防のための組成物の製造方法。     Including a step of mixing vacuum dried maitake extract B at a weight ratio of 0.05 to 10 with respect to heat-dried maitake extract A weight ratio of 1,
    A method for producing a composition for treatment and / or prevention of herpes virus infection.
  7. 　抽出物A重量比1に対し、抽出物Bを重量比1～5で混合する、請求項６に記載の方法。 The method according to claim 6, wherein the extract B is mixed at a weight ratio of 1 to 5 with respect to the extract A weight ratio of 1.
  8. 　抽出物A及び抽出物Bがいずれも水抽出物である、請求項６又は７に記載の方法。 The method according to claim 6 or 7, wherein both the extract A and the extract B are water extracts.
  9. 　前記組成物が外用剤である、請求項６～８のいずれか一項に記載の方法。 The method according to any one of claims 6 to 8, wherein the composition is an external preparation.
  10. 　前記組成物がヘルペスウイルスの回帰発症の治療及び／又は予防のためのものである、請求項６～９のいずれか一項に記載の方法。 The method according to any one of claims 6 to 9, wherein the composition is for the treatment and / or prevention of herpesvirus recurrent episodes.
  11. 　抽出物Aが、以下の工程：
    　マイタケを加熱乾燥する工程、
    　得られた加熱乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び
    　得られた抽出画分中の溶媒を除去し、乾燥物を得る工程
    により得られる、請求項６～１０のいずれか一項に記載の方法。     Extract A has the following steps:
    Heating and drying the maitake,
    A solvent is added to the obtained heat-dried maitake to obtain an extract fraction, and the solvent in the obtained extract fraction is removed to obtain a dried product. The method according to item.
  12. 　乾燥物を得る工程の前に、抽出画分にアルコールを添加し、析出する物質を除去する工程を含む、請求項１１に記載の方法。 The method according to claim 11, comprising a step of adding alcohol to the extracted fraction and removing a precipitated substance before the step of obtaining a dried product.
  13. 　抽出物Bが、以下の工程：
    　マイタケを真空乾燥する工程、
    　得られた真空乾燥マイタケに溶媒を加え、抽出画分を得る工程、及び
    　得られた抽出画分中の溶媒を除去し、乾燥物を得る工程
    により得られる、請求項６～１２のいずれか一項に記載の方法。     Extract B has the following steps:
    Vacuum drying the maitake,
    A solvent is added to the obtained vacuum-dried maitake to obtain an extract fraction, and the solvent in the obtained extract fraction is removed to obtain a dried product. The method according to item.
  14. 　マイタケを真空乾燥する工程が、凍結乾燥により行われる、請求項１３に記載の方法。 The method according to claim 13, wherein the step of vacuum drying the maitake is performed by freeze drying.
PCT/JP2017/038262 2016-10-28 2017-10-24 Composition of maitake extract for treatment and/or prevention of herpes infection WO2018079514A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0967269A (en) * 1995-08-31 1997-03-11 Beeta Shokuhin Kk Fruit body tablet of grifola frondosa s. f. gray
JP2007031665A (en) * 2005-07-29 2007-02-08 Yukiguni Maitake Co Ltd Glycoprotein extracted from grifola frondosa
WO2011108681A1 (en) * 2010-03-04 2011-09-09 株式会社雪国まいたけ Anti-allergic agent, and method for producing same
JP2014080373A (en) * 2012-10-12 2014-05-08 Yukiguni Maitake Co Ltd Herpes simplex virus infection preventive/therapeutic agent from grifola frondosa extract

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0967269A (en) * 1995-08-31 1997-03-11 Beeta Shokuhin Kk Fruit body tablet of grifola frondosa s. f. gray
JP2007031665A (en) * 2005-07-29 2007-02-08 Yukiguni Maitake Co Ltd Glycoprotein extracted from grifola frondosa
WO2011108681A1 (en) * 2010-03-04 2011-09-09 株式会社雪国まいたけ Anti-allergic agent, and method for producing same
JP2014080373A (en) * 2012-10-12 2014-05-08 Yukiguni Maitake Co Ltd Herpes simplex virus infection preventive/therapeutic agent from grifola frondosa extract

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
THE JOURNAL OF HYOGO WOMEN'S JUNIOR COLLEGE, vol. 25, 1992, pages 1 - 5 *

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