JP6080479B2 - Preventive and therapeutic agent for herpes simplex virus infection derived from maitake extract - Google Patents

Description

  The present invention relates to a preventive and therapeutic effect of a maitake extract on herpes simplex virus infection.

  As for the extract of Maitake, various actions are known by the development efforts of the present applicants. For example, Japanese Patent Application Laid-Open No. 7-69913 (Patent Document 1) for the effect of improving AIDS disease, Japanese Patent Application Laid-Open No. 9-238697 (Patent Document 2) for the anti-tumor action, and Japanese Patent Application Laid-Open No. 2000- No. 119650 (Patent Document 3), NO production inducing action is disclosed in JP-A No. 2001-172194 (Patent Document 4), and substances having anti-influenza virus activity are disclosed in JP-A No. 2005-145934 (Patent Document 5). ) And Japanese Patent Application Laid-Open No. 2008-106018 (Patent Document 6). In addition, Japanese Patent Application No. 2011-180642 (Patent Document 7) reports on a polymer α-glucan relating to prevention and treatment of colds such as influenza.

  There are type 1 and type 2 of herpes simplex viruses. In the case of initial infection, the infected site turns red 4 to 7 days after infection, and many blisters later appear. In herpes simplex virus infection, very small blisters appear in the skin and mucous membranes. Once cured, the virus remains dormant (hidden) in the ganglion where the nerve cells that supply nerve fibers to the site of infection gather, periodically reactivate, begin to proliferate, and It travels through the fibers, returns to the skin, and blisters appear at the same site as the previous infection site. Viruses may be present on the skin and mucous membranes even without apparent blisters. Herpes simplex virus infections include cold sores, sore throat, facial inflammation, genital herpes, corneal herpes, encephalitis, and neonatal herpes. When blisters develop, nearby lymph nodes are swollen and painful, and symptoms such as fever, malaise, and headache appear, and infants may have many stomatitis in the mouth, called herpetic gingiv stomatitis.

  Regardless of the type of virus, the first infection can infect any part of the body, and once infected, it lies in the sensory ganglion for the rest of its life. A red rash and small blisters appear, the blisters rupture and later becomes a scab, which heals in 1-2 weeks. Blisters and red rashes appearing on the face and body are very conspicuous and not cosmetically good.

  In patients with atopic dermatitis, since the protective function of the skin is reduced, herpes simplex virus is easily infected when attached to the skin, and blisters appear over a wide area of the face and body. This is called Kaposi's varicella-like eruption, which can occur in either primary infection or recurrence. In the case of primary infection, there is no immunity to the virus, and it may become severe and die.

  Viruses lurking in sensory ganglia are reactivated by triggers such as fever, overwork, gastrointestinal injury, mental stress, sunlight irradiation, coldness, sexual intercourse, and time difference, causing various inflammations in the skin through the nerves. The role of AIDS virus (HIV) as an infection spreading factor is also drawing attention. Also, in people with impaired immune function, when genital herpes or oral herpes recur, the erosion gradually widens and may take weeks to heal. Infection may progress in the body and spread from the esophagus to the lungs. If you have an ulcer in the esophagus, you will have pain when swallowing food, and if your lungs are infected, you will have pneumonia with cough and shortness of breath. This virus usually infects only the skin and body surface, but it rarely infects internal organs such as the brain (herpes encephalitis). Herpes encephalitis is a fatal disease that begins with confusion, fever, and seizures. Type 1 repeats recurrence mainly in the upper body, type 2 repeats in the lower body, type 2 has a higher recurrence frequency than type 1, and some people recur once or twice a month.

  There are no antiviral drugs that can eradicate herpes simplex virus infection, and oral and genital herpesvirus infections cannot prevent chronic infection of nerves even if treated at the time of initial infection. However, treatment with antiviral drugs can alleviate discomfort at the time of recurrence and shorten the duration of the disease by 1 to 2 days. If pain frequently occurs, it can be sustained by antiviral drugs. Treatment may be able to reduce the number of recurrences. However, there is a problem that antiviral drugs inevitably produce drug resistant viruses when used for a long time. In addition, acyclovir (ACV) has significant side effects such as acute renal failure, liver dysfunction, interstitial pneumonia, pneumonia, thrombocytopenia, delirium (consciousness disorder), seizures, agranulocytosis, respiratory depression, There are toxic epidermal necrosis.

  Herpes simplex virus is highly infectious, and due to contact infection, skin symptoms such as blisters and erosions are accompanied by intense pain, and once infected, the virus lies in the ganglion and repeats symptoms frequently, Symptoms are dramatic when immune function is compromised. Since there is no cure for this virus, search for effective substances for the prevention and treatment of herpes simplex virus infection from available natural products or their extracts without being satisfied with existing prevention and treatment methods. To do so, we have continued to research and search for substances effective in herpes simplex virus infections, which are contained in abundantly grown maitake.

  Prior literature on substances that are effective in preventing and treating herpes simplex virus infections in maitake include non-viral or pathogenic fungi derived from hot water extracts of mycelia of mushrooms. It has been described that the production of secretory IgA antibody is induced by an adjuvant to the activated antigen and exerts immune defense against pathogens such as herpes virus (Patent Document 8). However, the adjuvant described in Patent Document 8 can induce a large amount of secretory IgA antibody production specific to a virus or pathogen by administering it with an inactivated antigen (vaccine) derived from the virus or pathogen. As in the present invention, the substance itself is not effective in preventing or treating herpes simplex virus infection. Furthermore, only the influenza virus has been confirmed to be effective.

  In the prevention and treatment of herpes simplex virus infection, the present inventors have (1) a process of heat-extracting the mycelium or fruiting body of maitake with water, and (2) obtaining the extracted water-soluble fraction. Adding alcohol to a final volume concentration of 20 to 70% and leaving it at a temperature of 1 to 25 ° C. to remove the liquid surface or substances floating in the liquid or adhering to the wall of the container; (3) It was found that the extracted fraction of maitake produced by the process of removing alcohol from the extracted liquid and collecting the dried substance was effective for the prevention and treatment of herpes simplex virus infection, and completed the present invention.

Japanese Patent Laid-Open No. 7-69913 JP-A-9-238697 JP 2000-119650 A JP 2001-172194 A JP 2005-145934 A JP 2008-106018 A Japanese Patent Application No. 2011-180642 JP 2008-273446 A

  The present invention has found that a fraction extracted from maitake is effective for the prevention and treatment of herpes simplex virus infection, developed a method for extracting and purifying the same, searched for an effective action, and further infected with herpes virus. It is an object to be used as an external preparation applied to the skin and mucous membranes.

  The present inventors have (1) a process of thermally extracting the mycelium or fruiting body of maitake with maitake with water, and (2) a final volume concentration of alcohol of 20 to 70% in the extracted water-soluble fraction obtained. The step of removing substances adhering to the liquid surface or in the liquid or adhering to the wall surface of the container, (3) removing alcohol from the obtained extract and drying We found that the extract of maitake produced by the process of collecting the collected substances was effective for the prevention and treatment of herpes simplex virus infection, and solved the problem.

That is, the present invention includes (1) (i) a process of thermally extracting the mycelium or fruiting body of maitake with water, and (ii) a final volume concentration of alcohol of 20 to 70% in the obtained extracted water-soluble fraction. Step of removing substances adhering to the liquid surface or in the liquid or floating on the wall of the container, or (iii) removing alcohol from the resulting extract and drying A preventive / therapeutic agent for herpes simplex virus infection produced by the process of collecting the substance,
(2) An external preparation comprising the preventive / therapeutic agent described in (1) above,
(3) It relates to an external preparation characterized by containing 0.05 to 5% of the preventive / therapeutic agent described in (1) above.

In the present invention, a maitake is a narrowly defined genus of mushrooms ( Grifola frondosa ), white maitake ( Grifola albicans ), choreimaitake ( Grifola umbellatus ), and mushrooms ( Grifola gigantia ). Meaning and any can be used. In addition, fruit bodies and mycelia of these maitake mushrooms can be used. Recently, fruit bodies of maitake ( Grifola frondosa ) and white maitake ( Grifola albicans ) can be artificially cultivated, and it is preferable to use the above two fruit bodies from the viewpoint of securing a stable raw material.

  The equipment used may be appropriately selected in consideration of the performance of the equipment.

  Prior to extraction, removal of components and substances that interfere with extraction can be appropriately performed depending on the condition of the extraction object. For example, for the purpose of degreasing, treatment with ethanol, benzene, petroleum ether, acetone, ether or the like can be performed, but it is not always essential.

  As a method of heat extraction treatment, it is carried out at 50 to 135 ° C. for 15 minutes to 3 hours. In order to perform in a short time, extraction is performed at 100 ° C. or higher under pressure, for example, at about 120 ° C. under 1 to 2 atm using a pressure cooker for about 30 minutes to 1 hour. “Water” means tap water, deionized water (ion exchange water), distilled water, reverse osmosis water, and the like.

  As the extraction solution, methods using an organic solvent such as alcohol and acetone are known, and these methods are possible. In particular, alcohol is preferably used, and the alcohol is a lower alcohol such as methanol, ethanol, propanol, or butanol. Can be used. Add to the extract to a final volume concentration of 20-70%. At this time, an alcohol having a water content of 0 to 50% can be used. After the addition, if left at a temperature of 1 to 25 ° C. for 1 to 20 hours, a substance floating or adhering to the wall of the container appears in the liquid surface or the liquid. Since the effect of preventing and treating herpes simplex virus infection is enhanced by removing floating substances or adhering substances, the process of removing the substance is an extremely important process. For this purpose, it is essential that the final volume concentration of the alcohol is 20 to 70%, preferably 30 to 60%. Moreover, the alcohol is removed from the extract obtained at the end to complete the process by obtaining a dried substance.

  The properties of the object of the present invention obtained above will be described.

Appearance: Hygroscopic powder in brown color Solubility: Dissolved in water, alkaline solution and dimethylsulfoxide Color reaction: Anthrone reaction and ninhydrin reaction are positive aqueous solutions: Neutral to weakly acidic molecular weight: Several million Distributed around tens of millions

As a result of the analysis of the substance obtained in the present invention, it consists mainly of carbohydrates and proteins . The main component of the substance having the effect of preventing and treating herpes simplex virus infection obtained by the present invention is recognized to be a sugar-protein complex, and the ratio of sugar to protein is mainly in the range of 50:50 to 70:30. It is.

  The target substance of the present invention is a maitake-derived substance, a maitake-derived substance having a different production method, and acyclovir and mechabufucoidan. BALB / c mice, rabbits, herpes simplex virus type 2 (HSV-2) The target substance of the present invention was found to be effective for the prevention and treatment of herpes simplex virus infection.

  From the above results, the target substance of the present invention is effective for the prevention and treatment of herpes simplex virus infection, and can be used as an external preparation for preventing infection of viruses and the like or for reducing symptoms after illness.

  The topical preparation in the present invention is for herpes simplex virus infection in the skin or mucous membrane for prevention of infection or alleviation of symptoms after illness, and herpes virus infection or possibly There is no particular limitation as long as it can be applied to skin and mucous membranes. The position of the skin and mucous membrane is, for example, the opening or trauma of the human body such as the oral cavity, throat, nasal cavity, eyelids, anus, rectum, urethra, vagina and the like, as well as the periphery thereof. Accordingly, the dosage form includes all coating agents (creams, ointments, liquids, gels, etc.), patches (patches, plasters, tapes, patches, etc.), aerosols, etc. Also included are dosage forms that can be used as suppositories.

  The target substance of the present invention is an extract of maitake with rich food experience. In addition, as described above, it has been clarified by the inventors' research efforts that it has an effect such as an antitumor action, and is a safe substance without toxicity that has been sold as a health food. Based on these points, it can be applied as an external preparation for preventing herpes simplex virus infection or reducing symptoms after illness. In addition, the irritation of the target substance to the skin and mucous membranes was investigated, but its safety was confirmed.

The figure which shows the prevention and treatment effect of the herpes simplex virus infection by local administration of each extract prepared in Example 1, and acyclovir and mechabufucoidan using a BALB / c mouse. The figure which shows the viral load in the local 3 days after the herpes simplex virus infection by local administration of each extract prepared in Example 1, and acyclovir and mechabufucoidan using the BALB / c mouse. *: T-test (* p <0.05, ** p <0.01, *** p <0.001)

  EXAMPLES Examples will be shown below for specifically explaining the present invention, but the present invention is not limited to these examples.

[Example 1] Production of substances having preventive and therapeutic effects on herpes simplex virus infection (Test example)
3 kg of dried maitake powder is treated with 30 L of ion-exchanged water for 30 minutes at 121 ° C. under a pressure of 2 atm, and then filtered with a filter press to obtain 17 L of a black-brown liquid as a water-soluble fraction. The solution was concentrated to about 4.5 L under reduced pressure, 5 L of ethanol was added at room temperature, and the mixture was allowed to stand for about 16 hours. As a result, a brown substance that floated or adhered to the wall surface in the liquid was generated. The liquid surface, substances floating in the liquid or adhering to the wall surface are removed by a wire mesh to obtain a brown solution. The solution was freed from alcohol under reduced pressure and spray dried by spray drying to obtain 500 g of a brown powder. The resulting material was designated MD Fr.

(Comparative Example 1)
10 kg of freeze-dried 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. The extract was filtered using a filter press, the obtained extract solution was concentrated, and a dry powder was obtained by spray drying. The obtained substance was designated as YM-6A.

(Comparative Example 2)
13.5 kg of hot-air dried maitake powder was added to 180 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. The extract was filtered using a filter press, the obtained extract solution was concentrated, and a dry powder was obtained by spray drying. The obtained substance was designated as YM-18A.

[Example 2] Herpes simplex virus growth inhibitory effect: in vitro test In each extract prepared in Example 1, 50% cell growth inhibitory concentration (Cytotoxicity; CC ₅₀ ), 50% herpes simplex virus growth inhibitory concentration (Antiviral activity; IC ₅₀ ) was determined. Each extract was added at the same time as virus infection (A section) or immediately after virus infection (B group), and the selectivity index (CC ₅₀ / IC ₅₀ ) was calculated to determine antiviral activity.

<Result>
YM-6A and YM-18A showed a high herpes simplex virus growth inhibitory effect in in vitro tests, and MD Fr had a weak growth inhibitory effect (Table 1).

[Example 3] Preventive and therapeutic effects on herpes simplex virus infection: Infection test to mice
Using BALB / c mice, the effects of each extract prepared in Example 1, and acyclovir (ACV) and mechabufucoidan (mechabu FU) were verified.

<Method>
(1) BALB / c mice (♀, 6 weeks old) were subcutaneously injected with medroxyprogesterone (3 mg / 0.1 ml / mouse) for the purpose of increasing the susceptibility to virus 6 days and 1 day before infection.
(2) A virus (herpes simplex virus type 2: HSV-2, UW strain, 1 × 10 ⁴ PFU / 20 μl / mouse) was inoculated locally.
(3) Administration of each substance (local administration)
One hour before the infection, the sample was administered for the first time, and thereafter twice a day (am 9, pm 6) until 7 days after the infection.
(4) Three days later, the local area was washed with PBS, and the amount of virus therein was measured.
(5) The degree of onset (score) and death cases were recorded over 2 weeks (no deaths after 2 weeks). The degree of onset is represented by the score in Table 2, and the dosage and administration method are shown in Table 3.

<Result>
-Progression and mortality The average score is shown in FIG.
In the local administration group, YM-6A and MD Fr showed an onset-inhibitory effect, and MD Fr in particular showed a strong onset-inhibitory effect (FIG. 1). Table 4 shows the mortality rate of the mice. Three animals out of five survived the local administration of MD Fr.

-Virus production amount Local virus amount was measured 3 days after infection. MD Fr showed a virus growth inhibitory effect (FIG. 2).

3. Conclusion In the mouse infection test (Example 3), ACV showed a high onset prevention effect and a virus growth suppression effect. However, ACV, which is an antiviral agent, inevitably produces drug-resistant virus when used as described above, and ACV-resistant virus is actually a clinical problem. In addition, significant side effects of ACV include acute renal failure, liver dysfunction, interstitial pneumonia, pneumonia, thrombocytopenia, delirium (impaired consciousness), seizures, agranulocytosis, respiratory depression, toxic epidermis There is necrosis. MD Fr, which is the target substance of the present invention, showed a higher onset inhibitory effect and viral growth inhibitory effect than YM-6A or YM-18A in mouse infection tests. On the other hand, in the in vitro test (Example 2), MD Fr had a weaker virus growth inhibitory effect than YM-6A or YM-18A. From this, it is considered that MD Fr exhibits a higher onset-preventing effect and viral growth-inhibiting effect due to its action on the living body rather than a direct action on the virus, and no drug-resistant virus is produced. Furthermore, as described above, MD Fr is a safe substance that is not toxic and has a long history of being eaten as a food, and has been sold as a health food.

  The target substance of the present invention is an extract of maitake with a rich dietary experience. In addition, as described above, it has been clarified by the inventors' research efforts that it has an effect such as an antitumor action, and is a safe substance without toxicity that has been sold as a health food. Moreover, there is no irritation | stimulation with respect to skin and a mucous membrane, and the application as an external preparation for prevention of a herpes simplex virus infection, recurrence, or the symptom reduction after an illness is possible.

Claims (3)

An extract derived from maitake is an active ingredient, and the active ingredient is (1) a hygroscopic powder having a brown color tone,
(2) Dissolved in water, aqueous alkali solution and dimethyl sulfoxide,
(3) A prophylactic / therapeutic agent for herpes simplex virus infection, wherein the anthrone reaction and the ninhydrin reaction are positive, and (4) the aqueous solution is neutral to weakly acidic.   The herpes simplex virus infection according to claim 1, wherein the main component of the active ingredient is a sugar-protein complex, and the ratio of sugar to protein is mainly in the range of 50:50 to 70:30. Prophylactic / therapeutic agent.   An external preparation comprising the preventive / therapeutic agent according to claim 1 or 2.

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