WO2020026953A1 - Anti-herpes virus agent - Google Patents

Abstract

The present invention addresses the problem of providing a novel means that is effective in the treatment or the prevention of herpes virus infection. Provided is an anti-herpes virus agent containing a unicellular algae-derived substance as an active ingredient.

Description

Anti-herpes virus agent

(4) The present invention relates to an anti-herpes virus agent. More specifically, the present invention relates to an anti-herpes virus agent using a substance derived from microalgae, its use, and the like. This application claims the priority based on Japanese Patent Application No. 2018-143051 filed on Jul. 31, 2018, the entire content of which is incorporated by reference.

Herpes virus is a DNA virus having double-stranded DNA as its genome. Herpesviruses are classified into α-herpesvirinae, β-herpesvirinae and γ-herpesvirinae. Herpes simplex virus (HSV), which belongs to the alpha-herpesvirinae subfamily, causes various diseases in humans. Representative examples of HSV infection include herpes simplex virus type 1 (HSV-1), a pathogen herpes labialis, and herpes simplex virus type 2 (HSV-2), a genital herpes pathogen. Herpes virus is characterized by latent infection (persistent infection in the body) after primary infection, reactivated by various causes (ultraviolet rays, fever, various stresses, menstruation, immunosuppression, etc.) Causes (regression). There is no fundamental cure for herpes virus infection, and individuals infected with herpes virus will experience repeated local pathology / symptoms.

Nucleic acid analogs and DNA synthesis inhibitors (eg, acyclovir, valacyclovir) are used for the treatment of herpes virus infection (for example, see Non-Patent Documents 1 and 2), but there are many cases where a sufficient therapeutic effect cannot be obtained. The establishment of new treatment strategies is eagerly needed.

で Under the above background, an object of the present invention is to provide a new means effective for treating or preventing herpes virus infection.

鋭 As a result of intensive studies to solve the above-mentioned problems, an extract (monogalactosyldiacylglycerol-containing extract) of Coccomyxa microalgae was found to have an effect of suppressing the onset of herpes virus infection and an effect of suppressing virus growth. It was also found that the effect was dramatically enhanced by the combined use of thymidine (a synergistic effect was exhibited). The same effect was observed not only in the extract but also in the dry powder (algae) of the microalgae of the genus Komikusa. Similar to the extract, the effect was significantly enhanced by the combined use of thymidine. On the other hand, further studies revealed the structure of monogalactosyldiacylglycerol, the active ingredient in the extract. Further, further findings have been obtained which support that the utility value of the extract is extremely high.

On the other hand, monogalactosyldiacylglycerol-containing extracts derived from Chlorella vulgaris, Nannochloropsis occultata, Arthrospira platensis (spirulina) and Euglena gracilis, as well as monogalactosyldiacylglycerol-containing extracts derived from Coccomica microalgae It became clear that the effect of can be expected.
The following inventions are provided mainly based on the above results and considerations.
[1] An anti-herpesvirus agent comprising a substance derived from a single-cell algae as an active ingredient.
[2] The anti-herpesvirus agent according to [1], wherein the unicellular algae is a microalga belonging to the genus Kokkomikusa, Chlorella, Nannochloropsis, Arthrospira or Euglena.
[3] The anti-herpes virus agent according to [1], wherein the single-cell algae is a microalga belonging to the genus Kokkomikusa.
[4] The anti-herpes virus agent according to [3], wherein the microalgae is a strain of Kokko-mixa sp. KJ or a mutant thereof, or a strain of Kokko-mixa sp. MBIC11204 or a mutant thereof.
[5] The anti-herpes virus agent according to any one of [1] to [4], wherein the active ingredient is a monogalactosyldiacylglycerol-containing extract of the single-cell algae.
[6] The anti-herpes virus agent according to [5], wherein the main component is monogalactosyldiacylglycerol.
[7] The anti-herpes virus agent according to [6], wherein the monogalactosyldiacylglycerol is one or more monogalactosyldiacylglycerols selected from the group consisting of the following (1) to (6):
(1) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 3 and C18: 3;
(2) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 3;
(3) monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 3;
(4) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 2;
(5) Monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 2; and (6) Monogalactosyldiacylglycerol whose constituent fatty acids are C16: 1 and C18: 2.
[8] The method according to any one of [5] to [7], wherein the active ingredient is obtained by purifying an ethanol extract of the single-cell algae by chromatography to increase the content of monogalactosyldiacylglycerol. Anti-herpes virus agent.
[9] The anti-herpes according to any one of [5] to [8], wherein the monogalactosyldiacylglycerol content in the active ingredient is 70% (w / v) to 99% (w / v). Viral agent.
[10] The anti-herpes virus agent according to any one of [1] to [4], wherein the active ingredient is an algal body.
[11] The anti-herpes virus agent according to any one of [1] to [4], wherein the active ingredient is a dry powder of algal cells.
[12] An anti-herpes virus agent containing one or more monogalactosyldiacylglycerols as an active ingredient selected from the group consisting of the following (1) to (6):
(1) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 3 and C18: 3;
(2) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 3;
(3) monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 3;
(4) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 2;
(5) Monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 2; and (6) Monogalactosyldiacylglycerol whose constituent fatty acids are C16: 1 and C18: 2.
[13] The anti-herpes virus agent according to any one of [1] to [12], which is used in combination with thymidine.
[14] The anti-herpes virus agent according to [13], which is a combination drug containing the active ingredient and thymidine.
[15] The anti-herpes virus agent according to any one of [1] to [14], wherein the target herpes virus is herpes simplex virus type 1 or herpes simplex virus type 2.
[16] A composition comprising the anti-herpesvirus agent according to any one of [1] to [15].
[17] The composition according to [16], which is a medicament for herpes virus infection.
[18] The composition according to [16], which is a food or a feed.

Results of HSV-2 infection (genital herpes) experiment. The onset courses in the case of single administration were compared. Test plot # 1: control (distilled water), test plot # 2: ACV (acyclovir) (1 mg / day), test plot # 3: MGDG preparation (0.1 mg / day), test plot # 4: MGDG preparation (1 mg / day), test plot # 5: thymidine (1 mg / day), test plot # 6: thymidine (5 mg / day), test plot # 7: thymidine (20 mg / day). Results of HSV-2 infection (genital herpes) experiment. The onset course when MGDG preparation (0.1 mg administration) and thymidine were used together was compared. Test plot # 1: control (distilled water), test plot # 2: ACV (acyclovir) (1 mg / day), test plot # 3: MGDG preparation (0.1 mg / day), test plot # 8: MGDG preparation (0.1 mg / day) + thymidine (1 mg / day), test group # 9: MGDG preparation (0.1 mg / day) + thymidine (5 mg / day), test group # 10: MGDG preparation (0.1 mg / day) day) + thymidine (20 mg / day). Results of HSV-2 infection (genital herpes) experiment. The onset course when MGDG preparation (1 mg administration) and thymidine were used together was compared. Test plot # 1: control (distilled water), test plot # 2: ACV (acyclovir) (1 mg / day), test plot # 4: MGDG preparation (1 mg / day), test plot # 11: MGDG preparation (1 mg / day) + thymidine (1 mg / day), test plot # 12: MGDG preparation (1 mg / day) + thymidine (5 mg / day), test plot # 13: MGDG preparation (1 mg / day) day) + thymidine (20 mg / day). Results of HSV-2 infection (genital herpes) experiment. The onset course was compared when the alga body (dry powder) was administered alone or when the alga body and thymidine were used in combination. Test plot # 1: control (distilled water), test plot # 2: ACV (acyclovir) (1 mg / day), test plot # 14: algal (20 mg / day), test plot # 15: algal (20 mg / day) + thymidine (1 mg / day), test plot # 16: algal cells (20 mg / day) + thymidine (5 mg / day), test plot # 17: algal cells (20 mg / day) + thymidine (20 mg / day). Results of HSV-2 infection (genital herpes) experiment. The survival rates and cases of onset were compared. Results of HSV-2 infection (genital herpes) experiment. Three days after infection, the amount of virus was compared. * p <0.05 vs. control Results of HSV-1 infection (skin herpes) experiment. The onset course in the case of local administration (application) was compared. Test plot # 19: control (PBS) (applied), test plot # 20: 5% ACV (applied), test plot # 21: 5% MGDG preparation (applied), test plot # 22: 5% thymidine (applied) Test plot # 23: 5% {MGDG preparation + 5%} thymidine (applied). Results of HSV-1 infection (skin herpes) experiment. The course of onset in the case of oral administration was compared. Test plot # 24: control (distilled water), test plot # 25: ACV (1 mg / day), test plot # 26: MGDG preparation (1 mg / day), test plot # 27: thymidine (20 mg / day) ), Test plot # 28: ACV (1 mg / day) + MGDG preparation (1 mg / day). Results of HSV-1 infection (skin herpes) experiment. The survival rates and cases of onset were compared. Examination of the interaction between MGDG preparation and thymidine. Isobologram showing that the thymidine HSV-2 growth inhibitory effect is enhanced by MGDG preparation. Examination of the interaction between MGDG preparation and thymidine. Isobologram showing that the anti-HSV-2 action of MGDG preparation is enhanced by thymidine. Results of HSV-2 infection (genital herpes) experiment using algal cells (dry powder) of Kokkomixa sp. The onset course was compared when the alga body (dry powder) was administered alone or when the alga body and thymidine were used in combination. Test plot # 1: Control (distilled water), Test plot # 2: ACV (1 mg / day), Test plot # 3: Algae (dry powder) (5 mg / day), Test plot # 4: Algae ( Dry powder) (5 mg / day) + thymidine (0.5 mg / day), test group # 5: Algae (dry powder) (10 mg / day). Results of HSV-2 infection (genital herpes) experiment using algal cells (dry powder) of Kokkomixa sp. Three days after infection, the amount of virus was compared. * p <0.05, ** p <0.01 vs. control An example of a medium used for culturing Chlorella microalgae (C medium) and an example of a medium used for culturing Nannochloropsis microalgae (ESM medium). An example of a medium used for cultivation of microspheres of the genus Arthrospira (MA medium) and an example of a medium used for culture of algae of the genus Euglena (HUT medium). Effect on cold sores (patient 1: 45-year-old female). The cream containing the MGDG preparation was applied to the affected area three times daily, and the progress was observed. Effect on cold sores (patient 2: 25 year old male). The cream containing the MGDG preparation was applied to the affected area daily, and the progress was observed. Analysis result (GC / FID chromatogram) of the content component (MGDG1) of the MGDG preparation. Analysis result (GC / FID chromatogram) of the content component (MGDG2) of the MGDG preparation. Analysis result (GC / FID chromatogram) of the component (MGDG3) of the MGDG preparation. Analysis result (GC / FID chromatogram) of the content component (MGDG4) of the MGDG preparation. Analysis result (GC / FID chromatogram) of the content component (MGDG5) of the MGDG preparation. Virucidal activity of each MGDG fraction (MGDG1 to MGDG5). The virucidal activity of MGDG1 to MGDG5 and MGDG preparations (Mix product) against influenza virus, herpes simplex virus type 2, feline calicivirus and poliovirus was compared. HPLC chromatograms of MGDG preparations of various organisms (Cokkomixa @sp. @KJ strain, Chlorella vulgaris, Nannochloropsis oculata, Arsulospira platensis (spirulina)). MGDG-1 to MGDG-5 are peaks derived from the KDG strain MGDG preparation, and peaks presumed to have the same structure as these are indicated by *. HPLC chromatograms of MGDG preparations of various organisms (Comixa sp. KJ strain, Euglena gracilis (Euglena of life, Biozyme), spinach). MGDG-1 to MGDG-5 are peaks derived from the KDG strain MGDG preparation, and peaks presumed to have the same structure as these are indicated by *.

1. Terms, actions An anti-herpesvirus agent is an antiviral agent that targets a herpes virus. As shown in the Examples below, the anti-herpes virus agent of the present invention can be expected to have a therapeutic or preventive effect on herpes virus infection. Although not wishing to be bound by theory, it can be said that the anti-herpes virus agent of the present invention exerts its effects through suppression of herpes virus growth in view of the results of the experiments described in the examples below. As described above, herpesviruses are classified into three types of herpesvirinae (α-herpesvirinae, β-herpesvirinae, and γ-herpesvirinae). Alpha herpesvirus subfamily includes herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (HHV-3). Megalovirus (HHV-5), human herpesvirus 6 (HHV-6) and human herpesvirus 7 (HHV-7) belong to the subfamily γ-herpesvirinae, Epstein-Barr virus (HHV-4), human herpes Virus 8 (HHV-8, also known as Kaposi's sarcoma-associated herpesvirus (KSHV)) belongs. A particularly preferred target of the anti-herpes virus agent of the present invention is herpes simplex virus type 1 (HSV-1) or herpes simplex virus type 2 (HSV-2).

2. Active ingredient of anti-herpes virus agent The anti-herpes virus agent of the present invention contains a substance derived from a single-cell algae as an active ingredient. As long as the anti-herpes virus activity is observed, the unicellular algae (Trebouxia algae, Ephemeroptera, Cyanobacteria, Euglena algae, etc.) are not particularly limited. As preferred unicellular algae, Coccomyxa microalgae (specific examples are Coccomixa sp. KJ strain), Chlorella microalgae (specific examples are Chlorella vulgaris), Nannochloropsis microalgae ( Specific examples include Nannochloropsis oculata), Arthrospira microalgae (a specific example is Arthrospira platensis (spirulina)) and Euglena microalgae (a specific example is Euglena gracilis). Among these, microalgae of the genus Kokkomikusa are particularly preferred. The microalgae of the genus Kokkomikusa are not particularly limited, but preferred examples include Kokkomikusa sp. Strain KJ or a mutant thereof, or Kokkomikusa sp. MBIC11204 strain or a mutant thereof. Co., Ltd. sp. KJ shares (KJ Denso) was established on June 4, 2013 by the National Institute of Technology and Evaluation, National Institute of Technology and Evaluation Biotechnology Center Patent Organism Depositary Center (NITE-IPOD) (2-5 Kazusa Kamasa, Kisarazu-shi, Chiba) No. 8120) and deposited under accession number FERM BP-22254 on 2 June 2015 under the provisions of the Putavest Treaty on June 2, 2015. On the other hand, Kokkomixa sp. MBIC11204 strain (N1) was acquired by the Patent Organism Depositary Center (IPOD) of the National Institute of Advanced Industrial Science and Technology (IPOD) on January 18, 2006. Deposit center (NITE-IPOD) has been deposited internationally under the accession number FERM BP-10485 at Room 2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba. Strain MBIC11204 (N1) has been deposited with the National Institute of Technology and Evaluation Biotechnology Center (NBRC) NBRC Culture Collection on June 14, 2016 as NBRC No. NBRC 112354.

Mutants of the strain K. sp. KJ and the strains of strain S. cocoa M. sp. MBIC11204 can be irradiated with ultraviolet rays, X-rays, γ-rays, etc. Editing, etc.). As long as a mutant producing monogalactosyldiacylglycerol exhibiting anti-herpesvirus activity can be obtained, the method for obtaining the mutant, the characteristics, and the like are not particularly limited.

The method for cultivating Microalgae of the genus Kokkomushi is not particularly limited. The medium for cultivating the microalgae of the genus Kokkomikusa may be any of those commonly used for culturing microalgae.For example, various nutrients, trace metal salts, known media for freshwater microalgae containing vitamins, etc. Any medium for marine microalgae can be used. Examples of the medium include AF6 medium. The composition of AF6 medium (per 100 ml) is as follows.
NaNO ₃ 14mg
NH ₄ NO ₃ 2.2mg
MgSO ₄ · 7H ₂ O 3mg
KH ₂ PO ₄ 1mg
K ₂ HPO ₄ 0.5mg
CaC _l2 · 2H ₂ O 1mg
CaCO ₃ 1mg
Fe-citrate 0.2mg
Citric acid 0.2mg
Biotin 0.2μg
Thiamine HCl 1μg
Vitamin B ₆ 0.1μg
Vitamin B ₁₂ 0.1μg
Trace metals 0.5mL
Distilled water 99.5mL

Examples of the nutrients include nitrogen sources such as NaNO ₃ , KNO ₃ , NH ₄ Cl and urea, and phosphorus sources such as K ₂ HPO ₄ , KH ₂ PO ₄ and sodium glycerophosphate. The trace metals include iron, magnesium, manganese, calcium, zinc and the like, and the vitamins include vitamin B1, vitamin B12 and the like.

As for the culture method, stirring may be performed while supplying carbon dioxide under aeration conditions. At this time, the cells are cultured by irradiating with a fluorescent lamp for 12 hours, irradiating with light and dark cycles such as 12 hours of dark conditions, or irradiating with continuous light. The culture condition is not particularly limited as long as it does not adversely affect the growth of the microalgae of the genus Kompani. For example, the pH of the culture solution is 3 to 9, and the culture temperature is 10 to 35 ° C.

As for the method of cultivating the strain Kkkomixa sp. KJ, JP-A-2015-15918, WO 2015/190116 A1, Satoh, AA et al. . Jpn. Inst. Energy (2010) 89: 909-913. Similarly, WO 2006/109588 and the like can be referred to for the method of culturing Kokkomixa sp. MBIC11204 strain.

As the active ingredient, a monogalactosyldiacylglycerol-containing extract of a single-cell algae (particularly preferably a microalga belonging to the genus Kokkomikusa) (first embodiment), or an alga body or a dry powder thereof (second embodiment) is used. Hereinafter, each embodiment will be described. Usually, either the monogalactosyl diacylglycerol-containing extract, the alga body or its dry powder is the active ingredient, but this does not preclude the use of both as the active ingredient.

(1) Monogalactosyldiacylglycerol-containing extract In one embodiment of the present invention, a monogalactosyldiacylglycerol-containing extract of a single-cell algae (particularly preferably, a microalga belonging to the genus Kokkomikusa) is used as an active ingredient. The extraction method is not particularly limited as long as an extract containing monogalactosyldiacylglycerol can be obtained. For example, ethanol extraction can be adopted as the extraction method. Preferably, a product obtained by purifying after ethanol extraction and increasing the content of monogalactosyldiacylglycerol is used as "monogalactosyldiacylglycerol-containing extract of unicellular algae (particularly preferably microalgae belonging to the genus Kokkomikusa)". Examples of the purification method include column chromatography using a filler such as silica gel and alumina, gel filtration chromatography, and concentration.

先 Prior to extraction, the collected algal cells may be subjected to a drying treatment and / or a crushing treatment. In other words, a dried alga body, a dried and crushed alga body (typically, a dry powder), or a crushed alga body may be prepared, and an extraction operation may be performed using the prepared alga body. A single-cell algae that has been processed in advance (for example, a dried alga body, its powder / powder, or a tablet (which may contain an excipient, etc.)) is obtained and subjected to an extraction operation. You may. For example, processed products of Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (spirulina), Euglena gracilis and the like are commercially available.

The content of monogalactosyldiacylglycerol in the active ingredient is not particularly limited as long as the extract shows anti-herpesvirus activity, and is, for example, 70% (w / v) to 99% (w / v), preferably 80% (w / v). w / v) to 99% (w / v), more preferably 90% (w / v) to 99% (w / v), even more preferably 95% (w / v) to 99% (w / v). (Specific examples are 96% (w / v), 97% (w / v), and 98% (w / v)). In principle, the higher the content of monogalactosyldiacylglycerol, the higher the anti-herpesvirus activity can be expected. “Monogalactosyldiacylglycerol” is one of glyceroglycolipids and is known as a component of the chloroplast thylakoid membrane of plants. Monogalactosyldiacylglycerol has a skeleton in which galactose is β-linked to glycerol.

Preferably, the anti-herpesvirus agent of the present invention contains monogalactosyldiacylglycerol as a main component. Examples of the monogalactosyldiacylglycerol that the anti-herpesvirus agent of the present invention can contain include (1) constituent fatty acids C16: 3 and C18: 3 monogalactosyldiacylglycerol, and (2) constituent fatty acids C16: 2. C18: 3 monogalactosyldiacylglycerol, (3) constituent fatty acids are C18: 3 and C18: 3 monogalactosyldiacylglycerol, (4) constituent fatty acids are C16: 2 and C18: 2 monogalactosyldiacylglycerol, (5) The constituent fatty acids are C18: 3 and C18: 2 monogalactosyldiacylglycerol, and (6) the constituent fatty acids are C16: 1 and C18: 2 monogalactosyldiacylglycerol. These monogalactosyldiacylglycerols were identified as containing an extract of the genus Kokkomikusa, which is an anti-herpesvirus agent (for details, see “8. Identification 2 "). In consideration of the analysis results of “7. Identification of components in MGDG preparation 1” shown in the Examples section below, the constituent fatty acids C18: 3 of (1) above are preferably C18: 3 (n- 3), at least one of the constituent fatty acids C18: 3 of the above (3) is preferably C18: 3 (n-3), and the constituent fatty acids C18: 2 of the above (4) are preferably C18: 2 (n -6), and the constituent fatty acid C18: 3 of the above (5) is preferably C18: 3 (n-3), and the C18: 2 is preferably C18: 2 (n-6). Although it does not preclude the inclusion of a specific monogalactosyldiacylglycerol alone, usually, the anti-herpesvirus agent of the present invention contains two or more monogalactosyldiacylglycerols having different structures. The combination, content ratio, and the like are not particularly limited. The anti-herpes virus agent of the present invention is preferably two or more of the above (1) to (6), more preferably three or more of the above (1) to (6), still more preferably the above ( It contains four or more of the above (1) to (6), more preferably five or more of the above (1) to (6), and still more preferably all of the above (1) to (6). As shown in Examples below, monogalactosyldiacylglycerol (MGDG5) of (5) showed particularly high activity. Therefore, the anti-herpesvirus agent of a particularly preferred embodiment contains the monogalactosyldiacylglycerol (5) alone or in combination with one or more of (1), (2) to (4) and (6). Will be done.

(4) Monogalactosyldiacylglycerol, which has been identified as a major component of the extract of Microalgae belonging to the genus Aspergillus, exhibiting anti-herpesvirus activity, can be expected to have anti-herpesvirus activity itself. Thus, according to one aspect of the present invention, there is provided an anti-herpesvirus agent comprising one or more monogalactosyldiacylglycerols as an active ingredient selected from the group consisting of the above (1) to (6). When two or more types of monogalactosyldiacylglycerols are used as the active ingredient, the combination, content ratio, and the like are not particularly limited. Also in this embodiment, preferably two or more of the above (1) to (6), more preferably three or more of the above (1) to (6), further more preferably the above (1) to (6) It contains four or more of the above (6), more preferably five or more of the above (1) to (6), and still more preferably all of the above (1) to (6). As described above, monogalactosyldiacylglycerol (MGDG5) of (5) showed particularly high activity. Therefore, in a particularly preferred embodiment, the monogalactosyldiacylglycerol (5) is at least one of the active ingredients.

Here, there is monogalactosyldiacylglycerol containing various fatty acids (fatty acids are shown below) as constituent fatty acids. In view of the teachings of the present invention, it can be reasonably expected that monogalactosyldiacylglycerols, including known monogalactosyldiacylglycerols, will generally be able to exert anti-herpesvirus activity.
<Examples of fatty acids>
C12: 0, C13: 0, C14: 0, C14: 1, C14: 2, C15: 0, C15: 1, C16: 0, C16: 1, C16: 4, C17: 0, C17: 1, C18: 0, C18: 1, C18: 4, C19: 0, C19: 1, C20: 0, C20: 1, C20: 2, C20: 3, C20: 4, C20: 5, C22: 0, C22: 5, C24: 0

(2) Algae or its dry powder In this embodiment, an alga or a dry powder thereof is used as an active ingredient instead of an extract from the alga. The dry powder can be prepared by subjecting the collected algal cells to a drying treatment and a crushing (crushing) treatment. As the drying treatment, for example, drum drying, spray drying, freeze drying and the like can be employed. For the crushing treatment, a bead crusher, a homogenizer, a French press, a mixer / blender, a fine crusher, or the like can be used. The order of the drying treatment and the crushing treatment does not matter. Alternatively, a drying and crushing process may be performed simultaneously using an apparatus having drying and crushing functions.

粒子 The particle size of the dry powder is not particularly limited. For example, a dry powder having an average particle size of 0.2 μm to 2 mm, preferably 0.4 μm to 400 μm.

3. Combination of Thymidine In one embodiment of the present invention, thymidine is used in combination with the above-mentioned active ingredient (monogalactosyldiacylglycerol-containing extract of microalga belonging to the genus Kokkomikusa, alga body, and dry powder of alga body). In other words, the anti-herpesvirus agent of this embodiment contains, as active ingredients, a monogalactosyldiacylglycerol-containing extract of a microalga belonging to the genus Kokkomixa, an alga body or a dry powder of an alga body, and thymidine. By using thymidine in combination, a synergistic effect is exerted, and an anti-herpes virus agent having high anti-herpes virus activity (ie, enhanced therapeutic / prophylactic effect against herpes virus infection) is obtained. Thymidine (CAS No. 50-89-5) is one of DNA nucleosides and has a structure in which deoxyribose is connected to a pyrimidine base, thymine.

The amount of thymidine used in combination is not particularly limited.However, when using a monogalactosyldiacylglycerol-containing extract of microalgae belonging to the genus Kokkomikusa as the active ingredient, the amount of the active ingredient and thymidine is in a weight ratio, for example, 1 : 1 to 1: 500, preferably 1:10 to 1: 200. On the other hand, when an alga body of a microalga belonging to the genus Kokkomikusa or a dry powder thereof is used as the active ingredient, the amount of the active ingredient and thymidine is in a weight ratio (however, in the case of an alga body, the dry weight is used). For example, 100: 1 to 1:10, preferably 20: 1 to 1: 1.

Typically, the anti-herpes virus agent of this embodiment will be provided as a combination preparation obtained by mixing the above-mentioned active ingredient and thymidine. However, the anti-herpes virus agent of this embodiment can be provided in the form of a kit comprising the first component containing the active ingredient and the second component containing thymidine. In this case, the first component and the second component are used simultaneously. Simultaneous here does not require strict synchronism, and the subject of application is the active ingredient of the first component (monogalactosyldiacylglycerol-containing extract of microalga belonging to the genus Kokkomikusa, alga, drying of alga) The “simultaneous” condition is satisfied as long as a state in which the powder) and the active ingredient (thymidine) of the second component coexist is formed, and the synergistic effect of the combined use of thymidine is exhibited. For example, when a medicament is constituted by using the anti-herpesvirus agent of the present invention, for example, it is administered to a subject to be treated or prevented (typically a human) after mixing both components, After administration, the other may be administered immediately. In addition, as long as the effect of the combined use of thymidine can be obtained, the administration of one may be followed by administration of the other with a predetermined time difference. In this case, it is preferable to set the time difference as short as possible. For example, the other is administered within one hour (preferably within 30 minutes) after administration of one.

4. Use and usage of anti-herpes virus agent The anti-herpes virus agent of the present invention can be typically used as a composition containing the same for anti-herpes virus infection control. Examples of compositions here are pharmaceuticals, foods, feeds.

As evidenced by the examples described below, preferably, infection by HSV-1 (herpes labialis, herpes stomatitis, corneal herpes, herpes simplex encephalitis, etc.) or infection by HSV-2 (genital (genital) herpes, neonatal herpes) , Myelitis, etc.), the anti-herpesvirus agent of the present invention is used.

医 薬 The medicament of the present invention can exert a therapeutic effect or a preventive effect on herpes virus infection (these two effects are collectively referred to as “pharmaceutical effect”). Here, the medicinal effects include (1) prevention, suppression or delay of the onset of herpes virus infection, (2) alleviation of symptoms or accompanying symptoms characteristic of herpes virus infection (mildness), (3) It includes prevention, suppression or delay of deterioration of symptoms or accompanying symptoms characteristic of herpervirus infection, and the like. It should be noted that since the therapeutic effect and the preventive effect are partially overlapping concepts, it may be difficult to distinguish them clearly and the benefits of doing so are small.

Pharmaceutical formulation can be carried out according to a conventional method. When formulated, other pharmaceutically acceptable components (eg, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological Saline, etc.). As an excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As a disintegrant, starch, carboxymethyl cellulose, calcium carbonate and the like can be used. Phosphates, citrates, acetates and the like can be used as buffers. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As a suspending agent, glycerin monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate, and the like can be used. Benzyl alcohol, chlorobutanol, sorbitol and the like can be used as a soothing agent. As a stabilizer, propylene glycol, ascorbic acid, or the like can be used. As a preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methyl paraben and the like can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

剤 The dosage form in the case of formulating is not particularly limited. Examples of dosage forms include tablets, powders, fine granules, granules, capsules, syrups, injections, and external preparations (ointments, creams, lotions, solutions, gels, cataplasms, plasters, tapes) , Aerosols, etc.) and suppositories. Depending on the dosage form, the drug is administered orally or parenterally (local injection into the affected area, intravenous, intraarterial, subcutaneous, intradermal, intramuscular or intraperitoneal injection, transdermal, transnasal, transmucosal, etc.) Applied to the subject. Systemic administration and local administration are also adapted to the subject. These administration routes are not mutually exclusive, and arbitrarily selected two or more can be used in combination.

医 薬 The medicament of the present invention contains an active ingredient in an amount necessary for obtaining the expected effect (ie, a therapeutically or prophylactically effective amount). The amount of the active ingredient in the medicament of the present invention generally varies depending on the dosage form, but the amount of the active ingredient is set, for example, in the range of about 0.1% by weight to about 99% by weight to achieve a desired dose.

投 与 The dosage of the medicament of the present invention is set so as to obtain the expected effect. In setting a therapeutically or prophylactically effective dose, symptoms, the age, sex, and weight of the patient are generally considered. Those skilled in the art can determine an appropriate dose in consideration of these matters. As an example of the dosage, the dosage can be set so that the daily amount of the active ingredient is 1 mg to 20 mg, preferably 2 mg to 10 mg for an adult (body weight: about 60 kg). The administration schedule may be, for example, once to several times a day, once every two days, or once every three days. In preparing the administration schedule, the condition of the patient, the duration of the effect of the active ingredient, and the like can be considered.

As is apparent from the above description, the present application is to administer a therapeutic or prophylactically effective amount of a medicament containing the anti-herpes virus agent of the present invention to a subject suffering from or possibly suffering from herpes virus infection. Also provided is a method for treating or preventing a herpes virus infection characterized by the following. The target of treatment or prevention is typically a human, but a mammal other than a human (eg, monkey, cow, pig, horse, goat, sheep, dog, cat, rabbit, etc.), bird (chicken, quail, turkey) , Geese, ducks, ostriches, ducks, parakeets, birds, etc.).

As described above, one of the utilization forms of the anti-herpes virus agent of the present invention is a food or feed containing the anti-herpes virus agent of the present invention. Examples of the food of the present invention include general foods (cereals, vegetables, meat, various processed foods, confectionery and desserts, milk, soft drinks, fruit juice drinks, coffee drinks, vegetable juice drinks, alcoholic drinks, etc.), nutritional supplements ( Supplements, nutritional drinks, etc.), food additives, pet food for pets, and dietary supplements for pets. In the case of dietary supplements or food additives, they can be provided in the form of powders, granules, tablets, pastes, liquids and the like. By providing in the form of a food composition, it becomes easy to take the active ingredient of the present invention on a daily basis or continuously.

Examples of the feed of the present invention are feed (eg, feed for livestock and poultry) and pet food.

食品 The food or feed of the present invention preferably contains an active ingredient in an amount that can be expected to have a therapeutic or preventive effect. The amount to be added can be determined in consideration of the condition, age, sex, weight, and the like of the person to whom it is used.

1. Preparation of dry powder (algae) and extract of Kokkomikusa microalgae, and purification of extract 1-1. Kokkomikusa sp. KJ strain The Kokkomikusa sp. KJ strain was cultured according to the method described previously. Specifically, after inoculating the A. spp. KJ strain in the AF6 medium, 2% CO ₂ (v / v) was aerated and room temperature (25 ° C.) while irradiating with light (300 μmol / m ² / s). For 48 hours. Algae were collected from the culture solution by centrifugation. The collected algal cells were dried with a drum dryer and pulverized with a fine pulverizer to obtain a powder (dry powder of algal cells). Next, 1 l of ethanol was added to 100 g of the dry powder for dispersion, and the mixture was allowed to stand in a dark place for 3 days. After standing, the mixture was filtered to separate into a primary filtrate and a residue. To this residue, 1 l of ethanol was added and dispersed in the same manner as described above, allowed to stand for 3 days, and then filtered again to separate a secondary filtrate and a residue. This filtration operation was repeated once to obtain a tertiary filtrate and a residue.

The primary filtrate, the secondary filtrate, and the tertiary filtrate were mixed, ethanol was distilled off using an evaporator, and the residue was dried under reduced pressure to obtain an ethanol extract DE. Next, the DE was subjected to DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation, 3.5 × 57 cm) column chromatography. Elution was performed with H ₂ O, 50% ethanol (EtOH), EtOH and acetone in that order, and each eluted fraction was dried at room temperature under reduced pressure (DE1, 400.9 mg; DE2, 310.3 mg; DE3, 2.25 g; DE4, 4.86 g). The acetone fraction (DE4) was subjected to silica gel column (3 × 42 cm) chromatography. Hexane, hexane-ethyl acetate (AcOEt) (1: 1), AcOHt, AcOHt-acetone (1: 1), acetone and methanol (MeOH) were eluted sequentially.Eluted fractions, DE4A (55.4 mg), DE4B (2.0 g), DE4C (89.0 mg), DE4D (1.01 g), DE4E (95.5 mg) and DE4F (177.1 mg). Next, DE4D was subjected to chromatography on a silica gel column (1.5 x 35 cm) using AcOEt-acetone as a solvent system to obtain 3 fractions, DE4D1 (49.9 mg), DE4D2 (705.1 mg), and DE4D3 (16.2 mg). . Subsequently, subjected DE4D2 silica gel column (1.5 x 35 cm) _{chromatography, CHCl 3 -MeOH-H 2 0} DE4D2A by eluting with (10: 1: 0.1) and (15.6 mg) DE4D2B a (686.5 mg) Obtained. Among these, DE4D2B was subjected to LH-20 column (Sigma-Aldrich) chromatography using chloroform (CHCl ₃ ) -MeOH (3: 1) as a solvent system to obtain DE4D2Bl (11.6 mg) and DE4D2B2 (652.3 mg). Obtained. Subjected DE4D2B2 silica gel column (2 x 50 cm) _{chromatography, CHCl 3, CHCl 3 -MeOH (} 20: 1) and _{CHCl 3 -MeOH-H 2 0 (} 10: 3: 1) 4 fraction by eluting with , DE4D2B2A (4.8 mg), DE4D2B2B (3.5 mg), DE4D2B2C (25.1 mg) and DE4D2B2D (620.5 mg). Finally, CHCl ₃ -MeOH-acetic acid _{(AcOH) -H 2 0 (80} : 9: 12: 2) by preparative liquid chromatography and developed with (PLC) was purified DE4D2B2D, MGDG preparations and (578.9 mg) did.

1-2. Kokkomikusa sp. MBIC11204 strain 1-1. According to the method and operation described in the above, a dry powder of algal cells and an MGDG preparation were obtained from Kokkomixa sp. MBIC11204 strain.

2. Experiments on HSV-2 infection (genital herpes) and HSV-1 infection (skin herpes) (Effects of MGDG preparation of Algae sp. MBIC11204 strain and algal cells)
The efficacy of the MGDG preparation and the alga body (dry powder) of Kokkomixa sp. MBIC11204 strain against HSV-2 infection and HSV-1 infection was evaluated. Changes in efficacy when used in combination with thymidine were also investigated.

<Method>
The virus strain used HSV-2 (UW268 strain) and HSV-1 (HAY strain).
(A) Experimental treatment of genital herpes with HSV-2 infection (1) BALB / c mice (6 weeks old, female) were injected subcutaneously with medroxyprogesterone 17-acetate (3 mg / mouse) 6 days before and 1 day before virus inoculation I do.
(2) Set up the following test plots, and orally administer the sample twice a day (9:00 and 18:00) from 3 days before virus inoculation to 7 days after virus inoculation.
Test plot # 1: control (distilled water)
Test plot # 2: ACV (acyclovir) (1 mg / day)
Test plot # 3: MGDG preparation (0.1 mg / day)
Test plot # 4: MGDG preparation (1 mg / day)
Test plot # 5: thymidine (1 mg / day)
Test plot # 6: thymidine (5 mg / day)
Test plot # 7: thymidine (20 mg / day)
Test plot # 8: MGDG preparation (0.1 mg / day) + thymidine (1 mg / day)
Test plot # 9: MGDG preparation (0.1 mg / day) + thymidine (5 mg / day)
Test plot # 10: MGDG preparation (0.1 mg / day) + thymidine (20 mg / day)
Test plot # 11: MGDG preparation (1 mg / day) + thymidine (1 mg / day)
Test plot # 12: MGDG preparation (1 mg / day) + thymidine (5 mg / day)
Test plot # 13: MGDG preparation (1 mg / day) + thymidine (20 mg / day)
Test plot # 14: Algae (20 mg / day)
Test plot # 15: Algae (20 mg / day) + thymidine (1 mg / day)
Test plot # 16: Algae (20 mg / day) + thymidine (5 mg / day)
Test plot # 17: Algae (20 mg / day) + thymidine (20 mg / day)
(3) Local inoculation of HSV-2 (1 x 10 ³ PFU / 20 μl / mouse).
(4) Three days later, local washing is performed with cold PBS (100 μl), and the amount of the virus is measured by a plaque assay.
(5) Record the onset of herpes symptoms (lesion score) and deaths over 14 days.

(B) Treatment experiment of cutaneous herpes by HSV-1 infection (1) Under anesthesia, the left flank of a BALB / c mouse (6 weeks old, female) is dehaired (hair clipper + hair removal cream).
(2) The following test plots were set up, from 1 day to 14 days after virus inoculation in the application group (# 19 to # 22) and from 3 days to 7 days after virus inoculation in the oral group (# 23 to # 27) During the period, the sample is administered twice a day (9:00 and 18:00). (In the application group, the hair is removed with a cotton swab, and the amount of one application is about 50 to 60 mg / mouse as an emulsion).
Test plot # 19: control (PBS) (application)
Test plot # 20: 5% ACV (application)
Test plot # 21: 5% MGDG preparation (application)
Test plot # 22: 5% thymidine (applied)
Test plot # 23: 5% MGDG preparation + 5% thymidine (applied)
Test plot # 24: control (distilled water)
Test plot # 25: ACV (1 mg / day)
Test plot # 26: MGDG preparation (1 mg / day)
Test plot # 27: thymidine (20 mg / day)
Test plot # 28: thymidine (20 mg / day) + MGDG preparation (1 mg / day)
(3) Inject HSV-1 (2 × 10 ⁵ PFU / 50 μl / mouse) into the depilated area with a microsyringe.
(4) Record the onset degree (lesion score) of herpes symptoms (appearing in a band) and death cases over 14 days.

<Results and Discussion>
(A) HSV-2 genital herpes model (1) Onset and survival rate (Figs. 1 to 5)
The MGDG preparation administered alone significantly suppressed the onset in the 0.1 mg and 1 mg / day administration groups (# 3, # 4) (FIG. 1), and there were no deaths (FIG. 5). Thymidine dose-dependently suppressed the onset and mortality, and a remarkable therapeutic effect was observed particularly in the 20 mg / day administration group (# 7) (FIGS. 1 and 5).

When MGDG preparation (0.1 mg, 1 mg / day) was used in combination with thymidine, the onset was completely suppressed as compared to the case of each administration alone (FIGS. 2 and 3), and no deaths were observed (FIG. 5). .

At the time of single administration of 時 dry powder 20 mg / day (# 14), no significant suppression of onset was observed (FIG. 4), and two out of five animals died (FIG. 5). However, when used in combination with thymidine (# 15-17), onset and death were significantly suppressed (FIGS. 4, 5).

(2) Virus growth inhibitory effect (Fig. 6)
In general, a high effect was observed in both the single administration and the combined administration, and the effect of the combination (# 8 to # 13, # 15 to # 17) was particularly remarkable.

(4) The MGDG preparation administered alone showed a stronger virus growth inhibitory effect as the virus inoculation amount was lower, and a sufficient effect was obtained even at 0.1 mg / day. In contrast, in the case of thymidine (1 mg, 5 mg), there was no difference in the effect due to the difference in the amount of virus inoculated, and a marked suppression effect was observed in the 20 mg / day administration group. When the MGDG preparation and thymidine were used in combination, a remarkable combination effect was observed at a low inoculation dose, and a remarkable effect was observed even when a small amount of the MGDG preparation {0.1 mg} + {thymidine} 1 mg / day was administered.

Dry powder, when administered alone, was less effective than the MGDG preparation, but inhibited virus growth. When the amount of virus inoculated was reduced, a remarkable growth inhibitory effect was observed when used in combination with thymidine.

(B) HSV-1 cutaneous herpes model In the case of topical administration (application) (FIGS. 7 and 9), the MGDG preparation (# 21) and thymidine (# 22) were used together (# 23 ) Was superior in terms of the onset of the disease and the survival rate.

In the case of oral administration (FIGS. 8 and 9), MGDG preparation (# 26) significantly suppressed the onset, but thymidine (# 27) showed a significant onset-suppressing effect as compared with control (# 24). I couldn't. When both were used together (# 28), an onset suppression effect comparable to ACV (# 25) was observed, and there were no deaths.

3. Anti-HSV-2 activity of MGDG preparation and interaction with thymidine (In vitro experiment)
The anti-HSV-2 activity enhancing effect of the MGDG11204 MGC11204 sp. Strain combined with thymidine was examined.

<Method>
(1) Culture Vero cells in a 48-well plate.
(2) On the next day, HSV-2 (UW268 strain) is infected with 0.1 PFU / cell (room temperature, 1 hour).
(3) After washing twice with PBS, a medium to which MGDG preparation (0 to 20 μg / ml) and thymidine (0 to 2 μg / ml) are added, respectively, and culture is performed.
(4) After 24 hours, collect the plate, and perform freeze-thaw treatment (frozen at -80 ° C and then returned to room temperature) three times.
(5) A plaque assay is performed using Vero cells cultured in a monolayer on a 35 mm culture dish.
(6) to calculate IC ₅₀ values from each of the number of plaques, further creates an Isobologram based on the following calculation equation.

[Calculation formula of Isobologram shown in FIG. 10]
x-axis: concentration of thymidine / IC ₅₀ of thymidine in the absence of MGDG preparation (1.2 μg / ml)
_{y-axis: IC 50 (12 μg / ml} ) of MGDG preparation in IC ₅₀ / thymidine absence of MGDG preparation in thymidine presence of a fixed concentration

[Calculation formula of Isobologram shown in FIG. 11]
x-axis: concentration of MGDG preparation / IC ₅₀ of thymidine in the absence of MGDG preparation (12 μg / ml)
y-axis: IC thymidine in the presence of MGDG preparations constant concentration ₅₀ / MGDG preparation IC ₅₀ of MGDG preparation in the absence (1.2 ug / ml)

<Results and Discussion>
Each IC ₅₀ value was calculated from the average of two measurements. The IC ₅₀ values of MGDG preparation is 12μg / ml, IC ₅₀ of thymidine was 1.2 ug / ml (Table 1).

Examination of the change in activity caused by the combination of the two showed that the thromidine HSV-2 growth inhibitory effect was enhanced by the MGDG preparation (FIG. 10), and the anti-HSV-2 effect of the MGDG preparation was enhanced by thymidine (FIG. 11) A synergistic effect between the two substances was confirmed.

4. HSV-2 infection (genital herpes) experiment (Effect of Kokkomikusa sp. KJ strain)
It was verified that the strain KJ was similar to HSV-2 infectious disease, as was the case with S. spp. MBIC11204. In addition, changes in efficacy when combined with thymidine (0.5 mg / day) were examined.

<Method>
(1) BALB / c mice (6 weeks old, female) are injected subcutaneously with medroxyprogesterone 17-acetate (3 mg / mouse) 6 days before and 1 day before virus inoculation.
(2) Set up the following test plots and orally administer the sample twice a day (9:00 and 18:00) from 3 days before virus inoculation to 7 days after inoculation.
Test plot # 1: control (distilled water)
Test plot # 2: ACV (1 mg / day)
Test plot # 3: Algae (dry powder) (5 mg / day)
Test plot # 4: Algae (dry powder) (5 mg / day) + thymidine (0.5 mg / day)
Test plot # 5: Algae (dry powder) (10 mg / day)
(3) Local inoculation of HSV-2 (UW268 strain) (1 × 10 ³ PFU / 20 μl / mouse).
(4) Three days later, local washing is performed with cold PBS (100 μl), and the amount of the virus is measured by a plaque assay.
(5) Record the onset of herpes symptoms (lesion score) and deaths over 14 days.

<Results and Discussion>
Administration of the KJ strain alga alone alone suppressed onset (FIG. 12) and mortality at a low dose (5 mg or 10 mg / day), and also significantly reduced viral load (FIG. 13). There was no significant difference between the two doses. On the other hand, when thymidine was used in combination with the alga bodies of the KJ strain, the herpes treatment effect was improved as compared with the case of single administration.

5. Preparation of MGDG from Chlorella vulgaris, Nannochloropsis oculata, Arsulospira platensis (Spirulina) and Euglena gracilis Extract MGDG from Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (Spirulina) and Euglena gracilis I do. Cultivation of each algae is performed using a culture medium (for example, C medium shown in FIG. 14 for Chlorella vulgaris, ESM medium shown in FIG. 14 for Nannochloropsis oculata, Arthrospira platensis (spirulina). ), The MA medium shown in FIG. 15, and Euglena gracilis, the HUT medium shown in FIG. 15) may be used in the usual manner (using the same culture conditions as those of the above-mentioned strain of Kokkomixa sp. KJ). May be). Dried powder of each alga body can be obtained by drying the alga body collected from the culture solution by centrifugal separation with a drum dryer and pulverizing with a pulverizer. An MGDG preparation of each algae can be obtained from the dried powder by the same extraction operation as in the case of Kokkomixa sp. KJ. However, in this study, the processed products of each algae (Chlorella vulgaris "Chlorella vulgaris dried powder" (Aoi Seika Co., Ltd.), Nannochloropsis oculata "Nanochlorochloropsis oculata frozen" (Marin Tech Co., Ltd.), Arthrospira Platensis (Spirulina) purchased "Spirulina Powder" (DIC Lifetech Co., Ltd.), and Euglena Grasilis purchased "Euglena of Life" (Six Sense Lab Co., Ltd.) and Biozyme (Euglena). (The extraction procedure was the same as that for the strain Kkkomikusa sp. KJ.) The obtained MGDG preparations were used for the following examinations: Commercially available spinach (Spinacia oleracea) was also finely dried and freeze-dried. The MGDG preparation was obtained by crushing with a crusher and extracting MGDG.

6. Effect on cold sores The effect of MGDG preparation on cold sores was examined.
<Method>
(1) Prepare 30 g of hand cream (moina, manufactured by Denso).
(2) 0.165 g of a preparation of Kokkomixa sp. KJ strain MGDG is added to 0.610 g of olive oil (manufactured by J-Oil Mills), kneaded and dissolved (MGDG dissolved).
(3) To the hand cream prepared in (1), the MGDG solution is added little by little while kneading so as to be uniform, and the whole is added to obtain a coating sample.
(4) Apply an appropriate amount daily (1 to 3 times) to the affected area of a patient who developed herpes labialis (the following two patients were the subjects), and observe the state of the affected area over time.
Patient 1: 45-year-old female; experienced herpes labialis (several times). Usually, treatment is carried out using a commercial drug Aracena S (Sato Pharmaceutical Co., Ltd.) containing vidarabine as an active ingredient, and it takes 2 to 3 weeks to cure.
Patient 2: 25-year-old man; experience with onset of cold sores (multiple times). He usually does not use the drug (waits for healing spontaneously) and recovers to an inconspicuous level after 2-3 weeks.

<Results and Discussion>
The observation results are shown in FIG. 16 (patient 1) and FIG. 17 (patient 2). In Patient 1, a clear improvement was observed on the third day of application, and the patient was almost completely cured on the fifth day of application (FIG. 16). In addition, in the case of Patient 2, the therapeutic effect was recognized from the second day of application, and almost completely cured on the eighth day of application. Thus, the MGDG preparation exerted an excellent therapeutic effect on cold sores. What is remarkable is that a surprising therapeutic effect that can be said to surpass that of the over-the-counter drug was obtained (patient 1).

7. Identification of components in MGDG preparation 1
From the HPLC analysis, it was presumed that the MGDG preparation of the strain Kokkomixa sp. KJ contained five kinds of MGDG (referred to as MGDG1, MGDG2, MGDG3, MGDG4 and MGDG5). In order to elucidate the structure of these MGDG, fatty acid composition was analyzed using gas chromatography (GC / FID). A BPX90 column (length 100 m × inner diameter 0.25 mm, film thickness 0.25 μm) was used for analysis, and hydrogen was used as a carrier gas. FAME Standard (FAME Mix, C4-C24, manufactured by SUPELCO) and MGDG (MGDG plant, manufactured by Avanti) were used as standard samples for identification.

ク ロ Chromatograms of each sample (MGDG1, MGDG2, MGDG3, MGDG4, MGDG5) are shown in FIGS. Here, as for C6: 0, although the fatty acid composition of general lipids usually includes fatty acids with close carbon numbers (even numbers), these are hardly detected in this analysis. Therefore, it was considered that the peak was probably not a fatty acid. In addition, in the case of Unknown1 and Unknown2, the fatty acids detected during the retention time of these peaks are hardly assumed to be common lipids, and thus it is considered that there is a high possibility that the fatty acids are components other than the fatty acids. On the other hand, from the retention time, Unknown3 and Unknown4 were estimated to be C16: 2 and C18: 2 (other than n-6), respectively.

The results of the analysis are shown in Tables 2 and 3 below. Table 2 shows the fatty acid composition ratio of each sample by the area percentage method. Table 3 summarizes the calculation results excluding C6: 0 and unknown components 1 and 2 (Unknown 1 and 2). In MGDG3, the area ratio of the two components is extremely biased at 3: 1. This means that the combinations of fatty acid residues are (C18: 2 (other than n-6) and C18: 2 (other than n-6)) and (C18: 2 (other than n-6) and C18: 3 n3) It can be interpreted that the two types of MGDG are mixed at a ratio of about 1: 1.

"-" Represents non-detection.

"-" Represents non-detection.

As described above, in the MGDG preparation, (1) the constituent fatty acids are C16: 3 and C18: 3 (n-3) monogalactosyldiacylglycerol (MGDG1), and (2) the constituent fatty acids are C16: 2 and C18: 2. Monogalactosyldiacylglycerol (MGDG2), (3) monogalactosyldiacylglycerol (MGDG3) whose constituent fatty acids are C18: 2 and C18: 3 (n-3), (4) fatty acids of C18: 2 and C18: 2 Monogalactosyldiacylglycerol (MGDG3), (5) monogalactosyldiacylglycerol (MGDG4) whose constituent fatty acids are C16: 2 and C18: 2 (n-6), and (6) constituent fatty acid is C18: 2 (n-6) And C18: 3 (n-3) monogalactosyldiacylglycerol (MGDG5).

8. Identification of components in MGDG preparation 2
Using a triple quadrupole LC / MS system, the structure of MGDG contained in the preparation of Kokkomixa sp. KJ strain MGDG was examined in more detail.
<Analysis conditions>
Equipment name: Triple quadrupole LC / MS system LC column: YMC Triant C-18 (length 100 mm x inner diameter 2.1 mm, particle diameter 1.9 μm)
Mobile phase: methanol, acetonitrile, water, ammonium acetate Ion source: ESI
Measurement mode: MS2 scan, Product ion scan

<Result>
In addition to the five types of MGDG detected by preparative liquid chromatography (PLC) (see 7. Identification of components in MGDG preparation 1)), triple new LCDG analysis by LC / MS analysis Eye MGDG was detected. For these MGDGs (6 peaks), from the results of MS scan and product ion scan, each MGDG (peak 1: MGDG1, peak 2: MGDG2, peak 3: MGDG3, peak 4: MGDG4, peak 5: MGDG5, peak 6: MGDG6) could be identified as two fatty acid side chains. The following table shows the fatty acid side chains of each MGDG.

9. Virucidal activity of each MGDG fraction The virucidal activity of each MGDG (MGDG1 to MGDG5) constituting the MGDG preparation was compared.
<Method>
(1) The MGDG preparation derived from Kokkomixa sp. KJ strain was subjected to HPLC, and each peak was collected to obtain five kinds of purified MGDG (MGDG1, MGDG2, MGDG3, MGDG4, MGDG5). In addition, MGDG preparations (containing MGDG1 to MGDG5) before fractionation were prepared as Mix products.
(2) Each sample was mixed with each virus solution (2 × 10 ⁵ PFU / ml) to a final concentration of 50 μg / ml, and allowed to stand at 37 ° C. for 60 minutes. Table 5 shows the viruses and host cells used.

(3) After diluting the mixed solution of (2) with a MEM medium containing no fetal bovine serum, a plaque assay medium was overlaid and cultured at 37 ° C. for 2 to 3 days.
(4) After confirming the appearance of plaque, the cells were fixed and stained with neutral red solution, and the number of plaques was measured under a microscope. Using the number of appearances of the virus when no sample was added as a reference (100%), the virus remaining rate of each sample was calculated. The virucidal performance of each sample was evaluated from the virus residual rate.

<Results and Discussion>
Although the activity differs depending on the target virus, virus killing (inactivation) activity was observed for all of MGDG preparation (Mix product) and MGDG1 to MGDG5 (FIG. 23). MGDG5 showed a significant inactivation effect on influenza virus and herpes simplex virus type 2 as compared with MGDG preparation (Mix product). None of the feline caliciviruses and polioviruses without the envelope showed significantly higher activity than the other samples. The above results confirm that the MGDG preparation and each of the constituent MGDGs have virucidal activity, and that MGDG5 has particularly high virucidal activity against enveloped viruses such as influenza virus and herpes simplex virus type 2. To demonstrate.

10. Composition comparison of various MGDG preparations Kokkomakusa sp. KJ strain MGDG preparation, Chlorella vulgaris MGDG preparation, Nannochloropsis oculata MGDG preparation, Arthrospira platensis (spirulina) MGDG preparation, Euglena glaciris MGDG preparation and Spinach MGDG preparations were analyzed by reverse phase HPLC and the components (MGDG) were compared.
<HPLC analysis conditions>
Equipment name: Alliance2695
Column: YMC-Actus Triart C18
Mobile phase: mixed solution of methanol, water, and acetonitrile Detection wavelength: 205 nm

HPLC measurement results (chromatograms) are shown in FIGS. The vertical axis of the chromatogram is normalized so that the peak maximum value in each sample is 1.
-At least 5 peaks were detected in the MGJ preparation of the KJ strain. On the other hand, in the MGDG preparations of each organism other than the KJ strain, at least one peak whose retention time was the same as or very close to that of the MGJ preparation of the KJ strain was detected. It is presumed that the peak having the same retention time has the same chemical structure as MGDG derived from the KJ strain.
-Virucidal activity against HSV-2, IFV and FCV is observed in five peaks derived from the KJ strain MGDG preparation (Experiment 9; Fig. 23). As described above, since the MGDG preparation of each organism other than the KJ strain has at least one peak corresponding to the five peaks derived from the KJ strain MGDG preparation, it is presumed to have virusicidal activity as well. You. That is, these MGDG preparations can be expected to have the same effects as those of the KJ strain MGDG preparation. The fifth peak of the KDG strain MGDG preparation is known to have high virucidal activity against HSV-2 and IFV (FIG. 23).

The anti-herpes virus agent of the present invention contains a substance derived from a unicellular algae (preferably microalgae) as an active ingredient, and can exert anti-herpes virus activity by a different mechanism of action from existing anti-herpes virus agents. Therefore, it is also suitable for use in combination with existing anti-herpes virus agents, and provides a new therapeutic strategy. On the other hand, since the active ingredient of the anti-herpesvirus agent of the present invention is a single cell algae (preferably microalgae), high safety can be expected.

The present invention is not limited to the description of the embodiment and the example of the above invention. Various modifications are included in the present invention without departing from the scope of the claims and within the scope of those skilled in the art. The contents of articles, published patent gazettes, and patent gazettes specified in this specification are all incorporated by reference.

Claims (18)

1. (4) An anti-herpes virus agent containing a single-cell algae-derived substance as an active ingredient.
2. The anti-herpesvirus agent according to claim 1, wherein the unicellular algae is a microalga belonging to the genus Kokkomikusa, Chlorella, Nannochloropsis, Arthrospira or Euglena.
3. The anti-herpes virus agent according to claim 1, wherein the single-cell algae is a microalga belonging to the genus Kokkomikusa.
4. 4. The anti-herpes virus agent according to claim 3, wherein the microalgae is a Kokkomixa sp. Strain KJ or a mutant thereof, or a Kokkomixa sp. MBIC11204 strain or a mutant thereof.
5. The anti-herpes virus agent according to any one of claims 1 to 4, wherein the active ingredient is a monogalactosyldiacylglycerol-containing extract of the single-cell algae.
6. The anti-herpes virus agent according to claim 5, wherein the main component is monogalactosyldiacylglycerol.
7. The anti-herpes virus agent according to claim 6, wherein the monogalactosyldiacylglycerol is one or more monogalactosyldiacylglycerols selected from the group consisting of the following (1) to (6):
   (1) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 3 and C18: 3;
   (2) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 3;
   (3) monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 3;
   (4) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 2;
   (5) Monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 2; and (6) Monogalactosyldiacylglycerol whose constituent fatty acids are C16: 1 and C18: 2.
8. (8) The anti-herpes virus agent according to any one of (5) to (7), wherein the active ingredient is obtained by increasing the content of monogalactosyldiacylglycerol by chromatographic purification of the ethanol extract of the single-cell algae.
9. The anti-herpes virus agent according to any one of claims 5 to 8, wherein the monogalactosyldiacylglycerol content in the active ingredient is 70% (w / v) to 99% (w / v).
10. 抗 The anti-herpes virus agent according to any one of claims 1 to 4, wherein the active ingredient is an algal body.
11. (5) The anti-herpes virus agent according to any one of (1) to (4), wherein the active ingredient is a dry powder of algal cells.
12. An anti-herpesvirus agent comprising one or more monogalactosyldiacylglycerols as an active ingredient selected from the group consisting of the following (1) to (6):
    (1) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 3 and C18: 3;
    (2) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 3;
    (3) monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 3;
    (4) monogalactosyldiacylglycerol whose constituent fatty acids are C16: 2 and C18: 2;
    (5) Monogalactosyldiacylglycerol whose constituent fatty acids are C18: 3 and C18: 2; and (6) Monogalactosyldiacylglycerol whose constituent fatty acids are C16: 1 and C18: 2.
13. The anti-herpesvirus agent according to any one of claims 1 to 12, wherein thymidine is used in combination.
14. 14. The anti-herpes virus agent according to claim 13, which is a combination preparation containing the active ingredient and thymidine.
15. (15) The anti-herpes virus agent according to any one of (1) to (14), wherein the target herpes virus is herpes simplex virus type 1 or herpes simplex virus type 2.
16. A composition comprising the anti-herpesvirus agent according to any one of claims 1 to 15.
17. 17. The composition according to claim 16, which is a medicament for herpes virus infection.
18. 17. The composition according to claim 16, which is a food or a feed.

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