JP7378091B2 - Virucide - Google Patents

Description

IPOD FERM BP-22254

The present invention relates to virucides. In particular, the present invention relates to a virucidal agent containing an extract of microalgae or its components as an active ingredient, and its uses. This application claims priority based on Japanese Patent Application No. 2018-143049 filed on July 31, 2018, and the entire contents of the patent application are incorporated by reference.

Viral infections are diseases caused by pathogenic viruses such as HIV (human immunodeficiency virus) and influenza virus. Until now, measures against viral infections have focused on the development of vaccines and antiviral drugs. However, vaccines have only been developed for a limited number of infectious diseases, and if antigenic mutations occur on the virus side, they will lose their effectiveness. In addition, antiviral drugs are limited in their target infectious diseases, and also have problems such as side effects, emergence of drug-resistant viruses, and increased economic burden. In particular, the emergence of drug-resistant viruses is a serious problem that shortens the lifespan of the drugs. Resistance arises because most antiviral drugs target viral enzymes. In other words, it acts in an inhibitory manner on the stage of virus proliferation within cells. This suggests that in order to avoid drug resistance, it would be better to target the virus particles themselves that exist outside the cells, rather than the virus propagation inside the cells. Such an action causes the virus particles to lose their infectivity, and is called a virucidal effect or an inactivation effect. Examples of virucides that have been developed or proposed so far are shown below (Patent Documents 1 to 3). In addition, papers regarding the antiviral activity of monogalactosyldiacylglycerol are also shown below (Non-Patent Documents 1 to 3). Non-Patent Document 1 shows that monogalactosyldiacylglycerol contained in Chinese herbal medicine (Clinacanthus) exhibited anti-herpes activity. On the other hand, Non-Patent Document 2 shows that monogalactosyldiacylglycerol derived from seaweed has anti-HSV-1 activity and anti-HSV-2 activity. Furthermore, Non-Patent Document 3 reports the anti-HSV-1 activity and anti-HSV-2 activity of monogalactosyldiacylglycerol contained in Chinese herbal medicine (Clinacanthus).

JP2013-47196A Japanese Patent Application Publication No. 2014-55128 International Publication No. 2014/115860 pamphlet

Phytochemistry. 2003 Dec;64(7):1253-64. Mar Drugs. 2012 Apr; 10(4): 918-931. Asian Pac J Trop Biomed 2016; 6(3): 192-197

Although there have been reports of virucidal agents to date, there are still issues to be overcome and room for improvement in terms of activity, effectiveness, and safety, and the need for highly practical virucidal agents remains high. . Furthermore, there is a great need for the development of a virucidal agent that can kill not only a specific virus but also multiple viruses. Therefore, the main object of the present invention is to provide a virucidal agent that is highly practical and can be used for a wide range of purposes, including application to human infectious diseases.

While conducting research in view of the above issues, it was discovered that an extract containing monogalactosyldiacylglycerol from microalgae of the genus Coccomyxa exhibits excellent virucidal activity. Remarkably, the extract exhibited virucidal activity not only against a specific virus but also against multiple types of viruses. On the other hand, further studies revealed the structure of monogalactosyldiacylglycerol, the active ingredient in the extract. Furthermore, further findings were obtained that support the extremely high utility value of the extract.

On the other hand, monogalactosyldiacylglycerol-containing extracts derived from Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (Spirulina), and Euglena gracilis are similar to monogalactosyldiacylglycerol-containing extracts derived from microalgae of the genus Coccomyxa. It has become clear that this effect can be expected.
Mainly based on the above results and considerations, the following invention is provided.
[1] A virucidal agent containing a monogalactosyldiacylglycerol-containing extract of unicellular algae as an active ingredient.
[2] The virucide according to [1], wherein the unicellular algae is a microalgae belonging to the genus Coccomyxa, genus Chlorella, genus Nannochloropsis, genus Arthrospira, or genus Euglena.
[3] The virucide according to [1], wherein the unicellular algae are microalgae belonging to the genus Coccomyxa.
[4] The virucide according to [3], wherein the microalgae is Coccomyxa sp. KJ strain or a mutant strain thereof.
[5] The virucidal agent according to any one of [1] to [4], wherein the main component is monogalactosyldiacylglycerol.
[6] The virucide according to [5], 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.
[7] The active ingredient according to any one of [1] to [6], wherein the ethanol extract of the unicellular algae is purified by chromatography to increase the content of monogalactosyldiacylglycerol. Virucide.
[8] The virucidal according to any one of [1] to [7], wherein the content of monogalactosyldiacylglycerol in the active ingredient is 70% (w/v) to 99% (w/v). agent.
[9] A virucidal agent containing one or more monogalactosyldiacylglycerol as an active ingredient selected from the group consisting of (1) to (6) below:
(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.
[10] The virucidal agent according to any one of [1] to [9], wherein the target virus is an enveloped virus, such as a herpes virus or an influenza virus.
[11] The virucide according to any one of [1] to [9], wherein the target virus is a non-enveloped virus, such as Norovirus or Feline Calicivirus.
[12] The virucidal agent according to any one of [1] to [11], which exhibits virucidal activity through irreversible inactivation.
[13] A composition comprising the virucidal agent according to any one of [1] to [12].
[14] The composition according to [13], which is a medicament for viral infections.
[15] The composition according to [13], which is a disinfectant or a disinfectant.
[16] The composition according to [13], which is a food or bait.

Results of a time-of-addition experiment targeting herpes simplex virus type 2 (HSV-2). For each concentration of MGDG preparation (10 μg/ml, 25 μg/ml, 50 μg/ml), the virus proliferation rate for test zones 1 to 10 is shown in order from the left. Results of a time-of-addition experiment targeting influenza virus (IFV). For each concentration of MGDG preparation (10 μg/ml, 25 μg/ml, 50 μg/ml), the virus proliferation rate for test zones 1 to 10 is shown in order from the left. Results of a virucidal activity test targeting HSV-2. MGDG preparation of 0μg/ml (control), 0.01μg/ml, 0.025μg/ml, 0.1μg/ml, 0.25μg/ml, 1μg/ml, 2.5μg/ml, 10μg/ml, 25μg/ml, 50μg/ml The virus solution was treated for 0 to 360 minutes. Results of a virucidal activity test targeting influenza virus (IFV). MGDG preparation of 0μg/ml (control), 0.01μg/ml, 0.025μg/ml, 0.1μg/ml, 0.25μg/ml, 1μg/ml, 2.5μg/ml, 10μg/ml, 25μg/ml, 50μg/ml The virus solution was treated for 0 to 360 minutes. Transmission electron microscopy (TEM) image showing disruption of the influenza virus (IFV) envelope due to the action of the MGDG preparation. Left: before MGDG action (IFV treated with sterile water for 30 minutes at room temperature), right: after MGDG action (IFV treated with MGDG (50 μg/ml) for 30 minutes at room temperature). A Hitachi transmission electron microscope H-800 was used (acceleration voltage 200 kV). Results of a virucidal activity test targeting norovirus. The virus solution was treated with MGDG preparations at 1 μg/ml, 10 μg/ml, and 100 μg/ml for 0 to 30 minutes. Results of a virucidal activity test targeting feline calicivirus (FCV). The virus solution was treated with MGDG preparations at 10 μg/ml, 50 μg/ml, and 200 μg/ml for 0 to 60 minutes. Results (Lesion score) of virucidal activity test (in vivo experiment). The virus solution was treated with MGDG preparations of 10 μg/ml, 50 μg/ml, and 250 μg/ml for 30 minutes, and then inoculated into mice. Results of virucidal activity test (in vivo experiment) (viral load 3 days after infection). *p<0.05, **p<0.01, ***p<0.001 vs. control Transmission electron microscopy (TEM) image showing disruption of the herpesvirus envelope upon instillation of the MGDG preparation. 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 culturing microalgae of the genus Arthrospira (MA medium) and an example of a medium used for culturing algae of the genus Euglena (HUT medium). Results of virucidal activity test in the presence of organic matter. Feline calicivirus (FCV) was used as an alternative to norovirus. Effect on cold sores (Patient 1: 45-year-old female). A 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). A cream containing the MGDG preparation was applied to the affected area daily, and the progress was observed. Analysis results (GC/FID chromatogram) of the component (MGDG1) of the MGDG preparation. Analysis results (GC/FID chromatogram) of components (MGDG2) in MGDG preparation. Analysis results (GC/FID chromatogram) of components (MGDG3) in MGDG preparation. Analysis results (GC/FID chromatogram) of the component (MGDG4) of the MGDG preparation. Analysis results (GC/FID chromatogram) of components (MGDG5) in MGDG preparation. Viricidal activity of each MGDG fraction (MGDG1 to MGDG5). The virucidal activities of MGDG1 to MGDG5 and MGDG preparations (Mix product) against influenza virus, herpes simplex virus type 2, feline calicivirus, and poliovirus were compared. HPLC chromatograms of MGDG preparations of various organisms (Coccomyxa sp. KJ strain, Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (Spirulina)). MGDG-1 to MGDG-5 are peaks derived from the KJ strain MGDG preparation, and peaks presumed to have the same structure as these are indicated by *. HPLC chromatograms of MGDG preparations of various organisms (Koccomyxa sp. KJ strain, Euglena gracilis (Euglena of Life, Biozyme), spinach). MGDG-1 to MGDG-5 are peaks derived from the KJ strain MGDG preparation, and peaks presumed to have the same structure as these are indicated by *.

1. Terminology, Action A virucide is an agent that exhibits virucidal activity against a target virus. Virucidal activity is a direct inactivation effect on viruses, and exhibits excellent effects in preventing infection, inhibiting the spread of infection, and treating infectious diseases. Inactivation effects are broadly classified into reversible inactivation and irreversible inactivation. The latter, ie, irreversible inactivation, inactivates the virus to an irrecoverable state, and its effect, significance, etc. are definitely different from the former.

The virucidal agent of the present invention can be expected to have an irreversible inactivation effect by destroying virus particles (see Examples below). If the mechanism of action is destruction of virus particles, target specificity is considered to be low (that is, the target is not limited to a specific virus). In fact, the active ingredient of the virucidal agent of the present invention is effective against the non-enveloped RNA virus norovirus, the enveloped DNA virus herpes simplex virus type 2 (HSV-2), and the enveloped RNA virus influenza. The fact that it showed virucidal activity against viruses supports the high versatility of the virucidal agent of the present invention. On the other hand, the action of "destruction of virus particles" can kill viruses that are not in the reproduction cycle, and is not only extremely effective, but also effective in preventing viruses from becoming drug resistant.

2. Active ingredient of virucide The virucide of the present invention contains a monogalactosyldiacylglycerol-containing extract of unicellular algae as an active ingredient. Unicellular algae (such as Trebouxiaphyceae, Eophthalmophyceae, Cyanobacteria, Euglenaphyceae, etc.) are not particularly limited as long as the monogalactosyldiacylglycerol-containing extract has virucidal activity. Preferred unicellular algae include Coccomyxa microalgae (a specific example is Coccomyxa sp. KJ strain), Chlorella microalgae (a specific example is Chlorella vulgaris), and Nannochloropsis microalgae (a specific example is Chlorella vulgaris). Specific examples include Nannochloropsis oculata), microalgae of the genus Arthrospira (specific example is Arthrospira platensis (Spirulina)), and microalgae of the genus Euglena (specific example is Euglena gracilis). Among these, microalgae of the genus Coccomyxa are particularly preferred. In other words, in a particularly preferred embodiment of the present invention, a monogalactosyldiacylglycerol-containing extract of microalgae of the genus Coccomyxa is used as an active ingredient. Microalgae of the genus Coccomyxa are not particularly limited as long as their monogalactosyldiacylglycerol-containing extract has virucidal activity, but preferred examples include Coccomyxa sp. KJ strain or its mutants. Koccomyxa sp. KJ strain (KJ Denso) was registered as of June 4, 2013 at the National Institute of Technology and Evaluation, Biotechnology Center, Patent Organism Depositary (NITE-IPOD) (2-5 Kazusa Kamatari, Kisarazu City, Chiba Prefecture). 8 (Room 120) under accession number FERM P-22254, and as of June 2, 2015, it was transferred to the International Deposit under the provisions of the Ptabest Treaty under accession number FERM BP-22254. The former genus Pseudococcomyxa is now included in the genus Coccomyxa in the latest classification (Reference: PLoS One. 2015 Jun 16;10(6):e0127838. doi: 10.1371/journal. pone.0127838. eCollection 2015.).

Mutant strains of Coccomyxa sp. KJ strain can be obtained by irradiation with ultraviolet rays, X-rays, γ-rays, etc., mutagen treatment, heavy beam irradiation, genetic manipulation (introduction of foreign genes, gene destruction, genetic modification by genome editing, etc.). be able to. As long as a mutant strain that produces monogalactosyldiacylglycerol exhibiting virucidal activity can be obtained, the method for obtaining the mutant strain, its characteristics, etc. are not particularly limited.

The method for culturing Coccomyxa microalgae is not particularly limited. The medium for culturing Coccomyxa microalgae may be one that is normally used for culturing microalgae, such as a known medium for freshwater microalgae containing various nutrients, trace metal salts, vitamins, etc. , a culture medium for marine microalgae can be used. Examples of the medium include AF6 medium. The composition of AF6 medium (per 100ml) is as follows.
_NaNO3 14mg
_{NH4NO3 2.2mg
MgSO4}・_7H2O 3mg
_{KH2PO4 1mg
K2HPO4 0.5mg
CaCl2}・_2H2O 1mg
_CaCO3 1mg
Fe-citrate 0.2mg
Citric acid 0.2mg
Biotin 0.2μg
Thiamine HCl 1μg
Vitamin _B6 0.1μg
Vitamin B ₁₂ 0.1μg
Trace metals 0.5ml
Distilled water 99.5ml

Examples of nutrient salts 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. Further, examples of trace metals include iron, magnesium, manganese, calcium, zinc, etc., and examples of vitamins include vitamin B1, vitamin B12, etc.

The culture may be carried out by supplying carbon dioxide and stirring under aeration conditions. At this time, the cells are cultured by irradiation with a light/dark cycle such as 12 hours of light irradiation using a fluorescent lamp and 12 hours of darkness, or by continuous light irradiation. The culture conditions are not particularly limited as long as they do not adversely affect the growth of microalgae of the genus Coccomyxa, but for example, the pH of the culture solution is 3 to 9, and the culture temperature is 10 to 35°C.

Regarding the culture method of Coccomyxa sp. KJ strain, please refer to JP 2015-15918, WO 2015/190116 A1, Satoh, A. et al., Characterization of the Lipid Accumulation in a New Microalgal Species, Pseudochoricystis ellipsoidea (Trebouxiophyceae) J Jpn. Inst. Energy (2010) 89:909-913. etc. may be helpful.

Unicellular algae other than microalgae of the genus Coccomyxa can also be cultured by conventional methods. The medium used for culturing each algae is not particularly limited. For example, for culturing Chlorella microalgae, a medium commonly used for culturing freshwater algae or a common medium for culturing saltwater algae can be used, and an example of a suitable medium is C. Medium (Figure 11). Similarly, for culturing Nannochloropsis microalgae, a medium commonly used for culturing marine algae or a common medium for culturing brackish water algae can be used, and an example of a suitable medium is ESM medium. (FIG. 11). The same applies to the culture of Arthrospira microalgae, and a medium commonly used for culturing freshwater algae or a common medium for culturing saltwater algae can be used. An example of a suitable medium is MA medium ( Figure 12). The same applies to the culture of Euglena algae, and a medium commonly used for culturing freshwater algae or a common medium for culturing saltwater algae can be used. An example of a suitable medium is HUT medium (Fig. 12).

The method for extracting the active ingredient of the virucide of the present invention, that is, the "monogalactosyldiacylglycerol-containing extract of unicellular algae (particularly preferably microalgae belonging to the genus Coccomyxa)" is such that the extract containing monogalactosyldiacylglycerol is There are no particular limitations as long as it can be obtained. For example, ethanol extraction can be employed as an extraction method. Preferably, the extract is purified after ethanol extraction and has an increased content of monogalactosyldiacylglycerol, and is used as a "monogalactosyldiacylglycerol-containing extract of unicellular algae (particularly preferably microalgae belonging to the genus Coccomyxa)". Examples of purification methods include column chromatography using fillers such as silica gel and alumina, gel filtration chromatography, and concentration.

Prior to extraction, the collected algal bodies may be subjected to drying treatment and/or crushing treatment. In other words, dried algae, dried and crushed algae (typically dry powder), or crushed algae may be prepared and used for the extraction operation. It is decided to obtain pre-processed unicellular algae (for example, dried alga bodies, their powder/powder, or tablets (which may contain excipients, etc.)) and use them for the extraction operation. It's okay. For example, processed products of Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (Spirulina), Euglena gracilis, etc. are commercially available.

The content of monogalactosyldiacylglycerol in the active ingredient is not particularly limited as long as the extract exhibits virucidal activity, for example 70% (w/v) to 99% (w/v), preferably 80% (w/v). /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), 98% (w/v)). In principle, the higher the content of monogalactosyldiacylglycerol, the stronger the virucidal activity can be expected. "Monogalactosyldiacylglycerol" is one of the glyceroglycolipids and is known as a component of the chloroplast thylakoid membrane of plants. Monogalactosyldiacylglycerol has a backbone in which galactose is β-bonded to glycerol.

Preferably, the virucide of the present invention contains monogalactosyldiacylglycerol as the main component. Examples of monogalactosyldiacylglycerol that can be contained in the virucide of the present invention include (1) monogalactosyldiacylglycerol whose constituent fatty acids are C16:3 and C18:3, (2) monogalactosyldiacylglycerol whose constituent fatty acids are C16:2 and C18. :3 monogalactosyldiacylglycerol, (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) composition The fatty acids are monogalactosyldiacylglycerol with C18:3 and C18:2, and (6) the constituent fatty acids are monogalactosyldiacylglycerol with C16:1 and C18:2. These monogalactosyldiacylglycerols were identified as being contained in a microalgae extract of the genus Coccomyxa that exhibits virucidal activity (see 11. Identification of Components in MGDG Preparation in the Examples section below for details). 2”). Considering the analysis results of "10. Identification of components in MGDG preparation 1" shown in the Examples section below, the constituent fatty acid C18:3 in (1) above is preferably C18:3(n- 3), at least one of the constituent fatty acids C18:3 in (3) above is preferably C18:3(n-3), and the constituent fatty acids C18:2 in above (4) are preferably C18:2(n-3). -6), and the constituent fatty acid C18:3 in the above (5) is preferably C18:3(n-3), and the constituent fatty acid C18:2 is preferably C18:2(n-6). Although it is not excluded that a specific monogalactosyldiacylglycerol is contained alone, the virucidal agent of the present invention usually contains two or more types of monogalactosyldiacylglycerols with different structures, and in that case, the combination , content ratio, etc. are not particularly limited. The virucidal agent of the present invention preferably has two or more of the above (1) to (6), more preferably three or more of the above (1) to (6), and still more preferably the above (1). ) to (6), more preferably five or more of the above (1) to (6), even more preferably all of the above (1) to (6). As shown in the Examples below, monogalactosyldiacylglycerol (MGDG5) (5) was found to have particularly high virucidal activity. Therefore, a particularly preferred embodiment of the virucidal agent contains monogalactosyldiacylglycerol (5) alone or in combination with one or more of (1), (2) to (4), and (6). That will happen.

Monogalactosyldiacylglycerol, which has been identified as a major component of the Coccomyxa microalgae extract that exhibits virucidal activity, can be expected to have virucidal activity by itself. Accordingly, one aspect of the present invention provides a virucidal agent containing as an active ingredient one or more monogalactosyldiacylglycerols selected from the group consisting of (1) to (6) above. When two or more types of monogalactosyldiacylglycerol are used as active ingredients, the combination, content ratio, etc. 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), and even more preferably (1) to (6) above, It contains four or more of the above (1) to (6), more preferably five or more of the above (1) to (6), and even more preferably all of the above (1) to (6). As mentioned above, monogalactosyldiacylglycerol (MGDG5) (5) was found to have particularly high virucidal activity. Therefore, in a particularly preferred embodiment, monogalactosyldiacylglycerol (5) is at least one of the active ingredients.

Here, there is monogalactosyldiacylglycerol containing various fatty acids (examples of 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, can generally exhibit virucidal activity.
<Example 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

3. Target Virus As mentioned above, the mechanism of action of the virucidal agent of the present invention and the active ingredient of the virucidal agent of the present invention target viruses such as norovirus, feline calicivirus (FCV), and herpes simplex virus type 2 (HSV-2). The fact that the virucidal activity was shown against influenza virus (IFV) and influenza virus (IFV) supports the high versatility of the virucidal agent of the present invention. Target viruses for which the virucidal agent of the present invention is particularly effective include norovirus, feline calicivirus, herpesvirus, and influenza virus. Norovirus is an RNA virus without an envelope and belongs to the Caliciviridae family. Norovirus shows high resistance to surfactants, alcohol, etc. Feline calicivirus is a virus of the family Caliciviridae, and is known as the pathogen of feline calicivirus infection, which is an infectious disease in cats. It is an RNA virus without an envelope and exhibits characteristics similar to norovirus. Therefore, it is used in experiments as a substitute for norovirus.

Herpesvirus is a virus that has double-stranded DNA as its genome. Herpesviruses are classified into three types of herpesvirinae (α-herpesvirinae, β-herpesvirinae, and γ-herpesvirinae). The α-herpesvirinae subfamily includes herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV), and the β-herpesvirinae family includes cytomegalovirus. (CMV), human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and the gammaherpesvirus subfamily includes Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8). , also known as Kaposi's sarcoma-associated herpesvirus (KSHV).

Influenza viruses are single-stranded RNA viruses, and are broadly divided into three genera: influenza A viruses, influenza B viruses, and influenza C viruses, which differ in the proteins that make up virus particles, antigenicity, and morphology. Ru. Influenza A virus hosts humans, birds, horses, pigs, etc., and the symptoms (pathology) caused by its infection are generally more severe than those of other types. Influenza A causes an epidemic every year. Furthermore, there are concerns about a global pandemic, and countermeasures are urgently needed. Influenza A viruses are divided into two groups depending on the type of HA molecules: Group 1, which consists of H2, H5, H1, H6 (H1 cluster; H1a), H13, H16, and H11 (H1 cluster; H1b); , H8, H12, H9 (H9 cluster), H4, H14, H3 (H3 cluster), H15, H7, and H10 (H7 cluster). Examples of types of influenza A viruses include H1N1, H1N2, H2N2, H3N2, H5N1, H5N2, H6N1, H7N2, H7N3, H7N7, H7N9, H9N2, and H9N1. I can do it.

In addition to the viruses mentioned above, various enveloped DNA viruses (e.g. smallpox virus), various enveloped RNA viruses (e.g. measles virus, rubella virus, mumps virus, coronavirus), and various non-enveloped DNA viruses (e.g. Adenovirus, papillomavirus), various non-enveloped RNA viruses (eg poliovirus, rotavirus, norovirus), etc. can also be targeted by the virucidal agent of the present invention.

4. Applications and methods of using the virucidal agent The virucidal agent of the present invention can be used as a composition containing it for various purposes. Examples of compositions here are medicines (therapeutic or prophylactic), disinfectants, disinfectant cleaners, foods, feeds. Considering its use, the composition of the present invention includes acyclovir, ganciclovir, valacyclovir, vitarabine, foscarnet, trifluridine, oseltamivir phosphate (trade name Tamiflu), zanamivir hydrate (trade name Relenza), peramivir. Antiviral agents such as hydrate (trade name Rapiacta), laninamivir octanoate hydrate (trade name Inavir), baloxavir marboxil (trade name Xofluza), benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethyl Antibacterial or sterilizing ingredients such as phenol and chlorhexidine gluconate may also be included.

One specific example of the use of the virucidal agent of the present invention is the purification of oysters contaminated with norovirus (see Examples below). In such applications, oysters contaminated with norovirus (or oysters that may be contaminated) are typically immersed in a solution of a virucidal agent (e.g., after being raised for a period of several hours to several days). good).

(1) Pharmaceutical The pharmaceutical of the present invention is used for the treatment or prevention of viral infections. The medicament of the present invention can exert a therapeutic effect or a prophylactic effect (these two effects are collectively referred to as a "medicinal effect") against viral infections. The medicinal effects here include (1) prevention of viral infection, (2) prevention, suppression or delay of the onset of viral infection, and (3) alleviation of symptoms characteristic or accompanying symptoms of viral infection (mild symptoms). ), (4) prevention, suppression, or delay of the worsening of symptoms characteristic of viral infections or accompanying symptoms, etc. It should be noted that since therapeutic effects and preventive effects are partially overlapping concepts, it may be difficult to clearly distinguish them, and there is little practical benefit in doing so.

Pharmaceutical preparations can be carried out according to conventional methods. When formulating, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline solution, etc.). As excipients, lactose, starch, sorbitol, D-mannitol, white sugar, etc. can be used. As the disintegrant, starch, carboxymethyl cellulose, calcium carbonate, etc. can be used. Phosphates, citrates, acetates, etc. can be used as buffers. As the emulsifier, gum arabic, sodium alginate, tragacanth, etc. can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. can be used. As a soothing agent, benzyl alcohol, chlorobutanol, sorbitol, etc. can be used. As the stabilizer, propylene glycol, ascorbic acid, etc. can be used. As the preservative, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. can be used. As the preservative, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, etc. can be used.

The dosage form used for formulation is also not particularly limited. Examples of dosage forms are tablets, powders, fine granules, granules, capsules, syrups, injections, and external preparations (ointments, creams, lotions, liquids, gels, poultices, plasters, and tapes). , aerosols, etc.), and suppositories. Medications can be administered orally or parenterally depending on their dosage form (local injection into the affected area, intravenous, intraarterial, subcutaneous, intradermal, intramuscular, or intraperitoneal injection, transdermal, nasal, transmucosal, etc.) applied to the target by. Systemic administration and local administration are also indicated depending on the subject. These administration routes are not mutually exclusive, and two or more arbitrarily selected routes can also be used in combination.

The medicament of the present invention contains the active ingredient in an amount necessary to obtain 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, within the range of about 0.1% by weight to about 99% by weight so as to achieve the desired dosage.

The dosage of the medicament of the present invention is determined so as to obtain the expected effect. In setting a therapeutically or prophylactically effective dose, the symptoms, age, sex, and weight of the patient are generally taken into consideration. Those skilled in the art can take these matters into account and set an appropriate dosage. To give 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 5 mg to 10 mg, for adults (body weight approximately 60 kg). The administration schedule can be, for example, once to several times a day, once every two days, or once every three days. When creating a dosing schedule, the patient's condition and the duration of effect of the active ingredient can be taken into consideration.

In parallel with treatment or prophylaxis with the medicament of the present invention, other medicaments (e.g., acyclovir, ganciclovir, valacyclovir, vitarabine, foscarnet, trifluridine, oseltamivir phosphate (trade name Tamiflu), zanamivir hydrate (trade name Treatment with antiviral agents such as Relenza), peramivir hydrate (trade name Rapiacta), laninamivir octanoate hydrate (trade name Inavir), and baloxavir marboxil (trade name Xofluza) may be performed. . If the active ingredient of the present invention and drugs with different mechanisms of action are used together, a complex action/effect can be obtained, and for example, when applied to the treatment of viral infections, the therapeutic effect can be increased.

As is clear from the above description, the present application is characterized in that a therapeutically or prophylactically effective amount of a medicament containing the virucidal agent of the present invention is administered to a subject suffering from or at risk of suffering from a viral infection. Also provided are methods for treating or preventing various viral infections. Targets of treatment or prevention are typically humans, but may also include non-human mammals (e.g. monkeys, cows, pigs, horses, goats, sheep, dogs, cats, rabbits, etc.), birds (chickens, quail, turkeys, etc.). , geese, ducks, ostriches, ducks, parakeets, sparrows, etc.).

(2) Disinfectant, sterilizing cleaning agent The disinfecting agent or sterilizing cleaning agent of the present invention can be used, for example, to disinfect or sterilize cleaning living rooms (including hospital rooms), cooking rooms, toilets, washrooms, bathrooms, etc., tableware, cutlery (knives), etc. , forks, spoons, etc.), cooking utensils (knives, knives, pots, mixers, microwave ovens, ovens, etc.), medical instruments and equipment, etc., and used for disinfecting or sterilizing cleaning of hands, fingertips, oral cavity, etc. . The disinfectant or sterilizing cleaning agent of the present invention is configured, for example, in a liquid form (for example, a spray, a lotion), a gel form, or a solid form (for example, a powder), and is applied by coating, spraying, scattering, or the like. The disinfectant or sterilizing cleaning agent of the present invention may be supported or adhered to a carrier (for example, sheet-like) made of natural fibers or synthetic fibers, and may be used as a product used for wiping purposes, a mask for infection prevention, or the like.

Antibacterial or sterilizing ingredients such as benzalkonium chloride, cetylpyridinium chloride, phenoxyethanol, isopropylmethylphenol, and chlorhexidine gluconate, pH adjusters, surfactants, adsorbents, carriers, etc. are added to the disinfectant or sterilizing cleaning agent of the present invention. You may decide to add it.

The disinfectant or sterilizing cleaning agent of the present invention contains an amount of active ingredient that can be expected to have a sterilizing or sterilizing cleaning effect. The amount to be added can be determined by taking into consideration the use, form, etc.

(3) Foods, feed Examples of the foods of the present invention include general foods (cereals, vegetables, meat, various processed foods, confectionery/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, and pet nutritional supplements. In the case of nutritional supplements or food additives, they can be provided in the form of powders, granules, tablets, pastes, liquids, and the like. By providing the food composition 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 (for example, feed for livestock, poultry, etc.) and pet food.

The food or feed of the present invention contains an amount of active ingredient that can be expected to have a therapeutic or preventive effect. The amount added can be determined by considering the condition, age, sex, weight, etc. of the person to whom it will be used.

1. Preparation of extract of microalgae Coccomyxa sp. KJ strain and purification of extract Coccomyxa sp. KJ strain was cultured according to a previously reported method. Specifically, after planting Coccomyxa sp. KJ strain on AF6 medium, it was incubated at room temperature (25℃) while aerating with 2% CO ₂ (v/v) and irradiating with light (300 μmol/m ² /s). The cells were cultured for 48 hours. Algal bodies were collected from the culture solution by centrifugation. The collected algae were dried with a drum dryer and pulverized with a pulverizer to form a powder (dried algae powder). Next, 1 L of ethanol was added to 100 g of dry powder to disperse it, and the mixture was allowed to stand in the dark for 3 days. After standing still, it was filtered and separated into a primary filtrate and a residue. 1 L of ethanol was added to this residue in the same manner as above to disperse it, and after allowing it to stand for 3 days, it was filtered again to separate the secondary filtrate and the residue. This filtration operation was repeated once again to obtain a tertiary filtrate and a residue.

The primary filtrate, secondary filtrate, and tertiary filtrate were mixed, ethanol was distilled off using an evaporator, and the mixture was dried under reduced pressure to obtain an ethanol extract DE. Next, DE was subjected to DIAION HP-20 (manufactured by Mitsubishi Chemical Corporation, 3.5 x 57 cm) column chromatography. Elution was carried out sequentially with H ₂ O, 50% ethanol (EtOH), EtOH and acetone, and each eluted fraction was dried under reduced pressure at room temperature (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 x 42 cm) chromatography. Elution was carried out sequentially with hexane, hexane-ethyl acetate (AcOEt) (1:1), AcOHt, AcOHt-acetone (1:1), acetone and methanol (MeOH), and each eluted fraction, 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) were obtained. 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, DE4D2 was subjected to silica gel column (1.5 x 35 cm) chromatography and DE4D2A (15.6 mg) and DE4D2B (686.5 mg) were eluted with CHCl ₃ -MeOH-H ₂ 0 (10:1:0.1). Obtained. Among them, DE4D2B was subjected to LH-20 column chromatography (manufactured by Sigma-Aldrich) using chloroform (CHCl ₃ )-MeOH (3:1) as a solvent system, and DE4D2Bl (11.6 mg) and DE4D2B2 (652.3 mg) were separated. Obtained. DE4D2B2 was subjected to chromatography on a silica gel column (2 x 50 cm) and 4 fractions were collected by elution with CHCl ₃ , CHCl ₃ -MeOH (20:1) and CHCl ₃ -MeOH-H ₂ 0 (10:3:1). , DE4D2B2A (4.8 mg), DE4D2B2B (3.5 mg), DE4D2B2C (25.1 mg) and DE4D2B2D (620.5 mg) were obtained. Finally, DE4D2B2D was purified by preparative liquid chromatography (PLC) developed with CHCl ₃ -MeOH-acetic acid (AcOH)-H ₂ 0 (80:9:12:2) and combined with the MGDG preparation (578.9 mg). did.

2. Virucidal activity targeting herpes virus and influenza virus
Time-of-addition experiments and virucidal activity tests were performed to evaluate the virucidal activity of the MGDG preparation. The target viruses were HSV-2 and influenza A virus.

2-1. Time-of-addition experiment <method>
(1) Culture cells (Vero cells or MDCK cells) in a monolayer in a 48-well plate.
(2) Add the MGDG preparation (10 μg/ml, 25 μg/ml or 50 μg/ml) to the medium at the following addition times.
Test group 1: 3 hours before virus infection Test group 2: 1 hour before virus infection Test group 3: Only during virus infection (1 hour at room temperature) Test group 4: From 3 hours before virus infection to 24 hours after virus infection Test group 5: From 1 hour before to 24 hours after virus infection Test group 6: From 24 hours after virus infection Test group 7: From immediately after virus infection to 24 hours after virus infection Test group 8: From 1 hour to 24 hours after virus infection Test group 9: From 3 hours to 24 hours after virus infection Test group 10: From 6 hours to 24 hours after virus infection (3) Cultures were harvested 24 hours after infection, and cells were prepared separately in a 35 mm culture dish. Perform plaque assay using
(4) Using the number of plaques in a control in which a solvent (MEM medium) was added instead of the MGDG preparation as a standard (100%), calculate the virus proliferation rate for each test group.

<Results/Discussion>
The test results (average of two measurements) are shown in Figure 1 (targeting HSV-2) and Figure 2 (targeting influenza virus). HSV-2 proliferation was inhibited in a concentration-dependent manner of the MGDG preparation (Figure 1). At all concentrations, the effect in test group 3 (sample present only during virus infection) seemed to be strong. This seems to indicate that the MGDG preparation inhibits the adsorption/invasion stage of the virus into host cells, but in other test plots (test plots 4, 5, and 6) where samples were present at the same time, , as similar results were not obtained, this step cannot be considered as the target of action of the MGDG preparation. In addition, no significant inhibitory effect was observed during the period when viral protein and gene synthesis is taking place within cells (Test Groups 8, 9, and 10); It is unlikely that it acts selectively on the proliferation stage.

On the other hand, when targeting influenza virus (IFV), the concentration-dependent inhibitory effect on virus growth of the MGDG preparation was not as pronounced as compared to HSV-2 (Figure 2). In other respects, trends similar to those for HSV-2 were observed.

2-2. Viricidal activity test Virus inactivation effect (viricidal activity), which cannot be directly evaluated from the above-mentioned time-of-addition experiment, was investigated.
<Method>
(1) Add 0.1 ml of virus solution (2 x 10 ⁵ PFU/ml) and 0.1 ml of double-concentration sample solution (MGDG preparation with a concentration of 0.01 to 50 μg/ml after mixing) to a 1.5 ml tube and mix. and place it at 37℃.
(2) After 0 to 6 hours, collect 10 μl from each tube, transfer to ice, and add 990 μl of PBS to dilute 100 times.
(3) Immediately infect separately prepared host cells (35 mm culture dish) with 0.1 ml of this diluted solution (room temperature, 1 hour).
(4) Overlay the medium for plaque assay.
(5) After 2 days, confirm the appearance of plaques and fix and stain the cells with crystal violet solution.
(6) Measure the number of plaques.
(7) Calculate the relative number of plaques (%) for each test group using the number of plaques immediately after mixing the virus solution and diluent solution (PBS) (0 minutes) as the standard (100%).

<Results/Discussion>
The test results (average of two measurements) are shown in Figure 3 (targeting HSV-2) and Figure 4 (targeting influenza virus). When targeting HSV-2 (Figure 3), the control (MGDG preparation concentration 0 μg/ml) gradually reduced infectivity to approximately 40% during the 6 hour treatment period. The MGDG preparation killed more virus than the control in a concentration-dependent manner. In particular, the virucidal activity was extremely strong at concentrations of 10 μg/ml or higher, and at a concentration of 50 μg/ml, the infectivity was halved when mixed with the virus. Even at extremely low concentrations of 0.01 μg/ml (10 ng/ml), the MGDG preparation showed higher virus inactivation efficacy compared to the control.

On the other hand, the MGDG preparation also killed influenza virus (IFV) better than the control in a concentration-dependent manner (Figure 4). As with HSV-2, the MGDG preparation showed strong virucidal activity at concentrations above 10 μg/ml. However, compared to HSV-2, it took longer to inactivate the virus.

The above two test results revealed that the MGDG preparation had virucidal activity against both types of viruses (HSV-2 and IFV).

As shown in Figure 5, influenza virus (IFV) was treated with the MGDG preparation (50 μg/ml) (30 minutes, room temperature) and the morphology was observed. As a result, collapse of the viral envelope was observed, and the MGDG preparation It became clear that it exhibited an inert effect.

3. Virucidal activity targeting norovirus We evaluated whether the MGDG preparation exhibits virucidal activity against non-enveloped viruses. The MGDG preparation was evaluated for its inactivating effect on murine norovirus (MNV).
<Method>
(1) Dilute virus (MNV) stock with PBS (phosphate buffered saline) to 2×10 ⁵ PFU/ml.
(2) Add 0.5 ml of the sample (MGDG preparation at a predetermined concentration) and 0.5 ml of the virus solution to a 1.5 ml microtube and mix. As a control, add PBS instead of the sample.
(3) Place the sample and virus solution mixture at room temperature, collect 10 μl each at 0 minutes, 1 minute, 10 minutes, and 30 minutes and mix with 990 μl of PBS (100-fold dilution). Note that this dilution is to prevent the sample from affecting the measurement of the amount of residual virus, which will be performed below.
(4) Add 100 μl of the 100-fold diluted solution to host cells (RAW264.7 cells, which are mouse macrophage-like cells) that have been cultured in a monolayer in advance, and infect them for 1 hour at room temperature.
(5) Overlay with plaque assay medium and culture at 37°C for 2 to 3 days.
(6) After confirming the appearance of plaques, fix and stain the cells with neutral red solution, and measure the number of plaques under a microscope.
(7) Calculate the amount of remaining virus (relative plaque number) after each treatment time using the number of plaques at 0 hours as a reference (100%).

<Results/Discussion>
The MGDG preparation (1-100 μg/ml) exhibited virucidal activity against MNV in a concentration- and time-dependent manner (FIG. 6).

4. Viricidal activity targeting feline calicivirus We evaluated whether the MGDG preparation also exhibited virucidal activity against feline calicivirus (FCV), which is used as an alternative to norovirus.
<Method>
(1) Dilute virus (FCV) stock with PBS (phosphate buffered saline) to 2×10 ⁵ PFU/ml.
(2) Add 0.5 ml of the sample (MGDG preparation at a predetermined concentration) and 0.5 ml of the virus solution to a 1.5 ml microtube and mix. As a control, add PBS instead of the sample.
(3) Place the sample and virus solution mixture at room temperature, collect 10 μl each at 0 minutes, 1 minute, 10 minutes, 30 minutes, and 60 minutes and mix with 990 μl of PBS (100-fold dilution) . Note that this dilution is to prevent the sample from affecting the measurement of the amount of residual virus, which will be performed below.
(4) Add 100 μl of the 100-fold diluted solution to host cells (CRFK cells derived from cat kidney) that have been cultured in a monolayer in advance, and infect them for 1 hour at room temperature.
(5) Overlay with plaque assay medium and culture at 37°C for 2 to 3 days.
(6) After confirming the appearance of plaques, fix and stain the cells with neutral red solution, and measure the number of plaques under a microscope.
(7) Calculate the amount of remaining virus (relative plaque number) after each treatment time using the number of plaques at 0 hours as a reference (100%).

<Results/Discussion>
The MGDG preparation (10-200 μg/ml) exhibited virucidal (inactivating) activity against FCV in a concentration- and time-dependent manner (FIG. 7).

5. Examination of irreversibility of virucidal activity targeting herpesvirus (in vivo experiment using laboratory animals)
As mentioned above, it was confirmed by an in vitro assay using cultured cells that the MGDG preparation inactivates HSV-2. We evaluated whether this inactivation could be irreversibly maintained in vivo and suppress viral infection (genital herpes). Specifically, the virus treated with the MGDG preparation was inoculated into the genitals, and the viral load, lesion scores, and mortality rate were examined three days after infection.
<Method>
(1) BALB/c mice (6 weeks old, female) are injected subcutaneously with medroxyprogesterone (3 mg/0.1 ml/mouse) 6 days and 1 day before virus inoculation.
(2) On day 0, a virus solution (20 μl/mouse) treated as described below is inoculated into the genitals of the mouse.
Control: Mix the solvent (PBS) with the same amount of virus solution (HSV-2 (UW268 strain), 4 x 10 ⁴ PFU/20 μl) (final virus amount 2 x 10 ⁴ PFU/20 μl/mouse) and incubate for 30 Process for 1 minute at room temperature.
Test plot (MGDG preparation): Double concentration (20 μg/ml, 100 μg/ml, 500 μg/ml) of MGDG preparation was added to the same amount of virus solution (HSV-2 (UW268 strain), 4 x 10 ⁴ PFU/20 μl. ) (final virus amount 2 x 10 ⁴ PFU/20 μl/mouse) and incubate for 30 minutes at room temperature.
(3) Three days after infection, wash the genitals with PBS (100 μl/mouse), and quantify the virus in the wash by plaque assay.
(4) Observe the severity of the disease until 14 days after infection and evaluate it using a 6-level lesion score from 0 to 5.
(5) Record whether the mice are alive or dead until 14 days after infection.

<Results/Discussion>
(1) Time course of onset and death cases (Figure 8, Table 1)
In the control group, all animals (5 out of 5 animals) died by 10 days after infection. On the other hand, when the virus was pretreated with MGDG concentrations of 250 μg/ml and 50 μg/ml, no herpes symptoms were observed in any of the mice, and all of the mice survived. When treated with 10 μg/ml, 3 out of 5 animals developed symptoms and died, while 2 animals remained asymptomatic and survived.

(2) Viral load 3 days after infection (Figure 9)
No virus was detected in all mice when pretreated with virus at concentrations of 250 μg/ml and 50 μg/ml (***p<0.001). At a concentration of 10 μg/ml, virus was detected in 4 out of 5 animals, and the average value was significantly (*p<0.05) lower than that in the control group.

Considering the above results and the results of the in vitro virucidal activity test (item 2. above), it is clear that the MGDG preparation exerts an irreversible inactivating effect on HSV-2. Ta. This result is consistent with the result inferred from morphological observation that the virus envelope was destroyed (FIG. 10).

The results of evaluating the virucidal activity of the MGDG preparation in vitro and in vivo are summarized in Table 2 below.

6. Preparation of MGDG from Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (Spirulina) and Euglena gracilis Extraction of MGDG from Chlorella vulgaris, Nannochloropsis oculata, Arthrospira platensis (Spirulina) and Euglena gracilis do. Each algae is cultured on a medium suitable for each (for example, for Chlorella vulgaris, C medium shown in Figure 11, for Nannochloropsis oculata, ESM medium shown in Figure 11, Arthrospira platensis (Spirulina ), use the MA medium shown in Figure 12; for Euglena gracilis, use the HUT medium shown in Figure 12), and use the conventional method (adopt the same culture conditions as the above-mentioned Coccomyxa sp. KJ strain). ). Dry powder of each algae can be obtained by drying the algae collected from the culture solution by centrifugation using a drum dryer and pulverizing them using a pulverizer. MGDG preparations of each algae can be obtained from the dry powder by extraction operations similar to those for Coccomyxa sp. KJ strain. However, in this study, processed products of each algae (Chlorella vulgaris is "Chlorella vulgaris dried powder" (Aoi Seicha Co., Ltd.), Nannochloropsis oculata is "Nannochloropsis oculata frozen product" (Marine) Tech Co., Ltd.), Arthrospira platensis (Spirulina), ``Spirulina Powder'' (DIC Life Tech Co., Ltd.), and Euglena gracilis, ``Euglena Kiwami of Life'' (Six Sense Lab Co., Ltd.) and Biozyme (Euglena). , MGDG was extracted (the extraction procedure was the same as in the case of Coccomyxa sp. KJ strain). Each obtained MGDG preparation was used for the following studies. Commercially available spinach (Spinacia oleracea) was also used to extract finely divided MGDG after freeze-drying. MGDG preparation was obtained by grinding with a grinder and extracting MGDG.

7. Virucidal activity targeting norovirus concentrated in oysters (purification test of oysters contaminated with norovirus)
(1) Creation of contaminated oysters Live oysters are purchased, and after thoroughly cleaning the shell surface with a brush under running tap water, the shell surface is sterilized by leaving it in a clean bench and sterilizing it under ultraviolet light. Next, the oysters, whose shell surfaces have been treated with ultraviolet rays, are placed in filtered (pore diameter: 0.2 μm; manufactured by Millipore) sterilized seawater (water temperature: 10°C) and reared without food for a day and night while being aerated. Next, a stock of mouse norovirus prepared in advance is added to this filter-sterilized seawater at a final concentration of 2×10 ⁵ PFU/ml. The oysters are then raised for another day and night to produce oysters contaminated with mouse norovirus.

(2) Preparation of MGDG prepared seawater
MGDG preparation (Coccomyxa sp. KJ strain MGDG preparation, Chlorella vulgaris MGDG preparation, Nannochloropsis oculata MGDG preparation, Arthrospira platensis (Spirulina) MGDG preparation, Euglena gracilis MGDG preparation, Spinach MGDG preparation DMSO is added to MGDG or purified MGDG to homogeneously suspend it (MGDG suspension). Next, the MGDG suspension is added to 10 L of filtered (pore diameter: 0.2 μm; manufactured by Millipore) sterilized seawater so that the final concentration of MGDG is 100 ppm, to obtain cloudy MGDG-prepared sterile seawater.

(3) Purification test
In the MGDG treatment area, each contaminated oyster is stored in a beaker containing 1L of filter-sterilized seawater containing 100ppm of MGDG. In the control group (without MGDG treatment), one sample was placed in a beaker containing 1 L of filtered sterilized seawater to which MGDG was not added for comparison. The seawater temperature was set to 10℃ and the animals were kept for 48 hours with ventilation. After 48 hours have passed, remove the oysters from the seawater, remove them from their shells, place them on filter paper, and pat dry. Then, put it in a plastic bag and freeze it at -80℃. This operation preserves the mouse norovirus that accumulates in oysters without leaking out. Note that the number of experiments is set to be a sufficient number to find a significant difference through comparison.

(4) Evaluation of residual mouse norovirus Since mouse norovirus does not proliferate in the gastrointestinal tract of oysters, it is thought that basically all the added mouse norovirus is taken into the oysters and accumulated in the gastrointestinal tract. Thaw the oysters frozen in step (3), remove the digestive tract from the oyster, wash the inside of the digestive tract with a certain amount of phosphate buffered saline (PBS), and dilute the washing solution 100 times with PBS. 100 μl of this 100-fold dilution is added to host cells (RAW264.7 cells, which are mouse macrophage-like cells) that have been previously cultured in a monolayer, and the cells are infected at room temperature for 1 hour. Then, overlay with plaque assay medium and culture at 37°C for 2 to 3 days. After confirming the appearance of plaques, fix and stain the cells with neutral red solution, and measure the number of plaques under a microscope. Using the initial murine norovirus concentration of 2×10 ⁵ PFU/ml as a reference (100%), the amount of viable virus (relative plaque number) in the oyster digestive tract after 48 hours is calculated.

In the MGDG-treated area, the murine norovirus contaminated with oysters loses its ability to infect through contact with the ingested MGDG, and the results show that almost no plaques are generated from host cells.

8. Virucidal effect in the presence of organic matter Norovirus is contained in the vomit and feces of people infected with norovirus, and secondary infection due to these is a problem. Additionally, commonly used alcohol-based disinfectants are said to have low virucidal effects against norovirus, and chlorine-based disinfectants are mainly used. However, since chlorine is decomposed by organic matter contained in vomit, feces, etc., the virucidal effect of chlorine-based disinfectants is diminished. Therefore, we decided to investigate whether MGDG preparations exhibit virucidal effects even in the presence of organic substances.

<Virus, reagents, etc. used>
Virus: Feline calicivirus (FCV) is used as a substitute for norovirus Drugs used and concentration: Sodium hypochlorite or Coccomyxa sp. KJ strain MGDG preparation 1,000μg/ml each
Organic matter: Use 5% BSA (albumin) <Method>
(1) Prepare feline calicivirus (FCV) to 2×10 ⁵ PFU/ml.
(2) Prepare the MGDG preparation or sodium hypochlorite to a concentration 4 times the final assay concentration (1,000 μg/ml).
(3) Prepare bovine serum albumin (BSA) as an organic substance to a concentration 4 times the final concentration (5%).
(4) Mix virus solution: MGDG preparation or sodium hypochlorite: BSA = 2:1:1. MGDG preparations or samples with PBS added instead of sodium hypochlorite served as controls.
(5) Leave at room temperature for 1 minute, 10 minutes, and 60 minutes, then dilute 100 times with PBS. Add 100 μl/culture dish to CRFK cells cultured in a monolayer on a 35 mm culture dish and infect for 1 hour at room temperature.
(6) Overlay MEM medium supplemented with 1% SeaPlaque Agarose.
(7) After 2 days, the plaques that appear are fixed and stained with crystal violet solution.
(8) Calculate the number of plaques under a microscope.

<Results>
As shown in Figure 13, sodium hypochlorite inactivated 100% of the virus in 1 minute in the absence of organic matter (BSA). However, in the presence of 5% organic matter, the inactivation effect was significantly weakened. On the other hand, the inactivation effect of MGDG was almost unchanged even in the presence of organic matter, and showed a higher inactivation effect compared to sodium hypochlorite.

9. Effect on cold sores
The effect of MGDG preparation on cold sores was verified.
<Method>
(1) Prepare 30g of hand cream (Moina, manufactured by Denso).
(2) Add 0.165 g of Coccomyxa sp. KJ strain MGDG preparation to 0.610 g of olive oil (manufactured by J-Oil Mills Co., Ltd.), knead, and dissolve (MGDG dissolved product).
(3) Add the MGDG melt little by little to the hand cream prepared in (1) while kneading it until it becomes uniform, and use the mixture after all the addition as a sample for application.
(4) Apply an appropriate amount to the affected area of patients with cold sores (the following two patients were used as subjects) every day (1 to 3 times) and observe the condition of the affected area over time.
Patient 1: 45-year-old female; had experienced cold sores (multiple times). It is usually treated with the over-the-counter drug Aracena S (Sato Pharmaceutical Co., Ltd.), which contains vidarabine as the active ingredient, and it takes two to three weeks to heal.
Patient 2: 25-year-old male; had experienced cold sores (multiple times). Usually, no medication is used (you wait for it to heal naturally), and the condition recovers to an unnoticeable level after 2 to 3 weeks.

<Results/Discussion>
The observation results are shown in FIG. 14 (patient 1) and FIG. 15 (patient 2). In patient 1, a clear improvement was observed on the 3rd day of application, and the condition was close to complete recovery on the 5th day of application (Figure 14). In the case of patient 2, the therapeutic effect was observed from the second day of application, and the patient was almost fully cured on the eighth day of application. Thus, the MGDG preparation exhibited excellent therapeutic effects against cold sores. What is noteworthy is that a surprising therapeutic effect that can be said to surpass that of commercially available drugs was demonstrated (Patient 1).

10. Identification of components in MGDG preparations 1
HPLC analysis estimated that the MGDG preparation of Coccomyxa sp. KJ strain contained five types of MGDG (referred to as MGDG1, MGDG2, MGDG3, MGDG4, and MGDG5). In order to clarify the structure of these MGDGs, the fatty acid composition was analyzed using gas chromatography (GC/FID). A BPX90 column (length 100 m x inner diameter 0.25 mm, film thickness 0.25 μm) was used for the 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.

Chromatograms of each sample (MGDG1, MGDG2, MGDG3, MGDG4, MGDG5) are shown in Figures 16 to 20. Regarding C6:0, the fatty acid composition of common lipids usually includes fatty acids with adjacent even number of carbon atoms, but these were hardly detected in this analysis. Therefore, it was thought that there was a high possibility that the same peak was not a fatty acid. In addition, Unknown1 and Unknown2 were considered to be highly likely to be components other than fatty acids, since the fatty acids detected at the retention times of these peaks are difficult to imagine in common lipids. On the other hand, based on the retention time, Unknown3 and Unknown4 were estimated to be C16:2 and C18:2 (other than n-6), respectively.

「－」は不検出を表す。

「－」は不検出を表す。The results of the analysis are shown in Tables 3 and 4 below. Table 3 shows the fatty acid composition ratio of each sample using the area percentage method. Table 4 summarizes the results calculated by excluding C6:0 and unknown components 1 and 2 (Unknown1, 2). In addition, regarding 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 approximately 1:1.

"-" indicates non-detection.

"-" indicates non-detection.

As mentioned above, MGDG preparation contains (1) monogalactosyldiacylglycerol (MGDG1) whose constituent fatty acids are C16:3 and C18:3 (n-3), (2) constituent fatty acids are C16:2 and C18:2. (3) Monogalactosyldiacylglycerol (MGDG3) whose constituent fatty acids are C18:2 and C18:3 (n-3), (4) Monogalactosyldiacylglycerol (MGDG3) whose constituent fatty acids are C18:2 and C18:2. Monogalactosyldiacylglycerol (MGDG3), (5) Monogalactosyldiacylglycerol (MGDG4) whose constituent fatty acids are C16:2 and C18:2(n-6), and (6) Monogalactosyldiacylglycerol (MGDG4) whose constituent fatty acids are C18:2(n-6). and C18:3(n-3) monogalactosyldiacylglycerol (MGDG5).

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

<Results>
In addition to the five MGDGs detected by preparative liquid chromatography (PLC) (see section 10. Identification of components in MGDG preparations), six new MGDGs were detected by triple quadrupole LC/MS analysis. MGDG was detected in the eye. For these MGDGs (6 peaks), each MGDG (peak 1: MGDG1, peak 2: MGDG2, peak 3: MGDG3, peak 4: MGDG4, peak 5: MGDG5, peak We were able to identify two fatty acid side chains that constitute 6:MGDG6). The fatty acid side chains of each MGDG are shown in the table below.

（３）（２）の混合液をウシ胎児血清不含MEM培地で希釈後、プラークアッセイ用培地を重層し、37℃で2～3日間培養した。
（４）プラークの出現を確認後、ニュートラルレッド液で細胞を固定・染色し、顕微鏡下でプラーク数を測定した。サンプルを添加しなかった場合のウイルスの出現数を基準(100％)とし、各サンプルのウイルス残存率を算出した。ウイルス残存率から各サンプルの殺ウイルス性能を評価した。12. Viricidal activity of each MGDG fraction
The virucidal activity of each MGDG (MGDG1 to MGDG5) constituting the MGDG preparation was compared.
<Method>
(1) An MGDG preparation derived from Coccomyxa sp. KJ strain was subjected to HPLC, and each peak was fractionated to obtain five types of MGDG purified products (MGDG1, MGDG2, MGDG3, MGDG4, MGDG5). In addition, the MGDG preparation (containing MGDG1 to MGDG5) before fractionation was prepared as a Mix product.
(2) Each sample was mixed with each virus solution (2×10 ⁵ PFU/ml) so that the final concentration was 50 μg/ml, and the mixture was allowed to stand at 37° C. for 60 minutes. Table 6 shows the viruses and host cells used.

(3) After diluting the mixture of (2) with MEM medium containing fetal bovine serum, it was overlaid with a plaque assay medium and cultured at 37°C for 2 to 3 days.
(4) After confirming the appearance of plaques, the cells were fixed and stained with neutral red solution, and the number of plaques was measured under a microscope. The virus survival rate of each sample was calculated using the number of viruses that appeared when no sample was added as a standard (100%). The virucidal performance of each sample was evaluated based on the virus residual rate.

<Results/Discussion>
Although there were differences in activity depending on the target virus, virucidal (inactivating) activity was observed for the MGDG preparation (Mix product) and all of MGDG1 to MGDG5 (FIG. 21). MGDG5 showed a significant inactivating effect on influenza virus and herpes simplex virus type 2 compared to the MGDG preparation (Mix product). None of the non-enveloped feline caliciviruses and polioviruses showed significantly higher activity than other samples. The above results confirm that the MGDG preparation and each MGDG that constitutes it have virucidal activity, and that MGDG5 has particularly high virucidal activity against enveloped viruses such as influenza virus and herpes simplex virus type 2. Demonstrates the ability to demonstrate

13. Comparison of components of various MGDG preparations Coccomyxa sp. KJ strain MGDG preparation, Chlorella vulgaris MGDG preparation, Nannochloropsis oculata MGDG preparation, Arthrospira platensis (Spirulina) MGDG preparation, Euglena gracilis MGDG preparation and Spinach MGDG preparations were analyzed by reverse-phase HPLC and the content (MGDG) was compared.
<HPLC analysis conditions>
Device name: Alliance2695
Column: YMC-Actus Triart C18
Mobile phase: methanol, water, acetonitrile mixed solution Detection wavelength: 205nm

The HPLC measurement results (chromatograms) are shown in FIGS. 22 and 23. The vertical axis of the chromatogram is normalized so that the maximum peak value in each sample is 1.
-At least 5 peaks were detected in the MGDG preparation of 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 matched or was very close to that of the MGDG preparation of the KJ strain was detected. The peaks with matching retention times are estimated to have the same chemical structure as MGDG derived from KJ strain.
-Virucidal activity against HSV-2, IFV and FCV was observed in the five peaks derived from KJ strain MGDG preparation (Experiment 12, Figure 21). As mentioned above, in the MGDG preparations of each organism other than the KJ strain, at least one peak corresponding to the five peaks derived from the KJ strain MGDG preparation is observed, so it is presumed that they have virucidal activity as well. Ru. That is, these MGDG preparations can be expected to have the same effects as the KJ strain MGDG preparations. The fifth peak of the KJ strain MGDG preparation is known to have high virucidal activity against HSV-2 and IFV (Figure 21).

The virucidal agent of the present invention can be expected to have an irreversible inactivation effect by destroying virus particles, and exhibits a killing effect not only on a specific virus but also on a plurality of viruses. The virucidal agent of the present invention is expected to be used in a wide range of applications (eg, prevention and treatment of infectious diseases such as norovirus, herpes simplex virus, and influenza virus). On the other hand, the virucide of the present invention can be expected to have high safety because its active ingredient is derived from unicellular algae (preferably microalgae).

This invention is in no way limited to the description of the embodiments and examples of the invention described above. This invention includes various modifications that can be easily conceived by those skilled in the art without departing from the scope of the claims. All contents of articles, published patent publications, patent publications, etc. specified in this specification are cited by reference.

Claims (6)

Kokkomixa sp. KJ
Use of monogalactosyldiacylglycerol derived from a strain or a mutant strain thereof for the production of an influenza virus killer or a feline calicivirus killer, comprising:
The influenza virus killing agent or the feline calicivirus killing agent is used for the influenza virus killing agent or the feline calicivirus agent. A use characterized by containing monogalactosyldiacylglycerol of KJ strain or its mutant strain as an active ingredient. The use according to claim 1 , 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. The use according to claim 1 or 2 , wherein the influenza virus killing agent or feline calicivirus agent exhibits virucidal activity through irreversible inactivation. The use according to any one of claims 1 to 3 , wherein the influenza virus killing agent or feline calicivirus agent is a medicine for influenza virus infection or feline calicivirus infection . The use according to any one of claims 1 to 3 , wherein the influenza virucidal agent or feline caliciviricidal agent is a disinfectant or a germicidal cleaning agent. The use according to any one of claims 1 to 3, wherein the influenza virus killing agent or feline calicivirus killing agent is bait .

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