JP5971949B2 - Antiviral agent - Google Patents

Description

The present invention relates to antiviral agents, in particular, it relates to a neutral polysaccharide that certain lactic acid produced in including antiviral agents.

  Today, human beings are constantly being exposed to new virus threats, including the new influenza that is rampant around the world. In particular, respiratory infections such as influenza, including seasonal ones, are more likely to occur for those who have weakened immunity, such as elderly people, because the affected people are likely to be present in their daily lives. It's easy to do. In addition, there is a high possibility of falling seriously, and the mortality rate associated with complications is very high.

  Currently, antiviral drugs that act on the virus itself and are effective against such viral respiratory infections are used. These antiviral drugs are extremely effective against each viral infection, and the symptoms can be quickly alleviated by taking them early after onset. In recent years, neuraminidase inhibitors have been developed that inhibit neuraminidase proteins on the surface of viruses and prevent the growth of viruses in the body. Among them, as effective against influenza A and influenza B, osetamivir (trade name: Tamiflu; Tamiflu is a registered trademark) prescribed as an oral capsule and zanamivir (trade name: Relenza; Relenza) A registered trademark is used in practice. However, these drugs are drugs that suppress the growth of viruses and need to be taken early after infection. For example, it must be taken within 48 hours after symptoms appear. Also, these drugs must be taken continuously for a certain period.

  In addition, prevention by vaccination is also carried out. However, vaccines must be manufactured by specifying the target virus species in advance, and mass production is very time consuming. In addition, vaccination has the only effect of reducing the risk of increasing severity after infection. Furthermore, vaccination requires a certain period of time for the antibody to become established, must be vaccinated before it becomes epidemic, and if the type of vaccine inoculated differs from the type of virus actually infected, There is also a problem that the effect cannot be exhibited.

  Under such circumstances, it is urgent to develop antiviral agents that are not only safe but also effective when taken in a preventive manner, and that are effective against any virus when taken. Yes.

  On the other hand, in order to cope with the threat of viruses, it is also important to increase the natural immunity that normally prevents the invasion and proliferation of viruses into the living body at the forefront. White blood cells play a particularly important role in innate immunity. Among leukocytes, NK cells (also referred to as natural killer cells) spontaneously attack infected cells while the virus infection is small, and suppress the proliferation and spread of the virus.

In vivo, when a viral infection is detected, type I IFN including interferon (IFN) -α is produced. This type I IFN stimulation is essential for the activation of NK cells. It has been shown that NK cells are activated by type I IFN released from virus-infected cells. Therefore, by enhancing the degree of NK cell activity by virus detection, it is possible to suppress subsequent virus growth and prevent the spread of infection. In addition, NK cells, are known to attack the virus-infected cells. Therefore, it can be said that NK cells are effective not only for specific viruses like vaccines but also for a wide variety of viral infections.

  Under the circumstances described above, substances that promote the activation of NK cells are being searched for in various fields. Examples of what has been known to be effective to some extent include polysaccharides (eg, lentinan, chitin, chitosan, maitake D-fraction, nigerooligosaccharide, etc.) and bacterial cells (Lactobacillus casei shirota, Bifidobacterium lactis HN019, etc.) It is known (Non-patent Documents 1 to 6, Patent Document 1).

Also, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (hereinafter sometimes abbreviated as “1073R-1 strain”) is a neutral polysaccharide (NPS) and an acidic polysaccharide (APS) in which a phosphate group is added to the neutral polysaccharide. : Acidic polysaccharide). The present inventors have clarified that acidic polysaccharide (APS) has an effect of enhancing the activity of NK cells (Patent Document 2). For acidic polysaccharide (APS), spleen cells are used as effector cells, YAC-1 cells as target cells, and NK cell activity (hereinafter sometimes abbreviated as NK activity) by FACS (fluorescence activated cell sorter). Evaluating. However, for the neutral polysaccharide (NPS), neither the above-mentioned NK cell activity nor the NK cell activity upon stimulation with IFN-α corresponding to viral infection has been evaluated. Therefore, an immunostimulatory effect such as an enhancing effect of NK cell activity by neutral polysaccharide (NPS) has not been confirmed.
In addition, the isolation method of neutral polysaccharide (NPS) is reported in various literature (patent document 3).

JP 2001-31573 A JP 2005-194259 A JP 2000-247895 A

Akiyama Yukio, Hamuro Kaoru, Protein Nucleic Acid Enzyme, 26 (3), 208-224 (1981) Nishimura K, Nishimura S, Nishi N, Numata F, Tone Y, Tokuura S, Azuma I, Vaccine, 3 (5), 379-84 (1985) Kodama N, Komuta K, Sakai N, Nanba H, Biol Pharm Bull, 25 (12), 1647-50 (2002) Murosak S, Muroyama K, Yamamoto Y, Liu T, Yoshikai Y, Int Immunopharmacol, 2 (1), 151-9 (2002) Hori T, Kiyoshima J, Yasui H, Biosci Biotechnol Biochem, 67 (2), 420-2 (2003) Gil HS, Rutherfurd KJ, Cross ML, Gopal PK, Am J Clin Nutr, 74 (6), 833-9 (2001)

  As described above, various substances for promoting the activation of NK cells have been searched. However, it has not always been possible to obtain a sufficient activity promoting effect, and no evaluation study has been made on the increase in the activity of NK cells after stimulation with IFN-α.

  Therefore, an object of the present invention is to provide an antiviral agent that can be safely and easily ingested and enhances the increase in activity that occurs when NK cells responsible for a part of innate immunity obtain a viral stimulus. There is.

  In view of the above-mentioned conventional problems, the present inventors have conducted intensive research and have thought that they can be safely and easily ingested in daily life if they are derived from materials used as food. In particular, it is known that food-grade microorganisms used for yogurt fermentation can produce various useful substances. The present inventors pay attention to these food-use microorganisms, isolate a polysaccharide produced by a certain type of fungus, clarify the structure of the polysaccharide, and that the polysaccharide has antiviral activity. I found. That is, the present inventors have found that the increase in the activity of NK cells after stimulation with IFN-α is enhanced by orally administering this polysaccharide to mice before stimulation with IFN-α. The present invention has been completed.

  That is, one embodiment of the present invention is an antiviral agent containing a neutral polysaccharide produced by lactic acid bacteria as an active ingredient.

  Preferred examples of the lactic acid bacteria used for the preparation of the antiviral agent of the present invention include Lactobacillus delbrueckii ssp. bulgaricus. As a particularly preferred example of the lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1.

  The antiviral agent of the present invention is an antiviral agent that is a neutral polysaccharide produced by lactic acid bacteria, and that contains as an active ingredient a neutral polysaccharide that can increase the activity of NK cells in response to stimulation with IFN-α. It is preferable that it is an agent.

  The neutral polysaccharide has a structural unit represented by the following formula (1).

  Moreover, according to another aspect of this invention, the antiviral food-drinks composition containing the effective amount of neutral polysaccharide which lactic acid bacteria produce is provided.

  Preferred examples of the lactic acid bacteria used for the preparation of the antiviral food and beverage composition of the present invention include Lactobacillus delbrueckii ssp. bulgaricus. As a particularly preferred example of the lactic acid bacteria, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (may be abbreviated as “1073R-1 strain” in the present specification).

  The antiviral food and beverage composition of the present invention is a neutral polysaccharide produced by lactic acid bacteria, and an effective amount of a neutral polysaccharide that can increase the activity of NK cells in response to stimulation with IFN-α. It is preferable that it is an antiviral food / beverage composition containing.

  The antiviral food or drink composition of the present invention is preferably a fermented milk food or drink. Among fermented milk foods and drinks, yogurt foods and drinks such as yogurt and yogurt drink are particularly preferable.

  In the antiviral food and beverage composition of the present invention, the neutral polysaccharide is 0.002 to 0.014% (w / w), particularly 0.002 to 0.004, based on the total amount of the composition. It is preferably contained in an amount of% (w / w).

  The said neutral polysaccharide contained in the antiviral food-drinks composition of this invention has a structural unit shown by the said Formula (1).

  The antiviral agent and antiviral food / drink composition of the present invention can be safely and easily ingested, and when NK cells responsible for part of innate immunity have obtained viral stimulation, the activity of NK cells Increase the degree of increase.

It is a graph which shows the ratio (%) of the dead cancer cell to the whole cancer cell as a parameter | index of the degree of the activity of the NK cell in a spleen cell before the stimulation by IFN- (alpha). It is a graph which shows the ratio (%) of the dead cancer cell which occupies for the whole cancer cell as a parameter | index of the degree of the activity of the NK cell in a spleen cell after stimulation by IFN- (alpha).

  Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.

The lactic acid bacteria used in the present invention are not limited as long as they are lactic acid bacteria that produce neutral polysaccharides. Lactic acid bacteria may be used individually by 1 type, and may be used in combination of 2 or more type.
Examples of lactic acid bacteria that produce neutral polysaccharides include Lactobacillus delbrueckii ssp. Is mentioned. Among these, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (deposit number: FERM BP-10741) is preferred.
As for the neutral polysaccharide in the antiviral agent or antiviral food / beverage product composition of the present invention, one kind may be contained alone, or two or more kinds of different kinds may be contained.

  As the neutral polysaccharide used in the present invention, a culture of lactic acid bacteria containing the neutral polysaccharide may be used as it is without purification, or by using the method described in JP 2000-247895 A or the like. Alternatively, neutral polysaccharides isolated from a culture of lactic acid bacteria containing neutral polysaccharides or further purified as necessary may be used.

The isolation method or the purification method is not particularly limited, and examples thereof include an isolation and purification method according to the following procedure.
1. Trichloroacetic acid is added to the medium to a final concentration of 10% to denature the protein and obtain a culture containing the denatured protein.
2. The denatured protein and microbial cells are removed from the culture by centrifugation to obtain a polysaccharide-containing product.
3. The high molecular weight polysaccharide in the polysaccharide-containing material is precipitated by ethanol precipitation, and the precipitate is collected.
4). The acidic polysaccharide in the liquid obtained in 3 above is adsorbed with an anion exchange resin, and the neutral polysaccharide-containing material is recovered from the remaining eluate.
5. The neutral polysaccharide-containing material is treated with DNase and RNase to decompose the nucleic acid.
6). The neutral polysaccharide-containing material after the treatment in 5 is subjected to proteinase treatment to decompose the protein.
7). The neutral polysaccharide-containing material after the treatment of 6 is heated at 90 ° C. for 10 minutes to inactivate the enzyme.
8). The neutral polysaccharide-containing material after the treatment of 7 is subjected to ethanol precipitation and dialysis to purify the neutral polysaccharide.

  The neutral polysaccharide obtained from the culture of the 1073R-1 strain by the above isolation and purification method is a polysaccharide having a structural unit represented by the following formula (1) and an estimated molecular weight of about 4.3 MDa.

  The neutral polysaccharide used in the present invention is not only used as a culture of the lactic acid bacteria, but is also a concentrate of the culture, a pasted product processed into a paste, a spray-dried product, a freeze-dried product, a vacuum-dried product, a drum The present invention can be used as an object to be processed such as a dried product, a liquid material dispersed in a medium, a diluted product diluted with a diluent, or a crushed product obtained by crushing a dried product with a mill or the like. The said process process may be individual or may use multiple together.

The dosage of the antiviral agent of the present invention containing a neutral polysaccharide as an active ingredient can be appropriately set in consideration of various factors such as the route of administration and the age, weight, and symptoms of animals to be administered including humans. it can. The dosage of the antiviral agent of the present invention is not particularly limited, but is preferably 1 mg / kg / day (day) or more, more preferably 5 mg / kg / day or more, as the amount of the neutral polysaccharide as the active ingredient. Particularly preferably, it is 10 mg / kg / day or more. However, when it is taken for the purpose of prevention and / or treatment over a long period of time, the dose of the antiviral agent may be smaller than the above preferred amount. In addition, since the active ingredient used in the present invention is contained in yogurt as a food product and there is no problem in terms of safety, the dosage of the antiviral agent is that of the neutral polysaccharide that is the active ingredient. The amount may be an amount (for example, 100 mg / kg / day) that greatly exceeds the above amount (1 mg / kg / day).
In addition, each value (for example, 1 mg / kg / day or more) regarding the quantity of the said neutral polysaccharide is applied not only to an antiviral agent but also to the food / beverage product composition described later.
The antiviral agent of the present invention can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, transdermal, etc.).

  The dosage form of the antiviral agent of the present invention can be selected according to the therapeutic purpose, administration route, etc., for example, tablets, coated tablets, pills, capsules, granules, powders, liquids, suspensions. , Emulsion, syrup, injection, suppository, soaking agent, decoction, tincture and the like. These various preparations are prepared in accordance with conventional methods, as necessary, with respect to the active ingredient, fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, flavoring agents. It can be formulated using known adjuvants that can be generally used in the pharmaceutical formulation technical field, such as agents, solubilizers, suspensions, and coating agents. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.

The food / beverage product composition of the present invention contains an effective amount of a neutral polysaccharide produced by lactic acid bacteria.
Examples of the form of the food and drink composition include fermented milk such as yogurt, beverages, health functional foods, foods for the sick, and the like. Japan's health functional food system covers not only ordinary foods but also foods with shapes such as tablets and capsules, based on trends in Japan and overseas and consistency with conventional food systems for specified health use. It is provided and consists of two types of foods for specified health use (individual permission type) and functional foods for nutrition (standard specification type). In the present invention, it is possible to prevent and / or treat viral infection by directly ingesting a food or drink containing a neutral polysaccharide that activates NK cells as a food for specified health use or a food with nutritional function.

  As the food / beverage composition of the present invention, various food / beverage products (for example, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared powdered milk, liquid food, food for the sick, for infants Foods such as powdered milk, foods such as milk powder for breastfeeding women, and nutritional foods) to which neutral polysaccharides that activate NK cells are added. In this case, the neutral polysaccharide as an active ingredient may be added as it is, or may be added after mixing with other foods or food ingredients. Neutral polysaccharides can be used according to conventional methods in ordinary food compositions. Moreover, the property of the food / beverage product composition of the present invention may be a normal property of the food / beverage product, for example, solid (powder, granule, etc.), paste, liquid or suspension.

The main component of the food and drink composition of the present invention is not particularly limited, but water, protein (including degradation products), carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, and flavors. Etc. can be used.
Examples of the protein or its degradation product include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein. , Β-lactoglobulin, α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and vegetable proteins, hydrolysates thereof; whey, cream, non-protein nitrogen and other milk-derived components .
Examples of sugars include processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and lactose.
Examples of lipids include various milk-derived components such as butter, cream, and phospholipid; animal oils such as lard, fish oil, fractionated oils, hydrogenated oil, transesterified oil; palm oil, safflower oil, corn Examples thereof include vegetable oils such as oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils and transesterified oils.
Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And folic acid.
Examples of the minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey mineral.
Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid.
These components can be used in combination of two or more. Moreover, the main component of the food-drinks composition of this invention may be a synthetic product, and may contain many synthetic products.

  In the present invention, the content of the neutral polysaccharide can be arbitrarily determined according to its purpose, application (antiviral agent, food / beverage product composition) and the like, and is not particularly limited. Preferably it is 0.002-0.014% (w / w) with respect to the whole quantity of a thing, More preferably, it is 0.002-0.004% (w / w).

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by these Examples. In the present specification, “%” indicates “% by weight” unless otherwise indicated.

[Example of production of neutral polysaccharide (NPS)]
The skim milk powder was dissolved in MilliQ water at 10% (w / v) and sterilized at 120 ° C. for 7 minutes to prepare a medium. Using this medium, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (1073R-1 strain) was cultured at 37 ° C. for 18 hours under aerobic conditions.
100% trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) was added to the culture solution so that the final concentration was 10%, and the mixture was stirred well until the viscosity disappeared. The supernatant was recovered by centrifuging the obtained culture solution (12000 g, 4 ° C., 20 minutes). While stirring the supernatant well, an equal volume of cold ethanol was added. Thereafter, the resulting liquid was allowed to stand at 4 ° C. overnight, thereby precipitating a mixture of neutral polysaccharide (NPS) and acidic polysaccharide (APS). The precipitated mixture was recovered by centrifuging the precipitate-containing material. The collected mixture was dissolved in MilliQ water. The obtained aqueous solution was dialyzed against MilliQ water using a dialysis membrane (MWCO 3500, manufactured by SPECTRAM). Furthermore, the aqueous solution after dialysis was made into a 0.02M Tris-HCl (pH 8.6) solution.
This aqueous solution was charged into a column packed with an anion exchange resin DEAE Sepharose Fast Flow (trade name; manufactured by GE Healthcare) to adsorb the acidic polysaccharide (APS) to the column. The aqueous solution that passed through the column at this time contained neutral polysaccharide (NPS), and the aqueous solution containing this neutral polysaccharide (NPS) was recovered. The recovered aqueous solution containing neutral polysaccharide (NPS) was dialyzed against MilliQ water using a dialysis membrane. After completion of dialysis, an aqueous solution containing neutral polysaccharide (NPS) was used as a 0.05 M Tris-HCl (pH 8.0) solution containing 1 mM MgCl ₂ . To this solution, DNase I (Type II, manufactured by Sigma) and RNase A (manufactured by Sigma) are added so as to have a final concentration of 2 μg / ml, and the nucleic acid is digested by incubating at 37 ° C. for 6 hours. went. Thereafter, Proteinase K (manufactured by Sigma) was added so that the final concentration was 0.1 mg / ml, and the protein was digested by incubation at 37 ° C. for 16 hours. After completion of the enzyme treatment, the enzyme was inactivated by heating the solution at 90 ° C. for 10 minutes. The obtained aqueous solution containing the neutral polysaccharide (NPS) is cooled to 4 ° C., and then added with an equal amount of cold ethanol while stirring well, and then allowed to stand at 4 ° C. overnight, whereby the neutral polysaccharide ( NPS) was precipitated. The neutral polysaccharide (NPS) was recovered as a precipitate by centrifugation, and the recovered neutral polysaccharide (NPS) was dissolved in MilliQ water to obtain a neutral polysaccharide (NPS) aqueous solution. A neutral polysaccharide (NPS) aqueous solution was dialyzed against MilliQ water using a dialysis membrane and then freeze-dried.

<Example 1>
[Example of evaluation of NK activity by oral administration to mice]
Twenty BALB / c mice (female, 7 weeks old) were acclimated for 1 week, then weighed, and divided into two groups (average weight: about 21 g) where n = 10 and the average weight was almost the same. In one group, 0.4 ml / day of distilled water was administered to each individual as a control group, and in the other group, neutral polysaccharide (NPS) was administered to each individual at a dose of 20 μg (0.4 ml) / day. Oral administration was performed weekly. Thereafter, the mice were euthanized by cervical dislocation, and spleen cells were collected and prepared.
About the prepared spleen cell, the NK activity was measured using FACS by making YAC-1 cell into a target cell. The ratio of spleen cells to YAC-1 cells was set to 50: 1. The NK activity of spleen cells was measured before and after stimulation with IFN-α. Stimulation with IFN-α was performed by incubating spleen cells seeded in a 96-well (round bottom) microplate in a medium supplemented with IFN-α at 1000 units / ml for 4 hours.

  The obtained results were compared before and after stimulation with IFN-α. As a result, when the spleen cells were not stimulated with IFN-α, the NK activity of the NPS administration group was hardly changed compared to the distilled water administration group (control group) (FIG. 1). On the other hand, regarding the NK activity after spleen cells were stimulated with IFN-α, the NK activity in the NPS administration group was significantly increased compared to the distilled water administration group (control group) (FIG. 2). In FIGS. 1 and 2, “distilled water” means a distilled water administration group (control group). “NPS” means an NPS administration group.

<Example 2>
When 1073R-1 strain, which is a lactic acid bacterium producing NPS in which an effect of enhancing NK activity after stimulation with IFN-α in Example 1, was observed, yogurt was produced as a lactic acid bacterium food and drink by a conventional method. A desired antiviral yogurt food containing a neutral polysaccharide (NPS) corresponding to the food / beverage composition of the present invention could be obtained.

<Example 3>
A lactic acid bacterium concentrate was prepared by a conventional method using 1073R-1 strain, which is a lactic acid bacterium producing NPS, which was confirmed to have an effect of enhancing NK activity after stimulation with IFN-α in Example 1, and this lactic acid bacterium concentrate A desired antiviral tablet containing a neutral polysaccharide (NPS) corresponding to the antiviral agent of the present invention, wherein a tablet is produced using an appropriate pharmaceutical additive in combination with a conventional method. Got.
In addition, you may use the lactic acid bacteria dried material instead of the said lactic acid bacteria concentrate. Moreover, it can also formulate as a capsule or a syrup instead of the said tablet.

  As described above, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (1073R-1 strain) in which the neutral polysaccharide purified from the non-fat dry milk medium culture was administered, the NK activity of the spleen cells when stimulated with IFN-α was higher than that in the group administered with distilled water. Significantly increased. Therefore, it is suggested that the neutral polysaccharide produced by the 1073R-1 strain exhibits antiviral activity by enhancing the increase in NK activity in response to IFN-α produced when a living body is infected with a virus. Is done. Moreover, since the yogurt which used 1073R-1 strain | stump | stock for fermentation contains the said neutral polysaccharide, antiviral activity is obtained also about this yogurt. That is, by taking safe and inexpensive foods every day, it is possible to expect a prophylactic effect that is possible before the virus infection, in which natural immunity is normally increased, and there is no virus specificity like a vaccine.

Claims (3)

Lactobacillus delbrueckii ssp. neutral polysaccharide is produced of bulgaricus an anti-viral agent comprising as an active ingredient,
The neutral polysaccharide can increase the activity of NK cells in response to stimulation with IFN-α, compared to the case where the neutral polysaccharide is not given, and the following formula (1):

The antiviral agent which is a neutral polysaccharide which has a structural unit shown to.   The lactic acid bacterium is Lactobacillus delbrueckii ssp. The antiviral agent according to claim 1, which is bulgaricus OLL1073R-1 (deposit number: FERM BP-10741). The antiviral agent according to claim 1 or 2 , wherein the antiviral agent is for administration so that the amount of the neutral polysaccharide is an effective amount of 1 mg / kg / day or more.

Images