Patent Application
20120202771
The present invention relates to a therapeutic agent for an influenza virus infectious disease.
An influenza virus infectious disease causes symptoms such as sore throat, nasal discharge, high fever, headache, and arthralgia, and the symptoms may become serious in combination with bronchitis, pneumonia, or the like. In particular, the disease has a high mortality for elderly people or the like. In addition, group infection with the virus in schools and elderly facilities causes a social epidemic involving many people.
A conventional basic countermeasure for the influenza virus infectious disease is prevention by vaccination. However, antigenicity of the influenza virus is easily altered, and it has been difficult to completely control the epidemic only by vaccination. Further, it is difficult to produce a large amount of a vaccine in a short period of time, and there is a problem in that no countermeasure exists in a case of, for example, emergence of a novel influenza virus or an epidemic of an unexpected strain of the virus.
In recent years, to solve such problems, there have been used anti-influenza virus drugs such as oseltamivir (product name “Tamiflu,” Chugai Pharmaceutical Co., Ltd.), amantadine (product name “Symmetrel,” Novartis Pharma K.K.), and zanamivir (product name “Relenza,” GlaxoSmithKline). However, the drugs have been found to have problems such as unavailability because their effects may be less effective if the drugs are not administered within 48 hours of the onset of the disease, side effects including ones having unclear causal relationships, and appearance of a resistant virus.
On the other hand, Patent Literature 1 below describes that a culture containing β-1,3-1,6-glucan, obtained by culturing a bacterium belonging to Aureobasidium sp., is used as an active ingredient for an immunopotentiator.
A conventional antivirus composition utilizing an active ingredient derived from a natural product cannot be said to have sufficient effectiveness.
An object of the present invention is to provide a therapeutic agent for an influenza virus infectious disease, which utilizes an active ingredient derived from a natural product and has an excellent effect.
The inventors of the present invention have made extensive studies to achieve the object, and as a result, have completed the present invention. That is, the present invention is as follows.
[1] A therapeutic agent for an influenza virus infectious disease, including, as an active ingredient, a β-glucan-containing composition obtained from a culture of a microorganism belonging to Aureobasidium sp., the therapeutic agent being used for preventing an influenza virus infectious disease from becoming serious and for promoting healing of the influenza virus infectious disease.
[2] A therapeutic agent for an influenza virus infectious disease according to the above-mentioned item [1], in which the microorganism belonging to Aureobasidium sp. is Aureobasidium pullulans M-1 having an accession number of FERM BP-08615 or Aureobasidium pullulans M-2 having an accession number of FERM BP-10014.
[3] A therapeutic agent for an influenza virus infectious disease according to the above-mentioned item [1] or [2], further including, as another active ingredient, bacterial cells of a lactic acid bacterium.
[4] A therapeutic agent for an influenza virus infectious disease according to the above-mentioned item [3], in which the bacterial cells of a lactic acid bacterium are dead bacterial cells of Enterococcus faecalis.
[5] A therapeutic agent for an influenza virus infectious disease according to any one of the above-mentioned items [1] to [4], which is administered in combination with at least one kind selected from the group consisting of an influenza neuraminidase inhibitor, an antiviral drug, an antibiotic, and a steroid drug.
According to the therapeutic agent for an influenza virus infectious disease of the present invention, it is possible to prevent an influenza virus infectious disease from becoming serious and to promote the healing of the disease. The active ingredient in the agent is a β-glucan-containing composition obtained from a culture of a microorganism belonging to the Aureobasidium sp. Therefore, its excellent effect can be provided safely without a risk of side effects even if the agent is administered for a long period of time and even in an easy administration form. In addition, administration of the agent in combination with bacterial cells of a lactic acid bacterium can further enhance the effect. Further, administration of the agent in combination with at least one kind selected from the group consisting of an influenza neuraminidase inhibitor, an antiviral drug, an antibiotic, and a steroid drug can further enhance the effect.
A therapeutic agent for an influenza virus infectious disease of the present invention contains, as an active ingredient, a β-glucan-containing composition obtained from a culture of a microorganism belonging to Aureobasidium sp. (hereinafter, referred to as “β-glucan-containing composition derived from Aureobasidium”). The β-glucan-containing composition derived from Aureobasidium includes not only a culture itself obtained by culturing a microorganism that belongs to the Aureobasidium sp. and has a β-glucan producing ability (hereinafter, referred to as “Aureobasidium microorganism”), a culture solution obtained by separating and removing microorganism cells by centrifugation or the like, a concentrated solution of the culture solution, a diluted solution of the culture solution, solid matter obtained by removing water from the culture solution, or the like but also a product obtained by, for example, demineralizing any of the foregoing to increase the content of β-glucan or a purified β-glucan product obtained from any of the foregoing.
The β-glucan-containing composition derived from Aureobasidium to be used in the present invention contains β-glucan produced by culturing the Aureobasidium microorganism in an amount of preferably 50 to 3,000 mg, more preferably 100 to 2,000 mg in terms of a content relative to 100 g (mass) of the culture.
It should be noted that the content of β-glucan can be determined in accordance with, for example, the following method. Specifically, a culture solution is subjected to enzyme treatment with amylase, amyloglucosidase, protease, or the like, and proteins and α-glucan such as pullulan are removed, followed by ethanol precipitation. Further, the resultant is filtered using a glass filter, to thereby yield a high-molecular-weight sample. At this time, the sample is sufficiently washed with 80% ethanol in order to remove low-molecular-weight substances including monosaccharides. The washed high-molecular-weight sample is further washed with acetone, and sulfuric acid is added thereto for hydrolysis. After the hydrolysis, neutralization is carried out, and the filtrate is collected. Quantification of glucose is carried out by a glucose oxidase method, and a value calculated based on the following mathematical expression 1 is defined as the β-glucan amount.
β-glucan(g/100 g)=glucose(g/100 g)×0.9 Mathematical Expression 1
Moreover, the content of β-glucan may also be determined as an amount of a so-called sugar chain-containing high-molecular-weight substance (polysaccharide). In such case, a culture solution is subjected to enzyme treatment with amylase, amyloglucosidase, protease, or the like, and proteins and α-glucan such as pullulan are removed, followed by ethanol precipitation. Further, the resultant is filtered using a glass filter, to thereby yield a high-molecular-weight sample. At this time, the sample is sufficiently washed with 80% ethanol in order to remove low-molecular-weight substances including monosaccharides. The washed high-molecular-weight sample is further washed with acetone, and the weight of the resultant sample is determined as an amount of a sugar chain-containing high-molecular-weight substance (polysaccharide).
It should be noted that the β-glucan quantified as described above is quantified as a substance containing a functional group such as a sulfate group or a phosphate group. Therefore, in the case where the β-glucan is quantified as a broad-sense sugar chain-containing high-molecular-weight substance (polysaccharide) as described above, the content of the β-glucan produced by culturing the Aureobasidium microorganism is preferably 70 to 5,000 mg, more preferably 140 to 3,000 mg in terms of a content relative to 100 g (mass) of the culture.
The Aureobasidium microorganism to be used in the present invention has only to be a microorganism belonging to Aureobasidium sp. and having a β-glucan producing ability. For example, Aureobasidium pullulans M-1 (the National Institute of Advanced Industrial Science and Technology International Patent Organism Depositary, Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, accession number: FERM BP-08615, international deposit on Feb. 10, 2004) or, Aureobasidium pullulans M-2 (the National Institute of Advanced Industrial Science and Technology International Patent Organism Depositary, Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, accession number: FERM BP-10014, international deposit on Apr. 22, 2004) is suitably employed. It should be noted that, β-glucan produced by these strains of microorganisms has been shown to be β-1,3-1,6-glucan, which has a structure in which glucose is branched with a β-1,6 bond from a main chain in which glucose molecules are linked by a β-1,3 bond from a structural analysis by NMR measurement (13C NMR: Varian, UNITY INOVA500, 1H NMR: Varian, UNITY INOVA600).
The Aureobasidium microorganism can be cultured in accordance with a known method (see, for example, JP 57-149301 A). That is, the microorganism is inoculated in a medium (pH 5.2 to 6.0) supplemented with 0.5 to 5.0% by mass of a carbon source (sucrose), 0.1 to 5.0% by mass of an N source, and other trace substances (for example, vitamins or minerals). The microorganism is cultured at a temperature of 20 to 30° C. for 2 to 14 days with aeration, preferably with aeration and stirring. The viscosity of the culture solution becomes higher as β-glucan is produced, resulting in a gel-like composition having an increased viscosity. The culture solution thus obtained generally contains 0.6 to 10% by mass of solid matter, and the solid matter contains 5 to 80% by mass of β-glucan. Moreover, the solid matter contains not only β-glucan but also other useful ingredients such as phosphorus, potassium, magnesium, and vitamin C, which are ingredients for aiding an action of the glucan. Hence, a physiologically active effect of β-glucan can be exerted efficiently.
In the present invention, the culture thus obtained itself may be used after sterilization by heating without pressure, or after sterilization by heating under pressure. Alternatively, the culture solution may be sterilized and used after the separation and removal of microorganism cells by centrifugation or the like. Moreover, the culture may be used after concentration or after concentration and drying, if necessary. Further, only β-glucan may be extracted and used. It should be noted that the culture of a microorganism belonging to the Aureobasidium sp. is used as a food additive such as a thickener, and has high safety.
With regard to its application method, when the composition is prepared for oral administration, for example, it is possible to exert its physiologically active effect in the body through oral ingestion thereof.
The amount of the composition to be administered can be appropriately determined depending on, for example, the purpose of treatment or prevention, the degree of the symptom, the age of a patient, the administration method, the frequency of administration, and the administration period. For example, in the case of oral ingestion, a general effective dose is about 0.06 to 60 mg/kg (body weight) per day in terms of the amount of β-glucan. The therapeutic agent for an influenza virus infectious disease of the present invention is effective for prevention and treatment of complications caused after infection with an influenza virus. Examples of the complications include otitis media, sinusitis, bronchitis, exacerbation of chronic bronchitis, pneumonia, encephalopathy, and renal failure. In particular, the agent is effective for prevention of a disease caused by a so-called cytokine storm or a tissue-destroying disease (disease caused by TH1 enhancement).
The therapeutic agent for an influenza virus infectious disease of the present invention may further contain bacterial cells of a lactic acid bacterium as an active ingredient other than the culture of Aureobasidium. Such agent can be expected to have a synergistic action with a physiologically active effect of the lactic acid bacterium.
A lactic acid bacterium or the like applicable to food and having no problem in safety is employed as the lactic acid bacterium. Specific examples thereof may include Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Streptococcus cremoris, Streptococcus lactis, Streptococcus thermophilus, Bifidobacterium Longum, Bifidobacterium breve, and Bifidobacterium bifidum. The lactic acid bacteria may be used alone or in combination of two or more kinds thereof.
It should be noted that Enterococcus faecalis and Enterococcus faecium are lactic acid bacteria used in lactic acid bacterium preparations or the like, Lactobacillus casei and Lactobacillus acidophilus are lactic acid bacteria used in cheese, fermented milk, yogurt, lactic acid bacterium drinks, or the like, Streptococcus cremoris, Streptococcus lactis, and Streptococcus thermophilus are lactic acid bacteria used in cheese, yogurt, or the like, and Bifidobacterium Longum, Bifidobacterium breve, and Bifidobacterium bifidum are lactic acid bacteria used in fermented milk or the like. Thus, all of these lactic acid bacteria are easily obtainable for those skilled in the art.
In addition, as the lactic acid bacterium, not only viable cells but also heat-sterilized dead bacterial cells may be used. In the case where the dead bacterial cells are used, preservation stability is enhanced, and safety is enhanced when the cells are used as raw materials for drugs and functional foods. As the dead bacterial cells of the lactic acid bacterium, dead bacterial cells of Enterococcus faecalis and Lactobacillus brevis subspecies coagulans are commercially available and preferably used.
The lactic acid bacterium may be cultured in accordance with a conventional method. In addition, dead bacterial cells may be obtained by collecting bacterial cells from a culture of the lactic acid bacterium by means of a method such as filtration or centrifugation, washing the cells with water, and then suspending the cells in water or the like, followed by heat sterilization at 80 to 115° C. for 30 minutes to 3 seconds. The heat-sterilized lactic acid bacterium may be used after concentration or drying, if necessary.
The amount of the lactic acid bacterium to be administered can be appropriately determined depending on, for example, the purpose of treatment or prevention, the degree of the symptom, the age of a patient, the administration method, the frequency of administration, and the administration period. For example, in the case of oral ingestion, a general effective dose is about 1×109 to 4×1011 cells/kg (body weight), more preferably 2×109 to 1×1011 cells/kg (body weight) per day in terms of the amount of dry dead bacterial cells.
In the present invention, of the above-mentioned lactic acid bacteria, dead bacterial cells of Enterococcus faecalis are particularly preferably used. The dead bacterial cells of Enterococcus faecalis are known to have a strong immunopotentiating activity, and when the cells are used in combination with the culture of Aureobasidium, a synergistic effect of them may be expected in anti-influenza virus effect as well.
In the present invention, as a form of administration of the therapeutic agent, the agent may be administered in combination with at least one kind selected from the group consisting of an influenza neuraminidase inhibitor, an antiviral drug, an antibiotic, and a steroid drug. According to this form, a synergistic effect with the anti-influenza virus effect of such drug may be expected. Preferred examples of the influenza neuraminidase inhibitor may include oseltamivir and zanamivir. Preferred examples of the antiviral agent may include amantadine. Preferred examples of the antibiotic may include clarithromycin, cefaclor, cefdinir, cefpodoxime proxetil, cefotiam hydrochloride, cefcapene pivoxil hydrochloride, cefditoren pivoxil, cefinetazole sodium, cefotiam hydrochloride, cefozopran hydrochloride, pivmecillinam hydrochloride, sultamicillin tosilate, fosfomycin calcium, isepamicin sulfate, gentamicin sulfate, and kanamycinmonosulfate. Preferred examples of the steroid drug may include methylprednisolone.
In the present invention, in the case where, as a form of administration of the therapeutic agent, the agent is administered in combination with at least one kind selected from the group consisting of an influenza neuraminidase inhibitor, an antiviral drug, an antibiotic, and a steroid drug, the culture of Aureobasidium may be administered while the antiviral drugs are administered in suitable modes for the respective antiviral drugs. In this case, the culture of Aureobasidium shows no outstanding side effects, and hence the antiviral drugs may be administered for a relatively short period of time while the culture is administered continuously for a relatively long period of time.
Hereinafter, the present invention is specifically described by way of examples. However, these examples are not intended to limit the scope of the present invention.
A culture solution of Aureobasidium pullulans M-1 (FERM BP-08615) was prepared as follows.
An appropriate amount of a pre-culture was inoculated in a liquid medium (pH 5.3) containing 1% of sucrose, 0.1% of ascorbic acid, and 0.1% of rice bran, and cultured with aeration and stirring at 25° C. for 72 to 96 hours (varying depending on production batches). After completion of the culture, the culture solution was sterilized at 121° C. for 15 minutes. The resultant sterilized culture solution contained about 1.2% by mass of solid matter, and the content of β-glucan in 100 g of the solid matter was 16.7 g. In addition, the content of β-glucan was 0.2 g/100 g in terms of a content in 100 g (mass) of the culture solution itself. The culture solution was used in the following test.
Test solutions were prepared for the test groups and control group shown in Table 1 below, respectively, and effects of oral administration of the solutions on mice infected with an influenza virus were examined. It should be noted that “Tamiflu dry syrup 3%” (product name, Chugai Pharmaceutical Co., Ltd.) was used as an anti-influenza drug oseltamivir.
Aureobasidium (M-1)
As experimental animals, mice (C57BL/6NJcl, male, 6 weeks old) were used, and as the influenza virus, H1N1 subtype of influenza virus A/PR/8 was used. Infection of the mice with the influenza virus was carried out as follows. That is, the mice were placed in a container filled with isoflurane, and taken out after confirming that their breathing became slow. A diluted virus solution was prepared by serial dilution with PBS so that the solution had a virus titer of 2×104 pfu/mL, and injected from both nasal cavities alternately using a micropipette in an amount of 50 μL (1,000 pfu/mouse). After confirming that the mice recovered from anesthesia, the mice were returned to breeding cages.
The test solutions were administered in the forms and by the methods shown in Table 2 below. It should be noted that the culture solution of Aureobasidium (Test group 1) was orally administered every day during the test period by a sonde method once a day in an amount of 200 μL. The anti-influenza drug oseltamivir (Test group 2) was orally administered in accordance with a method which was considered to be the most effective as its administration method (see “DIRK B. MENDEL et al., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, March 1998, p. 640-646”) for five days after infection with the virus by the sonde method twice a day (morning and evening, in an amount of 200 μL per dose). The body weights of the mice after infection with the virus were measured every day, and transition of the body weights and survival rates were determined.
As illustrated in
As illustrated in
As illustrated in
The above-mentioned results are summarized as follows: administration of the culture solution of Aureobasidium suppressed a symptom caused by infection with the influenza virus, i.e., loss of the body weights of the mice and promoted recovery from the loss of the body weights. In addition, the effect of the culture solution was found to be equal to or larger than that of the anti-influenza virus drug oseltamivir (product name “Tamiflu,” Chugai Pharmaceutical Co., Ltd.). Therefore, it was found that the β-glucan-containing composition obtained from the culture of the microorganism belonging to the Aureobasidium is effective for treatment of an influenza virus infectious disease, in particular, for preventing the disease from becoming serious and promoting the healing of the disease after infection with the virus.
Although the mechanism of the β-glucan-containing composition derived from Aureobasidium to exert the effect is unknown, it was considered that the composition enhances not only actions caused by immunostimulation such as macrophage phagocytic capacity activation but also immune balance functions inherent in the body to maintain a balance between, for example, Th1 cell immunity and Th2 cell immunity, resulting in suppressing unnecessary attack on self-tissues by excessive immune responses.
A culture solution of Aureobasidium pullulans M-2 (FERM BP-10014) was prepared in the same manner as in Production Example 1. The culture solution contained about 1.2% by mass of solid matter, and the content of β-glucan in 100 g of the solid matter was 54.2 g. In addition, the content of β-glucan was 0.65 g/100 g in terms of a content in 100 g (mass) of the culture solution itself. The culture solution was used in the following test.
Test solutions were prepared for the test groups and control group as shown in Table 3 below, respectively, and effects of oral administration of the solutions on mice infected with an influenza virus were examined. It should be noted that the phrase “dead bacterial cells of EF lactic acid bacterium” in the table means a commercially available heat-sterilized bacterial cell powder of Enterococcus faecalis.
Infection of mice used as experimental animals with the influenza virus was carried out so that the virus titer was twice as large as that in Test Example 1. That is, the infection was carried out in the same manner as in Test Example 1 except that a diluted virus solution having a virus titer of 4×104 pfu/mL was injected from both nasal cavities alternately using a micropipette in an amount of 50 μL (2,000 pfu/mouse).
The test solutions were administered in the forms and by the methods shown in Table 4 below. That is, in the case of each of Test groups 3 to 5 and Control group 2, the test solution was orally administered every day during the test period by the sonde method once a day in an amount of 200 μL. In the case of Test group 6, to which the culture solution of Aureobasidium was administered in combination with oseltamivir, in order to administer oseltamivir effectively, in the same manner as in Test group 2 in Test Example 1, a suspension of oseltamivir in the culture solution of Aureobasidium was orally administered twice a day (morning and evening, in an amount of 200 μL per dose) for five days after infection with the virus, and during the remaining period, only the culture solution of Aureobasidium was orally administered once a day in an amount of 200 μL. Further, in the case of Test group 7, to which only oseltamivir was administered, in order to carry out comparison with the maximum effect of administration of only oseltamivir, oseltamivir was orally administered every day (morning and evening, in an amount of 200 μL per dose) continuously for 17 days after infection with the virus.
The body weights of the mice after infection with the virus were measured every day, and transition of the body weights and survival rates were determined.
In Test Example 2, as a result of infection of the mice carried out so that the virus titer was twice as large as that in Test Example 1, in the case of Control group 2, to which PBS was administered (
Moreover, in the cases of Test group 3 (
Further, in the case of Test group 7, to which oseltamivir was administered (
On the other hand, as illustrated in
The above-mentioned results show that the β-glucan-containing composition derived from Aureobasidium is effective for treating an influenza virus infectious disease, in particular, for preventing the disease from becoming serious and promoting the healing of the disease after virus infection, and the effect can be more enhanced by using bacterial cells of a lactic acid bacterium or oseltamivir in combination.
An effect of administration of the culture of Aureobasidium in combination with bacterial cells of a lactic acid bacterium was further examined. Specifically, test solutions were prepared for the test groups and control group shown in Table 5 below, respectively, and orally administered in the same manner as in Test Example 2, and transition of body weights and survival rates of mice infected with the influenza virus were examined. It should be noted that the phrase “dead bacterial cells of EF lactic acid bacterium” in the table means a commercially available heat-sterilized bacterial cell powder of Enterococcus faecalis.
The test solutions were administered in the forms and by the methods shown in Table 6 below. That is, in the case of Test group 8, the dead bacterial cells of the lactic acid bacterium (6.7×109 cells) were suspended in 200 μL of the culture solution of Aureobasidium pullulans M-2 (FERM BP-10014) prepared so as to have a β-glucan concentration of 0.2 g/100 g, and the suspension was orally administered every day during the test period by the sonde method once a day. In the case of Test group 9, the test was carried out in the same manner as in Test group 8 except that the dead bacterial cells of the lactic acid bacterium (10.1×109 cells) were suspended. In the case of Test group 10, the test was carried out in the same manner as in Test group 8 except that the dead bacterial cells of the lactic acid bacterium (3.4×109 cells) were suspended. In the case of Test group 11, the test was carried out in the same manner as in Test group 8 except that the culture solution of Aureobasidium was prepared so as to have a β-glucan concentration of 0.1 g/100 g. In the case of Test group 12, the dead bacterial cells of the lactic acid bacterium (6.7×109 cells) were suspended in PBS, and the suspension was orally administered every day during the test period by the sonde method once a day.
In Test Example 3, as in the case of Test Example 2, as a result of infection of the mice carried out so that the virus titer was twice as large as that in Test Example 1, in the case of Control group 3, to which PBS was administered (
Further, in the case of Test group 12, to which the heat-sterilized bacterial cell powder of the lactic acid bacterium was administered (
On the other hand, as illustrated in
The above-mentioned results show that the β-glucan-containing composition derived from Aureobasidium is effective for treating an influenza virus infectious disease, in particular, for preventing the disease from becoming serious and promoting the healing of the disease after virus infection, and the effect can be more enhanced by using bacterial cells of a lactic acid bacterium in combination.
In order to study mechanisms of the effects shown in Test Examples 1 to 3, influences on a virus titer in the lung of a mouse infected with the influenza virus were examined. Specifically, test solutions were prepared for the test groups and control group shown in Table 7 below, respectively, and orally administered, and the virus titer in the lung of each mouse infected with the influenza virus was examined. It should be noted that the phrase “dead bacterial cells of EF lactic acid bacterium” in the table means a commercially available heat-sterilized bacterial cell powder of Enterococcus faecalis.
Aureobasidium (M-2)
Aureobasidium (M-2) + dead
The test solutions were administered in the forms and by the methods shown in Table 8 below. That is, the mice were infected with the influenza virus in the same manner as in Test Example 2 except that the period after the beginning of administration of the test solutions was changed to one week. In the case of Test group 13, the test solution was prepared so that the culture solution of Aureobasidium pullulans M-2 (FERM BP-10014) had a β-glucan concentration of 0.2 g/100 g, and orally administered once a day in an amount of 200 μL every day during the test period by the sonde method. In the case of Test group 14, the test was carried out in the same manner as in Test group 13 except that the dead bacterial cells of the lactic acid bacterium (6.7×109 cells) were used in combination as a suspension in 200 μL of the culture solution of Aureobasidium. In addition, in the case of Test group 15, to which only oseltamivir was administered, in order to carry out comparison with the maximum effect of administration of only oseltamivir, oseltamivir was orally administered continuously everyday (morning and evening, in an amount of 200 μL per dose) after virus infection.
The virus titer in the lung of a mouse was measured as follows.
On day 1, day 3, and day 5 of virus infection, three mice of each group were dissected, and the lung washed with PBS to remove blood was collected. The collected lung was crushed, and the tissue was separated as a cell suspension and then centrifuged at 5,000 rpm for 10 minutes. The supernatant was collected and used as a sample. The collected sample was diluted serially in 10-fold steps with 1×MEM to concentrations ranging from 10−1 to 10−6. On the other hand, MDCK cells were cultured from the previous day, and a state in which the MDCK cells (one sample: one 12-well plate) were proliferated to confluency was confirmed. Then, the medium of the MDCK cells was removed, and the cells were washed with 1 ml of PBS. PBS used for washing was removed, and each of the samples diluted serially was added in an amount of 100 μL, and then the plate was allowed to stand still in a 5% CO2 incubator (37° C.) for one hour. During incubation, the plate was shaken every 15 minutes to prevent the cells from drying. Subsequently, 2×MEM was mixed with an equal amount of a 1.6% agar solution, and trypsin was added so as to reach a final concentration of 0.0005% to prepare a MEM-agar solution. The solution was warmed in a water bath at 42° C. One hour later, the virus solution was removed, and the cells were washed with 1 ml of PBS. The prepared MEM-agar solution was added to each well in an amount of 1 ml, and the plate was allowed to stand for a while until the agar was solidified. After that, the cells were cultured in a 5% CO2 incubator (35° C.), and 48 hours later, the number of plaques was counted.
The results show that on day 1 of infection, Control group 4, to which PBS was administered, had a mouse lung virus titer of 6.31 (log10PFU), Test group 13, to which the culture solution of Aureobasidium was administered, had a mouse lung virus titer of 6.36 (log10PFU), Test group 14, to which the culture solution of Aureobasidium was administered in combination with the lactic acid bacterium had a mouse lung virus titer of 5.59 (log10PFU), and Test group 15, to which oseltamivir (active ingredient in an anti-influenza drug Tamiflu) used as a positive control was administered, had a mouse lung virus titer of 5.88 (log10PFU). On day 3 and day 5 of infection, the virus titers of Control group 4, to which PBS was administered, were 8.80 (log10PFU) and 9.43 (log10PFU), respectively, and increased 154-fold and 2.057-fold, respectively, as compared to day 1 of infection. Further, on day 3 and day 5 of infection, the virus titers of Test group 13, to which the culture solution of Aureobasidium was administered, were 7.75 (log10PFU) and 7.18 (log10PFU), respectively, and increased 22.8-fold and 8.3-fold, respectively, as compared to day 1 of infection. In addition, the virus titers of Test group 14, to which the culture solution of Aureobasidium was administered in combination with the lactic acid bacterium were 7.61 (log10PFU) and 7.24 (log10PFU), respectively, and increased 81.4-fold and 40.1-fold, respectively, as compared to day 1 of infection, and increases in the virus titers of Test group 14 were suppressed as compared to Control group 4. On the other hand, when only oseltamivir, used as an active ingredient of the anti-influenza drug Tamiflu, was administered (Test group 15), on day 3 and day 5 of infection, the virus titers were 6.83 (log10PFU) and 7.76 (log10PFU), respectively, and increased 15.4-fold and 122-fold, respectively, as compared to day 1 of infection.
The results are collectively shown in Table 9. That is, the virus titer of Control group 4, to which PBS was administered, on each day after infection is defined as 1, and the virus titers of the test groups on each corresponding day after infection are tabulated.
As shown in Table 9, when the virus titer of Control group 4, to which PBS was administered, was defined as 1, the virus titers of Test group 13, to which the culture solution of Aureobasidium was administered, were 1/12 on day 3 and 1/425 on day 5, and the virus titers of Test group 14, to which the culture solution of Aureobasidium was administered in combination with the lactic acid bacterium, were 1/16 on day 3 and 1/442 on day 5. Therefore, administration of the culture solution of Aureobasidium (Test group 13) or administration of the culture solution of Aureobasidium in combination with the lactic acid bacterium (Test group 14) was considered to effectively suppress proliferation of the virus in the lung. It should be noted that as shown in the results of Test group 14, in this test example, when the culture solution of Aureobasidium was administered in combination with the bacterial cells of the lactic acid bacterium on day 1 of infection, an increase in the virus titer was further suppressed as compared to administration of only the culture solution of Aureobasidium.
In order to study mechanisms of the effects shown in Test Examples 1 to 3, activated T cells and activated NK cells in the lung of each mouse infected with the influenza virus were examined. Specifically, test solutions were prepared for the test groups and control group shown in Table 10 below, respectively, and orally administered, and the numbers of T cells and activated T cells among all the T cells and the numbers of NK cells and activated NK cells among all the NK cells in the lung of each mouse infected with the influenza virus were examined on day 5. It should be noted that the phrase “dead bacterial cells of EF lactic acid bacterium” in the table means a commercially available heat-sterilized bacterial cell powder of Enterococcus faecalis.
The test solutions were administered in the same manner as in Test Example 4 above in the forms and by the methods shown in Table 11 below. That is, the mice were infected with the influenza virus in the same manner as in Test Example 2 except that the period after the beginning of administration of the test solutions was changed to one week. In the case of Test group 16, the test solution was prepared so that the culture solution of Aureobasidium pullulans M-2 (FERM BP-10014) had a β-glucan concentration of 0.2 g/100 g, and orally administered once a day in an amount of 200 μL every day during the test period by the sonde method. In the case of Test group 17, the test was carried out in the same manner as in Test group 16 except that the dead bacterial cells of the lactic acid bacterium (6.7×109 cells) were used in combination as a suspension in 200 μL of the culture solution of Aureobasidium. In addition, in the case of Test group 17, to which only oseltamivir was administered, in order to carry out comparison with the maximum effect of administration of only oseltamivir, oseltamivir was orally administered every day (morning and evening, in an amount of 200 μL per dose) continuously after virus infection.
The numbers of the activated T cells and activated NK cells in the lung of each mouse were counted as follows. That is, three mice of each group were dissected on day 5 of virus infection, and the lung washed with PBS to remove blood was collected. 4.9 ml of HEPES buffer, 100 μl of collagenase D (100 mg/Ml in HEPES), and 10 μl of DNase 1 were added to the lung of one mouse, and the mixture was homogenized to crush the tissue in part. The homogenate was shaken at 37° C. for 30 minutes and treated enzymatically. The mixture was homogenized again to crush the tissue completely. The crushed lung tissues of three mice in each group were collected and passed through a mesh to remove extra components other than cells, and the cells were washed with PBS repeatedly, followed by counting the number of the cells.
The cell suspension was dispensed in 96 wells in an amount of 2×106 cells/well per sample, and “LEAF Purified anti-Mouse CD16/32” was added in an amount of 1 μl/well (in 100 μl FACS buffer) for blocking to prevent extra cells from adhering to antibodies. The plate was allowed to stand still on ice for 30 minutes. CD3 APC antibody against T cells, NK 1.1 FITC antibody against NK cells, PE Cy7-CD69 antibody against activated T cells, and PE Cy7-CD69 antibody against activated NK cells were added thereto in an amount of 1 μl/well, and the plate was allowed to stand still at 4° C. for 30 minutes while the plate was shielded from light. The plate was washed with FACS buffer several times to remove antibodies which were not bound to the cells, and 7-AAD for labeling and distinguishing dead cells was added in an amount of 10 μl per sample. An FACS analysis was carried out using a flow cytometer/cell sorter apparatus, and the ratio of the NK cells or T cells in viable cells was determined. In addition, the ratio of activated cells in the respective cells was determined.
On day 5 of infection, with regard to NK cells which play an important role in natural immunity, the ratios of the NK cells in viable cells in the lung of Control group 5, to which PBS was administered, Test group 16, to which the culture solution of Aureobasidium was administered, and Test group 17, to which the culture solution of Aureobasidium was administered in combination with the lactic acid bacterium, were found to be 7.5%, 6.4%, and 3.7%, respectively. The ratio in the case of Test group 18, to which oseltamivir (active ingredient in the anti-influenza drug Tamiflu) was administered, was found to be about 1.8%, which was only about one-quarter of that of Control group (the results are illustrated in
Next, with regard to T cells, which play an important role in acquired immunity, the number of T cells in lung viable cells of each group on day 5 of virus infection was very small (about 3%), and there were few differences among the numbers of the cells (the results are illustrated in
As mentioned above, it was suggested that administration of only the culture of Aureobasidium or administration of the culture of Aureobasidium in combination with the lactic acid bacterium increased the number of activated T cells or NK cells as compared to administration of oseltamivir (active ingredient in the anti-influenza drug Tamiflu) and not only suppressed invasion and proliferation of the influenza virus but also prevented symptoms caused by infection with the influenza virus from becoming serious. Further, it was suggested that administration of the culture of Aureobasidium or the culture of Aureobasidium in combination with the lactic acid bacterium possibly achieved smooth transition from natural immunity to acquired immunity, and as a result, contributed to reduction in the weight loss ratio or death rate.
It should be noted that it is considered that the culture of Aureobasidium and oseltamivir (active ingredient in the anti-influenza drug Tamiflu) have different mechanisms for protection from infection with the influenza virus, and hence as shown in Test group 6 in Test Example 2 above (
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-234023 | Oct 2009 | JP | national |
| 2009-265049 | Nov 2009 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2010/067680 | 10/7/2010 | WO | 00 | 4/3/2012 |
