WO2005034971A2

WO2005034971A2 - Antiviral agent

Info

Publication number: WO2005034971A2
Application number: PCT/JP2004/014811
Authority: WO
Inventors: Masami Moriyama
Original assignee: Masami Moriyama
Priority date: 2003-10-08
Filing date: 2004-09-30
Publication date: 2005-04-21
Also published as: WO2005034971A3; JP2007508233A

Abstract

Objectives of the present invention are to ascertain the existence of antiviral activity, which has not been known at the present time, in a culture supernatant of Bifidobacterium longum which is one species of Bifidobacteria, and to provide a novel antiviral agent based on that finding. An antiviral agent comprises, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium. More particularly, an antiviral agent comprises, as its active ingredient, a culture supernatant obtained by cultivation of Bifidobacterium longum in a medium containing corn starch as a gelling agent. Furthermore, a prophylactic or therapeutic agent against abnormal prion syndromes and Alzheimer's syndrome, comprises, as its active ingredient, the culture supernatant.

Description

DESCRIPTION

ANTIVIRAL AGENT

TECHNICAL FIELD The present invention relates to antiviral agents comprising as their active ingredient a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium.

BACKGROUND ART Bifidobacteria which are anaerobic bacteria grow in almost pure culture condition in the intestine of a breast-fed child. However, in tine intestine of a bottle-fed child, only a small number of Bifj-dobacteria exist in contrast to a great number of Escherichia coli , Enterococci, and the like, so that bottle-fed children are more susceptible to intestinal infection diseases. A breast-fed child is believed to have great resistance to infections by bacteria, viruses, and the like because of the immune strength acquired through breast milk from the mother. However, the great resistance is not irrelevant to the fact that

Bifidobacteria. are cultured in almost pure culture under the anaerobic conϋition in the intestine and significant amounts of culture fluid thereof exist in the intestine. Also in adults, Bifidobacteria are one of the dominant bacteria in ttie intestine in a healthy condition, however, it has been found that Bifidobacteria decrease or disappear in an unhealthy condition. For this reason, a variety of pharmaceuticals, health foods, and soft drinks such as fermented lactic drinks using Bifidobacteria are proposed and commercially available. Oral intake of Bif±dobacteria is utilized for prevention of diarrhea in adults or infants as well as for health maintenance. It is believed that Bifidobacteria produce large amounts of lactic acid and volatile acids in the intestine when orally taken, thereby lowering the intestinal pH, suppressing the growth of pathogenic bacteria such as Escherichia coli , promoting the peristaltic movement of intestines, and alleviating the constipation and diarrhea due to abnormal intestinal flora. As described above, Bifidobacteria are highly beneficial for human health maintenance, and in most cases provided in the form of health drink of fermented product prepared by using a cmlture fluid of Bifidobacteria and adding various kinds of additives . Bifidobacteria are also used as antiflatulents in powdei:, tablet, and the like forms prepared by collecting and drying viable cells of Bifidobacteria, and mixing them with appropriate excipients such as starch, lactose, sucrose, and the like. Among Bifidobacteria, Bifidobacterium longum has not been studied so actively. Quite recent years, various proposals are made concerning application of a culture of Bifidobactezrium longum to pharmaceuticals. However, they are foods and drinks (see, for example, Japanese Patent Laid-Open Publication No. 2003-250530), prophylactic, improving, therapeutic agents against complications associated with diabetes (see, for example, Japanese Patent Laid- Open Publication No. 2003-252770), cholesterol lowering drugs (see, for example, Japanese Patent Laid-Open Publication No. 2003-238423) and the like, comprising a culture containing bacterial cells of Bifidobacterium longum. In the pharmaceuticals proposed in these documents, however, the culture of Bifidobacterium longum is used in its entirety and pharmacological activities of individual components obtained by fractionation into bacterial cells and a supernatant are not studied. The inventors of the present invention took notice of the pharmacological activities included in a culture fluid of Bifidobacterium longum and particularly studied the pharmacological activity of a culture supernatant thereof. As a result, it was unexpectedly found that a culture supernatant of Bifidobacterium longum has very strong antiviral activity and finally completed the present invention. The existence of antiviral activity in a culture supernatant of Bifidobacterium longum has never been found up to now, which makes the present invention extremely specific. The studies made by the present inventors also demonstrated that a culture supernatant of Bifidobacterium longum has prophylactic or therapeutic efficacy against bovine spongiform encephalopathy (BSE) in bovine, Creutzfeldt Jakob disease (CJD) in human, Gerst ann-Staussler-Scheinker syndrome (GSS) which are diseases caused by proliferation of abnormal prion proteins or Alzheimer's syndrome which is a similar symptom occurring in the brain. Thus, the present invention was accomplished based on these new findings.

DISCLOSURE OF THE INVENTION Therefore, it is an object of the present invention to prove the existence of antiviral activity in a culture supernatant of Bifidobacterium which has not been known heretofore, to provide a novel antiviral agent based on that finding, and to provide a prophylactic or therapeutic agent against abnormal prion syndromes and Alzheimer's syndrome. In order to achieve the above object, in one basic aspect, the present invention provides an antiviral agent comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium. Bifidobacterium longum is an anaerobic bacterium belonging to the genus Bifidobacterium , and usually cultured in an oxygen free condition. In the present invention, when it was cultured in a medium containing a gelling agent for keeping the anaerobic condition, strong antiviral activity was found in the resultant culture supernatant. Therefore, more specifically, the present invention provides an antiviral agent comprising, as its active ingredient, a culture supernatant obtained from cultivation of Bifidobacterium longum in a medium containing a gelling agent. More specifically, the present invention provides an antiviral agent comprising, as its active ingredient, a culture supernatant obtained from cultivation of Bifidobacterium longum in a medium containing a gelling agent, the gelling agent being at least one selected from the group consisting of corn starch, alginic acid and agar, and preferably being corn starch among others . Bifidobacteria are anaerobic bacteria, and generally Bifidobacterium bifidium is a standard species. These are often found in feces and gastrointestinal tracts of breast- or bottle-fed infants and aged persons, and pathogenicity to human and other animals is not found. In genus Lactobacillus , Lactobacillus bifidus susp. Pennsylvanicus is also classified as one species of the Bifidobacteria. The present invention revealed that a culture supernatant of Bifidobacterium longum rather than a culture fluid of such a standard species of Bifidobacterium has an extremely strong antiviral activity. Therefore, in the most specific aspect, the present invention provides an antiviral agent comprising, as its active ingredient, a culture supernatant obtained from cultivation of Bifidobacterium longum cells in a medium containing a gelling agent, and also provides an antiviral agent comprising, as its active ingredient, the culture supernatant obtained from cultivation using corn starch as the gelling agent. In another aspect, the present invention provides a prophylactic or therapeutic agent against abnormal prion syndromes or Alzheimer's syndrome, wherein a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium serves as an active ingredient. More specifically, the present invention provides a therapeutic agent against the abnormal prion disease or a therapeutic agent against the Alzheimer's disease, wherein the culture supernatant of Bifidobacterium longum is a culture supernatant obtained from cultivation of Bifidobacterium longum in a medium containing corn starch as a gelling agent . As described above, the present invention provides an antiviral agent, as well as a prophylactic or therapeutic agent against abnormal prion syndromes or Alzheimer's syncϋrome, comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium. Pharmaceuticals, health drinks, soft drinks, and the like containing as their active ingredient, viable cells (dry cells) of Bifidobacteria or the whole culture fluid thereof have been conventionally known. They are effective in suppressing the growth of pathogenic bacteria such as Escherichia coli by producing a large amount of lactic acid and the like in the intestine and lowering the intestinal pH, however, an extremely strong antiviral activity is found for the first time in the present invention. Thus, since pathogenic viruses are killed or prevented from growing, an antiviral agent of the present invention is very efficacious in therapy of diseases caused by, for example, varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, SARS (severe acute respiratory syndrome) virus (one species of corona virus). West Nile virus, ATL (Adult T- cell Leukemia) virus, and HIV virus.

BEST MODES FOR CARRYING OUT THE INVENTION The present invention will be described by detailed description of features of the present invention. In a basic aspect, the present invention provides an antiviral agent comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum. Herein the term "antiviral agent" refers to an antiviral agent in an aspect capable of, for example, preventing virus infection, suppressing virus growth caused by virus infection, and making the viral titer due to virus infection disappear. Liquid cultivation of Bifidobacterium longum is usually conducted in a medium containing a reducing agent such as cysteine and ascorbic acid so as to maintain the anaerobic condition during the cultivation. However, the reducing agent is expensive and hence is unfavorable for industrial cultivation. In the trial for overcoming this drawback, we found that Bifidobacterium longum can be cultured in a very favorable fashion when an anaerobic condition is achieved by preparing the basal culture medium in gel state so as to prevent oxygen from entering the liquid medium. As the basal medium for σulturing Bifidobacterium longum, liquid media commonly used in cultivation of Bifidobacteria can be used without any restriction. For example, a liquid medium comprising carbon sources, nitrogen sources, inorganic substances, and other additives such as peptone, meat extract, yeast extract, glucose, potassium hydrogen phosphate, and purified water (neutrality or mild acidic pH) can be desirably used. In the present invention, in order to make the liquid medium into gel, a gelling agent is added. As such a gelling agent , at least one selected from the group consisting of corn starch, alginic acid, and agar, and among these, corn starch can be added, thereby increasing the viscosity of the medium. This prevents oxygen from entering the medium so as to achieve an anaerobic condition. As to gelation, a small amount of emulsifying agent (surfactant) such as Tween 80, for example, can be added for the purpose of keeping the uniform gel state of the liquid medium. Cultivation of Bifidobacterium longum for obtaining a culture supernatant of Bifidobacterium longum provided by the present invention can be conducted, for example, in the following manner. To the above liquid medium (pH 6.8) containing peptone, meat extract, yeast extract, glucose, potassium hydrogen phosphate, and purified water, for example, 10% corn starch aqueous solution is added, followed by adding purified water. Tlien, a small amount of Tween 80 is added, and the resultant solution is heated to about 80°C to gelatinize the medium with the aid o± corn starch. After cooling the medium, pH is adjusted (neutral or slightly acidic) , and then purified water is added again to pr-epare a culture liquid medium for Bifidobacterium longum. It goes without saying that the liquid medium may be prepared in various ways insofar as Bifidobacterium l ongum can be cultured in the resultant liquid medium. Concretely, Bifidobacterium longum can. be cultured using the liquid medium obtained above in the following manner. That is, after sterilizing the above liquid medium at high pressure, a culture fluid of Bifidobacterium longum is inoculated in an amount of about 5 mL per 100 mL medium, and cultured! in a normal condition. The cultivation is continued for about 72 hoiirs at 37°C while monitoring the degree of growth (cloudiness of liquid), for example. By the basic culturing procedure as described above, cultivation of Bifidobacterium longum completes. It goes without saying that the above culturing procedure is described just by way of example, and many variations are acceptable. An antiviral agent provided by the present invention is a culture supernatant of Bifidobacterium longum cultured in this manner. The culture supernatant can be obtained by removing bacterial cells from the liquid medium after cultivation by centrifugal separation. In conducting centrifugal separation of the culture fluid, when the viscosity of the medium after cultivation is large, centrifugal separation can be conducted after decomposing starch by adding amylase which is an amylolitic enzyme. Since the cultured bacterial cells remaining after collection of the culture supernatant also exhibit antiviral activity, an antiviral agent provided by the present invention may comprise as its active ingredient, the culture supernatant or the cultured bacterial cells. An antiviral agent of the present invention can be prepared as a liquid formulation by just diluting the culture supernatant obtained above in a pharmaceutically acceptable suitable diluent. Such a liquid formulation can be sprayed to oral mucosa or nasal mucosa, for example, in the form of spray, suspension, and the like. Also it can be administered through lung in the form of microparticles . Considering the fact that among virus infections, infections by e.g., influenza virus and SARS (severe acute respiratory syndrome) virus (one species of corona virus) occur through oral mucosa or nasal mucosa, these spray, suspension, and the like formulations are effective means for administering the antiviral agent of the present invention. Also administering microparticles through lung is effective administration means for prevention and therapy against these virus infections. The culture supernatant obtained above may be mixed with a pharmaceutically acceptable carrier, spray-dried, and orally administered in the form of powder. The powder formulation thus obtained may be mixed and kneaded together with an appropriate excipient or lubricant to form, e.g., granules or tablets. These dosage forms are efficacious against infections by, e.g., HIV virus and ATL virus through blood, and West Nile brain fever caused by West Nile virus mediated by mosquitoes, and the like. These formulations can be prepared in conformance with the methods described in the General Rules for Preparations in the Japanese Pharmacopoeia, and a variety of pharmaceutically acceptable carriers, excipients, lubricants, plasticizers, disintegrating agents, binders, isotonizing agents, and stabilizers can be selected and used as appropriate. The dose of the culture supernatant of Bifidobacterium longum which is an active ingredient in an antiviral agent provided by the present invention is not essentially limited. Any dose may be used insofar as the antiviral activity is exerted, and prevention of virus infection, suppression of infection, disappearance of viral titer and the like can be achieved, although it depends on the sex, age, body weight, symptom, and the like of the patient to whom the antiviral agent is administered. An antiviral agent provided by the present invention is especially efficacious in therapy of diseases caused by, concretely, varicella virus, measles virus, mumps virus, poliovirus, rotavirus, influenza virus , adenovirus , herpes virus , SARS ( severe acute respiratory syndrome) virus (one species of corona virus). West Nile virus, ATL (Adult T-cell Leukemia) virus, HIV virus, and the like. It is conceivable that it is especially valuable as a prophylactic agent against virus infection because it can effectively suppress the virus infection. Also the dose in a prophylactic or therapeutic agent against abnormal prion syndromes and Alzheimer's syndrome provided by the present invention, which comprises as its active ingredient a culture supernatant or cultured cells, is not essentially limited. In general, the dosage is as same level as in the above antiviral agent, although it depends on the sex, age, body weight, sympton, and the like of the patient to whom it is administered. In addition, the dosage form in a prophylactic or therapeutic agent against abnormal prion syndromes and Alzheimer's syndrome is not particularly limited, and various dosage forms can be employed.

EXAMPLES The present invention will be described in more detail by way of Examples, Test examples and so on, however the present invention is not limited to the description of these examples.

Example 1: Preparation of culture supernatant of Bifidobacterium (Part 1: Cultivation under gelling condition) As a basal medium, a liquid medium having the following composition (pH 6.8) was used. Peptone 10.0 g Meat extract 5.0 g Yeast extract 5.0 g Glucose 10.0 g Potassium hydrogen phosphate 3.0 g Tween 80 1.0 mL Purified water 1000 mL To 25 mL of the above liquid medium, 10 mL of 10% corn starch aqueous solution was added followed by adding purified water to obtain a total of 40 mL of liquid medium. This liquid medium was heated to 75 to 80° C to gelatinize the corn starch, thereby obtaining a homogeneous gel solution. After cooling, pH of the liquid medium was adjusted to 5.8. Then 20 mL of the liquid medium obtained above was put into a test tube, subjected to high-pressure sterilization at 115° C for 20 minutes, inoculated with 0.5 mL of culture medium of Bifidobacterium longum, and then cultured at 37° C for 72 hours. As the cultivation proceeded, the liquid medium became clouded. After completion of the cultivation, the pH of the culture medium was adjusted to between 5.0 and 6.0, and the medium was added with an appropriate amount of amylase. It was left at about 50° C for 30 minutes to cause decomposition of the starch. After cooling, the solution was centrifuged at 3000 rpm for 20 minutes, to obtain an objective culture supernatant.

Example 2: Preparation of culture supernatant of Bifidobacteria (Part 2: Cultivation under nongelling condition) As a basal medium, a liquid medium of the composition of Example 1 excluding Tween 80 was used. 20 mL of liquid medium was put into a test tube, subjected to high-pressure sterilization at 115° C for 20 minutes, inoculated with 0.5 mL of a culture fluid of Bifidobacterium longum, and cultured at 37° C for 72 hours in anaerobic condition. As the cultivation proceeded, the liquid medium became clouded. After completion of the cultivation, the solution was centrifuged at 3000 rpm for 20 minutes, to obtain an objective culture supernatant.

Test example 1: Antiviral activity ( in vivo) of culture supernatant Antiviral activity of the culture supernatant obtained in Example 1 was examined. A lactic acid bacteria culture fluid, a mixed culture fluid of butyric acid bacteria and lactic acid bacteria, and the like were used as controls for comparison.

(1) Test samples

Sample 1: Culture supernatant of Bifidobacterium longum according to the present invention, obtained above.

Sample 2 : Culture supernatant obtained from similar cultivation as

Example 1 using Bifidobacterium adolescent! as Bifidobacteria .

Sample 3 : Culture supernatant obtained from similar cultivation as

Example 1 using Lactobacillus acidophilus as lactic acid bacteria. Sample 4 : x 2 concentrated solution of Sample 3

Sample 5 : Cells deposited in obtaining Sample 3

Sample 6 : Mixed culture cells of butyric acid bacteria and lactic acid bacteria

Sample 7: Culture supernatant obtained by culturing Lactobacillus delbrueeki which is the standard species of lactic acid bacteria in the same manner as Example 1.

(2) Method Pretreatment was conducted by inoculating BALB/c mice (n=5 in each group) at either ear with 5 μL of each sample as described above. After 6 hours from the inoculation, they were inoculated with about 1.5 μL of 100 pfu (plaque forming unit) influenza virus PR8 to cause infection. After 3 days from infection, viral titer in wash fluid of nasal cavity was determined. As a negative control, a group not subjected to any treatment was provided.

(3) Result The result (average of 5 animals) is shown in Table 1 below. As is apparent from the result in the Table, proliferation of virus was not observed in the group having experienced the pretreatment with Sample 1 of the present invention, indicating strong antiprolifer tive effect against viruses.

TABLE 1

Test example 2: Bactericidal activity ( in vitro) of culture supernatant Using each of Samples 1 to 6 used in Test example 1, solutions having the concentrations listed in Table 2 below were incubated for 1 hour together with lxio³ CFU (colony forming unit) of S. typhimurium, and viable cells were allowed to grow in a nutrient agar medium, and then the bactericidal activity was quantified. Bacteriological assay was repeated three times. The result is shown in Table 2 below. The data is represented in percentage (100%: the count of killed cells from lxl0³ CFU of S. typhimurium cells in the positive control) of killed cells in the exposed cells. TABLE 2

As is apparent from the result shown in the Table, it was confirmed that the culture supernatant of Bifidobacterium longum according to the present invention has a bactericidal activity as is the same with culture fluids of other Bifidobacteria or lactic acid bacteria. From the results of Test example 1 and Test example 2, it was confirmed that the culture fluid of Bifidobacterium longum according to the present invention has very strong antiviral activity as well as the bactericidal activity. In contrast to this, in culture fluids of other Bifidobacteria or lactic acid bacteria, bactericidal activity but not antiviral activity can be observed. This confirms the specific effect of the culture supernatant of Bifidobacterium longum provided by the present invention. Formulation example 1: Spray formulation A spray formulation was prepared by using the culture supernatant obtained in Example 1.

Prescription: Culture supernatant obtained in Example 1 10 mL Ethylparaben 0.1 g Flavor trace amount Purified water balance According to the above prescription, a total of 100 mL of solution was prepared, sterilized at high pressure, and packed in a commonly used container, to produce a spray formulation.

Formulation example 2: Granule formulation A granule formulation was prepared by using the culture supernatant obtained in Example 1. Prescription: Culture supernatant obtained in Example 1 100 mL Lactose 200 g Corn starch 15 g Hydroxypropylcellulose 30 g Magnesium stearate 5 g According to the above prescription, lactose, corn starch, and hydroxypropylcellulose were mixed and granulated in a granulating machine. Then the resultant granules were sprayed with a culture supernatant obtained in Example 1, further kneaded, and dried to produce a granular formulation.

INDUSTRIAL APPLICABILITY As described above, the present invention provides an antiviral agent or a prophylactic or therapeutic agent against abnormal prion syndromes or Alzheimer's syndrome, comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium. This culture supernatant is very safe and has very strong antiviral activity. Considering the present state of art that no antiviral agent is available that is efficacious in prophylaxis or therapy of virus diseases caused by measles virus, mumps virus, poliovirus, rotavirus, influenza virus, adenovirus, herpes virus, SARS (severe acute respiratory syndrome) virus (one species of corona virus). West Nile virus, ATL (Adult T-cell Leukemia) virus, HIV virus, and the like, the present invention is very valuable in the medical field. Having prophylactic or therapeutic efficacy against bovine spongiform encephalopathy (BSE) in bovine, Creutzfeldt Jakob disease (CJD) in human, Gerstmann-Staussler-Sσheinker syndrome (GSS) which are diseases caused by proliferation of abnormal prion proteins, or Alzheimer's syndrome which is a similar symptom occurring in the brain, a culture supernatant of Bifidobacterium longum has enormous value in medical field.

Claims

1. An antiviral agent comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium.

2. The antiviral agent according to claim 1, wherein the culture supernatant is a culture supernatant obtained by cultivation of Bifidobacterium longum in a medium containing a gelling agent.

3. The antiviral agent according to claim 2, wherein the gelling agent is at least one selected from the group consisting of corn starch, alginic acid, and agar.

4. The antiviral agent according to claim 3, wherein the gelling agent is corn starch.

5. The antiviral agent according to any one of claims 1 to 4, wherein the antiviral agent exerts prophylactic effect against virus infection.

6. The antiviral agent according to any one of claims 1 to 4, wherein the antiviral agent exerts an effect of suppressing proliferation of virus due to virus infection.

7. The antiviral agent according to any one of claims 1 to 4, wherein the antiviral agent exerts an effect of making a viral titer due to virus infection disappear.

8. A prophylactic or therapeutic agent against abnormal prion syndromes, comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium.

9. A prophylactic or therapeutic agent against Alzheimer's syndrome, comprising, as its active ingredient, a culture supernatant of Bifidobacterium longum which is an anaerobic bacterium.

10. The prophylactic or therapeutic agent according to claim 8 or 9, wherein the culture supernatant is a culture supernatant obtained by cultivation of Bifidobacterium longum in a medium containing a gelling agent.

11. The prophylactic or therapeutic agent according to claim 8 or 9, wherein the gelling agent is at least one selected from the group consisting of corn starch, alginic acid, and agar.

12. The prophylactic or therapeutic agent according to claim 11, wherein the gelling agent is corn starch.