JP2911057B2 - Infection protective agents and functional foods having an infection protective effect - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-infection agent comprising a specific component which has a high ability to activate macrophages and recovers immunity and which enhances the defense of a living body against infection by pathogenic bacteria and viruses. And a functional food having an infection protective effect.

[0002]

2. Description of the Related Art Conventionally, antibiotics have been administered to prevent and treat infectious diseases caused by infection with pathogenic bacteria, which has been extremely effective. Its use is often restricted because it also exhibits side effects such as In addition, vaccines are administered for viral infectious diseases, but the target virus itself often mutates, so that there are many cases where the administration effect is not exhibited. In particular, influenza viruses often show little effect even if a vaccine is administered because the type of virus that spreads every year differs, and because they proliferate only in bronchiolar epithelial cells and do not exist in blood.

Therefore, it is desired to develop a method for preventing and treating infectious diseases that can be safely applied to allergic patients without any adverse effect and that does not lose its preventive effect even by mutation of the virus. In recent years, an anti-infective agent comprising a sulfated polysaccharide produced by a bacterium of the genus Arthrobacter as an active ingredient (Japanese Patent Publication No. 63-44128) and a sialic acid-binding protein or sialic acid-binding protein derived from milk Infection protective agents containing a peptide as an active ingredient (JP-A-63-284113) have been proposed.

[0004] On the other hand, the development of food has been in the pursuit of nutritional sufficiency and palatability, but as the relationship between diet and adult diseases becomes clear, health has become a major theme, Functional foods have been attracting attention as foods that have been elucidated and that are useful for preventing certain diseases. Those having the above-mentioned infection protective effect have also been proposed, for example, infant formula containing the above-mentioned sialic acid-binding protein and / or sialic acid-binding peptide
(Japanese Unexamined Patent Publication (Kokai) No. 64-55143). [Food and Development, 25 [3] pp. 24-31 (1990)].

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above-mentioned problems associated with the administration of antibiotics and vaccines which are conventional preventive and therapeutic agents for infectious diseases.
It is an object of the present invention to provide an infection protective agent that can be safely applied to allergic patients without any adverse effect and that does not lose its preventive effect even when the virus is mutated. Another object of the present invention is to provide a functional food having a protective effect against infection, which can easily incorporate its effects in the form of a functional food that is currently receiving attention.

[0006]

Means for Solving the Problems To achieve the above object, the present inventors have developed a herbal medicine, Sanyak (Dioscoreae Rhizom), which has been conventionally used as edible and is applied to nutritional tonic, antidiarrheal and the like.
a) (Dio), also known as the base plant of (a)
scoreaceae) A plant belonging to the genus Yamanoimo (Dioscorea L.), i.e.
ponica), potatoes (Dioscorea batatas), etc.
This rhizome was subjected to fermentation treatment, and it was found that the fraction having a specific molecular weight in the water-soluble fraction had high macrophage activating ability and immune recovery ability and increased the defense ability of the living body against infection with pathogenic bacteria and viruses. The present inventors have found that an infection protective agent containing the active ingredient as an active ingredient and a functional food containing the same solve the above problems.

[0007] That is, the present invention relates to Diosco family (Diosco
reaceae) Infection protective agent comprising a rhizome of a plant belonging to the genus Dioscorea L. fermented and containing, as an active ingredient, a water-soluble fraction having a molecular weight of from 1,000 to 2,000,000, The gist is a functional food having a protective effect against infection. Hereinafter, the present invention will be described in detail. Examples of plants belonging to the genus Yamaceae (Dioscoreaceae) genus Yam (Dioscorea L.) used in the present invention include yam (also known as dinnenjo, Dioscorea japonica), yam (D. batatas), and nagakashi (D. bulbifera).
Daijo (D. alata), Bark (D. esculeta), Yellow Guinea Yam (D. cayenensis), Kashuimo (D. bul)
bifera), honey beetle (D. trifida),
(D. pentaphylla), D. tokoro, D. nipponica, D. tenuipes,
Maple docolo (D. quinqueloba),
eptemloba), D. gracillima, D. asclepiadea, D. izuensis
These are all edible and have no adverse effect on the human body, but it is particularly preferable to use yam and yam, which are most generally widely used for food.

In the present invention, water is added to the rhizomes of the above-mentioned plants, ground, and inoculated with microorganisms for cultivation (fermentation treatment). As the microorganism used herein, B. subtilis belonging to the genus Bacillus and microorganisms of a closely related species are preferable. Examples of microorganisms related to Bacillus subtilis include Bacillus pumilus and B. licheniformis. Natto (B. natto), which has been used for the production of natto, which is conventionally known as a nutritional food, is currently classified as the same as Bacillus subtilis, and may be mentioned as a particularly preferred microorganism. it can. In addition, other microorganisms used in foods, such as Penicillium roqueforti used in cheese, and Rhizopus oligospora used in tempe
orus), the lactic acid bacterium Lactobacillus acidophilus (La
Ctobucillus acidophilus, Streptococcus lactis, Bifidobacterium longum and the like can also be used.

[0009] The time required for the culturing is preferably from 100 hours to 200 hours. The cells and insolubles were removed from the resulting culture to obtain a water-soluble fraction.
The active ingredient is obtained by performing ultrafiltration with an ultrafiltration membrane having a molecular weight of 1,000 or less and an ultrafiltration membrane having a molecular weight cut off of 1,000 or more. Further, it is preferable to freeze-dry the obtained effective fraction. The component thus obtained has a high ability to activate macrophages and an ability to recover immunity, and exhibits an excellent infection protective effect. A fraction having a molecular weight cut-off of less than 1,000 or a fraction having a molecular weight of more than 200,000 does not sufficiently exhibit the intended effect.

[0010] The active ingredient obtained by the above-mentioned treatment is used as an anti-infective agent as an oral agent such as powder, tablet, capsule, granule, liquid, etc .; subcutaneous, intramuscular or intravenous injection; rectal administration by infusion mixture or suppository. It can be administered by any of these methods. The above-mentioned active ingredient can be orally administered as it is, but can also be formulated by a known method. That is, powders, tablets, capsules or granules for oral administration may contain the active ingredient as an excipient such as starch, lactose and mannitol; binders such as carboxymethylcellulose and hydroxypropylcellulose; crystalline cellulose and calcium carboxymethylcellulose. Disintegrants; lubricating agents such as talc and magnesium stearate; and, if necessary, formulating appropriate combinations of wetting agents and the like. Oral liquid preparations are aqueous or oily emulsions Solution, syrup, etc .; suspending agents such as simple syrup, sorbitol, methylcellulose, sodium carboxymethylcellulose; emulsifiers such as egg yolk lecithin, sorbitan monofatty acid ester, lauromacrogol, castor oil; Add preservatives, stabilizers, etc. as appropriate Can. The injection may be in the form of an oil solution, an emulsion, or an aqueous solution, and these solvents are commonly used emulsifiers,
Stabilizers can be added as appropriate.

[0011] The dosage of the protective agent of the present invention is usually 200 mg to 1,000 mg for oral administration and 60 mg to 300 mg for parenteral administration once a day to several times a day. Is preferred. In addition, the active ingredient may be in the form of a functional food that can easily incorporate its effects. Various forms can be cited as the form of the functional food, for example, miso, soy sauce, mixed with ingredients for seasonings such as noodle soup, cooked food, desserts, confectionery,
It can be added to beverages and the like, and if necessary, sweeteners, coloring agents, flavors and the like can be appropriately added. In addition, known excipients for foods and other compounds may be added to form powders, tablets, capsules, syrups and the like. The amount of the active ingredient added to the food may be appropriately changed depending on the form of the food. For example, in the case of miso or noodle soup, it is preferably about 3%, and in the case of powder or tablet, it is preferably about 10 to 50%. .

The above-mentioned functional food of the present invention is usually used for adults.
The amount of the active ingredient per day is from 200mg to 1,000mg
It is preferable to take the food so that it is usually 5 per day.
About 00mg is enough. Next, production of the active ingredient of the present invention
Examples and test examples of effects will be described. [Production Example of Active Ingredient] To 100 g of rhizome of D. batatas with skin removed
Add 200 ml of distilled water and grind for 5 minutes with a homogenizer.
The dinate was obtained. Add 300g of this homogenate to 2L Sakaguchi
Put into a flask, cover with cotton, and put in an autoclave at 121 ° C20
It was sterilized for a minute. After cooling, add
Preculture in a yam homogenate medium at 30 ° C for 18 hours.
Was inoculated with 1 ml of a culture solution of B. subtilis.
The number of viable bacteria immediately after seeding is 1 × 10^FourPcs / ml). Shake at 28 ° C for 120 hours
After culturing (the viable cell count at the end of culturing is 3 × 10⁹Individual
/ Ml) and centrifuged at 12,000 rpm for 90 minutes at 5 ° C.
Was performed to separate into an insoluble matter containing bacterial cells and a supernatant. Obtained
The supernatant was filtered at 4 ° C with Ultrafilter UK-200 (ADVANTEC
Ultrafiltration using a
150 ml of the lower fraction were obtained. UH-1 (ADVANTEC)
Ultrafiltration using a filter with a molecular weight cut off of 1,000 or more and 2
Fractions of no more than 00,000 are freeze-dried and yellow-brown
2.4 g of a powder of the present invention was obtained (referred to as the effective fraction of the present invention) This one
The measured infrared absorption spectrum (IR) of
m^-1, 1620〜1670cm^-1, 1380cm^-1, 1120cm ^-1, 1025cm^-1
Met.

A fraction obtained simultaneously with a molecular weight of more than 200,000 (comparative fraction 1) and a fraction with a molecular weight of less than 1,000
The lyophilized powder (comparative fraction 2) was 0.4
g and 0.7 g. [Test Examples of Active Ingredients] Test Example 1: Macrophage activation test in vitro 1 × 10 ⁵ mouse macrophage-derived cell line J-774-1
10% fetal calf serum (FCS)
The cells were suspended in RPMI 1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and dispensed in 90 μl portions into a 96-well plate for cell culture. Next, each fraction obtained in the above-mentioned Preparation Example and unfermented yam homogenate
A 2% physiological saline solution (hereinafter referred to as unfermented fraction) was prepared by serially diluting a 2% physiological saline solution (solution dissolved in physiological saline to 2%, the same applies hereinafter) twice with physiological saline. , And sequentially add 10 μl to each well.
The cells were cultured for 72 hours under the conditions of% CO ₂ and 37 ° C. Activation of J-774-1 was caused by microscopic change of cells after 72 hours of culture, that is, the morphology of J-774-1 in the control group to which physiological saline was added instead of the sample solution (small spherical shape). ) Was determined to become a dreadnought, dreadnought or large sphere by culture with the addition of a sample solution [A. Amemura et al .; Agric. Bio
l. Chem., 51 , pp. 2649-2656 (1987)]. Table 1 shows the result of calculating the minimum activation concentration of each fraction from the maximum dilution factor of each sample solution in which this activation occurs.

[0014]

[Table 1]

According to Table 1, the fraction containing the active ingredient of the present invention has a very high activating ability against J-774-1,
It is clear that the fraction having a molecular weight of more than 200,000 (comparative fraction 1) and the fraction having a molecular weight of less than 1,000 (comparative fraction 2) have a weaker ability to activate J-774-1. Test Example 2: Immunity recovery test using mice Mice whose immunity was reduced by administration of cancerous ascites [I. Hrsak
J. Nat. Cancer Inst., 53 , pp.1113-1119 (1974).
Immunity recovery by administration of the sample
The hemolytic antibody titer using (SRBC) as an antigen was examined as an index.

Specifically, first, 0.2 ml of peritoneal cancerous ascites was administered into the tail vein 5 days or 2 days before the sensitization to SRBC, and 0.1% of each fraction obtained in the above-mentioned Preparation Example was administered. An 8 mg / ml saline solution was used as a sample, and 0.5, 4 days, 2 days, 1 day, 1 day, 3 days after SRBC sensitization per mouse.
Each ml was intraperitoneally administered. Schizophyllan as a positive control
(Manufactured by Kaken Pharmaceutical Co., Ltd.) Y. Numasaki et al .; Pharmacometri
Using a 0.1 mg / ml saline solution of cs, 39 , pp. 39-48 (1990), 0.5 ml was intraperitoneally administered in the same schedule as the sample administration. SRBC sensitization was performed by injecting 2 × 10 ⁴ mice into the tail vein, blood was collected on day 13 after sensitization, and anti-SRB in serum was collected.
The C antibody titer was measured by a hemolysis reaction. The mice were 6-week-old ICR male mice, and 36 mice were divided into 6 groups of 6 mice each. Six groups were administered physiological saline instead of cancerous ascites. Instead, saline was administered.
The third group was a group administered with schizophyllan, the fourth group was a group administered with an effective fraction of the present invention, the fifth group was a group administered with a comparative fraction 1, and the sixth group was a group administered with a comparative fraction 2. Table 2 shows the anti-SRBC hemolytic antibody titer of each group.

[0017]

[Table 2]

From Table 2, it can be seen that the administration of the fraction containing the active ingredient of the present invention has the same anti-S
Although RBC hemolytic antibody titer was shown and significant immune recovery ability was recognized, administration of the fraction having a molecular weight of more than 200,000 (comparative fraction 1) and the fraction having a molecular weight of less than 1,000 (comparative fraction 2) was not It is clear that almost no immune recovery ability is observed. Test Example 3: Lung Consolidation Test Due to Influenza Virus Infection (1) Degree of Lung Consolidation after Infecting Mice to which Samples were Intraperitoneally Infected with Influenza Virus
olidation score; Cs).

Specifically, 36 ICR male mice weighing 15 g were divided into four groups of 12 groups (group 1) and three groups of 8 mice (groups 2 to 4). The first group was a control group to which physiological saline was administered instead of the sample, and the second to fourth groups were sample administration groups. Group 1 was 0.5 ml of physiological saline once a day for 5 days before influenza virus infection, group 2 was the effective fraction of the present invention, group 3 was comparative fraction 1, and group 4 was comparative fraction 2
With 0.32% (w / v) saline solution, respectively
0.5 ml was administered intraperitoneally on the same schedule as the administration of the first group. Influenza viruses were径鼻infect 2 times the LD ₅₀ using the influenza A virus PR-8 strain (H1N1) (mouse-adapted strains several generations passaged in mouse lung). Seven days after infection, mice were dissected, and E. Shimomura et al.
The lung Cs was evaluated by the method described in Microbiol. Immunol., 26 (2), pp. 129-138 (1982). That is, the maximum value of Cs is 5, and the larger the value, the more consolidation is advanced. Table 3 shows the average Cs of each group.

[0020]

[Table 3]

According to Table 3, administration of the fraction containing the active ingredient of the present invention was 47.3% of Cs in the control group, and the administration effect was recognized, but the fraction having a molecular weight of more than 200,000 (comparative fraction) 1)
It is clear that the administration effect is hardly recognized in the fraction having a molecular weight of less than 1,000 (comparative fraction 2). Test Example 4: Lung Consolidation Test Due to Influenza Virus Infection (2) Cs in the lung after infecting a mouse to which a sample was orally administered with an influenza virus [influenza A virus PR-8 strain (HINI)] was evaluated.

Specifically, 0.8% (w / v) of a 0.8% (w / v) physiological saline solution of each sample solution was added to each of Groups 2 to 4 for 5 days before virus infection and 2 hours after infection. Cs was evaluated in exactly the same manner as in Test Example 3 except that oral administration was performed using an oral probe to the eyes. Table 4 shows the average Cs of each group.

[0023]

[Table 4]

From Table 4, administration of the fraction containing the active ingredient of the present invention was 59.6% of Cs in the control group, and the administration effect was recognized, but the fraction having a molecular weight of more than 200,000 (comparative fraction) 1)
It is clear that the administration effect is hardly recognized in the fraction having a molecular weight of less than 1,000 (comparative fraction 2). Test Example 5; Influenza virus infection protection test Regarding the infection protection effect after infecting a mouse to which the effective fraction of the present invention obtained in the above Production Example was orally administered with an influenza virus, the change in the body weight of the mouse, the average survival days and the survival The rate was evaluated.

Specifically, first, an ICR male weighing 15 g
The 16 mice were divided into 2 groups of 8 mice each.
Influenza A virus PR-8 strain (HINI)
LD₅₀1 day for 5 days before infection
Once, 2 hours after infection and once a day for 1-4 days after infection
Oral injection of 0.2 ml of physiological saline using an oral probe
Gave. The second group was a group to which the sample was administered, and 2.
0.2% of a 4% (w / v) saline solution was scheduled as in the first group
Orally administered with Of mouse weight after virus infection
Figure 1 shows the results of measurement of recovery, average survival days and survival rate.
2 and FIG. The average survival time of the control group 1 was 12.125.
Day, the survival rate is 25%, whereas the second group of sample administration group
Has an average survival time of 15.25 days and a survival rate of 87.5%
In addition, the recovery of the weight of the mice was also improved in the sample administration group of the second group.
Is 10 days after virus infection compared to control group 1?
The effect of the administration of the sample is clear. Test Example 6: Acute toxicity test ICR mice of 6 weeks old and weighing about 15 to 20 g
A total of 10 animals were intraperitoneally injected with 0.4 ml of a 10% aqueous solution of the effective fraction of the present invention.
Gave. The mortality after one week was 10% (1 out of 10)
Mice are healthy and no abnormalities are observed
Was. Therefore, LD ₅₀Is 2g / kg or more and extremely toxic
It is clear that it is weak.

[0026]

Next, the present invention will be described in detail with reference to examples. However, the present invention is not limited by these examples. The above components were mixed according to a conventional method, and the particle size was adjusted through a 60-mesh wire mesh. Then, one tablet was produced using a tableting machine.

[0027]

Since the present invention is constituted as described above, the infection-protecting agent of the present invention and the functional food having the infection-protecting effect containing the same are provided by the family Dioscore
aceae) The rhizome of a plant belonging to the genus Yams (Dioscorea L.) is subjected to fermentation treatment and the water-soluble fraction is administered by containing a fraction having a molecular weight of 1,000 or more and 200,000 or less as an active ingredient. Ingestion has the effect of providing high macrophage activating ability and immune recovery ability and increasing the defense ability of the living body against infection with pathogenic bacterium virus and the like. Functional foods that have no adverse effects on allergic patients and can be applied safely. It is a great place to improve.

[Brief description of the drawings]

FIG. 1 shows that influenza A virus PR-8 strain was administered to mice (Group 1) to which physiological saline was orally administered in Test Example 5.
In the body weight of mice after infection with (HINI)
(A) and the change in the number of survivors (B).

FIG. 2 shows the change in body weight of mice after infecting mice (Group 2) to which the effective fraction of the present invention was orally administered in Test Example 5 with influenza A virus PR-8 strain (HINI) (A) and It is a figure which shows the fluctuation | variation (B) of the number of survivors.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int.Cl. ⁶ , DB name) A61K 35/78

Claims (4)

(57) [Claims] 1. A method of fermenting a rhizome of a plant belonging to the genus Yamaceae of the family Yamaceae to produce a water-soluble fraction having a molecular weight of 1,000.
An agent for controlling infection, comprising as an active ingredient a fraction of at least 200,000. 2. The infection control agent according to claim 1, wherein the microorganism used for the fermentation treatment is a Bacillus subtilis belonging to the genus Bacillus and a microorganism of a closely related species thereof. 3. The rhizome of a plant belonging to the genus Yamaceae of the family Yamaceae is subjected to fermentation treatment, and the molecular weight in the water-soluble fraction is 1,000.
A food for controlling infection, comprising a fraction of at least 200,000 as an active ingredient. 4. The food for controlling infection according to claim 3, wherein the microorganisms used for the fermentation treatment are Bacillus subtilis belonging to the genus Bacillus and microorganisms of a closely related species thereof.