JP4683881B2 - Antitumor active - Google Patents

Description

  The present invention relates to an antitumor active agent using a metabolite of lactic acid bacteria.

Cancer prevention effects of lactic acid bacteria have been verified in various ways using mice, etc., all of which have been focused on the function as a carcinogenesis prevention action, especially regarding the cancer growth inhibition action of lactic acid bacteria metabolites However, the current situation is that it cannot always be said that a remarkable effect is obtained.
The growth inhibitory action using lactic acid bacteria is clarified in Table 8.4 on page 327 of “Science and Technology of Lactic Acid Bacteria” edited by Lactic Acid Bacteria Research Meeting. Casei was orally administered to mice. However, it has been clarified that even when lactic acid bacteria are directly administered, they hardly function as viable bacteria. Therefore, in the case of this conventional administration method, L.P. In order for casei to function as a living bacterium, there is a problem that its administration method must be devised, but no mention is made of its administration method.

Lactic Acid Bacteria Research Meeting, “Science and Technology of Lactic Acid Bacteria”, Academic Society Publishing Center, February 28, 1996, p. 327

  An object of the present invention is to provide a lactic acid bacterium metabolite that has a simple administration method and has a remarkable cancer growth inhibitory action.

The first invention is B.I. Longum (Bifidobacterium
longum), B.I. Bifidobacterium bifidum, B. Adrecentis (Bifidobacterium
adolescentis), L. Lactobacillus acidophilus, L. Brevis (Lactobacillus
brevis), L.M. Lactobacillus jensenii, L. Paracasei (Lactobacillus
paracasei subsp. paracasei), L. Gasserie (Lactobacillus
gasseri), L.M. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Helveticas (Lactobacillus)
helveticus), L.H. Casei (Lactobacillus casei subsp. Casei), L. Ramonausus (Lactobacillus)
rhamnosus), L. Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii), L. Lactococcus
lactis), E.I. Enterococcus faecium, S. Using 16 types of lactic acid bacteria consisting of thermophilus (Streptococcus thermophilus) , one or more types of lactic acid bacteria selected from these 3 genera were used to form multiple groups and subcultured for each group. The lactic acid bacteria culture solution obtained by maintaining the symbiotic state and further culturing the cultivated lactic acid bacteria of the subcultured group unit with each other after heat sterilization, and the filtrate obtained by filtering this filtrate It is assumed that the residue is mixed with powdered powder.

Of the 16 types of lactic acid bacteria , E. Fesium and L. Helveticas in the first group, E. Fesium and L. Acidophilus in the second group, E.I. Fesium and L. Gasseley is the third group, E.I. Fesium, L.C. Acidophilus and L. Brevis the 4th group, E. Fesium, L.C. Acidophilus and L. Brevis the 5th group, E. Fesium, L.C. Acidophilus, L.H. Brevis, L. Paracasei in the sixth group Adrecentis alone is group 7; Delbricky and L. Gasselie is in the 8th group, L.C. Delbricky alone is the ninth group, E.D. Fesium, L.C. Genseny, L. Paracasei and L. Brevis the 10th group, L. Acidophilus alone is group 11; Fesium and L. Gasserie was placed in the 12th group, L. Paracasei alone was added to the 13th group, L. Gassely, E.M. Fesium and B.I. Bifidum is the 14th group. Longum, S.M. Thermophilus and E. coli. Fecium was added to the 15th group, L.P. Gasserie alone, group 16; Bulgarix and S. Thermophilus in the 17th group, L. Gassely, L.C. Lactis, L. Gasserie and E.C. Fesium was added to the 18th group, L.C. Gassley, S.M. Thermophilus and L. Bulgarix was introduced in the 19th group, L.B. Lactis alone, 20th group, L. Gasserie and E.C. Fecium was added to the 21st group, L.P. In the 22nd group, L. Casei alone, group 23, B.I. Longum alone is the 24th group. And while producing | generating the culture solution subcultured for each group, among those culture solutions, 1st, 2nd group, 3rd, 4th group, 5th, 6th group, 7th, 8th group Between the 9th and 10th groups, between the 11th and 12th groups, between the 13th and 14th groups, between the 15th and 16th groups, between the 17th and 18th groups, between the 19th and 20th groups, between the 21st and 22nd groups First, 23 and 24 groups were cultured together, and further, the 1st, 2nd group, 3rd, 4th group, 5th, 6th group, 7th, 8th group, 9th, 10th group and 11th, 12th group Secondary culture between the 13th and 14th groups and the 15th and 16th groups, the 17th and 18th groups and the 19th and 20th groups, the 21st and 22nd groups and the 23rd and 24th groups, Et al in, characterized in that it tertiary culture as a mixture thereof each secondary culture.

The second invention is characterized in that the tertiary culture solution is heated to sterilize lactic acid bacteria and cooled to coagulate fat, and then naturally filtered to obtain the filtrate .

According to this invention, large tumor suppression effect, moreover, since the metabolites of the lactic acid bacteria can exert an effect of less side effects on the human body. In addition, since lactic acid bacteria are not used as viable bacteria but their metabolites are used, the administration method is simpler than when the live bacteria are introduced into the body.

  In order to produce the antitumor active agent in this embodiment, any of lactic acid bacteria belonging to the genus Lactobacillus, lactic acid bacteria belonging to the genus Bifidobacterium, and lactic acid bacteria belonging to the genus Streptococcus Several lactic acid bacteria belonging to were used.

  As a lactic acid bacterium belonging to the genus Lactobacillus, L. Lactobacillus acidophilus, L. Brevis (Lactobacillus brevis), L. Lactobacillus jensenii, L. Paracasei (Lactobacillus paracasei subsp. Paracasei), L. Gasseri (Lactobacillus gasseri), L. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Lactobacillus helveticus, L. Casei (Lactobacillus casei subsp. Casei), L. Lactobacillus rhamnosus, L. Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii), L. One kind or two or more kinds of Lactococcus lactis were used.

  As lactic acid bacteria belonging to the genus Bifidobacterium, B. Bongidobacterium longum, B. Bifidobacterium bifidum, B. One type or two or more types of Adrecentis (Bifidobacterium adolescentis) were used.

  As lactic acid bacteria belonging to the genus Streptococcus, S. Thermophilus (Streptococcus thermophilus), E.I. Enterococcus faecium, L. One or more of Lactococcus lactis was used.

And the above lactic acid bacteria were cultured as follows.
First, a plurality of lactic acid bacteria were divided into 24 groups, and they were subcultured for each group. The grouping is as follows. That is, as shown in FIG. Fesium and L. The second group consists of E. helveticas. Fesium and L. It consists of acidophilus. The third group is E.E. Fesium and L. The fourth group consists of E. Gasseley. Fesium, L.C. Acidophilus and L. Consists of Brevis.

  The fifth group is E.I. Fesium, L. Acidophilus and L. The sixth group consists of E.Brevis. Fesium, L. Acidophilus, L.H. Brevis, L. The seventh group consists of paracasei. It consists of Adrecentis alone.

  The eighth group is Delbricky and L. The 9th group consists of L. Gasseley. The 10th group consists of E.D. Fesium, L. Genseny, L. Paracasei and L. The eleventh group consists of L.B. It consists of Acidophilus alone.

  The twelfth group is Fesium and L. The thirteenth group consists of L. The 14th group is composed of L. Paracasei alone. Gassely, E.M. Fesium and B.I. The 15th group consists of B. Longum, S.M. Thermophilus and E. coli. It consists of fesium. 16th group, L.M. The 17th group consists of L. Bulgarix and S. It consists of thermophilus.

  The eighteenth group is Gassely, L.C. Lactis, L. Gasserie and E.C. The 19th group consists of L.F. Gassley, S.M. Thermophilus and L. The 20th group consists of L.B. Lactis is a simple substance. Gasserie and E.C. Fesium, 22nd group, L. It consists of Ramonausus alone, and the 23rd group Casei consists of a single case. It is composed of longum alone.

  While subculturing each group made as described above for each group, among those culture solutions, the first and second groups, the third and fourth groups, the fifth and sixth groups, the seventh, 8 groups, 9th, 10th group, 11th, 12th group, 13th, 14th group, 15th, 16th group, 17th, 18th group, 19th, 20th group, 21st, 22nd group Primary culture was performed between the groups and the 23rd and 24th groups.

Further, the first, second and third groups, the fourth and fifth groups, the fifth and sixth groups, the seventh and eighth groups, the ninth and tenth groups, the eleventh and twelfth groups, the thirteenth and fourteenth groups, and the fifteenth and sixteenth groups. Secondary culture was performed between the 17th and 18th groups and the 19th and 20th groups, the 21st and 22nd groups and the 23rd and 24th groups, and these secondary cultures were further mixed and subjected to tertiary culture.
Then, the tertiary culture solution obtained as described above is heated to sterilize lactic acid bacteria and filtered to obtain a filtrate, and the residue obtained by filtering the filtrate is pulverized to obtain the target powder. Obtained.

The concentrate obtained by concentrating the filtrate obtained as described above four times and the powder obtained from the filtrate residue were mixed, and the mixed substance was orally administered to a mouse transplanted with Meth-A sarcoma. However, sufficient antitumor activity was observed. That is, 8 weeks old BALB / c male mice were treated with Meth-A
While transplanting sarcoma, a certain amount of the above filtrate and powder were orally administered once a day for 28 days once a day from 14 days before transplantation. As a result, an antitumor activity with a tumor growth rate of 47.2% was observed.

  Each group of lactic acid bacteria was subcultured as follows. That is, each lactic acid bacterium in each group is assembled while predicting that a symbiotic state will be maintained even when they are subcultured.

As the culture medium of each lactic acid bacteria group, three types of medium consisting of GAM semi-fluid high layer medium, BL agar medium or modified GAM agar medium manufactured by Nissui Pharmaceutical Co., Ltd. are used according to the lactic acid bacteria, and these mediums are used. Incubated at 32 ° C. for 12 hours. Then, it culture | cultivated at 37 degreeC for 12 hours, and also cultured at 40 degreeC for 24 hours. The grouped lactic acid bacteria were subcultured as described above, and the culture solution was refrigerated at 5 ° C.
In the 24th group, B.I. As the longum, Morinaga BB536, a strain produced by Morinaga Milk Industry Co., Ltd., was used.

  In this way, subculture was performed and identification was made for each group. The identification test was requested from the Japan Food Analysis Center. The outline of the identification test is as follows. Each group of specimens is directly inoculated and cultured on an agar plate medium, and colonies with different shapes that have grown predominantly are isolated and lactic acid bacteria are separated for each group. Observation, physiological property test, and measurement of GC content of intracellular DNA were performed and identified with reference to the following literature.

1. Sneath, PHA, Mair, NS, Sharpe, ME and Holt, JG: “Bergey's Manual of Systematic Bacteriology” Vol.2, (1986) Williams & Wilkins.
2. Holt, JG, Krieg, NR, Sneath, PHA, Staley, JT and Williams, ST: “Bergey's Manual of Determinative Bacteriology” Ninth Edition (1994) Williams & Wilkins.
3. Tomokazu Mitsuoka: “The World of Enteric Bacteria”, (1984) Sobunsha.
4). Yoshino Kanno: Microorganisms 6,3-14 (1990).
5. Supervised by Ministry of Health and Welfare, Health Sanitation Bureau: “Food Sanitation Inspection Guidelines-Microorganisms” (1990) Japan Food Sanitation Association.
6). Schleifer, KH and Kilpper-Balz, R .: Int. J. Syst. Bacteriol., 34, 31-34 (1984).
As a result of the above identification, as shown in FIG. 1, it was confirmed that 34 strains were identified and maintained in a symbiotic state.

  The culture solution subcultured as described above was subjected to primary co-cultivation. In FIG. 1, (1) to (12) correspond to primary co-cultivation. And in this primary co-cultivation culture, while using three types of medium consisting of a GAM semi-fluid high layer medium, BL agar medium or modified GAM agar medium manufactured by Nissui Pharmaceutical Co., Ltd. as the primary co-culture medium, These primary co-cultivation culture media were selectively used according to lactic acid bacteria.

  Then, the grouped lactic acid bacteria were added to the medium for any one of the three types of primary co-cultivation, and the primary culture was performed. For example, the grouped lactic acid bacteria medium was fluidized as described above. When the lactic acid bacterium contained bifidobacteria in a high-quality medium, 10% lactic acid bacterium was added to the primary co-culturing medium. In the case of the group not containing bifidobacteria, 3% lactic acid bacteria were added to the medium for primary co-cultivation.

In addition, when the grouped lactic acid bacteria culture medium is a medium with high solidity, a single amount lifted with a platinum loop was added to the primary co-cultivation culture medium.
And in these culture media, it culture | cultivated for about 6 to 12 hours until it became pH 4.6 at 37 degreeC.

  In secondary co-cultivation, soy milk and skim milk are mixed in a ratio of 4: 1 as a medium, and 0.5% (w / w) of glucose, 0.4% (w / w) of yeast extract and pre-produced content are mixed with this mixture. 1% (w / w) of the third cultivated broth was added. Then, this medium was cultured at 37 ° C. until it reached pH 4.55 for about 6 to 10 hours. In this secondary culture, the medium and additive elements were all the same for all bacteria.

  In the third culture, soymilk: skim milk is mixed at a ratio of 8: 1, and 0.5% (w / w) of glucose, 0.4% (w / w) of yeast extract, and tertiary symbiosis that is already produced. 1% (w / w) of the culture solution was added. The medium thus prepared was first cultured at 32 ° C. for 24 hours, then cultured at 40 ° C. for 48 hours, and further cultured at 37 ° C. for 24 hours.

  Lactic acid bacteria were sterilized using the high-pressure steam sterilization method for the mixed culture liquid subjected to the third culture as described above. That is, in the sterilization apparatus heated to about 120 ° C., the tertiary culture solution was heated at 65 ° C. for 40 minutes to sterilize the lactic acid bacteria in the tertiary culture solution. The sterilized final culture solution was cooled to 15 ° C. to coagulate unnecessary substances such as fat. The reason why the fat is coagulated in this manner is to facilitate removal of the fat by filtration. If the fat is removed in this manner, the filtrate when filtered can be prevented from being suspended. Moreover, it can also prevent that an oxide increases in a filtrate, when fat content oxidizes.

  The culture broth cooled as described above was added with 12% absolute ethanol and crushed with a high-speed homogenizer. By crushing the culture broth in this way, the subsequent filtration can be performed in a short time. become.

And after naturally filtering the said sterilized culture solution using a 3 micrometer filter paper, while applying a 10-kg weight to it and further squeezing, this squeezed liquid is filtered with a 0.1-0.2 micrometer hollow fiber membrane, and impurities are removed. The filtrate consisting of metabolites of lactic acid bacteria was obtained.
In addition, although the said filtrate is heated for sterilization as mentioned above in the production | generation process, the component contained in lactic acid bacteria can be heat-extracted by performing heat processing in this way. .

Further, the residue filtered as described above is squeezed and solidified by applying a pressure of 1.2 tons, and the solidified residue is further sliced and then dried, and then the powder refiner To obtain a powder of 60 mesh particles.
And it mixed in the ratio of 2000 mg of powder with respect to 10 ml of filtrate, and produced | generated the antitumor active agent.

  The filtrate obtained as described above contained substances as shown in Tables 1 and 2. Table 2 shows the results of an amino acid analysis test, and an amino acid automatic analysis method was used as the amino acid analysis method. In Table 2, tryptophan was analyzed using a high-speed chromatographic method.

  Further, when the components of the above powder, which is a product of lactic acid bacteria, were examined, as shown in Table 3, various physiologically active substances such as amino acids as water-insoluble substances and γ-aminobutyric acid were present. found.

  It is speculated that the content of this amino acid is amazing and has a positive effect on the antitumor activity. This is because the amino acid is very important as a raw material for immune cells, and is also very important for carbon monoxide and immunoglobulin produced by these immune cells to attack cancer cells and bacteria.

In this example, it was found that, when the filtrate produced as described above was added to the culture medium in each of the above-described primary culture to tertiary culture, the growth of the target lactic acid bacteria was promoted. However, the following can be considered as the reason.
That is, as shown in Table 2 above, it can be seen that the filtrate contains a lot of microbial nutrient factors. Therefore, it is considered that the growth of the target lactic acid bacteria is promoted by further adding the filtrate to the culture medium of the target lactic acid bacteria.

  Furthermore, as shown in Table 2, this filtrate contains a large amount of amino acids. Therefore, it is considered that by adding this filtrate to the culture medium of the target lactic acid bacteria, the target lactic acid bacteria are grown by metabolizing amino acids.

Moreover, although it does not appear in the above analysis results, it can be said that, by co-culturing 16 types of lactic acid bacteria, this filtrate contains many unique metabolites produced by the action of various bacteria. Seem. The abundant types of these metabolites may have an effect on the promotion of the growth of the target lactic acid bacteria.
In any case, the above results are considered to be the result of coculturing 16 types of lactic acid bacteria. In other words, it can be inferred that such a result will not be obtained by coculturing about 4 types of lactic acid bacteria.

(Experimental example)
And the antitumor active agent which mixed the said filtrate and powder was orally administered to the mouse | mouth, This mouse | mouth uses a BALB / c type | system | group male mouse (8 weeks old: Nippon SLC Co., Ltd.), barrier system After being bred for 6 days in this environment, Meth-A sarcoma cells were transplanted subcutaneously in the buttocks and used for the experiment. The history of the experiment is as follows.

  The mice were divided into the non-administration group, the filtrate 5 ml / kg administration group, the filtrate 10 ml / kg administration group, the powder 2000 mg / kg administration group, the filtrate 10 ml / kg and the powder which are antitumor active agents of the examples. The mice were divided into 2000 mg / kg mixed administration groups, and 10 mice were used in each group.

Each mouse was orally administered with each test substance described above at a rate of 0.1 ml per 10 g of body weight once a day for 28 days once a day from 14 days before cancer cell transplantation.
On the 15th day (day 0) after administration, mouse Meth-A sarcoma cells subcultured in the peritoneal cavity of BALB / c male mice were aseptically collected with a syringe and 1.0 × 10 ⁷ using physiological saline. Per cell / ml of cell suspension. 0.2 ml (2.0 × 10 ⁶ cells / mouse) of this cell suspension was transplanted subcutaneously into the buttocks of BALB / c male mice.

The average tumor weight of each test substance administration group and non-administration group on the 15th day of transplantation (day 14) was determined, and the proliferation rate T / C (%) was determined using the following formula.
T / C (%) = (average tumor weight of each administration group / average tumor weight of non-administration group) × 100
When the growth rate was determined based on the above formula, as shown in Table 4, in the mixed administration group of the filtrate 10 ml / kg and the powder 2000 mg / kg which are antitumor active agents of the examples, the growth rate Of 47.2% was observed.

In contrast, the growth rate of the non-administered group was 100%, the growth rate of the administration group of 5 ml / kg of filtrate was 84.7%, and the growth rate of the administration group of 10 ml / kg of filtrate was 93. 0%, and the growth rate of the powder 2000 mg / kg administration group was 89.0%.
As is clear from the above results, a large significant difference was observed in the mixed administration group of the filtrate 10 ml / kg and the powder 2000 mg / kg, which are antitumor active agents of the examples.

  The significant difference was recognized as described above because the filtrate of the above example is a metabolite resulting from culturing 16 types of lactic acid bacteria. In addition, since more than 100 types of bacteria and fermented substances live in the intestines of the human body instead of a single type of bacteria, after metabolizing 16 types of lactic acid bacteria as described above, metabolites are those It is easily speculated that helps to strengthen the body's immunity.

  Further, as in this example, as many as 16 kinds of lactic acid bacteria are co-cultured, and various kinds of unique metabolites produced by the action of various bacteria are included in abundant kinds. It can also be assumed that the product affects the activation of lactic acid bacteria in the intestine.

  This invention is useful as an anticancer agent.

It is the figure which showed the process of primary culture to tertiary culture.

Claims (2)

B. Longum (Bifidobacterium
longum), B.I. Bifidobacterium bifidum, B. Adrecentis (Bifidobacterium
adolescentis), L. Lactobacillus acidophilus, L. Brevis (Lactobacillus
brevis), L.M. Lactobacillus jensenii, L. Paracasei (Lactobacillus
paracasei subsp. paracasei), L. Gasserie (Lactobacillus
gasseri), L.M. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus), L. Helveticas (Lactobacillus)
helveticus), L.H. Casei (Lactobacillus casei subsp. Casei), L. Ramonausus (Lactobacillus)
rhamnosus), L. Delbrickii (Lactobacillus delbrueckii subsp. Delbrueckii), L. Lactococcus
lactis), E.I. Enterococcus faecium, S. While using 16 kinds of lactic acid bacteria consisting of thermophilus (Streptococcus thermophilus), Fesium and L. Helveticas in the first group, E. Fesium and L. Acidophilus in the second group, E.I. Fesium and L. Gasseley is the third group, E.I. Fesium, L.C. Acidophilus and L. Brevis the 4th group, E. Fesium, L.C. Acidophilus and L. Brevis the 5th group, E. Fesium, L.C. Acidophilus, L.H. Brevis, L. Paracasei in the sixth group Adrecentis alone is group 7; Delbricky and L. Gasselie is in the 8th group, L.C. Delbricky alone is the ninth group, E.D. Fesium, L.C. Genseny, L. Paracasei and L. Brevis the 10th group, L. Acidophilus alone is group 11; Fesium and L. Gasserie was placed in the 12th group, L. Paracasei alone was added to the 13th group, L. Gassely, E.M. Fesium and B.I. Bifidum is the 14th group. Longum, S.M. Thermophilus and E. coli. Fecium was added to the 15th group, L.P. Gasserie alone, group 16; Bulgarix and S. Thermophilus in the 17th group, L. Gassely, L.C. Lactis, L. Gasserie and E.C. Fesium was added to the 18th group, L.C. Gassley, S.M. Thermophilus and L. Bulgarix was introduced in the 19th group, L.B. Lactis alone, 20th group, L. Gasserie and E.C. Fecium was added to the 21st group, L.P. In the 22nd group, L. Casei alone, group 23, B.I. Longum alone is used as the 24th group, and subcultured for each group to maintain the symbiotic state, and among these culture solutions, the 1st, 2nd group, 3rd, 4th group, 5th, 6th Groups, 7th, 8th group, 9th, 10th group, 11th, 12th group, 13th, 14th group, 15th, 16th group, 17th, 18th group, 19th, 20th group Primary cultures between the 21st and 22nd groups, the 23rd and 24th groups, the 1st and 2nd groups, the 3rd and 4th groups, the 5th and 6th groups, the 7th and 8th groups, the 9th, 10th group, 11th and 12th group, 13th and 14th group, 15th and 16th group, 17th and 18th group, 19th and 20th group, 21st and 22nd group, 23rd and 2nd group Was subcultured in a group with each other, further, filtrated lactic acid bacteria culture fluid obtained by 3 subcultured by mixing each of these secondary cultures and the filtrate obtained by filtration after heat sterilization, the filtrate An antitumor active agent obtained by mixing a powder obtained by pulverizing the residue. The antitumor active agent according to claim 1 , wherein the tertiary culture solution is heated to sterilize lactic acid bacteria, and cooled to coagulate fat, and then naturally filtered to obtain the filtrate .

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