JP5667642B2 - Endometriosis prevention and / or amelioration agent and food and beverage composition comprising the same - Google Patents

Description

  The present invention relates to an agent for preventing and / or improving endometriosis. In particular, the present invention relates to an agent for preventing and / or improving endometriosis comprising a specific lactic acid bacterium, and a food or beverage composition comprising the lactic acid bacterium.

  Endometriosis is a disease in which the endometrium or similar tissue detached during menstruation flows back in the fallopian tube and grows in the peritoneal cavity outside the uterus, affecting 6 to 10% of women in reproductive life It is one of the most serious gynecological diseases. Although there are many unclear points regarding the pathogenesis, many reports have revealed that this is caused by abnormalities in the immune surveillance system in the abdominal cavity. Usually, immunocompetent cells such as macrophages and NK cells eliminate endometrial tissue flowing into the abdominal cavity. However, if the function of these immunocompetent cells decreases for some reason, it is believed that endometrial tissue will engraft and grow in the abdominal cavity. In particular, it has been reported that a decrease in NK activity is significantly correlated with its severity (Non-Patent Document 1). Such a finding suggests the NK cell theory that a decrease in the function of removing ectopic endometrial cells by NK cells in the peritoneal cavity is involved in the onset and growth of endometriosis.

  On the other hand, endometriosis often accompanies pain and significantly impairs QOL (Quality of Life). In general, nonsteroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain during menstruation. However, NSAIDs are known to induce gastrointestinal disorders such as erosive lesions and ulcers due to their pharmacological properties of inhibiting cyclooxygenase to inhibit the production of prostaglandins having mucosal protective functions from arachidonic acid. . Furthermore, hormonal side effects have been observed in endometriosis therapy using danazol or gonadotropin-releasing hormone agonists.

  Therefore, substances that promote NK cell activation are being searched for in various fields. Examples of what has been known to be effective to some extent include polysaccharides (lentinan, chitin, chitosan, maitake D-fraction, nigerooligosaccharide, etc.), bacterial cells, and the like. However, studies on endometriosis were insufficient.

  On the other hand, IL-12 is a cytokine having an action of activating Th1 cells, NK cells and the like, and an ectopic endometrial cell growth inhibitory effect has been reported (Non-patent Document 2).

Osterynck DJ, Muleman C, Waer M, Vandputte M, and Koninckx PR, The natural killer activity of peri edenthe wisdom decim ediw edisw ediw edisw ediw ed mw edi w e s edi w e s e m e s s s s s s s s ation. Fertil. Steril. 58, 290-295 (1992). Somigliana E, Vigano P, Rossi G, Carinelli S, Vignali M, and Panina-Bordignon P. Endometricality to impulse in electrical sites can be presented by interleukin-12 in a murine model of endometriosis. Hum. Reprod. , 14, 2944-2950 (1999)

However, the compound for promoting the activation of NK cells and the compound capable of inducing IL-12 production for NK cell activation are still sufficiently related to the development of endometriosis. No serious consideration has been made.
Accordingly, an object of the present invention is to provide an agent for preventing and / or improving endometriosis based on the enhancement of NK activity. Moreover, this invention aims at provision of the food-drinks composition for prevention and / or improvement of endometriosis containing the said lactic acid bacteria. Furthermore, this invention aims at provision of the method of preventing and / or improving endometriosis. In particular, an object of the present invention is to provide a preventive and / or ameliorating agent for endometriosis that can be taken orally, has no side effects, and can obtain high safety and pain effects.

The inventors of the present invention have so far examined the IL-12 inducibility by probiotic lactic acid bacteria. In particular, various studies were conducted on the possibility that a lactic acid bacterium that promotes IL-12 production and enhances the cytotoxic activity of NK cells exhibits an improvement effect on endometriosis.
In view of the above-described conventional problems, the present inventors have conducted extensive research. As a result, it was recognized that certain types of lactic acid bacteria, in particular, Lactobacillus lactic acid bacteria, have excellent growth-inhibiting effects on endometriotic lesions, and the present invention has been completed.
Furthermore, when a lactobacillus lactic acid bacterium was ingested by a person suffering from endometriosis complaining of pelvic pain, which is a main factor that particularly lowers QOL, the lactic acid bacterium has an effect of improving endometriosis, particularly menstrual pain and dysmenorrhea. Admitted. Thus, the present inventors have found that lactic acid bacteria are effective in improving QOL in those suffering from endometriosis, and have completed the present invention.

  That is, one aspect of the present invention is an agent for preventing and / or improving endometriosis comprising lactic acid bacteria.

  The lactic acid bacteria contained in the agent for preventing and / or improving endometriosis is not limited in any way, but is preferably Lactobacillus genus, more preferably Lactobacillus gasseri, particularly Lactobacillus gasseri OLL2809 strain. Most preferably it is.

  Moreover, another one aspect | mode of this invention is the food-drinks composition for prevention and / or improvement of endometriosis which contains an effective amount of lactic acid bacteria.

  In the food and beverage composition for preventing and / or improving endometriosis, the lactic acid bacteria are not limited in any way, but are preferably Lactobacillus genus, more preferably Lactobacillus gasseri, and particularly Lactobacillus. Most preferred is gasseri OLL2809 strain.

  The food / beverage composition is preferably a fermented milk food / beverage product, and preferably a yogurt food / beverage product such as yogurt or yogurt drink, but is not limited thereto.

  Still another embodiment of the present invention is a method for preventing and / or improving endometriosis, comprising administering a desired amount of a composition containing an effective amount of lactic acid bacteria.

  Also in the method for preventing and / or improving endometriosis, the lactic acid bacteria are preferably of the genus Lactobacillus, more preferably Lactobacillus gasseri, and most preferably Lactobacillus gasseri OLL2809.

  In the method for preventing and / or improving endometriosis, the dose is preferably at least 2 mg / kg / day, but is not limited thereto.

Alternatively, the present invention relates to the following [1] to [19].
[1] A drug for preventing and / or improving endometriosis comprising lactic acid bacteria.
[2] The drug according to [1], wherein the lactic acid bacterium belongs to the genus Lactobacillus.
[3] The drug according to [1], wherein the lactic acid bacterium is Lactobacillus gasseri.
[4] The drug according to [1], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.
[5] A food and beverage composition for preventing and / or improving endometriosis comprising an effective amount of lactic acid bacteria.
[6] The food or beverage composition according to [5], wherein the lactic acid bacterium belongs to the genus Lactobacillus.
[7] The food or beverage composition according to [5], wherein the lactic acid bacterium is Lactobacillus gasseri.
[8] The food and beverage composition according to [5], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.
[9] The food / beverage composition according to [8], wherein the food / beverage composition is a fermented milk food / beverage product.
[10] The food or beverage composition according to [9], wherein the fermented milk food or beverage is yogurt.
[11] A method for preventing and / or improving endometriosis, which comprises administering a desired amount of a composition containing an effective amount of lactic acid bacteria.
[12] The method according to [11], wherein the lactic acid bacterium is a genus Lactobacillus.
[13] The method according to [11], wherein the lactic acid bacterium is Lactobacillus gasseri.
[14] The method according to [11], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.
[15] The method according to any one of [11] to [14], wherein the dose is at least 2 mg / kg / day.
[16] A lactic acid bacterium for use in the prevention and / or improvement of endometriosis.
[17] The lactic acid bacterium according to [16], wherein the lactic acid bacterium belongs to the genus Lactobacillus.
[18] The lactic acid bacterium according to [16], wherein the lactic acid bacterium is Lactobacillus gasseri.
[19] The lactic acid bacterium according to [16], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.

Alternatively, the present invention relates to the following [101] to [112].
[101] A preventive and therapeutic agent for endometriosis comprising lactic acid bacteria.
[102] The agent for preventing and treating endometriosis according to [101], wherein the lactic acid bacterium belongs to the genus Lactobacillus.
[103] The preventive and therapeutic agent for endometriosis according to [101], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.
[104] A food and drink composition for preventing and treating endometriosis, comprising an effective amount of lactic acid bacteria.
[105] The composition for preventing and treating endometriosis according to [104], wherein the lactic acid bacterium belongs to the genus Lactobacillus.
[106] The food and beverage composition for preventing and treating endometriosis according to [105], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.
[107] The food and drink composition for preventing and treating endometriosis according to [106], wherein the food and drink composition is a fermented milk food and drink.
[108] The food / beverage composition for prevention and treatment of endometriosis according to [107], wherein the fermented milk food / beverage product is a yogurt food / beverage product such as yogurt or yogurt beverage.
[109] A method for preventing and treating endometriosis, comprising administering a desired amount of a composition containing an effective amount of lactic acid bacteria.
[110] The method for preventing and treating endometriosis according to [109], wherein the lactic acid bacterium belongs to the genus Lactobacillus.
[111] The method for preventing and treating endometriosis according to [109], wherein the lactic acid bacterium is Lactobacillus gasseri OLL2809 strain.
[112] The method for preventing and treating endometriosis according to any one of [109] to [111], wherein the dose is at least 2 mg / kg / day.

  According to the present invention, a preventive and / or ameliorating agent for endometriosis comprising certain lactic acid bacteria, in particular, Lactobacillus lactic acid bacteria, is provided. The Lactobacillus lactic acid bacterium of the present invention is characterized by enhanced NK activity and enhances self-immunity, thereby exhibiting an unprecedented high improvement effect on endometriosis, particularly menstrual pain and dysmenorrhea Became clear. Accordingly, Lactobacillus lactic acid bacteria can be used for the prevention and / or improvement of these diseases. The agent for preventing and / or improving endometriosis of the present invention is effective in improving QOL in endometriosis sufferers.

  Moreover, the preventive and / or ameliorating agent for endometriosis of the present invention can be easily taken by oral ingestion. In addition, the prevention and improvement by hormone agents and the like may have side effects, but in the case of the present invention, there is no concern. Furthermore, it is safe enough to be handled as food. As described above, the preventive and / or ameliorating agent of the present invention is low in the ingestion by the patient, and can prevent and / or improve endometriosis without burdening life. Moreover, in order to provide such a characteristic, it is also easy to make this into a food-drinks composition, and simple intake can be further promoted.

  Moreover, since the preventive and / or ameliorating agent for endometriosis according to the present invention has an unprecedented alleviating effect on pain peculiar to the disease, it is particularly effective for improving QOL in patients suffering from endometriosis. .

It is a graph which compares the weight (FIG. 1 (A)) and magnitude | size (FIG.1 (B)) of an endometriosis lesion part after administering OLL2809 etc. with respect to an endometriosis model mouse. a−c: Significant difference between different signs (P <0.05). It is a graph which shows the enhancement of IL-2 (FIG. 2 (A)) and Ncr1 gene (FIG. 2 (B)) which exist in the abdominal cavity cell in an abdominal cavity washing | cleaning liquid. a, b: Significant difference between different signs (P <0.05). It is a graph which shows transition of the pain of the menstrual period by a visual analog scale. *, **: P <0.05 and 0.01, respectively. It is a graph which shows the variation | change_quantity before and behind ingestion about the pain of the menstrual period by a visual analog scale. **: P <0.01. It is a graph which shows transition of the dysmenorrhea score in the menstrual period. *: P <0.05. It is a graph which shows the variation | change_quantity before and behind ingestion about the dysmenorrhea score in menstruation. (*): P <0.1.

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

The present invention provides a preventive and / or ameliorating agent for endometriosis including lactic acid bacteria (hereinafter referred to as “preventive / improving agent of the present invention”). The preventive / ameliorating agent of the present invention can also be expressed as follows.
-Pharmaceutical composition for preventing and / or improving endometriosis containing lactic acid bacteria-Growth inhibitor for endometriotic lesions containing lactic acid bacteria-Pharmaceutical composition for inhibiting growth of endometriotic lesions containing lactic acid bacteria object

In the present specification, “prevention and / or improvement” can also be expressed as “prevention and / or improvement”. Therefore, “prevention and / or improvement” of the present invention includes
・ Prevention and improvement ・ Prevention ・ Improvement is included.
In the present specification, “improvement” can also be expressed as “treatment”.

  The preventive / ameliorating agent of the present invention contains lactic acid bacteria as its active ingredient. Lactic acid bacteria are not limited as long as they have a high ability to induce IL-12 production. Lactic acid bacteria have been classified into the genus Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Wissella, Tetragenococcus, Oenococcus, Enterococcus, and Vacococ. These lactic acid bacteria can be used for the preventive / ameliorating agent of the present invention. Preferred examples include Lactobacillus lactic acid bacteria, more preferred Lactobacillus gasseri, and particularly preferred Lactobacillus gasseri OLL2809 strain (accession number: NITE BP-72), but is not limited thereto. Moreover, you may use combining 2 or more types of lactic acid bacteria from which a kind differs.

The present inventors have deposited Lactobacillus gasseri OLL2809 strain to the Patent Microorganism Deposit Center, National Institute of Technology and Evaluation. The contents specifying the deposit are described below.
(1) Name of depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Deposit Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE BP-72
(4) Display for identification: Lactobacillus gasseri OLL2809
(5) Date of original deposit: February 1, 2005 (6) Date of transfer to deposit under the Budapest Treaty: January 18, 2006

Lactobacillus gasseri OLL2809
The strain (Accession No .: NITE BP-72) is a Gram-positive bacilli, and the colony morphology on Lactobacilli MRS Agar, Difcoo is round, pale yellow, and flat. Physiological characteristics include homolactic fermentation, growth at 45 ° C., fermentability to glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose and trehalose.

  As a medium for culturing Lactobacillus gasseri OLL2809 (Accession Number: NITE BP-72), a medium usually used for culturing lactic acid bacteria is used. That is, any medium can be used as long as it contains a nitrogen source, an inorganic substance and other nutrients in addition to the main carbon source. As the carbon source, lactose, glucose, sucrose, fructose, starch hydrolyzate, waste molasses and the like can be used according to the assimilation ability of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, whey protein hydrolyzate, and soy protein hydrolyzate can be used. In addition, meat extract, fish extract, yeast extract and the like are used as growth promoters.

  The culture is preferably performed under anaerobic conditions, but may be performed under microaerobic conditions such as liquid stationary culture that is usually used. For anaerobic culture, known methods such as a method of culturing under a carbon gas gas layer can be applied, but other methods may be used. In general, the culture temperature is preferably 30 to 40 ° C. However, other temperature conditions may be used as long as the temperature allows the bacteria to grow. The pH of the medium during the culture is preferably maintained at 6.0 to 7.0, but may be other pH conditions as long as the temperature allows the bacteria to grow. It can also be cultured under batch culture conditions. The culture time is usually preferably 10 to 24 hours, but may be any other culture time as long as the bacteria can grow.

  Other Lactobacillus gasseri that can be used in the present invention is also a gram-positive gonococcus and has other physiological characteristics similar to those of Lactobacillus gasseri OLL2809. Such Lactobacillus gasseri can be separated from human feces and the like based on physiological characteristics by those skilled in the art. Culturing can also be performed by the same method as described above for Lactobacillus gasseri OLL2809.

  The preventive / ameliorating agent of the present invention may contain lactic acid bacteria as they are, or may be contained as a processed product of lactic acid bacteria obtained by subjecting lactic acid bacteria to some kind of treatment. For example, lactic acid bacteria suspensions, lactic acid bacteria cultures (cells, culture supernatant (including medium components)), lactic acid bacteria fermentation products (lactic acid bacteria beverages, sour milk, yogurt, etc.), concentrates, pastes processed into pastes, Spray dried product, freeze dried product (at least one selected from spray dried product, freeze dried product, vacuum dried product, drum dried product), vacuum dried product, drum dried product, liquid dispersed in medium, diluted with diluent A diluted product obtained by crushing a diluted product or a dried product obtained by a mill or the like can be used as a processed product. As lactic acid bacteria, live cells, wet bacteria, dry bacteria and the like can be used as appropriate. It may be dead cells subjected to sterilization, that is, heat sterilization treatment, radiation sterilization treatment, or crushing treatment. These processing steps may be used alone or in combination.

The preventive / ameliorating agent of the present invention can be used alone for the prevention or improvement of endometriosis in humans and animals. Or it can also be orally administered by mixing with other ingredients that can be usually used in pharmaceuticals and foods. It can also be used in combination with other compounds, microorganisms and the like that are known to have an effect of preventing or improving endometriosis.
The prevention and treatment effects of endometriosis can also be confirmed by subjective evaluation using a visual analog scale (VAS) or a dysmenorrhea score, as shown in the examples.

The dose of the prophylactic / ameliorating agent of the present invention can be appropriately set in consideration of various factors such as the administration route, the age, weight, and symptoms of animals to be administered including humans. Although the present invention is not limited to this, it is preferable to administer at least 2 mg / kg / day as an active ingredient. However, when ingested for the purpose of prevention and / or improvement over a long period of time, the amount may be smaller than the above range. Alternatively, since the active ingredient of the preventive / ameliorating agent of the present invention has no problem with regard to food safety, it can be used in a larger amount than the above range.
The preventive / ameliorating agent of the present invention can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, transdermal, transgastric tube, etc.).

  The dosage form of the preventive / improving agent of the present invention can be selected according to the purpose of prevention, the purpose of improvement, the route of administration, etc., for example, tablets, coated tablets, pills, capsules, granules, powders , Liquids, suspensions, emulsions, syrups, injections, suppositories, dipping agents, decoction, tinctures and the like. These various preparations are prepared according to conventional methods as necessary with respect to the active ingredient, such as fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, flavoring agents. The pharmaceutical composition can be formulated using known adjuvants that can be usually used in the pharmaceutical preparation technical field, such as solubilizing agents, suspension agents, and coating agents. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.

In the present invention, the lactic acid bacteria can be used in the form of any medicine or food or drink. For example, it is expected to prevent and / or improve endometriosis by directly administering it as a pharmaceutical, or by directly ingesting it as a special-purpose food such as a food for specified health use or a nutritional functional food. That is, this invention provides the food-drinks composition for prevention and / or improvement of endometriosis which contains an effective amount of the lactic acid bacteria which comprise the prevention / amelioration agent of this invention. Examples of the form of the food and drink composition include prepared milk powder and fermented milk food and drink. Examples of fermented milk food and drink include, but are not limited to, yogurt and cheese.
The food / beverage composition of the present invention can be applied to fermented milk such as yogurt, beverages and the like as well as health functional foods and foods for the sick. The health functional food system was established not only for regular foods but also for foods in the form of tablets, capsules, etc., based on domestic and foreign trends and consistency with the conventional food system for specified health use. It consists of two types of food for specified health use (individual permission type) and functional food for nutrition (standard type). It is possible to prevent and / or improve endometriosis by directly ingesting it as a special-purpose food such as a food for specified health use or a nutritional functional food containing the lactic acid bacteria constituting the preventive / ameliorating agent of the present invention.

  The food / beverage composition of the present invention specifically includes various food / beverage products (milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crust, prepared milk powder, liquid food, food for the sick, Lactic acid bacteria constituting the preventive / improving agent of the present invention may be added to foods such as infant milk powder, foods such as lactating milk powder, and nutritional foods, and ingested. The active ingredient can be used as it is, or can be used in accordance with a conventional method for ordinary food and beverage compositions such as mixing with other foods or food ingredients. Moreover, about the property, the state of the food / beverage products normally used, for example, any of solid (powder, granule, etc.), paste, liquid or suspension may be sufficient.

  Although it does not specifically limit about another component, it is water, protein, saccharide | sugar, a lipid, vitamins, minerals, organic acid, organic in the food-drinks composition containing the lactic acid bacteria which comprise the prevention / improving agent of this invention. Bases, fruit juices, flavors and the like can be used as main components. Examples of the protein include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and plant proteins, hydrolysates thereof; butter, whey minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. Examples of the saccharide include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And minerals include, for example, calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey minerals. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. These components can be used in combination of two or more, and synthetic products and / or foods containing a large amount thereof may be used.

  The amount of the lactic acid bacteria constituting the preventive / improving agent or the food / beverage product composition of the present invention can be arbitrarily determined according to its purpose and application (drug, food / beverage product composition). Although this invention is not limited to this, As the content, 0.001 to 100% (w / w) is preferable normally with respect to the whole quantity, Especially 0.01 to 100% (w / w) is preferable.

  When lactic acid bacteria are used for the production of food / beverage product compositions and drugs, the production method can be carried out by methods well known to those skilled in the art. If it is a person skilled in the art, the process which mixes lactic acid bacteria or its processed product with other ingredients, a molding process, a sterilization process, a fermentation process, a baking process, a drying process, a cooling process, a granulation process, a packaging process, etc. are combined appropriately, It is possible to make food and drugs.

  Moreover, also when using lactic acid bacteria for manufacture of various dairy products, a desired dairy product can be manufactured by a method well-known to those skilled in the art. As an example of yogurt, yogurt can be manufactured through a step of preparing a starter using lactic acid bacteria, a step of adding the starter to pretreated milk, a step of culturing, a cooling step, a flavoring step, a filling step, and the like. If it is cheese, for example, a process of adding lactic acid bacteria of the present invention as a starter to milk that has been pretreated such as sterilization and the like, a process of adding rennet to produce a cheese curd, a card cutting process, whey discharge It can be manufactured through a process, a salting process, an aging process, and the like. Alternatively, in the production of the various dairy products, instead of a specific lactic acid bacterium (for example, Lactobacillus gasseri), another lactic acid bacterium is used as a starter, and the specific lactic acid bacterium (for example, Lactobacillus gasseri) or a processed product thereof is added during the production process. Also good.

The present invention also relates to a method for preventing and / or improving endometriosis, which comprises the step of orally administering lactic acid bacteria to animals. Mammals are examples of subjects to which lactic acid bacteria are administered. Mammals include humans and mammals other than humans, preferably humans and monkeys, more preferably humans.
The present invention also relates to a lactic acid bacterium for use in preventing and / or improving endometriosis. Alternatively, the present invention relates to the use of lactic acid bacteria in the manufacture of an agent for preventing and / or improving endometriosis. The present invention also relates to the use of lactic acid bacteria for the prevention and / or amelioration of endometriosis. The present invention also relates to a method for producing an agent for preventing and / or improving endometriosis, which comprises a step of blending lactic acid bacteria and a pharmaceutically acceptable carrier.
Examples of lactic acid bacteria include, but are not limited to, lactic acid bacteria belonging to the genus Lactobacillus, more preferably Lactobacillus gasseri, and particularly preferably Lactobacillus gasseri OLL2809.

Or this invention provides the growth inhibitor of the lesioned part of endometriosis containing lactic acid bacteria. The present invention also provides a method for inhibiting the growth of a lesion site of endometriosis comprising the step of administering lactic acid bacteria to an animal. Furthermore, the present invention relates to a lactic acid bacterium for use in the suppression of the growth of lesions of endometriosis. Or this invention relates to use of the lactic acid bacteria in manufacture of the growth inhibitor of the lesioned part of endometriosis. Or this invention relates to use of the lactic acid bacteria for suppressing the growth of the lesion part of endometriosis. Or this invention relates to the manufacturing method of the growth inhibitor of the lesioned part of endometriosis including the process of mix | blending lactic acid bacteria and a pharmaceutically acceptable carrier.
The growth inhibitor of the present invention can contain a carrier suitable for oral administration. The growth inhibitor of the present invention can be administered also as a food for the purpose of suppressing the growth of the lesion part of endometriosis.
Endometriosis occurs mainly in cells such as the peritoneum, ovary and uterus, Douglas fossa, the surface of organs such as the intestine and rectum, the inside of the ovary, and the muscle layer of the uterus. Whether or not the growth of the lesioned part is suppressed by the growth inhibitor of the present invention can be determined, for example, by measuring the area, volume, and weight of the lesioned part by a method well known to those skilled in the art. It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this. In addition, in this specification,% display shows weight%, when not showing clearly.

[Example 1]
Endometriosis model mice were prepared by the following method, and the prevention and improvement effects of the preventive / ameliorating agent of the present invention were examined.

[Experimental animals]
Six-week-old female BALB / c mice (Japan SLC Co., Ltd.) that had undergone ovariectomy at the age of 5 weeks were used. Mice were fed a CRF-1 diet (Oriental Yeast Co., Ltd.) and were housed in a room controlled at room temperature 21 ± 2 ° C., humidity 55 ± 15%, and light / dark cycle every 12 hours in cages divided into groups. . Food and water were ad libitum.

Preparation of endometriosis model mice is described in Somigliana et al. A method obtained by modifying the method of (Non Patent Literature 2) was used.
Specifically, a pair of donor mice described below is finely chopped and suspended in 0.6 ml of phosphate buffered saline (PBS; pH 7.2), and 0 per recipient mouse. 3 ml of endometrial suspension was injected intraperitoneally with an 18G needle (day 0). Recipient mice, which had undergone oophorectomy at the age of 5 weeks (7 days before the transplantation date), used estrogen dissolved in castor oil a total of 4 times on the 7th, 0th, 7th and 14th days. Kosei Pharmaceutical Co., Ltd. (100 μg / kg) was intramuscularly administered to the thigh. Donor mice were subjected to ovariectomy and estrogen administration 7 days before the transplantation day, similar to recipient mice. Ovariectomy and estrogen administration were performed to adjust the sexual cycle of all mice to the estrus.

  Mouse IL-12 recombinant (Wako Pure Chemical Industries, Ltd.) was suspended in PBS, and 0.15 μg / 0.4 ml was administered to the peritoneal cavity for 5 consecutive days from 2 days before to 2 days after transplantation. The other groups received only the solvent under the same conditions.

  Lactobacillus gasseri OLL2809 was subjected to activation culture (37 ° C., 18 hours) twice in Lactobacilli MRS broth (Difco). 1% of the activated cells were inoculated in the same medium and cultured at 37 ° C. for 18 hours. After collection, the cells were washed twice with physiological saline and once with sterilized distilled water. Sterilized by heating at 75 ° C. for 60 minutes and lyophilized. The lyophilized cells were suspended in water at a concentration of 2 mg / 0.5 ml, and at a dose of 2 mg / 0.5 ml / body, using a stomach tube for a period from the date of transplantation to the day before the dissection (day 20). The gastric tube was administered. The other groups received only the solvent under the same conditions.

  On day 21 after the endometrial transplantation, the mice were euthanized by cervical dislocation and the abdominal cavity lavage fluid was collected, followed by laparotomy. After measuring the size (long diameter, short diameter) of the endometrial lesion using calipers, it was extracted and weighed. When the lesion part formed a cyst, it was performed after the cyst fluid was aspirated.

  In addition, 1 ml of 1% FCS-containing PBS was injected into the abdominal cavity before abdominal laparotomy, and 0.5 ml of abdominal cavity lavage fluid was collected and used for measurement of cytokine concentration in ascites. Cytokine was measured using a Bio-plex cytokine assay kit (Bio-rad, Hercules, CA).

  After collecting 0.5 ml of peritoneal lavage fluid from the peritoneal cells, 4 ml of PBS containing 1% FCS was injected into the peritoneal cavity, and 3 ml of lavage fluid was collected. Cells in 0.5 ml of peritoneal lavage fluid collected for cytokine measurement were collected by centrifugation and combined into peritoneal cells.

  Trizol reagent (Invitrogen, Calsbad, CA) was used for total RNA from peritoneal cells. Using 1 μg of the extracted total RNA as a template, reverse transcription to cDNA was performed using PrimeScript RT reagent Kit (Takara Bio Inc.). Real-time PCR was measured with ABI Prism 7300 (Applied Biosystems, Foster City, Calif.) Using FastStart Universal SYBR Green Master reagent (Roche, Indianapolis, IN). Oligonucleotide primers for the IL-2 gene are described in Overbergb et al. (Overbergb L, Giulietti A, Valckx D, Decallonne B, Bouillon R., and Matebeu C. The use of real-time reverse transcribing. 33-43 (2003)). The NCR1 gene was measured using a TaqMan probe (Applied Biosystems). The expression level of each gene was corrected by the expression level of glyceraldehyde-3-phosphate dehydrogenase.

  The results are shown as mean ± standard error. Statistical analysis parametric data were subjected to multiple tests by one-way analysis of variance. If significant differences were observed, significant differences between groups were analyzed by Fisher's PLSD test. Non-parametric data was subjected to multiple comparison by the Steel-Dwash method. P <0.05 was considered significant.

Effect of Lactobacillus gasseri OLL2809 administration on endometriotic lesions No endometriotic lesions were observed in the normal (no treatment) group, but 2 to 7 lesions were detected per individual in the control group Both the weight and area were significantly higher than the normal group (FIG. 1). By administration of Lactobacillus gasseri OLL2809, both its weight and area were significantly lower than those of the control group, and the growth inhibitory effect on the lesion was observed. Moreover, the degree was the same as the IL-12 administration group set as a positive control. Therefore, the preventive and ameliorating effects on endometriosis could be proved by administering the prophylactic / ameliorating agent of the present invention.

Peritoneal cell gene expression analysis Most cytokine concentrations measured in the peritoneal lavage fluid were below the detection limit, and the significant effect of Lactobacillus gasseri OLL2809 could not be confirmed from the proportion of peritoneal cell types. Therefore, the gene expression level of peritoneal cells was measured. As a result, IL-2 was significantly lower in the control group than in the normal group (FIG. 2 (A)). In contrast, in the OLL2809 group and the IL-12 administration group, the expression level was recovered and showed a value similar to that in the normal group (FIG. 2 (B)). The IL-2 gene is a cytokine produced mainly by Th1 cells and promotes differentiation and proliferation of B cells, T cells, NK cells, etc., and has been shown to suppress endometriosis in the rat (Velasco). I, Quereda F, Bermejo R, Campos A, and Acien P. Intraperitoneal recombinant interleukin-2 activates leukocytes in ratentolisis.ol. The expression level of IL-2 gene was enhanced by administration of Lactobacillus gasseri OLL2809, and the effectiveness of the preventive / ameliorating agent of the present invention could be verified.

Furthermore, a significant increase in gene expression level was observed for NCR1 (Natural Cytotoxicity triggering receptor 1), which is one of the NK cell activation markers. NCR1 is also known as Ly94 or NKp46 in humans and has been shown to be expressed on the surface of activated NK cells. In measuring the gene expression level, the same number of peritoneal cells collected from each individual was used, and cDNA prepared from the same concentration of total RNA was used. Furthermore, the population analysis of peritoneal cells by FACS showed no difference between groups in the number of NK cells. Therefore, NK activity itself was not significantly increased by Lactobacillus gasseri OLL2809 administration, but the analysis at the gene level suggested that the activity of individual cells was enhanced.
Therefore, it was revealed that the preventive / ameliorating agent of the present invention including Lactobacillus gasseri OLL2809 has an effect of suppressing the growth of endometrial lesions.

[Example 2]
In Example 2 below, the improvement effect on menstrual pain and dysmenorrhea in patients suffering from endometriosis with the preventive / ameliorating agent of the present invention was examined.

Patients who were diagnosed with endometriosis, met the following selection criteria, and did not violate the exclusion criteria were included as subjects. Diagnostic criteria of endometriosis was carried out according to the ^{"6 th Obstetrics and Gynecology, 2003 (} published by People's Medical Publishing House) ". That is, when one of the following (1) to (5) and physical signs of either (6) or (7) were observed, it was determined to be affected.

Symptoms:
(1) Secondary dysmenorrhea that is gradually progressing,
(2) lower abdominal or pelvic pain,
(3) Sexual discomfort or pain,
(4) Intermittent diarrhea, constipation, anal pain,
(5) Infertility.
Physical signs:
(6) Rectal uterine fossa, nodule of uterine sacral ligament under the posterior wall of uterus,
(7) A cystic, fixed mass around the uterine appendage.
Selection criteria:
(1) Patients aged between 18 and 45 years,
(2) Patients who have menstruation every month and have a cycle of 28 ± 3 days.
Exclusion criteria:
(1) Patients who have received hormone therapy within the past 3 months,
(2) Patients with a history of severe liver damage, kidney damage, myocardial infarction or blood disease,
(3) A patient with or suspected of having a malignant tumor,
(4) A patient whose CA-125 is 100 IU / ml or more,
(5) A patient who has an endometrial lesion in the peritoneal incision,
(6) Patients who develop diarrhea after dairy consumption,
(7) Patients who are receiving allergic disease improvement,
(8) Patients taking drugs (such as constipation-improving drugs or gastrointestinal drugs) that may affect the test results,
(9) Patients taking foods (fermented milk and beverages containing lactic acid bacteria, etc.) that are thought to affect the test results for at least 4 days a week.
Cancellation / dropout criteria:
(1) Subjects who declined to continue testing due to free will,
(2) Subjects who are unable to keep track of their progress,
(3) Subjects who do not comply with compliance,
(4) Subjects who have experienced serious complications or side effects,
(5) Subjects whose study tablet intake is 80% or less or 120% or more of the specified amount.

  Based on the above, 66 people were diagnosed as having endometriosis and were taken as subjects. Of these, 62 patients completed the study, and finally 33 placebo groups and 29 active groups were analyzed.

For subjects who met the above selection / exclusion criteria, controllers who were not directly involved in the study were randomly assigned to the placebo group and the active group.
For each subject, the active group ingested two active tablets of the prophylactic / improving agent containing Lactobacillus gasseri OLL2809 of the present invention two times a day, and the placebo group ingested the placebo tablets for 12 weeks (three sexual cycles). . For each subject, the following evaluation items were recorded every month, along with follow-up by interviews, etc., before the start of the test, 1, 2, and 3 months after the start of intake.

Each score was set and evaluated for the following evaluation items.
First evaluation item: The degree of subjective pain by visual analog scale (VAS) and dysmenorrhea score during menstrual period.
Second evaluation item: VAS other than menstruation and pelvic pain score total.
Each score is shown in Table 1.

VAS during menstruation and other than menstruation was evaluated for the degree of pain due to endometriosis on a 10 cm straight line, with the left side being painless and the right side being the most severe pain experienced in the past.
As an objective evaluation item for endometriosis, serum CA-125 was measured before ingestion and 3 months after ingestion.
Blood safety tests (red blood cell count, white blood cell count, platelet count, hemoglobin) and blood biochemical tests (blood glucose level, total protein, alanine aminotransferase (ALT), asparagine aminotransferase (AST), lactate dehydrogenase ( LDH), total bilirubin, γ-transpeptidase, alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine, uric acid, total cholesterol, triglycerides, high density lipoprotein (HDL)) before intake and 3 months from the start of intake I went later.

  The test products were active tablets (active tablets, manufactured by Meiji Dairies) and placebo tablets as preventive / improving agents of the present invention containing Lactobacillus gasseri OLL2809. One active tablet was a 250 mg tablet, and the culture concentrate of Lactobacillus gasseri OLL2809 was heat-treated at 75 ° C. for 60 minutes, and 50 mg of lyophilized powder was blended. The placebo tablet was formulated with dextrin instead of the lyophilized powder.

Review of blinded results was performed before key opening, and handling of dropped cases was judged based on blinded data. This evaluation was performed on all subjects who completed the study.
The results are expressed as mean ± standard error (SE). Statistical analysis between the two groups was performed by Student's t-test or Mann-Whitney's U-test. P <0.05 was considered significant.

  Table 2 shows the background factors of the subjects recorded before ingestion. There was no significant difference between the two groups in all items.

  Each specific evaluation item will be described below with reference to the drawings. In the graphs of FIGS. 3 and 5, ● represents a placebo group (n = 33), and ■ represents an active group (n = 29).

  The result of the transition of VAS is shown in FIG. There was no significant difference between the two groups, 7.73 ± 0.26 in the placebo group and 7.51 ± 0.22 in the active group before the start of ingestion. Significant differences were observed in pain during menstruation 2 months and 3 months after the start of intake between the groups (P <0.05 and P <0.01, respectively). That is, the amount of change in VAS from the start of intake to 3 months after the intake was significantly improved to -3.28 ± 0.36 in the active group compared to -2.00 ± 0.29 in the placebo group. (P <0.01, FIG. 4). From the above results, it was revealed that the preventive / ameliorating agent of the present invention (active tablet in this example) is effective in reducing menstrual pain.

  The transition of menstrual dysmenorrhea score is shown in FIG. There was no significant difference between the two groups, 3.45 ± 0.20 in the placebo group and 3.14 ± 0.17 in the active group before ingestion, but 3 months later compared to placebo The active group showed a significantly low value (P <0.05). In addition, the change in dysmenorrhea score from the start of intake to 3 months after the intake was −1.03 ± 0.16 in the placebo group, while −1.44 ± 0.17 in the active group. The improvement tendency was shown (P <0.1, FIG. 6). That is, it was revealed that the preventive / ameliorating agent of the present invention (active tablet in this example) is effective in improving the menstrual dysmenorrhea score.

  After ingestion, there was no significant difference between groups regarding the number of red blood cells, white blood cells, platelets, and hemoglobin in the blood. There were no significant differences in blood glucose level, total protein, ALT, AST, LDH, total bilirubin, γ-transpeptidase, ALP, BUN, creatinine, uric acid, total cholesterol, triglycerides, and HDL in each group before and after intake. . Therefore, it was found that the preventive / ameliorating agent of the present invention is safe without side effects.

  As described above, Lactobacillus gasseri OLL2809 has been shown to improve pain and dysmenorrhea resulting from endometriosis in the menstrual period and is effective in improving QOL of affected individuals. In addition, since no side effects are observed, it is useful for improving QOL in patients with endometriosis, and is considered to be a highly safe preventive / ameliorating agent.

As described above, a preventive and / or ameliorating agent for endometriosis comprising certain lactic acid bacteria of the present invention, particularly Lactobacillus genus lactic acid bacteria, is characterized by enhancing NK activity and enhancing its own immunity, It shows a high improvement effect that has not been known so far for endometriosis, particularly menstrual pain and dysmenorrhea. Therefore, the preventive / ameliorating agent of the present invention is useful for preventing or ameliorating these diseases. It is also effective in improving QOL in patients with endometriosis.
Moreover, since the preventive / ameliorating agent of the present invention can be taken orally, it can be taken easily and endometriosis can be prevented or improved. In addition, since there are no side effects and safety is high, the ingestion by patients is low, and endometriosis can be prevented or improved without burdening life. Moreover, in order to provide such a characteristic, it is easy to use the preventive / improving agent of the present invention as a food / beverage product composition. By using a food / beverage product composition, simpler intake can be promoted. . The preventive / ameliorating agent of the present invention has an unprecedented alleviating effect on pain peculiar to endometriosis. Therefore, it is effective for improving QOL particularly in those suffering from endometriosis.

Claims (3)

A drug for the prevention and / or improvement of endometriosis (excluding those caused by obesity) comprising Lactobacillus gasseri lactic acid bacteria (excluding those containing at least one of drospirenone and estrogen). The drug according to claim 1, wherein the Lactobacillus gasseri lactic acid bacterium is Lactobacillus gasseri OLL2809 strain (Accession number: NITE BP-72). The agent according to claim 1 or 2 , for addition to food.

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