WO2023038350A1 - Composition for alleviating premenstrual syndrome, containing mixed lactobacillus strains as active ingredient - Google Patents

Abstract

The purpose of the present application is to provide a composition for alleviating premenstrual syndrome, containing, as an active ingredient, mixed strains of a Lactobacillus gasseri LM1065 strain (KCCM13018P) and a Lactobacillus reuteri LM1071 strain (KCCM12650P), or any one from among a culture product, a lysate, and an extract of the mixed strains. Mixed strains of a Lactobacillus gasseri LM1065 strain (KCCM13018P) and a Lactobacillus reuteri LM1071 strain (KCCM12650P), according to one implementation of the present application, reduce the production of blood NO and the expression of IL-6, iNOS and COX-2 genes and increase the production rate of blood prostaglandin E1/prostaglandin E2 and the levels of blood GLA and DGLA, so that premenstrual syndrome can be alleviated, and thus the mixed strains can be applied to food compositions, health functional food compositions, pharmaceutical compositions and the like.

Description

Composition for alleviating premenstrual syndrome containing a mixed strain of Lactobacillus as an active ingredient

In the present application, Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) LM1071 strain (KCCM12650P), or at least one of the culture, lysate, extract of the mixed strain is effective It relates to a composition for alleviating premenstrual syndrome comprising as components.

Premenstrual syndrome (PMS) is a group of symptoms characterized by recurrent emotional, behavioral, and physical symptoms that occur before menstruation. Accompanied by psychological changes. These symptoms gradually get worse after ovulation, are most severe one week before menstruation, and disappear within a few days after menstruation begins.

Among the characteristics of representative physical symptoms, dysmenorrhea is a common gynecological symptom experienced every month by women of childbearing age until they reach menopause, and is a very common symptom in the female menstrual cycle.

According to the results of a study targeting Korean women, 77-94% complained of menstrual cramps. Of these, 47% suffered menstrual cramps every month, and 53.2% experienced severe menstrual cramps. In the case of production workers, 46% responded that their activities were restricted due to menstrual cramps. In foreign countries, the prevalence of dysmenorrhea was as high as 60-93%, and 42% experienced severe dysmenorrhea. Between 10% and 50% of female students responded that menstrual cramps interfere with their daily activities, including school, and in the case of industrial sites, damage caused by menstrual cramps was estimated, resulting in 600 million hours of lost work and 2 billion dollars annually. There was a loss of productivity.

Therefore, the results of research on changes or mechanisms of factors related to dysmenorrhea can be used as sufficient evidence for the development of premenstrual syndrome-related food materials, health functional food materials, and pharmaceuticals.

As an example of developing a composition for alleviating premenstrual syndrome through changes in prolactin, there is a composition for improving symptoms of premenstrual syndrome containing a compound isolated from malt extract as an active ingredient (Korean Patent No. 10-2187335). . However, there is still a need for in-depth development and various studies on compositions exhibiting excellent effects in relation to premenstrual syndrome alleviation.

Accordingly, the present inventors have made diligent efforts to develop an excellent composition capable of alleviating premenstrual syndrome. As a result, NO, IL-6 gene expression is reduced, and prostaglandin E ₁ / prostaglandin E ₂ production rate and DGLA level are increased in the blood. A mixed strain was developed to complete the present invention.

[Prior art literature]

[Patent Literature]

(Patent Document 1) Korean Patent Registration No. 10-2187335

In the present application, Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) LM1071 strain (KCCM12650P), or at least one of the culture, lysate, extract of the mixed strain is effective It is intended to provide a composition for alleviating premenstrual syndrome comprising as components.

However, the problem to be solved by the present application is not limited to the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

The first aspect of the present application is a mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a pharmaceutical composition for preventing or treating premenstrual syndrome, comprising one or more as active ingredients.

The second aspect of the present application is a mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a food composition for alleviating dysmenorrhea, containing one or more as active ingredients.

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) according to an embodiment of the present application reduces the amount of NO production and the expression of IL-6, iNOS and COX-2 genes in the blood, By increasing the prostaglandin E ₁ /prostaglandin E ₂ production rate and the levels of GLA and DGLA in blood, premenstrual syndrome can be alleviated, and the mixed strain can be applied to food compositions, health functional food compositions, pharmaceutical compositions, and the like.

1 is an experiment to determine the symptom relieving effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome according to an embodiment of the present application. It is a diagram showing the results of comparing the NO producing ability between the strain treatment group and the control group and measuring the NO producing ability by mixing ratio of the mixed strains.

Figure 2 examines the symptom relieving effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome for premenstrual syndrome according to an embodiment of the present application. In the experiment for the present strain, it is a diagram showing the results of comparative measurement of IL-6, iNOS, and COX-2 gene expression ability between the treatment group and the control group.

Figure 3 is to determine the symptom relief effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome for premenstrual syndrome according to an embodiment of the present application. In the experiment for the experiment, it is a diagram showing the results of analyzing PGE ₁ / PGE ₂ values by ELISA analysis of blood collected from rats from 3 days before the last administration of the test substance.

Figure 4 is to determine the symptom relief effect of a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) for premenstrual syndrome for premenstrual syndrome according to an embodiment of the present application. In the experiment for the last test substance, serum obtained by collecting blood on the last day of administration was analyzed for concentrations of GLA and DGLA in blood using LC-MS/MS (Liquid Chromatograph-Tandem Mass Spectrometer; Liquid Chromatograph-Multiple Mass Spectrometry) method. is a drawing showing

Hereinafter, embodiments of the present application will be described in detail so that those skilled in the art can easily practice with reference to the accompanying drawings. However, the present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. And in order to clearly describe the present application in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the present specification, when a member is said to be located “on” another member, this includes not only a case where a member is in contact with another member, but also a case where another member exists between the two members.

Throughout the present specification, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

As used throughout this specification, the terms "about," "substantially," and the like are used at or approximating the value when manufacturing and material tolerances inherent in the stated meaning are given, and do not convey the understanding of this application. Accurate or absolute figures are used to help prevent exploitation by unscrupulous infringers of the disclosed disclosure. The term "step of (doing)" or "step of" as used throughout the present specification does not mean "step for".

Throughout this specification, the term "combination(s) of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means including one or more selected from the group consisting of the above components.

Throughout this specification, reference to "A and/or B" means "A or B, or A and B".

Hereinafter, embodiments and embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. However, the disclosure may not be limited to these embodiments and examples and drawings.

The first aspect of the present application is a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a pharmaceutical composition for preventing or treating premenstrual syndrome, comprising the above as an active ingredient.

In one embodiment of the present application, the mixed strain may reduce the amount of NO production in the blood.

The term "nitric oxide (NO)" used throughout the present specification is called "nitric oxide" or "nitrogen monoxide" and refers to a compound in which nitrogen is oxidized. It is formed from arginine, an amino acid, within cells, and is involved in various physiological activities such as immune action, vasodilation, and signal transmission as a kind of signaling substance. It is also known to induce inflammation and pain by promoting the secretion of inflammatory cytokines such as TNF-α and IL-6 (Hu et al., 2020).

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application reduces the amount of NO production, thereby exhibiting an effect of alleviating menstrual pain.

The term "Griess reagent" used throughout the present specification refers to a reagent used in a Griess test to determine the presence or absence of nitrite ions NO ₂ ^- . In general, it is prepared by mixing a solution of 0.5 g of sulfanilic acid in 150 ml of dilute acetic acid and a solution of 0.1 g of a-naphthylamine in 20 ml of water and 150 ml of acetic acid. The NO production ability measurement experiment is an experiment using the above grease reagent, and through this, the expression level of NO can be measured.

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application reduces the expression of IL-6, iNOS and COX-2 genes, thereby It may have a menstrual pain relief effect.

The term "IL-6 (InterLeukin-6)" used throughout the present specification refers to a physiologically active protein called a cytokine together with TNF-α (Tumor Necrosis Factor-α). IL-6 is secreted from various cells such as T lymphocytes and macrophages to promote immune responses. In particular, IL-6 is involved in inflammatory responses and causes inflammation and pain in the process of regulating high fever or acute phase proteins.

The term "iNOS (inducible NO synthase)" used throughout the present specification is an inducible NO synthase, an enzyme whose expression is induced by cytokine stimulation in cells of the immune system to synthesize nitric oxide (NO) using L-arginine as a substrate. it means. The gene expression of iNOS is closely related to the inflammatory response, and the gene is induced in cells such as macrophages, smooth muscle cells, and hepatocytes by stimulation of inflammatory cytokines to produce NO.

The term "cyclooxygenase-2 (COX-2)" used throughout the present specification refers to an enzyme that produces prostaglandin, and the expression level increases along with an inflammatory response.

The term "LPS (lipopolysaccharide)" used throughout the present specification refers to a representative pathogenic substance that induces macrophage-mediated inflammatory response. LPS induces an inflammatory response through the secretion of inflammatory cytokines and substances by binding to Toll-like receptor 4 (TLR4) expressed on the surface of macrophages. This inflammatory response is also induced by the inflammasome present in cells. When the inflammatory complex is activated, pyroptosis, a cell death by the inflammatory response, and interleukin-1 beta, an inflammatory cytokine, are induced. 1β, IL-1β) and interleukin-18 (IL-18) secretion is increased. In macrophage-mediated inflammatory responses, mouse caspase-11 and its human homologues caspase-4/5 are also known to induce pyroptosis and secretion of IL-1β and IL-18, which are derived from Gram-negative bacteria. It was found to induce an inflammatory response by directly recognizing LPS (intracellular LPS) within a cell (Lee Young-soo, 2017).

Therefore, the mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application reduces the expression of IL-6, iNOS and COX-2 genes, , it can be seen that it can be effective in relieving menstrual pain and treating or preventing premenstrual syndrome.

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application increases the production ratio of prostaglandin E ₁ / prostaglandin E ₂ in blood, thereby It can show the effect of alleviating menstrual cramps and premenstrual syndrome.

The term "prostaglandin" used throughout the present specification refers to a kind of hormone secreted during menstruation, which contracts the uterus and smoothly pushes menstrual blood out of the body when the endometrium is peeled off.

Prostaglandin is a process in which arachidonic acid is converted into prostaglandin using a cyclooxygenase (COX) system, and arachidonic acid is converted into prostaglandin G2 and then into prostaglandin H2, thereby producing prostaglandins with various physiological activities. In addition, prostaglandins have been reported as substances that cause inflammation and pain (Jayesh et al., 2020).

The term "PGE ₂ (prostaglandin E ₂ )" used throughout the specification is also called "dinoprostone" and refers to a naturally occurring prostaglandin with oxytocin properties.

PGE ₂ induces pain through inflammatory mediation in tissues or cells damaged by G protein bound to the PGE ₂ receptor (Treutlein et al., 2018), and stimulates the central nervous system and peripheral nervous system (Grφsch et al., 2017).

In one embodiment of the present application, the mixed strain may increase the blood prostaglandin E ₁ / prostaglandin E ₂ ratio, and specifically, the strain may increase the production ratio of prostaglandin E ₁ to prostaglandin E ₂ .

The term "PGE ₁ (prostaglandin E ₁ )" used throughout the present specification is also called "alprostadil" and refers to a naturally occurring prostaglandin that serves to dilate blood vessels in the body, relaxes smooth muscles and reduces secretion of inflammatory cytokines. It is known that there is a pain relieving effect (Gezginci-Oktayoglu et al., 2016).

The production ratio of PGE ₁ /PGE ₂ (PGE ₂ to PGE ₁ ) is a major index in the guidelines of the Ministry of Food and Drug Safety related to premenstrual syndrome in women, and if the ratio increases, it can be judged that it contains functions related to the treatment of premenstrual syndrome.

Therefore, the mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application increases the production ratio of prostaglandin E ₁ / prostaglandin E ₂ in blood, which Through this, it can be seen that it can be effective in relieving menstrual pain and relieving premenstrual syndrome.

In one embodiment of the present application, the mixed strain may increase the level of GLA or DGLA in blood.

The term "Gamma-linolenic acid (GLA)" used throughout the specification refers to a fatty acid found primarily in vegetable oils.

Prostaglandin metabolism disorders are found in women with premenstrual syndrome. Because prostaglandins are involved in central nervous system function, fluid balance, and regulation of uterine contractility, prostaglandin metabolism disorders can cause premenstrual syndrome. Therefore, it is known that allowing women with defects in the process of converting linoleic acid to gamma-linolenic acid in the prostaglandin synthesis process to take GLA can directly synthesize prostaglandin, which can help alleviate the symptoms of premenstrual syndrome.

The term "DGLA (Dihomo-gamma-linolenic acid)" used throughout the specification refers to an omega-6 fatty acid composed of 20 carbons and is mainly contained in plant seed oil. When linoleic acid, an essential fatty acid, is consumed, GLA is synthesized slowly, and then GLA is quickly converted to DGLA.

DGLA produces PGE ₁ and 15-OH-DGLA through metabolism. As described above, PGE ₁ is effective in suppressing inflammation, and 15-OH-DGL inhibits 5-lipoxygenase and 12-lipoxygenase to inhibit the production of inflammatory metabolites such as PGE ₂ and LTB4 produced from arachidonic acid. interfere

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application enhances the production of GLA and DGLA, thereby relieving menstrual pain. there is.

The term "treatment" used throughout the specification of the present application is a pharmaceutical composition comprising a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application as an active ingredient. It refers to all actions to improve or benefit the symptoms of premenstrual syndrome by administering the composition to a subject having premenstrual syndrome.

In one embodiment of the present application, the composition may be for treating or preventing premenstrual syndrome, and specifically, reduces blood NO production and IL-6, iNOS, and COX-2 gene expression, and blood prostaglandin E ₁ /prostaglandin and treating or preventing premenstrual syndrome by increasing the E ₂ production rate and the levels of GLA and DGLA.

As used throughout this specification, the term "premenstrual syndrome" refers to a series of symptoms characterized by emotional, behavioral, and physical symptoms that occur repeatedly before menstruation, such as edema, breast pain, digestive disorders, headache, back pain, lower abdominal pain, and abdominal pain. These include bloating, constipation, diarrhea, depression and insomnia.

In one embodiment of the present application, the pharmaceutical composition is in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. It may be formulated and used, but may not be limited thereto.

In one embodiment of the present application, when formulating the pharmaceutical composition, it may be prepared using diluents or excipients such as generally used fillers, extenders, binders, wetting agents, disintegrants, or surfactants, but limited thereto It may not be.

In one embodiment of the present application, solid preparations for oral administration include tablets, pills, powders, granules, or capsules, etc., and these solid preparations include at least one excipient in the dead cells of the strain, for example, It may be prepared by mixing starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, for example, lubricants such as magnesium stearate and talc may be used in addition to simple excipients, but may not be limited thereto.

In one embodiment of the present application, liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, Sweeteners, aromatics, preservatives, and the like may be included, but may not be limited thereto.

In one embodiment of the present application, preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories, but may not be limited thereto. For example, as the non-aqueous solvent or suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used, but may not be limited thereto. For example, as the suppository, witepsol, macrogol, tween 61, cacao paper, laurin paper, glycerogelatin, etc. may be used, but may not be limited thereto.

The pharmaceutical composition according to one embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.

The term "quasi-drugs" used throughout the present specification refers to items with a milder effect than pharmaceuticals among items used for the purpose of diagnosing, treating, improving, mitigating, treating or preventing diseases of humans or animals. According to the Act, quasi-drugs are items excluding items used for pharmaceutical purposes, and include products used for the treatment or prevention of human or animal diseases, products with minor or no direct action on the human body, etc.

The quasi-drug composition of the present application consists of body cleanser, disinfectant cleanser, detergent, kitchen detergent, cleaning detergent, toothpaste, gargling agent, wet tissue, detergent, soap, hand wash, hair detergent, hair softener, humidifier filler, mask, ointment and filter filler It can be prepared in a formulation selected from the group, but is not limited thereto.

In one embodiment of the present application, the pharmaceutical composition may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" herein refers to treating a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention Or means an amount sufficient to prevent, and the effective dose level is the severity of the disease, the activity of the drug, the age, weight, health, sex of the patient, the sensitivity of the patient to the drug, the administration time of the composition of the present invention used, the route of administration and excretion rate, treatment period, factors including drugs used in combination or simultaneous use with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present application may be administered alone or in combination with components known to exhibit therapeutic effects on known intestinal diseases. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors.

In one embodiment of the present application, the dosage of the pharmaceutical composition can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction of the disease, the patient's age, weight, sex, history, or the type of substance used as an active ingredient. there is. For example, the pharmaceutical composition of the present invention can be administered at about 0.1 ng to about 1,000 mg/kg, preferably 1 ng to about 100 mg/kg per adult, and the frequency of administration of the composition of the present application is particularly limited thereto. However, it can be administered once a day or divided into several doses. The dosage or frequency of administration is not intended to limit the scope of the present application in any way.

The pharmaceutical composition of the present application is not particularly limited thereto, but as desired, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, intrarectal administration, etc. It can be administered through the route of However, when administered orally, it can be administered in an unformulated form, and since the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain can be denatured or decomposed by gastric acid, the oral composition contains an active agent. It can also be administered orally in the form of a coating or formulated to protect it from degradation in the stomach or in the form of an oral patch. In addition, the composition may be administered by any device capable of transporting active substances to target cells.

The second aspect of the present application is a mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or a culture, lysate, or extract of the mixed strain It provides a food composition for alleviating dysmenorrhea, containing one or more as active ingredients. Content overlapping with the first aspect is also applied to the food composition of the second aspect.

The term "relief" used throughout the present specification refers to any activity in which menstrual pain is alleviated or related symptoms are improved by administration of the composition.

In one embodiment of the present application, the composition may alleviate menstrual pain, and specifically, reduce the amount of NO production in the blood and the expression of IL-6, iNOS and COX-2 genes, and the blood prostaglandin E ₁ / prostaglandin E ₂ production ratio and relieving menstrual cramps by increasing the levels of GLA and DGLA.

In one embodiment of the present application, the composition is a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P), or its live cells, dead cells, cultures, lysates and / or its It may contain extracts.

The term "dead cell body" used throughout the present specification is the opposite concept of live bacteria, and means a form in which the growth of bacteria is prevented from occurring by heat treatment or the like of live bacteria and metabolites obtained through fermentation. The dead cell body may include cytoplasm, cell wall, antibacterial active substances such as bacteriocin, polysaccharides, organic acids, and the like. Products using the dead cells have higher stability compared to live cell products, especially have excellent heat resistance and high stability to the external environment, so they are easier to store than existing live cell products and have the advantage of extending the shelf life. In addition, since regulations on the use of antibiotics are being strengthened, the marketability and growth potential are very high, as there are only a handful of companies that have entered into the production of dead cell products in earnest and usable as an alternative.

The term "culture" used throughout the specification refers to an object obtained by culturing the strain of the present application in a known liquid medium or solid medium, and may be used interchangeably with "culture medium".

The term "food" used throughout the present specification refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, health functional foods and health foods, etc., and include all foods in a conventional sense.

The term "functional health food" used throughout the present specification refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Act on Health Functional Foods No. 6727, and is referred to as 'functional'. It means to obtain useful effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological functions.

The food of the present application can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during the preparation. In addition, the formulation of the food may also be prepared without limitation as long as the formulation is recognized as a food. The composition for food of the present invention can be prepared in various types of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time using food as a raw material, and has excellent portability, so the present invention Of the foods can be consumed as supplements to enhance the effect of improving the intestinal environment.

The health food (health food) means food that has an active health maintenance or promotion effect compared to general food, and health supplement food (health supplement food) means food for the purpose of health supplement. In some cases, the terms health functional food, health food, and dietary supplement may be used interchangeably. Specifically, the health functional food is a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application is added to food materials such as beverages, teas, spices, gum, confectionery, It is a food manufactured in capsules, powders, suspensions, etc., which means that it brings specific health effects when ingested. there is

Since the food composition of the present application can be consumed on a daily basis, a high effect on depression improvement can be expected, so it can be used very usefully.

The food composition may further include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions and can improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr) may be included. In addition, amino acids such as lysine, tryptophan, cysteine, and valine may be included.

In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

The mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application may be added as is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, the food composition of the present invention may be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, based on the food or beverage when preparing food or beverage. However, when ingested for a long period of time for health and hygiene purposes, the amount below the above range may be included, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

An example of the food composition of the present application may be used as a health beverage composition, and in this case, as in conventional beverages, various flavoring agents or natural carbohydrates may be included as additional ingredients. The aforementioned natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame may be used. The ratio of the natural carbohydrates may be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health drink composition of the present invention.

In addition to the above, the health beverage composition includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, It may contain alcohol or a carbonating agent, and the like. In addition, it may contain fruit flesh for the manufacture of natural fruit juice, fruit juice beverages, or vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

The food composition of the present application may include a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) of the present application in various weight% if it can exhibit an intestinal environment improvement effect. A mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain may be included in 0.00001 to 100% by weight or 0.01 to 80% by weight based on the total weight of the food composition, but is not limited thereto.

In one embodiment of the present application, the food composition may be a health functional food composition.

Hereinafter, the present invention will be described in more detail through examples of the present application, but the following examples are only exemplified to aid understanding of the present application, and the content of the present application is not limited to the following examples.

Example 1: NO assay after LPS treatment

In order to confirm the ability to produce NO, which is an important biomarker in the immune response, the following experiment was conducted.

RAW 264.7 cells, which are mouse macrophages, were inoculated into a 96 well plate at a concentration of 1x10 ⁵ cell/well. The culture plate was cultured for 24 hours in an environment of 37°C and 5% CO ₂ .

Next, after removing the supernatant of the culture plate after incubation, a mixed strain of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) was treated at a concentration of 1x10 ⁸ CFU/ml for 2 hours cultured in an incubator.

Then, 1ug/ml concentration of LPS was treated and further incubated for 24 hours.

Finally, after dispensing 100ul of the supernatant of the culture plate to a new culture plate, the absorbance at 540 nm was measured with an ELISA reader machine after reaction with Griess reagent.

As a result, compared to the positive control (PC; Positive Control) treated with only LPS, when the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was additionally cultured, the amount of NO production at a concentration of 1x10 ⁸ CFU/ml was statistically It was confirmed that it significantly decreased (see Fig. 1).

In addition, in the mixing ratio of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain, experiments were conducted at a ratio of 5:5, 4:6, 3:7, 2:8, and 1:9, respectively. As a result, lactobacillus It was confirmed that NO production decreased as the ratio of the Bacillus reuteri LM1071 strain increased. However, no significant difference was observed when the mixing ratio of the Lactobacillus gasseri LM1065 strain and the Lactobacillus reuteri LM1071 strain was 3:7 or more. Therefore, it was confirmed that the optimal ratio was 3:7 to 1:9, and all subsequent experiments were performed with the ratio of the Lactobacillus gasseri LM1065 strain and the Lactobacillus leuteri LM1071 strain at 3:7 (see Fig. 1). ).

Through the above results, it was confirmed that the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain of the present application inhibits the production of NO, thereby exhibiting an effect of alleviating dysmenorrhea and premenstrual syndrome.

Example 2: Anti-inflammatory gene expression ability measurement experiment using real-time PCR (Real-time PCR)

In order to confirm the expression levels of anti-inflammatory genes IL-6, iNOS and COX-2, the following experiment was conducted.

Mouse macrophage RAW 264.7 cells were inoculated into a 24 well plate at a concentration of 1x10 ⁵ cell/well. The culture plate was cultured for 24 hours in an environment of 37°C and 5% CO ₂ .

Next, after removing the supernatant of the culture plate after incubation, the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was treated at a concentration of 1x10 ⁸ CFU/ml, and then cultured in an incubator for 2 hours.

Then, 1ug/ml concentration of LPS was treated and further incubated for 24 hours.

After that, RNA was extracted using an RNA extraction kit (RNA extraction kit [TaKaRa]), and cDNA was synthesized and synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Diluted to a final concentration of 5ng/ul.

Finally, real-time PCR was performed using SYBR Green PCR Master Mix [Applied Biosystems] to compare and confirm the expression levels of IL-6, iNOS, and COX-2 genes.

As a result, when the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was additionally cultured compared to the positive control (PC; Positive Control) treated only with LPS, IL ^-6 , IL-6, It was confirmed that iNOS and COX-2 gene expression levels decreased at a statistically significant level (see FIG. 2).

Based on the above results, it can be confirmed that the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus leuteri LM1071 strain of the present application suppresses IL-6, iNOS and COX-2 gene expression, thereby exhibiting an effect of alleviating menstrual pain and premenstrual syndrome. there was.

Example 3: PMS improvement effect evaluation experiment using SD Rat

The following experiment was performed to evaluate the effect of improvement when the mixed strain of the present invention was repeatedly administered daily for 4 weeks to a Sprague Dawley (SD) rat model in which premenstrual syndrome (PMS) was induced.

The rats used in this example are specific pathogen-free (SPF) rats produced and supplied by Coretech. For 63 6-week-old females, temperature 23 ± 3 ° C, relative humidity 55 ± 15%, ventilation frequency 10-20 times / hr, lighting time 1 hour (8:00 am on - pm on - 8:00 pm off) and Experiments were conducted in a laboratory set to 150 to 300 Lux.

In the case of test substance administration, after weighing an appropriate amount of test substance, it was diluted in sterile water for injection to prepare a prescribed concentration. In the case of the administration method, oral administration was administered once/day for 4 weeks, and the amount of administration was calculated based on 10 mL/kg of body weight measured on the latest weight measurement day.

A vaginal cornification (smear cytology) test was performed at 4 weeks after the start of test substance administration. Blood was collected once/day for 3 days (Day26, Day27, Day28) from 3 days before the last administration of the test substance. Blood collection was performed through the jugular vein, and the collected blood was injected into a vacutainer tube containing a clot activator, left at room temperature for about 15 minutes to coagulate, and then centrifuged at 3,000 rpm for 10 minutes. Serum was separated. Serum was stored in a deep freezer set at -70 ° C or lower until analysis, and used for ELISA analysis. At each blood collection point, a portion of serum obtained by centrifugation using an ELISA kit was used to analyze PGE ₁ and PGE ₂ concentrations and PGE ₁ /PGE ₂ concentrations. Analysis was performed using a commercially available ELISA kit.

The concentrations of GLA and DGLA in the blood were analyzed using the LC-MS/MS method using serum collected on Day 26 from the last test substance administration day.

Normality of the data was assumed for the results of this example, and analysis was performed using parametric multiple comparison procedures or non-parametric multiple comparison procedures. If the result of one-way ANOVA was significant, a post hoc test was performed using Dunnett's multiple comparison test, and the non-parametric Kruskal-Wallis' H-test If the analysis results were significant, a post hoc test was performed using the Mann-whitney test. Statistical analysis was performed using Prism 7.04 (GraphPad Software INC., San Diego, CA, USA), and a p value of less than 0.05 was determined to be statistically significant.

As a result of examination using vaginal cornification, no difference was observed in the pattern of keratinocyte change observed in estrus between all experimental groups.

As a result of ELISA analysis, the prostaglandin E ₁ /prostaglandin E ₂ ratio of the mixed strain-administered group of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain was significantly higher than that of the normal control group on Day 28 (p<0.05, FIG. 3 reference).

In addition, as a result of GLA and DGLA analysis, it was observed that the levels of GLA and DGLA were also significantly higher than those of the normal control group (p<0.05, see FIG. 4).

When the test substance was repeatedly administered to the PMS-induced SD rat model for 4 weeks under the present experimental conditions, the prostaglandin E ₁ /prostaglandin E ₂ ratio increased in the group administered with the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus reuteri LM1071 strain and increased blood levels of GLA and DGLA were observed. It is shown that the administration of the test substance induced an increase in PGE ₁ and a decrease in PGE ₂ to induce symptom improvement in the PMS model.

As such, the mixed strain of Lactobacillus gasseri LM1065 strain and Lactobacillus leuteri LM1071 strain increases the PGE ₁ / PGE ₂ ratio and promotes the production of GLA and DGLA, which are inflammatory substances, to contribute to relieving menstrual cramps and premenstrual symptoms. I was able to confirm that there is

Overall, the mixed strain of the present application, or a composition comprising a culture of the mixed strain, inhibits the production of NO, an inflammation and pain-inducing substance, and inhibits the expression of anti-inflammatory genes IL-6, iNOS and COX-2, , It was confirmed that PGE ₁ /PGE ₂ ratio, which is a major index related to premenstrual syndrome, was increased, and the production of GLA and DGLA, which are substances that relieve inflammation, was increased to show an effect in alleviating premenstrual syndrome.

The above description of the present application is for illustrative purposes, and those skilled in the art will understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present application. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present application is indicated by the following claims rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts thereof should be construed as being included in the scope of the present application.

[Accession number]

Name of depository organization: Korea Center for Microbial Conservation (overseas)

Accession number: KCCM13018P

Entrusted date: 20210622

Name of depository organization: Korea Center for Microbial Conservation (overseas)

Accession number: KCCM12650P

Entrusted date: 20191231

Claims (10)

1. Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) LM1071 strain (KCCM12650P) of the mixed strain or culture of the mixed strain, lysate, containing at least one of the extract as an active ingredient , A pharmaceutical composition for preventing or treating premenstrual syndrome.
2. According to claim 1,

   The mixed strain is to reduce the amount of production of NO (nitric oxide) in the blood, a pharmaceutical composition for preventing or treating premenstrual syndrome.
3. According to claim 1,

   Wherein the mixed strain reduces the expression of IL-6, iNOS and COX-2 genes, a pharmaceutical composition for preventing or treating premenstrual syndrome.
4. According to claim 1,

   The mixed strain increases the blood prostaglandin E ₁ / prostaglandin E ₂ (PGE ₁ / PGE ₂ ) ratio, a pharmaceutical composition for preventing or treating premenstrual syndrome.
5. According to claim 1,

   The mixed strain is to increase the level of DGLA in the blood, a pharmaceutical composition for preventing or treating premenstrual syndrome.
6. According to claim 1,

   The mixed strain is to increase the level of GLA in the blood, a pharmaceutical composition for preventing or treating premenstrual syndrome.
7. According to claim 1,

   The mixed strain is a mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) at a ratio of 3:7 to 1:9, premenstrual syndrome prevention or a pharmaceutical composition for treatment.
8. According to claims 1 to 7,

   The premenstrual syndrome is one or more selected from the group consisting of edema, breast pain, digestive disorders, headache, back pain, lower abdominal pain, abdominal bloating, constipation, diarrhea, depression and insomnia, premenstrual syndrome prevention or treatment pharmaceutical composition.
9. Lactobacillus gasseri ( Lactobacillus gasseri ) LM1065 strain (KCCM13018P) and Lactobacillus reuteri ( Lactobacillus reuteri ) LM1071 strain (KCCM12650P) mixed strain, or containing at least one of the culture, lysate, extract of the mixed strain as an active ingredient To, a food composition for relieving menstrual pain.
10. According to claim 9,

    The mixed strain is a mixture of Lactobacillus gasseri LM1065 strain (KCCM13018P) and Lactobacillus reuteri LM1071 strain (KCCM12650P) at a ratio of 3:7 to 1:9, a food for relieving menstrual pain composition.

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