Anti-inflammatory agent

Abstract

Provided is an anti-inflammatory agent containing at least one selected from a yeast deposited under Accession No. NITE BP-04309 and a yeast deposited under International Accession No. NITE BP-04308 in the National Institute of Technology and Evaluation. The yeast is preferably a yeast deposited under International Accession No. NITE BP-04309 in the National Institute of Technology and Evaluation. The anti-inflammatory agent is preferably for regulating cytokine production. More preferably, the production of cytokines in macrophage cells or skin keratinocytes is regulated.

Description

Anti-inflammatory agent

[0001] The present invention relates to an anti-inflammatory agent using yeast.

[0002] It has been pointed out that the immune system ages along with aging and other factors. As a result of immune system aging, the immune system's original mechanisms for eliminating non-self substances and controlling inflammatory responses decline, leading to increased susceptibility to infection and chronic inflammation that are commonly observed in the elderly. The decline of the immune system's regulatory mechanism specifically refers to the phenomenon where the immune system becomes unable to control itself and runs out of control. It is known that when the regulatory mechanism of the immune system declines, low-level inflammatory responses persist, leading to tissue damage, and this tissue damage is considered to affect the onset of diseases and the progression of aging. Macrophage cells are cells that serve as the first line of innate immunity. In addition to phagocytosis, macrophage cells play a role in transmitting antigen information to T cells, which act as coordinators. It is widely known that macrophage cells produce various cytokines when stimulated by pathogens and the like, and this production is important for immune responses. Furthermore, cells such as keratinocytes, which form the main body of the skin barrier, also produce and release inflammation- and barrier-related cytokines in response to external stimuli.

[0003] In living organisms such as the human body, inflammation is a local defensive response triggered by the immune system when adverse stimuli to biological tissues occur, such as invasion of foreign bodies, tissue damage, or oxidative stress. While it helps eliminate non-self substances from the body, it also has the property of causing a certain degree of damage and pain to the organism itself. Conditions where this damage and pain excessively harm the human body are known as inflammatory diseases including allergic diseases, rheumatoid arthritis, and inflammatory bowel disease. It has become clear that various physiologically active substances are involved in such inflammation. For example, IL-1α, IL-1β, IL-6, IL-8, and TNF-α are known as inflammatory cytokines involved in the induction or exacerbation of inflammation. For this reason, there is a demand for anti-inflammatory agents with high anti-inflammatory effects (see, for example, Non-Patent Documents 1 and 2). Furthermore, it has been conventionally known that yeast extract can be used as an anti-inflammatory agent (Patent Document 1).

[0004] Japanese Unexamined Patent Application Publication No. 2007-84444

[0005] Hiroshi Shibata et al., Vitamins (Japan), 84(8), 384-386 (2010), "Lactoferrin promotes keratinocyte differentiation and barrier function formation," National Agriculture and Food Research Organization (NARO) https: / / www.naro.go.jp / project / results / 4th_laboratory / nilgs / 2016 / nilgs16_s12.html, accessed February 11, 2024.

[0006] Under these circumstances, there is a need for methods that not only improve already aggravated inflammation but also prevent inflammation and suppress its exacerbation through daily lifestyle habits. In particular, there is a need for foods that can be consumed daily and have excellent anti-inflammatory effects. Therefore, the objective of this invention is to provide an anti-inflammatory agent with excellent anti-inflammatory effects by regulating cytokine production.

[0007] The inventors, after diligent research, discovered that a specific yeast strain possesses excellent anti-inflammatory properties, and thus completed the present invention. Based on the above findings, the present invention provides an anti-inflammatory agent comprising one or more yeasts selected from those deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04308 and those deposited under international deposit number NITE BP-04309.

[0008] Figure 1 is a graph showing the results of evaluating the relative expression level of IL-1β in human epidermal keratinocyte cells (HaCaT cells) using extracts from a specific yeast. Figure 2 is a graph showing the results of evaluating the relative expression level of IL-1β in human epidermal keratinocyte cells (HaCaT cells) using extracts from yeasts other than the specific yeast. Figure 3 is a graph showing the results of evaluating the amount of IL-6 produced in macrophage-like cells using powder from yeasts other than the specific yeast. Figure 4 is a graph showing the results of evaluating the amount of TNF-α produced in macrophage-like cells using powder from the specific yeast. Figure 5 is a graph showing the results of evaluating the amount of IL-1β produced in macrophage-like cells using powder from the specific yeast. Figure 6 is a graph showing the results of evaluating the amount of IL-8 produced in macrophage-like cells using powder from the specific yeast.

[0009] The following describes preferred embodiments of the present invention. The present invention is an anti-inflammatory agent comprising one or more yeasts (hereinafter also referred to as "specified yeasts") selected from those deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04309 and those deposited under international deposit number NITE BP-04308.

[0010] Regarding this specific yeast, the yeast deposited under international deposit number NITE BP-04309 was deposited with the National Institute of Technology and Evaluation (NITE) on February 20, 2025, under deposit number NITE P-04309, and was transferred to international deposit on December 22, 2025. Similarly, the yeast deposited under international deposit number NITE BP-04308 was deposited with the National Institute of Technology and Evaluation (NITE) on February 20, 2025, under deposit number NITE P-04308, and was transferred to international deposit on December 22, 2025. Both of these specific yeast strains are registered with the National Institute of Technology and Evaluation (NITE), and their scientific name is Saccharomyces cerevisiae. Their scientific properties are as follows.

[0011] 1. Chemical properties: Forms white to pale yellow colonies in a nutrient medium containing carbon and nitrogen sources. Under a light microscope, proliferation is observed by budding. Mother cells and daughter cells can be distinguished. 2. Taxonomic position: Yeast: Edible yeast (Saccharomyces genus) 3. Culture conditions: (1) Medium name: YPD medium (2) Medium composition: 10 g of yeast extract, 20 g of peptone, and 20 g of glucose per 1000 ml of medium.

[0012] (3) pH of the culture medium: 4-7 (optimal pH 5-6) (4) Sterilization conditions for the culture medium: 121°C, 10 minutes (5) Culture temperature: 30°C (6) Culture period: 2-3 days (7) Oxygen requirements: facultative anaerobic

[0013] 4. Storage conditions: Can be stored by freezing. (1) Freezing conditions: -80°C (2) Protective agent: 10-20% glycerin aqueous solution (15% is optimal) (3) Recovery rate after freezing: 40-80% in 2 years

[0014] 5. Conditions for the survival test (1) Restoration of microorganisms: 30°C (2) Inoculation, culture, and confirmation method: Same conditions as the culture conditions.

[0015] As described above, the specific yeast of the present invention is preferably cultured in a culture medium with a pH of 4 to 7, preferably 5 to 6. The culture atmosphere is preferably aerobic, such as under an atmospheric environment.

[0016] The form of yeast in this invention may be the yeast cell wall, the yeast contents, or the yeast cell itself. The yeast cell wall is a fraction insoluble in an aqueous liquid medium such as water, and is obtained by removing the contents from the yeast cell. The yeast contents are components that dissolve in an aqueous liquid medium, such as proteins, carbohydrates, amino acids, nucleic acids, and organic acids. There are no particular limitations on the method for preparing the yeast cell wall, but for example, the yeast cell may be crushed or destroyed to make the water-soluble contents elutable, then a water-insoluble fraction may be obtained by solid-liquid separation and dried. Methods of crushing or destroying include physical crushing methods such as ultrasonic treatment, grinding by bead milling, and pressurized liquid shearing methods such as French press, or chemical destruction methods using surfactants or lytic enzymes such as cell wall-degrading enzymes. Any drying method such as freeze-drying or spray-drying can be used.

[0017] Furthermore, the contents of yeast can be obtained by crushing or destroying the yeast cells to make the contents elutable in an aqueous solution, and then extracting the resulting crushed material in an aqueous solution. Instead of crushing or destroying the yeast cells, the contents may be made elutable in an aqueous solvent by dissolving the yeast cell wall with a cell wall-degrading enzyme. The contents of yeast are also called yeast extract. The yeast extract may be a solid dried by any method.

[0018] If the aqueous liquid medium is water, the water may contain components other than water, and the water content may be more than 50% by mass, preferably 60% by mass or more.

[0019] In the present invention, among yeast cell walls and yeast contents, it is preferable to use a yeast component containing yeast contents in order to obtain excellent anti-inflammatory effects. Yeast components containing yeast contents include not only the yeast contents themselves, but also a mixture obtained by crushing or destroying yeast cells, in which yeast cell walls and yeast contents are mixed together. By contacting such a mixture with an aqueous solution and performing solid-liquid separation, the yeast extract can be separated from the cell walls. The yeast component preferably contains 10% by mass or more, more preferably 20% by mass or more, even more preferably 50% by mass or more, and particularly preferably 60% by mass or more, in its dry mass, the proportion of yeast extract.

[0020] There are no strict restrictions on the dosage when using the specific yeast, which is the active ingredient of the anti-inflammatory agent of the present invention. Since the effects obtained will differ depending on the target person and the disease to be treated, it is desirable to set the dosage appropriately, but the preferred dosage for the specific yeast is preferably 10 mg to 100 g per day in terms of cell weight, more preferably 100 mg to 10 g. The agent of the present invention can be taken continuously, and can be taken continuously for one week or more, two weeks or more, or four weeks or more.

[0021] Furthermore, methods for ingesting the specific yeast of the present invention include oral ingestion, transdermal ingestion, transmucosal ingestion, and enteral ingestion, with oral ingestion being particularly preferred.

[0022] The anti-inflammatory agent of the present invention can be used as a pharmaceutical, quasi-drug, or food or beverage for animals, including humans, or for the manufacture of such products. The anti-inflammatory agent of the present invention may be directly administered to or ingested by animals, including humans, as a pharmaceutical, quasi-drug, or food or beverage, or it may be added to or blended with food or beverages or animal feed such as pet food to be used as an anti-inflammatory food or beverage or animal feed. In the latter case, the method of adding or blending the specific yeast into food or beverages or animal feed is not particularly limited. For example, the specific yeast may be directly blended into the raw materials before the manufacture of food or beverages or animal feed, added during the manufacturing process of food or beverages or animal feed, or added to the manufactured food or beverages or animal feed. The term "food or beverage" refers to anything that humans can ingest as food, and includes not only general food and beverages, including so-called health foods, but also, for example, health functional foods such as Foods for Specified Health Uses and Nutritional Functional Foods as defined in the Ministry of Health, Labour and Welfare's Health Functional Food System, and supplements. The term "animal feed" refers to anything given as feed to animals other than humans (animals raised by humans), such as livestock, poultry, and fish, and includes, for example, livestock feed and pet food.

[0023] When the anti-inflammatory agent of the present invention is used as a pharmaceutical or quasi-drug, it may contain the specific yeast as the active ingredient alone, or it may further contain a pharmaceutically acceptable carrier, or it may contain other active ingredients or pharmacological components to the extent that the anti-inflammatory effect of the specific yeast is not impaired. Examples of such carriers include excipients, coating agents, binders, bulking agents, disintegrants, surfactants, lubricants, diluents, dispersants, buffers, osmotic pressure adjusters, pH adjusters, emulsifiers, preservatives, stabilizers, antioxidants, colorants, UV absorbers, humectants, thickeners, activity enhancers, bactericides, flavoring agents, and odor-masking agents.

[0024] When the agent of the present invention is used as a pharmaceutical or quasi-drug, it can be administered in any dosage form. The dosage form may be oral or parenteral. For example, oral dosage forms include solid dosage forms such as tablets, coated tablets, granules, powders, and capsules, as well as liquid dosage forms such as elixirs, syrups, and suspensions. Parenteral dosage forms include injection, infusion, transdermal, transmucosal, nasal, enteral, inhalation, suppositories, boluses, and patches. Of these, oral dosage forms are preferred. The amount of specific yeast in the agent of the present invention is arbitrary as long as it can be an active ingredient, and the specific yeast may account for 5% or more by mass, or 10% or more by mass, or 20% or more by mass, or 30% or more by mass, or 40% or more by mass, or 50% or more by mass, or 70% or more by mass of the solid content in the agent. Here, solid content refers to the total amount excluding the solvent, and examples of solvents include water, ethanol, and organic solvents commonly used as solvents.

[0025] When the agent of the present invention is used as a food or beverage, it may contain the specific yeast, which is the active ingredient, alone, or it may also contain various additives used in the manufacture of food and beverages, to the extent that the anti-inflammatory effect of the specific yeast is not impaired. Examples of such additives include various oils and fats, herbal medicines, amino acids, polyhydric alcohols, natural polymers, vitamins, dietary fiber, surfactants, purified water, excipients, stabilizers, pH adjusters, antioxidants, sweeteners, flavoring components, acidulants such as organic acids, stabilizers, flavors, colorants, fragrances, and the like.

[0026] Examples of food and beverage products that may use it include oral medications (gum, candy, etc.), processed seafood products such as kamaboko and chikuwa, livestock products such as sausages and ham, bread, Western-style sweets, Japanese-style sweets, noodles such as fresh noodles, Chinese noodles, boiled noodles, and soba, seasonings such as sauces, soy sauce, dips, sugar, honey, powdered syrup, and starch syrup, spices such as curry powder, mustard powder, and pepper powder, jams, marmalades, chocolate spreads, pickles, side dishes, furikake, or processed vegetables and fruits such as canned and bottled vegetables and fruits, dairy products such as cheese, butter, and yogurt, beverages such as miso soup, soups, fruit juices, vegetable juices, whey drinks, soft drinks, and alcoholic beverages, and other general food and beverage products such as health foods.

[0027] The present invention includes a package comprising a packaging body and the aforementioned anti-inflammatory agent of the present invention contained in the packaging body, or food or animal feed containing the same. The packaging body only needs to be capable of containing the agent of the present invention, food or animal feed, and capable of printing ingredient information, etc., and its form and material are not particularly limited. Examples of the form of the packaging body include a box or a bag. Examples of the material of the packaging body include paper, plastic, woven fabric, metal, etc. Various information, such as the content of specific yeast in the agent of the present invention, food or animal feed contained in the packaging body, is clearly indicated on the packaging body. The method of presenting information in such packaging is not particularly limited. For example, 1) the information may be printed on the outer or inner surface of the packaging; 2) the anti-inflammatory use may be printed on a printing medium such as printed paper enclosed inside the packaging along with food or animal feed; or 3) the packaging or the printing medium enclosed therein may contain a QR code (registered trademark) that provides access to an internet URL or a site introducing the agent of the present invention, food or animal feed, etc., and the information may be presented by accessing that URL.

[0028] As shown in the examples described later, the specific yeast is excellent at regulating the production of pro-inflammatory cytokines from immune cells such as macrophages and non-immune cells such as skin keratinocytes. Mammals and their immune and non-immune cells are preferred targets for cytokine production regulation. Examples of mammals include humans, rats, mice, rabbits, cattle, pigs, dogs, cats, sheep, monkeys, and other non-human mammals. Besides macrophages, other immune cells that produce pro-inflammatory cytokines include dendritic cells, neutrophils, and mast cells. Besides skin keratinocytes, other non-immune cells that produce pro-inflammatory cytokines include epithelial cells, fibroblasts, vascular endothelial cells, mesenchymal stem cells, and nervous system cells.

[0029] The specific yeast of the present invention exhibits immunosuppressive effects on non-immune cells such as skin keratinocytes and immune cells such as macrophages, which are activated by stimulation with antigens (e.g., lipopolysaccharide (LPS)) or reactive oxygen species. For example, it can suppress the production of pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α, and IL-8 in macrophages.

[0030] IL-1β promotes inflammatory responses through various mechanisms, including inducing fever, recruiting and activating leukocytes, promoting the production of other inflammatory cytokines, increasing vascular permeability, and influencing tissue destruction and reconstruction. TNF-α actively promotes inflammatory responses at local inflammation sites through leukocyte recruitment, promotion of inflammatory cytokine production, increased vascular permeability, and induction of cell death. Therefore, it plays an important role in infection defense and tissue repair. IL-6 plays a crucial role in promoting inflammation through the induction of acute phase responses, differentiation and activation of immune cells, and interaction with other inflammatory cytokines. IL-8 effectively induces neutrophil accumulation and activation in acute inflammation, establishing an initial immune response and contributing to the rapid processing of pathogens and damaged sites. IL-8 is a chemokine.

[0031] IL-1β, IL-6, TNF-α, and IL-8 trigger acute and / or chronic inflammatory responses, activating immune defense mechanisms. However, excessive responses can contribute to tissue damage and disease progression. For example, excessive IL-1β production can cause autoinflammatory diseases, autoimmune diseases, systemic inflammatory responses, and sepsis. Excessive TNF-α secretion can contribute to chronic inflammatory diseases and conditions such as sepsis. Excessive IL-6 production may be involved in the progression of chronic inflammation and autoimmune diseases. Excessive or sustained secretion of IL-8 can lead to the spread of inflammation, chronic inflammatory states, and even tissue damage.

[0032] Based on the above, it can be expected that ingesting the specific yeast of the present invention will suppress the excessive production of pro-inflammatory cytokines caused by damage to immune cells or non-immune cells in the body or by antigen stimulation, thereby preventing or improving autoinflammatory diseases, autoimmune diseases, systemic inflammatory responses, and sepsis. For example, diseases to which the agent of the present invention may be applied include rheumatoid arthritis and inflammatory bowel disease related to the suppression of IL-1β and TNF-α, psoriasis and systemic lupus erythematosus related to the suppression of TNF-α and IL-6, allergic diseases such as asthma and atopic dermatitis related to the suppression of IL-6 and IL-8, chronic obstructive pulmonary disease related to the suppression of IL-8, sepsis related to the suppression of TNF-α and IL-6, excessive inflammatory responses due to infectious diseases such as COVID-19 and influenza, and Alzheimer's disease and Parkinson's disease related to the suppression of TNF-α and IL-6.

[0033] For example, LPS is known to have endotoxic activity. In response to large amounts of LPS, TNF-α and IL-1 produced by macrophage cells increase blood coagulation and vascular permeability in blood vessels, leading to a decrease in blood pressure, peripheral circulatory failure, and a state of shock (endotoxin shock). Furthermore, when IL-1, IL-6, TNF-α, etc., produced by various cells in response to the action of LPS are carried to the brain via the bloodstream, it is known that prostaglandin E2 is produced from brain cells, acting on the pituitary hypothalamus, which has thermoregulatory functions, and causing fever. Therefore, by using the specific yeast of the present invention, it may be possible to prevent or improve these intravascular shock effects and fevers.

[0034] Furthermore, it is known that when fat cells become too large due to obesity, they die, and immune cells such as macrophages that engulf them gather in the visceral adipose tissue, causing inflammation. This inflammation affects the entire body and is believed to lead to lifestyle-related diseases such as diabetes. Therefore, by using the specific yeast of the present invention, it is possible to prevent diseases such as inflammation caused by obesity.

[0035] As shown in Evaluation 1 of the Examples described later, the specific yeast of the present invention effectively suppresses the upregulation of IL-1β expression in evaluations using human epidermal keratinocytes (HaCaT cells), indicating that it exhibits a particularly high anti-inflammatory effect in the skin. For example, it is thought to inhibit the transcription and production of IL-1β by suppressing the activity of signaling pathways activated by reactive oxygen species (ROS). Therefore, it is expected that the inflammatory response caused by IL-1β will be suppressed by the intake of the agent of the present invention, and that this will lead to the alleviation of skin redness, swelling, pain, and itching, improvement of skin barrier function, improvement of wound healing, suppression of cellular aging, and suppression of skin aging.

[0036] In particular, as shown in Evaluation 1 or 2 of the Examples described later, by prophylactically administering the specific yeast of the present invention in a state where it can come into contact with immune cells and non-immune cells when causes of endotoxin stimulation such as oxidative stress or infection by pathogenic bacteria occur, it is expected that the increase of inflammatory cytokines can be suppressed. Therefore, the agent of the present invention can be used as an inflammation induction inhibitor or an inflammation provocation inhibitor.

[0037] The anti-inflammatory agent of the present invention is expected to provide an anti-inflammatory effect appropriate to the physical condition at the time of ingestion. For example, by administering the anti-inflammatory agent of the present invention when suffering from a cold, or by administering it to obese or elderly individuals, it is expected to exert an anti-inflammatory effect and reduce inflammation such as that caused by a cold or chronic inflammation. In mammals such as humans, methods for evaluating the degree and state of inflammation include, for example, inflammatory markers in the blood.

[0038] The agent of the present invention can also be used as an immunoinflammatory suppressant, an inhibitor of inflammation induced by endotoxins (such as LPS) or reactive oxygen species (ROS), an inhibitor of excessive cytokine production, an inhibitor of inflammatory responses resulting from innate immune responses, and an inhibitor of the induction or exacerbation of inflammation by suppressing the production of inflammatory cytokines (IL-6, TNF-α, IL-1β, IL-8); an inhibitor of inflammatory cytokine production, an inhibitor of the production of IL-6, TNF-α, IL-1β and / or IL-8, etc.

[0039] For example, the present invention provides the following. [1] An anti-inflammatory agent comprising at least one selected from the yeast deposited with the National Institute of Technology and Evaluation under the international deposit number NITE BP-04309 and the yeast deposited under the international deposit number NITE BP-04308. [2] The anti-inflammatory agent according to [1], wherein the yeast is the yeast deposited with the National Institute of Technology and Evaluation under the international deposit number NITE BP-04309. [3] The anti-inflammatory agent according to [1] or [2], which is for regulating cytokine production. [4] The anti-inflammatory agent according to [3], which regulates cytokine production in macrophage cells or skin keratinocytes. [5] The anti-inflammatory agent according to [3] or [4], wherein the cytokine is at least one selected from IL-1β, IL-6, IL-8, and TNF-α. [6] The anti-inflammatory agent according to any one of [1] to [5], which suppresses the production of at least one selected from IL-1β, IL-6, IL-8, and TNF-α under inflammatory conditions. [7] The anti-inflammatory agent according to any one of [1] to [6], comprising a cell wall component of said yeast. [8] The anti-inflammatory agent according to any one of [1] to [7], comprising contents of said yeast. [9] An anti-inflammatory food or beverage comprising the anti-inflammatory agent according to any one of [1] to [8].

[10] Use of at least one selected from the yeast deposited with the National Institute of Technology and Evaluation under the international deposit number NITE BP-04309 and the yeast deposited under the international deposit number NITE BP-04308 for producing an anti-inflammatory agent.

[0040] While the present invention has been appropriately described above based on its preferred embodiments, the present invention is not limited to the above description.

[0041] Hereinafter, the present invention will be described in further detail by way of examples. However, the scope of the present invention is not limited to these examples.

[0042] Evaluation 1: Evaluation of cytokine production amount in human epidermal keratinocyte cell line (HaCaT cells) (gene expression level) Regarding this test, the test procedures for the yeast with international deposit number NITE BP-04308 (also referred to as "Yeast 1" in the following examples) and the yeast with international deposit number NITE BP-04309 (also referred to as "Yeast 2" in the following examples) are shown below. The applicant also conducted the same test as described below using another 182 yeast strains (all data are not shown). Among the total 184 strains, Yeast 1 and Yeast 2 exhibited the most excellent effect, so the results are shown in Figure 1.

[0043] <Cultivation and Drying of Yeast> Yeast strains were cultivated by the following method. First, the inoculum was streaked onto Yeast extract-Peptone-Dextrose (YPD) agar medium. After culturing the YPD agar medium at 30°C for 3 days under atmospheric conditions, a plurality of colonies appearing on the agar medium were cultured under the above conditions. After cultivation, the yeast was dried by freeze-drying to obtain a dry powder.

[0044] <Preparation of Yeast Extract> 100 mg of the dry powder obtained in the above <Cultivation and Drying of Yeast> was suspended in 800 µL of water. This suspension was mixed with 600 µL of an enzyme solution obtained by dissolving Zymolyase (manufactured by Nacalai Tesque, Inc.) in water, and allowed to stand at 30°C for 60 minutes to dissolve the cell walls. The obtained treated product was freeze-dried to obtain a crushed product from which the contents were eluted. After the obtained crushed product was dissolved in water, it was centrifuged at 3,000 rpm for 5 minutes using a centrifuge. The supernatant was collected and freeze-dried to obtain the extract.

[0045] The human epidermal keratinocyte cell line (HaCaT cells) (Deutsches Krebsforschungszentrum) is an immortalized keratinocyte cell line established from adult male skin. It differs from normal culture conditions in terms of Ca ２＋ concentration and temperature conditions during isolation. HaCaT cells have not been introduced with an immortalizing gene during the immortalization process, and retain the ability to differentiate into each layer of the epidermis (stratum corneum, granular layer, basement membrane) when transplanted into thymic nude mice.

[0046] <Cell passage of human epidermal keratinocyte line (HaCaT cells)> Dulbecco's modified Eagle medium (DMEM, product name MG-30a, Cell Lines Service) was used as the culture medium. HaCaT cells were cultured for 3 days in medium A [containing DMEM (MG-30a), 10% by mass FBS (fetal bovine serum) (Biological Industries Ltd., Beit-Haemek, Israel) and 1% by mass Penicilin-Streptomycin (Nacalai Tesque)]. Medium A was removed by pipette, washed with DPBS (Dulbecco's phosphate buffer solution, Nacalai Tesque), removed by aspirate, ethylenediaminetetraacetic acid (EDTA, Nacalai Tesque) was added, and incubated in a 37°C, 5% CO2 incubator for 10 minutes. Subsequently, 2.5 mL of a 1:1 mass mixture of trypsin (Nacalai Tesque) and EDTA was added. After confirming that the cells had detached from the bottom, 2.5 mL of the aforementioned medium A was added to stop the activity of the trypsin. The mixture was collected in a 15 mL centrifuge tube and centrifuged at 1300 rpm for 3 minutes. The supernatant of the centrifuged sample was aspirated and removed, and the cells were resuspended in fresh medium A. The number of cells was then counted and transferred to a new flask. ４ cells / cm ２ The seeds were sown under these conditions.

[0047] <Method for preparing positive control> As a positive control, epicatechin gallate standard (ECg) (Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted with a 6% by mass ethanol aqueous solution to prepare a 10 mM (100 μM at the time of cell addition) dilution. Ethanol from Nacalai Tesque was used to dissolve the ECg (0.06% by mass at the time of cell addition).

[0048] <Anti-inflammatory Effect Evaluation System> The evaluation system consists of an untreated group (hereinafter also called "control"), a hydrogen peroxide exposure group (hereinafter also called "negative control"), an ECg pre-treated hydrogen peroxide exposure group (hereinafter also called "positive control"), and a yeast pre-treated hydrogen peroxide exposure group (hereinafter also called "yeast 1" and "yeast 2" depending on the yeast used). The yeast used was an extract prepared by the method described above. Specifically, the following was done. The test was conducted at 37°C under a 5% CO2 atmosphere. Each culture medium was used in a volume of 1 ml / well.

[0049] (Yeast 1, Yeast 2) HaCaT cells were placed in a 12-well plate at a rate of 1.0 × 10⁶ ４ cells / cm ２ Seeds were seeded under the specified conditions, incubated for 24 hours in the presence of medium A, washed with DPBS, and incubated in medium A supplemented with 1 v / v% yeast extract of yeast 1 or yeast 2. 47 hours after seeding, the seeds were washed with DPBS and incubated again for 1 hour in medium A without FBS, mixed with 1 v / v% yeast extract of the corresponding yeast. Subsequently (48 hours after seeding), the seeds were washed with DPBS and exposed for 1 hour in medium A supplemented with 0.0003% by mass hydrogen peroxide. After hydrogen peroxide exposure (49 hours after seeding), the seeds were washed with DPBS and incubated for 48 hours in normal medium A.

[0050] (Positive control) Instead of 1 v / v% yeast extract, a 1 v / v% solution of ECg diluted to 10 mM was used. Other than this, the treatment was the same as described above (Yeast 1, Yeast 2).

[0051] (Control) No yeast extract was added during each incubation stage, nor was hydrogen peroxide added. Other than these points, the treatment was the same as described above (Yeast 1, Yeast 2).

[0052] (Negative control) No yeast extract was added. Other than that, the treatment was the same as described above (Yeast 1, Yeast 2).

[0053] (Comparative Yeasts A and B) Two S. cerevisiae strains different from yeast 1 and yeast 2 were used as comparative yeasts. Comparative yeasts A and B were cultured and dried in the same manner as yeasts 1 and 2, except that the S. cerevisiae strains used were different, and yeast extracts were obtained in the same manner. The procedure was the same as in the above (yeast 1 and yeast 2), except that the yeast extract of comparative yeast A or B was used instead of the yeast extract of yeast 1 and yeast 2.

[0054] <Gene Expression Analysis> RNA was extracted from incubated HaCaT cells using Sepazol RNA I Super G (Nacalai Tesque), and the RNA concentration was measured and adjusted using a micro-spectrometer (ThermoFisher Scientific). The RNA was then reverse transcribed into cDNA using ReverTraAce qPCR Master Mix (Toyobo) and Thermals Cycler Dice (Takara Bio). Gene expression analysis of the synthesized cDNA was performed using real-time PCR with ThermalCycler Dice Real Time System II (Takara Bio) and Thunderbird SYBR qPCR Mix (Toyobo). All steps were performed according to the recommended protocol. Relative gene expression levels were calculated by comparative quantification using the ΔΔCt method against the Ct value, which is the expression level of the internal standard gene. The primer sequences are shown in Table 1. The test results for yeast 1 and yeast 2 are shown in Figure 1.

[0055]

[0056] When skin cells are exposed to ultraviolet light, reactive oxygen species (ROS) are involved in the mechanism that induces IL-1β. In this study, hydrogen peroxide was used as the ROS source to simulate this condition, and the expression level of IL-1β was evaluated as the evaluation system. As shown in Figure 1, by using specific yeasts, the upregulation of IL-1β expression caused by hydrogen peroxide exposure can be effectively suppressed. Figure 2 shows the results of a similar test conducted using comparative yeasts A and B instead of yeasts 1 and 2. The relative expression levels of the positive control and negative control differ between Figure 1 and Figure 2 because the experiments were conducted on different days. As shown in Figures 1 and 2, the inhibitory effect on the relative expression level of IL-1β compared to the negative control is higher for yeasts 1 and 2 than for comparative yeasts A and B. From this, it can be seen that yeasts 1 and 2 are particularly superior in inhibiting IL-1β expression. Based on the above tests, it can be seen that specific yeasts are effective as anti-inflammatory agents against inflammation caused by reactive oxygen species generated by ultraviolet light irradiation, etc.

[0057] Evaluation 2: Evaluation of cytokine production (ELISA analysis, macrophage-like cells) In the following tests, macrophage-like cells were used as a model for macrophage cells. Macrophage cells in this invention include macrophage-like cells.

[0058] <Preparation of Yeast Powder> 150 mg of the dried powder obtained in the <Culturing and Drying of Yeast> procedure described above was suspended in 1.5 mL of sterile water and placed in a tube. The cell walls were disrupted by pulverizing with a bead cell disruptor (Tommy Medico, Micro Smash® MS-100R), and then the contents were extracted by freeze-drying to obtain yeast powder (hereinafter sometimes simply referred to as "yeast powder"). The obtained yeast powder contained both yeast cell walls and yeast extract, with yeast extract accounting for approximately 60% by mass.

[0059] <Evaluation of Cytokine Production> Yeast deposited under international deposit number NITE BP-04309 was used as the yeast. THP-1 (human monocyte-derived cells) were cultured in growth medium (10% FBS (fetal bovine serum) - RPMI1640 medium), and then switched to macrophage-like differentiation induction medium (10% FBS, 0.5 μg / mL PMA (Phorbol 12-myristate 13-acetate) - RPMI1640 medium) 2-3 days after the start of culture, yielding 6.3 × 10⁶ cells. 4 Seeds were seeded in a 24-well plate at a concentration of 0.5 mL / well. (37°C, 5% CO2) ２ After culturing for 4 days under these conditions and confirming differentiation into macrophage-like cells, the culture medium was replaced with test medium containing yeast powder at concentrations of 0.08 w / v%, 0.4 w / v%, and 2 w / v% (10% FBS, 100 Units / mL Penicillin, 100 Units / mL Streptomycin-RPMI1640 medium), and cultured at 37°C and 5% CO2. ２ After culturing for 1 hour under these conditions, the culture medium was replaced with a test medium containing lipopolysaccharide (LPS) and yeast powder at the same concentration as above, and cultured for 24 hours. The amount of LPS used was 1 μg / mL in the medium. For the control, the same setup was used except that a corresponding amount of sterile water was used instead of yeast powder at a concentration of 2 w / v%.

[0060] The culture supernatant from each well was transferred to a 1.5 mL tube and centrifuged at 3,000 × g for 5 minutes. The supernatant was then used for ELISA analysis. In the ELISA analysis, IL-1β was measured using the IL-1β ELISA Kit, Human (ProteinTech), IL-6 was measured using the REVIS® Human IL-6 ELISA Kit (Fujifilm Wako Shibayagi Co., Ltd.), IL-8 was measured using the REVIS® Human IL-8 ELISA Kit (Fujifilm Wako Shibayagi Co., Ltd.), and TNF-α was measured using the Authenticine® TNF-alpha ELISA Kit, Human (ProteinTech).

[0061] The amount of cytokine production was evaluated (ELISA analysis, macrophage-like cells) by adding the yeast powder to macrophage-like cells, and the results of the ELISA analysis are shown in Figures 3 to 6. It was confirmed that the production of the inflammatory cytokines IL-6, TNF-α, IL-1β, and IL-8 was suppressed by the addition of the yeast powder.

[0062] According to the present invention, an anti-inflammatory agent exhibiting excellent anti-inflammatory effects can be provided.

Claims

1. An anti-inflammatory agent comprising one or more yeasts selected from those deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04309 and those deposited under international deposit number NITE BP-04308.

2. The anti-inflammatory agent according to claim 1, wherein the yeast is a yeast deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04309.

3. The anti-inflammatory agent according to claim 1, for regulating cytokine production.

4. The anti-inflammatory agent according to claim 3, which regulates cytokine production in macrophage cells or keratinocytes.

5. The anti-inflammatory agent according to claim 3, wherein the cytokine is at least one selected from IL-1β, IL-6, IL-8, and TNF-α.

6. An anti-inflammatory agent according to any one of claims 1 to 5, which suppresses the production of at least one selected from IL-1β, IL-6, IL-8, and TNF-α under inflammatory conditions.

7. The anti-inflammatory agent according to any one of claims 1 to 5, comprising the cell wall components of the yeast.

8. An anti-inflammatory agent according to any one of claims 1 to 5, comprising the contents of the yeast.

9. An anti-inflammatory food or beverage comprising the anti-inflammatory agent described in any one of claims 1 to 5.

10. Use of one or more yeasts selected from those deposited with the National Institute of Technology and Evaluation under international deposit number NITE BP-04309 and those deposited under international deposit number NITE BP-04308 for the manufacture of anti-inflammatory agents.

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