Anti-inflammatory agent and Anti-inflammatory composition, method for preparation thereof, and use thereof

Abstract

To provide a new application for an oral composition comprising vesicles separated from a food fermented from soybeans using a bacterium belonging to the genus Bacillus.SOLUTION: An oral composition containing vesicles separated from a food fermented from soybeans using a bacterium belonging to the genus Bacillus is used for at least one application selected from the group consisting of suppression of interleukin-1β production, improvement of diseases associated with interleukin-1β, and prevention of diseases associated with interleukin-1β.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to anti-inflammatory agents and compositions, their preparation methods and uses. [Background technology]

[0002] It has been known that ingesting nanovesicles (NVs), such as exosomes produced by plants and bacteria, can have various beneficial effects on the body.

[0003] For example, Patent Document 1 discloses that bacterial-derived vesicles are absorbed into the body and distributed throughout the body, and that Bacillus subtilis-derived vesicles suppressed the secretion of IL-6 and TNF-α by macrophage cell lines.

[0004] On the other hand, Non-Patent Document 1 describes that vesicles (NV) have been obtained from various plants such as onions and tomatoes, and that exosomes are their contents and are thought to protect miRNA, which may be a functional component, from degradation by digestive enzymes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 6839265 [Non-patent literature]

[0006] [Non-Patent Document 1] Masao Yamazaki et al. "Functional Study on Nanoparticles in Food" Chemistry and Biology Vol.61.2023 No.2 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the specific functions, applications, and uses of food-derived vesicles have not yet been fully developed.

[0008] An object of the present invention is to provide a novel use of an oral composition containing vesicles isolated from a food product obtained by fermenting soybeans with Bacillus bacteria. [Means for solving the problem]

[0009] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0010] [1] An oral composition containing vesicles isolated from a food product obtained by fermenting soybeans using Bacillus bacteria, the composition being intended for use in one or more applications selected from the group consisting of suppressing interleukin-1β production, improving diseases associated with interleukin-1β, and preventing diseases associated with interleukin-1β.

[0011] [2] The oral composition according to [1], wherein the Bacillus bacterium is Bacillus subtilis.

[0012] [3] The oral composition according to [2] or [3], wherein the Bacillus bacterium is Bacillus subtilis var. natto.

[0013] [4] The oral composition according to any one of [1] to [3], wherein the food is natto, and the vesicles are obtained as a precipitate by ultracentrifugation after grinding the natto.

[0014] [5] The oral composition according to [4], wherein the ultracentrifugation is a centrifugation treatment carried out at a centrifugal force of 10,000 to 500,000 × g.

[0015] [6] The oral composition according to any one of [1] to [5], wherein the vesicles contain 0.1 mg or more of RNA per 1 g of protein contained in the vesicles.

[0016] [7] The oral composition according to any one of [1] to [6], wherein the disease associated with interleukin-1β is a disease selected from the group consisting of coronary artery disease, alcoholic hepatitis, non-alcoholic hepatitis, and periodontal disease.

[0017] [8] The oral composition according to any one of [1] to [7], wherein the interleukin-1β is interleukin-1β produced by macrophages.

[0018] [9] The oral composition according to any one of [1] to [8], which is also for inhibiting nitric oxide production.

[0019]

[10] The oral composition according to any one of [1] to [9], which is also for preventing a disease involving nitric oxide.

[0020]

[11] The oral composition according to

[10] , wherein the disease involving nitric oxide is a disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, and sepsis.

[0021]

[12] The oral composition according to any one of [1] to

[11] , which is also for suppressing the production of interleukin-6 and tumor necrosis factor α.

[0022]

[13] The oral composition according to any one of [1] to

[12] , which is a food composition. [Effects of the Invention]

[0023] According to the present invention, it is possible to provide novel uses of an oral composition containing vesicles isolated from a food obtained by fermenting soybeans using Bacillus bacteria, such as the suppression of interleukin-1β production, the improvement of diseases associated with interleukin-1β, or the prevention of diseases associated with interleukin-1β. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is an image showing an ultracentrifuge tube containing 20Kp after ultracentrifugation at a centrifugal force of 20,000×g. [Figure 2] 1 is an image showing an ultracentrifuge tube containing 200Kp after ultracentrifugation at a centrifugal force of 200,000×g. [Figure 3] 1 is a graph showing the particle size distribution of 20Kp vesicles. [Figure 4] 1 is a graph showing the particle size distribution of 200Kp vesicles. [Figure 5] 1 is a graph showing the results of evaluating the cytotoxicity of vesicles in each group in which different amounts of vesicles were added to the medium within a relatively high concentration range. [Figure 6] 1 is a graph showing the concentration of nitric oxide in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range. [Figure 7] 1 is a graph showing the IL-1β concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range. [Figure 8] 1 is a graph showing the IL-6 concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range. [Figure 9] 1 is a graph showing the TNF-α concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range. [Figure 10] 1 is a graph showing the results of evaluating the cytotoxicity of vesicles in each group where different amounts of vesicles were added to the medium within a relatively low concentration range. [Figure 11] 1 is a graph showing the concentration of nitric oxide in the medium for each group in which different amounts of vesicles were added to the medium within a relatively low concentration range. [Figure 12] 1 is a graph showing the expression level of IL-1β in each group in which different amounts of vesicles were added to the medium within a relatively low concentration range. [Figure 13] 1 is a graph showing the expression level of IL-6 in each group in which different amounts of vesicles were added to the medium within a relatively low concentration range. DETAILED DESCRIPTION OF THE INVENTION

[0025] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings as needed.

[0026] <Oral Composition> An oral composition according to a preferred embodiment of the present invention comprises vesicles isolated from a food product obtained by fermenting soybeans using Bacillus bacteria, and is intended for use in one or more applications selected from the group consisting of suppressing interleukin-1β (hereinafter referred to as "IL-1β") production, ameliorating diseases associated with IL-1β, and preventing diseases associated with IL-1β. The vesicles are contained in the oral composition as an active ingredient for suppressing IL-1β production, ameliorating diseases associated with IL-1β, and preventing the diseases.

[0027] (Use of oral composition) IL-1β is a pro-inflammatory cytokine. As will be described in detail later in the Examples, adding nanovesicles isolated from soybean fermented with Bacillus bacteria to the culture medium of a macrophage-like cell line can reduce the amount of IL-1β produced (secreted) by the macrophage-like cell line. Furthermore, as disclosed in Patent Document 1, it is widely known that ingested vesicles are absorbed into the body and transported throughout the body. Therefore, vesicles isolated from foods obtained by fermenting soybeans with Bacillus bacteria can be suitably used, in particular, for suppressing IL-1β production by macrophages, ameliorating diseases associated with IL-1β, and preventing diseases associated with IL-1β. Hereinafter, the uses for suppressing IL-1β production, ameliorating diseases associated with IL-1β, and / or preventing diseases associated with IL-1β are also referred to as "uses related to IL-1β."

[0028] Nitric oxide is one of the components released by macrophages when they exhibit inflammatory activity. As will be described in detail later in the Examples, the amount of nitric oxide produced by macrophage-like cell lines can be reduced by adding vesicles isolated from soybean fermented with Bacillus bacteria to the culture medium of the macrophage-like cell lines. Therefore, vesicles isolated from foods in which soybeans are fermented using Bacillus bacteria can be particularly suitably used for suppressing nitric oxide production by macrophages, improving diseases associated with nitric oxide, and preventing diseases associated with nitric oxide.

[0029] Therefore, in addition to the above-mentioned uses related to IL-1β, the oral composition according to this embodiment can also be used for inhibiting nitric oxide production, ameliorating diseases associated with nitric oxide, and / or preventing diseases associated with nitric oxide. Hereinafter, uses related to inhibiting nitric oxide production, ameliorating diseases associated with nitric oxide, and / or preventing diseases associated with nitric oxide are also referred to as "uses related to nitric oxide." As described above, the oral composition according to this embodiment may be an oral composition for use in both uses related to IL-1β and uses related to nitric oxide.

[0030] Interleukin-6 (hereinafter referred to as "IL-6") and tumor necrosis factor α (hereinafter referred to as "TNF-α") are each a type of pro-inflammatory cytokine. As will be described in detail later in the Examples, the production of IL-6 and TNF-α by a macrophage-like cell line can be suppressed by adding vesicles isolated from a food product obtained by fermenting soybeans with Bacillus bacteria to the culture medium of the macrophage-like cell line. Therefore, vesicles isolated from a food product obtained by fermenting soybeans with Bacillus bacteria can be particularly suitably used for suppressing the production of IL-6 and TNF-α by macrophages.

[0031] Therefore, the oral composition according to this embodiment can be used for the above-mentioned IL-1β-related applications and / or nitric oxide-related applications, as well as for the suppression of IL-6 and TNF-α production. Thus, the oral composition according to this embodiment may be an oral composition for use in both IL-1β-related applications and the suppression of IL-6 and TNF-α production, or may be an oral composition for use in all of IL-1β-related applications, nitric oxide-related applications, and the suppression of IL-6 and TNF-α production.

[0032] As described above in detail, the oral composition according to this embodiment exhibits various anti-inflammatory effects.

[0033] The vesicles are thought to contain RNA and / or protein as direct active ingredients that suppress the production of IL-1β, nitric oxide, IL-6, and TNF-α inside the lipid bilayer membrane or between the components that make up the membrane. However, the above-mentioned vesicles contain RNAs with various base sequences and proteins with various amino acid sequences, and it is difficult or impractical to identify these RNAs and proteins one by one and confirm their activity.Furthermore, considering the possibility that the above-mentioned IL-1β production inhibitory effect and nitric oxide production inhibitory effect may be produced by the cooperative action of RNAs and / or proteins with several different sequences, it is difficult to directly identify "vesicles isolated from foods obtained by fermenting soybeans using Bacillus bacteria" based on their structure or properties.

[0034] (IL-1β-related diseases) Examples of diseases associated with IL-1β include one or more diseases selected from the group consisting of coronary artery disease, alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), and periodontal disease, all of which are diseases in which IL-1β is involved in the onset and exacerbation.

[0035] (Diseases involving nitric oxide) Examples of diseases involving nitric oxide include one or more diseases selected from the group consisting of Parkinson's disease, Alzheimer's disease, and sepsis, all of which are diseases in which nitric oxide is involved in the onset and exacerbation.

[0036] (Bacillus bacteria) The above-mentioned vesicles may be vesicles isolated from a food product in which soybeans are fermented using Bacillus bacteria, and the type of Bacillus bacteria that ferment soybeans is not particularly limited.

[0037] As the bacillus bacteria, Bacillus subtilis can be preferably used, and among Bacillus subtilis, Bacillus subtilis var. natto can be particularly preferably used.

[0038] (Fermented soybean food) The above-mentioned vesicles may be vesicles isolated from a food product obtained by fermenting soybeans (Glycine max) using Bacillus bacteria, and the type of food product from which the vesicles are isolated is not particularly limited, but is preferably natto.

[0039] (Separation method) The method for isolating vesicles from foods prepared by fermenting soybeans with Bacillus bacteria is not particularly limited, but examples thereof include ultracentrifugation and polymer precipitation.

[0040] In ultracentrifugation, a large centrifugal force is applied to the food to separate vesicles (a fraction containing vesicles) from the food.Ultracentrifugation allows for the production of highly pure vesicles, since the precipitate obtained by centrifugation contains few impurities other than vesicles.

[0041] The centrifugal force used in centrifugation is not particularly limited and may be, for example, 10,000 to 500,000 x g, 18,000 to 350,000 x g, 18,000 to 250,000 x g, 18,000 to 210,000 x g, 20,000 to 200,000 x g, 100,000 to 230,000 x g, 100,000 to 210,000 x g, or 100,000 to 290,000 x g. By using a centrifugal force of 100,000 x g or higher, the amount of impurities other than vesicles contained in the precipitate obtained by centrifugation can be reduced. The upper and lower limits of the above-mentioned ranges of centrifugal force can be combined in any manner.

[0042] The centrifugation time is not particularly limited, but may be 30 minutes or more, 40 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 5 hours or more, 8 hours or more, or 9 hours or more. Furthermore, the centrifugation time may be 1 hour or less, 1 hour 20 minutes or less, 1 hour 30 minutes or less, 2 hours or less, 4 hours or less, 6 hours or less, 10 hours or less, 11 hours or less, or 15 hours or less. The above lower and upper limits of the centrifugation time can be combined arbitrarily, and may be, for example, 30 minutes to 1 hour 30 minutes, 40 minutes to 1 hour 20 minutes, 2 to 4 hours, 5 to 6 hours, or 9 to 11 hours.

[0043] In contrast, the polymer precipitation method involves adding a superhydrophilic polymer to the food, followed by centrifugal separation of the vesicles. The addition of the superhydrophilic polymer removes water molecules from the vesicle surface, reducing the solubility of the vesicles in water, allowing for highly efficient separation of the vesicles even with a relatively small centrifugal force.

[0044] Regardless of the vesicle isolation method, it is preferable to pulverize (homogenize) the food before isolating the vesicles. This can increase the vesicle yield. Therefore, the vesicles according to this embodiment may be vesicles obtained as a precipitate by ultracentrifugation after pulverizing natto. Furthermore, it is preferable to filter and centrifugalize the liquid (natto extract) obtained by pulverizing natto for 5 to 10 minutes at a low centrifugal force of about 1,000 × g to remove larger debris before subjecting it to ultracentrifugation. Furthermore, when centrifuging at a centrifugal force of 100,000 × g or more, for example, the vesicles may be pre-centrifuged at a centrifugal force of about 10,000 to 50,000 × g (or 10,000 to 30,000 × g), and the resulting supernatant may then be centrifuged at a centrifugal force of 100,000 × g or more. In this way, centrifuging the supernatant obtained by pre-centrifugation at a higher centrifugal force can reduce impurities and increase the purity of the vesicle fraction finally obtained as a precipitate.

[0045] (Physical properties of vesicles) The average particle size of the vesicles according to this embodiment may be, for example, 30 to 1000 nm, 80 to 500 nm, 100 nm to 350 nm, or 100 to 200 nm, but is not limited to these ranges.

[0046] The vesicles of this embodiment may contain 0.1 mg or more, 1 mg or more, 5 mg or more, 10 mg or more, 15 mg or more, 17 mg or more, 30 mg or more, or 50 mg or more of RNA per gram of protein contained in the vesicles.

[0047] The amount of protein contained in the vesicles can be measured by known methods such as the BCA method and the Bradford method.

[0048] According to the BCA method, protein concentration (protein amount) can be measured by utilizing the fact that bicinchoninic acid and monovalent copper ions form a complex, which produces a purple color (maximum absorbance: 562 nm) whose intensity depends on the protein concentration.

[0049] According to the Bradford method, when Coomassie dye binds to a protein, the maximum absorbance shifts to 595 nm, and this can be used to measure the protein concentration (protein amount).

[0050] On the other hand, methods for measuring the amount of RNA contained in the above-mentioned vesicles include, but are not limited to, methods using a spectrophotometer or methods using a commercially available fluorescent dye that binds to RNA and a fluorometer.

[0051] (Animals taking oral compositions) Regardless of the type of oral composition (such as a food composition or pharmaceutical composition, which will be described in detail later), the type of animal that ingests (or orally administers) the oral composition containing the vesicles is not particularly limited as long as it is a mammal, and may be a human or a mammal other than a human. Examples of mammals that ingest the oral composition containing the vesicles include mice, rats, rabbits, guinea pigs, hamsters, hedgehogs, dogs, cats, monkeys, horses, cows, pigs, and sheep.

[0052] (Interval of oral composition intake) Regardless of the type of oral composition (such as a food composition or pharmaceutical composition, which will be described in detail later), it is preferable that the oral composition be ingested (orally administered) repeatedly. The interval between repeated ingestion can be determined appropriately depending on the symptoms, weight, age, and sex of the animal ingesting the composition. The interval between ingestion can be, for example, every few hours, 2-3 times a day, once a day, once every 2-3 days, or once a week.

[0053] (Duration of oral composition intake) The oral composition is preferably taken (administered orally) every day continuously for a long period of time. The specific period for continuously taking the oral composition is 3 days or more, preferably 1 week or more, and more preferably 1 month or more. There is no particular upper limit to the intake period of the oral composition, but examples of the intake (administration) period include 1 week to 1 month, 1 week to 3 months, 2 weeks to 3 months, 1 month to 3 months, and 1 month to 6 months.

[0054] (Intake of oral composition) Regardless of the type of oral composition, the intake amount of the oral composition is not particularly limited, and may be, for example, an amount that results in the intake of RNA contained in the above-mentioned vesicles of 0.03 μg / day or more, 1 μg / day or more, 15 μg / day or more, 50 μg / day or more, 100 μg / day or more, or 300 μg / day or more.

[0055] (Food composition) The oral compositions described above can, in one embodiment, be provided as food compositions.

[0056] (Form and type of food composition) The food composition may be in any form as long as it is a food that can contain (add) the above-mentioned vesicles, and may be in the form of, for example, a liquid, a paste, a solid, a semi-solid, a powder, etc.

[0057] Specific types of foods include, but are not limited to, bread, fermented foods, dried foods, fish paste products, frozen foods, retort foods, instant foods (instant noodles, dry foods), processed foods (processed fish products, processed livestock products), luxury foods such as confectionery, health foods (functional foods) such as supplements, foods for special dietary uses (foods for the sick, foods for children, foods for the elderly), foods with functional claims, foods for specified health uses, water, coffee, soft drinks, alcoholic beverages, tea, seasonings, etc. However, this does not include natto and processed foods containing natto.

[0058] In addition to the above-mentioned vesicles isolated from a food obtained by fermenting soybeans with Bacillus bacteria, the food composition may contain other ingredients such as seasonings, sugars, oils and fats, amino acids, proteins, dietary fiber, vitamins, minerals, thickeners, emulsifiers, flavorings, colorings, bulking agents, binders, excipients, food additives, etc. Suitable excipients include, for example, starch and dextrin.

[0059] (Pharmaceutical composition) In one embodiment, the oral composition described above may be a pharmaceutical composition (including quasi-drugs). The pharmaceutical composition of this embodiment contains the above-mentioned vesicles as an active ingredient, and may further contain a pharmaceutically acceptable carrier.

[0060] The term "pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of an active ingredient and is not substantially toxic to a recipient. "Not substantially toxic" means that the ingredient is not toxic to a recipient at a dose (amount ingested) typically used. In the pharmaceutical composition of this embodiment, a pharmaceutically acceptable carrier is a carrier that does not inhibit the inhibitory effect of the vesicles on the production of IL-1β, nitric oxide, IL-6, and / or TNF-α and is not substantially toxic to a recipient (an animal that ingests the composition).

[0061] Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered inactive ingredients. The types of pharmaceutically acceptable carriers are not particularly limited, but include, for example, solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. One type of pharmaceutically acceptable carrier may be used alone, or two or more types may be used in combination.

[0062] The pharmaceutical composition may contain one or more other ingredients in addition to the above ingredients. The other ingredients are not particularly limited, and any ingredients commonly used in the pharmaceutical field can be used without any particular restrictions. Other ingredients include, for example, pharmaceutical additives other than those mentioned above. Pharmaceutical additives include, but are not limited to, preservatives (e.g., antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, coloring agents, etc. The pharmaceutical composition may contain active ingredients other than the vesicles described above.

[0063] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used for pharmaceutical preparations. The pharmaceutical composition of this embodiment is an oral formulation. Examples of oral formulations include tablets, coated tablets, pills, powders, granules, capsules, syrups, fine granules, liquids, drops, and emulsions. Pharmaceutical compositions in these dosage forms can be formulated according to standard methods (for example, methods described in the Japanese Pharmacopoeia).

[0064] The pharmaceutical composition can be ingested or orally administered in a therapeutically effective amount of the vesicles. The term "therapeutically effective amount" refers to the amount of drug effective for improving or preventing the target disease, i.e., a disease involving IL-1β and / or a disease involving nitric oxide. The therapeutically effective amount may be determined appropriately depending on the patient's symptoms, weight, age, sex, etc. Examples of therapeutically effective amounts include the range of intake amounts of the oral composition described above (e.g., an amount that results in an intake of 0.03 μg / day or more of the RNA contained in the vesicles).

[0065] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the invention described in the claims, and it goes without saying that these modifications are also included within the scope of the present invention.

[0066] <Example of change> The oral composition according to the embodiment described above is an oral composition for use in applications related to at least IL-1β. In another preferred embodiment, the present invention can provide an oral composition for use in applications related to nitric oxide, which contains the above-mentioned vesicles isolated from a food product obtained by fermenting soybeans using Bacillus bacteria.

[0067] For example, in one embodiment, the present invention provides an oral composition containing vesicles isolated from a food product obtained by fermenting soybeans using Bacillus bacteria, for use in one or more applications selected from the group consisting of suppressing nitric oxide production, ameliorating diseases associated with nitric oxide, and preventing diseases associated with nitric oxide. Other aspects of the oral composition (such as the vesicles, type of oral composition, and intake amount) are the same as those of the oral composition according to the above-described embodiment. [Example]

[0068] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0069] <Experimental Method> (Method of preparing vesicles) Approximately 20 g of commercially available natto (Tannen Natto, Takenoshita Foods) was weighed out, 200 mL of saline was added, and the mixture was pulverized in a food processor. The resulting pulverized liquid (vesicle extract) was roughly filtered through a nonwoven fabric, and the filtrate was centrifuged in a 50 mL centrifuge tube at 1000 × g for 10 minutes at 4°C to remove any residue. After centrifugation, the supernatant was dispensed into ultracentrifuge tubes and subjected to ultracentrifugation at 20,000 × g for 1 hour at 4 ° C. Hereinafter, the precipitate obtained by this ultracentrifugation, which is a fraction containing vesicles, is referred to as "20Kp." The supernatant after ultracentrifugation was then collected and subjected to ultracentrifugation at 200,000 × g for 1 hour at 4°C. Hereinafter, the precipitate obtained by this ultracentrifugation, which is a fraction containing vesicles, will be referred to as "200Kp." Thereafter, 20Kp and 200Kp were suspended in PBS (Phosphate-Buffered Saline) or RNAlater solution and stored at -80°C.

[0070] In addition, the amount of protein contained in the vesicles was measured by the BCA method in preparation for cell experiments. Furthermore, total RNA was extracted from the vesicles stored in RNAlater solution using a NucleoSpin RNA kit (TaKaRa), and the amount of RNA contained in the 20Kp and 200Kp vesicles was measured using a Nanodrop® 2000 (Thermo Fisher Scientific). Furthermore, the particle size distribution of the 20Kp and 200Kp vesicles was analyzed using a dynamic light scattering photometer (Otsuka Electronics Co., Ltd.).

[0071] (Methods for evaluating anti-inflammatory activity and cytotoxicity) Murine macrophage-like cell line RAW264.7 cells (cell number: RCB0535) were obtained from the RIKEN BioResource Research Center. The cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (D-MEM) containing 10% fetal bovine serum at 37°C in 5% CO2. Specifically, 1.0 × 10 RAW264.7 cells were cultured at 1.0 × 10 5 cells / cm 2 The cells were seeded onto a 96-well adhesive plate so that the total volume was 1000 kJ / well and pre-cultured for 24 hours. After the pre-culture, the medium was changed to a phenol red-free medium, and the amount of 20 Kp or 200 Kp vesicles added to the medium was determined based on the protein amount measured by the BCA method, and the cells were then cultured for 24 hours.

[0072] After 24 hours of incubation, the supernatant was removed, and phenol red-free medium was added to the control group.To induce an inflammatory response in macrophage-like cells, 50 μL of 100 ng / mL LPS (Salmonella enterica serotype typhimurium, L6511, Merck) was added to the other groups, and the cells were incubated for 24 hours.

[0073] After the incubation, 50 μL of the culture supernatant was transferred to a 96-well plate, and the nitric oxide (NO) concentration was measured by the Griess method. Specifically, a calibration curve was prepared using NaNO2 as a standard substance. 50 μL of Griess reagent was added to 50 μL of the culture supernatant or calibration curve preparation solution to develop color. Thereafter, absorbance was measured at 550 nm using a microplate reader (Molecular Devices), and the nitric oxide concentration was calculated from a calibration curve.

[0074] After collecting the culture supernatant for measuring nitric oxide concentration, a Cell Counting Kit-8 (Dojindo Laboratories) solution was added to the cells remaining in the 96-well plate, and cytotoxicity was evaluated.

[0075] Cytotoxicity was assessed according to the manufacturer's recommended protocol, but the concentration of formazan dye produced by viable cells was measured as absorbance at 450 nm and converted to viable cell count. Cells were also cultured in 24-well plates under the same conditions, and the culture supernatants were collected. The production (concentrations) of three inflammatory cytokines, IL-1β, IL-6, and TNF-α, were measured by ELISA. The production levels of all cytokines were measured using DuoSet ELISA (R&D Systems) according to the manufacturer's protocol with some modifications.

[0076] (Method for measuring mRNA expression levels of inflammatory cytokines) The mRNA expression levels of two inflammatory cytokines, IL-1β and IL-6, in a mouse macrophage-like cell line were measured by real-time PCR after adding 200Kp vesicles to the culture medium. The cells and cell culture conditions used were the same as those described above in the "Method for evaluating anti-inflammatory activity," but 60mm dishes were used for culture. Specifically, RAW264.7 cells were washed with PBS, and total RNA was extracted using NucleoSpin RNA (TaKaRa) according to the protocol. The extracted RNA was dissolved in DEPC-treated water, and the RNA concentration was measured using Nanodrop® 2000.

[0077] Next, reverse transcription reaction was performed from the total RNA using ReverTra Ace (registered trademark, Toyobo Co., Ltd.) as a reverse transcription kit. 1 mg of RNA, nuclease-free water, and 0.2 mL of Master Mix were placed in a PCR tube and the reaction was carried out under the following conditions: 37°C for 15 minutes, 50°C for 5 minutes, 98°C for 5 minutes, and then 4°C. Real-time PCR was performed using the AriaMx Real-Time PCR System (Agilent Technologies). Primers with the base sequences shown in Table 1 below were used for real-time PCR.

[0078] [Table 1]

[0079] <Experimental Results> (Vehicle preparation results) Both 20Kp and 200Kp yielded sufficient amounts of vesicles, and as a result, the maximum vesicle concentration in the medium could be set at 200 μg / mL in cell culture experiments.

[0080] Figure 1 shows an image of an ultracentrifuge tube containing 20Kp after ultracentrifugation at a centrifugal force of 20,000 x g. Figure 2 shows an image of an ultracentrifuge tube containing 200Kp after ultracentrifugation at a centrifugal force of 200,000 x g.

[0081] As shown in Figure 1, precipitate 3, a 20Kp vesicle fraction, was observed at the bottom of ultracentrifuge tube 1 after ultracentrifugation at 20,000 x g. Furthermore, as shown in Figure 2, precipitate 4, a 200Kp vesicle fraction, was observed at the bottom of ultracentrifuge tube 2 after ultracentrifugation. The top row of Table 2 below shows the RNA and protein concentrations in the solution obtained by suspending precipitate 3 in ultracentrifuge tube 1 shown in Figure 1 in 1 mL of PBS. The bottom row of Table 2 shows the RNA and protein concentrations in the solution obtained by suspending precipitate 4 in ultracentrifuge tube 2 shown in Figure 1 in 200 μL of PBS.

[0082] [Table 2]

[0083] The vesicles obtained as precipitate 3 in ultracentrifuge tube 1 contained 17.3 mg of RNA per gram of protein contained in the vesicles. The vesicles obtained as precipitate 4 in ultracentrifuge tube 2 contained 57.3 mg of RNA per gram of protein contained in the vesicles.

[0084] Figure 3 is a graph showing the size distribution of 20Kp vesicles. Figure 4 is a graph showing the size distribution of 200Kp vesicles. As shown in Figures 3 and 4, the 200Kp vesicles tended to have smaller particle sizes than the 20Kp vesicles. The average particle size of the 20Kp vesicles was 305.1 nm, while the average particle size of the 200Kp vesicles was 146.8 nm.

[0085] (Evaluation results of cytotoxicity at high concentrations) Figure 5 is a graph showing the results of evaluating the cytotoxicity of vesicles in each group where different amounts of vesicles were added to the medium within a relatively high concentration range. In Figure 5 and subsequent figures, two asterisks (**) indicate a p-value of less than 0.01. In Figure 5 and subsequent figures, the values ​​shown in units of μg / mL on the horizontal axis indicate the concentration of protein derived from vesicles added to the medium of a macrophage-like cell line. In other words, these concentrations indirectly indicate the amount of vesicles added to the medium.

[0086] As shown in Figure 5, when 200Kp vesicles were added to the culture medium of a macrophage-like cell line, no significant difference in cell number was observed in any of the groups with vesicle-derived protein concentrations ranging from 25 to 200 μg / mL compared to the group with LPS but no vesicles (the second group from the left in Figure 5). In other words, no cytotoxicity of the vesicles was observed in any of the groups with different vesicle-derived protein concentrations (and therefore different amounts of vesicles added).

[0087] On the other hand, when 20Kp vesicles were added to the culture medium of a macrophage-like cell line, the group with a vesicle-derived protein concentration of 200 μg / mL had a significantly lower cell number (i.e., cytotoxicity was observed) than the group with LPS added but no vesicles added (the second group from the left in Figure 5). However, no significant difference in cell number was observed in the groups with vesicle-derived protein concentrations of 25 to 100 μg / mL.

[0088] (Anti-inflammatory activity evaluation results in the presence of high concentrations of vesicles) Figure 6 is a graph showing the nitric oxide concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range. In Figure 6, "μM" on the vertical axis means μmol / L.

[0089] As shown in Figure 6, it is easy to see that the amount of nitric oxide produced was suppressed in a protein concentration (i.e., vesicle amount)-dependent manner when 20Kp vesicles or 200Kp vesicles were added to the medium. Furthermore, in both the 20Kp and 200Kp groups, even the group with the lowest vesicle-derived protein concentration of 25 μg / mL showed significantly lower nitric oxide production than the group with LPS and no vesicle addition. These results revealed that the higher the concentration of vesicles isolated from fermented soybean foods (i.e., the greater the amount of vesicles consumed), the more effectively they were able to suppress nitric oxide production.

[0090] FIG. 7 is a graph showing the IL-1β concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range.

[0091] As shown in Figure 7, when 20Kp vesicles were added to the medium, the IL-1β production (concentration) was significantly reduced in the groups with vesicle-derived protein concentrations of 50-200 μg / mL compared to the group with LPS and no vesicles added. However, no significant difference was observed between the group with LPS and no vesicles added in the group with vesicle-derived protein concentration of 25 μg / mL.

[0092] On the other hand, when 200Kp vesicles were added to the culture medium, no significant difference was observed between the groups with vesicle-derived protein concentrations of 25-100μg / mL and the groups with and without LPS added, but the group with vesicle-derived protein concentrations of 200μg / mL showed a significant decrease in the amount (concentration) of IL-1β produced. These results demonstrated that vesicles isolated from fermented soybeans can suppress the production of IL-1β.

[0093] FIG. 8 is a graph showing the IL-6 concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range.

[0094] As shown in Figure 8, in both cases where 20Kp vesicles and 200Kp vesicles were added to the culture medium, the amount (concentration) of IL-6 production was significantly reduced in each group where the vesicle-derived protein concentration was 25 to 200 μg / mL compared to the group where LPS was added and no vesicles were added. These results demonstrated that vesicles isolated from fermented soybeans can suppress the production of IL-6.

[0095] FIG. 9 is a graph showing the TNF-α concentration in the medium for each group in which different amounts of vesicles were added to the medium within a relatively high concentration range.

[0096] As shown in Figure 9, in both cases where 20Kp vesicles and 200Kp vesicles were added to the culture medium, the amount (concentration) of TNF-α production was significantly reduced in each group where the vesicle-derived protein concentration was 25 to 200 μg / mL compared to the group where LPS was added and no vesicles were added. These results demonstrated that vesicles isolated from fermented soybeans can suppress the production of TNF-α.

[0097] (Evaluation results of cytotoxicity in the presence of low concentrations of vesicles) Figure 10 shows the results of evaluating the cytotoxicity of vesicles in each group where different amounts of vesicles were added to the medium within a relatively low concentration range. The figures after Figure 10 show data for cells to which 200Kp vesicles were added.

[0098] As shown in Figure 10, the cell counts in the groups with vesicle-derived protein concentrations of 1.56 to 12.5 μg / mL were not significantly different from the cell counts in the group with LPS and no vesicle addition, indicating that vesicles at low concentrations were not cytotoxic.

[0099] (Measurement of nitric oxide production in the presence of low concentrations of vesicles) FIG. 11 is a graph showing the concentration of nitric oxide in the medium for each group in which different amounts of vesicles were added to the medium within a relatively low concentration range.

[0100] As shown in Figure 11, in all groups where the vesicle-derived protein concentration was 1.56 to 12.5 μg / mL, the amount of nitric oxide produced was significantly lower than the number of cells in the group with added LPS but no added vesicles.

[0101] In addition, it was confirmed that the amount of nitric oxide produced was suppressed in a manner dependent on the concentration of vesicle-derived protein (i.e., the amount of vesicles added). These results revealed that even small amounts of vesicles isolated from fermented soybeans can suppress the production of nitric oxide, and that the higher the vesicle concentration (i.e., the more vesicles are consumed), the greater the suppression of nitric oxide production.

[0102] (Measurement of inflammatory cytokine expression levels in the presence of low concentrations of vesicles) FIG. 12 is a graph showing the expression level of IL-1β (transcription level of IL-1β mRNA) in each group where different amounts of vesicles were added to the medium within a relatively low concentration range.

[0103] As shown in Figure 12, in all groups where the vesicle-derived protein concentration was 1.56 to 12.5 μg / mL, the expression level of IL-1β was significantly and remarkably lower than in the group where LPS was added but no vesicles were added. These results revealed that vesicles isolated from fermented soybean foods can effectively suppress the expression (production) of IL-1β, even in very small amounts.

[0104] FIG. 13 is a graph showing the expression level of IL-6 (transcription level of IL-6 mRNA) in each group in which different amounts of vesicles were added to the medium within a relatively low concentration range.

[0105] As shown in Figure 13, in all groups where the vesicle-derived protein concentration was 1.56 to 12.5 μg / mL, the expression level of IL-6 was significantly and remarkably lower than in the group to which LPS was added but no vesicles were added. These results demonstrate that even small amounts of vesicles isolated from fermented soybeans can effectively suppress the expression (production) of IL-6.

[0106] Table 3 below shows the inhibitory effect on nitric oxide production, the inhibitory effect on IL-1β production, and the inhibitory effect on IL-1β mRNA transcription corresponding to each amount of vesicles added (protein concentration derived from vesicles) shown in Figures 6, 7, 11, and 12 above, as well as the RNA concentration derived from vesicles in the culture medium.

[0107] [Table 3]

[0108] *In Table 3, a double circle indicates a significant difference compared to the group with added LPS and no vesicles, a simple circle indicates no significant difference, and a hyphen (-) indicates that no data was obtained.

[0109] As shown by the double circles in Table 3, even at extremely low concentrations of RNA, 0.027 μg / mL for 20Kp and 0.089 μg / mL for 200Kp, an inhibitory effect on IL-1β production (inhibitory effect on IL-1β mRNA transcription) and nitric oxide production was observed.

[0110] As described in the above-mentioned non-patent document 1, it is generally believed that the RNA contained in vesicles may be a functional component, and based on the results shown in Table 3, it is expected that even small amounts of vesicles isolated from fermented soybean foods can be ingested to have an inhibitory effect on IL-1β production and nitric oxide production (and thus an effect of improving and preventing diseases involving IL-1β and diseases involving nitric oxide). [Industrial Applicability]

[0111] According to the present invention, an oral composition containing vesicles isolated from fermented soybean food exerts anti-inflammatory effects such as an inhibitory effect on IL-1β production, and therefore has industrial applicability. [Explanation of symbols]

[0112] 1,2...Ultracentrifuge tube, 3,4...Precipitate of vesicle fraction

Claims

1. It contains vesicles isolated from a food product obtained by fermenting soybeans using Bacillus bacteria, An oral composition for use in one or more applications selected from the group consisting of suppressing interleukin-1β production, ameliorating diseases associated with interleukin-1β, and preventing diseases associated with interleukin-1β.

2. The oral composition according to claim 1 , wherein the Bacillus bacterium is Bacillus subtilis.

3. The oral composition according to claim 2, wherein the Bacillus bacterium is Bacillus subtilis var. natto.

4. 2. The oral composition according to claim 1, wherein the food is natto, and the vesicles are obtained as a precipitate by ultracentrifugation after grinding the natto.

5. The oral composition according to claim 4, wherein the ultracentrifugation method is a centrifugation treatment carried out at a centrifugal force of 10,000 to 500,000 x g.

6. 5. The oral composition of claim 4, wherein the vesicles contain at least 0.1 mg of RNA per gram of protein contained in the vesicles.

7. The oral composition according to any one of claims 1 to 6, wherein the disease associated with interleukin-1β is a disease selected from the group consisting of coronary artery disease, alcoholic hepatitis, non-alcoholic hepatitis, and periodontal disease.

8. The oral composition according to any one of claims 1 to 6, wherein the interleukin-1β is interleukin-1β produced by macrophages.

9. The oral composition according to any one of claims 1 to 6, which is also used to inhibit nitric oxide production.

10. The oral composition according to any one of claims 1 to 6, which is also for the prevention of diseases in which nitric oxide is involved.

11. The oral composition of claim 10, wherein the disease involving nitric oxide is a disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, and sepsis.

12. The oral composition according to any one of claims 1 to 6, which is also used to inhibit the production of interleukin-6 and tumor necrosis factor α.

13. The oral composition according to any one of claims 1 to 6, which is a food composition.

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