JP6469394B2 - Apoptosis inhibitor - Google Patents

Description

  The present invention relates to an apoptosis inhibitor comprising a milk-derived component as an active ingredient.

  Exosomes are membrane vesicles with a diameter of 20-100 nm composed of lipid bilayers, such as blood (serum, plasma), breast milk, alveolar lavage fluid, malignant pleural effusion, synovial fluid, urine, amniotic fluid, semen, saliva, etc. Present in body fluids, and contains RNA and proteins. Exosomes are released from cells in vivo and are said to be involved in molecular transport to distant cells.

  For membrane vesicles in milk, membrane vesicles are prepared from milk (colostrum or mature milk), added to Raw 264.7 cells, a rodent macrophage system, and cytokines in the culture supernatant are measured. Although the results suggest that membrane vesicles promote inflammation, it has been reported that there was no significant difference in cell proliferation (Non-patent Document 1).

  By the way, it is known that milk with an increased amount of microRNA (miRNA) present in milk has a high immune enhancing action (Patent Document 1). And it has been reported that this miRNA exists in exosome (nonpatent literature 2).

  Also. It is known that the immune enhancement effect of milk can be enhanced by increasing the abundance of specific messenger RNA (mRNA) contained in milk (Patent Document 2).

  However, it has not been known that exosomes contained in milk or RNA contained therein have an inhibitory effect on apoptosis.

US Patent Publication No. 20120093874 US Patent Publication No. 20130280368

Sun, Q. et al., Protein Cell, 4 (3): 197-210, 2013 Kosaka, N., et al., Silence 1: 7, 2010

  An object of the present invention is to provide a highly safe apoptosis inhibitor.

  The present inventors have found that milk-derived exosomes and RNA contained therein have an action of suppressing cell apoptosis, and have completed the present invention.

That is, the present invention provides an apoptosis inhibitor containing, as an active ingredient, a milk-derived exosome or a fraction of the exosome containing at least RNA.
The present invention is also a method for producing an apoptosis inhibitor,
A step of preparing a whey fraction by removing a lipid fraction, a cell fraction and a casein fraction from milk;
Recovering an exosome fraction from the whey fraction; and
Formulating the exosome fraction or the exosome fraction containing at least RNA as an active ingredient,
A method comprising:

  The apoptosis inhibitor of the present invention has an apoptosis inhibitory action. The apoptosis inhibitor of the present invention uses milk, which is widely used as a food or drink, as a raw material, and is considered to be highly safe as a food or drink or a medicine.

The figure which shows the viable cell number (relative value) of the macrophage-like cell which carried out LPS processing. BSA: bovine serum albumin, Exo: exosome fraction, sonicated: sonicated exosome fraction, RNA: RNA purified from exosome fraction, LF: bovine lactoferrin. “*” And “**” indicate significance in p <0.05 and P <0.01, respectively, in the Dunnett test using the PBS group as a control.

  Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be freely changed within the scope of the present invention.

  The apoptosis inhibitor of the present invention contains milk-derived exosomes or a fraction of milk-derived exosomes containing at least RNA as an active ingredient. That is, the exosome used in the present invention is an exosome obtained from milk. The milk is not particularly limited as long as it is obtained from mammals, and examples thereof include milk obtained from humans, cows, goats, sheep, pigs, monkeys, dogs, cats, rats, mice, hamsters, guinea pigs and the like. Preferably it is milk obtained from humans and cows. The milk may be colostrum or mature milk, but mature milk is preferred.

The method for preparing exosomes from milk is not particularly limited, but an example is described below as a specific method.
A lipid fraction, a cell fraction and a casein fraction are removed from milk to prepare a whey fraction. The exosome fraction is collected from the obtained whey fraction. The exosome fraction is collected by a known method (International Publication No. 2014/030590) such as ultracentrifugation, filter method, density gradient centrifugation, sucrose cushion method, ultrafiltration, continuous flow electrophoresis, and chromatography. Can be used. Specifically, for example, the whey fraction is centrifuged at 30,000 to 1,000,000 × g, for example, 100,000 × g for 60 to 120 minutes, and the supernatant is removed to obtain the exosome fraction. .

  In addition, kits for isolating exosomes from biological fluids such as serum, plasma, urine, and cerebrospinal fluid are commercially available (for example, Takara Bio Exosome Isolation Kit, Life Technology Exosome Isolation Reagent). May be.

  The exosome fraction is sufficient if the exosome content is higher than the raw milk, and preferably the content (% by mass) relative to the total solid content is 2 times or more, more preferably 10 times or more, more preferably 25 times that of the raw milk. It is preferable that it is concentrated above. Alternatively, the exosome-derived RNA concentration contained in the exosome fraction is preferably 2 times or more, more preferably 10 times or more, and even more preferably 25 times or more higher than the exosome-derived RNA concentration in the raw milk. Moreover, the exosome fraction may consist essentially of exosomes or a solution or suspension thereof. The amount or concentration of the exosome can be measured by, for example, the Lowry method, the Bradford method, or a protein concentration quantification method obtained by improving these methods.

  By formulating the obtained exosome or a fraction of the exosome containing at least RNA (hereinafter sometimes simply referred to as “fraction”) as an active ingredient, an apoptosis inhibitor can be obtained. As shown in the Examples, milk-derived exosomes and RNA contained in exosomes have the effect of increasing the survival rate of macrophage-like cells derived from human monocytic leukemia cell lines supplemented with LPS. Macrophages are known to be induced by LPS (for example, Antonios, O. et al., SCIENCE, 285 (30): 736-739, 1999, Xaus, J. et al., Blood, 95 (12): 3823-3831, 2000), macrophage-like cells derived from the monocytic leukemia cell line are also considered to be induced to undergo apoptosis by LPS. Therefore, the above-mentioned effect of increasing the survival rate of cells is considered to be due to the apoptosis-suppressing effect. In addition, as described above, a composition containing milk-derived exosomes or a fraction thereof has an action of increasing cell survival rate, and may be a cell survival rate increasing agent. Moreover, since the increase in cell survival rate is also suppression of cell death, the composition containing the exosome derived from milk or its fraction may also be a cell death inhibitor.

  The effect of the apoptosis inhibitor of the present invention is examined based on the action of milk-derived exosomes and RNA contained in exosomes to increase the survival rate of macrophage-like cells derived from human monocytic leukemia cell lines supplemented with LPS. Is possible. These effects are caused by NADH produced in the mitochondrial respiration process in the cell via an electron transport reagent such as PES or PMS, and MTS (3- (4,5-dimethylthiazol-2-yl) -5- When (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) is reduced, formazan is formed, and the number of viable cells can be measured based on the phenomenon that the solution turns from yellow to brown. .

The exosome used in the present invention may be intact, or a disrupted product in which the membrane vesicle structure is partially or completely destroyed. As shown in the Examples, the apoptosis-suppressing action of milk-derived exosomes (hereinafter sometimes simply referred to as “exosomes”) is also exhibited by RNA extracted from exosomes. Therefore, the active ingredient of the apoptosis inhibitor of the present invention may be an exosome fraction containing at least RNA. The fraction containing RNA includes purified RNA.
Examples of RNA and purified RNA contained in the exosome fraction include micro RNA (miRNA) and messenger RNA (mRNA).

  In formulating the apoptosis inhibitor of the present invention, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, and solvents for injections can be used. Specific preparations include tablets (including sugar-coated tablets, enteric-coated tablets, buccal tablets), powders, capsules (including enteric capsules and soft capsules), granules (including those coated), pills, Illustrative examples include troches, encapsulated liposomes, solutions, and pharmaceutically acceptable sustained release formulations.

  Carriers and excipients used in these formulations include lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, licorice powder, gentian powder and the like as binders Examples thereof include starch, gelatin, syrup, polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, hydroxypropyl cellulose, ethyl cellulose, methyl cellulose, carboxymethyl cellulose and the like.

  Examples of the disintegrant include starch, agar, gelatin powder, sodium carboxymethyl cellulose, carboxymethyl cellulose calcium, crystalline cellulose, calcium carbonate, sodium bicarbonate, and sodium alginate.

Furthermore, as the lubricant, magnesium stearate, hydrogenated vegetable oil, macrogol, etc., and as the colorant, red No. 2, yellow No. 4, and blue No. 1, etc., which are allowed to be added to pharmaceuticals, etc. , Respectively.

  Tablets and granules are sucrose, hydroxypropylcellulose, purified shellac, gelatin, sorbitol, glycerin, ethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, cellulose phthalate acetate, hydroxypropylmethylcellulose phthalate, methyl methacrylate as necessary And a methacrylic acid polymer.

  The application target of the apoptosis inhibitor of the present invention is not particularly limited as long as it is a disease caused by or accompanied by apoptosis, and examples thereof include Parkinson's disease and cancer. These diseases can be treated or prevented by administering an apoptosis inhibitor.

One aspect of the present invention is the use of milk-derived exosomes or fractions thereof in the manufacture of a medicament for suppressing apoptosis. Here, suppression of apoptosis can be replaced with suppression of cell death or increase of cell viability.
Another embodiment of the present invention is a milk-derived exosome used for the treatment or prevention of a disease caused by or accompanied by apoptosis. Another aspect of the present invention is a method for treating or preventing a disease caused by or accompanied by apoptosis, wherein milk-derived exosomes or fractions thereof are administered to animals. Here, apoptosis can be replaced by cell death.

  The apoptosis inhibitor of the present invention may be administered either orally or parenterally, but oral administration is preferred. Parenteral administration includes intravenous injection, rectal administration, inhalation and the like. Examples of the dosage form for oral administration include tablets, capsules, troches, syrups, granules, powders, ointments and the like. Moreover, the medicine and pharmaceutical composition which have the apoptosis inhibitory effect known or discovered in the future can also be used together. The pharmaceutical composition to be used in combination may be contained as one of the active ingredients in the drug of the present invention, or may be combined and commercialized as a separate drug without being contained in the drug of the present invention.

In addition, milk-derived exosomes or fractions thereof can be contained in foods as active ingredients and processed as foods having an apoptosis-inhibiting action as one aspect of apoptosis-inhibiting agents.
Such foods may be in the form of liquids, pastes, solids, powders, etc., in addition to tablet confections, liquid foods, feeds (including for pets), etc., for example, bread, macaroni, spaghetti, noodles, cakes Flour products such as mix, fried flour, bread crumbs, instant noodles, cup noodles, retort / cooked food, cooking canned food, microwave food, instant soup / stew, instant miso soup / soup, canned soup, freeze-dried food, other instant foods Instant foods such as food; Canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed cereals (cereal processed products); canned marine products, fish ham and sausages, fish paste products, Processed marine products such as marine delicacies and simmered fish; livestock canned and pasted products, livestock processed products such as livestock ham and sausage; processed milk and milk drinks Milk and dairy products such as yogurt, lactic acid bacteria beverages, cheese, ice cream, prepared powdered milk, cream and other dairy products; fats and oils such as butter, margarine, vegetable oil; soy sauce, miso, sauces, tomato processing Basic seasonings such as seasonings, mirins, vinegars; mixed seasonings and foods such as cooking mixes, curry ingredients, sauces, dressings, noodle soups, spices, and other complex seasonings; frozen materials Frozen foods such as food, semi-cooked frozen food, cooked frozen food, etc .; caramel, candy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese confectionery, rice confectionery, bean confectionery, dessert confectionery, other confectionery Sweets such as carbonated drinks, natural fruit juices, fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruit granules, vegetable drinks, beans Taste drinks such as milk, soy milk drink, coffee drink, tea drink, powdered drink, concentrated drink, sports drink, nutrition drink, alcoholic drink, other taste drinks; other commercial products such as baby food, sprinkles, tea crush paste Examples include foods and the like; infant formulas; enteral nutrition; functional foods (special health foods, functional nutrition foods) and the like.

  Furthermore, milk-derived exosomes or fractions thereof can be contained in the feed as an active ingredient and processed as a feed having an apoptosis-inhibiting action as one aspect of the apoptosis-inhibiting agent.

  The form of the feed is not particularly limited. For example, milk-derived exosomes, cereals such as corn, wheat, barley, rye, and milo; vegetable oils such as soybean oil meal, rapeseed oil meal, coconut oil meal, and linseed oil meal; , Wheat straw, rice bran, defatted rice bran, etc .; corn gluten meal, corn jam meal, etc .; animal feed such as fish meal, skim milk powder, whey, yellow grease, tallow; torula yeast, brewer's yeast, etc. Yeasts; mineral feeds such as tribasic calcium phosphate and calcium carbonate; fats and oils; simple amino acids; sugars and the like. Examples of the form of the feed include pet food, livestock feed, and fish feed.

  In the apoptosis inhibitor of the present invention (pharmaceuticals, foods and drinks, feeds, quasi drugs, etc.), the exosome content is 0.01% by mass or more based on the final composition of the apoptosis inhibitor. It is preferably 0.1% by mass or more.

  The dosage of the apoptosis inhibitor of the present invention varies depending on the age, symptoms, etc., but is usually 1 mg to 10 g / day, preferably 100 mg to 1 g / day in terms of the amount of exosome-derived RNA, and 1 day per day. The dose may be divided into multiple doses. In addition, when the apoptosis inhibitor of the present invention is ingested or taken, the effects of the present invention are sufficiently exhibited at any timing before, between, and after a meal.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

(1) Preparation of milk-derived exosomes Raw milk used for dairy production is centrifuged at 1,200 xg for 10 minutes at 4 ° C to remove fat and cells contained in raw milk twice, and skim milk Got. To the obtained skim milk, 10% acetic acid was added dropwise to lower the pH to 4.6 to precipitate casein. The skim milk at pH 4.6 was centrifuged at 4,500 × g and 4 ° C. for 30 minutes to remove casein. Subsequently, centrifugation was performed at 20,000 × g and 4 ° C. for 30 minutes, and the operation of collecting the supernatant was performed twice to completely remove casein. The obtained sample was filtered through 0.45 μm and 0.22 μm filters (Millipore) to obtain whey. The obtained whey was subjected to ultracentrifugation at 100,000 × g, 4 ° C. for 90 minutes, the pellet-like mass containing exosomes was suspended in PBS, and exosomes were washed by performing ultracentrifugation in the same manner as described above. The same washing was performed again to obtain an exosome fraction.

(2) Confirmation of apoptosis inhibitory effect THP-1 (ATCC TIB-202), a human monocytic leukemia cell line, is treated with 10% inactivated fetal calf serum (HyClone) (extrasome in serum is removed by ultracentrifugation) ), 1% penicillin-streptomycin-glutamine solution (Gibco), 1% non-essential amino acid mixture (Gibco), 0.1% β-mercaptoethanol-containing medium (complete medium), 50 nM PMA (Phorbol 12-myristate 13-acetate) (Sigma) was added and cultured for 72 hours to differentiate the THP-1 strain into macrophages. A cell suspension obtained by adding 80 μl complete medium to 5 × 10 ⁴ cells, and a test substance solution ((i)) in which test substances (ii) to (vi) below are dissolved in 20 μl of PBS Were mixed with 20 μl of PBS only) to prepare test solutions (100 μl).

(I) PBS
(Ii) 10 μg bovine serum albumin (iii) 10 μg protein equivalent (12 ng RNA equivalent) exosome fraction (iv) 10 μg protein equivalent sonicated fraction * 1
(V) RNA purified from 10 μg exosome fraction in terms of protein (12 ng)
(Vi) 10 μg bovine lactoferrin
* 1: Ultrasonic treatment was performed 5 times for 20 seconds using a Nissei ultrasonic crusher US-300E.

The prepared test solution was cultured at 37 ° C. in a 5% CO ₂ environment for 24 hours, and then the culture supernatant was removed and the cells were washed twice with PBS. After washing, 100 μl of complete medium was added to the cells, and LPS (lipopolysaccharide) (manufactured by Sigma) was added to 100 ng / mL and cultured for 16 hours. After 16 hours, the culture supernatant containing LPS was removed, 100 μl of complete medium was added to the cells, and the cells were incubated at 37 ° C. for 10 minutes. Thereafter, 20 μl of Cell Titer 96 (registered trademark) AQueous Solution reagent (Promega) was added, and after further incubation for 1 hour 30 minutes, the number of viable cells was determined by measuring the absorbance at 492 nm (reduced amount of MTS). .

The results are shown in FIG. Milk-derived exosomes increased the number of surviving cells compared to PBS. In addition, exosomes whose membrane vesicles were disrupted by sonication increased the number of viable cells as well as intact exosomes. In addition, RNA extracted from exosomes increased the number of viable cells to the same level or higher than exosomes. From these results, it was confirmed that the effect of increasing the number of living cells of milk-derived exosomes does not require a membrane structure. It is also suggested that the action is due to RNA.
As described above, apoptosis of macrophage-like cells is considered to be induced by LPS. Therefore, it is considered that the increase in the number of viable cells observed above is due to the apoptosis-inhibiting action.

Claims (8)

An apoptosis inhibitor comprising, as an active ingredient, an exosome derived from bovine milk or a fraction of the exosome containing at least RNA.   The apoptosis inhibitor according to claim 1, wherein the exosome is a disrupted product in which a membrane vesicle structure is partially or completely destroyed.   The apoptosis inhibitor according to claim 2, wherein the crushed material is a sonication product. A method for producing an apoptosis inhibitor,
A step of preparing a whey fraction by removing a lipid fraction, a cell fraction and a casein fraction from bovine milk;
Recovering the exosome fraction from the whey fraction,
Partially or completely destroying the morphology of exosome membrane vesicles contained in the exosome fraction; and
Formulating the exosome fraction or the exosome fraction containing at least RNA as an active ingredient,
Including methods.   The method according to claim 4, wherein the step of partially or completely destroying the morphology of the membrane vesicle is a step of sonicating the exosome fraction. A food / beverage composition for inhibiting apoptosis containing an exosome derived from bovine milk, or a fraction of the exosome containing at least RNA as an active ingredient.   The food composition for inhibiting apoptosis according to claim 6, wherein the exosome is a crushed material in which the membrane vesicle structure is partially or completely destroyed.   The food / beverage product composition for suppressing apoptosis according to claim 7, wherein the crushed material is a sonicated product.

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