WO2019146669A1

WO2019146669A1 - mTOR INHIBITOR

Info

Publication number: WO2019146669A1
Application number: PCT/JP2019/002187
Authority: WO
Inventors: 博井ノ岡; 颯板谷; 礼訓八巻; 暢章奥村; 倫世結石
Original assignee: 株式会社山田養蜂場本社
Priority date: 2018-01-26
Filing date: 2019-01-24
Publication date: 2019-08-01
Also published as: TW201932128A; JPWO2019146669A1; CN111655226A; JP7256542B2

Abstract

Disclosed are: a composition comprising royal jelly and pomegranate pericarp or an extract thereof, said composition being a cosmetic, a food, a beverage, a drug or a quasi-drug; a composition comprising propolis and pomegranate or an extract thereof, said composition being a cosmetic, a food, a beverage, a drug or a quasi-drug; an mTOR inhibitor comprising royal jelly and/or propolis and pomegranate or an extract thereof; and a composition against aging, against fatigue, against stress, for improving metabolism, for dieting, for humidifying, for improving skin firmness or glossiness, for preventing cognitive impairment, for immunosuppression, against allergy, for preventing cancer or for inhibiting cancer progress, said composition comprising royal jelly and/or propolis and pomegranate or an extract thereof.

Description

mTOR inhibitor

The present invention relates to a composition having an mTOR inhibitory action. Furthermore, the present invention relates to mTOR inhibitors, anti-aging, anti-fatigue, anti-stress, metabolic improvement, diet, moisturizing, skin amelioration or gloss improvement, cognitive function deterioration prevention, immunosuppression The present invention relates to a composition for anti-allergy, for preventing cancer, or for suppressing the progression of cancer.

mTOR (mechanistic (mammalian) target of rapamycin) is a type of protein kinase involved in intracellular signal transduction in animals such as mammals, and important for protein translation, autophagy, immunoregulation, cytoskeleton regulation, gene transcription, etc. It works well and is deeply involved in fundamental life phenomena such as cell growth, division and death. mTOR is a protein found as an intracellular target of rapamycin that exhibits strong immunosuppressive action and anticancer action, and has been revealed to be a large serine-threonine kinase with a molecular weight of 290,000.

The difference in the interaction with rapamycin revealed that multiple proteins form two complexes, mTORC1 and mTORC2. mTORC1 and mTORC2 regulate multiple in vivo reactions, and in particular, mTORC1 plays an important role in life maintenance, such as biosynthesis regulation in ribosomes, biosynthesis of proteins as well as nucleic acids and lipids. . Rapamycin, which mainly controls mTORC1, has been scientifically confirmed to be effective in prolonging life in many organisms from nematodes to rodents, and clinical trials for life extension have been conducted in primates including humans. It is done. Furthermore, autophagy (autophagy) enhancement to recycle intracellular organs, browning of adipocytes to enhance metabolism, suppression of cognitive function decline such as reduction of β-amyloid deposition, immunosuppression such as suppression of inflammatory cytokine secretion, etc. It has various functions such as growth suppression of various cancer cells (Non-patent documents 1 and 2).

Recently, it was found that the inhibition of mTORC1 increases the production of filaggrin, a moisturizing regulator, through increased expression of cathepsin H. From this, by inhibiting mTOR, effects such as anti-skin aging and moisturizing can be expected (Non-Patent Document 3).

Pomegranate is the oldest cultivated fruit tree in the world, and there are many seeds in the fruit, and some kinds of clothing are consumed around the seeds. Pomegranate is eaten raw as well as juice and syrup. Patent Documents 1 to 3 report various effects of such pomegranate.

Patent Document 1 reports an antiaging agent containing coenzyme Q10 and a pomegranate compound. As described above, it is described that by using coenzyme Q10 in combination with a pomegranate compound, it is possible to obtain a synergistic effect of suppressing aging and to prevent quality deterioration.

In Patent Document 2, a composition for external use on the skin is reported using a compound herbal extract composed of stone peels, apricot kernel, yokjin kernel, three white herbs and shiso leaf as an active ingredient. And, the compound herbal extract is described to have skin moisturizing, strengthening of skin barrier function, and differentiation induction effect of skin keratinocytes.

Patent Document 3 reports a composition for preventing skin aging, which contains an ultraviolet ray damage inhibitor (such as loquat extract) and a near infrared ray damage inhibitor (such as pomegranate extract). It is described that the combination of the ultraviolet ray damage inhibitor and the near infrared ray damage inhibitor can make a significant contribution to the prevention of skin aging.

Japanese Patent Laid-Open Publication No. 2015-17081 Japanese Patent Application Publication No. 2011-505353 Japanese Unexamined Patent Publication No. 2015-86183

An object of the present invention is to provide a composition having excellent mTOR inhibitory activity. Furthermore, the present invention relates to mTOR inhibitors, anti-aging, anti-fatigue, anti-stress, metabolic improvement, diet, moisturizing, skin amelioration or gloss improvement, cognitive function deterioration prevention, immunosuppression It is an object of the present invention to provide a composition for anti-allergy, for preventing cancer, or for suppressing the progression of cancer.

As a result of intensive studies to achieve the above object, the present inventors added pomegranate material to royal jelly or propolis, and added each activity at least about 3 to 4 times stronger than the (additive average) activity. It has been found that mTOR inhibitory activity can be obtained, that is, synergistic effects can be obtained.

The present invention has been completed based on these findings and has been repeatedly studied, and provides the following composition, an mTOR inhibitor and the like.

Item 1. Cosmetics, food and drink, medicines, or quasi-drugs,
A composition comprising royal jelly and pomegranate peel or an extract thereof.
Item 2. Cosmetics, food and drink, medicines, or quasi-drugs,
A composition comprising propolis and pomegranate or an extract thereof.
Item 3. Royal jelly and / or propolis, and an mTOR inhibitor comprising pomegranate or an extract thereof.
Item 4. Containing royal jelly and / or propolis, and pomegranate or its extract, anti-aging, anti-fatigue, anti-stress, metabolic improvement, diet, moisturizing, skin amelioration or gloss improvement, cognitive function decline prevention Composition for immunosuppression, anti-allergy, cancer prevention, or cancer progression suppression.
Item 5. Item 5. The composition according to item 4, which is a cosmetic, food and drink, medicine, or quasi-drug.
Item 6. Item 6. The composition according to Item 2, 4 or 5, wherein the pomegranate is pomegranate peel, the mTOR inhibitor according to Item 3.
Item 7. Item 7. The composition according to Item 1, 4, 5, or 6, wherein the royal jelly is an enzyme-treated royal jelly, or the mTOR inhibitor according to Item 3 or 6.
Item 8. A composition for inhibiting mTOR comprising royal jelly and / or propolis, and pomegranate or an extract thereof.
Item 9. A method of inhibiting mTOR, comprising the step of administering an effective amount of royal jelly and / or propolis, and pomegranate or an extract thereof to a mammal.
Item 10. Aging suppression method, fatigue improvement method, stress improvement method, metabolism improvement method, diet method, moisturizing method, skin moisturizing method comprising a step of administering to a mammal an effective amount of royal jelly and / or propolis and pomegranate or an extract thereof Or the gloss improvement method, the cognitive function fall prevention method, the immunosuppression method, the antiallergic method, the cancer prevention method, or the cancer progression suppression method.
Item 11. Use of a composition comprising royal jelly and / or propolis, and pomegranate or an extract thereof in the manufacture of mTOR inhibitors.
Item 12. For anti-aging, anti-fatigue, anti-stress, for metabolic improvement, for dieting, for moisturizing, for improving skin agglutination or gloss, for preventing cognitive decline, for immune suppression, for anti-allergy, for cancer prevention, or Use of a composition containing royal jelly and / or propolis, and pomegranate or an extract thereof in the manufacture of a composition for suppressing cancer progression.
Item 13. Use of a composition comprising royal jelly and / or propolis as a mTOR inhibitor, and pomegranate or an extract thereof.
Item 14. Anti-aging agent, anti-fatigue agent, anti-stress agent, metabolism improving agent, diet agent, moisturizing agent, skin agglutination or gloss improving agent, cognitive function decrease preventive agent, immunosuppressant, antiallergic agent, cancer preventive agent, or Use of a composition containing royal jelly and / or propolis, and pomegranate or an extract thereof as a cancer progression inhibitor.
Item 15. The composition according to item 8, the method according to item 9 or 10, or the use according to any one of items 11 to 14, wherein the pomegranate is pomegranate peel.
Item 16. Item 16. The composition according to item 8 or 15, the method according to item 9, 10 or 15, and the use according to any one of items 11 to 15, wherein the royal jelly is an enzyme-treated royal jelly.

The composition of the present invention has excellent mTOR inhibitory activity due to the synergistic effect of royal jelly or propolis and pomegranate or its extract. Therefore, the combination of royal jelly or propolis with pomegranate or its extract is an mTOR inhibitor, and for anti-aging, anti-fatigue, anti-stress, metabolic improvement, diet, moisturizing, skin amelioration or gloss improvement It is useful as a component of a composition for preventing cognitive function deterioration, for immunosuppression, for anti-allergy, for cancer prevention, or for suppressing cancer progression.

In addition, extracts of royal jelly, propolis, and pomegranate are highly safe because they are components of natural origin.

It is a photograph which shows the result of the western blotting of a test example.

Hereinafter, the present invention will be described in detail.

In the present specification, "comprise" includes the meaning of "essentially consist of" and the meaning of "consist of".

The compositions and mTOR inhibitors according to the invention are characterized in that they comprise royal jelly and / or propolis and pomegranate or an extract thereof.

(Royal jelly)
Royal jelly is a milky white jelly-like substance formed by mixing secretions which are released from the hypopharyngeal gland and the major glands by working bees of 3 to 12 days among bees. The main physiologically active ingredients in royal jelly include, for example, organic acids such as 10-hydroxydecenoic acid (hereinafter referred to as "decenic acid") unique to royal jelly, proteins, lipids, saccharides, vitamin Bs, Vitamins such as folic acid, nicotinic acid and pantothenic acid, various minerals, and the like can be mentioned. The royal jelly in the present invention includes raw royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, royal jelly extract, royal jelly fermented product and the like. The royal jelly may be produced in any of European countries, Oceania countries, the United States, Brazil, Japan, China, and other Asian countries.

The dried royal jelly powder is obtained by drying and pulverizing raw royal jelly.

Enzyme-treated royal jelly is obtained by treating royal jelly with a protease (protease). Preferably, it is a hypoallergenic enzyme-treated royal jelly in which an allergic reaction caused by a protein contained in royal jelly is suppressed by protease treatment. Therefore, the enzyme-treated royal jelly may contain, in addition to the protease decomposition product of the protein contained in royal jelly, organic acids such as decenoic acid described above, lipids, saccharides, vitamins, and various minerals.

The royal jelly used for producing the enzyme-treated royal jelly is not limited and includes, for example, fresh royal jelly, royal jelly powder obtained by drying and powdered fresh royal jelly, or one obtained by extracting fresh royal jelly with water or water-containing ethanol or the like.

The enzyme-degraded royal jelly can be produced by treating a royal jelly raw material with at least an enzyme having endopeptidase activity, an enzyme having at least exopeptidase activity, and / or an enzyme having endopeptidase activity and exopeptidase activity.

As a proteolytic enzyme having at least endopeptidase activity, animal origin (for example, trypsin, chymotrypsin etc.), plant origin (eg papain etc.), microorganism origin (eg lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycete etc.) Endopeptidase and the like.

As a proteolytic enzyme having at least exopeptidase activity, carboxypeptidase, aminopeptidase, exopeptidase of microorganism origin (eg, lactic acid bacteria, Aspergillus genus bacteria, Rhizopus genus bacteria etc.), pancreatin, pepsin etc having endopeptidase activity also Can be mentioned.

Among these various enzymes, preferred examples of enzymes having both exopeptidase activity and endopeptidase activity include Streptomyces griseus-produced peptidase (trade name: Actinase AS), Aspergillus oryzae (Aspergillus) oryzae) The produced peptidase (trade name: Protease A, flavorzyme), Aspergillus melleus produced peptidase (trade name: Protease P) is also a preferred example of an enzyme having exoprotease activity. Aspergillus oryzae) Production peptidases (trade names: Umamizyme G, Promod 192P, Promod 194P, Sumizyme FLAP), Aspergillus sojae producing peptidases (trade name: Sternzyme B15024), Aspergillus sp. Hydrolase P), Rhizopus oryzae (Rhizopus oryzae) produce peptidase (trade name: peptidase R) can be mentioned. Furthermore, as a preferable example of an enzyme having endoprotease activity, Bacillus subtilis-produced peptidase (trade name: Orientase 22BF, nucleicin), Bacillus licheniformis-produced peptidase (trade name: Alcalase) ), Bacillus stearothermophilus produced peptidase (trade name: Protease S), Bacillus amyloliquefaciens produced peptidase (trade name: Neutrase), Bacillus genus produced peptidase (trade name) : Protamex) can be mentioned.

The enzyme treatment for reducing the allergenicity of royal jelly can be carried out according to, for example, the descriptions of JP-A-2007-295919 and JP-A-2007-295920.

The royal jelly extract is obtained by extracting royal jelly (including fresh, dried and ground products) with water or water-containing ethanol or the like.

The royal jelly fermented product can be produced by a conventional method using microorganisms such as yeast and lactic acid bacteria.

(Propolis)
Propolis is a resinous or waxy substance that constitutes the nest wall of a bee nest. In the present invention, propolis may be derived from any origin and plant, for example, Brazil, China, European countries, Oceania, America and the like. Therefore, any propolis such as alexrin, super green, eucalyptus, ultra green, etc. can be used.

The propolis in the present invention includes, for example, raw propolis, crushed product thereof, supercritical extract, extract with water or hydrophilic organic solvent, powder obtained by pulverizing the extract, granular form obtained by granulating the powder, etc. Is included. Among them, the hydrophilic organic solvent extract is preferable because the active ingredient of propolis is efficiently extracted in a short time and in a well-balanced manner, and large-scale equipment is not necessary when scaling up.

Examples of the hydrophilic organic solvent extract include lower alcohols such as ethanol, methanol and propanol, and ethanol is particularly preferable. For the extraction, propolis block, alcohol-washed propolis block, etc. can be used, and extraction efficiency can be improved by using a propolis block cut or crushed to a suitable size.

(Pomegranate or its extract)
Pomegranate (Sekigan) is a scientific name Punica granatum (Punica granatum L.), and is a deciduous tall tree belonging to the pomegranate. The site of the pomegranate to be used is not particularly limited, and includes, for example, a skin, a seed, a flesh, a leaf, a branch, a bark, a trunk, a stem, a flower, a root, a root bark and the like . Above all, it is desirable to use pomegranate peel. As a component contained in pomegranate peel, ellagic acid etc. are mentioned.

As the pomegranate, for example, those in a non-dried raw state, those in a dried state (by sun-drying etc.), those in a heat-dried state, etc. can be used. In addition, pomegranate can be used as a raw material in the original uncut or crushed state, and sliced or crushed can also be used as a raw material.

Pomegranate extract can be used in the present invention, and the extract may be extracted from pomegranate by any method. The method for obtaining the extract of pomegranate is not particularly limited, and examples thereof include a method of extraction using a polar solvent, and a method of extraction using supercritical extraction with carbon dioxide and the like. Extracts of pomegranate (especially pomegranate peel) may contain ellagic acid.

In the case of extraction with a polar solvent, examples of the extraction solvent to be used include water, lower alcohol (methanol, ethanol, n-propanol, isopropanol etc.), ketones (acetone, methyl ethyl ketone, methyl butyl ketone etc.), esters (acetic acid) Methyl, ethyl acetate and the like, glycols (ethylene glycol, propylene glycol and the like), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, dimethylsulfoxide and the like can be mentioned.

These extraction solvents may be used alone or in combination of two or more as a mixture. Preferably, water, lower alcohol (preferably ethanol) or a mixture thereof (hydroalcohol, preferably hydroethanol) is used. When a water-containing alcohol (preferably water-containing ethanol) is used as the extraction solvent, the alcohol concentration is preferably, but not limited to, 20 to 90% by volume, particularly 40 to 80% by volume.

Extraction is performed by adding part or all of pomegranate to a solvent, extracting from immersion for 30 minutes to about 3 days at 0 ° C. to a temperature at which the solvent boils, usually at a temperature of 100 ° C. or less, and then insoluble matter The removal can be easily performed. The solvent extract thus obtained can be used as it is, and if necessary, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography Purification may be further performed by (HPLC), dialysis methods, combinations thereof and the like. The solvent extract can also be concentrated and subjected to drying treatment such as lyophilization and spray drying. Here, concentration, lyophilization and spray drying can be performed according to a conventional method.

The ratio of the pomegranate or its extract contained in the composition of the present invention and the mTOR inhibitor is not particularly limited as long as the effects of the present invention can be obtained, and can be appropriately adjusted according to the final form etc. It is possible to suitably select from the range of preferably 1 to 80% by mass, more preferably 2 to 50% by mass.

In the composition of the present invention and the mTOR inhibitor, royal jelly is preferably 0.01 to 100 parts by mass, more preferably 0.1 to 70 parts by mass, still more preferably 1 to 10 parts by mass with respect to 1 part by mass of pomegranate or its extract. 50 parts by mass are included.

In the composition of the present invention and the mTOR inhibitor, propolis is preferably 0.01 to 100 parts by mass, more preferably 0.1 to 70 parts by mass, still more preferably 1 to 50 parts by mass with respect to 1 part by mass of pomegranate or its extract. 50 parts by mass are included.

In the composition of the present invention and the mTOR inhibitor, known components other than the above can be appropriately blended, as long as the effects of the present invention are not impaired.

The composition and the mTOR inhibitor of the present invention are cosmetic, food and drink (especially food and drink for health, health maintenance, promotion etc. (eg, health food, functional food, nutrition composition, nutrition) It can be used as a supplement, a supplement, a health food, a food for specified health use, a nutritive function food, or a functional indication food), a quasi-drug, a medicine and the like. In addition, the composition of the present invention and the mTOR inhibitor have mTOR inhibitory action, anti-aging action, anti-fatigue action, anti-stress action, metabolic improvement action, diet action, moisturizing action, skin ache or gloss improving action, cognitive function decline The meaning of additives for imparting a preventive action, an immunosuppressive action, an antiallergic action, a cancer preventive action, and a cancer progression suppressive action is also included.

In the above-mentioned cosmetics, in addition to the above-mentioned royal jelly, propolis and pomegranate or extracts thereof, ingredients usually used for cosmetics, for example, skin lightening agents, moisturizers, antioxidants, oil components, UV absorbers, surfactants, Adhesives, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as needed.

The cosmetics include all cosmetics applied to the skin of animals (including humans), mucous membranes, body hair, hair, scalp, nails, teeth, skins, lips and the like.

There are a wide range of cosmetic formulations, including aqueous solutions, solubilized, emulsifying, powder, oil-based, gel-based, ointment-based, aerosol-based, water-oil two-layer, water-oil-powder three-layer, etc. It can take a dosage form.

The use of cosmetics is also arbitrary, and for example, in the case of basic cosmetics, there may be mentioned face wash, lotion, milk, cream, gel, essence, essence, pack, mask etc., and in the case of makeup cosmetics, foundation, Lipsticks, blushers, eye shadows, eyeliners, mascaras, etc. may be mentioned, and in the case of nail cosmetics, manicures, base coats, top coats, light removal liquids etc. may be mentioned, others, cleansers, dentures or dentifrices , Mouthwash, Massage agent, Cleansing agent, After shave lotion, Pre shave lotion, Shaving cream, Body soap, Soap, Shampoo, Rinse, Hair treatment, Hair styling agent, Hair tonic agent, Hair restorer, Antiperspirant, Bath agent etc. Can be mentioned.

For the above food and drink, the above royal jelly, propolis and pomegranate or an extract thereof can be used as it is, and, if necessary, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential Fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, flavors, stabilizers, preservatives, sustained release modifiers, surfactants, solubilizers, wetting agents, etc. it can.

Food and drink include any food and drink that animals (including humans) can consume. The types of food and drink are not particularly limited, and, for example, dairy products; fermented foods (yogurt, royal jelly, etc.); beverages (coffee, juice, soft drinks such as tea beverages, milk drinks, lactic acid bacteria drinks, lactic acid bacteria-containing drinks, Yogurt drink, carbonated drink, sake, liquor, liquor such as fruit liquor) spreads (custard cream etc); pastes (fruit paste etc); Western confectionery (chocolate, donut, pie, shoe cream, gum, jelly, Candy, cookies, cakes, puddings, etc .; Japanese confectionery (Daifuku, rice cakes, buns, castellas, antels, lambs etc.); Ice confectionery (ice cream, ice candy, sherbet etc.); Foods (curry, beef porridge, risotto, Miso soup, soup, meat sauce, pasta, pickles, jam etc); seasonings (dressing, sprinkle, umami seasoning, soup ingredients etc) It is below.

The manufacturing method of the food-drinks of this invention is not specifically limited, either, It can follow the well-known method suitably.

The dosage unit form for use as a supplement is not particularly limited and can be appropriately selected, and examples thereof include tablets, capsules, granules, solutions, powders and the like.

The intake amount of the food and drink of the present invention can be appropriately set according to various conditions such as the weight, age, sex and symptoms of the user.

For the above-mentioned pharmaceutical products, only the above-mentioned royal jelly, propolis and pomegranate or their extract can be used, or they can be used in combination with other pharmaceutical ingredients described in Japanese Pharmacopoeia such as vitamins and herbal medicines.

When the composition of the present invention and the mTOR inhibitor are prepared as a medicament, the above royal jelly, propolis and pomegranate or an extract thereof is used together with a nontoxic carrier, diluent or excipient acceptable for Tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc., capsules, pills, powders (powders), fine granules, granules, solutions, suspensions, emulsions, It is possible to prepare in the form of syrup, paste and the like, for example, for preparation of pharmaceutical (for oral).

The dose of the pharmaceutical can be determined appropriately according to various conditions such as the weight, age, sex and symptoms of the patient.

The pharmaceuticals and cosmetics of the present invention also include quasi drugs.

The composition of the present invention and the mTOR inhibitor described above apply to mammals (preferably humans) including humans.

The composition of the present invention and the mTOR inhibitor have an excellent mTOR inhibitory action due to the synergistic effect of royal jelly or propolis and pomegranate or its extract.

It has been reported that as a result of mTOR inhibition, lifespan prolonging action, autophagy enhancement, white fat cell browning, skin moisturizing action, cognitive function improving action, immunosuppressive action, anticancer action and the like are derived. Anti-fatigue action, anti-stress action and the like as action based on autophagy enhancement, metabolism improvement action as the action based on browning of white fat cells, diet action and the like, and skin moistness, firmness or gloss as moisturizing action The improvement action etc. are mentioned.

Therefore, the combination of royal jelly or propolis with pomegranate or its extract is an mTOR inhibitor, and for anti-aging, anti-fatigue, anti-stress, metabolic improvement, diet, moisturizing, skin amelioration or gloss improvement It is useful as a component of a composition for preventing cognitive function deterioration, for immunosuppression, for anti-allergy, for cancer prevention, or for suppressing cancer progression.

In addition, the composition of the present invention and the mTOR inhibitor have high safety because they contain a naturally derived component as an active ingredient.

Types of cancer include gastric cancer, colon cancer (rectum cancer, colon cancer), small intestine cancer, liver cancer, pancreas cancer, lung cancer, pharyngeal cancer, esophageal cancer, renal cancer, gallbladder and cholangiocarcinoma, head and neck cancer, bladder cancer Prostate cancer, breast cancer, uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, brain cancer, leukemia, malignant lymphoma, multiple myeloma and the like.

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

Test Example <Material>
Raw royal jelly (non-enzyme treated), enzyme-treated royal jelly and lyophilized powder of propolis ethanol extract were dissolved in DMSO and used for the test. The enzyme-treated royal jelly is obtained by lyophilizing and grinding the ethanol extract of Alexin Propolis bulk (produced in Minas Gerais State, Brazil) by the method described in Example 1 of Patent No. 3994120, and the ethanol extract powder of propolis is obtained by lyophilization The

Pomegranate (A): A pomegranate purchased from the central market of Aracaju City, Sergipe, State of Northeast Brazil, was used. Pomegranate peel was placed in a mill and finely ground, and 25 ml of 80% EtOH (using specific alcohol traceable (95% EtOH)) was added to 5 g of pomegranate peel and shaken overnight with a shaker (room temperature). Then, it centrifuged at 3,000 rpm and 25 degreeC for 15 minutes, filtered the supernatant liquid, and collect | recovered to a 100 ml eggplant flask. Next, ethanol was removed by an evaporator, a small amount of milli-Q was added, and the mixture was frozen at −20 ° C. and lyophilized.

Pomegranate (B): Pomegranate ellagic acid (Pomegranate peel extract) purchased from Sabinsa Japan Corporation was used.

In addition, since an equivalent test result was obtained about pomegranate (A) and pomegranate (B), the result at the time of using pomegranate (B) is shown below.

<Reagent>
・ MTOR: eurofins # 14-770
ULight ^TM -4E-BP1 (Thr37 / 46) Peptide: PerkinElmer # TRF0128-D, # TRF0128-M
-Europium-anti-phospho-4E-BP1 (Thr 37/46) Antibody: PerkinElmer # TRF0216-D, # TRF0216-M
PI-103: Cayman Chemical (CAS 371935-74-9)
・ 10X LANCE Detection Buffer: PerkinElmer # CR97-100
・ Kinase buffer (KB): 50 mM HEPES (pH 7.5), 1 mM EGTA, 3 mM MnCl ₂ , 10 mM MgCl ₂ , 0.01% Tween-20, 2 mM DTT
Primary antibody: 4E-BP1 (T37 / 46) RABBIT-AB: Cell Signaling TECHNOLOGY # 9459S
・ Secondary antibody ・・・ Anti-rabbit Ig6, HRP-linked Antibody: Cell Signaling TECHNOLOGY # 7074S
B-Block-w (Blocking solution): Beacle, Inc. # BCL-BKBW-01

<Experimental method>
Raw royal jelly (raw RJ), enzyme-treated royal jelly (RJ), ethanol extract of propolis (PP), and pomegranate extract powder are dissolved in DMSO, and then the raw material alone and the equivalent mixture of raw RJ, RJ or PP and pomegranate are used. The evaluation of mTOR inhibitory activity in The evaluation of the mTOR inhibitory activity was carried out using the mTOR kinase inhibitory activity by the TR-FRET method and the Western blotting (dot plot) method.

[Test procedure]
· Common Operation KB mTOR 40 nM using, ATP 5 mM, PI-103 (Inhibitor) 40μM, was prepared ^{ULight TM -4E-BP1 (Thr37 /} 46) Peptide 200 nM. Europium-anti-phospho-4E-BP1 (Thr37 / 46) Antibody 80 μM (4 × Detection buffer) was prepared using 10 × LANCE Detection Buffer. PI-103 40 μM was diluted 5-fold by 8-step and used as a positive control (DMSO concentration was standardized to 4%). Royal jelly, propolis and pomegranate (B) were each dissolved in DMSO to be 3 mg / mL. An equivalent mixture of pomegranate (B) and DMSO, pomegranate (B) and royal jelly, pomegranate (B) and propolis, respectively, is prepared 5 times using DMSO, propolis, pomegranate (B) and propolis mixture are 4 stages (15 , 3, 0.6, 0.12 μg / mL), royal jelly, pomegranate (B), pomegranate (B) and royal jelly mixture are diluted in 6 steps (15, 3, 0.6, 0.12, 0.024, 0.0048 μg / mL) and used as test samples did.

Add 2.5 μL of mTOR, 1.25 μL of Inhibitor or test sample, 1.25 μL of ULight-4E-BP1 (Thr37 / 46) Peptide / ATP mix (ATP 80 μM) to MicroPlate (Greiner # 784075), attach Plate Seal and allow to stand at room temperature for 1 hour did. 2.5 μL of Stop Solution (32 mM EDTA in 1 × Detection Buffer) was added and allowed to stand at room temperature for 5 minutes.

-TR-FRET method 2.5 μL of Detection Mix (Eu-anti-phospho-4E-BP1 (Thr37 / 46) Antibody final concentration 2 nM) was added and a Plate Seal was attached, and allowed to stand at room temperature for 1 hour. EnVision ^TM Multilabel Reader of TR-FRET mode (excitation at 320 nm & emission at 665 nm) was measured using a.

Western blotting method: 2 μL / spot was applied to a nitrocellulose membrane (Trans-Blot, BIO-RAD # BR9316044) and air-dried. It was immersed in the blocking solution and reacted at room temperature for 1 hour. It was immersed in the primary antibody diluted 2000-fold with a blocking solution and allowed to stand overnight at 4 ° C. The cells were washed three times with 20-25 mL of PBS-T (0.1% Tween-20) for 5 minutes. It was immersed in the secondary antibody diluted 2000 folds with the blocking solution and left to stand at room temperature for 30 minutes. The cells were washed three times with 20-25 mL of PBS-T (0.1% Tween-20) for 5 minutes.

Chemiluminescent reagent 2 or ^{(Clarity TM Western ECL Substrate, BIO} -RAD # 170-5060) were mixed in equal amounts and then left to stand for 5 minutes and rub on the lap, Lumino image analyzer (LAS-3000 sandwiched hybridization bag It measured using mini, FujiFilm).

<Result>
Raw and enzyme-treated royal jelly showed no mTOR inhibitory activity in the range of 4.8 × 10 ^-3 to 75 μg / mL regardless of the concentration. The 50% inhibitory concentration (IC50) of propolis was 35.1 μg / mL. The IC50 of pomegranate alone was 9.7 μg / mL. When mixing RJ or PP with pomegranate, IC50 of Pomegranate + RJ = 2.9 μg / mL, IC50 of pomegranate + PP = 2.0 μg / mL, 3.3 times the theoretical value when assuming additive effect , Showed 3.7 times stronger inhibitory activity. In addition, the IC50 of pomegranate + raw RJ was 4.05 μg / mL, and showed a 3.4-fold stronger inhibitory activity than the theoretical value when it was assumed to be an additive effect.

In addition, the results of Western blotting are shown in FIG. In western blotting, the higher the mTOR activity, the darker the spots appear. The three side-by-side spots in the color frame are spots of the same condition. At the left end, the results of m-TOR inhibitor PI-103 are shown and compared. Taking a spot at 0.6 μg / mL as an example, compared to the spot with royal jelly, propolis and pomegranate (B) alone, the spot density is thinner in the sample in which the royal jelly or propolis is added to pomegranate, Western blotting The dot plot also confirmed a synergistic and dose dependent inhibition of mTOR.

Claims

Cosmetics, food and drink, medicines, or quasi-drugs,
A composition comprising royal jelly and pomegranate peel or an extract thereof.
Cosmetics, food and drink, medicines, or quasi-drugs,
A composition comprising propolis and pomegranate or an extract thereof.
Royal jelly and / or propolis, and an mTOR inhibitor comprising pomegranate or an extract thereof.
Containing royal jelly and / or propolis, and pomegranate or its extract, anti-aging, anti-fatigue, anti-stress, metabolic improvement, diet, moisturizing, skin amelioration or gloss improvement, cognitive function decline prevention Composition for immunosuppression, anti-allergy, cancer prevention, or cancer progression suppression.
The composition according to claim 4, which is a cosmetic, food and drink, medicine, or quasi-drug.