JP7623721B2 - mTOR inhibitors - Google Patents

Description

The present invention relates to a composition having an mTOR inhibitory effect. The present invention further relates to an mTOR inhibitor and a composition for use in anti-aging, anti-fatigue, anti-stress, improving metabolism, dieting, moisturizing, improving skin firmness or luster, preventing cognitive decline, immunosuppression, antiallergy, preventing cancer,
Or it relates to a composition for inhibiting the progression of cancer.

Mechanistic (mammalian) target of rapamycin (mTOR) is a type of protein kinase involved in intracellular signal transduction in mammals and other animals. It plays important roles in protein translation, autophagy, immune regulation, cytoskeleton regulation, gene transcription, and other fundamental life phenomena such as cell growth, division, and death. mTOR is a protein with a molecular weight of 29 that has been found to be the intracellular target of rapamycin, which has strong immunosuppressive and anticancer effects.
It was revealed that this is a giant serine-threonine kinase.

Two types of mTORC1 complexes composed of multiple proteins that differ in their interactions with rapamycin
It has been revealed that mTORC1 and mTORC2 each control multiple biological reactions, and mTORC1 in particular plays an important role in maintaining life, including controlling the ribosome biosynthetic system and the biosynthesis of not only proteins but also nucleic acids and lipids.
Rapamycin, which mainly regulates C1, has been scientifically confirmed to be effective in extending lifespan in many organisms, from nematodes to rodents, and clinical trials for extending lifespan are also being conducted in primates, including humans. In addition, it has a variety of functions, such as promoting autophagy (self-eating), which recycles intracellular organelles, browning fat cells by promoting metabolism, suppressing cognitive decline by reducing the deposition of β-amyloid, suppressing the immune system by suppressing the secretion of inflammatory cytokines, and inhibiting the proliferation of various types of cancer cells (Non-Patent Documents 1 and 2).

Recently, it has been found that inhibition of mTORC1 increases the production of filaggrin, a moisturizing regulator, through increased expression of cathepsin H. For this reason, inhibition of mTOR is expected to have anti-skin aging, moisturizing, and other effects (Non-Patent Document 3).

Mozuku (seaweed) is a type of seaweed found along the coasts of Japan, especially in inland bays. It is eaten raw, and in Japan it is mainly used as a vinegared dish. Mozuku is also known to contain a lot of useful components, such as polysaccharides alginic acid and fucoidan.

Regarding the effects of such fucoidan, Patent Document 1 reports that fucoidan derived from Mozuku has the effect of enhancing the action of anticancer drugs and suppressing the effect of anticancer drugs on normal gastric cells.

Propolis is a glue-like substance made by mixing tree sap, plant buds and exudates collected by honeybees with beeswax, and is known to have antibacterial, antioxidant, anti-inflammatory, anti-tumor, and anti-allergic effects.

Regarding such effects of propolis, Patent Document 2 reports that a micellar extract of propolis is useful as a medicine, food or drink for improving or preventing the decline in cognitive function associated with the NMDA (N-methyl-D-aspartate) receptor.

JP 2004-75595 A JP 2018-24654 A

Cold Spring Harb Perspect Med. 2016 6(5): Pii 1-19 Cell Metabolism 23, 2016, 990-1003 J Allergy Clin Immunol. 2017 Apr; 139(4): 1228-1241

An object of the present invention is to provide a composition having an excellent mTOR inhibitory effect.
The present invention aims to provide an mTOR inhibitor and a composition for anti-aging, anti-fatigue, anti-stress, metabolism improvement, dieting, moisturizing, improving skin firmness or luster, preventing cognitive decline, immunosuppression, anti-allergy, cancer prevention, or cancer progression inhibition.

As a result of extensive research to achieve the above-mentioned objective, the inventors have discovered that adding an extract of Mozuku to royal jelly or propolis results in mTOR inhibitory activity that is at least 1.7 times stronger than the sum (arithmetic mean) of the activities of each ingredient, i.e., a synergistic effect is obtained.

The present invention was completed based on these findings and through further investigation, and provides the following compositions, mTOR inhibitors, etc.

Item 1. Cosmetics, food and beverages, medicines, or quasi-drugs.
A composition comprising royal jelly and/or propolis, and mozuku seaweed or an extract thereof.
Item 2. An mTOR inhibitor comprising royal jelly and/or propolis, and mozuku seaweed or an extract thereof.
Item 3. A composition for anti-aging, anti-fatigue, anti-stress, metabolism improvement, dieting, moisturizing, improving skin firmness or luster, preventing cognitive decline, immunosuppression, anti-allergy, cancer prevention, or cancer progression inhibition, comprising royal jelly and/or propolis, and mozuku seaweed or an extract thereof.
Item 4. The composition according to Item 3, which is a cosmetic, a food or drink, a medicine, or a quasi-drug.

The composition of the present invention has excellent mTOR inhibitory activity due to the synergistic effect of royal jelly or propolis and mozuku or its extract.Therefore, the combination of royal jelly or propolis and mozuku or its extract is useful as an mTOR inhibitor and a component of a composition for anti-aging, anti-fatigue, anti-stress, metabolism improvement, diet, moisturizing, improving skin firmness or luster, preventing cognitive decline, immunosuppression, antiallergy, cancer prevention, or cancer progression suppression.

In addition, royal jelly, propolis, and mozuku are naturally derived ingredients and are therefore highly safe.

The following describes in detail the embodiments of the present invention.

In this specification, "comprise" means "essentially consisting of"
It also includes the meaning of "essentially consist of" and the meaning of "consist of only."

The composition and mTOR inhibitor of the present invention are characterized by containing royal jelly and/or propolis, and mozuku seaweed or an extract thereof.

(Royal Jelly)
Royal jelly is a milky white gel-like substance made by mixing secretions from the hypopharyngeal and mandibular glands of 3-12-day-old worker bees. The main physiologically active components in royal jelly include, for example, 10-hydroxy-2-decenoic acid (hereinafter referred to as "10-hydroxy-2-decenoic acid"), which is unique to royal jelly.
In addition to organic acids such as decenoic acid, proteins, lipids, sugars, B vitamins,
Examples of the royal jelly include vitamins such as folic acid, nicotinic acid, and pantothenic acid, and various minerals. The royal jelly of the present invention includes raw royal jelly, dried royal jelly, dried royal jelly powder, enzyme-treated royal jelly, royal jelly extract, royal jelly fermentation product, etc. The royal jelly may be produced in any of European countries, Oceanian countries, America, Brazil, Japan, China, and other Asian countries.

Dried royal jelly powder is made by drying and powdering fresh royal jelly.

The enzyme-treated royal jelly is royal jelly treated with a protein-degrading enzyme (protease). Preferably, the enzyme-treated royal jelly is hypoallergenic, in which allergic reactions caused by proteins contained in royal jelly are suppressed by protease treatment. Therefore, the enzyme-treated royal jelly contains, in addition to the protease decomposition products of proteins contained in royal jelly, the above-mentioned organic acids such as decenoic acid, lipids, sugars,
Vitamins and various minerals may be included.

The royal jelly used to produce the enzyme-treated royal jelly is not particularly limited, and examples thereof include raw royal jelly, royal jelly powder obtained by drying and powdering raw royal jelly, and raw royal jelly extracted with water or aqueous ethanol, etc.

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

As proteolytic enzymes having at least endopeptidase activity, animal-derived (
trypsin, chymotrypsin, etc.), plant-derived (e.g. papain, etc.), microbial-derived
(For example, endopeptidases from lactic acid bacteria, yeasts, molds, Bacillus subtilis, actinomycetes, etc.)

Examples of proteolytic enzymes having at least exopeptidase activity include carboxypeptidase, aminopeptidase, exopeptidases derived from microorganisms (e.g., lactic acid bacteria, Aspergillus, Rhizopus, etc.), and pancreatin and pepsin which also have endopeptidase activity.

Among these various enzymes, a preferred example of an enzyme having both exopeptidase activity and endopeptidase activity is the enzyme from Streptomyces griseus (Streptomyces griseus).
eus produced peptidase (product name: Actinase AS), Aspergillus oryzae
Examples of preferred enzymes having exoprotease activity include peptidases produced by Aspergillus oryzae (trade names: Protease A, Flavorzyme, Proteax), and peptidases produced by Aspergillus melleus (trade name: Protease P). Also preferred examples of enzymes having exoprotease activity include peptidases produced by Aspergillus oryzae (trade names: Umamizyme G, Proteax).
omod 192P, Promod 194P, Sumiteam FLAP), Aspergillus sojae
e) peptidase produced by Aspergillus sp. (trade name: Sternzyme B15024), peptidase produced by Aspergillus sp. (
Examples of preferred enzymes having endoprotease activity include peptidase produced by Bacillus subtilis (trade name: Orientase 22BF, Nucleisin), peptidase produced by Bacillus licheniform ...
Examples of peptidases that can be used include peptidases produced by Bacillus niformis (trade name: Alcalase), peptidases produced by Bacillus stearothermophilus (trade name: Protease S), peptidases produced by Bacillus amyloliquefaciens (trade name: Neutrase), and peptidases produced by the genus Bacillus (trade name: Protamex).

Enzyme treatment to reduce the allergenicity of royal jelly is described, for example, in JP 2007-29591 A.
This can be carried out according to the description in Japanese Patent Application Laid-Open No. 2007-295920.

Royal jelly extract is obtained by extracting royal jelly (including raw, dried and crushed royal jelly) with water or aqueous 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 wall of a beehive. In the present invention, the propolis may be derived from any place of origin or plant, for example, Brazil, China, European countries, Oceania, America, etc. Therefore, any propolis, such as Alecrin, Super Green, Eucalyptus, Ultra Green, etc., can be used.

The propolis in the present invention includes, for example, propolis original mass or residue, its pulverized product, supercritical extract, extract with water or hydrophilic organic solvent, powder obtained by powdering the extract, granule form obtained by granulating the powder, etc. Among them, hydrophilic organic solvent extract is preferable because it is an effective ingredient of propolis extracted efficiently and in a good balance in a short time, and does not require large-scale equipment when scaling up.

The hydrophilic organic solvent extractant may be a lower alcohol such as ethanol, methanol, or propanol, with ethanol being particularly preferred.
Propolis raw block or residue that has been washed with alcohol can be used, and the extraction efficiency can be improved by using propolis raw block or residue that has been cut or crushed to an appropriate size.

(Mozuku seaweed or its extract)
The mozuku used in the present invention is an algae belonging to the family Chordariaceae, order Chordariaceae, or the family Spermatochnaceae, order Chordariaceae, order Chordariaceae, class Chordariaceae. The mozuku used in the present invention is not particularly limited as long as it is edible, and preferably includes Sphaerotrichia firma, Sphaerotrichia di
varicata), Okinawan mozuku (Cladosiphon okamuranus), Futomozuku (Tinocladia crassa)
and Nemacystus decipiens, which belongs to the family Mozuku. Among them, Ishimozuku and Mozuku are preferred. In the present invention, both natural and cultured Mozuku can be used. In the present invention, any of fresh Mozuku immediately after harvesting, refrigerated Mozuku, frozen Mozuku, salted Mozuku, salted frozen Mozuku, dried Mozuku, etc. can be used.

The part of the mozuku seaweed to be used is not particularly limited, and any part can be used. In addition, the mozuku seaweed can be used as a raw material in its original form without being cut or crushed, and sliced or crushed mozuku seaweed can also be used.

In the present invention, an extract of Mozuku can be used, and the extract may be extracted from Mozuku by any method. The method for obtaining the extract of Mozuku is not particularly limited, and examples thereof include a method of extracting using a polar solvent and a method of extracting using a supercritical extraction method using carbon dioxide or the like.

When extracting with a polar solvent, the extraction solvent used may be, for example, water, lower alcohol (
Examples of suitable solvents include methanol, ethanol, n-propanol, isopropanol, etc., ketones (acetone, methyl ethyl ketone, methyl butyl ketone, etc.), esters (methyl acetate, ethyl acetate, etc.), glycols (ethylene glycol, propylene glycol, etc.), glycerin, tetrahydrofuran, acetonitrile, acetic acid, propionic acid, acetamide, dimethylformamide, and dimethyl sulfoxide.

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

The mozuku used for the extraction is not particularly limited, and examples thereof include fresh mozuku and mozuku powder obtained by drying and powdering fresh mozuku.

Extraction can be easily carried out by adding a part or the whole of the mozuku to a solvent, soaking and leaving it to stand or soaking and shaking it for about 30 minutes to 3 days at a temperature between 0°C and the temperature at which the solvent boils, usually 100°C or lower, and then removing insoluble matter.
It can also be extracted by soaking and standing or soaking and shaking in hot water at 0 to 90°C for about 30 minutes to 3 hours. The solvent extract thus obtained can be used as it is, or, if necessary, can be purified by ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography, etc.
Further purification may be carried out by high performance liquid chromatography (HPLC), dialysis, a combination thereof, etc. The solvent extract may also be
Concentration and drying treatment such as freeze-drying and spray-drying can also be carried out. Concentration, freeze-drying and spray-drying can be carried out according to conventional methods.

The content of fucoidan in the mozuku extract used in the present invention is, for example, 90% or less.
% or less by mass, 80% or less by mass, 70% or less by mass, 60% or less by mass, 50% or less by mass, 40% or less by mass, 30% or less by mass, 20% or less by mass, 10% or less by mass, or 5% or less by mass, or fucoidan-free. Fucoidan does not have mTOR inhibitory activity.

The proportion of mozuku or an extract thereof contained in the composition and mTOR inhibitor of the present invention is not particularly limited as long as the effects of the present invention can be obtained, and can be appropriately adjusted depending on the final form, etc., and can be appropriately selected from the range of preferably 1 to 80% by mass, more preferably 2 to 50% by mass.

The composition and mTOR inhibitor of the present invention contains royal jelly in an amount of preferably 0.01 to 100 parts by mass, more preferably 0.05 to 70 parts by mass, even more preferably 0.1 to 50 parts by mass, and particularly preferably 1 to 30 parts by mass, per part by mass of Mozuku or an extract thereof.

The composition and mTOR inhibitor of the present invention contains propolis in an amount of preferably 0.01 to 100 parts by mass, more preferably 0.05 to 70 parts by mass, even more preferably 0.1 to 50 parts by mass, even more preferably 1 to 30 parts by mass, particularly preferably more than 1 part by mass to 20 parts by mass, and most preferably 2.2 to 20 parts by mass, per part by mass of Mozuku or an extract thereof.

The composition and mTOR inhibitor of the present invention may contain, as appropriate, known ingredients other than those mentioned above, as long as they do not impair the effects of the present invention.

The composition and mTOR inhibitor of the present invention can be used in cosmetics, foods and beverages (particularly foods and beverages intended for health, health maintenance, and health promotion, etc., such as health foods, functional foods, and nutritional compositions).
n), nutritional supplements, supplements, health foods, foods for specified health uses, nutritionally functional foods, or foods with functional claims), quasi-drugs, medicines, etc. The composition and mTOR inhibitor of the present invention also encompasses the meaning of additives that impart mTOR inhibitory action, anti-aging action, anti-fatigue action, anti-stress action, metabolism improving action, diet action, moisturizing action, skin firmness or gloss improving action, cognitive function decline prevention action, immunosuppressive action, anti-allergic action, cancer prevention action, and cancer progression suppression action.

In addition to the royal jelly, propolis, and mozuku seaweed or extracts thereof, the above-mentioned cosmetics may contain, as necessary, ingredients typically used in cosmetics, such as whitening agents, moisturizing agents, antioxidants, oily ingredients, UV absorbers, surfactants, thickeners, alcohols, powdered ingredients, coloring materials, aqueous ingredients, water, various skin nutrients, etc.

Cosmetics include any cosmetic product that is applied to the skin, mucous membranes, body hair, head hair, scalp, nails, teeth, facial skin, lips, etc. of animals (including humans).

Cosmetics can take a wide variety of formulations, including aqueous solutions, solubilized solutions, emulsions, powders, oils, gels, ointments, aerosols, water-oil two-layer systems, and water-oil-powder three-layer systems.

The use of the cosmetics is also arbitrary. For example, basic cosmetics include face wash, lotion, milky lotion, cream, gel, essence, beauty serum, pack, mask, etc.; makeup cosmetics include foundation, lipstick, blusher, eye shadow, eyeliner, mascara, etc.; nail cosmetics include nail polish, base coat, top coat, nail polish remover, etc.; and other cosmetics include face wash, (paste or liquid) dentifrice, mouthwash, massage agent, cleansing agent, aftershave lotion, preshave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment,
Examples include hair styling products, hair tonics, hair growth products, antiperspirants, bath additives, etc.

In the above-mentioned foods and beverages, the above-mentioned royal jelly, propolis and mozuku seaweed or extracts thereof can be used as they are, and if necessary, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents, binders, sweeteners, disintegrants, lubricants, colorants, fragrances, stabilizers, preservatives, sustained-release regulators, surfactants, solubilizers, humectants, etc. can also be blended therein.

Food and drink includes any food and drink that can be ingested by animals (including humans). The types of food and drink are not particularly limited, and include, for example, dairy products; fermented foods (yogurt, royal jelly, etc.); beverages (soft drinks such as coffee, juice, and tea drinks, milk drinks, lactic acid bacteria drinks, drinks containing lactic acid bacteria, yogurt drinks, carbonated drinks, sake, Western liquor, and fruit liquor); spreads (custard cream, etc.); pastes (fruit paste, etc.); Western confectionery (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, puddings, etc.).
); Japanese sweets (daifuku, mochi, manju, castella, anmitsu, yokan, etc.); frozen desserts (ice cream,
Examples of such products include popsicles, sorbet, etc.; food products (curry, beef bowls, rice porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); and condiments (dressings, furikake, umami seasonings, soup bases, etc.).

The method for producing the food and beverage of the present invention is not particularly limited, and any known method can be used as appropriate.

The dosage unit form when used as a supplement is not particularly limited and can be selected appropriately. Examples include tablets, capsules, granules, liquids, powders, etc.

The intake amount of the food or drink of the present invention can be appropriately set depending on various conditions of the person taking it, such as the body weight, age, sex, and symptoms.

The above-mentioned pharmaceutical products may contain only the above-mentioned royal jelly, propolis and mozuku seaweed or extracts thereof, or may be used in combination with other medicinal ingredients such as vitamins and herbal medicines described in the Japanese Pharmacopoeia.

When the composition and mTOR inhibitor of the present invention are prepared as a pharmaceutical product, the above royal jelly,
Propolis and Mozuku or extracts thereof can be prepared, together with non-toxic carriers, diluents, or excipients acceptable for pharmaceutical use, into the form of tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (dispersed medicines), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, etc., to form pharmaceutical preparations (for example, for oral use).

The dosage of a pharmaceutical agent can be appropriately determined depending on various conditions such as the patient's weight, age, sex, and symptoms.

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

The composition and mTOR inhibitor of the present invention described above can be administered to mammals, including humans (preferably humans).
This applies to:

The composition and mTOR inhibitor of the present invention have excellent mTOR inhibitory activity due to the synergistic effect of royal jelly or propolis and mozuku or an extract thereof.

As a result of mTOR inhibition, lifespan extension, autophagy enhancement, browning of white adipocytes,
It has been reported that this leads to skin moisturizing effects, cognitive function improvement effects, immunosuppression effects, anti-cancer effects, etc. Effects based on increased autophagy include anti-fatigue and anti-stress effects, effects based on the browning of white fat cells include metabolism improvement effects and diet effects, and moisturizing effects include improving the moisture, firmness, and luster of the skin.

Therefore, the combination of royal jelly or propolis with mozuku or its extract is
It is useful as an mTOR inhibitor, and as a component of a composition for anti-aging, anti-fatigue, anti-stress, metabolism improvement, dieting, moisturizing, improving skin firmness or luster, preventing cognitive decline, immunosuppression, anti-allergy, cancer prevention, or cancer progression inhibition.

Types of cancer include stomach cancer, colon cancer (rectal cancer, colon cancer), small intestine cancer, liver cancer, pancreatic cancer, lung cancer, pharyngeal cancer, esophageal cancer, kidney cancer, gallbladder and bile duct cancer, head and neck cancer, bladder cancer, prostate cancer, breast cancer, uterine cancer (cervical cancer, endometrial cancer), ovarian cancer, brain tumors, leukemia, malignant lymphoma, multiple myeloma, etc.

In addition, the composition and mTOR inhibitor of the present invention are highly safe because they contain naturally derived active ingredients.

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

Test Example <Materials>
Fresh royal jelly (untreated), enzyme-treated royal jelly and freeze-dried powder of propolis ethanol extract were dissolved in DMSO and used for the test. Enzyme-treated royal jelly is patented under patent no. 3994.
According to the method described in Example 1 of Publication No. 120, the ethanol extract powder of propolis was obtained by freeze-drying and pulverizing the ethanol extract of Alecrin propolis raw material (produced in Minas Gerais, Brazil).

The mozuku used were Iwamozuku from Okinoshima Island (collected from the sea of Okinoshima Island) and mozuku from Sado Island (purchased from the Bandaijima Fresh Fish Center of Niigata Central Fisheries Market Co., Ltd.). The mozuku was washed with water, cut into pieces, and then freeze-dried. The freeze-dried material was put into a mill and crushed finely, and 10 ml of water was used for 1 g of mozuku.
Milli-Q (registered trademark) water was added, and the mixture was immersed in a water bath kept at 90° C. for 1 hour. Then, the mixture was centrifuged at 6,000 rpm and 25° C. for 15 minutes, the supernatant was filtered, the water was removed with an evaporator, and the mixture was centrifuged in a milli-Q (registered trademark) water bath for 1 hour.
A small amount of water was added, the mixture was frozen at -20°C, and then freeze-dried to obtain a powdered extract of Mozuku seaweed.

<Reagents>
・mTOR: eurofins #14-770
・ULight ^TM -4E-BP1 (Thr37/46) Peptide: PerkinElmer #TRF0128-D, #TRF0128-M
・Europium-anti-phospho-4E-BP1 (Thr37/46) Antibody：PerkinElmer #TRF0216-D, #TRF
0216-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
0.01% Tween-20, 2 mM DTT

<Experimental Method>
Raw royal jelly (raw RJ), enzyme-treated royal jelly (RJ), ethanol extract of propolis (PP), and extract powder of mozuku were dissolved in DMSO, and the mTOR inhibitory activity was evaluated in the materials alone and in an equal mixture of raw RJ, RJ, or PP and mozuku. mTOR inhibitory activity was evaluated by measuring mTOR kinase inhibitory activity using the TR-FRET method.

[Test procedure]
KB was used to incubate the cells with mTOR 40 nM, ATP 5 mM, PI-103 (Inhibitor) 40 μM, ULight ^TM -4E-BP1 (Th
r37/46) Peptide 200 nM was prepared. Europium-ant peptide was detected using 10X LANCE Detection Buffer.
i-phospho-4E-BP1 (Thr37/46) Antibody 80μM (4X Detection buffer) was prepared. PI-1
03 40μM was diluted 5-fold and 8-fold to serve as a positive control (DMSO concentration was standardized to 4%). Royal jelly, propolis, and mozuku were used as positive controls, respectively, at 3, 0.6, 0.12, and 0.024μg
The test samples were prepared by dissolving the solutions in DMSO at the above concentrations (30, 6, 1.2, 0.24μg/mL) and royal jelly/propolis (30, 6, 1.2, 0.24μg/mL).

In a MicroPlate (Greiner #784075), 2.5 μL of mTOR, 1.25 μL of inhibitor or test sample, and U
Add 1.25μL of Light-4E-BP1 (Thr37/46) Peptide/ATP mix (ATP 80μM), attach a plate seal, and leave at room temperature for 1 hour. Add 2.5μL of Stop Solution (32 mM EDTA in 1X Detection Buffer)
was added and allowed to stand at room temperature for 5 minutes.

2.5μL Detection Mix (Eu-anti-phospho-4E-BP1 (Thr37/46) Antibody final concentration 2 nM)
The ^plate was sealed and left to stand at room temperature for 1 hour.
Measurements were performed using the 320 nm excitation and 665 nm emission modes.

<Results>
The results of using Iwamozuku from Okinoshima and Mozuku from Sado Island are shown in Tables 1 and 2 below.

No mTOR inhibitory activity was observed in raw or enzyme-treated royal jelly, regardless of the concentration in the range of 0.24 to 30 μg/mL. The 50% inhibitory concentration (IC ₅₀ ) of propolis was 23.24 μg/mL. The IC ₅₀ of mozuku alone was 4.266 μg/mL for Iwamozuku from Oki Island and 1178 μg/mL for mozuku from Sado Island.
When RJ or PP was mixed with mozuku, in the case of Iwamozuku from Okinoshima, the _IC50 for mozuku + RJ was 2.44μg/mL, and the _IC50 for mozuku + PP was 1.812μg/mL, which were 1.7 and 1.9 times stronger inhibitory activity than the theoretical value when assuming an additive effect. In the case of mozuku from Sado Island, the _IC50 for mozuku + PP was 2.568μg/mL, which was 6.7 times stronger inhibitory activity than the theoretical value when assuming an additive effect.

Claims (1)

1. An mTOR inhibitor comprising royal jelly and/or propolis, and mozuku or an extract thereof, wherein the mozuku is Sphaerotrichia firma belonging to the family Chosanthellaceae of the order Chosanthellaceae .