JP7280015B2 - Composition for suppressing FABP5 production - Google Patents

Description

The present invention relates to a composition for suppressing production of FABP5 in living organisms.

In recent years, a family of proteins called fatty acid-binding proteins (FABPs) has attracted attention. In particular, the function as a "molecule" that connects mental illness and polyunsaturated fatty acids has been clarified. FABP is a low-molecular-weight protein of 14-15 kDa, mainly present in the cytoplasm, and is said to be involved in the uptake and transport of fatty acids, and the regulation of the expression of other genes by forming complexes with nuclear receptors ( Non-Patent Document 1).
Nine types of FABPs are known in mammals, and FABPs are generally found abundantly in tissues with high fatty acid metabolic activity, such as cardiac muscle, skeletal muscle, intestine, liver, and adipocyte. FABPs are named after the tissues or cells from which they were first isolated, such as heart-type (H-), brain-type (B-), liver-type (L-), and intestinal-type (I-). However, since each FABP is expressed in various tissues and cells, FABP3 (H-FABP), FABP5 (E-FABP), FABP7 (B-FABP), etc. have recently been numbered. This name is also used in the specification of the present application.

FABP5 is the first FABP reported in the epidermis. FABP5 is used as a wrinkle formation marker (Patent Document 1), an acne skin evaluation marker (Patent Document 2), and a biomarker common to degenerative diseases of the nervous system (Patent Document 3). Patent Document 4 also uses it as one of many markers for evaluating fatigue and stress conditions.

FABP5 suppresses the onset of atherosclerosis and diabetes in genetically mutated or knockout mice, and is therefore expected to be the cause of these diseases, but has not yet been clarified in humans. do not have.
In addition, fatty acids themselves damage the skin and cause inflammation, but this is because FABP5, which is abundant on the skin, aggravates skin conditions such as barrier function deterioration and inflammation via nuclear receptors. The presence of excess fatty acids has been shown to increase FABP5 on the skin and enhance skin inflammation. It is also known that palmitic acid, lauric acid, and oleic acid exhibit such behavior as fatty acids that cause inflammation.

As a substance that suppresses FABP5, Patent Document 5 discloses a triazolopyrimidinone derivative (5-((3-chloro-2-methylphenoxy)methyl)-2-phenyl-[1, 2,4]triazolo[1,5-α]pyrimidin-7(4H)-one, which is disclosed in Patent Document 5, has the effect of inhibiting FABP5 and inhibiting hair growth. and a method for evaluating hair growth inhibitors using FABP5 as an index.
In Patent Document 6, a cancer metastasis inhibitor and FABP5 gene are used as active ingredients, which are obtained by adsorbing an azuki bean water extract to an adsorbent or a carrier and eluting with a solvent such as methanol or ethanol. Inventions are described in which it is used as an anticancer agent such as an activity inhibitor and a cancer growth inhibitor.

Patent Document 7 discloses that FABP4 and FABP5 are expressed in intestinal cells that secrete GIP, that inhibition of FABP4/5 reduces blood GIP concentration, and that substances that inhibit FABP4/5 increase GIP. It is stated to be useful as an inhibitor.

JP 2010-164404 A JP 2016-95185 A JP 2014-071016 A JP 2013-150558 A JP 2016-214085 A JP 2017-155012 A JP 2017-215336 A

Nature Reviews Drug Discovery, 2008, 7:489-503

The present inventor searched for a compound that suppresses FABP5 production using a natural product-derived compound library as a search source, and found a compound that has FABP5 production suppression activity. A similar search was also conducted with respect to plant extracts, and it was found that among multiple plant extracts, an extract containing a specific compound has a strong FABP5 production inhibitory effect.
That is, an object of the present invention is to provide a composition having FABP5 production inhibitory action.

The main configuration of the present invention is as follows.
(1) A composition for suppressing FABP5 production containing as an active ingredient one or more substances selected from nobiletin, naringin, sulforaphane, homoorientin, and methyl jasmonate.
(2) The composition for suppressing FABP5 production according to (1), wherein the composition containing nobiletin is a citrus peel extract.
(3) The composition for inhibiting FABP5 production according to (2), wherein the citrus peel extract is chimp extract and/or kippi extract.
(4) The composition for suppressing FABP5 production according to (1), wherein the composition containing sulforaphane is a broccoli extract.
(5) The composition for suppressing FABP5 production according to (4), wherein the broccoli extract is a broccoli sprout extract.

The composition of the present invention has the effect of suppressing FABP5 production in human cells. Since the composition of the present invention can suppress the production of FABP5, which is abundantly present in the skin, it is expected to suppress inflammatory reactions derived from fatty acids in the skin.

It is a graph which shows the result of the FABP5 production suppression test of nobiletin. It is a graph which shows the result of the FABP5 production suppression test of naringin. It is a graph which shows the result of the FABP5 production suppression test of sulforaphane. It is a graph which shows the result of the FABP5 production suppression test of homoorientin. It is a graph which shows the result of the FABP5 production suppression test of methyl jasmonate. It is a graph which shows the result of the FABP5 production suppression test of chimpi extract. It is a graph which shows the result of the FABP5 production suppression test of a broccoli sprout.

The present invention relates to a FABP5 production-suppressing composition containing, as an active ingredient, one or more substances selected from nobiletin, naringin, sulforaphane, homoorientin, and methyl jasmonate.
As used herein, the term “FABP5” is an abbreviation for Fatty acid-binding protein 5, which means fatty acid binding protein 5.
Further, FABP5 production suppression means suppression of FABP5 gene transcription, suppression of FABP5 gene expression, suppression of FABP5 protein amount and/or suppression of FABP5 activity.

1. Nobiletin Nobiletin is a polymethoxyflavonoid (O-methylated flavonoid) and is abundantly contained in citrus peels and the like. It has a structure represented by the following chemical formula 1, and can be used in the present invention by extracting it from citrus peel or juice.
The IUPAC name for nobiletin can be identified as 2-(3,4-Dimethoxyphenyl)-5,6,7,8-tetramethoxychromen-4-one, the compound with CAS number 478-01-3.

（化学式１）

(Chemical Formula 1)

In the present invention, nobiletin may be highly purified or may be an extract from citrus fruits containing other flavonoid components. Chimpi Kippi extract can be exemplified as such an extract. It may also be a commercially available extract. As a commercially available product, Chimpi Extract K65B (NOF CORPORATION) can be exemplified.

2. Naringin Naringin is a flavanone glycoside that is abundantly contained near the pericarp of fruits such as grapefruit and hassaku, and is responsible for the bitterness and stimulation of citrus fruits.
Naringin (sometimes called naringin) has a structure represented by the following chemical formula 2, and can be used in the present invention when extracted from citrus peels and juices.

（化学式２）

(Chemical Formula 2)

In the present invention, naringin may be highly purified or may be an extract from citrus fruits containing other flavonoid components. Examples of such extracts include chimp extract, which is an extract of unshiu mandarin orange peel, and kippi extract, which is an extract of tachibana peel. Naringin is a compound with IUPAC name 4′,5-dihydroxy-7-[(2-O-α-L-rhamnopyranosyl-β-D-glucopyranosyl)oxy]flavan-4-one, CAS number 10236-47-2 can be identified as

3. Sulforaphane Sulforaphane is a kind of isothiocyanate and is contained in a large amount in cruciferous vegetables, especially in broccoli. It has a structure represented by the following chemical formula 3, and can be used in the present invention when extracted from broccoli or broccoli sprouts.
The IUPAC name for sulforaphane can be identified as 1-isothiocyanato-4-(methylsulfinyl)-butane, a compound with CAS number 4478-93-7.

（化学式３）

(Chemical formula 3)

In the present invention, sulforaphane may be highly purified or may be an extract containing other components. It may also be chemically synthesized. A method for extracting sulforaphane from broccoli or broccoli sprouts is disclosed in Japanese Patent Publication No. 2008-539261, and sulforaphane purified by this method can be used. Broccoli Sprout Extract PC (Oriza Yuka Co., Ltd.) can be exemplified as such an extract commercially available. Broccoli sprout extract PC is an extract extracted from sprouted broccoli (Brassica oleracea var. italica), and is a composition containing 2.0% by mass or more of sulforaphane.

4. Homoorientin Homoorientin is also known as isoorientin. It is a compound known to be contained in passion fruit flower extract. It can be represented by the following chemical formula 4. IUPAC name identified as 2-(3,4-Dihydroxyphenyl)-6-β-D-glucopyranosyl-5,7-dihydroxy-4H-1-benzopyran-4-one, compound with CAS number 4261-42-1 can.

（化学式４）

(Chemical Formula 4)

In the present invention, homoorientin may be highly purified or may be an extract containing other components. It may also be chemically synthesized. Homoorientin can be extracted from Passiflora flower extract (passion flower extract) by a conventional method. A commercially available Passiflora flower extract may also be used. Commercially available products include “Passion Flower Extract-PC” and “Passion Flower Extract-WSPC” (both manufactured by Oryza Yuka Co., Ltd.).

5. Methyl jasmonate Methyl jasmonate is a volatile organic compound that is used as a plant hormone in various developmental pathways such as plant defense, seed germination, root elongation, flowering, fruit ripening and senescence. be. It is a compound isolated from jasmine oil as an aromatic component and can be represented by the following chemical formula 5. The IUPAC name can be identified as Methyl (1R,2R)-3-Oxo-2-(2Z)-2-pentenyl-cyclopentaneacetate, the compound with CAS number 39924-52-2.

（化学式５）

(Chemical Formula 5)

Methyl jasmonate may be highly purified or may be an extract containing other components. It may also be chemically synthesized. Methyl jasmonate is contained in steam distilled essential oil from jasmine flowers, which can be used in the present invention.

The composition of the present invention can be used as foods, pharmaceuticals, cosmetics and the like. As for foods, in addition to ordinary foods, it may be used as dietary supplements, functional foods, health foods, foods for specified health uses, and the like. For example, it can be blended in beverages such as juice.

In pharmaceutical applications, for administration, the active ingredient is mixed with a non-toxic solid or liquid pharmaceutical carrier suitable for the administration method, such as oral ingestion, parenteral ingestion, rectal administration, injection, etc. It can be administered in the form of a formulation. Examples of forms include solid formulations such as powders, powders, granules, tablets and capsules, liquid formulations such as solutions, suspensions and emulsions, freeze-dried preparations, external preparations and the like. These formulations can be prepared by conventional means. It is preferably used as an external preparation. Examples of the above pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glycerides, polyethylene glycol, hydroxyethylene starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acids, gelatin, albumin, water. , physiological saline, and the like. Additives such as stabilizers, wetting agents, emulsifiers, binders, tonicity agents and the like can be added as necessary.

When the composition of the present invention is used as a cosmetic, various ingredients that are commonly used in cosmetics can be blended as appropriate within a range that does not impair the effects of the present invention. Such other ingredients that can be blended are not particularly limited as long as they do not interfere with the purpose of the present invention. Amino acids, vitamins, whitening agents, organic acids, inorganic salts, enzymes, antioxidants, stabilizers, preservatives, disinfectants, skin activators, blood circulation promoters, antiseborrheic agents, anti-inflammatory agents, etc. Sequestering agents, pH adjusters, astringents, cooling agents, perfumes, pigments, and water.

The form of the cosmetic containing the composition of the present invention is not particularly limited. can be used. In addition, the composition for external use on the skin of the present invention can be a product included in cosmetics, quasi-drugs, pharmaceuticals, etc. as defined by the Pharmaceutical Affairs Law. It is used in skin care products such as emulsions, creams, lotions, and ointments. Although not particularly limited, the dosage form is an aqueous solution system, a solubilization system, an emulsion system, a powder system, an oil system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, and a water-oil system. It can take a wide range of dosage forms, such as a powder three-layer system.

The dosage of the composition of the present invention is appropriately selected and determined according to the subject's age, body weight, symptoms, administration route, administration schedule, formulation form, and the like. For oral administration, 0.001-100 g per day is preferred. For external application, a composition containing 0.001 to 1% by weight is administered multiple times per day.

Test examples are shown below to specifically describe the present invention.
1. Primary Screening Test Using Natural Compound Library A test was conducted on 480 compounds of the natural compound library owned by the applicant.

(1) Test method 1) Cells used in test Normal human epidermal keratinocytes (NHEK passage4/Thermo Fisher Scientific).

2) Culture Conditions for Cells Cells were cultured at 37° C. in an atmosphere of 5% carbon dioxide and 95%. The medium used was medium DMEM (GIBCO/11995-073) supplemented with 10% inactivated fetal bovine serum (FBS) (Hyclone Laboratories) and 1% penicillin-streptomycin (Sigma Aldrich). For normal culture, cells were seeded in a 75 cm flask, and when the cells were 80 to 90% confluent, 0.05% Trypsin-EDTA (Sigma Aldrich) was added to detach the cells and passaging was performed.

3) Test sample Library compounds were tested by dissolving them in 100% dimethyl sulfoxide (DMSO) to a concentration of 0.4 mg/ml.

4) FABP Production Stimulation NHEK cells were seeded in a 24-well plate at 4×10 ⁴ cells/well and precultured for 24 hours. A test sample was added by diluting a solution dissolved in 100% DMSO with a medium to a final concentration of 0.1 to 1 μg/ml.

5) Protein Extraction Cells were lysed in Cell Lysis Buffer, shaken for 20 minutes, and then the cell lysate was collected. The protein amount of the recovered cell lysate was measured using the BCA protein kit. The protein amount was measured according to the protocol attached to the kit.

6) Measurement of FABP5 100 μl of anti-FABP5 antibody (Bio Vender) diluted with PBS was added to a 96-well EIA/RIA plate and stored at 4° C. to immobilize the antibody.
The following day, the cells were washed three times with 300 µl of wash buffer, excess liquid was removed, Reagent Diluent (R&D) was added, and blocking was performed at 25°C for 1 hour. After washing three times with 300 μl of PBS-T (0.05%) and removing excess liquid, 100 μl each of FABP5 (Funakoshi Co., Ltd.) diluted with Cell Lysis Buffer and the test sample were added.
After reacting at 37° C. for 1.5 hours, it was washed three times with 300 μl of PBS-T (0.05%), then excess liquid was removed and anti-FABP5 diluted with PBS-T (0.05%) 100 μl of antibody (R&D) was added and allowed to react at 37° C. for 1 hour.
After completion of the reaction, the mixture was washed three times with 300 μl of PBS-T (0.05%), excess liquid was removed, 100 μl of anti-Streptavidin-HRP2 diluted with PBS-T (0.05%) was added, and the mixture was heated at 37°C. It was reacted for 30 minutes. After washing three times with 300 μl of PBS-T (0.05%), excess liquid was removed, 100 μl of TMB One solution (Promega) was added, and the reaction was allowed to proceed at room temperature until the color changed. Next, 50 μl of 2N sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction, and the absorbance at 450 nm was measured. The production amount of FABP5 was measured in comparison with a previously prepared calibration curve. The obtained measured value of FABP5 was divided by the amount of cell protein and normalized to obtain the production amount of FABP5.

(2) Test Results Of the 480 compounds described above, the 12 compounds shown in Table 1 below showed changes in FABP5 production compared to the case where FABP5 production was not added.

2. FABP5 Production Suppression Test Compounds that were positive (changed FABP5 production) in the primary test were evaluated for their ability to suppress FABP5 production induced by LPS and fatty acid stimulation.

(1) Test method 1) Cell culture conditions Cells were cultured under an atmosphere of 37°C, 5% carbon dioxide, and 95%. The medium used was medium DMEM (GIBCO/11995-073) supplemented with 10% inactivated fetal bovine serum (FBS) (Hyclone Laboratories) and 1% penicillin-streptomycin (Sigma Aldrich). For normal culture, cells were seeded in a 75 cm flask, and when the cells were 80 to 90% confluent, 0.05% Trypsin-EDTA (Sigma Aldrich) was added to detach the cells and passaging was performed.

2) Test samples As in the primary screening, all test samples were dissolved in dimethyl sulfoxide (DMSO) and tested.

3) FABP5 Production Stimulation by LPS and Fatty Acid NHEK was seeded in a 24-well plate at 4×10 ⁴ cells/well and precultured for 24 hours. Then, the cells were cultured in a medium supplemented with 10 μg/ml LPS (lipopolysaccharide derived from E. coli 026:B6: Sigma Ardrich) for 8 hours. Thereafter, the test sample was added to a medium supplemented with 400 μM palmitic acid or 800 μM oleic acid and cultured for 20 hours. The test samples were added to a solution dissolved in 100% DMSO, and depending on the medium, the final concentration was 40 µg/ml for nobiletin, 0.25 µg/ml for naringin, 0.025 µg/ml for sulforaphane, and 2.5 µg/ml for homoorientin. ml, and methyl jasmonate was diluted to 1 μg/ml and added.

4) Extraction of Protein Cells were dissolved in Cell Lysis Buffer, shaken for 20 minutes, and then the cell lysate was collected. The protein amount of the recovered cell lysate was measured using the BCA protein kit. The protein amount was measured according to the protocol attached to the kit.

5) Measurement of FABP5 100 μl of anti-FABP5 antibody (Bio Vender) diluted with PBS was added to a 96-well EIA/RIA plate and stored at 4° C. to immobilize the antibody.
The following day, the cells were washed three times with 300 µl of wash buffer, excess liquid was removed, Reagent Diluent (R&D) was added, and blocking was performed at 25°C for 1 hour. After washing three times with 300 μl of PBS-T (0.05%) and removing excess liquid, 100 μl each of FABP5 (Funakoshi Co., Ltd.) diluted with Cell Lysis Buffer and the test sample were added.
After reacting at 37° C. for 1.5 hours, it was washed three times with 300 μl of PBS-T (0.05%), then excess liquid was removed and anti-FABP5 diluted with PBS-T (0.05%) 100 μl of antibody (R&D) was added and allowed to react at 37° C. for 1 hour.
After completion of the reaction, the mixture was washed three times with 300 μl of PBS-T (0.05%), excess liquid was removed, 100 μl of anti-Streptavidin-HRP2 diluted with PBS-T (0.05%) was added, and the mixture was heated at 37°C. It was reacted for 30 minutes. After washing three times with 300 μl of PBS-T (0.05%), excess liquid was removed, 100 μl of TMB One solution (Promega) was added, and the reaction was allowed to proceed at room temperature until the color changed. Next, 50 μl of 2N sulfuric acid (Wako Pure Chemical Industries, Ltd.) was added to stop the reaction, and the absorbance at 450 nm was measured. The production amount of FABP5 was measured in comparison with a previously prepared calibration curve. The obtained measured value of FABP5 was divided by the amount of cell protein and normalized to obtain the production amount of FABP5.

(2) Test Results Among the above 12 samples, compounds that inhibited FABP5 production were 5 samples of nobiletin, naringin, sulforaphane, homoorientin, and methyl jasmonate.
1 to 5 show the test results confirming the minimum concentration capable of suppressing FABP5 production.
As shown in FIGS. 1 to 5, nobiletin is 40 μg/ml, naringin is 0.25 μg/ml, sulforaphane is 0.025 μg/ml, homoorientin is 2.5 μg/ml, and methyl jasmonate is 1 μg/ml. concentration suppressed the production of FABP5.

3. FABP5 production inhibition test using extracts Tachibana pericarp extract (“Chimp Extract K65B” NOF Corporation) as an extract containing nobiletin, sprouted broccoli extract (“Broccoli Sprout Extract PC” Oryza Yuka Co., Ltd. as an extract containing sulforaphane) company) to: 1. The test was performed in the same manner as the test of The chimp extract used contained 0.8% nobiletin. A broccoli extract containing 2% sulforaphane was used.

The test results are shown in FIGS. 6 and 7. FIG.
It was found that chimp extract inhibited FABP5 production at a concentration of 0.4 μg/ml and broccoli sprout extract at a concentration of 0.00025% or higher.

Claims (3)

A composition for inhibiting FABP5 production containing nobiletin as an active ingredient (excluding skin inflammation-improving agent, skin roughening-improving agent, contact dermatitis-improving agent, and skin disease-improving agent) . The composition for suppressing FABP5 production according to claim 1, wherein the composition containing nobiletin is a citrus peel extract ( provided that it contains an agent for improving skin inflammation, an agent for improving rough skin, an agent for suppressing contact dermatitis, and an agent for improving skin diseases). except) . 3. The composition for suppressing FABP5 production according to claim 2, wherein the citrus peel extract is chimp extract and/or kippi extract ( provided that it is an agent for improving skin inflammation, an agent for improving rough skin, an agent for suppressing contact dermatitis , and an agent for improving skin diseases ) .

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