WO2023238910A1 - Skin health condition improving agent - Google Patents
Skin health condition improving agent Download PDFInfo
- Publication number
- WO2023238910A1 WO2023238910A1 PCT/JP2023/021367 JP2023021367W WO2023238910A1 WO 2023238910 A1 WO2023238910 A1 WO 2023238910A1 JP 2023021367 W JP2023021367 W JP 2023021367W WO 2023238910 A1 WO2023238910 A1 WO 2023238910A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- health condition
- skin health
- improving agent
- condition improving
- sterol ester
- Prior art date
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- 239000006072 paste Substances 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- OIGNJSKKLXVSLS-VWUMJDOOSA-N prednisolone Chemical group O=C1C=C[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OIGNJSKKLXVSLS-VWUMJDOOSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 206010037844 rash Diseases 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 235000021067 refined food Nutrition 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 235000012045 salad Nutrition 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- 238000006748 scratching Methods 0.000 description 1
- 230000002393 scratching effect Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 150000003431 steroids Chemical class 0.000 description 1
- 235000013547 stew Nutrition 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- TUNFSRHWOTWDNC-HKGQFRNVSA-N tetradecanoic acid Chemical compound CCCCCCCCCCCCC[14C](O)=O TUNFSRHWOTWDNC-HKGQFRNVSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
- A23L33/11—Plant sterols or derivatives thereof, e.g. phytosterols
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/63—Steroids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- Semaphorin 3A (Sema3A) is known to inhibit nerve outgrowth.
- Sema3A ointment has been reported to considerably suppress epidermal hypersensitivity and scratching behavior in animal tests (Non-Patent Document 1), but as a side effect, long-term use can cause contact dermatitis such as rash. there is a possibility.
- Oxidative stress is also cited as one of the factors that adversely affect the skin induced by external stimuli.
- oxidative stress is sometimes described as rust on the body and is known to accelerate skin aging.
- Arginase 1 (ARG1) is an enzyme that can suppress oxidative stress through its antioxidant effect and is expected to improve skin brightness and wrinkles. It has been reported that the expression is increased by 14-dehydroergosterol (Patent Document 1, Patent Document 2).
- An object of the present invention is to provide a skin health condition improving agent that has multiple skin health condition improving effects.
- the present invention includes the following inventions in order to solve the above problems.
- a skin health condition improving agent characterized by containing a sterol ester obtained from rice bran.
- the skin health condition improvement according to [1] above which has at least two selected from the group consisting of semaphorin 3A production promoting action, meionectin production promoting action, arginase 1 production promoting action and period 1 production promoting action. agent.
- a pharmaceutical product comprising the skin health condition improving agent according to [1] or [2] above.
- a cosmetic product comprising the skin health condition improving agent according to [1] or [2] above.
- a food or drink product comprising the skin health condition improving agent according to [1] or [2] above.
- a semaphorin 3A production promoter characterized by containing a sterol ester obtained from rice bran.
- the present invention provides a skin health condition improving agent.
- the skin health condition improving agent of the present invention may be any agent as long as it contains a sterol ester obtained from rice bran.
- the sterol ester obtained from rice bran in the present invention is not particularly limited as long as it is a sterol ester obtained from rice bran by a known method, and may contain esters of plant sterols and fatty acids, triterpene alcohols and fatty acids.
- Sterol refers to a compound having a hydroxyl group at the 3-position among compounds (steroids) having a cyclopentanoperhydrophenanthrene skeleton (sterane skeleton). Sterols are generally widely distributed in the animal and plant kingdoms in the form of free, ester, glycosides, and the like.
- the ester of a plant sterol and a fatty acid and/or the ester of a triterpene alcohol and a fatty acid contained in the skin health condition improving agent of the present invention is an ester type sterol with a fatty acid.
- the sterol may be an unsaturated compound having 27 to 30 carbon atoms and having one double bond at the 5/6 position, 7/8 position, 8/9 position or other position, It may also be a saturated compound obtained by hydrogenation.
- plant sterols that are abundant in plants include ⁇ -sitosterol, stigmasterol, campesterol, and the like.
- Triterpene alcohol is a sterol with 30 carbon atoms, and is a component abundantly contained in rice bran or rice oil.
- the ester of a plant sterol and a fatty acid and/or the ester of a triterpene alcohol and a fatty acid may be a sterol ester purified from rice bran using a known method, or a commercially available sterol ester.
- the method for obtaining sterol esters from rice bran is not particularly limited, and for example, a method in which by-products such as soup stock and deodorized scum produced in the process of producing rice oil from rice bran are used as raw materials, and extracted and purified with a solvent such as acetone or hexene. , a method described in a known document (Unexamined Japanese Patent Publication No.
- the sterol ester contained in the skin health condition improving agent of the present invention is a component that is also used for food, it is highly safe for living organisms and can be continuously used or ingested over a long period of time. Since the skin health condition improving agent of the present invention has a plurality of skin health condition improving effects, it is considered that the health condition of the skin surface or dermis can be comprehensively and efficiently improved or maintained in good condition.
- the skin health condition improving agent of the present invention is characterized by having a skin health condition improving effect.
- the skin health condition improving effect in the present invention is not particularly limited as long as it can improve the health condition of the skin surface or dermis, and includes, for example, semaphorin 3A production promoting effect, myonectin production promoting effect, arginase 1 production promoting effect, and It may have at least one selected from the group consisting of period 1 production promoting action, or it may have at least two selected from the group.
- Semaphorin 3A is a type of protein that suppresses the elongation of nerve fibers that feel itching.In atopic dermatitis, the production of Sema3A, which suppresses the elongation of nerve fibers near the epidermis, is reduced due to a decline in the skin barrier function. It is known that the nerve fibers decrease and elongate, making them more susceptible to itching stimuli.
- the skin health function improving agent of the present invention acts on the skin surface (particularly the epidermis) and promotes the production of Sema3A, a neurorepulsive factor, thereby suppressing itch and improving sensitive skin, thereby improving skin health. can be improved or maintained in good condition.
- the fact that the skin health condition improving agent of the present invention has a semaphorin 3A production promoting effect means that, for example, the skin health condition improving agent of the present invention has a semaphorin 3A production promoting effect. This can be confirmed by treating the skin with a health condition improving agent and measuring the amount of Sema3A protein expression or Sema3A gene expression in epidermal keratinocytes.
- the skin health condition improving agent of the present invention has an effect of promoting myonectin production
- the fact that the skin health condition improving agent of the present invention has a myonectin production promoting effect means that, for example, the skin health condition of the present invention can be transferred to muscle cells immediately after differentiation. This can be confirmed by treating the muscle cells with an improving agent and measuring the myonectin protein expression level or myonectin gene expression level in muscle cells.
- the fact that the skin health condition improving agent of the present invention has an effect of promoting arginase 1 production means, for example, that the skin health condition improving agent of the present invention has an effect of promoting arginase 1 production. This can be confirmed by treating the skin health condition improving agent and measuring the ARG1 protein expression level or ARG1 gene expression level of neonatal primary keratinocytes.
- Period 1 is a type of clock gene present in the skin that regulates the circadian rhythm by increasing its expression in the morning and decreasing its expression from evening to night due to light stimulation. It is known that the decrease in PERIOD1 expression slows down from night to night.
- the skin health condition improving agent of the present invention can promote the production of PERIOD1, increase the relative expression level of PERIOD1 during the day, and exert a circadian rhythm regulating effect on the skin. Skin health can be improved or maintained.
- the fact that the skin health condition improving agent of the present invention has a period 1 production promoting effect means that, for example, the skin health condition improving agent of the present invention has a period 1 production promoting effect.
- the expression levels of these proteins can be measured, for example, by extracting proteins from treated target cells and using known methods such as SDS-PAGE, Western blotting, ELISA, and immunoprecipitation. These gene expression levels can be targeted, for example, by extracting nucleic acids from treated cells and using known methods such as RT-PCR, quantitative PCR, and microarrays.
- the content of rice sterol ester in the skin health condition improving agent of the present invention is not particularly limited as long as it has an effect of improving skin health condition.
- it may be 0.001 (w/v)% or more, 0.0025 (w/v)% or more, and 0.5 (w/v)% or less, based on the entire agent. , 1 (w/v)% or less.
- it may be 0.05 (w/v)% or more, 0.1 (w/v)% or more, and 2.5 (w/v)% or less, 5 (w/v) % or less.
- the frequency of use or intake of the skin health condition improving agent of the present invention is not particularly limited, and includes, for example, once or more a day, 1 to 3 times a day, etc.
- the skin health condition improving agent of the present invention is used for the purpose of promoting period 1 production, it is preferably used or ingested once a day so that the effect of promoting period 1 production is obtained from early in the morning until the morning.
- the period of use or intake of the skin health condition improving agent of the present invention is not particularly limited, and may be, for example, one week or more, two weeks or more, or four weeks or more.
- the present invention provides pharmaceuticals, cosmetics, or food and drink products.
- the pharmaceutical products, cosmetics, or food and drink products of the present invention may be those containing the skin health condition improving agent of the present invention.
- the pharmaceutical, cosmetic, or food/beverage product of the present invention can be used in the same dosage form as the skin health condition improving agent described above.
- the food or drink of the present invention may be, for example, a health food, a functional food, a food for specified health uses, a supplement, a food for the sick, or the like.
- the form of the food and drink is not particularly limited, and may be, for example, a processed form such as a natural liquid food, a semi-digested nutritional food, an ingredient nutritional food, or a drink, including tea drinks, soft drinks, carbonated drinks, and nutritional drinks.
- the pharmaceutical products, cosmetics, or food and drink products of the present invention may contain pharmaceutically or physiologically acceptable ingredients in addition to the skin health condition improving agent of the present invention.
- Pharmaceutically or physiologically acceptable ingredients are not particularly limited, and include water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, and water-soluble Polymers, surfactants, alcohols, polyhydric alcohols, etc. can be used.
- the pharmaceuticals, cosmetics, or food/drinks of the present invention may further contain other active ingredients used for the purpose of improving skin health conditions.
- Other active ingredients can be used alone or in combination of two or more.
- the present invention includes semaphorin 3A production promoters, myonectin production promoters, arginase 1 production promoters, period 1 production promoters, etc., which contain sterol esters obtained from rice bran.
- the present invention includes a method for improving skin health, which comprises administering an effective amount of the skin health improving agent of the present invention to an animal in need of improvement.
- the animal is not particularly limited, and may be, for example, a human, a non-human mammal, or the like. Mammals other than humans include, but are not limited to, cows, horses, pigs, sheep, goats, llamas, alpacas, camels, rabbits, minks, foxes, chinchillas, geese, ducks, dogs, cats, and the like.
- Test Materials 1.1 Test Substances The following were used as test substances. ⁇ Rice sterol ester (manufacturer: Tsukino Foods Co., Ltd.)
- FBS Culture Medium 5 mL of penicillin/streptomycin solution and 55 mL of fetal bovine serum (FBS) were added to 500 mL of D-MEM (high glucose) to prepare a 10% FBS culture medium. Further, the 10% FBS culture medium was diluted 10 times using a mixed solution of D-MEM and penicillin/streptomycin solution to obtain a 1% FBS culture medium.
- D-MEM high glucose
- test medium 5 mL of penicillin/streptomycin solution and 10 mL of inactivated horse serum were added to 500 mL of D-MEM (high glucose) to prepare a medium for myoblast differentiation (test medium).
- Dedicated culture medium for primary keratinocytes 5 mL of penicillin/streptomycin solution and 1 mL of HKGS were added to 100 mL of EpiLife with 60 ⁇ M Calcium to prepare a culture medium exclusively for primary keratinocytes (special culture medium).
- the supernatant of the cultured cells was aspirated using a suction pump (Hitech).
- Cells were washed by dropping 5 mL of autoclaved Dulbecco's PBS (-) onto the dish using a 10 mL disposable pipette. The solution was then removed using a suction pump.
- 0.5 mL of trypsin/EDTA solution was added to the dish using a micropipette and left standing for 2 minutes.
- Cells were suspended by continuous suction and discharge operations using a micropipette, and 5 mL of 10% FBS culture medium was added using a disposable pipette to stop the reaction.
- the solution after the reaction was stopped was transferred to a 15 mL plastic tube and centrifuged at 1200 rpm for 5 minutes at room temperature using a bucket type centrifuge (Tomy Seiko Co., Ltd.) to accumulate cells.
- the solution was removed using a suction pump, and 10 mL of 10% FBS culture medium was added using a new disposable pipette to suspend the cells.
- Cell numbers were counted using a fully automatic cell counter (Bio-Rad). Specifically, 30 ⁇ L each of trypan blue and cell suspension supplied with the device were mixed, the mixed solution was added to two locations on a dedicated counting slide, and the added portion was inserted into the device to perform measurement. The dilution rate was calculated from the obtained concentration, and a cell suspension of 5.0 ⁇ 10 4 cells/mL was prepared.
- Mouse myoblasts (in the case of myonectin) were used as the test subject, and a cell suspension was prepared in the same manner as in 3.1, except that the concentration of the cell suspension to be prepared was 9.0 ⁇ 10 cells/mL. .
- Cells were seeded at 9.0 ⁇ 10 4 cells/well in a 24-well culture plate (Bio Lamo). The cells were cultured in a CO 2 incubator at 37° C. in a 5% CO 2 atmosphere until the cell density reached 80%.
- Neonatal primary human keratinocytes (in the case of ARG1) Neonatal primary human keratinocytes (HEKn) were used as the test subject, and a cell suspension was prepared in the same manner as in 3.1, except that a dedicated culture medium was used instead of the 10% FBS culture medium.
- RNA extraction for human epidermal keratinocytes (Sema3A, PERIOD1), mouse myoblasts (myonectin)
- RNA extraction was performed.
- the cultured medium in the 24-well culture plate was removed by suction using a suction pump, and 1 mL of sterile Dulbecco's PBS (-) was added per well to wash the medium residue with water. Thereafter, the solution was removed again using the suction pump.
- Centrifugation was performed at 12,000 ⁇ g for 15 minutes at room temperature using a high-speed microcentrifuge. The supernatant was removed by decantation, and the opening of the 1.5 mL tube was placed against a Kimtowel (Nippon Paper Crecia Co., Ltd.) for several seconds to remove drips. 500 ⁇ L of 75% ethanol solution was added and mixed by inversion. Centrifugation was performed at 12,000 ⁇ g for 5 minutes at room temperature using a high-speed microcentrifuge. The supernatant was removed by decantation, and the mouth of the tube was placed on a Kimtowel for a few seconds to remove drippings, and then 500 ⁇ L of 75% ethanol was added once again and mixed by inversion.
- cDNA synthesis was performed using the extracted RNA as a template.
- PrimeScript RT Reagent kit (Takara Bio Inc.) was used for cDNA synthesis.
- a modified version of M-MLV reverse transcriptase Roth MJ, Tanese N and Goff SP (1985) J. Biol. Chem., 260 (16); 9326-9335
- An enzymatic method was performed to convert the entire sequence starting from the complementary site of the primer and oligo-dT primer into DNA.
- Each extracted RNA was appropriately diluted with RNase-free distilled water to form a solution of 50 ⁇ g/ ⁇ L.
- a mixed solution was prepared in a 1.5 mL tube at the following doses. ⁇ 5 ⁇ PrimeScript Buffer (for Real Time) 26 ⁇ L ⁇ Oligo dT Primer 50 ⁇ M 6.5 ⁇ L ⁇ Random 6 mers 100 ⁇ M 6.5 ⁇ L ⁇ RNase free distilled water 84.5 ⁇ L Finally, add the following on ice. ⁇ PrimeScript RT Enzyme Mix I 6.5 ⁇ L 26 reactions total 130 ⁇ L
- Figure 1 shows the gene expression level of semaphorin 3A (Sema3A) in human epidermal keratinocytes (HaCaT) treated with the negative control and treated with 0.1% and 0.5% rice sterol ester.
- Sema3A semaphorin 3A
- HaCaT human epidermal keratinocytes
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Abstract
The present invention provides a skin health condition improving agent characterized by containing a sterol ester that is obtained from rice bran.
Description
本発明は、肌健康状態改善剤に関するものである。
The present invention relates to a skin health condition improving agent.
表皮は皮膚の最も外側に位置しており、紫外線や気温変化などの外部刺激に常に暴露されている。さらに加齢も加わることで肌の正常な機能は徐々に失われる。特に、長期にわたる外部刺激への暴露は、皮膚の神経過敏や肌の老化を促進させ、シミ、シワなどの様々な外見の変化をもたらす。
The epidermis is the outermost layer of the skin and is constantly exposed to external stimuli such as ultraviolet rays and temperature changes. Furthermore, as we age, our skin gradually loses its normal functions. In particular, long-term exposure to external stimuli accelerates skin hypersensitivity and skin aging, leading to various changes in appearance such as age spots and wrinkles.
表皮神経線維の密度の増加はかゆみの主な原因であり、アトピー性皮膚炎や敏感肌などのかゆみを主症状とする病因に顕著である。セマフォリン3A(Sema3A)は、神経の伸長を阻害することが知られている。実際に、Sema3A軟膏は、動物試験において表皮神経過敏およびひっかき行動をかなり抑制することが報告されているが(非特許文献1)、副作用として長期間の使用による、かぶれなどの接触皮膚炎を起こす可能性がある。
An increase in the density of epidermal nerve fibers is the main cause of itch, and is noticeable in pathologies where itch is the main symptom, such as atopic dermatitis and sensitive skin. Semaphorin 3A (Sema3A) is known to inhibit nerve outgrowth. In fact, Sema3A ointment has been reported to considerably suppress epidermal hypersensitivity and scratching behavior in animal tests (Non-Patent Document 1), but as a side effect, long-term use can cause contact dermatitis such as rash. there is a possibility.
酸化ストレスも外部刺激によって誘発される肌への悪影響を及ぼす要因の1つとして挙げられる。特に、酸化ストレスは、体の錆と形容されることもあり、肌の老化を促進することが知られている。アルギナーゼ1(ARG1)は、抗酸化作用によって酸化ストレスを抑制することができ、肌の明るさやシワの改善が期待されている酵素の1つであり、植物由来フィトステロールであるスチグマステロールや麹菌由来の14デヒドロエルゴステロールによって発現が上昇することが報告されている(特許文献1、特許文献2)。
Oxidative stress is also cited as one of the factors that adversely affect the skin induced by external stimuli. In particular, oxidative stress is sometimes described as rust on the body and is known to accelerate skin aging. Arginase 1 (ARG1) is an enzyme that can suppress oxidative stress through its antioxidant effect and is expected to improve skin brightness and wrinkles. It has been reported that the expression is increased by 14-dehydroergosterol (Patent Document 1, Patent Document 2).
本発明は、複数の肌健康状態改善作用を有する肌健康状態改善剤を提供することを課題とする。
An object of the present invention is to provide a skin health condition improving agent that has multiple skin health condition improving effects.
本発明は、上記の課題を解決するために、以下の各発明を包含する。
[1]米糠から得られるステロールエステルを含有することを特徴とする肌健康状態改善剤。
[2]セマフォリン3A産生促進作用、マイオネクチン産生促進作用、アルギナーゼ1産生促進作用およびピリオド1産生促進作用からなる群から選択される少なくとも2つを有する、前記[1]に記載の肌健康状態改善剤。
[3]前記ステロールエステルが、米糠から得られるステロールエステル混合物である、前記[1]または[2]に記載の肌健康状態改善剤。
[4]前記ステロールエステルが、植物ステロールと脂肪酸とのエステル、および/またはトリテルペンアルコールと脂肪酸とのエステルである、前記[1]または[2]に記載の肌健康状態改善剤。
[5]前記[1]または[2]に記載の肌健康状態改善剤を含むことを特徴とする医薬品。
[6]前記[1]または[2]に記載の肌健康状態改善剤を含むことを特徴とする化粧品。
[7]前記[1]または[2]に記載の肌健康状態改善剤を含むことを特徴とする飲食品。
[8]米糠から得られるステロールエステルを含有することを特徴とするセマフォリン3A産生促進剤。
[9]米糠から得られるステロールエステルを含有することを特徴とするマイオネクチン産生促進剤。
[10]米糠から得られるステロールエステルを含有することを特徴とするアルギナーゼ1産生促進剤。
[11]米糠から得られるステロールエステルを含有することを特徴とするピリオド1産生促進剤。 The present invention includes the following inventions in order to solve the above problems.
[1] A skin health condition improving agent characterized by containing a sterol ester obtained from rice bran.
[2] The skin health condition improvement according to [1] above, which has at least two selected from the group consisting of semaphorin 3A production promoting action, meionectin production promoting action, arginase 1 production promoting action and period 1 production promoting action. agent.
[3] The skin health condition improving agent according to [1] or [2] above, wherein the sterol ester is a sterol ester mixture obtained from rice bran.
[4] The skin health condition improving agent according to [1] or [2] above, wherein the sterol ester is an ester of a plant sterol and a fatty acid, and/or an ester of a triterpene alcohol and a fatty acid.
[5] A pharmaceutical product comprising the skin health condition improving agent according to [1] or [2] above.
[6] A cosmetic product comprising the skin health condition improving agent according to [1] or [2] above.
[7] A food or drink product comprising the skin health condition improving agent according to [1] or [2] above.
[8] A semaphorin 3A production promoter characterized by containing a sterol ester obtained from rice bran.
[9] A myonectin production promoter characterized by containing a sterol ester obtained from rice bran.
[10] An arginase 1 production promoter containing a sterol ester obtained from rice bran.
[11] A period 1 production promoter containing a sterol ester obtained from rice bran.
[1]米糠から得られるステロールエステルを含有することを特徴とする肌健康状態改善剤。
[2]セマフォリン3A産生促進作用、マイオネクチン産生促進作用、アルギナーゼ1産生促進作用およびピリオド1産生促進作用からなる群から選択される少なくとも2つを有する、前記[1]に記載の肌健康状態改善剤。
[3]前記ステロールエステルが、米糠から得られるステロールエステル混合物である、前記[1]または[2]に記載の肌健康状態改善剤。
[4]前記ステロールエステルが、植物ステロールと脂肪酸とのエステル、および/またはトリテルペンアルコールと脂肪酸とのエステルである、前記[1]または[2]に記載の肌健康状態改善剤。
[5]前記[1]または[2]に記載の肌健康状態改善剤を含むことを特徴とする医薬品。
[6]前記[1]または[2]に記載の肌健康状態改善剤を含むことを特徴とする化粧品。
[7]前記[1]または[2]に記載の肌健康状態改善剤を含むことを特徴とする飲食品。
[8]米糠から得られるステロールエステルを含有することを特徴とするセマフォリン3A産生促進剤。
[9]米糠から得られるステロールエステルを含有することを特徴とするマイオネクチン産生促進剤。
[10]米糠から得られるステロールエステルを含有することを特徴とするアルギナーゼ1産生促進剤。
[11]米糠から得られるステロールエステルを含有することを特徴とするピリオド1産生促進剤。 The present invention includes the following inventions in order to solve the above problems.
[1] A skin health condition improving agent characterized by containing a sterol ester obtained from rice bran.
[2] The skin health condition improvement according to [1] above, which has at least two selected from the group consisting of semaphorin 3A production promoting action, meionectin production promoting action, arginase 1 production promoting action and period 1 production promoting action. agent.
[3] The skin health condition improving agent according to [1] or [2] above, wherein the sterol ester is a sterol ester mixture obtained from rice bran.
[4] The skin health condition improving agent according to [1] or [2] above, wherein the sterol ester is an ester of a plant sterol and a fatty acid, and/or an ester of a triterpene alcohol and a fatty acid.
[5] A pharmaceutical product comprising the skin health condition improving agent according to [1] or [2] above.
[6] A cosmetic product comprising the skin health condition improving agent according to [1] or [2] above.
[7] A food or drink product comprising the skin health condition improving agent according to [1] or [2] above.
[8] A semaphorin 3A production promoter characterized by containing a sterol ester obtained from rice bran.
[9] A myonectin production promoter characterized by containing a sterol ester obtained from rice bran.
[10] An arginase 1 production promoter containing a sterol ester obtained from rice bran.
[11] A period 1 production promoter containing a sterol ester obtained from rice bran.
本発明により、複数の肌健康状態改善作用を有する肌健康状態改善剤を提供することができる。本発明の肌健康状態改善剤は、医薬品、化粧品または飲食品として好適に利用することができる。
According to the present invention, a skin health condition improving agent having multiple skin health condition improving effects can be provided. The skin health condition improving agent of the present invention can be suitably used as a pharmaceutical, cosmetic, or food/beverage product.
〔肌健康状態改善剤〕
本発明は、肌健康状態改善剤を提供する。本発明の肌健康状態改善剤は、米糠から得られるステロールエステルを含有するものであればよい。本発明における米糠から得られるステロールエステルは、米糠から公知の方法により得られるステロールエステルであれば特に限定されず、植物ステロールと脂肪酸とのエステルを含有するものであってもよく、トリテルペンアルコールと脂肪酸とのエステルを含有するものであってもよく、これらの混合物を含有するものであってもよく、ステロールエステルの他に米糠から得られる別の成分をさらに含有する、米糠から得られるステロールエステル混合物を含有するものであってもよい。 [Skin health condition improving agent]
The present invention provides a skin health condition improving agent. The skin health condition improving agent of the present invention may be any agent as long as it contains a sterol ester obtained from rice bran. The sterol ester obtained from rice bran in the present invention is not particularly limited as long as it is a sterol ester obtained from rice bran by a known method, and may contain esters of plant sterols and fatty acids, triterpene alcohols and fatty acids. A sterol ester mixture obtained from rice bran that further contains another component obtained from rice bran in addition to the sterol ester. It may contain.
本発明は、肌健康状態改善剤を提供する。本発明の肌健康状態改善剤は、米糠から得られるステロールエステルを含有するものであればよい。本発明における米糠から得られるステロールエステルは、米糠から公知の方法により得られるステロールエステルであれば特に限定されず、植物ステロールと脂肪酸とのエステルを含有するものであってもよく、トリテルペンアルコールと脂肪酸とのエステルを含有するものであってもよく、これらの混合物を含有するものであってもよく、ステロールエステルの他に米糠から得られる別の成分をさらに含有する、米糠から得られるステロールエステル混合物を含有するものであってもよい。 [Skin health condition improving agent]
The present invention provides a skin health condition improving agent. The skin health condition improving agent of the present invention may be any agent as long as it contains a sterol ester obtained from rice bran. The sterol ester obtained from rice bran in the present invention is not particularly limited as long as it is a sterol ester obtained from rice bran by a known method, and may contain esters of plant sterols and fatty acids, triterpene alcohols and fatty acids. A sterol ester mixture obtained from rice bran that further contains another component obtained from rice bran in addition to the sterol ester. It may contain.
ステロールとは、シクロペンタノペルヒドロフェナントレン骨格(ステラン骨格)を有する化合物(ステロイド)のうち、3位に水酸基を有する化合物をいう。ステロールは、通常、遊離状、エステル型、配糖体等の形で、動植物界に幅広く分布している。本発明の肌健康状態改善剤に含有される植物ステロールと脂肪酸とのエステルおよび/またはトリテルペンアルコールと脂肪酸とのエステルは、脂肪酸とのエステル型のステロールである。
Sterol refers to a compound having a hydroxyl group at the 3-position among compounds (steroids) having a cyclopentanoperhydrophenanthrene skeleton (sterane skeleton). Sterols are generally widely distributed in the animal and plant kingdoms in the form of free, ester, glycosides, and the like. The ester of a plant sterol and a fatty acid and/or the ester of a triterpene alcohol and a fatty acid contained in the skin health condition improving agent of the present invention is an ester type sterol with a fatty acid.
ステロールは、炭素数27~30であり、1個の二重結合を5/6位、7/8位、8/9位またはその他の位置に有している不飽和化合物であってもよく、水素化により得られる飽和化合物であってもよい。ステロールのうち植物に多く存在する植物ステロールとしては、β-シトステロール、スチグマステロール、カンペステロール等が挙げられる。トリテルペンアルコールは、炭素数30のステロールであり、米糠または米油中に多く含まれる成分である。
The sterol may be an unsaturated compound having 27 to 30 carbon atoms and having one double bond at the 5/6 position, 7/8 position, 8/9 position or other position, It may also be a saturated compound obtained by hydrogenation. Among sterols, plant sterols that are abundant in plants include β-sitosterol, stigmasterol, campesterol, and the like. Triterpene alcohol is a sterol with 30 carbon atoms, and is a component abundantly contained in rice bran or rice oil.
植物ステロールと脂肪酸とのエステルおよびトリテルペンアルコールと脂肪酸とのエステルにおけるエステルは、式(1):
R’CO-OH (1)
[式中、R’COは炭素数14~22であり、二重結合数0~3のいずれかの直鎖脂肪酸アシル基である。]
で示されるカルボン酸に由来し得る。式(1)で示されるカルボン酸としては、例えば、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレン酸、アラキジン酸、ベヘン酸等が挙げられる。 The ester of plant sterol and fatty acid and the ester of triterpene alcohol and fatty acid have the formula (1):
R'CO-OH (1)
[In the formula, R'CO is a straight chain fatty acyl group having 14 to 22 carbon atoms and 0 to 3 double bonds. ]
It can be derived from the carboxylic acid represented by Examples of the carboxylic acid represented by formula (1) include myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, and behenic acid.
R’CO-OH (1)
[式中、R’COは炭素数14~22であり、二重結合数0~3のいずれかの直鎖脂肪酸アシル基である。]
で示されるカルボン酸に由来し得る。式(1)で示されるカルボン酸としては、例えば、ミリスチン酸、パルミチン酸、ステアリン酸、オレイン酸、リノール酸、リノレン酸、アラキジン酸、ベヘン酸等が挙げられる。 The ester of plant sterol and fatty acid and the ester of triterpene alcohol and fatty acid have the formula (1):
R'CO-OH (1)
[In the formula, R'CO is a straight chain fatty acyl group having 14 to 22 carbon atoms and 0 to 3 double bonds. ]
It can be derived from the carboxylic acid represented by Examples of the carboxylic acid represented by formula (1) include myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, arachidic acid, and behenic acid.
植物ステロールと脂肪酸とのエステルおよび/またはトリテルペンアルコールと脂肪酸とのエステルは、米糠から公知の方法を用いて精製したステロールエステルであってもよく、市販のステロールエステルであってもよい。米糠からステロールエステルを得る方法は、特に限定されず、例えば、米糠から米油を製造する過程で生じるスープストックや脱臭スカム等の副産物を原料とし、アセトンやヘキセン等の溶媒で抽出、精製する方法、公知の文献(特開2014-47311号公報、谷口ら、日本食品科学工学会誌、2012年、59巻、7号、p301-318)に記載の方法、米糠から得られた植物ステロールおよびトリテルペンアルコールの少なくとも1種を、非特異的または特異的にエステル化する酵素を使用してエステル化する方法などが挙げられる。市販のステロールエステルとしては、例えば、築野食品工業株式会社製のライステロールエステル(商品名)等が挙げられる。
The ester of a plant sterol and a fatty acid and/or the ester of a triterpene alcohol and a fatty acid may be a sterol ester purified from rice bran using a known method, or a commercially available sterol ester. The method for obtaining sterol esters from rice bran is not particularly limited, and for example, a method in which by-products such as soup stock and deodorized scum produced in the process of producing rice oil from rice bran are used as raw materials, and extracted and purified with a solvent such as acetone or hexene. , a method described in a known document (Unexamined Japanese Patent Publication No. 2014-47311, Taniguchi et al., Journal of the Japanese Society of Food Science and Technology, 2012, Vol. 59, No. 7, p301-318), plant sterols and triterpene alcohols obtained from rice bran. Examples include a method of esterifying at least one of the above using an enzyme that esterifies non-specifically or specifically. Examples of commercially available sterol esters include Rice sterol ester (trade name) manufactured by Tsukino Foods Co., Ltd.
本発明の肌健康状態改善剤に含有されるステロールエステルは、食用にも利用される成分であるため、生体への安全性が高く、継続的に長期間の使用または摂取が可能である。本発明の肌健康状態改善剤は、複数の肌健康状態改善作用を有するので、肌表面または真皮の健康状態を総合的かつ効率的に改善、または良好に維持することができると考えられる。
Since the sterol ester contained in the skin health condition improving agent of the present invention is a component that is also used for food, it is highly safe for living organisms and can be continuously used or ingested over a long period of time. Since the skin health condition improving agent of the present invention has a plurality of skin health condition improving effects, it is considered that the health condition of the skin surface or dermis can be comprehensively and efficiently improved or maintained in good condition.
本発明の肌健康状態改善剤は、肌健康状態改善作用を有することを特徴とする。本発明における肌健康状態改善作用は、肌表面または真皮の健康状態を改善できるものであれば、特に限定されず、例えば、セマフォリン3A産生促進作用、マイオネクチン産生促進作用、アルギナーゼ1産生促進作用およびピリオド1産生促進作用からなる群から選択される少なくとも1つを有するものであってもよく、その群から選択される少なくとも2つを有するものであってもよい。
The skin health condition improving agent of the present invention is characterized by having a skin health condition improving effect. The skin health condition improving effect in the present invention is not particularly limited as long as it can improve the health condition of the skin surface or dermis, and includes, for example, semaphorin 3A production promoting effect, myonectin production promoting effect, arginase 1 production promoting effect, and It may have at least one selected from the group consisting of period 1 production promoting action, or it may have at least two selected from the group.
セマフォリン3A(Sema3A)は、かゆみを感じる神経線維の伸長を抑えるタンパク質の一種であり、アトピー性皮膚炎においては皮膚のバリア機能の低下により、表皮付近における神経線維の伸長を抑えるSema3Aの産生が減少し、神経線維が伸長することでかゆみの刺激を受けやすくなることが知られている。本発明の肌健康機能改善剤は、肌表面(特に、表皮)に働きかけ、神経反発因子であるSema3Aの産生を促進することで、かゆみの抑制作用や敏感肌の改善作用を奏し、肌健康状態を改善、または良好に維持することができる。
Semaphorin 3A (Sema3A) is a type of protein that suppresses the elongation of nerve fibers that feel itching.In atopic dermatitis, the production of Sema3A, which suppresses the elongation of nerve fibers near the epidermis, is reduced due to a decline in the skin barrier function. It is known that the nerve fibers decrease and elongate, making them more susceptible to itching stimuli. The skin health function improving agent of the present invention acts on the skin surface (particularly the epidermis) and promotes the production of Sema3A, a neurorepulsive factor, thereby suppressing itch and improving sensitive skin, thereby improving skin health. can be improved or maintained in good condition.
本発明の肌健康状態改善剤が、セマフォリン3A産生促進作用を有する場合、本発明の肌健康状態改善剤がセマフォリン3A産生促進作用を有することは、例えば、表皮角化細胞を本発明の肌健康状態改善剤で処理し、表皮角化細胞のSema3Aのタンパク質発現量またはSema3A遺伝子発現量を測定することにより、確認することができる。
When the skin health condition improving agent of the present invention has a semaphorin 3A production promoting effect, the fact that the skin health condition improving agent of the present invention has a semaphorin 3A production promoting effect means that, for example, the skin health condition improving agent of the present invention has a semaphorin 3A production promoting effect. This can be confirmed by treating the skin with a health condition improving agent and measuring the amount of Sema3A protein expression or Sema3A gene expression in epidermal keratinocytes.
マイオネクチン(myonectin)は、骨格筋から分泌される栄養応答性マイオカインであり、筋収縮により血流に放出され、血液成分として皮膚に運ばれることにより、シミのもとであるメラニンの生成を抑えられることや物理的刺激によって生じる炎症因子を抑制できることが知られている。本発明の肌健康状態改善剤は、骨格筋から分泌されるmyonectinの産生を促進することで、メラニンの生成抑制作用や炎症抑制作用を奏し、肌健康状態を改善、または良好に維持することができる。
Myonectin is a nutrient-responsive myokine secreted from skeletal muscles.It is released into the bloodstream by muscle contraction and transported to the skin as a blood component, thereby suppressing the production of melanin, which is the cause of age spots. It is known that inflammatory factors caused by physical stimulation can be suppressed. The skin health condition improving agent of the present invention promotes the production of myonectin secreted from skeletal muscles, exhibits melanin production suppressing effects and inflammation suppressing effects, and improves or maintains good skin health conditions. can.
本発明の肌健康状態改善剤が、マイオネクチン産生促進作用を有する場合、本発明の肌健康状態改善剤がマイオネクチン産生促進作用を有することは、例えば、分化直後の筋細胞を本発明の肌健康状態改善剤で処理し、筋細胞のmyonectinのタンパク質発現量またはmyonectin遺伝子発現量を測定することにより、確認することができる。
When the skin health condition improving agent of the present invention has an effect of promoting myonectin production, the fact that the skin health condition improving agent of the present invention has a myonectin production promoting effect means that, for example, the skin health condition of the present invention can be transferred to muscle cells immediately after differentiation. This can be confirmed by treating the muscle cells with an improving agent and measuring the myonectin protein expression level or myonectin gene expression level in muscle cells.
アルギナーゼ1(ARG1)は、皮膚の紫外線による酸化ストレスの防御に関係しているタンパク質であり、皮膚における紫外線からの酸化ストレスによるバリア機能の低下や炎症を抑制することができる。紫外線による酸化ストレスは、シワやシミだけでなく、皮膚のバリアを破壊して加齢による乾燥に影響することが知られている。本発明の肌健康状態改善剤は、皮膚中のアルギナーゼ1の産生を促進することで、抗酸化作用、肌の明るさ・シワの改善作用等を奏し、肌健康状態を改善、または良好に維持することができる。
Arginase 1 (ARG1) is a protein involved in protecting the skin from oxidative stress caused by ultraviolet rays, and can suppress the decline in barrier function and inflammation caused by oxidative stress from ultraviolet rays in the skin. It is known that oxidative stress caused by ultraviolet rays not only causes wrinkles and age spots, but also destroys the skin barrier and contributes to age-related dryness. The skin health condition improving agent of the present invention promotes the production of arginase 1 in the skin, thereby exhibiting antioxidant effects, skin brightening/wrinkle improvement effects, etc., thereby improving or maintaining the skin health condition. can do.
本発明の肌健康状態改善剤が、アルギナーゼ1産生促進作用を有する場合、本発明の肌健康状態改善剤がアルギナーゼ1産生促進作用を有することは、例えば、新生児由来初代角化細胞を本発明の肌健康状態改善剤で処理し、新生児由来初代角化細胞のARG1のタンパク質発現量またはARG1遺伝子発現量を測定することにより、確認することができる。
When the skin health condition improving agent of the present invention has an effect of promoting arginase 1 production, the fact that the skin health condition improving agent of the present invention has an effect of promoting arginase 1 production means, for example, that the skin health condition improving agent of the present invention has an effect of promoting arginase 1 production. This can be confirmed by treating the skin health condition improving agent and measuring the ARG1 protein expression level or ARG1 gene expression level of neonatal primary keratinocytes.
ピリオド1(PERIOD1)は、光の明暗刺激により、朝に発現増加し、夕方から夜にかけて発現減少することで概日リズムを調節する、皮膚に存在する時計遺伝子の一種であり、老化モデルでは昼から夜にかけてPERIOD1の発現減少が鈍くなることが知られている。本発明の肌健康状態改善剤は、朝に使用することで、ピリオド1の産生を促進することができ、相対的な日中のPERIOD1発現量を高め、皮膚における概日リズム調節作用を奏し、肌健康状態を改善、または良好に維持することができる。
Period 1 (PERIOD1) is a type of clock gene present in the skin that regulates the circadian rhythm by increasing its expression in the morning and decreasing its expression from evening to night due to light stimulation. It is known that the decrease in PERIOD1 expression slows down from night to night. When used in the morning, the skin health condition improving agent of the present invention can promote the production of PERIOD1, increase the relative expression level of PERIOD1 during the day, and exert a circadian rhythm regulating effect on the skin. Skin health can be improved or maintained.
本発明の肌健康状態改善剤が、ピリオド1産生促進作用を有する場合、本発明の肌健康状態改善剤がピリオド1産生促進作用を有することは、例えば、表皮角化細胞を本発明の肌健康状態改善剤で処理し、表皮角化細胞のPERIOD1のタンパク質発現量またはPERIOD1遺伝子発現量を測定することにより、確認することができる。
When the skin health condition improving agent of the present invention has a period 1 production promoting effect, the fact that the skin health condition improving agent of the present invention has a period 1 production promoting effect means that, for example, the skin health condition improving agent of the present invention has a period 1 production promoting effect. This can be confirmed by treating the epidermal keratinocytes with a condition improving agent and measuring the PERIOD1 protein expression level or PERIOD1 gene expression level in epidermal keratinocytes.
本発明の肌健康状態改善剤が、セマフォリン3A産生促進作用、マイオネクチン産生促進作用、アルギナーゼ1産生促進作用およびピリオド1産生促進作用からなる群から選択される少なくとも1つ、または少なくとも2つの肌健康状態改善作用を有する場合、本発明の肌健康状態改善剤がこれらの肌健康状態改善作用を有することは、表皮角化細胞、分化直後の筋細胞または新生児由来初代角化細胞を本発明の肌健康状態改善剤で処理し、Sema3A、myonectin、ARG1またはPERIOD1のタンパク質発現量またはSema3A、myonectin、ARG1またはPERIOD1の遺伝子発現量を測定することにより確認することができる。これらのタンパク質発現量は、例えば、処理後の標的細胞からタンパク質を抽出して、SDS-PAGE、ウェスタンブロッティング、ELISA、免疫沈降等の公知の方法を用いて測定することができる。これらの遺伝子発現量は、例えば、処理後の細胞から核酸を抽出して、RT-PCR、定量的PCR、マイクロアレイ等の公知の方法を用いて標的することができる。
The skin health condition improving agent of the present invention has at least one or at least two skin health conditions selected from the group consisting of semaphorin 3A production promoting action, meionectin production promoting action, arginase 1 production promoting action and period 1 production promoting action. In the case where the skin health condition improving agent of the present invention has the skin health condition improving effect, it means that the skin health condition improving agent of the present invention has these skin health condition improving effects. This can be confirmed by treating with a health condition improving agent and measuring the protein expression level of Sema3A, myonectin, ARG1 or PERIOD1 or the gene expression level of Sema3A, myonectin, ARG1 or PERIOD1. The expression levels of these proteins can be measured, for example, by extracting proteins from treated target cells and using known methods such as SDS-PAGE, Western blotting, ELISA, and immunoprecipitation. These gene expression levels can be targeted, for example, by extracting nucleic acids from treated cells and using known methods such as RT-PCR, quantitative PCR, and microarrays.
本発明の肌健康状態改善剤におけるライステロールエステルの含有量は、肌健康状態改善作用が奏される範囲であれば、特に限定されず、ライステロールエステルの含有量は、例えば、肌健康状態改善剤全体に対して、細胞組織に使用する場合、0.001(w/v)%以上、0.0025(w/v)%以上であってもよく、0.5(w/v)%以下、1(w/v)%以下であってもよい。生体に使用する場合、0.05(w/v)%以上、0.1(w/v)%以上であってもよく、2.5(w/v)%以下、5(w/v)%以下であってもよい。
The content of rice sterol ester in the skin health condition improving agent of the present invention is not particularly limited as long as it has an effect of improving skin health condition. When used for cell tissue, it may be 0.001 (w/v)% or more, 0.0025 (w/v)% or more, and 0.5 (w/v)% or less, based on the entire agent. , 1 (w/v)% or less. When used in living organisms, it may be 0.05 (w/v)% or more, 0.1 (w/v)% or more, and 2.5 (w/v)% or less, 5 (w/v) % or less.
本発明の肌健康状態改善剤の投与対象は、動物であれば特に限定されず、例えば、ヒト、ヒト以外の哺乳動物(ウシ、ウマ、ブタ、ヒツジ、ヤギ、リャマ、アルパカ、ラクダ、ウサギ、ミンク、キツネ、チンチラ、ガチョウ、アヒル等の家畜動物、イヌ、ネコ等の愛玩動物)等が挙げられる。
The subject to which the skin health condition improving agent of the present invention can be administered is not particularly limited as long as it is an animal; for example, humans, non-human mammals (cows, horses, pigs, sheep, goats, llamas, alpacas, camels, rabbits, Domestic animals such as mink, fox, chinchilla, goose, duck, pet animals such as dogs and cats), etc.
本発明の肌健康状態改善剤の使用または摂取頻度としては、特に限定されず、例えば、1日1回以上、1日1~3回の範囲内等が挙げられる。本発明の肌健康状態改善剤を、ピリオド1産生促進作用を目的として使用する場合、早朝から朝にかけてピリオド1産生促進作用が得られるように、1日1回、使用または摂取することが好ましい。
The frequency of use or intake of the skin health condition improving agent of the present invention is not particularly limited, and includes, for example, once or more a day, 1 to 3 times a day, etc. When the skin health condition improving agent of the present invention is used for the purpose of promoting period 1 production, it is preferably used or ingested once a day so that the effect of promoting period 1 production is obtained from early in the morning until the morning.
本発明の肌健康状態改善剤の使用または摂取期間としては、特に限定されず、例えば、1週間以上、2週間以上、4週間以上であってもよい。
The period of use or intake of the skin health condition improving agent of the present invention is not particularly limited, and may be, for example, one week or more, two weeks or more, or four weeks or more.
本発明の肌健康状態改善剤の剤形は、特に限定されず、例えば、低粘度液体、ローション等の液剤、懸濁剤、乳剤、ゲル剤、ペースト剤、クリーム剤、フォーム剤、シート剤、軟膏、粉剤、エアゾール剤、吸入剤、点眼剤、点鼻剤、坐剤、貼付剤等の外用剤であってもよく、錠剤、顆粒剤、カプセル剤、内服用液剤等の内服剤であってもよく、筋肉、血管内に直接投与する注射剤であってもよい。
The dosage form of the skin health condition improving agent of the present invention is not particularly limited, and examples thereof include low-viscosity liquids, liquids such as lotions, suspensions, emulsions, gels, pastes, creams, foams, sheets, They may be external preparations such as ointments, powders, aerosols, inhalants, eye drops, nasal drops, suppositories, patches, etc., or internal preparations such as tablets, granules, capsules, or oral liquid preparations. Alternatively, it may be an injection that is administered directly into muscles or blood vessels.
〔医薬品、化粧品または飲食品〕
本発明は、医薬品、化粧品または飲食品を提供する。本発明の医薬品、化粧品または飲食品は、本発明の肌健康状態改善剤を含有するものであればよい。本発明の医薬品、化粧品または飲食品の形態は、上述の肌健康状態改善剤の剤形と同様の形態で使用することができる。 [Pharmaceuticals, cosmetics, or food and beverages]
The present invention provides pharmaceuticals, cosmetics, or food and drink products. The pharmaceutical products, cosmetics, or food and drink products of the present invention may be those containing the skin health condition improving agent of the present invention. The pharmaceutical, cosmetic, or food/beverage product of the present invention can be used in the same dosage form as the skin health condition improving agent described above.
本発明は、医薬品、化粧品または飲食品を提供する。本発明の医薬品、化粧品または飲食品は、本発明の肌健康状態改善剤を含有するものであればよい。本発明の医薬品、化粧品または飲食品の形態は、上述の肌健康状態改善剤の剤形と同様の形態で使用することができる。 [Pharmaceuticals, cosmetics, or food and beverages]
The present invention provides pharmaceuticals, cosmetics, or food and drink products. The pharmaceutical products, cosmetics, or food and drink products of the present invention may be those containing the skin health condition improving agent of the present invention. The pharmaceutical, cosmetic, or food/beverage product of the present invention can be used in the same dosage form as the skin health condition improving agent described above.
本発明が飲食品である場合、本発明の飲食品は、例えば、健康食品、機能性食品、特定保健用食品、サプリメント、病者用食品等であってもよい。飲食品の形態は、特に限定されず、例えば、自然流動食、半消化態栄養食、成分栄養食、ドリンク剤等の加工形態であってもよく、茶飲料、清涼飲料、炭酸飲料、栄養飲料、果実飲料、乳酸飲料等の飲料、そば、うどん、中華麺、即席麺等の麺類、飴、キャンディー、ガム、チョコレート、スナック菓子、ビスケット、ゼリー、ジャム、クリーム、焼き菓子、パン等の菓子およびパン類、かまぼこ、ハム、ソーセージ等の水産・畜産加工食品、加工乳、発酵乳等の乳製品、サラダ油、てんぷら油、マーガリン、マヨネーズ、ショートニング、ホイップクリーム、ドレッシング等の油脂および油脂加工食品、ソース、たれ等の調味料、カレー、シチュー、丼、お粥、雑炊等のレトルトパウチ食品、アイスクリーム、シャーベット、かき氷等の冷菓などであってもよい。
When the present invention is a food or drink, the food or drink of the present invention may be, for example, a health food, a functional food, a food for specified health uses, a supplement, a food for the sick, or the like. The form of the food and drink is not particularly limited, and may be, for example, a processed form such as a natural liquid food, a semi-digested nutritional food, an ingredient nutritional food, or a drink, including tea drinks, soft drinks, carbonated drinks, and nutritional drinks. , beverages such as fruit drinks and lactic acid drinks, noodles such as soba, udon, Chinese noodles, and instant noodles, sweets and breads such as candies, candies, gum, chocolate, snack foods, biscuits, jellies, jams, creams, baked goods, and bread. Processed marine and livestock foods such as kamaboko, ham, and sausages, dairy products such as processed milk and fermented milk, fats and oils and processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings, sauces, It may also be seasonings such as sauce, retort pouch foods such as curry, stew, rice bowls, porridge, rice porridge, etc., frozen desserts such as ice cream, sherbet, shaved ice, etc.
本発明の医薬品、化粧品または飲食品は、本発明の肌健康状態改善剤の他に、薬学的もしくは生理学的に許容される成分を含んでいてもよい。薬学的もしくは生理学的に許容される成分としては、特に限定されず、水、油脂類、ロウ類、炭化水素類、脂肪酸類、高級アルコール類、エステル類、植物抽出エキス類、ビタミン類、水溶性高分子、界面活性剤、アルコール、多価アルコール等を使用することができる。
The pharmaceutical products, cosmetics, or food and drink products of the present invention may contain pharmaceutically or physiologically acceptable ingredients in addition to the skin health condition improving agent of the present invention. Pharmaceutically or physiologically acceptable ingredients are not particularly limited, and include water, oils and fats, waxes, hydrocarbons, fatty acids, higher alcohols, esters, plant extracts, vitamins, and water-soluble Polymers, surfactants, alcohols, polyhydric alcohols, etc. can be used.
本発明の医薬品、化粧品または飲食品は、必要に応じて、医薬品、医薬部外品、化粧品、飲食品等の組成物に使用される公知の添加剤、例えば、酸化防止剤、増粘剤、保存剤、pH調整剤、安定化剤、刺激軽減剤、防腐剤、着色剤、香料等を添加することができる。添加剤は、1種単独、または2種以上を組み合わせて使用することができる。
The pharmaceuticals, cosmetics, or food/beverage products of the present invention may contain known additives used in compositions of pharmaceuticals, quasi-drugs, cosmetics, food/beverage products, etc., such as antioxidants, thickeners, etc., as necessary. Preservatives, pH adjusters, stabilizers, irritation reducers, preservatives, colorants, fragrances, and the like can be added. The additives can be used alone or in combination of two or more.
本発明の医薬品、化粧品または飲食品は、肌健康状態改善作用を目的として使用される他の有効成分をさらに添加することができる。他の有効成分は、1種単独、または2種以上を組み合わせて使用することができる。
The pharmaceuticals, cosmetics, or food/drinks of the present invention may further contain other active ingredients used for the purpose of improving skin health conditions. Other active ingredients can be used alone or in combination of two or more.
本発明には、米糠から得られるステロールエステルを含有する、セマフォリン3A産生促進剤、マイオネクチン産生促進剤、アルギナーゼ1産生促進剤、ピリオド1産生促進剤等が含まれる。
The present invention includes semaphorin 3A production promoters, myonectin production promoters, arginase 1 production promoters, period 1 production promoters, etc., which contain sterol esters obtained from rice bran.
本発明は、本発明の肌健康状態改善剤の有効量を、肌健康状態改善を必要とする動物に投与することを特徴とする肌健康状態改善方法を包含する。動物は、特に限定されないが、例えば、ヒト、ヒト以外の哺乳動物等であってもよい。ヒト以外の哺乳動物は、特に限定されないが、ウシ、ウマ、ブタ、ヒツジ、ヤギ、リャマ、アルパカ、ラクダ、ウサギ、ミンク、キツネ、チンチラ、ガチョウ、アヒル、イヌ、ネコ等が挙げられる。
The present invention includes a method for improving skin health, which comprises administering an effective amount of the skin health improving agent of the present invention to an animal in need of improvement. The animal is not particularly limited, and may be, for example, a human, a non-human mammal, or the like. Mammals other than humans include, but are not limited to, cows, horses, pigs, sheep, goats, llamas, alpacas, camels, rabbits, minks, foxes, chinchillas, geese, ducks, dogs, cats, and the like.
以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。
Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.
〔実施例1:肌健康状態関連遺伝子発現量の確認〕
[試験材料および方法]
1 試験材料
1.1 被験物質
被験物質として以下を使用した。
・ライステロールエステル(製造元:築野食品工業株式会社) [Example 1: Confirmation of expression levels of genes related to skin health status]
[Test materials and methods]
1 Test Materials 1.1 Test Substances The following were used as test substances.
・Rice sterol ester (manufacturer: Tsukino Foods Co., Ltd.)
[試験材料および方法]
1 試験材料
1.1 被験物質
被験物質として以下を使用した。
・ライステロールエステル(製造元:築野食品工業株式会社) [Example 1: Confirmation of expression levels of genes related to skin health status]
[Test materials and methods]
1 Test Materials 1.1 Test Substances The following were used as test substances.
・Rice sterol ester (manufacturer: Tsukino Foods Co., Ltd.)
1.2 試薬
試薬として以下を使用した。
・D-MEM(高グルコース)(製造元:富士フィルム和光純薬株式会社)
・ウシ胎児血清(製造元:Bosera)
・非働化ウマ血清(製造元:Sigma-Aldrich)
・Gibco EpiLife with 60μM Calcium(製造元:Thermo Fisher Scientific)
・Gibco Human Keratinocyte Growth Supplement (HKGS)(製造元:Thermo Fisher Scientific)
・0.25w/v%トリプシン-1mmol/1EDTA・4Na Solution with phenol Red (トリプシン/EDTA溶液)(製造元:富士フィルム和光純薬株式会社)
・ペニシリン/ストレプトマイシン溶液(×100)(製造元:富士フィルム和光純薬株式会社)
・ダルベッコPBS(-)「ニッスイ」(製造元:日水製薬株式会社)
・ジメチルスルホキシド(DMSO)(製造元:富士フィルム和光純薬株式会社)
・アイソジェンII(製造元:富士フィルム和光純薬株式会社(株式会社ニッポンジーン))
・2-プロパノール(製造元:富士フィルム和光純薬株式会社)
・エタノール(99.5)(製造元:富士フィルム和光純薬株式会社)
・Distilled Water, Deionized, Sterile(RNase free蒸留水)(製造元:株式会社ニッポンジーン)
・PrimeScript RT Reagent kit(製造元:タカラバイオ株式会社)
・PowerUp SYBR Green Master Mix(製造元:applied biosystems) 1.2 Reagents The following were used as reagents.
・D-MEM (high glucose) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Fetal bovine serum (manufacturer: Bosera)
・Inactivated horse serum (manufacturer: Sigma-Aldrich)
・Gibco EpiLife with 60μM Calcium (Manufacturer: Thermo Fisher Scientific)
・Gibco Human Keratinocyte Growth Supplement (HKGS) (Manufacturer: Thermo Fisher Scientific)
・0.25w/v% trypsin-1mmol/1EDTA・4Na Solution with phenol Red (trypsin/EDTA solution) (manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Penicillin/streptomycin solution (x100) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Dulbecco PBS (-) “Nissui” (Manufacturer: Nissui Pharmaceutical Co., Ltd.)
・Dimethyl sulfoxide (DMSO) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Isogen II (manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd. (Nippon Gene Co., Ltd.))
・2-Propanol (manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Ethanol (99.5) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Distilled Water, Deionized, Sterile (RNase free distilled water) (Manufacturer: Nippon Gene Co., Ltd.)
・PrimeScript RT Reagent kit (Manufacturer: Takara Bio Inc.)
・PowerUp SYBR Green Master Mix (manufacturer: applied biosystems)
試薬として以下を使用した。
・D-MEM(高グルコース)(製造元:富士フィルム和光純薬株式会社)
・ウシ胎児血清(製造元:Bosera)
・非働化ウマ血清(製造元:Sigma-Aldrich)
・Gibco EpiLife with 60μM Calcium(製造元:Thermo Fisher Scientific)
・Gibco Human Keratinocyte Growth Supplement (HKGS)(製造元:Thermo Fisher Scientific)
・0.25w/v%トリプシン-1mmol/1EDTA・4Na Solution with phenol Red (トリプシン/EDTA溶液)(製造元:富士フィルム和光純薬株式会社)
・ペニシリン/ストレプトマイシン溶液(×100)(製造元:富士フィルム和光純薬株式会社)
・ダルベッコPBS(-)「ニッスイ」(製造元:日水製薬株式会社)
・ジメチルスルホキシド(DMSO)(製造元:富士フィルム和光純薬株式会社)
・アイソジェンII(製造元:富士フィルム和光純薬株式会社(株式会社ニッポンジーン))
・2-プロパノール(製造元:富士フィルム和光純薬株式会社)
・エタノール(99.5)(製造元:富士フィルム和光純薬株式会社)
・Distilled Water, Deionized, Sterile(RNase free蒸留水)(製造元:株式会社ニッポンジーン)
・PrimeScript RT Reagent kit(製造元:タカラバイオ株式会社)
・PowerUp SYBR Green Master Mix(製造元:applied biosystems) 1.2 Reagents The following were used as reagents.
・D-MEM (high glucose) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Fetal bovine serum (manufacturer: Bosera)
・Inactivated horse serum (manufacturer: Sigma-Aldrich)
・Gibco EpiLife with 60μM Calcium (Manufacturer: Thermo Fisher Scientific)
・Gibco Human Keratinocyte Growth Supplement (HKGS) (Manufacturer: Thermo Fisher Scientific)
・0.25w/v% trypsin-1mmol/1EDTA・4Na Solution with phenol Red (trypsin/EDTA solution) (manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Penicillin/streptomycin solution (x100) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Dulbecco PBS (-) “Nissui” (Manufacturer: Nissui Pharmaceutical Co., Ltd.)
・Dimethyl sulfoxide (DMSO) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Isogen II (manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd. (Nippon Gene Co., Ltd.))
・2-Propanol (manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Ethanol (99.5) (Manufacturer: Fuji Film Wako Pure Chemical Industries, Ltd.)
・Distilled Water, Deionized, Sterile (RNase free distilled water) (Manufacturer: Nippon Gene Co., Ltd.)
・PrimeScript RT Reagent kit (Manufacturer: Takara Bio Inc.)
・PowerUp SYBR Green Master Mix (manufacturer: applied biosystems)
2 試薬の調製
2.1 FBS培養培地
D-MEM(高グルコース)500mLに5mLのペニシリン/ストレプトマイシン溶液および55mLのウシ胎児血清(FBS)を加え10%FBS培養培地とした。また、D-MEMおよびペニシリン/ストレプトマイシン溶液の混合溶液を用いて10%FBS培養培地を10倍希釈し、1%FBS培養培地とした。 2 Preparation of Reagents 2.1 FBS Culture Medium 5 mL of penicillin/streptomycin solution and 55 mL of fetal bovine serum (FBS) were added to 500 mL of D-MEM (high glucose) to prepare a 10% FBS culture medium. Further, the 10% FBS culture medium was diluted 10 times using a mixed solution of D-MEM and penicillin/streptomycin solution to obtain a 1% FBS culture medium.
2.1 FBS培養培地
D-MEM(高グルコース)500mLに5mLのペニシリン/ストレプトマイシン溶液および55mLのウシ胎児血清(FBS)を加え10%FBS培養培地とした。また、D-MEMおよびペニシリン/ストレプトマイシン溶液の混合溶液を用いて10%FBS培養培地を10倍希釈し、1%FBS培養培地とした。 2 Preparation of Reagents 2.1 FBS Culture Medium 5 mL of penicillin/streptomycin solution and 55 mL of fetal bovine serum (FBS) were added to 500 mL of D-MEM (high glucose) to prepare a 10% FBS culture medium. Further, the 10% FBS culture medium was diluted 10 times using a mixed solution of D-MEM and penicillin/streptomycin solution to obtain a 1% FBS culture medium.
2.2 筋芽細胞分化用培地(試験培地)
D-MEM(高グルコース)500mLに5mLのペニシリン/ストレプトマイシン溶液および非働化ウマ血清10mLを加え、筋芽細胞分化用培地(試験培地)とした。 2.2 Myoblast differentiation medium (test medium)
5 mL of penicillin/streptomycin solution and 10 mL of inactivated horse serum were added to 500 mL of D-MEM (high glucose) to prepare a medium for myoblast differentiation (test medium).
D-MEM(高グルコース)500mLに5mLのペニシリン/ストレプトマイシン溶液および非働化ウマ血清10mLを加え、筋芽細胞分化用培地(試験培地)とした。 2.2 Myoblast differentiation medium (test medium)
5 mL of penicillin/streptomycin solution and 10 mL of inactivated horse serum were added to 500 mL of D-MEM (high glucose) to prepare a medium for myoblast differentiation (test medium).
2.3 初代角化細胞専用培養培地(専用培養培地)
EpiLife with 60μM Calcium100mLに5mLのペニシリン/ストレプトマイシン溶液およびHKGS1mLを加え、初代角化細胞専用培養培地(専用培養培地)とした。 2.3 Dedicated culture medium for primary keratinocytes (dedicated culture medium)
5 mL of penicillin/streptomycin solution and 1 mL of HKGS were added to 100 mL of EpiLife with 60 μM Calcium to prepare a culture medium exclusively for primary keratinocytes (special culture medium).
EpiLife with 60μM Calcium100mLに5mLのペニシリン/ストレプトマイシン溶液およびHKGS1mLを加え、初代角化細胞専用培養培地(専用培養培地)とした。 2.3 Dedicated culture medium for primary keratinocytes (dedicated culture medium)
5 mL of penicillin/streptomycin solution and 1 mL of HKGS were added to 100 mL of EpiLife with 60 μM Calcium to prepare a culture medium exclusively for primary keratinocytes (special culture medium).
2.4 ダルベッコPBS(-)
電子天秤で秤量した粉末のダルベッコPBS(-)「ニッスイ」9.8gを蒸留水1000mLに溶解した後、オートクレーブ滅菌し、細胞培養用のダルベッコPBS(-)を得た。 2.4 Dulbecco PBS (-)
After dissolving 9.8 g of Dulbecco's PBS (-) "Nissui" powder weighed using an electronic balance in 1000 mL of distilled water, it was sterilized in an autoclave to obtain Dulbecco's PBS (-) for cell culture.
電子天秤で秤量した粉末のダルベッコPBS(-)「ニッスイ」9.8gを蒸留水1000mLに溶解した後、オートクレーブ滅菌し、細胞培養用のダルベッコPBS(-)を得た。 2.4 Dulbecco PBS (-)
After dissolving 9.8 g of Dulbecco's PBS (-) "Nissui" powder weighed using an electronic balance in 1000 mL of distilled water, it was sterilized in an autoclave to obtain Dulbecco's PBS (-) for cell culture.
2.5 75%エタノール溶液
30mLのエタノール(99.5)および10mLのRNase free蒸留水を混合し、40mLの75%エタノール溶液を得た。 2.5 75% ethanol solution 30 mL of ethanol (99.5) and 10 mL of RNase free distilled water were mixed to obtain 40 mL of 75% ethanol solution.
30mLのエタノール(99.5)および10mLのRNase free蒸留水を混合し、40mLの75%エタノール溶液を得た。 2.5 75% ethanol solution 30 mL of ethanol (99.5) and 10 mL of RNase free distilled water were mixed to obtain 40 mL of 75% ethanol solution.
2.6 ライステロールエステル含有培養培地
微量電子天秤(ザルトリウス社)を用いてライステロールエステルを100mg、500mgをそれぞれ1.5mL容チューブに秤量した後、マイクロピペット(サーモフィッシャーサイエンティフィック社)にてそれぞれ1mLのDMSOを添加した。小型ヒートブロック(アズワン社)で50℃に加温し、転倒混和により澄明な10%、50%ライステロールエステル溶液を得た。450μLのDMSOを1.5mL容チューブに添加したものを50℃に加温し、そこに10%ライステロールエステル溶液を50μL加え溶解し、1%ライステロールエステル溶液を得た。15mL容プラスチックチューブ(ビオラモ社)にそれぞれ1%FBS培養培地または専用培養培地をディスポーザブルピペット(ビオラモ社)にて4.95mL添加し、マイクロピペットにて1%、10%、50%ライステロールエステル溶液を各50μL添加して、それぞれ0.01%、0.1%、0.5%ライステロールエステルを含有するFBS培養培地または専用培養培地を得た。なお、それぞれの陰性コントロールとして、1%FBS培養培地または専用培養培地4.95mLにライステロールエステルを含まないDMSOを50μL添加、溶解した溶液を使用した。 2.6 Rice sterol ester-containing culture medium Weighed 100 mg and 500 mg of rice sterol ester into 1.5 mL tubes using an electronic microbalance (Sartorius), and then added them using a micropipette (Thermo Fisher Scientific). 1 mL of DMSO was added each. The mixture was heated to 50° C. using a small heat block (As One Corporation) and mixed by inversion to obtain clear 10% and 50% rice sterol ester solutions. 450 μL of DMSO was added to a 1.5 mL tube and heated to 50° C., and 50 μL of 10% rice sterol ester solution was added and dissolved therein to obtain a 1% rice sterol ester solution. Add 4.95 mL of 1% FBS culture medium or dedicated culture medium to a 15 mL plastic tube (Violamo Co., Ltd.) using a disposable pipette (Biolamo Co., Ltd.), and add 1%, 10%, and 50% rice sterol ester solutions using a micropipette. 50 μL of each were added to obtain FBS culture medium or dedicated culture medium containing 0.01%, 0.1%, and 0.5% rice sterol ester, respectively. As a negative control, a solution prepared by adding and dissolving 50 μL of DMSO containing no rice sterol ester in 4.95 mL of 1% FBS culture medium or dedicated culture medium was used.
微量電子天秤(ザルトリウス社)を用いてライステロールエステルを100mg、500mgをそれぞれ1.5mL容チューブに秤量した後、マイクロピペット(サーモフィッシャーサイエンティフィック社)にてそれぞれ1mLのDMSOを添加した。小型ヒートブロック(アズワン社)で50℃に加温し、転倒混和により澄明な10%、50%ライステロールエステル溶液を得た。450μLのDMSOを1.5mL容チューブに添加したものを50℃に加温し、そこに10%ライステロールエステル溶液を50μL加え溶解し、1%ライステロールエステル溶液を得た。15mL容プラスチックチューブ(ビオラモ社)にそれぞれ1%FBS培養培地または専用培養培地をディスポーザブルピペット(ビオラモ社)にて4.95mL添加し、マイクロピペットにて1%、10%、50%ライステロールエステル溶液を各50μL添加して、それぞれ0.01%、0.1%、0.5%ライステロールエステルを含有するFBS培養培地または専用培養培地を得た。なお、それぞれの陰性コントロールとして、1%FBS培養培地または専用培養培地4.95mLにライステロールエステルを含まないDMSOを50μL添加、溶解した溶液を使用した。 2.6 Rice sterol ester-containing culture medium Weighed 100 mg and 500 mg of rice sterol ester into 1.5 mL tubes using an electronic microbalance (Sartorius), and then added them using a micropipette (Thermo Fisher Scientific). 1 mL of DMSO was added each. The mixture was heated to 50° C. using a small heat block (As One Corporation) and mixed by inversion to obtain clear 10% and 50% rice sterol ester solutions. 450 μL of DMSO was added to a 1.5 mL tube and heated to 50° C., and 50 μL of 10% rice sterol ester solution was added and dissolved therein to obtain a 1% rice sterol ester solution. Add 4.95 mL of 1% FBS culture medium or dedicated culture medium to a 15 mL plastic tube (Violamo Co., Ltd.) using a disposable pipette (Biolamo Co., Ltd.), and add 1%, 10%, and 50% rice sterol ester solutions using a micropipette. 50 μL of each were added to obtain FBS culture medium or dedicated culture medium containing 0.01%, 0.1%, and 0.5% rice sterol ester, respectively. As a negative control, a solution prepared by adding and dissolving 50 μL of DMSO containing no rice sterol ester in 4.95 mL of 1% FBS culture medium or dedicated culture medium was used.
3 細胞培養
3.1 ヒト表皮角化細胞の培養(Sema3A、PERIOD1の場合)
ヒト表皮角化細胞(Human Skin Keratinocyte:HaCaT)を試験対象とした。
キャビネット下で無菌状態を維持しつつ、液体窒素中のバイアルに保管されている細胞を解凍し、10cmカルチャーディッシュ(ビオラモ社)に播種した。10%FBS培養培地を10mL使用した。以降細胞取り扱いの一切の操作はキャビネット下にてエタノール除菌および火炎滅菌のうえ実施した。CO2インキュベーター(PHC社)により37℃、5%CO2雰囲気下で3日間培養した。 3 Cell culture 3.1 Culture of human epidermal keratinocytes (for Sema3A, PERIOD1)
The test subject was human skin keratinocytes (HaCaT).
While maintaining sterile conditions under a cabinet, cells stored in a vial in liquid nitrogen were thawed and seeded in a 10 cm culture dish (Biolamo). 10 mL of 10% FBS culture medium was used. Thereafter, all cell handling operations were performed under the cabinet after sterilization with ethanol and flame sterilization. The cells were cultured for 3 days at 37° C. in a 5% CO 2 atmosphere using a CO 2 incubator (PHC).
3.1 ヒト表皮角化細胞の培養(Sema3A、PERIOD1の場合)
ヒト表皮角化細胞(Human Skin Keratinocyte:HaCaT)を試験対象とした。
キャビネット下で無菌状態を維持しつつ、液体窒素中のバイアルに保管されている細胞を解凍し、10cmカルチャーディッシュ(ビオラモ社)に播種した。10%FBS培養培地を10mL使用した。以降細胞取り扱いの一切の操作はキャビネット下にてエタノール除菌および火炎滅菌のうえ実施した。CO2インキュベーター(PHC社)により37℃、5%CO2雰囲気下で3日間培養した。 3 Cell culture 3.1 Culture of human epidermal keratinocytes (for Sema3A, PERIOD1)
The test subject was human skin keratinocytes (HaCaT).
While maintaining sterile conditions under a cabinet, cells stored in a vial in liquid nitrogen were thawed and seeded in a 10 cm culture dish (Biolamo). 10 mL of 10% FBS culture medium was used. Thereafter, all cell handling operations were performed under the cabinet after sterilization with ethanol and flame sterilization. The cells were cultured for 3 days at 37° C. in a 5% CO 2 atmosphere using a CO 2 incubator (PHC).
培養後の培養細胞の上清をサクションポンプ(ハイテック社)で吸引した。オートクレーブ滅菌したダルベッコPBS(-)5mLを10mL容ディスポーザブルピペットで、ディッシュ上に滴下して細胞を洗浄した。その後サクションポンプにより溶液を除いた。0.5mLのトリプシン/EDTA溶液をマイクロピペットにてディッシュに添加し、2分静置した。マイクロピペットの連続的な吸引排出操作により細胞を懸濁し、ディスポーザブルピペットにて10%FBS培養培地5mLを加えて反応を停止した。
After culturing, the supernatant of the cultured cells was aspirated using a suction pump (Hitech). Cells were washed by dropping 5 mL of autoclaved Dulbecco's PBS (-) onto the dish using a 10 mL disposable pipette. The solution was then removed using a suction pump. 0.5 mL of trypsin/EDTA solution was added to the dish using a micropipette and left standing for 2 minutes. Cells were suspended by continuous suction and discharge operations using a micropipette, and 5 mL of 10% FBS culture medium was added using a disposable pipette to stop the reaction.
15mL容プラスチックチューブに反応停止後の溶液を移し、バケットタイプ遠心機(トミー精工社)にて、1200rpm、5分、室温で遠心して細胞を集積した。サクションポンプにより溶液を除き、新しいディスポーザブルピペットにて10%FBS培養培地10mLを加え細胞を懸濁した。全自動セルカウンター(バイオラッド社)にて細胞数を計測した。具体的には機器に付属のトリパンブルーと細胞懸濁液各30μLを混合し、専用カウンティングスライドの2か所に混合溶液を添加し、添加部分を機器内に挿入して計測を実施した。得られた濃度から希釈率を算出し、5.0×104細胞/mLの細胞懸濁液を作製した。
The solution after the reaction was stopped was transferred to a 15 mL plastic tube and centrifuged at 1200 rpm for 5 minutes at room temperature using a bucket type centrifuge (Tomy Seiko Co., Ltd.) to accumulate cells. The solution was removed using a suction pump, and 10 mL of 10% FBS culture medium was added using a new disposable pipette to suspend the cells. Cell numbers were counted using a fully automatic cell counter (Bio-Rad). Specifically, 30 μL each of trypan blue and cell suspension supplied with the device were mixed, the mixed solution was added to two locations on a dedicated counting slide, and the added portion was inserted into the device to perform measurement. The dilution rate was calculated from the obtained concentration, and a cell suspension of 5.0×10 4 cells/mL was prepared.
24ウェルカルチャープレート(ビオラモ社)に5.0×104細胞/ウェルとなるよう細胞を播種した。CO2インキュベーターにより37℃、5%CO2雰囲気下で一晩培養した。0.01%、0.1%、0.5%ライステロールエステル含有FBS培養培地またはその陰性コントロールを1ウェルあたり1mL添加し、CO2インキュベーターにより37℃、5%CO2雰囲気下で24時間培養した。各群4ウェルを使用した(n=4)。
Cells were seeded at 5.0×10 4 cells/well in a 24-well culture plate (Bio Lamo). The cells were cultured overnight at 37° C. in a 5% CO 2 atmosphere in a CO 2 incubator. Add 1 mL of FBS culture medium containing 0.01%, 0.1%, 0.5% rice sterol ester or its negative control per well, and culture in a CO 2 incubator at 37°C in a 5% CO 2 atmosphere for 24 hours. did. Four wells were used for each group (n=4).
3.2 マウス筋芽細胞の培養(myonectinの場合)
マウス筋芽細胞(C2C12)を試験対象とし、作製する細胞懸濁液の濃度を9.0×104細胞/mLとすること以外は、3.1と同様にして細胞懸濁液を作製した。 3.2 Culture of mouse myoblasts (in the case of myonectin)
Mouse myoblasts (C2C12) were used as the test subject, and a cell suspension was prepared in the same manner as in 3.1, except that the concentration of the cell suspension to be prepared was 9.0 × 10 cells/mL. .
マウス筋芽細胞(C2C12)を試験対象とし、作製する細胞懸濁液の濃度を9.0×104細胞/mLとすること以外は、3.1と同様にして細胞懸濁液を作製した。 3.2 Culture of mouse myoblasts (in the case of myonectin)
Mouse myoblasts (C2C12) were used as the test subject, and a cell suspension was prepared in the same manner as in 3.1, except that the concentration of the cell suspension to be prepared was 9.0 × 10 cells/mL. .
24ウェルカルチャープレート(ビオラモ社)に9.0×104細胞/ウェルとなるように細胞を播種した。CO2インキュベーターにより37℃、5%CO2雰囲気下で細胞密度が80%になるまで培養した。
Cells were seeded at 9.0×10 4 cells/well in a 24-well culture plate (Bio Lamo). The cells were cultured in a CO 2 incubator at 37° C. in a 5% CO 2 atmosphere until the cell density reached 80%.
培養後、24ウェルカルチャープレートから培養上清のみサクションポンプで取り除き、試験培地1mLを添加してCO2インキュベーター内にて37℃、5%CO2雰囲気下で分化を開始(day0)した。分化開始の翌日(day1)、再度、培養上清を取り除き試験培地1mLに交換した。この操作を分化終了まで2、3日おきに計3回行った。
After culturing, only the culture supernatant was removed from the 24-well culture plate using a suction pump, 1 mL of the test medium was added, and differentiation was started (day 0) in a CO 2 incubator at 37° C. under a 5% CO 2 atmosphere. The day after the start of differentiation (day 1), the culture supernatant was removed again and replaced with 1 mL of the test medium. This operation was performed a total of 3 times every 2 to 3 days until the differentiation was completed.
分化開始から6~7日後、顕微鏡BZ-X800(KEYENCE社)下にて十分な分化細胞を確認してから分化終了とした。分化終了後、0.1%、0.5%ライステロールエステル含有FBS培養培地またはその陰性コントロールで1ウェルあたり1mL置換し、CO2インキュベーターにより37℃、5%CO2雰囲気下で24時間培養した。各群4ウェルを使用した(n=4)。
Six to seven days after the start of differentiation, sufficient differentiated cells were confirmed under a microscope BZ-X800 (KEYENCE), and differentiation was terminated. After differentiation was completed, 1 mL of FBS culture medium containing 0.1% or 0.5% rice sterol ester or its negative control was substituted per well, and cultured in a CO 2 incubator at 37°C in a 5% CO 2 atmosphere for 24 hours. . Four wells were used for each group (n=4).
3.3 新生児由来初代ヒト角化細胞の培養(ARG1の場合)
新生児由来初代ヒト角化細胞(HEKn)を試験対象とし、10%FBS培養培地の代わりに専用培養培地を使用すること以外は、3.1と同様にして細胞懸濁液を作製した。 3.3 Culture of newborn-derived primary human keratinocytes (in the case of ARG1)
Neonatal primary human keratinocytes (HEKn) were used as the test subject, and a cell suspension was prepared in the same manner as in 3.1, except that a dedicated culture medium was used instead of the 10% FBS culture medium.
新生児由来初代ヒト角化細胞(HEKn)を試験対象とし、10%FBS培養培地の代わりに専用培養培地を使用すること以外は、3.1と同様にして細胞懸濁液を作製した。 3.3 Culture of newborn-derived primary human keratinocytes (in the case of ARG1)
Neonatal primary human keratinocytes (HEKn) were used as the test subject, and a cell suspension was prepared in the same manner as in 3.1, except that a dedicated culture medium was used instead of the 10% FBS culture medium.
24ウェルカルチャープレート(ビオラモ社)に5.0×104細胞/ウェルとなるよう細胞を播種した。CO2インキュベーターにより37℃、5%CO2雰囲気下で一晩培養した。0.01%、0.1%ライステロールエステル含有専用培養培地またはその陰性コントロールを1ウェルあたり1mL添加し、CO2インキュベーターにより37℃、5%CO2雰囲気下で24時間培養した。各群4ウェルを使用した(n=4)。
Cells were seeded at 5.0×10 4 cells/well in a 24-well culture plate (Bio Lamo). The cells were cultured overnight at 37° C. in a 5% CO 2 atmosphere in a CO 2 incubator. 1 mL of a dedicated culture medium containing 0.01% and 0.1% rice sterol ester or its negative control was added per well, and cultured in a CO 2 incubator at 37° C. in a 5% CO 2 atmosphere for 24 hours. Four wells were used for each group (n=4).
4 遺伝子発現量解析
4.1 RNA抽出(ヒト表皮角化細胞(Sema3A、PERIOD1)、マウス筋芽細胞(myonectin)の場合)
3にて得られた培養細胞を用い、グアニジンチオイソシアネート-フェノールクロロホルム変法(Chomczynski P and Sacchi N (1987) Anal. Biochem., 162(1): 156-159)に準じ、以下の操作にてRNA抽出を実施した。
培養した24ウェルカルチャープレートの培地をサクションポンプにて吸引除去し、1ウェルあたり1mLの滅菌ダルベッコPBS(-)を添加して培地残渣を水洗した。その後、再度サクションポンプにて溶液を除去した。 4 Gene expression level analysis 4.1 RNA extraction (for human epidermal keratinocytes (Sema3A, PERIOD1), mouse myoblasts (myonectin))
Using the cultured cells obtained in step 3, perform the following procedure according to the modified guanidine thioisocyanate-phenol chloroform method (Chomczynski P and Sacchi N (1987) Anal. Biochem., 162(1): 156-159). RNA extraction was performed.
The cultured medium in the 24-well culture plate was removed by suction using a suction pump, and 1 mL of sterile Dulbecco's PBS (-) was added per well to wash the medium residue with water. Thereafter, the solution was removed again using the suction pump.
4.1 RNA抽出(ヒト表皮角化細胞(Sema3A、PERIOD1)、マウス筋芽細胞(myonectin)の場合)
3にて得られた培養細胞を用い、グアニジンチオイソシアネート-フェノールクロロホルム変法(Chomczynski P and Sacchi N (1987) Anal. Biochem., 162(1): 156-159)に準じ、以下の操作にてRNA抽出を実施した。
培養した24ウェルカルチャープレートの培地をサクションポンプにて吸引除去し、1ウェルあたり1mLの滅菌ダルベッコPBS(-)を添加して培地残渣を水洗した。その後、再度サクションポンプにて溶液を除去した。 4 Gene expression level analysis 4.1 RNA extraction (for human epidermal keratinocytes (Sema3A, PERIOD1), mouse myoblasts (myonectin))
Using the cultured cells obtained in step 3, perform the following procedure according to the modified guanidine thioisocyanate-phenol chloroform method (Chomczynski P and Sacchi N (1987) Anal. Biochem., 162(1): 156-159). RNA extraction was performed.
The cultured medium in the 24-well culture plate was removed by suction using a suction pump, and 1 mL of sterile Dulbecco's PBS (-) was added per well to wash the medium residue with water. Thereafter, the solution was removed again using the suction pump.
250μLのISOGEN II(ニッポンジーン社)を各ウェルに加え、ピペッテイングで細胞を溶解し、溶解液を1.5mL容チューブに回収した。100μLのRNase free蒸留水(ニッポンジーン社)を加えボルテックスで混合した。室温で15分静置した後、微量高速遠心機(トミー精工社)を用い、12,000×gで15分間、室温で遠心した。沈殿を吸引しないように250μLを目安に上清を回収し、新しい1.5mL容チューブに移した。250μLの2-プロパノール(富士フィルム和光純薬社)を加え、転倒混和した後、室温で15分静置した。
250 μL of ISOGEN II (Nippon Gene) was added to each well, the cells were lysed by pipetting, and the lysate was collected into a 1.5 mL tube. 100 μL of RNase-free distilled water (Nippon Gene) was added and mixed by vortexing. After standing at room temperature for 15 minutes, the mixture was centrifuged at 12,000×g for 15 minutes at room temperature using a high-speed microcentrifuge (Tomy Seiko Co., Ltd.). The supernatant was collected in an amount of 250 μL so as not to aspirate the precipitate, and transferred to a new 1.5 mL tube. 250 μL of 2-propanol (Fuji Film Wako Pure Chemical Industries, Ltd.) was added, mixed by inversion, and then allowed to stand at room temperature for 15 minutes.
微量高速遠心機を用い、12,000×gで15分間、室温で遠心した。デカントにより上清を除き、1.5mL容チューブの口をキムタオル(日本製紙クレシア社)に数秒当てて液だれを除去した。500μLの75%エタノール溶液を加え、転倒混和した。微量高速遠心機を用い、12,000×gで5分間、室温で遠心した。デカントにより上清を除き、チューブの口をキムタオルに数秒当てて液だれを除去した後、もう一度500μLの75%エタノールを加え、転倒混和した。微量高速遠心機を用い、12,000×gで5分間、室温で遠心した。デカントにより上清を除き、チューブの口をキムタオルに数秒当てて水分を切り、再度軽い遠心を行うことで1.5mL容チューブ底面に溶液を集積した。底面の溶液をマイクロピペットで除去し、その後キムワイプ(日本製紙クレシア社)の上に口が下になるように1.5mL容チューブを立て、15分間風乾した。各1.5mL容チューブに10μLのRNase free蒸留水を加え、RNAを溶解した。マイクロプレートリーダーEpoch(BioTek社)を用い、吸光度にてRNAの濃度を算出した。
Centrifugation was performed at 12,000×g for 15 minutes at room temperature using a high-speed microcentrifuge. The supernatant was removed by decantation, and the opening of the 1.5 mL tube was placed against a Kimtowel (Nippon Paper Crecia Co., Ltd.) for several seconds to remove drips. 500 μL of 75% ethanol solution was added and mixed by inversion. Centrifugation was performed at 12,000×g for 5 minutes at room temperature using a high-speed microcentrifuge. The supernatant was removed by decantation, and the mouth of the tube was placed on a Kimtowel for a few seconds to remove drippings, and then 500 μL of 75% ethanol was added once again and mixed by inversion. Centrifugation was performed at 12,000×g for 5 minutes at room temperature using a high-speed microcentrifuge. The supernatant was removed by decantation, the mouth of the tube was placed on a Kimtowel for a few seconds to drain water, and the solution was collected at the bottom of the 1.5 mL tube by performing light centrifugation again. The solution at the bottom was removed with a micropipette, and then a 1.5 mL tube was placed on top of a Kimwipe (Nippon Paper Crecia Co., Ltd.) with the mouth facing down and air-dried for 15 minutes. 10 μL of RNase-free distilled water was added to each 1.5 mL tube to dissolve the RNA. The concentration of RNA was calculated from absorbance using a microplate reader Epoch (BioTek).
4.2 RNA抽出(新生児由来初代ヒト角化細胞(ARG1)の場合)
ISOGEN IIの添加量、RNase free蒸留水の添加量、上清の回収量、および2-プロパノールの添加量をそれぞれ倍量とすること以外は、4.1と同様にしてRNA抽出を行い、RNAの濃度を算出した。 4.2 RNA extraction (for neonatal primary human keratinocytes (ARG1))
RNA was extracted in the same manner as in 4.1, except that the amount of ISOGEN II added, the amount of RNase free distilled water added, the amount of supernatant recovered, and the amount of 2-propanol added were doubled. The concentration of was calculated.
ISOGEN IIの添加量、RNase free蒸留水の添加量、上清の回収量、および2-プロパノールの添加量をそれぞれ倍量とすること以外は、4.1と同様にしてRNA抽出を行い、RNAの濃度を算出した。 4.2 RNA extraction (for neonatal primary human keratinocytes (ARG1))
RNA was extracted in the same manner as in 4.1, except that the amount of ISOGEN II added, the amount of RNase free distilled water added, the amount of supernatant recovered, and the amount of 2-propanol added were doubled. The concentration of was calculated.
4.3 cDNA合成
抽出されたRNAを鋳型にcDNA合成を行った。cDNA合成はPrimeScript RT Reagent kit(タカラバイオ社)を用いた。具体的にはM-MLV逆転写酵素(Roth MJ,Tanese N and Goff SP(1985)J.Biol.Chem.,260(16);9326-9335)の改変体の活性を利用し、ランダムヘキサマープライマーおよびオリゴdTプライマーの相補部位を起点とする全配列をDNAに変換する酵素法を実施した。
各抽出RNAをRNase free蒸留水にて適宜希釈し、50μg/μLの溶液とした。マイクロピペットを用い、1.5mL容チューブにて以下の用量で混合液を調製した。
・ 5 × PrimeScript Buffer (for Real Time) 26μL
・ Oligo dT Primer 50μM 6.5μL
・ Random 6 mers 100μM 6.5μL
・ RNase free蒸留水 84.5μL
最後に氷上で以下を添加する。
・ PrimeScript RT Enzyme Mix I 6.5μL
26反応分 total 130μL 4.3 cDNA synthesis cDNA synthesis was performed using the extracted RNA as a template. PrimeScript RT Reagent kit (Takara Bio Inc.) was used for cDNA synthesis. Specifically, we utilize the activity of a modified version of M-MLV reverse transcriptase (Roth MJ, Tanese N and Goff SP (1985) J. Biol. Chem., 260 (16); 9326-9335) to generate random hexamers. An enzymatic method was performed to convert the entire sequence starting from the complementary site of the primer and oligo-dT primer into DNA.
Each extracted RNA was appropriately diluted with RNase-free distilled water to form a solution of 50 μg/μL. Using a micropipette, a mixed solution was prepared in a 1.5 mL tube at the following doses.
・5 × PrimeScript Buffer (for Real Time) 26μL
・Oligo dT Primer 50μM 6.5μL
・Random 6 mers 100μM 6.5μL
・RNase free distilled water 84.5μL
Finally, add the following on ice.
・PrimeScript RT Enzyme Mix I 6.5μL
26 reactions total 130μL
抽出されたRNAを鋳型にcDNA合成を行った。cDNA合成はPrimeScript RT Reagent kit(タカラバイオ社)を用いた。具体的にはM-MLV逆転写酵素(Roth MJ,Tanese N and Goff SP(1985)J.Biol.Chem.,260(16);9326-9335)の改変体の活性を利用し、ランダムヘキサマープライマーおよびオリゴdTプライマーの相補部位を起点とする全配列をDNAに変換する酵素法を実施した。
各抽出RNAをRNase free蒸留水にて適宜希釈し、50μg/μLの溶液とした。マイクロピペットを用い、1.5mL容チューブにて以下の用量で混合液を調製した。
・ 5 × PrimeScript Buffer (for Real Time) 26μL
・ Oligo dT Primer 50μM 6.5μL
・ Random 6 mers 100μM 6.5μL
・ RNase free蒸留水 84.5μL
最後に氷上で以下を添加する。
・ PrimeScript RT Enzyme Mix I 6.5μL
26反応分 total 130μL 4.3 cDNA synthesis cDNA synthesis was performed using the extracted RNA as a template. PrimeScript RT Reagent kit (Takara Bio Inc.) was used for cDNA synthesis. Specifically, we utilize the activity of a modified version of M-MLV reverse transcriptase (Roth MJ, Tanese N and Goff SP (1985) J. Biol. Chem., 260 (16); 9326-9335) to generate random hexamers. An enzymatic method was performed to convert the entire sequence starting from the complementary site of the primer and oligo-dT primer into DNA.
Each extracted RNA was appropriately diluted with RNase-free distilled water to form a solution of 50 μg/μL. Using a micropipette, a mixed solution was prepared in a 1.5 mL tube at the following doses.
・5 × PrimeScript Buffer (for Real Time) 26μL
・Oligo dT Primer 50μM 6.5μL
・Random 6 mers 100μM 6.5μL
・RNase free distilled water 84.5μL
Finally, add the following on ice.
・PrimeScript RT Enzyme Mix I 6.5μL
26 reactions total 130μL
冷却したラック上に8連PCRチューブ(サーモフィッシャーサイエンティフィック社)を準備し、前記混合液を5μLずつ加えた。混合液を加えた8連PCRチューブの蓋に50μg/μL RNA溶液を5μLずつスポットした。蓋を閉め、転倒混和またはタッピングで溶液を混合後、スイング型プレート遠心機(久保田製作所社)にて溶液をチューブ底面に集積した。サーマルサイクラー(アプライドバイオシステムズ社)を用い、以下のプログラムで逆転写反応を実施した後、反応液に50μLの蒸留水を加え希釈した。
I) 37℃ 15分
II) 85℃ 5秒
III) 4℃ 保温 Eight PCR tubes (Thermo Fisher Scientific) were prepared on a cooled rack, and 5 μL of the above mixture was added to each tube. 5 μL each of the 50 μg/μL RNA solution was spotted on the lid of the 8 consecutive PCR tubes to which the mixture had been added. After closing the lid and mixing the solution by inversion or tapping, the solution was collected on the bottom of the tube using a swing-type plate centrifuge (Kubota Seisakusho Co., Ltd.). After performing a reverse transcription reaction using a thermal cycler (Applied Biosystems) according to the following program, the reaction solution was diluted by adding 50 μL of distilled water.
I) 37℃ 15 minutes II) 85℃ 5 seconds III) 4℃ Keep warm
I) 37℃ 15分
II) 85℃ 5秒
III) 4℃ 保温 Eight PCR tubes (Thermo Fisher Scientific) were prepared on a cooled rack, and 5 μL of the above mixture was added to each tube. 5 μL each of the 50 μg/μL RNA solution was spotted on the lid of the 8 consecutive PCR tubes to which the mixture had been added. After closing the lid and mixing the solution by inversion or tapping, the solution was collected on the bottom of the tube using a swing-type plate centrifuge (Kubota Seisakusho Co., Ltd.). After performing a reverse transcription reaction using a thermal cycler (Applied Biosystems) according to the following program, the reaction solution was diluted by adding 50 μL of distilled water.
I) 37℃ 15 minutes II) 85℃ 5 seconds III) 4℃ Keep warm
4.4 リアルタイム定量PCR(RT-qPCR)
4.3にて合成されたcDNAライブラリーを用い、各目的遺伝子の発現量をリアルタイム定量PCR(Higuchi R et al. (1993) Biotechnology, 11: 1026-1030)で評価した。PowerUp SYBR Green Master Mix(アプライドバイオシステムズ社)による、サイバーグリーン法(Kitahara T et al. (2004) Hear Res., 196: 39-48)を実施した。プライマーは過去の文献(Kim J et al. (2017) J. Microbiol. Biotechnol., 27(2): 242-250、Seo GY et al. (2019) Int. Mol. Sci., 20(3) 536、Buommino E et al. (2018) Med. Myco., 56(8): 987-993、Yang M et al., (2013), Lipids Health Dis, 12, Article number 104、Susana B et al., (2000), J. Invest. Dermatol, 115(4), 757-760、非特許文献1)を参考に以下の配列をデザインし、サーモフィッシャーサイエンティフィック社より購入した。 4.4 Real-time quantitative PCR (RT-qPCR)
Using the cDNA library synthesized in 4.3, the expression level of each target gene was evaluated by real-time quantitative PCR (Higuchi R et al. (1993) Biotechnology, 11: 1026-1030). The Cyber Green method (Kitahara T et al. (2004) Hear Res., 196: 39-48) was performed using PowerUp SYBR Green Master Mix (Applied Biosystems). The primers were based on previous literature (Kim J et al. (2017) J. Microbiol. Biotechnol., 27(2): 242-250, Seo GY et al. (2019) Int. Mol. Sci., 20(3) 536 , Buommino E et al. (2018) Med. Myco., 56(8): 987-993, Yang M et al., (2013), Lipids Health Dis, 12, Article number 104, Susana B et al., ( 2000), J. Invest. Dermatol, 115(4), 757-760, the following sequence was designed with reference to Non-Patent Document 1) and purchased from Thermo Fisher Scientific.
4.3にて合成されたcDNAライブラリーを用い、各目的遺伝子の発現量をリアルタイム定量PCR(Higuchi R et al. (1993) Biotechnology, 11: 1026-1030)で評価した。PowerUp SYBR Green Master Mix(アプライドバイオシステムズ社)による、サイバーグリーン法(Kitahara T et al. (2004) Hear Res., 196: 39-48)を実施した。プライマーは過去の文献(Kim J et al. (2017) J. Microbiol. Biotechnol., 27(2): 242-250、Seo GY et al. (2019) Int. Mol. Sci., 20(3) 536、Buommino E et al. (2018) Med. Myco., 56(8): 987-993、Yang M et al., (2013), Lipids Health Dis, 12, Article number 104、Susana B et al., (2000), J. Invest. Dermatol, 115(4), 757-760、非特許文献1)を参考に以下の配列をデザインし、サーモフィッシャーサイエンティフィック社より購入した。 4.4 Real-time quantitative PCR (RT-qPCR)
Using the cDNA library synthesized in 4.3, the expression level of each target gene was evaluated by real-time quantitative PCR (Higuchi R et al. (1993) Biotechnology, 11: 1026-1030). The Cyber Green method (Kitahara T et al. (2004) Hear Res., 196: 39-48) was performed using PowerUp SYBR Green Master Mix (Applied Biosystems). The primers were based on previous literature (Kim J et al. (2017) J. Microbiol. Biotechnol., 27(2): 242-250, Seo GY et al. (2019) Int. Mol. Sci., 20(3) 536 , Buommino E et al. (2018) Med. Myco., 56(8): 987-993, Yang M et al., (2013), Lipids Health Dis, 12, Article number 104, Susana B et al., ( 2000), J. Invest. Dermatol, 115(4), 757-760, the following sequence was designed with reference to Non-Patent Document 1) and purchased from Thermo Fisher Scientific.
各目的遺伝子を増幅するためのフォワードおよびリバースプライマー溶液(濃度100μM)のそれぞれ10μLを使用し、80μLの蒸留水で希釈し、100μLの各プライマーが10μMとなるプレミックス溶液を調製した。以下の溶液を1.5mL容チューブにて混合し、反応溶液を調製した(一種のプライマーセットにつき1本作製した)。
Power Up SYBR Green Master Mix(2×) 130μL
プライマー 10μM プレミックス溶液 20.8μL
滅菌精製水 57.2μL
Total 208μL 10 μL each of forward and reverse primer solutions (concentration 100 μM) for amplifying each target gene were used and diluted with 80 μL of distilled water to prepare a premix solution in which 100 μL of each primer was 10 μM. The following solutions were mixed in a 1.5 mL tube to prepare a reaction solution (one primer set was prepared for each type of primer set).
Power Up SYBR Green Master Mix (2×) 130μL
Primer 10μM premix solution 20.8μL
Sterile purified water 57.2μL
Total 208μL
Power Up SYBR Green Master Mix(2×) 130μL
プライマー 10μM プレミックス溶液 20.8μL
滅菌精製水 57.2μL
Total 208μL 10 μL each of forward and reverse primer solutions (concentration 100 μM) for amplifying each target gene were used and diluted with 80 μL of distilled water to prepare a premix solution in which 100 μL of each primer was 10 μM. The following solutions were mixed in a 1.5 mL tube to prepare a reaction solution (one primer set was prepared for each type of primer set).
Power Up SYBR Green Master Mix (2×) 130μL
Primer 10μM premix solution 20.8μL
Sterile purified water 57.2μL
Total 208μL
cDNA合成により得られたcDNAを2μLずつPCR専用プレートの各ウェルに加えた。cDNAを加えたPCR専用プレートの各ウェルにマイクロピペットにて8μLの反応溶液を加え、吸引排出を繰り返すことで混合した。専用のシールで専用プレートを密閉した。以下のプログラムで反応させた。各目的遺伝子の発現量は、ハウスキーピング遺伝子と標的遺伝子のサイクル数の差から比較CT法(Livak KJ and Schmittgen TD (2001) Methods., 25(4): 402-408)により算出した。統計解析は、大阪大学遺伝情報実験センターの提供する医薬学データ用統計解析プログラムMEPHASを用い、Dunnettの多重検定を実施した。
2 μL of cDNA obtained by cDNA synthesis was added to each well of a PCR-specific plate. Using a micropipette, 8 μL of the reaction solution was added to each well of the PCR plate containing cDNA, and the mixture was mixed by repeating suction and discharge. The special plate was sealed with a special seal. I reacted with the following program. The expression level of each target gene was calculated by the comparative CT method (Livak KJ and Schmittgen TD (2001) Methods., 25(4): 402-408) from the difference in cycle numbers between the housekeeping gene and the target gene. For statistical analysis, Dunnett's multiple test was performed using MEPHAS, a statistical analysis program for pharmaceutical data provided by the Genetic Information Experiment Center, Osaka University.
[結果]
図1は、陰性コントロールで処理したヒト表皮角化細胞(HaCaT)におけるセマフォリン3A(Sema3A)の遺伝子発現量を1とした場合に、0.1%、0.5%ライステロールエステルで処理したHaCaTにおけるSema3A遺伝子の相対遺伝子発現量を示す。p<0.05で有意差ありとした。0.1%および0.5%ライステロールエステル処理で陰性コントロールに比べてSema3A遺伝子発現の上昇が確認できた。 [result]
Figure 1 shows the gene expression level of semaphorin 3A (Sema3A) in human epidermal keratinocytes (HaCaT) treated with the negative control and treated with 0.1% and 0.5% rice sterol ester. The relative gene expression level of Sema3A gene in HaCaT is shown. A significant difference was determined at p<0.05. An increase in Sema3A gene expression was confirmed when treated with 0.1% and 0.5% rice sterol ester compared to the negative control.
図1は、陰性コントロールで処理したヒト表皮角化細胞(HaCaT)におけるセマフォリン3A(Sema3A)の遺伝子発現量を1とした場合に、0.1%、0.5%ライステロールエステルで処理したHaCaTにおけるSema3A遺伝子の相対遺伝子発現量を示す。p<0.05で有意差ありとした。0.1%および0.5%ライステロールエステル処理で陰性コントロールに比べてSema3A遺伝子発現の上昇が確認できた。 [result]
Figure 1 shows the gene expression level of semaphorin 3A (Sema3A) in human epidermal keratinocytes (HaCaT) treated with the negative control and treated with 0.1% and 0.5% rice sterol ester. The relative gene expression level of Sema3A gene in HaCaT is shown. A significant difference was determined at p<0.05. An increase in Sema3A gene expression was confirmed when treated with 0.1% and 0.5% rice sterol ester compared to the negative control.
図2は、陰性コントロールで処理した分化後のマウス筋芽細胞(C2C12)におけるマイオネクチン(myonectin)の遺伝子発現量を1とした場合に、0.1%、0.5%ライステロールエステルで処理した分化後のC2C12におけるmyonectin遺伝子の相対遺伝子発現量を示す。p<0.05で有意差ありとした。0.5%ライステロールエステル処理で陰性コントロールに比べてmyonectin遺伝子発現の上昇が確認できた。
Figure 2 shows that the gene expression level of myonectin in differentiated mouse myoblasts (C2C12) treated with the negative control was set to 1, and the amount of myonectin gene expression in mouse myoblasts (C2C12) treated with 0.1% and 0.5% rice sterol ester was 1. The relative gene expression level of myonectin gene in C2C12 after differentiation is shown. A significant difference was determined at p<0.05. An increase in myonectin gene expression was confirmed when treated with 0.5% rice sterol ester compared to the negative control.
図3は、陰性コントロールで処理した新生児由来初代ヒト角化細胞(HEKn)におけるアルギナーゼ1(ARG1)遺伝子発現量を1とした場合に、0.01%、0.1%ライステロールエステルで処理したHEKnにおけるARG1遺伝子の相対遺伝子発現量を示す。p<0.05で有意差ありとした。0.1%ライステロールエステル処理で陰性コントロールに比べてARG1遺伝子発現の上昇が確認できた。
Figure 3 shows the expression level of arginase 1 (ARG1) gene in newborn-derived primary human keratinocytes (HEKn) treated with negative control and treated with 0.01% and 0.1% rice sterol ester. The relative gene expression level of ARG1 gene in HEKn is shown. A significant difference was determined at p<0.05. An increase in ARG1 gene expression was confirmed when treated with 0.1% rice sterol ester compared to the negative control.
図4は、陰性コントロールで処理したHaCaTにおけるピリオド1(PERIOD1)遺伝子発現量を1とした場合に、0.01%、0.1%ライステロールエステルで処理したHaCaTにおけるPERIOD1遺伝子の相対遺伝子発現量を示す。p<0.05で有意差ありとした。0.1%ライステロールエステル処理で陰性コントロールに比べてPERIOD1遺伝子発現の上昇が確認できた。
Figure 4 shows the relative gene expression level of PERIOD1 gene in HaCaT treated with 0.01% and 0.1% rice sterol ester, when the expression level of period 1 (PERIOD1) gene in HaCaT treated with the negative control is set to 1. shows. A significant difference was determined at p<0.05. An increase in PERIOD1 gene expression was confirmed when treated with 0.1% rice sterol ester compared to the negative control.
なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。
It should be noted that the present invention is not limited to the embodiments and examples described above, and various changes can be made within the scope of the claims, and technical means disclosed in different embodiments can be combined as appropriate. The resulting embodiments also fall within the technical scope of the present invention. Additionally, all academic and patent documents mentioned herein are incorporated by reference herein.
Claims (11)
- 米糠から得られるステロールエステルを含有することを特徴とする肌健康状態改善剤。 A skin health condition improving agent characterized by containing sterol ester obtained from rice bran.
- セマフォリン3A産生促進作用、マイオネクチン産生促進作用、アルギナーゼ1産生促進作用およびピリオド1産生促進作用からなる群から選択される少なくとも2つを有する、請求項1に記載の肌健康状態改善剤。 The skin health condition improving agent according to claim 1, which has at least two selected from the group consisting of semaphorin 3A production promoting action, meionectin production promoting action, arginase 1 production promoting action and period 1 production promoting action.
- 前記ステロールエステルが、米糠から得られるステロールエステル混合物である、請求項1または2に記載の肌健康状態改善剤。 The skin health condition improving agent according to claim 1 or 2, wherein the sterol ester is a sterol ester mixture obtained from rice bran.
- 前記ステロールエステルが、植物ステロールと脂肪酸とのエステル、および/またはトリテルペンアルコールと脂肪酸とのエステルである、請求項1または2に記載の肌健康状態改善剤。 The skin health condition improving agent according to claim 1 or 2, wherein the sterol ester is an ester of a plant sterol and a fatty acid, and/or an ester of a triterpene alcohol and a fatty acid.
- 請求項1または2に記載の肌健康状態改善剤を含むことを特徴とする医薬品。 A pharmaceutical product comprising the skin health condition improving agent according to claim 1 or 2.
- 請求項1または2に記載の肌健康状態改善剤を含むことを特徴とする化粧品。 A cosmetic product comprising the skin health condition improving agent according to claim 1 or 2.
- 請求項1または2に記載の肌健康状態改善剤を含むことを特徴とする飲食品。 A food or drink product comprising the skin health condition improving agent according to claim 1 or 2.
- 米糠から得られるステロールエステルを含有することを特徴とするセマフォリン3A産生促進剤。 A semaphorin 3A production promoter characterized by containing a sterol ester obtained from rice bran.
- 米糠から得られるステロールエステルを含有することを特徴とするマイオネクチン産生促進剤。 A myonectin production promoter characterized by containing a sterol ester obtained from rice bran.
- 米糠から得られるステロールエステルを含有することを特徴とするアルギナーゼ1産生促進剤。 An arginase 1 production promoter characterized by containing a sterol ester obtained from rice bran.
- 米糠から得られるステロールエステルを含有することを特徴とするピリオド1産生促進剤。 A period 1 production promoter characterized by containing a sterol ester obtained from rice bran.
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