WO2021084665A1 - Platelet-derived growth factor (pdgf)-bb production promoter, stem cell stabilizer containing this, and skin anti-aging agent containing these - Google Patents
Platelet-derived growth factor (pdgf)-bb production promoter, stem cell stabilizer containing this, and skin anti-aging agent containing these Download PDFInfo
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- WO2021084665A1 WO2021084665A1 PCT/JP2019/042689 JP2019042689W WO2021084665A1 WO 2021084665 A1 WO2021084665 A1 WO 2021084665A1 JP 2019042689 W JP2019042689 W JP 2019042689W WO 2021084665 A1 WO2021084665 A1 WO 2021084665A1
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- 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)
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- 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)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
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- 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)
- A61K36/185—Magnoliopsida (dicotyledons)
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- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
Definitions
- the present invention relates to a platelet-derived growth factor-BB (PDGF-BB) production enhancer, a stem cell stabilizer containing the PDGF-BB production enhancer, and a skin anti-aging agent containing them.
- PDGF-BB platelet-derived growth factor-BB
- mesenchymal stem cells differentiate into various cells belonging to the mesenchymal system (osteocytes, muscle cells, chondrocytes, tendon cells, adipocytes, etc.), their application to regenerative medicine is being studied. For example, by stabilizing mesenchymal stem cells, it is effective for various purposes such as blood vessel stabilization, maintenance of tissue homeostasis, prevention / improvement of various states such as repair / regeneration of damaged tissue, and mesenchymal stem cells in the skin. It has been reported that stabilization of mesenchymal stem cells is effective for skin activation and anti-aging (Patent Documents 1 and 2).
- Platelet-derived growth factor-BB (PDGF-BB) has been reported as a factor that stabilizes mesenchymal stem cells (Patent Documents 1 and 2). Therefore, if an effective component for enhancing the production of PDGF-BB can be found, it can be used to stabilize mesenchymal stem cells, and thus can be effectively used for applications such as skin activation and anti-aging. ..
- Patent Documents 1 and 2 Retinoic acid, amla extract, lingonberry extract and the like have been reported as effective components for enhancing the production of PDGF-BB (Patent Documents 1 and 2). It is expected to search for various substances that have a high PDGF-BB production enhancing effect.
- the present invention has been made in view of the above background, and the subject thereof is to provide an agent effective for enhancing the production of PDGF-BB, and to use the agent for stabilizing mesenchymal stem cells in the skin.
- the purpose is to provide an agent effective for activation and anti-aging of the skin.
- the present application includes the following inventions: [1] A platelet-derived growth factor-BB (PDGF-BB) production enhancer containing at least one of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient. [2] A stem cell stabilizer comprising the PDGF-BB production enhancer according to [1]. [3] A skin anti-aging agent comprising the PDGF-BB production enhancer according to [1], which suppresses skin aging by stabilizing stem cells in the skin.
- PDGF-BB platelet-derived growth factor-BB
- an agent effective for enhancing the production of PDGF-BB is provided, and by using this agent, an agent effective for stabilizing stem cells and the like is also provided. If stem cells are stabilized in the skin, it is effective in suppressing skin aging.
- FIG. 1 shows the screening results of Experiment 2 and shows the results of the control (0.5% DSMO) as relative values of 100.0.
- FIG. 2 shows the results of comparing the PDGF-BB production enhancing ability of various samples with the 15 ⁇ g / mL lingon berry extract and the 2 ⁇ g / mL amla extract in Experiment 3, and the results of the control (15 ⁇ g / mL lingon berry extract). The result is shown as a relative value with 100.0.
- FIG. 1 shows the screening results of Experiment 2 and shows the results of the control (0.5% DSMO) as relative values of 100.0.
- FIG. 2 shows the results of comparing the PDGF-BB production enhancing ability of various samples with the 15 ⁇ g / mL lingon berry extract and the 2 ⁇ g / mL amla extract in Experiment 3, and the results of the control (15 ⁇ g / mL lingon berry extract). The result is shown as a relative value
- the present inventors have found that wasabi extract, chamomile extract, L-theanine, and hibiscus extract exhibit remarkable PDGF-BB production enhancing action and stem cell stabilizing action, and the present invention has made such a discovery. Based on.
- Wasabi extract has been reported to have bactericidal action, antioxidant action, moisturizing action, lipase inhibitory activity, etc. (Japanese Patent Laid-Open No. 2016-124845). It has been reported that the fermented product obtained by fermenting the chamomile extract has a cell differentiation promoting effect (Japanese Patent Laid-Open No. 2015-157772). In addition, the chamomile extract has a stem cell growth factor (SCF) mRNA expression increase inhibitory effect (Japanese Patent Laid-Open No. 2011-1327) and a basic fibroblast growth factor (bFGF) mRNA expression increase inhibitory effect (Japanese Patent Laid-Open No. 2011-1328).
- SCF stem cell growth factor
- bFGF basic fibroblast growth factor
- SCF stem cell factor
- Japanese Patent Laid-Open No. 2003-194809 Japanese Patent Laid-Open No. 2003-194809
- theanine has an effect of improving epidermal stem cell functionality (Japanese Patent Laid-Open No. 2015-178485), an effect of suppressing parakeratosis (Japanese Patent Laid-Open No. 2007-204417), and the like.
- the hibiscus extract has an inhibitor of hyaluronidase activity, an inhibitory effect on the expression of stem cell growth factor mRNA (Japanese Patent Laid-Open No. 2014-218476), and the like.
- these substances have the PDGF-BB production promoting action and the stem cell stabilizing action of the present invention.
- PDGF-BB production enhancing action refers to the action of improving the production of platelet-derived growth factor-BB (PDGF-BB) protein.
- PDGF-BB platelet-derived growth factor-BB
- the PDGF-BB production-enhancing effect can be evaluated, for example, by measuring the amount of PDGF-BB to determine the amount of protein.
- This measurement uses PDGF-BB-specific antibodies and is known in the art, such as immunostaining, Western blotting, immunoassays, such as ELISA, using fluorescent substances, dyes, enzymes, etc. It can be implemented by various methods such as RIA method. It can also be determined indirectly by, for example, extracting total RNA in mesenchymal stem cells and measuring the amount of mRNA encoding PDGF-BB, but the method of measuring the actual amount of protein is more direct. Can be evaluated.
- the stem cell stabilizing action refers to an action of attracting and localizing a stem cell to a target site and / or an action of retaining the stem cell at the target site to maintain a localized state.
- the stem cell stabilizing action is distinguished from the action of promoting the proliferation of stem cells and / or the action of maintaining the stem cells in an undifferentiated state, and may not include these actions.
- the stem cell stabilizing action is not limited, but can be measured by, for example, the method described in Patent Document 1 for measuring migration ability or localization in the skin.
- Skin activation includes, but is not limited to, promoting metabolism and turnover of animal cells including humans, such as skin tissue, improving function, promoting proliferation, suppressing oxidation, and improving resistance to fatigue and external stimuli. Examples include suppression of deterioration of function and activity.
- the skin is activated, it is expected to have effects such as prevention and improvement of wrinkles, age spots, skin aging, photoaging and the like.
- [Wasabi extract] Wasabi (Saltwater cress) is a perennial plant of the Brassicaceae genus Wasabi.
- this wasabi (Eutrema japonicum (Miq.) Koidz.) Is preferable, but other species such as Eutrema pneumonia and related species (Eutrema yunnanense) may be used. ..
- wasabi leaf extract is preferable, but since seeds, stems, flowers, roots and the like also contain an active ingredient, any one or more of these extracts can be used.
- the wasabi extract is commercially available from Kinjirushi Co., Ltd., and such a commercially available product can also be used.
- Chamomile (scientific name: Matricaria chamomilla) is an annual plant belonging to the genus Mayweed in the family Asteraceae.
- the chamomile extract used in the present invention the chamomile head flower extract is preferable, but since the chamomile seeds, leaves, stems, flowers, roots and the like also contain active ingredients, any of these Or one or more extracts can also be used.
- the chamomile extract is commercially available from Maruzen Pharmaceuticals Co., Ltd. and the like, and such a commercially available product can also be used.
- L-Theanine is a type of amino acid represented by the following structural formula.
- L-theanine is abundantly contained in tea leaves such as green tea and black tea
- L-theanine contained in these tea leaves may be used, or may be used in the form of an extract of these tea leaves, but artificially. It may be synthesized, or a commercially available product commercially available from Taiyo Kagaku or the like can be used.
- Hibiscus is an annual or perennial sub-shrub belonging to the genus Confederate rose in the Malvales family Malvales.
- an extract of roselle Hibiscus sabdariffa
- Hibiscus flower extracts are preferred, but since hibiscus fruits, seeds, leaves, stems, flowers, roots, etc. also contain active ingredients, use one or more of these extracts. You can also do it.
- the hibiscus extract is commercially available from Nikken Foods Co., Ltd., and such a commercially available product can also be used.
- the extraction method is not particularly limited, but an extraction method using a solvent is preferable.
- the plant body can be used as it is, but it is better to crush it into granules or powder and use it for extraction in a short time under mild conditions with high extraction efficiency. It can be carried out.
- the extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, and the like. Usually, it is set in the range from room temperature to the boiling point of the solvent.
- the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.
- the type of solvent is not particularly limited, but water, lower alcohols such as hydrous ethanol and ethanol, organic solvents such as hexane, and mixed solvents such as hexane / ethanol are preferable. Extraction may be carried out at room temperature, but may be carried out under heating (for example, using a heated solvent such as hot water or hot water). Alternatively, the extraction process may be carried out by adding an enzyme to the solvent. By adding the enzyme, the cell tissue of the plant can be disrupted, which can further increase the extraction efficiency. As the enzyme, it is preferable to use a cell tissue disrupting enzyme.
- Examples of such an enzyme include pectinase, cellulase, hemicellulase, ⁇ -amylase, and phytase. One of these enzymes may be used alone, or two or more of these enzymes may be mixed and used.
- the active ingredient is extracted and dissolved in the solvent.
- the solvent containing the extract may be used as it is, or may be used after undergoing conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. Further, it may be used after being concentrated or diluted if necessary. Further, the solvent may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
- the squeezed liquid obtained by squeezing the raw material plant also contains the same active ingredient as the extract, the squeezed liquid can be used instead of the extract.
- the PDGF-BB production enhancer of the present invention contains at least one of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient.
- the stem cell stabilizer of the present invention contains the PDGF-BB production enhancer of the present invention containing the above-mentioned active ingredient.
- the stem cell stabilizer of the present invention can enhance the production of PDGF-BB, and the enhanced production of PDGF-BB can act on stem cells such as mesenchymal stem cells, resulting in stabilization of the stem cells. ..
- the skin anti-aging agent of the present invention contains the PDGF-BB production enhancer of the present invention containing the above-mentioned active ingredient.
- the skin anti-aging agent of the present invention enhances the production of PDGF-BB, and the enhanced production of PDGF-BB acts on stem cells such as mesenchymal stem cells, thereby stabilizing the stem cells and activating the skin. By making it, it suppresses the aging of the skin.
- the PDGF-BB production enhancer, stem cell stabilizer and skin anti-aging agent of the present invention are any one of the above active ingredients. It may be contained alone, or two or more kinds may be contained in any combination and ratio.
- the agent of the present invention may also be a composition in which the above-mentioned active ingredient is combined with one or more other ingredients such as excipients, carriers and / or diluents.
- the composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as the active ingredient and use.
- the composition can be produced by a conventional method with a formulation appropriately combined with an excipient, a carrier and / or a diluent and the like and other components according to the dosage form.
- the agent of the present invention can be blended with various foods and drinks and feeds and ingested by humans and animals. Further, it may be blended in cosmetics or the like and used for humans and animals, or may be administered to humans and animals as a pharmaceutical preparation.
- the blending amount (dry mass) of the plant or its extract is appropriately determined according to their types, purposes, forms, usage methods, and the like. be able to.
- the daily intake of the plant or its extract for an adult is about 0.5 mg to 3 g (dry residue).
- 10 mg to 1.5 g (dry residue) of the extract can be ingested per adult per day so that the prescribed effect of the active ingredient of the present invention can be sufficiently exerted. It is preferable to contain it in.
- the form of food and drink and feed can be any form, for example, granules, granules, pastes, gels, solids, or liquids. These forms include various known substances that are approved to be contained in foods and drinks, such as binders, disintegrants, thickeners, dispersants, reabsorption promoters, emulsifiers, buffers, and surfactants. Excipients such as activators, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers and pH adjusters can be appropriately contained.
- the blending amount (dry mass) of the plant or its extract depends on the type, purpose, form, usage method and the like. , Can be decided as appropriate.
- the wasabi extract can contain 0.00001% to 50% (dry mass conversion) of chamomile extract, L-theanine, and hibiscus extract, respectively, and 0.0001% to 5% (dry mass conversion). ) Is preferable.
- ingredients usually used for external skin preparations such as cosmetics, pharmaceuticals, quasi-drugs, etc., such as antioxidants, oils, UV protection agents, within the range that does not impair the effects of the present invention.
- ingredients usually used for external skin preparations such as cosmetics, pharmaceuticals, quasi-drugs, etc., such as antioxidants, oils, UV protection agents, within the range that does not impair the effects of the present invention.
- Surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as needed.
- metal ion blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, preservatives such as methylparaben, ethylparaben, and butylparaben, caffeine, tannin, Bellapamil, tranexamic acid and its derivatives, licorice extract, glabridin, hot water extract of carin fruit, various crude drugs, tocopherol acetate, glycyrrhizinic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbic acid phosphate, Whitening agents such as ascorbic acid glucoside, arbutin, and kodiic acid, and sugars such as glucose, fructose, mannose, sucrose, and trehalose can also be appropriately added.
- preservatives such as methylparaben, ethylparaben, and butylparaben, caffeine, tannin, Bellapamil
- the external preparation for skin of the present invention can be applied as a cosmetic, a non-medicinal product, etc. applied to the outer skin, particularly preferably as a cosmetic, and the dosage form is not limited as long as it can be applied to the skin.
- Any dosage form such as solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc. is applied.
- the agent of the present invention when used as cosmetics, it is used in lotions, emulsions, foundations, lipsticks, lip balms, cleansing creams, massage creams, facial masks, hand creams, hand powders, body shampoos, body lotions, body creams, bath cosmetics, etc. It may be used as a form.
- the preparation is appropriately used orally or parenterally (intravenous administration, intraperitoneal administration, etc.).
- the dosage form is also arbitrary, for example, oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as oral liquids and syrups, and parenteral liquid preparations such as injections.
- the form of the above can be appropriately prepared by a known method. If it is an external preparation, it can be used in various forms such as a lotion, a suspension / emulsion, a liquid, an ointment, and a patch.
- formulations include commonly used binders, disintegrants, thickeners, dispersants, reabsorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents. , Excipients such as stabilizers and pH adjusters may be used as appropriate.
- the possible forms of the agent of the present invention are not limited to the above-mentioned dosage forms and forms.
- Experiment 1 Preparation of sample The following samples were used as the samples to be evaluated for the effect of enhancing the production of PDGF-BB.
- Experiment 2 Evaluation of PDGF-BB production-enhancing effect Measurement was performed using an ELISA kit (product name: EHCSRP2) manufactured by Thermo Fisher Scientific. The contents and amount of the kit are shown below.
- reagents Preparation of reagents 1. Before measurement, all reagents and samples were returned to room temperature (18-25 ° C). 2. The sample diluent (10 mL ⁇ 50 mL) and the assay diluent (6 mL ⁇ 30 mL) were diluted 5-fold with deionized or distilled water before measurement. Cell lysate buffer was diluted 2-fold with deionized or distilled water. 3. Sample dilution: A measurement sample was prepared by diluting the reaction solution of HUVEC (human umbilical vein endothelial cells) with the sample to be evaluated at least 5 times with 1 ⁇ sample dilution (PDGF-BB level is sample). The optimum dilution factor for each sample was determined as appropriate). 4.
- HUVEC human umbilical vein endothelial cells
- Standard preparation 280 ⁇ L of 1 ⁇ sample diluent was added to a lyophilized standard vial to prepare a standard solution of 50 ng / mL. Gently mixed to completely dissolve the powder.
- 4 ⁇ L PDGF-BB standard solution was added from a reconstituted standard vial to a tube containing 496 ⁇ L sample diluent. Each tube was pipetted with 400 ⁇ L of 1 ⁇ sample diluent. A dilution series was prepared using the stock solution standard solution as shown below. Each tube was thoroughly mixed before the next transfer. The 1 ⁇ sample diluent was used as the zero standard solution (0 pg / mL).
- wash buffer contained visible crystals, warm to room temperature and mix gently until dissolved.
- a concentrated solution of 20 mL of wash buffer was diluted with deionized water or distilled water to obtain 400 mL of 1 ⁇ wash buffer.
- 100 ⁇ L of 1 ⁇ assay diluent was added to the vial to prepare a biotinylated antibody concentrate. Lightly mixed up and down with a pipette. The biotinylated antibody concentrate was diluted 80-fold with 1 ⁇ assay diluent (180 ⁇ L ⁇ 14400 ⁇ L) and used in step 4 of the assay procedure described below.
- Streptavidin-HRP reagent was diluted 800-fold with 1 ⁇ assay diluent.
- 20 ⁇ L of HRP-streptavidin concentrate was added to a tube containing 16 mL of 1 ⁇ assay diluent to prepare an 800-fold diluted HRP-streptavidin solution.
- Assay procedure 1 Before use, all reagents and samples were returned to room temperature (18-25 ° C). All standards and samples were run in at least two runs. 2. 100 ⁇ L of each standard solution was added (see step 3 of the reagent preparation procedure) and the sample was dispensed into the appropriate wells. Wells were covered and incubated for 2.5 hours at room temperature with gentle shaking. 3. The solution was discarded and washed 4 times with 1 x wash buffer. Each well was flushed with wash buffer (300 ⁇ L) using a multi-channel pipette. After the final wash, all remaining wash buffer was removed by suction or decanting. The plate was turned upside down and a clean paper towel was used to thoroughly remove the water. 4.
- Sensitivity 1 pg / mL
- LLD lower limit of sensitivity or detection
- FIG. 1 and Table 3 The ratio to the amount of protein obtained for the negative control (0.5% DMSO without sample) is shown in FIG. 1 and Table 3 below. From the results of FIGS. 1 and 3, it was found that the wasabi extract, chamomile extract, L-theanine, and hibiscus extract were particularly effective among the 52 types of samples, and these components were found to be particularly effective in PDGF-. It can be seen that it has the effect of enhancing the production of BB. In the figure, samples A to C show three samples out of 47 kinds of substances other than wasabi extract, chamomile extract, L-theanine, and hibiscus extract, but other substances are also sample A to C. Similarly, it did not meet the criteria, such as being lower than the negative control (DMSO) or showing no significant difference.
- DMSO negative control
- Experiment 3 Comparison with Amla extract and lingon berry extract
- wasabi extract, chamomile extract, L-theanin, and hibiscus extract which showed a significant PDGF-BB production-enhancing effect in Experiment 2.
- the amount of PDGF-BB protein was measured according to the above, and compared with the 15 ⁇ g / mL lingon berry extract and the 2 ⁇ g / mL Amla extract, which are known to have a PDGF-BB production-enhancing effect.
- the amount of protein obtained for the control (15 ⁇ g / mL lingon berry extract) was 100.0 and the ratio to it was shown in FIG. 2, and the amount of protein obtained for the negative control (0.5% DMSO without sample) was 100.0.
- the ratio is shown in FIG. From FIGS. 2 and 3, PDGF-BB is significantly higher than that of lingonberry extract and amla extract (Patent Document 2), which were previously known to have a high PDGF-BB production enhancing effect as well as a negative control. It was found that it exerts a production-enhancing ability.
- wasabi extract, chamomile extract, L-theanine, and hibiscus extract are particularly effective in enhancing PDGF-BB production. These substances are expected to stabilize mesenchymal stem cells by enhancing PDGF-BB production, thereby activating the skin and suppressing aging.
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Abstract
An agent effective in promoting production of PDGF-BB, and an agent which, using that agent, is effective for activation and anti-aging of the skin through stabilization of mesenchymal stem cells in the skin are provided. The PDGF-BB production promoter, which contains, as an effective component, any of wasabi extract, chamomile extract, L-theanine, and hibiscus extract, is effective for skin activation and anti-aging through stabilization of mesenchymal stem cells.
Description
本発明は、血小板由来成長因子-BB(PDGF-BB)産生亢進剤、及び、当該PDGF-BB産生亢進剤を含んでなる幹細胞安定化剤、並びにそれらを含む皮膚抗老化剤に関する。
The present invention relates to a platelet-derived growth factor-BB (PDGF-BB) production enhancer, a stem cell stabilizer containing the PDGF-BB production enhancer, and a skin anti-aging agent containing them.
間葉系幹細胞は、間葉系に属するさまざまな細胞(骨細胞、筋細胞、軟骨細胞、腱細胞、脂肪細胞など)に分化することから、再生医療への応用が研究されている。例えば、間葉系幹細胞の安定化を図ることにより、血管安定化、組織恒常性維持、損傷組織の修復・再生等の各種状態の予防・改善等、各種の用途に有効であり、皮膚における間葉系幹細胞の安定化は、皮膚の賦活化や抗老化に有効であることが報告されている(特許文献1,2)。
Since mesenchymal stem cells differentiate into various cells belonging to the mesenchymal system (osteocytes, muscle cells, chondrocytes, tendon cells, adipocytes, etc.), their application to regenerative medicine is being studied. For example, by stabilizing mesenchymal stem cells, it is effective for various purposes such as blood vessel stabilization, maintenance of tissue homeostasis, prevention / improvement of various states such as repair / regeneration of damaged tissue, and mesenchymal stem cells in the skin. It has been reported that stabilization of mesenchymal stem cells is effective for skin activation and anti-aging (Patent Documents 1 and 2).
間葉系幹細胞を安定化する因子として血小板由来成長因子-BB(PDGF-BB)が報告されている(特許文献1,2)。よって、PDGF-BBの産生亢進に有効な成分を見出すことができれば、これを用いて間葉系幹細胞の安定化を図ることができ、ひいては皮膚の賦活化や抗老化といった用途に有効に使用できる。
Platelet-derived growth factor-BB (PDGF-BB) has been reported as a factor that stabilizes mesenchymal stem cells (Patent Documents 1 and 2). Therefore, if an effective component for enhancing the production of PDGF-BB can be found, it can be used to stabilize mesenchymal stem cells, and thus can be effectively used for applications such as skin activation and anti-aging. ..
PDGF-BBの産生亢進に有効な成分としては、レチノイン酸、アムラ抽出物及びリンゴンベリー抽出物等が報告されている(特許文献1,2)。PDGF-BB産生亢進効果が高い様々な物質の探索が期待される。
Retinoic acid, amla extract, lingonberry extract and the like have been reported as effective components for enhancing the production of PDGF-BB (Patent Documents 1 and 2). It is expected to search for various substances that have a high PDGF-BB production enhancing effect.
本発明は、上記背景に鑑みてなされたもので、その課題は、PDGF-BBの産生亢進に有効な剤を提供すると共に、これを用いて、皮膚における間葉系幹細胞の安定化を介し皮膚の賦活化や抗老化に有効な剤を提供することにある。
The present invention has been made in view of the above background, and the subject thereof is to provide an agent effective for enhancing the production of PDGF-BB, and to use the agent for stabilizing mesenchymal stem cells in the skin. The purpose is to provide an agent effective for activation and anti-aging of the skin.
本発明者らは、多種多様な素材について検討を重ね、PDGF-BBの産生を亢進させる薬剤をスクリーニングした結果、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物が、顕著なPDGF-BB産生亢進作用を示すことを見出し、本発明を為すに至った。
As a result of repeated studies on a wide variety of materials and screening for drugs that enhance PDGF-BB production, the wasabi extract, chamomile extract, L-theanine, and hibiscus extract are prominent PDGF. -It has been found that it exhibits a BB production-enhancing effect, and the present invention has been made.
したがって、本願は下記の発明を包含する:
[1]ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物の少なくともいずれかを有効成分として含んでなる血小板由来成長因子-BB(PDGF-BB)産生亢進剤。
[2][1]に記載のPDGF-BB産生亢進剤を含んでなる、幹細胞安定化剤。
[3][1]に記載のPDGF-BB産生亢進剤を含んでなる皮膚抗老化剤であって、皮膚における幹細胞を安定化することにより皮膚の老化を抑制する、皮膚抗老化剤。 Therefore, the present application includes the following inventions:
[1] A platelet-derived growth factor-BB (PDGF-BB) production enhancer containing at least one of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient.
[2] A stem cell stabilizer comprising the PDGF-BB production enhancer according to [1].
[3] A skin anti-aging agent comprising the PDGF-BB production enhancer according to [1], which suppresses skin aging by stabilizing stem cells in the skin.
[1]ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物の少なくともいずれかを有効成分として含んでなる血小板由来成長因子-BB(PDGF-BB)産生亢進剤。
[2][1]に記載のPDGF-BB産生亢進剤を含んでなる、幹細胞安定化剤。
[3][1]に記載のPDGF-BB産生亢進剤を含んでなる皮膚抗老化剤であって、皮膚における幹細胞を安定化することにより皮膚の老化を抑制する、皮膚抗老化剤。 Therefore, the present application includes the following inventions:
[1] A platelet-derived growth factor-BB (PDGF-BB) production enhancer containing at least one of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient.
[2] A stem cell stabilizer comprising the PDGF-BB production enhancer according to [1].
[3] A skin anti-aging agent comprising the PDGF-BB production enhancer according to [1], which suppresses skin aging by stabilizing stem cells in the skin.
本発明によれば、PDGF-BBの産生亢進に有効な剤が提供されるとともに、これを用いることにより、幹細胞の安定化等に有効な剤も提供される。皮膚において幹細胞が安定化されれば、皮膚の老化抑制に有効である。
According to the present invention, an agent effective for enhancing the production of PDGF-BB is provided, and by using this agent, an agent effective for stabilizing stem cells and the like is also provided. If stem cells are stabilized in the skin, it is effective in suppressing skin aging.
本発明者らは、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物が、顕著なPDGF-BB産生亢進作用及び幹細胞安定化作用を示すことを見出し、本発明は、かかる発見に基づく。
The present inventors have found that wasabi extract, chamomile extract, L-theanine, and hibiscus extract exhibit remarkable PDGF-BB production enhancing action and stem cell stabilizing action, and the present invention has made such a discovery. Based on.
ワサビ抽出物は、殺菌作用、抗酸化作用、保湿作用、リパーゼ阻害活性等が報告されている(特開2016-124845号公報)。カミツレ抽出物を発酵させて得られた発酵物には、細胞分化促進作用があることが報告されている(特開2015-157772号公報)。また、カミツレ抽出物には、幹細胞増殖因子(SCF)mRNA発現上昇抑制作用(特開2011-1327号公報)、塩基性線維芽細胞増殖因子(bFGF)mRNA発現上昇抑制作用(特開2011-1328号公報)、幹細胞因子(以下「SCF」)の産生及び/又は放出の抑制作用(特開2003-194809号公報)等が報告されている。テアニンには、表皮幹細胞機能性向上作用(特開2015-178485号公報)、不全角化抑制作用(特開2007-204417号公報)等が報告されている。ハイビスカス抽出物には、ヒアルロニダーゼ活性阻害剤、幹細胞増殖因子mRNA発現上昇抑制作用(特開2014-218476号公報)等が報告されている。しかしながら、これらの物質が、本発明のPDGF-BB産生促進作用や幹細胞安定化作用を有することは、本発明者らによって今回初めて見出された。
Wasabi extract has been reported to have bactericidal action, antioxidant action, moisturizing action, lipase inhibitory activity, etc. (Japanese Patent Laid-Open No. 2016-124845). It has been reported that the fermented product obtained by fermenting the chamomile extract has a cell differentiation promoting effect (Japanese Patent Laid-Open No. 2015-157772). In addition, the chamomile extract has a stem cell growth factor (SCF) mRNA expression increase inhibitory effect (Japanese Patent Laid-Open No. 2011-1327) and a basic fibroblast growth factor (bFGF) mRNA expression increase inhibitory effect (Japanese Patent Laid-Open No. 2011-1328). No.), an action of suppressing the production and / or release of stem cell factor (hereinafter referred to as “SCF”) (Japanese Patent Laid-Open No. 2003-194809) and the like have been reported. It has been reported that theanine has an effect of improving epidermal stem cell functionality (Japanese Patent Laid-Open No. 2015-178485), an effect of suppressing parakeratosis (Japanese Patent Laid-Open No. 2007-204417), and the like. It has been reported that the hibiscus extract has an inhibitor of hyaluronidase activity, an inhibitory effect on the expression of stem cell growth factor mRNA (Japanese Patent Laid-Open No. 2014-218476), and the like. However, it has been discovered for the first time by the present inventors that these substances have the PDGF-BB production promoting action and the stem cell stabilizing action of the present invention.
PDGF-BB産生亢進作用とは、血小板由来成長因子-BB(PDGF-BB)タンパク質の産生を向上する作用のことを言う。例えば、産生の亢進されたPDGF-BBは間葉系幹細胞に作用し、その結果間葉系幹細胞を安定化させ皮膚を賦活することで、皮膚の老化を抑制することができる。PDGF-BB産生亢進作用は、例えばPDGF-BBの量を測定してタンパク質量を決定し、評価することができる。この測定は、PDGF-BBに特異的な抗体を利用し、当業界において周知の方法、例えば蛍光物質、色素、酵素等を利用する免疫染色法、ウェスタンブロット法、免疫測定方法、例えばELISA法、RIA法等、様々な方法により実施できる。また、例えば、間葉系幹細胞中の総RNAを抽出し、PDGF-BBをコードするmRNAの量を測定することにより間接的に決定することもできるが実際のタンパク質量を測定する方法のほうが直接的に評価できる。
PDGF-BB production enhancing action refers to the action of improving the production of platelet-derived growth factor-BB (PDGF-BB) protein. For example, PDGF-BB with enhanced production acts on mesenchymal stem cells, and as a result, stabilizes mesenchymal stem cells and activates the skin, thereby suppressing skin aging. The PDGF-BB production-enhancing effect can be evaluated, for example, by measuring the amount of PDGF-BB to determine the amount of protein. This measurement uses PDGF-BB-specific antibodies and is known in the art, such as immunostaining, Western blotting, immunoassays, such as ELISA, using fluorescent substances, dyes, enzymes, etc. It can be implemented by various methods such as RIA method. It can also be determined indirectly by, for example, extracting total RNA in mesenchymal stem cells and measuring the amount of mRNA encoding PDGF-BB, but the method of measuring the actual amount of protein is more direct. Can be evaluated.
幹細胞安定化作用とは、幹細胞を標的部位に誘引して局在化させる作用、及び/又は幹細胞を標的部位に留めて局在化状態を維持する作用を言う。本明細書において、幹細胞安定化作用は、幹細胞の増殖を促進する作用、及び/又は幹細胞を未分化状態に維持する作用とは区別され、これらの作用を含まない場合がある。幹細胞安定化作用は、限定されないものの、例えば、特許文献1に記載の遊走能や皮膚における局在を測定する方法等によって測定できる。
The stem cell stabilizing action refers to an action of attracting and localizing a stem cell to a target site and / or an action of retaining the stem cell at the target site to maintain a localized state. In the present specification, the stem cell stabilizing action is distinguished from the action of promoting the proliferation of stem cells and / or the action of maintaining the stem cells in an undifferentiated state, and may not include these actions. The stem cell stabilizing action is not limited, but can be measured by, for example, the method described in Patent Document 1 for measuring migration ability or localization in the skin.
皮膚の賦活とは、限定されないものの、ヒトを含む動物の細胞、例えば、皮膚組織の新陳代謝やターンオーバーの促進、機能の向上、増殖の促進、酸化の抑制、疲労や外部刺激に対する耐性の向上、機能や活性の低下の抑制などが挙げられる。皮膚が賦活されると、しわ、シミ、皮膚老化、光老化等の予防・改善といった効果が期待される。
Skin activation includes, but is not limited to, promoting metabolism and turnover of animal cells including humans, such as skin tissue, improving function, promoting proliferation, suppressing oxidation, and improving resistance to fatigue and external stimuli. Examples include suppression of deterioration of function and activity. When the skin is activated, it is expected to have effects such as prevention and improvement of wrinkles, age spots, skin aging, photoaging and the like.
[ワサビ抽出物]
ワサビ(Eutrema属)は、アブラナ科ワサビ属の多年草である。本発明に用いられるワサビの抽出物としては、本ワサビ(Eutrema japonicum (Miq.) Koidz.)が好ましいが、ユリワサビ(Eutrema tenue)や近縁種(Eutrema yunnanense)といった他の種を用いてもよい。また、ワサビの葉の抽出物が好ましいが、種、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。ワサビの抽出物は、金印株式会社等から市販されており、このような市販品を用いることもできる。 [Wasabi extract]
Wasabi (Saltwater cress) is a perennial plant of the Brassicaceae genus Wasabi. As the wasabi extract used in the present invention, this wasabi (Eutrema japonicum (Miq.) Koidz.) Is preferable, but other species such as Eutrema tenue and related species (Eutrema yunnanense) may be used. .. Further, wasabi leaf extract is preferable, but since seeds, stems, flowers, roots and the like also contain an active ingredient, any one or more of these extracts can be used. The wasabi extract is commercially available from Kinjirushi Co., Ltd., and such a commercially available product can also be used.
ワサビ(Eutrema属)は、アブラナ科ワサビ属の多年草である。本発明に用いられるワサビの抽出物としては、本ワサビ(Eutrema japonicum (Miq.) Koidz.)が好ましいが、ユリワサビ(Eutrema tenue)や近縁種(Eutrema yunnanense)といった他の種を用いてもよい。また、ワサビの葉の抽出物が好ましいが、種、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。ワサビの抽出物は、金印株式会社等から市販されており、このような市販品を用いることもできる。 [Wasabi extract]
Wasabi (Saltwater cress) is a perennial plant of the Brassicaceae genus Wasabi. As the wasabi extract used in the present invention, this wasabi (Eutrema japonicum (Miq.) Koidz.) Is preferable, but other species such as Eutrema tenue and related species (Eutrema yunnanense) may be used. .. Further, wasabi leaf extract is preferable, but since seeds, stems, flowers, roots and the like also contain an active ingredient, any one or more of these extracts can be used. The wasabi extract is commercially available from Kinjirushi Co., Ltd., and such a commercially available product can also be used.
[カミツレ抽出物]
カミツレ(学名:Matricaria chamomilla)は、キク科シカギク属に属する一年草である。本発明に用いられるカミツレの抽出物としては、カミツレの頭花の抽出物が好ましいが、カミツレの種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。カミツレの抽出物は、丸善製薬株式会社等から市販されており、このような市販品を用いることもできる。 [Chamomile extract]
Chamomile (scientific name: Matricaria chamomilla) is an annual plant belonging to the genus Mayweed in the family Asteraceae. As the chamomile extract used in the present invention, the chamomile head flower extract is preferable, but since the chamomile seeds, leaves, stems, flowers, roots and the like also contain active ingredients, any of these Or one or more extracts can also be used. The chamomile extract is commercially available from Maruzen Pharmaceuticals Co., Ltd. and the like, and such a commercially available product can also be used.
カミツレ(学名:Matricaria chamomilla)は、キク科シカギク属に属する一年草である。本発明に用いられるカミツレの抽出物としては、カミツレの頭花の抽出物が好ましいが、カミツレの種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。カミツレの抽出物は、丸善製薬株式会社等から市販されており、このような市販品を用いることもできる。 [Chamomile extract]
Chamomile (scientific name: Matricaria chamomilla) is an annual plant belonging to the genus Mayweed in the family Asteraceae. As the chamomile extract used in the present invention, the chamomile head flower extract is preferable, but since the chamomile seeds, leaves, stems, flowers, roots and the like also contain active ingredients, any of these Or one or more extracts can also be used. The chamomile extract is commercially available from Maruzen Pharmaceuticals Co., Ltd. and the like, and such a commercially available product can also be used.
[L-テアニン]
L-テアニンは、以下の構造式で表されるアミノ酸の一種である。
[L-Theanine]
L-Theanine is a type of amino acid represented by the following structural formula.
L-テアニンは、以下の構造式で表されるアミノ酸の一種である。
L-Theanine is a type of amino acid represented by the following structural formula.
L-テアニンは、緑茶、紅茶などの茶葉に多く含まれるため、これらの茶葉に含まれるL-テアニンを用いてもよく、これらの茶葉の抽出物の形態で用いてもよいが、人工的に合成してもよく、太陽化学等から市販されている市販品を用いることもできる。
Since L-theanine is abundantly contained in tea leaves such as green tea and black tea, L-theanine contained in these tea leaves may be used, or may be used in the form of an extract of these tea leaves, but artificially. It may be synthesized, or a commercially available product commercially available from Taiyo Kagaku or the like can be used.
[ハイビスカス抽出物]
ハイビスカス(Hibiscus属)は、アオイ目アオイ科フヨウ属に属する一年生または多年生の亜灌木である。本発明に用いられるハイビスカスの抽出物としては、ローゼル(Hibiscus sabdariffa)の抽出物が好ましい。ハイビスカスの花の抽出物が好ましいが、ハイビスカスの果実、種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。ハイビスカスの抽出物は、日研フード株式会社等から市販されており、このような市販品を用いることもできる。 [Hibiscus extract]
Hibiscus (genus Hibiscus) is an annual or perennial sub-shrub belonging to the genus Confederate rose in the Malvales family Malvales. As the hibiscus extract used in the present invention, an extract of roselle (Hibiscus sabdariffa) is preferable. Hibiscus flower extracts are preferred, but since hibiscus fruits, seeds, leaves, stems, flowers, roots, etc. also contain active ingredients, use one or more of these extracts. You can also do it. The hibiscus extract is commercially available from Nikken Foods Co., Ltd., and such a commercially available product can also be used.
ハイビスカス(Hibiscus属)は、アオイ目アオイ科フヨウ属に属する一年生または多年生の亜灌木である。本発明に用いられるハイビスカスの抽出物としては、ローゼル(Hibiscus sabdariffa)の抽出物が好ましい。ハイビスカスの花の抽出物が好ましいが、ハイビスカスの果実、種、葉、茎、花、根等にも有効成分が含まれているので、これらのうちいずれか1又は2以上の抽出物を使用することもできる。ハイビスカスの抽出物は、日研フード株式会社等から市販されており、このような市販品を用いることもできる。 [Hibiscus extract]
Hibiscus (genus Hibiscus) is an annual or perennial sub-shrub belonging to the genus Confederate rose in the Malvales family Malvales. As the hibiscus extract used in the present invention, an extract of roselle (Hibiscus sabdariffa) is preferable. Hibiscus flower extracts are preferred, but since hibiscus fruits, seeds, leaves, stems, flowers, roots, etc. also contain active ingredients, use one or more of these extracts. You can also do it. The hibiscus extract is commercially available from Nikken Foods Co., Ltd., and such a commercially available product can also be used.
抽出物を用いる場合、抽出方法は特に限定されるものではないが、溶媒を用いた抽出法が好ましい。抽出を行う際には、植物体をそのまま使用することもできるが、顆粒状や粉末状に粉砕して抽出に供した方が、穏和な条件で短時間に高い抽出効率で有効成分の抽出を行うことができる。抽出温度は特に限定されるものではなく、粉砕物の粒径や溶媒の種類等に応じて適宜設定すればよい。通常は、室温から溶媒の沸点までの範囲内で設定される。また、抽出時間も特に限定されるものではなく、粉砕物の粒径、溶媒の種類、抽出温度等に応じて適宜設定すればよい。さらに、抽出時には、撹拌を行ってもよいし、撹拌せず静置してもよいし、超音波を加えてもよい。
When an extract is used, the extraction method is not particularly limited, but an extraction method using a solvent is preferable. When extracting, the plant body can be used as it is, but it is better to crush it into granules or powder and use it for extraction in a short time under mild conditions with high extraction efficiency. It can be carried out. The extraction temperature is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, and the like. Usually, it is set in the range from room temperature to the boiling point of the solvent. Further, the extraction time is not particularly limited, and may be appropriately set according to the particle size of the pulverized product, the type of solvent, the extraction temperature, and the like. Further, at the time of extraction, stirring may be performed, the mixture may be allowed to stand without stirring, or ultrasonic waves may be applied.
溶媒の種類は特に限定されるものではないが、水、含水エタノール、エタノール等の低級アルコール、ヘキサン等の有機溶媒、又はヘキサン/エタノールといったこれらの混合溶媒が好ましい。抽出は常温で行ってもよいが、加熱下で(例えば温水や熱水等の加熱した溶媒を用いて)行ってもよい。また、溶媒に酵素を加えて抽出処理を行ってもよい。酵素を加えることによって、植物の細胞組織を崩壊させることができ、これにより抽出効率をより高めることができる。酵素としては、細胞組織崩壊酵素を用いることが好ましい。このような酵素としては、例えば、ペクチナーゼ、セルラーゼ、ヘミセルラーゼ、α-アミラーゼ、フィターゼが挙げられる。これらの酵素は1種類を単独で用いてもよいし、2種以上を混合して用いてもよい。
The type of solvent is not particularly limited, but water, lower alcohols such as hydrous ethanol and ethanol, organic solvents such as hexane, and mixed solvents such as hexane / ethanol are preferable. Extraction may be carried out at room temperature, but may be carried out under heating (for example, using a heated solvent such as hot water or hot water). Alternatively, the extraction process may be carried out by adding an enzyme to the solvent. By adding the enzyme, the cell tissue of the plant can be disrupted, which can further increase the extraction efficiency. As the enzyme, it is preferable to use a cell tissue disrupting enzyme. Examples of such an enzyme include pectinase, cellulase, hemicellulase, α-amylase, and phytase. One of these enzymes may be used alone, or two or more of these enzymes may be mixed and used.
このような抽出操作により、有効成分が抽出され、溶媒に溶け込む。抽出物を含む溶媒は、そのまま使用してもよいが、滅菌、洗浄、濾過、脱色、脱臭等の慣用の精製処理を加えてから使用してもよい。また、必要により濃縮又は希釈してから使用してもよい。さらに、溶媒を全て揮発させて固体状(乾燥物)としてから使用してもよいし、該乾燥物を任意の溶媒に再溶解してから使用してもよい。
By such an extraction operation, the active ingredient is extracted and dissolved in the solvent. The solvent containing the extract may be used as it is, or may be used after undergoing conventional purification treatments such as sterilization, washing, filtration, decolorization, and deodorization. Further, it may be used after being concentrated or diluted if necessary. Further, the solvent may be completely volatilized to form a solid (dried product) before use, or the dried product may be redissolved in an arbitrary solvent before use.
また、原料の植物を圧搾することにより得られる圧搾液にも抽出物と同様の有効成分が含まれているので、抽出物の代わりに圧搾液を使用することもできる。
Further, since the squeezed liquid obtained by squeezing the raw material plant also contains the same active ingredient as the extract, the squeezed liquid can be used instead of the extract.
本発明のPDGF-BB産生亢進剤は、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物の少なくともいずれかを有効成分として含有する。また、本発明の幹細胞安定化剤は、上記の有効成分を含む本発明のPDGF-BB産生亢進剤を含有する。例えば、本発明の幹細胞安定化剤は、PDGF-BBの産生を亢進し、産生の亢進されたPDGF-BBが間葉系幹細胞などの幹細胞に作用し、その結果幹細胞を安定化することができる。本発明の皮膚抗老化剤は、上記の有効成分を含む本発明のPDGF-BB産生亢進剤を含有する。例えば、本発明の皮膚抗老化剤は、PDGF-BBの産生を亢進し、産生の亢進されたPDGF-BBが間葉系幹細胞などの幹細胞に作用し、その結果幹細胞を安定化させ皮膚を賦活化することで、皮膚の老化を抑制する。本発明のPDGF-BB産生亢進剤、幹細胞安定化剤及び皮膚抗老化剤(以降これらを総称して「本発明の剤」という場合がある。)は、上記の有効成分の何れか1種を単独で含有してもよく、2種類以上を任意の組み合わせ及び比率で含有してもよい。
The PDGF-BB production enhancer of the present invention contains at least one of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient. In addition, the stem cell stabilizer of the present invention contains the PDGF-BB production enhancer of the present invention containing the above-mentioned active ingredient. For example, the stem cell stabilizer of the present invention can enhance the production of PDGF-BB, and the enhanced production of PDGF-BB can act on stem cells such as mesenchymal stem cells, resulting in stabilization of the stem cells. .. The skin anti-aging agent of the present invention contains the PDGF-BB production enhancer of the present invention containing the above-mentioned active ingredient. For example, the skin anti-aging agent of the present invention enhances the production of PDGF-BB, and the enhanced production of PDGF-BB acts on stem cells such as mesenchymal stem cells, thereby stabilizing the stem cells and activating the skin. By making it, it suppresses the aging of the skin. The PDGF-BB production enhancer, stem cell stabilizer and skin anti-aging agent of the present invention (hereinafter, these may be collectively referred to as "agent of the present invention") are any one of the above active ingredients. It may be contained alone, or two or more kinds may be contained in any combination and ratio.
本発明の剤は、上記の有効成分を、1種又は2種以上の他の成分、例えば賦形剤、担体及び/又は希釈剤等と組み合わせた組成物とすることもできる。組成物の組成や形態は任意であり、有効成分や用途等の条件に応じて適切に選択すればよい。当該組成物は、その剤形に応じ、賦形剤、担体及び/又は希釈剤等及び他の成分と適宜組み合わせた処方で、常法を用いて製造することができる。
The agent of the present invention may also be a composition in which the above-mentioned active ingredient is combined with one or more other ingredients such as excipients, carriers and / or diluents. The composition and form of the composition are arbitrary, and may be appropriately selected according to conditions such as the active ingredient and use. The composition can be produced by a conventional method with a formulation appropriately combined with an excipient, a carrier and / or a diluent and the like and other components according to the dosage form.
本発明の剤は、各種の飲食品、飼料に配合してヒト及び動物に摂取させることができる。また、化粧品等に配合してヒト及び動物に使用し、或いは医薬製剤としてヒト及び動物に投与してもよい。
The agent of the present invention can be blended with various foods and drinks and feeds and ingested by humans and animals. Further, it may be blended in cosmetics or the like and used for humans and animals, or may be administered to humans and animals as a pharmaceutical preparation.
具体的に、本発明の剤を飲食品や飼料等に配合する場合、植物体又はその抽出物の配合量(乾燥質量)は、それらの種類、目的、形態、利用方法等に応じて適宜決めることができる。例えば、成人一日当たり植物又はその抽出物の摂取量が、0.5mg~3g(乾燥残分)程度になるように配合できる。特に、保健用飲食品等として利用する場合には、本発明の有効成分による所定の効果が十分発揮されるように、成人一日当たり、抽出物が10mg~1.5g(乾燥残分)摂取できるように含有させることが好ましい。
Specifically, when the agent of the present invention is blended in foods and drinks, feeds, etc., the blending amount (dry mass) of the plant or its extract is appropriately determined according to their types, purposes, forms, usage methods, and the like. be able to. For example, it can be blended so that the daily intake of the plant or its extract for an adult is about 0.5 mg to 3 g (dry residue). In particular, when it is used as a food or drink for health use, 10 mg to 1.5 g (dry residue) of the extract can be ingested per adult per day so that the prescribed effect of the active ingredient of the present invention can be sufficiently exerted. It is preferable to contain it in.
飲食品や飼料の形態としては、任意の形態とすることが可能であり、例えば、顆粒状、粒状、ペースト状、ゲル状、固形状、又は、液体状にすることができる。これらの形態には、飲食品等に含有することが認められている公知の各種物質、例えば、結合剤、崩壊剤、増粘剤、分散剤、再吸収促進剤、矯味剤、緩衝剤、界面活性剤、溶解補助剤、保存剤、乳化剤、等張化剤、安定化剤やpH調製剤等の賦形剤を適宜含有させることができる。
The form of food and drink and feed can be any form, for example, granules, granules, pastes, gels, solids, or liquids. These forms include various known substances that are approved to be contained in foods and drinks, such as binders, disintegrants, thickeners, dispersants, reabsorption promoters, emulsifiers, buffers, and surfactants. Excipients such as activators, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers and pH adjusters can be appropriately contained.
本発明を化粧品、医薬品、医薬部外品等の皮膚外用剤に適用する場合、植物体又はその抽出物の配合量(乾燥質量)は、それらの種類、目的、形態、利用方法などに応じて、適宜決めることができる。例えば、化粧料全量中に、ワサビ抽出物では、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物それぞれ0.00001%~50%(乾燥質量換算)を配合でき、中でも0.0001%~5%(乾燥質量換算)が好ましい。
When the present invention is applied to external preparations for skin such as cosmetics, pharmaceuticals, quasi-drugs, etc., the blending amount (dry mass) of the plant or its extract depends on the type, purpose, form, usage method and the like. , Can be decided as appropriate. For example, in the total amount of cosmetics, the wasabi extract can contain 0.00001% to 50% (dry mass conversion) of chamomile extract, L-theanine, and hibiscus extract, respectively, and 0.0001% to 5% (dry mass conversion). ) Is preferable.
上記成分に加えて、さらに必要により、本発明の効果を損なわない範囲内で、通常化粧品、医薬品、医薬部外品等の皮膚外用剤に用いられる成分、例えば酸化防止剤、油分、紫外線防御剤、界面活性剤、増粘剤、アルコール類、粉末成分、色材、水性成分、水、各種皮膚栄養剤等を必要に応じて適宜配合することができる。
In addition to the above ingredients, if necessary, ingredients usually used for external skin preparations such as cosmetics, pharmaceuticals, quasi-drugs, etc., such as antioxidants, oils, UV protection agents, within the range that does not impair the effects of the present invention. , Surfactants, thickeners, alcohols, powder components, coloring materials, aqueous components, water, various skin nutrients and the like can be appropriately blended as needed.
さらに、エデト酸二ナトリウム、エデト酸三ナトリウム、クエン酸ナトリウム、ポリリン酸ナトリウム、メタリン酸ナトリウム、グルコン酸等の金属イオン封鎖剤、メチルパラベン、エチルパラベン、ブチルパラベン等の防腐剤、カフェイン、タンニン、ベラパミル、トラネキサム酸及びその誘導体、甘草抽出物、グラブリジン、カリンの果実の熱水抽出物、各種生薬、酢酸トコフェロール、グリチルリチン酸及びその誘導体又はその塩等の薬剤、ビタミンC、アスコルビン酸リン酸マグネシウム、アスコルビン酸グルコシド、アルブチン、コウジ酸等の美白剤、グルコース、フルクトース、マンノース、ショ糖、トレハロース等の糖類なども適宜配合することができる。
Furthermore, metal ion blockers such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid, preservatives such as methylparaben, ethylparaben, and butylparaben, caffeine, tannin, Bellapamil, tranexamic acid and its derivatives, licorice extract, glabridin, hot water extract of carin fruit, various crude drugs, tocopherol acetate, glycyrrhizinic acid and its derivatives or salts thereof, vitamin C, magnesium ascorbic acid phosphate, Whitening agents such as ascorbic acid glucoside, arbutin, and kodiic acid, and sugars such as glucose, fructose, mannose, sucrose, and trehalose can also be appropriately added.
本発明の皮膚外用剤は、外皮に適用される化粧料、医薬部外品等、特に好適には化粧料として適用可能であり、その剤型も皮膚に適用できるものであれば限定されず、溶液系、可溶化系、乳化系、粉末分散系、水-油二層系、水-油-粉末三層系、軟膏、化粧水、ゲル、エアゾール等、任意の剤型が適用される。
The external preparation for skin of the present invention can be applied as a cosmetic, a non-medicinal product, etc. applied to the outer skin, particularly preferably as a cosmetic, and the dosage form is not limited as long as it can be applied to the skin. Any dosage form such as solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc. is applied.
本発明の剤を化粧品として用いる場合は、化粧水、乳液、ファンデーション、口紅、リップクリーム、クレンジングクリーム、マッサージクリーム、パック、ハンドクリーム、ハンドパウダー、ボディシャンプー、ボディローション、ボディクリーム、浴用化粧品等の形態として用いてもよい。
When the agent of the present invention is used as cosmetics, it is used in lotions, emulsions, foundations, lipsticks, lip balms, cleansing creams, massage creams, facial masks, hand creams, hand powders, body shampoos, body lotions, body creams, bath cosmetics, etc. It may be used as a form.
本発明の剤を医薬品や医薬部外品等の製剤として用いる場合であれば、該製剤は経口的にあるいは非経口的(静脈投与、腹腔内投与、等)に適宜使用される。剤型も任意で、例えば錠剤、顆粒剤、散剤、カプセル剤等の経口用固形製剤や、内服液剤、シロップ剤等の経口用液体製剤、又は、注射剤などの非経口用液体製剤など、いずれの形態にも公知の方法により適宜調製することができる。外用製剤であれば、ローション剤、懸濁剤・乳剤、液剤、軟膏剤、貼付剤等の各種形態として使用できる。これらの製剤には、通常用いられる結合剤、崩壊剤、増粘剤、分散剤、再吸収促進剤、矯味剤、緩衝剤、界面活性剤、溶解補助剤、保存剤、乳化剤、等張化剤、安定化剤やpH調整剤などの賦形剤を適宜使用してもよい。
When the agent of the present invention is used as a preparation for a drug, a quasi drug, etc., the preparation is appropriately used orally or parenterally (intravenous administration, intraperitoneal administration, etc.). The dosage form is also arbitrary, for example, oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as oral liquids and syrups, and parenteral liquid preparations such as injections. The form of the above can be appropriately prepared by a known method. If it is an external preparation, it can be used in various forms such as a lotion, a suspension / emulsion, a liquid, an ointment, and a patch. These formulations include commonly used binders, disintegrants, thickeners, dispersants, reabsorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonic agents. , Excipients such as stabilizers and pH adjusters may be used as appropriate.
しかしながら、本発明の剤の採り得る形態は、上述の剤型や形態に限定されるものではない。
However, the possible forms of the agent of the present invention are not limited to the above-mentioned dosage forms and forms.
次に実施例によって本発明をさらに詳細に説明する。なお、本発明はこれにより限定されるものではない。
Next, the present invention will be described in more detail by way of examples. The present invention is not limited thereto.
実験1:試料の調製
PDGF-BBの産生亢進作用の評価対象試料として以下を用いた。 Experiment 1: Preparation of sample The following samples were used as the samples to be evaluated for the effect of enhancing the production of PDGF-BB.
PDGF-BBの産生亢進作用の評価対象試料として以下を用いた。 Experiment 1: Preparation of sample The following samples were used as the samples to be evaluated for the effect of enhancing the production of PDGF-BB.
その他に、動植物の抽出物といった天然由来成分や合成成分を47種類調製した。実験2ではアムラ抽出物及びリンゴンベリー抽出物を除く上記試料と合わせて合計52種類の試料を用い、更に、陰性対照として0.5%DMSO、陽性対照としてトレチノインを使用した。実験3では、陰性対照として0.5%DMSO、比較対照としてアムラ抽出物及びリンゴンベリー抽出物を使用した。抽出物は乾燥した状態で冷蔵庫に保存し、培地に対して(抽出物の乾燥重量換算で)10μg/mLとなるように使用したが、リンゴンベリー抽出物及びアムラ抽出物については、特許文献2の実施例で効果が見られた値とほぼ同等である15μg/mLおよび2μg/mLとなるようにそれぞれ調製した。L-テアニン、トレチノイン等の化合物は、培地に対して10μg/mLとなるように使用した。
In addition, 47 kinds of naturally derived ingredients such as animal and plant extracts and synthetic ingredients were prepared. In Experiment 2, a total of 52 types of samples were used, including the above samples excluding Amla extract and Ringonberry extract, and 0.5% DMSO was used as a negative control and tretinoin was used as a positive control. In Experiment 3, 0.5% DMSO was used as a negative control, and Amla extract and lingonberry extract were used as comparative controls. The extract was stored in a refrigerator in a dry state and used so as to be 10 μg / mL with respect to the medium (in terms of dry weight of the extract). The values were adjusted to 15 μg / mL and 2 μg / mL, which are almost the same as the values obtained in the examples of. Compounds such as L-theanine and tretinoin were used so as to be 10 μg / mL with respect to the medium.
実験2:PDGF-BBの産生亢進作用の評価
Thermo Fisher Scientific社のELISAキット(製品名EHCSRP2)を使用し測定を行った。当該キットの内容および量を以下に示す。
Experiment 2: Evaluation of PDGF-BB production-enhancing effect Measurement was performed using an ELISA kit (product name: EHCSRP2) manufactured by Thermo Fisher Scientific. The contents and amount of the kit are shown below.
Thermo Fisher Scientific社のELISAキット(製品名EHCSRP2)を使用し測定を行った。当該キットの内容および量を以下に示す。
試薬の調製
1. 測定前に、すべての試薬およびサンプルを室温(18~25℃)に戻した。
2. サンプル希釈液(10mL⇒50mL)とアッセイ希釈液(6mL⇒30mL)は、測定前に脱イオン水または蒸留水で5倍に希釈した。細胞ライセート緩衝液は、脱イオン水または蒸留水で2倍に希釈した。
3. 試料の希釈:HUVEC(ヒト臍帯静脈内皮細胞)と評価対象試料との反応液を1×サンプル希釈液で少なくとも5倍に希釈して測定試料を調製した(PDGF-BBのレベルは、サンプルによって異なるので、各サンプルの最適希釈係数は適宜決定した)。
4. 標準の調製:凍結乾燥した標準バイアルに280μLの1×サンプル希釈液を加えて50ng/mLの標準液を調製した。穏やかに混合して粉末を完全に溶解させた。400pg/mLのストック標準溶液を調製するために、496μLのサンプル希釈液を含むチューブに、再構成標準のバイアルから4μLのPDGF-BB標準液を加えた。各チューブに400μLの1×サンプル希釈液をピペットで注入した。下記のように原液標準液を使用して希釈系列を作成した。次の移送の前に各チューブを十分に混合した。1×サンプル希釈液をゼロ標準液(0pg/mL)とした。
Preparation of reagents
1. Before measurement, all reagents and samples were returned to room temperature (18-25 ° C).
2. The sample diluent (10 mL ⇒ 50 mL) and the assay diluent (6 mL ⇒ 30 mL) were diluted 5-fold with deionized or distilled water before measurement. Cell lysate buffer was diluted 2-fold with deionized or distilled water.
3. Sample dilution: A measurement sample was prepared by diluting the reaction solution of HUVEC (human umbilical vein endothelial cells) with the sample to be evaluated at least 5 times with 1 × sample dilution (PDGF-BB level is sample). The optimum dilution factor for each sample was determined as appropriate).
4. Standard preparation: 280 μL of 1 × sample diluent was added to a lyophilized standard vial to prepare a standard solution of 50 ng / mL. Gently mixed to completely dissolve the powder. To prepare a 400 pg / mL stock standard solution, 4 μL PDGF-BB standard solution was added from a reconstituted standard vial to a tube containing 496 μL sample diluent. Each tube was pipetted with 400 μL of 1 × sample diluent. A dilution series was prepared using the stock solution standard solution as shown below. Each tube was thoroughly mixed before the next transfer. The 1 × sample diluent was used as the zero standard solution (0 pg / mL).
1. 測定前に、すべての試薬およびサンプルを室温(18~25℃)に戻した。
2. サンプル希釈液(10mL⇒50mL)とアッセイ希釈液(6mL⇒30mL)は、測定前に脱イオン水または蒸留水で5倍に希釈した。細胞ライセート緩衝液は、脱イオン水または蒸留水で2倍に希釈した。
3. 試料の希釈:HUVEC(ヒト臍帯静脈内皮細胞)と評価対象試料との反応液を1×サンプル希釈液で少なくとも5倍に希釈して測定試料を調製した(PDGF-BBのレベルは、サンプルによって異なるので、各サンプルの最適希釈係数は適宜決定した)。
4. 標準の調製:凍結乾燥した標準バイアルに280μLの1×サンプル希釈液を加えて50ng/mLの標準液を調製した。穏やかに混合して粉末を完全に溶解させた。400pg/mLのストック標準溶液を調製するために、496μLのサンプル希釈液を含むチューブに、再構成標準のバイアルから4μLのPDGF-BB標準液を加えた。各チューブに400μLの1×サンプル希釈液をピペットで注入した。下記のように原液標準液を使用して希釈系列を作成した。次の移送の前に各チューブを十分に混合した。1×サンプル希釈液をゼロ標準液(0pg/mL)とした。
1. Before measurement, all reagents and samples were returned to room temperature (18-25 ° C).
2. The sample diluent (10 mL ⇒ 50 mL) and the assay diluent (6 mL ⇒ 30 mL) were diluted 5-fold with deionized or distilled water before measurement. Cell lysate buffer was diluted 2-fold with deionized or distilled water.
3. Sample dilution: A measurement sample was prepared by diluting the reaction solution of HUVEC (human umbilical vein endothelial cells) with the sample to be evaluated at least 5 times with 1 × sample dilution (PDGF-BB level is sample). The optimum dilution factor for each sample was determined as appropriate).
4. Standard preparation: 280 μL of 1 × sample diluent was added to a lyophilized standard vial to prepare a standard solution of 50 ng / mL. Gently mixed to completely dissolve the powder. To prepare a 400 pg / mL stock standard solution, 4 μL PDGF-BB standard solution was added from a reconstituted standard vial to a tube containing 496 μL sample diluent. Each tube was pipetted with 400 μL of 1 × sample diluent. A dilution series was prepared using the stock solution standard solution as shown below. Each tube was thoroughly mixed before the next transfer. The 1 × sample diluent was used as the zero standard solution (0 pg / mL).
5. 20×洗浄緩衝液に目に見える結晶が含まれている場合は、室温まで暖め、溶解するまで静かに混合した。20mLの洗浄緩衝液の濃縮液を脱イオン水または蒸留水に希釈して、400mLの1×洗浄緩衝液を得た。
6. 使用前に、100μLの1×アッセイ希釈液をバイアルに加えて、ビオチン化抗体濃縮物を調製した。ピペットで上下に軽く混ぜた。ビオチン化抗体濃縮物は、1×アッセイ希釈液で80倍に希釈し(180μL⇒14400μL)、以下に記載のアッセイ手順のステップ4で使用した。
7. ストレプトアビジン-HRP試薬は、1×アッセイ希釈液で800倍に希釈した。HRP-ストレプトアビジン濃縮液20μLを16mLの1×アッセイ希釈液の入ったチューブに加え、800倍希釈HRP-ストレプトアビジン溶液を調製した。 5. If the wash buffer contained visible crystals, warm to room temperature and mix gently until dissolved. A concentrated solution of 20 mL of wash buffer was diluted with deionized water or distilled water to obtain 400 mL of 1 × wash buffer.
6. Prior to use, 100 μL of 1 × assay diluent was added to the vial to prepare a biotinylated antibody concentrate. Lightly mixed up and down with a pipette. The biotinylated antibody concentrate was diluted 80-fold with 1 × assay diluent (180 μL ⇒ 14400 μL) and used instep 4 of the assay procedure described below.
7. Streptavidin-HRP reagent was diluted 800-fold with 1 × assay diluent. 20 μL of HRP-streptavidin concentrate was added to a tube containing 16 mL of 1 × assay diluent to prepare an 800-fold diluted HRP-streptavidin solution.
6. 使用前に、100μLの1×アッセイ希釈液をバイアルに加えて、ビオチン化抗体濃縮物を調製した。ピペットで上下に軽く混ぜた。ビオチン化抗体濃縮物は、1×アッセイ希釈液で80倍に希釈し(180μL⇒14400μL)、以下に記載のアッセイ手順のステップ4で使用した。
7. ストレプトアビジン-HRP試薬は、1×アッセイ希釈液で800倍に希釈した。HRP-ストレプトアビジン濃縮液20μLを16mLの1×アッセイ希釈液の入ったチューブに加え、800倍希釈HRP-ストレプトアビジン溶液を調製した。 5. If the wash buffer contained visible crystals, warm to room temperature and mix gently until dissolved. A concentrated solution of 20 mL of wash buffer was diluted with deionized water or distilled water to obtain 400 mL of 1 × wash buffer.
6. Prior to use, 100 μL of 1 × assay diluent was added to the vial to prepare a biotinylated antibody concentrate. Lightly mixed up and down with a pipette. The biotinylated antibody concentrate was diluted 80-fold with 1 × assay diluent (180 μL ⇒ 14400 μL) and used in
7. Streptavidin-HRP reagent was diluted 800-fold with 1 × assay diluent. 20 μL of HRP-streptavidin concentrate was added to a tube containing 16 mL of 1 × assay diluent to prepare an 800-fold diluted HRP-streptavidin solution.
アッセイ手順
1. 使用前に、すべての試薬およびサンプルを室温(18~25℃)に戻した。すべての標準とサンプルは、少なくとも2連で実行した。
2. 各標準液100μLを添加し(試薬調製手順のステップ3を参照)、適切なウェルにサンプルを分注した。ウェルを覆い、穏やかに振盪しながら室温で2.5時間インキュベートした。
3. 溶液を捨て、1×洗浄緩衝液で4回洗浄した。マルチチャンネルのピペットを使用して各ウェルに洗浄緩衝液(300μL)を満たして洗浄した。最後の洗浄の後、吸引またはデカントすることによって残りの洗浄緩衝液をすべて除去した。プレートを逆さまにし、きれいな紙タオルで水分を十分に除去した。
4. 100μLの1×調製ビオチン化抗体(試薬調製手順のステップ6を参照)を各ウェルに加えた。穏やかに振とうしながら室温で1時間インキュベートした。
5. 溶液を捨て、ステップ3の洗浄を再び行った。
6. 調製したストレプトアビジン-HRP溶液(試薬調製手順のステップ7を参照)100μLを各ウェルに加えた。穏やかに振とうしながら室温で45分間インキュベートした。
7. 溶液を捨て、ステップ3の洗浄を再び行った。
8. 各ウェルに100μLのTMB基質を加えた。穏やかに振とうしながら暗所・室温で30分間インキュベートした。
9. 50μLの停止液を各ウェルに加えた。
10. プレートは反応を停止してから30分以内に評価した。450nmおよび550nmに設定したELISAプレートリーダーで吸光度を測定した。550nm値を450nm値から差し引いて、マイクロプレートの光学的不完全性を補正した。 Assay procedure
1. Before use, all reagents and samples were returned to room temperature (18-25 ° C). All standards and samples were run in at least two runs.
2. 100 μL of each standard solution was added (see step 3 of the reagent preparation procedure) and the sample was dispensed into the appropriate wells. Wells were covered and incubated for 2.5 hours at room temperature with gentle shaking.
3. The solution was discarded and washed 4 times with 1 x wash buffer. Each well was flushed with wash buffer (300 μL) using a multi-channel pipette. After the final wash, all remaining wash buffer was removed by suction or decanting. The plate was turned upside down and a clean paper towel was used to thoroughly remove the water.
4. 100 μL of 1 × prepared biotinylated antibody (see step 6 of reagent preparation procedure) was added to each well. Incubated for 1 hour at room temperature with gentle shaking.
5. Discard the solution and repeat the wash in step 3.
6. 100 μL of the prepared streptavidin-HRP solution (see step 7 of the reagent preparation procedure) was added to each well. Incubated for 45 minutes at room temperature with gentle shaking.
7. The solution was discarded and the wash in step 3 was repeated.
8. 100 μL of TMB substrate was added to each well. Incubated for 30 minutes in the dark at room temperature with gentle shaking.
9. 50 μL of stop solution was added to each well.
10. Plates were evaluated within 30 minutes of stopping the reaction. Absorbance was measured with an ELISA plate reader set at 450 nm and 550 nm. The 550 nm value was subtracted from the 450 nm value to correct for the optical imperfections of the microplate.
1. 使用前に、すべての試薬およびサンプルを室温(18~25℃)に戻した。すべての標準とサンプルは、少なくとも2連で実行した。
2. 各標準液100μLを添加し(試薬調製手順のステップ3を参照)、適切なウェルにサンプルを分注した。ウェルを覆い、穏やかに振盪しながら室温で2.5時間インキュベートした。
3. 溶液を捨て、1×洗浄緩衝液で4回洗浄した。マルチチャンネルのピペットを使用して各ウェルに洗浄緩衝液(300μL)を満たして洗浄した。最後の洗浄の後、吸引またはデカントすることによって残りの洗浄緩衝液をすべて除去した。プレートを逆さまにし、きれいな紙タオルで水分を十分に除去した。
4. 100μLの1×調製ビオチン化抗体(試薬調製手順のステップ6を参照)を各ウェルに加えた。穏やかに振とうしながら室温で1時間インキュベートした。
5. 溶液を捨て、ステップ3の洗浄を再び行った。
6. 調製したストレプトアビジン-HRP溶液(試薬調製手順のステップ7を参照)100μLを各ウェルに加えた。穏やかに振とうしながら室温で45分間インキュベートした。
7. 溶液を捨て、ステップ3の洗浄を再び行った。
8. 各ウェルに100μLのTMB基質を加えた。穏やかに振とうしながら暗所・室温で30分間インキュベートした。
9. 50μLの停止液を各ウェルに加えた。
10. プレートは反応を停止してから30分以内に評価した。450nmおよび550nmに設定したELISAプレートリーダーで吸光度を測定した。550nm値を450nm値から差し引いて、マイクロプレートの光学的不完全性を補正した。 Assay procedure
1. Before use, all reagents and samples were returned to room temperature (18-25 ° C). All standards and samples were run in at least two runs.
2. 100 μL of each standard solution was added (see step 3 of the reagent preparation procedure) and the sample was dispensed into the appropriate wells. Wells were covered and incubated for 2.5 hours at room temperature with gentle shaking.
3. The solution was discarded and washed 4 times with 1 x wash buffer. Each well was flushed with wash buffer (300 μL) using a multi-channel pipette. After the final wash, all remaining wash buffer was removed by suction or decanting. The plate was turned upside down and a clean paper towel was used to thoroughly remove the water.
4. 100 μL of 1 × prepared biotinylated antibody (see step 6 of reagent preparation procedure) was added to each well. Incubated for 1 hour at room temperature with gentle shaking.
5. Discard the solution and repeat the wash in step 3.
6. 100 μL of the prepared streptavidin-HRP solution (see step 7 of the reagent preparation procedure) was added to each well. Incubated for 45 minutes at room temperature with gentle shaking.
7. The solution was discarded and the wash in step 3 was repeated.
8. 100 μL of TMB substrate was added to each well. Incubated for 30 minutes in the dark at room temperature with gentle shaking.
9. 50 μL of stop solution was added to each well.
10. Plates were evaluated within 30 minutes of stopping the reaction. Absorbance was measured with an ELISA plate reader set at 450 nm and 550 nm. The 550 nm value was subtracted from the 450 nm value to correct for the optical imperfections of the microplate.
感受性:1pg/mL
感度または検出の下限(LLD)は、ゼロおよび測定毎の標準曲線によって決定した。標準曲線から読み取ったゼロ+2標準偏差の値がLLDである(95%信頼度でゼロでない最小線量)。 Sensitivity: 1 pg / mL
The lower limit of sensitivity or detection (LLD) was determined by zero and a standard curve per measurement. The value of zero + 2 standard deviation read from the standard curve is LLD (minimum dose that is not zero with 95% confidence).
感度または検出の下限(LLD)は、ゼロおよび測定毎の標準曲線によって決定した。標準曲線から読み取ったゼロ+2標準偏差の値がLLDである(95%信頼度でゼロでない最小線量)。 Sensitivity: 1 pg / mL
The lower limit of sensitivity or detection (LLD) was determined by zero and a standard curve per measurement. The value of zero + 2 standard deviation read from the standard curve is LLD (minimum dose that is not zero with 95% confidence).
上記評価手順に従い、上記52種類の各サンプル(n=4)について得られたPDGF-BBタンパク量を測定し、スクリーニングを行った。陽性対照として10μg/mLのトレチノインを、陰性対照として0.5%DMSOを使用した。
According to the above evaluation procedure, the amount of PDGF-BB protein obtained for each of the above 52 types of samples (n = 4) was measured and screened. 10 μg / mL tretinoin was used as the positive control and 0.5% DMSO was used as the negative control.
結果:
陰性対照(試料無添加の0.5%DMSO)について得られたタンパク量に対する割合を、図1及び下記表3に示す。図1、表3の結果から、52種類のサンプルのうち、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物にとりわけ高い効果がみられることが判明し、これらの成分はPDGF-BBの産生を亢進させる作用を有することが分かる。図中、試料A~Cは、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物以外の47種類の物質のうち3種の試料を示すが、他の物質も試料A~Cと同様に陰性対照(DMSO)より低いか又は有意な差が見られない等、基準を満たすものではなかった。 result:
The ratio to the amount of protein obtained for the negative control (0.5% DMSO without sample) is shown in FIG. 1 and Table 3 below. From the results of FIGS. 1 and 3, it was found that the wasabi extract, chamomile extract, L-theanine, and hibiscus extract were particularly effective among the 52 types of samples, and these components were found to be particularly effective in PDGF-. It can be seen that it has the effect of enhancing the production of BB. In the figure, samples A to C show three samples out of 47 kinds of substances other than wasabi extract, chamomile extract, L-theanine, and hibiscus extract, but other substances are also sample A to C. Similarly, it did not meet the criteria, such as being lower than the negative control (DMSO) or showing no significant difference.
陰性対照(試料無添加の0.5%DMSO)について得られたタンパク量に対する割合を、図1及び下記表3に示す。図1、表3の結果から、52種類のサンプルのうち、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物にとりわけ高い効果がみられることが判明し、これらの成分はPDGF-BBの産生を亢進させる作用を有することが分かる。図中、試料A~Cは、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物以外の47種類の物質のうち3種の試料を示すが、他の物質も試料A~Cと同様に陰性対照(DMSO)より低いか又は有意な差が見られない等、基準を満たすものではなかった。 result:
The ratio to the amount of protein obtained for the negative control (0.5% DMSO without sample) is shown in FIG. 1 and Table 3 below. From the results of FIGS. 1 and 3, it was found that the wasabi extract, chamomile extract, L-theanine, and hibiscus extract were particularly effective among the 52 types of samples, and these components were found to be particularly effective in PDGF-. It can be seen that it has the effect of enhancing the production of BB. In the figure, samples A to C show three samples out of 47 kinds of substances other than wasabi extract, chamomile extract, L-theanine, and hibiscus extract, but other substances are also sample A to C. Similarly, it did not meet the criteria, such as being lower than the negative control (DMSO) or showing no significant difference.
実験3:アムラ抽出物及びリンゴンベリー抽出物との比較
実験2において有意なPDGF-BB産生亢進作用が見られたワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物について、上記評価手順に従ってPDGF-BBタンパク量を測定し、PDGF-BB産生亢進作用を有することが知られている15μg/mLリンゴンベリー抽出物および2μg/mLアムラ抽出物と比較した。 Experiment 3: Comparison with Amla extract and lingon berry extract The above evaluation procedure for wasabi extract, chamomile extract, L-theanin, and hibiscus extract, which showed a significant PDGF-BB production-enhancing effect in Experiment 2. The amount of PDGF-BB protein was measured according to the above, and compared with the 15 μg / mL lingon berry extract and the 2 μg / mL Amla extract, which are known to have a PDGF-BB production-enhancing effect.
実験2において有意なPDGF-BB産生亢進作用が見られたワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物について、上記評価手順に従ってPDGF-BBタンパク量を測定し、PDGF-BB産生亢進作用を有することが知られている15μg/mLリンゴンベリー抽出物および2μg/mLアムラ抽出物と比較した。 Experiment 3: Comparison with Amla extract and lingon berry extract The above evaluation procedure for wasabi extract, chamomile extract, L-theanin, and hibiscus extract, which showed a significant PDGF-BB production-enhancing effect in Experiment 2. The amount of PDGF-BB protein was measured according to the above, and compared with the 15 μg / mL lingon berry extract and the 2 μg / mL Amla extract, which are known to have a PDGF-BB production-enhancing effect.
結果:
対照(15μg/mLリンゴンベリー抽出物)について得られたタンパク量を100.0としてそれに対する割合を図2に示し、陰性対照(試料無添加の0.5%DMSO)について得られたタンパク量を100.0としてそれに対する割合を図3に示す。図2,3より、陰性対照のみならず、従前に高いPDGF-BB産生亢進作用を有することが知られていたリンゴンベリーエキスやアムラエキス(特許文献2)と比較しても有意に高いPDGF-BB産生亢進能を奏することが分かった。 result:
The amount of protein obtained for the control (15 μg / mL lingon berry extract) was 100.0 and the ratio to it was shown in FIG. 2, and the amount of protein obtained for the negative control (0.5% DMSO without sample) was 100.0. The ratio is shown in FIG. From FIGS. 2 and 3, PDGF-BB is significantly higher than that of lingonberry extract and amla extract (Patent Document 2), which were previously known to have a high PDGF-BB production enhancing effect as well as a negative control. It was found that it exerts a production-enhancing ability.
対照(15μg/mLリンゴンベリー抽出物)について得られたタンパク量を100.0としてそれに対する割合を図2に示し、陰性対照(試料無添加の0.5%DMSO)について得られたタンパク量を100.0としてそれに対する割合を図3に示す。図2,3より、陰性対照のみならず、従前に高いPDGF-BB産生亢進作用を有することが知られていたリンゴンベリーエキスやアムラエキス(特許文献2)と比較しても有意に高いPDGF-BB産生亢進能を奏することが分かった。 result:
The amount of protein obtained for the control (15 μg / mL lingon berry extract) was 100.0 and the ratio to it was shown in FIG. 2, and the amount of protein obtained for the negative control (0.5% DMSO without sample) was 100.0. The ratio is shown in FIG. From FIGS. 2 and 3, PDGF-BB is significantly higher than that of lingonberry extract and amla extract (Patent Document 2), which were previously known to have a high PDGF-BB production enhancing effect as well as a negative control. It was found that it exerts a production-enhancing ability.
以上の結果により、ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物には、PDGF-BB産生を亢進する効果がとりわけ高いことが分かった。これらの物質は、PDGF-BB産生を亢進することにより、間葉系幹細胞を安定化させ、ひいては皮膚を賦活化し老化を抑制することが期待される。
From the above results, it was found that wasabi extract, chamomile extract, L-theanine, and hibiscus extract are particularly effective in enhancing PDGF-BB production. These substances are expected to stabilize mesenchymal stem cells by enhancing PDGF-BB production, thereby activating the skin and suppressing aging.
Claims (3)
- ワサビ抽出物、カミツレ抽出物、L-テアニン、及びハイビスカス抽出物の少なくともいずれかを有効成分として含んでなる血小板由来成長因子-BB(PDGF-BB)産生亢進剤。 A platelet-derived growth factor-BB (PDGF-BB) production enhancer containing at least one of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient.
- 請求項1に記載のPDGF-BB産生亢進剤を含んでなる、幹細胞安定化剤。 A stem cell stabilizer comprising the PDGF-BB production enhancer according to claim 1.
- 請求項1に記載のPDGF-BB産生亢進剤を含んでなる皮膚抗老化剤であって、皮膚における幹細胞を安定化することにより皮膚の老化を抑制する、皮膚抗老化剤。 A skin anti-aging agent comprising the PDGF-BB production enhancer according to claim 1, which suppresses skin aging by stabilizing stem cells in the skin.
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JPWO2021084665A1 (en) | 2021-05-06 |
JP2024036562A (en) | 2024-03-15 |
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