JP2025170060A - Platelet-derived growth factor (PDGF)-BB production enhancer, stem cell stabilizer containing same, and skin anti-aging agent containing same - Google Patents

Abstract

[Problem] To provide an agent effective in enhancing PDGF-BB production, and an agent effective in revitalizing and anti-aging skin by stabilizing mesenchymal stem cells in the skin using such an agent. [Solution] A PDGF-BB production enhancer containing any of wasabi extract, chamomile extract, L-theanine, and hibiscus extract as an active ingredient is effective in revitalizing and anti-aging skin by stabilizing mesenchymal stem cells. [Selected Figure] Figure 3

Description

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.

Mesenchymal stem cells differentiate into a variety of mesenchymal cells (bone cells, muscle cells, chondrocytes, tendon cells, adipocytes, etc.), and their application in regenerative medicine is being studied. For example, stabilizing mesenchymal stem cells is effective for a variety of applications, including stabilizing blood vessels, maintaining tissue homeostasis, and preventing and improving various conditions such as repairing and regenerating damaged tissue. It has also been reported that stabilizing mesenchymal stem cells in the skin is effective for skin revitalization 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 a component effective in enhancing PDGF-BB production can be found, it could be used to stabilize mesenchymal stem cells, which could then be effectively used for skin revitalization and anti-aging purposes.

Retinoic acid, amla extract, and lingonberry extract have been reported as ingredients effective in increasing PDGF-BB production (Patent Documents 1 and 2). It is anticipated that various substances with a high PDGF-BB production-enhancing effect will be explored.

Patent No. 5933443 Patent No. 5496951

The present invention was made in light of the above background, and its objective is to provide an agent that is effective in enhancing PDGF-BB production, and to use this agent to provide an agent that is effective in revitalizing the skin and preventing aging through the stabilization of mesenchymal stem cells in the skin.

The inventors conducted extensive research into a wide variety of materials and screened for drugs that enhance PDGF-BB production. As a result, they discovered that wasabi extract, chamomile extract, L-theanine, and hibiscus extract exhibited significant PDGF-BB production-enhancing effects, leading to the creation of the present invention.

Therefore, the present application encompasses the following inventions:
[1] A platelet-derived growth factor-BB (PDGF-BB) production enhancer comprising 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 described in [1].
[3] A skin anti-aging agent comprising the PDGF-BB production enhancer described in [1], which inhibits skin aging by stabilizing stem cells in the skin.

The present invention provides an agent that is effective in increasing PDGF-BB production, and by using this agent, an agent that is effective in stabilizing stem cells, etc. is also provided. Stabilizing stem cells in the skin is effective in inhibiting skin aging.

FIG. 1 shows the screening results of Experiment 2, and is shown as a relative value with the result of the control (0.5% DSMO) set at 100.0. Figure 2 shows the results of Experiment 3, comparing the PDGF-BB production-enhancing ability of various samples with 15 μg/mL lingonberry extract and 2 μg/mL amla extract, with the results for the control (15 μg/mL lingonberry extract) set at 100.0, and shows the relative values. Figure 3 shows the results of Experiment 3, comparing the PDGF-BB production-enhancing ability of various samples with that of the negative control (0.5% DSMO), 15 μg/mL lingonberry extract, and 2 μg/mL amla extract. The results are shown as relative values, with the result of the negative control (0.5% DSMO) set at 100.0.

The inventors have discovered that wasabi extract, chamomile extract, L-theanine, and hibiscus extract exhibit significant PDGF-BB production-enhancing and stem cell stabilizing effects, and the present invention is based on this discovery.

Wasabi extract has been reported to have antibacterial, antioxidant, moisturizing, and lipase inhibitory activities (Japanese Patent Publication No. 2016-124845). Fermented products obtained by fermenting chamomile extract have been reported to promote cell differentiation (Japanese Patent Publication No. 2015-157772). Chamomile extract has also been reported to inhibit the elevation of stem cell growth factor (SCF) mRNA expression (Japanese Patent Publication No. 2011-1327), basic fibroblast growth factor (bFGF) mRNA expression (Japanese Patent Publication No. 2011-1328), and inhibit the production and/or release of stem cell factor (hereinafter referred to as "SCF") (Japanese Patent Publication No. 2003-194809). Theanine has been reported to improve epidermal stem cell functionality (JP 2015-178485 A) and inhibit parakeratosis (JP 2007-204417 A). Hibiscus extract has been reported to inhibit hyaluronidase activity and inhibit the increase in stem cell growth factor mRNA expression (JP 2014-218476 A). However, the present inventors have now discovered for the first time that these substances also have the PDGF-BB production-promoting and stem cell-stabilizing effects of the present invention.

The PDGF-BB production-enhancing effect refers to the effect of increasing the production of platelet-derived growth factor-BB (PDGF-BB) protein. For example, increased PDGF-BB production acts on mesenchymal stem cells, stabilizing them and activating 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 protein amount. This measurement can be performed using a PDGF-BB-specific antibody and various methods well known in the art, such as immunostaining using fluorescent substances, dyes, enzymes, etc., Western blotting, and immunoassays such as ELISA and RIA. It can also be determined indirectly, for example, by extracting total RNA from mesenchymal stem cells and measuring the amount of mRNA encoding PDGF-BB, but measuring the actual protein amount provides a more direct assessment.

Stem cell stabilizing activity refers to the activity of attracting and localizing stem cells to a target site, and/or the activity of retaining stem cells at a target site and maintaining that localized state. In this specification, stem cell stabilizing activity is distinguished from the activity of promoting stem cell proliferation and/or the activity of maintaining stem cells in an undifferentiated state, and may not include these activities. Stem cell stabilizing activity can be measured, for example, by methods such as those for measuring migration ability and localization in the skin, as described in Patent Document 1, without limitation.

Skin revitalization includes, but is not limited to, promoting the metabolism and turnover of cells in animals, including humans, such as skin tissue, improving function, promoting proliferation, inhibiting oxidation, improving resistance to fatigue and external stimuli, and inhibiting decline in function and activity. Skin revitalization is expected to have effects such as preventing and improving wrinkles, age spots, skin aging, photoaging, etc.

[Wasabi extract]
Wasabi (genus Eutrema) is a perennial plant of the Brassicaceae family. The wasabi extract used in the present invention is preferably from Eutrema japonicum (Miq.) Koidz., but other species such as lily wasabi (Eutrema tenue) and related species (Eutrema yunnanense) may also be used. Extracts of wasabi leaves are preferred, but since active ingredients are also contained in the seeds, stems, flowers, roots, etc., extracts of one or more of these may also be used. Wasabi extracts are commercially available from Kinjirushi Co., Ltd., etc., and such commercially available products may also be used.

[Chamomile extract]
Chamomile (scientific name: Matricaria chamomilla) is an annual plant belonging to the Asteraceae family, genus Matricaria. The chamomile extract used in the present invention is preferably an extract of chamomile flower heads. However, since active ingredients are also contained in chamomile seeds, leaves, stems, flowers, roots, etc., extracts of one or more of these may also be used. Chamomile extracts are commercially available from Maruzen Pharmaceutical Co., Ltd., etc., and such commercially available products may also be used.

[L-theanine]
L-theanine is a type of amino acid represented by the following structural formula.

L-theanine is found in large amounts in tea leaves such as green tea and black tea, so the L-theanine contained in these tea leaves can be used, or it can be used in the form of an extract of these tea leaves. However, it can also be artificially synthesized, and commercially available products from companies such as Taiyo Kagaku can also be used.

[Hibiscus extract]
Hibiscus (genus Hibiscus) is an annual or perennial subshrub belonging to the family Malvaceae, order Malvaceae. The hibiscus extract used in the present invention is preferably an extract of roselle (Hibiscus sabdariffa). While an extract of hibiscus flowers is preferred, hibiscus fruits, seeds, leaves, stems, flowers, roots, etc. also contain active ingredients, so extracts of one or more of these can also be used. Hibiscus extracts are commercially available from Nikken Foods Co., Ltd., etc., and such commercially available products can also be used.

When using an extract, the extraction method is not particularly limited, but extraction methods using a solvent are preferred. When performing extraction, the plant body can be used as is, but crushing it into granules or powder before extraction allows for extraction of active ingredients under milder conditions, in a shorter time, and with higher extraction efficiency. The extraction temperature is not particularly limited and can be set appropriately depending on the particle size of the crushed material, the type of solvent, etc. It is usually set within the range from room temperature to the boiling point of the solvent. The extraction time is also not particularly limited and can be set appropriately depending on the particle size of the crushed material, the type of solvent, and the extraction temperature, etc. Furthermore, during extraction, stirring may be performed, the mixture may be left to stand without stirring, or ultrasound may be used.

The type of solvent is not particularly limited, but water, lower alcohols such as hydrous ethanol and ethanol, organic solvents such as hexane, or mixed solvents of these, such as hexane/ethanol, are preferred. Extraction can be performed at room temperature or under heat (using a heated solvent, such as warm water or hot water). Alternatively, enzymes can be added to the solvent for extraction. Adding enzymes can disrupt plant cell tissue, thereby further increasing extraction efficiency. Cell tissue-disintegrating enzymes are preferred. Examples of such enzymes include pectinase, cellulase, hemicellulase, α-amylase, and phytase. These enzymes can be used alone or in combination.

By this extraction procedure, the active ingredients are extracted and dissolved in the solvent. The solvent containing the extract may be used as is, or may be subjected to conventional purification processes such as sterilization, washing, filtration, bleaching, and deodorization before use. It may also be concentrated or diluted as necessary before use. Furthermore, the solvent may be completely evaporated to form a solid (dried product) before use, or the dried product may be redissolved in any solvent before use.

In addition, the juice obtained by pressing the raw plant material contains the same active ingredients as the extract, so the juice can also 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. Furthermore, the stem cell stabilizer of the present invention contains the PDGF-BB production enhancer of the present invention, which contains the above-mentioned active ingredient. For example, the stem cell stabilizer of the present invention enhances PDGF-BB production, and the enhanced PDGF-BB production acts on stem cells such as mesenchymal stem cells, thereby stabilizing the stem cells. The skin anti-aging agent of the present invention contains the PDGF-BB production enhancer of the present invention, which contains the above-mentioned active ingredient. For example, the skin anti-aging agent of the present invention enhances PDGF-BB production, and the enhanced PDGF-BB production acts on stem cells such as mesenchymal stem cells, thereby stabilizing the stem cells and revitalizing the skin, thereby suppressing skin aging. The PDGF-BB production enhancer, stem cell stabilizer, and skin anti-aging agent of the present invention (hereinafter sometimes collectively referred to as "the agent of the present invention") may contain any one of the above active ingredients alone, or may contain two or more of them in any combination and ratio.

The agent of the present invention can also be a composition in which the active ingredient described above is combined with one or more other ingredients, such as excipients, carriers, and/or diluents. The composition may have any composition and form, and may be appropriately selected depending on the active ingredient, intended use, and other conditions. The composition can be manufactured using conventional methods in a formulation in which excipients, carriers, and/or diluents, etc., and other ingredients are appropriately combined depending on the dosage form.

The agent of the present invention can be incorporated into various foods, beverages, and feeds and ingested by humans and animals. It can also be incorporated into cosmetics and other products for use by humans and animals, or administered to humans and animals as a pharmaceutical preparation.

Specifically, when the agent of the present invention is incorporated into foods, beverages, feed, etc., the amount of plant matter or its extract (dry mass) to be incorporated can be determined appropriately depending on the type, purpose, form, method of use, etc. For example, it can be incorporated so that the daily intake of the plant matter or its extract by an adult is approximately 0.5 mg to 3 g (dry residue). In particular, when used in health foods, beverages, etc., it is preferable to include an extract so that the daily intake of an adult is 10 mg to 1.5 g (dry residue) of the extract, so that the desired effects of the active ingredients of the present invention can be fully exerted.

Food, beverage, or feed can be in any form, such as granules, particles, paste, gel, solid, or liquid. These forms can contain various known substances approved for inclusion in foods, beverages, etc., such as excipients such as binders, disintegrants, thickeners, dispersants, resorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, tonicity agents, stabilizers, and pH adjusters, as appropriate.

When applying this invention to external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, the amount (dry mass) of the plant or its extract can be determined appropriately depending on its type, purpose, form, and method of use. For example, wasabi extract, chamomile extract, L-theanine, and hibiscus extract can each be blended in an amount of 0.00001% to 50% (dry mass equivalent) of the total amount of the cosmetic, with 0.0001% to 5% (dry mass equivalent) being preferred.

In addition to the above ingredients, if necessary, ingredients typically used in external skin preparations such as cosmetics, pharmaceuticals, and quasi-drugs, such as antioxidants, oils, UV protection agents, surfactants, thickeners, alcohols, powder ingredients, colorants, aqueous ingredients, water, various skin nutrients, etc., can be appropriately blended as needed within limits that do not impair the effects of the present invention.

Further ingredients may include sequestering agents such as edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, and gluconic acid; preservatives such as methylparaben, ethylparaben, and butylparaben; caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, glabridin, hot water extract of quince fruit, various herbal medicines, pharmaceuticals such as tocopherol acetate, glycyrrhizic acid and its derivatives or salts; whitening agents such as vitamin C, magnesium ascorbyl phosphate, ascorbic acid glucoside, arbutin, and kojic acid; and sugars such as glucose, fructose, mannose, sucrose, and trehalose.

The topical skin preparation of the present invention can be applied to the outer skin as a cosmetic or quasi-drug, and is particularly suitable as a cosmetic. There are no limitations on its formulation, so long as it can be applied to the skin. Any formulation can be used, including solution, solubilized, emulsified, powder dispersion, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc.

When the agent of the present invention is used as a cosmetic, it may be used in the form of a lotion, emulsion, foundation, lipstick, lip balm, cleansing cream, massage cream, pack, hand cream, hand powder, body shampoo, body lotion, body cream, bath cosmetics, etc.

When the agent of the present invention is used as a pharmaceutical or quasi-drug formulation, the formulation may be administered orally or parenterally (intravenously, intraperitoneally, etc.), as appropriate. Any dosage form may be used, including oral solid formulations such as tablets, granules, powders, and capsules; oral liquid formulations such as oral liquids and syrups; and parenteral liquid formulations such as injections, all of which can be prepared appropriately by known methods. Topical formulations may be used in various forms, including lotions, suspensions/emulsions, solutions, ointments, and patches. These formulations may contain, as appropriate, commonly used excipients such as binders, disintegrants, thickeners, dispersants, resorption promoters, flavoring agents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonicity agents, stabilizers, and pH adjusters.

However, the forms that the agent of the present invention can take are not limited to the dosage forms and shapes described above.

The present invention will now be described in more detail using examples. However, the present invention is not limited to these examples.

Experiment 1: Sample preparation
The following samples were used to evaluate the effect of enhancing PDGF-BB production.

In addition, 47 types of natural 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 lingonberry extract. Additionally, 0.5% DMSO was used as a negative control and tretinoin 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 extracts were stored in a dried state in a refrigerator and used at a concentration of 10 μg/mL (calculated as the dry weight of the extract) in the medium. The lingonberry extract and amla extract were prepared at 15 μg/mL and 2 μg/mL, respectively, which are approximately the same values as the effects observed in the examples of Patent Document 2. Compounds such as L-theanine and tretinoin were used at a concentration of 10 μg/mL in the medium.

Experiment 2: Evaluation of the effect of enhancing PDGF-BB production
Measurements were performed using an ELISA kit (product name EHCSRP2) from Thermo Fisher Scientific. The contents and amounts of the kit are shown below.

Preparation of reagents
1. Before measurement, all reagents and samples were allowed to return to room temperature (18-25°C).
2. Sample diluent (10 mL → 50 mL) and 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: The reaction solution of HUVEC (human umbilical vein endothelial cells) and the sample to be evaluated was diluted at least 5-fold with 1x sample dilution solution to prepare the measurement sample (since the PDGF-BB level varies depending on the sample, the optimal dilution factor for each sample was determined appropriately).
4. Standard Preparation: A 50 ng/mL standard solution was prepared by adding 280 μL of 1x sample diluent to a lyophilized standard vial. Gently mix to completely dissolve the powder. To prepare a 400 pg/mL stock standard solution, 4 μL of PDGF-BB standard solution from the reconstituted standard vial was added to a tube containing 496 μL of sample diluent. 400 μL of 1x sample diluent was pipetted into each tube. A dilution series was made using the stock standard as follows: Each tube was mixed thoroughly before the next transfer. The 1x sample diluent served as the zero standard (0 pg/mL).

5. If the 20x Wash Buffer contained visible crystals, warm it to room temperature and mix gently until dissolved. Dilute 20 mL of the Wash Buffer concentrate into deionized or distilled water to obtain 400 mL of 1x Wash Buffer.
6. Before use, prepare the biotinylated antibody concentrate by adding 100 μL of 1x Assay Diluent to the vial. Mix gently by pipetting up and down. The biotinylated antibody concentrate was diluted 80-fold (180 μL to 14,400 μL) with 1x Assay Diluent and used in Step 4 of the assay procedure described below.
7. The streptavidin-HRP reagent was diluted 800-fold in 1x Assay Diluent. 20 μL of HRP-streptavidin concentrate was added to a tube containing 16 mL of 1x Assay Diluent to prepare an 800-fold diluted HRP-streptavidin solution.

Assay procedure
1. All reagents and samples were allowed to warm to room temperature (18-25°C) before use. All standards and samples were run in at least duplicate.
2. Add 100 μL of each standard (see step 3 of the reagent preparation procedure) and dispense the samples into the appropriate wells. Cover the wells and incubate at room temperature with gentle shaking for 2.5 hours.
3. The solution was discarded and the plate was washed four times with 1x wash buffer. Each well was filled with wash buffer (300 μL) using a multichannel pipette. After the final wash, all remaining wash buffer was removed by aspiration or decanting. The plate was then inverted and thoroughly dried on a clean paper towel.
4. Add 100 μL of 1x prepared biotinylated antibody (see step 6 of the reagent preparation procedure) to each well. Incubate at room temperature with gentle shaking for 1 hour.
5. Discard the solution and repeat the wash in step 3.
6. Add 100 μL of the prepared streptavidin-HRP solution (see step 7 of the reagent preparation procedure) to each well. Incubate at room temperature with gentle shaking for 45 minutes.
7. Discard the solution and repeat the wash in step 3.
8. Add 100 μL of TMB substrate to each well. Incubate for 30 minutes at room temperature in the dark 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 using 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 optical imperfections in the microplate.

Sensitivity: 1pg/mL
The sensitivity or lower limit of detection (LLD) was determined by zero and the standard curve for each run. The value of zero plus two standard deviations read from the standard curve is the LLD (the lowest non-zero dose with 95% confidence).

Following the evaluation procedure described above, the amount of PDGF-BB protein obtained from each of the 52 samples (n=4) was measured and screening was performed. 10 μg/mL tretinoin was used as a positive control, and 0.5% DMSO was used as a negative control.

result:
The percentage of protein obtained for the negative control (0.5% DMSO without sample) is shown in Figure 1 and Table 3 below. The results in Figure 1 and Table 3 indicate that, of the 52 samples, wasabi extract, chamomile extract, L-theanine, and hibiscus extract were particularly effective, demonstrating that these components have the ability to enhance PDGF-BB production. In the figure, samples A to C represent three samples from the 47 substances other than wasabi extract, chamomile extract, L-theanine, and hibiscus extract. However, like samples A to C, the other substances either had lower levels than the negative control (DMSO) or showed no significant difference, and therefore did not meet the criteria.

Experiment 3: Comparison with Amla Extract and Lingonberry Extract The PDGF-BB protein levels of wasabi extract, chamomile extract, L-theanine, and hibiscus extract, which were shown to have a significant effect of enhancing PDGF-BB production in Experiment 2, were measured according to the evaluation procedure described above, and compared with 15 μg/mL lingonberry extract and 2 μg/mL amla extract, which are known to have the effect of enhancing PDGF-BB production.

result:
The protein amount obtained for the control (15 μg/mL lingonberry extract) is set to 100.0, and the percentages relative to this are shown in Figure 2, while the protein amount obtained for the negative control (0.5% DMSO with no sample added) is set to 100.0 and the percentages relative to this are shown in Figure 3. Figures 2 and 3 demonstrate that the extract exhibits a significantly higher ability to enhance PDGF-BB production than not only the negative control, but also lingonberry extract and amla extract (Patent Document 2), which have previously been known to have a high PDGF-BB production-enhancing effect.

These results demonstrate that wasabi extract, chamomile extract, L-theanine, and hibiscus extract are particularly effective in enhancing PDGF-BB production. By enhancing PDGF-BB production, these substances are expected to stabilize mesenchymal stem cells, thereby revitalizing the skin and suppressing aging.

Claims (3)

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. A stem cell stabilizing agent comprising the PDGF-BB production enhancer described in claim 1. A skin anti-aging agent comprising the PDGF-BB production enhancer of claim 1, which inhibits skin aging by stabilizing stem cells in the skin.