Skin dullness improvement agent

Abstract

To provide a loricrin expression promoter, a horny layer formation promoter and a skin darkening improver which promote loricrin expression.SOLUTION: A loricrin expression promoter contains Eucalyptus globulus or its extract, and Prunus persica or its extract as active ingredients.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a loricrin expression promoter, a stratum corneum formation promoter, and a skin dullness improvement agent. [Background technology]

[0002] To improve the skin's moisture retention ability, prevent and improve rough skin, and prevent and improve skin aging such as wrinkle formation and reduction of skin texture, it is considered beneficial to act on the epidermal cells of the skin, promote keratinization of epidermal cells, encourage the formation of a healthy stratum corneum, and maintain a healthy stratum corneum barrier function that protects the body from external stimuli. Furthermore, the formation of a healthy stratum corneum also leads to an improvement in skin dullness (Non-Patent Literature 1). Skin dullness is a phenomenon that occurs on the entire face or in areas such as around the eyes and cheeks, and is a condition in which the skin's transparency is reduced and it appears dark, with indistinct boundaries. Several factors are thought to cause skin dullness, but one of them is a decrease in transparency (light transmittance) due to thickening of the stratum corneum, etc. (Non-Patent Literature 2).

[0003] Thickening of the stratum corneum refers to a condition in which the thickness of the stratum corneum becomes thicker than normal, and is also called stratum corneum stratification. Causes of stratum corneum thickening include disruption of turnover due to the effects of aging, dryness, and ultraviolet rays, which leads to abnormalities in the formation and exfoliation of stratum corneum cells. Here, turnover refers to the continuous repetition of proliferation of keratinocytes (epidermal keratinocytes) in the basal layer, the process of keratinization, and the exfoliation of the stratum corneum. These keratinocytes change sequentially outward during the keratinization process, forming each layer as basal cells, spinous cells, granular cells, and stratum corneum cells. Proteins that constitute the cornified envelope (CE) are synthesized from the upper layer of the stratum spinosum to the granular layer. Furthermore, in the process leading to the stratum corneum, substrate proteins such as involucrin, loricrin, and cystatin bind to the cell membrane of keratinocytes by the enzyme transglutaminase, forming insoluble CE. Ceramides and other proteins then covalently bind to the insoluble CE, forming the basis of the stratum corneum barrier function. Ultimately, as the keratinocytes reach the surface of the stratum corneum, their adhesive strength decreases, causing them to flake off as dead skin cells. Therefore, disruptions in cell turnover lead to abnormal formation and shedding of the stratum corneum, causing abnormal incomplete keratinization and contributing to stratum corneum thickening. Traditionally, skin problems caused by disruptions in cell turnover have been addressed with creams containing ceramides. However, these have not sufficiently promoted keratinization of epidermal cells and improved the health of the stratum corneum, and there is a need for the development of ingredients that promote the formation of a healthy stratum corneum.

[0004] Among the substrate proteins mentioned above, loricrin is a major component of CE and is involved in various stratum corneum functions, including the formation of natural moisturizing factors. Diseases caused by loricrin gene mutations are collectively called loricrin keratosis, and symptoms such as diffuse keratinization and hyperkeratosis accompanied by abnormal keratinization are observed (Non-Patent Literature 3). Transfection of HaCaT cells, a human epidermal keratinocyte cell line, with the loricrin gene leads to programmed cell death associated with terminal keratinization, and it has been reported that loricrin is an important molecule for normal keratinization (Non-Patent Literature 4). Therefore, it is thought that promoting loricrin expression in epidermal cells can promote normal keratinization of keratinocytes and the formation of a healthy stratum corneum, thereby improving stratum corneum thickening and, as a result, improving skin dullness. In recent years, loricrin has attracted attention as a differentiation marker for epidermal cells.

[0005] On the other hand, eucalyptus globulus, a member of the Myrtaceae family, has been reported to have an extract that promotes stratum corneum formation by stimulating involucrin expression, and is useful for improving the skin's moisture retention ability (moisturizing effect) and reducing the appearance of pores (Patent Document 1). Furthermore, regarding the peach (Prunus persica) of the Rosaceae family, it has been reported that its extract has an involucrin production-promoting effect and is useful in preventing and improving skin conditions such as dry skin, rough skin, atopic dermatitis, and psoriasis by normalizing epidermal keratinocytes (Patent Document 2). However, there have been no reports on the effect of combining eucalyptus and peach on loricrin expression. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2007-277149 [Patent Document 2] Japanese Patent Publication No. 2008-7412 [Non-patent literature]

[0007] [Non-Patent Document 1] T. Ishida et al. J. Soc. Cosmet. Chem. Jpn. 54 (3): 258-63 (2020) [Non-Patent Document 2] Masako Naganuma, Journal of the Japanese Society of Cosmetic Scientists, Vol. 39, No. 4, pp. 275-285 (2015) [Non-Patent Document 3] A. I-Yamamoto. J. Dermatol. Sci. 31 (1): 3-8 (2003) [Non-Patent Document 4] K. Yoneda et al. J. Dermatol. 37 (11): 956-964 (2010) [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention relates to providing a loricrin expression promoter, a stratum corneum formation promoter, and a skin dullness improving agent that promotes the expression of loricrin. [Means for solving the problem]

[0009] In view of the above problems, the inventors conducted thorough research and found that combining eucalyptus extract and peach extract synergistically enhances the expression of loricrin protein, and that these are useful for promoting stratum corneum formation and improving skin dullness.

[0010] In other words, the present invention relates to the following 1) to 3). 1) A lorriculin expression promoter containing eucalyptus or its extract, and peach or its extract as active ingredients. 2) A stratum corneum formation promoter containing eucalyptus or its extract, and peach or its extract as active ingredients. 3) A skin dullness improver containing eucalyptus or its extract, and peach or its extract as active ingredients.

Advantages of the Invention

[0011] According to the present invention, by promoting the expression of lorriculin, the formation of a healthy stratum corneum can be promoted, and skin dullness can also be improved.

Modes for Carrying Out the Invention

[0012] In this specification, "eucalyptus" refers to Eucalyptus globulus of the genus Eucalyptus in the Myrtaceae family. "Peach" refers to Prunus persica of the genus Prunus in the Rosaceae family. Examples of the parts of the above plants to be used include the whole herb, whole tree, leaves, stems, buds, flowers, flower buds, trees, xylem, bark, roots, rhizomes, pseudobulbs, tuberous roots, thalli, thalloids, seeds, fruits, resins, nuclei, or mixtures thereof. In the case of eucalyptus, the preferred parts to be used are leaves, flowers, and fruits, and more preferably leaves. In the case of peach, the preferred parts to be used are leaves, flowers, fruits, and nuclei, and more preferably leaves.

[0013] Such plants can be used as they are, or as juice obtained by squeezing them, dried products obtained by drying the plant bodies themselves or their pulverized products, or extracts extracted from these, but it is preferable to use them as extracts.

[0014] Examples of the extracts include various solvent extracts obtained by extracting the above plants at normal temperature or under heating, or by using an extraction device such as a Soxhlet extractor, extracts obtained by supercritical extraction such as supercritical carbon dioxide, dilutions thereof, concentrated solutions thereof, or dried powders thereof. The extraction means is not particularly limited, and for example, ordinary means such as immersion, decoction, leaching, reflux extraction, ultrasonic extraction, microwave extraction, stirring, etc. can be used.

[0015] For the solvent for extraction, either a polar solvent or a non-polar solvent can be used. Specific examples of the solvent include, for example, water; monohydric, dihydric or polyhydric alcohols; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; chain or cyclic ethers such as diethyl ether and tetrahydrofuran; polyethers such as polyethylene glycol; saturated or unsaturated hydrocarbons such as hexane; aromatic hydrocarbons such as benzene and toluene; halogenated hydrocarbons such as dichloromethane, chloroform, dichloroethane and carbon tetrachloride; pyridines; dimethyl sulfoxide; acetonitrile; carbon dioxide, supercritical carbon dioxide; oils, waxes and other oils; and mixtures thereof. Preferably, water, alcohols and their aqueous solutions are included. Examples of alcohols include methanol, ethanol, 1,3-butylene glycol, n-propanol, isopropanol, n-butanol, isobutanol, sec-butanol, t-butanol, etc. Preferably, ethanol and 1,3-butylene glycol are used, and more preferably 1,3-butylene glycol.

[0016] The concentration of the alcohols in the aqueous solution of the above-mentioned alcohols (volume percentage at 25 °C, hereinafter expressed as %(v / v)) is preferably 12% (v / v) or more, more preferably 15% (v / v) or more, still more preferably 18% (v / v) or more, and is also preferably 99.8% (v / v) or less, more preferably 90% (v / v) or less, still more preferably 85% (v / v) or less. In the case of eucalyptus extract, the concentration of the alcohols is preferably 30% (v / v) or more, more preferably 40% (v / v) or more, still more preferably 45% (v / v) or more, and is also preferably 99.8% (v / v) or less, more preferably 90% (v / v) or less, still more preferably 85% (v / v) or less. In the case of peach extract, the concentration of alcohols is preferably 12%(v / v) or higher, more preferably 15%(v / v) or higher, even more preferably 18%(v / v) or higher, and also preferably 85%(v / v) or lower, more preferably 55%(v / v) or lower, and even more preferably 45%(v / v) or lower.

[0017] The amount of solvent used in the extraction is preferably 1 to 100 mL per 1 g of the above-mentioned plant (based on dry weight), and more preferably 3 to 50 mL. The extraction conditions are not particularly limited as long as they allow for sufficient extraction, but for example, the extraction time is preferably 1 hour or more, more preferably 3 hours or more, and preferably 2 months or less, more preferably 5 weeks or less, and more preferably 3 weeks or less. The extraction temperature is preferably 0°C or higher, more preferably 5°C or higher, and preferably below the solvent boiling point, more preferably 90°C or lower. Typically, extraction is performed for a long time at low temperatures and for a short time at high temperatures.

[0018] The extract may be a crude product as long as it conforms to the standards acceptable for cosmetics and pharmaceuticals and exhibits the effects of the present invention. Furthermore, if necessary, it can be treated to remove inert impurities, deodorize, decolorize, etc., by known techniques such as liquid-liquid partitioning, solid-liquid partitioning, filtration membranes, activated carbon, adsorption resins, ion exchange resins, and sedimentation. Furthermore, the purity of these materials may be further increased by appropriately combining known separation and purification methods. Examples of purification methods include organic solvent precipitation, centrifugation, limiting filtration, high-performance liquid chromatography, and column chromatography.

[0019] The extract may be used as is, diluted with an appropriate solvent, or prepared as a concentrated extract, dried powder, or paste. Alternatively, it can be freeze-dried and diluted at the time of use with solvents commonly used for extraction, such as water, ethanol, 1,3-butylene glycol, water-ethanol mixture, or water-1,3-butylene glycol mixture. It can also be encapsulated in vesicles such as liposomes or microcapsules.

[0020] As shown in the examples below, the combination of eucalyptus extract and peach extract promotes the expression of loricrin. Moreover, this promoting effect is superior to that of eucalyptus extract and peach extract individually, and it is a synergistic effect that exceeds the additive effect of adding each individually. As mentioned above, if the expression of loricrin is promoted, normal keratinization of keratinocytes and healthy stratum corneum formation are promoted. As a result, an improvement in skin dullness can be expected. Therefore, a combination of eucalyptus or its extract and peach or its extract can act as a loricrin expression promoter, a stratum corneum formation promoter, and a skin dullness improvement agent (hereinafter also referred to as "loricrin expression promoter, etc."), and can be used to promote loricrin expression, promote stratum corneum formation, and improve skin dullness, and can also be used to manufacture loricrin expression promoters, etc. Here, "use" can be use on humans or non-human animals, and may be therapeutic or non-therapeutic use. "Non-therapeutic" is a concept that does not include medical procedures, that is, methods of surgery, treatment, or diagnosis on humans, and more specifically, methods of surgery, treatment, or diagnosis performed on humans by a physician, medical professional, or person under the direction of a physician. In the present invention, non-therapeutic uses include the use of the above-mentioned plants or their extracts for cosmetic or aesthetic purposes.

[0021] In this specification, "promotion of loricrin expression" includes both the promotion of loricrin expression at the gene level and the promotion of loricrin expression at the protein level. Promotion of expression at the gene level includes the promotion of transcription to mRNA in the expression of mRNA encoding loricrin, while promotion of expression at the protein level includes the promotion of mRNA translation. Of these, promotion of expression at the protein level is preferred in the present invention. Human loricrin is composed of 315 amino acids, and its molecular weight is approximately 26 kDa.

[0022] "Promotion of stratum corneum formation" means that the normal keratinization of keratinocytes is promoted, and the formation of a healthy stratum corneum is accelerated. Here, a healthy stratum corneum refers to a stratum corneum in which the skin's turnover is healthy, and specifically refers to a stratum corneum in which there is a balance between the proliferation of keratinocytes in the basal layer and the shedding of the stratum corneum from the surface, and in which no thickening of the stratum corneum is observed.

[0023] "Skin dullness" refers to a condition where the skin appears dark due to a decrease in transparency caused by factors such as thickening of the stratum corneum. Areas where skin dullness can occur include, for example, the entire face, around the eyes, cheeks, forehead, neck, back of the hands, and tops of the feet. "Improvement" refers to a rise in the condition, prevention or delay of deterioration, or a reversal, prevention, or delay of the progression of deterioration.

[0024] In the present invention, eucalyptus or its extract and peach or its extract may be administered either first or simultaneously. If the two agents are not administered simultaneously, the interval between administrations can be appropriately selected as long as it enhances the loricrin expression-promoting effect of eucalyptus or its extract, or peach or its extract. A preparation comprising eucalyptus or its extract and peach or its extract may be formulated as a single dosage form containing these combined preparations, or as a kit in which the preparations are individually formulated and can be used simultaneously or separately at intervals.

[0025] The loricrin expression promoter of the present invention may itself be a pharmaceutical, quasi-drug, or cosmetic product for promoting loricrin expression, promoting stratum corneum formation, or improving skin dullness, or it may be a material or formulation used in such a pharmaceutical, quasi-drug, or cosmetic product.

[0026] The pharmaceutical product (including quasi-drugs, hereinafter the same) contains eucalyptus or its extract, and peach or its extract as active ingredients to promote the expression of loricrin, promote the formation of the stratum corneum, and improve skin dullness. Furthermore, the pharmaceutical product may contain, as necessary, pharmaceutically acceptable carriers or other active ingredients, pharmacoactive ingredients, etc., as long as the function of the active ingredients is not lost. The form of administration of a drug is optional, but parenteral administration is preferred. Dosage forms for parenteral administration include topical, transdermal, transmucosal, nasal, enteral, injection, suppository, inhalation, and patch preparations. Of these, topical preparations are preferred, specifically in the form of ointments, emulsions, creams, lotions, gels, and aerosols.

[0027] The cosmetic contains eucalyptus or its extract, and peach or its extract, as active ingredients to promote the expression of loricrin, promote the formation of the stratum corneum, and improve skin dullness. Furthermore, the cosmetic may contain, as necessary, carriers permitted for cosmetics, or other active ingredients, cosmetic ingredients, etc., as long as the function of the active ingredients is not lost. Preferred examples of cosmetics include cosmetics for the face and body (e.g., lotions, gels, creams, masks, etc.), makeup cosmetics, and cleansers for the face or body.

[0028] Such pharmaceutical and cosmetic formulations can be manufactured in accordance with conventional methods by combining eucalyptus or its extract, and peach or its extract, as necessary, with a pharmaceutically or cosmetically acceptable carrier, the other active ingredients, medicinal ingredients, cosmetic ingredients, etc. mentioned above. Examples of pharmaceutically or cosmetically acceptable carriers include various oils, surfactants, gelling agents, buffers, preservatives, antioxidants, solvents, dispersants, chelating agents, thickeners, UV absorbers, emulsifying stabilizers, pH adjusters, pigments, fragrances, and the like. Other active ingredients, medicinal ingredients, and cosmetic ingredients include, for example, plant extracts, disinfectants, moisturizers, anti-inflammatory agents, antibacterial agents, keratolytic agents, cooling agents, anti-seborrheic agents, cleansing agents, and makeup ingredients.

[0029] The content of eucalyptus or its extract in the above-mentioned pharmaceutical and cosmetic formulations varies depending on the form of the formulation and cannot be stated in general terms. However, for example, based on the total amount of the formulation, in terms of solid content, it is preferably 0.00006% by mass or more, more preferably 0.0001% by mass or more, and even more preferably 0.0002% by mass or more, and also preferably 0.006% by mass or less, more preferably 0.002% by mass or less, and even more preferably 0.0006% by mass or less. Furthermore, the content of peach or its extract in the above-mentioned pharmaceutical and cosmetic formulations varies depending on the form of the formulation and cannot be stated in general terms, but for example, based on the total amount of the formulation, in terms of solid content, it is preferably 0.00003% by mass or more, more preferably 0.00006% by mass or more, even more preferably 0.0003% by mass or more, and also preferably 0.006% by mass or less, more preferably 0.0018% by mass or less, and even more preferably 0.0006% by mass or less.

[0030] In the loricrin expression promoter of the present invention, the ratio of eucalyptus or its extract to peach or its extract is preferably 0.01 or more, more preferably 0.1 or more, and even more preferably 0.8 or more in terms of mass ratio [based on the solid content of eucalyptus or its extract / based on the solid content of peach or its extract], from the viewpoint of promoting loricrin expression and promoting stratum corneum formation. Furthermore, the mass ratio [based on the solid content of eucalyptus or its extract / based on the solid content of peach or its extract] is preferably 0.01 to 300, more preferably 0.1 to 30, and even more preferably 0.8 to 4.

[0031] The dosage or amount of use of eucalyptus or its extract can be an amount that can achieve the effects of the present invention. The dosage or amount of use may vary according to the species, body weight, sex, age, condition, or other factors of the subject. However, in the case of parenteral administration such as topical skin preparations, for an adult (60 kg), per administration, as the mass of use in terms of solid content of eucalyptus or its extract, preferably 0.0012 μg / cm 2 or more, more preferably 0.002 μg / cm 2 or more, even more preferably 0.004 μg / cm 2 or more, and preferably 0.12 μg / cm 2 or less, more preferably 0.04 μg / cm 2 or less, even more preferably 0.012 μg / cm 2 or less. Also, the dosage or amount of use of peach or its extract is, for an adult (60 kg), per administration, as the mass of use in terms of solid content of peach or its extract, preferably 0.0006 μg / cm 2 or more, more preferably 0.0012 μg / cm 2 or more, even more preferably 0.006 μg / cm 2 or more, and preferably 0.12 μg / cm 2 or less, more preferably 0.036 μg / cm 2 or less, even more preferably 0.012 μg / cm 2 or less. In the present invention, such an amount can be divided into one to multiple times a day and administered or used repeatedly and continuously for one day or more, preferably 7 days or more, more preferably 14 days or more, even more preferably 42 days or more.

[0032] . . The subject to which the lolipklin expression promoter or the like of the present invention is administered or used is not particularly limited as long as it is a human or non-human animal that needs or desires it. Preferred examples of the subject include humans who desire lolipklin expression promotion, stratum corneum formation promotion, and improvement of skin dullness. Non-human animals include non-human mammals such as anthropoid apes and other primates. Furthermore, the site on which the loricrin expression promoter of the present invention is administered or used is not particularly limited, but preferred sites include the face, neck, arms, back of the hands, fingers, legs, and back of the feet. [Examples]

[0033] Example 1: Promoting loricrin expression plant extract The eucalyptus extract used was obtained by extracting eucalyptus leaves with an 80% (v / v) 1,3-butylene glycol aqueous solution (Eucalyptus Extract BG-KA, manufactured by Maruzen Pharmaceutical Co., Ltd., solid content concentration 0.2% (w / v)). The peach leaf extract was prepared by extracting peach leaves with a 20% (v / v) 1,3-butylene glycol aqueous solution, and finally preparing a 40% (v / v) 1,3-butylene glycol aqueous solution (Peach Leaf Liquid B®, manufactured by Ichimaru Falcos Co., Ltd., solid content concentration 0.6% (w / v)).

[0034] Cell culture for experiments Normal human epidermal keratinocytes (derived from neonatal foreskin; Kurabo) were used. For cell proliferation culture, EpiLife Medium, with 60 μM calcium (Thermo Fisher) was used with the addition of HKGS (Thermo Fisher). Additionally, for culture with added material, EpiLife Medium, with 60 μM calcium was used with the addition of HKGS Kit (BPE and EGF-free, Thermo Fisher). Cell culture was performed at 37°C and 5% CO2 according to standard procedures.

[0035] Protein extraction and loricrin expression analysis Normal human epidermal keratinocytes were placed in a 6-well plate (collagen-coated; Corning) in a 1.5 × 10⁶ arrangement. 5Cells were seeded at a density of cells / well and cultured in growth medium for 1 day, then transferred to a medium containing added material and cultured for another day. The following day, eucalyptus extract dissolved in 80% (v / v) 1,3-butylene glycol (added at a concentration of 0.3% (v / v)) and peach leaf extract dissolved in 40% (v / v) 1,3-butylene glycol (added at a concentration of 0.1% (v / v)) were added, and the cells were cultured for 48 hours. After washing once with PBS, the cells were recovered by dissolving them in RIPA buffer (SIGMA) with a 100-fold dilution of Protease / Phosphatase Inhibitor Cocktail (Cell Signaling). The cell suspension was homogenized (sonic disruption), centrifuged (15,000 × g, 10 min), and the supernatant was collected. Subsequently, protein quantification was performed using the Pierce® BCA Protein Assay Kit (Thermo Fisher). 8-10 μg of protein was required for detection. Samples were separated by electrophoresis using NuPAGE® 4-12% SDS (sodium dodecyl sulfate)-polyacrylamide gel (Thermo Fisher), transferred to a PVDF membrane, blocked for 1 hour with TBS-T (Tween20 0.1% (w / v)) containing 5% (w / v) skim milk, and incubated overnight at 4°C with primary antibody (Anti-Loricrin antibody; abcam) diluted 2000-fold with blocking solution. After washing three times with PBS-T for 15 minutes each, the substrate was incubated for 1 hour at room temperature with secondary antibody (Anti-rabbit IgG antibody (HRP labeled); Dako) diluted 4000-fold with blocking solution. After washing three times with PBS-T for 10 minutes each, imaging was performed using the luminescence reagent SuperSignal® West Dura Extended Duration substrate (Thermo Fisher) and a detector (Amersham Imager 600). Band concentrations were quantified using the Amersham Imager 600. The measurement data was used to relatively evaluate the protein expression level of loricrin, with the control group's value set to 1. The results are shown in Table 1.

[0036] [Table 1]

[0037] As is clear from Table 1, in the group to which eucalyptus extract and peach leaf extract were added in combination, loricrin expression was confirmed to be synergistically promoted compared to the group to which eucalyptus extract and peach leaf extract were added individually.

Claims

1. A loricrin expression promoter containing eucalyptus leaf extract and peach leaf extract as active ingredients.

2. The loricrin expression promoter according to claim 1, wherein the mass ratio of eucalyptus leaf extract to peach leaf extract (calculated as solid content of eucalyptus leaf extract / calculated as solid content of peach leaf extract) is 0.01 or more and 300 or less.

3. The loricrin expression promoter according to claim 1 or 2, wherein the formulation is a topical skin preparation.

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