Plant-derived extract composition

Abstract

To provide a plant-derived extract composition that can enhance expression levels of transglutaminase (TGM-1) and loricrin (LOR) in one aspect.SOLUTION: The present disclosure relates, in one aspect, to a plant-derived extract composition comprising rosmarinic acid, wherein a mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to plant-derived extract compositions. [Background technology]

[0002] To improve the skin's moisture retention ability, prevent and improve rough skin, and prevent and improve skin aging such as the formation of wrinkles and reduction of texture, it is believed that it is effective to act on the epidermal cells of the skin, promote keratinization of the epidermal cells, encourage the formation of a healthy stratum corneum, and improve the stratum corneum barrier function to protect the body from external stimuli and other factors.

[0003] It is said that maintaining the stratum corneum's moisture content and flexibility is important for the health of this barrier function. Aging, dryness, ultraviolet rays, and other factors can disrupt the cell turnover process, leading to abnormalities in the formation of stratum corneum cells and the structure of intercellular lipids. This is known to lead to various skin disorders and skin problems, such as rough skin.

[0004] Here, turnover refers to the continuous repetition of keratinocyte (epidermal keratinocyte) proliferation in the basal layer, the cornification process, and the peeling of the stratum corneum. These keratinocytes form each layer, from basal membrane cells, spinous cells, granular cells, and corneocytes, cornifying sequentially outward. Proteins that make up the cornified envelope (CE) are synthesized from the upper spinous layer to the granular layer. Furthermore, as they reach the stratum corneum, transglutaminase (TGM) binds matrix proteins such as involucrin (IVL), loricrin (LOR), and cystatin to the keratinocyte cell membrane, forming insolubilized CE. Furthermore, ceramides and other proteins covalently bind to the insolubilized CE, forming the basis of the stratum corneum's barrier function.

[0005] Conventionally, skin problems such as rough skin caused by a decrease in the stratum corneum barrier function have been resolved by supplementing the stratum corneum barrier function with creams containing ceramides, etc. However, the improvement in CE of stratum corneum cells is insufficient, and there is a demand for ingredients (agents, compositions, etc.) that can promote the formation of healthy CE. For example, Patent Document 1 proposes a composition for improving skin viscoelasticity, which contains an inhibitor of an increase in extracellular ATP concentration, in which the weight ratio of (A) rosmarinic acid to (B) luteolin glucuronide is (A):(B) = 1:0.2 to 1.5. Patent Document 2 proposes a filaggrin production promoter containing rosmarinic acid and / or eriodictyol 7-O-rutinoside as active ingredients to promote the mRNA expression of profilaggrin, a precursor of filaggrin that is involved in improving the skin's moisturizing function. Patent Document 3 proposes an involucrin expression promoter and a stratum corneum formation promoter that contain one or more plants or extracts thereof selected from Japanese pepper, peppermint, eucalyptus, saxifrage, rosemary, asparagus, comfrey, shiitake mushroom, calamus, thyme, burnet, gambir, ginkgo, kudzu, Chinese quince, gardenia, loquat, cinchona, hawthorn, clove, mugwort, and Phellodendron amurense as agents capable of maintaining healthy skin barrier function. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2020 / 158912 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-90037 [Patent Document 3] Japanese Patent Application Laid-Open No. 2007-277149 Summary of the Invention [Problem to be solved by the invention]

[0007] As mentioned above, transglutaminase (TGM) is known to catalyze the cross-linking reaction of basic proteins such as involucrin (IVL) and loricrin (LOR), thereby constructing the cornified envelope (CE). There is a need for an extract composition that can increase the expression levels of transglutaminase (TGM) and loricrin (LOR) more than conventional plant extract compositions and promote the formation of healthy CEs.

[0008] Therefore, the present disclosure provides a plant-derived extract composition that can increase the expression levels of transglutaminase 1 (TGM-1) and loricrin (LOR), which are representative transglutaminases (TGMs). [Means for solving the problem]

[0009] In one aspect, the present disclosure relates to a plant-derived extract composition containing rosmarinic acid, wherein the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less.

[0010] In one aspect, the present disclosure relates to a composition containing a plant extract component, the composition containing rosmarinic acid, and the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) being 0.25 or less.

[0011] In one aspect, the present disclosure relates to a method for producing a plant-derived extract composition, which includes an extraction step of extracting so that the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less.

[0012] In one aspect, the present disclosure relates to a cosmetic product containing the plant-derived extract composition of the present disclosure. [Effects of the Invention]

[0013] According to one aspect of the present disclosure, a plant-derived extract composition that can increase the expression levels of transglutaminase (TGM-1) and loricrin (LOR) can be provided. DETAILED DESCRIPTION OF THE INVENTION

[0014] Rosmarinic acid has attracted attention as a component for improving (promoting) keratinization. Rosmarinic acid is a type of polyphenol found in large amounts in Lamiaceae plants such as rosemary. A known example of an extract (plant-derived composition) containing rosmarinic acid is rosemary extract extracted from rosemary (Japanese name: Mannenrou). Rosemary extract contains rosmarinic acid as the main component and usually also contains other components such as betulinic acid. As a result of extensive research, the present inventors have found that the coexistence of rosmarinic acid and betulinic acid in a plant-derived extract composition such as rosemary extract inhibits the effect of rosmarinic acid in promoting the expression of transglutaminase (TGM-1) and loricrin (LOR). Furthermore, the present inventors have found that by extracting so that the mass ratio of betulinic acid to rosmarinic acid is reduced to a predetermined value or less, the inhibitory effect of betulinic acid on the expression of transglutaminase (TGM-1) and loricrin (LOR) can be reduced and the amount of expression promoted by the extract can be increased.

[0015] That is, in one aspect, the present disclosure relates to a plant-derived extract composition (hereinafter also referred to as the "extract composition of the present disclosure") that contains rosmarinic acid and has a mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) of 0.25 or less.

[0016] Although the details of the mechanism by which the effects of the present disclosure are manifested are not clear, it is presumed as follows. Betulinic acid, which is contained in plant-derived extract compositions such as rosemary extract, is thought to inhibit rosmarinic acid. In the present disclosure, by setting the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) to 0.25 or less, the inhibitory effect of betulinic acid on the action of rosmarinic acid can be drastically reduced, which is thought to have led to an increase in the expression levels of transglutaminase (TGM-1) and loricrin (LOR). However, the present disclosure need not be construed as being limited to these mechanisms.

[0017] In one or more embodiments, the plant of the present disclosure may be a plant containing rosmarinic acid. The plant containing rosmarinic acid is not particularly limited as long as it contains rosmarinic acid, and examples thereof include Lamiaceae plants. Examples of Lamiaceae plants include herb plants of the Lamiaceae family, such as rosemary (Rosmarinus officinalis), perilla, lemon balm, and common sage. The plant part used for extraction may be the whole or a part of the plant, and can be appropriately selected depending on the purpose. Suitable plant parts for extraction include leaves, stems, flowers, fruits, pericarp, pits, above-ground parts, and mixtures thereof. Among these, leaves are preferred from the viewpoint of efficient extraction of rosmarinic acid.

[0018] [Rosmarinic acid] In one or more embodiments, the extraction composition of the present disclosure contains rosmarinic acid. The concentration of rosmarinic acid in the extraction composition of the present disclosure can be appropriately set. In one or more embodiments, the concentration (content, ppm) of rosmarinic acid in the extract composition of the present disclosure is preferably 0.5 ppm or more, more preferably 0.7 ppm or more, even more preferably 0.9 ppm or more, and even more preferably 1 ppm or more, from the viewpoint of improving the expression levels of TGM-1 and LOR. In this disclosure, "ppm" is based on mass, and 1 ppm is 0.0001 mass% (the same applies hereinafter).

[0019] [Betulinic acid] In one or more embodiments, the extraction composition of the present disclosure may include betulinic acid, and in one or more other embodiments, may be betulinic acid-free. From the viewpoint of improving the expression levels of TGM-1 and LOR, the concentration (content, ppm) of betulinic acid in the extract composition of the present disclosure preferably satisfies the following mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid), and in one or more embodiments, is more preferably 0.25 ppm or less, even more preferably 0.20 ppm or less, even more preferably 0.10 ppm or less, even more preferably 0.09 ppm or less, and even more preferably 0.005 ppm or less.

[0020] [Mass ratio (betulinic acid / rosmarinic acid)] The mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) in the extract composition of the present disclosure is 0.25 or less, preferably 0.20 or less, more preferably 0.15 or less, even more preferably 0.10 or less, even more preferably 0.09 or less, and even more preferably 0.005 or less, from the viewpoint of improving the expression levels of TGM-1 and LOR. In the present disclosure, the concentrations of rosmarinic acid and betulinic acid can be measured using high performance liquid chromatography (HPLC), for example, by the method described in the Examples.

[0021] [Method of producing the extract composition] In one or more embodiments, the extract composition of the present disclosure can be obtained by extraction from a plant. Thus, in one aspect, the present disclosure relates to a method for producing an extract composition (hereinafter also referred to as a "method for producing the extract composition of the present disclosure"), which includes a step of obtaining the extract composition from a plant (an extraction step). In one or more embodiments, the method for producing an extract composition of the present disclosure is a method for producing a plant-derived extract composition, which includes an extraction step of extracting betulinic acid to rosmarinic acid so that the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less. Since rosmarinic acid and betulinic acid can be considered to be relatively hydrophilic and hydrophobic components, the mass ratio of betulinic acid to rosmarinic acid can be controlled within a predetermined range by utilizing this difference. In one or more embodiments, the content of rosmarinic acid in the extract composition extracted in the extraction step is preferably 0.5 ppm or more. The preferred values ​​of the content of rosmarinic acid, the content of betulinic acid (ppm), and the mass ratio of betulinic acid to rosmarinic acid in the extract composition extracted in the extraction step are the same as those of the extract composition of the present disclosure described above. Examples of plants used for extraction include those mentioned above, with rosemary being preferred. Examples of plant parts used for extraction include those mentioned above, with leaves being preferred.

[0022] Examples of extraction methods used in the extraction step include soaking, decoction, percolation, reflux extraction, supercritical fluid extraction, ultrasonic extraction, and microwave extraction. A preferred method involves soaking a plant in an extraction solvent. For example, a method for extracting rosmarinic acid involves placing a certain amount of rosemary in a kettle, adding a soaking solvent (e.g., water), and then soaking the mixture at 25°C for 24 hours. Stirring may or may not be performed during soaking. Therefore, in one or more embodiments, the extraction step is a step of soaking a plant in an extraction solvent to obtain an extract composition.

[0023] <Extraction solvent> The extraction solvent used in the extraction step may be either a polar solvent or a non-polar solvent. Examples of the extraction solvent include water, monohydric alcohols such as methanol, ethanol, propanol, and butanol, polyhydric alcohols such as 1,3-propanediol, dipropylene glycol, pentanediol, hexanediol, propylene glycol, butylene glycol, and glycerin, and mixtures thereof. Among these, at least one selected from water, ethanol, glycerin, 1,3-propanediol, butylene glycol, and mixtures thereof is preferred from the viewpoint of efficiently extracting rosmarinic acid. In one or more embodiments, the extraction solvent is preferably water from the viewpoint of efficiently extracting rosmarinic acid. When an aqueous solution of alcohol (monohydric alcohol and / or polyhydric alcohol) is used as the extraction solvent, its concentration is preferably more than 0% by mass and not more than 20% by mass, from the viewpoint of efficiently extracting rosmarinic acid and extracting it so that the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less.

[0024] In one or more embodiments, the extraction solvent used in the extraction step may further contain other components as needed, such as a preservative to prevent decay and a spreading agent to promote penetration into the plant body.

[0025] From the viewpoint of efficient extraction of rosmarinic acid, the amount of the extraction solvent used in the extraction step is preferably 3 times or more, more preferably 5 times or more, and even more preferably 10 times or more, based on the weight or dry weight of the plant. From the same viewpoint, the amount of the extraction solvent used in the extraction step is preferably 3 to 100 times, more preferably 5 to 75 times, and even more preferably 10 to 50 times.

[0026] <Extraction temperature> The temperature of the extraction solvent used for extraction (extraction temperature) or the temperature at which the plant is immersed in the extraction solvent (immersion temperature) is preferably 5°C or higher, more preferably 15°C or higher, and even more preferably 20°C or higher, from the viewpoint of efficient extraction of rosmarinic acid. From the same viewpoint, the extraction temperature or the immersion temperature is preferably 5°C or higher and 35°C or lower, more preferably 15°C or higher and 30°C or lower, and even more preferably 20°C or higher and 30°C or lower ...

[0027] <Extraction time> The extraction time or the time for soaking the plant in the extraction solvent (soaking time) is preferably 5 hours or more, more preferably 12 hours or more, and even more preferably 24 hours or more, from the viewpoint of efficient extraction of rosmarinic acid, and from the same viewpoint, it is preferably 120 hours or less, more preferably 72 hours or less, and even more preferably 36 hours or less. From the same viewpoint, the extraction time or the soaking time is preferably 5 hours or more and 120 hours or less, more preferably 12 hours or more and 72 hours or less, and even more preferably 24 hours or more and 36 hours or less.

[0028] In one or more embodiments, the extract composition of the present disclosure can be obtained by filtering using a filter. Therefore, in one or more embodiments, the extraction step is a step of soaking a plant in an extraction solvent and then filtering using a filter to obtain an extract composition. Examples of filters used for filtration include filter paper, membrane filters, cartridge filters, and disposable filters.

[0029] <Pre-extraction treatment> In one or more embodiments, the plant before extraction or the plant before soaking in the extraction solvent may be blanched. Thus, in one or more embodiments, the extraction step may include a step of blanching rosemary before extraction. A common method can be used for the blanching treatment. For example, a plant such as rosemary can be steamed, boiled, or microwaved. The steam blanching treatment temperature can be, for example, 100 to 150°C, and the blanching treatment time can be, for example, 5 to 60 minutes.

[0030] <Drying process> In one or more embodiments, the plant before extraction or the plant before soaking in the extraction solvent may be dried after pre-extraction treatment. Therefore, in one or more embodiments, the extraction step may include a step of blanching hydroponically grown rosemary and then drying it before extraction. The drying method may be a common method, such as natural drying, hot air drying, freeze drying, reduced pressure drying, or spray drying. The hot air drying temperature may be, for example, 30°C to 100°C, and the drying time may be, for example, 1 to 24 hours.

[0031] In one or more embodiments, the extraction composition of the present disclosure may be further purified to produce a purified product. Therefore, in one or more embodiments, the method for producing the extraction composition of the present disclosure may further include a step of purifying the extract composition.

[0032] In one or more embodiments, the extraction composition of the present disclosure may be used as is, or may be diluted, concentrated, or dried before use. That is, the extract composition of the present disclosure includes the extract obtained in the extraction step, as well as a diluted product thereof, a concentrated product thereof, a dried product thereof, or a purified product thereof. Examples of the extract composition of the present disclosure include an extract obtained by soaking a plant in water and then adjusting the extract using only an extracting solvent other than water so that the rosmarinic acid concentration and solvent concentration in the extract reach predetermined concentrations, or an extracting composition prepared using an extracting solvent other than water and water.

[0033] [Uses of the extract composition] In one or more embodiments, the extract composition of the present disclosure can be used to promote transglutaminase expression in human epidermal keratinocytes, promote loricrin expression in human epidermal keratinocytes, promote stratum corneum formation, or promote epidermal turnover. Thus, in another aspect, the present disclosure relates to a method for promoting transglutaminase expression in human epidermal keratinocytes using the extract composition of the present disclosure. In another aspect, the present disclosure relates to a method for promoting loricrin expression in human epidermal keratinocytes using the extract composition of the present disclosure. In another aspect, the present disclosure relates to a method for promoting stratum corneum formation using the extract composition of the present disclosure. In another aspect, the present disclosure relates to a method for promoting epidermal turnover using the extract composition of the present disclosure. In one or more embodiments, the extract composition of the present disclosure can be used to improve uneven redness of the skin. Thus, in another aspect, the present disclosure relates to a method for improving uneven redness of the skin using the extract composition of the present disclosure. In one or more embodiments, the extract composition of the present disclosure can be used as an active ingredient of a composition for promoting the expression of transglutaminase in human epidermal keratinocytes, promoting the expression of loricrin in human epidermal keratinocytes, promoting stratum corneum formation, or promoting epidermal turnover, or can be used to produce such a composition. In one or more embodiments, the extract composition of the present disclosure can be used as an active ingredient in a composition for improving uneven redness of the skin, or can be used to produce such a composition. Therefore, in one aspect, the present disclosure relates to a composition containing a plant extract, the composition containing rosmarinic acid, and the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) being 0.25 or less. In one or more embodiments, the content of rosmarinic acid in the composition of this aspect is preferably 0.5 ppm or more. Preferred values ​​for the content of rosmarinic acid, the content of betulinic acid, and the mass ratio of betulinic acid to rosmarinic acid in the composition of this aspect are the same as those for the extract composition of the present disclosure described above. In one or more embodiments, the composition of this aspect is a composition for promoting transglutaminase expression in human epidermal keratinocytes, promoting loricrin expression in human epidermal keratinocytes, promoting stratum corneum formation, or promoting epidermal turnover. In one or more embodiments, the composition of this aspect is a composition for improving uneven redness of the skin. In one or more embodiments, the composition of this aspect is a cosmetic, pharmaceutical, or quasi-drug for improving uneven redness of the skin. In another aspect, the present disclosure relates to a cosmetic, pharmaceutical, or quasi-drug for improving uneven redness of the skin, comprising the composition of the present disclosure. In one or more embodiments, the composition of this aspect can be suitably used as an external product or food. In the present disclosure, examples of external products include cosmetics, pharmaceuticals, quasi-drugs, bath additives, and perfumed products such as toothpaste. In another aspect, the present disclosure relates to an external product or food containing the composition of the present disclosure. In one or more embodiments, the extract composition of the present disclosure can be suitably used in cosmetics. That is, in one aspect, the present disclosure relates to a cosmetic containing the extract composition of the present disclosure. Examples of the cosmetic include hair cosmetics and skin cosmetics. Examples of product forms of hair cosmetics include hair shampoo, hair conditioner, and hair treatment. Examples of product forms of skin cosmetics include facial cleanser, moisturizer, emulsion, and serum. In another aspect, the present disclosure relates to a cosmetic containing the extract composition of the present disclosure. When the extract composition of the present disclosure or the composition of this embodiment is incorporated into a skin topical preparation such as a cosmetic, effects such as improving the skin's moisture retention ability, preventing and improving rough skin, reducing the formation of wrinkles and texture patterns, improving firmness, and improving uneven redness of the skin can be expected. Examples of the form of topical products include creams, liquid lotions, milky lotions, sprays, moisturizers, moisturizing liquids, and skin lotions. In one or more embodiments, the content of the extract composition of the present disclosure or the composition of this aspect in an external product or food is preferably 0.0001% or more, more preferably 0.01% or more, even more preferably 1% or more, and preferably 10% or less, more preferably 5% or less. In one or more embodiments, the content of the extract composition of the present disclosure or the composition of this aspect in an external product or food is preferably 0.0001% or more and 10% or less, more preferably 0.01% or more and 5% or less, and even more preferably 1% or more and 5% or less. [Example]

[0034] The present disclosure will be described in more detail below with reference to examples, but these are merely illustrative examples and the present disclosure is not limited to these examples.

[0035] (1) Production of rosemary extracts 1 to 4 Rosemary Extract 1 Rosemary extract 1 was prepared as follows. [Pre-extraction processing] Fresh rosemary leaves were blanched (steamed) to obtain dried plants. <Branching process> Equipment: Steam convection oven (CSVH-E10-T, Comet Kato) Blanching temperature: 110℃ Blanching time: 15 minutes [Drying process] After pre-extraction treatment, the rosemary leaves were dried. Equipment: Constant temperature dryer (DRR420DA, ADVANTEC) Drying temperature: 60℃ Drying time: 18 hours [Extraction Process] The dried plant was immersed in water and extracted for 24 hours at 25° C. to obtain an extract. The amount of extraction solvent used was 25 times the dry weight of the rosemary. The above extract was adjusted with butylene glycol and water so that the rosmarinic acid concentration was 300 ppm and the solvent concentration was 50% (v / v) butylene glycol aqueous solution, and this was designated as rosemary extract 1. The rosemary extract 1 thus obtained contained 300 ppm of rosmarinic acid and 1 ppm of betulinic acid. Rosemary Extract 2 Rosemary Extract 2 was obtained by immersing the plant in a 50% (v / v) ethanol solution after the same pre-extraction and drying processes as those for Rosemary Extract 1 and extracting it at 25°C for 24 hours. The amount of extraction solvent used was 25 times the dry weight of the rosemary. Rosemary Extract 2 thus obtained contained 312 ppm of rosmarinic acid and 94 ppm of betulinic acid. Rosemary Extract 3 Rosemary Extract 3 was prepared by diluting Rosemary Extract 2 with 50% (v / v) ethanol to a betulinic acid concentration of 27 ppm, and then adding reagent rosmarinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) to the diluted solution to a rosmarinic acid concentration of 300 ppm. Rosemary Extract 3 thus obtained contained 300 ppm of rosmarinic acid and 27 ppm of betulinic acid. Rosemary Extract 4 Rosemary Extract 4 was prepared by diluting Rosemary Extract 2 with 50% (v / v) ethanol to a betulinic acid concentration of 54 ppm, and then adding reagent rosmarinic acid (Fujifilm Wako Pure Chemical Industries, Ltd.) to the diluted solution to a rosmarinic acid concentration of 300 ppm. Rosemary Extract 4 thus obtained contained 300 ppm of rosmarinic acid and 54 ppm of betulinic acid. The concentrations of rosmarinic acid and betulinic acid were measured as follows.

[0036] [Measurement of rosmarinic acid and betulinic acid concentrations] The concentrations of rosmarinic acid and betulinic acid were measured using high performance liquid chromatography (HPLC) under the following measurement conditions. <Measurement conditions> Equipment: Ultimate 3000 (HPLC), Corona Ultra (detector) (manufactured by Dionex) Column: InertSustain ODS-3 5um 3.0x150mm (GL Sciences) Eluent: 0.1% formic acid (Solution A) and acetonitrile (Solution B) Inclination conditions: from 95:5 for A liquid to 10:90 for B liquid Flow rate: 0.5mL / min Column temperature: 40℃ Detector: UV (quantitation of rosmarinic acid), CAD (quantitation of betulinic acid)

[0037] (2) Transglutaminase 1 (TGM-1) and loricrin (LOR) expression analysis (Examples 1 to 3, Comparative Examples 1 and 2) [Preparation of additives] Using rosemary extracts 1 to 4 or an aqueous ethanol solution (50% (v / v)), additives containing rosmarinic acid and betulinic acid at the concentrations shown in Table 1 below (Examples 1 to 3, Comparative Examples 1 and 2) were prepared.

[0038] [Experimental cell culture] Normal human epidermal keratinocytes (derived from neonatal foreskin; Kurabo Industries) were used. Cell proliferation and culture were performed using EpiLife Medium with 60 μM calcium (Thermo Fisher) supplemented with HKGS (Thermo Fisher). For material supplementation, EpiLife Medium with 60 μM calcium supplemented with the HKGS Kit (BPE- and EGF-free; Thermo Fisher) was used. Cell culture was performed according to standard procedures at 37°C in a 5% CO2 atmosphere.

[0039] [Protein extraction and transglutaminase 1 (TGM-1) and loricrin (LOR) expression analysis] Normal human epidermal keratinocytes were cultured in a 6-well plate (collagen-coated; Corning) at 1.5 × 10 5 After culturing in growth medium for one day, the medium was replaced with material-supplemented medium and cultured for another day. The next day, the additives shown in Table 1 were added, and the mixture was cultured for 48 hours. Thereafter, the cells were washed once with PBS, lysed in RIPA buffer (SIGMA) supplemented with 100-fold diluted Protease / Phosphatase Inhibitor Cocktail (Cell Signaling), and the cells (cell suspension) were collected. The cell suspension was homogenized (sonicated) and centrifuged (15,000 × g, 10 min), and the supernatant was collected. Protein was then quantified using the Pierce® BCA Protein Assay Kit (Thermo Fisher). Detection required 8–10 μg of protein. Samples were separated by electrophoresis using NuPAGE® 4-12% SDS (sodium dodecyl sulfate)-polyacrylamide gels (Thermo Fisher). After transfer to a PVDF membrane, the gels were blocked for 1 hour with TBS-T (Tween 20 0.1% (w / v)) containing 5% (w / v) skim milk. They were then incubated overnight at 4°C with primary antibodies (anti-transglutaminase 1 antibody; Novus; anti-loricrin antibody; Abcam) diluted 2000-fold in blocking solution. After three 15-minute washes with PBS-T, the gels were incubated for 1 hour at room temperature with secondary antibodies (anti-rabbit IgG antibody (HRP-conjugated; Dako) diluted 4000-fold in blocking solution. After washing three times with PBS-T for 10 minutes, images were taken using the luminescence reagent SuperSignal® West Dura Extended Duration substrate (Thermo Fisher) and a detector (Amersham Imager 600). Band density was quantified using the Amersham Imager 600. The measurement data (mean, SD) were evaluated relative to the control group value of 100 to determine the protein expression levels of transglutaminase 1 (TGM-1) and loricrin (LOR). The results are shown in Table 1. In addition, since the expression level of LOR was not evaluated for Example 3, it is shown as "-" in Table 1.

[0040] [Table 1]

[0041] As shown in Table 1, when rosemary extracts 1, 3, and 4, each having a mass ratio (betulinic acid / rosmarinic acid) of 0.25 or less (Examples 1 to 3), were used, the expression level of transglutaminase (TGM-1) was increased compared to when rosemary extract 2, each having a mass ratio (betulinic acid / rosmarinic acid) of 0.3, was used (Comparative Example 2). Also, as shown in Table 1, when rosemary extracts 1 and 3, each having a mass ratio (betulinic acid / rosmarinic acid) of 0.25 or less (Examples 1 and 2), the expression level of loricrin (LOR) was increased compared to when rosemary extract 2, each having a mass ratio (betulinic acid / rosmarinic acid) of 0.3, was used (Comparative Example 2).

[0042] (3) Improvement effect of stratum corneum function (Example 4, Comparative Example 3, Reference Examples 1 and 2) Moisturizing liquids having the compositions and blending amounts (active ingredient, mass %) shown in Tables 2 and 3 were prepared, and their effects were evaluated. (Manufacturing method) Xanthan gum, 1,3-butylene glycol, and 30% by mass of purified water were heated to 80°C, mixed uniformly with stirring, and then cooled to 25°C. The remaining ingredients (rosemary extract, citric acid, sodium citrate, and phenoxyethanol) were added and mixed uniformly with stirring at 25°C to obtain each moisturizing liquid (Example 4, Comparative Example 3, and Reference Examples 1 and 2). (Evaluation method) The effect of continuous application for 4 weeks on improving stratum corneum function was evaluated in 32 healthy female subjects. Each subject's face was covered in a half-face pattern, with one side receiving a moisturizer (Example 4, Comparative Example 3) containing each rosemary extract (Example 1, Comparative Example 2), and the other side receiving a placebo moisturizer (Reference Examples 1, 2) not containing rosemary extract, each at approximately 0.2 g per application, twice daily. Measurements were taken before and after four weeks of application, and the number of subjects who showed greater improvement on the side receiving the placebo moisturizer and the side receiving the moisturizer containing each rosemary extract was compared. Evaluations were made based on moisture retention ability and visual observation of improvement in skin condition. The skin's moisture retention ability was confirmed by measuring changes in stratum corneum moisture content. Measurements of stratum corneum moisture content were performed using a Corneometer (Courage+Khazaka). Measurements were taken five times on each cheek, and the average of three measurements, excluding the lowest and highest values, was calculated. Measurements were compared before and after four weeks of application, and the greater the change, the greater the improvement. Visual improvement in skin condition was assessed by one expert evaluator who is involved in the development of cosmetics and regularly conducts evaluations. Facial images were evaluated before and after four weeks of application, and the number of people who judged their skin condition to have improved was shown. Uneven redness of the skin was confirmed by image analysis. Using a VISIA-CR (Canfield Scientific), facial photographs with surface light removed were taken before and after four weeks of application. Pigment components derived from hemoglobin were separated from the acquired images according to the method described in Patent 6977744, and hemoglobin-extracted images (color images obtained by multiplying the amount of hemoglobin component by the hemoglobin vector and then converting it to RGB) were created. Image analysis software Image J was used to calculate the standard deviation (StdDev) of red on the cheeks, and the value after four weeks of application minus the value before application was used to determine the change in uneven redness. The smaller the change in uneven redness, the greater the improvement in uneven redness of the skin. The results are shown in Tables 2 and 3.

[0043] [Table 2]

[0044] [Table 3]

[0045] The results in Table 2 show that the moisturizer containing the rosemary extract of Example 1 (Example 4) was more effective in improving stratum corneum function than the placebo moisturizer not containing rosemary extract (Reference Example 1).The moisturizer of Example 4 was also found to be more effective in improving uneven redness of the skin than the placebo moisturizer (Reference Example 1). On the other hand, the results in Table 3 show that the moisturizer containing rosemary extract of Comparative Example 2 (Comparative Example 3) did not show any effect of improving stratum corneum function compared to the placebo moisturizer not containing rosemary extract (Reference Example 2).Furthermore, the moisturizer of Comparative Example 3 did not show any effect of improving uneven redness of the skin compared to the placebo moisturizer (Reference Example 2). [Industrial Applicability]

[0046] According to one aspect of the present disclosure, a plant-derived extract composition capable of increasing the expression levels of transglutaminase (TGM-1) and loricrin (LOR) can be provided.

Claims

1. A plant-derived extract composition containing rosmarinic acid, wherein the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less.

2. The extract composition according to claim 1, wherein the content of rosmarinic acid is 0.5 ppm or more.

3. 2. The extract composition of claim 1, wherein the plant is rosemary.

4. A composition containing a plant extract component, which contains rosmarinic acid and has a mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) of 0.25 or less.

5. The composition according to claim 4, for promoting loricrin expression in human epidermal keratinocytes, promoting stratum corneum formation, or promoting epidermal turnover.

6. The composition according to claim 4 for promoting the expression of transglutaminase in human epidermal keratinocytes.

7. The composition according to claim 4 for improving uneven redness of the skin.

8. The composition of claim 4, wherein the plant is rosemary.

9. A method for producing a plant-derived extract composition, comprising an extraction step of extracting so that the mass ratio of betulinic acid to rosmarinic acid (betulinic acid / rosmarinic acid) is 0.25 or less.

10. The method of claim 9, wherein the plant is rosemary.

11. A cosmetic comprising the plant-derived extract composition according to claim 1.

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