CDP choline production promoter

Abstract

To acquire a component promoting epidermis reproduction.SOLUTION: The inventors found that CDP choline exerts actions such as promotion of epidermis reproduction, migration promotion of keratinocyte, wound healing, enhancement of a cell membrane, and improvement of a skin barrier function, and provide a CDP choline production promoter, or an epidermis reproduction promotor, a migration promotor of keratinocyte, a wound healing promotor, a cell membrane enhancer of keratinocyte, or a skin barrier function promotor which contain CDP choline. Further, also provided is lindane extract as the CDP choline production promoter.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This paper relates to the technical field of promoting the production of CDP choline. By promoting the production of CDP choline, epidermal regeneration can be promoted, and effects such as promoting keratinocyte migration, wound healing, strengthening cell membranes, and improving skin barrier function can be exhibited. [Background technology]

[0002] CDP choline is a substance in which choline is bound to cytidine diphosphate (CDP), a nucleotide, via a phosphate ester bond. CDP choline is one of the intermediates in the biosynthetic pathway of phosphatidylcholine and also one of the intermediates in sphingomyelin biosynthesis. CDP choline has been reported to improve cognitive function and have neuroprotective effects (Patent Document 1: JP 2023-011800 A, Patent Document 2: JP 2022-107631 A). It is sold as a prescription drug in Japan and as a supplement overseas. CDP choline has also been reported to act on vascular endothelial cells and affect the expression of tight junction factors ZO-1 and occludin (Non-Patent Document 1: PLoS One. 2013).

[0003] Linden extract, obtained from the common linden tree, is known to exhibit various beneficial effects, including elastase inhibition, hyaluronidase inhibition, collagenase inhibition, SOD-like activity, DPPH radical scavenging activity, PGE2 production inhibition, MMP1 activity inhibition, AGE production inhibition, and estrogen-like activity. These activities primarily affect the dermis layer, and it is used in cosmetics for various purposes, such as improving sagging, promoting collagen production, and whitening, as well as in foods such as herbal teas and supplements. However, there have been few reports on its effects on epidermal cells, and its effects on wound healing were unknown. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2023-011800 [Patent Document 2] Japanese Patent Publication No. 2022-107631 [Non-patent literature]

[0005] [Non-Patent Document 1] Ma X, et al. Plops One. 2013. https: / / doi.org / 10.1371 / journal.pone.0082604 Summary of the Invention [Problem to be solved by the invention]

[0006] The purpose is to obtain components that promote epidermal regeneration. [Means for solving the problem]

[0007] The present inventors have found that the addition of CDP-choline to cultured keratinocytes increases the expression of tight junction-associated proteins and promotes keratinocyte migration in scratch assays. Focusing on the epidermal regeneration effect of CDP-choline, they screened for ingredients that act on the epidermis by promoting the production of CDP-choline in vivo. Surprisingly, they found that linden extract increases the production of CDP-choline in keratinocytes, leading to the present invention. Thus, the present invention relates to: [1] Contains linden extract, a CDP choline production enhancer. [2] The CDP-choline production promoter according to Item 1, which promotes CDP-choline production in keratinocytes. [3] An epidermal regeneration promoter comprising the CDP choline production promoter according to item 1 or CDP choline. [4] A keratinocyte migration promoter comprising the CDP-choline production promoter according to item 1 or CDP-choline. [5] A wound healing promoter comprising the CDP choline production promoter according to item 1 or CDP choline. [6] A keratinocyte cell membrane enhancer comprising the CDP-choline production promoter according to item 1 or CDP-choline. [7] A skin barrier function promoter comprising the CDP choline production promoter according to item 1 or CDP choline. [8] A composition for transdermal or oral administration containing the CDP choline production promoter or CDP choline described in item 1. [9] The composition according to item 8, which is a cosmetic composition or a pharmaceutical composition.

[10] A food product containing the CDP choline production promoter described in Item 1.

[11] A wound healing method comprising applying a CDP choline production promoter containing linden extract to a wound area of a subject having a wound.

[12] A cosmetic method comprising applying a CDP choline production promoter containing linden extract to a subject having rough skin.

[13] Linden extract for use in wound healing.

[14] The linden extract according to Item 13, which promotes the production of CDP-choline, thereby healing wounds.

[15] The linden extract according to Item 14 promotes the production of CDP-choline, thereby promoting epidermal regeneration, promoting keratinocyte migration, strengthening the keratinocyte cell membrane, or improving skin barrier function.

[16] CDP choline for use in wound healing.

[17] The CDP choline according to Item 16, which heals wounds by promoting epidermal regeneration, promoting keratinocyte migration, strengthening the keratinocyte cell membrane, or improving skin barrier function.

[18] Use of linden extract for producing a CDP choline production promoter.

[19] Use of linden extract or CDP choline for the manufacture of an epidermal regeneration promoter, a keratinocyte migration promoter, or a wound healing promoter. [Effects of the Invention]

[0008] Linden extract promotes the production of CDP choline, which contributes to epidermal regeneration, promotion of keratinocyte migration, wound healing, strengthening of cell membranes, and improvement of skin barrier function. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 shows the results of metabolome analysis of keratinocytes cultured with linden extract. Compared to the control group (no linden extract added), metabolites whose production increased are shown in black, while metabolites whose production decreased are shown in red (A). The vertical axis shows the negative common logarithm of the P-value of the significant difference test in the t-test between the production levels of the linden extract-added group and the control group, while the horizontal axis shows the base 2 logarithm of the ratio of the change in production level of the linden extract-added group to the control group. Focusing on CDP choline, the change in production level is shown graphically (B). The vertical axis shows the relative area value of CDP choline to the internal standard. [Figure 2] FIG. 2 shows changes in the expression of claudin (A), occludin (B), and ZO-1 (C) when keratinocytes were cultured with the addition of CDP-choline (0.05% and 0.1%). [Figure 3] Figure 3 shows (A) a fluorescent immunostaining photograph showing the localization of occludin protein in keratinocytes cultured under hypoxic conditions (1% oxygen) without (Control) or with (citicoline) CDP-choline, and (B) a graph showing the results of quantification from the fluorescent immunostaining photograph. [Figure 4] Figure 4 shows a photograph (A) of a scratch assay of keratinocytes cultured under hypoxic conditions (1% oxygen) without (Control) or with (citicoline) CDP-choline added, and a graph (B) showing changes in cell density. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to a CDP-choline production promoter containing linden extract. When CDP-choline production is promoted in keratinocytes, it contributes to epidermal regeneration, promotion of keratinocyte migration, wound healing, strengthening of cell membranes, and improvement of skin barrier function.

[0011] CDP choline has the following formula: [ka] CDP choline is a compound represented by the formula (I) and is also known as citicoline. CDP choline is produced in any cell and contributes to the production of phosphatidylcholine and sphingomyelin. Phosphatidylcholine is a major component of cell membranes, and in nerve cells, it is the source of the neurotransmitter acetylcholine. Increasing the production of CDP choline increases the amount of phosphatidylcholine and / or sphingomyelin in the cell membrane. Sphingomyelin is thought to be involved in the formation of raft regions, and increasing the amount of these phospholipids increases the stability of lipid bilayer membranes. Thus, the present invention may relate to a cell membrane enhancer comprising CDP choline.

[0012] A CDP choline production promoter is a component that, when applied, promotes the production of CDP choline in any cell, particularly keratinocytes. The promotion of CDP choline production in keratinocytes contributes to epidermal regeneration, promotion of keratinocyte migration, wound healing, strengthening of cell membranes, and improvement of skin barrier function.

[0013] When the skin is wounded, bleeding occurs first. Over time, the blood coagulates and a scab forms. Within the scab, keratinocytes around the wound proliferate and migrate to seal the wound, while differentiation into the stratum corneum occurs, forming tight junctions as they differentiate. The formation of tight junctions improves the skin's barrier function and regenerates the epidermis.

[0014] Keratinocytes typically divide repeatedly in the basal layer. As the divided cells migrate toward the surface, they differentiate to form the spinous layer, granular layer, and stratum corneum. Meanwhile, when skin is injured, keratinocytes around the wound and their progenitor epidermal stem cells begin migrating to the wound. Migration is induced, for example, by hypoxia, and migration is promoted by keratinocytes inside the wound that are isolated from the outside air by a scab or other barrier. Keratinocyte migration can be measured by scratch assays in cultured cells. Scratch assays involve scraping cells from a portion of the surface of a confluently cultured dish, and then determining migration ability based on the cells that migrate to that area. Higher migration ability was demonstrated in cultures supplemented with CDP-choline (Figures 4A and 4B). Thus, the present invention may relate to a keratinocyte migration promoter, wound healing promoter, or epidermal regeneration promoter for keratinocytes, containing CDP-choline.

[0015] Tight junctions are formed between cells in the granular layer of the stratum corneum, and their formation provides a skin barrier function. This prevents the loss of components and moisture from within the skin and also prevents the invasion of allergens and pathogens from the outside. Claudins, occludins, and ZO family proteins (ZO-1 to ZO-3) are known to be tight junction factors involved in tight junctions. Increased expression of these proteins strengthens tight junctions, enhancing skin barrier function. Furthermore, tight junctions are formed not only in keratinocytes but also in epithelial cell systems, such as intestinal epithelial cells and vascular endothelial cells. CDP-choline is known to enhance the expression of tight junction factors in vascular endothelial cells under hypoxic conditions (Non-Patent Document 1). The present inventors have shown that CDP-choline enhances the expression of tight junction factors in keratinocytes under hypoxic conditions (FIGS. 2A to 2C), and have also shown that CDP-choline enhances the expression of occludin, which is localized in the cell membrane, in keratinocytes cultured under hypoxic conditions (FIGS. 3A and 3B). Thus, the present invention can be said to be a promoter of tight junction formation in keratinocytes or a skin barrier function enhancer, which contains CDP-choline.

[0016] Linden extract is an extract extracted from plants belonging to the genus Tilia in the family Tiliaceae. Examples of plants belonging to the genus Tilia include American linden (T. americana L.), summer linden (T. platyphyllos Scop.), tilia cordata (T. cordata Mill.), and hybrids thereof, such as Tilia x europaea. Linden extract is also known as linden extract or tilia cordata extract. These plant extracts can be obtained by a common method for producing plant extracts from at least one part of the plant, particularly buds, leaves, flowers, branches, trunks, roots, fruits, and seeds. For example, linden extract can be obtained by immersing or refluxing the flowers or buds, either whole or dried, with an extraction solvent at room temperature or under heating, followed by filtration and concentration. Any solvent commonly used in extraction can be used as the extraction solvent. For example, aqueous solvents such as water, physiological saline, phosphate buffer, borate buffer, or organic solvents such as alcohols (e.g., ethanol, propylene glycol, 1,3-butylene glycol, glycerin), hydrous alcohols, chloroform, dichloroethane, carbon tetrachloride, acetone, ethyl acetate, hexane, etc. can be used alone or in combination. A mixed solvent of water and alcohol, such as 1,3-butylene glycol, is preferably used. The extract obtained by extraction with the above solvent can be used directly or after concentration, for example, by freeze-drying. If necessary, it can also be used after removing impurities using an adsorption method, such as an ion exchange resin, or after adsorption on a porous polymer (e.g., Amberlite XAD-2) column, followed by elution with a desired solvent and further concentration. Commercially available linden extracts can also be used. More specifically, they can be prepared by extraction with water, alcohol, or a mixture thereof. The alcohol used can be ethanol, propylene glycol, or butylene glycol. More preferably, extraction can be performed with a mixture of water and alcohol in any ratio, for example, a mixture of 10:90 to 90:10, preferably a mixture of 30:70 to 70:30, and more preferably a mixture of 50:50.More preferably, a mixture of water and 1,3-butylene glycol is used. Linden extract is blended into cosmetics at a concentration of 0.005% to 0.1%, more preferably 0.01% to 0.05%.

[0017] Linden extract can promote the production of CDP-choline in cultured keratinocytes (FIGS. 1A and 1B), thereby enabling use of linden extract as a CDP-choline production promoter. Furthermore, CDP-choline can promote the expression of claudins, occludin, and ZO-1 in keratinocytes (FIGS. 2A-C, 3A and 3B), and further promotes keratinocyte migration in a scratch assay (FIGS. 4A and 4B). Therefore, linden extract can also exert its effects via the production of CDP-choline. Thus, the present invention may relate to an epidermal regeneration promoter, a keratinocyte migration promoter, a wound healing promoter, a cell membrane enhancer, and a skin barrier function improver, each containing linden extract.

[0018] The CDP-choline production promoter, epidermal regeneration promoter, keratinocyte migration promoter, wound healing promoter, cell membrane enhancer, and skin barrier function improver of the present invention may be incorporated into cosmetics, pharmaceuticals, or quasi-drugs, or into foods, such as dietary supplements or functional foods. Foods, which can also be called food compositions, can be administered to subjects suffering from rough skin, wounds, scratches, or impaired skin barrier function. These agents may be administered orally or parenterally, for example, transdermally. When administered transdermally, they can be formulated as topical skin preparations. The topical skin preparations are not particularly limited as long as they are applicable to the skin, and any formulation may be used, such as a solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, or stick. When formulated into a topical skin preparation, bases and excipients commonly used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used. Alternatively, the composition may be formulated as a patch to cover a wound. When incorporated into cosmetics, the composition may be incorporated into face or body cosmetics such as lotions, emulsions, serums, creams, lotions, packs, essences, and gels, makeup cosmetics such as foundations, makeup bases, and concealers, as well as bath additives. The use of cosmetics, pharmaceuticals, and quasi-drugs containing the components of the present invention can exhibit at least one effect selected from the group consisting of promoting epidermal regeneration, promoting keratinocyte migration, promoting wound healing, strengthening cell membranes, and improving skin barrier function through the promotion of CDP-choline production in cells, particularly keratinocytes.

[0019] The concentrations of the CDP choline production promoter, epidermal regeneration promoter, keratinocyte migration promoter, wound healing promoter, cell membrane enhancer, and skin barrier function improver of the present invention can be selected arbitrarily to achieve the desired effect, such as improving skin barrier function and promoting skin regeneration. When used as an external skin preparation, the extract of the present invention can be incorporated at 0.0005% to 0.5%. To fully exert its effects, it can be incorporated at a concentration of preferably 0.001% or more, more preferably 0.005% or more. To avoid a strong odor, it can be incorporated at a concentration of preferably 0.1% or less, more preferably 0.05% or less.

[0020] In yet another aspect of the present invention, the present invention may relate to a method for promoting epidermal regeneration, keratinocyte migration, wound healing, cell membrane enhancement, and skin barrier function through the promotion of CDP-choline production. More specifically, the method of the present invention relates to a method comprising the step of applying a composition containing CDP-choline according to the present invention or a composition containing linden extract to the skin of a subject. Such a method may be a therapeutic method or a non-therapeutic cosmetic method. Such a subject may be one who desires the promotion of epidermal regeneration, keratinocyte migration, wound healing, cell membrane enhancement, and improvement of skin barrier function. For example, the composition may be administered to a subject suffering from rough skin, skin inflammation, or scratches.

[0021] All documents mentioned in this specification are incorporated herein by reference in their entirety. The examples of the present invention described below are for illustrative purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Modifications of the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, can be made without departing from the spirit of the present invention. [Example]

[0022] Example 1: Metabolomic analysis A culture of human keratinocytes (Kurabo Industries) seeded in a growth medium for human epithelial cells (CELLnTEC) was supplemented with 0.1% linden extract (Koei Kogyo Co., Ltd.) and cultured for 6 hours. After the culture, the medium was removed, a methanol solution was added and stirred, and then Milli-Q water was added and stirred, followed by centrifugation (2,300 × g, 4 °C, 5 minutes). After ultrafiltration using an ultrafiltration tube (Ultrafree MC PLHCC, HMT, centrifugal filter unit 5 kDa), the filtrate was dried and dissolved in Milli-Q water again for measurement. 1.26 × 10 per sample 6 Metabolomic measurements were performed using cells with N=5.

[0023] Metabolite detection A CE-MS system combining capillary electrophoresis (Agilent CE system) and mass spectrometry (MS) was used to comprehensively detect cationic and anionic metabolites. Peaks with a signal-to-noise (S / N) ratio of 3 or greater were automatically extracted using automatic integration software, and their mass-to-charge ratios (m / z), peak areas, and migration times (MT) were obtained. Peaks with a signal-to-noise (S / N) ratio of 3 or greater were automatically extracted, and their mass-to-charge ratios (m / z), peak areas, and migration times (MT) were obtained. Metabolites were identified by matching and searching all substances registered in the HMT metabolite library based on the m / z and MT values. The results are shown in Figure 1(A). Components whose production increased with the addition of linden extract are written in black, while components whose production decreased are written in gray. Focusing on CDP-choline, the change in production is shown (Figure 1(B)).

[0024] Example 2: Effect of adding CDP-choline Human keratinocytes (Kurabo) were seeded in human epithelial cell growth medium (CELLnTEC) and cultured in human epithelial cell differentiation medium (CELLnTEC). CDP-choline (Sigma-Aldrich, final concentration 0.1 mM) was added to the culture and incubated at 1% oxygen for 24 hours. Total RNA was extracted and cDNA was prepared using the AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies). qPCR was performed using the LightCycler® FastStart DNA MasterPLUS SYBR Green I and a LightCycler (Roche Molecular Systems, Inc., Pleasanton). Gene expression of claudins, occludin, and ZO-1 was measured using the primers listed below and quantified by the ΔCt method using GAPDH as a standard, according to the accompanying instructions. The results are shown in Figure 2. [Table 1]

[0025] Cell immunostaining Human keratinocytes (Kurabo Industries) were seeded in human epithelial cell medium (CELLnTEC) and cultured in human epithelial cell differentiation medium (CELLnTEC) with the addition of CDP-choline (final concentration 0.1 mM) at 1% oxygen for 24 hours, followed by methanol fixation. After blocking with 4% skim milk, the cells were incubated with anti-occludin monoclonal antibody (Invitrogen) overnight at 4°C, and finally incubated with Alexa Fluor 594-conjugated anti-mouse IgG antibody (Invitrogen) and detected using a confocal microscope LSM880 (Leica). Fluorescence intensity of the images was quantified using ImageJ image analysis software. The results are shown in Figure 3(A) and (B).

[0026] Scratch assay The assay was performed according to the instructions for the Incucyte® 96-Well Scratch Wound Cell Migration and Invasion Assays module (Sartorius). Human keratinocytes were cultured on an Incucyte® Imagelock 96-Well Plate (Sartorius) until confluent. Then, cells were scraped at a uniform width using the Incucyte® 96-Well Woundmaker Tool (Sartorius) to create grooves in the wells. CDP-choline (final concentration 0.1 mM) was added and the plate was cultured at 1% oxygen for 36 hours. The filling of the cell-free areas in the wells was analyzed using the Incucyte Live Cell Analysis System. The results are shown in Figure 4(A). The cell density at the scratch site was calculated (Figure 4(B)).

Claims

1. A CDP choline production promoter containing linden extract.

2. The CDP choline production promoter according to claim 1, which promotes CDP choline production in keratinocytes.

3. A keratinocyte migration promoter comprising the CDP choline production promoter according to claim 1 or CDP choline.

4. A skin barrier function promoter comprising the CDP choline production promoter according to claim 1 or CDP choline.

5. A composition for transdermal or oral administration, which contains the CDP choline production promoter according to claim 1 or CDP choline.

6. The composition according to claim 5, which is a cosmetic or pharmaceutical composition.

7. A food comprising the CDP choline production promoter according to claim 1.

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