Ceramide production promoter and ceramide synthase gene expression enhancer

Abstract

To provide novel ceramide production promoters and expression enhancers for the ceramide synthase gene.SOLUTION: The present invention provides a ceramide production promoter containing Hippophae rhamnoides L. seed oil as an active ingredient, and an expression enhancer for the ceramide synthase gene containing Hippophae rhamnoides L. seed oil as an active ingredient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a ceramide production promoter and an agent for enhancing the expression of a ceramide synthase gene. [Background technology]

[0002] Ceramide is the main intercellular lipid component in the skin, and plays a particularly important role in the barrier function. A lack of ceramide causes the skin to become dry, unable to retain moisture, and the barrier function is weakened, making the skin more susceptible to external stimuli, which is thought to cause various skin problems. In addition, in recent years, it has been reported that ceramide is involved in various functions in the body in addition to the skin's barrier function.

[0003] Several enzymes are involved in the biosynthesis and metabolism of ceramide, one of which is ceramide synthase. For example, when the activity of ceramide synthase declines due to factors such as aging, the amount of ceramide decreases, disrupting the balance of intercellular lipids, and it is said that this reduces the skin's barrier function and moisture retention ability, causing dry skin. By promoting ceramide production, it is expected that the skin's barrier function and moisture retention ability will be improved. Patent Document 1 and other documents are known to be related to enhancing the expression of this ceramide synthase. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2022-170780 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel ceramide production promoter and a novel agent for enhancing the expression of a ceramide synthase gene. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems and have found that the following inventions meet the above objectives, thereby completing the present invention. <1> A ceramide production promoter containing sea buckthorn seed oil as an active ingredient. <2> The sea buckthorn seed oil is a supercritical carbon dioxide extract and / or a subcritical carbon dioxide extract of sea buckthorn seeds. <1> 2. The ceramide production promoter according to claim 1. <3> An agent for enhancing the expression of ceramide synthase genes, containing sea buckthorn seed oil as an active ingredient. <4> The ceramide synthase gene is one or more selected from the group consisting of CERS2, CERS3, and CERS4. <3> 2. An expression enhancer for the ceramide synthase gene described in 1. [Effects of the Invention]

[0007] According to the present invention, a novel ceramide production promoter and a novel agent for enhancing the expression of a ceramide synthase gene are provided. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of a test to evaluate the expression of a ceramide synthase gene. DETAILED DESCRIPTION OF THE INVENTION

[0009] The following describes in detail an embodiment of the present invention, but the following description of the constituent elements is one example (typical example) of an embodiment of the present invention, and the present invention is not limited to the following content unless the gist of the present invention is changed. Note that when the expression "to" is used in this specification, it is used as an expression including the numerical values ​​or physical property values ​​before and after it.

[0010] The present invention relates to a ceramide production promoter containing seabuckthorn seed oil as an active ingredient. The present invention also relates to a ceramide synthase gene expression enhancer containing seabuckthorn seed oil as an active ingredient. Hereinafter, the ceramide production promoter and the ceramide synthase gene expression enhancer of the present invention will be collectively referred to as "the agent of the present invention."

[0011] (Sea Buckthorn Seed Oil) Seabuckthorn seed oil is an oil (hydrophobic component) extracted from seabuckthorn seeds. Seabuckthorn (scientific name: Hippophae rhamnoides L., English name: Sea buckthorn) is a plant of the genus Hippophae in the family Elaeaceae native to Eurasia. Seabuckthorn seeds can be extracted from the fruit itself or from the residue left after processing the fruit. For example, when seabuckthorn fruit is pressed or strained to process it into juice such as liquid or puree, seeds can be selected from the residue removed and used to extract oil. The seeds selected from the residue can be washed with water and dried as appropriate before being used to extract oil.

[0012] The method for extracting the oil is not particularly limited, and known methods can be used. Sea buckthorn seeds can be crushed or pulverized as appropriate, and then extracted using an extraction method such as supercritical carbon dioxide extraction, subcritical carbon dioxide extraction, steam distillation, organic solvent extraction, or compression extraction, and the resulting extract can be used as sea buckthorn seed oil.

[0013] Among these, supercritical carbon dioxide extraction and subcritical carbon dioxide extraction are preferred, as they allow extraction at low temperatures and do not destroy heat-sensitive components. Seabuckthorn seed oil is preferably a supercritical carbon dioxide extract and / or subcritical carbon dioxide extract of seabuckthorn seeds. Specifically, seabuckthorn seed oil is preferably obtained by a method that uses carbon dioxide in a supercritical or subcritical state as an extraction solvent, extracts oil from seabuckthorn seeds, and then removes the carbon dioxide. The extraction conditions may be any temperature and pressure that allows carbon dioxide to reach a supercritical or subcritical state, such as a temperature of 20 to 80°C and a pressure of 10 to 60 MPa, a temperature of 30 to 80°C and a pressure of 10 to 50 MPa, a temperature of 31 to 60°C and a pressure of 15 to 40 MPa, or a temperature of 31 to 40°C and a pressure of 20 to 30 MPa.

[0014] (ceramide synthase gene) The agent of the present invention can promote ceramide synthesis. Furthermore, the agent of the present invention can enhance the expression of the ceramide synthase gene, one of the enzymes involved in ceramide biosynthesis. Enhanced gene expression refers to an increase in the gene (DNA) transcription product and / or the protein encoded by the gene. Specifically, the agent of the present invention can induce or promote the transcription of the ceramide synthase gene into mRNA, and induce or promote the translation of mRNA into ceramide synthase.

[0015] Six types of ceramide synthase genes, CERS1 to CERS6, have been reported. Among these, the agent of the present invention is preferably used to enhance the expression of one or more genes selected from the group consisting of CERS2, CERS3, and CERS4. CERS2 is a gene that is expressed in large amounts in many cell tissues, such as the kidney, liver, and intestine. CERS3 is a gene that is expressed in keratinocytes, and CERS4 is a gene that is expressed not only in the skin but also in leukocytes, the heart, and the liver. The genes are listed by their abbreviations according to their official symbols listed in NCBI (http: / / www.ncbi.nlm.nih.gov / ).

[0016] The agent of the present invention can be used in various forms, such as liquids such as aqueous solutions, emulsions, dispersions, and emulsions; semi-solids such as gels, ointments, pastes, and creams; and solids. For example, it can be in any form of topical preparation, such as lotion, cosmetic liquid, cream, emulsion, sunscreen, cleanser, pack, cosmetic oil, body rinse, massage agent, scalp agent, hair wash, hair rinse, and lip care cosmetic. In the case of topical preparations, their effects can be achieved by using a typical amount for each topical preparation form once to several times a day.

[0017] Depending on the form, etc., the agent of the present invention may be composed solely of sea buckthorn seed oil, or may contain, in addition to sea buckthorn seed oil, other ingredients such as base materials and additives known in the technical fields of pharmaceuticals, quasi-drugs, cosmetics, etc. The content of sea buckthorn seed oil in the agent of the present invention is not particularly limited as long as the effects of the present invention can be achieved, but is preferably 0.5 v / v% or more, and may be 0.6 v / v% or more, 0.8 v / v% or more, 1 v / v% or more, etc. The upper limit of the content of sea buckthorn seed oil in the agent of the present invention is not particularly limited, and may be 90 v / v% or less, 50 v / v% or less, 10 v / v% or less, 5 v / v% or less, etc., depending on the form, etc. [Example]

[0018] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples as long as the gist of the present invention is not changed.

[0019] 1. Test sample (sea buckthorn seed oil) The seeds were selected from the residue (including seeds and parts of the peel) after processing sea buckthorn juice using H. rhamnoides fruit (produced in Inner Mongolia) and washed with drinking water. The dried seeds were then crushed and the oil was dissolved and separated using supercritical carbon dioxide extraction (pressure 24-26 MPa, temperature 31.25°C, time 3.5 hours), yielding a brown, transparent extract. The product was packaged and sealed under aseptic conditions.

[0020] 2. Test Method (1) Evaluation sample Sea buckthorn seed oil Final concentration: 0.2v / v%, 0.4v / v%, 0.5v / v% ·control Ethanol, final concentration: 0.5 v / v%

[0021] (2) Experimental method ·Cell culture Human epidermal keratinocytes (HaCaT cells) were precultured in Dulbecco's Modified Eagle Medium (DMEM) (high glucose) containing 1 v / v% penicillin-streptomycin and 10 v / v% fetal bovine serum (FBS) in a 10 cm dish until confluent. After washing with phosphate-buffered saline (PBS), the cells were resuspended in medium and plated at 1.0 × 10 cells per well in a 24-well plate. 5 The cells were seeded at a density of 1000 cells / well and cultured overnight in a CO2 incubator (37°C, 5% CO2).

[0022] Adding sample 24 hours after seeding of HaCaT cells, 1 mL of serum-free DMEM (high glucose) (containing 1 v / v% penicillin-streptomycin) containing the sample was added to each well, and the cells were cultured in a CO2 incubator (37°C, 5% CO2) for 48 hours.

[0023] Changes in gene expression levels by real-time PCR HaCaT cells were harvested 48 hours after sample addition, and total RNA was extracted using an RNeasy Mini kit (Qiagen). cDNA was synthesized from the extracted total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Real-time PCR was performed using the synthesized cDNA as a template on an AriaMX (Agilent) system. THUNDERBIRD SYBR qPCR Mix (Toyobo) was used for the real-time PCR reaction. For the ceramide synthase gene CERS2 primers, a forward primer represented by SEQ ID NO: 1 (5'-CCGATTACCTGCTGGAGTCAG-3') and a reverse primer represented by SEQ ID NO: 2 (5'-GGCGAAGACGATGAAGATGTTG-3') were used as primers, and a forward primer represented by SEQ ID NO: 3 (5'-ACATTCCACAAGGCAACCATTG-3') and a reverse primer represented by SEQ ID NO: 4 (5'-CTCTTGATTCCGCCGACTCC-3') were used as primers for the ceramide synthase gene CERS3. The forward primer represented by SEQ ID NO: 5 (5'-GGAGGCCTGTAAGATGGTCA-3') and the reverse primer represented by SEQ ID NO: 6 (5'-GAGGACCAGTCGGGTGTAGA-3') were used as primers for the ceramide synthase gene CERS4, and the forward primer represented by SEQ ID NO: 7 (5'-GGGTCAGAAGGACTCCTATG-3') and the reverse primer represented by SEQ ID NO: 8 (5'-GTAACAATGCCATGTTCAAT-3') were used as primers for the internal standard β-actin. The real-time PCR reaction conditions were an initial denaturation at 95°C for 60 seconds, followed by denaturation at 95°C for 15 seconds, and annealing / extension at 60°C for 60 seconds, followed by 40 cycles of a two-step PCR reaction.

[0024] 3. Evaluation Results Figure 1 shows the results of the ceramide synthase gene expression evaluation test. Figure 1 shows relative values ​​(n=3, mean ± SD, ** indicates p<0.01) with the control set at 1, and shows the evaluation results for CERS2, CERS3, and CERS4 (from left to right) at each concentration of control and sea buckthorn seed oil. As shown in Figure 1, the addition of 0.5 v / v% sea buckthorn seed oil confirmed an approximately two-fold increase in expression of all CERS2, CERS3, and CERS4 compared to the control. Based on these results, it can be expected that sea buckthorn seed oil will activate ceramide synthase genes and increase ceramide production.

Claims

1. An agent for enhancing the expression of ceramide synthase genes, containing sea buckthorn seed oil as an active ingredient.

2. The ceramide synthase gene expression enhancer according to claim 1, wherein the ceramide synthase gene is one or more selected from the group consisting of CERS2, CERS3 and CERS4.

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