Novel application of codium cylindricum extract

Abstract

To discover new applications of Codium cylindricum in order to obtain a novel marine resource, and to provide a novel anti-aging agent, Neu-1 gene expression promoter, antioxidant, and cosmetic.SOLUTION: Provided are an anti-aging agent, a Neu-1 gene expression promoter, an antioxidant, and cosmetic, each comprising Codium cylindricum extract, the Codium cylindricum extract being either an alkali-treated product of crude extract of Codium cylindricum, or a product obtained by subjecting the alkali-treated product to an organic solvent treatment for removal of insoluble components.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to novel uses of Nagamil extract, specifically, to an anti-aging agent, a Neu-1 gene expression promoter, an antioxidant, and a cosmetic composition using Nagamil extract. [Background technology]

[0002] As we age, aging symptoms such as loss of skin elasticity and wrinkles progress. The main causes of these aging symptoms include a decrease in elastin, which is an extracellular matrix (ECM), and denaturation due to cross-linking of collagen. For example, elastin is a fibrous protein that mainly functions to bind collagen together, and a decrease in elastin leads to a decrease in skin elasticity.

[0003] In addition, active oxygen generated by external stress such as ultraviolet rays is deeply involved in the progression of aging symptoms. For example, it is said that extracellular matrices such as elastin and collagen are oxidized by active oxygen, and when they are denatured, the firmness and elasticity of the skin decreases.

[0004] In recent years, with the growing trend towards anti-aging, anti-aging care products that address skin aging have been attracting attention. Various natural products that exhibit physiologically active effects targeting the above-mentioned phenomenon have been reported. For example, Patent Document 1 discloses that an extract of Rosea japonica, which belongs to the genus Rosa and the family Rosaceae, exhibits an elastase activity inhibitory effect that suppresses the decomposition of elastin. Patent Document 2 discloses that an extract of Acanthus nigra, a deciduous tall tree, exhibits antioxidant effects.

[0005] By the way, Codium cylindricum is known as a type of green algae that includes sea grapes (Japanese name: Kubirezuta) and sea lettuce. Sea grapes and sea lettuce are widely used as food, but Codium cylindricum is only eaten in limited areas and is hardly used as a fishery resource. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2021-107349 [Patent Document 2] JP 2006-321730 A Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel anti-aging agent, a Neu-1 gene expression promoter, an antioxidant, and a cosmetic. [Means for solving the problem]

[0008] In order to find new uses for Nagamil, the present inventors have conducted extensive research on Nagamil extract, focusing particularly on its anti-aging effect, and have found that Nagamil extract has anti-aging effects, Neu-1 (neuraminidase-1) gene expression promoting effect, and antioxidant effect, and can be used for specific purposes, etc. That is, the present invention has the following configuration.

[0009] The anti-aging agent, Neu-1 gene expression promoter, and antioxidant of the present invention are characterized in that they contain a Nagamil extract, which is an alkali-treated product of a crude Nagamil extract.

[0010] The cosmetic preparation of the present invention is characterized in that it contains a Nagamil extract. The Nagamil extract is an alkali-treated product of a crude Nagamil extract. Effect of the Invention

[0011] The anti-aging agent of the present invention contains Nagamil extract, and therefore exhibits excellent anti-aging activity. This anti-aging activity is exerted based on the Neu-1 gene expression promoting activity and antioxidant activity, but is not limited to these activities. Since the above-mentioned Nagamil extract has excellent anti-aging activity, it can be used as an active ingredient of an anti-aging agent, for example, and the Nagamil extract can be effectively used in a wide range of applications such as food and beverages, medicines, and cosmetics.

[0012] In addition, the above-mentioned Nagamil extract has an excellent Neu-1 gene expression promoting effect, and therefore can be used as an active ingredient of, for example, a Neu-1 gene expression promoting agent. By promoting the expression of the Neu-1 gene, it activates the ECM remodeling of elastin, promotes the incorporation of old and decomposed elastin into fibroblasts, and promotes the production of new elastin, leading to the construction of firm skin. Thus, the Neu-1 gene expression promoting agent has the effect of promoting the incorporation of decomposed elastin into cells, and also has the effect of activating ECM remodeling.

[0013] In addition, the Nagamil extract has an excellent antioxidant effect, and can be used, for example, as an active ingredient of an antioxidant. The antioxidant effect can eliminate active oxygen generated in the skin, thereby suppressing the denaturation of elastin and collagen, and thus preventing the loss of firmness and elasticity of the skin.

[0014] Furthermore, since the above-mentioned Nagamil extract is an alkali-treated product of the Nagamil crude extract, impurities in the extract can be efficiently removed, and the physiological activity can be enhanced.

[0015] The cosmetic composition of the present invention contains Nagamil extract, and is therefore particularly suitable as an anti-aging care product due to its various physiologically active effects. [Brief description of the drawings]

[0016] [Figure 1] This is a photo of raw Nagamil algae. [Diagram 2]FIG. 1 is a process schematic diagram of an example of a method for producing a Nagamil extract. [Diagram 3] FIG. 1 shows an overview of the ECM remodeling mechanism in the dermis. [Figure 4] 1 is a graph showing the evaluation results of the promoting effect on Neu-1 gene expression. [Diagram 5] The figures include graphs showing the evaluation results of the intracellular ROS scavenging effect. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The anti-aging agent, Neu-1 gene expression promoter, and antioxidant agent, as well as the cosmetic composition of the present invention, contain Nagamil extract.

[0018] Codium cylindricum is a type of green algae belonging to the Codiales order and Codiaceae family. Codium cylindricum grows in the bathyal zone (subtidal zone) of inlets close to the open sea, and its habitat is the central to southern Pacific coast of Honshu, Shikoku, Kyushu, etc. As shown in the photograph in Figure 1, the algae of Codium cylindricum are long, thin, string-like, and branched, measuring 0.5m to 3.0m or more in length.

[0019] Figure 2 shows an example of a method for producing the Nagamil extract used in the various agents and cosmetics of the present invention. The method in Figure 2 has (a) an extraction step and (b) a post-treatment step. The "Nagamil extract" used in the various agents and cosmetics of the present invention is a concept that comprehensively includes the "crude extract" obtained in the extraction step and the "Nagamil extract" obtained in the post-treatment step. Each step will be described in detail below.

[0020] [(a) Extraction process] The raw material is Nagamil algae. The Nagamil algae may be in its raw (raw) state, but it is preferable to use the dried algae. The drying method is not particularly limited, and natural drying, heat drying, freeze drying, etc. can be used. From the viewpoint of extraction efficiency, it is preferable to use the dried algae in powder form. For example, a well-known crushing device such as a crusher can be used to crush the algae into powder form.

[0021] The extraction method in the extraction step is not particularly limited, but for example, solvent extraction can be performed. Examples of the extraction solvent include water, organic solvents, and mixtures thereof. Examples of the organic solvent include alcohol-based solvents such as monohydric alcohol solvents having 1 to 4 carbon atoms, such as methanol, ethanol, and isopropanol, and polyhydric alcohol solvents, such as 1,3-butylene glycol and glycerin; ketone-based solvents, such as acetone and methyl ethyl ketone; ester-based solvents, such as ethyl acetate; hydrocarbon-based solvents, such as hexane; ether-based solvents, such as diethyl ether; and halogenated hydrocarbon-based solvents, such as chloroform. These extraction solvents may be used alone or in combination of two or more. In the present invention, the water-containing alcohol refers to a mixture of water and an alcohol-based solvent.

[0022] As the extraction solvent, for example, in the case of a skin external preparation, it is preferable to use acetone or aqueous ethanol in consideration of safety to the skin, etc. The ethanol concentration in the aqueous ethanol is, for example, 10 v / v% to 99.5 v / v%, preferably 50 v / v% to 80 v / v%, and may be 60 v / v% or more.

[0023] Nagamil is immersed in the above extraction solvent and allowed to stand or stir at room temperature or under heating to elute the components contained in Nagamil. Thereafter, the extraction residue can be removed by centrifugation or filtration as necessary to obtain a crude extract.

[0024] The amount of the solvent used for extraction is not particularly limited, but is, for example, 10 to 100 times, and preferably 10 to 60 times, the dry weight of Nagamil.

[0025] The extraction conditions are not particularly limited, but the extraction temperature is appropriately set within the range of, for example, 20° C. to 100° C., preferably 20° C. to 80° C., and may be 20° C. to 50° C. The extraction time is appropriately set within the range of 10 minutes to 24 hours, preferably 1 hour to 12 hours. Furthermore, the number of extractions may be one, but it is preferable to perform multiple extractions (preferably 2 to 3 times) to improve the extraction efficiency.

[0026] The extraction method in the extraction step is not limited to solvent extraction, and may be a method using a supercritical fluid such as water or carbon dioxide.

[0027] [(b) Post-treatment process] In this step, the crude extract obtained in the extraction step is subjected to post-treatment to remove impurities, etc. This post-treatment step preferably includes at least one of a step (b1) of treating the solution of the crude extract with an alkali, and a step (b2) of removing insoluble matters using an organic solvent. In this case, the post-treatment step may include only the step (b1), or may include both the step (b1) and the step (b2).

[0028] The alkali used in step (b1) is not particularly limited, and examples thereof include hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as magnesium and calcium; carbonates of alkali metals; carbonates of alkaline earth metals; hydrogen carbonates of alkali metals; hydrogen carbonates of alkaline earth metals. Preferred are hydroxides of alkali metals. These alkalis may be added as solids or as solutions such as aqueous solutions.

[0029] The amount of alkali added to the solution of the crude extract is not particularly limited, but the amount of alkali is, for example, 0.1 to 10 times the weight of the solid content (evaporation residue) of the crude extract.

[0030] The solvent used in step (b1) is not particularly limited, and the solvents listed in the above extraction step can be used appropriately. Among them, water or a mixture of water and an organic solvent is preferable, and in the case of a mixture, an alcohol solvent or a ketone solvent is preferably used as the organic solvent. The treatment temperature is appropriately set, for example, in the range of 20°C to 100°C, and the treatment time is appropriately set, for example, in the range of 10 minutes to 5 hours.

[0031] In step (b1), an acid such as hydrochloric acid or sulfuric acid is added to the reaction mixture after the alkali treatment to neutralize it to about pH 7.

[0032] In step (b2), insoluble matters are removed using various solvents. In this step, removal of insoluble matters may be performed multiple times using different solvents.

[0033] For example, an equivalent or more amount of organic solvent is added to the neutralized solution neutralized with an acid in the above step (b1) to precipitate insoluble matter, which can then be removed by filtration or the like. In this case, it is preferable to use an aprotic polar solvent such as a ketone solvent, an ester solvent, or an ether solvent, for example, acetone, as the organic solvent. After removing the insoluble matter, the solvent is distilled off to obtain a concentrated residue. The same operation may be repeated multiple times.

[0034] After removing the insoluble matter, if necessary, a different solvent may be added to precipitate the insoluble matter, which may then be removed by filtration or the like. As the solvent used here, it is preferable to use a protic polar solvent such as water-containing alcohol (e.g., 60 v / v% ethanol). After removing the insoluble matter, the solvent can be distilled off to obtain a concentrated residue.

[0035] In addition to or instead of the above steps (b1) and (b2), in the post-treatment step (b), an ion exchange resin treatment or an activated carbon treatment may be carried out. These treatments can also remove impurities.

[0036] The Nagamil extract used in the various agents and cosmetics of the present invention is preferably an alkali-treated product of the Nagamil crude extract. Here, the alkali-treated product may be one that has been subjected to at least the above-mentioned step (b1) in the (b) post-treatment step, and may also be one that has been subjected to a subsequent step (e.g., the above-mentioned step (b2)). By the alkali treatment, impurities and the like can be efficiently removed, and the physiological activity of the Nagamil extract can be enhanced.

[0037] The Nagamil extract used in the present invention contains multiple components derived from Nagamil and is not purified into a single component.

[0038] The Neu-1 gene expression promoter of the present invention contains the above-mentioned Nagamil extract and exhibits an action of promoting Neu-1 gene expression. The relationship between this action of promoting Neu-1 gene expression and anti-aging will be explained with reference to FIG.

[0039] Figure 3 shows the remodeling mechanism of the extracellular matrix (ECM) in the dermis, with elastin shown as an example of the ECM. As shown in Figure 3, old elastin is transformed into new elastin through a cycle of "formation," "decomposition," and "uptake and production." This maintains the firmness of the skin. However, as skin cells deteriorate with age, this function also declines, and as a result, it becomes difficult for decomposed old elastin to be taken up by fibroblasts. As a result, old elastin accumulates in the dermis, making it difficult for new elastin to be produced, which is thought to lead to a decrease in skin firmness.

[0040] Elastin is taken up into fibroblasts via the elastin receptor complex (ERC). ERC is a receptor that takes up fragmented elastin into fibroblasts, and is composed of three proteins: EBP (elastin-binding protein), PPCA (protective protein), and Neu-1 (receptor). Of these, the Neu-1 gene expression promoter of the present invention focuses on Neu-1, which is thought to play a central role in elastin remodeling, and enhances the expression level of the gene encoding this protein. This expression promoting effect can be evaluated by the method described in the Examples below.

[0041] The Neu-1 gene expression promoter of the present invention promotes the expression of the Neu-1 gene and enhances the activity of Neu-1, so that the fragmented elastin accumulated around the fibroblasts is easily taken up into the fibroblasts. The taken up elastin is then decomposed in the cells to become the material for elastin, which results in the regeneration of new elastin and promotes the rebirth of firm skin. In this way, the Neu-1 gene expression promoting effect activates ECM remodeling, which ultimately leads to an anti-aging effect.

[0042] Various studies have been conducted to inhibit the activity of elastase, the enzyme that breaks down elastin, an ECM, in order to suppress its degradation; however, very little research has been conducted focusing on the uptake and production of elastin in elastin remodeling.

[0043] The antioxidant of the present invention contains the above-mentioned Nagamil extract and exhibits antioxidant activity. This antioxidant activity can be evaluated by a test using, for example, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical, superoxide radical (SOD), intracellular reactive oxygen species (ROS), etc. Intracellular ROS is a highly reactive oxygen species generated during the ATP synthesis process in mitochondria, and is said to act as an oxidant against DNA and proteins, causing various diseases and aging.

[0044] The antioxidant of the present invention can eliminate active oxygen generated in the skin and other areas, thereby preventing and improving skin aging such as wrinkle formation and loss of elasticity, as well as diseases caused by such oxidative stress.

[0045] The various agents of the present invention can be used as foods and beverages, medicines, cosmetics, etc., or as ingredients to be blended therein.

[0046] The various agents of the present invention are not particularly limited in terms of dosage form or application method, and can be, for example, oral or topical skin agents. In the case of oral use, the dosage form can be, for example, tablets, liquids, powders, capsules, etc. In the case of topical skin agents, the dosage form can be, for example, liquids, lotions, emulsions, creams, ointments, etc. The content of the Nagamil extract (in terms of solid content) is not particularly limited, and is, for example, 0.01% by mass to 50% by mass (preferably 0.05% by mass to 20% by mass) relative to the total amount of the various agents or cosmetics.

[0047] The cosmetic of the present invention is applied to the skin and can be in the form of, for example, a liquid, a lotion, an emulsion, a cream, an ointment, etc. The content of the Nagamil extract (converted into solid content) is not particularly limited, but is, for example, 0.01% by mass to 50% by mass (preferably 0.05% by mass to 20% by mass) based on the total amount of various agents or cosmetics.

[0048] Furthermore, the cosmetic of the present invention contains the above-mentioned Nagamil extract and can be made into an anti-aging product touting its physiological activity, etc. The cosmetic of the present invention can be appropriately blended with various oils, other anti-aging ingredients, surfactants, UV absorbers, powders, pigments, polyhydric alcohols, thickeners, antioxidants, fragrances, preservatives, etc. EXAMPLES

[0049] [1. Production of Nagamil Extract] After obtaining a dry powder from Nagamil, a crude Nagamil extract was obtained by supercritical extraction. Potassium hydroxide was added to the solution of this crude extract, and it was heated in a boiling bath for 1 hour, after which HCl was added to neutralize it. The reaction solution was then mixed with an equal amount of acetone, filtered through filter paper, and the process of concentration and drying was repeated to obtain the Nagamil extract (Codium cylindricum extract; CCE).

[0050] [2. Evaluation of the effect of promoting Neu-1 gene expression] Normal human dermal fibroblasts (NHDF) that had been subcultured twice were seeded in a 6-well culture plate containing Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and cultured at 37°C in a 5% CO2 atmosphere until the cells reached 60% confluence. After that, the NHDF was replaced with serum-free DMEM and cultured at 37°C in a 5% CO2 atmosphere for 24 hours, and then replaced with DMEM containing different concentrations of CCE (final concentrations 6, 60, and 600 ng / mL) and cultured at 37°C in a 5% CO2 atmosphere for 48 hours. After culture, total RNA was collected using an RNA recovery reagent, and cDNA was synthesized using reverse transcriptase. Next, the expression level of elastin receptor complex subunit Neu-1 mRNA was analyzed by quantitative RT-PCR using the synthesized cDNA. The primers used were 5'-GCGATGGAGCTTCAGCAATG-3' (SEQ ID NO: 1) as the forward primer and 5'-CAGGGTTGCCAGGGATGAAT-3' (SEQ ID NO: 2) as the reverse primer, and GAPDH mRNA was used as the internal standard gene, with 5'-TGCACCACCAACTGCTTAGC-3' (SEQ ID NO: 3) as the forward primer and 5'-GAGGAGCCAGTCTTGGATGAG-3' (SEQ ID NO: 4) as the reverse primer. Comparison of gene expression was analyzed by the ΔΔCT method using the SYBR Green method with the measuring device QuantStudio3. This test was performed five times independently, and the expression level without CCE (control) was calculated as a relative value when it was set to 1.0.

[0051] Figure 4 shows the expression level of Neu-1 mRNA. The results are shown as the mean ± standard deviation (SD; n = 5). Significance was evaluated using one-way analysis of variance (ANOVA). As shown in Figure 4, the higher the concentration of Nagamil extract, the more the expression level of Neu-1 mRNA was enhanced, and concentration dependency was confirmed. Here, for example, Reference 1 (JP Patent Publication No. 2021-006043) reports that increasing the expression level of Neu-1 mRNA promotes the uptake of elastin, normalizes ECM remodeling, and maintains the homeostasis of the extracellular matrix. Reference 2 (Scandolera et al., 2015, PLOS ONE (https: / / doi.org / 10.1371 / journal.pone.0129994) reports that the activity of the elastin receptor complex (ERC), which contains Neu-1, decreases with age and affects the degradation of elastin in ECM remodeling. From these reports, it can be said that promoting the expression level of the Neu-1 gene activates ECM remodeling, which in turn leads to anti-aging effects.

[0052] [3. Evaluation of antioxidant activity] (a) DPPH radical scavenging effect The DPPH radical scavenging activity of Nagamil extract was measured by spectrophotometry (visible light).The result was 0.32±0.02μmolTE / g, confirming that Nagamil extract has the DPPH radical scavenging activity.

[0053] (b) Intracellular ROS scavenging effect First, the amount of reactive oxygen species (ROS) was compared and evaluated between NHDFs (normal cells) that had been subcultured twice and cells (senescent cells) created by adding a senescence-inducing agent to NHDFs. The senescent cells were created by treating normal cells with DMEM containing 30 μmol / L 5-bromo-2-deoxyuridine (BrdU) and culturing them at 37°C in a 5% CO2 atmosphere for 96 hours. To confirm whether the normal cells had become senescent cells, we observed the cell morphology under an optical microscope and stained the cells with senescence-associated β-galactosidase (SA-β-gal), a senescence marker. Senescent cells are characterized by being larger than normal cells, slower cell division, and stained with senescence markers. After confirming that the normal cells had become senescent cells through appearance observation and cell staining, the normal cells and senescent cells were seeded on 96-well culture plates and cultured at 37°C in a 5% CO2 atmosphere until they reached 80% confluence. After that, we measured the ROS in the normal and senescent cells using an intracellular ROS detection reagent. For the measurement, we used a fluorescent plate reader to calculate the fluorescence intensity (excitation wavelength 490 nm, fluorescence wavelength 530 nm). The results are shown in Figure 5(a).

[0054] The fluorescence intensity shown in Figure 5(a) is shown as the mean ± standard deviation (Mean ± SD; n = 3). Significance was evaluated using Student's t-test. As shown in Figure 5(a), the amount of intracellular reactive oxygen species was significantly increased in senescent cells compared to normal cells.

[0055] Next, the inhibitory effect of Nagamil extract on the increase in the amount of reactive oxygen species in senescent cells was evaluated. Senescent cells were seeded on a 35 mm glass bottom dish and cultured at 37°C under a 5% CO2 atmosphere until they reached 60% confluence. After that, NHDF was replaced with serum-free DMEM and cultured at 37°C under a 5% CO2 atmosphere for 24 hours, and then replaced with DMEM containing CCE (final concentration 600 ng / mL) and cultured at 37°C under a 5% CO2 atmosphere for 48 hours. After that, ROS in senescent cells and senescent cells treated with CCE (CCE) was measured using an intracellular ROS detection reagent. For the measurement, a fluorescent plate reader was used to calculate the fluorescence intensity (excitation wavelength 490 nm, fluorescence wavelength 530 nm). The results are shown in Figure 5(b).

[0056] The fluorescence intensity shown in Figure 5(b) is shown as the mean ± standard deviation (Mean ± SD; n = 3). The significance was evaluated using Student's t-test. As shown in Figure 5(b), Nagamil extract inhibited the increase in the amount of intracellular reactive oxygen, indicating that it has an intracellular ROS scavenging effect.

[0057] According to the present invention, Nagamil extract has been found to exhibit various physiological activities, and new uses have been found. In particular, regarding its effectiveness on the skin, it has shown anti-aging and antioxidant effects focusing on the ECM remodeling system, and has found a complex anti-aging care effect. In addition, according to the present invention, Nagamil has been found to be a new marine resource, which may lead to advanced use of Nagamil in the future.

Claims

1. An anti-aging agent characterized by containing Nagamil extract.

2. A Neu-1 gene expression promoter comprising a Nagamil extract.

3. An antioxidant characterized by containing Nagamil extract.

4. The agent according to any one of claims 1 to 3, characterized in that the Nagamil extract is an alkali-treated product of a crude Nagamil extract.

5. A cosmetic preparation characterized by containing a Nagamil extract.

6. 6. The cosmetic preparation according to claim 5, wherein the Nagamil extract is an alkali-treated product of a crude Nagamil extract.

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