JP2023053512A - Anti-aging agent, anti-aging cosmetic composition, elastin gene expression promoter, and collagen gene expression promoter - Google Patents

Abstract

To provide an anti-aging agent, anti-aging cosmetic composition, elastin gene expression promoter, and collagen gene expression promoter that promote the production of dermis, containing materials derived from natural products.SOLUTION: Provided are an anti-aging agent, anti-aging cosmetic composition, elastin gene expression promoter, and collagen gene expression promoter, containing an Elaeagnaceae plant. The Elaeagnaceae plant is one or more plants selected from Elaeagnus pungens, Elaeagnus multiflora, Elaeagnus umbellata, Elaeagnus multiflora var. hortensis, and Elaeagnus glabra.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to an anti-aging agent, an anti-aging cosmetic composition, an elastin gene expression enhancer, and a collagen gene expression enhancer containing a Gummiaceae plant.

The Gumaceae plant is a general term for the genus Elaeagnus of the Gumaceae family, which is widely distributed in Japan from Eurasia to Southeast Asia in Honshu, Shikoku, Kyushu, and Okinawa generally west of Fukushima Prefecture. Gumaceous plants grow from lowlands to hilly areas, and although there are many species, they are not widely used commercially.

In Japan, only in the Amami archipelago, the branches of Elaeagnus glabra Thunb. Yoke tea is said to be effective for detoxification, tetanus, neuralgia, colds, barnyard removal, liver disease, kidney disease, hypotension, antidote, antipyretic, gonorrhea, and postpartum anemia. However, in other regions, it is almost unknown that Gumaceous plants are used as food or medicine, so only a small part of the details of the ingredients and efficacy has been elucidated (Patent Document 1, etc.).

On the other hand, in the development of cosmetics, in addition to skin and hair moisturizing, softness, and cleansing properties, skin whitening, UV protection, anti-wrinkle properties, etc. Moisturizing ingredients, softening ingredients, cleansing ingredients, whitening ingredients, UV inhibitors, surfactants, etc. , antioxidants, preservatives, etc. are added. At this time, it is desirable to use materials derived from natural products in terms of the burden on human skin and safety. , chemically synthesized materials are often used.

Patent Literature 2 describes that consideration has been given to partially blending materials derived from natural products into cosmetics, and that dry skin is improved as a softening lotion. However, the results were based on a three-level sensory evaluation of the effect of improving skin feel on subjects with dry skin, indicating that there is a clear relationship between improvement in skin dryness and improvement in skin feel. I don't know until

Furthermore, in the age of an aging society, there is an increasing need for young people to have fresh skin with less wrinkles and sagging even as they age. Here, in order to keep the skin fresh like a young person, it is not enough to temporarily penetrate the skin with a moisturizer, etc., and dermal fibroblasts produce dermal components such as collagen, elastin, and hyaluronic acid. It is necessary to let anti-aging (anti-aging). That is, since wrinkles and sagging are said to be caused by functional deterioration of dermal components, there is a demand for fundamental cosmetics that promote the production of dermal components.

JP-A-2004-75638 Japanese Patent Application Laid-Open No. 2001-226218

The present invention has been made in view of the above circumstances, and provides an anti-aging agent that promotes the production of components in the dermis, the anti-aging agent comprising a material derived from a natural product, and the material derived from a natural product. Although it is widely distributed in Japan, the details of its ingredients and efficacy are only limitedly known. , to provide a collagen gene expression promoter.

In order to solve the above problems, the anti-aging agent of the present invention is characterized by containing a gummy plant.

The anti-aging cosmetic composition of the present invention is characterized by containing a gummy plant.

The elastin gene expression promoter of the present invention is characterized by containing a gummy plant.

The collagen gene expression promoter of the present invention is characterized by containing a gummy plant.

The Gumaceae plant is preferably one or more plants selected from Nawashirogumi, Natsugumi, Akigumi, Togumi, and Tsurugumi.

The anti-aging agent of the present invention promotes the expression of genes encoding dermal components, promotes the production of dermal components, prevents wrinkles and sagging, maintains fresh skin, and is anti-aging (anti-aging). )can do.
In addition, by including a gummy plant in the anti-aging agent, it is possible to increase the blending ratio of materials derived from natural products, and it is possible to gently anti-aging the human skin compared to the case of blending chemically synthesized materials. becomes possible.

It is a graph showing the elastin gene expression level. It is a graph showing a collagen gene expression level. It is a graph showing the collagenase gene expression level.

The anti-aging agent, anti-aging cosmetic composition, elastin gene expression promoter, and collagen gene expression promoter of the present invention are described below.

(Anti-aging agent)
The anti-aging agent of the present invention contains gummy plants.

The gummy family plant is a general term for plants belonging to the genus Gummy (Elaeagnus) in the family Gummiaceae. About 50 to 70 kinds of species such as Nawashigumi, Natsugumi, Akigumi, Tougumi, and Tsurugumi exist.
Depending on the species, it is an evergreen or deciduous shrub, some of which are climbing. The evergreen ones are less cold-hardy, while the deciduous ones are more cold-tolerant. The leaves are alternate and the leaves and stems are hairy. It blooms and bears fruit on new shoots growing from the nodes of the branches.

Examples of types of Gummiaceae plants are shown below.
Nawashirogumi flowers in autumn and bears fruit in early summer of the following year. It has wavy leaf margins and thorny branches.
Natsugumi blooms in spring and bears fruit in early summer. It is characterized by a mixture of silvery white and brown scales on the underside of the leaves.
Akigumi flowers bloom in early summer and bear fruit in autumn.
Togumi blooms in spring and bears fruit in early summer. It is characterized by having no spines on its branches.
Tsurugumi blooms in spring and bears fruit in early summer. The branches have no thorns and grow like a vine. The branches are ridged and densely covered with reddish-brown scaly hairs. It grows as if leaning against other plants with reverse branches (downward-facing branches). The leaves are alternate and oblong to ovate oblong, with reddish-brown scales densely growing on the underside. It is characterized by hanging flowers on the leaf axils and reddish-brown scales densely growing on the long and narrow calyx tube.

Gumaceous plants are widely distributed in Honshu, Shikoku, Kyushu, and Okinawa west of Fukushima Prefecture in Japan, but they are used only for extracting tea from the branches. However, flowers, fruits, leaves, branches (stems), roots, or the whole Gumaceous plant can be used for the anti-aging agent.

Gumaceous plants can be extracted by splitting branches like small clappers or small firewoods like yoke tea and boiling them in water (hot water) to obtain active ingredients. Here, not only water but also a water-soluble organic solvent, a poorly water-soluble organic solvent, and the like can be used as the extract. Examples thereof include alcohols (ethanol, butanediol, etc.), aqueous alcohol solutions (ethanol+water, butanediol+water, etc.), organic solvents (hexane, chloroform, toluene, etc.), and the like. Furthermore, the extract may contain various ions (Fe ions, Ca ions, K ions, etc.).

Alternatively, the extract can be phase-separated with an organic solvent and an aqueous solvent, and the organic layer and the aqueous layer can be respectively concentrated to obtain the active ingredient. For example, a pulverized Gummiaceae plant or a dried (heat-dried, freeze-dried, etc.) Gummiaceae plant is separated into a hexane layer and an aqueous layer, and each of these is concentrated.
Alternatively, steam distillation may be performed to separate the oil and steam-distilled water, and each may be concentrated.

Furthermore, not only the branches but also the flowers, leaves, fruits, roots, or the whole Gumaceous plant can be appropriately treated in the same manner as described above to obtain the active ingredient from the Gumaceae plant.

An anti-aging agent is an agent containing a component having an anti-aging effect.
Human skin undergoes aging due to wrinkles and sagging, and such wrinkles and sagging are caused mainly by a decrease in dermal components such as collagen, elastin, and hyaluronic acid, and a decline in function. Conversely, if the expression of genes encoding each dermal component is promoted in dermal fibroblasts and the production of dermal components is promoted, wrinkles and sagging can be improved and aging can be prevented. Become.

Collagen is one of the proteins that constitute the dermis, ligaments, tendons, bones, cartilage, etc. of vertebrates, and is a major component of the extracellular matrix of multicellular animals such as humans. The amino acid residues constituting the peptide chain of the collagen protein are characterized by repeating every three glycine residues. Type I collagen has a molecular weight of about 100,000, and the gene corresponding to type I collagen production is COL1A1, which is involved in skin firmness and elasticity.
The gene encoding the collagenase is MMP1. Therefore, the smaller the expression level of MMP1, the more difficult it is for collagen to decompose.

Elastin (elastic fiber) is the elastic fiber that supports the fibers of collagen. Elastin is first biosynthesized as a precursor protein, tropoelastin, in fibroblasts and the like, gathered around and in the interstices of microfibrils and crosslinked to form elastic fibers. The gene corresponding to elastin production is ELN, which is involved in skin firmness and elasticity.
The gene encoding elastin degrading enzyme is MME. Therefore, the lower the expression level of MME, the more difficult it is for elastin to decompose.

Hyaluronic acid has a structure in which two kinds of sugars, N-acetylglucosamine and D-glucuronic acid, are linked in a straight chain, and has a molecular weight of 800,000 to 1,200,000. Hyaluronic acid is abundant in the skin, joints, and vitreous of the eyeball of humans and vertebrates, and in humans, half of the hyaluronic acid is present in the skin. Genes corresponding to hyaluronic acid synthase production by dermal fibroblasts are HAS1 and HAS2, and are involved in skin moisture retention. When hyaluronic acid is reduced from the epidermis due to aging, fine wrinkles are formed.
The gene encoding the hyaluronan degrading enzyme is HYAL1. Therefore, the lower the expression level of HYAL1, the more difficult it is for hyaluronic acid to decompose.

Gummaceae plants contain anti-aging components that can promote gene expression corresponding to the production of components of the dermis. The anti-aging effect can be further improved by appropriately treating the Gummiaceae plant to concentrate the active ingredient serving as the anti-aging ingredient.

(Anti-aging cosmetic composition)
The anti-aging cosmetic composition of the present invention contains a gummy plant. That is, it contains the above-mentioned anti-aging agent.

The anti-aging cosmetic composition can be optionally blended with additives and components as necessary, in addition to the gummy plant containing the anti-aging agent.

Additives and ingredients that can be optionally added include water-soluble moisturizing ingredients (e.g., polyhydric alcohols such as glycerin, pentylene glycol, and dipropylene glycol), oil-soluble emollient ingredients (e.g., vegetable oil, ester oil, mineral oil, etc.). oils and fats), polymeric materials (e.g., ionic polymers, polymers such as thickeners, or natural polymers), fatty acids, alcohols, surfactants, vitamins, amino acids, antioxidants , antiseptic or antibacterial agents, ultraviolet absorbers and ultraviolet scattering agents, fragrances, essential oils, and the like.

Fats and oils include fatty acid glycerides such as soybean oil, rapeseed oil, castor oil, cottonseed oil, coconut oil, coconut oil, safflower oil, beef tallow, and lard, POE (polyoxyethylene) adducts thereof, hydrogenation hardening Things etc. are exemplified.

Examples of mineral oils include liquid paraffin, paraffin, petrolatum, and microcrystalline wax.

Examples of fatty acids include saturated fatty acids and unsaturated fatty acids having about 6 to 24 carbon atoms, such as lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, and behenic acid.

Examples of alcohols include linear alcohols and branched alcohols having about 2 to 24 carbon atoms such as ethanol, isopropanol, lauryl alcohol and stearyl alcohol, ethylene glycol, butylene glycol, ethylene glycol monomethyl ether, glycerin, pentaerythritol, sorbitol and the like. A polyhydric alcohol etc. are illustrated.

Examples of ester oils include compounds having an ester bond such as isopropyl myristate, isopropyl palmitate, decyl oleate, and ethylene glycol monostearate.

Examples of surfactants include anionic surfactants (alkylbenzene sulfonate, etc.), cationic surfactants (alkylamine salts, etc.), nonionic surfactants (ether type, ester type, etc.), and the like. .

Vitamins include vitamin C, cholecalciferol, vitamin E, ubiquinones, γ-oryzanol, rutin, carotene, folic acids, pantothenic acids, biotins, lycopene, thiamine sulfate, riboflavin, pyridoxine, cyanocobalamin, choline, and inositol. and the like are exemplified.

Examples of amino acids include asparagine, glutamine, glycine, alanine, serine, arginine, cysteine, cystine, valine, lysine, tyrosine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, proline, hydroxyproline, ornithine, histidine, and the like. be done.

Examples of antioxidants include tocopherol, dibutylhydroxytoluene, ascorbic acid, sodium hydrogen sulfite and the like.

Examples of preservatives include phenoxyethanol, parahydroxybenzoic acid esters, benzoic acids, phenol, dehydroacetic acids and the like.

Examples of antibacterial agents include isopropylmethylphenol, salicylic acid, dichlorobenzyl alcohol, thiobischlorophenol, chloramine T and the like.

UV absorbers include ethyl para-aminobenzoate, glyceryl para-aminobenzoate, ethylene glycol salicylate, phenyl salicylate, p-tert-butylphenyl salicylate, benzyl cinnamate, isopropyl para-methoxycinnamate, 2-(2-hydroxy- 5-methylphenyl)benzotriazole and the like are exemplified.
Examples of ultraviolet scattering agents include titanium oxide, zinc oxide, cerium oxide, and silica.

Cosmetics include lotions, milky lotions, creams, packs, foundations, lip balms, sunscreens, cleansers, shampoos, rinses, conditioners, treatments, facial cleansers, body soaps, hair restorers, hair tonics, bath agents, etc., dermal fibroblasts It is not particularly limited as long as it is a dosage form that works on

(Elastin gene expression promoter)
The elastin gene expression promoter of the present invention contains plants of the Gummy family.

As described above, elastin (elastic fiber) is an elastic fiber that supports collagen fibers, and the gene encoding elastin is ELN (Human Elastin).
Gummies contain active ingredients capable of promoting expression of the ELN gene, which encodes elastin. Further enhancement of ELN gene expression can be achieved by appropriately treating the Gumaceae plant to concentrate the active ingredient. If the presence of active ingredients of gummy family plants promotes ELN gene expression by dermal fibroblasts and promotes the production of elastin, it becomes possible to improve skin elasticity (tension) again.

The gene encoding the elastinase is MME (Human Membrane Metalloendopeptidase). The lower the expression level of MME, the lower the production of elastin-degrading enzymes, the less elastin is decomposed, and the more elastic skin can be maintained.

(Collagen gene expression promoter)
The collagen gene expression promoter of the present invention contains a gummy plant.

As described above, collagen is one of the proteins that constitute the dermis, ligaments, tendons, bones, cartilage, etc. of vertebrates, and is a major component of the extracellular matrix of multicellular animals such as humans. The gene corresponding to type I collagen production is COL1A1.
Gummiaceous plants contain active ingredients capable of promoting COL1A1 (Human Collagen Type 1 Alpha 1) gene expression that encodes collagen. The COL1A1 gene expression can be further promoted by appropriately treating the Gumaceae plant to concentrate the active ingredient. If the presence of the active ingredient of the gummy family plant promotes the expression of the COL1A1 gene by dermal fibroblasts and promotes the production of collagen, it becomes possible to improve the elasticity (tension) of the skin again.

The gene encoding the collagenase is MMP1 (Human Matrix Metallopeptidase 1). The lower the expression level of MMP1, the lower the production of collagen-degrading enzymes, the less likely collagen is to be degraded, and the firmer skin can be maintained.

The anti-aging agent, elastin gene expression promoter, and collagen gene expression promoter of the present invention are applicable not only to cosmetics, but also to quasi-drugs and over-the-counter drugs ( (refers to Classes 1 to 3), and can also be applied to ethical pharmaceuticals and the like. For example, quick bandage, wound medicine, disinfectant cotton, hand/skin disinfectant, anti-itch, antipruritic anti-inflammatory medicine for insect bites, itching, heat rash, athlete's foot remedy, corn remedy, skin disease remedy acne remedy), pharmaceutical hand cream, crack, chapped remedies, xeroderma/keratosis remedies, hair growth stimulants/hair growth agents, emollients, external anti-inflammatory analgesics, stiff shoulders/ lumbago medicine etc. are mentioned.

The following examples and evaluations illustrate the present invention, but the present invention is not limited to the following examples.

(Evaluation of elastin gene expression)
A branch of Tsurugi was split into small clappers and soaked in hot water at 80° C. or higher for 7 days for extraction to obtain an extract. The extract was concentrated and dried and used as a test substance.
A total of 3 concentrations (1%, 10%, 100%) were prepared by serially diluting the above test substance with a 30% BG (butylene glycol) solution at a common ratio of 10 before use. The prepared test substance was further diluted 100-fold to prepare a total of 3 concentrations (0.01%, 0.1%, 1%) before use and used in the test. A 30% BG solution was used as a negative control.

Human neonatal-derived dermal fibroblast cell line NB1RGB cells (RIKEN BRC, Japan) were cultured using a CO ₂ incubator (CO ₂ concentration 5%, 37do) and used as cultured cells for this test.

10.0% (v/v) Fetal Bovine Serum (FBS, Cat# SH30071.03, Hyclone, UK) and 1.0% (v/v) Antimycotic (Antibiotic-Antimycotic 100X, Cat#15240-062, Invitrogen, USA) containing Eagle's Minimal Essential Medium (EMEM, model number 051-07615, Wako, Japan) was used as a medium.

Gene expression analysis of Human Elastin (ELN), a gene encoding elastin, was performed using TaqMan Assay (Applied Biosystems, USA). Human Glyceraldehyde 3 Phosphate Dehydrogenase (GAPDH) was used as an internal standard gene.
The average value of three 35 mm dishes (model number 150318, Thermo Scientific, USA) per treatment group was used for gene expression level analysis.

- Cell culture and addition of test substance 5.0 x ¹⁰⁵ cells/2 mL of NB1RGB cells were seeded in a 35 mm dish and cultured in a _CO2 incubator for 24 hours.
After 24 hours, the medium was removed from the 35 mm dish, medium containing each concentration of test substance and control was added, and cultured in a CO ₂ incubator for 24 hours.

- RNA extraction, generation, and quantification The following RNA extraction and purification were performed using PureLink ^™ RNA Mini Kit (model number 12183018A, Invitrogen, USA).
After culturing for 24 hours, the 35 mm dish from which the medium was removed was washed twice with 2 mL of PBS(-) heated to 37°C.
Cells were lysed by adding 600 μL of Lysis Buffer containing 2M Dithiothreitol (Model No. 27565-41-9, Invitrogen, USA), and the lysate was collected.
Cells in the harvested lysate were disrupted using a Homogenizer (Model No. 12183-026, Invitrogen, USA).
After adding 600 μL of 70% ethanol (model number 64-17-5, Japan Alcohol, Japan) solution to the cell lysate, it was transferred to a column with a silica membrane. After centrifugation at 12,000 xg for 15 seconds at room temperature, the filtrate was discarded.
The silica membrane was washed by adding 700 μL of guanidine isothiocyanate-containing Wash Buffer I and 500 μL of ethanol-containing Wash Buffer II.
After washing the silica membrane with Wash Buffer II containing ethanol, it was centrifuged at 12,000×g at room temperature for 15 seconds and dried.
30 μL of RNase-Free Water was added, left to stand at room temperature for 1 minute, and then centrifuged at 12,000×g at room temperature for 15 seconds. RNA was eluted from the membrane by repeating the procedure twice.
A portion of the eluted RNA was aliquoted into a UV-permeable 96-well plate (model number 8404, Thermo Scientific, USA) and added with Tris-EDTA Buffer (TE (pH 8.0), model number 310-90023, NIPPON GENE, Japan). and measured the absorbance at 230 nm, 260 nm and 280 nm (OD ₂₃₀ , OD ₂₆₀ and OD ₂₈₀ ) using a microplate reader (SPARK (registered trademark) 10M TECAN, Switzerland).
Using OD ₂₆₀ , the RNA concentrations of the control and test substances were calculated according to the following formula and diluted with TE Buffer to adjust the RNA concentration to 10 μg/mL.

RNA concentration (μg/mL) = A × K × 0.3 × 10 (dilution factor)

A is OD ₂₆₀ of control or test article, K=40 (extinction coefficient of RNA), path length was 0.3 cm.

- Gene expression analysis by real-time PCR method Reverse transcription of the following RNA was performed using SuperScript™ IV VILO™ Master Mix with ezDNase (model number 11766050, Invitrogen, USA).
1 μL of 10×ezDNase Buffer, 1 μL of ezDNase enzyme, 6 μL of Nuclease-free Water and 2 μL of 10 μg/mL RNA were added per well to an 8-tube tube (AB1182, Thermo Scientific, USA) and incubated at 37° C. for 2 minutes. .
After 2 minutes, 4 μL of SuperScript™ IV VILO™ Master Mix and 6 μL of Nuclease-free Water were added per well to the 8-tube tube and incubated at 25° C. for 10 minutes using real-time PCR (QuantStudio (registered trademark) 3, Applied Biosystems, USA). cDNA was synthesized by heating at 50° C. for 10 minutes and at 85° C. for 5 minutes.
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The solution was spun down using a plate centrifuge to remove foam.
Real-Time qPCR was performed using a real-time PCR system, and the Threshold Cycle (Ct) value, which is the cycle number at which the fluorescence signal of each gene in the control and test substances reaches an arbitrary threshold, was calculated. The Ct value was corrected using an internal standard gene to obtain the ΔCt value. The ΔCt value was corrected by the average of the ΔCt values of the controls, and this was taken as the ΔΔCt value. Assuming that the difference in detection per cycle by the ΔΔCt method is double the difference, the gene expression level of the test substance was analyzed by substituting it into 2-ΔΔCt and setting the gene expression level of the control to 1. Significant differences were tested by a paired t-test between the gene expression levels of the control and test substance-added. All tests were two-sided with a significance level of less than 5% (P<0.05, P<0.01, P<0.001).

Table 1 and FIG. 1 show the measured Ct values, the average values of the measurement results, the standard deviations, and the results of gene expression. In addition, the vertical axis of FIG. 1 is a. u. (arbitrary unit).

From Table 1 and FIG. 1, it was found that the addition of thrush significantly increased the gene expression of the elastin gene (ELN) compared to the control.

(Evaluation of collagen gene expression)
Gene expression analysis of Human Collagen Type 1 Alpha 1 (COL1A1), a gene encoding type I collagen, was performed using TaqMan Assay (Applied Biosystems, USA). The test conditions are the same except that the ELN is replaced by COLIA1 in evaluating elastin gene expression.

Table 2 and FIG. 2 show the measured Ct values, the average values of the measurement results, the standard deviations, and the results of gene expression. In addition, the vertical axis of FIG. 2 is a. u. (arbitrary unit).

From Table 2 and FIG. 2, it was found that the addition of Tsurugumi significantly increased the gene expression of the type I collagen gene (COLIA1) compared to the control.

(Evaluation of collagenase gene expression)
Gene expression analysis of Human Matrix Metallopeptidase 1 (MMP1), a gene encoding a collagenase, was performed using TaqMan Assay (Applied Biosystems, USA). The test conditions are the same except that ELN is replaced by MMP1 in evaluating elastin gene expression.

Table 3 and FIG. 3 show the measured Ct values, the average values of the measurement results, the standard deviations, and the results of gene expression. In addition, the vertical axis of FIG. 3 is a. u. (arbitrary unit).

From Table 3 and FIG. 3, it was found that the addition of Tsurugumi significantly decreased the gene expression of the collagenase gene (MMP1) compared to the control.

Claims (6)

An anti-aging agent comprising gummy bears. 2. The anti-aging agent according to claim 1, wherein the Gumaceae plant is one or more plants selected from Nawashirogumi, Natsugumi, Akigumi, Togumi, and Tsurugumi. An anti-aging cosmetic composition comprising a gummy plant. 4. The anti-aging cosmetic composition according to claim 3, wherein the Gummiaceae plant is one or more plants selected from Nawashirogumi, Nathogumi, Akigumi, Togumi, and Tsurugumi. Elastin gene expression promoters, including Gumaceae plants. A collagen gene expression promoter containing a gummy plant.

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