JP2019210212A - Hyaluronic acid production promoter, collagen production promoter, mmp inhibitor, wrinkle improver, medicines or food compositions - Google Patents

Abstract

To provide novel external or internal preparations that are excellent in hyaluronic acid production promoting action, collagen production promoting action, and MMP inhibitory effect.SOLUTION: The extract of Ecklonia kurome and Sargassum fulvellum have excellent hyaluronic acid production promoting action, collagen production promoting action, and MMP inhibitory effect, as well as also have excellent stability. The extract of Ecklonia kurome and Sargassum fulvellum can be used not only in the esthetic field such as skin aging prevention, but in the medical field such as the suppression of functional deterioration due to aging, cancer prevention and treatment, and can be expected to be applied to foods, cosmetics, quasi-drugs and pharmaceuticals.SELECTED DRAWING: None

Description

  The present invention relates to a hyaluronic acid production promoter, a collagen production promoter, an MMP inhibitor, a wrinkle improving agent, a pharmaceutical product or a food composition.

  Skin is exposed to various physical and chemical stresses such as ultraviolet rays, dryness, coldness, heat, and drugs every day. As a result, the skin function is lowered, and various skin aging phenomena become apparent. One of the skin aging phenomena is wrinkles. It is known that there are two types of wrinkles: epidermal wrinkles and dermal wrinkles. Epidermal wrinkles are called fine wrinkles, and are wrinkles that are temporarily generated due to a decrease in the amount of water in the epidermal stratum corneum due to dry skin. On the other hand, dermal wrinkles are wrinkles formed by ultraviolet rays contained in sunlight or aging. The formation mechanism includes a decrease in the ability to synthesize collagen in dermal fibroblasts due to ultraviolet rays and aging, and an acceleration of collagen degradation due to an increase in matrix metalloproteinase (MMP).

  Histological morphology, onset mechanism and treatment method differ between epidermal wrinkles and dermal wrinkles due to dryness, and dermal wrinkles caused by ultraviolet rays and aging are improved by the use of cosmetics with a moisturizing effect It is difficult.

  So far, for the purpose of improving dermal wrinkles caused by ultraviolet rays, an extract of skin wrinkle formation preventing / improving agent containing hydrolyzed almond as an active ingredient (Patent Document 1) An agent for improving wrinkles caused by ultraviolet irradiation (Patent Document 2) has been reported.

  Collagen is a major structural protein occupying about 1/3 of mammalian tissue, and is an essential component of many matrix tissues such as cartilage, bone, tendon, and skin. When one site is cleaved by collagenase (MMP-1) belonging to MMP, a stable collagen molecule in a normal tissue is denatured to become single-chain gelatin and is degraded by various other proteases. Become. As a result, the structural integrity of the matrix structure is lost.

  Gelatinase (MMP-2) belonging to MMP is an enzyme produced by fibroblasts, endothelial cells, cancer cells, etc., and is a structural protein that forms a special component of elastic tissues such as collagen, gelatin, and elastin (arteries, tendons, skin, etc.) ) And other substrates. Therefore, a substance having an inhibitory activity on gelatinase is expected to have an effect of suppressing angiogenesis and cancer metastasis in cancer tissue, and is considered useful for the prevention and treatment of cancer diseases. Furthermore, inhibition of MMP is useful not only for cancer diseases, but also for prevention, treatment and improvement of various diseases caused by increased MMP, such as ulcer formation, rheumatoid arthritis, osteoporosis and periodontitis.

  As a material having a collagenase inhibitory activity, for example, a cacao husk extract (Patent Document 3), a rose family Onistrawberry extract (Patent Document 4), a lactoferrin (Patent Document 5), etc., which are cacao bean hulls have been proposed. Under the circumstances of increasing interest in skin aging and oral hygiene, it is required to find a material having no side effect, high safety, and excellent collagenase activity inhibitory action.

  In addition, fibroblasts produce proteins such as collagen and glycosaminoglycans such as hyaluronic acid to form a dermal connective tissue and keep the skin firm. It is believed that this connective tissue loses contraction and loses elasticity, resulting in skin wrinkles and sagging.

  In particular, hyaluronic acid is known as a high-molecular polysaccharide widely distributed in connective tissue, and has a gel-like form in the dermis and maintains the elasticity of the skin. Therefore, alteration and reduction of hyaluronic acid are considered to be important in skin aging. Further, since hyaluronic acid is a polymer, there is a problem that it is difficult to absorb even if a cosmetic containing it is directly applied to the skin. So far, an external preparation for skin that can promote the production of collagen and hyaluronic acid by activating fibroblasts has been sought (Patent Document 6). Hyaluronic acid is also present in joints, and is known to play a function of reducing the impact of joint loads and smoothing the movement of joints. In the case of joint diseases such as osteoarthritis, rheumatoid arthritis, pyogenic arthritis, gouty arthritis, traumatic arthritis and osteoarthritis, the amount of hyaluronic acid in the joint fluid may decrease or decrease with age It has been known. In such joint diseases, it is effective to increase the amount of hyaluronic acid in joint fluid in order to improve lubrication function, cover and protect articular cartilage, suppress pain, and improve or normalize pathological joint fluid. It is believed that there is. For example, it is known that improvement of the above symptoms is observed when sodium hyaluronate joint injection is performed in patients with rheumatoid arthritis, traumatic arthritis, osteoarthritis and osteoarthritis. However, these treatments are long-lasting. Therefore, foods and pharmaceuticals containing hyaluronic acid production promoters are desired so that they can be easily prevented or treated in daily life.

  Flying mosquito disease is a symptom in which a thin shadow that looks like lint and mosquito appears in the field of view, and it occurs when the intravitreal opacity that fills the inside of the eye casts a shadow on the retina. Flying mosquitoes can be roughly divided into two types. Physiological flying mosquitoes and retinal detachment, retinal tears, vitreous hemorrhage, uveitis and other diseases that develop due to the effects of aging, ultraviolet rays, active oxygen, etc. There is morbid mosquito disease that manifests as symptoms. Physiological fly mosquito disease is caused by liquefaction caused by a decrease in hyaluronic acid, which is a major component of the vitreous body, and turbidity in the vitreous body due to the degradation of collagen fibers associated therewith. There are vitrectomy surgery and laser treatment as treatment methods, but there is a fact that these treatments are not performed in Japan from the viewpoint of safety, and it requires a large amount of money to treat in foreign countries . Therefore, in order to prevent and improve physiological flying mosquito disease, foods and pharmaceuticals containing hyaluronic acid production promoters that can be used on a daily basis are desired.

  It is known that an extract of Ecklonia kurome belonging to the order of the Compositae Lessoniaceae (Compositae) Kajime has testosterone-5α-reductase activity inhibitory action and is applied to a hair restorer (Patent Document 7). . Moreover, it is known that the extract of Sargassum fulvalum of the genus Hondamatidae, the genus Honda walla (Sargassum fulvelum), has a tyrosinase activity inhibitory action, and it is proposed to use it as a whitening agent (Patent Document 8). However, it has not been known that these chrome and Honda straw have hyaluronic acid production promotion, collagen production promotion and MMP inhibitory effects.

JP 2000-119125 A JP 2006-199611 A JP-A-3-44331 JP 2003-137801 A JP-A-5-186368 Japanese Patent Laid-Open No. 2007-1924 JP 2012-229167 A JP-A-2015-86187

  A material that is safe and excellent in stability and excellent in hyaluronic acid production promotion, collagen production promotion, and MMP inhibitory effect is desired, but no material that can be fully satisfied has yet been provided.

  Under such circumstances, as a result of intensive studies, the present inventors have found that the extract of chrome and hondawald has excellent hyaluronic acid production promotion, collagen production promotion and MMP inhibitory effects, and is also excellent in stability. It was. Furthermore, the external preparation or the internal preparation containing the extract is safe and stable, and has excellent hyaluronic acid production promotion, collagen production promotion and MMP inhibitory effect, and has multifunctional beauty / health materials / pharmaceuticals. It has been found that it can be realized, and the present invention has been completed.

  The black seaweed (Ecklonia kurome) used in the present invention is a seaweed belonging to the genus Crested genus (Lepidoptera) (Combraceae), mainly from the middle south of the Pacific coast of Honshu to Kyushu, and the Seto Inland Sea and the Sea of Japan side south of Niigata Prefecture. Distributed. The size of the alga body is 40 to 50 cm, and the large one is 1 m, which is mainly used for food.

  The Honda Walla (Sargassum fulvellum) used in the present invention is a seaweed belonging to the genus Honda Walla belonging to the order of the Hidamatidae, and is distributed mainly from Kyushu to the entire Pacific coast of Honshu, and to the Echigo vicinity in the Sea of Japan. The alga body has a lanceolate shape and is characterized by notches.

  Kurome and Honda Walla can be obtained from the above growing areas. In the present invention, it is preferable to use the whole seaweed (whole algae) as an extraction raw material for the seaweed, but some of them, for example, a leaf part (leaf body / adult leaf), a stem part (core / medium cocoon). ), Spores, roots, and the like. Moreover, the seaweed main body may be used for extraction as it is, and treatments such as drying, pulverization, and shredding may be performed.

  The extraction method is not particularly limited, and can be performed by stirring or column extraction using water or hot water, or a mixed solvent of water and an organic solvent. Examples of the extraction solvent include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol). , Glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether) Etc.). Preferred are polar solvents such as water, lower alcohols and liquid polyhydric alcohols, and particularly preferred are water, ethanol, 1,3-butylene glycol and propylene glycol. These solvents may be used alone or in combination of two or more. Particularly preferred extraction solvents include water or water-ethanol mixed polar solvents. The amount of the solvent used is not particularly limited. For example, it may be 10 times or more, preferably 20 times or more of the seaweed (dry weight). For convenience of operation, it is preferably 100 times or less. The extraction temperature and time can be appropriately selected depending on the type of solvent used and the pressure during extraction.

  The above extract may be used as it is, but if necessary, within the range where the effects of the present invention are exhibited, concentration (concentration by reduced pressure concentration, membrane concentration, etc.), dilution, filtration, decolorization by activated carbon, etc. , Deodorization, ethanol precipitation, etc. may be used after treatment. Further, the extracted solution may be subjected to a treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product. The extract of chrome and Honda walla may be used alone or in combination of two. When using the extract of chrome and Honda straw together, the mixing ratio is not limited.

  In the present invention, the extract may be used as it is, and within the range not impairing the effect of the extract, oils and fats, waxes, carbonized components used in cosmetics, quasi drugs, pharmaceuticals, foods, etc. Hydrogens, fatty acids, alcohols, esters, surfactants, metal soaps, pH adjusters, preservatives, fragrances, moisturizers, powders, UV absorbers, thickeners, pigments, antioxidants, whitening agents In addition, components such as chelating agents, excipients, film agents, sweeteners, and sour agents may be contained.

  The present invention can be used for cosmetics, quasi-drugs, pharmaceuticals, and foods. Examples of the dosage form include skin lotion, cream, emulsion, gel, aerosol, essence, pack, and detergent. , Bath preparation, foundation, powder, lipstick, ointment, cataplasm, tablet confection, capsule, chocolate, gum, candy, beverage, powder, granule, tablet, sugar-coated tablet, capsule, syrup, pill, suspension Agents, solutions, emulsions, suppositories, injection solutions, and the like.

  In the case of external use, the content of the extract used in the present invention is preferably 0.0001% by weight or more, more preferably 0.001 to 10% by weight in terms of solid matter. Furthermore, 0.01 to 5% by weight is most preferable. If it is less than 0.0001% by weight, a sufficient effect is hardly expected. If it exceeds 10% by weight, the enhancement of the effect is hardly recognized and it is uneconomical.

  In the case of internal use, the intake varies depending on age, weight, symptoms, therapeutic effect, administration method, treatment time and the like. Usually, the daily intake per adult is preferably 5 mg or more, more preferably 10 mg to 5 g. Furthermore, 20 mg to 2 g is most preferable.

  Next, in order to describe the present invention in detail, examples of production of the extract used in the present invention, experimental examples and formulation examples will be given as examples, but the present invention is not limited thereto. In the production examples, “%” means “% by weight”, and “content” shown in the formulation examples means “parts by weight”.

Production Example of Extracts of Chrome and Honda Walla Extracts of each seaweed of Chrome and Honda Walla were produced as follows. In Production Examples 1 to 8, seaweed whole algae was used as the extraction material.

(Production Example 1) Preparation of hot water extract of chrome 200 ml of water was added to 10 g of a dried product of chrome and extracted at 95-100 ° C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.1 g of a hot water extract of chrome.

(Production Example 2) Preparation of 50% ethanol extract of chrome 10g of dried chrome was immersed in 200mL of 50% ethanol aqueous solution at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 1.1 g of a 50% ethanol extract of chrome.

(Manufacture example 3) Preparation of the ethanol extract of a chrome 10 g of dried chrome was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 0.2 g of an ethanol extract of chrome.

(Production Example 4) Preparation of 1,3-butylene glycol extract of chrome: 10 g of a dried product of chrome was immersed in 200 mL of 1,3-butylene glycol at room temperature for 7 days for extraction. The obtained extract was filtered to obtain 192 g of a chrome 1,3-butylene glycol extract.

(Manufacture example 5) Preparation of hot water extract of Honda Walla 200 ml of water was added to 10 g of dried material of Honda Walla, and extracted at 95-100 ° C for 2 hours. The obtained extract was filtered, and the filtrate was concentrated and freeze-dried to obtain 2.0 g of a hot water extract of Honda walla.

(Production Example 6) Preparation of Honda Walla 50% Ethanol Extract 10 g of dried Honda walla was immersed in 200 mL of a 50% aqueous ethanol solution at room temperature for 7 days for extraction. The obtained extract was filtered and concentrated to dryness with an evaporator to obtain 1.0 g of 50% ethanol extract of Honda Walla.

(Manufacture example 7) Preparation of the ethanol extract of Honda straw 10 g of dried Honda straw was immersed in 200 mL of ethanol at room temperature for 7 days for extraction. The obtained extract was filtered and then concentrated to dryness with an evaporator to obtain 0.2 g of an ethanol extract of Honda straw.

(Production Example 8) Preparation of Honda Walla 1,3-butylene glycol extract 10 g of dried Honda Walla was immersed in 200 mL 1,3-butylene glycol at room temperature for 7 days for extraction. The obtained extract was filtered to obtain 185 g of 1,3-butylene glycol extract of Honda walla.

(Formulation Example 1) Lotion 1
Prescription Content (parts)
1. Chlome hot water extract (Production Example 1) 2.0
2. 1,3-butylene glycol 8.0
3. Glycerin 2.0
4). Xanthan gum 0.02
5. Citric acid 0.01
6). Sodium citrate 0.1
7). Ethanol 5.0
8). Methyl paraoxybenzoate 0.1
9. Polyoxyethylene hydrogenated castor oil (40E.O.) 0.1
10. Perfume proper amount11. [Manufacturing method] Components 1 to 6 and 11 and components 7 to 10 are uniformly dissolved in purified water, and both are mixed and filtered to obtain a product.

(Formulation Example 2) Lotion 2
In Formulation Example 1, Formula 1 was obtained by replacing the hot water extract of chrome with the hot water extract of Honda Walla (Production Example 5).

(Comparative Formulation Example 1) Conventional lotion In Formulation example 1, a hot water extract of chrome was replaced with purified water to obtain a conventional lotion.

(Formulation example 3) Cream 1
Prescription Content (parts)
1. 50% ethanol extract of chrome (Production Example 2) 1.0
2. Squalane 5.5
3. Olive oil 3.0
4). Stearic acid 2.0
5. Beeswax 2.0
6). Octyldodecyl myristate 3.5
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Behenyl alcohol 1.5
9. Glycerol monostearate2.5
10. Fragrance 0.1
11. Methyl paraoxybenzoate 0.2
12.1,3-Butylene glycol 8.5
13. [Manufacturing method] Components 2 to 9 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C to obtain an oil phase. Ingredients 1 and 11 to 13 are heated and dissolved and mixed, and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring.

(Formulation example 4) Cream 2
Formulation Example 4 was obtained by replacing 50% ethanol extract of chrome with 50% ethanol extract of Honda Walla (Production Example 6) in Formulation Example 3.

(Comparative Formulation Example 2) Conventional Cream In Formulation Example 3, a 50% ethanol extract of chrome was replaced with purified water to obtain a conventional cream.

(Formulation Example 5) Latex 1
Prescription Content (parts)
1. Ethanol extract of chrome (Production Example 3) 0.01
2. Squalane 5.0
3. Olive oil 5.0
4). Jojoba oil 5.0
5. Cetanol 1.5
6). Glycerol monostearate 2.0
7). Polyoxyethylene cetyl ether (20E.O.) 3.0
8). Polyoxyethylene sorbitan monooleate (20E.O.) 2.0
9. Fragrance 0.1
10. Propylene glycol 1.0
11. Glycerin 2.0
12 Methyl paraoxybenzoate 0.2
13. [Manufacturing method] Components 1 to 8 are heated and dissolved and mixed with purified water, and the mixture is kept at 70 ° C. to obtain an oil phase. Ingredients 10 to 13 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled while stirring.

(Formulation Example 6) Emulsion 2
Formulation Example 6 was obtained by replacing the ethanol extract of chrome with the ethanol extract of Honda Walla (Production Example 7) in Formulation Example 5.

(Formulation example 7) Gel agent Formulation Content (parts)
1. 1,3-butylene glycol extract of chrome (Production Example 4) 1.0
2. Ethanol 5.0
3. Methyl paraoxybenzoate 0.1
4). Polyoxyethylene hydrogenated castor oil (60 EO) 0.1
5. Perfume appropriate amount 6.1,3-butylene glycol 5.0
7). Glycerin 5.0
8). Xanthan gum 0.1
9. Carboxyvinyl polymer 0.2
10. Potassium hydroxide 0.2
11. [Production method] Ingredients 2 to 5 and ingredients 1 and 6 to 11 are uniformly dissolved in purified water, and the two are mixed to obtain a product.

Formulation Example 8 Gel Agent Formulation Example 8 was obtained by replacing Chlome's 1,3-butylene glycol extract with Honda Walla 1,3-butylene glycol extract (Production Example 8) in Formulation Example 7.

(Formulation example 9) Pack prescription Content (parts)
1. Chlome hot water extract (Production Example 1) 1.0
2. 1,3-butylene glycol extract of Honda Walla (Production Example 8) 5.0
3. Polyvinyl alcohol 12.0
4). Ethanol 5.0
5.1,3-Butylene glycol 8.0
6). Methyl paraoxybenzoate 0.2
7). Polyoxyethylene hydrogenated castor oil (20 EO) 0.5
8). Citric acid 0.1
9. Sodium citrate 0.3
10. Perfume proper amount11. [Production Method] Components 1 to 11 are uniformly dissolved in purified water to make a total amount of 100 to obtain a product.

(Formulation Example 10) Foundation Formulation Content (parts)
1. Honda Walla 50% ethanol extract (Production Example 6) 1.0
2. Stearic acid 2.4
3. Polyoxyethylene sorbitan monostearate (20E.O.) 1.0
4). Polyoxyethylene cetyl ether (20E.O.) 2.0
5. Cetanol 1.0
6). Liquid lanolin 2.0
7). Liquid paraffin 3.0
8). Isopropyl myristate 6.5
9. Sodium carboxymethylcellulose 0.1
10. Bentonite 0.5
11. Propylene glycol 4.0
12 Triethanolamine 1.1
13. Methyl paraoxybenzoate 0.2
14 Titanium dioxide 8.0
15. Talc 4.0
16. Bengala 1.0
17. Yellow iron oxide 2.0
18. Perfume proper amount19. [Manufacturing method] Components 2 to 8 are heated and dissolved in purified water to a total amount of 100, and kept at 80 ° C to obtain an oil phase. Swell component 9 well in component 19, then add components 1 and 10-13 and mix uniformly. To this, components 14 to 17 pulverized and mixed with a pulverizer are added, and the mixture is stirred with a homomixer and kept at 75 ° C. to obtain an aqueous phase. The aqueous phase is added to this oil phase while stirring and emulsified. Then, it cools, the component 18 is added at 45 degreeC, and it cools to 30 degreeC, stirring, and makes it a product.

(Formulation example 11) Bath agent Formulation Content (parts)
1. Chromome ethanol extract (Production Example 3) 1.0
2. Sodium bicarbonate 50.0
3. Yellow No. 202 (1) Appropriate amount 4. Perfume appropriate amount 5. [Manufacturing method] Ingredients 1 to 5 are mixed uniformly with sodium sulfate to make a product.

(Formulation Example 12) Ointment Formulation Content (parts)
1. 1,3-butylene glycol extract of chrome (Production Example 4) 1.0
2. Honda Walla Hot Water Extract (Production Example 5) 5.0
3. Polyoxyethylene cetyl ether (30E.O.) 2.0
4). Glycerol monostearate 10.0
5. Liquid paraffin 5.0
6). Cetanol 6.0
7). Methyl paraoxybenzoate 0.1
8). Propylene glycol 10.0
9. [Production Method] Components 3 to 6 are heated and dissolved and mixed with purified water to a total amount of 100, and kept at 70 ° C. to obtain an oil phase. Ingredients 1, 2, and 7-9 are dissolved by heating and mixed, and kept at 75 ° C. to form an aqueous phase. The aqueous phase is added to the oil phase to emulsify, and the mixture is cooled to 30 ° C. with stirring to obtain a product.

(Prescription Example 13) Powder 1
Prescription Content (parts)
1. Chlome hot water extract (Production Example 1) 1.0
2. Dried corn starch 39.0
3. Microcrystalline cellulose 60.0
[Production method] Components 1 to 3 are mixed to obtain a powder.

(Prescription Example 14) Powder 2
In Formulation Example 13, Powder 2 was obtained by replacing the hot water extract of chrome with the hot water extract of Honda Walla (Production Example 5).

(Prescription Example 15) Tablet Formulation Content (parts)
1. Ethanol extract of Honda Walla (Production Example 7) 5.0
2. Dried corn starch 25.0
3. Carboxymethylcellulose calcium 20.0
4). Microcrystalline cellulose 40.0
5. Polyvinylpyrrolidone 7.0
6). Talc 3.0
[Production method] Components 1 to 4 are mixed, and then an aqueous solution of component 5 is added as a binder to form granules. Ingredient 6 is added to the molded granules and compressed. One tablet is 0.52 g.

(Prescription Example 16) Tablet Confection Prescription Content (parts)
1. Chromome ethanol extract (Production Example 3) 2.0
2. Dried corn starch 49.8
3. Erythritol 40.0
4). Citric acid 5.0
5. Sucrose fatty acid ester 3.0
6). Fragrance 0.1
7). Purified water 0.1
[Production method] Components 1 to 4 and 7 are mixed and granulated. Ingredients 5 and 6 are added to the molded granules and tableted. One tablet is 1.0 g.

(Prescription Example 17) Beverage Formulation Content (parts)
1. Hot water extract of Honda Walla (Production Example 5) 0.05
2. Stevia 0.05
3. Malic acid 5.0
4). Fragrance 0.1
5. Purified water 94.8
[Production Method] Components 2 and 3 are dissolved in a small amount of water. Components 1, 4 and 5 are then added and mixed.

  Next, experimental examples will be given to explain the effects of the present invention in detail.

Experimental Example 1 Measurement of Hyaluronic Acid Synthase 2 (HAS2), Type I Collagen (COL1A), MMP-1 and MMP-2 mRNA Expression Levels 1 × 10 ⁵ human fibroblasts NB1RGB were seeded in a 60 mm dish and confluent. At that point, the sample was added to a final concentration of 10 μg / mL. As a control, a solvent in which the sample was diluted was added. After culturing for 24 hours, total RNA was extracted. Extraction of total RNA from the cells was performed using TRIZOL Reagent (Invitrogen), and the total RNA amount was determined by absorbance at 260 nm using a spectrophotometer (NanoDrop). Measurement of mRNA expression level was performed by real-time RT-PCR method based on total RNA extracted from cells. For the real-time RT-PCR method, SuperScriptIII Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen) was used. Specifically, 500 ng of total RNA was subjected to a reverse transcription reaction, followed by a PCR reaction (95 ° C .: 15 seconds, 60 ° C .: 30 seconds, 40 cycles). For other operations, the expression levels of HAS2, COL1A, MMP-1 and MMP-2 mRNA were determined as a ratio with respect to the expression level of β-actin mRNA as an internal standard in accordance with a predetermined method. The HAS2 expression rate was calculated as a ratio of the HAS2 mRNA expression level in the sample addition group to the control HAS2 mRNA expression level. The COL1A expression rate and the MMP expression rate were similarly calculated. The primers used for measuring the expression level of each gene are as follows.

Primer set TGGATGACCCTACGAAGCGATTA for HAS2 (SEQ ID NO: 1)
GCTGGATTACTGTGGCAATGAG (SEQ ID NO: 2)
Primer set AGGACAAGAGGGCATGTCTGGTT for COL1A (SEQ ID NO: 3)
TTGCAGTGGGTAGGTGATGTCTG (SEQ ID NO: 4)
Primer set GGGAGATCATCGGGGACAACTC for MMP-1 (SEQ ID NO: 5)
TGAGCATCCCCCCTCAATAC (SEQ ID NO: 6)
Primer set CCGTCGCCCATCATCAA for MMP-2 (SEQ ID NO: 7)
CTTCTGCCATCTTCTTTAGTGGTCCTT (SEQ ID NO: 8)
Primer set CACTCTCCCAGCCCTTCCTTC for β-actin (SEQ ID NO: 9)
GTGTTGCGCGTACAGGTCTTTG (SEQ ID NO: 10)

  The results of these experiments are shown in Tables 1-4. As a result, the chrome extract and Honda walla extract of the present invention have excellent HAS2 expression promoting effect (hyaluronic acid production promoting effect), COL1A expression promoting effect (collagen production promoting effect) and MMP expression suppressing effect (MMP inhibition). Effect). In particular, HAS2 expression promotion and MMP-2 expression suppression of the hot water extract of Chromome (Production Example 1) and MMP-1 expression suppression of the 50% ethanol extract of Honda Walla (Production Example 6) were remarkably high.

Experimental Example 2 Use Test Using the lotion of Formulation Example 1 and the conventional lotion of Comparative Formulation Example 1, a 1 month use test was conducted on five people (26 to 64 years old) with wrinkles and sagging. . After use, the degree of wrinkles and sagging was determined by a questionnaire.

  As a result, wrinkles and sagging were reduced by the external preparation for skin containing the extract of the present invention. During the test period, there was no skin problem and there was no problem with safety. There was also no problem with the deterioration of the prescription ingredients.

  From the above, the chrome and honda wall extract of the present invention had excellent hyaluronic acid production promoting action, collagen production promoting action and MMP inhibitory effect, and was also excellent in stability. Therefore, the extract of chrome and hondawala according to the present invention can be used not only in the beauty field such as skin aging, but also in the medical field such as suppression of functional deterioration due to aging, prevention of cancer, treatment, etc. Expected to be applied to foreign products and pharmaceuticals.

Claims (6)

An MMP inhibitor comprising an extract of chrome and / or Honda walla. A collagen production promoter characterized by containing an extract of chrome and / or Honda walla. A hyaluronic acid production promoter characterized by containing an extract of chrome and / or Honda walla. A wrinkle improving agent characterized by containing an extract of chrome and / or honda. A pharmaceutical comprising an extract of chrome and / or Honda walla. A food composition for preventing and improving various diseases, joint diseases and physiological fly mosquito disease caused by increased MMP, comprising an extract of chrome and / or Honda walla.