JP2013006815A - Matrix metalloprotease inhibitor, elastase inhibitor, hyaluronidase inhibitor, and skincare preparation for external use for preventing wrinkle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a matrix metalloprotease inhibitor, elastase inhibitor, hyaluronidase inhibitor, and skincare preparation for external use for preventing wrinkles, each characterized in containing extract of Ardisia crenata Sims of the Myrsinaceae family.SOLUTION: There are provided the matrix metalloprotease inhibitor, elastase inhibitor, hyaluronidase inhibitor, and skincare preparation for external use, each characterized in containing extract of Ardisia crenata Sims of the Myrsinaceae family. The extract has excellent matrix metalloprotease inhibitory, elastase inhibitory and hyaluronidase inhibitory actions, and the skincare preparation for external use applied thereof is highly safe, and has excellent wrinkles-preventing effects.

Description

  The present invention relates to a matrix metalloprotease inhibitor, an elastase inhibitor, a hyaluronidase inhibitor, and a skin external preparation for preventing wrinkles containing an extract of a specific plant. More specifically, matrix metalloproteases (MMPs) inhibition containing an extract obtained by extracting Ardisia creena Sims from the family Myrsinaceae with water, an organic solvent, or a mixed solvent thereof. The present invention relates to an agent, an elastase inhibitor, a hyaluronidase inhibitor, and a skin external preparation for preventing wrinkles.

  Skin aging manifests itself in various forms, including increased wrinkles, decreased elasticity, and pigmentation. The most typical aging phenomenon is wrinkles. The causes of wrinkling skin aging can be roughly divided into two. Aging due to internal and external factors of natural aging due to aging. External factors include ultraviolet radiation, pollution, illness, drinking, smoking, etc. Internal factors include free radicals and inflammation, stress, decreased cellular activity and mutation, and cellular metabolic imbalance (See Non-Patent Document 1). The skin can be roughly divided into epidermis and dermis. The cause of the occurrence of wrinkles in the epidermis is that the stratum corneum layer becomes thick due to a decrease in the turnover of the epidermis cells, and the moisture content of the stratum corneum decreases, so that the entire stratum corneum is cured and wrinkles are generated. The boundary between the epidermis and the dermis is involved in epidermal cell support, adhesion, nutrient transport, and regulation of epidermal differentiation. Due to various aging factors, wrinkles are generated because the dermis support function is reduced, the selective permeation function is weakened, and harmful components easily affect the skin. (See Non-Patent Document 2) As a factor for inducing these changes, reduction or denaturation of an extracellular matrix (ECM) component composed of a fibrous protein such as collagen and elastin and a polysaccharide such as hyaluronic acid. Is known to be involved.

  In recent years, research has progressed and the involvement of MMPs has been pointed out as a factor inducing these changes. MMPs is a generic name for a group of metalloproteases whose main substrate is ECM. It is currently known that there are 20 types of MMPs, and their structures and actions are various. For example, collagenase, that is, MMP-1, is known as an enzyme that degrades collagen type I and type III, which are the main components of the dermal matrix of the skin. MMP-2 and MMP-9 are known as enzymes that degrade base IV membrane components such as type IV collagen and laminin, and dermal matrix component elastin.

  Furthermore, the expression of each of these enzymes is promoted by ultraviolet irradiation, and may be a major factor such as the formation of wrinkles on the skin. Therefore, MMPs inhibition, elastase inhibition and hyaluronidase inhibition are important in preventing skin aging.

  In addition, as a conventional technique of a Myraceaceae plant according to the present invention, cosmetics characterized by containing a bean root having both a highly stable whitening action and an anti-inflammatory action (see Patent Document 1) ), Anti-aging effect by eliminating active oxygen, anti-inflammatory effect and whitening effect, which is an extract of Ardilia japonica Blue, A. pusilla Blume, and Ardisia Sieboldi Miq Skin external preparation characterized by containing 1 type or 2 types or more selected from (refer patent document 2), 1 type or 2 types or more selected from the extract of 3 types of the above-mentioned Azalea family plant Skin external preparation that has both cell activation and antioxidant effects Patent Document 3), β-1,3-glucan sulfated at one or two or more species selected from the above-mentioned extracts of three species of Asteraceae, and glucose 2 or 6 as a constituent sugar A skin external preparation (see Patent Document 4) characterized by having an anti-inflammatory action, a wound healing promoting action and an anti-allergic action by containing one or more selected from the above is disclosed. However, these literature-known inventions do not describe and are not known about the MMPs inhibitory action, elastase inhibitory action, and hyaluronidase action of the plant extract of the genus Myrsinaceae (Ardisia credata Sims). Moreover, the utilization to the skin external preparation for wrinkle prevention using these effect | actions was not assumed.

  Physiology Of The Skin, Allured Public Corporation, Peter T .; PG MD, p61-72   Keene, J .; Cell Biol, 104, p611-612, 1987   JP-A-4-34912   Japanese Patent Laid-Open No. 10-1438   Japanese Patent Publication No. 3734222   Japanese Patent Laid-Open No. 11-171784

Problems to be solved by the invention

  Naturally derived components are known to have various pharmacological and cosmetic effects, and so far many plants have been widely applied to skin external preparations. However, there are many naturally-derived components whose effects are not yet known, and the development of naturally-derived components having excellent ECM protection action has been expected. However, there are a limited number of conventional anti-wrinkle skin external preparations that focus on the activity inhibitors of MMPs against ECM, and those that are not yet fully satisfactory are not provided. In addition, although it is strongly desired to provide naturally-derived components using these actions including an elastase inhibitor and a hyaluronidase inhibitor, there is not yet a satisfactory one yet.

Problems to be solved by the invention

  Therefore, the present invention has an action to surely inhibit the activities of MMPs, elastase and hyaluronidase, which are closely related to wrinkles due to skin aging, and through these actions, the decrease and degeneration of ECM components are suppressed. An object of the present invention is to provide a skin external preparation for preventing wrinkles useful for prevention.

Means for solving the problem

  In order to solve the above-mentioned problems, the present inventors have studied the ECM degradation inhibitory action for various substances widely, and as a result, surprisingly, the plant of the Myrrhinaceae family Ardilia credata Sims, Alternatively, by containing an extract obtained from a hydrophilic organic solvent or a mixed solvent thereof, the activities of MMPs, elastase and hyaluronidase can be reliably inhibited, and through MMPs inhibitory action, elastase inhibitory action and hyaluronidase inhibitory action, The inventors have found that an external preparation for skin having a wrinkle-preventing action can be obtained, and have completed the present invention.

  Hereinafter, embodiments of the present invention will be described in detail. Mandyo (Ardisia creena Sims) used in the present invention is a plant species belonging to the genus Myrsinaceae of the Myrsinaceae family. It is an evergreen small shrub with a height of 50-60 cm inhabiting in warm mountain forests such as Japan, the Korean Peninsula, South Korea, Taiwan, and China. These extracts can use any part of leaves, bark, flowers, fruits, and roots of these plants, but it is preferable to use leaves.

  As the extraction solvent, it is preferable to use water, an organic solvent, or a mixed solvent thereof. Specific examples of suitable extraction solvents include water, alcohols such as ethanol, methanol, propanol, butanol, propylene glycol, and glycerin, hydrous alcohols, and organic solvents such as acetone and ethyl acetate. Preferably, ethanol is good. These solvents may be used alone or in combination of two or more.

  The manly extract can be obtained by a conventional method. For example, it can be obtained by immersing or heating under reflux with a manly extract, followed by filtration and concentration.

  For example, the whole plant of ginseng is dried to 20 to 200 ° C., preferably 50 to 50 times by weight of water, an organic solvent or a mixed solvent thereof in a weight ratio of 5 to 20 times, preferably 8 to 10 times the weight (KG). It is obtained by extraction at 100 ° C. for 30 minutes to 20 hours, preferably 2 to 5 hours, or at 5 to 37 ° C., preferably 2 hours to 15 days at room temperature, preferably 1 to 3 days to obtain. be able to.

  The extract obtained as described above has an MMPs inhibitory action, an elastase inhibitory action, and a hyaluronidase inhibitory action. Moreover, when applied to the skin, it is suitable for blending into an external preparation for preventing wrinkles due to the above-mentioned action.

  The ginseng extract of the present invention can be administered in various dosage forms. The amount of the active ingredient in the preparation is not particularly limited, but is 0.0001 to 20% by weight, preferably 0.0001 to 1% by weight. If the amount is less than 0.0001% by weight, the effect of the present invention is not sufficiently exhibited. On the other hand, if the amount exceeds 20% by weight, no significant improvement in the effect is observed, and preparation becomes difficult, which is not preferable.

When formulating, components that can usually be incorporated into cosmetics, quasi-drugs and pharmaceuticals, such as whitening agents, moisturizers, antioxidants, oily ingredients, UV absorbers, interfaces, within the range that does not impair the effect. Activators, detergents, thickeners, drugs, alcohols, fragrances, resins, alcohols, powder components, coloring materials, pigments, aqueous components, water, various skin nutrients, etc. may be appropriately blended as necessary. it can. In addition, other MMPs inhibitors, elastase inhibitors, and hyaluronidase inhibitors can be used in combination as long as the effects of the present invention are not impaired.
The blending component that may be added is not limited to this, and any of the above components can be blended within a range that does not impair the object and effect of the present invention.

  The topical skin preparation of the present invention means a composition applied to the skin in the fields of pharmaceuticals, quasi drugs, cosmetics and the like. The dosage form is not limited as long as the effect of the present invention is exhibited. Arbitrary dosage forms such as solution, solubilization system, emulsification system, powder dispersion system, water-oil two-phase system, water-oil-powder three-phase system, solid, ointment, gel, aerosol, mousse and the like are applied. The use form is also arbitrary, for example, basic cosmetics such as lotion, milky lotion, pack, cosmetic liquid, gel, cream, makeup cosmetics such as foundation, cosmetics for hair, aromatic cosmetics, bath preparations, etc. However, it is not limited to these.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the technical scope of this invention is not limited at all by these Examples.

  Prior to the examples, the method for producing the ginseng extract used in the present invention will be described.

(Adjustment Example 1) Manryo Leaf Ethyl Acetate Extract The dried mantis (Ardisia credata Sims) leaves were extracted twice with a 95% (v / v) aqueous ethanol solution at room temperature for 24 hours. The obtained ginseng extract was filtered, the solvent was distilled off under vacuum at 60 ° C., and then redissolved in water to obtain an ethyl acetate extract. This was designated as Adjustment Example 1.

(Adjustment Example 2) Manryo Leaf Hexane Extract A dried mantis (Ardisia crena Sims) leaf was extracted twice with a 95% (v / v) aqueous ethanol solution at room temperature for 24 hours. The obtained ginseng extract was filtered, the solvent was distilled off under vacuum at 60 ° C., and then redissolved in water to obtain a hexane extract. This was designated as Adjustment Example 2.

About the extract obtained in MMP-1 and MMP-3 inhibition test adjustment example 1, the MMP-1 and MMP-3 inhibition test was done. Specifically, the test was conducted as follows.

  In order to measure the expression level of the MMP-1 and 3 genes, the above-mentioned Manryo extract was subjected to RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and MMP-1 and MMP-3 mRNA as described below. The expression level was measured.

For the manly extract obtained in the above example, normal human fibroblast cell line (ATCC, CRL-2076) was added to Dulbecco containing 1 × 10 ⁶ 10% fetal calf serum in a 60 mm culture dish. The cells were cultured in a modified Eagle medium (Gibco / BRL, Invitrogen) at 37 ° C. and 5% CO ₂ . The next day, after removing the medium, the cells were washed with HBSS and replaced with physiological saline. After irradiation with 6 J / cm ² of UVA, the cells were cultured in a serum-free medium for 24 hours. Thereafter, total RNA was extracted from the cells by the guanidinium thiocyanate-phenol-chloroform extraction method. The RNA obtained by the above method was treated with a DNA degrading enzyme (RNase-free DNase, Promega) at 37 ° C. for 30 minutes, and pure RNA was obtained by phenol / chloroform extraction method and ethanol precipitation method. PCR was performed. The reaction conditions for MMP-1 were 95 cycles of 95 ° C. for 1 minute, 48 ° C. for 1 minute, and 72 ° C. for 1 minute for 29 cycles. The reaction conditions for MMP-3 were 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 30 cycles of 45 ° C. as one set were performed, and the reaction conditions of β-actin were 22 cycles with 95 ° C. for 1 minute, 62 ° C. for 1 minute and 72 ° C. for 1 minute as one set. The PCR product was electrophoresed on a 1.5% (w / v) agarose (Sigma) gel and quantified with a gel image analyzer (BIO-RAD). Β-actin was used for the control group, and the primers used for RT-PCR were those having the nucleotide sequences shown in the sequence numbers in Table 1 below.

  Expression rate of MMP-1 and MMP-3 in a reaction system containing no manly extract (control UVA irradiation and MMP expression), and a reaction system containing manli extract (UVA irradiation and MMP expression added to a sample) (%) Was calculated. The results are shown in Tables 2 and 3.

  As a reference example, epigallocatechin gallate (EGCG), which is a component well known to inhibit MMPS expression, was tested in the same manner as described above. The results are also shown in Tables 2 and 3.

  From the results of Tables 2 and 3, it was confirmed that the manly extract has an excellent MMP-1 and MMP-3 inhibitory action. Moreover, when the concentration of the ginseng extract was 50 μg / ml, it was almost the same as the positive control EGCG 2.29 μg / ml, and the effect was extremely high.

MMP-2, MMP-9 Inhibition Test The extract obtained in Preparation Example 1 was subjected to an MMP-2, MMP-9 inhibition test. Specifically, the test was conducted as follows.

Normal human fibroblasts (human fibroblast cell line: ATCC, CRL-2076) were seeded at 1 × 10 ⁶ cells in a 60 mm culture dish and cultured at 37 ° C. under 5% CO ₂ conditions. The next day, after removing the medium, the cells were washed twice with HBSS, replaced with serum-free medium, added with 10 ng / ml TNF-α and a sample and exposed for 24 hours. The medium was collected and the amount of protein was determined by the BCA method. The sample was applied to a polyacrylamide gel containing 10% gelatin under non-reducing conditions and subjected to SDS-PAGE. The gel after electrophoresis was washed with 2.5% Triton-X 100 (pH 7.5), and further at 37 ° C. for 16 hours, regeneration buffer (50 mM Tris-HCl 200 mM, NaCl 5 mM, CaCl ₂ 0.0006%, Reaction with Triton-X 100), staining with 0.1% Coomassie brilliant blue, decolorization with 10% acetic acid / 50% methanol, and quantification of the intensity of the band appearing after decolorization with an image analyzer (BIO-RAD) did.

  Expression rate of MMP-2 and MMP-9 in a reaction system containing no manly extract (added with only control TNF-α 10 μg / ml) and a reaction system containing manly extract (adding a sample with TNF-α 10 μg / ml) (%) Was calculated. The results are shown in Tables 4 and 5.

  As a reference example, EGCG, which is a well-known component for inhibiting MMPs activity, was tested in the same manner as described above. The results are also shown in Tables 4 and 5.

From the results of Tables 4 and 5, it was confirmed that the manly extract has an excellent MMP-2 and MMP-9 activity inhibitory action.
The strength of the action is about the same as that of EGCG 2.29 μg / ml of the positive control when the concentration of Munryo extract is 50 μg / ml in MMP-2, and the concentration of Munryo extract is 50 μg / ml in MMP-9. In the case of ml, it was the same as the positive control EGCG 2.29 μg / ml, and the effect was extremely high.

Test for Elastase Inhibitory Action The elastase inhibitory action was tested on the extracts obtained in Preparation Examples 1-2. Specifically, the test was conducted as follows.

The substrate (N-SUCCINYL-ALA-ALA-ALA-p-NITROANILIDE, Sigma) was prepared with 0.1232M Tris-HCl buffer so that the concentration was 1.015 mM, and 1300 μl of this solution and final concentrations of 100, 50, 10 μg The sample prepared to be / ml was mixed.
They were preincubated with vortexing at 25 ° C. for 10 minutes. 5 μl of porcine pancreatic elastase (0.025 units / ml Sigma) was added to 100 μl of this solution. After vortexing, the sample was allowed to stand in a 25 ° C. water bath for 10 minutes, and the absorbance at 410 nm was measured.

  The inhibition rate was measured by reducing the sample concentration to 300, 150, and 50 μg / ml, and the concentration of the sample solution that inhibits the activity of elastase by 50% was determined by interpolation. The results are shown in Table 6.

  From the results in Table 6, it was confirmed that the manly extract has an elastase inhibitory action.

Hyaluronidase inhibition test The extracts obtained in Preparation Examples 1 and 2 were tested for hyaluronidase inhibitory action. Specifically, the test was conducted as follows.

Hyaluronidase activity was determined by measuring the amount of N-acetylglucosamine, which is the main component of sodium hyaluronate, with a spectrophotometer. 50 ml (7900 units / ml) of bovine-derived hyaluronidase dissolved in 0.1 M acetate buffer (pH 3.5) was mixed with 100 ml of a sample having a predetermined concentration and incubated in a 37 ° C. water bath for 20 minutes. A control treated with 100 ml of 40% BG instead of the sample was set as a control. 100 ml of 12.5 mM CaCl ₂ aqueous solution was added to the reaction solution and incubated in a 37 ° C. hot water bath for 20 minutes. 250 ml of sodium hyaluronate aqueous solution (1.2 mg / ml) was added and incubated in a 37 ° C. water bath for 40 minutes. Further, 100 ml of 0.4N NaOH and 0.4N potassium borate were further added, and incubated for 3 minutes in hot water. After cooling to room temperature, 3 ml DMAB solution was added and incubated in a 37 ° C. water bath for 20 minutes. Absorbance at 585 nm was measured with a spectrophotometer to determine the concentration of the reaction mixture.

The formula for calculating the inhibition rate is
Inhibition rate (%) = [(ODc−ODs) / ODc × 100]
Here, ODc is the OD at 585 nm of the control, and ODs is the OD at 585 nm of the sample.

  From the results of Table 7, it was confirmed that the manly extract has an excellent hyaluronidase inhibitory action.

  From the above results, the MMP-1, MMP-2, MMP-3, MMP-9 activity inhibitory effect, elastase and hyaluronidase activity effect of the ginseng extract used in the present invention were extremely excellent. Therefore, wrinkles can be effectively prevented using the ginseng extract.

Then, the formulation example of this invention is given as Examples 5-9.

Gel for skin (1) 1,3-butylene glycol 1.0 (% by weight)
(2) Cyclohexane-1,4-dicarboxylic acid bisethoxydiglycol 3.8
(3) Carboxyvinyl polymer (2% by weight aqueous solution 28.0
(4) Potassium hydroxide (10% amount aqueous solution) 3.0
(5) Methylparaben 0.1
(6) Purified water 63.9
(7) Dipropylene glycol 0.2
(8) Manryo leaf ethyl acetate extract (conditioning agent 1) 0.2
Production method: After uniformly mixing the mixture from (1) to (6), add (8) well dissolved in (7) to obtain a product.

Cream for skin (1) Liquid paraffin 6.0 (wt%)
(2) Isopropyl myristate 3.0
(3) Cyclohexane 2.5
(4) Dimethicone 2.0
(5) Behenyl alcohol 3.4
(6) Vaseline 2.0
(7) Sorbitan stearate 1.5
(8) Seths-20 3.4
(9) Tocopherol 0.02
(10) Citric acid 0.05
(11) 1,3-butylene glycol 1.0
(12) Methylparaben 0.15
(13) Propylparaben 0.07
(14) Purified water 72.91
(15) Manryo leaf ethyl acetate extract (Preparation Example 1) 0.1
(16) Dipropylene glycol 1.9
Production method: The oil phase components (1) to (9) are heated and dissolved at 80 ° C. On the other hand, the aqueous phase components (10) to (16) are dissolved by heating at 80 ° C. This is added to the oil phase component with stirring and uniformly emulsified with a homogenizer. After the emulsification, cooling is started, and a solution obtained by adding (15) to (16) is added and mixed.

Cream for skin (1) Cetyl dimethicone polyol 3.0 (% by weight)
(2) Lauryl PEG-9 polydimethylsiloxyethyl dimethicone 1.0
(3) Liquid paraffin 7.44
(4) Hydrogenated castor oil 1.0
(5) Microclostein wax 0.56
(6) Diethylhexanoic acid neopentylene glycol 11.8
(7) Manryo leaf ethyl acetate extract (Preparation Example 1) 0.1
(8) Dipropylene glycol 0.1
(9) Sodium chloride 1.0
(10) Purified water 68.9
(11) 1,3-butylene glycol 5.0
(12) Methylparaben 0.1
Production: The oil phase components (1) to (6) are mixed and dissolved by heating at 80 ° C. On the other hand, the aqueous phase components (7) to (12) are mixed and dissolved by heating at 80 ° C. The aqueous phase component is added to the oil phase component with stirring, and the mixture is uniformly emulsified with a homogenizer. Cool after the emulsification.

Lotion (1) Manryo leaf ethyl acetate extract (Preparation Example 1) 0.1 (% by weight)
(2) Dipropylene glycol 0.1
(3) 1,3-butylene glycol 1.0
(4) Methylparaben 0.2
(5) Xanthan gum 0.2
(6) Purified water 98.4
Production: (1) is dissolved in (2). After dissolution, (3) to (6) are sequentially added, and then sufficiently stirred and mixed uniformly.

A use test was conducted on Example 6 to evaluate the effect of preventing wrinkles. At that time, in Example 6, the blended ginseng leaf ethyl acetate extract was replaced with purified water, and a use test was simultaneously conducted as Comparative Example 1.
Specifically, the test was conducted as follows.

  In order to evaluate the preventive effect of wrinkles, it was conducted in the winter with low water content, which would worsen if nothing was done. In Example 6, the moisturizing agent was hardly added so that the effects of the components other than the ginseng leaf ethyl acetate extract were not obtained. Eight male and female panelists in their 20s and 50s are grouped together, and each group uses Example 2 and Comparative Example 1 in blinds twice a day for 12 weeks, 4 weeks, 8 weeks, and 12 weeks later. The condition of wrinkles was observed and compared with the value before use. Wrinkle symptoms were scored according to the criteria shown in Table 8. When it did not meet the standard of each grade, it was set as the score of the intermediate value.

  Table 9 summarizes the average values of the degree of change in wrinkle scores at 4 weeks, 8 weeks and 12 weeks. In addition, the wrinkle improvement degree of 8 people in 12 weeks is summarized in FIG. As for the degree of change of the wrinkle score, “−” means that wrinkle formation is recognized and “+” means that wrinkle improvement is recognized.

  A replica of the corner of the eye was collected with a silicone replica (Silflo, FLEXICO DE VELOPMENTS), and the volume of wrinkles and the number of wrinkles were measured. The results are shown in FIG.

  From the results shown in Table 9 and FIGS. 1 and 2, it was confirmed that the skin cosmetic containing the salmon extract has a very remarkable effect on the wrinkle-preventing action. Moreover, there was no skin trouble and it was safe.

  The ginseng extract according to the present invention has excellent MMPs inhibitory action, elastase inhibitory action, hyaluronidase inhibitory action, and the anti-wrinkle skin external preparation of the present invention to which these are applied has a very remarkable effect in preventing wrinkles. .

It is a figure which shows the wrinkle score change degree by the manly extract extract cream by this invention. It is a figure which shows the change of the wrinkle area rate and the number of wrinkles by the gypsum extract cream by this invention.

Claims (4)

Matrix metalloproteases (MMPs) characterized by containing extracts obtained by extracting Ardisia creena Sims of the family Myrsinaceae with water or a hydrophilic organic solvent or a mixed solvent thereof. Inhibitor. An elastase inhibitor characterized by containing an extract obtained by extracting Ardisia creena Sims of Myrsinaceae with water, an organic solvent or a mixed solvent thereof. A hyaluronidase inhibitor characterized by containing an extract obtained by extracting Ardisia creena Sims of Myrsinaceae with water, an organic solvent or a mixed solvent thereof. A skin external preparation for preventing wrinkles, characterized by containing an extract obtained by extracting Ardisia creneta Sims of the family Myrsinaceae with water, an organic solvent or a mixed solvent thereof.

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