TW201701895A - Matrix metalloproteinase production inhibitor - Google Patents

Abstract

It is an object of the present invention to provide an inhibitor of MMP-1 production. The solution of the present invention is an MMP-1 production inhibitor containing, as an active ingredient, one or more plant extracts selected from the group consisting of Ashwagandha, Passiflora, and Robinia.

Description

Matrix metalloproteinase production inhibitor

The present invention relates to novel matrix metalloproteinase production inhibitors and skin external preparations comprising the same.

The skin is composed of the stratum corneum, epidermis, basement membrane, and dermis from the outside. The dermis is filled with cellular components and the giant molecular mesh structure called extracellular matrix produced by these cells. As a cellular component, the presence of cells composed of fibroblasts, macrophages, obese cells, blood vessels, or nerves is known. As an extracellular matrix, fibrous proteins such as collagen or elastin, and hyaluronic acid or proteoglycan are known. The presence of sugars, etc. Collagen fibers, which are extracellular matrices, are said to account for about 70% of the dry weight of the dermis and play an important role in maintaining firmness or elasticity of the skin. There are 20 subtypes in the collagen molecule, and most of the collagen fibers present in the dermis are type I collagen.

Matrix metalloproteinase (hereinafter referred to as "MMP") is a proteolytic enzyme involved in catabolism of extracellular matrices. MMPs are classified into several subtypes from the viewpoints of molecular structure or matrix specificity and the like. For example, MMP-1 decomposes type I collagen eggs. The white 3-helix structure, MMP-2 or MMP-9 is decomposed to form type IV collagen or gelatin of the basement membrane. MMP not only regulates the metabolism of dermal extracellular matrix, but also involves the activation of physiologically active molecules such as cell wetting or cytokines. It has been reported to be related to various diseases. In particular, in the skin, it has been known that MMP is excessively produced by ultraviolet light exposure or various types of inflammation, and the MMP decomposes/denatures collagen or elastin, which causes wrinkles or sagging of the skin.

In this context, various reviews have been conducted from the viewpoint of controlling MMP, and many reports have been made to indicate plant extracts that inhibit the activity of MMP (Patent Documents 1 and 2). In addition, it has been reported that MMP-1 production inhibitors which are intended to inhibit the production of the enzyme itself and which substantially inhibit the action of MMP, which have a yeast culture solution as an active ingredient (Patent Document 3), have a compound having a mercapto group, and have two A compound of a sulfur bond is used as an MMP-1 production inhibitor of an active ingredient (Patent Document 4). On the other hand, in recent years, Peroxisome Proliferator-activated receptor (Peroxisome Proliferator-activated receptor), which is a nuclear receptor responsible for the expression of a gene group responsible for maintaining lipid or sugar metabolism, has been suggested. An agonist of PPAR δ of one subtype of "PPAR" inhibits the production of MMP-1 by UVB exposure in fibroblasts (Non-Patent Document 1).

[Previous Technical Literature] [Patent Literature]

[Patent Document 1] Japanese Patent Laid-Open No. Hei 7-196526

[Patent Document 2] Japanese Patent Laid-Open No. Hei 7-291873

[Patent Document 3] Japanese Patent Laid-Open Publication No. 2009-24638

[Patent Document 4] Japanese Patent Laid-Open Publication No. 2002-47178

[Non-patent literature]

[Non-Patent Document 1] SA Ham et al: Journal of Investigative Dermatology 133, 2593-2600 (2013)

However, as described in the article, "Since the short-wavelength ultraviolet rays up to the UVB hardly penetrate into the dermis, the ultraviolet rays that affect the dermis are mainly UVA" (Imakawa Takataro et al., "The effect of light on the skin", Japan Ray In the general environment, even if the skin is exposed to ultraviolet light, ultraviolet rays other than UVA can hardly reach the dermis. . Thus, it is generally believed that the production of MMP-1 by dermal fibroblasts in a normal environment is due to the direct action of UVA on dermal fibroblasts or the inflammatory cytokines produced by skin cells exposed to UVB. Or reactive oxygen species (ROS). Non-Patent Document 1 directly irradiates UVB to fibroblasts derived from dermis, and the skin system differs from the environment exposed to UVB. That is, it is unknown that the ligand for PPAR δ inhibits the production of MMP-1 which may occur under normal circumstances. It is an object of the present invention to provide an inhibitor of MMP-1 production.

Under the above circumstances, the inventors of the present case and others proceeded to explore various materials and found out the activity of inhibiting the production of MMP-1. As a result, it was found that the plant extract prepared by the specific plant effectively inhibited the production of MMP-1. Based on this kind of insight, the present invention has been completed.

The present invention provides the following.

(1) A MMP-1 production inhibitor comprising, as an active ingredient, one or more plant extracts selected from the group consisting of Ashwagandha, Passiflora, and Robinia pseudoacacia.

(2) The MMP-1 production inhibitor according to (1), wherein the plant extract is a plant extract obtained by solvent extraction.

(3) The MMP-1 production inhibitor according to (2), wherein the solvent is a solvent containing an alcohol.

(4) The MMP-1 production inhibitor according to any one of (1) to (3), which is applied before or after the start of the production of the MMP-1.

(5) The MMP-1 production inhibitor according to any one of (1) to (4) wherein the MMP-1 is produced by cells constituting the skin.

(6) The MMP-1 production inhibitor according to (5), wherein the cell is a dermal fibroblast.

(7) The MMP-1 production inhibitor according to any one of (1) to (6) wherein the MMP-1 is produced by exposure to ultraviolet rays or inflammation.

(8) The MMP-1 production inhibitor according to (7), wherein the purple The outside line is UVB.

(9) The MMP-1 production inhibitor according to any one of (1) to (8), which is applied to the epidermis.

(10) The MMP-1 production inhibitor according to any one of (1) to (8), which is applied to the skin.

(11) The MMP-1 production inhibitor according to any one of (1) to (10), which is for preventing or improving aging of the skin.

(12) The MMP-1 production inhibitor according to (11), wherein the skin aging is wrinkles or slack.

(13) A skin external preparation comprising the MMP-1 production inhibitor according to any one of (1) to (9).

(14) Use of the MMP-1 production inhibitor according to any one of (1) to (9), which is used in the production of an external preparation for skin.

(15) A non-medical treatment method for skin, which comprises the use of the MMP-1 production inhibitor according to any one of (1) to (12) or the skin external preparation according to (13).

According to the present invention, MMP-1 production can be remarkably suppressed by using an extract obtained from a specific plant of Ashwagandha, Passionflower, and Robinia pseudoacacia. This inhibitory effect was found by the inventors of the present invention to effectively effect the induction of MMP-1 production in a general environment such as ultraviolet exposure or inflammation. According to the present invention, the extracellular matrix of the dermis originating from MMP-1, in particular, the breakdown of collagen is affected It suppresses and can exert an excellent aging prevention effect. That is, it is possible to provide a skin external preparation excellent in the aging prevention effect. A higher aging prevention effect can be expected by combining other agents such as a whitening agent or an antioxidant, an anti-inflammatory agent, a cell activating agent, a moisturizing agent, an ultraviolet ray preventing agent, and the like. The excellent effects as described above cannot be predicted by the prior art.

Fig. 1 is a graph showing the production of IL-1β induced by epidermal keratinocytes irradiated with UVB. The full human human epidermal keratinocytes were irradiated with UVB for 0 to 60 seconds, and the concentration of IL-1β in the medium after 24 hours (◆) and 72 hours (■) was quantified.

Fig. 2 is a diagram showing the production of MMP-1 induced by dermal fibroblasts in the presence of IL-β. The full human dermal fibroblasts were treated with IL-1β for 24 hours, and the proMMP-1 in the medium was quantified and used as the amount of MMP-1 produced.

Fig. 3 is a graph showing the effect of plant extracts on the inhibition of the production of reactive oxygen species (ROS) induced by hydrogen peroxide. AG: Extract of Ashwagandha, PF: Passionflower extract, NAC: N-acetylcysteine. **: P < 0.01 for the control group, #: P < 0.05 for the addition of only hydrogen peroxide, ##: P < 0.01 for the addition of only hydrogen peroxide.

<MMP-1 production inhibitor>

The present invention provides an MMP containing a plant extract as an active ingredient. 1 produces an inhibitor.

In the present invention, the plant extract means a plant-derived extract which inhibits the production of MMP-1. The plant which is a raw material of the plant extract can be selected from the group consisting of a variety of plants and selected from the group consisting of South African stalked ( Withania somnifera ), Passiflora incarnata , and locust ( One or more of the group consisting of Robinia pseudoacasia). Hereinafter, each plant will be described.

The South African drunken eggplant is a Solanaceae plant whose leaves or roots have been used as the longevity drug Sarayan in the traditional Indian medical Ayurveda since ancient times. In the present invention, it is preferred to use a root. Passionflower is a passionflower plant whose upper part is called a passion flower and is used in Europe for restlessness or insomnia. In the present invention, it is preferred to use an aerial part. Hedgehog is a leguminous plant also known as artichoke, and in the present invention, it is preferred to use flowers.

The plant used as the raw material of the plant extract used in the present invention is preferably a site described above for each plant, but is not limited thereto, and buds, flowers, fruits, peels, seeds, leaves, branches may be used. , dry, bark, root, root bark, aboveground, whole grass and other parts. In addition, one or two or more selected from the above may be used in combination with the above-mentioned preferred portions.

As the raw material, a fresh or dried product can be used, and if necessary, it can be processed by pulverization, fine cutting, powdering or the like. In addition, it is also possible to use a person who can obtain it in the form of a crude drug.

Plant extracts can be directly used by plants as raw materials by using solvents. Obtained by extraction. The extraction solvent is not particularly limited, and examples thereof include water, alcohol, liquid polyol (for example, 1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (for example, acetone, methyl ethyl ketone), and esters. Classes (such as ethyl acetate, butyl acetate, etc.) and the like. Here, as the alcohol, a lower alcohol having a carbon number of 1 to 4 is preferable, and methanol, ethanol, propanol, butanol or the like can be exemplified. These extraction solvents may be used singly or in combination of two or more. Further, in the solvent used in the present invention, any other components may be contained without significantly impairing the extraction efficiency of the obtained plant extract or the MMP-1 production inhibitory action.

As one aspect of the preparation of the plant extract via a solvent, a plant extract can be obtained by treating a plant as a raw material with a solvent containing an alcohol. The concentration of the alcohol is not particularly limited until the obtained plant extract has an effect of suppressing the production of MMP-1. The concentration of the alcohol in the extraction solvent is from about 10 to 100% by volume, preferably from about 10 to 70% by volume. As the solvent containing an alcohol, an aqueous alcohol solution can be suitably used. In the present invention, ethanol is preferably used as the alcohol from the viewpoint of safety.

The extraction method of the raw material plant is not particularly limited, and can be appropriately set according to a known means. For example, the solvent is immersed in a solvent or the like to bring the solvent into contact with the raw material plant, and if necessary, it may be left to be stored, stirred, or heated to reflux. The extraction temperature is not particularly limited, and may be appropriately set depending on the temperature of the solvent, and may be set to be equal to or lower than the boiling point of the solvent from the viewpoint of handling. Further, conditions such as pressurization or decompression may be set as needed.

The extraction time may be appropriately set depending on the type of the plant material to be used, the type and amount of the extraction solvent, and the like. The amount of use can be usually from 1 to 1000 times, preferably from 1 to 100 times, more preferably from 1 to 10 times, relative to 1 part by weight of the raw material plant, and the extraction time can be usually set to about 10 minutes to 1 time. About month, it is preferably about 10 minutes to 7 days. Further, in the case where hot water is used as the solvent, the amount thereof can be usually 1 to 1000 times, preferably 1 to 100 times, more preferably 1 to 10 times, based on 1 part by weight of the raw material, and extraction time. Usually, it can be set to about 10 minutes to 7 days, preferably about 10 minutes to 1 day, more preferably about 10 minutes to 1 hour.

The plant extract of the present invention is obtained by separating the extraction residue after the above extraction operation to form an extract. As the separation means, a known method can be used, and for example, filtration, centrifugation, or the like can be used. In the present invention, the above extract may also be used as it is. Alternatively, it is also possible to obtain a concentrate or a dried product of the above extract (concentrated dry solids) from various viewpoints such as adjustment of inhibition of MMP-1 production, convenience of transportation, and convenience of storage. ) and use that. The concentration can be carried out under normal pressure or reduced pressure, and the volume of the obtained concentrated liquid can be adjusted to about 10 to 50% by volume, preferably about 10 to 30% by volume, based on the volume of the extraction liquid before concentration. The dried solid can be obtained by drying the extract under reduced pressure and removing the solvent.

Further, the plant extract may be prepared by a method other than the method of using the above solvent. For example, by performing steam distillation, or carbon dioxide extraction using supercritical extraction technology, and further adding a solvent to the press liquid (squeezing) and/or residue obtained after the pressing treatment, and extracting The above-mentioned press liquid itself is also included in the definition of the plant extract of the present invention. Further, the plant extract of the present invention may be further purified by column chromatography or the like as needed.

The MMP-1 production inhibitor of the present invention can be applied to all subjects producing MMP-1, thereby inhibiting the production of MMP-1. Here, the inhibition of MMP-1 production is not limited to the complete inhibition of the production of MMP-1, but it is sufficient to suppress partial inhibition of MMP-1 production such as excessive production of MMP-1. In the present specification, "subject" means a mammal, preferably a human.

The tissue in which MMP-1 is produced is not limited, and the skin can be exemplified. Therefore, the MMP-1 production inhibitor of the present invention can be applied to inhibit the production of MMP-1 in the skin. Further, the skin is composed of the stratum corneum, the epidermis, the basement membrane, and the dermis, and the MMP-1 system is mainly produced in the dermis. Therefore, the MMP-1 production inhibitor of the present invention can be applied to inhibit the production of MMP-1 in the dermis. Furthermore, the production of MMP-1 in the dermis is performed by cells present in the dermis. Therefore, the MMP-1 production inhibitor of the present invention can be applied to inhibit the production of MMP-1 caused by cells present in the dermis. Furthermore, the cells that produce MMP-1 in the dermis are mainly fibroblasts. Therefore, the MMP-1 production inhibitor of the present invention can be applied to inhibit the production of MMP-1 induced by fibroblasts present in the dermis.

In the skin, MMP-1 is produced constantly and involves metabolic control of the dermis. On the other hand, it is also known that MMP-1 is excessively produced by stimulation and involves a wide variety of disorders. Here, the stimulation is exemplified by ultraviolet rays, inflammation, and the like, if it is exemplified in relation to the skin. Therefore, this The MMP-1 production inhibitor of the invention can be applied to inhibit MMP-1 production in the dermis induced by ultraviolet exposure of the skin or inflammation of the skin. Here, as ultraviolet rays, the presence of UVA and UVB having different wavelengths is known. It is generally believed that UVA reaches the dermis through the epidermis and acts on fibroblasts of the dermis to induce the production of MMP-1. On the other hand, it is considered that since UVB hardly reaches the dermis, it is impossible to directly act on fibroblasts of the dermis to induce MMP-1 production. That is, it is suggested that if the production of MMP-1 in the dermis is induced by exposing the skin to UVB, another factor is involved. The MMP-1 production inhibitor of the present invention can be preferably applied to inhibit MMP-1 production in the dermis induced by skin exposure to UVB.

The influence of MMP-1 production by UVB exposure has been reported in Non-Patent Document 1. In this report, a review of fibroblasts that directly irradiate UVB to the dermis has been performed. However, as described above, even if UVB is applied to the skin, since UVB hardly reaches the dermis, the report of Non-Patent Document 1 does not mean that the skin is exposed to UVB under a normal environment. On the other hand, the inventors of the present invention found that skin epidermal cells (e.g., epidermal keratinocytes) produce IL-1β and ROS by UVB exposure. Furthermore, it was also found that exogenous IL-1 β stimulates MMP-1 production by fibroblasts of the dermis. Furthermore, it is known that if ROS is produced in the skin, a signal cascade (MAPK) that induces the production of MMP-1 is activated (Hyeon Ho Kim et al, Journal of Lipid Research 46, 1712-1720 (2005)). This and other matters suggest that in the case of exposing the skin to UVB, IL-1 β is produced by epidermal cells. And/or ROS, the IL-1 β and/or ROS system induces the production of MMP-1 induced by fibroblasts of the dermis. Therefore, the MMP-1 production inhibitor of the present invention can be applied to inhibit MMP-1 production in the dermis mediated by IL-1 β and/or ROS produced by UVB exposure of the skin. Further, the IL-1 β and/or ROS are produced by epidermal cells, and epidermal keratinocytes can be exemplified as the epidermal cells.

There is no particular limitation on the period in which the MMP-1 producing inhibitor of the present invention is used. For example, as long as the MMP-1 production inhibitor of the present invention is applied before the initiation of the production of MMP-1, the production of MMP-1 can be prevented prophylactically. As another example, as long as the MMP-1 production inhibitor of the present invention is applied after the initiation of the production of MMP-1, the degree of MMP-1 production can be suppressed. By the inhibition of the production of MMP-1, collagen, in particular, the decomposition of type I collagen can be prevented.

The application of the MMP-1 production inhibitor of the present invention may be carried out by any method as long as it exhibits an effect of suppressing the production of MMP-1. It is preferably applied by, for example, coating, attaching, or spraying the inhibitor. As another preferred embodiment, it can also be applied by injecting the inhibitor into the skin. However, it is not limited to these examples. The amount of the inhibitor to be used may be an amount effective to suppress the production of MMP-1. The application of the inhibitor can be set once a day, or can be divided into several times. In addition, the application period can be set to the number of days of one day or more. Those skilled in the art can consider the state (age, sex, symptoms, etc.) of the subject (eg, human) to which the inhibitor is applied, and other circumstances, and determine the appropriate application amount, number of applications, and application period of the inhibitor.

The MMP-1 production inhibitor of the present invention, although not limited, can be applied to prevention or improvement of aging of the skin. MMP-1 is involved in the breakdown of type I collagen in the skin and involves the breakdown or denaturation of the extracellular matrix. Decomposition or denaturation of the extracellular matrix of the skin is responsible for wrinkles or sagging of the skin. Since the MMP-1 production inhibitor of the present invention can inhibit the production of MMP-1, it can be applied to prevention or improvement of, for example, wrinkles or sagging of the skin.

<For topical application>

According to the present invention, a skin external preparation containing the above-described MMP-1 production inhibitor can be provided. The skin external preparation is not limited, but can be applied to prevention or improvement of aging of the skin, and prevention or improvement of wrinkles or slack of the skin.

The application of the external preparation for skin of the present invention may be carried out by any method as long as it exhibits an effect of suppressing the production of MMP-1. It is preferably applied by, for example, applying, attaching, or spraying the external preparation for skin to the skin or the like. As another preferred example, it may be applied by injecting the external preparation for skin into the skin. However, it is not limited to these examples. The amount of the external preparation for skin may be an amount effective to suppress the production of MMP-1. The application of the external preparation for skin can be set once a day or divided into several times. In addition, the application period can also be set to the number of days of 1 day or more. Those skilled in the art may consider the state (age, sex, symptoms, etc.) of the subject (such as a human) to which the skin external preparation is applied, and other circumstances, and determine the appropriate application amount, application frequency, and application period of the external preparation for the skin. .

The skin external preparation of the present invention may further comprise an allowable substrate. Here, as the substrate which can be tolerated, a component which can be used for humans or animals can be cited as a preferred example. The substrate may be any one which does not significantly impair the MMP-1 production inhibitory action, and examples thereof include vitamins such as vitamin E and vitamin C, water, alcohols, sugars, excipients, disintegrators, binders, and lubricants. Agent, emulsifier, tonicity agent (isoterizing agent), buffering agent, dissolution aid, preservative, absorption enhancer, stabilizer, antioxidant, colorant, flavoring agent, perfume, coagulant, pH adjuster, Tackifier, extract powder, crude drug, various whitefly, chelating agent, inorganic salt, oil agent, surfactant, blood circulation promoter, antibacterial/bactericide, various animal and plant/microbial extracts, etc. From among these substrates, those suitable for the intended use can be selected and used. For example, in the case where the external preparation for skin of the present invention is used as a cosmetic, the user can be selected from the above-mentioned base materials and can be blended in a cosmetic or a medical product. For example, when the external preparation for skin of the present invention is used as a pharmaceutical product, a user who can be used in a pharmaceutical for external use can be selected from the above-mentioned base materials and blended.

The external preparation for skin of the present invention can be made into a suitable form according to the intended use. For example, in the case where the external preparation for skin is used as a cosmetic, it may be in the form of an emulsion, a cream, a lotion, a mask, a film, a cleansing, a foundation, an ointment, an aerosol, or the like. As another example, when the external preparation for skin of the present invention is used as a pharmaceutical, it may be in the form of an emulsion, a cream, an aqueous solution, a patch, a film, a cleansing material, an ointment, an aerosol, or the like.

The skin external preparation of the present invention may be used in combination with an additional component having an action of inhibiting the production of MMP-1. Examples of the additional component include an example. Such as whitening agents, antioxidants, anti-inflammatory agents, cell activators, moisturizers, UV inhibitors. Here, the combined use means not only the blending of the additional component to the external preparation for skin of the present invention, but also the constitution of the additional component which is different from the external preparation for skin of the present invention, and the application of the present invention The additional component is applied before, after, or at the same time as the external preparation for skin.

<Other>

The present invention provides the use of plant extracts for use in the manufacture of MMP-1 production inhibitors. The present invention further provides the use of an MMP-1 production inhibitor which is used in the manufacture of a skin external preparation. The present invention provides a method for treating non-medical skin comprising applying the MMP-1 production inhibitor of the present invention to the skin of a subject in need thereof. Here, the MMP-1 production inhibitor, the plant extract, the external preparation for skin, and the subject are as described above. In addition, the term "non-medical" as used in the present specification means that it is not intended to prevent or improve the appearance of aging, such as wrinkles or slack, for the purpose of medical treatment such as treatment of diseases or diseases. The appearance of the skin is beautiful, or beautiful, such as cosmetic or cosmetic treatment.

[Examples]

In the present embodiment, the present invention will be further described by way of specific examples. This example is not intended to limit the scope of the invention.

[Test Example 1] IL-1 induced by epidermal keratinocytes irradiated by UVB Production of β

The production of IL-1β in epidermal keratinocytes induced by UVB was evaluated using normal human epidermal keratinocytes derived from adults (NHEK-AD, purchased from Kurabo Co., Ltd.). The cells were seeded in a 35 mm petri dish and cultured to fullness at 37 ° C, a carbon dioxide concentration of 5 vol%. The cells were irradiated with UVB of 0.130 mJ/cm ² for 0 to 60 seconds. The culture supernatant was recovered from the medium 24 hours and 72 hours after the UVB irradiation, and IL-1β secreted into the medium was quantified. The IL-1 β quantitative ELISA Kit (manufactured by R&D Systems, Inc.) was used to quantify IL-1 β according to the attached instructions (Fig. 1).

The amount of IL-1β produced by epidermal keratinocytes is shown to increase as the irradiation time of UVB becomes longer. Specifically, in the case where the irradiation time of UVB exceeds 20 seconds, the increase in the amount of IL-1 β produced becomes clear. This tendency was observed 24 hours and 72 hours after UVB irradiation. Further, 72 hours after the UVB irradiation, it was shown that a large amount of IL-1 β was accumulated.

[Test Example 2] Production of MMP-1 by dermal fibroblasts of IL-β

Normal human skin fibroblasts derived from newborns (NHDF-NB, purchased from Kurabo Co., Ltd.) were used as dermal fibroblasts, and the amount of MMP-1 produced was used as an index. The dermal fibroblasts were seeded in a 24-well plate and cultured to fullness at 37 ° C, a carbon dioxide concentration of 5 vol%. IL-1β was added to the medium at a final concentration of 0, 30, 100, 300, 1000 pg/mL, and cultured for 24 hours. After training, The culture supernatant was recovered, and the proMMP-1 secreted into the medium was quantified. Quantification of proMMP-1 (Fig. 2) was carried out using a quantitative ELISA kit (manufactured by R&D Systems, Inc.) in accordance with the attached instructions, and was used as the amount of MMP-1 produced.

As a result, it was revealed that MMP-1 was produced in dermal fibroblasts due to the presence of IL-1β. The amount of MMP-1 produced is accompanied by an increase in the concentration of IL-1β. If this result is combined with the results of Test Example 1, it is suggested that if the skin is exposed to UVB, IL-1β is produced by epidermal keratinocytes, which induces MMP-induced by dermal fibroblasts. The production of 1 is a series of processes in which MMP-1 decomposes collagen.

[Modulation Example 1] Preparation of plant-derived extract

Eight plants selected by screening for a wide variety of plants were subjected to ethanol extraction to obtain an extract. Immerse the pieces of Ashwagandha, Passiflora, Robinia pseudoacacia, Longevity Grass, Heather, Fennel, Bright-leaved Poplar, and Western Sambucus in 70% by volume ethanol with a weight of 10 (w/v). At room temperature, extraction was carried out for 7 days in addition to the stirring operation once a day. Then, the residue was removed by filtration to obtain an extract. After concentrating the extract, the ethanol extract of each plant was obtained by freeze-drying. This plant extract was used in this example.

[Test Example 3] MMP-1 production inhibitory effect

The effect of plant-derived extracts on the inhibition of MMP-1 production was reviewed. PPAR δ ligand was used as a positive control group, similarly Conduct a review. Normal human skin fibroblasts derived from newborns (NHDF-NB, purchased from Kurabo Co., Ltd.) were used as dermal fibroblasts, and the amount of MMP-1 produced was used as an index.

The dermal fibroblasts were seeded in a 24-well plate and cultured to fullness at 37 ° C, a carbon dioxide concentration of 5 vol%. Then, the following components were added: ‧ PPAR δ ligand GW501516 (made by EnzoLife Science Co., Ltd.) dissolved in DMSO, ‧ plant extract prepared by modulating DMSO in DMSO (see Preparation Example 1) Or ‧ no addition (DMSO solution only). The concentration of the component to be added in the culture solution was set to 25 nM for GW501516, 10 μg/mL for the plant extract system, and 0.1% for the DMSO system. After 24 hours of culture, IL-1β was added to the medium at a final concentration of 100 pg/mL. Here, one of the wells to which only the DMSO solution was added was set to have no IL-1β added, and this was used as a control group. After 48 hours of culture, the culture supernatant was recovered, and proMMP-1 secreted into the medium was quantified and used as the amount of MMP-1 produced. The quantification of proMMP-1 was carried out using a quantitative ELISA kit (manufactured by R&D Systems, Inc.) in accordance with the attached instructions. The quantitative results are expressed in the form of a relative value (%) in which the amount of production of MMP-1 for the control group is set to 100% (Table 1).

When only IL-1 β was added to dermal fibroblasts, the amount of MMP-1 produced was approximately 220% (2.2 times that of the control group). Further, when the cells were treated with GW501516 before the addition of IL-1β, the amount of MMP-1 produced was 165.2±6.8%. Thus, it was confirmed that the production of MMP-1 by IL-1β was inhibited by GW501516.

On the other hand, among the 8 plant-derived extracts supplied to the experiment, they were treated with 5 plant extracts prepared from longevity, heather, fennel, euonymus, and western elderberry. In the case of cells, the amount of MMP-1 produced was increased as compared with the case of only IL-1β. On the other hand, in the case where cells were treated with three plant extracts prepared from Ashwagandha, Passionflower, and Robinia pseudoacacia, the production of MMP-1 by IL-1β was strongly inhibited. The amount of MMP-1 produced in the case of treatment with these three plant extracts was 147.1 ± 1.5%, 200.2 ± 1.3%, and 205.6 ± 1.8% were significantly inhibited compared with those who only added IL-1 β. It can be confirmed that the inhibitory effect of MMP-1 production by plant extracts, especially prepared by South African swallow eggplant, is higher than that caused by GW501516. Among the plant extracts tested, only the three plant extracts prepared from Ashwagandha, Passiflora, and Robinia pseudoacacia strongly inhibited the production of MMP-1.

[Test Example 4] MMP-1 production inhibition effect by plant extract

In the test example 3, it was confirmed that the three plant extracts which inhibited the production of MMP-1 were further examined.

The test was carried out under the same conditions as in Test Example 3 except that the concentrations of these plant extracts in the medium were 2.5 μg/mL, 5 μg/mL, and 10 μg/mL. The amount of production of MMP-1 is expressed in the form of a relative value (%) in which the amount of production of MMP-1 for the control group is set to 100% (Table 2).

When only IL-1 β was added to dermal fibroblasts, the amount of MMP-1 produced was approximately446% (4.5 times that of the control group). In addition, the three plant extracts supplied to the test significantly inhibited the production of MMP-1 at all concentrations tested. As a result of the above, it was revealed that the plant extract prepared by Ashwagandha, Passionflower, and Robinia pseudoacacia has an excellent MMP-1 production inhibitory effect.

From the above, it has been found that the production of MMP-1 induced by dermal fibroblasts which are inflammatory cytokines produced by skin cells exposed to UVB in the skin exposed to ultraviolet rays under normal circumstances is effectively effective by the present invention. inhibition.

[Test Example 5] Effect of plant extract on inhibition of production of active oxygen species (ROS) induced by hydrogen peroxide (H ₂ O ₂ )

It is known that if human skin is exposed to UVB, hydrogen peroxide (H ₂ O ₂ ) is produced (Hyeon Ho Kim et al, Journal of Lipid Research 46, 1712-1720 (2005)). In this test, the generation of reactive oxygen species (ROS) caused by stimulation of hydrogen peroxide and the inhibition of the production of ROS by plant extracts were reviewed.

The dermal fibroblasts were seeded in a 24-well plate and cultured to fullness at 37 ° C, a carbon dioxide concentration of 5 vol%.

Then, the following components were added: ‧ an antioxidant N-acetyl cysteine (manufactured by Nacalai Tesque Co., Ltd.) dissolved in DMSO, ‧ PPAR δ ligand GW501516 (manufactured by Enzo Life Science Co., Ltd.) dissolved in DMSO, ‧ dissolved Plant extract in DMSO (see Preparation Example 1), or ‧ no addition (DMSO solution only). The concentration of the component to be added in the culture solution was 30 μM and 300 μM for the N-acetylcysteine system, 25 nM for the GW501516 system, and 10 μg/mL and 20 μg/mL for the plant extract system. The DMSO system was set to 0.1%. After 24 hours of culture, the medium was replaced with a new medium, and hydrogen peroxide was added to the medium at a final concentration of 10 μM, and the level of active oxygen species in the cells after 10 minutes was quantified and used as the amount of ROS production. Quantification of ROS was performed by using a DCFDA analysis kit (manufactured by Abcam) in accordance with the attached instructions. One of the wells to which only the DMSO solution was added was set to have no H ₂ O ₂ added, and this was used as a control group. The quantitative results are expressed in the form of a relative value (%) in which the amount of ROS produced for the control group is set to 100% (Fig. 3).

As a result of adding hydrogen peroxide to the cells and the control group without adding the components, it was confirmed that the cells were stimulated by hydrogen peroxide to induce the production of ROS. Further, by comparing the results with cells in which hydrogen peroxide was added to the unadded components, the following items were revealed: ‧ The production of ROS was inhibited by the extract of Ashwagandha and the extract of Passiflora. All extracts significantly inhibited the production of ROS by the addition of 20 μg/ml; ‧ The production of ROS was also inhibited by N-acetylcysteine, which is known as an antioxidant. In the addition amount of 300 μM, the production of ROS was remarkably suppressed. In addition, GW501516 does not show the production of ROS Inhibitory effect.

It can be understood from the above that the extract of Ashwagandha and the extract of Passiflora inhibit the production of ROS induced by the stimulation of hydrogen peroxide. The effect is comparable to or above the effect of N-acetylcysteine known as an antioxidant.

In Hyeon Ho Kim et al. (Journal of Lipid Research 46, 1712-1720 (2005)), it is suggested that if human skin is exposed to ultraviolet light, hydrogen peroxide will be produced rapidly and remarkably, and the rising ROS involves signals. Activation of the cascade (MAPK) results in the induction of MMP-1 production. It is further taught herein that an antioxidant such as N-acetylcysteine inhibits the production of MMP-1 by capturing ROS and interfering with the activation of the cascade. If these matters are combined with the above results, it is suggested that Ashwagandha and Passionflower also inhibit the production of MMP-1 by inhibiting the production of ROS induced by hydrogen peroxide.

Claims (15)

An inhibitor of MMP-1 production, which comprises, as an active ingredient, one or more plant extracts selected from the group consisting of Ashwagandha, Passiflora, and Robinia. The MMP-1 producing inhibitor of claim 1, wherein the plant extract is a plant extract obtained by solvent extraction. The MMP-1 producing inhibitor of claim 2, wherein the solvent is a solvent containing an alcohol. The MMP-1 production inhibitor according to any one of claims 1 to 3, which is applied before or after the start of the production of the aforementioned MMP-1. The MMP-1 production inhibitor according to any one of claims 1 to 4, wherein the aforementioned MMP-1 is produced by cells constituting the skin. The MMP-1 producing inhibitor of claim 5, wherein the aforementioned cells are dermal fibroblasts. The MMP-1 production inhibitor according to any one of claims 1 to 6, wherein the production of the aforementioned MMP-1 is caused by exposure to ultraviolet rays or inflammation. The MMP-1 producing inhibitor of claim 7, wherein the ultraviolet ray is UVB. The MMP-1 production inhibitor according to any one of claims 1 to 8, which is applied to the epidermis. The MMP-1 production inhibitor according to any one of claims 1 to 8, which is applied subcutaneously. The MMP-1 producing inhibitor according to any one of claims 1 to 10, which is for preventing or improving aging of the skin. The MMP-1 of claim 11 produces an inhibitor wherein the aforementioned skin is aged or wrinkled or relaxed. A skin external preparation comprising the MMP-1 production inhibitor of any one of claims 1 to 9. Use of the MMP-1 production inhibitor according to any one of claims 1 to 9, which is for use in the manufacture of a skin external preparation. A non-medical treatment method for skin comprising the MMP-1 production inhibitor of any one of claims 1 to 12 or the skin external preparation of claim 14.