JP6877332B2 - Matrix metalloproteinase production inhibitor - Google Patents

Description

The present invention relates to a novel matrix metalloproteinase production inhibitor and an external preparation for skin containing the inhibitor.

The skin is composed of the stratum corneum, epidermis, basement membrane, and dermis from the outside. The dermis is filled with cellular components and a network of macromolecules called extracellular matrix produced by these cells. The existence of fibroblasts, macrophages, mast cells, and constituent cells of blood vessels and nerves is known as cell components, and fibrous proteins such as collagen and elastin, and polysaccharides such as hyaluronic acid and proteoglycan are known as extracellular matrix. The existence of such is known. Collagen fibers as extracellular matrix are said to account for about 70% of the dry weight of the dermis and play an important role in maintaining the firmness and elasticity of the skin. There are 20 subtypes of collagen molecules, but 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 the degradation and metabolism of extracellular matrix. MMPs are classified into several subtypes in terms of molecular structure, substrate specificity, and the like. For example, MMP-1 decomposes the triple helix structure of type I collagen, and MMP-2 and MMP-9 decompose type IV collagen and gelatin constituting the basement membrane. MMP not only controls the metabolism of the extracellular matrix of the dermis, but is also deeply involved in cell moisturization and activation of bioactive molecules such as cytokines, and its association with various diseases has been reported. .. Especially in the skin, it has been clarified that MMP is excessively produced by exposure to ultraviolet rays and various inflammations, and the MMP decomposes and denatures collagen and elastin, causing wrinkles and sagging of the skin.

Against this background, various studies have been conducted from the viewpoint of controlling MMP, and many plant extracts that inhibit the activity of MMP have been reported (Patent Documents 1 and 2). Furthermore, an MMP-1 production inhibitor (Patent Document 3) containing a yeast culture solution as an active ingredient, a compound having a sulfhydryl group, and a compound having a sulfhydryl group intended to suppress the production of the enzyme itself and fundamentally suppress the action of MMP. An MMP-1 production inhibitor (Patent Document 4) containing a compound having a disulfide bond as an active ingredient has been reported. On the other hand, in recent years, a subtype of peroxisome proliferator-activated receptor (hereinafter referred to as "PPAR"), which is a nuclear receptor responsible for controlling the expression of genes that maintain lipid and glucose metabolism. It has been suggested that an agonist of PPARδ, which is one of the above, suppresses the production of MMP-1 by exposure to UVB in fibroblasts (Non-Patent Document 1).

Japanese Unexamined Patent Publication No. 7-196526 Japanese Unexamined Patent Publication No. 7-291873 Japanese Unexamined Patent Publication No. 2009-24638 JP-A-2002-47178

SA Ham et al: Journal of Investigative Dermatology 133, 2593-2600 (2013)

However, "Because short-wavelength ultraviolet rays up to UVB hardly penetrate to the dermis, the ultraviolet rays that affect the dermis are mainly UVA" (Kotaro Imakawa et al., "Effects of light on the skin", Nikkei Medical Journal (JJSLSM) As described in Vol. 32, No. 4 (2012), p. 448, pp. 22-24), even if the skin is exposed to ultraviolet rays under normal circumstances, most of it except UVA is in the dermis. It is believed to be unreachable. Therefore, the production of MMP-1 by dermal fibroblasts under normal circumstances is either the direct action of UVA on dermal fibroblasts or the inflammatory cytokines and activity produced by skin cells exposed to UVB. It is believed to be due to indirect action via the oxygen species (ROS). Non-Patent Document 1 directly irradiates dermis-derived fibroblasts with UVB, which is different from the environment in which the skin is exposed to UVB. That is, it is not known that the ligand agent of PPARδ suppresses the production of MMP-1 which may occur under normal environment. An object of the present invention is to provide an agent for suppressing MMP-1 production.

Under the above circumstances, the inventors of the present application have begun to search for various materials and find an activity that suppresses MMP-1 production. As a result, it was found that a plant extract prepared from a specific plant effectively suppresses MMP-1 production. Based on such findings, the present invention has been completed.

The present invention provides:
(1) An MMP-1 production inhibitor containing one or more plant extracts selected from the group consisting of Ashwagandha, Passionflower, and Black locust as an active ingredient.
(2) The MMP-1 production inhibitor according to (1), wherein the plant extract is a plant extract extracted using a solvent.
(3) The MMP-1 production inhibitor according to (2), wherein the solvent is a solvent containing alcohol.
(4) The MMP-1 production inhibitor according to any one of (1) to (3), which is applied before or after the production of MMP-1 is started.
(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 production of MMP-1 is caused by exposure to ultraviolet rays or inflammation.
(8) The MMP-1 production inhibitor according to (7), wherein the ultraviolet rays are 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 subcutaneously.
(11) The MMP-1 production inhibitor according to any one of (1) to (10), which is used for preventing or improving skin aging.
(12) The MMP-1 production inhibitor according to (11), wherein the aging of the skin is wrinkles or sagging.
(13) An external preparation for skin containing 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) in the production of an external preparation for skin.
(15) A non-medical treatment method for skin comprising applying the MMP-1 production inhibitor according to any one of (1) to (12) or the external preparation for skin according to (13).

According to the present invention, it is possible to remarkably suppress MMP-1 production by using extracts obtained from specific plants such as Ashwagandha, Passionflower, and Black locust. Such an inhibitory effect also effectively acted on the induction of MMP-1 production under normal circumstances such as UV exposure and inflammation, which was found by the inventor of the present application. According to the present invention, the degradation of the extracellular matrix of the dermis due to MMP-1 production, particularly collagen, is suppressed, and an excellent antiaging effect is exhibited. That is, an external preparation for skin having an excellent antiaging effect is provided. By combining this with other agents such as whitening agents, antioxidants, anti-inflammatory agents, cell activators, moisturizers, ultraviolet rays inhibitors, etc., a higher antiaging effect can be expected. The above-mentioned excellent effects cannot be predicted from the prior art.

FIG. 1 shows the production of IL-1β by UVB-irradiated epidermal keratinocytes. Confluent 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 shows the production of MMP-1 by dermal fibroblasts in the presence of IL-β. Confluent human dermal fibroblasts were treated with IL-1β for 24 hours, and proMMP-1 in the medium was quantified to determine the amount of MMP-1 produced. FIG. 3 shows the effect of plant extracts on hydrogen peroxide-induced suppression of reactive oxygen species (ROS) production. AG: Ashwagandha extract, PF: Passionflower extract, NAC: N-acetylcysteine. **: P <0.01 vs. control, #: P <0.05 vs. hydrogen peroxide only, ##: P <0.01 vs. hydrogen peroxide only.

<MMP-1 production inhibitor>
The present invention provides an MMP-1 production inhibitor containing a plant extract as an active ingredient.

In the present invention, the plant extract means a plant-derived extract capable of suppressing the production of MMP-1. The plant used as a raw material for the plant extract is selected from the group consisting of Ashwagandha (Withania somnifera), Passiflora incarnata, and Harienju (Robinia psiudoacasia), which were found by screening various plants. It can be a species or two or more species. Hereinafter, each plant will be described.

Ashwagandha is a plant of the Solanaceae family, and its leaves and roots have long been used as a longevity medicine Sarayan in the traditional Indian medicine Ayurveda. In the present invention, roots can be preferably used. Passionflower is a plant of the Passionflower family, and its above-ground part is called passionflower, which is an herb used for nervous anxiety and insomnia in Europe. In the present invention, a above-ground portion can be preferably used. Harienju is a leguminous plant also called black locust, and flowers are preferably used in the present invention.

As the plant used as a raw material of the plant extract used in the present invention, it is preferable to use the part described above for each plant, but the present invention is not limited to this, and buds, flowers, fruits, bark, seeds and leaves. , Branches, trunks, bark, roots, root bark, above-ground parts, whole plants, etc. can be used. Moreover, in addition to the above-mentioned preferable part, one kind or two or more kinds selected from all the above contents may be used together.

As the raw material plant, a raw or dried plant can be used, and if necessary, it can be processed by crushing, shredding, powdering or the like. In addition, those available as crude drugs may be used.

The plant extract can be obtained by directly extracting from the raw material plant using a solvent. The extraction solvent is not particularly limited, but is water, alcohol, liquid polyhydric alcohol (for example, 1,3-butylene glycol, propylene glycol, glycerin, etc.), ketones (for example, acetone, methyl ethyl ketone), esters (for example, ethyl acetate). , Butyl acetate, etc.). Here, as the alcohol, a lower alcohol having 1 to 4 carbon atoms is preferable, and methanol, ethanol, propanol, butanol, and the like can be exemplified. These extraction solvents can be used alone or in combination of two or more. In addition, the solvent used in the present invention may contain any other component as long as the extraction efficiency of the obtained plant extract and the effect of suppressing MMP-1 production are not significantly impaired.

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

The method for extracting the raw material plant is not particularly limited, but can be appropriately set as desired according to known means. For example, the solvent may be brought into contact with the raw material plant by immersing the raw material plant in the solvent, and if necessary, it may be stored statically, stirred, or heated to reflux. The extraction temperature is not particularly limited and may be appropriately set according to the temperature of the solvent, but can be set to be equal to or lower than the boiling point of the solvent from the viewpoint of operation. In addition, conditions such as pressurization and depressurization can be set as needed.

The extraction time may be appropriately set depending on the type of plant raw material used, the type of extraction solvent, the amount used, and the like. The amount used can be usually 1 to 1000 times, preferably 1 to 100 times, more preferably 1 to 10 times with respect to 1 part by weight of the raw material plant, and the extraction time is usually about 10 minutes to. It can be about 1 month, preferably about 10 minutes to 7 days. When hot water is used as the solvent, the amount used can be usually 1 to 1000 times, preferably 1 to 100 times, more preferably 1 to 10 times with respect to 1 part by weight of the raw material, and extraction can be performed. The time is usually about 10 minutes to 7 days, preferably about 10 minutes to 1 day, and more preferably about 10 minutes to 1 hour.

In the plant extract of the present invention, after the above extraction operation, the extraction residue is separated into an extract form. 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 be used as it is. Alternatively, from various viewpoints such as adjustment of MMP-1 production inhibitory action, convenience of transportation, convenience of storage, etc., a concentrated or dried product (concentrated dry product) of the extract is obtained, if necessary. , You may use this. Concentration can be carried out under normal pressure or reduced pressure, and the volume of the obtained concentrate may be adjusted to about 10 to 50% by volume, preferably about 10 to 30% by volume of the volume of the extract before concentration. .. The dry matter can be obtained by drying the extract under reduced pressure and removing the solvent.

The plant extract can also be prepared by a method other than the above-mentioned method using a solvent. For example, those obtained by steam distillation, carbon dioxide extraction using supercritical extraction technology, and extraction by adding a solvent to the squeezed liquid (squeezed juice) and / or the residue obtained after the squeezing treatment. The squeezed 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 fractionally purified from its active ingredient by column chromatography or the like, if necessary.

The MMP-1 production inhibitor of the present invention can be applied to any subject in which MMP-1 is produced, thereby suppressing the production of MMP-1. Here, suppression of MMP-1 production is not limited to completely suppressing the production of MMP-1, but partial suppression of MMP-1 production such as suppression of overproduction of MMP-1. Sufficient. As used herein, the term "subject" means a mammal, preferably a human.

The tissue from which MMP-1 is produced is not limited, but skin can be exemplified. Therefore, the MMP-1 production inhibitor of the present invention can be applied to suppress the production of MMP-1 in the skin. The skin is composed of the stratum corneum, epidermis, basement membrane, and dermis, and MMP-1 is mainly produced in the dermis. Therefore, the MMP-1 production inhibitor of the present invention can be applied to suppress the production of MMP-1 in the dermis. Furthermore, the production of MMP-1 in the dermis is carried out by cells present in the dermis. Therefore, the MMP-1 production inhibitor of the present invention can be applied to suppress the production of MMP-1 by cells existing in the dermis. In addition, 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 suppress the production of MMP-1 by fibroblasts present in the dermis.

In the skin, MMP-1 is constantly produced and is involved in the metabolic regulation of the dermis. On the other hand, MMP-1 is also known to be overproduced by stimulation and involved in various diseases. Here, examples of irritation with respect to the skin include ultraviolet rays and inflammation. Therefore, the MMP-1 production inhibitor of the present invention can be applied to suppress MMP-1 production in the dermis, which is induced by exposure to ultraviolet rays of the skin or inflammation of the skin. Here, the existence of UVA and UVB having different wavelengths is known as ultraviolet rays. UVA is thought to pass through the epidermis and reach the dermis, acting on fibroblasts in the dermis to induce the production of MMP-1. On the other hand, since UVB hardly reaches the dermis, it is considered that it cannot directly act on fibroblasts in the dermis to induce MMP-1 production. That is, if exposure of the skin to UVB induces the production of MMP-1 in the dermis, it is suggested that yet another factor is involved. The MMP-1 production inhibitor of the present invention can be preferably applied to suppress MMP-1 production in the dermis, which is induced by exposure of the skin to UVB.

The effect of UVB exposure on MMP-1 production has been reported in Non-Patent Document 1. In this report, a study was made in which UVB was directly irradiated to dermal fibroblasts. However, as explained above, even if UVB is irradiated to the skin, UVB hardly reaches the dermis, so the report of Non-Patent Document 1 reflects the condition that the skin is exposed to UVB under normal environment. I can't say that I'm doing it. On the other hand, the inventor of the present application has found that epidermal cells of the skin (for example, epidermal keratinocytes) produce IL-1β and ROS upon exposure to UVB. We also found that exogenous IL-1β stimulates MMP-1 production by dermal fibroblasts. It is also known that the development of ROS in the skin activates the signal cascade (MAPK) that induces the production of MMP-1 (Hyeon Ho Kim et al, Journal of Lipid Research 46, 1712-1720 (Hyeon Ho Kim et al, Journal of Lipid Research 46, 1712-1720). 2005)). From these matters, when the skin is exposed to UVB, IL-1β and / or ROS is produced by epidermal cells, and the IL-1β and / or ROS induces the production of MMP-1 by dermal fibroblasts. It was suggested to do. Therefore, the MMP-1 production inhibitor of the present application can be applied to suppress MMP-1 production in the dermis mediated by IL-1β and / or ROS produced by UVB exposure of the skin. The IL-1β and / or ROS are produced by epidermal cells, and epidermal keratinocytes are exemplified as the epidermal cells.

The timing of applying the MMP-1 production inhibitor of the present invention is not particularly limited. For example, if the MMP-1 production inhibitor of the present invention is applied before the production of MMP-1 is started, the production of MMP-1 can be prophylactically suppressed. As another example, if the MMP-1 production inhibitor of the present invention is applied after the production of MMP-1 is started, the degree of MMP-1 production can be suppressed. By such an action of suppressing the production of MMP-1, it is possible to prevent the decomposition of collagen, particularly type I collagen.

The application of the MMP-1 production inhibitor of the present invention may be carried out by any method as long as the effect of suppressing the production of MMP-1 is exhibited. Preferably, for example, the inhibitor can be applied by coating, pasting, spraying, or the like. As another preferred example, it can also be applied by injecting the inhibitor subcutaneously. However, it is not limited to these examples. The applied amount of the inhibitor may be an amount effective for suppressing the production of MMP-1. The inhibitor may be applied once a day or divided into several times. In addition, the applicable period may be one day or more. Those skilled in the art will consider the condition (age, gender, symptoms, etc.) of the subject (for example, human) to which the inhibitor is applied, and other circumstances, and the appropriate amount, frequency of application, and duration of application of the inhibitor. Can be determined.

The MMP-1 production inhibitor of the present invention can be applied to prevent or improve skin aging without limitation. MMP-1 is involved in the degradation or degeneration of extracellular matrix through the degradation of type I collagen in the skin. Degradation or degeneration of the extracellular matrix of the skin causes wrinkles and sagging of the skin. Since the MMP-1 production inhibitor of the present invention can suppress the production of MMP-1, it can be applied to, for example, the prevention or improvement of wrinkles or sagging of the skin.

<External skin preparation>
According to the present invention, an external preparation for skin containing the MMP-1 production inhibitor described above is provided. The external preparation for skin can be applied to prevent or improve skin aging and prevent or improve wrinkles or sagging of skin, without limitation.

The application of the external preparation for skin of the present invention may be carried out by any method as long as the effect of suppressing the production of MMP-1 is exhibited. Preferably, for example, the external preparation for skin can be applied by applying, sticking, or spraying on the skin. As another preferred example, it can also be applied by injecting the external preparation for skin subcutaneously. However, it is not limited to these examples. The applied amount of the external preparation for skin may be an amount effective for suppressing the production of MMP-1. The application of the external preparation for skin may be applied once a day or may be divided into several times. In addition, the applicable period may be one day or more. Those skilled in the art will consider the condition (age, gender, symptoms, etc.) of the subject (for example, human) to which the external preparation for skin is applied, and other circumstances, and consider the appropriate amount, number of times, and number of applications of the external preparation for skin. The application period can be determined.

The external preparation for skin of the present invention may further contain an acceptable base material. Here, as an acceptable base material, a component that is permitted to be used for humans and animals can be mentioned as a preferable example. The base material may be any as long as it does not significantly impair the MMP-1 production inhibitory action, for example, vitamins such as vitamin E and vitamin C, water, alcohol, sugars, excipients, disintegrants, binders, and abundance. Agents, emulsifiers, tensioning agents (isotonic agents), buffers, solubilizers, preservatives, absorption promoters, stabilizers, antioxidants, colorants, flavoring agents, fragrances, coagulants, pH regulators , Thickeners, extract powders, crude drugs, various powders, chelating agents, inorganic salts, oils, surfactants, blood circulation promoters, antibacterial / bactericidal agents, various animal / plant / microbial extracts and the like. From these base materials, those suitable for the intended use mode can be selected and used. For example, when the external preparation for skin of the present invention is used as a cosmetic, a material that can be used in cosmetics and quasi-drugs can be selected from the above-mentioned base materials and blended. For example, when the skin external preparation of the present invention is used as a pharmaceutical product, those that can be used in the external preparation can be selected from the above-mentioned base materials and blended.

The external preparation for skin of the present invention can be in an appropriate form according to the intended use mode. For example, when the external preparation for skin is used as a cosmetic, it can be in the form of a milky lotion, a cream, a lotion, a pack, a film, a cleansing agent, 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 product, it can be in the form of a milky lotion, a cream, an aqueous solution, a patch, a film, a cleaning agent, an ointment, an aerosol or the like.

The external preparation for skin of the present invention may be used in combination with an additional component having an action of suppressing the production of MMP-1. Examples of the additional component include whitening agents, antioxidants, anti-inflammatory agents, cell activators, moisturizers, UV inhibitors and the like. Here, to use in combination means not only blending the additional component with the external preparation for skin of the present invention, but also making the additional component different from the external preparation for skin of the present invention for external use with the skin of the present invention. It also includes applying the additional ingredient before, after, or at the same time as applying the agent.

<Others>
The present invention provides the use of plant extracts in the production of MMP-1 production inhibitors. The present invention further provides the use of MMP-1 production inhibitors in the manufacture of topical skin preparations. The present invention also provides a non-medical method of treating skin comprising applying the MMP-1 production inhibitor of the present invention to the skin of a subject in need. Here, the MMP-1 production inhibitor, the plant extract, the external preparation for skin, and the mode of the subject are as described above. The term "non-medical" as used herein is not intended for medical treatment such as treatment of diseases or diseases, but prevents or improves aging in appearance, such as wrinkles and sagging. Thereby, cosmetic or cosmetic treatments such as beautifying the appearance of the skin and maintaining the beauty are intended.

In this embodiment, the present invention will be further described with reference to specific examples. The present embodiment is not intended to limit the scope of the present invention.

[Test Example 1] Production of IL-1β by epidermal keratinocytes by UVB irradiation Using adult-derived normal human epidermal keratinocytes (NHEK-AD, purchased from Keratinocyte Co., Ltd.), IL by UVB in epidermal keratinocytes The production of -1β was evaluated. Cells were seeded in a 35 mm culture dish and cultured at 37 ° C. in a carbon dioxide concentration of 5 vol% until confluent. Cells were irradiated with 0.130 mJ / cm ² UVB for 0-60 seconds. Culture supernatants were collected from the medium 24 hours and 72 hours after UVB irradiation, and IL-1β secreted into the medium was quantified. IL-1β quantification Using an ELISA kit (manufactured by R & D Systems), IL-1β was quantified according to the attached instructions (Fig. 1).

It was shown that the amount of IL-1β produced by epidermal keratinocytes increased as the UVB irradiation time increased. In particular, it was clarified that the amount of IL-1β produced increased when the UVB irradiation time exceeded 20 seconds. Such a tendency was observed both 24 hours and 72 hours after UVB irradiation. It was also shown that more IL-1β was accumulated 72 hours after UVB irradiation.

[Test Example 2] Production of MMP-1 by dermal fibroblasts by IL-β MMP-1 using neonatal-derived normal human skin fibroblasts (NHDF-NB, purchased from Kurabou Co., Ltd.) as dermal fibroblasts The study was carried out using the amount of production of. Dermal fibroblasts were seeded on a 24-well plate and cultured at 37 ° C. at a carbon dioxide concentration of 5 vol% until confluent. IL-1β was added to the medium to a final concentration of 0, 30, 100, 300, 1000 pg / mL and cultured for 24 hours. After culturing, the culture supernatant was collected, and proMMP-1 secreted into the medium was quantified. Quantitative ELISA Kit (manufactured by R & D Systems) was used to quantify proMMP-1 according to the attached instructions (Fig. 2), and the amount of MMP-1 produced was determined.

From this result, it was shown that the presence of IL-1β produces MMP-1 in dermal fibroblasts. The amount of MMP-1 produced increased with the concentration of IL-1β. Considering this result and the result of Test Example 1 together, when the skin is exposed to UVB, IL-1β is produced by epidermal keratinocytes, and the IL-1β is produced by dermal fibroblasts. Is suggested, and a series of processes in which the MMP-1 decomposes collagen is suggested.

[Preparation Example 1] Preparation of plant-derived extracts Eight kinds of plants selected by screening of various plants were extracted with ethanol to obtain extracts. Soak each crushed material of Ashwagandha, Passionflower, Harienju, Sakuna, Gyoryumodoki, Anise, Black locust, and Elderberry in 70% by volume ethanol by 10 (w / v) times the weight, and stir once a day at room temperature. The operation was added and extraction was performed for 7 days. Then, the residue was removed by filtration to obtain an extract. The extract was concentrated and then freeze-dried to obtain an ethanol extract of each plant. The plant extract was used in this example.

[Test Example 3] Effect of suppressing MMP-1 production The effect of a plant-derived extract on suppressing MMP-1 production was examined. The PPARδ ligand was also examined as a positive control. Using normal human skin fibroblasts derived from newborns (NHDF-NB, purchased from Kurabou Co., Ltd.) as dermal fibroblasts, the amount of MMP-1 produced was used as an index for examination.

Dermal fibroblasts were seeded on a 24-well plate and cultured at 37 ° C. at a carbon dioxide concentration of 5 vol% until confluent. Then the following ingredients were added:
PPARδ ligand agent GW501516 dissolved in DMSO (manufactured by EnzoLife Science),
A plant extract prepared from each of the eight plants dissolved in DMSO (see Preparation Example 1), or no addition (DMSO solution only).
The concentration of the added component in the culture solution was 25 nM for GW501516, 10 μg / mL for the plant extract, and 0.1% for DMSO. After culturing for 24 hours, IL-1β was added to the medium at a final concentration of 100 pg / mL. Here, IL-1β was not added to a part of the wells to which only the DMSO solution was added, and this was used as a control. After culturing for 48 hours, the culture supernatant was collected, and proMMP-1 secreted into the medium was quantified and used as the amount of MMP-1 produced. Quantification of proMMP-1 was performed using a quantitative ELISA Kit (manufactured by R & D Systems) according to the attached instructions. The quantitative results are shown as relative values (%) with the amount of MMP-1 produced for the control as 100% (Table 1).

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

On the other hand, when cells were treated with 5 types of plant extracts prepared from Sakuna, Gyoryumodoki, Anise, Sekigacha, and Elderberry among the 8 types of plant-derived extracts used in this test, only IL-1β was used. It was shown that the amount of MMP-1 produced was rather high as compared with the one to which was added. On the other hand, when the cells were treated with three types of plant extracts prepared from Ashwagandha, Passionflower, and Black locust, the production of MMP-1 by IL-1β was strongly suppressed. The amount of MMP-1 produced when treated with these three types of plant extracts was 147.1 ± 1.5%, 200.2 ± 1.3%, and 205.6 ± 1.8%, respectively. , IL-1β alone was significantly suppressed as compared with the one to which only IL-1β was added. In particular, it was confirmed that the inhibitory effect of MMP-1 production by the plant extract prepared from Ashwagandha exceeds the effect of GW501516. It was completely unexpected that among the plant extracts used in the test, only three types of plant extracts prepared from Ashwagandha, Passionflower, and Black locust strongly suppressed the production of MMP-1.

[Test Example 4] Effect of suppressing MMP-1 production by plant extracts In Test Example 3, three types of plant extracts confirmed to suppress MMP-1 production were further investigated.

The test was conducted 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 MMP-1 produced is shown as a relative value (%) with the amount of MMP-1 produced for the control as 100% (Table 2).

Addition of IL-1β alone to dermal fibroblasts resulted in approximately 446% MMP-1 production (4.5-fold increase over control). All three types of plant extracts used in this test markedly suppressed the production of MMP-1 at all the concentrations tested. From these results, it was shown that the plant extract prepared from Ashwagandha, Passionflower, and Black locust has an excellent effect of suppressing MMP-1 production.

From the above, in the skin exposed to ultraviolet rays under normal environment, the production of MMP-1 by dermal fibroblasts via inflammatory cytokines produced by skin cells exposed to UVB is effective according to the present invention. It turned out to be suppressed.

[Test Example 5] Effect of plant extract on suppression of reactive oxygen species (ROS) production induced by _{hydrogen peroxide (H 2} O _{2 ) When human skin is exposed to UVB, hydrogen peroxide (H 2} O ₂₎ ) Is known to occur (Hyeon Ho Kim et al, Journal of Lipid Research 46, 1712-1720 (2005)). In this test, the production of reactive oxygen species (ROS) by stimulation with hydrogen peroxide and the suppression of ROS production by plant extracts were investigated.

Dermal fibroblasts were seeded on a 24-well plate and cultured at 37 ° C. at a carbon dioxide concentration of 5 vol% until confluent.
Then the following ingredients were added:
-Antioxidant N-acetylcysteine (manufactured by Nacalai Tesque) dissolved in DMSO,
PPARδ ligand agent GW501516 dissolved in DMSO (manufactured by EnzoLife Science),
-Plant extract dissolved in DMSO (see Preparation Example 1), or-No addition (DMSO solution only).
The concentrations of the added components in the culture medium were 30 μM and 300 μM for N-acetylcysteine, 25 nM for GW501516, 10 μg / mL and 20 μg / mL for plant extracts, and 0.1% for DMSO. After culturing for 24 hours, the medium was replaced with a new medium, hydrogen peroxide was added to the medium at a final concentration of 10 μM, and the intracellular reactive oxygen species level after 10 minutes was quantified to obtain the amount of ROS produced. ROS was quantified by using the DCFDA assay kit (manufactured by Abcam) according to the attached instructions. _{H 2} O ₂ was not added to a part of the wells to which only the DMSO solution was added, and this was used as a control. The quantitative results are shown as relative values (%) with the ROS production amount for the control as 100% (Fig. 3).

From the results of the control and the cells to which hydrogen peroxide was added without adding the component, it was confirmed that the production of ROS was induced by stimulating the cells with hydrogen peroxide. Then, by comparing with the results for cells to which hydrogen peroxide was added without adding the component, the following items were shown:
-The production of ROS was suppressed by the extract of Ashwagandha and the extract of Passionflower. Both extracts significantly suppressed ROS production with the addition of 20 μg / ml;
-N-Acetylcysteine, also known as an antioxidant, also suppressed ROS production. At the addition amount of 300 μM, the production of ROS was significantly suppressed. GW501516 did not show the effect of suppressing the production of ROS.

From the above, it was clarified that the extract of Ashwagandha and the extract of Passionflower suppress the production of ROS induced by the stimulation of hydrogen peroxide. Its effect is comparable to or greater than that of N-acetylcysteine, known as an antioxidant.

Hyeon Ho Kim et al. (Journal of Lipid Research 46, 1712-1720 (2005)) found that when human skin was exposed to UV light, hydrogen peroxide was produced rapidly and significantly, and ROS was elevated. It has been suggested that it is involved in the activation of the signal cascade (MAPK) and, as a result, induces the production of MMP-1. It is also taught here that antioxidants such as N-acetylcysteine may capture the ROS and interfere with the activation of the cascade, thereby suppressing the production of MMP-1. Considering these matters together with the above results, it is suggested that Ashwagandha and Passionflower also suppress the production of MMP-1 by suppressing the production of ROS induced by hydrogen peroxide.

Claims (11)

An MMP-1 production inhibitor containing a passionflower plant extract as an active ingredient.
The MMP-1 production is due to exposure of the epidermis to UV light or inflammation of the epidermis, and the UV light is UVB.
The MMP-1 production inhibitor. The MMP-1 production inhibitor according to claim 1, wherein the plant extract is a solvent extract. The MMP-1 production inhibitor according to claim 2, wherein the solvent is a solvent containing alcohol. The MMP-1 production inhibitor according to any one of claims 1 to 3, which is applied before or after the production of MMP-1 is started. The MMP-1 production inhibitor according to any one of claims 1 to 4, which suppresses the production of MMP-1 by cells constituting the skin. The MMP-1 production inhibitor according to claim 5, wherein the cells are dermal fibroblasts. The MMP-1 production inhibitor according to any one of claims 1 to 6, further containing an extract of Ashwagandha root as an active ingredient. The MMP-1 production inhibitor according to any one of claims 1 to 7, wherein the production of MMP-1 is caused by IL-1β produced by exposure to ultraviolet rays of the epidermis or inflammation of the epidermis. An external preparation for skin used for preventing or ameliorating skin aging, which is applied to an epidermis irradiated with ultraviolet rays or an epidermis in an inflamed state, containing the MMP-1 production inhibitor according to claim 7. Use of the MMP-1 production inhibitor according to claim 7 in the manufacture of an external preparation for skin used for preventing or ameliorating skin aging, which is applied to an epidermis irradiated with ultraviolet rays or an inflamed epidermis. The skin external preparation according to MMP-1 production inhibitor or claim 9 according to claim 7, applied to the skin of the UV irradiated skin, or inflammatory conditions, include preventing or improving skin aging A non-medical treatment of the skin.

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