CN114617907B

CN114617907B - Inhibitors of granzyme B

Info

Publication number: CN114617907B
Application number: CN202210276176.4A
Authority: CN
Inventors: 山崎纮子; 山野美怜; 横田真穗; 龟井大嗣; 伊藤良树
Original assignee: Sato Pharmaceutical Co Ltd
Current assignee: Sato Pharmaceutical Co Ltd
Priority date: 2021-04-22
Filing date: 2022-03-21
Publication date: 2023-08-15
Anticipated expiration: 2042-03-21
Also published as: JP6988016B1; CN114617907A; JP2022166987A

Abstract

The present invention provides a granzyme B inhibitor with high effect. It contains geranium extract as an active ingredient.

Description

Inhibitors of granzyme B

Technical Field

The present invention relates to a granzyme B inhibitor comprising geranium extract as an active ingredient.

Background

Granzyme B blocking (inhibiting) agents suggest an immune induction effect and an effect on autoimmune/chronic inflammatory diseases (patent documents 1 to 2). When applied to the skin, the composition exhibits a promoting effect on healing burns and wounds and an appearance improving effect on skin aging (patent documents 3 to 5).

As inhibitors of granzyme B activity, known low molecular peptides and the like are known (patent documents 1 to 3 and non-patent documents 1 to 2). However, there is no report on an inhibitor of granzyme B activity which is easily available as a raw material and is inexpensive from a natural substance.

In addition, granzyme B is induced to be expressed by differentiation-inducing cytokines, and is involved in apoptosis (non-patent document 3). In the skin, the expression level is low in a normal state, and in an autoimmune/chronic inflammatory skin state, the expression level is increased, and the expression is induced by ultraviolet irradiation (non-patent documents 4 to 6). However, there is no report at all on the raw materials inhibiting the expression of granzyme B.

Prior art literature

Patent literature

Patent document 1: international publication No. 2003/065987;

patent document 2: U.S. patent application publication No. 2003/0148511;

patent document 3: international publication No. 2017/132771;

patent document 4: japanese patent No. 6134268;

patent document 5: japanese patent No. 5746813;

non-patent literature

Non-patent document 1: bird et al mol, cell, biol, 1998, 18, 6387-6398;

non-patent document 2: kam et al, biochim, biophys, acta, 2000, 1477 (1-2), 307-23;

non-patent document 3: tamang et al, cytokine, 2006 November, 36 (3-4), 148-159;

non-patent document 4: turner et al, J.Invest. Dermatol., 2021, jan, 141 (1), 36-47;

non-patent document 5: hiebert et al, exp. Gerontol, 2011 Jun, 46 (6), 489-99;

non-patent document 6: parkinson et al, aging Cell, 2015 Feb, 14 (1), 67-77.

Disclosure of Invention

Problems to be solved by the invention

Raw materials that inhibit the increase in the expression of granzyme B and also have an inhibitory effect on granzyme B activity suggest that the skin deterioration caused by granzyme B is effectively inhibited.

Therefore, a natural substance having both an inhibitory effect on the expression of granzyme B and an activity-inhibiting effect is expected to be used as an inhibitor of granzyme B action in a wider range of applications.

Means for solving the problems

The inventors found that: in a wide range of plant extracts, geranium extract has both effects of inhibition of granzyme B expression and inhibition of activity, thereby completing the present invention. In addition, the present inventors further showed that: by adding various plant extracts, the inhibition of granzyme B activity is enhanced. That is, the present invention relates to [1] to [23] described below.

[1] A granzyme B inhibitor comprising geranium extract as an active ingredient.

[2]Above [1]]The granzyme B inhibitor contains 1 to 100 percentμg/mL geranium extract.

[3]Above [ 2]]The granzyme B inhibitor contains 1.8-55 percentμg/mL geranium extract.

[4]Above [ 3]]The granzyme B inhibitor contains 18 to 55 percentμg/mL geranium extract.

[5] The granzyme B inhibitor according to any one of [1] to [4], wherein the granzyme B inhibitor further comprises 1 or more plant extracts having granzyme B inhibitory activity.

[6] The granzyme B inhibitor according to [5] above, wherein, in 1:1 to 1:4 comprises the above plant extract and the above herba Erodii seu Geranii extract.

[7] The granzyme B inhibitor according to the above [5] or [6], wherein the plant extract is selected from the group consisting of: pomegranate (Punica granatum) pericarp extract, himalayan Huang Shumei (Yellow himalayan raspberry) root extract, eucalyptus (Eucalyptus globulus) extract, garden burnet (Sanguisorba officinalis) extract, acer palmatum (Acer palmatum) leaf extract, spiraea (Spiraea) extract, witch hazel (Hamamelis virginiana) extract, rose (Rose) extract, avocado (Persea gratissima) extract, sweet tea (Rubus Suavissimus) extract, evening primrose (Oenothera biennis) extract, oolong tea extract, cinnamon bark extract (Cinnamomum Cassia Bark Extract), saxifraga (Saxifraga stolonifera) extract, bee flower (Melissa officinalis) extract, potentilla (Potentilla erecta) extract, rosa damascana (Rosa damascana) flower extract the extract of Artemisia princeps (Artemisia princeps), isodon japonicus (Isodon japonica) extract, lithospermum erythrorhizon (Comfrey) extract, hypericum perforatum (Hypericum erectum) extract, mentha arvensis extract, vitis vinifera leaf extract, tilia japonica (Tilia japonica) extract, rosa multiflora) extract, eriobotrya japonica leaf extract, sargassum extract, paeonia (Paeonia albiflora) extract, houttuynia cordata (Houttuynia Cordata) extract, crataegus pinnatifida (Crataegus cuneata) extract, perilla frutescens (Perilla ocymoides) extract, lavender (Lavandula Angustifolia) extract, rosa multiflora (Rosa canina) extract, uncaria catechu (Uncaria gambir) extract, xiang Liangbu seaweed (Eisenia arborea) extract, peony (Paeonia suffruticosa) extract, filiform aspartame (Aspalathus Linearis) extract, licorice (Glycyrrhiza glabra) extract powder and kuh-seng (Sophora angustifolia) extract.

[8] The granzyme B inhibitor according to the above [7], wherein at least 1 of the plant extracts is selected from the group consisting of an extract of Hypericum perforatum and an extract of sweet tea.

[9] The granzyme B inhibitor according to any one of the above [1] to [8], wherein granzyme B inhibition is inhibition of expression of granzyme B gene.

[10] The granzyme B inhibitor according to any one of the above [1] to [8], wherein granzyme B inhibition is an inhibition of granzyme B activity.

[11] The granzyme B inhibitor according to any one of the above [1] to [8], wherein granzyme B inhibition is inhibition of granzyme B gene expression and inhibition of granzyme B activity.

[12] A cosmetic composition comprising only the granzyme B inhibitor according to any one of [1] to [11 ].

[13] Collagen degradation inhibitor comprising geranium extract as an active ingredient.

[14]Above [13]]The collagen degradation inhibitor contains 1-100 parts ofμg/mL geranium extract.

[15]Above [14 ]]The saidWherein the collagen degradation inhibitor comprises 1.8 to 55μg/mL geranium extract.

[16]Above [15 ]]The collagen degradation inhibitor contains 18-55 percentμg/mL geranium extract.

[17] The collagen degradation inhibitor according to any one of the above [13] to [16], wherein the collagen degradation inhibitor further comprises 1 or more plant extracts having a collagen degradation inhibiting effect.

[18] The collagen degradation inhibitor according to [17] above, wherein 1:1 to 1:4 comprises the above plant extract and the above herba Erodii seu Geranii extract.

[19] The collagen degradation inhibitor according to the above [17] or [18], wherein the plant extract is selected from the group consisting of: pomegranate peel extract, himalayan Huang Shumei root extract, eucalyptus extract, garden burnet extract, acer palmatum leaf extract, spiraea ulmaria extract, witch hazel extract, rose extract, avocado extract, sweet tea extract, evening primrose extract, oolong tea extract, cinnamon bark extract, saxifraga extract, bee balm extract, potentilla extract, rosa damascena flower extract, artemisia kuhaui extract, coriander extract, lithospermum extract, hypericum perforatum extract, peppermint extract, grape leaf extract, tilia extract, rose hip extract, loquat leaf extract, seaweed extract, paeonia lactiflora extract, houttuynia cordata extract, crataegus cuneata extract, perilla extract, lavender extract, wild rose extract, catechol gambir extract, xiang Liangbu seaweed extract, peony extract, filiform mesquite extract, licorice extract powder and kuh-seng extract.

[20] The collagen degradation inhibitor according to the above [19], wherein at least 1 of the above plant extracts is selected from the group consisting of Hypericum perforatum extract and sweet tea extract.

[21] The collagen degradation inhibitor according to any one of the above [13] to [20], wherein the collagen degradation inhibition is inhibition of the expression of granzyme B gene.

[22] The collagen degradation inhibitor according to any one of the above [13] to [20], wherein the collagen degradation inhibition is inhibition of the activity of granzyme B.

[23] The collagen degradation inhibitor according to any one of the above [13] to [20], wherein the collagen degradation inhibition is inhibition of expression of granzyme B gene and inhibition of granzyme B activity.

Effects of the invention

As shown in examples described later, the geranium extract according to the present invention can be used as a granzyme B inhibitor since it has granzyme B expression inhibitory effect and activity inhibitory effect.

Drawings

FIG. 1 is a graph showing the experimental results of the inhibition of the expression of granzyme B gene by geranium extract.

FIG. 2A is a graph showing the experimental results of the inhibition of granzyme B activity by geranium extract.

FIG. 2B is a graph showing the experimental results of the inhibition of granzyme B activity by geranium extract.

FIG. 3 shows a plant extract having an inhibitory effect on the activity of granzyme B.

FIG. 4A is a graph showing the inhibitory effect of Hypericum perforatum extract on the activity of granzyme B.

FIG. 4B is a graph showing the inhibitory effect of sweet tea extract on the activity of granzyme B.

Fig. 5A is a graph showing the synergistic effect of inhibition of degradation of decorin by inhibition of granzyme B of geranium extract and hypericum perforatum extract.

Fig. 5B is a graph showing the synergistic effect of the geranium extract and the herba Hyperici perforati extract in inhibiting degradation of decorin by inhibiting enzyme B.

FIG. 6A is a graph showing the synergistic effect of inhibition of degradation of decorin by inhibition of granzyme B of geranium extract and sweet tea extract.

Fig. 6B is a graph showing the synergistic effect of inhibition of degradation of decorin by inhibition of enzyme B in geranium extract and sweet tea extract.

Detailed Description

Embodiments of the present invention will be specifically described below. The preferred embodiments and more preferred embodiments described below may be appropriately combined with each other, regardless of the expression "preferred" or "more preferred" and the like. The numerical ranges are described as examples, and ranges in which the numerical values of the respective ranges are appropriately combined with the upper and lower limits and the numerical values of the embodiments can be preferably used. Moreover, the terms "comprising" or "including" and the like may be understood as "consisting essentially of" or "consisting of only of".

[ granzyme B inhibitor ]

Hereinafter, the granzyme B inhibitor of the present invention will be described in detail.

Granzyme B is a serine protease released by cytoplasmic granules within cytotoxic T cells and natural killer cells, degrading various extracellular matrices, involved in skin disorders (Turner et al, matrix biol. (2019) 75-76, 126-140). Among them, granzyme B is known to degrade decorin protecting type I collagen, thereby promoting type I collagen degradation by MMP-1 (matrix metalloproteinase-1) (Parkinson et al, aging cell.2015 Feb; 14 (1): 67-77). Granzyme B is expressed in a low amount under normal conditions, and is induced to express by irradiation of skin with ultraviolet rays or the like (Hernandez-Pigeon et al, J Biol chem 2006 May 12; 281 (19): 13525-32).

As a scheme of inhibition of granzyme B by the granzyme B inhibitor (hereinafter, abbreviated as "inhibitor") of the present invention, there can be mentioned: inhibition of granzyme B activity and inhibition of granzyme B gene expression.

Collagen is protected by the inhibition of granzyme B activity. For example, while not being bound by theory, if the activity of granzyme B is hindered, degradation of decorin is inhibited, with the result that degradation of type I collagen by MMP-1 is inhibited.

Collagen is also protected by inhibition of granzyme B gene expression. While not being bound by theory, by inhibiting the increase in expression of granzyme B, the activity of granzyme B can be more effectively inhibited.

However, it is apparent that inhibition of the activity of granzyme B and inhibition of the expression of granzyme B gene by the inhibitor of the present invention, and thus the protection of collagen obtained are not necessarily limited to the above-mentioned substances associated with inhibition of degradation of decorin.

Granzyme B is known to degrade various extracellular matrices in addition to decorin (Russo et al, sci rep., 2018 Jun 26.8 (1), 9690, turner et al, matrix biol 2019 Jan, 75-76, 126-140, pardo et al, cell Death differ., 2007 Oct, 14 (10), 1768-79). For example, examples of the substrate for granzyme B other than decorin include: collagen VII, collagen XVII, α6/β4 integrin, fibronectin, vitronectin, laminin, biglycan, plasminogen, VE-cadherin, ZO-1 and von Willebrand factor.

< Geranium extract >

The inhibitor of the present invention contains geranium extract as an active ingredient.

The herba Erodii seu Geranii extract is extracted from herba Erodii seu Geranii (Geranium thunbergii Siebold et Zuccarini (Geraniaceae). Geranium is a perennial plant of Geranium genus of Geraniaceae family, also called as Miao コ, di Lara etc., and is used as raw material of crude drug (Chinese herbal medicine) having intestinal tract regulating effect. Geranium extract is a known substance, which is readily available or modulatable on the market. In preparing the geranium extract, a method known in the art can be used, and for example, an ethanol solution or a 1, 3-butanediol solution can be used to extract the aerial parts of geranium, thereby obtaining a substance satisfying the standards of geranium extract described in the medical department external material standard 2006.

The inhibitor of the present invention preferably contains 1 to 100 relative to the total volume of the granzyme B inhibitorμg/mL geranium extract. The inhibitor of the present invention preferably contains 1.8 relative to the total volume of the granzyme B inhibitorμMore preferably, the geranium extract contains more than 5.5 g/mLμThe content of the catalyst is preferably at least 18 g/mLμg/mL or more. The upper limit of the content is not particularly limited, and for example, 80μg/mL or less, 90μg/mL or less, 95μg/mL or less. Because the geranium extract has special odor, the geranium extract has the effect of acting as a granzyme B inhibitorThe content of the odor of the geranium extract can be suppressed while the fruits are, for example, desirably 18 to 55μg/mL。

< additional plant extract >)

The inhibitor of the present invention may further contain, as an active ingredient, 1 or more plant extracts having a granzyme B inhibitory effect (hereinafter, referred to as "additional plant extracts") in addition to the geranium extract.

The plant extract to be added having the granzyme B inhibitory activity is not limited, and for example, the evaluation system of granzyme B activity inhibitory effect using a fluorogenic substrate can be used according to the examples]Selection showed granzyme B blocking activity (IC ₅₀ Value of<3μg/mL).

Examples of the additional plant extract include: is selected from the group consisting of pomegranate peel extract, himalayan Huang Shumei root extract, eucalyptus extract, garden burnet extract, acer palmatum leaf extract, elm extract, witch hazel extract, rose extract, avocado extract, sweet tea extract, evening primrose extract, oolong tea extract, cinnamon bark extract, saxifraga extract, melissa extract, potentilla extract, rosa damascena flower extract, artemisia kuhakui extract, coriander extract, lithospermum extract, hypericum perforatum extract, peppermint extract, grape leaf extract, tilia japonica extract, rose fruit extract, loquat leaf extract, seaweed extract, paeonia lactiflora extract, houttuynia cordata extract, crataegus cuneata extract, perilla extract, lavender extract, wild rose extract, catechol gambir extract, xiang Liangbu seaweed extract, peony extract, filiform mesquite extract, licorice extract and kuh-seng extract. These extracts, either alone or in combination, can be used as additional plant extracts.

The additional plant extract is preferably: plant extracts which, although also having an inhibitory effect on granzyme B alone, exert a synergistic granzyme B inhibitory effect when combined with geranium extract. Examples of the additional plant extract that exhibits a synergistic effect with the geranium extract include: herba Hyperici perforati extract and/or folium hydrangeae strigosae extract.

< Hypericum perforatum extract >)

The herba Hyperici perforati extract is extracted from herba Hyperici perforati (Hypericum perforatum Linne) or herba Hyperici perforati (Hypericum erectum Thunberg (Guttiferae, guttiferae)) of Guttiferae. Hypericum perforatum and Hypericum perforatum are perennial plants of Hypericum genus of Hypericum of Hyperiaceae. Hypericum perforatum extract is a known substance that is readily available or modulatable on the market. In preparing the Hypericum perforatum extract, a method known in the art can be used, and for example, water, ethanol, propylene glycol, 1, 3-butanediol, diethylene glycol, or a mixture thereof can be used to extract Hypericum perforatum or aerial parts of Hypericum perforatum, thereby obtaining a material satisfying the standards of Hypericum perforatum extract described in the medical department external raw material standard 2006.

< sweet tea extract >)

The sweet tea extract is an extract extracted from sweet tea raspberry (Rubus suavissimus Shugan lee (Rosaceae)). The Rubus suavissimus seed is cold-resistant fallen leaf shrub of Rubus genus of Rosaceae family. Sweet tea extracts are known substances that are readily available or modulatable on the market. For example, the sweet tea extract can be prepared by immersing leaves of sweet tea in boiled water (hot water), drying the leaves, heating the dried leaves, and extracting the dried leaves with boiled water (hot water), thereby obtaining a substance satisfying the standard of the sweet tea extract described in the medical department external raw material standard 2006. In addition, 1, 3-butanediol may be used as the extraction solvent.

In addition to the Hypericum perforatum extract and the sweet tea extract, additional plant extracts are also known materials and are readily available on the market or can be prepared by methods well known in the art.

The amount of the additional plant extract to be blended in the inhibitor of the present invention is not particularly limited, for example, in the case where the additional plant extract and geranium extract are mixed in an amount of 1:1 to 1:4, it is possible to inhibit granzyme B with a particularly high efficiency. The mass ratio of the additional plant extract to the geranium extract is, for example, 1:1.5, preferably 1: 2. further preferably 1: 3. particularly preferably 1:4.

in the case of blending the extract of Hypericum perforatum as an additional plant extract, the mass ratio of Hypericum perforatum extract to Geranium extract is preferably 1:1 to 1:4, a high synergistic effect can be obtained at this concentration ratio.

In the case of blending sweet tea extract as additional plant extract, the mass ratio of sweet tea extract to geranium extract is preferably 1:1 to 1:4, a high synergistic effect can be obtained at this concentration ratio.

The inhibitor of the present invention contains, for example, 1 to 100% relative to the total volume of the inhibitorμg/mL of additional plant extract.

In the case of containing an extract of Hypericum perforatum as a supplemental plant extract, it is preferably contained in an amount of 1.5 relative to the total volume of the inhibitorμMore preferably, the extract of Hypericum perforatum contains more than 4.6 g/mLμThe content of the catalyst is more than g/mL, particularly preferably 15μg/mL or more. The upper limit of the content is not particularly limited, and for example, 80μg/mL or less, 90μg/mL or less, 95μg/mL or less. If blended in this concentration range, the inhibitor can exert a higher inhibition effect of granzyme B activity.

In the case of containing sweet tea extract as additional plant extract, it is preferable to contain 3.1 relative to the total volume of the inhibitorμMore preferably, the sweet tea extract contains 10 g/mL or moreμThe content of the catalyst is more than g/mL, particularly preferably 31μg/mL or more. The upper limit of the content is not particularly limited, and for example, 80μg/mL or less, 90μg/mL or less, 95μg/mL or less. If blended in this concentration range, the inhibitor can exert a higher inhibition effect of granzyme B activity.

< arbitrary component >)

In the inhibitor of the present invention, 1 or more components may be optionally blended in addition to the geranium extract and the additional plant extract within a range not to impair the effects of the present invention. Further, any component may be a component which is applicable to a cosmetic composition described later. The following components may be mentioned as optional components.

[ basic agent component ]

Water (e.g., purified water), lower alcohols (e.g., ethanol), polyols (e.g., propylene glycol), oils, surfactants, ultraviolet absorbers, ultraviolet scattering agents, thickeners, pigments, and the like.

[ others ]

Emulsifying agents, fragrances, preservatives, and the like.

[ moisture-keeping ingredient ]

Tocopherol, sodium hyaluronate, akebia trifoliata (Akebia trifoliata) stem extract, hydrolyzed elastin, hydrolyzed collagen, rehmannia glutinosa (Rehmannia chinensis root) extract, sodium hyaluronate, arnica (Arnica montana flower) extract, aloe vera (Aloe barbadatsis) leaf extract, scutellaria baicalensis (Scutellaria baicalensis) extract, orchid (Orchid) extract, ginseng (Panax gineng) root extract, chamomile (Matricaria chamomilla) flower extract, cucumber (cufmis sativus) fruit extract, pueraria (purearia lobata) extract, chlorella (Chlorella) extract, hedera helix (Hedera helix) leaf/stem extract, salix alba (Salix alba) bark extract, sambucus nigra (Sambucus nigra) flower extract Malva sylvestris (Malva sylvestris) extract, swertia (Swertia) extract, soybean extract, calendula (Calendula officinalis) extract, pyridoxine trihexyldecanoate, wall grass (Parietaria officinalis) extract, retinol palmitate, tilia cordata (Tilia cordata) flower extract, purple soybean (Macroptilium atropurpureum) flower/leaf/stem extract, cornflower (Centaurea cyanus) flower extract, chamomile (Anthera nobilis) flower extract, royal jelly (Royal jelly) extract, hydrolyzed coix seed, coptis (Coptis japonica) extract, artemisia capillaris (Artemisia capillaris) extract, phellodendron (Phellodendron amurense) bark extract, gardenia (Gardenia jasminoides) fruit extract, dipotassium glycyrrhizinate, rhizoma Dioscoreae Bulbiferae (root/stem extract, rhizoma Ligustici Chuanxiong root extract, radix Angelicae sinensis root extract, herba Houttuyniae extract, fructus Jujubae extract, coicis semen extract, betaine, jojoba esters, hydrolyzed hyaluronic acid, hydrogenated retinol, and water-soluble proteoglycan.

Dosage form >, dosage form

The dosage form of the inhibitor of the present invention is not particularly limited. Examples include: solutions, solubilizations, emulsions, powder dispersions, water-oil two-layer, water-oil-powder three-layer, ointments, gels, aerosols, and the like. The inhibitor of the present invention can be formulated into a desired dosage form by appropriately combining any of the above-described components and blending.

< manufacturing method >)

The inhibitors of the present invention may be manufactured according to manufacturing methods commonly employed in the art. Examples of the production method include: the conventional manufacturing method of skin care products (skin cosmetics) is not limited thereto. For example, the composition can be produced by adding the active ingredient to the base material component and mixing the same.

< method of use >)

The inhibitor of the present invention can be applied to a subject by application or the like. Suitable subjects for the inhibitors of the present invention are generally mammals, and are specifically contemplated as being human beings.

[ collagen degradation inhibitor ]

As described above, geranium extract inhibits collagen degradation by inhibition of granzyme B activity and inhibition of granzyme B gene expression. Thus, the granzyme B inhibitors of the present invention can also be understood as collagen degradation inhibitors.

As a scheme for inhibition of collagen degradation caused by the collagen degradation inhibitor of the present invention, there can be mentioned: inhibition of granzyme B activity and inhibition of granzyme B gene expression.

Description of granzyme B inhibitors the active ingredient, optional ingredient, dosage form, manufacturing method and method of use of the collagen degradation inhibitor suitable for use in the present invention.

[ cosmetic composition ]

The cosmetic composition of the present invention consists only of the granzyme B inhibitor. The mode of use of the cosmetic composition of the present invention is not particularly limited. Examples include: lotions, creams, lotions, essences, gels, ointments, foundations, masks, and the like.

The cosmetic composition of the present invention can be applied to the skin of a subject in need of maintenance of collagen by application or the like. Suitable subjects for the granzyme B inhibitors of the present invention are generally mammals, particularly contemplated as humans.

Description of granzyme B inhibitors the present invention is applicable to the active ingredient, optional ingredient, formulation and manufacturing method of the cosmetic composition of the present invention.

[ pharmaceutical composition ]

The granzyme B inhibitor of the present invention can also be used for medical use. The diseases to be treated include: the pharmaceutical composition comprising the granzyme B inhibitor of the present invention can be administered to a subject by applying or the like to a subject, such as wounds, burns, autoimmune dermatitis, chronic wounds, keloids (keloids), hypertrophic scars, discoid lupus erythematosus (Discoid lupus erythematosus), alopecia areata, scleroderma, stevens-Johnson syndrome, toxic epidermal necrosis and the like, thereby improving the symptoms of the disease. Suitable subjects for pharmaceutical compositions are generally mammals, particularly contemplated as being humans.

The description of the granzyme B inhibitor applies to the active ingredient, optional ingredient, dosage form and manufacturing method of the pharmaceutical composition.

Examples

Next, the effects of the present invention will be specifically described by examples, but the present invention is not limited to the examples.

[ preparation of plant extract ]

Each plant extract was prepared as follows.

The geranium extract was prepared by the method described in the pharmaceutical sector external material standard 2006 and used in the following experiments. In the extraction, the aerial parts (flowers, leaves and stems) of geranium were used, and a 1, 3-butanediol solution was used as an extraction solvent.

The Hypericum perforatum extract was prepared by the method described in the medical department external raw material standard 2006 and used in the following experiments. In the extraction, aerial parts (flowers, leaves and stems) of Hypericum perforatum were used, and 1, 3-butanediol solution was used as the extraction solvent.

The sweet tea extract was prepared by the method described in the pharmaceutical sector external material standard 2006 and used in the following experiments. In the extraction, sweet tea leaves are used, and boiled water (hot water) is used as an extraction solvent.

[ measurement System Using human epidermal keratinocytes ]

Human epidermal keratinocytes were cultured in accordance with the following procedure to prepare a human epidermal monolayer model.

At 37℃with 5% CO ₂ In an incubator at 75cm ² Human epidermal keratinocytes were cultured in a Flask (flash, flask) using D-MEM (high glucose) (manufactured by Fuji film and Wako pure chemical industries, ltd., hereinafter referred to as D-MEM medium) containing 10% FBS and 1% PS mix, which had been mixed with L-glutamine and phenol red, until 70 to 90% confluence. Cells were peeled off using Ackutase solution (manufactured by Sigma-Aldrich Co.), and inoculated on a 24-well transparent plate (manufactured by Corning Co.) to 1.5X10 ⁴ Individual cells/cm ² . D-MEM Medium was used at 37℃with 5% CO ₂ The cells were cultured in the incubator for 3 days, and the cultured cells were used as a human epidermis monolayer model for measurement.

[ evaluation System for inhibition effect of expression of granzyme B Gene ]

It is known that the expression level of granzyme B gene is low in skin in a normal state, and the expression is induced by ultraviolet (UV-B) irradiation. Therefore, geranium extract was added to the human epidermis monolayer model after UV-B irradiation, and the inhibition effect of granzyme B gene expression induction by UV-B irradiation was evaluated.

UV-B (wavelength: 302nm, cumulative irradiation amount: 10 mJ/cm) was irradiated to the human epidermis monolayer model ² ) Then, geranium extract dissolved in D-MEM was added, and the gene was extracted from the test cells after 16 hours of culture, and the mRNA amount was measured by a real-time PCR method. The amount of expressed granzyme B gene was calculated by the ΔΔct method using GAPDH as an internal standard substance.

The expression inhibition effect of geranium extract on granzyme B gene was evaluated as follows: the UV-B non-irradiated group was used as a control, and the control value was calculated, and the UV-B irradiated geranium extract non-treated group and treated group were compared by a significant difference test (Dunnett multiple comparison test), thereby performing evaluation.

[ inhibition effect of Geranium extract on expression of granzyme B Gene ]

The expression inhibition effect of the geranium extract on the granzyme B gene is shown in figure 1.

As shown in FIG. 1, the geranium extract inhibited the increase in the expression of granzyme B gene induced by UV-B stimulation.

[ evaluation System for inhibition of granzyme B Activity Using decorin ]

Granzyme B degrades various extracellular matrices and participates in skin disorders. Among them, granzyme B is known to degrade decorin, thereby promoting type I collagen degradation caused by MMP-1. Therefore, a plant extract and granzyme B were added to decorin, and the granzyme B activity blocking effect by the plant extract was evaluated.

Recombinant granzyme B (final concentration 100ng +.μL、R&D systems Co.), cathepsin C recombinant (final concentration 10ng +.μL、R&D systems Co., ltd.), CHAPS (final concentration 0.01%), DTT (final concentration 5 mM), naCl (final concentration 50 mM), MES (final concentration 50mM, pH 5.5) were mixed and reacted at 37℃for 16 hours to activate granzyme B. Recombinant decorin (final concentration 10ng +.μL, abcam), plant extract, activated granzyme B (final concentration 2.5ng +.μL) and Tris-HCl (final concentration 50mM, pH 7.5) were mixed and reacted at 37℃for 20 to 22 hours. The reacted solution was subjected to protein separation by SDS-PAGE, and then, proteins were detected using silver staining reagent (manufactured by Cosmo Bio Inc.), and the protein amount of the degradation product fragment of decorin was quantified.

The inhibition effect of granzyme B activity was evaluated as follows: the control value was calculated using the plant extract untreated group as a control, and the plant extract untreated group and the treated group were compared by a significant difference test (Dunnett multiple comparison test) to evaluate.

[ inhibition effect of granzyme B Activity by Geranium extract ]

In the above evaluation system using the effect of blocking granzyme B activity of decorin, geranium extract as a plant extract was treated and granzyme B activity was evaluated. The evaluation results are shown in FIG. 2.

As shown in fig. 2, geranium extract blocks the activity of granzyme B, thereby inhibiting degradation of decorin.

[ evaluation System for inhibition effect of granzyme B Activity Using fluorogenic substrate ]

Granzyme B activity blocking effect was evaluated on various plant extracts in addition to the plant extracts shown in fig. 3, using N-acetyl-Ile-Glu-Pro-Asp-7-amino-4-methylcoumarin (Ac-IEPD-AMC, sigma-Aldrich) as a fluorogenic substrate.

The granzyme B recombinant was activated in the same manner as in the evaluation system for the inhibition effect of granzyme B activity using decorin. Ac-IEPD-AMC (final concentration 50) was added to 384 Kong Heiban (manufactured by Corning Co., ltd.)μM), plant extract, activated granzyme B (final concentration 0.3ng +.μL), CHAPS (final concentration 0.01%) and HEPES (final concentration 50mM, pH 7.5), at 30℃for 60 minutes. Fluorescence intensity was measured immediately after addition of the solution (R1) and after reaction (R2) (ex/em=380/460 nm).

The granzyme B activity was calculated using the following calculation formula.

Control% (%) =fluorescence value of plant extract treated group (R2-R1)/fluorescence value of plant extract untreated group (R2-R1) ×100

By calculating 50% blocking concentration (IC) from% of control value ₅₀ Value), and the inhibition effect of the granzyme B activity was evaluated.

[ inhibition effect of granzyme B Activity produced by various plant extracts ]

In the above evaluation system using fluorogenic substrate, the granzyme B inhibitory activity (IC ₅₀ Value of<3μg/mL) is shown in FIG. 3.

As shown in fig. 3, various plant extracts other than geranium extract block the activity of granzyme B.

[ inhibitory Effect of granzyme B Activity produced by Hypericum perforatum extract and sweet tea extract ]

In the above evaluation system using decorin, the inhibition effect of granzyme B activity was evaluated by treating the extract of hypericum perforatum and the extract of rubus suavissimus as plant extracts, respectively. The evaluation results are shown in FIG. 4.

As shown in fig. 4, the extracts of Hypericum perforatum and sweet tea, like the extracts of Geranium, also inhibit degradation of decorin by blocking the activity of granzyme B.

[ evaluation System of synergistic Effect of inhibition of degradation of decorin based on granzyme B inhibition ]

The test was performed in the same manner as in the evaluation system for the inhibition effect of the activity of granzyme B using decorin.

The inhibition effect of degradation of decorin based on granzyme B inhibition was evaluated as follows: the control value was calculated using the untreated plant extract group as a control, and each group was compared by a significant difference test (Tukey multiple comparison test) to evaluate the plant extract.

Regarding the synergistic effect, the degradation inhibition ratio of decorin (100% of control value) (%) was calculated, and the ratio of the theoretical value obtained by adding the degradation inhibition ratios of decorin after the individual treatment of the plant extracts was calculated from the actual measurement value of the degradation inhibition ratio of decorin after the simultaneous treatment of the plant extracts, and evaluated.

Examples:

synergistic effect = decorin degradation inhibition ratio when plant extracts a and B were treated simultaneously (measured value)/[ decorin degradation inhibition ratio when plant extract a was treated alone + decorin degradation inhibition ratio when plant extract B was treated alone ] (theoretical value)

[ synergistic Effect of Geranium extract and Hypericum perforatum extract in inhibition of degradation of decorin ]

The evaluation results of the synergistic effect of degradation inhibition of decorin produced by geranium extract and Hypericum perforatum extract are shown in FIG. 5.

As shown in fig. 5, the simultaneous treatment of the Hypericum perforatum extract and the Geranium extract enhances the decorin degradation inhibition effect compared to the theoretical value obtained by adding the decorin degradation inhibition effects alone. In addition, the ratio of the adding amount of the herba Hyperici Erecti extract to the adding amount of the herba Erodii seu Geranii extract is 1:4, a higher synergistic effect is obtained.

[ synergistic Effect of Geranium extract and sweet tea extract in inhibition of degradation of decorin ]

The evaluation results of the synergistic effect of degradation inhibition of decorin produced by geranium extract and sweet tea extract are shown in fig. 6.

As shown in fig. 6, the simultaneous treatment of sweet tea extract and geranium extract enhanced the decorin degradation inhibition effect compared to the theoretical value obtained by adding the decorin degradation inhibition effects alone. In addition, the addition amount ratio of sweet tea extract to geranium extract is 1:4, a higher synergistic effect is obtained.

Industrial applicability

The present invention can be used in cosmetic compositions, pharmaceutical compositions, etc.

Claims

1. Use of geranium extract and herba Hyperici perforati extract as active ingredients only for the manufacture of a composition for inhibiting skin disorders due to collagen degradation, wherein collagen degradation is collagen degradation promoted by degradation of decorin based on granzyme B, the composition comprising only components in a mass ratio of 1:1 to 1:4 and herba Erodii seu Geranii extract as effective components.

2. Use of geranium extract and sweet tea extract as active ingredients only for the manufacture of a composition for inhibiting skin disorders due to collagen degradation, wherein collagen degradation is collagen degradation promoted by degradation of a granzyme B-based decorin, the composition comprising only components in a mass ratio of 1:1 to 1:4 and herba Erodii seu Geranii extract as effective components.

3. The use according to claim 1 or 2, wherein the concentration of geranium extract in the composition is 1-100 μg/mL.

4. The use according to claim 3, wherein the concentration of geranium extract in the composition is 1.8-55 μg/mL.

5. The use according to claim 4, wherein the concentration of geranium extract in the composition is 18-55 μg/mL.

6. The use according to any one of claims 1, 2, 4 and 5, wherein the inhibition of degradation of the granzyme B-based decorin is inhibition of expression of granzyme B gene.

7. The use of any one of claims 1, 2, 4 and 5, wherein inhibition of degradation of granzyme B-based decorin is a block in granzyme B activity.

8. The use according to any one of claims 1, 2, 4 and 5, wherein inhibition of degradation of the granzyme B-based decorin is inhibition of granzyme B gene expression and inhibition of granzyme B activity.