JP2021176855A - Composition for protein l-isoaspartate methyltransferase activation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To evaluate a composition for activating PIMT capable of activating protein L-isoaspartate methyltransferase (PIMT), a new index for evaluating skin restoration or repair activity, and skin anti-aging activity. To provide a new index for this purpose, a method for evaluating skin restoration or repair activity using this index, a method for evaluating skin anti-aging activity, and the like.
SOLUTION: Kabanoki family Kabanoki genus plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus plant, Shobu family Shobu genus plant, Kiku family Arnica genus plant and A composition for activating protein L-isoaspartate methyl transferase (PIMT), which comprises an extract of one or more plants selected from the group consisting of plants of the genus Peach of the family Rose family.
[Selection diagram] None

Description

The present invention relates to a composition for activating protein L-isoaspartate methyltransferase (PIMT). The present invention also relates to compositions for promoting restoration or repair of proteins containing isomerized amino acids. The present invention further relates to a method for activating PIMT and a method for promoting restoration or repair of a protein containing an isomerized amino acid. The present invention also relates to a method for evaluating skin restoration or repair activity, a method for evaluating skin anti-aging activity, and a method for screening an active ingredient for activating PIMT present in skin.

Keeping the skin healthy and youthful is a matter of great concern to many. However, internal factors such as deterioration of various skin functions due to aging and external factors such as exposure to ultraviolet rays cause skin aging phenomena such as wrinkles, sagging, lack of firmness, dullness, and skin roughness. Is done. In particular, skin damage caused by ultraviolet rays, which is an external factor, is accumulative and has a great effect on the skin, such as accelerating the progress of skin aging due to aging and making aging more serious.

L-type aspartic acid (L-Asn) residues and aspartic acid (L-Asp) residues in proteins are prone to isomerization, resulting in D-type aspartic acid (D-Asp) residues, D-type or D-type or It is known that L-type isoaspartic acid (D- or L-isoAsp) residues are produced. It has been suggested that the isomerization of amino acids in this protein impairs the function of the protein and may cause the onset of diseases such as Alzheimer's disease and various aging phenomena. Non-Patent Document 1 describes that D-Asp is detected in tissues such as the skin of the elderly, the crystalline lens of the eye, bones, and teeth, and that D-Asp is detected in the skin tissue from an ultraviolet-exposed area. ing.

Protein L-isoaspartic acid methyltransferase (PIMT) has been found in animal brain tissues, erythrocyte cells, and the like. PIMT specifically recognizes the free α-carboxyl group of L-isoAsp or the free β-carboxyl group of D-Assp, and the methyl group transfer from the coenzyme S-adenosyl-L-methionine (SAM). It is an enzyme that catalyzes the reaction and promotes the conversion of L-isoAsp and D-Asp in proteins to L-Asp. It has been reported that isomerized aspartic acid residue-containing proteins are increased in the brain, heart, liver and erythrocytes in PIMT knockout mice (Non-Patent Document 2).

Fujii N. Biol Pharm Bull 28 (9): 1585-1589 (2005) Kim E. et al., Proc. Natl. Acad. Sci. U.S.A., 94, 6132-6137 (1997)

An increase in proteins (isomerized proteins) containing isomerized aspartic acid (isomerized Aspart) such as D-Asp and L-isoAsp (L-β-Asp) in the body due to aging and exposure to ultraviolet rays, that the amino acids Since the three-dimensional structure of the protein is changed by isomerization and the original function is impaired, it is considered that the isomerization of amino acids in the protein is also related to the aging of skin and the like. For example, if the conversion of an isomerized amino acid residue in a skin protein to a normal L-amino acid can be promoted, the function of the protein denatured by the isomerization can be restored, and by extension, the isomerization can restore the function of the denatured protein. It is considered that the impaired various functions of the skin can be restored.

However, the repair mechanism of isomerized proteins in the skin has not been clarified. In addition, no substance having an action of promoting restoration or repair of a protein containing the isomerized amino acid in the skin has been reported.

An object of the present invention is to provide a composition for activating PIMT capable of activating protein L-isoaspartate methyltransferase (PIMT).

Another object of the present invention is a new index for evaluating skin restoration or repair activity, a new index for evaluating skin anti-aging activity, and a new index for evaluating skin restoration or repair activity using this index. It is to provide the evaluation method and the evaluation method of the anti-aging activity of the skin.

As a result of diligent research to solve the above problems, the present inventor has conducted protein L-isoaspartate methyltransferase (hereinafter, also referred to as PIMT), which is an enzyme that promotes restoration or repair of isomerized protein in human skin cells. ) Was expressed and contributed to the mechanism of isomerized protein repair in the skin. The expression of PIMT in skin cells is the first finding found by the present inventor. The present inventor has also found that when a skin-derived protein is irradiated with ultraviolet rays, isomerization of Asp in the protein is induced and the isomerized protein increases. Furthermore, the present inventor has found that the expression of PIMT in skin cells decreases due to factors such as aging and exposure to ultraviolet rays. From these findings, it was clarified that the activity of PIMT in the skin decreases due to factors that cause skin aging such as aging and ultraviolet rays, and by activating PIMT existing in the skin, the skin denatured by the isomerization of amino acids. We came up with the idea that protein restoration or repair can be promoted. Based on these findings, we conducted research on substances that enhance the activity of PIMT, and found that extracts of specific plants are effective in activating the PIMT.

The present inventor has also come up with the idea that the skin restoration or repair activity and the skin anti-aging activity can be evaluated using the expression level of PIMT in the skin or its activity as an index. Furthermore, by using the expression level or activity of PIMT in skin cells as an index, it was conceived that substances effective for skin activation, skin restoration or repair, and skin anti-aging can be screened.

That is, the present invention includes the following composition for activating PIMT, a method for evaluating skin restoration or repair activity, and the like.
The composition for activating the protein L-isoaspartate methyl transferase (PIMT) of the present invention is a plant of the genus Kabanoki of the family Kabanoki, a plant of the genus Vineaceae, a plant of the genus Soybean of the family Mame, a plant of the genus Karahanasou of the family Asa, a plant of the family Perilla, and a citrus. Includes extracts of one or more plants selected from the group consisting of family Mikan plants, Shobu family Shobu plants, Kiku family Arnica plants and Rose family peach genus plants.
The above-mentioned plant extract has an action of activating PIMT.
The PIMT activation composition of the present invention is preferably a PIMT activation composition present on the skin. The plant extracts are useful for the activation of PIMT (in the skin) present in the skin.
The above plant extract is preferably an extract of one or more plants selected from the group consisting of white birch, grape, soybean, hop, lavender, sage, enmeisou, yuzu, perilla, iris, arnica and peach. .. Extracts of these plants are preferable because they have a high effect of activating PIMT. Since the action of activating PIMT is particularly high, the extract of the above plant is more likely to be an extract of one or more plants selected from the group consisting of birch, grape, soybean, lavender, sage and emmeiso. preferable.

The composition for activating PIMT of the present invention is preferably a skin external preparation, more preferably a cosmetic. Moreover, as an example of another preferable embodiment in the present invention, it is also preferable that the composition for activating PIMT is a food or drink.

The composition for promoting restoration or repair of a protein containing an isomerized amino acid of the present invention includes a plant belonging to the genus Kabanoki, a plant belonging to the genus Grape, a plant belonging to the genus Soybean, a plant belonging to the genus Karahanasou, a plant belonging to the family Shiso, and a family of Mikan. It contains an extract of one or more plants selected from the group consisting of plants of the genus Mikan, plants of the genus Shobu of the family Shobu, plants of the genus Arnica of the family Kiku, and plants of the genus Peach of the family Rose.
The composition for promoting restoration or repair of a protein containing an isomerized amino acid of the present invention is preferably a composition for promoting restoration or repair of a protein containing an isomerized amino acid in the skin.
As the extract of the above-mentioned plant, an extract of one or more plants selected from the group consisting of birch, grape, soybean, hop, lavender, sage, emmeisou, yuzu, perilla, iris, arnica and peach is preferable. An extract of one or more plants selected from the group consisting of, grape, soybean, lavender, sage and birch is more preferred. Since the extracts of these plants have a high effect of activating PIMT, they have a high effect of promoting restoration or repair of proteins containing isomerized amino acids.
The protein restoration or repair promoting composition of the present invention is preferably a skin external preparation, more preferably a cosmetic.
Moreover, as an example of another preferable aspect in the present invention, it is also preferable that the composition for promoting restoration or repair of the protein of the present invention is a food or drink.

The present invention relates to plants of the genus Kabanoki of the family Kabanoki, plants of the genus Vineaceae, plants of the genus Soybean of the family Mame, plants of the genus Karahanasou of the family Asa, plants of the family Perilla, plants of the genus Mikan of the family Mikan, plants of the genus Shobu of the family Shobu, plants of the genus Arnica of the family Kiku. It also includes a method of activating protein L-isoaspartate methyl transferase (PIMT), which applies an extract of one or more plants selected from the group consisting of plants of the genus Peach of the family Rose family to animals.

The present invention relates to plants of the genus Kabanoki of the family Kabanoki, plants of the genus Vineaceae, plants of the genus Soybean of the family Mame, plants of the genus Karahanasou of the family Asa, plants of the family Perilla, plants of the genus Mikan of the family Mikan, plants of the genus Shobu of the family Shobu, plants of the genus Arnica of the family Kiku. It also includes methods for promoting restoration or repair of proteins containing isomerized amino acids by applying an extract of one or more plants selected from the group consisting of plants of the genus Peach of the family Rose family to animals.

The present invention also includes methods for promoting restoration or repair of proteins containing isomerized amino acids in the skin, which activate the protein L-isoaspartate methyltransferase (PIMT) present in human skin.

The method for evaluating skin restoration or repair activity of the present invention uses the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index.
In the method for evaluating skin restoration or repair activity of the present invention, it is preferable that the skin cells are epidermal cells and / or dermis cells.
Examples of the skin restoration or repair activity include skin restoration or repair activity against aging of the skin due to ultraviolet rays and / or aging. The evaluation method of the present invention is suitably used for evaluating the restoration or repair activity of the skin against the isomerization of amino acids due to ultraviolet rays and / or aging.

The method for evaluating the anti-aging activity of the skin of the present invention uses the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index.
In the method for evaluating the anti-aging activity of the skin of the present invention, it is preferable that the anti-aging activity is the anti-aging activity against the aging of the skin due to ultraviolet rays and / or aging. The method for evaluating the anti-aging activity of the skin of the present invention is suitably used for evaluating the anti-aging activity against aging of the skin due to ultraviolet rays and / or aging.

The present invention uses the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index to activate PIMT present in the skin, promote skin restoration or repair, or anti-aging of the skin. Also includes methods for screening active ingredients for.
In the screening method of the present invention, by using the expression level or activity of PIMT in skin cells as an index, a substance having an act of activating PIMT present in the skin, a substance having an action of promoting restoration or repair of the skin, or skin. Substances having an anti-aging effect can be screened.

The screening method of the present invention comprises contacting the skin cells with a test substance to measure the expression level or activity of PIMT in the skin cells, and
When the expression level or activity of PIMT in the skin cells contacted with the test substance is larger than the expression level or activity of PIMT in the skin cells not contacted with the test substance, the test substance is used as an active ingredient. It is preferable to include a step of selection.
The skin cells are preferably cultured skin cells.

The use of the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index of skin restoration or repair activity or skin anti-aging activity is also one of the present inventions. The expression level or activity of PIMT in the skin is useful as an index of skin restoration or repair activity and an index of skin anti-aging activity.

The present invention also includes the following uses.
For activation of protein L-isoaspartate methyl transferase (PIMT), Kabanoki family Kabanoki plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus Use of an extract of one or more plants selected from the group consisting of plants, plants of the family Shobu, plants of the genus Arnica of the family Kiku and plants of the genus Peach of the family Rose.
To restore or promote the restoration or repair of proteins containing isomerized amino acids, plants of the genus Kabanoki, plants of the genus Grape, plants of the genus Soybeans of the family Asa, plants of the genus Karahanasou of the family Asa, plants of the genus Mikan of the family Mikan, Use of an extract of one or more plants selected from the group consisting of the plants of the genus Shobu, the genus Arnica of the family Kiku, and the plants of the genus Peach of the rose family.

According to the present invention, it is possible to provide a composition for activating PIMT, which can activate PIMT. By activating PIMT present in the skin according to the present invention, it is possible to promote the restoration or repair of proteins containing isomerized amino acids in the skin.

Further, according to the present invention, it is possible to provide a new index for evaluating the restoration or repair activity of the skin and a new index for evaluating the anti-aging activity of the skin. According to the present invention, it is possible to provide a method capable of evaluating skin restoration or repair activity and a method capable of evaluating skin anti-aging activity using the expression level or activity of PIMT in skin cells as an index. can. INDUSTRIAL APPLICABILITY According to the present invention, it is possible to objectively evaluate the restoration or repair activity of the skin or the anti-aging activity of the skin, and it may contribute to the selection of an appropriate treatment for the symptoms related to the aging of the skin. .. In addition, by using the expression level or activity of PIMT in skin cells as an index, a substance having an activating effect of PIMT existing in the skin, a substance having an effect of promoting restoration or repair of the skin, and a substance having an anti-aging effect of the skin. Can be screened.

FIG. 1 is a graph showing the expression level of PIMT in skin cells. FIG. 2 is a graph showing the amount of SAH produced per amount of protein by irradiation with ultraviolet B wave (UVB). FIG. 3 is a graph showing the expression level of PIMT in skin fibroblasts having different passage numbers. FIG. 4 is a graph showing changes in the expression level of PIMT in skin fibroblasts due to stress loading.

In the present specification, the isomerized amino acid (also referred to as an isomerized amino acid) is an isomer of L-aspartic acid produced by isomerization of L-aspartic acid (L-Assp) and L-asparagine (L-Asn). It means (isomerized aspartic acid).
The isomerized aspartic acid (isomerized Aspartic acid) is more specifically D-aspartic acid (D-Assp), D-type or L-type isoaspartic acid (D- or L-isoAsp), and is preferable. D-Asp and L-isoAsp. D-isoAsp and L-isoAsp are also referred to as D-β-Asp and L-β-Asp, respectively.

As used herein, isomerization of amino acids means that L-Asp or L-Asn becomes isomerized aspartic acid. A protein containing an isomerized amino acid (amino acid residue) is also called an isomerized protein. The isomerized amino acid (isomerized amino acid residue) in the isomerized protein may be one kind or two or more kinds.

When an amino acid in a protein is isomerized, the three-dimensional structure of the protein is changed and the original function is impaired. Therefore, the isomerized protein can be said to be a protein denatured by isomerization of amino acids. The isomerization of amino acids in this protein is thought to be associated with various aging phenomena and the like. By converting (restoring or repairing) the isomerized amino acids in the isomerized protein to L-Asp, the protein denatured by isomerization is restored to its original state (pre-isomerization state, pre-denaturation state), or , It can be restored to a state close to the original state. Restoration or repair of the isomerized protein in the present invention is to convert the isomerized Asp in the isomerized protein to L-Asp, or to bring the protein denatured by amino acid isomerization into the original state (before isomerization). It refers to restoration to a state (state, state before denaturation), or restoration to a state close to the original state. Further, the conversion of the isomerized Asp in the isomerized protein to L-Asp means that at least a part of the isomerized Asp in the isomerized protein is converted to L-Asp, and all the isomerized Asps are converted to L-Asp. It is not limited to converting to -Asp.

PIMT has the effect of promoting the conversion (restoration or repair) of isomerized amino acids in proteins to normal L-amino acids. More specifically, PIMT has an action of promoting the conversion of isomerized Asp L-isoAsp and D-Asp to L-Asp. Therefore, activation of PIMT can promote the restoration or repair of isomerized proteins. For example, by activating PIMT present in the skin, restoration or repair of isomerized proteins in the skin can be promoted. Thereby, the function of the skin protein impaired by the isomerization of L-Asp and / or L-Asn can be restored to the original state or a state close to the original state, and by extension, due to the isomerization of the amino acid. It is considered that the deterioration of various skin functions can be suppressed or the reduced skin functions can be restored.
The activation of PIMT is any action that enhances or promotes the action of PIMT, such as enhancement of expression of PIMT, promotion of expression of PIMT, suppression of decrease in expression of PIMT, recovery of expression of PIMT, and any action that suppresses the decrease in the action. Including.

<Composition for activating PIMT and composition for promoting restoration or repair of proteins containing isomerized amino acids>
The composition for activating PIMT of the present invention is a plant of the genus Kabanoki of the family Kabanoki, a plant of the genus Vineaceae, a plant of the genus Soybean of the family Mame, a plant of the genus Karahanasou of the family Asa, a plant of the genus Shiso, a plant of the genus Mikan of the family Mikan, a plant of the genus Shobu of the family Shobu. Includes extracts of one or more plants selected from the group consisting of plants of the genus Arnica of the family Kiku and plants of the genus Peach of the rose family.
The above-mentioned plant extract has an action of activating PIMT. Therefore, the extracts of these plants are preferably used as the active ingredient of the composition for activating PIMT. The plant extract is also effective for promoting restoration or repair of a protein containing an isomerized amino acid, and also as an active ingredient of a composition for promoting restoration or repair of a protein containing the isomerized amino acid. Suitable for use.
A composition for promoting restoration or repair of a protein containing an isomerized amino acid (hereinafter, also referred to as a composition for promoting restoration or repair of an isomerized protein) containing an extract of one or more of the above plants is also one of the present invention. Is one. The composition for activating PIMT and the composition for promoting restoration or repair of an isomerized protein of the present invention usually contain the above-mentioned plant extract as an active ingredient.
The composition for activating PIMT is suitable as the composition for activating PIMT present in the skin. The composition for promoting restoration or repair of an isomerized protein is suitable as a composition for promoting restoration or repair of a protein containing an isomerized amino acid in the skin.

In the composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention, the above-mentioned extracts of one or more kinds of plants can be used. When the composition for activating PIMT and the composition for promoting restoration or repair of an isomerized protein of the present invention contain extracts of two or more kinds of plants, they may belong to the same genus, and may belong to different genera. It may be a plant to which it belongs.
Examples of Birchaceae (Betulaceae Betula) plants include birch (Betula pendula or Betula alba).
Examples of Vitaceae Vitis plants include grapes (Vitis spp.). The grape varieties are not particularly limited, and examples thereof include black grapes such as Cabernet Sauvignon, Concord, Merlot, and Muscat Berry A; and white grapes such as Chardonnay and Koshu. Among them, black grapes are preferable, and Cabernet Sauvignon, Merlot and the like are more preferable.

Examples of the leguminous soybean genus (Fabaceae Glycine) plant include soybean (Glycine max).
Examples of the Cannabaceae Humulus plant of the Cannabaceae family include hops (Humulus lupulus).
Labiatae (Lamiaceae) plants include Lavandula such as Lavandula angustifolia; Salvia such as Salvia officinalis; Rabdosia japonica (Isodon japonicus) or Rabdosia trichocarpa (Isodon) trichocarpa (Isodon) ) And other Isodon; Perilla frutescens var. Crispa and other Perilla plants.
Examples of Rutaceae Citrus plants include Yuzu (Citrus junos).
Examples of Acoraceae Acorus plants in the Acoraceae family include Acorus calamus.
Examples of Asteroideae Arnica plants include Arnica montana.
Examples of Rosaceae Amygdalus plants include peaches (Amygdalus persica).

As the plant extract in the present invention, the plant of the genus Kabanoki of the family Kabanoki, the genus of grapes of the family of grapes, the plant of the genus Soybean of the family Mame, the plant of the genus Karahanasou of the family Asa, and the plant of the family Shiso (preferably plants belonging to the genus Lavandra, the genus Akigiri or the genus Yamahakka). , Mikan family Mikan genus plant, Shobu family Shobu genus plant, Rose family peach genus plant, and one or more plant extracts are preferable. Extracts of Kalahanasou plants and Perilla family plants are more preferable, and extracts of Kabanoki family Kabanoki spp.
Among them, the extracts of white birch, grape, soybean, hop, lavender, sage, emmeisou, yuzu, perilla, shobu, arnica, and peach are preferable because they have a high effect of activating PIMT existing in the skin and the like. Extracts of grapes, soybeans, hops, lavender, sage, emmeiso, shobu are more preferred, extracts of white birch, grapes, soybeans, hops, lavender, sage, emmeisou are more preferred, white birch, grapes, soybeans, lavender, sage, Extracts of sage are even more preferred, and extracts of sage, grapes, soybeans and sage are even more preferred. Among them, as plant extracts, birch extract, grape extract and soybean extract are particularly preferable, and birch extract and grape extract are most preferable. It is preferable to use extracts of one or more of these plants.

For the preparation of plant extracts, any part of the above plant, for example, roots, rhizomes, stems, leaves, branches, bark, flowers, seeds (including seed coat, germ, endosperm, hypocotyl, cotyledon, etc.), fruits (Including flesh and bark), or multiple combinations thereof can be used. A plant extract can be produced by extracting any part of the plant with a solvent.
Preferred sites for producing plant extracts include, for example, the following sites. As the plant extract in the present invention, an extract of the following part of the plant is preferable.
The bark of the birch of the genus Birch in the family Betulaceae is preferable.
The grapes of the genus Grapes are preferably fruits, seed coats and / or seeds, more preferably peels and seeds.
For soybeans of the genus Soybean of the leguminous family, it is preferable to use seeds, and germs (soybean buds) are more preferable. Hops of the genus Hops of the family Cannabaceae are preferably flowers (more preferably female spikes).
In the case of Labiatae plants, it is preferable to use flowers for lavender of the genus Lavandra, leaves for sage of the genus Sage and perilla of the genus Perilla, and leaves and / or stems for the perilla of the genus Isodon.
For Yuzu of the genus Citrus of the Rutaceae family, it is preferable to use fruits. For Acorus genus Acorus, it is preferable to use roots. For Arnica of the genus Arnica of the Asteraceae family, it is preferable to use flowers. Seeds are preferably used for peaches of the genus Rosaceae.
The plant parts may be subjected to the extraction step as they are, or may be subjected to the extraction step after being crushed, cut or dried.

The method for extracting the plant is not particularly limited, and the plant can be obtained by a usual extraction method used for extracting the plant component. The extraction method can be set as appropriate, and the extraction conditions are not particularly limited. For example, it is preferable to obtain each part of the plant by extracting it at room temperature or under heating. At the time of extraction, an operation such as stirring may be performed as appropriate.

The solvent (extraction solvent) used for extracting the plant can be appropriately selected, and the solvent usually used for extracting the plant component can be used. Examples of the extraction solvent include water; lower monohydric alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propanol, butanol, and pentanol; ethylene glycol, propylene glycol, 1,2-butylene glycol, and 1,3-butylene glycol. , 1,4-butylene glycol, 2,3-butylene glycol and other polyhydric alcohols; acetone, methyl ethyl ketone and other ketones; methyl acetate, ethyl acetate and other esters; tetrahydrofuran, diethyl ether and other chain and cyclic ethers; polyethylene glycol Etc., such as polyether; squalane and the like. These may be used alone, or a mixed solvent in which two or more kinds are combined may be used. The lower monohydric alcohol is preferably a monohydric alcohol having 1 to 4 carbon atoms, more preferably 2 to 4 carbon atoms, and even more preferably ethanol or the like. As the polyhydric alcohol, for example, a divalent or trivalent alcohol is preferable, and a divalent alcohol is more preferable. Further, as the polyhydric alcohol, for example, a polyhydric alcohol having 2 to 4 carbon atoms is preferable, and 1,3-butylene glycol and the like are more preferable. Among them, as the extraction solvent, water, a lower monohydric alcohol, a polyhydric alcohol, a squalane, and a mixed solvent of two or more of these are preferable, and water, a lower monohydric alcohol, a polyhydric alcohol, and a mixed solvent of these two or more kinds are preferable. More preferred. In one embodiment, as the extraction solvent, for example, water, a lower monohydric alcohol or an aqueous solution thereof, a polyhydric alcohol or an aqueous solution thereof, squalane and the like are more preferable, and water, ethanol, an aqueous ethanol solution, 1,3-butylene glycol, 1 , 3-butylene glycol aqueous solution, squalane and the like are more preferable. For example, the lower monohydric alcohol aqueous solution (preferably an ethanol aqueous solution or the like) and the polyhydric alcohol aqueous solution preferably have an alcohol concentration of 10 to 98% by volume, more preferably 30 to 95% by volume, still more preferably 50 to 95% by volume. .. As the plant extract in the present invention, the above solvent extract is preferable.
In one embodiment, Kabanoki family Kabanoki plant, Vine family grape genus plant, Asa family Karahanasou genus plant, Labiatae plant (preferably a plant belonging to Lavandra genus, Akigiri genus or Yamahakka genus), Mikan family Mikan genus plant, Acorus family As the extract of the genus Acorus plant and the genus Plant of the Labiatae family, an extract of a lower monovalent alcohol aqueous solution is preferable. The extract of the legume Soybean plant is preferably a water extract. For the Labiatae plant of the genus Perilla, an extract of an aqueous solution of polyhydric alcohol is preferable.
Acid or alkali may be added at the time of extraction to adjust the pH of the extraction solvent.
After extraction, it is preferable to remove the plant residue (plant body after extraction or a portion thereof) from the extract. The method for removing the plant residue from the extract is not particularly limited, and examples thereof include known separation means such as filtration and centrifugation.

As an example of the plant extraction method, the following methods can be used, for example. The plant substance or the dried product is crushed, and the extraction solvent is added in an amount of 5 to 20 times by mass, preferably at room temperature for 10 minutes to 15 days, more preferably 30 minutes to 10 days, and further preferably 1 hour under normal pressure. Extraction is performed for about 7 days, or extraction is preferably performed for about 10 minutes to 1 day (more preferably 10 minutes to 2 hours) near the boiling point of the extraction solvent, and then filtered to obtain a filtrate. At the time of extraction, it may be allowed to stand or stirred as appropriate. Further, the obtained filtrate (plant extract) may be used as it is as a plant extract, and may be concentrated or dried as needed.

In the present invention, the plant extract obtained by extraction can be used as it is as a plant extract. Further, the plant extract may be diluted with an appropriate solvent to obtain a plant extract, or may be used as a concentrate or a dry powder by performing a concentration or drying step, or may be used as a paste. The concentration method and the drying method are not particularly limited, and known methods such as freeze-drying and spray-drying can be used. In addition, the dry powder can be used by dissolving it in a solvent or suspending it.
Further, the above-mentioned plant extract, its concentrate and dry powder may be further purified by deodorization, decolorization and the like, if necessary, as long as the effects of the present invention are not impaired. Such a purification method may be carried out by arbitrarily selecting an ordinary means. For example, a method using various chromatographies such as filtration; column chromatography using a carrier such as an ion exchange resin, a synthetic adsorption resin or activated carbon may be used. Can be mentioned. Examples of carriers used for chromatography include Diaion HP-20 15L (product name, manufactured by Mitsubishi Chemical Corporation), Sephadex LH-20 (product name, manufactured by GE Healthcare Co., Ltd.) and the like.
The plant extract in the present invention includes various solvent extracts obtained by the above-mentioned extraction methods, diluted solutions thereof, concentrates thereof or dry powders thereof, and purified products thereof.
In addition, a commercially available product can also be used as the extract of the above plant.

Activation of PIMT promotes restoration or repair of isomerized proteins. The above plant extracts can be used to activate PIMT and promote restoration or repair of proteins containing isomerized amino acids. For example, the above plant extracts are used for activating PIMT and promoting restoration or repair of isomerized proteins, such as cosmetics (cosmetic compositions), pharmaceuticals (pharmaceutical compositions), and quasi-drugs (quasi-drugs). It is suitably used for producing quasi-drug compositions), foods and drinks (food and drink compositions), and the like. The above-mentioned plant extract may be blended in cosmetics, pharmaceuticals, quasi-drugs, foods and drinks, etc., and may be an active ingredient for activating PIMT, restoring isomerized protein, or promoting repair. The activation of PIMT is preferably activation of PIMT present in the skin. Restoration or repair of isomerized proteins is preferably restoration or repair of isomerized proteins in the skin.

When the above plant extract is used, PIMT can be activated as compared with the case where the plant extract is not used. For example, the above plant extract can be used to increase the expression level of PIMT in the skin. In addition, for example, it is possible to suppress a decrease in the expression of PIMT in the skin due to factors such as exposure to ultraviolet rays. The above plant extracts can restore the expression of PIMT in the skin, which has been reduced due to factors such as UV exposure. In one embodiment of the present invention, the above-mentioned plant extract has reduced PIMT in the skin in order to promote the expression of PIMT in the skin and to suppress the decrease in the expression of PIMT in the skin (for example, the decrease in expression due to exposure to ultraviolet rays or aging). Can be used as an active ingredient for recovery of expression.
Irradiation of skin-derived proteins with ultraviolet light as described above increases isomerized proteins. On the other hand, the expression level of PIMT in the skin decreases due to factors such as aging and ultraviolet rays. From these findings, it is presumed that the accumulation of isomerized proteins in the skin is promoted by factors such as aging and ultraviolet rays. Degeneration of skin proteins due to amino acid isomerization is also thought to be associated with skin aging.
Activation of PIMT present in the skin promotes restoration or repair of isomerized proteins in the skin, and is therefore effective for promoting recovery of aged skin (skin condition or skin function), preventing or improving skin aging, and the like. Is considered to be. Aging includes one or both of aging due to aging (physiological aging associated with aging) and aging due to ultraviolet light (photoaging due to exposure to ultraviolet light). Prevention includes prevention and delay of the onset of symptoms. Improvement includes relief of symptoms, suppression of progression of symptoms and complete recovery.
The composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention containing the above plant extract are effective for activating PIMT present in the skin and promoting restoration or repair of isomerized protein in the skin. Is. The above plant extract is applied to, for example, a person who is concerned about skin aging or a person who desires prevention or improvement of skin aging symptoms, for promoting skin recovery, preventing or improving skin aging, etc. It can be preferably used.
The use of the plant extracts for the above purposes may be therapeutic or non-therapeutic. "Non-therapeutic" is a concept that does not include medical practice, that is, a concept that does not include methods of surgery, treatment, or diagnosis of humans.

The form of the composition for activating PIMT and the composition for promoting restoration or repair of the isomerized protein of the present invention is not particularly limited, and may be in the form of powder, granules, paste, solid, etc., and is liquid. It may be selected as appropriate. For example, the extract of the above plant is used alone as a composition for activating PIMT (also referred to as a PIMT activator), a composition for promoting restoration or repair of an isomerized protein (a composition for promoting restoration or repair of an isomerized protein). Can also be used). Further, as long as the effect of the present invention is not impaired, other components may be blended in addition to the above-mentioned plant extract to prepare a composition for activating PIMT and a composition for promoting restoration or repair of isomerized protein. Examples of other ingredients include ingredients that can be used in pharmaceuticals, quasi-drugs, cosmetics, foods and drinks, which will be described later. Such pharmaceuticals, quasi-drugs, cosmetics, foods and drinks can be produced by a conventional method according to the compounding ingredients.
The composition for activating PIMT and the composition for promoting restoration or repair of an isomerized protein of the present invention can be provided, for example, in the form of an agent, but are not limited to this form. The agent can be provided as it is as a composition or as a composition containing the agent.

The composition for activating PIMT and the composition for promoting restoration or repair of isomerized proteins of the present invention can be used for various purposes such as pharmaceuticals, quasi-drugs, cosmetics, foods and drinks. The composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention are themselves pharmaceuticals and quasi-drugs for activating PIMT and promoting restoration or repair of proteins containing isomerized amino acids. It may be an external product, a cosmetic or a food or drink, or may be a material or a preparation used in combination with the drug, a quasi drug, a cosmetic or a food or drink.

The composition for activating PIMT and the composition for promoting restoration or repair of an isomerized protein of the present invention are suitably used, for example, as an external preparation for skin. The external skin preparation includes pharmaceuticals, quasi-drugs, and cosmetics (cosmetics), and is preferably a cosmetic. In one embodiment, the composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein, when used for activating PIMT in the skin or for promoting restoration or repair of isomerized protein in skin. It is preferable that the product is an external preparation for the skin, and cosmetics are more preferable. The skin external preparation containing the above plant extract is preferably used as a skin external preparation for activating PIMT existing in the skin; a skin external preparation for restoring or promoting repair of isomerized proteins in the skin.
The composition for activating PIMT and the composition for promoting restoration or repair of isomerized proteins of the present invention can also be used as pharmaceuticals or quasi-drugs other than external skin preparations.
In another embodiment, it is also preferable to use the composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein as foods and drinks. Foods and drinks containing the above plant extracts are preferably used, for example, for PIMT activation in the skin or for promoting restoration or repair of isomerized proteins in the skin. Foods and drinks containing the above plant extracts can be used as foods and drinks for activating PIMT existing in the skin, and foods and drinks for promoting restoration or repair of isomerized proteins in the skin.

The case where the composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention is used as a pharmaceutical product, a quasi-drug, a skin external preparation such as a cosmetic, a food or drink, etc. will be described below.

When the composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention are used as a skin external agent (external agent for activating PIMT, external agent for restoring or promoting restoration of isomerized protein). , The dosage form and the like are not particularly limited, and may be in any form such as a solution, a milky lotion, a cream, a gel, a powder, an aerosol, a mist, a capsule and a sheet. The external skin preparation is preferably a cosmetic. The product form of cosmetics is not particularly limited, and for example, skin care cosmetics such as wash pigments, makeup removers, lotions, beauty liquids, packs, milky lotions, creams, and sunscreens; foundations, makeup bases, lipsticks, eye shadows, and eyes. Makeup cosmetics such as liner, mascara, eyebrow, cheek red, nail enamel; hair cosmetics such as hair shampoo, hair rinse, hair conditioner, hair dye, hair restorer; cleaning agents such as soap and body soap; bathing agents, etc. Be done.

The extract of the plant may be used alone in the cosmetic. In addition, such cosmetics include components such as carriers and additives that are acceptable for cosmetics, such as water, alcohols, oils, surfactants, and thickeners, as long as the effects of the present invention are not impaired. , Metal soaps, gelling agents, powders, chelating agents, water-soluble polymers, film-forming agents, resins, inclusion compounds, antibacterial agents, deodorants, salts, pH adjusters, ultraviolet absorbers, animals and plants other than the above -One or two or more of microbial-derived extracts, keratolytic agents, enzymes, hormones, other vitamins, moisturizers, bactericides, anti-inflammatory agents, fragrances, etc. can be appropriately contained. Cosmetics can be manufactured by a general manufacturing method. For example, it can be obtained by mixing the above-mentioned plant extract with one or two or more components that can be used in the above-mentioned cosmetics, and then processing the mixture into a desired form.

When the skin external preparation is used as a drug or quasi-drug, the above-mentioned plant extract may be used alone, and the plant extract and a carrier, additive, etc. permitted for the drug or quasi-drug, etc. It may be used in combination with the ingredients of. Examples of such a component include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants and the like, which can be used as needed. Pharmaceuticals and quasi-drugs can be manufactured by a general manufacturing method. For example, it can be obtained by mixing the above-mentioned plant extract with one or two or more of the components that can be used in pharmaceutical products or quasi-drugs, and then processing them into a desired form.

The content of the extract of the above plant in the external preparation for skin such as cosmetics is not particularly limited. For example, the mass of the extract in terms of dry matter is preferably 0.000000001 to 100% by mass in the external preparation for skin. , 0.000001 to 100% by mass, more preferably 0.00001 to 99.9% by mass, and particularly preferably 0.00001 to 10% by mass.

The composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention can also be pharmaceuticals or quasi-drugs other than the above-mentioned external preparations for skin. Such drugs or quasi-drugs may be administered orally or parenterally. The drug or quasi-drug may be in the form of an orally-administered preparation (oral preparation) or a parenteral-administered preparation. Examples of the dosage form of the orally administered preparation include liquid preparations, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewable agents and the like. Examples of the dosage form of the parenteral preparation include injections, infusions and the like. The above-mentioned plant extract may be used alone for such a drug or quasi-drug, and the plant extract and components such as a carrier and an additive permitted for the drug or quasi-drug may be used. May be used in combination with. Examples of such components include one or more of excipients, binders, disintegrants, lubricants, antioxidants, colorants, flavoring agents and the like, which can be used as needed. Pharmaceuticals and quasi-drugs can be manufactured by a general manufacturing method. For example, it can be obtained by mixing the above-mentioned plant extract with one or two or more of the components that can be used in pharmaceutical products or quasi-drugs, and then processing them into a desired form.

The content of the above-mentioned plant extract in a drug or quasi-drug other than an external preparation for skin is not particularly limited. For example, the mass of the extract in terms of dry matter is 0. It is preferably 0000001 to 100% by mass, more preferably 0.000001 to 100% by mass, further preferably 0.00001 to 99.9% by mass, and particularly preferably 0.00001 to 10% by mass.

When the composition for activating PIMT and the composition for promoting restoration or repair of isomerized protein of the present invention are used as foods and drinks, the foods and drinks are not particularly limited, and for example, general foods and drinks, health foods, and functional claims. Examples include foods and foods for specified health use. The form of food and drink is not particularly limited. The above-mentioned health foods, foods with functional claims, and foods for specified health use include various preparations such as liquid preparations, tablets, powders, fine granules, granules, sugar-coated tablets, capsules, suspensions, emulsions, chewables, and liquid foods. It can also be in the form.

The extract of the plant may be used alone in the food or drink, and components permitted in the food or drink, for example, other food or drink materials, additives to be blended in the food or drink, or the like may be blended. It may be a food or drink for activating PIMT (food and drink composition for activating PIMT), or a food or drink for promoting restoration or repair of isomerized protein (food or drink composition for restoring or promoting restoration of isomerized protein). Such foods and drinks can be produced by a general production method. For example, in the production of food and drink, the above-mentioned plant extract may be added to the food and drink material.

The content of the extract of the above plant in the food or drink is not particularly limited. For example, the mass of the extract in terms of dry matter is preferably 0.000000001 to 100% by mass, preferably 0.000001 to 100% by mass in the food or drink. The mass% is more preferable, 0.00001 to 99.9 mass% is further preferable, and 0.00001 to 10 mass% is particularly preferable.

The amount of the above-mentioned pharmaceutical products, quasi-drugs, cosmetics or foods and drinks used is not particularly limited and can be appropriately set according to the condition, weight, gender, age or other factors of the subject. In the case of a skin external preparation such as a cosmetic, for example, the amount of the above-mentioned plant extract (dry matter equivalent) is preferably 0.01 to 500 mg per day per adult (60 kg). When the drug or quasi-drug is orally administered, the dose is preferably, for example, 0.01 to 500 mg per day for an adult (60 kg) as a plant extract (dry matter equivalent). .. In the case of food and drink, the intake is preferably, for example, 0.01 to 500 mg of the above-mentioned plant extract (dry matter equivalent) per adult (60 kg) per day. The above amount of plant extract can be applied, ingested or administered in a single dose or in multiple doses. The timing of applying the external skin preparation containing the above plant extract to the skin and the timing of ingesting or administering foods and drinks, pharmaceuticals or quasi-drugs are not particularly limited.

The applicable person of the above-mentioned drug, quasi-drug, cosmetics or food or drink is not particularly limited and can be applied to humans or non-human mammals, but humans are preferable. Applicants include, for example, those who desire or require activation of PIMT (preferably activation of PIMT in the skin), restoration or promotion of repair of isomerized proteins (preferably restoration of isomerized proteins in the skin or Those who desire or need (promotion of restoration) are preferred. In one embodiment, for example, a person who is concerned about skin aging, a person who desires or needs prevention or improvement of skin aging symptoms, a person who recovers aged skin, and a person who prevents or improves skin aging. Those who desire or need are preferred. The part of the body to which the external skin preparation such as cosmetics is applied is not particularly limited, but when used for the above purpose, for example, the skin of the part where aging is anxious, the part where prevention or improvement of aging symptoms is desired. It is preferable to apply it to the skin of the skin, the skin of the part where prevention or improvement of aging symptoms is necessary.

<Method of activating PIMT and method of promoting restoration or repair of proteins containing isomerized amino acids>
Also included in the present invention are methods of promoting the restoration or repair of proteins containing isomerized amino acids in the skin by activating PIMT present in human skin.
When the isomerized protein is restored or repaired, the function of the protein impaired by isomerization can be restored. Therefore, the method of the present invention is effective, for example, for promoting recovery of aged skin (skin condition or skin function), preventing or ameliorating skin aging, and the like.
Examples of the method for activating PIMT present in human skin include a method of applying the above plant extract to humans. More specifically, for example, a method of applying the plant extract to human skin, a method of applying or ingesting the plant extract to humans, and the like can be mentioned. Preferably the plant extract is applied to human skin. The plant extract is as described in the PIMT activation composition described above. The plant extract can be applied in the form of the above-mentioned external skin preparations, pharmaceuticals, quasi-drugs, foods and drinks, and the like. By activating PIMT present in the skin, restoration or repair of proteins containing isomerized amino acids in the skin can be promoted.

Kabanoki family Kabanoki genus plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus plant, Shobu family Shobu genus plant, Kiku family Arnica genus plant and Rose family peach genus A method for activating PIMT, applying an extract of one or more plants selected from the group consisting of plants to an animal; comprising an isomerized amino acid, applying the extract of one or more plants described above to an animal. Methods for promoting protein restoration or repair are also included in the present invention.
As the animal, the above-mentioned human or non-human mammal is preferable, and human is more preferable.
The method for activating PIMT is preferably a method for activating PIMT present in the skin. The method for promoting restoration or repair of a protein containing an isomerized amino acid is preferably a method for promoting restoration or repair of a protein containing an isomerized amino acid in the skin. In activating PIMT and promoting restoration or repair of isomerized proteins in the skin, it is preferable to bring the plant extract into contact with the skin. In a preferred embodiment of the present invention, the above plant extract is applied to animal skin. For example, by applying a skin external preparation containing the above plant extract to the skin of an animal such as a human, the extract of the above plant can be brought into contact with the skin cells. The plant extract, its preferred mode, preferred amount used and the like are as described above. The external skin preparation is also as described above. In one aspect, the method is preferably a non-therapeutic method (a method that does not include medical practice).

<Skin restoration or repair activity evaluation method and skin anti-aging activity evaluation method>
Using the expression level or activity of PIMT present in the skin as an index, the restoration or repair activity of the skin and the anti-aging activity of the skin can be evaluated.
The method for evaluating skin restoration or repair activity of the present invention uses the expression level or activity of PIMT in skin cells as an index. The method for evaluating the anti-aging activity of the skin of the present invention uses the expression level or activity of PIMT in skin cells as an index.
In the present specification, the method for evaluating the skin restoration or repair activity of the present invention and the method for evaluating the anti-aging activity of the skin of the present invention are also referred to as the evaluation method of the present invention.

The skin restoration or repair activity in the present invention is usually obtained by isomerization (conversion to L-isoAsp or D-Asp) of amino acids (specifically, L-Asp or L-Asn) in skin proteins. It refers to the activity of restoring or repairing a denatured protein (isomerized protein), and the activity of promoting the restoration or repair of the isomerized protein in the skin. The activity of promoting the restoration or repair of the isomerized protein in the skin includes an activity of suppressing a decrease in the restoration or repair activity of the isomerized protein in the skin due to factors such as ultraviolet rays and aging. Since PIMT has an action of promoting restoration or repair of isomerized protein, the expression level or activity of PIMT in skin cells is useful as an index for evaluating the restoration or repair activity of the skin. The skin restoration or repair activity can also be rephrased as the skin restoration or repair power, the skin restoration or repair action. Skin restoration or repair activity can also contribute to skin recovery.
In addition, as described above, denaturation of skin proteins due to amino acid isomerization is considered to be associated with skin aging. It is considered that, for example, the function of aged skin is restored by restoring or repairing the isomerized protein in the skin. Therefore, the expression level or activity of PIMT in skin cells is also useful as an index of skin anti-aging activity. The anti-aging in the present invention is the prevention or amelioration of aging, preferably skin aging. Aging includes age-related aging and / or UV aging.
In addition, for example, the expression of PIMT in skin cells decreases with ultraviolet rays and / or aging, but in the skin where the decrease in expression of PIMT with ultraviolet rays and / or aging is small, the protein of the skin with ultraviolet rays and / or aging High activity to restore or repair isomerization. Using the expression level or activity of PIMT in skin cells as an index, the restoration or restoration of the skin due to aging, which suppresses the decrease in the restoration or repair activity of the skin due to ultraviolet rays and / or aging. Alternatively, the repair activity can be evaluated.
The method for evaluating skin restoration or repair activity of the present invention can be used as a method for evaluating skin restoration or repair activity against UV and / or age-related skin aging. The method for evaluating the anti-aging activity of the skin of the present invention can be used as a method for evaluating the anti-aging activity against aging of the skin due to ultraviolet rays and / or aging.

In the evaluation method of the present invention, the expression level or activity of PIMT in skin cells is used as an index. More specifically, the degree or change in skin restoration or repair activity and the degree or change in skin anti-aging activity are evaluated using the expression level or activity level of PIMT as an index.
For example, when the expression level or activity of PIMT in skin cells is high, the activity of converting isomerized Asp in the protein to L-Asp is high. That is, it has a high activity of restoring or repairing isomerized proteins. Further, when the expression level or activity of PIMT in skin cells is high, the activity of suppressing deterioration of various functions of the skin due to isomerization of amino acids is high. Therefore, it can be said that when the expression level or activity of PIMT in skin cells is high, the activity (action) of restoring or repairing the skin is high, and the activity (action) of preventing or improving skin aging is high.

Since skin having a high expression level or activity of PIMT has high restoration or repair activity of isomerized protein, it can be said that skin restoration or repair activity and skin anti-aging activity are high. In addition, it can be said that the skin having a high expression level or activity of PIMT has a low degree of aging of skin cells (aging has not progressed). Conversely, it can be said that skin having a low expression level or activity of PIMT has low skin restoration or repair activity and skin anti-aging activity. In addition, when the expression or activity of PIMT is low, it can be said that the skin has advanced aging.
Therefore, by using the expression level or activity of PIMT in skin cells as an index, the restoration or repair activity of the skin and the anti-aging activity of the skin can be objectively evaluated. The expression level or activity of PIMT in skin cells can also be used as an index for evaluating the progress of skin aging.

By using the expression level or activity of PIMT in skin cells as an index, the skin restoration or repair activity of the subject or the anti-aging activity of the skin can be compared with the activity of standard skin, for example, or the skin by skin care. It is also possible to evaluate changes in the restoration or repair activity of the skin or the anti-aging activity of the skin. Therefore, based on the above evaluation, for example, an appropriate treatment for the symptoms associated with skin aging can be selected.

The skin cells used in the evaluation method of the present invention may be any skin cells of a subject who needs the evaluation or a subject who desires the evaluation, and examples thereof include human or non-human mammalian skin cells. Skin cells are preferred. Skin cells are usually skin cells taken from a subject. As the skin cells, epidermal cells and / or dermis cells are preferable. The expression level or activity of PIMT in epidermal cells and / or dermis cells is preferable as an index for evaluating skin restoration or repair activity and skin anti-aging activity. Among them, dermal cells are more preferable. Since damage due to aging, ultraviolet rays, etc. accumulates in the dermis tissue, the skin restoration or repair activity of the subject and the anti-aging activity of the skin are more appropriate when the expression level or activity of PIMT in the dermis cells is used as an index. Can be evaluated. Moreover, epidermal cells are preferable from the viewpoint of easy collection.

The method for collecting skin cells is not particularly limited. Epidermal cells can be collected, for example, by the tape stripping method. Dermis cells can be collected with an injection needle or the like. Further, for example, when a part of the skin is collected for the purpose of regenerative medicine or the like, or when extra cells from the skin are used at the time of fat resection, cells from the skin can be used.

The site of the skin is not particularly limited, and for example, a site where an evaluation of skin restoration or repair activity or an anti-aging activity of the skin is required or a desired site is preferable. For example, the skin of the face (forehead, cheeks, around the eyes, around the mouth, chin), behind the ears, neck, arms, hands, feet, buttocks, etc. can be mentioned. In one aspect, the skin cells of the exposed portion (ultraviolet exposed portion) are preferable, and the skin of the exposed portion includes the face (particularly the cheek portion and the like), the neck, the upper arm portion, the back of the hand and the like. The exposed skin cells are suitably used for evaluation of skin restoration or repair activity against skin aging caused by ultraviolet rays and evaluation of anti-aging activity against skin aging caused by ultraviolet rays. In the present invention, it is preferable to evaluate using facial skin cells for which evaluation of skin restoration or repair activity and anti-aging activity is considered to be important.

The evaluation method of the present invention preferably includes a step of measuring the expression level of PIMT or the activity of PIMT in skin cells.
The expression level of PIMT can be measured by measuring the amount of PIMT (protein amount) in skin cells or the amount of mRNA encoding PIMT. Since the operation is simple, it is preferable to measure the amount of PIMT as the expression level of PIMT.
When measuring the amount of PIMT, a protein may be extracted from the collected skin cells to prepare a sample, and the amount of PIMT in the sample may be measured. The method for extracting the protein and the method for measuring the amount of PIMT are not particularly limited. For example, a commercially available protein extraction solvent (for example, M-PER (registered trademark) Mammalian Protein Extraction Regent manufactured by Thermo, product name Mammalian Cell Lysis Buffeter manufactured by GE Healthcare, etc.) is used to be soluble in the extraction solvent from skin cells. Protein can be extracted and used as a sample for PIMT measurement. Further, for extracting the protein from the skin cells, a method such as an osmotic shock method, a freeze-thaw method, an ultrasonic treatment method, or a homogenization method can be adopted.
The amount of PIMT can be measured by a known method such as an immunoassay method using an antibody specific for PIMT (for example, an ELISA method using an enzymatic reaction, a Western blotting method). As the ELISA method, for example, a commercially available kit (ELISA Kit for Homo sapiens (Huma) Protein L-Isoasepartate-O-Methyltransferase (PCMT1), manufactured by CLOUD-CLONE) can be used.
The method for extracting mRNA and measuring the amount thereof may be performed by a known method. The measurement of mRNA can be carried out by a method such as the PCR method. The DNA sequence of human PIMT is known (NCBI Accession No. NM_005389). A person skilled in the art can easily design and synthesize a primer for measuring the amount of mRNA encoding PIMT, amplify DNA, and the like. In addition, primers that can be used for quantification of the human PIMT gene are commercially available (OriGene, product number HK25454, HK205453, etc.). Such commercially available primers can also be obtained to measure the amount of mRNA encoding PIMT.

Measurement of the activity of PIMT in skin cells can be performed quantitatively or qualitatively according to any method capable of measuring the enzymatic activity of PIMT. Specifically, it serves as a method and substrate for extracting a protein from skin cells and measuring the amount of S-adenosyl homocysteine (SAH) produced from S-adenosyl-L-methionine (SAM) by the reaction of PIMT by HPLC. A method in which an isoAsp-containing peptide is fluorescently labeled with nitrobenzoxaziazole (NBD), reacted with PIMT, methylated, and then separated and measured by HPLC (Furuchi et al., "YAKUGAKU ZASSHI", Vol. 127, No. 12, 1927-1936). (2007)) and the like, the activity of PIMT can be measured.
As the measured values of the expression level of PIMT and the activity of PIMT, values that are easy to use may be used depending on the measuring method. For example, when the PIMT amount (protein amount) is used as the expression level of PIMT, the measured value of the PIMT expression level can be converted into the PIMT amount per protein amount.
The expression level of PIMT or the measured value of PIMT activity can be used to evaluate the degree of skin restoration or repair activity, the degree of skin anti-aging activity, and the change in the activity.

The method for evaluating the restoration or repair activity of the skin and the method for evaluating the anti-aging activity of the skin are not particularly limited using the expression level or activity of PIMT in the skin cells as an index.
For example, as an example of an embodiment, a step of measuring the expression level or activity of PIMT in the skin cells of a subject, a group in which the expression level or activity of PIMT in the skin cells of the subject is measured in advance (for example, the same generation). A step can be performed to compare the expression level or activity distribution in the skin cells of (population). For example, if the expression level or activity of PIMT in the skin cells of a subject is high compared to the distribution of the expression level or activity of PIMT in a pre-measured population of skin cells, the subject restores or repairs the skin. It is evaluated (determined) that the activity and the anti-aging activity of the skin are high.

In addition, since it is considered that the skin restoration or repair activity and the skin anti-aging activity vary from person to person, for example, in the same subject, the expression level of PIMT in the exposed and unexposed skin cells or The above activities can also be evaluated by comparing the activities. As an example of such an evaluation method, a step of measuring the expression level or activity of PIMT in the exposed and unexposed skin cells of the same subject, and the expression level of PIMT in the exposed and unexposed skin cells of the same subject. Alternatively, an evaluation method including a step of comparing the activities can be mentioned. Ultraviolet light reduces the expression or activity of PIMT in skin cells. In addition, since the damage caused by ultraviolet rays accumulates, it is considered that the expression or activity of PIMT is usually lower in the exposed skin than in the non-exposed skin in the same subject. Therefore, when the expression level or activity of PIMT in the exposed and unexposed skin cells is compared and the difference is small, it is evaluated that the skin restoration or repair activity of the subject and the anti-aging activity of the skin are high. NS. Examples of the skin of the exposed portion include the above-mentioned skin such as the face. The non-exposed portion is not particularly limited, and examples thereof include a buttocks and the like.

Comparing the expression level or activity of PIMT in the exposed and unexposed areas in the same subject can be used to evaluate the skin restoration or repair activity against UV-induced skin aging and the anti-aging activity against UV-induced skin aging. Suitable for evaluation. The smaller the difference between the PIMT expression level or activity in the unexposed skin cells and the PIMT expression level or activity in the exposed skin cells, the more the skin restoration or repair activity against UV-induced skin aging, and the UV-induced skin restoration or repair activity. It is evaluated as having high anti-aging activity against aging.

It is also possible to irradiate skin cells with ultraviolet rays and compare the expression level or activity of PIMT before and after the irradiation. In this case, for example, the step of measuring the expression level or activity of PIMT in the skin cells of the subject, the step of irradiating the skin cells of the subject with ultraviolet rays, and the expression level or activity of PIMT in the skin cells of the subject after irradiation with ultraviolet rays are determined. It is preferable to include a step of measuring and a step of comparing the expression level or activity of PIMT before and after irradiation with ultraviolet rays.
Comparing the expression level or activity of PIMT before and after irradiation with ultraviolet rays is suitable for evaluating the skin restoration or repair activity against skin aging caused by ultraviolet rays and the anti-aging activity against skin aging caused by ultraviolet rays. It is evaluated that the smaller the decrease in PIMT expression level or activity after irradiation is compared with that before UV irradiation, the higher the skin restoration or repair activity against UV-induced skin aging and the higher the anti-aging activity against UV-induced skin aging. Will be done.

Further, as an example of another embodiment, the PIMT expression level or activity in the skin cells when the skin is subjected to the predetermined skin care can be compared with the PIMT expression level or activity when the skin care is not performed. This comparison may be a comparison of PIMT expression levels or activity before and after skin care. This makes it possible to evaluate changes in skin restoration or repair activity due to skin care, or changes in skin anti-aging activity. For example, when a certain skin care increases the expression level or activity of PIMT as compared with the case where the care is not performed, the skin care enhances the skin restoration or repair activity and the anti-aging activity of the skin. Can be evaluated.

Furthermore, the PIMT expression level or activity in the skin cells when a certain food or ingredient is ingested can be compared with the PIMT expression level or activity when the food or ingredient is not ingested. This makes it possible to evaluate changes in skin restoration or repair activity or changes in skin anti-aging activity due to the food or ingredient. For example, when the ingestion of a certain food or ingredient increases the expression level or activity of PIMT as compared with the case where the food or ingredient is not ingested, the ingestion of the food or ingredient increases the skin restoration or repair activity, and , It can be evaluated that the anti-aging activity of the skin is enhanced.

Substances present in the skin that have an act of activating PIMT, substances having an action of promoting restoration or repair of the skin, and substances having an anti-aging action of the skin can be screened by the method described later.

<Method of screening active ingredients for activation of PIMT present in skin, promotion of skin restoration or repair, or anti-aging of skin>
The screening method of the present invention is a method for screening an active ingredient for activation of PIMT present in the skin, promotion of restoration or repair of the skin, or anti-aging of the skin. In the screening method of the present invention, the expression level or activity of PIMT in skin cells is used as an index.
Using the expression level or activity of PIMT in skin cells as an index, the active ingredient for activation of PIMT present in the skin, promotion of skin restoration or repair, or anti-aging of the skin, in other words, PIMT present in the skin It is possible to screen for substances having an activating effect, substances having a skin restoration or repair promoting effect, or substances having an anti-aging effect on the skin.
The screening method of the present invention is usually a method performed in vitro (in vitro screening method).

Skin cells used in the screening method of the present invention include human or non-human mammalian skin cells. The skin cells may be cells collected from the above-mentioned subject, but cultured skin cells are preferably used. For example, cultured skin cells such as human epidermal keratinocytes and human dermal fibroblasts can be used. The skin cells used are preferably those that have been proliferated until they become confluent. The culture conditions for growing the cells are not particularly limited, and known conditions may be adopted depending on the cells.

The screening method of the present invention comprises a step of contacting a test substance with the skin cells to measure the expression level or activity of PIMT in the skin cells, and the expression level of PIMT or the expression level of PIMT in the skin cells contacted with the test substance. When the activity is large compared to the expression level or activity of PIMT in the skin cells not contacted with the test substance, it is preferable to include a step of selecting the test substance as an active ingredient.

The test substance is not particularly limited, and examples thereof include plant extracts, cell extracts, fermentation products, proteins, peptides, vitamins, and synthetic compounds. These may be known substances or new substances.
The method of contacting the test substance with the skin cells is not particularly limited as long as the test substance can come into contact with the cells. For example, in the case of cultured skin cells, the test substance may be added to the medium for culturing the skin cells. In this case, it is preferable to culture the skin cells in the medium to which the test substance is added for a predetermined time, and then measure the expression level or activity of PIMT in the cells. The medium, culture conditions, etc. can be appropriately set according to the type of cells and the like. The culturing time is not particularly limited, and may be appropriately set depending on the culturing conditions and the like. For example, skin cells may be cultured in a medium containing a test substance for preferably 5 to 72 hours, more preferably 12 to 48 hours. For example, an α-MEM medium can be mentioned as an example of the medium. Serum (eg, fetal bovine serum) may be added to the medium. As the medium, a commercially available product may be used.
The expression level or activity of PIMT in skin cells can be measured by the method described above.
Further, as described above, since the expression level or activity of PIMT in the skin cells is reduced by the ultraviolet rays, for example, the skin cells may be irradiated with the ultraviolet rays before or after the test substance is brought into contact with the skin cells. When the expression level or activity of PIMT in the skin cells contacted with the test substance is larger than the expression level or activity of PIMT in the skin cells not contacted with the test substance when irradiated with ultraviolet rays, the test It can be said that the substance has a high PIMT activating effect existing in the skin. In addition, the test substance is considered to have a high effect of suppressing a decrease in isomerized protein restoration or repair activity in the skin due to ultraviolet rays, that is, a skin restoration or repair effect on skin aging caused by ultraviolet rays. In addition, the test substance is considered to have a high anti-aging effect on skin aging caused by ultraviolet rays.

Skin cells that have not been contacted with the test substance are controls. As a control, skin cells contacted with an active ingredient of a known anti-aging agent or the like can be used instead of the test substance. When skin cells contacted with an active ingredient of a known anti-aging agent or the like are used as a control, a test substance existing in the skin having a higher PIMT activating effect and a test having a higher skin restoration or repairing effect than the known active ingredient. A substance or a test substance having a high anti-aging effect on the skin can be selected.

The active ingredient obtained by the screening method of the present invention can activate PIMT present in the skin. This can promote skin restoration or repair. In addition, the active ingredient obtained by the screening method of the present invention can prevent or improve the above-mentioned skin aging. More specifically, skin aging can be prevented or ameliorated based on the activity of promoting the conversion of isomerized Asp to L-Asp in skin cells.

The present invention also includes the use of the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index of skin restoration or repair activity or skin anti-aging activity. More specifically, the expression level or the degree of activity of PIMT in skin cells can be used as an index of the degree of activity. The expression level or activity of PIMT in skin cells is preferably used as an index of skin restoration or repair activity. In addition, the expression level or activity of PIMT in skin cells is preferably used as an index of skin anti-aging activity.

The present invention also includes the following uses and the like.
For activation of protein L-isoaspartate methyl transferase (PIMT), Kabanoki family Kabanoki plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus Use of an extract of one or more plants selected from the group consisting of plants, plants of the family Shobu, plants of the genus Arnica of the family Kiku and plants of the genus Peach of the family Rose.
To restore or promote the restoration or repair of proteins containing isomerized amino acids, plants of the genus Kabanoki, plants of the genus Grape, plants of the genus Soybeans of the family Asa, plants of the genus Karahanasou of the family Asa, plants of the genus Mikan of the family Mikan, Use of an extract of one or more plants selected from the group consisting of the plants of the genus Shobu, the genus Arnica of the family Kiku, and the plants of the genus Peach of the rose family.
The above use is preferably in human or non-human mammals. The use may be therapeutic or non-therapeutic use. The above use may be for non-therapeutic cosmetic purposes. Preferred embodiments of the plant extract are as described above. The plant extract may be used as it is, or may be used as a composition containing the plant extract. The plant extract can be used in the form of the above-mentioned external skin preparation, food and drink, and the like.
The present invention uses the plant extract to produce a composition for activating PIMT; the use of the plant extract to produce a composition for promoting restoration or repair of a protein containing an isomerized amino acid. Also includes. Preferred embodiments of the plant extract are as described above.

Hereinafter, examples will be shown in which the present invention will be described in more detail. The present invention is not limited to these examples.

The protein extraction reagent used in the following experiments is a product name M-PER (registered trademark) Mammalian Protein Exhibition Reagent manufactured by Thermo. In the test example, the test of the significant difference was performed by the Student's t-test.

<Test Example 1>
Measurement of PIMT expression level in skin cells Adult-derived normal human epidermal keratinized cells (NHEK-AD: purchased from Kurabou Co., Ltd.), adult-derived normal human dermal fibroblasts (NHDF-AD: purchased from Kurabou Co., Ltd. and NHDF-Ad) : Methyl transferase for protein L-isoaspartate in human skin using neonatal-derived human skin fibroblasts (NHDF-NB: purchased from Kurabou Co., Ltd. and NHDF-Neo: purchased from Lonza). The protein expression level of PIMT) was evaluated. The medium in which each cell was suspended was seeded in a 100 mm culture dish and cultured at 37 ° C. in a carbon dioxide concentration of 5 vol% until it became confluent. Proteins soluble in the extraction reagent were recovered from cells that reached confluence using the protein extraction reagent, and the amount of PIMT per amount of protein was measured. The amount of PIMT per amount of this protein was taken as the expression level of PIMT. The amount of PIMT was measured using ELISA Kit for Homo sapiens (Huma) Protein L-Isoaspartate-O-Methyltransferase (PCMT1) (manufactured by CLOUD-CLONE) according to the attached instructions. In addition, a cytolytic solution (manufactured by Santa Cruz) of fetal-derived human kidney-derived cells (HEK293), which has already been reported to express PIMT as an index of expression level, was used as a positive control.

The results are shown in FIG. FIG. 1 is a graph showing the expression level of PIMT in skin cells (the amount of PIMT per protein amount).
It was shown that PIMT was expressed in all skin-derived NHEK cells and NHDF cells, although it was lower than that of HEK293 cells.

The neonatal-derived normal human skin fibroblasts (NHDF-NB) used in Test Examples 2 to 8 below are the same as the NHDF-NB used in Test Example 1. Unless otherwise specified, the cells were cultured at 37 ° C. and a carbon dioxide concentration of 5 vol%.

<Test Example 2>
Examination of the repair effect of isomerized aspartic acid in skin proteins produced by ultraviolet B wave (UVB) irradiation by PIMT Neonatal-derived normal human skin fibroblasts (NHDF-NB) that induced isomerization of aspartic acid by UVB irradiation It was examined whether PIMT also has a repairing effect on isomerized aspartic acid in skin proteins using the above proteins. Specifically, a medium in which NHDF-NB cells were suspended was seeded in a culture dish and cultured at 37 ° C. in a carbon dioxide concentration of 5 vol% until it became confluent. After recovering the protein from the cells that have reached the confluence using a protein extraction reagent, the protein is irradiated with UVB for a certain period of time, and the amount of L-β-Asp as isomerized aspartic acid is adjusted to ISOQUANT® Isoaspertate Detection Kit (Promega). Quantified according to the attached instructions. This kit transfers the methyl group of the coenzyme S-adenosyl-L-methionine (SAM) to the α-carboxyl group of isoaspartic acid when PIMT acts on isoaspartic acid in a protein or peptide, and the SAM This is a method for indirectly quantifying isoaspartic acid residues by quantifying SAH by utilizing the production of the demethylated S-adenosyl homocystein (SAH). In addition, a delta sleep-inducing peptide (DSIP), which is known to contain isoaspartic acid at one site in one molecule, was used as a positive control. From the results of the positive control, it was confirmed that the above reaction occurred and the generated SAH could be detected. The quantitative result was calculated as the amount of SAH per protein amount of each test sample.

The results are shown in FIG. FIG. 2 is a graph showing the amount of SAH produced per amount of protein by UVB irradiation. The data in FIG. 2 is shown as mean ± standard deviation (n = 3). In FIG. 2, * is a significant difference with respect to the UVB irradiation time of 0 minutes (*: p <0.05). The larger the amount of SAH produced, the larger the amount of isomerized aspartic acid produced.
A certain amount of SAH was also detected in the NHDF-NB cell-derived protein that had not been irradiated with UVB, but the amount of SAH increased depending on the UVB irradiation time (40 minutes, 60 minutes). NHDF-NB is a dermal fibroblast. From this result, it was shown that aspartic acid isomerization is induced by UVB irradiation in the dermal fibroblast-derived protein, and PIMT acts on aspartic acid in the isomerized skin protein. Was shown.

<Test Example 3>
Evaluation of PIMT Expression Level in Dermal Fibroblasts with Different Passage Numbers It is known that when cells are repeatedly subcultured and cultured, the cells age and eventually lose their division function. Therefore, neonatal-derived normal human skin fibroblasts (dermal fibroblasts) that have been repeatedly subcultured were used as an aging model.
In order to verify how the expression level of PIMT fluctuates with aging, the protein expression level of PIMT was used using normal human skin fibroblasts derived from newborns (NHDF-NB, purchased from Kurabo Industries Ltd.) with repeated passages. I verified the change in. NHDF-NB cells (passage number (P): 3, 6, 10, 12, 15 and 17) that were repeatedly subcultured were seeded and cultured at 37 ° C. at a carbon dioxide concentration of 5 vol% until they became confluent. .. Using a protein extraction reagent from cells that reached confluence, proteins soluble in the extraction reagent were recovered and the amount of PIMT per protein amount was measured. The amount of PIMT was measured by the same method as in Test Example 1.

FIG. 3 is a graph showing the expression level of PIMT (the amount of PIMT per protein amount) in skin fibroblasts having different passage numbers. In NHDF-NB cells in which subculture was repeated 6 times or more, it was confirmed that the expression level of PIMT per protein amount tended to decrease. It has been confirmed that isomerized aspartic acid is accumulated in aged skin tissue, but in aging cells, the expression of PIMT, which can repair isomerized aspartic acid in proteins, is reduced, resulting in isomerization. It is expected that the accumulation of aspartic acid will increase.

<Test Example 4>
Changes in PIMT expression levels under various stress loads In order to investigate whether PIMT expression levels are affected by the external environment, neonatal-derived normal human skin fibroblasts (NHDF-NB) are exposed to UVB, induce inflammation, and deplete nutrients. , Active oxygen and other stimuli were given, and changes in the protein expression level of PIMT were verified. The cells were seeded in a 100 mm culture dish and cultured at 37 ° C. in a carbon dioxide concentration of 5 vol%. An α-MEM medium containing 10% fetal bovine serum (FBS) (manufactured by SIGMA) was used for culturing. Various stimuli were applied to the cells that reached the confluence as follows.
(1) Verification of the effect of exposure to active oxygen species (hydrogen peroxide (H ₂ O ₂ )) on the expression level of PIMT Remove the culture supernatant and adjust the final concentration of hydrogen peroxide solution (manufactured by Nacalai Tesque) to 10 μM. Was replaced with fresh FBS-containing α-MEM medium added to.
(2) Verification of Effect of UVB Exposure on PIMT Expression Level The culture supernatant was removed, replaced with a Hanks balanced salt solution (manufactured by Nacalai Tesque, Inc.), and then irradiated with ^{UVB (integrated light intensity of about 5.7 mJ / cm 2).} After irradiation, the medium was replaced with fresh FBS-containing α-MEM medium.
(3) Verification of Effect on PIMT Expression Level in Undernutrition The α-MEM medium containing 10% FBS was replaced with α-MEM medium containing no FBS.

After stimulating by the above methods (1) to (3), a protein soluble in a protein extraction reagent was recovered from cells cultured for 48 hours, and the amount of PIMT per protein amount was measured. The PIMT amount was measured by the same method as in Test Example 1. In addition, as a control, cells replaced with α-MEM medium containing 10% FBS without applying the stimuli of (1) to (3) above were used.

FIG. 4 is a graph showing changes in the PIMT expression level (PIMT level per protein amount) of skin fibroblasts due to stress loading.
The data in FIG. 4 is shown as mean ± standard deviation (n = 3). * Is a significant difference from the control (*: p <0.05). The expression level of PIMT was significantly reduced as compared with the control cells under hydrogen peroxide (H ₂ O _{2) exposure, UVB exposure, and undernutrition.} From these facts, the expression of PIMT decreases in the skin exposed to ultraviolet rays, the skin exposed to reactive oxygen species, and the skin whose nutritional status decreases with aging, resulting in isomerized aspartic acid. It is expected that acid accumulation will increase.

<Preparation Example 1> Preparation of plant-derived extract The plants and their parts used for extraction are as follows.
(1) Isodon japonicus: Stems and leaves (2) Perilla (Perilla frutescens var. Crispa): Leaves (3) Betula pendula: Bark (4) Yuzu (Citrus junos): Fruits (5) Sage (5) Salvia officinalis): Leaf (6) Yuzu (Glycine max): Stem (Yuzu bud)
(7) Arnica montana: flowers (8) apricots (Prunus armeniaca): seeds (9) shobu (Acorus calamus): roots (10) lavandula angustifolia: flowers (11) peaches (Amygdalus persica): seeds (12) Hop (Humulus lupulus): Female flower spike (13) Grape (Vitis spp.) (Caberne sauvignon): Peel and seed

Each of the above-mentioned crushed plants (1), (3) to (5) and (7) to (13) is immersed in a 30 to 90 vol% ethanol aqueous solution having a mass of 10 times the mass, and once a day at room temperature. The mixture was stirred for 10 minutes to 7 days. Then, the residue was removed by filtration to obtain an extract. The extract was concentrated and then freeze-dried to obtain an extract of each plant (ethanol extract) as a powder. The powdered plant extract was used for the evaluation of Test Examples 5-8.

(2) Perilla was extracted by the above method using an aqueous solution of 1,3-butylene glycol instead of an aqueous solution of ethanol. After extraction, the residue was removed by filtration to obtain an extract (perilla butylene glycol extract). The extract was concentrated and then freeze-dried to obtain a perilla extract (butylene glycol extract) as a powder. The powdered perilla extract was used for the evaluation of Test Examples 5-6.

For the soybean of (6) above, the germ of soybean was crushed and extracted with hot water in an amount of 10 times the mass. Then, the residue was removed by filtration to obtain an extract (soybean water extract). This extract was concentrated and then freeze-dried to obtain a soybean extract (water extract) as a powder. This powdered soybean extract was used for the evaluation of Test Examples 5-6.

<Test Example 5>
Changes in PIMT expression level in skin cells due to addition of plant extract Humans when the plant extract prepared in Preparation Example 1 was added using neonatal-derived human dermal fibroblasts (NHDF-NB: purchased from Kurabou Co., Ltd.). The protein expression level of PIMT in the skin was evaluated.
Cells were seeded in a 100 mm culture dish and cultured at 37 ° C. in a carbon dioxide concentration of 5 vol% until confluent. Then, each plant extract shown in Table 1 or Table 2 was added to the obtained cells at a concentration of 10 μg / mL and cultured for 24 hours. As the medium, α-MEM medium without FBS was used, and the plant extract was dissolved in dimethyl sulfoxide (DMSO) and added so that the final concentration of DMSO in the medium was 0.1% by mass. A medium containing 0.1% by mass DMSO was used as a control.

Twenty-four hours after the addition of the plant extract, the culture supernatant was removed, replaced with a Hanks balanced salt solution (manufactured by Nacalai Tesque, Inc.), and irradiated with ^{UVB (integrated light intensity of about 5.7 mJ / cm 2).} After irradiation, the medium was replaced with a fresh 10% FBS-containing α-MEM medium containing the DMSO solution of the above plant extract (final concentration of plant extract was 10 μg / mL, final concentration of DMSO was 0.1% by mass). The cells were further cultured for 48 hours. After this culture, a sample obtained by recovering a protein soluble in a protein extraction reagent from cells was used as a sample, and the amount of PIMT per amount of protein (PIMT mass / mass of protein) was measured. The amount of PIMT was measured using ELISA Kit for Homo sapiens (Huma) Protein L-Isoaspartate-O-Methyltransferase (PCMT1) (manufactured by CLOUD-CLONE) according to the attached instructions.
In this experiment, a sample was prepared at n = 3 for each plant extract according to the above procedure. The measurement of the amount of PIMT was performed twice for each sample, and the average value was used as the measured value. For each plant extract, the average value of the measured values (n = 3) of each sample was used as the quantitative result of the amount of PIMT.

The results are shown in Tables 1 and 2. The plant extracts of (1) to (6) shown in Table 1 and the plant extracts of (7) to (12) shown in Table 2 were tested in the same test system.
In Tables 1 and 2, "control" is a control that is not irradiated with UVB, and "control (+ UV)" is a control that is irradiated with UVB. In Tables 1 and 2, "-" of UV irradiation means that UVB was not irradiated, and "+" means that UV irradiation was performed. In the table, "*" and "**" indicate a significant difference from "control (+ UV)" (*: p <0.05, **: p <0.01).

The quantification result of PIMT was shown as a relative value (%) (PIMT expression level (%)) with the PIMT amount (PIMT expression level) per protein amount of the UVB-irradiated control (control (+ UV)) as 100%. In addition, the degree of recovery effect of the plant extract on the decrease in PIMT expression level due to UVB irradiation was shown as "recovery rate (%)". The recovery rate (%) is as follows from the PIMT expression level (C0) of the control without UV irradiation, the PIMT expression level (C1) of the UV-irradiated control, and the PIMT expression level (S) of the cells to which each plant extract was added. It was obtained by the calculation of.
Recovery rate (%) = 100 × ((S)-(C1)) / ((C0)-(C1))

In the cells (control (+ UV)) irradiated with UVB without adding the plant extract, the expression level of PIMT was remarkably decreased. In addition, in the cells to which the extracts of Enmeisou, Perilla, Shirakaba, Yuzu, Sage, Soybean, Arnica, Shobu, Lavender, Peach, and Hop were added, the cells subjected to UVB irradiation without adding these plant extracts Compared with the result of, the expression level of PIMT was higher. In particular, the effects of extracts obtained from birch, soybean, iris, lavender, peach, hop, sage, and emmeisou were remarkable. Extracts of birch, soybean, emmeisou, sage, and lavender showed a significant difference from the control (+ UV).

<Test Example 6>
Changes in PIMT expression level in skin cells due to addition of plant extract The following evaluations were carried out using the white birch extract, sage extract, soybean extract, emmeiso extract, and lavender extract prepared in Preparation Example 1.
In the same manner as in Test Example 5, neonatal-derived human dermal fibroblasts (NHDF-NB: purchased from Kurabo Industries Ltd.) were cultured until they became confluent.
Then, each plant extract was added to the obtained cells at a concentration of 10 μg / mL, and the cells were cultured for 24 hours. As the medium, α-MEM medium without FBS was used, and the plant extract was dissolved in DMSO and added so that the final concentration of DMSO in the medium was 0.1% by mass. A medium containing 0.1% by mass DMSO was used as a control.
Twenty-four hours after the addition of the plant extract, the culture supernatant was removed, and a fresh 10% FBS-containing α-MEM medium containing the DMSO solution of the above plant extract (final concentration of the plant extract was 10 μg / mL, final DMSO). The concentration was changed to 0.1% by mass), and the mixture was further cultured for 24 hours. After this culture, a sample obtained by recovering a protein soluble in a protein extraction reagent from cells was used as a sample, and the amount of PIMT per amount of protein (PIMT mass / mass of protein) was measured by the same method as in Test Example 5.
In this experiment, a sample was prepared at n = 3 or n = 4 for each plant extract according to the above procedure. The amount of PIMT was measured twice for each sample, and the average value was taken as the measured value of each sample. For each plant extract, the average value of the measured values (n = 3 or n = 4) of each sample was used as the quantitative result of the amount of PIMT.

The results are shown in Table 3. Table 3 shows the relative value (%) (PIMT expression level (%)) when the PIMT expression level of the control was 100% when each plant extract was added. In Table 3, "*" indicates a significant difference from the control (*: p <0.05).

As shown in Table 3, addition of each extract of birch, sage, soybean, emmeisou and lavender to the skin cells increased the expression level of PIMT as compared with the control. This result indicates that the extracts of these plants have an activating effect on PIMT in the skin. The birch extract was significantly different from the control.

<Test Example 7>
Changes in PIMT expression level in skin cells due to addition of plant extract In human skin when the grape extract prepared in Preparation Example 1 was added to neonatal-derived human dermal fibroblasts (NHDF-NB: purchased from Kurabou Co., Ltd.) The protein expression level of PIMT was evaluated.

Cells were cultured until confluent in the same manner as in Test Example 5. Then, the grape extract was added to the obtained cells at a concentration of 2.5 μg / mL, 5 μg / mL or 10 μg / mL, and cultured for 24 hours. As the medium, α-MEM medium without FBS was used, and the grape extract was dissolved in dimethyl sulfoxide (DMSO) and added so that the final concentration of DMSO in the medium was 0.1% by mass. A medium containing 0.1% by mass DMSO was used as a control.

Twenty-four hours after the addition of the grape extract, the culture supernatant was removed, replaced with a Hanks balanced salt solution (manufactured by Nacalai Tesque, Inc.), and irradiated with ^{UVB (cumulative light intensity of about 5.7 mJ / cm 2).} After irradiation, fresh 10% FBS-containing α-MEM medium containing a DMSO solution of grape extract (final concentration of grape extract is 2.5 μg / mL, 5 μg / mL or 10 μg / mL, final concentration of DMSO is 0. It was replaced with 0.1% by mass) and cultured for another 48 hours. After this culture, a sample obtained by recovering a protein soluble in a protein extraction reagent from cells was used as a sample, and the amount of PIMT per amount of protein (PIMT mass / mass of protein) was measured by the same method as in Test Example 5.
In this experiment, a sample was prepared at n = 4 for each plant extract according to the above procedure. The amount of PIMT was measured twice for each sample, and the average value was taken as the measured value of each sample. For each plant extract, the average value of the measured values (n = 4) of each sample was used as the quantitative result of the amount of PIMT.

The results are shown in Table 4. In Table 4, "control" is a control that is not irradiated with UVB, and "control (+ UV)" is a control that is irradiated with UVB. In Table 4, "-" of UV irradiation means that UVB was not irradiated, and "+" means that UV irradiation was performed. “**” indicates a significant difference from “control (+ UV)” (**: p <0.01).

The quantification result of PIMT was shown as a relative value (%) (PIMT expression level (%)) with the PIMT amount (PIMT expression level) per protein amount of the UVB-irradiated control (control (+ UV)) as 100%. In addition, the degree of recovery effect of the grape extract on the decrease in PIMT expression level due to UVB irradiation was shown as "recovery rate (%)". The recovery rate (%) was determined by the same method as in Test Example 5.

In the cells to which the grape extract was added, the expression level of PIMT was higher than that in the cells to which the extract was not added and UVB irradiation was performed. It was shown that the grape extract suppressed the decrease in PIMT expression level due to UVB irradiation.

<Test Example 8>
In the same manner as in Test Example 5, neonatal-derived human dermal fibroblasts (NHDF-NB: purchased from Kurabo Industries Ltd.) were cultured until they became confluent.
Then, the grape extract prepared in Preparation Example 1 was added to the obtained cells at a concentration of 2.5 μg / mL or 5 μg / mL, and the cells were cultured for 24 hours. As the medium, α-MEM medium without FBS was used, and the grape extract was dissolved in DMSO and added so that the final concentration of DMSO in the medium was 0.1% by mass. A medium containing 0.1% by mass DMSO was used as a control.
Twenty-four hours after the addition of the grape extract, the culture supernatant was removed and a fresh 10% FBS-containing α-MEM medium containing the DMSO solution of the grape extract (final concentration of the grape extract was 2.5 μg / mL or 5 μg / g /). The final concentration of mL and DMSO was 0.1% by mass), and the mixture was further cultured for 24 hours. After this culture, a sample obtained by recovering a protein soluble in a protein extraction reagent from cells was used as a sample, and the amount of PIMT per amount of protein (PIMT mass / mass of protein) was measured by the same method as in Test Example 5.
In this experiment, a sample was prepared at n = 5 for each concentration of grape extract according to the above procedure. The amount of PIMT was measured twice for each sample, and the average value was taken as the measured value of each sample. The average value of the measured values (n = 5) of each sample was used as the quantitative result of the PIMT amount.

The results are shown in Table 5. Table 5 shows the relative value (%) (PIMT expression level (%)) with the PIMT expression level of the control as 100% when the grape extract was added. In Table 5, "*" indicates a significant difference from the control (*: p <0.05). Addition of grape extract to skin cells significantly increased the expression level of PIMT compared to the control.

Examples of manufacturing cosmetics and foods and drinks containing extracts of plants such as white birch, grape, soybean, hop, lavender, sage, emmeisou, yuzu, perilla, iris, arnica and peach are shown below. These cosmetics and foods and drinks can be used as cosmetics or foods and drinks for activating PIMT, and are particularly suitable as cosmetics or foods and drinks for activating PIMT present on the skin. In addition, these cosmetics and foods and drinks can also be used as cosmetics or foods and drinks for promoting restoration or repair of isomerized proteins, and also as cosmetics or foods and drinks for promoting restoration or repair of isomerized proteins in the skin. It can be preferably used.

The plant extracts used in Production Examples 1 to 3 are ethanol extracts of each plant (white birch, grape, hop, lavender, sage, emmeiso, yuzu, shobu, arnica or peach) obtained in Preparation Example 1, and butylene of perilla. Glycol extract or soybean water extract is concentrated to a solid content concentration of 0.12% by volume / volume, respectively. As the plant extract of Production Example 4, each plant extract obtained in Preparation Example 1 (ethanol extract of each of the above plants, butylene glycol extract of perilla or freeze-dried powder of soybean water extract) was used. .. In Production Examples 1 to 4, each extract of perilla, grape, hop, lavender, sage, emmeiso, yuzu, perilla, iris, arnica, peach and soybean was used as the plant extract, but two or more of these plants It is also possible to use the extracts of the above in combination.

<Manufacturing example 1>
Toner The raw materials shown in Table 6 were uniformly stirred to prepare a Toner for activating PIMT.

<Manufacturing example 2>
Emulsion A emulsion for activating PIMT was prepared by uniformly stirring the raw materials shown in Table 7.

<Manufacturing example 3>
Cream The ingredients shown in Table 8 were uniformly stirred to prepare a cream for activating PIMT.

<Manufacturing example 4>
Powder for dissolution during use The raw materials shown in Table 9 were uniformly stirred to prepare a powder for activating PIMT. The powder for dissolution at the time of use can be used, for example, by dissolving it in a beverage such as water, coffee, tea or juice, or by mixing it with a food such as yogurt.

<Test Example 9>
Using the expression level or activity of PIMT in skin cells as an index, a substance having an activating effect of PIMT present in the skin, a substance having an effect of promoting restoration or repair of the skin, or a substance having an anti-aging effect of the skin is screened. be able to.
In the evaluation method according to any one of Test Examples 5 to 8, an arbitrary test substance is used instead of the plant extract, and the expression level of PIMT in the skin cells when the test substance is added is evaluated.
When the PIMT expression level (PIMT amount per protein amount) of the cells cultured with the addition of the test substance is higher than the PIMT expression level of the cells (control) cultured without the addition of the test substance, the test is performed. Select the substance as the active ingredient. The test substance selected as the active ingredient can be used as an active ingredient for activating PIMT present in the skin, promoting restoration or repair of the skin, or anti-aging of the skin.

The present invention is useful in the fields of pharmaceuticals, quasi-drugs, cosmetics, foods and drinks, and the like.

Claims (28)

Kabanoki family Kabanoki genus plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus plant, Shobu family Shobu genus plant, Kiku family Arnica genus plant and Rose family peach genus A composition for activating protein L-isoaspartate methyl transferase (PIMT), which comprises an extract of one or more plants selected from the group consisting of plants. The composition for activating PIMT according to claim 1, which is a composition for activating PIMT present on the skin. Claim 1 that the plant extract is an extract of one or more plants selected from the group consisting of birch, grape, soybean, hop, lavender, sage, emmeisou, yuzu, perilla, iris, arnica and peach. Or the composition for activating PIMT according to 2. The PIMT activation according to any one of claims 1 to 3, wherein the plant extract is an extract of one or more plants selected from the group consisting of birch, grape, soybean, lavender, sage and emmeisou. Composition. The composition for activating PIMT according to any one of claims 1 to 4, which is an external preparation for skin. The composition for activating PIMT according to any one of claims 1 to 5, which is a cosmetic. The composition for activating PIMT according to any one of claims 1 to 4, which is a food or drink. Kabanoki family Kabanoki genus plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus plant, Shobu family Shobu genus plant, Kiku family Arnica genus plant and Rose family peach genus A composition for promoting restoration or repair of a protein containing an isomerized amino acid, which comprises an extract of one or more plants selected from the group consisting of plants. The composition for promoting restoration or repair of a protein according to claim 8, which is a composition for promoting restoration or repair of a protein containing an isomerized amino acid in the skin. 8. The plant extract is an extract of one or more plants selected from the group consisting of white birch, grape, soybean, hop, lavender, sage, emmeisou, yuzu, perilla, iris, arnica and peach. Or the composition for promoting restoration or repair of the protein according to 9. Restoration of the protein according to any one of claims 8 to 10, wherein the plant extract is an extract of one or more plants selected from the group consisting of birch, grape, soybean, lavender, sage and emmeiso. Composition for promoting restoration. The composition for promoting restoration or repair of the protein according to any one of claims 8 to 11, which is an external preparation for skin. The composition for promoting restoration or repair of the protein according to any one of claims 8 to 12, which is a cosmetic. The composition for promoting restoration or repair of the protein according to any one of claims 8 to 11, which is a food or drink. Kabanoki family Kabanoki genus plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus plant, Shobu family Shobu genus plant, Kiku family Arnica genus plant and Rose family peach genus A method for activating protein L-isoaspartate methyl transferase (PIMT), which applies an extract of one or more plants selected from the group consisting of plants to an animal. Kabanoki family Kabanoki genus plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus plant, Shobu family Shobu genus plant, Kiku family Arnica genus plant and Rose family peach genus A method for promoting restoration or repair of a protein containing an isomerized amino acid, which applies an extract of one or more plants selected from the group consisting of plants to an animal. A method for promoting restoration or repair of a protein containing an isomerized amino acid in the skin, which activates the protein L-isoaspartate methyltransferase (PIMT) present in human skin. A method for evaluating skin restoration or repair activity using the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index. The evaluation method according to claim 18, wherein the skin cells are epidermal cells and / or dermis cells. The evaluation method according to claim 18 or 19, wherein the skin restoration or repair activity is a skin restoration or repair activity against aging of the skin due to ultraviolet rays and / or aging. A method for evaluating the anti-aging activity of skin, using the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index. The evaluation method according to claim 21, wherein the anti-aging activity is an anti-aging activity against aging of the skin due to ultraviolet rays and / or aging. Effective for activation of PIMT present in the skin, promotion of skin restoration or repair, or anti-aging of the skin, using the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index. Ingredient screening method. A step of contacting the skin cells with a test substance to measure the expression level or activity of PIMT in the skin cells, and
When the expression level or activity of PIMT in the skin cells contacted with the test substance is larger than the expression level or activity of PIMT in the skin cells not contacted with the test substance, the test substance is used as an active ingredient. The screening method according to claim 23, which comprises a step of selection. The screening method according to claim 23 or 24, wherein the skin cells are cultured skin cells. Use of the expression level or activity of protein L-isoaspartate methyltransferase (PIMT) in skin cells as an index of skin restoration or repair activity or skin anti-aging activity. For activation of protein L-isoaspartate methyl transferase (PIMT), Kabanoki family Kabanoki plant, Vine family grape genus plant, Bean family Soybean genus plant, Asa family Karahanasou genus plant, Shiso family plant, Mikan family Mikan genus Use of an extract of one or more plants selected from the group consisting of plants, plants of the family Shobu, plants of the genus Arnica of the family Kiku and plants of the genus Peach of the family Rose. To restore or promote the restoration or repair of proteins containing isomerized amino acids, plants of the genus Kabanoki, plants of the genus Grape, plants of the genus Soybeans of the family Asa, plants of the genus Karahanasou of the family Asa, plants of the genus Mikan of the family Mikan, Use of an extract of one or more plants selected from the group consisting of the plants of the genus Shobu, the genus Arnica of the family Kiku, and the plants of the genus Peach of the rose family.

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