JP2019207232A - Screening method using, as index, expression level of lox gene in hypoxia condition, increase inhibitor of expression of lox gene, fibrosis inhibitor of subcutaneous fat cell, and improvement or preventive agent of sagging - Google Patents

Abstract

To provide a new technology for screening a component having an effect of improving or preventing an aging phenomenon of a skin, especially sagging.SOLUTION: A reduction effect of the expression increase of a lox gene in a cell cultivated in a hypoxia condition is used as an index.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a screening method for components that suppress tissue fibrosis.

  In recent years, research on anti-aging for the purpose of suppressing the deterioration of the function of each tissue of the human body accompanying aging, that is, the aging phenomenon, has been actively conducted. In particular, in the technical fields of beauty and cosmetics, search for active ingredients that suppress aging phenomena such as wrinkles, sagging, and spots accompanying aging has been conducted.

For example, Patent Document 1 discloses a method for screening an active ingredient having effects such as wrinkle improvement, sagging improvement, prevention of reduction of elasticity, prevention of reduction of skin elasticity, and the like, using the expression level of skin stroma components in cells as an index. Is disclosed.
Patent Document 2 discloses a method for screening a component having an effect of preventing or improving wrinkles using an abnormal state of the nuclear membrane of cells as an index.

  In addition, research aimed at improving sagging has been conducted by focusing on subcutaneous fat cells present in subcutaneous tissue. Patent Document 3 discloses a method for screening a sag improving component using the activity of adhesion molecules of subcutaneous fat cells as an index (Patent Document 3).

By the way, it is known that pO ₂ (oxygen partial pressure) of arterial blood is reduced by analysis by blood gas analysis (Non-patent Document 1). This indicates that each tissue of the human body is placed in a hypoxic state with aging.
These findings suggest a relationship between hypoxia and aging, but it is not clear what kind of physiological change occurs under hypoxic conditions to cause aging. Absent.

JP 2017-1112918 A JP 2013-257347 A JP, 2006-187324, A

Journal of the Japan Geriatrics Society / Vol. 15 (1978) No. 1, “Analytical blood gas analysis values of healthy elderly people”, Reiko Taguchi et al., 1978

  The problem to be solved by the present invention is to provide a new technique for screening a component having an effect of improving or preventing skin aging, particularly sagging.

  As a result of intensive studies by the present inventors, it has been clarified that the relative viscoelasticity of the subcutaneous fat layer decreases with aging. A more detailed analysis based on this finding revealed that collagen fibers encapsulating subcutaneous fat cells present in the subcutaneous tissue become fibrotic with aging. Since it is known that arterial oxygen concentration decreases with age (Non-patent Document 1), the present inventors focused on the possibility that fibrosis of subcutaneous adipocytes is caused by hypoxic conditions . As a result of further analysis, the present inventors have found that an increase in the expression level of lox, a gene involved in collagen fiber crosslinking, is observed in subcutaneous adipocytes placed under hypoxic conditions. Was completed.

The present invention that solves the above problems uses as an index the effect of reducing the increase in lox gene expression in cells cultured under hypoxic conditions,
This is a screening method for a component that suppresses fibrosis of subcutaneous fat cells due to hypoxic conditions and / or aging.

  According to the method of the present invention, components that suppress fibrosis of subcutaneous adipocytes due to hypoxic conditions and / or aging can be easily screened.

  In a preferred form of the invention, the cells are adipocytes. Thereby, the component which suppresses fibrosis of a subcutaneous fat cell can be screened more accurately.

  The present invention is preferably applied to a screening method for anti-aging components.

  Moreover, it is preferable to apply this invention to the improvement method of the sagging accompanying aging, or the screening method of a preventive component.

In a preferred embodiment of the present invention, the amount of lox gene expression in cells cultured under hypoxic conditions in the presence of the test component is low compared to cells cultured under hypoxic conditions in the absence of the test component. In this case, the test component is determined to be a component that suppresses fibrosis of subcutaneous fat cells due to hypoxic conditions and / or aging.
By conducting a control experiment in this way, screening can be performed with higher accuracy.

In a preferred embodiment of the present invention, the low oxygen condition is a condition where the oxygen concentration in the cell culture atmosphere is 5% or less.
Screening can be performed more effectively by culturing cells under such conditions.

In a preferred embodiment of the present invention, an increase in the expression level of the vegf gene is used as an indicator that a hypoxic response occurs in cells cultured under hypoxic conditions.
By placing such an index, the suitability of the test system can be confirmed, and screening can be performed more accurately.

  The present invention also relates to an active ingredient found by the above screening method. Specifically, the present invention contains an extract of a plant belonging to the genus Caryophyllaceae Saponaria as an active ingredient, and suppresses the increase in lox gene expression due to hypoxic conditions and / or aging. The present invention also relates to an agent and an inhibitor of fibrosis of subcutaneous fat cells, and an agent for improving or preventing sagging with aging.

According to the present invention, an anti-aging component, specifically, a component that suppresses tissue fibrosis due to hypoxic conditions and / or aging can be easily screened.
Further, an agent containing an extract of a plant belonging to the genus Cervaceae, which is confirmed to be effective by the screening method of the present invention, as an active ingredient is an inhibitory effect on the increase in lox gene expression due to hypoxic conditions and / or It exerts an effect of suppressing fibrosis of subcutaneous fat cells and an effect of improving or preventing sagging associated with aging.

It is the reference | standard photograph acquired by the electron microscope used in order to evaluate the grade of fibrosis of a subcutaneous fat cell. A photograph with the lowest degree of progression of fibrosis is a photograph with a score of 1, and a photograph with the highest degree of progression of fibrosis is a photograph with a score of 5. It is a scatter diagram showing the result of observing the subcutaneous fat cells of nine donors and assigning scores based on a reference photograph. It is a microscope picture which shows the difference in the fiber structure of a subcutaneous fat cell in the cell cultured on normal oxygen concentration conditions and low oxygen conditions. It is a bar graph showing the expression levels of the vegf gene, col1a1 gene, col3a1 gene, tgf-β gene, and lox gene in cells cultured under normal oxygen concentration conditions and low oxygen conditions. It is a bar graph showing the expression level of the lox gene in the cell which added the sorghum leaf extract or the grape leaf extract, and was culture | cultivated on hypoxic conditions.

  The present invention is a method for screening for a component that suppresses fibrosis of subcutaneous adipocytes that progress under hypoxic conditions. In addition, since the tissue is placed under hypoxic conditions with aging, the present invention can also target components that suppress fibrosis of subcutaneous adipocytes due to aging.

Here, “fibrosis” refers to a phenomenon in which a tissue becomes hard due to an abnormal increase in collagen surrounding the tissue or cross-linking of collagen fibers.
As a factor of fibrosis, an increase in the expression level of collagen itself and an increase in the expression level of genes involved in the cross-linking reaction of the collagen fiber structure can be assumed. It became clear that the increase in the expression level of the lox gene is deeply involved in fibrosis.

  LOX (lysine oxidase), which is a product of the lox gene, is an extracellular enzyme that catalyzes the reaction of forming an aldehyde group at the lysine residue of collagen and elastin precursor. These aldehydes are highly reactive, and spontaneously react with other oxidized lysine-derived aldehyde groups and unmodified lysine residues. The result is collagen and elastin cross-linking, which is important for collagen fiber stabilization and mature elastin integrity and elasticity.

  The lox gene itself plays an important role in tissue stabilization, but it causes fibrosis due to increased expression. As described above, fibrosis of subcutaneous adipocytes under hypoxia is caused not by an increase in collagen expression but by an increase in lox gene expression. Therefore, it can be said that the component that suppresses the increase in lox gene expression under hypoxic conditions is a component that suppresses fibrosis of subcutaneous fat cells due to hypoxic conditions or aging.

  As mentioned above, this invention makes it an essential structure to use as a parameter | index the reduction effect of the expression increase of lox gene in the cell culture | cultivated on hypoxic conditions.

  As described above, fibrosis of the subcutaneous fat cells progresses with aging (decrease in oxygen concentration in arterial blood). Therefore, the present invention can be suitably applied to a screening method for a component that suppresses fibrosis of subcutaneous fat cells due to hypoxic conditions and / or aging.

In addition, as the fibrosis of the collagen fiber structure surrounding fat cells in the subcutaneous fat layer progresses, the physical properties of the subcutaneous tissue deteriorate, making it difficult to support the weight of the skin, and aging represented by sagging skin Advances.
Therefore, the present invention is preferably applied to a screening method for an anti-aging component, more specifically, an improvement in sagging associated with aging or a preventive component.

Hereinafter, a specific configuration of the present invention will be described.
The cell used in the present invention may be an established cultured cell line or a primary cultured cell.
The type of cells is not particularly limited, but it is preferable to use fat cells. Brown adipocytes or white adipocytes may be used, but white adipocytes are preferably used.
Mature subcutaneous fat cells obtained by differentiating subcutaneous fat precursor cells may be used in the present invention.

  As a medium used for cell culture, a known medium suitable for the cells to be used can be used without limitation. The growth medium, differentiation medium, and maintenance medium used when differentiating subcutaneous fat precursor cells into subcutaneous fat cells may be any known ones, and commercially available kits may be used.

In the present invention, it is essential to culture cells under hypoxic conditions. Since the oxygen concentration is about 18 to 19% in a CO ₂ incubator in normal cell culture, cells are cultured at an oxygen concentration lower than this. Specifically, the cells are cultured at an oxygen concentration of preferably 15% or less, more preferably 10% or less, more preferably 7% or less, still more preferably 5% or less, and even more preferably 3% or less.
The lower limit of the oxygen concentration is not particularly limited as long as the cultured cells do not die, but the cells are preferably cultured at an oxygen concentration of 0.1% or more, more preferably 0.5% or more.

In cell culture under low oxygen conditions, a CO ₂ incubator for low oxygen concentration culture (for example, manufactured by Ikemoto Rika Kogyo Co., Ltd.) equipped with a cylinder such as nitrogen gas or mixed gas in addition to carbon dioxide gas may be used. You may use the hypoxic culture instrument (for example, the product made from a Sugiama Giken) which consists of a gas-barrier pouch provided with a gas concentration regulator.

In the present invention, a test component is added to the medium of cells cultured under hypoxic conditions, and the expression level of the lox gene is measured after culturing for a predetermined period.
The method for measuring the expression level of the lox gene is not limited, and a method of measuring the mRNA level of the lox gene in the collected cells by RT-PCR is preferable.

  When cells are cultured under hypoxic conditions, the expression level of lox gene increases. In the present invention, when the test component is added to the medium and the degree of increase in the expression of the lox gene under hypoxic conditions is reduced, the test component is a hypoxic condition and / or aging of subcutaneous adipocytes due to aging. A component that suppresses fibrosis is determined and selected.

In order to accurately determine whether the test component has an effect of reducing the increase in lox gene expression under hypoxic conditions, a control experiment is also preferably performed.
That is, the expression level of the lox gene in the cells cultured under the hypoxic condition in the presence of the test component is compared with the cells cultured under the hypoxic condition in the absence of the test component. When the lox gene expression level is lower in the latter than in the former, it is preferable to determine that the test component is a component that suppresses tissue fibrosis due to hypoxic conditions and / or aging.

In addition to the measurement of the expression level of the lox gene, it is also preferable to measure the expression level of the vegf gene, which is a marker for the hypoxia response reaction, in order to confirm that the cell has undergone a hypoxia response reaction.
If it can be confirmed that the expression level of the vegf gene is higher than when cells are cultured at a normal oxygen concentration, the test system can be evaluated as appropriate, and the accuracy of screening can be improved.

  According to the screening method of the present invention, a component that suppresses fibrosis of subcutaneous adipocytes due to hypoxic conditions and / or aging can be screened. And this invention relates also to the agent which contains the component discovered by the said screening method as an active ingredient.

  As a component that suppresses fibrosis of subcutaneous fat cells due to hypoxic conditions and / or aging, an extract of a plant belonging to the genus Caryophyllaceae Saponaria can be mentioned.

  Although it does not specifically limit as a plant which belongs to Nadesicoaceae genus Saponaria, Saponaria (Saponaria officinalis) can be illustrated.

  The plant extract according to the present invention can be prepared using plants grown or grown in Japan or abroad, such as Chinese herbal medicine raw materials, or plant extracts such as Maruzen Co., Ltd. It is also possible to purchase and use a commercial extract sold by a company that handles

  The plant extract means not only the plant extract itself but also the extract fraction, the purified fraction, the extract or fraction, and the solvent-removed product of the purified product. In addition, an extract using a plant grown as a native or grown plant, a herbal medicine raw material or the like, a commercially available extract, and the like can be mentioned.

For the extraction operation, the whole plant part can be used, and other parts such as the plant body, the above-ground part, the rhizome part, the tree trunk part, the leaf part, the stem part, the flower ear, and the flower bud can be used. It is preferable to improve the extraction efficiency by cutting.
Extraction solvents include water, alcohols such as ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, ethers such as diethyl ether and tetrahydrofuran. 1 type or 2 types or more selected from polar solvents, such as these, can be illustrated as a suitable thing.

  As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a part used for extraction of a plant or the like or a dried product thereof. For example, after immersion for several hours and cooling to room temperature, the insoluble matter and / or solvent may be removed if desired, and fractional purification may be performed by column chromatography or the like.

It is preferable that the agent of the present invention is appropriately combined with an arbitrary component used for formulation to form an external preparation or an oral preparation.
Examples of the external preparation include cosmetics, quasi-drugs, skin external medicines and the like. Moreover, those dosage forms are not particularly limited. Among them, the form of cosmetics that can be used continuously is preferable from the viewpoint of the use of improving or preventing skin sag associated with aging, and among them, lotion, cosmetic liquid, milky lotion, cream, gel, suncare product. Etc. are preferable.

  Moreover, when setting it as an oral preparation, it is preferable to set it as the form of the composition for foodstuffs which contains the agent of this invention as an active ingredient. More specifically, it is preferable to use a supplement form having dosage forms such as general foods, tablets, granules, and drinks.

  The content of the plant extract in the external preparation (dry mass in the case of the extract) is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, Usually, it is 80 mass% or less, Preferably it is 30 mass% or less, More preferably, it is 10 mass% or less.

  In the case of oral preparations, the amount of intake per dose is usually 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, depending on the dosage form. It is 2000 mg or less, preferably 1000 mg or less, more preferably 500 mg or less.

  When setting it as the form of cosmetics, you may contain the arbitrary component normally used with cosmetics. As optional components, polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4- Anionic surfactants such as polyols such as hexylene glycol, 1,2-hexanediol, 1,2-octanediol, fatty acid soaps (sodium laurate, sodium palmitate, etc.), potassium lauryl sulfate, alkylsulfuric acid triethanolamine ether, etc. , Cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide, imidazoline-based amphoteric surfactants (2-cocoyl-2-imidazoli Umhydroxide-1-carboxyethyloxy disodium salt, etc.), betaine surfactants (alkyl betaines, amide betaines, sulfobetaines, etc.), amphoteric surfactants such as acylmethyltaurine, sorbitan fatty acid esters (sorbitan mono) Stearates, sorbitan sesquioleate, etc.), glycerin fatty acids (glyceryl monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hardened castor oil derivatives, glycerin alkyl ethers, POE sorbitan fatty acid esters ( POE sorbitan monooleate, polyoxyethylene sorbitan monostearate, etc.), POE sorbite fatty acid esters (POE-sorbite monolaurate, etc.), POE glycerin fatty acid ester (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether) ), Pluronic types, POE / POP alkyl ethers (POE / POP2-decyltetradecyl ether, etc.), Tetronics, POE castor oil / hardened castor oil derivatives (POE castor oil, POE hardened castor oil, etc.), Sho Nonionic surfactants such as sugar fatty acid esters and alkyl glucosides, moisturizing ingredients such as sodium pyrrolidone carboxylate, lactic acid and sodium lactate, surface treated, mica, talc, kaolin, synthetic mica, carbonic acid carbonate Powders such as calcium, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, etc., may be surface treated, inorganic pigments of cobalt oxide, ultramarine, bitumen, zinc oxide, surface treated Further, composite pigments such as iron oxide titanium dioxide sintered body, surface treatment, pearl agents such as titanium mica, fish phosphorus foil, bismuth oxychloride, red 202 which may be raked, Red 228, Red 226, Yellow 4, Blue 404, Yellow 5, Red 505, Red 230, Red 223, Orange 201, Red 213, Yellow 204, Yellow 203, Blue 1 , Organic dyes such as green 201, purple 201, red 204, polyethylene powder, polymethyl methacrylate, nylon powder, organopolysiloxane elastomer, etc. Powders, lower alcohols such as ethanol and isopropanol, vitamin A or derivatives thereof, vitamin B6 hydrochloride, vitamin B6 tripalmitate, vitamin B6 dioctanoate, vitamin B2 or derivatives thereof, vitamin B12, vitamin B15 or derivatives thereof, etc. Vitamin E such as vitamin B, α-tocopherol, β-tocopherol, γ-tocopherol, vitamin E acetate, vitamin D, vitamin H, pantothenic acid, panthetin, pyrroloquinoline quinone, and the like.

<Test Example 1> Observation of change in collagen structure with aging Subcutaneous fat cells in subcutaneous tissues provided by nine donors 20 years of age or older were photographed with a scanning electron microscope. This electron micrograph was evaluated by a skilled evaluator and given a score of 1 to 5 for the degree of fibrosis of the subcutaneous fat cells. The evaluation was performed based on a five-step reference photograph (FIG. 1) with different degrees of fibrosis. The results are shown in FIG.

As shown in FIG. 2, the age of the donor and the degree of fibrosis of the subcutaneous fat cells were significantly correlated. This result shows that fibrosis of subcutaneous fat cells progresses with aging.
Considering that oxygen concentration in arterial blood decreases with age, the fact that subcutaneous adipose tissue is put into hypoxic conditions with age is a factor in the progression of fibrosis of subcutaneous fat cells with age Conceivable.

<Test Example 2> Observation of change in collagen structure due to hypoxic condition Human subcutaneous fat precursor cells (HPAD) were seeded in a 24-well multiwell plate with a growth medium (2.0 × 10 ⁴ cells / well), and then confluent. Incubated until. Thereafter, the culture medium was changed to a differentiation medium, and cultured for 14 days while changing the differentiation medium every 3 days to mature into subcutaneous fat cells. Thereafter, the medium was replaced with a maintenance medium, and the plate was sealed in a low oxygen culture apparatus (BIONIX, manufactured by Sugiyama Giken) equipped with an oxygen scavenger, and the culture was continued after adjusting the oxygen concentration to 1%. At this time, as a control, the plate was sealed in a hypoxic culture instrument excluding the oxygen scavenger, and cells that were continuously cultured were also prepared. After culturing, the structure of the collagen fiber was photographed with a scanning electron microscope. The results are shown in FIG.

  As shown in FIG. 3, it was found that the cells cultured under low oxygen conditions have a non-uniform collagen fiber thickness and form an irregular structure as compared with cells cultured under normal oxygen concentration.

<Test Example 3> Cell Culture under Hypoxic Conditions Human subcutaneous fat precursor cells (HPAd) are seeded in a 24-well multiwell plate with a growth medium (2.0 × 10 ⁴ cells / well) and then cultured until confluent. did. Thereafter, the culture medium was changed to a differentiation medium, and cultured for 14 days while changing the differentiation medium every 3 days to mature into subcutaneous fat cells. Thereafter, the medium was replaced with a maintenance medium, and the plate was sealed in a low oxygen culture apparatus (BIONIX, manufactured by Sugiyama Giken) equipped with an oxygen scavenger, and the culture was continued after adjusting the oxygen concentration to 1%. At this time, as a control, the plate was sealed in a hypoxic culture instrument excluding the oxygen scavenger, and cells that were continuously cultured were also prepared. One day after the replacement with the maintenance medium, the culture was terminated, and mRNA was extracted by the following procedure.

After washing the cells in the well with PBS, QIAzol Lysis Reagent of RNeasy Lipid Tissue Kit (QIAGEN) was added (1 mL / well) to extract mRNA from the cells.
The extracted mRNA was defined as cDNA by Superscript VILO cDNA Synthesis Kit (Life Technologies).
Real-time PCR was performed using QuantTect Primer Assay, and the expression levels of col1a1 gene, col3a1 gene, tgf-β gene, and lox gene were measured. At this time, the expression level of the vegf gene, which is a hypoxic response marker, was also measured. The results are shown in FIG.

  As shown in FIG. 4, an increase in the expression level of the vegf gene was observed in cells cultured under hypoxic conditions. That is, it was confirmed that the cells had a hypoxic response reaction by culturing under hypoxic conditions.

Moreover, as shown in FIG. 4, it was found that the expression levels of the col1a1 gene and the col3a1 gene did not change when cultured under hypoxic conditions.
On the other hand, the expression levels of the tgf-β gene and lox gene were significantly increased by culturing under hypoxic conditions (FIG. 4).

  LOX, which is a product of the lox gene, is an enzyme involved in cross-linking collagen fibers. TGF-β is a factor involved in the production of collagen fibers. That is, the results shown in FIG. 4 indicate that the production amount of LOX and TGF-β related to collagen fibers is increased under hypoxic conditions, and fibrosis can be promoted.

<Discussion>
The results of Test Example 1 and Test Example 3 show that when subcutaneous adipocytes are placed under hypoxic conditions due to aging, the expression of lox gene increases, and the gene product LOX promotes the crosslinking of collagen fibers. Indicates that fibrosis proceeds. This indicates that a component capable of suppressing the increase in lox gene expression under hypoxic conditions can suppress fibrosis of subcutaneous fat cells.
In other words, the results of Test Examples 1 and 3 indicate that fibrosis of subcutaneous adipocytes due to hypoxic conditions or aging is suppressed by using the effect of reducing the increase in lox gene expression in cells cultured under hypoxic conditions as an index. It shows that the component can be screened.

Test Example 4 Screening Test Human subcutaneous fat precursor cells (HPAd) were seeded (1.5 × 10 ⁴ cells / well) in a 24-well multiwell plate together with a growth medium. After 48 hours, the medium was changed using the growth medium.
After 48 hours, the culture medium was changed to a differentiation medium and differentiation was started. Differentiation culture was performed for 17 days while changing the medium once every two days.
The culture medium was replaced with a maintenance medium, and the sorghum leaf extract and grape leaf extract to be screened were added thereto. A plate without the extract was prepared as a control.
The plate was sealed in a hypoxic culture apparatus (BIONIX, manufactured by Sugiama Giken) equipped with an oxygen scavenger, and the culture was continued after adjusting the oxygen concentration to 1%.
After culturing for 72 hours, mRNA was collected by the same method as in Test Example 3, a cDNA library was prepared by reverse transcription, and the expression level of the LOX gene was analyzed by real-time PCR. The results are shown in FIG.

As shown in FIG. 5, the Soybean leaf extract exerted an effect of significantly suppressing an increase in the expression level of the lox gene under hypoxic conditions. On the other hand, the grape leaf extract did not have such a significant effect.
This test example confirms that a component that suppresses fibrosis of subcutaneous fat cells due to hypoxic conditions or aging can be screened by using the effect of reducing the increase in lox gene expression in cells cultured under hypoxic conditions as an index. did it.

  In addition, this result shows that an agent containing an extract of a plant belonging to the genus Cerambyceae as an active ingredient has an effect of suppressing the increase in lox gene expression due to hypoxic conditions and / or aging, and the effect of inhibiting fibrosis of subcutaneous fat cells In addition, it shows that the effect of improving or preventing sagging associated with aging is exhibited.

  The present invention can be applied to search for active ingredients relating to anti-aging.

Claims (10)

Using the reduction effect of increased expression of lox gene in cells cultured under hypoxic conditions as an index,
A screening method for a component that suppresses fibrosis of subcutaneous fat cells under hypoxic conditions and / or aging.   The screening method according to claim 1, wherein the cells are fat cells.   The screening method according to claim 1, wherein the screening method is an anti-aging component screening method.   The screening method according to any one of claims 1 to 3, which is a screening method for an ingredient for improving or preventing sagging associated with aging.   When the expression level of the lox gene in cells cultured under hypoxic conditions in the presence of the test component is low compared to cells cultured under hypoxic conditions in the absence of the test component, the test component The screening method according to any one of claims 1 to 4, characterized in that it is a component that suppresses fibrosis of subcutaneous adipocytes due to hypoxic conditions and / or aging.   The screening method according to claim 1, wherein the low oxygen condition is a condition in which an oxygen concentration in a cell culture atmosphere is 5% or less.   The method for screening according to any one of claims 1 to 6, wherein an increase in the expression level of the vegf gene is used as an indicator that a hypoxic response has occurred in cells cultured under hypoxic conditions. .   An agent for suppressing an increase in the expression of a lox gene due to hypoxic conditions and / or aging, comprising an extract of a plant belonging to the genus Caryophyllaceae Saponaria as an active ingredient.   An agent for suppressing fibrosis of subcutaneous adipocytes due to hypoxic conditions and / or aging, comprising an extract of a plant belonging to the genus Caryophyllaceae Saponaria as an active ingredient.   An agent for improving or preventing sagging associated with aging, comprising an extract of a plant belonging to the genus Caryophyllaceae Saponaria as an active ingredient.