JP7328793B2 - Screening method using the expression level of lox gene under hypoxic conditions as an index, inhibitor of increase in lox gene expression, inhibitor of subcutaneous adipocyte fibrosis, and agent for improving or preventing sagging - Google Patents

Description

The present invention relates to a method of screening for a component that suppresses tissue fibrosis.

BACKGROUND ART In recent years, research on anti-aging has been actively conducted with the aim of suppressing the functional deterioration of each tissue of the human body due to aging, that is, the aging phenomenon. In particular, in the technical fields of beauty and cosmetics, active ingredients for suppressing aging phenomena such as wrinkles, sagging, and spots on the skin due to aging are being searched for.

For example, Patent Document 1 discloses a method of screening active ingredients having effects such as wrinkle improvement, slackness improvement, reduction in firmness, prevention of reduction in skin elasticity, etc., using the expression level of skin retinaculum components in cells as an index. is disclosed.
Patent Literature 2 discloses a method of screening for a component having an effect of preventing or improving wrinkles, using the abnormal state of the nuclear membrane of cells as an index.

In addition, research aimed at improving sagging is also being conducted by focusing on subcutaneous adipocytes present in subcutaneous tissue. Patent Document 3 discloses a method of screening for a sagging-improving component using the activity of adhesion molecules in subcutaneous adipocytes as an indicator (Patent Document 3).

By the way, it is known that the pO ₂ (oxygen partial pressure) of arterial blood is lowered 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 hypoxic conditions and aging phenomena, but the full picture of the mechanism of how aging is caused by what physiological changes occur under hypoxic conditions has not been elucidated. do not have.

JP 2017-112918 A JP 2013-257347 A JP 2016-187324 A

Journal of the Geriatrics Society/Volume 15 (1978) No. 1, "Arterial blood gas analysis values of healthy elderly people", Etsuko Taguchi et al., 1978

The problem to be solved by the present invention is to provide a new technique for screening for ingredients that are effective in improving or preventing skin aging, particularly sagging.

As a result of intensive research by the present inventors, it has become clear that the relative viscoelasticity of the subcutaneous fat layer decreases with aging. Further detailed analysis based on this finding revealed that collagen fibers that wrap subcutaneous adipocytes present in the subcutaneous tissue become fibrous with aging. Since it is known that the oxygen concentration of arterial blood decreases with aging (Non-Patent Document 1), the present inventors focused on the possibility that fibrosis of subcutaneous adipocytes is induced by hypoxic conditions. . As a result of further analysis, the present inventors found that an increase in the expression level of lox, a gene involved in cross-linking of collagen fibers, was observed in subcutaneous adipocytes placed under hypoxic conditions. completed.

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

INDUSTRIAL APPLICABILITY According to the method of the present invention, it is possible to easily screen for a component that suppresses subcutaneous adipocyte fibrosis due to hypoxic conditions and/or aging.

In a preferred form of the invention, said cells are adipocytes. Thereby, it is possible to more accurately screen for a component that suppresses fibrosis of subcutaneous adipocytes.

The present invention is preferably applied to screening methods for anti-aging ingredients.

Moreover, it is preferable to apply the present invention to a method of screening for ingredients for improving or preventing age-related sagging.

In a preferred embodiment of the present invention, the lox gene expression level in cells cultured under hypoxic conditions in the presence of the test component is lower than in cells cultured under hypoxic conditions in the absence of the test component. In some cases, the test component is determined to be a component that suppresses subcutaneous adipocyte fibrosis 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 hypoxic conditions are conditions in which the oxygen concentration in the cell culture atmosphere is 5% or less.
By performing cell culture under such conditions, screening can be performed more effectively.

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

The present invention also relates to active ingredients found by the screening method described above. Specifically, the present invention is characterized by containing, as an active ingredient, an extract of a plant belonging to the genus Caryophyllaceae Saponaria of the family Caryophyllaceae, suppressing elevation of lox gene expression due to hypoxic conditions and/or aging. It also relates to an agent, an agent for suppressing fibrosis of subcutaneous adipocytes, and an agent for improving or preventing age-related sagging.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to easily screen anti-aging components, specifically components that suppress tissue fibrosis due to hypoxic conditions and/or aging.
In addition, the agent containing, as an active ingredient, an extract of a plant belonging to the genus Caryophyllaceae, which has been confirmed to be effective by the screening method of the present invention, has an effect of suppressing an increase in lox gene expression due to hypoxic conditions and/or aging, It exerts an effect of suppressing fibrosis of subcutaneous adipocytes and an effect of improving or preventing age-related sagging.

1 is a reference photograph obtained by an electron microscope and used for evaluating the degree of fibrosis of subcutaneous adipocytes. The photograph with a score of 1 has the lowest degree of progression of fibrosis, and the photograph with a score of 5 has the highest degree of progression of fibrosis. Fig. 10 is a scatter diagram showing the results of observing subcutaneous adipocytes of 9 donors and scoring them based on reference photographs. Fig. 4 is a micrograph showing differences in fibrous structure of subcutaneous adipocytes in cells cultured under normal oxygen concentration conditions and hypoxic conditions. 1 is a bar graph showing expression levels of vegf gene, col1a1 gene, col3a1 gene, tgf-β gene, and lox gene in cells cultured under normal oxygen concentration conditions and hypoxic conditions. 2 is a bar graph showing the expression level of lox genes in cells cultured under hypoxic conditions with the addition of soapwort leaf extract or grape leaf extract.

The present invention is a method of screening for a component that suppresses subcutaneous adipocyte fibrosis that progresses under hypoxic conditions. In addition, since tissues are exposed to hypoxic conditions with aging, the present invention can also be used to screen for components that suppress fibrosis of subcutaneous adipocytes due to aging.

Here, "fibrosis" refers to a phenomenon in which tissue hardens due to an abnormal increase in collagen surrounding 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 was revealed that the increase in the expression level of the lox gene is deeply involved in the fibrosis of .

LOX (lysine oxidase), the product of the lox gene, is an extracellular enzyme that catalyzes the formation of aldehyde groups on lysine residues of collagen and elastin precursors. These aldehydes are highly reactive and undergo spontaneous chemical reactions with other oxidized lysine-derived aldehyde groups and unmodified lysine residues. The result is cross-linking of collagen and elastin, which is important for the stabilization of collagen fibers and the integrity and elasticity of mature elastin.

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

As described above, the essential configuration of the present invention is to use the effect of reducing the increase in lox gene expression in cells cultured under hypoxic conditions as an indicator.

As described above, fibrosis of subcutaneous adipocytes progresses with aging (decrease in oxygen concentration in arterial blood). Therefore, the present invention can be suitably applied to screening methods for components that suppress fibrosis of subcutaneous adipocytes due to hypoxic conditions and/or aging.

In addition, as the fibrosis of the collagen fiber structure that wraps the adipocytes in the subcutaneous fat layer progresses, the physical properties of the subcutaneous tissue deteriorate, making it difficult to support the weight of the skin, leading to aging typified by skin sagging. advances.
Therefore, the present invention is preferably applied to an anti-aging ingredient, more specifically, a screening method for an ingredient that improves or prevents age-related sagging.

The specific configuration of the present invention will be described below.
Cells used in the present invention may be established cultured cell lines or primary cultured cells.
Although the type of cells is not particularly limited, it is preferable to use adipocytes. Either brown adipocytes or white adipocytes may be used, but white adipocytes are preferably used.
Mature subcutaneous adipocytes obtained by differentiating subcutaneous preadipocytes may be used in the present invention.

A known medium suitable for the cells to be used can be used without limitation for the medium used for cell culture. The growth medium, differentiation medium, and maintenance medium used for differentiating subcutaneous preadipocyte cells into subcutaneous adipocytes 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 in a CO ₂ incubator for normal cell culture is about 18-19%, the 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, even more preferably 5% or less, still 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 cultured at an oxygen concentration of preferably 0.1% or more, more preferably 0.5% or more.

In cell culture under hypoxic conditions, a CO ₂ incubator for low oxygen concentration culture (for example, manufactured by Ikemoto Rika Kogyo Co., Ltd.) equipped with cylinders such as nitrogen gas and mixed gas in addition to carbon dioxide gas may be used. A hypoxic culture device (for example, manufactured by Sugiyama Giken Co., Ltd.) consisting of a gas barrier pouch containing a gas concentration adjusting agent may be used.

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 of time.
The method for measuring the expression level of the lox gene is not limited, and a preferred example is a method of measuring the mRNA level of the lox gene in the collected cells by RT-PCR.

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

It is preferable to also perform a control experiment in order to accurately determine whether the test component has the effect of reducing the increased expression of the lox gene under hypoxic conditions.
Specifically, the lox gene expression levels in cells cultured under hypoxic conditions in the presence of the test component are compared with cells cultured under hypoxic conditions in the absence of the test component as controls. 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 measuring the expression level of the lox gene, it is also preferable to measure the expression level of the vegf gene, which is a hypoxic response marker, in order to confirm that the cells are undergoing a hypoxic response.
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 subcutaneous adipocyte fibrosis due to hypoxic conditions and/or aging can be screened. The present invention also relates to an agent containing, as an active ingredient, the ingredient found by the screening method.

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

Examples of plants belonging to the genus Saponaria of the family Caryophyllaceae include, but are not particularly limited to, Saponaria officinalis.

The plant extract in the present invention can be prepared by using a plant that is naturally grown or grown in Japan or a foreign country, an extract that is sold as a raw material for Chinese herbal medicine, etc., or a plant extract such as Maruzen Co., Ltd. It is also possible to purchase and use commercially available extracts sold by companies that deal with

The plant extract shall mean not only the plant extract itself, but also the fraction of the extract, the purified fraction, the extract or the fraction, and the solvent-removed product of the refined product. , wild or grown plants, extracts using those sold as raw materials for Chinese herbal medicines, commercially available extracts, and the like.

In the extraction operation, in addition to using the whole plant part, parts such as the plant body, the ground part, the rhizome part, the tree trunk, the leaf part, the stem part, the flower spike, and the flower bud can be used. It is preferable to cut to improve the extraction efficiency.
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, and ethers such as diethyl ether and tetrahydrofuran. One or more selected from polar solvents such as polar solvents can be exemplified as suitable.

As a specific extraction method, for example, 1 to 30 parts by mass of a solvent is added to 1 mass of the part used for extraction of the plant or the like or its dry matter, and if it is at room temperature, it will be several days, even if it is at a temperature near the boiling point. For example, a method of immersing for several hours, cooling to room temperature, optionally removing insoluble matter and/or solvent, and fractionating and purifying by column chromatography or the like can be mentioned.

The agent of the present invention is preferably in the form of an external preparation or an oral preparation by appropriately combining with optional ingredients used for formulation.
Examples of external preparations include cosmetics, quasi-drugs, skin external medicines, and the like. Moreover, their dosage forms are not particularly limited. Among them, the form of cosmetics that can be used continuously is preferable from the relationship with the application of improving or preventing skin sagging due to aging. etc. is preferable.

In the case of an oral formulation, it is preferably in the form of a food composition containing the agent of the present invention as an active ingredient. More specifically, it is preferably in the form of supplements having dosage forms such as general foods, tablets, granules, and drinks.

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

In the case of an oral formulation, the dry mass of the extract per intake is usually 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, depending on the dosage form. 2000 mg or less, preferably 1000 mg or less, more preferably 500 mg or less.

When it is in the form of cosmetics, it may contain optional ingredients that are usually used in cosmetics. Optional ingredients include polyethylene glycol, glycerin, 1,3-butylene glycol, erythritol, sorbitol, xylitol, maltitol, propylene glycol, dipropylene glycol, diglycerin, isoprene glycol, 1,2-pentanediol, 2,4- Hexylene glycol, polyols such as 1,2-hexanediol and 1,2-octanediol, fatty acid soaps (sodium laurate, sodium palmitate, etc.), anionic surfactants such as potassium lauryl sulfate and triethanolamine ether of alkyl sulfates cationic surfactants such as stearyltrimethylammonium chloride, benzalkonium chloride, laurylamine oxide, imidazoline amphoteric surfactants (2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium salt etc.), betaine surfactants (alkylbetaine, amidobetaine, sulfobetaine, etc.), amphoteric surfactants such as acylmethyl taurine, sorbitan fatty acid esters (sorbitan monostearate, sorbitan sesquioleate, etc.), glycerin fatty acid (glyceryl monostearate, etc.), propylene glycol fatty acid esters (propylene glycol monostearate, etc.), hydrogenated castor oil derivatives, glycerin alkyl ether, POE sorbitan fatty acid esters (POE sorbitan monooleate, polyoxyethylene monostearate) sorbitan, etc.), POE sorbitol fatty acid esters (POE-sorbitol monolaurate, etc.), POE glycerin fatty acid esters (POE-glycerin monoisostearate, etc.), POE fatty acid esters (polyethylene glycol monooleate, POE distearate, etc.), POE alkyl ethers (POE 2-octyldodecyl ether, etc.), POE alkyl phenyl ethers (POE nonylphenyl ether, etc.), Pluronic types, POE/POP alkyl ethers (POE/POP 2-decyltetradecyl ether, etc.), Tetronic , POE castor oil, hydrogenated castor oil derivatives (POE castor oil, POE hydrogenated castor oil, etc.), nonionic surfactants such as sucrose fatty acid esters, alkyl glucosides, sodium pyrrolidone carboxylate, lactic acid, sodium lactate, etc. Ingredients, powders such as mica, talc, kaolin, synthetic mica, calcium carbonate, magnesium carbonate, silicic anhydride (silica), aluminum oxide, barium sulfate, which may be surface-treated, even if surface-treated Inorganic pigments such as cobalt oxide, ultramarine blue, Prussian blue, and zinc oxide May be surface-treated Composite pigments such as sintered iron oxide titanium dioxide May be surface-treated Titanium mica, fish phosphorus foil , Pearl agents such as bismuth oxychloride, Red No. 202, Red No. 228, Red No. 226, Yellow No. 4, Blue No. 404, Yellow No. 5, Red No. 505, Red No. 230, Red which may be laked Organic pigments such as No. 223, Orange No. 201, Red No. 213, Yellow No. 204, Yellow No. 203, Blue No. 1, Green No. 201, Purple No. 201, Red No. 204, polyethylene powder, polymethyl methacrylate, nylon powder , organic powders such as organopolysiloxane elastomers, 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 its derivatives, vitamin E such as α-tocopherol, β-tocopherol, γ-tocopherol, vitamin E acetate, vitamin D, vitamin H, pantothenic acid, pantethine, pyrroloquinoline quinone, etc. Examples include vitamins.

<Test Example 1> Observation of age-related changes in collagen structure Subcutaneous adipocytes in subcutaneous tissue provided by 9 donors aged 20 or over were photographed with a scanning electron microscope. The electron micrographs were evaluated by a trained evaluator and scored from 1 to 5 for the degree of subcutaneous adipocyte fibrosis. The evaluation was performed based on five-stage reference photographs (Fig. 1) with different degrees of progression of fibrosis. The results are shown in FIG.

As shown in FIG. 2, there was a significant correlation between the age of the donor and the degree of subcutaneous adipocyte fibrosis. This result indicates that fibrosis of subcutaneous adipocytes progresses with aging.
Considering that the oxygen concentration in arterial blood decreases with aging, hypoxic conditions in the subcutaneous adipose tissue are a factor in the progression of age-related subcutaneous adipocyte fibrosis. Conceivable.

<Test Example 2> Observation of changes in collagen structure under hypoxic conditions Human subcutaneous adipose precursor cells (HPAd) were seeded in a 24-well multiwell plate (2.0 × 10 ⁴ cells/well) together with a growth medium, and then confluent. cultured until Thereafter, the medium was changed to a differentiating medium, and cultured for 14 days while changing the differentiating medium every 3 days to mature into subcutaneous adipocytes. Thereafter, the medium was replaced with a maintenance medium, the plate was placed in a hypoxic culture device (BIONIX, manufactured by Sugiyama Giken Co., Ltd.) equipped with an oxygen scavenger, and the oxygen concentration was adjusted to 1%, and the culture was continued. At this time, as a control, cells were also prepared by encapsulating the plate in a hypoxic culture device from which the oxygen scavenger was removed and continuing the culture. After culturing, the structure of collagen fibers was photographed with a scanning electron microscope. The results are shown in FIG.

As shown in FIG. 3, in the cells cultured under hypoxic conditions, the thickness of the collagen fibers was uneven compared to the cells cultured under normal oxygen concentration, and it was found that the collagen fibers formed amorphous structures.

<Test Example 3> Cell culture under hypoxic conditions Human subcutaneous adipose precursor cells (HPAd) were seeded in a 24-well multiwell plate (2.0 × 10 ⁴ cells/well) together with a growth medium, and then cultured until confluent. did. Thereafter, the medium was changed to a differentiating medium, and cultured for 14 days while changing the differentiating medium every 3 days to mature into subcutaneous adipocytes. Thereafter, the medium was replaced with a maintenance medium, the plate was placed in a hypoxic culture device (BIONIX, manufactured by Sugiyama Giken Co., Ltd.) equipped with an oxygen scavenger, and the oxygen concentration was adjusted to 1%, and the culture was continued. At this time, as a control, cells were also prepared by encapsulating the plate in a hypoxic culture device from which the oxygen scavenger was removed and continuing the culture. 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 wells 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 used as cDNA using Superscript VILO cDNA Synthesis Kit (Life Technologies).
Real-time PCR was performed using QuantiTect Primer Assay to measure the expression levels of col1a1 gene, col3a1 gene, tgf-β gene, and lox gene. 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. In other words, it was confirmed that the cells induced a hypoxic 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 the lox gene were remarkably increased by culture under hypoxic conditions (Fig. 4).

LOX, the product of the lox gene, is an enzyme involved in the cross-linking of collagen fibers. Also, TGF-β is a factor involved in the production of collagen fibers. In other words, the results shown in FIG. 4 indicate that the production of LOX and TGF-β associated with collagen fibers increases under hypoxic conditions, and fibrosis can be promoted.

<Discussion>
The results of Test Examples 1 and 3 show that when subcutaneous adipocytes are placed under hypoxic conditions due to aging, the expression of the lox gene increases, and the gene product, LOX, promotes cross-linking of collagen fibers. indicates that fibrosis progresses. This indicates that a component capable of suppressing an increase in lox gene expression under hypoxic conditions can suppress fibrosis of subcutaneous adipocytes.
In other words, the results of Test Examples 1 and 3 show that the effect of reducing the increased expression of the lox gene in cells cultured under hypoxic conditions is used as an index to suppress fibrosis of subcutaneous adipocytes due to hypoxic conditions or aging. It shows that the ingredients can be screened.

<Test Example 4> Screening Test Human subcutaneous adipose 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, a medium change was performed using growth medium.
After 48 hours, the differentiation medium was changed to initiate differentiation. Differentiation culture was performed for 17 days while replacing the medium once every two days.
The medium was replaced with a maintenance medium, and the soapwort leaf extract and grape leaf extract to be screened were added thereto. A plate without added extract was prepared as a control.
The plate was sealed in a hypoxic culture apparatus (BIONIX, manufactured by Sugiyama Giken Co., Ltd.) 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 in the same manner as in Test Example 3, cDNA library was prepared by reverse transcription, and the LOX gene expression level was analyzed by real-time PCR. The results are shown in FIG.

As shown in FIG. 5, the soapwort leaf extract exerted the effect of significantly suppressing the increase in the lox gene expression level under hypoxic conditions. On the other hand, grape leaf extract did not show such a significant effect.
This test example confirms that it is possible to screen for components that suppress fibrosis of subcutaneous adipocytes due to hypoxic conditions or aging by using the effect of reducing the increase in lox gene expression in cells cultured under hypoxic conditions as an indicator. did it.

In addition, this result shows that the agent containing as an active ingredient an extract of a plant belonging to the genus Soapwort of the family Caryophyllaceae has an effect of suppressing the increase in lox gene expression due to hypoxic conditions and/or aging, and an effect of suppressing fibrosis of subcutaneous adipocytes. , and shows that it exerts an improving effect or a preventive effect on sagging associated with aging.

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

Claims (10)

A screening method for a component that suppresses subcutaneous adipocyte fibrosis under hypoxic conditions and/or age-related hypoxic conditions , using as an index the effect of reducing lox gene expression elevation in cells cultured under hypoxic conditions. 2. The screening method according to claim 1, wherein said cells are adipocytes. 3. The screening method according to claim 1 or 2, which is a screening method for an anti-aging component. The screening method according to any one of claims 1 to 3, which is a screening method for improving or preventing sagging due to hypoxia associated with aging. When the lox gene expression level in cells cultured under hypoxic conditions in the presence of the test component is lower than in cells cultured under hypoxic conditions in the absence of the test component, the test component The screening according to any one of claims 1 to 4, characterized in that it is determined to be a component that suppresses fibrosis of subcutaneous adipocytes due to hypoxic conditions and / or hypoxic conditions associated with aging Method. The screening method according to any one of claims 1 to 5, wherein the hypoxic condition is a condition in which the oxygen concentration in the cell culture atmosphere is 5% or less. The screening method according to any one of claims 1 to 6, characterized in that an increase in the expression level of the Vegf gene is used as an indicator of the occurrence of a hypoxic response in cells cultured under hypoxic conditions. . An agent for suppressing elevation of lox gene expression due to hypoxic conditions and/or hypoxic conditions associated with aging, characterized by containing a soapwort leaf extract as an active ingredient. An agent for suppressing fibrosis of subcutaneous adipocytes caused by hypoxic conditions and/or hypoxic conditions associated with aging, characterized by containing a soapwort leaf extract as an active ingredient. An agent for improving or preventing age-related sagging due to hypoxia , characterized by containing a soapwort leaf extract as an active ingredient.