JP2015028051A - Skin-beautifying method by heparanase inhibitor and evaluation method of substance having skin-beautifying effect - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new skin-beautifying method, and to provide an evaluation method of a substance having skin-beautifying effect.SOLUTION: This invention relates to: skin-beautifying method that includes inhibiting activities of heparanase existing in the skin; and an evaluation method of a skin-beautifying effect of a subject substance, which includes bringing the subject substance into contact with a skin, a skin tissue or cells of a human or an animal, measuring enzyme activities of heparanase in the skin, and evaluating skin-beautifying effects of the subject substance using the changes in the enzyme activities of heparanase as an index.

Description

  The present invention relates to a whitening method using a heparanase inhibitor and a method for evaluating a substance having a whitening effect.

  It is thought that pigmentation such as skin spots and freckles causes excessive melanin formation due to hormonal abnormalities, ultraviolet rays, and local inflammation of the skin, which deposits in the skin. This melanin, which causes skin pigmentation, is synthesized in pigment cells (melanocytes) in the basal layer of the epidermis and stored in vesicles called melanosomes. This melanosome moves into the melanocyte, is taken up by surrounding keratinocytes (keratinocytes), and the skin becomes black. Melanin in the melanocytes is produced by converting tyrosine into black melanin through an enzymatic or non-enzymatic oxidation reaction via dopaquinone by the action of the enzyme tyrosinase. Thus, it has been considered that the suppression of tyrosinase activity, which is the first stage reaction, is important in suppressing the production of melanin, and substances that inhibit tyrosinase activity have been developed.

  However, the compounds that suppress the activity of tyrosinase exhibit very slow effects except for hydroquinone, so that the effect of improving skin pigmentation is not sufficient (Japanese Patent Laid-Open No. 58-154507 (Patent Document 1)). . On the other hand, hydroquinone has an effect, but its general use is limited because of its sensitization. Therefore, development of a whitening agent by an action mechanism other than tyrosinase activity inhibition is desired.

JP 58-154507 A WO 2005/042495 A1

Semin Cancer Biol., 2002; 12 (2): 121-129 Mol Cancer Ther., 2004; 3 (9): 1069-1077 Bioorg. Med. Chem. Lett., 2006; (16): 409-412.

  An object of the present invention is to provide a more effective whitening method. Furthermore, an object of the present invention is to provide a method capable of evaluating the whitening effect of a test substance more efficiently and simply.

  As a result of biochemical analysis of the wrinkle formation process induced by the fine wrinkle model, the present inventor previously showed that the expression of heparanase increased in the wrinkle formation process, and heparan sulfate of perlecan, a heparan sulfate proteoglycan in the basement membrane. We have found that wrinkle formation can be suppressed by applying a substance that inhibits the activity of heparanase by finding that the chain is degraded (PCT / JP2009 / 056717). Therefore, as a result of investigating the relationship between the inhibition of heparanase activity and stains, new knowledge was obtained that heparan sulfate was specifically decomposed at the stain site. Then, the compound 1- [4- (1H-benzimidazol-2-yl) -phenyl] -3- [4- (1H-benzimidazol-2-yl) -phenyl] -urea known as a heparanase inhibitor is whitened. It was found that by acting on a skin model for effect verification (a skin model including melanocytes), the skin blackening was significantly suppressed as compared with the control, and the present invention was completed. 1- [4- (1H-Benzimidazol-2-yl) -phenyl] -3- [4- (1H-benzimidazol-2-yl) -phenyl] -urea is known to have a heparanase inhibitory effect. There is no known whitening effect.

Therefore, this application includes the following inventions.
(1) A whitening method characterized by suppressing the activity of heparanase present in the skin.
(2) The whitening method according to (1), wherein a substance that suppresses heparanase gene expression is applied as a method for suppressing the activity of heparanase present in the skin.
(3) The whitening method according to (1), wherein a substance that suppresses translation of the heparanase gene is applied as a method for suppressing the activity of heparanase present in the skin.
(4) The whitening method according to (1), wherein a substance that inhibits the enzyme activity of heparanase is applied as a method of suppressing the activity of heparanase present in the skin.
(5) The whitening method according to (1), wherein a substance that inhibits activation of the enzyme activity of heparanase is applied as a method of suppressing the activity of heparanase present in the skin.
(6) The whitening method according to (2), wherein a substance that suppresses the activity of heparanase present in the skin is administered by means such as oral, injection, or external application.
(7) A whitening agent for oral administration containing a substance that suppresses the activity of heparanase present in the skin as an active ingredient.
(8) A whitening agent for injection containing a substance that suppresses the activity of heparanase present in the skin as an active ingredient.
(9) A skin whitening agent containing a substance that suppresses the activity of heparanase present in the skin as an active ingredient.
(10) The whitening agent according to any one of (7) to (9), wherein the substance that suppresses the activity of heparanase is 4- (1H-benzoimidazol-2-yl) -phenylamine.
(11) A test substance is brought into contact with human or animal skin, skin tissue or cells, and the enzyme activity, gene expression level or heparan sulfate chain of heparanase in the skin is measured, and the enzyme activity, gene expression level or heparan sulfate of heparanase is measured. A method for evaluating a whitening effect of a test substance, wherein the whitening effect of the test substance is evaluated using a change in chain as an index.
(12) The method according to (11), wherein epidermal keratinocytes are used.
(13) The method according to (11), wherein dermal fibroblasts are used.

  By applying the oral, injection and skin external preparations of the present invention, and by the cosmetic method of the present invention, it is possible to suppress heparanase present in the skin and obtain a whitening effect very effectively. Moreover, it is possible to specify the substance which has the whitening effect which has a higher effect efficiently and simply by the evaluation method of this invention.

An appearance photograph of a skin model containing melanocytes (MEL-FT, manufactured by MatTeK, USA) is shown. The comparison result of the melanin amount in the epidermis of each skin model is shown. The results of immunostaining of senile pigment spots and normal tissues of perlecan and heparan sulfate in the vicinity thereof are shown. The results of immunostaining with an anti-CD31 antibody, which is a blood vessel marker, and the results of image analysis are shown. The results of immunostaining with an anti-LYVE-1 antibody, which is a lymphatic marker, and image analysis results are shown. The result of the in situ bFGF binding assay in a senile pigment spot tissue is shown. The result of the in situ bFGF binding assay in a seborrheic pigment spot tissue is shown. The evaluation result of heparanase activity inhibition of 4- (1H-benzimidazol-2-yl) -phenylamine is shown.

  Heparanase is an enzyme that exists in various cells and specifically degrades the heparan sulfate chain of various heparan sulfate proteoglycans. In the skin, epidermis keratinocytes constituting the epidermis, dermal fibroblasts, vascular endothelial cells and the like are produced. It is known that the production is also increased in various cancer cells, and a relationship with the malignancy of cancer is also suggested. High production of heparanase in cancer cells is known to be highly metastatic and to induce angiogenesis (Vlodavsky I., et. Al., Semin Cancer Biol., 2002; 12 (2): 121 -129 (see Non-Patent Document 2)).

  In the present invention, it has been clarified that heparan sulfate in the basement membrane is degraded in the senile pigmented spot tissue as compared with the exposed skin. With the degradation of heparan sulfate, the regulation of vascular endothelial growth factor-A (VEGF-A) expressed in the epidermis is disrupted, causing inflammation due to changes in vascular and lymphatic vessels of the dermis and activating melanocytes Promotes melanin storage in melanosomes. In addition, disruption of fibroblast growth factor-7 (FGF-7) expressed in the dermis promotes delivery of melanosomes from melanocytes to epidermal cells. That is, it is considered that the degradation of heparan sulfate accompanying heparanase activation synergistically accumulates in keratinocytes by activation of melanocytes due to inflammation and promotion of melanosome delivery due to failure of FGF-7 control.

  Substances showing the whitening effect of the present invention include substances that inhibit the activity of heparanase existing in the skin, substances that inhibit the expression of heparanase, substances that inhibit the translation of heparanase, and substances that inhibit the activation of the enzyme activity of heparanase It contains a heparanase activity inhibitory substance such as as an active ingredient.

  The whitening method of the present invention is characterized in that the heparanase activity-suppressing substance is administered to heparanase present in the skin by an oral, injection or external method to suppress heparanase in the skin.

In this specification, “suppression of heparanase activity” means not only inhibiting the enzyme activity of heparanase, but also inhibiting gene expression and protein biosynthesis, inhibiting activation of the enzyme activity of heparanase, etc. It includes any action that suppresses the action of heparanase. Examples of substances that inhibit the enzyme activity of heparanase include 1- [4- (1H-benzoimidazol-2-yl) -phenyl] -3- [4- (1H-benzimidazol-2-yl) -phenyl] -Urea (WO 2005/042495 A1 (Patent Document 2), various compounds and herbal medicines including suramin are known.
The measurement of the activity of heparanase according to the present invention can be carried out quantitatively or qualitatively according to any method capable of measuring heparanase itself or its enzyme activity. Specifically, it measures the degradation product of the heparanase substrate heparan sulfate, or an immunoassay method using an antibody specific to heparanase to measure the amount of heparanase biosynthesis, for example, an ELISA method using an enzyme label Various methods such as an RIA method using a radioactive label, an immunoturbidimetric method, a Western blot method, a latex agglutination method, and an erythrocyte agglutination method can be mentioned. Examples of immunoassay methods include competitive methods and sandwich methods. In addition, it can also be performed by measuring the amount of a gene encoding heparanase. In this case, preferably, heparanase expression is determined by measuring the amount of mRNA encoding heparanase in the cell. Extraction of mRNA and quantitative or qualitative measurement of the amount thereof are also well known in the art, and can be performed by various well-known methods such as PCR, 3SR, NASBA, and TMA. In addition, heparanase can be qualitatively determined through in situ hybridization and measurement of its biological activity.

  If the activity of heparanase is significantly lower than that of, for example, control skin, it is determined that the activity is suppressed. “Significantly reduced compared to control skin” means that the measured activity of heparanase is statistically significant compared to “control skin cells”, ie, skin cells not treated with a substance that inhibits the activity of heparanase. For example, 95% or less, or 90% or less, or 80% or less, or 70% or less, or 60% or less, or 50% or less, or 30% or less, or 10% or less. Say.

  The method for evaluating the whitening effect of a test substance of the present invention comprises contacting a test substance with human or animal skin, skin tissue, cells or enzymes, and enzyme activity, gene expression level or heparan sulfate of heparanase in the skin, tissues or cells. It is characterized by evaluating the whitening effect using the change of the chain as an index.

  In this specification, “whitening” refers to the suppression of skin darkening due to melanosome accumulation in keratinocytes resulting from the activation of melanocytes associated with the degradation of heparan sulfate in the basement membrane, and improves spots, freckles, dullness, etc. It means to do. The “whitening method” as used in the present invention means a cosmetic purpose unless otherwise specified, but in some cases, it may be a medical purpose.

  In the whitening method of the present invention, the above-mentioned substance that suppresses heparanase can be applied in any form as long as the object of the present invention can be achieved, and can be applied alone or together with other optional components. You may mix and apply. When applied to the skin, the location of the skin is not limited, and includes any skin on the body surface including the scalp.

  The method for evaluating the whitening effect of the present invention comprises a step of bringing a test substance into contact with human or animal skin, skin tissue, cells or enzymes.

  The human or animal skin that can be used in the evaluation method is not particularly limited as long as the object of the present invention can be achieved. In the evaluation method of the present invention, for example, by specifying a substance that decreases gene expression enhanced in a whitening model or a substance that inhibits degradation of heparan sulfate chains, a substance that exhibits a whitening effect can be efficiently identified. It is done.

  The primary evaluation using heparanase activity as an index is, for example, biotinylated heparan sulfate was immobilized in 96 wells, then heparanase was allowed to act in the presence of drugs or crude drugs, and the amount of biotinylated heparan sulfate was labeled with peroxidase. It can be carried out by evaluating heparanase activity by allowing avidin to act and color it. A drug having an inhibitory effect on heparanase activity in the primary evaluation can be evaluated for reproducibility and concentration dependency in a secondary evaluation system using heparanase activity different from the primary evaluation as an index. The secondary evaluation can be performed using HT1080 cells expressing heparanase. HT1080 cells are known to migrate depending on heparanase activity when the cells are scratched after culturing confluently (Ishida K., et. Al., Mol Cancer Ther., 2004; 3 (9) : 1069-1077. (Non-Patent Document 4)). Therefore, by adding an evaluation agent, heparanase inhibitory activity is evaluated from the degree of migration (recovery) of the scratch site.

  As a result, 4- (1H-benzimidazol-2-yl) -phenylamine was found as a compound that significantly suppresses heparanase activity. It is not known in the prior art that 4- (1H-benzoimidazol-2-yl) -phenylamine and its derivatives show heparanase activity inhibitory action or whitening action.

  4- (1H-Benzimidazol-2-yl) -phenylamine and plant extracts having heparanase activity can be used in the whitening method of the present invention. In particular, plant extracts are highly safe and useful for the treatment, prevention, improvement and the like of various symptoms, diseases, and pathologies caused by heparanase activity. In particular, it is suitably applied to prevention of melanocyte activation based on inhibition of heparanase activity in the skin. More specifically, the prevention and improvement of synergistic melanosome accumulation by the activation of melanocytes associated with the degradation of the heparan sulfate chain of the basement membrane proteoglycan due to aging and photoaging and the promotion of melanosome delivery due to the breakdown of FGF-7 control, It is suitably applied to maintaining a skin state free from spots, freckles, and dullness.

  When the heparanase activity inhibitor of the present invention is blended in a skin external preparation, the amount of each of the above heparanase activity inhibitors is preferably 0.0001 to 1% by mass, particularly 0.0001 to dry mass (solid content mass) in the total amount of the external preparation. 0.2% by mass. If the amount is less than 0.0001% by mass, the effect of the present invention is not sufficiently exerted. On the other hand, if the amount exceeds 1% by mass, no significant improvement in the effect is observed, and formulation becomes difficult.

  When the heparanase activity inhibitor of the present invention is applied to, for example, an external preparation for skin, in addition to the above essential components, components that are usually used for external preparations such as cosmetics and pharmaceuticals within the range not impairing the effects of the present invention, , Whitening agents, moisturizers, antioxidants, oil components, UV absorbers, surfactants, thickeners, alcohols, powder components, colorants, aqueous components, water, various skin nutrients, etc. as required It can mix | blend suitably.

  Furthermore, edetate disodium, edetate trisodium, sodium citrate, sodium polyphosphate, sodium metaphosphate, sequestering agents such as gluconic acid, methylparaben, ethylparaben, butylparaben, phenoxyethanol and other preservatives, caffeine, Tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, vitamin C, ascorbic acid phosphate Whitening agents such as magnesium, ascorbic acid glucoside, arbutin, kojic acid, 4-methoxysalicylic acid, sugars such as glucose, fructose, mannose, sucrose, trehalose, retinoic acid, retinol, retinol acetate, Vitamin A derivatives such as palmitic acid retinol can also be appropriately blended. When the heparanase activity inhibitor of the present invention is used in combination with a whitening agent, a further whitening effect is expected.

  The external preparation for skin can be applied to cosmetics, quasi-drugs, etc., particularly preferably cosmetics applied to the outer skin, and the dosage form can also be applied to the skin. Any dosage form such as solution system, solubilization system, emulsification system, powder dispersion system, water-oil two-layer system, water-oil-powder three-layer system, ointment, lotion, gel, aerosol, etc. is applicable. Is done.

  The use form is also arbitrary, and for example, it can be used for facial cosmetics such as lotions, emulsions, creams and packs, makeup cosmetics such as foundations, lipsticks and eye shadows, aromatic cosmetics, bath preparations and the like.

  In addition, makeup cosmetics can be widely applied to foundations and the like, and toiletries such as body soaps and soaps. Furthermore, if it is a quasi-drug, it can be widely applied to various ointment forms. And the form which can take the heparanase activity inhibitor of this invention is not limited to these dosage forms and forms.

  When the heparanase activity inhibitor of the present invention is used as a pharmaceutical preparation, the preparation is appropriately used orally or parenterally (intravenous administration, intraperitoneal administration, etc.). The dosage form is also arbitrary, for example, oral solid preparations such as tablets, granules, powders and capsules, oral liquid preparations such as internal liquids and syrups, and parenteral liquid preparations such as injections. These forms can also be appropriately prepared by known methods. For these pharmaceutical preparations, commonly used binders, disintegrants, thickeners, dispersants, reabsorption accelerators, corrigents, buffers, surfactants, solubilizers, preservatives, emulsifiers, isotonicity Excipients such as agents, stabilizers and pH adjusters may be used as appropriate.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to the following Example.
Experiment 1: Evaluation of whitening effect with heparanase inhibitor Using a skin model containing melanocytes, heparanase inhibitor 1- [4- (1H-benzoimidazol-2-yl) -phenyl] -3- [4- ( The whitening effect of 1H-benzoimidazol-2-yl) -phenyl] -urea was investigated.
Culture of a skin model containing melanocytes (MEL-FT, manufactured by MatTeK, USA) was started in a special medium (MEL-FT-NMM-113, manufactured by MatTeK, USA). From the second day of the culture, 1- [4- (1H-benzoimidazol-2-yl) -phenyl] -3- [4- (1H--) was prepared so that the control group had DMSO and the heparanase inhibitor group had a final concentration of 50 μM Benzimidazol-2-yl) -phenyl] -urea was added for culture, and the medium was changed every 2 or 3 days. On the 10th and 14th days of culture, skin models were collected and external appearance photographs were taken. As a result, it was found that the color of the appearance of the heparanase inhibitor group was lighter than that of the control group.

Further, only the epidermis of the skin model was collected, added with 300 μL of 0.2N sodium hydroxide solution, stirred, allowed to stand at room temperature for 24 hours, and heated at 95 ° C. for 30 minutes to completely solubilize the epidermis. When the amount of melanin contained in the epidermis was examined by measuring the 475 nm absorbance of the solution after solubilization, the heparanase inhibitor group had a significantly lower OD475 nm value than the control group, that is, the amount of melanin was small. It became clear.
FIG. 1 shows an appearance photograph of the MEL-FT skin model. It is clear that the heparanase inhibitor group is clearly white on the 10th and 14th day of culture. FIG. 2 shows a comparison result of the amount of melanin in the epidermis of each skin model. It can be seen that the absorbance value at OD475 nm, which is an indicator of melanin in the heparanase inhibitor group, is significantly low on the 10th and 14th days of culture. Therefore, it was proved that the heparanase inhibitor has a whitening effect.

Experiment 2: Immunostaining of frozen human tissue Frozen tissue blocks of senile pigment spots and nearby normal site skin were newly sectioned, and 8 μm sections were prepared. The tissue section cut to 8 μm was fixed with cold acetone, dried, and then de-OCTed with PBS. The tissue endogenous peroxidase was inactivated by treatment with 3% hydrogen peroxide solution, blocked with 10% normal goat serum, and reacted in the order of the primary antibody and secondary antibody in Table 1. The tissue labeled with HRP was washed 5 times with PBS and then developed with AEC. The tissue after color development was sufficiently washed with running water and then encapsulated with a water-soluble mounting agent.

Experiment 3: in situ bFGF assay
25 μg of bFGF was bound to 200 μL of swollen heparin-Sepharose (CL-6B; Pharmacia Biotech), NH ₂ -reactive-biotin (Dojindo molecular tech.) Dissolved in DMSO was reacted at room temperature for 5 minutes, and 800 μL washed Wash with buffer (20mmol / L HEPES, pH7.4, 400mmol / L NaCl) and elute twice with 200μL elution buffer (20mmol / L HEPES, pH7.4, 0.2% BSA, 3mol / L NaCl). High salt concentration biotinylated bFGF was recovered. Thereafter, it was placed in an Ultra free C3LGC column (Amicon) and washed 3 times with PBS (0.25 g / L) to obtain biotinylated bFGF.

  A paraffin tissue section (senile, seborrheic keratosis site and its normal part in the vicinity) cut into 5 μm was deparaffinized with xylene and replaced with ethanol (100% → 70%) and 3% peroxidation Tissue endogenous peroxidase was inactivated by hydrogen water treatment. Subsequently, the endogenous heparan sulfate binding factor was released by washing with a pH 5 buffer (containing 0.5 M NaCl) and a pH 10 buffer (containing 0.5 M NaCl). Blocked with 10% serum, reacted with biotinylated bFGF (10 nmol / L) for 1 hour at room temperature, and washed 3 times with PBS. Thereafter, peroxidase-labeled streptavidin (Nichirei, Japan) was allowed to act at room temperature for 15 minutes, washed 3 times with PBS, and developed with DAB. The tissue after color development was thoroughly washed with running water, stained with hematoxylin, and substituted with ethanol (70% → 100%) and xylene, and then sealed.

Experiment 4: Blood vessel and lymphatic vessel image analysis
For CD31-stained and LYVE1-stained tissues, three photographs were taken per section, and the number and area of stained blood vessels and lymphatic vessels were calculated by image analysis using win roof (Mitani Corporation). Furthermore, the density and size of blood vessels and lymphatic vessels were calculated by calculating the total dermis area of the dermal papilla layer area by image analysis.

  FIG. 3 shows immunostaining results of senile pigment spots and normal tissues of perlecan and heparan sulfate in the vicinity thereof. In normal tissues, the basement membrane is stained for both perlecan and heparan sulfate, whereas in senile pigmented spots, only perlecan staining is stained, and the staining for heparan sulfate is significantly reduced. From this result, it can be seen that heparan sulfate is specifically decomposed at the senile pigment spot site.

  FIG. 4 shows the results of immunostaining with an anti-CD31 antibody, which is a blood vessel marker, and the results of image analysis. By calculating the number, thickness, and area of blood vessels stained with cd31 using the image analysis software win roof, each stained tissue was analyzed for changes in blood vessels in the senile pigmented spot region and its nearby normal region. It was revealed that the size of the blood vessel and the blood vessel area were significantly higher at the senile pigment spot. From these results, it became clear that vasodilation occurred in the senile pigment spot.

  FIG. 5 shows the results of immunostaining with anti-LYVE-1 antibody, which is a lymphatic marker, and image analysis results. Analyzing changes in lymphatic vessels in senile pigmented spots and nearby normal sites by calculating the number, thickness, and area of lymphatic vessels stained with LYVE-1 using analysis software win roof did. It was revealed that the size of the lymphatic vessel and the lymphatic vessel area were significantly higher at the senile pigment spot. From this result, it became clear that lymphangiectasia occurred in the senile pigment spot.

  FIG. 6 shows the results of an in situ bFGF binding assay in senile pigment spot tissue. In the normal tissue near the senile pigment spot, the basement membrane is stained brown, indicating that bFGF binds, but in the senile pigment spot part, no staining of the basement membrane is seen, That is, this indicates that bFGF cannot be bound, and it is thought that bFGF could not be bound due to decomposition of heparan sulfate.

  FIG. 7 shows the results of an in situ bFGF binding assay in seborrheic pigmented tissue. In normal tissues near the seborrheic pigment spot, the basement membrane is stained brown, indicating that bFGF binds, but at the seborrheic pigment spot site, the basement membrane is stained. This indicates that bFGF cannot be bound, and that bFGF could not be bound due to degradation of heparan sulfate.

Screening for heparanase activity inhibition
4- (1H-benzimidazole, a derivative of 1- [4- (1H-benzimidazol-2-yl) -phenyl] -3- [4- (1H-benzimidazol-2-yl) -phenyl] -urea Heparanase activity was examined for -2-yl) -phenylamine.
A431 cells (invasive human epithelial cancer cells) were cultured in DMEM medium containing 10% serum. The cultured cells were solubilized with Lysis Buffer (50 mM Tris, 0.5% TritonX-100, 0.15 M NaCl, pH 4.5), collected with a scraper, pipetted, and allowed to stand on ice for 30 minutes. Thereafter, centrifugation was performed at 10,000 rpm for 10 minutes to remove insoluble matters, and the supernatant was used as a cell extract. The amount of protein in the cell extract was measured with BCA Protein Assay Kit (PIERCE, CA46141).

The cell extract is diluted with assay buffer (50 mM HEPES, 50 mM CH ₃ COONa, 150 mM NaCl, 9 mM CaCl ₂ , 0.1% BSA) to a predetermined concentration, and 4- (1H-benzoimidazol-2-yl) -phenylamine is diluted. Were added and mixed, and then seeded on a biotinylated heparan sulfate-immobilized plate at 100 μL / well. Incubate for 2 hours at 37 ° C, wash 3 times with PBS-T, inoculate 10,000-fold diluted HRP-avidin (Vector, A-2004) / PBS-T at 100 µL / well, and react at 37 ° C for 1 hour I let you. Washed again with PBS-T three times, seeded and reacted with TMB reagent (BIO-RAD, 172-1066) at 100 μL / well, stopped with 1NH ₂ SO ₄ , and then measured OD475nm (See Special Table 2003-502054 (Patent Document 3)).
The heparanase activity was calculated from the calibration curve of the A431 cell extract, and showed the inhibition rate (%) with a value relative to the data (control) to which no drug extract was added.

As a result, it was revealed that 4- (1H-benzoimidazol-2-yl) -phenylamine effectively suppresses heparanase activity in a concentration-dependent manner. The result is shown in FIG. In the control, DMSO was allowed to act instead of the candidate drug.
IC50 = 256μM

Claims (8)

  A whitening method characterized by suppressing the activity of heparanase present in the skin.   The whitening method according to claim 1, wherein a substance that inhibits the enzyme activity of heparanase is applied as a method of suppressing the activity of heparanase present in the skin.   The whitening method according to claim 1 or 2, wherein a substance that suppresses the activity of heparanase present in the skin is administered by means selected from oral, injection, and external application.   A whitening agent containing, as an active ingredient, a substance that suppresses the activity of heparanase present in the skin.   The whitening agent of Claim 4 which is a skin external preparation.   The test substance is brought into contact with human or animal skin, skin tissue or cells, the enzyme activity of heparanase in the skin is measured, and the whitening effect of the test substance is evaluated using changes in the enzyme activity of heparanase as an index. Evaluation method of whitening effect of test substance.   The method according to claim 6, wherein epidermal keratinocytes are used.   The method according to claim 6, wherein dermal fibroblasts are used.