Composition for improving skin pigmentation

Abstract

The present invention relates to a composition for improving skin pigmentation and an information providing method for selecting a custom-made substance for improving skin pigmentation, the method comprising a step of confirming a base of a polymorphic site of a gene polymorphic marker which is significant in association with the degree of skin pigmentation. Furthermore, the present invention relates to: a composition for diagnosing the type of skin in which skin pigmentation is likely to occur, the composition comprising a probe capable of detecting a gene polymorphism marker having significance in association with the degree of skin pigmentation or a preparation capable of amplifying the gene polymorphism marker; a kit or microarray comprising the composition; and a method for providing information on the type of skin in which skin pigmentation is likely to occur, using the gene polymorphism marker or the combination of markers.

Description

Technical Field

[0001] This invention relates to compositions for improving skin pigmentation and a method for providing information on selecting a customized substance for improving skin pigmentation, the method comprising the step of identifying bases at polymorphic sites of gene polymorphic markers that are significantly associated with the degree of skin pigmentation. Additionally, this invention relates to compositions for diagnosing skin types prone to pigmentation, comprising probes capable of detecting gene polymorphic markers significantly associated with the degree of skin pigmentation or preparations capable of amplifying such gene polymorphic markers; kits or microarrays comprising the above compositions; and methods for providing information on skin types prone to pigmentation using the aforementioned gene polymorphic markers or combinations of markers. The aforementioned skin pigmentation includes scattered dark spots, dull skin tone, or patchy pigmentation. Background Technology

[0002] The skin is located on the outermost part of our body and occupies the largest area (1.5-2 m²). 2 The skin is an organ responsible for defending against external environmental factors (ultraviolet radiation, blue light emitted by various electronic devices) and foreign substances (air pollutants). Pigmentation is one aspect of this defense mechanism. When the skin is exposed to ultraviolet radiation, melanocytes produce melanin, which migrates to the area around the nucleus of keratinocytes, forming a melanin cap that protects the DNA within the nucleus, thus preventing skin aging and skin cancer.

[0003] However, excessive melanin production due to aging or UV exposure can lead to pigmentation, such as dark spots (scars), dull skin tone, and freckles.

[0004] Dark spots on the skin can naturally appear due to exposure to ultraviolet rays or the effects of pregnancy and childbirth. However, once dark spots appear, skin evenness decreases, luster diminishes, and gives a lifeless, loose, and listless impression. Dark spots are difficult to remove once they appear, and the risk of recurrence is high. Therefore, prevention is crucial after predicting the risk of developing dark spots. In contrast, current products only focus on removing existing dark spots and are composed of standardized formulas, resulting in minimal effectiveness and low consumer satisfaction.

[0005] Furthermore, it is known that dull skin tone gradually develops with exposure to ultraviolet radiation or aging. These external factors may alter the expression of specific genes and the activity of proteins that regulate melanin formation, and the resulting improvement in skin brightness is time-consuming and difficult. To date, skin-brightening products have been uniformly marketed as improving not only skin tone but also freckles, melasma, and sunspots; therefore, in reality, satisfaction is low for products that focus solely on improving skin tone. In particular, research accompanying studies on the genes and mechanisms of action that cause specific skin darkening is rare, making the development of customized materials virtually nonexistent.

[0006] Naturally occurring pigmentation on the skin, caused by factors such as aging or UV exposure, may include age spots, freckles, or sunspots. Unlike scattered pigmentation, such as melasma, which results from the aggregation of multiple pigment deposits, isolated pigmentation that differs from its surroundings can cause the skin to appear noticeably darker and out of sync with its surroundings, creating a tired and unhealthy impression. Once pigmentation appears, it is difficult to remove and has a high risk of recurrence. Therefore, there is a need for formulas that predict the risk of pigmentation and take preventative measures. However, current products focus on removing existing pigmentation. Even methods for removing pigmentation often only offer simple formulas that inhibit melanin production. As a result, existing formulas with insufficient effectiveness have low satisfaction rates for improving pigmentation.

[0007] Previous studies have investigated genetic polymorphic markers that determine skin color across ethnic groups, but there are very few examples of research identifying genetic polymorphic markers that determine differences in skin pigmentation levels within the same ethnic group. Naturally, there has also been no research on developing customized materials based on the analysis of genetic polymorphic markers related to individual skin pigmentation. Summary of the Invention

[0008] Technical issues

[0009] The inventors aim to establish a skin characteristic classification system by constructing big data related to genetic information and skin type information. By understanding the genetic characteristics that determine individual skin characteristics, they seek to concretize scientific skin classification standards. Based on this, they are dedicated to discovering personalized effective ingredients to develop customized cosmetics. As part of the results, specific single nucleotide polymorphism (SNP) markers significantly associated with skin pigmentation were screened, and it was confirmed that these markers can be used to provide consumers with personalized formulations that help improve skin pigmentation.

[0010] Problem-solving methods

[0011] One object of the present invention is to provide a composition for improving skin pigmentation.

[0012] One object of the present invention is to provide a composition for improving skin dispersible dark spots.

[0013] Another object of the present invention is to provide a cosmetic composition or quasi-pharmaceutical composition comprising the above-described composition as an active ingredient.

[0014] Another object of the present invention is to provide an information provision method for selecting a custom substance for improving diffuse melasma, comprising the step of identifying the bases of polymorphic sites of significant single nucleotide polymorphism markers associated with the degree of diffuse melasma.

[0015] One object of the present invention is to provide a single nucleotide polymorphism (SNP) marker for diagnosing whether a skin type is prone to developing scattered dark spots.

[0016] Another object of the present invention is to provide a composition for diagnosing skin types prone to developing scattered dark spots, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing skin types prone to developing scattered dark spots, or an agent capable of amplifying the single nucleotide polymorphism (SNP) marker.

[0017] Another object of the present invention is to provide a kit or microarray comprising the above-described composition for diagnosing skin types prone to developing scattered dark spots.

[0018] Another object of the present invention is to provide a method for providing information on whether a skin type is prone to developing diffuse dark spots, comprising the step of identifying the polymorphic sites of the aforementioned single nucleotide polymorphism markers.

[0019] Another object of the present invention is to provide a method for providing information on customized formulations of substances for high-risk groups prone to developing scattered dark spots on the skin, which includes the step of identifying the polymorphic sites of the aforementioned single nucleotide polymorphism markers.

[0020] One object of the present invention is to provide a composition for improving dull skin tone.

[0021] Another object of the present invention is to provide a cosmetic composition or quasi-pharmaceutical composition comprising the above-described composition as an active ingredient.

[0022] Another object of the present invention is to provide an information provision method for selecting a custom substance for improving dull skin tone, comprising the step of identifying the bases of polymorphic sites of a significant single nucleotide polymorphism marker associated with dull skin tone.

[0023] One object of the present invention is to provide a single nucleotide polymorphism (SNP) marker for diagnosing whether a skin type is prone to developing dull skin.

[0024] Another object of the present invention is to provide a composition for diagnosing skin types prone to dull skin, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing skin types prone to dull skin or an agent capable of amplifying the single nucleotide polymorphism (SNP) marker.

[0025] Another object of the present invention is to provide a kit or microarray comprising the above-described composition for diagnosing skin types prone to developing dull skin tone.

[0026] Another object of the present invention is to provide a method for providing information on whether a skin type is prone to developing dull skin tone, comprising the step of identifying the polymorphic sites of the aforementioned single nucleotide polymorphism markers.

[0027] Another object of the present invention is to provide a method for providing information on customized substance formulations for high-risk groups prone to developing dull skin, which includes the step of identifying the polymorphic sites of the aforementioned single nucleotide polymorphism markers.

[0028] One object of the present invention is to provide a composition for improving spot-type pigmentation.

[0029] Another object of the present invention is to provide a cosmetic composition or quasi-pharmaceutical composition comprising the above-described composition as an active ingredient.

[0030] Another object of the present invention is to provide an information provision method for selecting a custom substance for improving freckle pigmentation, comprising the step of identifying the bases of polymorphic sites associated with freckle pigmentation as a significant single nucleotide polymorphism marker.

[0031] One object of the present invention is to provide a single nucleotide polymorphism (SNP) marker for diagnosing whether a skin type is prone to developing freckle-like pigmentation.

[0032] Another object of the present invention is to provide a composition for diagnosing skin types prone to freckle pigmentation, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing skin types prone to freckle pigmentation or an agent capable of amplifying the single nucleotide polymorphism (SNP) marker.

[0033] Another object of the present invention is to provide a kit or microarray comprising the above-described composition for diagnosing skin types prone to developing freckle-type hyperpigmentation.

[0034] Another object of the present invention is to provide a method for providing information on whether a skin type is prone to developing freckle-like pigmentation, comprising the step of identifying the polymorphic sites of the aforementioned single nucleotide polymorphism markers.

[0035] Another object of the present invention is to provide a method for providing information on customized substance formulations for high-risk groups prone to developing speckled pigmentation, which includes the step of identifying the polymorphic sites of the aforementioned single nucleotide polymorphism markers.

[0036] Invention Effects

[0037] Information about an individual's skin pigmentation level can be provided through the gene polymorphic markers of the present invention that are significantly associated with the degree of skin pigmentation. Furthermore, customized ingredients or products that can alleviate the degree of skin pigmentation can be developed based on the information of the gene polymorphic markers observed in an individual. Attached Figure Description

[0038] Figure 1 The dispersed black spot gene expression material of the present invention is shown.

[0039] Figure 2 The dark skin color gene expression material of the present invention is shown.

[0040] Figure 3 The spotted pigmentation gene expression material of the present invention is shown.

[0041] Figure 4 The results of the analysis show the efficacy and synergistic effects of bisabolol, nutmeg extract and DIPA in reducing melanin production according to the present invention.

[0042] Figure 5 The efficacy and synergistic analysis results of ferulic acid, oil-soluble licorice extract and / or psoralen in reducing melanin production according to the present invention are shown.

[0043] Figure 6 The efficacy and synergistic analysis results of the present invention in reducing melanin production by polydipsia glycoside and / or quercetin are shown.

[0044] Figure 7 The efficacy and synergistic analysis results of hexyldecyl alcohol, tocopherol and / or hexylresorcinol in reducing melanin production of the present invention are shown.

[0045] Figure 8 The pigmentation improvement rate of the dispersive dark spot improvement synergistic combination of the present invention is shown.

[0046] Figure 9The efficacy of γ-oryzanol, Aspergillus ferment broth, or isostearic acid in inhibiting protein carbonylation of the present invention has been confirmed.

[0047] Figure 10 A representative diagram of the dispersion-type black spot improvement of the present invention is shown.

[0048] Figure 11 The pigmentation improvement rate of the dispersible dark spot customized formulation of the present invention is shown.

[0049] Figure 12 A representative image of the improvement of dull skin tone according to the present invention is shown.

[0050] Figure 13 The pigmentation improvement rate of the customized formula for dull skin tone of the present invention is shown.

[0051] Figure 14 A representative graph showing the improvement of spot-type pigmentation in the high-risk group of the present invention is shown.

[0052] Figure 15 The improved spot recognition rate of the AI ​​based on the spot-type pigmentation customized formulation of the present invention is shown. Detailed Implementation

[0053] The specific details are explained below. Furthermore, the various descriptions and embodiments disclosed in this invention can also be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Additionally, the scope of this invention should not be considered limited by the specific descriptions below.

[0054] Furthermore, those skilled in the art can recognize or identify multiple equivalents to specific aspects of the invention described herein using only conventional experiments. Moreover, such equivalents are intended to be included in this invention.

[0055] As another aspect of achieving the objectives of the present invention, the present invention provides a composition for improving skin pigmentation. As an example, it may contain a selection from myristica fragrans (Nutmeg) extract, Polygonum cuspidatum root extract, bisabolol, diisopropyl adipate (DIPA), cysteine, cysteamine hydrochloride, sodium hyaluronate, hyaluronic acid, niacinamide, glycolic acid, gluconolactone, salicylic acid, polydatin, ferulic acid, bakuchiol, genistein, oil-soluble licorice extract, and Aspergillus ferment broth. Fermented, quercetin, tannic acid, zinc PCA (ZnPCA), gamma oryzanol, chrysin, tranexamic acid, sunflower oil, isostearic acid, ascorbyl glucoside, 3-O-ethyl ascorbic acid, ascorbic acid, licorice extract (Glycyrrhiza uralensis (Licorice) Extract), tocopherol, sodium pyruvate, kelp extract (Ecklonia cava extract). The active ingredient may be one or more substances in the group consisting of extract, hexyldecanol, Asiaticoside and hexylresorcinol, but is not limited thereto.

[0056] As an example, the aforementioned skin pigmentation can be scattered dark spots, dull skin tone, or spot-like pigmentation.

[0057] As another aspect of achieving the objectives of the present invention, the present invention provides a composition for improving dispersible dark spots on the skin. As an example, the composition comprises one or more substances selected from the group consisting of Myristica Fragrans (Nutmeg) Extract, Polygonum Cuspidatum Extract, Bisabolol, Diisopropyl Adipate (DIPA), Cysteine, Cysteine ​​Hydrochloride, Sodium Hyaluronate, Hyaluronic Acid, Niacinamide, Glycolic Acid, Gluconolactone, and Salicylic Acid as active ingredients. As another example, the above composition may contain (i) cysteine, cysteine ​​hydrochloride, myristica extract, high molecular weight hyaluronic acid and ultra-low molecular weight hyaluronic acid; (ii) bisabolol, Polygonum cuspidatum root extract and diisopropyl adipate; or (iii) nicotinamide, glycolic acid, gluconolactone and salicylic acid as active ingredients, but is not limited thereto.

[0058] The term "Myristica Fragrans (Nutmeg) Extract" refers to the extraction of nutmeg, which is the dried fruit of the nutmeg tree (Myristica fragrans Houtt), belonging to the Myristicaceae family. Nutmeg is mainly produced in Malaysia, Indonesia, and Sri Lanka, and is harvested approximately from April to June or November to December. The fruit is then peeled and used. Nutmeg has effects that improve blood circulation and lung function, as well as antibacterial properties. Therefore, it has been used to improve blood flow, treat respiratory disorders, and treat skin diseases. It also has aromatic components and is used as a spice.

[0059] Nutmeg extract contains lignans such as Nectandrin B, tetrahydrofurogualacin B, and fragransin C1, as well as flavonoids such as myricetin and myricitrin. Additionally, nutmeg extract is known to also contain phenylpropanoids, which are toxic to humans, including myristicin, elemicin, and safrole. The extraction method for nutmeg extract is not limited to this one; methods known in the art can be used.

[0060] The term "Polygonum cuspidatum extract" refers to the extraction of Polygonum cuspidatum root. Polygonum cuspidatum is a perennial herbaceous plant belonging to the Polygonaceae family, which grows in the mountains of Korea and is distributed in Korea, Japan, and China. In traditional medicine, Polygonum cuspidatum root has been used as a diuretic, emmenagogue, and sedative. It has also been utilized in traditional medicine in East Asian regions, including Korea, to treat and alleviate various ailments, including purulent dermatitis, urethritis, cystitis, hypertension, cancer, and arteriosclerosis. The extraction method for Polygonum cuspidatum root extract is not limited to any specific extraction method; methods known in the art can be used.

[0061] The term "bisabolol" is a natural monocyclic sesquiterpene alcohol, also known as α-bisabolol or levomenol. α-bisabolol has the structure of Formula 1 and is chemically named 6-methyl-2-(4-methylcyclohex-3-en-1-yl)hept-5-en-2-ol, with a molecular weight of 222.372 g·mol⁻¹. Bisabolol is also known as a major component of the essential oils of Brazilian candelia (Eremanthus erythropappus or Vanillosmopsis erythoropappa) or German chamomile (Matricaria recutita), i.e., a colorless, viscous oil. Most commercially available bisabolol is produced by distillation of the bark of the Candelia tree. Bisabolol can also be chemically synthesized and obtained using a variety of extraction methods known in the art, with the option of additional purification processes if necessary.

[0062] [Chemical Formula 1]

[0063]

[0064] The term "diisopropyl adipate (DIPA)" refers to a compound having the structure of the following chemical formula 2, which acts as a lubricant on the skin surface, thereby making the skin soft and smooth. It can be used as a carrier for other cosmetic ingredients and can also reduce brittleness and cracking, thus making the synthesized compound soft. For example, it is used in various applications such as the production of moisturizers, shaving lotions, and cleansers. The method of obtaining diisopropyl adipate is not limited to its preparation; it can be chemically synthesized using methods known in the art, or using commercially available substances.

[0065] [Chemical Formula 2]

[0066]

[0067] The term "cysteine" refers to a sulfur-containing α-amino acid with the structure HS-CH2CH(NH2)-COOH, possessing a sulfhydryl group, and therefore exhibiting the characteristic of forming disulfide bonds with other cysteines. The method of obtaining cysteine ​​is not limited; it can be chemically synthesized using methods known in the art, or using commercially available substances.

[0068] The term "cysteamine hydrochloride" refers to a compound having the structure HSCH2CH2NH2HCl, used as a reducing agent in cosmetic compositions. The method of obtaining cysteamine hydrochloride is not limited to its specific preparation method; it can be chemically synthesized using methods known in the art, or using commercially available substances.

[0069] The term "hyaluronic acid" refers to a naturally occurring biosynthetic substance widely found in the skin of animals. Due to its high hydroxyl (-OH) content, it is hydrophilic and plays a moisturizing role in animal skin. It is also known to exist in human skin, particularly in earthworm skin. It is primarily formulated as a moisturizer in cosmetics. It reacts with the CD44 protein in epithelial cells, thereby regulating various physiological functions. Hyaluronic acid is a linear anionic natural polysaccharide composed of alternating β-DN-acetylglucosamine and β-D-glucuronic acid, existing in salt form. The molecular structure of hyaluronic acid can be represented by the following chemical formula 3.

[0070] [Chemical Formula 3]

[0071]

[0072] In addition, the aforementioned hyaluronic acid can be divided into molecules with a molecular weight of 1.5 ~ 2.0 * 10⁻⁶. 6 Da's high molecular weight hyaluronic acid (sodium hyaluronate) and hyaluronic acid with a molecular weight of 0.5 ~ 1 * 10 4 Da's ultra-low molecular weight hyaluronic acid. High molecular weight hyaluronic acid has a high viscosity, forming a moisture film on the skin to prevent moisture loss. Ultra-low molecular weight hyaluronic acid has a low viscosity similar to water, allowing it to be deeply absorbed into the dermis, thus aiding in skin hydration when used in products. For the purposes of this invention, the hyaluronic acid can be either high molecular weight or ultra-low molecular weight hyaluronic acid. The aforementioned hyaluronic acid is not limited to its acquisition method; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0073] The term "niacinamide" refers to a water-soluble derivative of niacin, which is a water-soluble vitamin B3. It is a white powder, readily soluble in water, with the chemical formula C6H6N2O and a molecular weight of 122. It is known to exhibit whitening effects by inhibiting the migration of melanin from melanocytes to keratinocytes, and is a food and drug safety ingredient recognized for its whitening function when contained in cosmetics at concentrations of 2–5%. The method of obtaining niacinamide is not limited to its specific preparation method; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0074] [Chemical Formula 4]

[0075]

[0076] The term "glycolic acid" refers to a colorless, odorless, hygroscopic crystalline solid that is readily soluble in water. Its chemical formula is C₂H₄O₃. It is used in various skincare products. Glycolate is a salt or ester form of glycolic acid. Glycolic acid is a type of AHA (alpha-hydroxy acid). AHAs are mainly found in fruits and refer to organic acids derived from lactose. Besides glycolic acid, other substances with exfoliating and moisturizing effects include lactic acid, citric acid, and malic acid. The methods of obtaining glycolic acid are not limited to those known in the art; it can be synthesized using methods known in the art or by using commercially available substances.

[0077] The term "gluconolactone" is also known as glucono-delta-lactone (GDL), a food additive used as a chelating agent, acidifying agent or curing agent, pickling agent or leavening agent. Pure gluconolactone is a white, odorless crystalline powder. When added to water, gluconolactone partially decomposes into gluconic acid; the balance between the lactone and acid forms is achieved through chemical equilibrium. Gluconolactone is a type of PHA (polyhydroxy acid). The advantages of PHA are its large molecular weight, low absorption through the skin, and low irritation. Besides gluconolactone, lactobionic acid derived from corn starch is also available. The methods of obtaining gluconolactone are not limited to those described above; they can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0078] The term "salicylic acid" refers to an organic acid also known as salicylic acid, widely used in organic acid synthesis. It is also frequently used in pharmaceuticals as a plant hormone. It has a pH of 2.4 and is highly acidic. Its chemical formula is C7H6O3. Salicylic acid is a type of BHA (beta-hydroxy acid). BHAs are fat-soluble and used to remove sebum and waste. A representative example is salicylic acid extracted from the bark of the white willow tree. The method of obtaining salicylic acid is not limited to its specific method; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0079] Based on the total weight of the composition, the content of the substances of the present invention can be from about 0.0001% by weight to 10% by weight, but is not limited thereto. As an example, based on the total weight of the composition, the skin-dispersible melasma-improving material of the present invention can be from about 0.0001% by weight to 10% by weight, from about 0.0005% by weight to 5% by weight, from about 0.001% by weight to 5% by weight, from about 0.01% by weight to 5% by weight, from about 0.0001% by weight to 3% by weight, from about 0.0005% by weight to 3% by weight, from about 0.001% by weight to 3% by weight, from about 0.01% by weight to 3% by weight, from about 0.0001% by weight to 1% by weight, from about 0.0005% by weight to 1% by weight, from about 0.001% by weight to 1% by weight, or from about 0.01% by weight to 1% by weight.

[0080] For the purposes of this invention, the above-mentioned substance has the effect of improving scattered dark spots on the skin.

[0081] For the purposes of this invention, the term "melasma" refers to pigmentation in certain areas of an individual's skin caused by excessive melanin production, melanin accumulation, keratin accumulation, and the accumulation of oxidized proteins and lipids, resulting in lower chroma than other areas of the skin and the appearance of micro-black spots. When these spots are symmetrically distributed across the entire face, such as the cheeks, cheekbones, and under the eyes, it is called "dispersed melasma." "Dispersed melasma" can be representative of melasma, but is not limited to it, and can include post-inflammatory spots, moles (black spots, age spots, lentigo), freckles, etc., but is not limited to these.

[0082] Typically, to determine the severity of dark spots on the skin, methods are used to measure the lightness or color of the skin, or to measure the unevenness of skin color. However, it is practically impossible to measure the entirety of the lightness or color of a single "dispersed dark spot" that is small and distributed over a wide area. Methods for measuring the unevenness of skin color also struggle to distinguish wrinkles, raised keratin, pores, etc., from dark spots. Therefore, defining "dispersed dark spots" using the lightness, color, or unevenness of skin color has limitations. While visual assessment is best suited for expressing a subject's perceived skin concerns, it suffers from poor objectivity and the limitation that a small number of evaluators cannot effectively assess the majority of subjects. To address this issue, this invention constructs a diagnostic AI trained on visual assessment results, defining "dispersed dark spots." Evaluation using the diagnostic AI has the following advantages: it is better suited to displaying the subjectively perceived severity of "dispersed dark spots" than objective quantitative evaluation, and it can consistently evaluate a wide range of subjects compared to visual assessment.

[0083] For the purposes of this invention, the aforementioned substances are characterized by reducing melanin production. Specifically, treatment of the aforementioned substances with bisabolol, myristica extract, and diisopropyl adipic acid can promote melanin reduction.

[0084] As an example, the results of applying a cream containing the substances of the present invention (including bisabolol, myristica extract, and diisopropyl adipate, which are effective in reducing melanin production) to the face confirmed that it showed a superior rate of improvement in pigmentation compared to vitamin C, which served as a control group.

[0085] For the purposes of this invention, the aforementioned substances are characterized by inhibiting the expression of AKAP1 or promoting the expression of RAB11FIP2 or PPARGC1B. Specifically, treatment with cysteine, cysteine ​​hydrochloride, myristica extract, high molecular weight hyaluronic acid, and / or ultra-low molecular weight hyaluronic acid in the aforementioned substances can inhibit the expression of AKAP1; treatment with bisabolol, Polygonum cuspidatum root extract, and / or diisopropyl adipate can promote the expression of RAB11FIP2; and treatment with nicotinamide, glycolic acid, gluconolactone, and / or salicylic acid can promote the expression of PPARGC1B, but is not limited thereto. It can be seen that the responsiveness to the substances of this invention varies according to specific gene polymorphism types, indicating that by providing customized formulations to high-risk groups with a higher risk level of developing diffuse melasma, the efficiency of improving diffuse melasma can be improved.

[0086] In this invention, the term "about" may be given before a specific numerical value. As used in this application, the term "about" includes not only the exact number stated after the term, but also a range that is almost or close to that number. Whether it is close to or almost is that number can be determined by considering the context in which the number is given. As one example, the term "about" may refer to a range of -10% to +10% of a numerical value. As another example, the term "about" may refer to a range of -5% to +5% of a given numerical value. However, it is not limited to these examples.

[0087] The composition described above is characterized by being personalized. Specifically, it can be provided to individuals with a significant single nucleotide polymorphism (SNP) marker associated with a specific degree of diffuse melasma.

[0088] As an example, the significant single nucleotide polymorphism (SNP) markers associated with the degree of diffuse melasma can be selected from any one or more of those in Table 1, but are not limited thereto.

[0089] In this invention, the term "polymorphism" refers to the presence of two or more alleles at a single locus, and at polymorphic sites, differences of only a single base between individuals are called single nucleotide polymorphisms (SNPs). Preferred polymorphic markers have two or more alleles exhibiting a frequency of 1% or more, more specifically 10% or 20% or more, in a selected population. "Gene polymorphic markers" generally refer to the observation of two or more alleles at the same gene locus (bases), typically exhibiting major allele / major allele, major allele / minor allele, or minor allele / minor allele depending on the individual. In this invention, it can be used interchangeably with "polymorphism marker" and refers to the bases and base sites of minor alleles, or it can be defined together with chromosome number and base position, but is not limited thereto.

[0090] In this invention, the term "allele" refers to multiple types of a gene present at the same locus on homologous chromosomes. Alleles are also used to represent polymorphism; for example, an SNP has two types of bialleles. Additionally, it refers to a combination of two or more bases with the same chromosome number and base position, including major alleles that occur frequently in individuals within a specific population and minor alleles that occur less frequently than the major allele.

[0091] Specifically, the gene polymorphism marker of the present invention is significantly associated with diffuse melasma of the skin and can hold two alleles, which can be divided into cases of holding major allele / minor allele, minor allele / minor allele, and major allele / major allele.

[0092] When an allele is present that is a significant polymorphic marker associated with diffuse melasma, the effect of improving diffuse melasma by the substance of the present invention may vary depending on the allele possessed by the individual. For example, when a major allele is present, a major allele is present, a minor allele is present, or a minor allele is present, the reactivity induced by the substance of the present invention can be measured, i.e., the skin characteristic of having a high or low degree of improvement in diffuse melasma. Therefore, information can be provided for selecting a custom substance to enhance the improvement of diffuse melasma in individuals who possess one or more minor alleles.

[0093] In this invention, the term "rs_id" refers to the unique identifier, or rs-ID, assigned by NCBI to all SNPs initially registered since 1998 when SNP information was accumulated. The rs_id recorded in this table refers to the polymorphism marker, or SNP marker, of this invention.

[0094] The single nucleotide polymorphism (SNP) markers mentioned above can be selected from one or more SNP markers shown in Table 1. The SNP markers shown in Table 1 can be used to determine the degree of correlation with the severity of diffuse melasma.

[0095] The degree of skin-dispersed melasma marked by single nucleotide polymorphisms of the present invention is determined by measuring the frequency of each marker. This significance is characterized by, but not limited to, p-values, such as p-values ​​less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.00000001, less than 0.00000001, or less than 0.000000001. Specifically, the p-value can be less than 0.01, more specifically, the p-value can be less than 0.001, and even more specifically, it can be less than 0.0001, but is not limited thereto.

[0096] The single nucleotide polymorphism (SNP) markers of the present invention can be selected from any one or more of the markers shown in Table 1, but are not limited thereto. The above-mentioned single nucleotide polymorphism (SNP) markers can be one or more, and can be used in combination of two or more, three or more, four or more, five or more, or six or more, etc., which can determine the number of scattered dark spots on the skin, but are not limited thereto.

[0097] The aforementioned markers may be SNPs themselves, or polynucleotides consisting of 5-100 consecutive DNA sequences containing the aforementioned SNP sites, or polynucleotides consisting of their complementary sequences, but are not limited thereto.

[0098] As an example, the single nucleotide polymorphism (SNP) markers that significantly correlate the degree of the aforementioned diffuse melasma can be one or more SNP markers present in the genes encoding proteins of AKAP1, RAB11FIP2, PPARGC1B, BNC2, MC1R, or MITF. Specifically, they can be selected from any one or more of the group consisting of rs11079281, rs10444110, rs251466, rs16935073, rs2228479, and rs7430957, but are not limited thereto.

[0099] As an example, when the SNP ID is rs11079281, Chr.Position (GRCh ver. 37) is recorded as "17:55231787". If the allele is publicly disclosed as T>C, this means that the 55231787th base on human chromosome 17 is either T or C. Furthermore, the base to the left of the ">" in the allele can indicate the major allele, while the base to the right can indicate the minor allele.

[0100] As a specific example, the markers selected from Table 1 may consist of one or more polynucleotides selected from the group consisting of the following polynucleotides; and one or more polynucleotides selected from the group consisting of their complementary polynucleotides, but not limited thereto. The polynucleotides are: 5-100 consecutive DNA sequences comprising the 55,231,787th base of human chromosome 17, wherein the 55,231,787th base is T or C (rs11079281); 5-100 consecutive DNA sequences comprising the 119,563,401st base of human chromosome 10, wherein the 119,563,401st base is T or C (rs10444110); 5-100 consecutive DNA sequences comprising the 149,195,603rd base of human chromosome 5. A polynucleotide consisting of 0 consecutive DNA sequences, wherein the 149,195,603rd base is C or G (rs251466); a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 16,795,790th base of human chromosome 9, wherein the 16,795,790th base is A or C (rs16935073); a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 89,985,940th base of human chromosome 16, wherein the 89,985,940th base is G or A (rs2228479); a polynucleotide consisting of 5-100 consecutive DNA sequences comprising the 69,978,466th base of human chromosome 3, wherein the 69,978,466th base is A or G (rs7430957).

[0101] The compositions of the present invention can be used as cosmetic compositions for relieving or improving dispersible dark spots on the skin and can be formulated in various forms. The cosmetic compositions of the present invention can be prepared into dosage forms selected from, but not limited to, solutions, ointments, creams, foams, nourishing lotions, softening lotions, masks, softening waters, emulsions, makeup bases, serums, soaps, liquid cleansers, bath additives, sunscreens, sunscreen oils, suspensions, emulsions, pastes, gels, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays.

[0102] In addition, the cosmetic composition of the present invention may further include one or more cosmetically acceptable carriers incorporated into general skin cosmetics. As conventional ingredients, such as oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, pigments, preservatives, fragrances, etc., may be appropriately incorporated, but are not limited thereto.

[0103] The cosmetically acceptable carriers contained in the cosmetic compositions of the present invention vary depending on the dosage form.

[0104] When the dosage form of the present invention is an ointment, paste, cream, or gel, the carrier component may be animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or mixtures thereof.

[0105] When the dosage form of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder or mixtures thereof can be used as the carrier component. In particular, when it is a spray, a propellant such as chlorofluorocarbon, propane / butane or dimethyl ether can be additionally included.

[0106] When the dosage form of the present invention is a solution or emulsion, the carrier component utilizes a solvent, solubilizer, or emulsifier, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, and in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic esters, polyethylene glycol or fatty acid esters of sorbitol.

[0107] When the dosage form of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester and polyoxyethylene dehydrated sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar or tragacanth gum, etc., can be used as the carrier component.

[0108] When the dosage form of the present invention is soap, the carrier component can be alkali metal salts of fatty acids, fatty acid half-ester salts, fatty acid protein hydrolysates, hydroxyethyl sulfonates, lanolin derivatives, fatty alcohols, vegetable oils, glycerin, sugars, etc.

[0109] In another respect, the above composition provides a topical pharmaceutical composition for relieving or improving dispersible dark spots on the skin.

[0110] In this invention, the term "quasi-medicine" refers to an article whose effect is milder than that of a medicine and is used for the purpose of diagnosing, treating, improving, alleviating, treating or preventing diseases in humans or animals. For example, according to the Korean Pharmaceutical Affairs Act, quasi-medicine does not include articles used for the purpose of medicine, but includes products used for the treatment or prevention of diseases in humans or animals, and products that have a mild or indirect effect on the human body.

[0111] As an example, the quasi-pharmaceutical composition of the present invention can be prepared in the form selected from, but not limited to, shower gels, shampoos, conditioners, foams, soaps, face masks, ointments, creams, lotions, serums and sprays.

[0112] In another aspect, the present invention provides a method for providing information on selecting a custom substance for improving diffuse melasma, comprising the following steps: (a) obtaining a biological sample from an individual; (b) amplifying or hybridizing a polymorphic site of one or more single nucleotide polymorphism markers selected from the group consisting of rs11079281, rs10444110, rs251466, rs16935073, rs2228479 and rs7430957 from the biological sample; and (c) confirming the bases of the polymorphic site amplified or hybridized in step (b).

[0113] Dispersed melasma can be improved by treating individuals with specific polymorphic sites with the aforementioned customized substances.

[0114] In this invention, the term "individual" refers to a subject used to diagnose the degree of diffuse melasma on the skin. DNA can be obtained from samples such as hair, urine, blood, various bodily fluids, isolated tissues, isolated cells, or saliva, but is not limited thereto.

[0115] The method for obtaining genomic DNA in step (a) above can be any method known to those skilled in the art.

[0116] The amplification of the polymorphic sites marked with the single nucleotide polymorphism markers or hybridization with the probes in the DNA obtained from step (a) above can be performed using any method known to those skilled in the art. For example, the target nucleic acid can be obtained by PCR amplification and purification. In addition, ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)), self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)), and nucleic acid-based sequence amplification (NASBA) can be used.

[0117] In the methods described above, the determination of the bases at the polymorphic sites in step (b) includes, but is not limited to, sequencing analysis, hybridization via microarrays, allele-specific PCR, dynamic allele-specific hybridization (DASH), PCR extension analysis, SSCP, PCR-RFLP analysis or TaqMan technology, SNPlex platforms (Applied Biosystems), mass spectrometry (e.g., Sequenom's MassARRAY system), mini-sequencing methods, Bio-Plex systems (BioRad), CEQ and SNPstream systems (Beckman), molecular inversion probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium analysis). Using the methods described above or other methods available to those skilled in the art to which this invention pertains, one or more alleles of a polymorphic marker, including microsatellites, SNPs, or other types of polymorphic markers, can be identified. Specifically, the determination of the bases at such polymorphic sites can be performed using SNP microarrays.

[0118] The aforementioned substances may be selected from one or more of the following groups: phloretin, myristica extract, Polygonum cuspidatum root extract, bisabolol, diisopropyl adipate, cysteine, cysteine ​​hydrochloride, high molecular weight hyaluronic acid, ultra-low molecular weight hyaluronic acid, nicotinamide, glycolic acid, gluconolactone, and salicylic acid, but are not limited thereto.

[0119] For the purposes of this invention, the aforementioned gene significantly associated with the severity of diffuse melasma can provide information that allows for the selection of specific substances for each individual. As an example, for individuals possessing one or more single nucleotide polymorphism markers present in genes encoding AKAP1, RAB11FIP2, PPARGC1B, BNC2, MC1R, or MITF proteins, treatment with one or more substances selected from the group consisting of nutmeg extract, Polygonum cuspidatum root extract, bisabolol, diisopropyl adipate, cysteine, cysteine ​​hydrochloride, high molecular weight hyaluronic acid, ultra-low molecular weight hyaluronic acid, nicotinamide, glycolic acid, gluconolactone, and salicylic acid can enhance the improvement of diffuse melasma. However, an optimal combination of substances can be provided based on the genetic characteristics of each individual.

[0120] This suggests that information can be provided to screen for specific single nucleotide polymorphism (SNP) markers that are significantly associated with disseminated melasma and to select, based on genetic information, personalized substances that can maximize the effect of improving disseminated melasma.

[0121] Specifically, if the risk score obtained by multiplying and summing the genotypes of rs11079281, rs10444110, rs251466, rs16935073, rs2228479, and rs7430957 by their effect size (β) is less than 0.28, the risk of developing diffuse melasma can be classified as low-risk; if the risk score is above 0.28, the risk of developing diffuse melasma can be classified as high-risk. As an example, an information provision method can be provided where, in cases where the risk of developing diffuse melasma is classified as high-risk, bisabolol and nutmeg extract are selected as customized substances, but this method is not limited to these.

[0122] In this invention, the term "SNP chip" refers to one of the DNA microarrays capable of identifying the bases of hundreds of thousands of SNPs at once.

[0123] The TaqMan method includes: (1) designing and fabricating primers and TaqMan probes to amplify the desired DNA fragment; (2) labeling probes with different alleles using FAM dye and VIC dye (Applied Biosystems); (3) performing PCR using the DNA as a template and the primers and probes; (4) analyzing and confirming the TaqMan analysis plate using a nucleic acid analyzer after the PCR reaction is completed; and (5) determining the genotype of the polynucleotide from step (1) based on the analysis results.

[0124] In the above, sequencing analysis can be performed using conventional methods for determining base sequences and can be performed using automated gene analyzers. Additionally, allele-specific PCR refers to a PCR method that amplifies the DNA fragment containing the SNP using a primer set including primers designed with the SNP base at its 3' end. The principle of this method utilizes the following: for example, when a specific base is replaced by G, when a PCR reaction is performed using primers designed with A as the 3' end base and reverse primers capable of amplifying DNA fragments of appropriate size, if the SNP base is A, the amplification reaction proceeds normally, and the desired band is observed. If the base is replaced by G, the primers can bind complementaryally to the template DNA, but because complementary binding does not occur at the 3' end, the amplification reaction cannot proceed normally. DASH can be performed using conventional methods, specifically the method of Prince et al.

[0125] On the other hand, PCR extension analysis is achieved through the following process: First, a DNA fragment containing the base of the single nucleotide polymorphism (SNP) is amplified using primer pairs. Then, all nucleotides added to the reaction are dephosphorylated to inactivate them, and SNP-specific extension primers, a dNTP mixture, dideoxynucleotides, reaction buffer, and DNA polymerase are added to perform a primer extension reaction. In this case, the extension primers have the 3' end of the base directly adjacent to the SNP at the 5' direction. The dNTP mixture does not contain nucleic acids with the same base as the dideoxynucleotides, and the dideoxynucleotides are selected from one of the base types displaying the SNP. For example, in the case of A being replaced by G, when a mixture of dGTP, dCTP, and dTTP, along with ddATP, is added to the reaction, the primers are extended by DNA polymerase at the base where the substitution occurs, and the primer extension reaction is terminated by ddATP at the position where the A base first appears after several bases. If the substitution does not occur, the extension reaction terminates at that position. Therefore, the base type displaying the SNP can be determined by comparing the lengths of the extended primers.

[0126] At this point, as a detection method, when the extension primer or dideoxynucleotide is fluorescently labeled, the above-mentioned SNP can be detected by detecting fluorescence using a conventional gene analyzer used to determine the base sequence (e.g., ABI Model 3700, etc.), and when unlabeled extension primer and dideoxynucleotide are used, the above-mentioned SNP can be detected by measuring the molecular weight using MALDI-TOF (matrix-assisted laser desorption ionization-time offlight) technology.

[0127] As one aspect of achieving the objectives of this invention, the present invention provides a single nucleotide polymorphism (SNP) marker for diagnosing the degree of diffuse melasma on the skin.

[0128] In another aspect, the present invention provides a composition for diagnosing the degree of diffuse melasma, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing the degree of diffuse melasma or an agent capable of amplifying the single nucleotide polymorphism (SNP) marker.

[0129] Dispersed melasma refers to the uneven skin color in specific areas of the skin, which appears relatively darker compared to the surrounding skin, resulting from the sum of all target areas measured relative to the total number of measured areas, but is not limited to this. Specifically, it refers to using one or more single nucleotide polymorphism (SNP) markers selected from Table 1 to determine the susceptibility to the formation of dispersed melasma. More specifically, skin types prone to dispersed melasma are those with a higher rate of melasma formation per unit area.

[0130] For the alleles described above in this invention, each individual has the same chromosome number, which includes major and minor alleles of the SNP. As the bases at the polymorphic sites of the polymorphic markers increase one by one to become minor alleles, the number of major alleles can decrease one by one. However, the range within which minor and major alleles can increase or decrease is within three types: i) major allele / major allele, ii) major allele / minor allele, and iii) minor allele / minor allele. Furthermore, alleles can decrease or increase within these three types, but are not limited to these.

[0131] In this invention, the term "probe capable of detecting markers used to diagnose the degree of diffuse melasma" refers to a composition capable of diagnosing the degree of diffuse melasma through specific hybridization with polymorphic sites of the aforementioned genes. Furthermore, the specific method of such gene analysis is not particularly limited and can be performed using any gene detection method known in the technical field to which this invention pertains. Additionally, the above term may be used interchangeably with "for diagnosing whether a skin type is prone to developing diffuse melasma" or "for diagnosing whether a high-risk skin type is prone to developing diffuse melasma."

[0132] In this invention, the term "preparation capable of amplifying markers used to diagnose the degree of diffuse melasma" refers to a composition capable of diagnosing the degree of diffuse melasma by amplifying and confirming the polymorphic sites of the aforementioned genes. Specifically, it refers to primers capable of specifically amplifying the polynucleotides used to diagnose the degree of diffuse melasma. Furthermore, the term may be used interchangeably with "for diagnosing whether a skin type is prone to developing diffuse melasma" or "for diagnosing whether a high-risk skin type is prone to developing diffuse melasma."

[0133] Primers used to amplify the aforementioned polymorphic markers are single-stranded oligonucleotides that function as templates—the starting points for directing DNA synthesis—under appropriate conditions in a suitable buffer (e.g., four different nucleoside triphosphates and polymerases such as DNA, RNA polymerase, or reverse transcriptase) and at an appropriate temperature. The appropriate length of these primers can vary depending on the intended use, but is typically 15 to 30 nucleotides. Shorter primer molecules usually require lower temperatures to form stable hybrids with the template. The primer sequence does not need to be perfectly complementary to the template, but must be sufficiently complementary to hybridize with it.

[0134] In this invention, the term "primer," as a short base sequence with a free 3' hydroxyl group, refers to a short sequence that can form a base pair with a complementary template and act as the starting point for template strand replication. Primers can initiate DNA synthesis in the presence of reagents used for polymerization reactions (i.e., DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at appropriate buffer solutions and temperatures. PCR amplification is performed, allowing prediction of skin type based on the desired level of product formation. PCR conditions, and the lengths of the positive and negative primers, can be varied based on principles known in the art.

[0135] The probes or primers of the present invention can be chemically synthesized using the phosphoramidite solid support method or other widely known methods. Such nucleic acid sequences can also be modified using many techniques known in the art. Non-limiting examples of such modifications include methylation, capping, substitution with one or more homologues of a natural nucleotide, and modifications between nucleotides, such as modification to uncharged linkers (e.g., methyl phosphonate, phosphotriester, phosphoroamidate, carbamate, etc.) or charged linkers (e.g., phosphorothioate, phosphorodithioate, etc.).

[0136] In another aspect, the present invention provides a kit for diagnosing the degree of diffuse melasma, comprising the composition described above for diagnosing the degree of diffuse melasma. The kit may be an RT-PCR kit or a DNA microarray kit, but is not limited thereto.

[0137] The kit of the present invention can diagnose the degree of diffuse melasma by amplifying and confirming SNP polymorphic markers used for the diagnosis of the degree of diffuse melasma, or by confirming the expression levels of SNP polymorphic markers and their mRNA expression levels. As a specific example, in this invention, the kit for measuring the mRNA expression level of the diagnostic markers for the degree of diffuse melasma can be a kit containing the necessary elements for RT-PCR. In addition to the respective gene-specific primer pairs for the diagnostic markers for the degree of diffuse melasma, the RT-PCR kit may also include test tubes or other suitable containers, reaction buffers (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC-water, sterile water, etc. Furthermore, it may include gene-specific primer pairs used as quantitative controls. Specifically, the kit of the present invention can be a kit for diagnosing the degree of diffuse melasma containing the necessary elements for DNA microarray analysis. DNA microarray kits typically consist of nucleic acid species attached in a gridded array to a flat, solid support plate, usually no larger than a microscope slide. The nucleic acids are uniformly arranged on the chip surface to enable multiple hybridization reactions between the nucleic acids on the DNA chip and complementary nucleic acids contained in the solution treated on the chip surface, thus enabling large-scale parallel analysis.

[0138] In another aspect, the present invention provides a microarray for diagnosing the degree of diffuse melasma on the skin, comprising the above-described composition for diagnosing the degree of diffuse melasma on the skin.

[0139] The microarrays described above may contain DNA or RNA polynucleotides. Except for the inclusion of the polynucleotides of the present invention in the probe polynucleotides, the microarrays described above consist of conventional microarrays.

[0140] The method of preparing microarrays by immobilizing probe polynucleotides on a substrate is well known in the art. The aforementioned probe polynucleotides refer to hybridizable polynucleotides, which are oligonucleotides capable of binding sequence-specifically to the complementary strand of a nucleic acid. The probes of the present invention are allele-specific probes, where the polymorphic site exists in nucleic acid fragments from two members of the same species, hybridizing with a DNA fragment from one member but not with a fragment from the other. In this case, the hybridization conditions show significant differences in hybridization intensity between alleles, and therefore must be sufficiently stringent to hybridize only with one allele. By doing so, good hybridization differences between different allele forms can be induced. The probes of the present invention can be used in methods for diagnosing diffuse melasma by detecting alleles, etc. The aforementioned diagnostic methods include nucleic acid hybridization-based detection methods, such as Southern blot hybridization, etc., and in methods utilizing DNA chips, they can also be provided in the form of pre-bonded onto a DNA chip substrate. The aforementioned hybridization can typically be performed under stringent conditions, such as salt concentrations below 1 M and temperatures above 25°C. For example, conditions of 5x SSPE (750mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and 25–30°C are suitable for allele-specific probe hybridization.

[0141] The process of immobilizing the probe polynucleotides of the present invention, which are related to the degree of diffuse dark spots on the skin, onto a substrate can also be easily prepared using this prior art. Furthermore, the hybridization of nucleic acids on a microarray and the detection of the hybridization results are well known in the art. The above detection can be performed by the following process: for example, labeling the nucleic acid sample with a labeling material capable of generating a detectable signal (including fluorescent materials, such as Cy3 and Cy5), then hybridizing on a microarray and detecting the signal generated by the labeling material.

[0142] In another aspect, the present invention provides a method for providing information about the degree of diffuse dark spots on the skin, comprising the following steps: (a) amplifying or hybridizing the polymorphic site of the single nucleotide polymorphism marker in DNA obtained from a sample isolated from an individual; and (b) confirming the bases of the polymorphic site amplified or hybridized in step (a).

[0143] The terms “individual, (a) step, (b) step” are as described above.

[0144] In the above method, additionally (c) if the risk score obtained by multiplying and adding the effect sizes (β) of the amplified or hybridized single nucleotide polymorphism markers is 0.28 or higher, it can be judged as a high-risk group with a higher risk of developing diffuse melasma, but is not limited thereto.

[0145] As one aspect of achieving the objectives of this invention, the present invention provides a composition for improving dull skin tone. As an example, the composition comprises one or more substances selected from the group consisting of polydatin, ferulic acid, bakuchiol, genistein, oil-soluble licorice extract, Aspergillus ferment, quercetin, tannic acid, zinc PCA (ZnPCA), gamma oryzanol, chrysin, tranexamic acid, sunflower oil, and isostearic acid as active ingredients. As another example, the above composition may contain (i) ferulic acid, polygalactoside, zinc PCA, tranexamic acid, quercetin, genistein, tannic acid, Aspergillus ferment, γ-oryzanol, sunflower seed oil, and isostearic acid; (ii) ferulic acid, polygalactoside, psoralen, genistein, oil-soluble licorice extract, and Aspergillus ferment; (iii) ferulic acid, quercetin, genistein, tannic acid, polygalactoside, zinc PCA, and γ-oryzanol; or (iv) apigenin, tannic acid, and zinc PCA as active ingredients, but is not limited thereto.

[0146] The term "polydatin," also known as Picid or 3,4',5-trihydroxy-3-β-mono-D-glucoside, is a stilbene polyphenol isolated from the root of *Polygonum cuspidatum*. It can be derived from naturally sourced compounds, chemically synthesized compounds, or mixtures thereof. Furthermore, the aforementioned polydatin can be trans-polydatin, cis-polydatin, or mixtures thereof. Chemical formula 5 below represents the trans form of polydatin, and chemical formula 6 represents the cis form. The method of obtaining the aforementioned polydatin is not limited; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0147] [Chemical Formula 5]

[0148]

[0149] [Chemical Formula 6]

[0150]

[0151] The term "ferulic acid" refers to a polyphenolic compound found in many food crops, including whole grains, fruits, and seaweed, also known as 4-hydroxy-3-methoxycinnamicacid. Studies on ferulic acid have focused on its anti-diabetic and anti-hyperglycemic effects in peripheral tissues, but its effects on the central nervous system remain unclear. The methods of obtaining ferulic acid are not limited; it can be chemically synthesized using methods known in the art, or commercially available substances can be used. The INCI name of the aforementioned substance is ferulic acid.

[0152] [Chemical Formula 7]

[0153]

[0154] The term "bakuchiol" refers to a plant-based retinol extracted from wild plants used in Ayurveda and Chinese medicine in Asia. As a plant-based retinol that does not exhibit skin irritation or side effects, bakuchiol avoids stability issues compared to conventional retinol, which may induce skin irritation, sensitization, and teratogenicity. It exhibits high photostability and demonstrates similar efficacy to retinol on aging skin. Furthermore, compared to retinol, bakuchiol exhibits superior collagen synthesis and wrinkle improvement effects. Therefore, cream-type cosmetic compositions containing bakuchiol as an active ingredient can effectively improve problem skin such as acne without skin irritation, and also provide skin whitening and moisturizing effects. Bakuchiol can be chemically synthesized and obtained using various extraction methods known in the art, and may be further purified if necessary.

[0155] [Chemical Formula 8]

[0156]

[0157] The term "genistein" refers to one of the isoflavones isolated from soybeans, known to have various effects such as anti-tumor, anti-osteoporosis, and antioxidant properties. It is a substance that, due to its phytoestrogenous activity, is particularly effective in alleviating various menopausal symptoms in postmenopausal women, and is therefore widely used as an ingredient in pharmaceuticals and health foods. The method of obtaining genistein is not limited to its specific methods; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0158] [Chemical Formula 9]

[0159]

[0160] The term "oil-soluble licorice extract," as one of the ingredients advertised for skin whitening, refers to an oil-soluble extract derived from the roots of licorice (Glycyrrhiza glabra L. var. glandulifera Regal et Herder), Glycyrrhiza uralensis Fisher, or other leguminous plants. It is primarily used in powdered form, but is not limited to this.

[0161] The term "Aspergillus ferment" refers to substances obtained through fermentation by microorganisms, specifically Aspergillus (Aspergillus mold), which can be used directly or filtered. Aspergillus ferment contains large amounts of ferulic acid and phenolic acids, which have DPPH free radical scavenging functions, thus exhibiting antioxidant effects. It can also inhibit tyrosinase and elastase activity, thereby contributing to skin whitening and wrinkle improvement.

[0162] The term "quercetin" refers to a plant-derived flavonoid (polyphenolic flavonoid) compound extracted from the flavonoid group of polyphenols. Quercetin is found in many fruits, vegetables, leaves, seeds, and grains. It has a bitter taste and is used as an ingredient in health supplements, beverages, and foods. Quercetin is also known as quercetin extract. The method of obtaining quercetin is not limited; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0163] [Chemical Formula 10]

[0164]

[0165] The term "tannic acid" refers to one of the most abundant polyphenols found in fruits, vegetables, olives, cocoa, and other plants. In recent years, tannic acid has been used as a multifunctional coating molecule. Furthermore, it is known to have excellent affinity for biomacromolecules, including DNA and proline-rich proteins such as thrombin, gelatin, collagen, and mucin. The phenolic hydroxyl moiety of tannic acid (five galloyl groups (three -OH groups attached to the aromatic ring) and five catechol groups (two -OH groups attached to the aromatic ring via covalent bonds)) can bind to proteins through multiple hydrogen bonds and hydrophobic interactions with target proteins. The tannic acid described above is not limited to its acquisition methods and can be chemically synthesized using methods known in the art, or commercially available substances can be used. The INCI name of the aforementioned substances is tannic acid.

[0166] [Chemical Formula 11]

[0167]

[0168] The term "zinc PCA (ZnPCA)" refers to a raw material synthesized from glutamic acid obtained from sugar beets. Zinc (Zinc) is an essential mineral for physiological activities in the body and is known to have various functions (synthesis of cell differentiation proteins, antibacterial activity). In particular, zinc inhibits 5-α reductase, thereby reducing sebum secretion. The aforementioned zinc PCA can be used in combination with zinc PCA (Zinc PCA) and zinc pyrrolidone carboxylic acid. The aforementioned zinc PCA is not limited to its acquisition method and can be synthesized by methods known in the art or by using commercially available substances.

[0169] The term "γ-oryzanol" refers to a growth-promoting substance found in cereals, isolated from rice bran. It possesses strong antioxidant properties, thus helping to prevent damage from radiation exposure or chemotherapy. Furthermore, this component increases cerebral blood flow by regulating blood pressure and strengthening blood vessel walls, thereby enhancing brain cell metabolism, improving autonomic nervous system disorders, preventing stroke and dementia, improving memory, and improving sleep quality—all beneficial for cardiovascular and cerebrovascular diseases. Additionally, due to its ability to promote liver function and alcohol metabolism, it is also used in hangover remedies. Moreover, it is known to have effects in reducing obesity and promoting kidney function. The method of obtaining γ-oryzanol is not limited to its specific form; it can be chemically synthesized using methods known in the art, or commercially available substances can be used. The INCI name of the aforementioned substance is oryzanol.

[0170] The term "Chrysin," also known as 5,7-dihydroxyflavone, is one of the flavonoids identified in honey and propolis. Chrysin can be derived from C... 15 H 10 The chemical formula O4 and the following chemical formula 12 are used. The above-mentioned salicylic acid is not limited to its acquisition method; it can be chemically synthesized by methods known in the art, or using commercially available substances.

[0171] [Chemical Formula 12]

[0172]

[0173] The term "tranexamic acid (TXA)" refers to trans-4-(aminomethyl)cyclohexanecarboxylic acid, a substance having a C8H group. 15The molecular formula of NO2 has previously been known to have anti-fibrinolytic effects, and therefore it has been used as a hemostatic agent or thrombolytic agent. In recent years, with the validation of its inhibitory effects on the proliferation of skin pigment cells (melanocytes) and melanin production induced by ultraviolet radiation, it has proven to have industrial value as a skin-whitening ingredient. The aforementioned tranexamic acid is not limited to its acquisition method; it can be chemically synthesized using methods known in the art, or commercially available substances can be used. The INCI name of the aforementioned substance is tranexamic acid.

[0174] [Chemical Formula 13]

[0175]

[0176] The term "sunflower oil" refers to oil extracted from sunflower seeds. Sunflower oil is rich in beneficial nutrients such as protein, unsaturated fatty acids like linoleic acid and oleic acid, lecithin, carotenoids, tocopherols, vitamins A, C, and D, and dietary fiber. Sunflower oil is particularly high in vitamin E. Vitamin E, as an antioxidant, promotes healthy and beautiful skin, for example, by preventing damage from harmful substances like ultraviolet rays and alleviating scars and wrinkles. It is also known to be rich in beta-carotene, which is converted into the antioxidant vitamin A, thus delaying aging and keeping the skin youthful and firm. The method of obtaining sunflower oil is not limited to its extraction method; oil extracted using methods known in the art or commercially available substances may be used.

[0177] The term "isostearic acid" refers to a mixture of fatty acids produced through a multi-stage separation and purification process under high temperature and pressure. This multi-stage process requires high purity of the resulting mixed isostearic acid. Rather than being used as isostearic acid itself, it is typically formulated as isostearyl alcohol and used primarily in cosmetics, lubricants, solvents, and metal treatment agents. The method of obtaining this isostearic acid is not limited to its specific method; it can be chemically synthesized using methods known in the art or by using commercially available substances. The INCI name of this substance is isostearic acid.

[0178] Based on the total weight of the composition, the content of the substances of the present invention may be from about 0.0001 to 10% by weight, but is not limited thereto. As an example, based on the total weight of the composition, the dull skin tone improving material of the present invention may be from about 0.0001 to 10% by weight, from about 0.0005 to 5% by weight, from about 0.001 to 5% by weight, from about 0.01 to 5% by weight, from about 0.0001 to 3% by weight, from about 0.0005 to 3% by weight, from about 0.001 to 3% by weight, from about 0.01 to 3% by weight, from about 0.0001 to 1% by weight, or from about 0.01 to 1% by weight.

[0179] For the purposes of this invention, the above-mentioned substance has the effect of improving dull skin tone.

[0180] For the purposes of this invention, the term "dull skin tone" refers to skin pigmentation caused by excessive melanin production, melanin accumulation, keratin accumulation, and the accumulation of oxidized proteins and lipids, resulting in a skin color that is darker than the individual's original skin color. The term "skin tone" can be used interchangeably with "skin color," and can refer to all skin colors categorized by brightness (skin lightness), redness, and yellowness, but is not limited to this. Additionally, it can include skin texture, including the degree of unevenness in skin color.

[0181] For the purposes of this invention, the aforementioned substances are characterized by reducing melanin production. Specifically, when i) ferulic acid, oil-soluble licorice extract, and psoralen; or ii) resveratrol and quercetin are treated with the aforementioned substances, melanin reduction can be promoted.

[0182] As an example, applying a cream containing a mixture of the substances of the present invention (i) ferulic acid, oil-soluble licorice extract and psoralen (which are effective in reducing melanin production; or ii) resveratrol and quercetin) to the face confirmed a synergistic effect in reducing melanin production compared to treating the substances separately.

[0183] For the purposes of this invention, the aforementioned substances are characterized by reducing protein carbonylation. Specifically, when γ-oryzanol, Aspergillus ferment broth, or isostearic acid in the aforementioned substances are treated, carbonylation reduction can be promoted.

[0184] For the purposes of this invention, the aforementioned substances are characterized by inhibiting the expression of OCA2, BNC2, RAB32, or KITLG. Specifically, when treating ferulic acid, polygalactoside, zinc PCA, tranexamic acid, quercetin, genistein, tannic acid, Aspergillus ferment, γ-oryzanol, sunflower seed oil, and / or isostearic acid, the expression of OCA2 can be inhibited; when treating polygalactoside, ferulic acid, psoralen, genistein, oil-soluble licorice extract, and / or Aspergillus ferment, the expression of BNC2 can be inhibited; when treating ferulic acid, quercetin, genistein, tannic acid, polygalactoside, zinc PCA, and / or γ-oryzanol, the expression of RAB32 can be inhibited; and when treating salicumin, tannic acid, or zinc PCA, the expression of KITLG can be inhibited, but these are not limited to these. It can be seen that the reactivity to the substances of this invention varies according to specific gene polymorphism types, which suggests that the efficiency of improving dull skin tone can be improved by providing customized formulations to high-risk groups with a higher risk level of developing dull skin tone.

[0185] In this invention, the term "about" is used as described above.

[0186] The composition described above is characterized by being personalized. Specifically, it can be provided to individuals with a single nucleotide polymorphism (SNP) marker that is significantly associated with a specific degree of skin darkening.

[0187] As an example, the significant single nucleotide polymorphism (SNP) markers associated with the degree of dull skin tone can be selected from any one or more of the markers in Table 2, but are not limited thereto.

[0188] In this invention, the terms "polymorphism", "allele" and "rs_id" are as described above.

[0189] Specifically, the gene polymorphism marker of the present invention is significantly associated with dark skin color and can hold two alleles, which can be divided into cases of holding a major allele / minor allele, a minor allele / minor allele, and a major allele / major allele.

[0190] When an allele possesses a significant polymorphic marker associated with dull skin tone, the effect of improving dull skin tone through reactivity induced by the substance of the present invention may vary depending on the allele possessed by the individual. For example, when possessing a major allele / major allele, a major allele / minor allele, or a minor allele / minor allele, the reactivity induced by the substance of the present invention can be measured, i.e., the skin characteristic of having a high or low degree of improvement in dull skin tone. Therefore, information can be provided for selecting customized substances to enhance the improvement of dull skin tone for individuals containing more than one minor allele.

[0191] The single nucleotide polymorphism (SNP) markers mentioned above can be selected from one or more SNP markers shown in Table 2. The SNP markers shown in Table 2 can determine the degree of correlation between the markers and the degree of skin darkening.

[0192] The degree of skin darkening indicated by the single nucleotide polymorphism markers of this invention is determined by measuring the frequency of each marker. This significance is characterized by, but is not limited to, p-values, such as p-values ​​less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, or less than 0.000000001. Specifically, the p-value can be less than 0.01, more specifically, the p-value can be less than 0.001, and even more specifically, it can be less than 0.0001, but is not limited thereto.

[0193] The single nucleotide polymorphism (SNP) markers of the present invention can be selected from any one or more of the markers shown in Table 2, but are not limited thereto. The above-mentioned single nucleotide polymorphism (SNP) markers can be one or more, and can be used in combination of two or more, three or more, four or more, or five or more, etc., which can determine the number of dark skin tones, but are not limited thereto.

[0194] The aforementioned markers may be SNPs themselves, or polynucleotides consisting of 5-100 consecutive DNA sequences containing the aforementioned SNP sites, or polynucleotides consisting of their complementary sequences, but are not limited thereto.

[0195] As an example, the single nucleotide polymorphism (SNP) markers that significantly correlate with the degree of dull skin tone can be one or more SNP markers present in genes encoding proteins of OCA2, BNC2, RAB32, KITLG, or ASIP. Specifically, they can be selected from any one or more of the group consisting of rs74653330, rs16935073, rs72620727, rs77310623, and rs6059711, but are not limited thereto.

[0196] As an example, when the SNP ID is rs74653330, Chr.Position (GRCh ver. 37) is recorded as "15:28228553". If the allele is publicly listed as T>C, this means that the 28228553rd base on human chromosome 15 is either T or C. Furthermore, the base to the left of the ">" in the allele can indicate the major allele, while the base to the right can indicate the minor allele.

[0197] As a specific example, the markers selected from Table 2 may consist of one or more polynucleotides selected from the following polynucleotides; and one or more polynucleotides from the group consisting of their complementary polynucleotides, but not limited to: polynucleotides consisting of 5-100 consecutive DNA sequences containing the 28,228,553rd base of human chromosome 15, wherein the 28,228,553rd base is T or C (rs74653330); polynucleotides consisting of 5-100 consecutive DNA sequences containing the 16,795,790th base of human chromosome 9, wherein the 16,795,790th base is C or A (rs16935073). ); a polynucleotide consisting of 5-100 consecutive DNA sequences from the 89110410th base of human chromosome 12, wherein the 89110410th base is C or A (rs72620727); a polynucleotide consisting of 5-100 consecutive DNA sequences from the 146871819th base of human chromosome 6, wherein the 146871819th base is A or G (rs77310623); a polynucleotide consisting of 5-100 consecutive DNA sequences from the 32780822nd base of human chromosome 20, wherein the 32780822nd base is T or C (rs6059711).

[0198] The compositions of the present invention can be used as cosmetic compositions for alleviating or improving dull skin tone, as described above.

[0199] In another aspect, the above composition provides a quasi-medicinal composition for relieving or improving dull skin tone. In this invention, the term "quasi-medicinal" is used as described above.

[0200] In another aspect, the present invention provides a method for providing information on selecting a custom substance for improving dull skin tone, comprising the following steps: (a) obtaining a biological sample from an individual; (b) amplifying or hybridizing a polymorphic site of one or more single nucleotide polymorphism markers selected from the group consisting of rs74653330, rs16935073, rs72620727, rs77310623 and rs6059711 from the biological sample; and (c) confirming the bases of the polymorphic site amplified or hybridized in step (b).

[0201] Dull skin tone can be improved by treating individuals with specific polymorphic sites with the customized substances selected above.

[0202] In this invention, the term "individual" refers to a subject used to diagnose the degree of skin darkening. DNA can be obtained from samples such as hair, urine, blood, various bodily fluids, isolated tissues, isolated cells, or saliva, but is not limited thereto. Steps (a) and (b) above are also as described above.

[0203] The aforementioned substances may be one or more selected from the group consisting of polygalactoside, ferulic acid, psoralen, genistein, oil-soluble licorice extract, Aspergillus ferment, quercetin, tannic acid, zinc PCA, γ-oryzanol, succinate, tranexamic acid, sunflower seed oil, and isostearic acid, but are not limited thereto.

[0204] For the purposes of this invention, the aforementioned genes significantly associated with the degree of dull skin tone can provide information that allows for the selection of specific substances for each individual. As an example, for individuals possessing one or more single nucleotide polymorphism markers present in genes encoding OCA2, BNC2, RAB32, KITLG, or ASIP proteins, treatment with one or more substances selected from the group consisting of polygalactoside, ferulic acid, psoralen, genistein, oil-soluble licorice extract, Aspergillus ferment broth, quercetin, tannic acid, zinc PCA, γ-oryzanol, succinate, tranexamic acid, sunflower seed oil, and isostearic acid can enhance the improvement of dull skin tone; however, an optimal combination of substances can be provided based on the genetic characteristics of each individual.

[0205] This suggests that information can be provided to screen for specific single nucleotide polymorphism (SNP) markers that are significantly associated with dull skin tone and to select personalized substances based on genetic information that can maximize the effect of improving dull skin tone.

[0206] Specifically, if the risk score obtained by multiplying and summing the genotypes of rs74653330, rs16935073, rs72620727, rs77310623, and rs6059711 by their effect sizes (β) is less than -0.193, the risk of developing dull skin can be classified as low-risk; if the risk score is above -0.193, the risk of developing dull skin can be classified as high-risk. As an example, an information provision method could be provided where, in cases where the risk of developing dull skin is classified as high-risk, isostearic acid, sunflower seed oil, and polygalactoside are selected as custom-formulated substances, but this method is not limited to these.

[0207] In this invention, the term "SNP chip" is used as described above.

[0208] As one aspect of achieving the objectives of this invention, the present invention provides a single nucleotide polymorphism (SNP) marker for diagnosing the degree of dull skin tone.

[0209] In another aspect, the present invention provides a composition for diagnosing the degree of dull skin tone, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing the degree of dull skin tone, or a formulation capable of amplifying the single nucleotide polymorphism (SNP) marker.

[0210] Dull skin tone refers to skin that is darker than an individual's natural skin color due to factors such as ultraviolet radiation, hormones, and aging, but it is not limited to this. Specifically, it refers to determining whether a person is prone to developing dull skin tone by using one or more single nucleotide polymorphism (SNP) markers selected from Table 2. More specifically, skin types prone to developing dull skin tone are those with a higher rate of overall darkening due to factors such as ultraviolet radiation, hormones, and aging.

[0211] For the alleles described above in this invention, each individual has the same chromosome number, which includes major and minor alleles of the SNP. As the bases at the polymorphic sites of the polymorphic markers increase one by one to become minor alleles, the number of major alleles can decrease one by one. However, the range within which minor and major alleles can increase or decrease is within three types: i) major allele / major allele, ii) major allele / minor allele, and iii) minor allele / minor allele. Furthermore, alleles can decrease or increase within these three types, but are not limited to these.

[0212] In this invention, the term "probe capable of detecting markers used to diagnose the degree of dull skin tone" refers to a composition capable of diagnosing the degree of dull skin tone through a specific hybridization reaction with the polymorphic sites of the aforementioned genes. Furthermore, the specific method of such gene analysis is not particularly limited and can be performed using any gene detection method known in the technical field to which this invention pertains. Additionally, the above term can be used interchangeably with the terms "for diagnosing whether a skin type is prone to dull skin tone" or "for diagnosing whether a high-risk skin type is prone to dull skin tone."

[0213] In this invention, the term "preparation capable of amplifying markers for diagnosing the degree of dull skin tone" refers to a composition capable of diagnosing the degree of dull skin tone by amplifying and confirming the polymorphic sites of the aforementioned genes. Specifically, it refers to primers capable of specifically amplifying the polynucleotides of the aforementioned markers for diagnosing the degree of dull skin tone. Furthermore, the term may be used interchangeably with "for diagnosing whether a skin type is prone to developing dull skin tone" or "for diagnosing whether a high-risk skin type is prone to developing dull skin tone."

[0214] Primers used to amplify the aforementioned polymorphic markers are single-stranded oligonucleotides that function as templates—the starting points for directing DNA synthesis—under appropriate conditions in a suitable buffer (e.g., four different nucleoside triphosphates and polymerases such as DNA, RNA polymerase, or reverse transcriptase) and at an appropriate temperature. The appropriate length of these primers can vary depending on the intended use, but is typically 15 to 30 nucleotides. Shorter primer molecules usually require lower temperatures to form stable hybrids with the template. The primer sequence does not need to be perfectly complementary to the template, but must be sufficiently complementary to hybridize with it.

[0215] In this invention, the term "primer" is used as described above.

[0216] In another aspect, the present invention provides a kit for diagnosing the degree of dull skin tone, comprising the above-described composition for diagnosing the degree of dull skin tone. The kit may be an RT-PCR kit or a DNA microarray kit, but is not limited thereto.

[0217] In another aspect, the present invention provides a microarray for diagnosing the degree of dull skin tone, comprising the above-described composition for diagnosing the degree of dull skin tone.

[0218] The process of immobilizing the probe polynucleotides related to the degree of skin darkening of the present invention on a substrate can also be easily prepared using this prior art. Furthermore, the hybridization of nucleic acids on a microarray and the detection of the hybridization results are well known in the art. The above detection can be performed by the following process: for example, labeling nucleic acid samples with a labeling material capable of generating a detectable signal (including fluorescent materials, such as Cy3 and Cy5), then hybridizing on a microarray and detecting the signal generated by the labeling material.

[0219] In another aspect, the present invention provides a method for providing information about the degree of skin darkening, comprising the steps of: (a) amplifying or hybridizing the polymorphic site of the single nucleotide polymorphism marker in DNA obtained from a sample isolated from an individual; and (b) confirming the bases of the polymorphic site amplified or hybridized in step (a).

[0220] The terms “individual, (a) step, (b) step” are as described above.

[0221] In the above method, additionally (c) if the risk score obtained by multiplying and adding the effect sizes (β) of the amplified or hybridized single nucleotide polymorphism markers is -0.193 or higher, it can be judged as a high-risk group with a higher risk of developing dark skin, but is not limited to this.

[0222] As one aspect of achieving the objectives of this invention, the present invention provides a composition for improving spot-type pigmentation. As an example, the composition comprises one or more substances selected from the group consisting of ascorbyl glucoside, 3-O-Ethyl ascorbic acid, ascorbic acid, licorice extract, tocopherol, sodium pyruvate, kelp extract, hexyldecanol, Asiaticoside, and hexylresorcinol as active ingredients. As another example, the above composition may contain (i) tocopherol, licorice extract and senna extract; (ii) sodium pyruvate, hexyldecyl alcohol, asiaticoside and hexylresorcinol; or (iii) ascorbate glucoside, 3-O-ethyl ascorbic acid and ascorbic acid as active ingredients, but is not limited thereto.

[0223] The term "ascorbyl glucoside," as a representative ascorbic acid derivative, is mostly synthesized through enzymatic reactions. However, this requires optimized reaction conditions or high-purity purification processes, leading to increased production costs. Therefore, it is used as a high-value-added cosmetic ingredient. Ascorbyl glucosides can be broadly classified into α-type and β-type, with β-ascorbyl glucoside (AA-β-2G) exhibiting superior physiological activity due to differences in its absorption mechanism within the human body. The methods for obtaining the aforementioned ascorbyl glucosides are not limited to those known in the art; they can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0224] [Chemical Formula 14]

[0225]

[0226] The term "3-O-ethyl ascorbic acid" is a whitening active ingredient with excellent antioxidant and wrinkle-improving effects. Due to the ether formation between the ethyl group and the 3-hydroxyl group of ascorbic acid, 3-O-ethyl ascorbic acid exhibits exceptional stability due to its unique chemical structure. The method of obtaining 3-O-ethyl ascorbic acid is not limited to its specific preparation method; it can be chemically synthesized using methods known in the art or by using commercially available substances.

[0227] [Chemical Formula 15]

[0228]

[0229] The term "ascorbic acid" refers to vitamin C, a water-soluble vitamin. It is also an important component that plays a role in preventing skin aging by exerting skin-whitening, antioxidant, and collagen-promoting effects. Ascorbic acid has a sugar-like structure that readily oxidizes to dihydro-L-ascorbic acid by accepting two electrons, thus preventing the oxidation of biological components such as proteins by highly reactive free radicals and peroxides in the body. Ascorbic acid can be chemically synthesized and obtained using various extraction methods known in the art, with additional purification processes possible if necessary.

[0230] [Chemical Formula 16]

[0231]

[0232] The term "Glycyrrhiza Uralensis (Licorice) Extract" encompasses extracts obtained from the roots, fruits, stems, or whole licorice plant, which has long been used as a Korean medicinal herb in the East; diluted or concentrated extracts of the aforementioned extracts; dried extracts obtained by drying the aforementioned extracts; crude or purified extracts of the aforementioned extracts; mixtures thereof; the extract itself; and extracts of all dosage forms that can be formed using the extract. There are no particular limitations on the method for obtaining the aforementioned licorice extract; it can be extracted using methods commonly used in the relevant technical field.

[0233] The term "Ecklonia cava extract" refers to a plant belonging to the Laminariaceae family of brown algae, primarily found in waters around 10 meters deep along the Jeju coast in South Korea, and also inhabiting Japan. It is extracted from Ecklonia cava, a plant with a length of 1-2 meters, a cylindrical stem, and a root-like base. There are no particular restrictions on the method for obtaining this extract; it can be extracted using methods commonly used in the relevant technical field.

[0234] As non-limiting examples of the above extraction methods, hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc. can be cited. These methods can be performed individually or in combination with two or more other methods.

[0235] In this invention, there are no particular limitations on the type of solvent used for the extraction, and any solvent known in the relevant art can be used. Examples of non-limiting extraction solvents include water, alcohol, or mixtures thereof, which can be used alone or in combination; specifically, water can be used. When using an alcohol as a solvent, alcohols with 1 to 4 carbon atoms can be used.

[0236] The term “tocopherol” as used above refers to a group of tocopherols that are fat-soluble vitamin E derivatives. Any one or more of α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol, or a mixture thereof, may be used, and any product isolated from the extract, synthetic or commercially available products may be used without restriction.

[0237] The term "sodium pyruvate" as used above refers to sodium pyruvate, which is a salt of the conjugated anionic form of pyruvate, known as pyruvate. Sodium pyruvate possesses antioxidant properties, energy-generating effects, and the ability to cross the blood-brain barrier. The method of obtaining sodium pyruvate is not limited to its specific preparation; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0238] The term "hexyldecanol" is a synthetic non-drying fatty alcohol used as an emollient and skin conditioning agent. The method of obtaining hexyldecanol is not limited; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0239] The term "Asiaticoside" refers to a natural organic compound that reduces skin irritation, enhances elasticity, and has antioxidant properties. As a triterpenoid saponin compound, asiaticoside exhibits skin moisturizing and antioxidant effects. The method of obtaining asiaticoside is not limited; it can be synthesized using methods known in the art or by using commercially available substances.

[0240] The term "hexylresorcinol" refers to an organic compound with properties as a local anesthetic, antiseptic, and anthelmintic. The method of obtaining hexylresorcinol is not limited to its specific preparation method; it can be chemically synthesized using methods known in the art, or commercially available substances can be used.

[0241] [Chemical Formula 17]

[0242]

[0243] Based on the total weight of the composition, the content of the substances of the present invention may be from about 0.0001 to 10% by weight, but is not limited thereto. As an example, based on the total weight of the composition, the spot-type pigmentation improvement material of the present invention may be from about 0.0001 to 10% by weight, from about 0.0005 to 5% by weight, from about 0.001 to 5% by weight, from about 0.01 to 5% by weight, from about 0.0001 to 3% by weight, from about 0.0005 to 3% by weight, from about 0.001 to 3% by weight, from about 0.01 to 3% by weight, from about 0.0001 to 1% by weight, or from about 0.01 to 1% by weight.

[0244] For the purposes of this invention, the above-mentioned substances have the effect of improving spot-type pigmentation.

[0245] For the purposes of this invention, the term "freckle-like pigmentation" refers to pigmentation in certain areas of an individual's skin caused by factors such as aging and ultraviolet radiation exposure, resulting in excessive melanin production, melanin accumulation, keratin accumulation, and the accumulation of oxidized proteins and lipids. This leads to lower chroma than other areas of the skin, resulting in micro-dark spots. Furthermore, unlike scattered pigmentation such as melasma, which is caused by the aggregation of multiple pigment deposits, freckle-like pigmentation, which is distinct from its surroundings, causes the skin to appear noticeably darker and out of sync with its surroundings, creating a lifeless and unkempt impression. Freckle-like pigmentation is not limited to spots caused by melanin; it can also include post-inflammatory spots, moles (lentigo), melanocytic lentigines (black warts, age spots), etc.

[0246] Specifically, the aforementioned freckle-type pigmentation can be selected from one or more of the following groups: senile lentigines, age spots, solar lentigines, melanocytic lentigines, and moles, but is not limited to these.

[0247] Typically, to determine the degree of freckle-like hyperpigmentation, methods are used to measure the lightness or color of the skin in the freckled area, or to measure the unevenness of the skin color in the freckled area. However, it is impossible to measure the lightness or color of all areas of the face, and methods for measuring the unevenness of skin color in freckled areas are difficult to distinguish from dark spots such as wrinkles, raised keratin, and pores. Therefore, defining "skin tone in freckled areas" using the lightness, color, or unevenness of skin color in freckled areas has limitations. While visual evaluation is best suited for expressing the subject's perceived skin concerns, it suffers from poor objectivity and the limitation that a few evaluators cannot evaluate the majority of subjects. To address this issue, this invention constructs a diagnostic AI trained on visual evaluation results, defining "freckle-like hyperpigmentation." Evaluation through the diagnostic AI has the following advantages: compared to objective quantitative evaluation, it is more suitable for displaying the subjectively perceived degree of "freckle-like hyperpigmentation," and compared to visual evaluation, it can consistently evaluate various subjects.

[0248] For the purposes of this invention, the aforementioned substances are characterized by reducing melanin production. Specifically, when treating i) hexylresorcinol and hexyldecyl alcohol; or ii) hexyldecyl alcohol and tocopherol with the aforementioned substances, melanin reduction can be promoted.

[0249] As an example, applying a cream containing a mixture of the substances of the present invention (i) hexylresorcinol and hexyldecyl alcohol, which are effective in reducing melanin production; or ii) hexyldecyl alcohol and tocopherol) to the face confirmed a synergistic effect in reducing melanin production compared to treating the substances separately.

[0250] For the purposes of this invention, the aforementioned substances are characterized by inhibiting the expression of ASCL2 or TP63, or promoting the expression of PPARGC1B. Specifically, treatment with tocopherol, licorice extract, and / or senna extract can inhibit the expression of ASCL2; treatment with sodium pyruvate, hexyldecyl alcohol, asiaticoside, and / or hexylresorcinol can inhibit the expression of TP63; and treatment with ascorbate glucoside, 3-O-ethyl ascorbic acid, and / or ascorbic acid can promote the expression of PPARGC1B, but is not limited thereto. It can be seen that the responsiveness to the substances of this invention varies according to specific gene polymorphism types, indicating that by providing customized formulations to high-risk groups with a higher risk level of speckled pigmentation, the efficiency of speckled pigmentation improvement can be improved.

[0251] In this invention, the term "about" is used as described above.

[0252] The composition described above is characterized by being personalized. Specifically, it can be provided to individuals with a significant single nucleotide polymorphism (SNP) marker associated with a specific degree of speckled pigmentation.

[0253] As an example, the significant single nucleotide polymorphism (SNP) markers associated with the degree of speckled pigmentation can be selected from any one or more of the markers in Table 3, but are not limited thereto.

[0254] In this invention, the terms "polymorphism", "allele" and "rs_id" are as described above.

[0255] Specifically, the gene polymorphism marker of the present invention is significantly associated with freckle pigmentation and can hold two alleles, which can be divided into cases of holding a major allele / minor allele, a minor allele / minor allele, and a major allele / major allele.

[0256] When an allele is present that is a significant polymorphic marker associated with freckle pigmentation, the effect of improving freckle pigmentation based on the reactivity induced by the substance of the present invention may vary depending on the allele possessed by the individual. For example, when a major allele is present, a major allele is present, a minor allele is present, or a minor allele is present, the reactivity induced by the substance of the present invention can be measured, i.e., the skin characteristic of having a high or low degree of improvement in freckle pigmentation. Therefore, information can be provided for selecting a custom substance to enhance the improvement of freckle pigmentation for individuals containing more than one minor allele.

[0257] The single nucleotide polymorphism (SNP) markers mentioned above can be selected from one or more SNP markers shown in Table 3. The SNP markers shown in Table 3 can be used to determine the degree of correlation with the severity of speckled pigmentation.

[0258] The degree of speckled pigmentation from single nucleotide polymorphism markers of the present invention is determined by measuring the frequency of each marker. This significance is characterized by, but not limited to, p-values, such as p-values ​​less than 0.05, less than 0.01, less than 0.001, less than 0.0001, less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001, or less than 0.000000001. Specifically, the p-value can be less than 0.01, more specifically, the p-value can be less than 0.001, and even more specifically, it can be less than 0.0001, but is not limited thereto.

[0259] The single nucleotide polymorphism (SNP) markers of the present invention can be selected from any one or more of the markers shown in Table 3, but are not limited thereto. The above-mentioned single nucleotide polymorphism (SNP) markers can be one or more, and can be used in combination of two or more, three or more, etc., that can determine the number of spots of pigmentation, but are not limited thereto.

[0260] The aforementioned markers may be SNPs themselves, or polynucleotides consisting of 5-100 consecutive DNA sequences containing the aforementioned SNP sites, or polynucleotides consisting of their complementary sequences, but are not limited thereto.

[0261] As an example, the single nucleotide polymorphism markers associated with the degree of the above-mentioned spot-type pigmentation can be one or more single nucleotide polymorphism markers present in the genes encoding proteins of ASCL2, TP63 or PPARGC1B. Specifically, they can be selected from any one or more of the group consisting of rs251466, rs12222609 and rs9865771, but are not limited thereto.

[0262] As an example, when the SNP ID is rs251466, Chr.Position (GRCh ver. 37) is recorded as "5:149195603". If the allele is publicly known as C>G, this means that the 149195603rd base on human chromosome 5 is either C or G. Furthermore, the base to the left of the ">" in the allele can indicate the major allele, while the base to the right can indicate the minor allele.

[0263] As a specific example, the markers selected from Table 3 may consist of one or more polynucleotides selected from the following polynucleotides; and one or more polynucleotides from the group consisting of their complementary polynucleotides and their complementary polynucleotides, but are not limited to: a polynucleotide consisting of 5-100 consecutive DNA sequences containing the 149,195,603rd base of human chromosome 5, wherein the 149,195,603rd base is C or G (rs251466); a polynucleotide consisting of 5-100 consecutive DNA sequences containing the 229,7093rd base of human chromosome 11, wherein the 229,7093rd base is C or T (rs12222609); a polynucleotide consisting of 5-100 consecutive DNA sequences containing the 189,491,453rd base of human chromosome 3, wherein the 189,491,453rd base is A or G (rs9865771).

[0264] The compositions of the present invention can be used as cosmetic compositions for relieving or improving spot-type hyperpigmentation, as described above.

[0265] In another aspect, the above composition provides a quasi-medicinal composition for relieving or improving spot-type pigmentation. In this invention, the term "quasi-medicinal" is used as described above.

[0266] In another aspect, the present invention provides a method for providing information on selecting a custom substance for improving freckle pigmentation, comprising the following steps: (a) obtaining a biological sample from an individual; (b) amplifying or hybridizing a polymorphic site of one or more single nucleotide polymorphism markers selected from the group consisting of rs251466, rs12222609 and rs9865771 from the biological sample with a probe; and (c) confirming the bases of the polymorphic site amplified or hybridized in step (b).

[0267] Spot-like pigmentation can be improved by treating individuals with specific polymorphic sites with the aforementioned customized substances.

[0268] In this invention, the term "individual" refers to a subject used to diagnose the degree of skin pigmentation. DNA can be obtained from samples such as hair, urine, blood, various bodily fluids, isolated tissues, isolated cells, or saliva, but is not limited thereto. Steps (a) and (b) above are also as described above.

[0269] The aforementioned substances may be one or more selected from the group consisting of ascorbate glucoside, 3-O-ethyl ascorbic acid, ascorbic acid, licorice extract, tocopherol, sodium pyruvate, glycoside extract, hexyldecyl alcohol, asiaticoside and hexylresorcinol, but are not limited thereto.

[0270] For the purposes of this invention, the aforementioned gene associated with significant freckle pigmentation can provide information for selecting specific substances for each individual. As an example, for individuals possessing one or more single nucleotide polymorphism markers present in genes encoding ASCL2, TP63, or PPARGC1B proteins, treatment with one or more substances selected from the group consisting of ascorbate glucoside, 3-O-ethyl ascorbic acid, ascorbic acid, licorice extract, tocopherol, sodium pyruvate, senna extract, hexyldecyl alcohol, asiaticoside, and hexylresorcinol can enhance the improvement of freckle pigmentation; however, an optimal combination of substances can be provided based on the genetic characteristics of each individual.

[0271] This suggests that information can be provided to screen for specific single nucleotide polymorphism (SNP) markers that are significantly associated with freckle pigmentation and to select personalized substances based on genetic information that can maximize the effect of improving freckle pigmentation.

[0272] Specifically, when the hazard score obtained by multiplying and summing the genotypes of rs251466, rs12222609, and rs9865771 by their effect sizes (β) is less than -0.009, the risk of freckle pigmentation can be classified as low-risk; when the hazard score is above 0.000, the risk of freckle pigmentation can be classified as high-risk. As an example, an information provision method can be provided where, in cases where the risk of freckle pigmentation is classified as high-risk, tocopherol, hexylresorcinol, and hexyldecyl alcohol are selected as custom-formulated substances, but this method is not limited to these.

[0273] In this invention, the term "SNP chip" is used as described above.

[0274] As one aspect of achieving the objectives of this invention, the present invention provides a single nucleotide polymorphism (SNP) marker for diagnosing the degree of freckle pigmentation.

[0275] In another aspect, the present invention provides a composition for diagnosing the degree of freckle pigmentation, comprising a probe capable of detecting a single nucleotide polymorphism (SNP) marker for diagnosing the degree of freckle pigmentation or a formulation capable of amplifying the single nucleotide polymorphism (SNP) marker.

[0276] Spotty hyperpigmentation refers to a condition where the skin color in specific areas is darker than an individual's natural skin color due to factors such as ultraviolet radiation, hormones, and aging, but it is not limited to these. Specifically, it refers to using one or more single nucleotide polymorphism (SNP) markers selected from Table 3 to determine whether a person is prone to spotty hyperpigmentation. More specifically, skin types prone to spotty hyperpigmentation are those where the overall skin color in the affected areas darkens more significantly due to factors such as ultraviolet radiation, hormones, and aging.

[0277] For the alleles described above in this invention, each individual has the same chromosome number, which includes major and minor alleles of the SNP. As the bases at the polymorphic sites of the polymorphic markers increase one by one to become minor alleles, the number of major alleles can decrease one by one. However, the range within which minor and major alleles can increase or decrease is within three types: i) major allele / major allele, ii) major allele / minor allele, and iii) minor allele / minor allele. Furthermore, alleles can decrease or increase within these three types, but are not limited to these.

[0278] In this invention, the term "probe capable of detecting markers used to diagnose the degree of lentigines" refers to a composition capable of diagnosing the degree of lentigines through a specific hybridization reaction with the polymorphic sites of the aforementioned genes. Furthermore, the specific method of such gene analysis is not particularly limited and can be performed using any gene detection method known in the technical field to which this invention pertains. Additionally, the above term can be used interchangeably with the terms "for diagnosing whether a skin type is prone to lentigines" or "for diagnosing whether a high-risk skin type is prone to lentigines."

[0279] In this invention, the term "preparation capable of amplifying markers for diagnosing the degree of lentigines" refers to a composition capable of diagnosing the degree of lentigines by amplifying and confirming the polymorphic sites of the aforementioned genes. Specifically, it refers to primers capable of specifically amplifying the polynucleotides of the aforementioned markers for diagnosing the degree of lentigines. Furthermore, the above term may be used interchangeably with the terms "for diagnosing whether a skin type is prone to lentigines" or "for diagnosing whether a high-risk skin type is prone to lentigines."

[0280] Primers used to amplify the aforementioned polymorphic markers are single-stranded oligonucleotides that function as templates—the starting points for directing DNA synthesis—under appropriate conditions in a suitable buffer (e.g., four different nucleoside triphosphates and polymerases such as DNA, RNA polymerase, or reverse transcriptase) and at an appropriate temperature. The appropriate length of these primers can vary depending on the intended use, but is typically 15 to 30 nucleotides. Shorter primer molecules usually require lower temperatures to form stable hybrids with the template. The primer sequence does not need to be perfectly complementary to the template, but must be sufficiently complementary to hybridize with it.

[0281] In this invention, the term "primer" is used as described above.

[0282] In another aspect, the present invention provides a kit for diagnosing the degree of freckle pigmentation, comprising the above-described composition for diagnosing the degree of freckle pigmentation. The kit may be an RT-PCR kit or a DNA microarray kit, but is not limited thereto.

[0283] In another aspect, the present invention provides a microarray for diagnosing the degree of freckle pigmentation, comprising the above-described composition for diagnosing the degree of freckle pigmentation.

[0284] The process of immobilizing the probe polynucleotides related to the degree of speckled pigmentation of the present invention on a substrate can also be easily prepared using this prior art. Furthermore, the hybridization of nucleic acids on a microarray and the detection of the hybridization results are well known in the art. The above detection can be performed by the following process: for example, labeling the nucleic acid sample with a labeling material capable of generating a detectable signal (including fluorescent materials, such as Cy3 and Cy5), then hybridizing on a microarray and detecting the signal generated by the labeling material.

[0285] In another aspect, the present invention provides a method for providing information about the degree of speckled pigmentation, comprising the steps of: (a) amplifying or hybridizing the polymorphic site of the single nucleotide polymorphism marker in DNA obtained from a sample isolated from an individual; and (b) confirming the bases of the polymorphic site amplified or hybridized in step (a).

[0286] The terms “individual, (a) step, (b) step” are as described above.

[0287] In the above method, additionally (c) if the risk score obtained by multiplying and adding the effect sizes (β) of the amplified or hybridized single nucleotide polymorphism markers is 0.000 or higher, it can be judged as a high-risk group with a high risk of developing freckle pigmentation, but is not limited thereto.

[0288] Implementation

[0289] The present invention will now be described in more detail through embodiments. These embodiments are for illustrative purposes only, and the scope of the invention should not be construed as being limited to these embodiments.

[0290] Example 1: Deriving a significant gene polymorphism marker associated with skin pigmentation

[0291] This invention aims to identify genomic loci (genetic variations) in Korean women that show differences in the presence and degree (numerical value) of facial skin pigmentation based on genetic information. To discover genomic loci (genetic variations) associated with facial skin pigmentation, this invention utilizes a microarray genotyping chip (Illumina product) capable of screening for full-length genome-wide variations without pre-screening candidate genes.

[0292] In this invention, to evaluate the presence and extent of diffuse melasma and / or freckle-like pigmentation, an image-based diagnostic AI was constructed, novel phenotypes were extracted, and a high correlation (0.876) with visual evaluation was confirmed. The novel AI-based phenotypes were used, and to minimize external effects that might influence the presence and extent (numerical) of diffuse melasma, they were corrected for age, sex, BMI, and cluster principal component values.

[0293] In this invention, to evaluate the presence and degree of dull skin tone, a skin diagnostic device (Janus III, PIE) was used to extract skin color brightness (L*). The extracted skin color brightness value was used as a phenotype, and was used after correction for age, gender, BMI, and cluster principal component values ​​to minimize external effects that might influence the dull skin tone and its degree (numerical value).

[0294] To confirm the association between genomic loci (genetic variations) and the presence and extent (numerical) of diffuse melasma, the extent (numerical) of dark skin tone, and / or the presence and extent (numerical) of freckle-like hyperpigmentation, Bolt-LMM was used to quantify the significance and genetic effects of the associations. The Bolt-LMM method minimizes the influence of inter-individual genetic associations in a study while maximizing the analytical effectiveness.

[0295] 1-1. Evaluation methods for scattered dark spots, dull skin tone, and spot-type pigmentation.

[0296] To derive genetic polymorphic markers indicating the degree of diffuse dark spots, dull skin tone, and / or freckle-like pigmentation on the cheeks and cheekbones under the eyes, healthy Koreans aged 20-70 were recruited. For skin measurements, all participants washed their faces with facial cleanser or soap and waited 20 minutes in a temperature- and humidity-controlled chamber without applying any products to allow their skin to acclimatize to the measurement environment. Skin pigmentation was then evaluated, and high-resolution cameras from a specialized skin diagnostic instrument (PIE Corporation Janus3) were used for facial imaging. Measurements and analysis were performed according to the instrument manufacturer's manual. Specifically, participants were instructed to wear a headband and a black cloth wrapped around their clothing, then fix their faces to the forehead and chin supports built into the instrument, close their eyes, and have high-resolution images of their faces captured using the camera built into the Janus3 instrument. For the high-resolution images between the eyes and cheekbones, facial landmark detection technology was used to extract landmarks in a way that minimized interference from other areas and prevented individual identification.

[0297] To extract the degree of scattered dark spots on the skin, the AI ​​used a ResNet (Residual Network) structure from CNNs (Convolutional Neural Networks, the basic architecture of image deep learning), and selected it after performance comparison with other well-known existing structures. The constructed AI further evaluated images flipped horizontally, and the average value was used as the final result, thereby improving accuracy and stability. To confirm the reliability of the AI's analysis values, they were compared with human evaluation results, showing a very high correlation coefficient of 0.876. Therefore, it was applied to a dataset of 50,000 people to extract the degree of scattered dark spots as a novel phenotype.

[0298] To extract the degree of dull skin tone, images of the cheek area were extracted from the aforementioned images using a built-in algorithm. If the cheek area was not accurately selected, it was manually adjusted by the evaluator. The average RGB value of the skin color and the conversion to the CIE-LAB color space were performed using a built-in algorithm.

[0299] To extract the degree of freckle-like pigmentation in the skin and to build an AI for diagnosing freckle-like OX, a tool was created that generates one data point with a single click, producing numerous freckle-like and non-freckle-like images. This was used to train the AI ​​initially, and after application to real images, images similar to false positives and false negatives were re-included in the training data, thus achieving maximum performance improvement with minimal data appending. The AI ​​was implemented by scanning the entire face with images of the same size as the training data. Since freckle-like pigmentation usually presents with about one freckle or none in most cases on the face, the maximum value was given as the final diagnostic result.

[0300] For a subset of images, the AI ​​employed the ResNet (Residual Network) architecture from CNNs (Convolutional Neural Networks, the basic structure for deep image learning), selecting it through performance evaluation against existing well-known architectures. The constructed AI was further evaluated on horizontally flipped images, and the average of these evaluations was used as the final result, thereby improving accuracy and evaluation stability. The final result showed a very high accuracy of over 99%, and was therefore applied to a dataset of 50,000 individuals to extract the degree of speckled pigmentation as a novel phenotype.

[0301] 1-2. Gene Collection and Analysis Methods

[0302] For genetic analysis, genomic DNA was extracted from the subjects' saliva using the QIAmp mini prep kit (QIAGEN). The quality of the DNA was confirmed by absorbance (OD 260 / 280) > 1.7, concentration 50 ng / ul, and DNA gel bands on 1X TAE 1% agarose gel. Genetic analysis was limited to those that passed the quality test.

[0303] Genetic analysis was performed using Illumina's microarray genotyping chip; specifically, the genes of the subjects were analyzed using the same company's global screening array product.

[0304] The gene analysis experiments using Illumina's microarray genotyping chip were conducted according to the provided manual, using the provided reagents, and involved the processes of genomic DNA amplification, DNA fragmentation, precipitation, hybridization, staining, washing, coating, and scanning.

[0305] The microarray genotyping chip completed in the above experimental process was scanned using iScan Control Software (Illumina). After scanning, the automatically generated idat file was checked for data quality management level (sample call rate > 98%, marker call rate > 98%) and gene information using GenomeStudio (Illumina) program.

[0306] In this invention, only data that has undergone gene analysis and passed data quality management is used.

[0307] 1-3. Extract gene polymorphism markers associated with the degree of scattered dark spots, dull skin tone, and freckle-like pigmentation.

[0308] To derive gene polymorphic markers that significantly correlated with the severity of diffuse melasma, dull skin tone, and freckle-like hyperpigmentation, a linear-mixed model (linear regression plus inter-sample genetic association penalty model) was performed using these markers. The BOLT-LMM v2.3.5 program was employed for the analysis. Age, sex, BMI, and 10^-10 cases (adjusted for genetic differences from ethnic groups) were used as covariates.

[0309] To ensure the inclusion of additional gene polymorphism markers, an imputation analysis was performed using existing gene polymorphism markers (using the Beagle v 5.1 program), and information registered in the Genomes project, an international open-access genome database, was used as the standard reference data.

[0310] Imputation is a statistical technique that infers information about novel gene polymorphic markers based on experimentally confirmed gene polymorphic marker information.

[0311] The significance of gene polymorphic markers associated with the severity of diffuse melasma was evaluated by the chi-square statistic of the BOLT-LMM results, with the standard set as p-value < 1.0 * e-4.

[0312] As a result, many gene polymorphic markers that were significantly associated with the degree of diffuse melasma were derived, as shown in Table 1 below.

[0313] [Table 1]

[0314] Are there any associated gene polymorphic markers for scattered dark spots?

[0315]

[0316] 1) The National Institutes of Health (NIH) ID can be verified on the relevant website.

[0317] 2) Meaning of (major allele) > (minor allele)

[0318] 3) Minor allele frequency = (2 mm + Mm) / 2(MM + Mm + mm)

[0319] 4) Statistical significance of phenotypic differences among the three genotypes (M / M, M / m, m / m) (M: major allele, m: minor allele)

[0320] 5) A numerical value indicating how many times the risk of having a case (dispersed macular degeneration sensitivity) is increased when holding a minor allele compared to not holding a minor allele.

[0321] The significance of gene polymorphism markers associated with skin brightness was evaluated using the chi-square statistic of the BOLT-LMM results, with the standard set as p-value < 5e-08.

[0322] The results confirmed many gene polymorphism markers that were significantly associated with the degree of dark skin tone, as shown in Table 2 below.

[0323] [Table 2]

[0324] Dark skin color associated gene polymorphism markers

[0325]

[0326] 1) The National Institutes of Health (NIH) ID can be verified on the relevant website.

[0327] 2) Meaning of (major allele) > (minor allele)

[0328] 3) Minor allele frequency = (2 mm + Mm) / 2(MM + Mm + mm)

[0329] 4) Statistical significance of phenotypic differences among the three genotypes (M / M, M / m, m / m) (M: major allele, m: minor allele)

[0330] 5) A numerical value indicating that the risk of having a case (having dull skin tone) is several times higher when holding a minor allele compared to not holding a minor allele.

[0331] The significance of gene polymorphic markers associated with the degree of skin lentigines was evaluated by the chi-square statistic of the BOLT-LMM results, with the standard set as p-value < 5e-8.

[0332] As a result, many gene polymorphic markers that were significantly associated with the degree of freckle pigmentation were derived, as shown in Table 3 below.

[0333] [Table 3]

[0334] Spot-related pigmentation-associated gene polymorphism markers

[0335]

[0336] 1) The National Institutes of Health (NIH) ID can be verified on the relevant website.

[0337] 2) Meaning of (major allele) > (minor allele)

[0338] 3) Minor allele frequency = (2 mm + Mm) / 2(MM + Mm + mm)

[0339] 4) Statistical significance of phenotypic differences among the three genotypes (M / M, M / m, m / m) (M: major allele, m: minor allele)

[0340] 5) The degree of change in phenotypic risk as the number of minor alleles increases (-: decreased risk of freckle pigmentation, +: increased risk of freckle pigmentation).

[0341] Presence or absence of freckle-type pigmentation = AI-based facial image analysis

[0342] Example 2: Analysis of the regulatory effects of various substances on the expression of skin pigmentation-related genes

[0343] 2-1. Analysis of the regulatory effects of various substances on the expression of genes associated with dispersible melasma in the skin

[0344] Among various substances with skin-improving effects, a screening process was conducted to identify those that influenced the expression of genes associated with diffuse melanoma. Changes in the expression of these genes were confirmed in cells treated with UVB to increase melanin synthesis, and substances that restored expression to pre-UVB irradiation levels were identified.

[0345] Mouse melanoma cells (B16F10; mouse melanoma cell, CRL-6475) were purchased from ATCC and used. The purchased cells were cultured in DMEM (Dulbecco Modified Eagle Medium-GIBCO) medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator.

[0346] To analyze the efficacy of changes in mRNA expression of genes associated with diffuse melanoma in the skin, mouse melanoma cells were cultured at a concentration of 2 * 10-1. 5 Cells were aliquoted per well into 6-well plates and incubated at 37°C for one day in a 5% CO2 incubator. After replacing the culture medium with PBS, the cells were irradiated with UVB (312 nm) at 30 mJ / cm² using an RMX 3W (Vilber Lourmat) instrument. 2The substances were added to the culture medium at appropriate concentrations and then cultured under the same conditions for one day. Subsequently, total RNA was obtained using the AccuPrep Universal RNA Extraction Kit (BIONEER, K-3140), and cDNA was synthesized using the AccuPower® RocketScript™ Cycle (BIONEER, K-2202). The expression levels of each gene were analyzed by qPCR using the synthesized cDNA and TaqMan probes (assay IDs: AKAP1-Mm00501539_m1, RAB11FIP2-Mm01261268_m1, PPARGC1B-Mm00504730_m1).

[0347] The result, such as Figure 1 As shown, UVB led to increased AKAP1 expression and decreased RAB11FIP2 and PPARGC1B expression. Substances that decreased AKAP1 expression included cysteine, cysteine ​​hydrochloride, myristica extract, high molecular weight hyaluronic acid, and ultra-low molecular weight hyaluronic acid. Substances that increased RAB11FIP2 expression included bisabolol, Polygonum cuspidatum root extract, and diisopropanol (DIPA). Furthermore, nicotinamide, glycolic acid (AHA), gluconolactone (PHA), and salicylic acid (BHA) were confirmed to increase PPARGC1B expression.

[0348] 2-2. Analysis Results of the Regulatory Effects of Various Substances on the Expression of Genes Related to Dark Skin Color

[0349] Among various substances with skin-improving effects, a screening process was conducted to identify those that affected the expression of genes associated with dull skin tone. In cells treated with α-MSH to increase melanin synthesis, changes in the expression of genes associated with dull skin tone were confirmed, and substances that restored expression to pre-α-MSH treatment levels were identified.

[0350] Mouse melanoma cells (B16F10; mouse melanoma cell, CRL-6475) were purchased from ATCC and used. The purchased cells were cultured in DMEM (Dulbecco Modified Eagle Medium-GIBCO) medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator.

[0351] To analyze the effects of changes in mRNA expression of genes associated with dull skin tone, an RT-qPCR experiment was performed.

[0352] The cells were cultured at 37°C and 5% CO2 for one day. To evaluate the effect of UVB on KITLG expression, the culture medium for human keratinocytes (HaCaT) was replaced with PBS, and the cells were irradiated with UVB (312 nm) at 30 mJ / cm² using an RMX 3W (Vilber Lourmat) instrument. 2 The cells were then cultured in fresh medium for one day. To evaluate the effect of the substance treatment on KITLG expression, the cells were treated with fresh medium and an appropriate concentration of the substance, and cultured for another day. Additionally, to evaluate the expression of OCA2, BNC2, and RAB32, melanocytes were treated with α-MSH (10 nM) and an appropriate concentration of the substance in fresh medium, and cultured for one day. Subsequently, total RNA was obtained using the AccuPrep Universal RNA Extraction Kit (BIONEER, K-3140), and cDNA was synthesized using the AccuPower® RocketScript™ Cycle (BIONEER, K-2202). The expression levels of each gene were analyzed by qPCR using synthesized cDNA and TaqMan probes (analysis IDs: OCA2-Mm00498969_m1, BNC2-Mm00556284_m1, RAB32-Mm00458960_m1, KITLG-Hs00241497_m1).

[0353] The result, such as Figure 2 As shown, α-MSH leads to an increase in OCA2, BNC2, and RAB32, and UVB also leads to an increase in KITLG expression.

[0354] Specifically, substances that reduce OCA2 expression include ferulic acid, polygalactoside, zinc PCA, tranexamic acid, quercetin, genistein, Aspergillus ferment broth, γ-oryzanol, sunflower seed oil, and isostearic acid. Substances that reduce BNC2 expression include polygalactoside, ferulic acid, psoralen, genistein, oil-soluble licorice extract, and a combination of oil-soluble licorice extract and Aspergillus ferment broth.

[0355] In addition, substances that reduce RAB32 expression were identified as ferulic acid, quercetin, genistein, tannic acid, polygalactoside, zinc PCA, and γ-oryzanol, while substances that reduce KITLG expression were identified as succinic acid, zinc PCA, and tannic acid.

[0356] 2-3. Results of the analysis of the regulatory effects of various substances on the expression of genes associated with skin pigmentation.

[0357] Among various substances with skin-improving effects, a screening process was conducted to identify those that affected the expression of genes associated with melanin pigmentation. In cells treated with α-MSH to increase melanin synthesis, changes in the expression of genes associated with melanin pigmentation were confirmed, and substances that restored expression to pre-α-MSH treatment levels were identified.

[0358] Mouse melanoma cells (B16F10; mouse melanoma cell, CRL-6475) were purchased from ATCC and used. The purchased cells were cultured in DMEM (Dulbecco Modified Eagle Medium-GIBCO) medium supplemented with 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin in a 37°C, 5% CO2 incubator.

[0359] To analyze the effects of changes in mRNA expression of genes associated with melanoma pigmentation, mouse melanoma cells were injected at a rate of 2 × 10⁻⁶ mcg / mL. 5 Cells / well were aliquoted into 6-well plates and incubated at 37°C with 5% CO2 for one day. The medium was then replaced with fresh medium, treated with 10 nM α-MSH, and the substance was added to the medium at an appropriate concentration. The plates were then incubated under the same conditions for one day. Total RNA was then obtained using the AccuPrep Universal RNA Extraction Kit (BIONEER, K-3140), and cDNA was synthesized using the AccuPower® RocketScript™ Cycle (BIONEER, K-2202). qPCR was performed using the synthesized cDNA and TaqMan probes (detection ID: ASCL2-Mm01268891_m1, PPARGC1B-Mm00504730_m1, TP63-Hs00978349_m1) to analyze the expression levels of each gene.

[0360] The result, such as Figure 3As shown, in B16F10 cells, α-MSH led to increased ASCL2 expression and decreased PPARGC1B expression. In human keratinocyte HaCaT cells, UVB 10 mJ / cm 2 This led to an increase in TP63 expression. Tocopherol, licorice extract, and senna extract were found to further reduce ASCL2 expression, while AA2G, ascorbic acid, and SINOWHITE EA (3-O-ethyl ascorbic acid) again increased PPARGC1B expression. Treatment of HaCaT cells with sodium pyruvate, hexyldecyl alcohol, asiaticoside, and hexylresorcinol confirmed that these substances reduced TP63 expression. Figure 3 ).

[0361] Example 3: Melanin-reducing efficacy and synergistic effect analysis of customized materials for improving skin pigmentation

[0362] 3-1. Analysis of the melanin-reducing efficacy and synergistic effect of customized materials for improving scattered dark spots on the skin.

[0363] It was confirmed whether the combination of substances screened in Example 2 for improving scattered dark spots on the skin has a synergistic effect in inhibiting melanin production increased by UVB.

[0364] Specifically, mouse melanoma cells were divided into 2*10... 5 Cells / well were dispensed into 6-well plates and incubated at 37°C in a 5% CO2 incubator for one day, followed by UVB (312 nm) irradiation at 30 mJ / cm². 2 The cells were placed in a culture medium containing appropriate concentrations and combinations of substances and incubated again for one day at 37°C in a 5% CO2 incubator to confirm whether the substances inhibited melanin production increased by UVB. Cells adhering to the plate were recovered in PBS and centrifuged to obtain a cell pellet. The cell pellet was visually assessed and photographed. A 1N NaOH solution containing 10% DMSO (dimethyl sulfoxide) was added to the cell pellet and dissolved at 80°C for 10 minutes. The pellet was then placed in a 96-well plate, and the absorbance was measured at 405 nm using an Epoch (BioteK) instrument. Melanin was quantified using the measured absorbance, standardized to the protein concentration of the sample, to measure the melanin concentration. The melanin production inhibition rate of the sample was calculated by dividing the melanin concentration of the sample treatment group by the melanin concentration increased by UVB.

[0365] The results confirmed that treatment with 100 ppm nutmeg extract and 10 ppm bisabolol reduced melanin production by approximately 5.2% (X), while treatment with 25 ppm DIPA alone reduced melanin production by approximately 1.5% (Y). Furthermore, the combined treatment with 100 ppm nutmeg extract, 10 ppm bisabolol, and 25 ppm DIPA reduced melanin production by approximately 32.6% (T, effect measurement). This is greater than the Colby prediction (E) of 6.6%, thus indicating a synergistic effect in reducing melanin production. Figure 4 ).

[0366] Colby's formula: E = (X + Y) - (X * Y / 100)

[0367] 3-2. Analysis of the melanin-reducing efficacy and synergistic effects of customized materials for improving dull skin tone

[0368] It was confirmed whether the combination of substances screened in Example 2 for improving dull skin tone has a synergistic effect in inhibiting melanin production increased by α-MSH treatment.

[0369] Specifically, mouse melanoma cells and human keratinocytes were aliquoted at 2 × 10^5 cells / well into 6-well plates and cultured for one day at 37°C in a 5% CO2 incubator. Then, they were treated with 10 nM α-MSH. The plates were then placed in a culture medium supplemented with appropriate concentrations and combinations of substances and cultured again for two days at 37°C in a 5% CO2 incubator to confirm whether the substances inhibited melanin production increased by α-MSH. Cells adhering to the plates were recovered in PBS and centrifuged to obtain cell pellets. The cell pellets were visually assessed and photographed. A 1N NaOH solution containing 10% DMSO (dimethyl sulfoxide) was added to the cell pellets and dissolved at 80°C for 10 minutes. The pellets were then transferred to 96-well plates, and the absorbance at 405 nm was measured using an Epoch (BioteK) instrument. Melanin was quantified using measured absorbance and standardized to the protein concentration of the sample to measure melanin concentration. The melanin production inhibition rate of the sample was calculated by dividing the melanin concentration of the sample treatment group by the increase in melanin concentration due to α-MSH treatment.

[0370] The results confirmed that treatment with 10 ppm ferulic acid, 5 ppm oil-soluble licorice extract, and 5 ppm psoralen reduced melanin production by approximately 5.04% (X), 6.47% (Y), and 2.07% (Z), respectively. Furthermore, when these concentrations of ferulic acid, oil-soluble licorice extract, and psoralen were combined, melanin production was reduced by approximately 35.07% (T, effect measurement). This is greater than the Colby prediction (E1) of 13.03% for the combination of the three substances, thus indicating a synergistic effect in reducing melanin production. Figure 5 ).

[0371] Colby's formula E1 = (X + Y) - (XY + XZ + YZ / 100) + XYZ / 10000

[0372] Furthermore, when treated with different combinations of 10 ppm resveratrol and 20 ppm quercetin, melanin production was reduced by approximately 23.8% and 33.9%, respectively. However, when treated simultaneously with these concentrations of resveratrol and quercetin, melanin production was reduced by 57.9% (T, effect measurement). This is greater than the Colby prediction (E2) of 49.5% for the combination of the two substances, therefore a synergistic effect in reducing melanin production is indicated. Figure 6 ).

[0373] Colby's formula: E² = X + Y - (XY / 100)

[0374] 3-3. Analysis of the melanin-reducing efficacy and synergistic effect of customized materials for improving freckle-type pigmentation.

[0375] It was confirmed whether the combination of substances screened in Example 2 for improving spot-type pigmentation has a synergistic effect in inhibiting melanin production increased by UVB.

[0376] Mouse melanoma cells B16F10 were used at a rate of 2×10⁻⁶. 5Cells / well were aliquoted into 6-well plates and incubated at 37°C with 5% CO2 for one day, followed by treatment with 10 nM α-MSH. The plates were then transferred to culture medium supplemented with appropriate concentrations and combinations of substances and incubated again at 37°C with 5% CO2 for one day to confirm whether the substances inhibited melanin production increased by α-MSH. Cells adhering to the plates were recovered along with PBS and centrifuged to obtain a cell pellet. A 1N NaOH solution containing 10% DMSO (dimethyl sulfoxide) was added to the cell pellet and dissolved at 80°C for 10 minutes. The pellet was then transferred to a 96-well plate, and the absorbance was measured at 405 nm using an Epoch (BioteK) instrument. Melanin was quantified using the measured absorbance, standardized to the protein concentration of the sample, to determine melanin levels. The melanin production inhibition rate of the samples was calculated as the ratio of melanin produced by the treated group to melanin produced by α-MSH. Conversely, the melanin production increase inhibition rate was calculated as the ratio of the increased melanin production to the degree of inhibition in the untreated α-MSH control group.

[0377] The results showed that simultaneous treatment with 1 ppm hexyldecyl alcohol and 10 ppm hexylresorcinol resulted in a 53.7% inhibition rate of melanin production, exhibiting a synergy higher than the Colby prediction of 48.5%. Simultaneous treatment with 5 ppm hexyldecyl alcohol and 1 ppm tocopherol resulted in a 45.1% inhibition rate of increased melanin production, also exhibiting a synergistic effect higher than the Colby prediction of 27.1%. Figure 7 ).

[0378] Colby's formula: E = (X + Y) - (X × Y / 100)

[0379] Example 4: Human applicability trial to confirm the degree of improvement in diffuse melasma relative to vitamin C

[0380] The aim was to confirm whether the combination obtained through Example 3, which demonstrated a synergistic effect of reducing melanin to improve scattered dark spots in the skin, also showed excellent efficacy in humans.

[0381] 4-1. Selecting Subjects

[0382] Following approval by the Bioethics Committee (Institutional Review Board, IRB, Korea) (LG-SKEV-2022-09-A), the trial was conducted in accordance with the standard operating guidelines and clinical trial standards of the Bioethics Committee of LG Household & Health Corporation, and 10 healthy subjects with no major abnormalities in skin appearance among adults aged 20 to 50 were selected.

[0383] 4-2. Exclusion criteria for subjects

[0384] Among the above subjects, those who 1) suffer from eczema / infectious skin diseases, 2) have allergic constitution or allergies, 3) are pregnant or lactating, 4) have obvious nutritional deficiencies, 5) are poisoned by drugs or alcohol, 6) have moles, acne, tattoos, erythema, burn marks, etc. at the test site, 7) have not been in the same test for more than 1 month, and 8) have hypertension or diabetes are excluded from the subjects.

[0385] 4-3. Instruments and methods for measuring and analyzing scattered dark spots on the skin

[0386] The control group received an 8% Vitamin C serum, applied twice daily (morning and evening) to the right cheek and under the eyes for four weeks. The experimental group received a serum containing 0.5% bisabolol (Alpha-Melight), 2% nutmeg extract (Nutmeg Extract L1), and 1% DIPA (CRODAMOL DA-LQ), applied twice daily (morning and evening) to the left cheek and under the eyes.

[0387] Facial skin was photographed from the front and side using a PIE Janus3 camera under normal light, polarized light, and ultraviolet light. Pigmentation values ​​under polarized light were obtained by specifying areas under the eyes and cheeks. The reduction rate of pigmentation values ​​after 4 weeks of use was calculated compared to the pre-use polarized pigmentation values, thus determining the pigmentation improvement rate (%).

[0388] 4-4. Confirm the degree of improvement in scattered dark spots on the skin relative to vitamin C.

[0389] The three substances of the present invention (bisabolol, myristica extract, and DIPA), which have the effect of inhibiting melanin production to improve scattered dark spots of the skin, obtained through Example 3, were mixed and applied to the left side of the face, while the control group essence (vitamin C 8%) was applied to the right side of the face.

[0390] The results confirmed that after 4 weeks of application, compared with vitamin C, the application of a serum containing bisabolol, nutmeg extract, and DIPA significantly increased the rate of improvement in pigmentation. (The improvement rate increased by approximately 14%, p-value = 0.03, Student's t-test) Figure 8 ).

[0391] Example 5: Protein of dull skin material used to confirm the degree of improvement in dull skin tone relative to vitamin C Confirmation experiment on carbonylation inhibition

[0392] Existing research indicates that dullness and yellowing of the skin are promoted by protein carbonylation in keratinocytes. Therefore, this study used a protein carbonylation assay to confirm whether customized materials for dull skin tone could also improve dullness and yellowing of the skin.

[0393] Human keratinocytes (HaCaT) were seeded at 1 × 10^4 in black clear-bottom 96-well plates. After 24 hours, customized materials were treated according to concentration. After another 24 hours, the cells were irradiated with UVA at 10 J / cm, washed with PBS, and then incubated with fluorescein-5-thioaminothiourea in MES buffer at RT for 1 hour. The cells were washed again with PBS, and fluorescence measurements were performed using a plate reader at an excitation wavelength of 492 nm and an emission wavelength of 562 nm. Furthermore, the reduction in protein carbonylation of other active ingredients was measured against the UVA-treated group alone.

[0394] As a result, when γ-oryzanol, Aspergillus ferment broth, and isostearic acid, which were used as the above materials, were treated at 2.5 ppm, 1%, and 50 ppm, respectively, protein carbonylation was significantly reduced by 39%, 43%, and 31%, respectively. Figure 9 ).

[0395] Example 6: Human-appropriate formulation for developing customized pigmentation improvement formulas for high-risk groups with scattered melasma. Experiment

[0396] Example 2 screened for substances that modulate the expression of significant polymorphic markers associated with diffuse melasma, dull skin tone, and / or freckle-like pigmentation. Therefore, it is sought to provide substances that can improve the efficiency of diffuse melasma improvement based on the risk level of diffuse melasma, dull skin tone, and / or freckle-like pigmentation.

[0397] 6-1. Selecting Subjects

[0398] Following approval by the Bioethics Committee (Institutional Review Board, IRB, Korea) (LG-SKEV-2022-09-A), the trial was conducted in accordance with the standard operating guidelines and clinical trial standards of the Bioethics Committee of LG Household & Health Corporation, and 10 healthy subjects with no major abnormalities in skin appearance among adults aged 20 to 50 were selected.

[0399] Based on the presence or absence of associated gene polymorphic markers for scattered melasma in Table 1, subjects were classified into low-risk and high-risk groups according to the risk level of developing scattered melasma, as shown below.

[0400] Risk scores (the sum of genotype and effect size (β)) of the six gene markers less than 0.28 indicate a low-risk group (n=5).

[0401] A risk score (the sum of the product of genotype and effect size (β)) of 0.28 or higher for the six gene markers indicates a high-risk group (n=5).

[0402] 6-2. Exclusion criteria for subjects

[0403] Same as Example 4-2

[0404] 6-3. Instruments and methods for measuring and analyzing scattered dark spots on the skin

[0405] The control group received an 8% Vitamin C serum, applied twice daily (morning and evening) to the right cheek and under the eyes for four weeks. The experimental group received a serum containing 0.5% bisabolol (Alpha-Melight), 5% nutmeg extract (Nutmeg Extract L1), and 2% niacinamide, applied twice daily (morning and evening) to the left cheek and under the eyes.

[0406] Facial skin was photographed from the front and side using a PIE Janus3 camera under normal light, polarized light, and ultraviolet light. Pigmentation values ​​under polarized light were obtained by specifying areas under the eyes and cheeks. The reduction rate of pigmentation values ​​after 4 weeks of use was calculated compared to the pre-use polarized pigmentation values, thus determining the pigmentation improvement rate (%).

[0407] 6-4. Confirm the pigmentation improvement rate of the substance treatment according to the present invention.

[0408] When the serum containing the three substances of this invention (bisabolol, nutmeg extract, and niacinamide) (customized formula) was applied, the improvement rate of pigmentation in the high-risk group was significantly better than that in the low-risk group (approximately 13.9%, p-value 0.03, Student's t-test). Unlike the customized formula, vitamin C alone did not improve pigmentation in either the low-risk or high-risk groups. While the customized formula did not significantly increase the improvement rate of pigmentation in the low-risk group, it did significantly increase the improvement rate of pigmentation in the high-risk group (an increase of approximately 24%, p-value 0.03, Student's t-test).

[0409] Therefore, it can be seen that the responsiveness to a substance varies depending on whether the individual possesses the allele of a specific gene SNP. Furthermore, it can be seen that providing customized formulations to high-risk groups with a higher risk level of disseminated melasma can improve the efficiency of disseminated melasma treatment. Figure 10 and Figure 11 ).

[0410] Example 7: Human applicability trial for developing a customized pigmentation improvement formula for a high-risk group with dull skin tone

[0411] Example 2 screened for substances that modulate the expression of significant polymorphic markers associated with dull skin tone. Therefore, it is sought to provide substances that can improve the efficiency of dull skin tone improvement based on the risk level of dull skin tone occurrence.

[0412] 7-1. Selecting Subjects

[0413] Following approval by the Bioethics Committee (Institutional Review Board, IRB, Korea) (LG-SKEV-2022-09-A), the trial was conducted in accordance with the standard operating guidelines and clinical trial standards of the Bioethics Committee of LG Household & Health Corporation, and nine healthy subjects aged 20 to 50 with no major abnormalities in their skin appearance were selected.

[0414] Based on the skin color brightness-related gene polymorphism markers in Table 2, the subjects were classified into low-risk and high-risk groups according to the risk level of developing dull skin color, as shown below.

[0415] Risk scores for the five gene markers (the sum of the product of genotype and effect size (β)) less than -0.193 indicate a low-risk group (n=4).

[0416] A risk score (the sum of the product of genotype and effect size (β)) of -0.193 or higher for the five gene markers indicates a high-risk group (n=5).

[0417] 7-2. Exclusion criteria for subjects

[0418] Same as Example 4-2

[0419] 7-3. Skin tone brightness measurement instruments and measurement / analysis methods

[0420] The control group received an 8% Vitamin C serum, applied twice daily (morning and evening) to the right cheek and under the eyes for four weeks. The experimental group received a serum containing 1% isostearic acid, 1% sunflower seed oil, and 0.2% polygalactoside, applied twice daily (morning and evening) to the left cheek and under the eyes.

[0421] Facial skin was photographed from the front and side using a PIE Janus3 camera under normal light, polarized light, and ultraviolet light. Skin tone brightness values ​​under normal light were obtained by specifying areas under the eyes and cheeks. The increase in skin tone brightness value (ΔL) after 4 weeks of use compared to the initial value was calculated to determine the degree of improvement in pigmentation.

[0422] 7-4. Confirm the pigmentation improvement rate of the substance treatment according to the present invention.

[0423] When the serum containing the three substances of this invention (isostearic acid, sunflower seed oil, and polygalactoside) (customized formula) was applied, the improvement rate of pigmentation in the high-risk group was significantly better than that in the low-risk group (ΔL increased by approximately 1.3, p-value 0.01, Student's t-test). Vitamin C alone, unlike the customized formula, showed a slight improvement in the low-risk group, but no improvement in pigmentation in the high-risk group. While the customized formula did not significantly increase the improvement rate of pigmentation in the low-risk group, it significantly increased the improvement rate of pigmentation in the high-risk group (ΔL increased by approximately 2.1, p-value 0.03, Student's t-test).

[0424] Therefore, it can be seen that the reactivity to a substance varies depending on whether the individual possesses the allele of a specific gene SNP. Furthermore, it can be understood that providing customized formulations to high-risk groups with a higher risk of developing dull skin can improve the efficiency of dull skin improvement. Figure 12 , Figure 13 ).

[0425] Example 8: For developing customized pigmentation improvement formulas for high-risk groups with melasma. In vivo applicability test

[0426] Example 2 screened for substances that modulate the expression of significant polymorphic markers associated with speckled pigmentation. Therefore, it is sought to provide substances that can improve the efficiency of speckled pigmentation improvement based on the risk level of speckled pigmentation occurrence.

[0427] 4-1. Selecting Subjects

[0428] Following approval by the Bioethics Committee (Institutional Review Board, IRB, Korea) (LG-SKEV-2022-09-A), the trial was conducted in accordance with the standard operating guidelines and clinical trial standards of the Bioethics Committee of LG Household & Health Corporation, and nine healthy subjects aged 20 to 50 with no major abnormalities in their skin appearance were selected.

[0429] Based on the presence or absence of gene polymorphic markers associated with freckle pigmentation in Table 3, the subjects were classified into low-risk and high-risk groups according to the risk level of freckle pigmentation, as shown below.

[0430] The sum of the minor allele effect sizes (β) of the three gene markers is below -0.009 → low-risk group (n=5)

[0431] The sum of the minor allele effect sizes (β) of the three gene markers is greater than 0.000 → high-risk group (n=4)

[0432] 4-2. Exclusion criteria for subjects

[0433] Same as Example 4-2

[0434] 4-3. Instruments and methods for measuring and analyzing skin pigmentation (including spot-type pigmentation)

[0435] The experimental group's serum consisted of 0.2% tocopherol (raw material name: COVIOX-70), 0.9% hexylresorcinol (raw material name: hexylresorcinol), and 5% hexyldecanol (raw material name: hexyldecanol), which was applied to the affected areas twice daily, morning and evening.

[0436] Facial skin was photographed from the front and side using a PIE Janus3 camera under normal light, polarized light, and ultraviolet light. In the side photos with freckles, areas near the freckles were identified, and AI analysis was used to confirm the degree of freckle recognition and to confirm the decrease in freckle recognition caused by the use of the experimental serum.

[0437] 4-4. Confirmation of improved AI pigmentation recognition based on the material treatment according to the present invention.

[0438] The three substances of the present invention (tocopherol, hexylresorcinol and hexyldecyl alcohol) that have the effect of inhibiting melanin production, obtained through Example 3, are mixed and applied to the spots.

[0439] like Figure 14 and Figure 15As shown, after 8 weeks of applying the serum to the experimental group, the AI ​​significantly improved the recognition of spots in the high-risk group compared to the low-risk group (the AI's spot recognition improvement rate increased by approximately 53% compared to the low-risk group, p-value below 0.01, Student's t-test).

[0440] Based on the foregoing description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering the technical concept or essential features of the invention. Relatedly, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the invention should be interpreted to include the meaning and scope of the following claims and all modifications or variations derived from their equivalents, and should not be construed as including only the detailed description above.

Claims

1. A composition for improving skin pigmentation, comprising one or more substances selected from the group consisting of: Myristica Fragrans (Nutmeg) Extract, Polygonum Cuspidatum Extract, Bisabolol, Diisopropyl Adipate (DIPA), Cysteine, Cysteine ​​Hydrochloride, Sodium Hyaluronate, Hyaluronic Acid, Niacinamide, Glycolic Acid, Gluconolactone, Salicylic Acid, Polydatin, Ferulic Acid, Bakuchiol, Genistein, Oil-soluble Licorice Extract, Aspergillus Fermentation Product. Fermented, quercetin, tannic acid, zinc PCA (ZnPCA), gamma oryzanol, chrysin, tranexamic acid, sunflower oil, isostearic acid, ascorbyl glucoside, 3-O-Ethyl ascorbic acid, ascorbic acid, licorice extract, tocopherol, sodium pyruvate, ecklonia cava extract, hexyldecanol, Asiaticoside, and hexylresorcinol.

2. The composition according to claim 1, wherein, The aforementioned skin pigmentation includes scattered dark spots, dull skin tone, or freckle-like pigmentation.

3. A composition for improving dispersible dark spots on the skin, comprising one or more substances selected from the group consisting of: Myristica Fragrans (Nutmeg) Extract, Polygonum Cuspidatum Extract, Bisabolol, Diisopropyl Adipate (DIPA), Cysteine, Cysteine ​​Hydrochloride, Sodium Hyaluronate, Hyaluronic Acid, Niacinamide, Glycolic Acid, Gluconolactone, and Salicylic Acid.

4. The composition according to claim 3, wherein, The above composition contains the following substances as active ingredients: (i) Cysteine, cysteine ​​hydrochloride, myristica extract, high molecular weight hyaluronic acid and ultra-low molecular weight hyaluronic acid; (ii) Bisabolol, Polygonum cuspidatum root extract, and diisopropyl adipate; or (iii) Nicotinamide, glycolic acid, gluconolactone and salicylic acid.

5. The composition according to claim 3, characterized in that, Based on the total weight of the composition, the content of the above substances is from 0.0001 to 10 by weight.

6. The composition according to claim 3, characterized in that, The above composition reduces melanin production.

7. The composition according to claim 6, wherein, The substances used to reduce melanin production mentioned above are bisabolol, myristica extract, or diisopropyl adipate.

8. The composition according to claim 3, characterized in that, The aforementioned substances inhibit the expression of AKAP1 or promote the expression of RAB11FIP2 or PPARGC1B.

9. The composition according to claim 8, wherein, Substances used to inhibit AKAP1 expression include cysteine, cysteine ​​hydrochloride, myristica extract, high molecular weight hyaluronic acid, or ultra-low molecular weight hyaluronic acid.

10. The composition according to claim 8, wherein, The substances used to promote RAB11FIP2 expression are bisabolol, Polygonum cuspidatum root extract, or diisopropyl adipate.

11. The composition according to claim 8, wherein, Substances used to promote PPARGC1B expression include nicotinamide, glycolic acid, gluconolactone, or salicylic acid.

12. The composition according to claim 3, wherein, The above composition is a customized product designed for individuals who have one or more single nucleotide polymorphism markers in the genes encoding proteins of AKAP1, RAB11FIP2, PPARGC1B, BNC2, MC1R, or MITF.

13. The composition according to claim 12, wherein, The single nucleotide polymorphism markers mentioned above are selected from any one or more of the group consisting of rs11079281, rs10444110, rs251466, rs16935073, rs2228479 and rs7430957.

14. The composition according to claim 13, wherein, The single nucleotide polymorphism markers used for the diagnosis of the aforementioned diffuse melasma are composed of one or more polynucleotides selected from the group consisting of: [list of polynucleotides] and their complementary polynucleotides, wherein the polynucleotides are: A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 55,231,787th base of human chromosome 17, wherein the 55,231,787th base is either T or C (rs11079281). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 119,563,401st base of human chromosome 10, wherein the 119,563,401st base is either T or C (rs10444110). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 149,195,603rd base of human chromosome 5, wherein the 149,195,603rd base is C or G (rs251466). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 16,795,790th base of human chromosome 9, wherein the 16,795,790th base is either A or C (rs16935073). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 89,985,940th base of human chromosome 16, wherein the 89,985,940th base is either G or A (rs2228479). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 69,978,466th base of human chromosome 3, wherein the 69,978,466th base is either A or G (rs7430957).

15. A method for providing information on selecting a custom-made substance for improving scattered dark spots on the skin, comprising the following steps: (a) Obtaining biological samples from individuals; (b) Amplify or hybridize the polymorphic sites of any one or more single nucleotide polymorphism markers selected from the group consisting of rs11079281, rs10444110, rs251466, rs16935073, rs2228479, and rs7430957 from the above biological samples; and (c) Confirm the bases of the polymorphic sites amplified or hybridized in step (b) above.

16. The information providing method according to claim 15, wherein, The above-mentioned substances are selected from one or more of the following groups: nutmeg extract, Polygonum cuspidatum root extract, bisabolol, diisopropyl adipate, cysteine, cysteine ​​hydrochloride, high molecular weight hyaluronic acid, ultra-low molecular weight hyaluronic acid, nicotinamide, glycolic acid, gluconolactone, and salicylic acid.

17. The information providing method according to claim 15, wherein, When the risk score obtained by multiplying and summing the genotypes of rs11079281, rs10444110, rs251466, rs16935073, rs2228479, and rs7430957 by their effect size (β) is less than 0.28, the risk of developing diffuse melasma is classified as low-risk. When the risk score is greater than 0.28, the risk of developing diffuse melasma is classified as high-risk.

18. The information providing method according to claim 17, wherein, In cases where the risk of developing scattered melasma is classified as high, a customized formulation of the substance will be prescribed.

19. The information providing method according to claim 18, wherein, The substances mentioned above are bisabolol, myristica extract, and nicotinamide.

20. The information providing method according to claim 15, wherein, The above-mentioned samples are hair, urine, blood, various body fluids, isolated tissues, isolated cells, or saliva.

21. The information providing method according to claim 15, wherein, The amplification and confirmation of the aforementioned polymorphic sites were performed using SNP chips.

22. A composition for improving dull skin tone, comprising one or more substances selected from the group consisting of polydatin, ferulic acid, bakuchiol, genistein, oil-soluble licorice extract, Aspergillus ferment, quercetin, tannic acid, zinc PCA (ZnPCA), gamma oryzanol, chrysin, tranexamic acid, sunflower oil, and isostearic acid as active ingredients.

23. The composition according to claim 22, wherein, The above composition contains the following as active ingredients: (i) Ferulic acid, Polygonum cuspidatum glycoside, zinc PCA, tranexamic acid, quercetin, genistein, tannic acid, Aspergillus ferment, γ-oryzanol, sunflower seed oil and isostearic acid; (ii) Ferulic acid, Polygonum cuspidatum glycoside, Psoralen, Gentiana tinctoria, oil-soluble licorice extract and Aspergillus ferment; (iii) Ferulic acid, quercetin, genistein, tannic acid, polygalactoside, zinc PCA, and γ-oryzanol; or (iv) Glutamin, tannic acid and zinc PCA.

24. The composition according to claim 22, characterized in that, Based on the total weight of the composition, the content of the above substances is from 0.0001 to 10 by weight.

25. The composition according to claim 22, characterized in that, The above composition reduces melanin production.

26. The composition according to claim 25, wherein, The substances used to reduce melanin production mentioned above are i) ferulic acid, oil-soluble licorice extract, and psoralen; or ii) polygalactoside and quercetin.

27. The composition according to claim 22, characterized in that, The above composition reduces protein carbonylation.

28. The composition according to claim 27, wherein, The substances used to reduce protein carbonylation mentioned above are γ-oryzanol, Aspergillus ferment, or isostearic acid.

29. The composition according to claim 22, characterized in that, The above substances inhibit the expression of OCA2, BNC2, RAB32, or KITLG.

30. The composition according to claim 29, wherein, Substances used to inhibit OCA2 expression include ferulic acid, polygalactoside, zinc PCA, tranexamic acid, quercetin, genistein, tannic acid, Aspergillus ferment, γ-oryzanol, sunflower seed oil, or isostearic acid.

31. The composition according to claim 29, wherein, Substances used to inhibit BNC2 expression include polygalactoside, ferulic acid, psoralen, genistein, oil-soluble licorice extract, or Aspergillus ferment.

32. The composition according to claim 29, wherein, Substances used to inhibit RAB32 expression include ferulic acid, quercetin, genistein, tannic acid, polygalactoside, zinc PCA, or γ-oryzanol.

33. The composition according to claim 29, wherein, The substances used to inhibit KITLG expression are apigenin, tannic acid, or zinc PCA.

34. The composition according to claim 22, wherein, The above composition is a customized design for individuals who have one or more single nucleotide polymorphism markers present in genes encoding proteins of OCA2, BNC2, RAB32, KITLG, or ASIP.

35. The composition according to claim 34, wherein, The single nucleotide polymorphism markers mentioned above are selected from any one or more of the group consisting of rs74653330, rs16935073, rs72620727, rs77310623 and rs6059711.

36. The composition according to claim 35, wherein, The single nucleotide polymorphism markers used for diagnosing dull skin tone described above consist of one or more polynucleotides selected from the group consisting of: [list of polynucleotides], and their complementary polynucleotides. The polynucleotides are: A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 28,228,553rd base of human chromosome 15, wherein the 28,228,553rd base is either T or C (rs74653330). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 16,795,790th base of human chromosome 9, wherein the 16,795,790th base is either C or A (rs16935073). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 89110410th base of human chromosome 12, wherein the 89110410th base is either C or A (rs72620727). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 146,871,819th base of human chromosome 6, wherein the 146,871,819th base is either A or G (rs77310623). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 32,780,822nd base of human chromosome 20, where the 32,780,822nd base is either T or C (rs6059711).

37. A method for providing information on selecting a custom-made substance for improving dull skin tone, comprising the following steps: (a) Obtaining biological samples from individuals; (b) Amplify or hybridize the polymorphic sites of any one or more single nucleotide polymorphism markers selected from the group consisting of rs74653330, rs16935073, rs72620727, rs77310623, and rs6059711 in the above biological samples with the probe; and (c) Confirm the bases of the polymorphic sites amplified or hybridized in step (b) above.

38. The information providing method according to claim 37, wherein, The aforementioned substances are selected from one or more of the following groups: polygalactoside, ferulic acid, psoralen, genistein, oil-soluble licorice extract, Aspergillus ferment, quercetin, tannic acid, zinc PCA, γ-oryzanol, succinate, tranexamic acid, sunflower seed oil, and isostearic acid.

39. The information providing method according to claim 37, wherein, If the risk score obtained by multiplying and summing the genotypes of rs74653330, rs16935073, rs72620727, rs77310623, and rs6059711 by their effect size (β) is less than -0.193, the risk of developing dull skin is classified as low risk; if the risk score is greater than -0.193, the risk of developing dull skin is classified as high risk.

40. The information providing method according to claim 39, wherein, In cases where the risk of developing dull skin tone is considered high, a customized formula of the substance will be prescribed.

41. The information providing method according to claim 40, wherein, The substances mentioned above are isostearic acid, sunflower seed oil, and polygalactoside.

42. The information providing method according to claim 37, wherein, The above-mentioned samples are hair, urine, blood, various body fluids, isolated tissues, isolated cells, or saliva.

43. The information providing method according to claim 37, wherein, The amplification and confirmation of the aforementioned polymorphic sites were performed using SNP chips.

44. A composition for improving spot-type pigmentation, comprising one or more substances selected from the group consisting of ascorbyl glucoside, 3-O-Ethyl ascorbic acid, ascorbic acid, licorice extract, tocopherol, sodium pyruvate, kelp extract, hexyldecanol, Asiaticoside, and hexylresorcinol as active ingredients.

45. The composition according to claim 44, wherein, The above composition contains the following as active ingredients: (i) Tocopherol, licorice extract and glycoside extract; (ii) Sodium pyruvate, hexyldecyl alcohol, asiaticoside, and hexylresorcinol; or (iii) Ascorbate glucoside, 3-O-ethyl ascorbic acid and ascorbic acid.

46. ​​The composition according to claim 44, characterized in that, Based on the total weight of the composition, the content of the above substances is from 0.0001 to 10 by weight.

47. The composition according to claim 44, wherein, The above-mentioned freckle-type pigmentation is selected from one or more of the following groups: senile lentigines, age spots, solar lentigines, melanocytic lentigines, and moles.

48. The composition according to claim 44, characterized in that, The above composition reduces melanin production.

49. The composition according to claim 48, wherein, The substances used to reduce melanin production mentioned above are i) hexylresorcinol and hexyldecyl alcohol; or ii) hexyldecyl alcohol and tocopherol.

50. The composition according to claim 44, characterized in that, The above substances inhibit the expression of ASCL2 or TP63, or promote the expression of PPARGC1B.

51. The composition according to claim 50, wherein, The substances used to inhibit ASCL2 expression are tocopherol, licorice extract, or glycoside extract.

52. The composition according to claim 50, wherein, The substances used to inhibit TP63 expression are sodium pyruvate, hexyldecyl alcohol, asiaticoside, or hexylresorcinol.

53. The composition according to claim 50, wherein, The substances used to promote PPARGC1B expression are ascorbate glucoside, 3-O-ethyl ascorbic acid, or ascorbic acid.

54. The composition according to claim 44, wherein, The above composition is a customized design for individuals who have one or more single nucleotide polymorphism markers present in genes encoding proteins of ASCL2, TP63, or PPARGC1B.

55. The composition according to claim 54, wherein, The single nucleotide polymorphism markers mentioned above are selected from any one or more of the group consisting of rs251466, rs12222609 and rs9865771.

56. The composition according to claim 55, wherein, The single nucleotide polymorphism markers used for the diagnosis of the above-mentioned lentigines consist of one or more polynucleotides selected from the group consisting of the following polynucleotides and their complementary polynucleotides, wherein the polynucleotides are: A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 149,195,603rd base of human chromosome 5, wherein the 149,195,603rd base is C or G (rs251466). A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 2297093rd base of human chromosome 11, wherein the 2297093rd base is C or T (rs12222609); A polynucleotide consisting of 5-100 consecutive DNA sequences containing the 189,491,453rd base of human chromosome 3, wherein the 189,491,453rd base is either A or G (rs9865771).

57. A method for providing information on selecting a custom-made substance for improving spot-type hyperpigmentation, comprising the following steps: (a) Obtaining biological samples from individuals; (b) Amplify or hybridize the polymorphic sites of any one or more single nucleotide polymorphism markers selected from the group consisting of rs251466, rs12222609, and rs9865771 in the above biological samples with the probe; and (c) Confirm the bases of the polymorphic sites amplified or hybridized in step (b) above.

58. The information providing method according to claim 57, wherein, The above-mentioned substances are selected from one or more of the group consisting of ascorbate glucoside, 3-O-ethyl ascorbic acid, ascorbic acid, licorice extract, tocopherol, sodium pyruvate, glycoside extract, hexyldecyl alcohol, asiaticoside and hexylresorcinol.

59. The information providing method according to claim 57, wherein, When the risk score obtained by multiplying and summing the genotypes of rs251466, rs12222609, and rs9865771 by the effect size (β) is below -0.009, the risk of freckle pigmentation is classified as low risk; when the risk score is above 0.000, the risk of freckle pigmentation is classified as high risk.

60. The information providing method according to claim 59, wherein, In cases where the risk of developing spot-type pigmentation is classified as high, a custom-made substance formulation will be prescribed.

61. The information providing method according to claim 60, wherein, The substances mentioned above are tocopherol, hexylresorcinol, and hexyldecyl alcohol.

62. The information providing method according to claim 57, wherein, The above-mentioned samples are hair, urine, blood, various body fluids, isolated tissues, isolated cells, or saliva.

63. The information providing method according to claim 57, wherein, The amplification and confirmation of the aforementioned polymorphic sites were performed using SNP chips.

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