Use of o-tolylbiguanide in the preparation of skin lightening products, lightening products and methods of formulation

Abstract

The application discloses application of o-tolyl biguanide in preparation of skin whitening products, a whitening product and a preparation method, and belongs to the technical field of skin care. It is found for the first time that o-tolyl biguanide can effectively inhibit generation of late glycosylation end product carboxymethyl lysine (CML), lipofuscin and melanin when serving as a whitening active ingredient, thereby achieving a multi-pathway whitening effect. The application further provides a whitening product which comprises o-tolyl biguanide and an anionic surfactant with similar electric charge quantity; the o-tolyl biguanide is positively charged, and the anionic surfactant is negatively charged; the two can form a complex without net electric charge or with weak net electric charge, thereby effectively improving stability of the o-tolyl biguanide in the whitening product system, avoiding irreversible aggregation, flocculation or precipitation phenomenon with commonly used thickening agents such as carbomer, and improving formulation compatibility and stability of the o-tolyl biguanide.

Description

Technical Field

[0001] This invention relates to the field of skin care technology, and in particular to the application of o-tolyl biguanide in the preparation of skin whitening products, whitening products and their preparation methods. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Evenness of skin tone is a key factor influencing the apparent age of the skin, while age spots and pigmentation are the main causes of uneven skin tone. With age and increased UV exposure, the skin experiences increased darkness and yellowing, accompanied by the formation of various age spots. These changes directly affect the skin's visual youthfulness. Currently, most mainstream whitening active ingredients work by inhibiting tyrosinase activity to block melanin production. While these ingredients can reduce melanin synthesis to some extent, their effectiveness in removing stubborn melanin already formed in the skin is limited, especially in targeting pigment particles deposited in the dermis.

[0004] Skin pigmentation problems have diverse causes. Besides melanin, lipofuscin and glycated pigments are also important factors leading to dull and sallow skin. Lipofuscin, a persistent pigment particle formed by the oxidation and accumulation of cellular metabolic waste, accumulates more rapidly in the deeper layers of the skin with age, causing the skin to lose its translucency. Glycated pigments, on the other hand, are advanced glycation end products formed by non-enzymatic reactions between sugars and skin proteins. They not only have a brownish-yellow color themselves but also indirectly exacerbate pigmentation by promoting oxidative stress and inflammation. Existing whitening ingredients lack specific targeting for these two types of pigments produced through non-tyrosinase pathways, resulting in whitening solutions that only target melanin being less effective in improving the yellowing and dullness common in Asian skin types.

[0005] Furthermore, current solutions for various pigmentation problems often rely on the combination of multiple ingredients to achieve comprehensive improvement by combining active substances targeting different points. This approach not only increases the complexity of formulation design but may also raise raw material management and production costs, while making it difficult to ensure the synergistic stability between the ingredients. In terms of simultaneously regulating melanin, lipofuscin, and glycated pigments with a single component, existing technologies still have significant gaps, lacking effective means to simplify formulations and achieve multi-dimensional skin tone improvement. Summary of the Invention

[0006] In view of this, the present invention provides the application of o-tolyl biguanide in the preparation of skin whitening products, whitening products and preparation methods. The present invention finds that o-tolyl biguanide can significantly reduce the levels of melanin, glycated pigments and lipofuscin, thereby effectively improving skin brightness, lightening skin tone and achieving a good whitening effect.

[0007] In a first aspect, the present invention provides the application of o-tolyl biguanide in the preparation of skin whitening products; wherein o-tolyl biguanide is used as a whitening active ingredient in the skin whitening products.

[0008] Preferably, the skin whitening product is used to prevent, alleviate and / or reduce symptoms associated with hyperpigmentation.

[0009] Furthermore, symptoms associated with hyperpigmentation include freckles, sunspots, age spots, melasma, or post-inflammatory hyperpigmentation.

[0010] Furthermore, the pigments include melanin, lipofuscin, and glycated pigments.

[0011] Preferably, the concentration of o-tolyl biguanide in the skin whitening product is 0.01~2.0 wt%.

[0012] Secondly, the present invention provides a skin whitening product, comprising at least o-tolyl biguanide, an anionic surfactant, a thickener and water; wherein the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolyl biguanide is (0.9~1.2):1.

[0013] Preferably, the mass fraction of the o-tolyl biguanide is 0.01~2.0 wt%; the anionic surfactant is an anionic surfactant in its unsalted protonated form.

[0014] Preferably, the thickener is one or more of polyacrylic acid thickeners, sodium carboxymethyl cellulose, or sodium alginate.

[0015] Furthermore, the thickener is carbomer, and the mass fraction of the thickener is 0.1~3wt%.

[0016] Thirdly, the present invention provides a method for preparing the above-mentioned whitening product, comprising the following steps:

[0017] o-Tolyl Biguanide and anionic surfactant are pre-mixed and emulsified in an aqueous phase to form a pre-dispersion;

[0018] The thickener is stirred in water to swell or dissolve; then the pre-dispersed material is added under stirring conditions, stirred until well mixed, and allowed to stand to obtain the final product.

[0019] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0020] (1) This invention first discovered that o-tolyl biguanide, when used as a whitening active ingredient, can effectively inhibit the production of advanced glycation end products carboxymethyl lysine (CML), lipofuscin and melanin, thereby achieving a multi-pathway whitening effect.

[0021] (2) The present invention provides a whitening product comprising o-tolyl biguanide and an anionic surfactant. The o-tolyl biguanide carries a positive charge, and the anionic surfactant carries a negative charge. When the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to that of o-tolyl biguanide is (0.9~1.2):1, the two can form a complex with no net charge or a weak net charge, thereby effectively improving the stability of o-tolyl biguanide in the whitening product system, avoiding irreversible aggregation, flocculation or precipitation, and improving the formulation compatibility and stability of o-tolyl biguanide. Attached Figure Description

[0022] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0023] Figure 1 These are appearance images of three parallel samples (labeled 1#, 2# and 3#) from different treatment groups in Embodiment 1 of the present invention;

[0024] Figure 2 These are fluorescence imaging images of carboxymethyl lysine from three parallel samples (labeled 1#, 2# and 3#) in different treatment groups in Example 1 of the present invention.

[0025] Figure 3 These are photographs of melanin distribution in three parallel samples (labeled 1#, 2# and 3#) from different treatment groups in Example 1 of the present invention.

[0026] Figure 4 These are lipofuscin distribution photographs of three parallel samples (labeled 1#, 2# and 3#) from different treatment groups in Example 1 of the present invention. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] In this invention, the terms "comprising," "including," "having," "containing," and any other variations thereof are all non-exclusive inclusions. For example, a composition, step, method, article, or apparatus that includes the listed elements is not limited to including only these elements, but may also include other elements not expressly listed, or elements inherent to such compositions, steps, methods, articles, or apparatus.

[0029] In this invention, "and / or" is used to indicate that one or both of the described situations may occur. For example, "A and / or B" includes "A and B", "A" or "B".

[0030] In this invention, when mass fraction, concentration, temperature, or other values ​​or parameters are expressed as ranges, preferred ranges, or ranges defined by a series of upper and lower preferred values, it should be understood that all combinations of ranges formed by any pairing of upper (or preferred) and lower (or preferred) values ​​are included, regardless of whether the range is listed separately. For example, when the range "1~5" is disclosed, the described range should be interpreted as including ranges "1~4", "1~3", "1~2", "1~2 and 4~5", "1~3 and 5", etc. When numerical ranges are described in this invention, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0031] In this invention, the term "inhibition" may be used interchangeably with "reduction," "lowering," "downregulation," and other similar terms, and includes any level of inhibition. Inhibition can be assessed by a reduction in the absolute or relative level of one or more variables compared to a control level. This control level can be any type of control level used in the art, such as a baseline level before administration or a level determined from an untreated or controlled (e.g., a buffer-only control or an inert agent control) subject, cell, or sample.

[0032] In this invention, unless otherwise stated, "optional" or "preferred" means that the events or circumstances described below may but do not have to occur, including the situations in which the events or circumstances occur or do not occur.

[0033] This invention provides the application of o-tolyl biguanide in the preparation of skin whitening products; wherein o-tolyl biguanide is used as the whitening active ingredient.

[0034] o-Tolyl Biguanide, also known as 1-o-Tolyl Biguanide, has the CAS number 93-69-6 and its structural formula is shown below:

[0035] .

[0036] The skin whitening products described in this invention preferably include cosmetics and pharmaceuticals. Cosmetics may include serums, face creams, face masks, lotions, toners, sunscreens, facial cleansers, facial soaps, foundations, concealers, or serums, etc.

[0037] In an optional embodiment of the present invention, the skin whitening product is used to prevent, alleviate and / or reduce symptoms associated with hyperpigmentation.

[0038] In optional embodiments of the present invention, symptoms associated with hyperpigmentation include freckles, sunspots, age spots, melasma, or post-inflammatory hyperpigmentation.

[0039] In an optional embodiment of the present invention, the pigments include melanin, lipofuscin, and glycated pigments. The present invention has found that o-tolyl biguanide can simultaneously prevent, alleviate, and / or reduce the accumulation and pigmentation of melanin, lipofuscin, and glycated pigments.

[0040] Melanin is a natural biological pigment synthesized by melanocytes, divided into eumelanin and pheomelanin. Its main function is to absorb ultraviolet rays and protect the DNA of dermal cells from damage. Abnormal synthesis of melanin can lead to problems such as age spots, freckles, and uneven skin tone. Lipofuscin is a brownish-yellow, non-degradable fluorescent pigment particle formed by the oxidation and accumulation of cellular metabolic waste in lysosomes. With age and ultraviolet exposure, it accumulates in the deep epidermis and superficial dermis, and is the core cause of sallow, dull, and less translucent skin. Glycated pigments are pigmentation problems caused by the deposition of advanced glycation end products (AGEs) in the skin. When reducing sugars such as glucose undergo non-enzymatic glycation reactions with collagen and elastin in the skin, AGEs are generated, such as carboxymethyl lysine (CML) and pentasaccharides. These substances are brownish-yellow and not only directly cause skin pigmentation but also indirectly induce pigmentation by exacerbating oxidative stress and inflammatory responses, eventually forming age spots on aging areas such as nasolabial folds and the forehead. This invention is the first to discover that o-tolyl biguanide can achieve a multi-pathway whitening effect by simultaneously inhibiting the production of melanin, lipofuscin, and glycated pigments.

[0041] In an optional embodiment of the present invention, the concentration of o-tolyl biguanide in the skin whitening product is 0.01~2.0wt%, more preferably 0.1~0.5wt%, for example, it can be 0.1wt%, 0.15wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.4wt%, 0.5wt%, etc.

[0042] In optional embodiments of the present invention, the dosage form of the skin whitening product includes pastes, ointments, sprays, gels, liniments, coatings, films, patches, plasters, or films. Those skilled in the art can prepare products with suitable dosage forms as needed.

[0043] In an optional embodiment of the present invention, the skin whitening product further includes excipients, which include one or more of the following: solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, gelling agents, osmotic pressure regulators, stabilizers, flow aids, anti-caking agents, flavoring agents, antibacterial agents, suspending agents, coating agents, film-forming agents, fragrances, preservatives, thickeners, anti-adhesion agents, antioxidants, antioxidant synergists, chelating agents, pH adjusters, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protectants, moisturizers, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, empty capsules, matrix or drug carrier materials.

[0044] In optional embodiments of the present invention, o-tolyl biguanide can be used alone as a whitening active ingredient or in combination with other whitening active ingredients, such as arbutin and kojic acid.

[0045] The present invention also provides a skin whitening product, comprising at least o-tolyl biguanide, an anionic surfactant, a thickener and water; wherein the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolyl biguanide is (0.9~1.2):1.

[0046] In an optional embodiment of the present invention, the mass fraction of the o-tolyl biguanide is 0.01~2.0wt%.

[0047] o-Tolyl biguanide carries a significant positive charge due to the protonation of the guanidinium group in its molecular structure in conventional weakly acidic or neutral aqueous systems. This characteristic leads to strong electrostatic interactions with carbomer-type thickeners commonly used in formulations, which become negatively charged after dissociation in the aqueous phase. These interactions readily induce irreversible aggregation, flocculation, or precipitation, causing severe compatibility problems such as loss of physical stability, inactivation of the active ingredient, or uneven local concentrations, greatly limiting the application of o-tolyl biguanide in formulations containing this type of anionic polymer. It is worth noting that, because this invention limits the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolyl biguanide to (0.9~1.2):1, a charge neutralization effect exists between the anion of the anionic surfactant and the protonated guanidinium group. For example, for anionic surfactants where each molecule carries one negative charge after dissociation, such as palmitic acid, sodium dodecyl sulfate, sodium lauryl ether sulfate, and sodium N-lauroyl sarcosinate, the molar ratio of the anionic surfactant to o-tolyl biguanide is (0.9~1.2):1. For anionic surfactants where each molecule carries multiple negative charges after dissociation, such as potassium hexadecyl phosphate where each molecule carries two negative charges after complete dissociation, the molar ratio of the anionic surfactant to o-tolyl biguanide is calculated to be (0.45~0.6):1. Therefore, the complex formed by the anionic surfactant and o-tolyl biguanide in this invention typically exhibits near-electroneutral or weakly electrostatic characteristics, reducing the possibility of electrostatic interactions with anionic thickeners such as carbomer, and effectively improving the system stability of the final product.

[0048] In this invention, the anionic surfactant is preferably a protonated form of anionic surfactant that has not formed a salt, such as a sulfonic acid anionic surfactant or a carboxylic acid anionic surfactant. More specifically, the protonated form of the anionic surfactant that has not formed a salt includes, but is not limited to, palmitic acid, lauric acid, stearic acid, myristic acid, etc. The acidic group (-COOH) of these surfactants can provide hydrogen ions, which can undergo a protonation reaction with the amino group in the o-tolyl biguanide molecule to form a stable salt compound; at the same time, its hydrophobic segment (alkyl part) can generate a hydrophobic interaction with the phenyl structure of o-tolyl biguanide. This dual effect promotes the formation of a stable dispersion of the two complexes in water.

[0049] In an optional embodiment of the present invention, the thickener is one or more selected from polyacrylic acid thickeners, sodium carboxymethyl cellulose, or sodium alginate. Further, the thickener is carbomer, which belongs to the polyacrylic acid thickener class, and is highly efficient in thickening while being mild and non-irritating. It can also effectively encapsulate active ingredients and prolong the duration of efficacy. The mass fraction of the thickener is 0.1~3wt%, and can be selected according to the specific dosage form and application scenario.

[0050] The whitening products of this invention may further include antioxidants, moisturizers, chelating agents, pH adjusters, etc., to further optimize the stability, safety, and user comfort of the whitening products. This invention does not impose special limitations on the dosage form of the whitening products; for example, they can be pastes, ointments, gels, liniments, coatings, films, patches, plasters, etc.

[0051] The present invention also provides a method for preparing the above-mentioned whitening product, comprising the following steps:

[0052] o-Tolyl Biguanide and anionic surfactant are pre-mixed and emulsified in an aqueous phase to form a pre-dispersion;

[0053] The thickener is stirred in water to swell or dissolve; then the pre-dispersed material is added under stirring conditions, stirred until well mixed, and allowed to stand to obtain the final product.

[0054] In the above-described scheme of the present invention, after the addition of o-tolyl biguanide, the anionic surfactant and o-tolyl biguanide rapidly combine through electrostatic interaction to form an ionic complex or ion pair. This process is carried out under stirring conditions, thereby forming a uniform emulsion. The obtained pre-dispersion tends to be neutral or carries only a weak charge. When it is added to the thickener system, there is no longer a strong electrostatic attraction between the two, effectively avoiding the occurrence of aggregation and precipitation, and successfully solving the compatibility problem.

[0055] This invention does not impose special restrictions on the mixing and emulsification process, and heating and stirring emulsification can be carried out as needed.

[0056] In the preparation of the pre-dispersion, this invention can also add high-melting-point hydrophobic or hydrophobic-group-containing ingredients, such as solid waxes, long-chain fatty alcohols, phospholipids, etc., preferably waxes. The high melting point of the components allows the pre-dispersion to remain in a solid state over a wider temperature range and reduces the escape of o-tolyl biguanide. Waxes can be selected from palm wax, candelilla wax, jojoba wax, Japanese wax, beeswax, etc.

[0057] The technical solution of the present invention will be further described below with reference to specific embodiments. The present invention does not have any special restrictions on the source of reagents used in the following embodiments; commercially available products well known to those skilled in the art can be used. In the following embodiments, the 3D melanin full-thickness skin model (MelaFulKutis™) and model culture medium were provided by the Shaanxi Branch of Guangdong Boxi Biotechnology Co., Ltd.

[0058] In the following examples, AGEs represent advanced glycation end products. MGO represents methylglyoxal, which promotes the formation of AGEs. UV represents ultraviolet light. UVA represents long-wave ultraviolet light, which has strong penetrating power and can reach deep into the dermis, mainly causing skin aging and pigmentation. UVB represents medium-wave ultraviolet light, which has higher energy and mainly acts on the epidermis, and is a major cause of sunburn, erythema, and melanin synthesis. * The value reflects the brightness of skin tone, L * The higher the value, the brighter the skin tone. * The lower the value, the darker the skin tone. * The value reflects the reddish-green tint of skin tone; a positive value indicates a reddish skin tone, and a negative value indicates a greenish skin tone. * The value reflects the yellow-blue tendency of skin tone; a positive value indicates a yellowish skin tone, and a negative value indicates a bluish skin tone. The ITA° value represents the individual type angle; the larger the ITA° value, the lighter the skin tone. CML represents carboxymethyl lysine. DAB represents 3,3'-diaminobenzidine.

[0059] Example 1

[0060] This embodiment provides a test of the inhibitory effect of o-tolyl biguanide on melanin, lipofuscin, and carboxymethyl lysine.

[0061] This embodiment uses the 3D melanin full-thickness skin model (MelaFulKutis™) as a testing tool and employs a surface drug delivery method to construct skin models of different treatment groups and evaluate the in vitro whitening effect of different treatment groups.

[0062] The grouping of different treatment groups is shown in Table 1.

[0063] Table 1 Grouping of different treatment groups

[0064]

[0065] Note: In Table 1, kojic acid is a typical tyrosinase inhibitor that reduces melanin content in the skin model by blocking the melanin synthesis pathway. Aminoguanidine sulfate is a methylglyoxal (MGO) scavenger that binds to MGO via a nucleophilic reaction, blocking its non-enzymatic glycosylation reaction with proteins. TGF-β1 is a key regulator of skin repair, promoting fibroblast proliferation, increasing type I / III collagen synthesis, and inhibiting UV-induced matrix metalloproteinase expression, thereby combating collagen degradation caused by photoaging.

[0066] The specific steps are as follows:

[0067] 1. Preparation before testing: Transfer the 3D melanin full-thickness skin model to a 6-well plate, add 2 mL of model culture medium to each well, and group the models according to the treatment groups in Table 1, with 3 replicates per group.

[0068] 2. Drug administration: The drug administration was carried out according to the concentration of whitening active ingredients in Table 1: (1) BC group: The model culture medium was replaced with fresh model culture medium, and no additional treatment was given. (2) NC group: The model culture medium was replaced with model culture medium containing 3mM MGO. (3) PC1 group: The model culture medium was replaced with model culture medium containing 3mM MGO, and kojic acid working solution was added to the surface. (4) PC2 group: The model culture medium was replaced with model culture medium containing aminoguanidine sulfate and 3mM MGO. (5) PC3 group: The model culture medium was replaced with model culture medium containing TGF-β1 and 3mM MGO. (6) Sample group: The model culture medium was replaced with model culture medium containing 3mM MGO, and the drug administration method was surface administration.

[0069] 3. Combined UVA and UVB irradiation: irradiation dose of 12 J / cm² 2 UVA and 50mJ / cm 2 UVB.

[0070] 4. Model Culture: The model was transferred to a 6-well plate and incubated in a CO2 incubator (37℃, 5% CO2). Groups BC did not receive drug administration or UVA+UVB irradiation, while the other groups received drug administration and UVA+UVB irradiation. The specific steps were as follows: drug administration was performed only on day 1; UVA+UVB irradiation was performed from day 2 to day 8, once a day for a total of 7 times; drug administration was performed after irradiation from day 2 to day 7, once a day; after the last irradiation, the sample was collected, and any remaining test substance was washed with a sterile PBS solution in a wash bottle, and any residual liquid was wiped away with a sterile cotton swab.

[0071] 5. Test Analysis:

[0072] (1) Apparent skin color: The model used for testing was photographed and observed under a stereomicroscope, and the images were collected and analyzed. For example... Figure 1 The images shown are microscope images of groups BC, NC, PC1, PC2, PC3, and the sample group, with three parallel samples in each group (labeled 1#, 2#, and 3#). As can be seen from the images, the skin tone in group NC is darker. Compared to group NC, the skin tones in groups PC1, PC2, PC3, and the sample group are lighter to varying degrees.

[0073] (2) L * a * b * Value detection and ITA° value calculation: After the apparent color detection is completed, the L value is calculated. * value, a * value and b *ITA° value determination and calculation. The specific testing procedure is as follows: Place the model on a flat, hard white surface with the stratum corneum facing upwards. Align the detection aperture of the skin color meter vertically with the model surface to perform the test and read the L value. * value, a * value and b * The value is calculated by taking the reading three times for each model and averaging the results as the reading for a single model; the calculation is performed according to the following formula:

[0074] .

[0075] The data for different treatment groups are summarized in Table 2.

[0076] As can be seen from Table 2, in terms of L, which reflects skin brightness... * In terms of skin tone, the NC group showed a highly significant decrease compared to the BC group. The three positive control groups and the o-tolyl biguanide sample group all showed highly significant increases compared to the NC group, indicating that all four whitening active ingredients (kojic acid, aminoguanidine sulfate, TGF-β1, and o-tolyl biguanide) could significantly improve skin brightness. The a* value reflecting red-green and the b* value reflecting blue-yellow skin tones showed little difference between the groups. Regarding the ITA° value, which reflects skin tone depth, the NC group showed a significant decrease, indicating a darker skin tone. The three positive control groups and the o-tolyl biguanide sample group showed highly significant increases compared to the NC group, indicating that all four whitening active ingredients could significantly reduce skin tone. The o-tolyl biguanide sample group's value was significantly higher than PC1 (kojic acid) and PC2 (aminoguanidine sulfate), and slightly higher than PC3 (TGF-β1), suggesting that o-tolyl biguanide was more effective than kojic acid (a commonly used whitening agent) and aminoguanidine sulfate (a glycation inhibitor) in this model.

[0077] Table 2 L of different treatment groups * a * b * Value detection and ITA° value

[0078]

[0079] Note: In Table 2, results are expressed as mean ± standard deviation. t-tests were used for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant. Compared to group BC, significance is indicated by # (P < 0.05 = #, P < 0.01 = ##); compared to group NC, significance is indicated by * (P < 0.05 = *, P < 0.01 = **); subsequent tables have the same meaning.

[0080] (3) Immunofluorescence assay of carboxymethyl lysine (CML), an advanced glycosylation product: The model used for detection was fixed with 4 wt% paraformaldehyde for 24 h. Immunofluorescence was then performed, and images were taken and analyzed quantitatively under a microscope. The formula for calculating the IOD (Integrated Optical Density) value is as follows:

[0081] IOD = Average optical density × Measurement area.

[0082] Relative IOD value = Sample IOD / Group BC IOD.

[0083] The results are shown in Table 3 and Figure 2 As shown.

[0084] From Table 3 and Figure 2 As can be seen, compared with the BC group, the AGEs (CML) content in the NC group was significantly increased, indicating that the stimulation condition of MGO was effective. Compared with the NC group, the AGEs (CML) content in the PC2 (aminoguanidine sulfate) group was significantly decreased, indicating that the positive control aminoguanidine sulfate was effective in this test; compared with the NC group, the AGEs (CML) content of o-tolyl biguanide in the sample was significantly decreased, with an inhibition rate of 35.89%.

[0085] Table 3. CML test results of advanced glycation end products in different treatment groups

[0086]

[0087] (4) Melanin detection: After fixation with 4wt% paraformaldehyde solution for 24 h, the sections were embedded and stained according to the silver staining kit instructions. Images were then photographed under a microscope, and the images were quantitatively analyzed. The results are summarized in Table 4 and... Figure 3 .

[0088] Table 4. Melanin test results of different treatment groups

[0089]

[0090] Note: In Table 4, the relative area of ​​melanin granules is obtained by the following method: the area of ​​melanin granules per unit area is statistically analyzed, the area of ​​group BC is defined as 1, and the area of ​​other groups is converted based on the area of ​​group BC, which is the relative area of ​​melanin granules.

[0091] As can be seen, compared with the BC group, the number of melanin granules in the NC group increased significantly, indicating that the stimulation conditions in this embodiment were effective. Compared with the NC group, the number of melanin granules in the PC1 (kojic acid) group decreased significantly, indicating that the positive control kojic acid was effective in this test. Compared with the NC group, the number of melanin granules in the o-tolyl biguanide sample group decreased significantly, with an inhibition rate of 34.97%.

[0092] (5) Detection of lipofuscin: The model used for detection was fixed with 4wt% paraformaldehyde for 24 hours, then embedded, sectioned, dewaxed, and hydrated. Blocking was performed using a biotin detection blocking kit. Working solution was prepared with lipofuscin staining agent GL13 and incubated at 37℃ for 10 minutes. Primary antibody was incubated overnight at 4℃, followed by secondary antibody incubation at 37℃ for 1.5 hours. DAB staining was performed for 2-10 minutes, then staining was stopped, counterstained, mounted, and photographed under a microscope. Images were collected and analyzed.

[0093] The formula for calculating the IOD (Integrated Optical Density) value is as follows:

[0094] IOD = Average optical density × Measurement area.

[0095] Relative IOD value = Sample IOD / Group BC IOD.

[0096] The results are summarized in Table 5 and Figure 4 .

[0097] Table 5. Results of lipofuscin test

[0098]

[0099] As can be seen, the lipofuscin content in the NC group was significantly higher than that in the BC group, indicating that the stimulation conditions in this embodiment were effective. Compared with the NC group, the lipofuscin content in the o-tolyl biguanide sample group was significantly lower, with an inhibition rate of 61.94%.

[0100] The above data indicate that using only o-tolyl biguanide, a single-component drug, can significantly reduce the levels of melanin, lipofuscin, and advanced glycation end products induced by UV+MGO stimulation in a 3D full-thickness skin model, and significantly improve the skin's L... * The values ​​of ITA° and ITA° have the effect of reducing skin pigmentation and brightening skin tone.

[0101] Example 2

[0102] This embodiment provides a skin whitening gel and its preparation method.

[0103] (1) Preparation of predispersant: Weigh 8.00 g of purified water into a beaker and heat to about 85°C. While maintaining the temperature on a magnetic stirrer and stirring continuously, add palmitic acid (0.410 g, 1.60 mmol) and stir until homogeneous. Then add o-tolyl biguanide (0.30 g, 1.57 mmol) and stir continuously until a uniform white emulsion is formed. Continue stirring for about 20 min to ensure thorough mixing and dispersion. Allow the emulsion to cool naturally to room temperature (about 25°C) with stirring to obtain a predispersant with complete charge preneutralization, which is ready for use.

[0104] (2) Preparation of the gel matrix: Weigh 90.00 g of purified water and place it in another beaker. While stirring continuously, slowly and evenly sprinkle 0.50 g of carbomer 940 onto the water surface. Continue stirring until the carbomer powder is completely wetted and evenly dispersed, forming a turbid suspension. Let it stand for about 40 minutes or until the carbomer particles are fully hydrated and swollen into a nearly transparent gel. Slowly add triethanolamine (TEA) while stirring until the carbomer gel forms a transparent, viscous gel with a pH of about 6.5. Then add 1.0 g of phenoxyethanol (preservative) and stir until homogeneous.

[0105] (3) Mixing: Slowly add the pre-dispersion from step (1) while continuously stirring the gel matrix from step (2); after the addition is complete, continue stirring for 10 minutes and let stand to obtain the whitening gel.

[0106] The whitening gel system prepared in this embodiment is homogeneous, without any visible particles, flocculent material, or precipitates. The system viscosity is stable, and no precipitation, layering, or significant viscosity change was observed after standing at 25°C for one month.

[0107] Ten participants used the whitening gel of this embodiment for four weeks, and then conducted a subjective evaluation of their experience. Nine participants reported whiter and brighter skin, and seven of them reported improvement in pigmentation.

[0108] Comparative Example

[0109] The difference between this comparative example and Example 2 is that the whitening gel in this comparative example is prepared using the following method:

[0110] (1) Preparation of o-tolyl biguanide malate solution: Weigh 8.00 g of purified water into a beaker and heat it to about 45 °C. While keeping it warm on a magnetic stirrer and stirring continuously, add 0.107 g (0.80 mmol) of DL-malic acid and stir until completely dissolved. Then add o-tolyl biguanide (0.30 g, 1.57 mmol) and continue stirring for about 20 min to obtain o-tolyl biguanide malate solution.

[0111] (2) Preparation of the gel matrix: Weigh 90.00 g of purified water and place it in another beaker. While stirring continuously, slowly and evenly sprinkle 0.50 g of carbomer 940 onto the water surface. Continue stirring until the carbomer powder is completely wetted and evenly dispersed, forming a turbid suspension. Let it stand for about 40 minutes until the carbomer particles are fully hydrated and swollen into a nearly transparent gel. Slowly add triethanolamine (TEA) while stirring until the carbomer gel forms a transparent, viscous gel with a pH of about 6.5. Then add 1.0 g of phenoxyethanol and stir until homogeneous.

[0112] (3) Mixing: Under the condition of continuous stirring of the gel matrix in step (2), the solution in step (1) is slowly added, resulting in white flocculent precipitation, and the system becomes thinner and no longer has viscosity.

[0113] In this comparative example, DL-malic acid (a dicarboxylic acid) is added. Its carboxyl group provides hydrogen ions to protonate the amino group in the o-tolyl biguanide molecule, forming water-soluble o-tolyl biguanide malate. The protonation of the terminal amino group of the biguanide group (-NH3) + This imparts a strong positive charge to the molecule, while carbomer in the system, as an anionic polymer, has a carboxyl anion (-COO). - It can form a stable ion pair with the positive charge of the guanidine group through electrostatic attraction; the two combine and flocculate through electrostatic and hydrophobic interactions, causing the system to lose the desired viscosity.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of o-tolyl biguanide in the preparation of skin whitening products, characterized in that, In the skin whitening product, o-tolyl biguanide is used as the whitening active ingredient, which achieves the whitening effect by inhibiting melanin, lipofuscin and carboxymethyl lysine.

2. The application as described in claim 1, characterized in that, The skin whitening products are used to prevent, alleviate and / or reduce symptoms associated with hyperpigmentation.

3. The application as described in claim 2, characterized in that, Symptoms associated with hyperpigmentation include freckles, sunspots, age spots, melasma, or post-inflammatory hyperpigmentation.

4. The application as described in claim 1, characterized in that, The concentration of o-tolyl biguanide in the skin whitening product is 0.01~2.0wt%.

5. The application as described in claim 1, characterized in that, The skin whitening product includes at least o-tolyl biguanide, anionic surfactant, thickener and water, wherein the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolyl biguanide is (0.9~1.2):

1.

6. The application as described in claim 5, characterized in that, In the skin whitening product, the mass fraction of o-tolyl biguanide is 0.01~2.0wt%; the anionic surfactant is an anionic surfactant in its protonated form without salt formation.

7. The application as described in claim 5, characterized in that, The thickener is one or more of polyacrylic acid thickeners, sodium carboxymethyl cellulose, or sodium alginate.

8. The application as described in claim 7, characterized in that, The thickener is carbomer, and the mass fraction of the thickener is 0.1~3wt%.

9. The application as described in any one of claims 5 to 8, characterized in that, The preparation method of the skin whitening product includes the following steps: o-Tolyl Biguanide and anionic surfactant are pre-mixed and emulsified in an aqueous phase to form a pre-dispersion; The thickener is stirred in water to swell or dissolve; then the pre-dispersed material is added under stirring conditions, stirred until well mixed, and allowed to stand to obtain the final product.

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