Application of o-tolyl biguanide in preparation of skin whitening product, skin whitening product and preparation method

Abstract

The invention discloses application of o-tolyl biguanide in preparation of a skin whitening product, the skin 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 advanced glycosylation end products, namely carboxy methyl lysine (CML), lipofuscin and melanin when serving as a whitening active ingredient, and therefore the multi-channel whitening effect is achieved. The invention further provides a whitening product, the whitening product comprises the o-tolyl biguanide and the anionic surfactant which are similar in charge quantity, the o-tolyl biguanide is positively charged, the anionic surfactant is negatively charged, and the o-tolyl biguanide and the anionic surfactant can form a complex without net charge or with weak net charge. Furthermore, the stability of the o-tolyl biguanide in a whitening product system can be effectively improved, irreversible aggregation, flocculation or precipitation with common thickening agents such as carbomer is avoided, and the formula compatibility and stability of the o-tolyl biguanide are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of skin care, and in particular to application of o-tolylbiguanide in the preparation of skin whitening products, the whitening products and a preparation method. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Skin tone evenness is a key factor influencing the skin's apparent age, while spots and pigmentation are the main causes of uneven skin tone. With aging and UV exposure, the skin develops increased darkness and yellowness, accompanied by the formation of various spots. These changes directly impact the skin's visual youthfulness. Currently, mainstream whitening actives mostly block melanin production by inhibiting tyrosinase activity. While these ingredients can reduce melanin synthesis to a certain extent, they are limited in their effectiveness at clearing stubborn melanin already formed in the skin, and are particularly difficult to target pigment particles deposited in the dermis.

[0004] The causes of skin pigmentation problems are diverse. In addition to melanin, lipofuscin and glycated pigments are also important factors that lead to dull and sallow skin. Lipofuscin, as a difficult-to-degrade pigment granule formed by the oxidation and accumulation of cellular metabolic waste, will accelerate its deposition deep in the skin with age, causing the skin to lose its transparency; glycated pigments are advanced glycation end products generated by the non-enzymatic reaction of sugars and skin proteins. Not only are they brownish-yellow in color, they also indirectly aggravate pigmentation by promoting oxidative stress and inflammatory responses. Existing whitening ingredients lack a targeted effect on these two types of pigments produced by non-tyrosinase pathways, resulting in whitening solutions that only target melanin being ineffective in improving the yellowing and dullness of the skin, which is common in Asians.

[0005] Furthermore, current solutions for multi-type pigmentation issues often rely on the combination of multiple ingredients, combining active ingredients with different targets to achieve comprehensive improvements. This approach not only increases the complexity of formulation design, but can also increase raw material management and production costs, while also making it difficult to ensure the synergistic stability of the various ingredients. When it comes to simultaneously regulating melanin, lipofuscin, and glycated pigments with a single component, existing technology still has 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 use of o-tolylbiguanide in the preparation of skin whitening products, whitening products and preparation methods. The present invention finds that o-tolylbiguanide can significantly reduce the levels of melanin, glycated pigments and lipofuscin, thereby effectively improving skin brightness, lightening skin color, and achieving a good whitening effect.

[0007] In a first aspect, the present invention provides a use of o-tolylbiguanide in the preparation of a skin whitening product; in the skin whitening product, o-tolylbiguanide serves as a whitening active ingredient.

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

[0009] Further, hyperpigmentation-related symptoms include freckles, sun spots, age spots, melasma, or post-inflammatory hyperpigmentation.

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

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

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

[0013] Preferably, the mass fraction of the o-tolylbiguanide is 0.01-2.0 wt %; and the anionic surfactant is an anionic surfactant in a protonated form without forming a salt.

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

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

[0016] In a third aspect, the present invention provides a method for preparing the above-mentioned whitening product, comprising the following steps: The 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-dispersion is added under stirring, and the mixture is stirred and mixed, and then allowed to stand to obtain the product.

[0017] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The present invention discovered for the first time that o-tolylbiguanide, when used as a whitening active ingredient, can effectively inhibit the production of advanced glycation end products carboxymethyllysine (CML), lipofuscin, and melanin, thereby achieving a multi-pathway whitening effect.

[0018] (2) The present invention provides a whitening product, which includes o-tolylbiguanide and an anionic surfactant, wherein o-tolylbiguanide is positively charged and the anionic surfactant is negatively charged. When the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolylbiguanide 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-tolylbiguanide in the whitening product system, avoiding irreversible aggregation, flocculation or precipitation, and improving the formula compatibility and stability of o-tolylbiguanide. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute undue limitations thereon. It is obvious that one of ordinary skill in the art could derive other drawings based on these drawings without inventive effort.

[0020] Figure 1 These are the appearance pictures of three parallel samples (labeled as 1#, 2# and 3#) from different treatment groups in Example 1 of the present invention; Figure 2 These are fluorescence imaging photos of carboxymethyllysine of three parallel samples (labeled 1#, 2#, and 3#) from different treatment groups in Example 1 of the present invention; Figure 3 These are photos of melanin distribution of three parallel samples (labeled 1#, 2#, and 3#) from different treatment groups in Example 1 of the present invention; Figure 4 These are photos of lipofuscin distribution in three parallel samples (labeled 1#, 2#, and 3#) from different treatment groups in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0022] As used herein, the terms "comprise," "include," "have," "contain," and any other variations thereof are intended to be non-exclusive. For example, a composition, process, method, article, or apparatus comprising the listed elements is not limited to comprising only those elements but may also include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0023] In the present invention, "and / or" is used to indicate that one or both of the situations described may occur, for example, "A and / or B" includes "A and B", "A" or "B".

[0024] In the present invention, when mass fraction, concentration, temperature or other values ​​or parameters are expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, it should be understood that all combined ranges formed by pairing any upper limit (or preferred value) with a lower limit (or preferred value) are included, regardless of whether the range is listed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in the present invention, unless otherwise specified, the range is intended to include its end values ​​and all integers and fractions within the range.

[0025] In the present invention, the term "inhibit" can be used interchangeably with "reduce," "lower," "downregulate," 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 of the variables compared to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from an untreated or control-treated subject, cell, or sample (e.g., a buffer-only control or an inert agent control).

[0026] In the present invention, unless otherwise stated, "optional" or "preferred" means that the subsequently described event or environment can but need not occur, and the description includes occasions where the event or environment occurs or does not occur.

[0027] The present invention provides the use of o-tolyl biguanide in preparing skin whitening products, wherein o-tolyl biguanide is used as a whitening active ingredient.

[0028] o-Tolylbiguanide is also known as 1-o-tolylbiguanide, its CAS number is 93-69-6, and its structural formula is as follows: .

[0029] The skin whitening products of the present invention preferably include cosmetics and medicines. Cosmetics may include essences, creams, masks, lotions, toners, sunscreens, cleansers, soaps, foundations, concealers or stock solutions, etc.

[0030] In an optional embodiment of the present invention, the skin whitening product is used to prevent, alleviate and / or reduce symptoms associated with hyperpigmentation.

[0031] In an alternative embodiment of the present invention, the symptoms associated with hyperpigmentation include freckles, sun spots, age spots, melasma, or post-inflammatory hyperpigmentation.

[0032] In an optional embodiment of the present invention, the pigments include melanin, lipofuscin and glycated pigments. The present invention has found that o-tolylbiguanide can simultaneously prevent, alleviate and / or reduce the accumulation and pigmentation of melanin, lipofuscin and glycated pigments.

[0033] Melanin is a natural biological pigment synthesized by melanocytes and is divided into eumelanin and pheomelanin. Its primary function is to absorb ultraviolet light and protect the DNA of dermal cells from damage. Abnormal synthesis can lead to dark spots, freckles, and uneven skin tone. Lipofuscin is a brownish-yellow, fluorescent pigment granule that is oxidized and accumulated in lysosomes by cellular metabolic waste products. With aging and UV exposure, it accumulates in the deep epidermis and superficial dermis, contributing to sallow, dull skin and decreased translucency. Glycated pigments are pigmentation problems caused by the accumulation of advanced glycation end products (AGEs) in the skin. AGEs, such as carboxymethyllysine (CML) and pentosidine, are formed when reducing sugars such as glucose undergo non-enzymatic glycation reactions with collagen and elastin in the skin. These substances, which are brownish-yellow in color, not only directly cause skin pigmentation but also indirectly induce hyperpigmentation by exacerbating oxidative stress and inflammation, ultimately forming dark spots on aging skin areas such as the nasolabial folds and forehead. The present invention discovers for the first time that o-tolyl biguanide can achieve a multi-pathway whitening effect by simultaneously inhibiting the production of melanin, lipofuscin and glycated pigments.

[0034] In an optional embodiment of the present invention, the concentration of o-tolylbiguanide 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.

[0035] In an optional embodiment of the present invention, the dosage form of the skin whitening product includes a paste, an ointment, a spray, a gel, a liniment, a paint, a film, a patch, a plaster or a film. Those skilled in the art can prepare a product in a suitable dosage form as needed.

[0036] In an optional embodiment of the present invention, the skin whitening product further includes excipients, which include solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, gelling agents, osmotic pressure regulators, stabilizers, glidants, anti-caking agents, flavoring agents, antibacterial agents, suspending agents, coating agents, film-forming agents, fragrances, preservatives, viscosity enhancers, anti-adhesive agents, antioxidants, antioxidant synergists, chelating agents, pH regulators, adsorbents, plasticizers, surfactants, thickeners, inclusion agents, protective agents, moisturizers, softeners, absorbents, diluents, release regulators, pressure-sensitive adhesives, hardeners, hollow capsules, matrices or drug carrier materials. One or more.

[0037] In an optional embodiment of the present invention, o-tolylbiguanide can be used alone as a whitening active ingredient, or can be used in combination with other whitening active ingredients, such as arbutin, kojic acid, and the like.

[0038] The present invention also provides a whitening product comprising at least o-tolylbiguanide, an anionic surfactant, a thickener, and water; the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolylbiguanide is (0.9-1.2):1.

[0039] In an optional embodiment of the present invention, the mass fraction of o-tolylbiguanide is 0.01-2.0 wt %.

[0040] Because the guanidine group in its molecular structure is protonated in conventional weakly acidic or neutral aqueous systems, it carries a significant positive charge. This characteristic leads to strong electrostatic interactions with carbomer thickeners commonly used in formulations, which carry a negative charge after dissociation in the aqueous phase. This interaction can easily lead to irreversible aggregation, flocculation, or precipitation, resulting in serious compatibility issues such as loss of system physical stability, inactivation of active ingredients, or uneven local concentrations. This greatly limits the application of o-tolylbiguanide in formulations containing such anionic polymers. It is worth noting that since the present invention limits the molar ratio of the negative charge generated by the dissociation of the anionic surfactant to o-tolylbiguanide to (0.9-1.2):1, there is a charge neutralization effect between the anion of the anionic surfactant and the protonated guanidine group. For example, for anionic surfactants with a single negative charge per molecule after dissociation, such as palmitic acid, sodium lauryl sulfate, sodium laureth sulfate, and sodium N-lauroyl sarcosinate, the molar ratio of the anionic surfactant to o-tolylbiguanide is (0.9-1.2):1. For anionic surfactants with multiple negative charges per molecule after dissociation, such as potassium cetyl phosphate, which has two negative charges per molecule after complete dissociation, the molar ratio of the anionic surfactant to o-tolylbiguanide is (0.45-0.6):1, calculated based on the ratio. Therefore, the complex formed by the anionic surfactant and o-tolylbiguanide in the present invention generally exhibits near-neutral or weakly charged characteristics, reducing the possibility of electrostatic interaction with anionic thickeners such as carbomer, and effectively improving the system stability of the final product.

[0041] In the present invention, the anionic surfactant is preferably a protonated, unsalted anionic surfactant, such as a sulfonic acid anionic surfactant or a carboxylic acid anionic surfactant. More specifically, the protonated, unsalted anionic surfactant includes, but is not limited to, palmitic acid, lauric acid, stearic acid, and myristic acid. The acidic group (-COOH) of such surfactants can provide hydrogen ions, reacting with the amino group in the o-tolylbiguanide molecule to form a stable salt compound. Simultaneously, the hydrophobic chain segment (alkyl portion) can interact hydrophobically with the phenyl structure of o-tolylbiguanide. This dual action enables the complex to form a stable dispersion in water.

[0042] In an optional embodiment of the present invention, the thickener is one or more of a polyacrylic acid thickener, sodium carboxymethyl cellulose, or sodium alginate. Furthermore, the thickener is carbomer, a polyacrylic acid thickener that is highly effective in thickening, mild, and non-irritating, and can effectively encapsulate the active ingredient, extending the duration of efficacy. The mass fraction of the thickener is 0.1 to 3 wt %, and can be selected based on the specific dosage form and application scenario.

[0043] The whitening product of the present invention may also include antioxidants, moisturizers, chelating agents, pH regulators, etc. to further optimize the stability, safety, and comfort of use of the whitening product. The present invention also imposes no particular limitation on the dosage form of the whitening product, and may be, for example, a paste, ointment, gel, liniment, paint, film coating, patch, or plaster.

[0044] The present invention also provides a method for preparing the above-mentioned whitening product, comprising the following steps: The 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-dispersion is added under stirring, and the mixture is stirred and mixed, and then allowed to stand to obtain the product.

[0045] In the above embodiment of the present invention, after the addition of o-tolylbiguanide, the anionic surfactant and o-tolylbiguanide rapidly combine through electrostatic interaction to form an ionic complex or ion pair. This process occurs under stirring, thereby forming a uniform emulsion. The resulting pre-dispersion has a neutral or weakly charged electrical property. When added to the thickener system, the strong electrostatic attraction between the two is eliminated, effectively preventing aggregation and precipitation, and successfully solving the compatibility problem.

[0046] The present invention does not impose any particular limitation on the mixing and emulsification process, and heating, stirring and emulsification can be performed as needed.

[0047] During the preparation of the pre-dispersion, high-melting-point hydrophobic ingredients or ingredients containing hydrophobic groups, such as solid waxes, long-chain fatty alcohols, and phospholipids, may be added. Waxes are preferred because high-melting-point ingredients can maintain the pre-dispersion in a solid state over a wider temperature range and reduce the release of o-tolylbiguanide. Waxes may include palm wax, candelilla wax, jojoba wax, Japan wax, and beeswax.

[0048] The technical solutions of the present invention are further illustrated below with reference to specific examples. The sources of the reagents used in the following examples are not particularly limited; commercially available products familiar to those skilled in the art may be used. In the following examples, the 3D melanin-rich full-thickness skin model (MelaFulKutis™) and model culture medium were provided by the Shaanxi Branch of Guangdong Boxi Biotechnology Co., Ltd.

[0049] In the following examples, AGEs stands for advanced glycation end products. MGO stands for methylglyoxal, which is used to promote the formation of advanced glycation end products AGEs. UV stands for ultraviolet light. UVA stands for long-wave ultraviolet light, which has strong penetrating power and can penetrate deep into the dermis, mainly causing skin aging and pigmentation. UVB stands for medium-wave ultraviolet light, which has higher energy and mainly acts on the epidermis. It is the main cause of sunburn, erythema and melanin synthesis.* The value reflects the brightness of the skin color, L * The higher the value, the brighter the skin color. * The lower the value, the darker the skin tone. * The value reflects the red-green tendency of the skin color, a positive value indicates that the skin color is reddish, and a negative value indicates that the skin color is greenish. * The value reflects the yellow-blue tendency of skin color; positive values ​​indicate a yellowish complexion, and negative values ​​indicate a bluish complexion. The ITA° value stands for Individual Type Angle; a larger ITA° value indicates a lighter complexion. CML stands for carboxymethyllysine. DAB stands for 3,3'-diaminobenzidine.

[0050] Example 1 This example provides a test of the inhibitory effect of o-tolylbiguanide on melanin, lipofuscin and carboxymethyllysine.

[0051] In this example, a 3D melanin full-thickness skin model (MelaFulKutis™) was used as a testing tool. Surface drug administration was adopted to construct skin models of different treatment groups and evaluate the in vitro whitening effects of the different treatment groups.

[0052] The grouping of different treatment groups is shown in Table 1.

[0053] Table 1 Grouping of different treatment groups

[0054] Note: In Table 1, kojic acid is a typical tyrosinase inhibitor that reduces melanin content in skin models by blocking the melanin synthesis pathway. Aminoguanidine sulfate is a scavenger of methylglyoxal (MGO), which binds to MGO via a nucleophilic reaction, blocking its non-enzymatic glycosylation 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 counteracting collagen degradation caused by photoaging.

[0055] The specific steps are as follows: 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.

[0056] 2. Administration: The administration treatment 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 performed. (2) NC group: the model culture medium was replaced with a model culture medium containing 3 mM MGO. (3) PC1 group: the model culture medium was replaced with a model culture medium containing 3 mM MGO, and kojic acid working solution was added to the surface. (4) PC2 group: the model culture medium was replaced with a model culture medium containing aminoguanidine sulfate and 3 mM MGO. (5) PC3 group: the model culture medium was replaced with a model culture medium containing TGF-β1 and 3 mM MGO. (6) Sample group: the model culture medium was replaced with a model culture medium containing 3 mM MGO, and the administration method was surface administration.

[0057] 3. UVA+UVB combined irradiation: irradiation dose is 12J / cm 2 UVA and 50mJ / cm 2 UVB.

[0058] 4. Model Culture: The models were transferred to a 6-well plate and incubated in a CO2 incubator (37°C, 5% CO2). The BC group did not receive medication or UVA+UVB irradiation, while the other groups received medication and UVA+UVB irradiation. The specific steps were as follows: only medication was administered on day 1, and UVA+UVB irradiation was performed on days 2 to 8, with a frequency of once per day for a total of 7 times. After irradiation on days 2 to 7, medication was administered once per day. After the last irradiation, samples were collected, and the residual test substance was washed with a sterile PBS solution wash bottle, and the residual liquid was wiped away with a sterile cotton swab.

[0059] 5. Test analysis: (1) Apparent skin color: Take the model to be tested, take photos and observe under a stereo microscope, collect pictures and analyze them. Figure 1 Shown are microscopic images of the BC, NC, PC1, PC2, PC3, and sample groups, with three replicates per group (labeled 1#, 2#, and 3#). The images show that the skin tone in the NC group darkened, while the skin tone in the PC1, PC2, PC3, and sample groups all lightened to varying degrees compared to the NC group.

[0060] (2) L * 、a * 、b * Value detection and ITA ° value calculation: After the apparent color detection, the model is L * value, a * Value and b * The specific test operation is as follows: Place the model on a flat and hard white surface with the stratum corneum facing upwards, align the detection hole of the skin color measuring instrument vertically with the model surface for testing, and read the L * value, a* Value and b * The readings of each model were repeated three times, and the average value was taken as the reading of a single model; it was calculated according to the following formula: .

[0061] The data for the different treatment groups are summarized in Table 2 .

[0062] As can be seen from Table 2, in the L that reflects the skin brightness * Compared with the BC group, the NC group showed a highly significant decrease in values, while the three positive control groups and the o-tolylbiguanide sample group showed highly significant increases relative to the NC group, indicating that the four whitening active ingredients (kojic acid, aminoguanidine sulfate, TGF-β1, and o-tolylbiguanide) all significantly improved skin brightness. There were small differences between the groups in a* values, which reflect the red-green and blue-yellow scales of the skin, and b* values, which reflect the lightness and redness of the skin. The ITA° value, which reflects skin tone, showed a significant decrease in the NC group, indicating a darkening of the skin. The three positive control groups and the o-tolylbiguanide sample group showed highly significant increases relative to the NC group, indicating that the four whitening active ingredients all significantly reduced skin tone. The o-tolylbiguanide sample group showed values ​​significantly higher than those for PC1 (kojic acid) and PC2 (aminoguanidine sulfate), and slightly higher than PC3 (TGF-β1), indicating that o-tolylbiguanide is more effective than kojic acid (a commonly used whitening agent) and aminoguanidine sulfate (a glycation inhibitor) in this skin tone model.

[0063] Table 2 L in different treatment groups * 、a * 、b * Value detection and ITA° value

[0064] Note: In Table 2, the results are expressed as mean ± standard deviation. Comparisons between groups were analyzed using the t-test. All statistical analyses were two-tailed. P < 0.05 was considered a significant difference, and P < 0.01 was considered a highly significant difference. Compared with the BC group, significance is indicated by #, P < 0.05 is indicated by #, and P < 0.01 is indicated by ##. Compared with the NC group, significance is indicated by *, P < 0.05 is indicated by *, and P < 0.01 is indicated by **. The meanings of the data in the subsequent tables are the same as above.

[0065] (3) Immunofluorescence test for advanced glycation end product carboxymethyllysine (CML): The model to be tested was fixed with 4 wt% paraformaldehyde for 24 h, followed by immunofluorescence detection. The images were taken under a microscope for observation, and quantitative analysis was performed. The IOD (Integrated Optical Density) value was calculated using the following formula: IOD = average optical density × area of ​​measurement area.

[0066] Relative IOD value = sample IOD / BC group IOD.

[0067] The results are shown in Table 3 and Figure 2 shown.

[0068] From Table 3 and Figure 2 As can be seen, the NC group showed a significant increase in AGEs (CML) compared to the BC group, indicating that MGO stimulation was effective. The PC2 (aminoguanidine sulfate) group showed a significant decrease in AGEs (CML) compared to the NC group, demonstrating the effectiveness of the positive control aminoguanidine sulfate. The o-tolylbiguanide sample also showed a significant decrease in AGEs (CML) compared to the NC group, with an inhibition rate of 35.89%.

[0069] Table 3 Advanced glycation end products CML test results in different treatment groups

[0070] (4) Melanin detection: Fix with 4 wt% paraformaldehyde solution for 24 h, embed the slices, and stain according to the instructions of the silver staining kit. Take pictures under a microscope and analyze the images quantitatively. The results are summarized in Table 4 and Figure 3 .

[0071] Table 4 Melanin test results of different treatment groups

[0072] Note: In Table 4, the relative area of ​​melanin granules was obtained by the following method: the area of ​​melanin granules per unit area was counted, the area of ​​the BC group was defined as 1, and the areas of other groups were converted based on the BC group, which is the relative area of ​​melanin granules.

[0073] As can be seen, compared to the BC group, the NC group showed a significant increase in melanin granules, demonstrating the effectiveness of the stimulation conditions in this example. Compared to the NC group, the PC1 (kojic acid) group showed a significant decrease in melanin granules, demonstrating the effectiveness of the positive control kojic acid in this test. Compared to the NC group, the o-tolylbiguanide sample group showed a significant decrease in melanin granules, with an inhibition rate of 34.97%.

[0074] (5) Lipofuscin detection: The model to be tested was fixed with 4 wt% paraformaldehyde for 24 h, then embedded, sectioned, dewaxed, and hydrated. Blocking was performed using a biotin detection blocking kit, and a working solution was prepared with the lipofuscin stain GL13. The cells were incubated at 37°C for 10 min. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated at 37°C for 1.5 h. The cells were then stained with DAB for 2–10 min, counterstained, and mounted. The cells were photographed under a microscope, and images were collected and analyzed.

[0075] The calculation formula of IOD (Integrated Optical Density) value is as follows: IOD = average optical density × area of ​​measurement area.

[0076] Relative IOD value = sample IOD / BC group IOD.

[0077] The results are summarized in Table 5 and Figure 4 .

[0078] Table 5 Lipofuscin test results

[0079] It can be seen that the lipofuscin content in the NC group increased significantly compared to the BC group, indicating that the stimulation conditions of this embodiment are effective. Compared to the NC group, the lipofuscin content in the o-tolylbiguanide sample group decreased significantly, with an inhibition rate of 61.94%.

[0080] The above data show that the use of only a single ingredient, o-tolyl biguanide, can significantly reduce the levels of melanin, lipofuscin, and advanced glycation end products caused by UV+MGO stimulation in a 3D full-thickness skin model, and significantly increase the skin's L * The value and ITA° value can reduce skin pigmentation and brighten skin tone.

[0081] Example 2 This embodiment provides a whitening gel and a preparation method thereof.

[0082] (1) Preparation of pre-dispersion: Weigh purified water (8.00 g) into a beaker and heat to approximately 85°C. Add palmitic acid (0.410 g, 1.60 mmol) while maintaining the temperature and stirring continuously on a magnetic stirrer and stir evenly. Then add o-tolylbiguanide (0.30 g, 1.57 mmol) and continue stirring until a uniform white emulsion forms. Continue stirring for approximately 20 min to allow the two to be thoroughly mixed and dispersed. Cool the resulting emulsion naturally to room temperature (approximately 25°C) while stirring to obtain a pre-dispersion with charge pre-neutralization, which is then set aside.

[0083] (2) Preparation of gel matrix: Weigh purified water (90.00 g) and place it in another beaker. Slowly and evenly sprinkle carbomer 940 (0.50 g) onto the water surface while stirring continuously. Continue stirring until the carbomer powder is completely wetted and evenly dispersed to form a turbid suspension. Let it stand for about 40 minutes or until the carbomer particles are fully hydrated and swell to form a nearly transparent gel. Slowly add triethanolamine (TEA) dropwise while stirring until the carbomer gel forms a transparent, viscous gel with a system pH of approximately 6.5. Then add 1.0 g of phenoxyethanol (preservative) and stir until uniform.

[0084] (3) Mixing: Slowly add the pre-dispersion of step (1) while continuously stirring the gel matrix of step (2); after the addition is completed, continue stirring for 10 minutes and let it stand to obtain the whitening gel.

[0085] The whitening gel system prepared in this example was uniform, without visible particles, flocculation, or sediment. The system had stable viscosity, and no precipitation, stratification, or significant viscosity change was observed after being stored at 25°C for one month.

[0086] Ten subjects used the whitening gel of this example for four weeks and then conducted a subjective evaluation of their experience. Nine subjects reported that their skin became whiter and brighter, and seven subjects reported that their pigmentation was improved.

[0087] Comparative Example Compared with Example 2, this comparative example is different in that the preparation method of the whitening gel in this comparative example is as follows: (1) Preparation of o-tolylbiguanide malate solution: Weigh purified water (8.00 g) into a beaker and heat to approximately 45°C. Add 0.107 g (0.80 mmol) of DL-malic acid while maintaining the temperature on a magnetic stirrer and stirring continuously. Stir until completely dissolved. Then, add o-tolylbiguanide (0.30 g, 1.57 mmol) and continue stirring for approximately 20 min to obtain the o-tolylbiguanide malate solution.

[0088] (2) Preparation of gel matrix: Weigh purified water (90.00 g) and place it in another beaker. Slowly and evenly sprinkle carbomer 940 (0.50 g) onto the water surface while stirring continuously. Continue stirring until the carbomer powder is completely wetted and evenly dispersed to form a turbid suspension. Let it stand for about 40 minutes until the carbomer particles are fully hydrated and swell to form a nearly transparent gel. Slowly add triethanolamine (TEA) dropwise while stirring until the carbomer gel forms a transparent, viscous gel with a system pH of approximately 6.5. Then add 1.0 g of phenoxyethanol and stir until uniform.

[0089] (3) Mixing: Slowly add the solution of step (1) while continuously stirring the gel matrix of step (2), producing a white flocculent precipitate, and the system becomes thinner and no longer viscous.

[0090] In this comparative example, DL-malic acid (dicarboxylic acid) is added, and its carboxyl group can provide hydrogen ions to react with the amino group in the o-tolylbiguanide molecule to form water-soluble o-tolylbiguanide malate. The amino group at the end of the biguanide group is protonated (-NH3 + ) gives the molecule a strong positive charge, while the carbomer in the system acts as an anionic polymer, and its carboxyl anion (-COO -) can form a stable ion pair with the positive charge of the guanidine group through electrostatic attraction; the two combine and flocculate through electrostatic interaction and hydrophobic interaction, causing the system to lose the desired viscosity.

[0091] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. The use of o-tolylbiguanide in preparing skin whitening products, characterized in that: In the skin whitening product, o-tolylbiguanide is used as a whitening active ingredient.

2. The use according to claim 1, characterized in that The skin lightening product is used to prevent, alleviate and / or reduce symptoms associated with hyperpigmentation.

3. The use according to claim 2, characterized in that Symptoms associated with hyperpigmentation include freckles, sun spots, age spots, melasma, or post-inflammatory hyperpigmentation.

4. The use according to claim 2, characterized in that The pigments include melanin, lipofuscin and saccharified pigments.

5. The use according to claim 1, characterized in that The concentration of o-tolylbiguanide in the skin whitening product is 0.01-2.0 wt %.

6. A whitening product, characterized in that: The invention comprises at least o-tolylbiguanide, 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-tolylbiguanide is (0.9-1.2):

1.

7. The whitening product according to claim 6, characterized in that The mass fraction of the o-tolylbiguanide is 0.01-2.0 wt %; the anionic surfactant is an anionic surfactant in a protonated form without forming a salt.

8. The whitening product according to claim 6, characterized in that The thickener is one or more of polyacrylic acid thickener, sodium carboxymethyl cellulose or sodium alginate.

9. The whitening product according to claim 8, characterized in that The thickener is carbomer, and the mass fraction of the thickener is 0.1-3 wt %.

10. The method for preparing a whitening product according to any one of claims 6 to 9, wherein: The steps include: The 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-dispersion is added under stirring, and the mixture is stirred and mixed, and then allowed to stand to obtain the product.

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