Application of oxidized resveratrol derivative in preparation of whitening or freckle-removing product

Abstract

The invention discloses an application of an oxyresveratrol derivative in preparation of a whitening or freckle-removing product in the technical field of cosmetics, and particularly relates to any one of the following applications: (1) an application of the oxyresveratrol derivative as a tyrosinase inhibitor; (2) application of the oxidized resveratrol derivative as a PAR-2 inhibitor; and (3) application of the oxidized resveratrol derivative as an MNT-1 melanin synthesis inhibitor. The oxidized resveratrol derivative provided by the invention shows a core advantage far superior to that of a parent compound oxidized resveratrol in the field of whitening skin care products, efficient whitening regulation and control are realized from melanin synthesis and transport whole links, meanwhile, the problem of stability pain points of traditional whitening components is solved, and the oxidized resveratrol derivative is more prominent in effect, more excellent in stability and better in application prospect. The requirements of industrial production and marketization application of skin care products are better met.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to the application of oxidized resveratrol derivatives in the preparation of whitening or freckle-removing products. Background Technology

[0002] Skin whitening and blemish removal is a major category in skincare cosmetics. Finding compounds with multi-faceted whitening effects is a crucial direction in the research of cosmetic active ingredients. Tyrosinase is a key rate-limiting enzyme in melanin synthesis; inhibiting tyrosinase activity can fundamentally reduce melanin synthesis. Therefore, most mainstream whitening ingredients on the market currently have varying degrees of tyrosinase inhibition. This is also a core target in the development of whitening ingredients. Simultaneously, current research has found that PAR-2 ​​plays a vital role in the transport of melanosomes. Therefore, finding compounds that can both strongly inhibit tyrosinase activity and reduce melanin synthesis, while also possessing PAR-2 ​​inhibitory activity, will create a significant competitive advantage over existing whitening ingredients on the market.

[0003] Stilbene compounds, such as resveratrol and oxidized resveratrol, not only have a strong inhibitory effect on human tyrosinase, but also a strong inhibitory effect on melanin synthesis, which can reduce melanin production at the source; in addition, they also have a strong inhibitory effect on the transport of melanosomes, making them highly competitive in the field of skin whitening and spot removal.

[0004] Oxidized resveratrol shares a similar molecular skeleton with resveratrol, and its chemical structure can be considered a derivative of resveratrol with hydroxylation repair at the 2-position of the benzene ring. The multiple phenolic hydroxyl groups of oxidized resveratrol are central to its biological activity, but they also make it susceptible to oxidation (e.g., degradation upon exposure to light, heat, and oxygen) and discoloration, thus affecting its efficacy. Therefore, screening based on the structure of oxidized resveratrol to find chemical components that possess multi-dimensional whitening effects, good stability, and applicability, meeting the practical application needs of cosmetics, pharmaceuticals, and other fields, is of positive significance for enhancing the industrial application value of stilbene compounds. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned technical problems by providing the application of oxidized resveratrol derivatives in the preparation of whitening or freckle-removing products.

[0006] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, the use of oxidized resveratrol derivatives in the preparation of whitening or freckle-removing products.

[0007] As a further optimization of the present invention, it can be applied in any of the following ways: (1) Application of oxidized resveratrol derivatives as tyrosinase inhibitors; (2) Application of oxidized resveratrol derivatives as PAR-2 ​​inhibitors; (3) Application of oxidized resveratrol derivatives as MNT-1 melanin synthesis inhibitors.

[0008] As a further optimization of the present invention, the product is a skin care product.

[0009] As a further optimization of the present invention, the structure of the oxidized resveratrol derivative is shown in formula (Ⅰ): Equation (I) R1, R2, and R3 are independently selected from at least one of hydrogen, hydroxyl, or methoxy.

[0010] As a further optimization of the present invention, the oxidized resveratrol derivative is any one of the following: 2,4-dihydroxy-3'5'-dimethoxystilbene as shown in formula (II), 2,4-dihydroxy-3'4'-dimethoxystilbene as shown in formula (III), or 2,4-dihydroxy-4'-methoxystilbene as shown in formula (IV). Formula (II) Formula (III) Equation (Ⅳ) The "oxidized resveratrol derivative" described in this invention refers to a trans-stilbene derivative containing a benzene ring modification. This trans-stilbene derivative can be represented by two benzene rings named A ring and B ring, and a vinyl group connecting the A ring and B ring. The A ring represents a benzene ring with hydroxyl substituents at positions 2 and 4, and the B ring represents a benzene ring where one or more sites at positions 3, 4, and 5 can be substituted with a methoxy group, or where positions 3, 4, and 5 are not substituted with any substituent. Preferably, the oxidized resveratrol derivative can include, but is not limited to, structures shown in formulas (II), (III), and (IV).

[0011] A method for preparing oxidized resveratrol derivatives includes the following steps: (1) Using 1,3-bis(benzyloxy)benzene as raw material, (E)-2-(2,4-bis(benzyloxy)styryl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane was synthesized via halogenation, Sonogashira cross-coupling reaction, deprotection, and hydroboration reaction as intermediate I. The structure of intermediate I is shown in formula (V): Formula (V) (2) Using intermediate I obtained in step (1) as raw material, the oxidized resveratrol derivative is obtained by coupling reaction and debenzylation protection reaction with halomethoxybenzene compounds and purification.

[0012] Step (1) specifically includes the following steps: (1.1) At 20~30℃, silver salt (1.5-2.0 eq.) and halogen element (1.0-1.5 eq.) are added to an organic solvent containing 1,3-bis(benzyloxy)benzene and stirred. The reaction is carried out, and the resulting product is concentrated and purified to obtain (((4-halo-1,3-phenylene)bis(oxy))bis(methylene))diphenyl, wherein the silver salt is preferably silver acetate or silver sulfate, the halogen element is preferably elemental iodine, and the (((4-halo-1,3-phenylene)bis(oxy))bis(methylene))diphenyl is (((4-iodine-1,3-phenylene)bis(oxy))bis(methylene))diphenyl. The stirring time is preferably 14-18 hours, the organic solvent is preferably chloroform, and the purification is preferably carried out by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1). (1.2) At 20~30℃, ethynyltrimethylsilane (9.0-12.0 eq), triethylamine (2.0-4.0 eq), bis(triphenylphosphine)palladium dichloride (0.1-0.2 eq) and cuprous iodide (0.1-0.2 eq) are added to an organic solvent containing ((4-halo-1,3-phenylene)bis(oxy)bis(methylene)diphenyl chloride. Under nitrogen protection, the reaction is stirred, and the resulting product is concentrated and purified to obtain 2,4-bis(benzyloxy)phenylethynyltrimethylsilane. The stirring time is preferably 14-18 hours, the organic solvent is preferably tetrahydrofuran, and the purification is preferably carried out by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1). (1.3) At 20~30℃, potassium carbonate (1.5-3.0 eq) is added to an organic solvent containing 2,4-bis(benzyloxy)phenylethynyltrimethylsilane, the mixture is stirred, and the resulting product is concentrated and purified to obtain ((4-ethynyl-1,3-phenylene)bis(oxy)bis(methylene))diphenyl. The stirring time is preferably 2-5 hours, the organic solvent is preferably a mixture of methanol and tetrahydrofuran, and the purification method is preferably silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1). (1.4) At 20~30℃, copper powder (0.1-0.2 eq) and sodium methoxide (1.0-2.0 eq) are added to an organic solution containing ((4-ethynyl-1,3-phenylene)bis(oxy)bis(methylene))diphenyl and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborane) (1.0-2.0 eq). The reaction is carried out under nitrogen protection and the product is concentrated and purified to obtain intermediate I. The stirring time is preferably 14-18 hours, the organic solvent is preferably ethanol, and the purification method is preferably silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1).

[0013] Step (2) specifically includes the following steps: (2.1) At 20~30℃, add halomethoxybenzene compound (1.0-2.0 eq), potassium carbonate (2.0-4.0 eq), and palladium salt (0.1-0.2 eq) to an organic solvent containing intermediate I. Heat to 90-95℃ under nitrogen protection and stir the reaction. Concentrate and purify the product to obtain intermediate II. The stirring time is preferably 14-18 hours. The organic solvent is preferably a mixture of dioxane and water. The palladium salt is preferably diphenylphosphine-palladium dichloride. The purification method is preferably silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1). (2.2) At -75~-80℃, pentamethylbenzene (9.0-12.0 eq) and boron trichloride (1.5-3.0 eq) (in 1.0 M hexane solution) are added to an organic solvent containing intermediate II. The mixture is stirred under nitrogen protection. The resulting reaction mixture is quenched with ice water and extracted with ethyl acetate. The combined organic layers are dried with anhydrous sodium sulfate, filtered, concentrated, and purified to obtain oxidized resveratrol derivatives. The preferred stirring temperature is -75~-80℃, the preferred stirring time is 1-2 hours, and the preferred purification method is silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1).

[0014] The halomethoxybenzene compound is any one of 4-bromo-1,2-dimethoxybenzene, 1-bromo-3,5-dimethoxybenzene, or 1-bromo-4-methoxybenzene.

[0015] The beneficial effects of this invention are as follows: The three oxidized resveratrol derivatives provided by this invention exhibit core advantages far exceeding those of the parent compound, oxidized resveratrol, in the field of whitening skincare products. They achieve highly efficient whitening regulation across the entire melanin synthesis and transport chain, while simultaneously addressing the stability issues of traditional whitening ingredients. They possess extremely high market development potential and application value, as detailed below: At the core whitening activity level, the three derivatives exhibit dual and potent inhibition of key steps in melanin production and transport, demonstrating significantly superior whitening efficacy compared to oxidized resveratrol. On one hand, they exhibit more prominent human tyrosinase inhibitory activity. Tyrosinase, as the rate-limiting enzyme in melanin synthesis, is the core target of whitening ingredients. The derivatives' potent inhibition of this enzyme directly blocks the initiation of melanin production, reducing melanin synthesis at its source. On the other hand, the derivatives possess excellent melanin synthesis inhibitory activity, further inhibiting melanin production within melanocytes and enhancing the whitening effect. Simultaneously, they also possess significant PAR-2 ​​inhibitory activity. PAR-2, a key receptor regulating melanosome transport, effectively blocks the transport of melanosomes from melanocytes to keratinocytes when its activity is inhibited, preventing melanin deposition on the skin surface. This achieves end-to-end whitening, better meeting the skincare product's demand for efficient and comprehensive whitening. Regarding the stability and applicability of ingredients, this invention addresses the core pain point of traditional stilbene compounds (such as oxidized resveratrol) being easily oxidized and having poor stability, which leads to reduced activity and weakened efficacy. The oxidized resveratrol derivative provided by this invention improves molecular stability through chemical structure optimization. It can maintain good activity and chemical stability during the production, storage and use of skin care products, and is not prone to degradation. This ensures the long-lasting whitening effect and reduces the risk of irritation caused by ingredient degradation, making it more compatible.

[0016] In summary, the three oxidized resveratrol derivatives provided by this invention have more prominent efficacy and better stability than oxidized resveratrol, and are more in line with the requirements of industrial production and market application of skin care products. Attached Figure Description

[0017] Figure 1 The NMR spectrum of intermediate I; Figure 2 The NMR spectrum of JK-55 is shown below. Figure 3 The C-NMR spectrum of JK-55; Figure 4 The NMR spectrum of JK-56 is shown below. Figure 5 The C-NMR spectrum of JK-56; Figure 6 The NMR spectrum of JK-23 is shown below. Figure 7 The C-NMR spectrum of JK-23; Figure 8 Photographs of water, 1% aqueous solution of JK-23, 1% aqueous solution of JK-55, 1% aqueous solution of JK-56, 1% aqueous solution of resveratrol, and 1% aqueous solution of oxidized resveratrol (from left to right) on day 1 for stability testing. Figure 9 Photographs of water, 1% aqueous solution of JK-23, 1% aqueous solution of JK-55, 1% aqueous solution of JK-56, 1% aqueous solution of resveratrol, and 1% aqueous solution of oxidized resveratrol (from left to right) after two months of stability testing. Detailed Implementation

[0018] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0019] I. Materials and Reagents MNT-1 cells were purchased from Zhongqiao Xinzhou, catalog number ZQ1100; DMEM high-glucose medium (phenol red-free), purchased from Procell, catalog number PM150223; Fetal bovine serum (FBS), purchased from Ausbian, catalog number Ws500T; Triple antibody (penicillin-streptomycin-amphomycin B solution, 100X), purchased from Beyotime, product number C0224-100ml; NEAA-100X, purchased from Gibco, part number 11140050; Triton X-100, purchased from Sigma, part number T9284; PMSF was purchased from Beyotime, product number ST506; BCA test kit, purchased from Thermo Scientific, catalog number A55860; L-DOPA, purchased from Sigma, product number D9628; Peptide Amide, purchased from MCE, product number HY-W251181; Kojic acid, purchased from MCE, product number HY-W050154.

[0020] Unless otherwise specified, all reagents and materials used in the following examples are commercially available products, and all methods used are conventional methods known to those skilled in the art.

[0021] II. Methods This invention provides a novel method for synthesizing oxidized resveratrol derivatives. By synthesizing a new intermediate I, and using it as a raw material, the three oxidized resveratrol derivatives are obtained through coupling reaction, debenzylation protection reaction, and purification. Intermediate I has a simple structure and can be used as a precursor to synthesize a series of oxidized resveratrols, which can reduce the difficulty of synthesis, improve the synthesis efficiency, realize the efficient utilization of raw materials, and has better applicability.

[0022] 1. Synthesis of oxidized resveratrol derivatives 1.1 Synthesis of Intermediate I (1) Synthesis of (((4-iodo-1,3-phenylene)bis(oxy))bis(methylene))diphenyl, the synthetic route is shown in formula (1): Equation (1) At 25°C, silver acetate (4.32 g, 25.86 mmol, 1.5 eq.) and elemental iodine (5.26 g, 20.69 mmol, 1.2 eq.) were added to a chloroform (100 mL) solution containing 1,3-bis(benzyloxy)benzene (5.0 g, 17.24 mmol, 1.0 eq.). The resulting mixture was stirred at room temperature for 16 hours. The mixture was then concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain (((4-iodo-1,3-phenylene)bis(oxy))bis(methylene))diphenyl (6.6 g, yellow solid, yield: 92.0%). LCMS analysis of the product showed LCMS: m / z = 417.0 [M+H]+. (2) Synthesis of 2,4-bis(benzyloxy)phenylethynyltrimethylsilane, the synthetic route is shown in formula (2): Equation (2) At 25°C, to a solution of tetrahydrofuran (100 mL) containing (((4-iodo-1,3-phenylene)bis(oxy))bis(methylene))diphenyl (7.75 g, 18.63 mmol, 1.0 eq.), ethynyltrimethylsilane (18.26 g, 186.30 mmol, 10.0 eq.), triethylamine (5.64 g, 55.89 mmol, 3.0 eq.), bis(triphenylphosphine)palladium dichloride (1.31 g, 1.86 mmol, 0.1 eq.), and cuprous iodide (353 mg, 1.86 mmol, 1.86 eq.) were added. The resulting mixed reaction solution was stirred at room temperature for 16 hours under nitrogen protection (mmol, 0.1 eq.). The resulting mixed reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain 2,4-bis(benzyloxy)phenylethynyltrimethylsilane (7.58 g, brown solid, crude product, yield 75.6%). The product was analyzed by LCMS, and the LCMS value was found to be m / z = 387.2 [M+H]+. (3) Synthesis of ((4-ethynyl-1,3-phenylene)bis(oxy)bis(methylene))diphenyl, the synthetic route is shown in formula (3): Equation (3) At 25°C, potassium carbonate (5.41 g, 39.8 mmol, 2.0 eq.) was added to a methanol / tetrahydrofuran (50 mL / 50 mL) solution containing 2,4-bis(benzyloxy)phenylethynyltrimethylsilane (7.58 g, 19.59 mmol, 1.0 eq.). The resulting mixture was stirred at room temperature for 3 hours, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30 / 1) to obtain ((4-ethynyl-1,3-phenylene)bis(oxy)bis(methylene))diphenyl (5.5 g, yellow solid, yield 89.4%). LCMS analysis showed that the product had an LCMS value of m / z = 315.1 [M+H]+. (4) Synthesis of (E)-2-(2,4-bis(benzyloxy)styryl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane, the synthetic route is shown in formula (4): Equation (4) At 25°C, copper powder (63 mg, 0.99 mmol, 0.1 eq.) and sodium methoxide (53 mg) were added to an ethanol (50 mL) solution containing ((4-ethynyl-1,3-phenylene)bis(oxy)bis(methylene))diphenyl (3.1 g, 9.87 mmol, 1.0 eq.) and 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bis(1,3,2-dioxaborane) (3.76 g, 14.81 mmol, 1.5 eq.). (mg, 9.87mmol, 1.0eq.), the resulting mixed reaction solution was stirred at room temperature for 16 hours under nitrogen protection. The resulting mixed reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20 / 1) to obtain (E)-2-(2,4-bis(benzyloxy)styryl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (2.8g, yellow solid, yield: 64.1%), referred to as intermediate I; the product was analyzed by LCMS, and the LCMS values ​​were found to be: m / z = 443.2 [M+H]+. Nuclear magnetic resonance (NMR) analysis of intermediate I ( Figure 1 ): 1 H NMR (400 MHz, DMSO-d6): δ7.58-7.54 (m, 2 H), 7.45-7.33 (m, 10 H), 6.75 (d, J=2.4 Hz 1 H), 6.63 (dd, J=8.4 Hz, 2.0 Hz, 1 H), 5.96 (d, J=18.4 Hz1 H), 5.18 (s, 2 H), 5.11 (s, 2 H), 1.21 (s, 2 H). 1.2 Synthesis of 2,4-dihydroxy-4'-methoxystilbene (abbreviated as JK-55) (1) Synthesis of (E)-(((4-(4-methoxystyrene)-1,3-benzene)di(oxy))di(methylene))dibenzene, the synthetic route is shown in formula (5): Equation (5) At 25°C, potassium carbonate (468 mg, 3.39 mmol, 3.0 eq) and diphenylphosphino-palladium dichloride (83 mg, 0.113 mmol, 0.1 eq) were added to a solution of dioxane / water (10 mL / 1 mL) containing intermediate I (500 mg, 1.13 mmol, 1.0 eq) and 1-bromo-4-methoxybenzene (211 mg, 1.13 mmol, 1.0 eq). The resulting mixture was heated to 90°C and stirred for 16 hours under nitrogen protection. The resulting reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to obtain (E)-(((4-(4-methoxystyrene)-1,3-benzene)di(oxo))di(methylene))dibenzene (380 mg, white solid, yield 79.6%). The product was analyzed by LCMS, and the LCMS m / z was 423.1. [M+H]+. (2) Synthesis of 2,4-dihydroxy-4'-methoxystilbene (abbreviated as JK-56), the synthetic route is shown in formula (6): Equation (6) At -78°C, pentamethylbenzene (696 mg, 9.0 mmol, 10.0 eq.) and boron trichloride (1.8 mL, 1.0 M hexane solution, 1.0 M hexane solution, 1.8 mL, 1.8 mmol, 2.0 eq.) were added to a solution of dichloromethane (380 mg, 0.90 mmol, 1.0 eq.) containing (E)-(((4-(4-methoxystyrene)-1,3-benzene)di(oxy)di(methylene)di(methyl) ether (380 mg, 0.90 mmol, 1.0 eq.). The resulting mixture was stirred at -78°C for 1 hour under nitrogen protection. The resulting reaction mixture was quenched with ice water (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3 / 1) to give JK-55 (47.74 mg, white solid, yield: 21.9%). LCMS spectral analysis of JK-55 revealed the following: LCMS m / z: 243.0 [M+H]+. Nuclear magnetic resonance (NMR) spectroscopy analysis of JK-55 Figure 2-3 ): 1H NMR (400 MHz, DMSO-d6): 7.40 (d, J=8.4 Hz, 2 H), 7.33 (d, J=9.2 Hz1 H), 7.21 (d, J=16.8 Hz, 1 H), 6.93-6.86 (m, 3 H), 6.31-6.30 (m, 2 H), 3.79 (s, 3 H). 13 C NMR (100 MHz, DMSO-d6): 158.8, 157.5, 155.7, 131.5, 126.8, 124.7,121.4, 116.9, 113.6, 107.0, 102.2, 54.3. Synthesis of 1,3,2,4-dihydroxy-3'4'-dimethoxystilbene (abbreviated as JK-56) (1) Synthesis of (E)-((4-(3,4-dimethoxystyryl)-1,3-phenylene)bis(oxy)bis(methylene))diphenyl, the synthetic route is shown in formula (7): Equation (7) At 25°C, potassium carbonate (373 mg, 2.7 mmol, 3.0 eq) and diphenylphosphino-palladium dichloride (66 mg, 0.09 mmol, 0.1 eq) were added to a solution of dioxane / water (10 mL / 2 mL) containing intermediate I (400 mg, 0.9 mmol, 1.0 eq) and 4-bromo-1,2-dimethoxybenzene (195 mg, 0.9 mmol, 1.0 eq). The resulting mixture was heated to 90°C and stirred for 16 hours under nitrogen protection. The resulting reaction mixture was then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give (E)-((4-(3,4-dimethoxystyryl)-1,3-phenylene)bis(oxy)bis(methylene))diphenyl (258 mg, white solid, yield 63.4%). The product was analyzed by LCMS, and the LCMS values ​​were m / z: 453.3 [M+H]+. (2) Synthesis of 2,4-dihydroxy-3'4'-dimethoxystilbene (abbreviated as JK-56), the synthetic route is shown in formula (8): Equation (8) Pentamethylbenzene (844 mg, 5.7 mmol, 10.0 eq.) and boron trichloride (1.14 mL, 1.14 mmol, 2.0 eq.) in a solution of (E)-((4-(3,4-dimethoxystyryl)-1,3-phenylene)bis(oxy)bis(methylene))diphenyl (258 mg, 0.57 mmol, 1.0 eq.) in dichloromethane (15 mL). The mixture was stirred at -78 °C for 1 hour under nitrogen protection. The resulting reaction mixture was quenched with ice water (10 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give JK-56 (39.48 mg, white solid, yield 25.5%).

[0023] LCMS spectral analysis of JK-56 revealed the following: LCMS m / z: 273.1 [M+H]+. Nuclear magnetic resonance (NMR) spectroscopy analysis of JK-56 Figure 4-5 ): 1 HNMR (400 MHz, DMSO-d6): 9.56 (s, 1 H), 9.39 (s, 1H), 7.32 (d, J=8.8Hz, 1 H), 7.14 (d, J=16.4 Hz 1 H), 7.08 (d, J=1.2 Hz 1 H), 6.98 (dd, J=8.4Hz, 1.6 Hz, 1 H), 6.93-6.89 (m, 2 H), 6.33 (d, J=2.0 Hz 1 H), 6.24 (dd, J=8.4Hz, 2.4 Hz, 1 H), 3.80 (s, 3 H), 3.75 (s, 3 H). 13 C NMR (100 MHz, DMSO-d6): 158.4, 156.4, 149.4, 148.4, 131.8, 127.6,124.9, 122.5, 119.2, 116.1, 112.4, 109.3, 107.6, 103.1, 55.9. Synthesis of 1,4,2,4-dihydroxy-3'5'-dimethoxystilbene (abbreviated as JK-23) (1) Synthesis of (E)-((4-(3,5-dimethoxystyryl)-1,3-phenylene)bis(oxy)bis(methylene))diphenyl, the synthetic route is shown in formula (9): Equation (9) At 25°C, potassium carbonate (468 mg, 3.39 mmol, 3.0 eq) and diphenylphosphino-palladium dichloride (83 mg, 0.113 mmol, 0.1 eq) were added to a solution of dioxane / water (10 mL / 2 mL) containing intermediate I (500 mg, 1.13 mmol, 1.0 eq) and 1-bromo-3,5-dimethoxybenzene (245 mg, 1.13 mmol, 1.0 eq). The resulting mixture was heated to 90°C and stirred for 16 hours under nitrogen protection, and the resulting reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10 / 1) to give (E)-((4-(3,5-dimethoxystyryl)-1,3-phenylene)bis(oxy)bis(methylene))diphenyl (342 mg, yellow solid, yield 66.7%). The product was analyzed by LCMS, and the LCMS m / z was 453.3 [M+H]+. (2) Synthesis of 2,4-dihydroxy-3'5'-dimethoxystilbene, the synthetic route is shown in formula (10): Equation (10) At -78°C, pentamethylbenzene (1.11 g, 7.5 mmol, 10.0 eq.) and boron trichloride (1.5 mL, 1.50 mmol, 2.0 eq.) were added to a solution of (E)-(((4-(3,5-dimethoxystyryl)-1,3-phenylenephenylene)bis(oxy))bis(methylene))diphenyl (342 mg, 0.75 mmol, 1.0 eq.) in dichloromethane (10 mL, 1.0 M, 1.0 M, 1.50 mmol, 2.0 eq.). The resulting mixture was stirred at -78°C for 1 hour under nitrogen protection. The reaction mixture was quenched with ice water (30 mL), extracted with ethyl acetate (10 mL × 2), and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 1) to give JK-23 (18.37 mg, white solid, yield 8.9%).

[0024] LCMS spectrum analysis of JK-23 revealed the following LCMS values: m / z: 273.0 [M+H]+. Nuclear magnetic resonance (NMR) spectroscopy analysis of JK-23 ( Figure 6-7 ): 1H NMR (400 MHz, DMSO-d6): δ 9.61 (s, 1 H), 9.43 (s, 1 H), 7.34 (d, J=8.8 Hz, 1 H), 7.26 (d, J=16.4 Hz, 1 H), 6.92 (d, J=16.4 Hz, 1 H), 6.63 (d, J=2.4 Hz, 2 H), 6.34 (dd, J=5.2 Hz, 2.0 Hz, 2 H), 6.25 (dd, J=8.4 Hz, 2.0 Hz, 1 H), 3.76 (s, 6 H). 13 C NMR (100 MHz, DMSO-d6): δ 161.1, 158.8, 156.7, 140.9, 128.1,124.99, 124.96, 115.6, 107.8, 104.2, 103.1, 99.4, 55.6. 2. Human tyrosinase inhibition experiment and evaluation Melanin synthesis is influenced by tyrosinase, which catalyzes the hydroxylation of L-tyrosine to dopa and then oxidizes dopa to dopaquinone, which forms the final product, melanin. Test substances that inhibit tyrosinase activity can suppress the tyrosinase-catalyzed conversion of L-tyrosine to dopaquinone. The inhibitory effect of the test substance on tyrosinase activity is evaluated by measuring the absorbance of dopaquinone at 475 nm and analyzing the changes in absorbance.

[0025] The relevant experimental groups and experimental procedures are as follows: Total intracellular protein extraction from MNT-1 cells: MNT-1 cells were cultured in T150 flasks using DMEM (containing 20% ​​FBS, 1X NEAA, and 1X triple antibodies); after confluence, cells were collected, and an appropriate amount of sodium phosphate buffer (containing 1% Triton X-100 and 1% PMSF) was added. Cells were lysed by repeated freeze-thaw cycles in liquid nitrogen; the cells were centrifuged at 12000g for 15 min at 4°C, and the supernatant protein was collected; protein quantification was performed using a BCA assay kit, and the protein concentration was adjusted to 1 mg / ml.

[0026] Experimental Groups: Negative control group (Ctl): 110 μL sodium phosphate buffer + 90 μL L-DOPA (5 mM); Background experimental group (Mdl): 90ul MNT-1 intracellular total protein (1mg / ml) + 90ul L-DOPA (5 mM) + 20ul sodium phosphate buffer; Positive control group (P): 90ul MNT-1 intracellular total protein (1mg / ml) + 90ul L-DOPA (5mM) + 20ul positive control (kojic acid, final concentrations per well were 0.0128μM, 0.064μM, 0.32μM, 1.6μM, 8μM, 40μM, 200μM, and 1000μM, respectively). Sample group (IBT): 90ul MNT-1 intracellular total protein (1mg / ml) + 90ul L-DOPA (5mM) + 20ul sample; A. Sample group 1 (JK-23), the final reaction concentrations per well were 28.29, 5.66, 1.13, 0.23, 0.05, and 0.01 μM, respectively; B. Sample group 2 (JK-55), the final reaction concentrations per well were 178.06, 35.61, 7.12, 1.42, 0.28, 0.06, and 0.01 μM, respectively; C. Sample group 3 (JK-56), with final reaction concentrations of 151.03, 30.21, 6.04, 1.21, 0.24, 0.05, and 0.01 μM per well.

[0027] For each concentration, three parallel wells were prepared for the positive control group and the sample group for detection. The total volume of each well was 200 μL, and the reaction was carried out at 37°C for 60 min. The OD value was then measured at 475 nm using a microplate reader.

[0028] Calculate the tyrosinase inhibition rate using the following formula: A curve was plotted with the concentration of the test samples (including the positive control group and the sample group) on the x-axis and the corresponding tyrosinase inhibition rate on the y-axis. A regression equation was obtained by fitting the curve (R>0.9). Based on the regression equation, the sample concentration (IC50) corresponding to a tyrosinase inhibition rate of 50% was calculated. 50 IC 50 The smaller the value, the stronger the inhibitory activity. The statistical results are shown in Table 1.

[0029] Table 1. Results of human tyrosinase inhibition experiment Kojic acid is a widely used active ingredient in current skin whitening products. As shown in Table 1, in the human tyrosinase inhibition experiment, JK-23, JK-55, and JK-56 exhibited significantly lower IC50 values ​​compared to kojic acid. 50 Value, i.e., more significant tyrosinase inhibitory activity.

[0030] 3. MNT-1 melanin synthesis inhibition model and evaluation MNT-1 cells are a commonly used cell model for studying melanin production. By measuring the changes in melanin synthesis after whitening substances act on melanocytes, the whitening activity of the raw materials can be evaluated.

[0031] The relevant experimental groups and experimental procedures are as follows: MNT-1 cells were seeded at 7.5 weeks / 500 μL / well in 24-well plates and incubated overnight. The supernatant was discarded before testing. Each test required a negative control group (NC), a test sample group, a positive control group, and a blank control group. The concentrations of JK-23, JK-55, and JK-56 were pre-screened to select concentrations that did not affect cell growth; these were designated as the non-toxic concentrations. Cells were dissolved in PBS to a high concentration, filtered for sterilization, and then diluted to the appropriate concentration with phenol red cell-free medium.

[0032] Blank group (BK): Culture medium; Negative control group (NC): MNT-1 cells + culture medium; Positive control group: MNT-1 cells + peptide amantadine, with final concentrations of peptide amantadine per well of 0.08, 0.25, 0.74, 2.22, and 6.67 μM, respectively; Sample group (IBT): MNT-1 cells + sample; A. Sample group 1 (JK-23), the final reaction concentrations of each well were 0.48, 1.45, 4.36, and 13.09 μM, respectively; B. Sample group 2 (JK-55), the final reaction concentrations of each well were 0.61, 1.83, 5.5, and 16.49 μM, respectively; C. Sample group 3 (JK-56), the final reaction concentrations of each well were 0.52, 1.55, 4.66, 13.98, and 41.95 μM, respectively; D. Sample group 4 (oxidized resveratrol), the final reaction concentrations of each well were 0.54, 1.63, 4.9, 14.71, and 44.13 μM, respectively; Each well had a total volume of 500 μL, with three replicates. Cells were incubated for 5 days. The intracellular melanin content of MNT-1 cells was detected using the sodium hydroxide lysis method. Cell phenotypes were photographed under a light microscope before detection. The supernatant was discarded, and the cells were rinsed with 500 μL of PBS and discarded. The lower cell layer was incubated with 150 μL of 1M NaOH solution (containing 10% DMSO) at 60°C for 30 min. 100 μL of the supernatant was transferred to a 96-well plate, and melanin content was measured using a microplate reader at a wavelength of 405 nm.

[0033] The MNT-1 melanin synthesis inhibition rate is calculated using the following formula: ; A curve was plotted with the concentration of the tested samples (including the positive control group and the sample group) on the x-axis and the corresponding MNT-1 melanin synthesis inhibition rate on the y-axis. A regression equation was obtained by fitting the curve (R>0.9). Based on the regression equation, the sample concentration (IC50) corresponding to a 50% MNT-1 melanin synthesis inhibition rate was calculated. 50 IC 50 The smaller the value, the stronger the inhibitory activity. The statistical results are shown in Table 2.

[0034] Table 2. Results of MNT-1 Melanin Synthesis Inhibition Experiment Peptide-Amide is currently one of the most effective ingredients in whitening skincare products and is used as a positive control here. The experimental results above show that in the MNT-1 melanin synthesis inhibition experiment, JK-23, JK-55, and JK-56 exhibited significantly lower IC50 values ​​compared to oxidized resveratrol. 50 The value, i.e., more significant melanin synthesis inhibition activity, shows that JK-55 exhibits the best melanin synthesis inhibition activity, which is close to that of peptide Ammido.

[0035] 4. Inhibition experiments and evaluation of PAR-2 HaCaT-PAR2-OE cells were seeded at a rate of 3-3.5 weeks / well / 100 μL in black 96-well plates and cultured for 24 h. After 24 h, the supernatant was aspirated, and each well was washed twice with 200 μL of 1×HHBS (containing 20 mM HEPES). Each well was then incubated with 100 μL of staining solution (Cal-520® AM working solution: 50 μL of Cal-520® AM stock solution (1 mg / ml) diluted with 6 mL of 1×HHBS) and incubated in a CO2 incubator for 60 min ± 5 min. After staining, the supernatant was discarded, and 200 μL of 1×HHBS was added to each well for washing twice. 75 μL of 1×HHBS was added to each well. 25 μL of 1×HHBS was added to the model group, 25 μL of different concentrations of sample were added to the sample group, and 25 μL of positive control was added to the positive control group. The cells were incubated in a cell culture incubator for 10 min. 25 μL of SLIGRL-NH2 (PAR-2 ​​agonist, final concentration 2 μM) was automatically added to each well of the FlexStation3 cell culture device, and the signal was collected. The excitation wavelength (Ex) was 485 nm, and the emission wavelength (Em) was 525 nm.

[0036] The agonist, analyte, and positive control were all prepared using 1×HHBS. The positive control used the PAR-2 ​​antagonist AZ3451.

[0037] The HaCaT-PAR2-OE cells mentioned above are PAR-2 ​​overexpressing stable cell lines constructed by Shanghai Jiakai Biotechnology Co., Ltd. The human-PAR2 gene CDS sequence was transferred into HaCaT cells (human immortalized keratinocytes) through lentiviral transfection.

[0038] Model group: 100 μL 1×HHBS + 25 μL SLIGRL-NH2 (PAR-2 ​​agonist, final concentration 2 μM); Negative control group (NC): 150 μL 1×HHBS; Positive control group (AZ3451): The final concentrations of AZ3451 per well were 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256, and 0.0000512 μM, respectively. Sample group (IBT): A. Sample group 1 (JK-23), the final reaction concentrations of each well were 39.28, 13.09, 4.36, and 1.45 μM, respectively; B. Sample group 2 (JK-55), the final reaction concentrations of each well were 148.39, 49.46, 16.49, 5.5, and 1.83 μM, respectively; C. Sample group 3 (JK-56), the final reaction concentrations of each well were 125.86, 41.95, 13.98, 4.66, and 1.55 μM, respectively; D. Sample group 4 (oxidized resveratrol), the final reaction concentrations of each well were 397.13, 132.38, 44.13, 14.71, 4.9, and 1.63 μM, respectively; ; A curve was plotted with the concentration of the test samples (including the positive control group and the sample group) on the x-axis and the corresponding PAR-2 ​​inhibition rate on the y-axis. A regression equation was obtained by fitting the curve (R>0.9). Based on the regression equation, the sample concentration (IC50) corresponding to a PAR-2 ​​inhibition rate of 50% was calculated. 50 IC 50 The smaller the value, the stronger the inhibitory activity. The statistical results are shown in Table 3.

[0039] Table 3 Results of PAR-2 ​​Inhibition Experiment As can be seen from the experimental results in Table 3, in the PAR-2 ​​inhibition experiment, JK-23, JK-55, and JK-56 showed significantly lower IC50 values ​​compared to oxidized resveratrol. 50 The value indicates more significant PAR-2 ​​inhibitory activity.

[0040] 4. Stability test To investigate the stability of the prepared samples, oxidized resveratrol, resveratrol, JK-23, JK-55, and JK-56 were prepared into 1% aqueous solutions and observed at 45°C for two months. Results were then observed after the experiment. Figure 8 As shown, from left to right, the following are water, a 1% aqueous solution of JK-23, a 1% aqueous solution of JK-55, a 1% aqueous solution of JK-56, a 1% aqueous solution of resveratrol, and a 1% aqueous solution of oxidized resveratrol. Figure 8 To display the sample photos from day 1, Figure 9 This is a photo of the sample after it has been stored for two months.

[0041] The experimental results showed that after two months of stability testing at 45℃, the 1% aqueous solutions prepared from JK-23, JK-55, and JK-56 were all clear and transparent; while the 1% aqueous solutions prepared from oxidized resveratrol and resveratrol both showed varying degrees of discoloration. This indicates that the 1% aqueous solutions of JK-23, JK-55, and JK-56 have better stability.

[0042] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Use of an oxidized resveratrol derivative in the preparation of a product for whitening or removing freckles.

2. Use according to claim 1, characterized in that, Specifically, any of the following uses: (1) Use of an oxidized resveratrol derivative as a tyrosinase inhibitor; (2) Use of an oxidized resveratrol derivative as a PAR-2 inhibitor; (3) Use of an oxidized resveratrol derivative as a MNT-1 melanin synthesis inhibitor.

3. Use according to any one of claims 1 or 2, characterized in that, The product is a skin care product.

4. Use according to any one of claims 1 or 2, characterized in that: The structure of the oxidized resveratrol derivative is shown in formula (I): Formula (I) wherein R1, R2 and R3 are independently selected from at least one of hydrogen, hydroxyl or methoxyl.

5. Use according to claim 4, characterized in that: The oxidized resveratrol derivative is any one of 2,4-dihydroxy-3',5'-dimethoxy stilbene with the structure shown in formula (II), 2,4-dihydroxy-3',4'-dimethoxy stilbene with the structure shown in formula (III), or 2,4-dihydroxy-4'-methoxy stilbene with the structure shown in formula (IV): Formula (II) Formula (III) Formula (IV).

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