Phenylethyl resorcinol tannic acid energy whitening lightening ternary co-assembly and preparation method and application thereof

Abstract

The present application belongs to the technical field of biological medicine, cosmetics, personal care and health, and discloses a phenylethyl resorcinol tannic acid energy whitening and freckle lightening ternary co-assembly body as well as a preparation method and application thereof. The method comprises the following steps: dissolving tannic acid and a cyclodextrin compound in water, adding a phenylethyl resorcinol solution into the water phase, performing ultrasonic dispersion, centrifugation, rotary evaporation and drying to obtain the ternary co-assembly body. The assembly body can destroy the crystal structure of phenylethyl resorcinol through non-covalent interaction, and can improve the water solubility and storage stability of phenylethyl resorcinol. The apparent solubility of the assembly body can be increased by 153.6 times compared with that of the monomer. The assembly body can realize the synergistic delivery of phenylethyl resorcinol and tannic acid, and the 2h penetration effect can be increased by 5.78 times compared with that of the monomer. The assembly body can cover the whitening links of melanin production, inflammatory pigmentation and cell energy metabolism, and can improve the comprehensive whitening effect. The method has a mild process and low equipment requirement. The obtained assembly body can be used as a whitening cosmetic raw material, and can be applied to skin care products such as essence, emulsion, facial mask and cream.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine, beauty and personal care health technology, and particularly relates to a phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly, its preparation method and application. Background Technology

[0002] Phenethyl resorcinol, commonly known as 377, is a potent tyrosinase inhibitor used in cosmetic whitening products. It reduces melanin formation at its source by inhibiting tyrosinase activity, thus being widely used in whitening and spot-fading products. However, 377 is a hydrophobic crystalline small molecule with low aqueous solubility and is sensitive to light, heat, oxidation, and metal ions. In high-aqueous-phase systems such as serums, lotions, and gels, it is prone to problems such as insufficient dissolution, crystallization, discoloration, and decreased content. Increasing its dosage or enhancing penetration may increase irritation and formulation compatibility risks, limiting its application in transparent, refreshing, and highly stable whitening and spot-fading products.

[0003] Regarding the solubility and stability of 377, various improvement schemes have been developed in the industry. For example, CN103860389A discloses a 377-loaded nanostructured lipid carrier, CN104523442B discloses 377 lipid nanoparticles, CN103536454A discloses a 377 hydroxypropyl-β-cyclodextrin inclusion complex, and CN108743430B discloses a 377 co-delivery nanocomposition. These schemes can improve the water dispersibility, stability, irritation, or delivery efficiency of 377 to some extent, but they generally rely on a high proportion of oils, emulsifiers, polyols, or large amounts of cyclodextrin. Some also require high-shear and high-pressure homogenization processes, resulting in complex systems, a heavy burden of excipients, and most of them involve external encapsulation, emulsification dispersion, or single host-guest inclusion, which are still difficult to fully change the crystalline state and recrystallization tendency of 377 itself.

[0004] Tranexamic acid, also known as aminomethylformamide or tranexamic acid, is a commonly used water-soluble whitening and spot-fading ingredient. Unlike whitening agents that target tyrosinase alone, it primarily works by inhibiting the plasminogen / plasmin system, interfering with abnormal signal transduction between keratinocytes and melanocytes induced by ultraviolet radiation, inflammation, and hormones. This reduces the release of melanin-promoting mediators such as arachidonic acid, prostaglandins, and related cytokines, thereby inhibiting excessive melanocyte activation and melanin production. Simultaneously, tranexamic acid can alleviate pigmentation and dullness induced by inflammation and oxidative stress, aligning with the principles of energy-based whitening that emphasize improving the cellular microenvironment, reducing stress signals, and maintaining skin homeostasis. Therefore, it is suitable as a gentle, long-term, synergistic whitening and spot-fading ingredient.

[0005] From the perspective of skin whitening pathways, 377 and tranexamic acid are complementary: 377 focuses on inhibiting tyrosinase activity, reducing melanin synthesis; tranexamic acid focuses on inhibiting the plasmin-inflammatory mediator-melanocyte activation pathway, reducing the pigmentation deepening and recurrence caused by exogenous stimulation and the inflammatory microenvironment. The combination of the two helps to form a multi-stage skin whitening and spot-fading strategy, and reduces the irritation and stability pressure caused by relying solely on high concentrations of 377. CN104721064A has disclosed a skin whitening composition and cosmetic containing tetrahydrocurcumin, tranexamic acid, and phenylethyl resorcinol, indicating that the combination of 377 and tranexamic acid already has an application basis in the industry. However, 377 is hydrophobic and easily crystallizes, while tranexamic acid is hydrophilic and mainly distributed in the aqueous phase. When the two are simply physically mixed, it is difficult to form a stable homogeneous system. 377 may still undergo crystal rearrangement and long-term crystallization, and tranexamic acid is also difficult to achieve uniform binding and synergistic delivery with 377 at the microscale. Summary of the Invention

[0006] To address the technical problems existing in the prior art, the present invention aims to provide a ternary co-assembly of phenylethyl resorcinol and tranexamic acid for skin whitening and fading spots, its preparation method, and its application, thereby solving the aforementioned technical problems. This invention addresses the issue of the significant differences in the physicochemical properties of phenylethyl resorcinol and tranexamic acid, and the difficulty in achieving efficient co-delivery of the two in conventional water-based systems, by providing a ternary co-amorphous assembly composed of phenylethyl resorcinol, tranexamic acid, and a cyclodextrin-like compound.

[0007] The system of this invention is based on the complementary effects of tyrosinase inhibition by phenylethyl resorcinol and anti-inflammatory pigmentation and melanin regulation by tranexamic acid. The alcoholic pre-solution of phenylethyl resorcinol is added dropwise to the aqueous pre-solution of tranexamic acid-cyclodextrin. After water-alcohol solvent replacement and multiple non-covalent interactions, the phenylethyl resorcinol ternary amorphous assembly is obtained after drying.

[0008] According to a first aspect of the present invention, the present invention provides a method for preparing a phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly, comprising the following steps: (1) Add tranexamic acid and cyclodextrin compounds to water and stir to dissolve them to obtain a tranexamic acid-cyclodextrin aqueous solution; (2) Dissolve phenylethyl resorcinol (i.e., 377) in an alcohol solvent to obtain a phenylethyl resorcinol alcohol phase pre-solution; (3) Under light-protected conditions, the phenylethyl resorcinol alcohol phase pre-solution described in step (2) is added to the tranexamic acid-cyclodextrin aqueous phase co-solution described in step (1), ultrasonically treated, centrifuged to obtain the supernatant, the alcohol solvent is removed by rotary evaporation, and the mixture is freeze-dried to obtain the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly (ternary co-amorphous assembly).

[0009] In some embodiments, the cyclodextrin compound in step (1) is at least one of α-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and hydroxypropyl-α-cyclodextrin.

[0010] In some embodiments, the cyclodextrin compound in step (1) is hydroxypropyl-β-cyclodextrin or methyl-β-cyclodextrin.

[0011] In some embodiments, the mass-to-volume ratio of tranexamic acid to water in step (1) is 0.005-0.20:1 g / mL.

[0012] In some embodiments, the mass-to-volume ratio of tranexamic acid to water in step (1) is 0.01-0.1:1 g / mL.

[0013] In some embodiments, the mass ratio of tranexamic acid to cyclodextrin in step (1) is (1-40):(2-80).

[0014] In some embodiments, the temperature of stirring and dissolving in step (1) is 40-65°C.

[0015] In some embodiments, in step (2), phenylethyl resorcinol is dissolved in an alcohol solvent under light-protected conditions to obtain a phenylethyl resorcinol alcohol phase pre-solution; In some embodiments, the alcohol solvent in step (2) is at least one of ethanol, methanol, and isopropanol; In some embodiments, the alcohol solvent in step (2) is ethanol; In some embodiments, the mass-to-volume ratio of phenylethyl resorcinol to alcohol solvent in step (2) is 0.005-0.08:1 g / mL.

[0016] In some embodiments, in the preparation method provided by the present invention, the mass ratio of phenylethyl resorcinol, tranexamic acid, and cyclodextrin compounds is 1:(1-20):(2-30). In some embodiments, in the preparation method provided by the present invention, the mass ratio of phenylethyl resorcinol, tranexamic acid, and cyclodextrin compounds is 1:(2-10):(3-15).

[0017] In some embodiments, the ultrasonic treatment in step (3) takes 10-60 minutes.

[0018] In some embodiments, the frequency of the ultrasonic treatment in step (3) is 10-50 kHz.

[0019] In some embodiments, the temperature of the ultrasonic treatment in step (3) is 30-40°C.

[0020] In some embodiments, the centrifugation speed in step (3) is 2000-5000 rpm and the centrifugation time is 5-15 min; the purpose of centrifugation is to remove undissolved matter, coarse crystals and large particles; the supernatant needs to be collected after centrifugation.

[0021] In some embodiments, the temperature of the rotary evaporation in step (3) is 40-60°C.

[0022] In some embodiments, in step (3), after the phenethyl resorcinol alcohol phase pre-solution is mixed with the tranexamic acid-cyclodextrin aqueous phase co-solution, a mixture is obtained and then subjected to ultrasonic treatment; in the mixture, the volume percentage of alcohol solvent is 30-75%.

[0023] In some embodiments, the volume percentage of alcohol solvent in the mixture is 40-60%.

[0024] In some embodiments, the pre-freezing temperature of the freeze-drying in step (3) is -80±10℃, and the freeze-drying time is 24-72h.

[0025] According to a second aspect of the present invention, the present invention provides a phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly prepared by the above-described preparation method.

[0026] According to a third aspect of the present invention, the present invention provides the application of a phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly in the preparation of topical pharmaceuticals or cosmetics.

[0027] The phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly provided by the present invention can be used as a cosmetic raw material or a raw material for cosmetic active composition, for developing cosmetics with the effects of brightening skin tone, fading pigmentation, inhibiting melanin production and assisting in improving cell energy metabolism; the cosmetics include serums, lotions, gels, emulsions, creams, masks or lotions.

[0028] The phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly provided by this invention forms a single-phase or near-single-phase amorphous co-assembly system through non-covalent forces such as hydrogen bonding. This system can reduce the crystallization tendency of phenylethyl resorcinol and improve the synergistic dispersion stability of tranexamic acid and low water-soluble whitening active ingredients.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Improve water solubility and dispersibility: The phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly provided by the present invention can destroy the crystal structure of phenylethyl resorcinol, improve its molecular dispersion, and improve the performance of aqueous phase dispersion and resolubility; (2) Improve system stability: The phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly provided by the present invention restricts the migration of active ingredients through cyclodextrin inclusion and multiple non-covalent interactions, reduces oxygen contact, and avoids problems such as crystallization, aggregation, discoloration and decreased storage stability. (3) Enhance the synergistic effect of whitening: The phenylethyl resorcinol and tranexamic acid energy whitening and spot-fading ternary co-assembly provided by the present invention can work together to act on the melanin generation, transport and inflammatory pigmentation through the synergistic delivery of phenylethyl resorcinol and tranexamic acid, thereby improving the overall whitening effect. (4) Mild process and convenient application: The preparation conditions of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly provided by the present invention are mild and the equipment requirements are low, making it suitable for the stabilization treatment of poorly soluble whitening active ingredients and cosmetic formulation applications. Attached Figure Description

[0030] Figure 1 A comparison of the solubility of the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly and the control sample is shown in the figure. Figure 2 A comparison of the stability of the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly and the control sample; Figure 3 Raman spectroscopy comparison results of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 4 A polarizing microscope comparison of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 5 XRD comparison results of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 6 DLS spectrum of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly; Figure 7 TEM comparison images of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 8 SEM comparison images of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 9 FTIR comparison results of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 10 Figure showing the intermolecular interaction analysis results of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 11ESP analysis results for the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly and the control sample; Figure 12 The image shows the comparison results of FMO between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample. Figure 13 A comparison of melanin content between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 14 A comparison of tyrosinase activity results between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 15 The image shows the comparison of ET-1 expression levels between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample. Figure 16 A comparison of ATP content between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 17 A comparison of mitochondrial membrane potential between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 18 A comparison of ROS content between the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 19 A comparison of the autophagy index LC3Ⅱ / LC3Ⅰ of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 20 A comparison of the autophagy index p62 of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample; Figure 21 The image shows the comparison results of PGE2 in the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly and the control sample. Detailed Implementation

[0031] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1 A method for preparing a phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly includes the following steps: (1) Add 3.0 g of tranexamic acid and 6.0 g of cyclodextrin compound to 180 mL of pure water, wherein the cyclodextrin compound is hydroxypropyl-β-cyclodextrin, and stir to dissolve at 50 °C to obtain a tranexamic acid-cyclodextrin aqueous solution. (2) Under light-protected conditions, 1.0 g of phenylethyl resorcinol was dissolved in 120 mL of anhydrous ethanol to obtain a phenylethyl resorcinol alcohol phase pre-solution; (3) Under light-proof and stirring conditions, the phenylethyl resorcinol alcohol phase pre-solution obtained in step (2) is added to the tranexamic acid-cyclodextrin aqueous phase co-solution obtained in step (1), and after mixing, a mixture is obtained, wherein the volume fraction of ethanol in the mixture is 40%, and then ultrasonic treatment is performed. The ultrasonic treatment temperature is 35℃, the frequency is 40 kHz, and the time is 20 min to obtain the ultrasonically treated mixture. (4) The ultrasonic mixture obtained in step (3) is centrifuged to remove undissolved matter, coarse crystals and large particles. The centrifugation speed is 3000 rpm and the centrifugation time is 10 min. After centrifugation, the supernatant is collected. The supernatant is removed by rotary evaporation at 50°C, then pre-frozen at -80°C, and then freeze-dried for 48 h to obtain the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly (labeled as assembly 1).

[0033] Meanwhile, to screen for better processing conditions, this invention also conducted a series of experiments on the preparation method of the phenylethyl resorcinol tranexamic acid energy-based whitening and spot-fading ternary co-assembly. The processing conditions for each group were adjusted based on the preparation method of the phenylethyl resorcinol tranexamic acid energy-based whitening and spot-fading ternary co-assembly provided in Example 1. The adjusted parameters for each group are shown in Table 1 below. All other treatments were the same as in Example 1 and can be referred to as shown in Example 1. Except for the factors listed in Table 1, the amounts of other raw materials, cyclodextrin types, solvent types, order of addition, and centrifugation conditions were consistent with those in Example 1. The apparent solubility of each sample was measured according to the method in subsequent test example 1, i.e., the apparent solubility was converted into the concentration of phenylethyl resorcinol.

[0034] Table 1 Table 1 shows that the mass ratio of the three components, the ratio of each raw material to the solvent, the ultrasonic treatment conditions, the rotary evaporation temperature, and the freeze-drying time all significantly affect the apparent solubility of the obtained ternary co-assembled compound. Comparing the results of each group, the combination of process parameters used in Example 1 performed best overall, achieving an apparent solubility of phenylethyl resorcinol of 48.83 mg / mL. When the raw material ratio, solvent dosage, or treatment intensity deviated from these conditions, varying degrees of solubility decrease occurred, indicating that suitable component ratios and process conditions are beneficial for the full assembly of the three components and for improving the aqueous dispersion and solubilization capacity of the system. Therefore, the conditions described in Example 1 were determined to be the optimal preparation process.

[0035] Example 2 Example 2 is basically the same as Example 1, except that in step (1), 6.0 g of hydroxypropyl-β-cyclodextrin is replaced by 6.0 g of γ-cyclodextrin; the other raw material amounts, solvent composition, ultrasonic treatment, centrifugation, rotary evaporation and freeze drying conditions are the same as in Example 1.

[0036] Example 3 Example 3 is basically the same as Example 1, except that in step (1), 6.0 g of hydroxypropyl-β-cyclodextrin is replaced with 6.0 g of methyl-β-cyclodextrin; the other raw material amounts, solvent composition, ultrasonic treatment, centrifugation, rotary evaporation and freeze drying conditions are the same as in Example 1.

[0037] Example 4 Example 4 is basically the same as Example 1, except that in step (1), 6.0 g of hydroxypropyl-β-cyclodextrin is replaced by 6.0 g of sulfobutyl ether-β-cyclodextrin; the other raw material amounts, solvent composition, ultrasonic treatment, centrifugation, rotary evaporation and freeze drying conditions are the same as in Example 1.

[0038] Example 5 Example 5 is basically the same as Example 1, except that in step (1), 6.0 g of hydroxypropyl-β-cyclodextrin is replaced with 6.0 g of hydroxypropyl-α-cyclodextrin; the other raw material amounts, solvent composition, ultrasonic treatment, centrifugation, rotary evaporation and freeze drying conditions are the same as in Example 1.

[0039] Comparative Example 1 This comparative example uses a conventional solution inclusion method to prepare cyclodextrin inclusion complexes, which are then used to evaluate the differences in effectiveness between traditional cyclodextrin inclusion methods and the preparation methods of the embodiments of this invention.

[0040] This comparative example uses a conventional solution inclusion method to prepare phenylethyl resorcinol-cyclodextrin inclusion complexes, specifically including the following steps: (1) Weigh 0.2 g of phenylethyl resorcinol, add it to 10 g of anhydrous ethanol, shake and mix until a clear solution is formed, and obtain phenylethyl resorcinol ethanol solution; (2) Weigh 10 g of hydroxypropyl-β-cyclodextrin and add it to the phenylethyl resorcinol ethanol solution obtained in step (1). Stir thoroughly until the mixture is homogeneous to obtain a mixture. (3) The mixture obtained in step (2) is stirred for 60 min at a speed of 300 rpm under the condition of 50°C and protection from light to obtain the stirred mixture; (4) Place the stirred mixture in a disc and vacuum dry it at 50°C for 120 min to obtain a white, loose, porous solid. Grind it into powder to obtain phenylethyl resorcinol-cyclodextrin inclusion complex (abbreviated as cyclodextrin inclusion complex).

[0041] Comparative Example 2 This comparative example prepares a physical mixture to compare the effects of a simple physical mixing method with the preparation method of the present invention.

[0042] This comparative example provides a physical mixture in which the components are fed in the same proportions as in Example 1. The main difference is that no processing is performed. Instead, the components are weighed in the same proportions as in Example 1. Tranexamic acid, cyclodextrin compounds (hydroxypropyl-β-cyclodextrin), and phenylethyl resorcinol are mixed together and stirred evenly to obtain the physical mixture (referred to as physical mixture in some of the accompanying drawings).

[0043] Comparative Example 3 This comparative example provides a binary co-processed product lacking tranexamic acid, used to evaluate the contribution of tranexamic acid as a third co-forming component to apparent solubility and amorphous stabilization.

[0044] The preparation method of this comparative example is the same as that of Example 1 except that tranexamic acid is not added in step (1). The amount of raw materials, solvent composition, ultrasonic treatment, centrifugation, rotary evaporation and freeze drying conditions are the same. The resulting product is a phenylethyl resorcinol-cyclodextrin binary co-treated product (a binary assembly with the first component removed).

[0045] Comparative Example 4 This comparative example provides a binary co-treated product lacking cyclodextrin to evaluate the contribution of cyclodextrin host-guest interaction and hydrophilic stabilization to the solubilization performance of the system.

[0046] The preparation method of this comparative example is the same as that of Example 1, except that no cyclodextrin compound is added in step (1) (hydroxypropyl-β-cyclodextrin is selected). The other raw material amounts, solvent composition, ultrasonic treatment, centrifugation, rotary evaporation and freeze drying conditions are the same, and phenylethyl resorcinol-tranexamic acid binary co-treated product (the binary assembly with the second component deleted).

[0047] Effect verification In the following efficacy verification tests, if the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly prepared in Example 1 is used as the test sample, it refers to assembly 1 prepared in Example 1.

[0048] Test Example 1: Solubility Test 12 g of the products from Examples 1-5 (phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly), 12 g of the products from Comparative Examples 1-4 (cyclodextrin inclusion complex, physical mixture, and binary assembly with the first and second components removed, respectively), and 3 g of phenylethyl resorcinol were added to 10 mL of pure water. The mixtures were stirred for 24 h at 25°C and 300 rpm. The filtrates were filtered, and the equilibrium solubility of phenylethyl resorcinol was determined by HPLC. The experiment was performed in triplicate, and the results are as follows: Figure 1 As shown. Figure 1 The active pharmaceutical ingredient (API) in the text represents phenylethyl resorcinol. (The rest of the text appears to be a Figure 1 As can be seen, in Comparative Example 2, the solubility of phenylethyl resorcinol was improved to a certain extent when simply physically mixed, approximately 3.3 times that of the monomer; while in Comparative Example 1, when phenylethyl resorcinol was encapsulated with cyclodextrin compounds, its solubility was significantly improved, reaching 9.0 times that of the monomer; if the binary assembly (a binary assembly with reduced components) was prepared according to the methods described in Comparative Examples 3 and 4, its solubility was improved by a maximum of 10.5 times; therefore, none of the comparative examples could bring phenylethyl resorcinol to its upper limit of 0.5% for whitening addition, and its application in water-based formulations remains limited. In the embodiments of this invention, the ternary co-amorphous formation of phenylethyl resorcinol, tranexamic acid, and cyclodextrin compounds can further improve the solubility of phenylethyl resorcinol, and the improvement effect varies with different processes; when the mass ratio of phenylethyl resorcinol, tranexamic acid, and cyclodextrin compounds is 1:3:6 (assembly 1 in Example 1), the solubility of phenylethyl resorcinol is improved the most, by 153.6 times that of the monomer.

[0049] Test Example 2: Stability Test To investigate the effects of different implementation methods on the stability of phenylethyl resorcinol, the following samples were selected as test samples: phenylethyl resorcinol monomer, the phenylethyl resorcinol-tranexamic acid ternary co-assembly prepared in Example 1, the cyclodextrin inclusion complex prepared in Comparative Example 1, and the physical mixture prepared in Comparative Example 2. The corresponding test samples were weighed and prepared into an aqueous solution of phenylethyl resorcinol with a concentration of 0.4 mg / ml using pure water. These solutions were then placed in sealed containers and stored at a constant temperature of 45°C for 3 months. After storage, the samples were removed and their appearance was observed under the same conditions to evaluate the effect of different implementation methods on the thermal stability of the active ingredient. The results are as follows: Figure 2 As shown, Figure 2 The monomer in the text represents phenylethyl resorcinol monomer.

[0050] After being stored at 45°C for 3 months, the phenylethyl resorcinol monomer turned a deep yellowish-brown and became turbid. While Comparative Examples 2 and 1 showed some improvement compared to the monomer, they still exhibited varying degrees of yellowing and deposition, indicating that physical mixing or conventional inclusion methods have limited effect on improving the stability of the active ingredient. In contrast, the sample in Example 1 remained clear and transparent, without significant yellowing, turbidity, or precipitation, demonstrating that the phenylethyl resorcinol-tranexamic acid ternary co-assembly can effectively improve the color stability and system homogeneity of phenylethyl resorcinol under high-temperature storage conditions.

[0051] Test Example 3: Transdermal Absorption Efficacy Test The phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly (assembly 1) and monomer phenylethyl resorcinol prepared in Example 1 were used as test samples. The test was carried out according to the method described in T / SHRH064-2024 "Transdermal Penetration Test of Cosmetic Ingredients - In Vivo Raman Spectroscopy" to obtain Raman penetration images of the two on isolated pig skin. Figure 3 These are Raman osmosis images of the phenylethyl resorcinol-tranexamic acid ternary co-assembly and the monomer phenylethyl resorcinol from porcine skin. Figure 3 The co-amorphous assembly in the text represents the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly. For example... Figure 3 As shown, compared to the monomeric phenylethyl resorcinol, the phenylethyl resorcinol-tranexamic acid ternary co-assembly for whitening and fading spots exhibited a 5.78-fold increase in permeability after 2 hours, with the maximum penetration depth also increasing from 10 μm to 80 μm. Furthermore, it reached the active epidermis within 2 hours, significantly improving bioavailability. The increased permeability, penetration depth, and penetration rate of phenylethyl resorcinol all corroborate the improved effect of the co-amorphous technology.

[0052] Test Example 4: Observation under a polarizing microscope The cyclodextrin, phenylethyl resorcinol, tranexamic acid, a physical mixture thereof (the product of Comparative Example 2), and the phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly prepared in Example 1 were observed under a polarizing microscope. The results are as follows: Figure 4 As shown, Figure 4 The term "co-amorphous assembly" refers to the phenylethyl resorcinol tranexamic acid energy-enhancing and spot-fading ternary co-assembly, while "physical mixture" refers to a physical mixture of the three components. Figure 4 It can be seen that both phenylethyl resorcinol and tranexamic acid monomers exhibit relatively obvious crystal morphology and birefringence under polarized light conditions, indicating that they have certain crystallization characteristics. Crystal morphology and polarized light signals originating from each monomer are still visible in the physical mixture of the three, indicating that simple physical mixing did not effectively destroy the original crystal structure of each monomer. In contrast, the regular crystal morphology in the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots basically disappeared, and no obvious bright crystal points or birefringence were observed under polarized light, exhibiting a relatively irregular and uniform dispersion. These results indicate that the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots is not a simple superposition of phenylethyl resorcinol, tranexamic acid, and cyclodextrin, but rather that the crystallinity of the monomers was effectively reduced during the assembly process, forming a novel amorphous assembly state distinct from physical mixtures.

[0053] Test Example 5: Crystal Structure Test Figure 5 The image shows a comparative XRD pattern of the phenylethyl resorcinol-tranexamic acid ternary co-assembly prepared in Example 1, a physical mixture (the product of Comparative Example 2), cyclodextrin (hydroxypropyl-β-cyclodextrin), phenylethyl resorcinol, and tranexamic acid. The amorphous assembly in the image represents the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading dark spots, and 377 represents phenylethyl resorcinol. Figure 5 It can be seen that both phenylethyl resorcinol and tranexamic acid have obvious sharp diffraction peaks, exhibiting crystallization characteristics; the characteristic peaks of the monomers are still visible in the physical mixture, indicating that simple mixing did not change its crystal structure. In contrast, the phenylethyl resorcinol-tranexamic acid ternary co-assembly did not show obvious sharp crystal peaks, but only broad diffuse peaks, indicating that the original crystal structure of the monomers was significantly weakened during the assembly process, forming an amorphous assembly state that is different from that of the monomers and the physical mixture.

[0054] Test Example 6 Microstructure Characterization DLS Test: 10g of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly (assembly 1) from Example 1 was added to 100mL of deionized water. The mixture was stirred continuously for 30min at 25°C and 300rpm, followed by sonication for 10min to ensure complete dissolution. The particle size distribution was then measured using DLS. The results are as follows: Figure 6 As shown. By Figure 6 The particle size distribution of the phenylethyl resorcinol-tranexamic acid ternary co-assembly solution showed an average particle size of approximately 239.5 nm and a PDI (polydispersity index) of 0.1764, demonstrating that the phenylethyl resorcinol-tranexamic acid ternary co-assembly possesses nanoscale particle size. This nanoscale assembly may enhance the structural stability of phenylethyl resorcinol during storage and transdermal delivery through a spatial shielding effect.

[0055] Transmission electron microscopy (TEM) observation: The phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly prepared in Example 1 and the physical mixture obtained in Comparative Example 2 were observed under a TEM. The results are as follows: Figure 7 As shown in the figure, the co-amorphous assembly represents the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly, and the physical mixture represents the physical mixture obtained in Comparative Example 2. From Figure 7 It can be seen that the physical mixture contains obvious irregular aggregates and local blocky particles with relatively clear particle boundaries, indicating that the components mainly exist in a simple mixed and locally aggregated state. In contrast, the phenylethyl resorcinol-tranexamic acid ternary co-assembly exhibits a more continuous morphology, without obvious independent crystals or monomer particles, indicating that the components formed a more compact composite structure during the assembly process. The above results show that the phenylethyl resorcinol-tranexamic acid ternary co-assembly is not a simple physical superposition of phenylethyl resorcinol, tranexamic acid, and cyclodextrin, but rather a novel assembly morphology that is different from the physical mixture, formed through the interaction between the components.

[0056] Scanning electron microscopy (SEM) observation: The phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly prepared in Example 1 and the physical mixture obtained in Comparative Example 2 were observed under a scanning electron microscope. The results are as follows: Figure 8 As shown in the figure, the co-amorphous assembly represents the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly, and the physical mixture represents the physical mixture obtained in Comparative Example 2. From Figure 8 It can be seen that the particles in the physical mixture have irregular morphology, a wide particle size distribution, and many blocky or flaky particles, indicating poor overall dispersibility. This suggests that the components mainly exist in a simple mixed state. In contrast, the phenylethyl resorcinol tranexamic acid ternary co-assembled assembly has a more uniform particle morphology, smaller overall particle size, denser distribution, and better dispersibility, without the obvious large, irregular particles found in the physical mixture. These results indicate that the phenylethyl resorcinol tranexamic acid ternary co-assembled assembly is not a simple physical superposition of the components, but rather forms a new microstructure distinct from the physical mixture during the assembly process.

[0057] Fourier transform infrared spectroscopy analysis: The phenylethyl resorcinol-tranexamic acid energy whitening and spot-fading ternary co-assembly prepared in Example 1 and the physical mixture obtained in Comparative Example 2 were dried and pulverized, and then subjected to Fourier transform infrared spectroscopy. The results are as follows: Figure 9 As shown in the figure, the co-amorphous assembly represents the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly, and the physical mixture represents the physical mixture obtained in Comparative Example 2. From Figure 9 It can be seen that the physical mixture still retains many sharp characteristic absorption peaks, indicating that the original functional group vibrational characteristics of each component are relatively obvious. In contrast, in the infrared spectrum of the phenylethyl resorcinol tranexamic acid ternary co-assembly for skin whitening and spot removal, several characteristic absorption peaks show peak position shifts and peak broadening. Among them, the peaks at approximately 1600–1700 cm⁻¹ are particularly prominent. -1 The absorption band attributable to the carbonyl vibration of phenylethyl resorcinol broadens and shifts towards lower wavenumbers, indicating strong hydrogen bonding between the components. Furthermore, at approximately 2200 cm⁻¹... -1 The characteristic absorption peaks related to the vibrations of tranexamic acid groups in the vicinity also changed, further indicating the existence of intermolecular interactions between different components. The above-mentioned peak position changes and peak shape adjustments indicate that a stable non-covalent interaction network has been formed between phenylethyl resorcinol, tranexamic acid, and cyclodextrin, supporting the formation of a ternary co-assembled structure.

[0058] Test Example 7: Molecular Simulation Test Intermolecular interaction analysis: IGMH weak interaction analysis was performed on the phenylethyl resorcinol-tranexamic acid ternary co-assembly of Example 1 and the cyclodextrin inclusion complex of Comparative Example 1. The results are as follows: Figure 10 As shown in the figure, the cyclodextrin inclusion complex represents the cyclodextrin inclusion complex of Comparative Example 1, and the ternary amorphous assembly represents the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly of Example 1. Figure 10 It can be seen that the interactions in the cyclodextrin inclusion system are mainly concentrated near the cavity between phenylethyl resorcinol and cyclodextrin, with a relatively limited interaction area. However, a richer network of weak interactions and hydrogen bond sites can be observed in the phenylethyl resorcinol-tranexamic acid ternary co-assembly, indicating that a tighter non-covalent network is formed after the multi-component amorphous assembly. Binding energy calculations show that the binding energy of the phenylethyl resorcinol-tranexamic acid ternary co-assembly is -85.82 kcal / mol, significantly higher than the -34.21 kcal / mol of the cyclodextrin inclusion system, indicating stronger inter-component binding and higher structural stability. These results suggest that the multiple non-covalent interactions formed in the phenylethyl resorcinol-tranexamic acid ternary co-assembly are beneficial for stabilizing the assembled structure and may further improve the system's stability, dispersibility, and delivery performance.

[0059] Surface electrostatic potential analysis: The surface electrostatic potential of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly prepared in Example 1, phenylethyl resorcinol, and the cyclodextrin inclusion complex of Comparative Example 1 were analyzed. Specifically, molecular geometry optimization and single-point wave function calculation were performed using Orca 5.0.4, the surface electrostatic potential of the molecules was calculated using the Multiwfn program and the grid file was output, and the surface distribution map of the electrostatic potential colored molecules was obtained by rendering using VMD software. Figure 11 The surface electrostatic potential (ESP) analysis results of the phenylethyl resorcinol, cyclodextrin inclusion system (the cyclodextrin inclusion complex of Comparative Example 1), and the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots are presented. In the figure, the cyclodextrin inclusion complex represents the cyclodextrin inclusion complex of Comparative Example 1, and the ternary amorphous co-assembly represents the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots prepared in Example 1. Figure 11 It is evident that the potential distribution of phenylethyl resorcinol monomer is relatively limited. After inclusion with cyclodextrin or ternary co-amorphous assembly, the surface potential distribution of the system is rearranged, indicating changes in the local charge environment and interaction modes. Furthermore, the molecular polarity index of the phenylethyl resorcinol-tranexamic acid ternary co-assembled system increased from 0.53 eV for the phenylethyl resorcinol monomer to 0.72 eV, and the polar surface area ratio increased from 43.52% to 62.87%. These results indicate that the ternary co-amorphous assembly significantly increases the polar region on the system surface, resulting in a richer distribution of positive and negative potentials, which is beneficial for enhancing its interaction with water molecules and the polar environment. In summary, the phenylethyl resorcinol-tranexamic acid ternary co-assembled system, by introducing tranexamic acid and cyclodextrin, improves the original surface charge distribution and hydrophilic-hydrophobic balance of phenylethyl resorcinol, contributing to improved aqueous dispersibility, structural stability, and delivery adaptability of the system.

[0060] Molecular front orbital analysis: Molecular front orbital comparison analysis was performed on the phenylethyl resorcinol-tranexamic acid ternary co-assembly prepared in Example 1, the cyclodextrin inclusion complex prepared in Comparative Example 1, phenylethyl resorcinol, and tranexamic acid. Specifically, the geometry optimization and frequency analysis of each molecule and assembly were completed at the B3LYP-D3 (BJ) / 6-31G level using the Orca 5.0.4 program. Single-point calculations were performed at the M06-2X / def2-TZVP level to obtain wavefunction files, and the HOMO and LUMO orbital energies were extracted and the orbital band gaps were calculated. The front orbital grid file was exported using the Multiwfn program, and the HOMO and LUMO orbital isosurface distribution maps were rendered using VMD software. Finally, energy, band gap, and sample labels were added using plotting software to obtain a front orbital comparison map. Figure 12The frontier molecular orbital (FMO) analysis results of the phenylethyl resorcinol, tranexamic acid, and cyclodextrin inclusion system (the cyclodextrin inclusion complex prepared in Comparative Example 1) and the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots are presented. In the figure, the cyclodextrin inclusion complex represents the cyclodextrin inclusion complex of Comparative Example 1, the ternary amorphous co-assembly represents the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots prepared in Example 1, and 377 represents phenylethyl resorcinol. Figure 12 It can be seen that the HOMO / LUMO distributions of phenylethyl resorcinol and tranexamic acid monomers are mainly concentrated in their respective local molecular structures, with a relatively limited electron distribution range. After inclusion by cyclodextrin, the frontier orbitals begin to expand towards the periphery of the inclusion cavity, indicating that the inclusion effect has a certain influence on the molecular electronic environment. Further comparison of the band gap results shows that the band gap ΔE of the phenylethyl resorcinol-tranexamic acid ternary co-assembly is 3.30 eV, lower than that of the phenylethyl resorcinol monomer (3.79 eV), the tranexamic acid monomer (4.12 eV), and the cyclodextrin inclusion system (3.49 eV). The results indicate that the electron distribution of the system after ternary co-amorphous assembly is more delocalized, and the intermolecular electronic interactions are enhanced, indicating that it forms a stable assembly structure that is different from the monomer and simple cyclodextrin inclusion systems, which helps to improve the dispersibility, stability, and delivery adaptability of the system.

[0061] Test Example 8: Whitening Efficacy Test To evaluate the skin-whitening effect of the phenylethyl resorcinol-tranexamic acid energy-based skin-whitening and spot-fading ternary co-assembly, this test case established an α-MSH-induced A375 cell melanin production model and an ultraviolet-induced HaCaT cell ET-1 expression model, respectively. The regulatory effects of the samples on melanin production and ultraviolet-induced melanin-promoting signal release were evaluated by detecting melanin content, tyrosinase activity, and ET-1 expression levels. Melanin content reflects the final pigment production level; tyrosinase is the rate-limiting enzyme in melanin synthesis, and its activity changes reflect the intensity of melanin synthesis; ET-1 is an important melanin-promoting signaling factor released by keratinocytes after ultraviolet stimulation, which can act on melanocytes through paracrine signals and promote melanin production. Its expression level was used to evaluate the inhibitory effect of the samples on ultraviolet-induced melanin-promoting signal release.

[0062] 1. Experimental Materials and Equipment Experimental materials: A375 human malignant melanoma cells (CL-0014, Wuhan Pronosei Life Science Technology Co., Ltd.); phenylethyl resorcinol; physical mixture (prepared in Comparative Example 2); phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly (assembly 1 prepared in Example 1); kojic acid (K105452-25g, Shanghai Aladdin Biochemical Technology Co., Ltd.); melanin (M329644-25mg, Shanghai Aladdin Biochemical Technology Co., Ltd.); tyrosinase activity assay kit (BC4055, Beijing Solarbio Science & Technology Co., Ltd.).

[0063] Experimental equipment: Ensight PerkinElmer full-function microplate reader; ELISA reader (800TS BioTek Instruments, Inc.); NanoDrop2000 Thermo spectrophotometer; Clean bench (SW-CJ-1FD Sujing Antai).

[0064] In the following experiments, the negative control group was denoted as NC, the model control group as MC, and the positive control group as PC. All experiments were independently repeated at least three times, and data are expressed as mean ± standard deviation. In all sample groups of this test case, the effective concentration of phenylethyl resorcinol was 30 ppm, and the effective concentration of tranexamic acid was 60 ppm.

[0065] 2. Melanin content detection After resuscitating A375 cells, passaged them, and then took cells in good condition, digested, centrifuged and resuspended them. After counting, the cells were seeded into cell culture plates and cultured at 37°C and 5% CO2 until the cells were completely adhered.

[0066] The experimental groups are shown in Table 2 below. Except for the negative control group (NC), all other groups used α-MSH to establish a melanin production enhancement model, and the final concentration of α-MSH was 2 μg / mL; the positive control group (PC) also added kojic acid at a concentration of 100 μg / mL.

[0067] Table 2 Experimental Groups After the cells have fully adhered to the culture plate, the original culture medium is aspirated, and culture medium containing the appropriate inducer and sample is added. The amount added per well can be adjusted according to the culture plate specifications. After culture, the cells from each group are collected and washed twice with PBS. Each group of cells is divided into two aliquots: one aliquot is used to determine the total protein concentration according to the BCA protein concentration assay method; the other aliquot is resuspended in 1 mol / L NaOH solution containing 10% dimethyl sulfoxide, heated at 80℃ for 1 h to fully dissolve the melanin, and after cooling, the absorbance is measured at a wavelength of 405 nm.

[0068] The formula for fitting the standard curve of BCA protein concentration is shown below.

[0069] y1=a1X1 2 +b1X1+c1 X1: OD536; y1: protein concentration (mg / mL); where a1 is the quadratic regression coefficient, used to correct the curve deviation of absorbance from linearity in the high concentration range; b1 is the linear regression coefficient, characterizing the main linear response relationship between absorbance and analyte concentration; c1 is the curve intercept, representing the background interference value brought by blank reagent and instrument baseline.

[0070] The formula for fitting the standard curve of melanin is shown below.

[0071] y2=a2X2 2 +b2X2+c2 X2: OD405; y2: melanin concentration (μg / ml); where a2 is the quadratic regression coefficient, used to correct the curve deviation of absorbance from linearity in the high concentration range; b2 is the linear regression coefficient, characterizing the main linear response relationship between absorbance and analyte concentration; c2 is the curve intercept, representing the background interference value brought by blank reagent and instrument baseline.

[0072] The formula for calculating the relative concentration of melanin is shown below.

[0073] 3. Tyrosinase activity detection After resuscitating A375 cells, passaged them, and then digested, centrifuged, and resuspended the cells in the logarithmic growth phase. After counting, the cells were seeded into cell culture plates and cultured until the cells were completely adherent.

[0074] The experimental groups are shown in Table 3 below. Except for NC, all other groups used α-MSH to establish a melanin production enhancement model, with a final concentration of α-MSH of 2 μg / mL; kojic acid was added to PC at a concentration of 100 μg / mL.

[0075] Table 3 Experimental Groups After culture, cells from each group were collected and washed twice with PBS buffer. Each group of cells was divided into two aliquots: one aliquot was used for protein concentration determination by the BCA method; the other aliquot was added with the extraction solution of the tyrosinase activity assay kit, sonicated and lysed under ice bath conditions, centrifuged at 4°C and 12000 g for 10 min, and the supernatant was collected and placed on ice for analysis.

[0076] Follow the instructions for the tyrosinase activity assay kit: Add 200 μL of Reagent 1, 40 μL of Reagent 2, and 27 μL of sample to the test tube; add 200 μL of Reagent 1, 40 μL of Reagent 2, and 27 μL of distilled water to the blank tube. Mix thoroughly and react at 37℃ for 20 min. Then add 13 μL of Reagent 3, mix well, and centrifuge at 4℃ and 12000 g for 3 min. Add 200 μL of the supernatant to a 96-well plate and measure the absorbance at 505 nm. Record these results as Atest and Ablank, respectively. Reagents 1, 2, and 3 are all reagents included in the tyrosinase activity assay kit.

[0077] The formula for fitting the standard curve of BCA protein concentration is shown below.

[0078] y3=a3X3 2 +b3X3+c3 X3: OD536; y3: protein concentration (mg / mL); where a3 is the quadratic regression coefficient, used to correct the curve deviation of absorbance from linearity in the high concentration range; b3 is the linear regression coefficient, characterizing the main linear response relationship between absorbance and analyte concentration; c3 is the curve intercept, representing the background interference value brought by blank reagent and instrument baseline.

[0079] The formula for calculating tyrosinase activity is shown below.

[0080] : =A test sample - A blank; : Sample protein concentration (mg / ml); F: Dilution factor.

[0081] 4. ET-1 expression level detection After resuscitating HaCaT cells, passaged them, and then digested, centrifuged, and resuspended the logarithmic growth phase cells. After counting, the cells were seeded into cell culture plates and cultured until the cells were completely adherent.

[0082] The experimental groups are shown in Table 4 below. Except for NC, all other groups used ultraviolet irradiation to establish a melanin production enhancement model; kojic acid was added to PC at a concentration of 100 μg / mL.

[0083] Table 4 Experimental Groups After culturing, discard the culture medium, wash the cells three times with PBS buffer, add RNA extraction buffer, pre-cool on ice, and then lyse by pipetting. Add chloroform and mix thoroughly, let stand, centrifuge at 4°C, collect the supernatant, add isopropanol, mix well, and incubate at low temperature to precipitate RNA. After centrifugation, discard the supernatant, wash the RNA precipitate with 75% ethanol, air dry, and dissolve in nuclease-free water.

[0084] RNA concentration and purity were determined using an ultra-micro spectrophotometer. After adjusting the RNA concentration to a suitable range, reverse transcription was performed to obtain cDNA. ET-1 was used as the target gene, and GAPDH or β-actin was used as an internal reference gene. Quantitative real-time PCR was used to detect the Ct values ​​of each group. At least three technical replicates were set up for each sample.

[0085] RNA expression levels: Rate of change detection: 5. Experimental Results 5.1 Comparison of melanin content Figure 13 The results show the effects of a phenylethyl resorcinol-tranexamic acid ternary co-assembly and a physical mixture on melanin content in melanocytes. The co-amorphous component in the figure represents the phenylethyl resorcinol-tranexamic acid ternary co-assembly of Example 1, and the physical mixture represents the physical mixture of Comparative Example 2. Figure 13 The results showed that the melanin content of the phenylethyl resorcinol tranexamic acid energy-based whitening and spot-fading ternary co-assembly decreased by 41.84% compared with the model control group, and by 22.18% compared with the physical mixture group. These results indicate that the phenylethyl resorcinol tranexamic acid energy-based whitening and spot-fading ternary co-assembly can more effectively reduce melanin production under induced conditions, demonstrating a superior whitening effect compared with the physical mixture.

[0086] 5.2 Comparison of tyrosinase activity Figure 14 The effects of the phenylethyl resorcinol-tranexamic acid ternary co-assembly and the physical mixture on tyrosinase activity in melanocytes are shown. The co-amorphous component represents the phenylethyl resorcinol-tranexamic acid ternary co-assembly of Example 1, and the physical mixture represents the physical mixture of Comparative Example 2. Figure 14The results showed that the tyrosinase activity of the phenylethyl resorcinol-tranexamic acid energy-based whitening and spot-fading ternary co-assembly decreased by 49.65% compared to the model control group, and by 37.77% compared to the model control group in the physical mixture group. These results indicate that the phenylethyl resorcinol-tranexamic acid energy-based whitening and spot-fading ternary co-assembly can more effectively inhibit tyrosinase activity, reducing melanin production at the key rate-limiting enzyme stage of melanin synthesis, demonstrating a whitening effect superior to that of simple physical mixtures.

[0087] 5.3 Comparison of ET-1 expression levels Figure 15 The results show the effects of the phenylethyl resorcinol-tranexamic acid ternary co-assembly and tranexamic acid on ET-1 expression levels in cells. The co-amorphous component in the figure represents the phenylethyl resorcinol-tranexamic acid ternary co-assembly of Example 1, and the physical mixture represents the physical mixture of Comparative Example 2. Figure 15 The results showed that the ET-1 expression level of the phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly decreased by 60.16% compared with the model control group, and by 21.22% compared with the model control group in the tranexamic acid group. These results indicate that the phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly can more effectively downregulate UV-induced ET-1 expression, reduce the release of melanin-promoting signals from keratinocytes, and thus inhibit melanin production-related processes, demonstrating a significantly superior whitening effect compared with tranexamic acid monomer.

[0088] Test Example 9: Energy Boosting Efficacy Test 1. Experimental Objective and Principle Using HaCaT immortalized human keratinocytes as an in vivo epidermal cell model, a UV-induced mitochondrial functional damage model was established by combined UVA and UVB irradiation. The effects of the samples of this invention on cellular energy metabolism, mitochondrial oxidative stress, mitochondrial membrane potential, and expression of mitochondrial autophagy-related proteins after UV damage were evaluated.

[0089] Ultraviolet (UV) irradiation can induce an increase in reactive oxygen species (ROS) within keratinocytes, leading to a decrease in mitochondrial membrane potential, reduced ATP synthesis capacity, and affecting the expression of autophagy-related proteins such as LC3. Comparison of these changes in the negative control group, model control group, and sample group demonstrates that the samples of this invention have a protective or repairing effect against UV-induced mitochondrial damage. All experiments were independently repeated at least three times, and data are expressed as mean ± standard deviation. In all samples of this test, the effective concentration of tranexamic acid was 60 ppm.

[0090] 2. Experimental Materials and Equipment Experimental materials: HaCaT immortalized human keratinocytes (CL-0090, Wuhan Pronosei Biotechnology Co., Ltd.); reactive oxygen species detection kit (S0033M, Shanghai Beyotime Biotechnology Co., Ltd.); mitochondrial membrane potential detection kit (JC-10) (C2002S, Shanghai Beyotime Biotechnology Co., Ltd.); enhanced ATP detection kit (S0027, Shanghai Beyotime Biotechnology Co., Ltd.); BCA protein concentration assay kit (P0010, Shanghai Beyotime Biotechnology Co., Ltd.); PVDF membrane; ECL chemiluminescent substrate (P0018AM, Beyotime Biotechnology); LC3 antibody (14600-1-AP Wuhan Sanying Co., Ltd.); p62 antibody (YM8025 ImmunoWayBiotechnology Company); internal control antibody (10494-1-AP Wuhan Sanying Co., Ltd.); and HRP-labeled secondary antibody (SA00001-2 Wuhan Sanying Co., Ltd.).

[0091] Experimental equipment: Ensight microplate reader (PerkinElmer), ELISA reader (800TS, BioTek Instruments, Inc.), NanoDrop 2000 spectrophotometer (Thermo), laminar flow hood (SW-CJ-1FD, Suzhou Jingantai), Western blotting and developing system (Tanon 5200, Shanghai Tianneng Technology Co., Ltd.), electrophoresis power supply (DYY-7C, Beijing Liuyi Biotechnology Co., Ltd.), decolorizing shaker (TS-1, Haimen Qilinbei Instrument Manufacturing Co., Ltd.), and ultra-high resolution laser confocal microscope (STELLARIS, Leica Microsystems (Shanghai) Trading Co., Ltd.).

[0092] 3. Experimental Grouping After cell resuscitation, cells were passaged and cultured until their condition stabilized before being used in experiments. Cells were seeded into cell culture plates or laser confocal microscopy dishes according to the detection requirements. After complete cell adhesion, cells were grouped as shown in Table 5. The experiment consisted of a negative control group, a model control group, and a sample group. The negative control group received no UV irradiation but only the same volume of culture medium or solvent; the model control group received UV irradiation and the same volume of culture medium or solvent; the sample group received UV irradiation and the corresponding concentration of the test sample was added. The UV irradiation conditions were UVA 350 mJ / cm². 2 and UVB 30 mJ / cm 2In the negative control group, the irradiation was performed by covering the sample with aluminum foil to protect it from light. After irradiation, the appropriate treatment solution was added, and the sample was cultured for another 24 hours. Subsequently, ATP, mitochondrial membrane potential, ROS, and autophagy-related indicators were measured. The test samples used in the sample group were the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly solution prepared in Example 1, the physical mixture solution prepared in Comparative Example 2, or the tranexamic acid solution. The effective concentration of tranexamic acid in the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly solution prepared in Example 1, the physical mixture solution prepared in Comparative Example 2, or the tranexamic acid solution was 60 ppm.

[0093] Table 5 Cell Experiment Grouping 4. Cell Experiment 1: ATP Content Detection HaCaT cells were seeded in 6-well plates and, after cell attachment, were grouped according to the method described above. The model control group (MC) and the sample group were irradiated with combined UVA and UVB. After irradiation, the sample group was added with a predetermined concentration of sample and cultured for another 24 h. The negative control group (NC) was treated under the same culture conditions but without irradiation.

[0094] After treatment, discard the culture medium, wash the cells with PBS buffer, and add the ATP lysis buffer from the enhanced ATP assay kit to each well to lyse the cells (follow the kit instructions). After lysing the cells, obtain cell lysates, centrifuge at 4°C and 12000g for 5 min, and use the supernatant for ATP detection; simultaneously, use a portion of the cell lysate for BCA protein concentration determination. Prepare the ATP detection working solution according to the enhanced ATP assay kit instructions and establish a standard curve using ATP standards. Add the ATP detection working solution to the wells, incubate at room temperature for 5 min, then add the sample or standard, mix well, and measure the chemiluminescence (RLU) value.

[0095] The formula for fitting the standard curve of BCA protein concentration is shown below.

[0096] y4=a4X4 2 +b4X4+c4 X4: OD536; y4: protein concentration (mg / mL); where a4 is the quadratic regression coefficient, used to correct the curve deviation of absorbance from linearity in the high concentration range; b4 is the linear regression coefficient, characterizing the main linear response relationship between absorbance and analyte concentration; c4 is the curve intercept, representing the background interference value brought by blank reagent and instrument baseline.

[0097] The formula for fitting the ATP standard curve is shown below.

[0098] y5 = a5 x 52 +b5X5+c5 X5: ATP (nM); y5: Luminescence (RLU); where a5 is the quadratic regression coefficient, used to correct the curve deviation of absorbance from linearity in the high concentration range; b5 is the linear regression coefficient, characterizing the main linear response relationship between absorbance and analyte concentration; c5 is the curve intercept, representing the background interference value brought by blank reagent and instrument baseline.

[0099] ATP content in the samples was calculated based on the ATP standard curve and normalized to total protein concentration. The relative ATP content of each group was calculated with the ATP level of the model control group as 100%. The increased ATP content in the sample groups relative to the model group indicates that the samples can improve UV-induced cellular energy deficiency.

[0100] 5. Cell Experiment 2: Detection of Mitochondrial Membrane Potential HaCaT cells were seeded in laser confocal culture dishes, and after cell adhesion, they were grouped according to the above method. The MC group and the sample group were irradiated with combined UVA and UVB, while the NC group was shielded from light with aluminum foil during irradiation. After irradiation, the sample group was added with a predetermined concentration of the present invention, and cultured for another 24 h.

[0101] After incubation, retain 500 μl of culture medium in each culture dish, add an equal volume of JC-10 staining working solution, and incubate at 37°C in the dark for 50 min. After incubation, observe and acquire images using a fluorescence microscope or laser confocal microscope. JC-10 monomers exhibit green fluorescence (recommended excitation / emission wavelengths are 490 / 530 nm), and JC-10 aggregates exhibit red fluorescence (recommended excitation / emission wavelengths are 560 / 585 nm). The ratio of red to green fluorescence intensity is calculated using image analysis software, and the red / green fluorescence ratio characterizes the mitochondrial membrane potential level. A higher red / green fluorescence ratio indicates a higher mitochondrial membrane potential level.

[0102] The relative mitochondrial membrane potential of each group was calculated with the mitochondrial membrane potential level of the model control group as 100%. The increased red / green fluorescence ratio in the sample group indicates that the sample can improve the ultraviolet-induced decrease in mitochondrial membrane potential and maintain mitochondrial functional stability.

[0103] 6. Mitochondrial reactive oxygen species (ROS) detection HaCaT cells were seeded in 96-well black cell culture plates. After cell adhesion, the cells were grouped according to the above method. The sample group was added with a predetermined concentration of sample and incubated for 24 h. After incubation, part of the culture medium (50 μL) was discarded and PBS buffer was added. The model control group (MC) and the sample group were irradiated with UVA and UVB in combination according to the above method. The NC group was shielded from light with aluminum foil during irradiation.

[0104] After irradiation, fluorescent probes were added according to the instructions of the reactive oxygen species (ROS) detection kit, and the cells were incubated at 37°C in the dark for 30 minutes. The fluorescence intensity was then detected using a multi-functional microplate reader. Higher fluorescence intensity indicates higher intracellular ROS levels.

[0105] The relative ROS levels of each group were calculated with the ROS fluorescence intensity of the model control group as 100%. The relative ROS levels in the sample group were lower, indicating that the sample could inhibit UV-induced cellular oxidative stress and mitochondrial-related reactive oxygen species accumulation.

[0106] 7. Autophagy-related expression HaCaT cells were seeded in 6-well cell culture plates and grouped according to the above method after cell adhesion. The model control group (MC) and the sample group were subjected to combined UVA and UVB irradiation as described above. After irradiation, the sample group was added with a predetermined concentration of the sample, and cultured for another 24 h. After treatment, cells were washed with PBS buffer and protein samples were collected. Protein concentration was determined using the BCA method. Equal amounts of total protein were subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% skim milk. After blocking, LC3, p62, and internal control protein primary antibodies were added for incubation. After washing with TBST, the corresponding HRP-labeled secondary antibodies were added for incubation. After washing again, the membrane was developed using ECL chemiluminescence. The obtained band images were analyzed for grayscale using ImageJ and other image analysis software. The autophagy level was evaluated by the LC3-II / LC3-I ratio and the relative expression level of p62; an increased LC3-II / LC3-I ratio generally indicated increased autophagosome formation, while decreased p62 expression generally indicated enhanced autophagic degradation flux. The relative expression levels of each group were calculated with the model control group as 100%. If the LC3-II / LC3-I ratio increased and p62 expression decreased in the sample group, it indicates that the sample can promote the autophagy process after UV damage and help clear damaged organelles and restore mitochondrial homeostasis.

[0107] 8. Experimental Results Each experiment should have at least three replicates. Experimental results are expressed as mean ± standard deviation.

[0108] 8.1 ATP content Figure 16 This study demonstrates the effects of phenylethyl resorcinol tranexamic acid ternary co-assemblies and physical mixtures on cellular ATP levels. Figure 16 The term "co-amorphous" in the text refers to the sample corresponding to the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly prepared in Example 1. (The remaining text appears to be a fragment and doesn't translate coherently.) Figure 16The results showed that the ATP content of the phenylethyl resorcinol tranexamic acid energy-enhancing and spot-fading ternary co-assembly was 140.21% higher than that of the model control group, and 82.45% higher than that of the physical mixture group. These results indicate that the phenylethyl resorcinol tranexamic acid energy-enhancing and spot-fading ternary co-assembly can more effectively increase the ATP level of damaged cells and improve cellular energy production, demonstrating a superior mitochondrial energy-enhancing effect compared to simple physical mixtures.

[0109] 8.2 Mitochondrial membrane potential Figure 17 This study demonstrates the effects of phenylethyl resorcinol tranexamic acid ternary co-assembly for skin whitening and spot removal, and the influence of tranexamic acid on mitochondrial membrane potential. Figure 17 The term "co-amorphous" in the text refers to the sample corresponding to the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly prepared in Example 1. (The remaining text appears to be a fragment and doesn't translate coherently.) Figure 17 The results showed that the mitochondrial membrane potential of the phenylethyl resorcinol-tranexamic acid energy-enhancing and spot-fading ternary co-assembly was increased by 55.12% compared with the model control group, and by 53.83% compared with the model control group in the tranexamic acid group. These results indicate that the phenylethyl resorcinol-tranexamic acid energy-enhancing and spot-fading ternary co-assembly can effectively restore the mitochondrial membrane potential of damaged cells, improve mitochondrial function, and enhance the mitochondrial energy-enhancing effect of phenylethyl resorcinol.

[0110] 8.3 Comparison of ROS content Figure 18 This study demonstrates the effects of the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and spot removal, and tranexamic acid on intracellular ROS levels. Figure 18 The term "co-amorphous" in the text refers to the sample corresponding to the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly prepared in Example 1. (The remaining text appears to be a fragment and doesn't translate coherently.) Figure 18 The fluorescence intensity of the phenylethyl resorcinol-tranexamic acid ternary co-assembled group decreased by 18.14% compared to the model control group, and by 18.03% compared to the model control group in the tranexamic acid group. These results indicate that the ternary co-amorphous assembly can reduce cellular ROS accumulation under induced conditions, improve oxidative stress, and introduce antioxidant and mitochondrial protective effects into phenylethyl resorcinol.

[0111] 8.4 Autophagy-related indicators Figure 19 and Figure 20 The effects of the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots and tranexamic acid on autophagy-related indicators LC3-II / LC3-I and p62 are shown in the figures. The co-amorphous components in the figures represent the samples corresponding to the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading spots prepared in Example 1. Figure 19It can be seen that the LC3-II / LC3-I ratio of the phenylethyl resorcinol tranexamic acid energy-whitening and spot-fading ternary co-assembly was increased by 203.65% compared with the model control group, and the tranexamic acid group was increased by 91.79% compared with the model control group; at the same time, from Figure 20 It was found that the p62 expression level of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly decreased by 42.50% compared with the model control group, and decreased by 21.88% compared with the model control group in the tranexamic acid group.

[0112] An elevated LC3-II / LC3-I ratio suggests increased autophagosome formation, while a decreased p62 level indicates enhanced degradation of autophagy substrates. Both results together demonstrate that the phenylethyl resorcinol-tranexamic acid ternary co-assembly not only promotes autophagy-related processes but may also improve autophagy flux, aiding in the clearance of damaged organelles and the restoration of mitochondrial homeostasis, exhibiting superior autophagy regulation and mitochondrial quality maintenance effects compared to tranexamic acid monomers.

[0113] Test Example 10: Soothing Efficacy Test To evaluate the soothing and anti-inflammatory effects of the phenylethyl resorcinol-tranexamic acid three-component co-assembly for skin whitening and pigmentation reduction, this test case used lipopolysaccharide (LPS) to induce RAW264.7 mouse macrophages to establish an in vitro inflammatory response model. The inhibitory effect of the sample on the release of inflammatory mediators was evaluated by detecting the content of prostaglandin E2 (PGE2) in the cell culture supernatant. PGE2 is an important lipid pro-inflammatory mediator in the inflammatory response; elevated PGE2 levels usually indicate enhanced cellular inflammatory response. The sample was able to reduce the PGE2 release level after LPS induction, indicating that it has a soothing effect by inhibiting the release of inflammatory mediators and alleviating the stimulation response. All experiments were independently repeated at least three times, and data are expressed as mean ± standard deviation.

[0114] 1. Experimental Materials and Equipment The experimental cells were RAW264.7 mouse mononuclear macrophage leukemia cells (iCell-m047, Mirror Image (Shanghai) Cell Technology Co., Ltd.). The main reagents included lipopolysaccharide (LPS), a PGE2 ELISA kit (E-EL-0034, Elabscience Biotechnology Co., Ltd.), and PBS buffer. Dexamethasone was used as a positive control. The main equipment included a full-function microplate reader, an ELISA reader, a quantitative PCR instrument, an ultra-micro spectrophotometer, and a clean bench.

[0115] 2. Experimental grouping and modeling After resuscitation, RAW264.7 cells were passaged twice and cultured to a larger scale. After digestion, centrifugation, resuspending and counting, the cells were seeded into 24-well cell culture plates and cultured in a cell culture incubator until the cells were completely adherent.

[0116] The experiment was divided into a negative control group (NC), a model control group (MC), a positive control group (PC), and a sample group. The negative control group received no LPS, only the same volume of culture medium or solvent. The model control group received LPS for inflammatory stimulation, with a final LPS concentration of 0.5 μg / mL. The positive control group received LPS and dexamethasone, with a final LPS concentration of 0.5 μg / mL and a dexamethasone concentration of 50 μg / mL. The sample group received LPS and the corresponding concentration of the test sample, with a final LPS concentration of 0.5 μg / mL. The test samples were the phenylethyl resorcinol-tranexamic acid energy whitening and spot-fading ternary co-assembly solution and tranexamic acid solution prepared in Example 1. In all samples of this test example, the effective concentration of tranexamic acid was set to 60 ppm.

[0117] After the cells have fully adhered to the culture medium, the original medium was aspirated, and medium containing LPS and the corresponding test substance was added at a rate of 500 μL per well. The cells were incubated for 24 h. Then, the medium was replaced with one containing the corresponding test substance but without LPS, and incubation continued for another 24 h. After the culture was complete, the cell culture supernatant was carefully collected. If precipitation occurred during storage or processing, the supernatant could be collected again after centrifugation for PGE2 content detection.

[0118] 3. PGE2 content detection The PGE2 content in cell supernatant was detected using a PGE2 ELISA kit. Blank wells, standard wells, and sample wells were prepared. Blank wells contained no sample or enzyme-labeled reagent; the remaining steps were the same. Sample diluent and cell supernatant were added to the sample wells. When adding the sample, it should be placed at the bottom of the well, avoiding contact with the well walls, and gently shaken to mix.

[0119] After adding the samples, seal the plate with sealing film and incubate at 37°C for 30 min. After incubation, discard the liquid in the wells, add washing buffer, and wash for 30 s each time, then discard the washing buffer. Repeat the washing process 5 times and pat dry. Then add 50 μL of enzyme-labeled reagent to each well, except for the blank wells, seal the plate again, and incubate at 37°C for 30 min. After incubation, repeat the washing process 5 times and pat dry.

[0120] For color development, add 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B to each well sequentially, gently shake to mix, and incubate at 37°C in the dark for 15 min. After color development, add 50 μL of stop solution to each well to terminate the reaction; the solution color will change from blue to yellow. Zero the instrument using a blank well and measure the absorbance of each well sequentially at a wavelength of 450 nm. The measurement should be completed within 15 min after adding the stop solution.

[0121] A standard curve was established based on PGE2 standards, and the curve was fitted using software. The PGE2 content in the samples was calculated based on the absorbance values ​​of each well. The relative PGE2 level of each group was calculated with the PGE2 content of the model control group as 100%.

[0122] The formula for calculating PGE2 concentration is shown below.

[0123] x OD450; y : Sample concentration (pg / mL); where A is the asymptote on the standard curve, representing the maximum absorbance corresponding to zero PGE2 concentration; B is the curve slope factor, characterizing the steepness of the immune binding response; D is the asymptote below the standard curve, representing the lowest absorbance baseline under high-concentration PGE2 saturation conditions.

[0124] The formula for calculating the rate of change is shown below.

[0125] 4. Experimental Results Figure 21 This figure illustrates the effect of the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading dark spots, and tranexamic acid on cellular PGE2 release levels. The co-amorphous components in the figure represent the samples corresponding to the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and fading dark spots prepared in Example 1. Figure 21 The results showed that the PGE2 release level of the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and spot removal decreased by 34.99% compared with the model control group, and by 33.51% compared with the model control group in the tranexamic acid group. These results indicate that the phenylethyl resorcinol-tranexamic acid ternary co-assembly for skin whitening and spot removal can effectively inhibit PGE2 release, reduce inflammatory mediator levels, and has a certain soothing and anti-inflammatory effect, while also reducing the irritation of phenylethyl resorcinol.

[0126] In summary, this invention constructs a stable multi-component amorphous interaction network through the ternary co-amorphous assembly of phenylethyl resorcinol, tranexamic acid, and cyclodextrin compounds. Polarizing microscopy, XRD, and molecular simulation results show that the assemblies of this invention can reduce the crystalline characteristics of the active ingredients and improve the water solubility and storage stability of phenylethyl resorcinol through non-covalent interactions such as hydrogen bonds and van der Waals forces. Cellular testing further demonstrates that the system of this invention can enhance the overall whitening effect by synergistically delivering substances across multiple stages, including melanin production, inflammatory pigmentation, and cellular energy metabolism. This method is mild, requires minimal equipment, and is suitable for the stabilization and efficient application of poorly soluble whitening active ingredients.

[0127] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a phenylethyl resorcinol-tranexamic acid energy-whitening and spot-fading ternary co-assembly, characterized in that, Includes the following steps: (1) Add tranexamic acid and cyclodextrin compounds to water and stir to dissolve them to obtain a tranexamic acid-cyclodextrin aqueous solution; (2) Dissolve phenylethyl resorcinol in an alcohol solvent to obtain a phenylethyl resorcinol alcohol phase pre-solution; (3) Under light-protected conditions, the phenylethyl resorcinol alcohol phase pre-solution of step (2) is added to the tranexamic acid-cyclodextrin aqueous phase co-solution of step (1), ultrasonically treated, centrifuged to collect the supernatant, rotary evaporated and freeze-dried to obtain the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly.

2. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The cyclodextrin compound in step (1) is at least one of α-cyclodextrin, γ-cyclodextrin, hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, and hydroxypropyl-α-cyclodextrin.

3. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The mass-to-volume ratio of tranexamic acid to water in step (1) is 0.005-0.20:1 g / mL.

4. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The mass ratio of tranexamic acid and cyclodextrin compounds in step (1) is (1-40):(2-80).

5. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The temperature for stirring and dissolving in step (1) is 40-65℃.

6. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The alcohol solvent in step (2) is at least one of ethanol, methanol, and isopropanol; the mass-volume ratio of phenylethyl resorcinol to the alcohol solvent is 0.005-0.08:1 g / mL.

7. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The mass ratio of phenylethyl resorcinol in step (2) to tranexamic acid in step (1) is 1:(1-40).

8. The preparation method of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 1, characterized in that, The ultrasonic treatment in step (3) lasts for 10-60 min, at a temperature of 30-40 °C, and at a frequency of 10-50 kHz; the rotary evaporation temperature is 40-60 °C; and the freeze-drying time is 24-72 h.

9. A phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly prepared by the preparation method according to any one of claims 1-8.

10. The use of the phenylethyl resorcinol tranexamic acid energy whitening and spot-fading ternary co-assembly according to claim 9 in the preparation of topical medicines or cosmetics.

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