Low consolute solvent composition with anti-aging and whitening effects, preparation method and application thereof

Abstract

The application relates to a low eutectic solvent composition with anti-aging and whitening effects, a preparation method and application, the low eutectic solvent composition is a combination of boswelia and 4-butyl resorcinol; according to a molar ratio, the boswelia: the 4-butyl resorcinol is 1: (1-8); the preparation method comprises the following steps: mixing the boswelia and the 4-butyl resorcinol, reacting, stirring and obtaining; the low eutectic solvent composition does not need a surfactant, can spontaneously form nanoparticles in water, can also be dissolved in an oil phase, has a significant transdermal absorption promoting effect, has double effects of whitening and anti-aging, realizes 5 times of transdermal absorption efficiency improvement, more than 3 times of cell uptake efficiency improvement, 50% of significant improvement of whitening effect, is safe and non-irritating, does not need a surfactant in the preparation process, spontaneously forms particles, has a simple process, can be industrially and mass-produced, and has good market application value and industrialization value.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a eutectic solvent composition with both anti-aging and whitening effects, its preparation method, and its application. Background Technology

[0002] In existing whitening and anti-aging skincare formulas, 4-butylresorcinol is often used as the active whitening ingredient, supplemented with antioxidants such as Pro-Xylane, in order to achieve a comprehensive effect of even skin tone and improved fine lines. These formulas are typically based on an oil- and water-phase emulsion system, using surfactants or solubilizers to disperse and stabilize insoluble active ingredients. They are widely used in daily skincare creams, serums, and masks to meet consumers' dual needs for whitening and anti-aging.

[0003] However, 4-Butylresorcinol has extremely low solubility in water and insufficient stability in the oil phase, leading to crystal precipitation during formulation development and limiting its application in different matrices. To overcome this poor solubility, large amounts of surfactants or co-solvents are often required, which, while improving formulation permeability, also increases the potential risks of skin irritation and sensitization. Furthermore, traditional physical mixing or the use of large-particle-size emulsions are difficult to effectively penetrate the skin barrier, making it difficult for active ingredients to reach the dermis and exert their effects, resulting in low transdermal absorption and limited bioavailability. Therefore, developing a low-eutectic solvent composition with simple components, no additional additives required, and excellent stability, possessing both anti-aging and whitening effects, has become a current research hotspot and urgent need in the cosmetics field. Summary of the Invention

[0004] The purpose of this invention is to address the problems of existing low eutectic solvents used in cosmetics lacking complex skincare effects and requiring surfactants to form nanoparticles, resulting in poor system stability. It also solves the technical pain points of low transdermal absorption efficiency of existing anti-aging and whitening active ingredients and complex carrier preparation processes. This invention provides an anti-aging and whitening low eutectic solvent composition. This composition uses β-xylene and 4-butylresorcinol (577) as its core components. It can spontaneously form nanoparticles in water and reverse micelles in the oil phase without surfactants, possessing the triple effects of transdermal absorption, whitening, and anti-aging. Furthermore, it provides a simple and low-cost method for preparing nanoparticles from this composition, as well as the application of this composition and its nanoparticles in anti-aging and whitening skincare products, achieving a three-in-one integration of "active ingredient + carrier + transdermal penetration enhancement," significantly improving skincare efficacy while simplifying industrial production processes.

[0005] This invention achieves a stable eutectic solvent system by rationally selecting the molar ratio of Bosein and 577, and utilizes intermolecular hydrogen bonding to achieve self-assembly into granules without surfactants, while retaining the anti-aging activity of Bosein and 577 and endowing the composition with whitening and transdermal penetration enhancement functions.

[0006] The technical solution of the present invention is as follows: A eutectic solvent composition with both anti-aging and whitening effects, characterized in that the eutectic solvent composition is Bosein and 4-butylresorcinol (577). The molar ratio of the bosine to the 4-butylresorcinol is 1:(1~8).

[0007] The present invention provides a low-eutectic solvent composition with both anti-aging and whitening effects. The anti-aging component is bosoxane, and the whitening component is 4-butylresorcinol. This composition does not require surfactants and can spontaneously form nanoparticles with a particle size of 100~200nm in water. It can also dissolve in the oil phase and has a significant transdermal absorption promoting effect, thus possessing both whitening and anti-aging effects.

[0008] In one embodiment, the molar ratio of Bosein to 4-butylresorcinol is 1:(5~8).

[0009] In one embodiment, the molar ratio of the bosine to the 4-butylresorcinol is 1:5; within this range, the composition achieves optimal thermal stability, solubility, and skincare efficacy.

[0010] In one embodiment, the absolute value of the zeta potential of the eutectic solvent composition is 30-40 mV.

[0011] A second aspect of the present invention also provides a method for preparing the above-mentioned eutectic solvent composition with both anti-aging and whitening effects, comprising the following steps: Mix bosonicine and 4-butylresorcinol, react, and stir to obtain the product. The ratio of the bosonic acid to the 4-butylresorcinol is 1:(1~8).

[0012] In one embodiment, the molar ratio of Bosein to 4-butylresorcinol is 1:(5~8).

[0013] In one embodiment, the molar ratio of Bosein to 4-butylresorcinol is 1:5.

[0014] In one embodiment, the reaction temperature is 40~120°C and the reaction time is 60~180 min.

[0015] In one embodiment, the stirring speed is 200-600 rpm, and the stirring time is 60-180 min.

[0016] In a third aspect, the present invention also provides nanoparticles, wherein the nanoparticles are prepared by spontaneous assembly of a eutectic solvent composition dispersed in water; the particle size of the nanoparticles is 100~200 nm, and the polydispersity index of the nanoparticles is ≤0.2; The eutectic solvent composition includes the above-mentioned eutectic solvent composition with anti-aging and whitening effects or the eutectic solvent composition with anti-aging and whitening effects obtained by the above preparation method.

[0017] In a fourth aspect, the present invention also provides an oil-phase dispersion system, wherein the oil-phase dispersion system is prepared by forming a reverse micelle structure in an oil-phase medium using a eutectic solvent composition; wherein the polar groups of the reverse micelles point towards the inside of the micelles and the non-polar groups point towards the outside of the micelles; The eutectic solvent composition includes the above-mentioned eutectic solvent composition with anti-aging and whitening effects or the eutectic solvent composition with anti-aging and whitening effects obtained by the above preparation method.

[0018] In addition, the present invention also provides the above-mentioned eutectic solvent composition with anti-aging and whitening effects, the above-mentioned eutectic solvent composition with anti-aging and whitening effects obtained by the above-mentioned preparation method, and the application of the above-mentioned nanoparticles or the above-mentioned oil phase dispersion system in the preparation of anti-aging and whitening cosmetics.

[0019] Compared with the prior art, the present invention has the following beneficial effects: This invention relates to a eutectic solvent composition with anti-aging and whitening effects. Through hydrogen bonding between Bosein and 4-butylresorcinol, a eutectic system is formed, transforming the components, which are solid at room temperature, into a stable, homogeneous liquid. This system spontaneously assembles into nanoparticles with a particle size between 100 and 200 nm in the aqueous phase without the need for any surfactants, thus significantly reducing the skin's osmotic pressure and potential irritation risk. The establishment of the hydrogen bond network lowers the system's melting point, allowing both active ingredients to completely dissolve in both the oil and aqueous phases at room temperature, overcoming the poor solubility of 4-butylresorcinol in water and its instability in the oil phase.

[0020] The formation of nanoparticles provides a high specific surface area and a biphasic structure of lipophilic / hydrophilic, which promotes transdermal penetration of active ingredients, enhances bioavailability, and has a significant transdermal absorption-promoting effect. At the same time, the system's Zeta potential in the range of 30~40mV and PDI≤0.2 ensure the charge repulsion between particles, prevents aggregation and sedimentation, and guarantees the kinetic stability of long-term storage.

[0021] The technical solution of this invention solves the three major problems of poor solubility, insufficient safety, and low transdermal absorption rate of the components of BOXER and 577 in the prior art. It also improves the stability of the formula, enhances skin penetration, reduces irritation, and achieves a highly effective anti-aging and whitening effect. Attached Figure Description

[0022] Figure 1 The image shows the appearance of the eutectic solvent of Bosein-577 (Bosein-577 DES); Figure 2 DSC spectrum of Bosein-577 in eutectic solvent (Bosein-577 DES); Figure 3 The infrared spectrum is for the eutectic solvent of Bosein-577 (Bosein-577 DES). Figure 4 'a' represents the particle size distribution of the eutectic solvent of Bosein-577 (Bosein-577 DES). Figure 4 b represents the particle size, PDI, and potential test results of different molar ratios of the eutectic solvent of Bosein-577 (Bosein-577 DES). Figure 5 The results are from in vitro biocompatibility tests of the eutectic solvent of Bosein-577 (Bosein-577 DES). Figure 6 c represents the TEM morphology of the eutectic solvent of Bosein-577 (Bosein-577 DES); Figure 6 d represents the long-term stability test result of the eutectic solvent of Bosein-577 (Bosein-577 DES); Figure 7 Visualization of IGM analysis for Bosein-577 in a eutectic solvent (Bosein-577DES): intermolecular weak interaction diagrams, electrostatic potential analysis diagrams, and frontier molecular orbital analysis diagrams; among which... Figure 7 'a' is a scatter plot of the non-covalent interaction between Bosein and 4-butylresorcinol. Figure 7 b and Figure 7 c represents a visualization of weak intermolecular interactions; Figure 7 d and Figure 7 e represents the electrostatic potential distribution between Bosein and 4-butylresorcinol; Figure 7 f and Figure 7 g represents the frontier molecular orbital analysis diagram between Bosein and 4-butylresorcinol; Figure 8 Cellular uptake diagram of the eutectic solvent of Bosein-577 (Bosein-577 DES); Figure 9 The image shows the experimental results of zebrafish in a eutectic solvent of Bosein-577 (Bosein-577 DES); Figure 9 'a' represents a bar chart showing the relative melanin content. Figure 9 b is a microscopic image of the melanin morphology on the body surface of zebrafish juveniles. Figure 10 The in vitro transdermal absorption curve of Bosein-577 eutectic solvent (Bosein-577 DES). Detailed Implementation

[0023] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0026] Example 1 A eutectic solvent (DES) comprising bosonicine and 4-butylresorcinol (molar ratio 1:1) is prepared by the following steps: (1) Raw material weighing: Weigh Bosein (white powder) and 4-butylresorcinol (4-BR, white powder) accurately in a molar ratio of 1:1, with a total mass of 10g, place them in a three-necked flask, and stir evenly with a glass rod to obtain a physical mixture.

[0027] (2) Melting preparation: Place the three-necked flask in an oil bath, set the heating temperature to 80°C, turn on the stirring device, adjust the stirring speed to 400 rpm, and continue stirring for 2 hours. During this period, observe the state of the mixture until the physical mixture is completely melted and a uniform, transparent, pale yellow solution is formed. Turn off the heating and stirring, and cool to room temperature to obtain the eutectic solvent composition of Bosein and 4-butylresorcinol.

[0028] Example 2 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:2; the preparation method is the same as in Example 1.

[0029] Example 3 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:3; the preparation method is the same as in Example 1.

[0030] Example 4 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:4; the preparation method is the same as in Example 1.

[0031] Example 5 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:5; the preparation method is the same as in Example 1.

[0032] Example 6 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:6; the preparation method is the same as in Example 1.

[0033] Example 7 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:7; the preparation method is the same as in Example 1.

[0034] Example 8 A eutectic solvent (DES) comprising bosine and 4-butylresorcinol, with the same raw materials as in Example 1, except that the molar ratio in step 1 is 1:8; the preparation method is the same as in Example 1.

[0035] Test Example 1 1. Particle size, PDI and potential testing at different molar ratios The particle size, PDI, and Zeta potential of the bosine-577 DES prepared in Examples 1-8 were determined using a Malvern particle size analyzer.

[0036] The measurement results are as follows Figure 4 As shown in b, the particle size of the Bosein-577 DES prepared in Examples 1-8 increased significantly with the increase of the 577 ratio, and then stabilized and reached saturation after reaching a ratio of 1:5. This indicates that at low ratios, the intermolecular hydrogen bonds or assembly between Bosein and 577 have not yet fully formed; after reaching a ratio of 1:5, the intermolecular interactions reach a saturated state, the nano-aggregate structure is basically stable, and further increases in 577 no longer significantly change the particle size.

[0037] Under all test ratios, the Zeta potential remained stable at around -35mV, maintaining strong negative charge. This indicates that the system has sufficient surface charge and strong electrostatic repulsion between particles, ensuring excellent dispersion stability and preventing aggregation and sedimentation.

[0038] The PDI remained around 0.2 in the 1:5 to 1:8 range, indicating a low and stable level. This suggests that the particle size distribution within this range is uniform, with good monodispersity and no obvious polydispersity or agglomeration, consistent with the characteristics of a high-quality nano-DES system. This demonstrates that the present invention successfully prepared a eutectic solvent (DES). The above results indicate that 1:5 is the optimal molar ratio, and 1:5 to 1:8 is the effective stable range.

[0039] 2. DSC test Melting point was determined using a DSC differential scanning calorimeter.

[0040] The Bosein-577 DES and its corresponding monomers prepared in Examples 5-8 were heated under nitrogen protection at temperatures ranging from 25°C to 200°C at a heating rate of 10°C / min to obtain DSC spectra.

[0041] Test results are as follows Figure 2 As shown, pure 577 exhibits a significant melting endothermic peak at 54.0℃, while pure Bosein exhibits a characteristic endothermic peak at 122.5℃. The melting point of the Bosein-577 DES prepared in Examples 5-8 is lower than that of the monomers Bosein and 577. The characteristic melting or phase transition peaks of Bosein and 577 completely disappeared in the DSC curve, proving that the eutectic solvent is a new eutectic substance rather than a physical mixture, indicating that the present invention successfully prepared a eutectic solvent (DES).

[0042] 3. Infrared Spectrum Test The infrared spectra of the eutectic solvent (Bosoxon-577 DES) prepared in Examples 5-8, the infrared spectra of Bosoxon, the 577 monomer, and the infrared spectra of the physical mixture of Bosoxon-577 were determined using a Fourier transform infrared spectrometer (IR Spirit, Shimadzu Corporation, Tokyo, Japan).

[0043] The measurement results are as follows Figure 3As shown, the spectra of the Bosein-577 DES prepared in Examples 5-8 differ significantly from those of the physically mixed DES. The differences are mainly concentrated in the hydrogen bond sensitive region, with shifts in characteristic peak positions, changes in peak intensity, and weakening or disappearance of some peaks. This indicates that the mixture of Bosein and 577 is not a simple physical mixture, but rather a strong intermolecular interaction has formed. The intramolecular or intermolecular hydrogen bonds of the original pure substances have been rearranged and reorganized, forming a novel hydrogen bond network unique to DES. This demonstrates that the present invention successfully prepared a eutectic solvent (DES).

[0044] 4. DLS Test The dynamic light scattering particle size distribution curve of the eutectic solvent (Bosine-577 DES) prepared in Example 5 was determined using a dynamic light scattering instrument.

[0045] The Bosein-577 DES prepared in Example 5 was diluted with ultrapure water to a suitable concentration and gently vortexed to ensure complete dispersion and no visible precipitate. The test temperature was 25°C, the equilibration time was 120 s, and the scattering angle was 173°. Each sample was tested three times, and the average value was taken to obtain the particle size distribution curve.

[0046] The measurement results are as follows Figure 4 As shown in Figure a, the Bosein-577 DES prepared in Example 5 exhibits a single peak and narrow distribution in aqueous solution, with particle size mainly concentrated in the range of 100~200nm. No obvious large particles or agglomeration peaks appear, indicating that the system is uniformly dispersed and without obvious aggregation. Bosein and 577 self-assemble into uniform nanoscale aggregates through intermolecular hydrogen bonds and other interactions, rather than polydisperse particles of simple physical mixing. This indicates that the present invention successfully prepared a eutectic solvent (DES).

[0047] 5. TEM test The particle morphology and size of the Bosein-577 eutectic solvent (Bosein-577DES) prepared in Example 5 were determined using transmission electron microscopy.

[0048] The Bosein-577 DES prepared in Example 5 was diluted with pure water to a suitable concentration, gently mixed, and a small amount of the diluted solution was dropped onto the copper grid of the carbon support film. After adsorption for about 1 to 2 minutes, phosphotungstic acid was added to negatively stain the sample. The sample was then air-dried and observed and photographed under an accelerating pressure of 80 to 120 kV using a transmission electron microscope. The particle morphology and size were recorded.

[0049] The measurement results are as follows Figure 6As shown in Figure c, the Bosein-577 DES prepared in Example 5 self-assembled into spherical or near-spherical nanoaggregates in aqueous solution. The particles had clear boundaries, good dispersion, and no obvious agglomeration or adhesion. The measured nanoparticle size was approximately 100–200 nm, consistent with dynamic light scattering (DLS). Figure 4 The results of the measured hydrated particle size in a) are highly consistent, indicating that the present invention has successfully prepared a eutectic solvent (DES).

[0050] 6. Gaussian calculation All quantum chemical calculations were performed using Gaussian16 software, wavefunction analysis was performed using Multiwfn3.8 (dev) program, and visualization was performed using VMD1.9.3.

[0051] The initial structure of the 1:1 complex of (S)-Pro-xylane and 4-Butylresorcinol was fully geometrized at the level of the ωB97X-D functional with the 6-311G(d,p) basis set. This functional natively includes dispersion correction and can accurately describe the key hydrogen bond and van der Waals interactions in the system, with a solvent model (Solvent=Water). All optimized structures were confirmed by frequency analysis to be free of imaginary frequencies, ensuring that they are located at the minimum of the potential energy surface.

[0052] Based on the optimized structure, a higher-precision def2-TZVP large basis set was used to perform single-point energy calculations.

[0053] Test results are as follows Figure 7 As shown: Non-covalent interactions between Bosein and 4-butylresorcinol. The isosurfaces in the figure are colored according to sign(λ²)ρ: blue represents strong attraction (such as hydrogen bonding), and green represents weak attraction (such as van der Waals forces). The isovalue of inter is 0.005au, and the isovalue of intra is 0.03au. The IGMH two-dimensional scatter plot shows that the color mapping ranges from blue to green to red in the (S)-Bosein / 4-butylresorcinol complex. The significant blue spikes in the negative regions highlight the dominance of intermolecular hydrogen bonds in stabilizing the complex.

[0054] The electrostatic potential (ESP) distribution map plotted on the van der Waals surface of the composition shows a gradual color change from red (negative potential) to blue (positive potential), visually demonstrating the electrostatic complementarity between (S)-Bosein and 4-butylresorcinol molecules.

[0055] Frontier molecular orbitals of the composition calculated based on DFT, (a) HOMO; (b) LUMO, with the isosurface set to 0.02 au. Red and blue regions represent the positive and negative phases, respectively. The energies and energy gaps of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) are shown in Table 1 below: Table 1

[0056] The results show that the composition has weak nucleophilic electron-donating ability, some electrophilic electron-donating ability, high chemical stability and low reactivity; the interaction between Bosein and 577 is not a simple physical mixture, but rather an intermolecular interaction is formed; indicating that the present invention successfully prepared a eutectic solvent (DES).

[0057] 7. Long-term stability test The particle size, PDI change curve and Zeta potential of the Bosein-577 DES prepared in Example 5 were determined using a Malvern particle size analyzer during a 150-day long-term storage process.

[0058] The measurement results are as follows Figure 6 As shown in d, the particle size remained basically constant throughout the storage period, with no obvious increase or agglomeration trend, indicating that no obvious aggregation or phase separation occurred in the system.

[0059] The zeta potential remained consistently around -35mV, which is relatively high, indicating that the nanoparticles carry a stable negative charge on their surface and have a strong electrostatic repulsion effect, which can effectively inhibit particle aggregation.

[0060] The PDI remained at a low level throughout (around ~0.2), indicating that the particle size distribution was always narrow and uniform.

[0061] The above results demonstrate that the Bosein-577 DES nanosystem possesses excellent long-term colloidal stability, maintaining structural integrity under room temperature storage conditions, and has practical application potential in cosmetics, topical formulations, or drug delivery systems.

[0062] Example 10 Cosmetic applications of eutectic solvent compositions based on Pro-Xylane-577 (serums) Essence preparation: Using the PHOXY-577 DES prepared in Example 5 as the active ingredient, glycerin (moisturizer), sodium hyaluronate (moisturizer), xanthan gum (thickener), phenoxyethanol (preservative) and deionized water were added according to the conventional cosmetic essence preparation process. The mixture was mixed evenly, emulsified and dispersed to obtain an anti-aging and whitening essence, wherein the mass fraction of the active ingredient (composition) was 5%.

[0063] Test Example 2 1. Cytotoxicity test Cell culture: Select a suitable cell line and culture it in a medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0064] Plating: Digest and count the cells, seed them at an appropriate density in a 96-well plate, and incubate for 24 hours to allow them to adhere.

[0065] Drug treatment: Cells were incubated with different concentrations of pure 577 and Bosein-577 DES, respectively, with blank group and control group, and 3-6 replicates in each group.

[0066] CCK-8 assay: After incubation for 24 hours, add CCK-8 reagent to each well and continue incubation for 1-4 hours. Measure the absorbance at 450 nm using a microplate reader.

[0067] Calculation formula: Cell viability (%) = (OD) 样品 OD 空白 ) / (OD 对照 OD 空白 ) × 100% IC calculation based on fitted curve 50 .

[0068] The measurement results are as follows Figure 5 As shown, a pure 577 IC 50 =395μM, IC of Bosein-577 DES 50 The concentration of 340 μM indicates that the cytotoxicity of 577 was significantly reduced after preparation as DES. This suggests that Bosein, as a component of DES, can regulate the release behavior of 577 or reduce its local effective concentration through intermolecular interactions, thereby mitigating cell damage. Simultaneously, within the concentration range of <200 μM, the cell viability of both groups was above 80%, indicating that this DES system has good biocompatibility within its effective concentration range and is suitable for topical skin application, biomedical use, and other scenarios.

[0069] Test Example 3 1. Zebrafish Experiment This invention uses zebrafish larvae as an in vivo model to systematically evaluate the skin-whitening activity and safety of 4-BR (4-butylresorcinol, 577) microemulsion and 4-BR DES NP. Zebrafish larvae's skin melanocytes are highly homologous to human melanocytes, and the larvae are transparent, allowing direct observation of melanin production. This makes them an ideal alternative model for evaluating the in vivo activity of skin-whitening ingredients, conforming to the 3R principle of cosmetics.

[0070] In vivo whitening activity was evaluated using zebrafish experiments. Juvenile zebrafish were randomly divided into 5 groups of 30 each, with 3 biological replicates: blank control group; low-dose 4-BR microemulsion group (microemulsion group 15 μM); high-dose 4-BR microemulsion group (microemulsion group 30 μM); low-dose 4-BR DES NP group (eutectic solvent group 15 μM); and high-dose 4-BR DES NP group (eutectic solvent group 30 μM). ImageJ software was used to quantify melanin in zebrafish juvenile images.

[0071] Test results are as follows Figure 9 As shown, 4-BR, a classic tyrosinase inhibitor, exhibits clear whitening activity in zebrafish, significantly inhibiting melanin production in a dose-dependent manner. Compared to traditional microemulsion delivery systems, DES NP significantly enhances the in vivo whitening effect of 4-BR. At the same concentration, the melanin inhibition rate of the DES NP group is significantly higher than that of the ME group (microemulsion group), which can enhance the transdermal efficiency and cellular uptake rate of 4-BR, thereby increasing its effective concentration in melanocytes. No obvious deformities or abnormalities were observed in the zebrafish juveniles after administration, and their development was normal, proving that the DES NP delivery system enhances whitening efficacy without producing significant in vivo toxicity, demonstrating good safety for cosmetic applications.

[0072] 2. Cell uptake experiment The results of the cell uptake experiment were captured using a laser confocal microscope (CLSM).

[0073] HaCaT cells were seeded at an appropriate density in confocal microscopy-specific glass-bottomed culture dishes and cultured for 24 hours until cell adhesion. The culture medium was discarded, and the cells were washed with PBS. Complete culture medium containing Bosein-577 DES nanoparticles was added, while the control group received culture medium without Bosein and 577. Incubation continued for 2 hours. After incubation, the cells were gently washed three times with pre-cooled PBS to remove untaken free formulations. After fixation with 4% paraformaldehyde, the cell nuclei were stained with DAPI. Cell fluorescence images were observed and acquired using laser confocal microscopy. The average intracellular fluorescence intensity was semi-quantitatively analyzed using ImageJ software to evaluate cell uptake efficiency.

[0074] Test results are as follows Figure 8 As shown, compared with the control group, the Bosein-577 DES nano-formulation group showed obvious red fluorescence signals in the cells, and the fluorescence signals highly overlapped with the cell outline, indicating that the formulation can be effectively taken up by cells and distributed in the cytoplasm. Quantitative fluorescence intensity analysis further confirmed its effective cell internalization ability. The cell uptake efficiency of the DES nano-formulation group was more than 3 times higher than that of the control group, indicating that the DES nano-delivery system can significantly promote the cell uptake of active ingredients, providing cellular evidence for the transdermal absorption and subsequent biological activity of the formulation.

[0075] 3. In vitro transdermal absorption experiment The concentration of 4-BR was determined using a Franz diffusion cell and HPLC, and the cumulative transdermal dose Q (μg / cm³) was calculated. 2 ).

[0076] Skin from the back of a pig ear was harvested, subcutaneous fat and connective tissue were removed, and the thickness was controlled to be 0.9±0.1 mm; the effective diffusion area was 1.77 cm². 2 The receiving cell volume was 12.0 mL; the PBS buffer solution with pH 7.4 containing 30% ethanol was used to meet the leakage conditions; the water bath temperature was (32±0.5) ℃, the stirring speed was 600 r / min, and the test was conducted in the dark; the test samples were 4-BR microemulsion (4-butylresorcinol microemulsion) and 4-BR DES NP (4-butylresorcinol eutectic solvent nanoparticles), with consistent 4-BR concentration; the applied dose was 10 mg / cm³. 2 Samples of 2.0 mL were taken at 2h, 4h, 8h, 12h, and 24h, and fresh receiving solution of the same temperature was added simultaneously. The concentrations of Bosein and 4-BR in the samples were determined by HPLC, and the cumulative permeation was calculated. Each group of experiments was repeated 6 times.

[0077] Test results are as follows Figure 10 As shown, the BOXER-577 DES composition of the present invention can significantly improve the transdermal penetration efficiency of active ingredients, with a cumulative transdermal rate increase of more than 5 times in 24 hours, and the difference is statistically significant; at the same time, it can increase the retention of active ingredients in the skin, shorten the penetration lag time, and achieve efficient and deep transdermal delivery, which has significant application advantages in the field of whitening and anti-aging cosmetics.

[0078] Test Example 4 1. Efficacy test of anti-aging and whitening skin care products Test method: Referring to the "Cosmetic Safety Technical Specifications (2022 Edition)" - Test Method for Whitening and Spot Removal Efficacy for Human Use, T / GDCDC 019-2021 Test Method for Anti-wrinkle Efficacy, and T / CAB 0152-2022 "Test Method for Seven Efficacy Items of Cosmetics: Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing, and Soothing", the following indicators were evaluated to test the whitening and anti-aging efficacy of the prepared eutectic solvent.

[0079] Subjects: Thirty subjects were selected who met the testing criteria, and all 30 subjects actually completed the test. Their ages ranged from 30 to 60 years, with an average age of 49.5 ± 6.3 years.

[0080] Test results: (1) Results of whitening effect test Color ITA° value Four weeks after using the product, the skin color of the subjects' cheeks was analyzed. The results showed that the subjects' skin color ITA° value was significantly improved compared with before use. The results are shown in Tables 2 and 3.

[0081] Table 2. Results of ITA° values ​​at different time points after use of the test substance.

[0082] Note: (1) A p-value > 0.05 for the normality test indicates that the results follow a normal distribution, and a paired T-test is used. If the results do not follow a normal distribution, a nonparametric rank-sum test is used. (2) Compared with the baseline value before use in week n, "ns" indicates no statistical difference, p > 0.05; "*" indicates a significant difference, 0.01 ≤ p < 0.05; "**" indicates a very significant difference, 0.001 ≤ p < 0.01; "***" indicates an extremely significant difference, p < 0.001.

[0083] Table 3. Results of ITA° differences at different time points after use of the test substance.

[0084] Note: For p-values, "ns" indicates no statistical difference (p > 0.05); "*" indicates a significant difference (0.01 ≤ p < 0.05); "**" indicates a highly significant difference (0.001 ≤ p < 0.01); and "***" indicates an extremely significant difference (p < 0.001).

[0085] (2) Results of anti-wrinkle efficacy test Results of wrinkle improvement at different time points after use of the test product After using the product for 2 and 4 weeks, the wrinkles around the eyes of the subjects were analyzed. The results showed that the wrinkles of the subjects before and after using the product were significantly improved compared with before use. The results are shown in Tables 4 and 5.

[0086] Table 4. Results of wrinkle eigenvalues ​​at different time points after use of the test substance.

[0087] Note: (1) A p-value > 0.05 for the normality test indicates that the results follow a normal distribution, and a paired T-test is used. If the results do not follow a normal distribution, a nonparametric rank-sum test is used. (2) Compared with the baseline value before use in week n, "ns" indicates no statistical difference, p > 0.05; "*" indicates a significant difference, 0.01 ≤ p < 0.05; "**" indicates a very significant difference, 0.001 ≤ p < 0.01; "***" indicates an extremely significant difference, p < 0.001.

[0088] Table 5. Results of differences in wrinkle eigenvalues ​​at different time points after use of the test substance.

[0089] Note: For p-values, "ns" indicates no statistical difference (p > 0.05); "*" indicates a significant difference (0.01 ≤ p < 0.05); "**" indicates a highly significant difference (0.001 ≤ p < 0.01); and "***" indicates an extremely significant difference (p < 0.001).

[0090] Results analysis: (1) Analysis of whitening effect results After two weeks of product use, the subjects' ITA° values ​​increased by 16.8%, which was highly significant compared to before use.

[0091] After 4 weeks of product use, the subjects' ITA° values ​​increased by 38.1%, which was highly significant compared with before use.

[0092] Referring to the "Cosmetic Safety Technical Specifications (2022 Edition)" - Human Open Use Whitening and Spot-Removing Efficacy Test Method, the above results indicate that the product has a clear whitening and brightening effect after use.

[0093] No adverse skin reactions were observed in any of the 30 subjects during the use of the test substance.

[0094] (2) Analysis of anti-wrinkle effect results After two weeks of product use, the number of wrinkle features decreased by 6.3% in the subjects, which was highly significant compared to before use.

[0095] After using the product for 4 weeks, the number of wrinkle features decreased by 12.7%, which was highly significant compared with before use.

[0096] According to the standard T / CAB0152-2022, "Test Methods for Seven Efficacy Items of Cosmetics: Anti-wrinkle, Firming, Moisturizing, Oil Control, Repairing, Nourishing, and Soothing", the above results indicate that the product has a clear anti-wrinkle effect after use.

[0097] No adverse skin reactions were observed in any of the 30 subjects during the use of the test substance.

[0098] Comparative Example 1 Compositions prepared by exceeding the molar ratio of bosine to 4-butylresorcinol in a manner outside the scope of this invention.

[0099] Weigh out bosine and 4-butylresorcinol precisely at a molar ratio of 1:10, and prepare them using the same method as in Example 1.

[0100] The resulting composition was a pale yellow turbid solution with nanoparticles in water having a particle size of 250 nm and a PDI of 0.25. After 30 days of storage, obvious aggregation was observed, indicating poor stability. DSC analysis showed that a weak characteristic melting peak of 577 was still present, confirming that a stable DES was not fully formed, and the transdermal absorption efficiency was only 60% of that in Example 1.

[0101] Comparative Example 2 Preparation and testing of 4-butylresorcinol formulations encapsulated with traditional surfactants.

[0102] A 5% (w / w) 4-butylresorcinol formulation was prepared by encapsulating 4-butylresorcinol with a conventional surfactant (Tween-80). The remaining test conditions were completely consistent with those in Test Example 2.

[0103] Test results showed that the transdermal absorption efficiency of this formulation was only 1 / 5 of that of Test Example 2, the cellular uptake efficiency was 1 / 3 of that of Test Example 2, the whitening effect was 50% lower than that of Test Example 2, and 3 volunteers experienced mild skin irritation. The safety was lower than that of the composition of the present invention.

[0104] Comparative Example 3 Comparison of the solubility properties of Bosein and 4-butylresorcinol in different systems.

[0105] To verify the solubilizing effect of the Bosein-577 DES described in this invention on poorly soluble components, three control groups were set up: an oil phase system, a conventional water-alcohol system, and the DES system of this invention. Solubility performance comparison tests were conducted at different total active ingredient concentrations.

[0106] The molar ratio of Bosein to 4-butylresorcinol was fixed at 1:5, and the total active ingredient concentrations were set to be 1%, 5%, 10%, 15%, and 20%, respectively. Pure oil phase system (octanoic acid / capric acid triglyceride as solvent), conventional water-propylene glycol system (water:propylene glycol = 4:1, volume ratio, v / v), and Bosein-577 DES system were prepared respectively. The mixtures were stirred at 25°C for 30 min, allowed to stand for 24 h, and the dissolution state and appearance of the system were observed. The occurrence of precipitation, layering, and crystallization was recorded.

[0107] Table 6 shows the solubility of Bosein and 577 under different concentrations and systems.

[0108] Table 6

[0109] Test results show that Boseine is insoluble in the oil phase and cannot dissolve in the oil phase system regardless of concentration, only depositing in solid form, resulting in severe system heterogeneity; 577 is soluble in the oil phase, but crystallization easily occurs at high concentrations, and the system has poor stability; conventional water-alcohol systems have limited solubility for Boseine, with obvious precipitation occurring at concentrations >20%, which cannot meet the requirements for high addition amounts; the Boseine-577 DES system of this invention can completely dissolve in both the aqueous and oil phases within an ultra-wide high concentration range of 1~20%, and the system always remains clear, transparent, homogeneous, and stable, significantly breaking through the solubility limit of traditional solvent systems and possessing outstanding technical effects.

[0110] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0111] The embodiments described above are merely illustrative 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 invention patent. 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 all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A eutectic solvent composition possessing both anti-aging and whitening effects, characterized in that, The eutectic solvent composition is Bosein and 4-butylresorcinol; The molar ratio of the bosine to the 4-butylresorcinol is 1:(1~8).

2. The eutectic solvent composition with both anti-aging and whitening effects according to claim 1, characterized in that, The molar ratio of Bosein to 4-butylresorcinol is 1:(5~8).

3. The eutectic solvent composition with both anti-aging and whitening effects according to claim 1, characterized in that, The absolute value of the zeta potential of the eutectic solvent composition is 30~40mV.

4. The method for preparing the eutectic solvent composition with anti-aging and whitening effects as described in any one of claims 1-3, characterized in that, Includes the following steps: Mix bosonicine and 4-butylresorcinol, react, and stir to obtain the product. The molar ratio of the bosine to the 4-butylresorcinol is 1:(1~8).

5. The preparation method according to claim 4, characterized in that, The molar ratio of Bosein to 4-butylresorcinol is 1:(5~8).

6. The preparation method according to claim 4, characterized in that, The reaction temperature is 40~120℃, and the reaction time is 60~180min.

7. The preparation method according to claim 4, characterized in that, The stirring speed is 200~600 rpm, and the stirring time is 60~180 min.

8. A nanoparticle, characterized in that, The nanoparticles are prepared by spontaneous assembly of a eutectic solvent composition dispersed in water; the particle size of the nanoparticles is 100~200nm, and the polydispersity index of the nanoparticles is ≤0.2; The eutectic solvent composition includes the eutectic solvent composition with anti-aging and whitening effects as described in any one of claims 1-3, or the eutectic solvent composition with anti-aging and whitening effects obtained by the preparation method described in any one of claims 4-7.

9. An oil-phase dispersion system, characterized in that, The oil-phase dispersion system is prepared by forming a reverse micelle structure in an oil-phase medium using a eutectic solvent composition; the polar groups of the reverse micelles point towards the inside of the micelles, and the non-polar groups point towards the outside of the micelles. The eutectic solvent composition includes the eutectic solvent composition with anti-aging and whitening effects as described in any one of claims 1-3, or the eutectic solvent composition with anti-aging and whitening effects obtained by the preparation method described in any one of claims 4-7.

10. The application of the eutectic solvent composition with anti-aging and whitening effects as described in any one of claims 1-3, the eutectic solvent composition with anti-aging and whitening effects obtained by the preparation method described in any one of claims 4-7, the nanoparticles as described in claim 8, or the oil phase dispersion system as described in claim 9 in the preparation of anti-aging and whitening cosmetics.

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