An ergothioneine supramolecule, and a preparation method and application thereof

Abstract

The present application relates to the field of daily chemical products, and specifically discloses a supermolecule of ergothioneine, which is a co-crystal of ergothioneine and carnosine, wherein the molar ratio of ergothioneine to carnosine is 3:1 to 1:2. The supermolecule of the present application not only solves the problems of instability and easy degradation of ergothioneine, but also improves the skin permeability of ergothioneine, and synergistically enhances the antioxidant effect and has the anti-glycosylation effect. Therefore, it has the effects of anti-glycosylation, skin lightening, whitening, antioxidant, anti-UV, anti-early aging or anti-aging, etc., and can be used for preparing skin external agents, foods, food raw materials, daily chemical raw materials or pharmaceutical raw materials. In addition, the preparation method of the supermolecule of the present application is simple, has the advantages of high yield, low cost and suitability for large-scale production.

Description

Technical Field

[0001] This invention relates to the field of daily chemical products, and specifically discloses an ergothioneine supramolecular, its preparation method, and its application. Background Technology

[0002] Ergothioneine is a long-acting natural antioxidant with antioxidant, anti-inflammatory, and anti-aging effects; it can only be obtained externally and cannot be synthesized by the human body. Ergothioneine molecules enter the mitochondria via the transport protein OCTN-1, intelligently targeting the mitochondria and cell nucleus to scavenge free radicals and protect the mitochondria.

[0003] Ergothioneine, with its structure shown in Formula a, possesses a unique thione-thiol tautomer structure and quaternary ammonium inner salt characteristics. It exhibits high polarity and strong water solubility at physiological pH, while also possessing ultra-stable antioxidant capabilities. It can effectively scavenge strong oxidants such as hydroxyl radicals, singlet oxygen, and HOCl without undergoing self-oxidation. This "non-sacrificial" antioxidant property makes it more suitable in cosmetics as a long-term photoaging protection and cellular defense molecule, especially for anti-photodamage, anti-inflammatory, and sensitive skin repair formulas in high-exposure environments. However, it also has limitations. Ergothioneine molecules are excited by light, especially ultraviolet light, triggering photochemical reactions that lead to structural damage and degradation product formation, resulting in a yellowing color and the production of trimethylamine with a fishy odor. Furthermore, due to its strong hydrophilicity, its transdermal efficiency is relatively limited.

[0004] Carnosine is a dipeptide found in skin and muscles. Its molecule contains peptide bonds, an imidazole ring, and charged amino and carboxyl groups, giving it good water solubility and significant pH buffering capacity. The structure of carnosine is shown in formula b. It is a highly effective anti-aging ingredient. Its core efficacy in cosmetics stems from its ability to capture active carbonyl groups and inhibit protein glycation, thereby reducing collagen cross-linking and yellowing. It is a typical anti-glycation, anti-yellowing, and anti-early aging ingredient.

[0005] Carnosine, with its structure shown in Formula b, exerts powerful anti-glycation and antioxidant effects by scavenging free radicals, chelating metal ions, and inhibiting the formation of protein carbonylation and glycation end products (AGEs). It reduces dullness, roughness, and fine lines caused by glycation and oxidation, while protecting collagen and elastin from degradation, thus improving skin firmness and radiance. Furthermore, it possesses soothing and anti-inflammatory properties, reduces damage from external stimuli, and maintains skin homeostasis, effectively improving dullness and sagging, delaying skin aging, and leaving skin radiant, smooth, and plump. The multi-site hydrogen bonding of the carnosine molecule also facilitates the formation of a stable aqueous network, enhancing the structural stability of the system. However, carnosine readily attracts volatile molecules from the environment in formulations, potentially causing odors, and there are certain contraindications for its use.

[0006] CN119235688 A discloses a composition of ergothioneine and carnosine. By combining ergothioneine and carnosine, the degradation rate of ergothioneine is reduced, the odor of the composition is lessened, and yellowing is avoided. After the composition is placed at room temperature, under light, and at pH 5-8.5 for one month, the degradation rate of ergothioneine in the composition is less than 10%. Although this technology solves the problem of ergothioneine storage stability, products containing ergothioneine still exhibit degradation and unpleasant odor when applied to the skin under sunlight and high temperatures. Furthermore, the mixture does not solve the problem of low transdermal efficiency.

[0007] Therefore, ergothioneine needs to be improved to address its stability and transdermal efficiency, and further enhance its skincare efficacy. Summary of the Invention

[0008] The present invention aims to provide an ergothioneine supramolecular, its preparation method and application.

[0009] The first embodiment of the present invention is an ergothioneine supramolecular crystal, which is an ergothioneine-carnosine cocrystal, wherein the molar ratio of ergothioneine to carnosine is 3:1-1:2.

[0010] In some embodiments of the present invention, the molar ratio of ergothioneine to carnosine is 1:1.

[0011] In ergothioneine supramolecular structures, ergothioneine and carnosine undergo non-covalent intermolecular interactions, leading to the reconstruction of the original crystal structure and the formation of a new ordered structure, thus creating an ergothioneine-carnosine cocrystal supramolecular composition.

[0012] Differential scanning calorimetry (DSC) results showed melting or crystal transformation of its own lattice structure, indicating that ergothioneine and carnosine formed a new molecular stacking mode through multi-site hydrogen bonding and charge-assisted interactions, disrupting their original lattice structures and constructing a co-crystal supramolecular composition with independent thermal stability. Powder X-ray diffraction (XRD) confirmed significant crystal rearrangement or local disorder, disrupting the long-range order of the original lattice; this indicates that ergothioneine-carnosine reconstructs the original crystal structure through intermolecular non-covalent interactions, thus forming an ergothioneine-carnosine co-crystal supramolecular composition. Fourier transform infrared spectroscopy also confirmed changes in the characteristic infrared absorption peaks, indicating the existence of intermolecular interactions such as hydrogen bonds between ergothioneine and carnosine, which altered the local chemical environment and caused a shift in the infrared absorption peaks, demonstrating that ergothioneine and carnosine can form a stable supramolecular structure through non-covalent interactions. The results prove that the above-mentioned ergothioneine supramolecular structure is an ergothioneine-carnosine co-crystal supramolecular structure with a stable supramolecular co-crystal structure, rather than a simple physical mixture.

[0013] The present invention also provides a method for preparing the above-mentioned ergothioneine supramolecular, which can be prepared by solid-state grinding or solution synthesis.

[0014] Solid-state grinding methods include dry grinding with solids or solvent-assisted grinding.

[0015] When using solvent-assisted grinding, the added grinding solvent is water, an organic solvent, or any mixture thereof. Preferably, the organic solvent is ethanol. In some embodiments of the present invention, the grinding solvent is water.

[0016] During solvent-assisted grinding, the total weight ratio of ergothioneine and carnosine to the added grinding solvent is 6-15:1, preferably 9-12:1.

[0017] Solution synthesis refers to the synthesis of eutectic crystals in solution, including slow evaporation, cooling crystallization, suspension crystallization, or dissolution crystallization. When using solution synthesis, the total weight ratio of ergothioneine and carnosine to the added solvent is 1:0.5-10. The solvent is water, an organic solvent, or any mixture thereof. Preferably, the organic solvent is an alcohol, more preferably ethanol.

[0018] The present invention also provides the use of the above-mentioned ergothioneine supramolecular.

[0019] The ergothioneine supramolecular of this invention can penetrate the active epidermis and reach the dermis, where it continues to permeate. Skin permeability and penetration depth are significantly improved, resulting in a markedly enhanced transdermal effect compared to ergothioneine, thus providing better skincare benefits. Furthermore, the ergothioneine supramolecular has better stability, is less prone to degradation under high temperatures and sunlight, and does not produce an unpleasant odor when applied to the skin, making the application of ergothioneine more widespread.

[0020] In addition, the ergothioneine supramolecular of the present invention can significantly reduce the content of carboxymethyl lysine (CML) in human primary fibroblasts, and has a strong inhibitory effect on CML. It has an anti-glycation effect and has significant effects on inhibiting skin glycation, brightening skin tone, whitening, anti-early aging or anti-aging.

[0021] The ergothioneine supramolecular of this invention further enhances its antioxidant effect, effectively inhibiting the level of reactive oxygen species (ROS) in skin cells, especially UVB-induced ROS. Therefore, the ergothioneine supramolecular has antioxidant, anti-ultraviolet, whitening, anti-early aging, and anti-aging effects.

[0022] Therefore, the ergothioneine supramolecular of the present invention can be used to prepare food, health food, food ingredients, daily chemical ingredients, skin care products, cosmetics, pharmaceuticals or pharmaceutical ingredients.

[0023] The medicines mentioned include external or internal dosage forms.

[0024] The present invention also includes: daily chemical raw materials, food, health food, food raw materials, pharmaceuticals or pharmaceutical raw materials containing the above-mentioned ergothioneine supramolecular molecules.

[0025] Another aspect of the present invention is a skin care product or cosmetic containing ergothioneine supramolecular or the aforementioned daily chemical raw materials.

[0026] The skincare or cosmetic products mentioned above have the effects of anti-glycation, brightening skin tone, whitening, anti-oxidation, anti-ultraviolet, anti-early aging, or anti-aging.

[0027] Skincare or cosmetic products include, but are not limited to, sunscreen, makeup base, toner, lotion, serum, facial oil, lotion, cream, facial cleanser, facial cleanser milk, face mask, makeup remover, makeup remover oil, shampoo, conditioner, body wash, soap, liquid foundation, pressed powder, loose powder, eyeshadow, and lipstick.

[0028] Compared with existing technologies, the ergothioneine-carnosine cocrystal supramolecular of the present invention not only solves the problems of light and heat instability of ergothioneine and carnosine, and the generation of strong amine odor and degradation during long-term storage or exposure to light and high temperatures, but also improves skin permeability, enabling it to penetrate into the dermis and continue to penetrate. Simultaneously, it can synergistically enhance antioxidant and whitening effects; in addition, it has excellent anti-glycation effects, making it suitable for use in the food, cosmetic, and pharmaceutical fields. This supramolecular material can be prepared by conventional methods such as grinding, offering advantages such as high yield, low cost, and suitability for large-scale production. Attached Figure Description

[0029] Figure 1 DSC curves of ergothioneine, carnosine, and ergothioneine-carnosine supramolecular (raw material molar ratio 1:1) in Example 1; Figure 2 The DSC curves for different raw material molar ratios of ergothioneine-carnosine supramolecular and ergothioneine and carnosine 1:1 molar ratio mixture are shown in Example 1. Figure 3 The XRD patterns of ergothioneine, carnosine, and ergothioneine-carnosine supramolecular (raw material molar ratio 1:1) in Example 1 are shown below. Figure 4 The infrared spectra of ergothioneine, carnosine, and ergothioneine-carnosine supramolecular (raw material molar ratio 1:1) in Example 1 are shown. Figure 5 Raman permeation image of ergothioneine-carnosine supramolecular (raw material molar ratio 1:1) in Example 2; Figure 6 Raman permeation image of a simple mixture of ergothioneine and carnosine (molar ratio of raw materials 1:1) in Example 2; Figure 7Immunofluorescence image showing the inhibition of carboxymethyl lysine (CML) content in human primary fibroblasts by ergothioneine-carnosine supramolecular (raw material molar ratio 1:1) in Example 3; Figure 8 Immunofluorescence images of reactive oxygen species (ROS) levels in HaCaT cells after UVB stimulation using ergothioneine-carnosine supramolecular (raw material molar ratio 1:1) in Example 4. Detailed Implementation

[0030] Example 1 Ergothioneine was mixed with carnosine at different molar ratios, placed in a grinder, and water was added as an auxiliary solvent. The mixture was ground at 5000 rpm and 25-35°C. After grinding, the material was discharged and allowed to stand. The raw material amounts (parts by weight) are shown in Table 1.

[0031] Alternatively, ergothioneine and carnosine can be added to an auxiliary solvent, placed in a ball mill, and ground at 20-40 Hz to obtain ergothioneine-carnosine cocrystal supramolecular.

[0032] In addition to the solvent-assisted grinding mentioned above, ergothioneine and carnosine can be dispersed in water or 75%-95% ethanol solution, and then slowly evaporated, cooled to crystallize, suspended to crystallize, or dissolved to crystallize to obtain a co-crystal supramolecular.

[0033] Table 1 The obtained supramolecular products were subjected to structural characterization tests.

[0034] 1. Differential Scanning Calorimetry (DSC) Test Experimental Methods: The melting points of ergothioneine, carnosine raw materials, and ergothioneine-carnosine supramolecular compositions were determined using a simultaneous thermal analyzer (HCT-1, Beijing Hengjiu Experimental Equipment Co., Ltd.). 10 mg of sample was placed in an aluminum crucible and sealed for analysis. During the experiment, the temperature was increased from 25 °C to 300 °C at a rate of 10 °C / min.

[0035] Test results: Figure 1The DSC curves for ergothioneine, carnosine, and the ergothioneine-carnosine combination (raw material molar ratio 1:1) are shown. Pure ergothioneine exhibits a sharp endothermic peak at 291.7 °C, corresponding to the melting or crystal transformation of its own lattice structure. Pure carnosine also shows a clear and sharp endothermic peak at 284.4 °C, reflecting its stable molecular packing structure and well-defined thermal transition behavior. In contrast, the DSC curve of the ergothioneine-carnosine combination does not show the characteristic endothermic peaks of either raw material; instead, a new, single, and sharp endothermic peak is observed at 263.9 °C. This endothermic peak appears significantly lower than the thermal transition temperatures of the two raw materials and does not exhibit simple peak superposition or peak broadening. This indicates that this thermal behavior is not due to the physical mixing of ergothioneine and carnosine, but rather corresponds to a thermal transition process of a new ordered structure. This significant change in thermal transition temperature indicates that, in the composite system, ergothioneine and carnosine form a new molecular stacking mode through multi-site hydrogen bonds and charge-assisted interactions, which disrupts their original crystal structures and constructs a eutectic supramolecular composite structure with independent thermal stability.

[0036] like Figure 2 As shown, after simple physical mixing of ergothioneine and carnosine, the melting point of the mixture is 285.6℃, which is between the melting points of the two raw materials and closer to the melting point of carnosine. The DSC curves of the ergothioneine-carnosine compositions prepared by mixing ergothioneine and carnosine at different molar ratios all showed a single endothermic peak, with melting points ranging from 262.7℃ to 263.9℃. This single DSC endothermic peak indicates that ergothioneine and carnosine in different molar ratios can form stable supramolecular compositions.

[0037] 2. Powder X-ray diffraction (XRD) test Experimental Methods: The crystal structures of ergothioneine, carnosine raw materials, and ergothioneine-carnosine supramolecular compositions were determined using a powder X-ray diffractometer (Rigaku D / max-2550, Rigaku Corporation, Japan). Cu / Kα (λ=0.154 nm) rays were used as the light source at a wavelength of 1.5406 Å, tube voltage of 40 kV, tube current of 40 mA, a scanning angle of 2θ, a scanning range of 10°–80°, a scanning wavelength of 0.02°, and a scanning speed of 5° / min.

[0038] Test results: such as Figure 3As shown, the XRD pattern of the ergothioneine-carnosine composition (raw material molar ratio 1:1) shows that, compared with carnosine, the two peaks at 11.24° and 14.40° completely disappear; compared with ergothioneine, the characteristic peak at 38.58° also no longer appears. This result indicates that the crystal structure of the ergothioneine-carnosine composition is not a simple superposition or mechanical mixing, but rather a significant crystal rearrangement or local disorder, disrupting the long-range order of the original lattice. Furthermore, the ergothioneine-carnosine composition exhibits a series of diffraction peaks at 2θ = 15.50°, 17.26°, 18.98°, 19.86°, 20.48°, 21.68°, 22.54°, and 24.84°, and these peak positions can all be found in the single-component XRD patterns of ergothioneine or carnosine. This phenomenon further illustrates that ergothioneine-carnosine induces the reconstruction of the original crystal structure through intermolecular non-covalent interactions, thereby forming an ergothioneine-carnosine cocrystal supramolecular composition.

[0039] 3. Fourier transform infrared spectroscopy test 1. Experimental Method: Fourier transform infrared spectroscopy was used to study the hydrogen bonding interactions between the components of the ergothioneine-carnosine composition (raw material molar ratio 1:1). Figure 7 As shown, ergothioneine is at 3160.81 cm. -1 A distinct absorption peak appears at 2996.89 cm⁻¹, mainly attributed to the NH₄⁺ / OH stretching vibration; -1 The infrared absorption peak at 1639.22 cm⁻¹ corresponds to the CH stretching vibration of the alkyl side chain. -1 The infrared absorption peak at 1089.60 cm⁻¹ originates from the C=N stretching vibration; while the peak at 1089.60 cm⁻¹... -1 The infrared absorption peak at 3241.81 cm⁻¹ corresponds to the CN or CO stretching vibration. For carnosine, the peak is at 3241.81 cm⁻¹. -1 The infrared absorption peak at 1660.43 cm⁻¹ is attributed to the N–H / O–H stretching vibration peak. -1 and 1565.94 cm -1 The infrared absorption peaks at 1093.58 cm⁻¹ originate from the amide I band and the amide II band (C=O stretching vibration), respectively. -1 The infrared absorption peak at that location corresponds to the C–N stretching vibration.

[0040] 2. Experimental Results: like Figure 7 As shown, ergothioneine is at 3160.81 cm. - A distinct absorption peak appears at ¹, mainly attributed to the NH / OH stretching vibration; at 2996.89 cm⁻¹... -The infrared absorption peak at ¹ corresponds to the CH stretching vibration of the alkyl side chain; 1639.22 cm⁻¹ - The infrared absorption peak at ¹ originates from the C=N stretching vibration; while at 1089.60 cm⁻¹... -1 The infrared absorption peak at 3241.81 cm⁻¹ corresponds to the CN or CO stretching vibration. For carnosine, the peak is at 3241.81 cm⁻¹. -1 The infrared absorption peak at 1660.43 cm⁻¹ is attributed to the NH₄⁺ / OH stretching vibration peak. -1 and 1565.94 cm -1 The infrared absorption peaks at 1093.58 cm⁻¹ originate from the amide I band and the amide II band (C=O stretching vibration), respectively. -1 The infrared absorption peak at that location corresponds to the CN stretching vibration.

[0041] When the ergothioneine-carnosine supramolecular composition was formed, the positions of the characteristic peaks in the infrared spectra of the two raw materials shifted to a certain extent. In the infrared spectrum of the ergothioneine-carnosine supramolecular composition, ergothioneine showed a peak position at 3400 cm⁻¹. - ¹The hydrogen bond peak near the wavelength shows significant broadening; the N–H / O–H stretching vibration peak of carnosine increases from 3241.81 cm⁻¹. - ¹Slightly redshifted to 3239.88 cm - ¹; The absorption peaks of carnosine's amide I and amide II bands showed significant changes, with the original peak at 1660.43 cm⁻¹ decreasing. - The infrared absorption peak at ¹ redshifts to 1643.08 cm⁻¹. - ¹ This indicates that ergothioneine has a significant impact on the C=O bond of the amide group of carnosine; while the C–N / C–O stretching vibration peak changes from 1093.58 cm⁻¹. - ¹Moved to 1091.53 cm - ¹. The changes in the above characteristic infrared absorption peaks indicate that there are intermolecular interactions such as hydrogen bonds between ergothioneine and carnosine in the ergothioneine-carnosine supramolecular composition, which alter the local chemical environment and cause a shift in the infrared absorption peaks. The infrared spectroscopy results demonstrate that ergothioneine and carnosine can form a stable supramolecular structure through non-covalent interactions.

[0042] The efficacy was then tested using a 1:1 ergothioneine-carnosine cocrystal supramolecular structure.

[0043] Example 2 The permeability of supramolecular molecules was evaluated using human body Raman spectroscopy permeation testing.

[0044] Experimental Methods: Raman spectroscopy was performed on the anterior region of the human forearm. The samples used for testing were supramolecular ergothioneine-carnosine and a mixture of ergothioneine and carnosine. A 1×1 cm sample was selected. 2 A square area is taken as one region, and a total of 6 regions of equal size are taken from one arm. Five points are taken from each region using the vertex method and the center method. The average of three scans of each point is taken as a valid data. One person is tested, and tests are conducted at different time points of 0h, 0.5h, 1h, 2h, 4h, 6h and 8h.

[0045] For specific methods, please refer to T / SHRII "4-20γ "Cosmetic Ingredient Transdermal Penetration Testing: In Vivo Raman Spectroscopy". The experimental samples were supramolecular ergothioneine-carnosine (1:1 molar ratio) and ordinary mixed ergothioneine + carnosine (1:1 molar ratio); Usage: Topical application, dosage according to human Raman spectroscopy standards is 2 mg / cm². 2 .

[0046] Raman permeation images of supramolecular molecules and mixtures are as follows: Figure 5 , 6 As shown, the relative permeability (%) and maximum permeation depth (μm) detection results are shown in Tables 2 and 3: Table 2. Relative permeability (%) of ergothioneine Table 3. Maximum penetration depth of ergothioneine (μm) In-depth analysis of Raman images can reveal the distribution of ergothioneine in supramolecular ergothioneine-carnosine at different depths in human skin, as shown in the following diagram. Figure 5 As shown, it can be seen that: it did not penetrate to the stratum corneum within 0 h; it penetrated to the stratum corneum within 0.5 h; it penetrated to the active epidermis within 1 h, 2 h, and 4 h; and it broke through the active epidermis and entered the dermis within 6 h and 8 h, continuing to penetrate. The relative permeability of ergothioneine-carnosine on human skin after in vivo application was calculated to be 0%, 2.82%, 3.15%, 5.86%, 6.91%, 9.38%, and 10.56% at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h, respectively; the maximum penetration depths were 0 μm, 20 μm, 30 μm, 50 μm, 50 μm, 110 μm, and 120 μm, respectively.

[0047] Depth analysis of Raman images can reveal the distribution of ergothioneine in a simple mixture of ergothioneine and carnosine at different depths in human skin. The distribution is shown in the figure below. Figure 6As shown, it can be seen that: it did not penetrate the stratum corneum within 0 h; and it broke through the stratum corneum and penetrated into the active epidermis within 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h. The relative penetration rates of ergothioneine after applying a simple mixture of ergothioneine and carnosine to human skin at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 8 h were calculated to be 0%, 1.45%, 2.86%, 3.71%, 5.05%, 7.01%, and 7.25%, respectively; the maximum penetration depths were 0 μm, 30 μm, 40 μm, 60 μm, 100 μm, 80 μm, and 90 μm, respectively.

[0048] In summary, the test results show that, using ergothioneine as a marker, the human body penetration ability of the ergothioneine-carnosine supramolecular is superior to that of the ordinary mixed ergothioneine + carnosine. The relative penetration rate at 8 hours is 1.46 times that of the ordinary mixed ergothioneine + carnosine, and the maximum penetration depth at 8 hours is also significantly higher than that of the ordinary mixed ergothioneine + carnosine.

[0049] Therefore, the ergothioneine-carnosine cocrystal supramolecular has a good transdermal effect, which solves the problem of low transdermal efficiency of ergothioneine.

[0050] Example 3 The antiglycemic properties of ergothionein-carnosine supramolecular molecules were evaluated by assessing the carboxymethyl lysine (CML) content in human primary fibroblasts after stimulation with methylglyoxal (MGO) using immunofluorescence staining.

[0051] Non-enzymatic glycation refers to the Maillard reaction between the aldehyde or ketone groups of reducing sugars and the free amino groups of macromolecules such as proteins, lipids, and nucleic acids under enzyme-free conditions, resulting in advanced glycation end products (AGEs). AGEs are a significant factor contributing to skin aging and dull, yellowish skin tone. The main structural components of AGEs include carboxymethyl lysine (CML), carboxyethyl lysine (CEL), and methylacetaldehyde-lysine dimer (MOLD), among others. Carboxymethyl lysine is currently the most abundant known form of AGEs in the body.

[0052] Methylglyoxal (MGO) is a dicarbonyl compound and a highly reactive intermediate in non-enzymatic glycosylation reactions. It rapidly binds to intracellular proteins, undergoing a series of reactions to ultimately form advanced glycation end products (AGEs). Aminoguanidine hydrochloride is a classic AGEs inhibitor. It competitively blocks the cross-linking of proteins and sugars by chemically adding to the reactive carbonyl intermediates produced in early glycosylation reactions, thereby inhibiting AGEs formation. Aminoguanidine hydrochloride was used as a positive control in this experiment.

[0053] Immunofluorescence technology utilizes antigen-antibody reactions to locate antigens within tissues or cells or on cell membranes. First, a known, unlabeled specific antibody (primary antibody) reacts with the test substance (antigen). Unbound antibodies are washed away with PBS. Then, a labeled antibody (secondary antibody) reacts with the primary antibody-antigen sample to form an antigen-primary-secondary antibody complex. Unreacted labeled antibodies are washed away with PBS, and the sample is dried, mounted, and examined under a microscope. The intensity of the immunofluorescence can qualitatively indicate the content or expression level of the labeled substance.

[0054] The experimental method and steps are as follows: 1. Cell Culture Thaw the frozen cells in a 37°C water bath. Add the cell suspension to 9 mL of pre-warmed complete culture medium and centrifuge at 500 g / min for 5 min. Remove the supernatant, resuspend the cells in complete culture medium, and count them. Seed the cells into culture dishes as needed and incubate at 37°C with 5% CO2. Change the medium every 1-2 days, removing the old medium and adding fresh medium, until the cells reach over 80% confluence.

[0055] 2. Cell seeding on 8-well chamber slides When the cells in the cell culture dish reach 80% confluence, remove the culture medium and wash twice with PBS. Add an appropriate amount of trypsin-EDTA to the culture dish and incubate for 2 min. Add an equal volume of complete culture medium to terminate digestion, transfer the liquid to a centrifuge tube, and centrifuge at 500 g / min for 5 min. Prepare a cell suspension with complete culture medium and seed 20,000 cells / well into 8-well slides. Incubate the cells to allow them to recover, adhere, and enter the logarithmic growth phase. Observe the cells during this period to ensure relatively uniform cell growth throughout the well plate. 3. Modeling and sample preparation After 24 h of cell incubation, the original culture medium was discarded, and the cells were washed twice with PBS. Sample addition was performed according to Table 4. For the experimental group samples: the sample powder was weighed, dissolved in PBS to prepare a 1% stock solution, and then diluted with complete culture medium containing 400 μM MGO to the final concentration shown in Table 4. The cells were then cultured at 37 ℃ and 5% CO2 for another 48 h. A blank control group (NT), a negative control group (NC), and a positive control group (PC) were set up. No MGO was added to the blank control group, and 10 mM aminoguanidine hydrochloride was used as a control in the positive control group.

[0056] 4. Immunofluorescence staining and photography After 48 h of cell incubation, the original culture medium was discarded, and the cells were rinsed twice with PBS. Immunofluorescence staining was performed following the steps of fixation-permeability-blocking-primary antibody incubation-secondary antibody incubation-DAPI staining-mounting. The cells were then observed and photographed under a fluorescence microscope.

[0057] 5. Data Processing Fluorescence intensity was analyzed using ImageJ image processing software, and bar charts were plotted and significance analysis was performed using Graphpad plotting software. Relative fluorescence intensity (%) = fluorescence intensity of negative control well, positive control well, or test sample group / fluorescence intensity of blank control group × 100% 6. Verification of experimental validity The standard deviation (SD) between parallel wells in each group was statistically measured, and the coefficient of variation (CV) was calculated. If the CV value is ≤20%, the parallelism of the experiment is considered to be valid.

[0058] The test results are shown in Table 4 and Figure 7. Compared with the control group, supramolecular ergothionein-carnosine significantly reduced the carboxymethyl lysine (CML) content in human primary fibroblasts, with an inhibition rate of 19.35%.

[0059] Simple mixing of ergothioneine and carnosine had no significant effect on the carboxymethyl lysine (CML) content in human primary fibroblasts.

[0060] Table 4: Summary of CML Immunofluorescence Analysis Results Example 4 The following study investigated the antioxidant efficacy of ergothioneine-carnosine supramolecular molecules, and assessed the reactive oxygen species (ROS) levels in HaCaT cells after UVB stimulation using immunofluorescence staining.

[0061] Ultraviolet radiation (UVR) is divided into three categories: UVC (100-280 nm) is mainly blocked by the atmospheric ozone layer and usually does not reach the skin surface; UVB (280-320 nm) has a shorter wavelength, higher energy, and shallower penetration depth, and is mainly absorbed by the epidermis; UVA (320-400 nm) has the longest wavelength, the strongest penetration, and can penetrate deep into the dermis.

[0062] Reactive oxygen species (ROS) are a collective term for oxygen-containing, chemically active, and highly oxidizing free radicals and their oxygen-containing non-free radical products. UVB can generate ROS by activating photosensitizing substances and NADPH oxidase in epidermal keratinocytes, leading to mitochondrial dysfunction and causing excess electrons to leak from the mitochondrial electron transport chain, reacting with oxygen to generate ROS and resulting in elevated intracellular ROS levels. L-ascorbic acid has a low redox potential and a strong electron / hydrogen atom donor capacity, enabling it to directly scavenge intracellular ROS free radicals and effectively reduce the increase in ROS levels caused by UVB irradiation. L-ascorbic acid was used as a positive control in this experiment.

[0063] CELLROX™ GREEN reagent is a fluorescent probe for measuring oxidative stress in living cells. This cell-permeable dye emits weak fluorescence in its reduced state and, upon oxidation by reactive oxygen species, binds to DNA, emitting bright green fluorescence.

[0064] The experimental steps and methods are as follows: 1. Cell Culture Thaw the frozen cells in a 37°C water bath. Add the cell suspension to 9 mL of pre-warmed complete culture medium and centrifuge at 500 g / min for 5 min. Remove the supernatant, resuspend the cells in complete culture medium, and count them. Seed the cells into culture dishes as needed and incubate at 37°C with 5% CO2. Change the medium every 1-2 days, removing the old medium and adding fresh medium, until the cells reach over 80% confluence.

[0065] 2. Seeding cells in 24-well plates When the cells in the cell culture dish reach 80% confluence, remove the culture medium and wash twice with PBS. Add an appropriate amount of trypsin-EDTA to the culture dish and incubate for 8 min. Add an equal volume of complete culture medium to stop digestion, transfer the liquid to a centrifuge tube, and centrifuge at 500 g / min for 5 min. Prepare a cell suspension with complete culture medium and seed 50,000 cells / well in a 24-well plate. Incubate the cells to allow them to recover, adhere, and enter the logarithmic growth phase. Observe the cells during this period to ensure relatively uniform cell growth throughout the plate.

[0066] 3. Sample addition treatment After 24 h of cell incubation, the original culture medium was discarded, the cells were rinsed twice with PBS, and the complete culture medium prepared according to Table 5 was added. The positive control was 0.02% L-ascorbic acid. The cells were then cultured for another 24 h at 37 ℃ and 5% CO2.

[0067] 4. UVB irradiation and dye incubation After 24 h of cell incubation, the original culture medium was discarded, and the cells were washed twice with PBS. 500 μL of PBS was added to each well. Cells in the blank control group were covered with aluminum foil, while the other groups were irradiated with ultraviolet light at 20 mJ / cm². 2 UVB irradiation was performed at the prescribed dose. After irradiation, the PBS was discarded, and ELLROX was added immediately. TM The GREEN working solution was incubated at 37 °C and 5% CO2 for 30 min.

[0068] 5. Photographing fluorescence images After dye incubation, the cells were rinsed three times with PBS, and fresh complete culture medium was added again. The cells were then observed and photographed using a fluorescence microscope.

[0069] 6. Data Processing Fluorescence intensity was analyzed using ImageJ image processing software, and bar charts were plotted and significance analysis was performed using Graphpad plotting software. Relative fluorescence intensity (%) = fluorescence intensity of negative control well, positive control well, or test sample group / fluorescence intensity of blank control group × 100% 7. Verification of experimental validity The standard deviation (SD) between parallel wells in each group was statistically measured, and the coefficient of variation (CV) was calculated. If the CV value is ≤20%, the parallelism of the experiment is considered to be valid.

[0070] Test Results Table 5: Summary of ROS fluorescence analysis results Example 5 The stability of ergothioneine supramolecular (ergothioneine to carnosine molar ratio 1:1) was tested.

[0071] The testing method was as follows: Ergothioneine 0.1 wt% and supramolecular ergothioneine (to make the ergothioneine content equal to 0.1 wt%) were added to the basic toner system, and a solar simulator test was conducted to simulate a month of long-term high temperature and light exposure test.

[0072] After a month of processing using a solar simulator, the fragrance was evaluated by experts, and the results are shown in Table 6: Table 6 The results showed that preparing ergothioneine into eutectic and eutectic-containing compositions could improve the photostability, thermal stability, and long-term stability of ergothioneine and inhibit the formation of amine odor.

Claims

1. An ergothioneine supramolecular, characterized in that, It is an ergothioneine-carnosine cocrystal, wherein the molar ratio of ergothioneine to carnosine is 3:1-1:

2.

2. The method for preparing the ergothioneine supramolecular according to claim 1, characterized in that, Ergothioneine and carnosine were mixed in a certain ratio and prepared by solution synthesis or solid-state grinding to obtain ergothioneine-carnosine cocrystal.

3. The preparation method according to claim 2, characterized in that, Solid-state grinding is a solvent-assisted grinding method, in which a grinding solvent is added for grinding and mixing.

4. The preparation method according to claim 3, characterized in that, The added grinding solvent is water or an organic solvent, or any mixture thereof.

5. The application of the ergothioneine supramolecular as described in claim 1 in the preparation of food, health food, food raw materials, daily chemical raw materials, skin care products, cosmetics, pharmaceuticals or pharmaceutical raw materials.

6. A daily chemical raw material, characterized in that, Contains the ergothioneine supramolecular as described in claim 1.

7. A skincare or cosmetic product, characterized in that, Contains the ergothioneine supramolecular as described in claim 1 or the daily chemical raw material as described in claim 6.

8. The skincare or cosmetic product according to claim 7, characterized in that, The skincare or cosmetic products mentioned above have the effects of anti-glycation, brightening skin tone, whitening, anti-oxidation, anti-ultraviolet, anti-early aging, or anti-aging.

9. A food, health food, or food ingredient, characterized in that, Contains the ergothioneine supramolecular as described in claim 1.

10. A pharmaceutical product or pharmaceutical raw material, characterized in that, Contains the ergothioneine supramolecular as described in claim 1.

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