Zinc-based nanoscale enzyme and preparation method and application thereof

Abstract

This invention relates to a zinc-based nanozyme, its preparation method, and its applications. The zinc-based nanozyme is prepared by coordination complexation of a zinc-containing compound and a polyphenolic compound as precursors. The preparation method includes the following steps: mixing and stirring the zinc-containing compound and the polyphenolic compound in water to obtain a uniformly dispersed solution; adjusting the pH of the solution to 5.0-7.4, followed by heating; cooling to obtain a zinc-based nanozyme solution, and then centrifuging and drying to obtain zinc-based nanozyme powder. Compared with existing technologies, the zinc-based nanozyme preparation process of this invention is simple and efficient, requiring only one aqueous phase reaction, and is suitable for large-scale preparation of zinc-based nanozymes, with broad application prospects in skin care, scalp and hair health, and medical health fields.

Description

Technical Field

[0001] This invention belongs to the field of nanoenzyme technology, and in particular relates to a zinc-based nanoenzyme, its preparation method and application. Background Technology

[0002] Enzymes are protein molecules that catalyze chemical reactions in living organisms. They possess advantages such as high efficiency, specificity, and mild reaction conditions, and are widely used in fields such as biosynthesis, biocatalysis, and biosensors. However, natural enzymes face many limitations in practical applications: their poor stability makes them susceptible to inactivation due to factors such as temperature, pH, and organic solvents; high separation and purification costs make them difficult to reuse, severely restricting their industrial application.

[0003] In recent years, the rise of nanozymes has brought about revolutionary breakthroughs. Nanozymes are a class of nanomaterials with enzyme-like catalytic activity, combining the high catalytic efficiency of natural enzymes with the unique physicochemical properties of nanomaterials (such as high specific surface area, tunable morphology, and strong stability). Since the discovery of peroxidase-like activity in iron oxide nanoparticles in 2007, nanozyme research has covered a variety of materials, including gold, copper, and cerium dioxide, and has been widely applied in fields such as biosensing, disease treatment, and environmental protection. Despite the enormous potential shown by nanozymes, their development still faces challenges: their catalytic efficiency lags behind that of natural enzymes, and their biocompatibility and targeting are insufficient. Therefore, developing novel nanozyme materials and improving their performance through functional design has become a key research focus.

[0004] Zinc (Zn) is an essential trace element for living organisms; more than 300 enzymes depend on zinc ions (Zn). 2+ These enzymes maintain their structure and function, including carbonic anhydrase, alcohol dehydrogenase, and DNA polymerase. For example, in carbonic anhydrase-catalyzed reactions, Zn... 2+ By coordinating with histidine residues and water molecules, Zn significantly accelerates the rate of enzyme-catalyzed reactions through the polarization of water molecules; in the alcohol dehydrogenase catalysis reaction, Zn... 2+ It coordinates with cysteine ​​and histidine residues to form catalytic sites, stabilizing the intermediate state and lowering the activation energy of the reaction by binding to the hydroxyl group of the ethanol molecule, thus assisting the hydrogen transfer process and achieving high efficiency and specificity in enzyme catalysis. In summary, Zn 2+ Zinc can act as an electron acceptor to polarize substrate molecules, thereby lowering the activation energy of reactions. Furthermore, zinc can form flexible coordination structures with nitrogen-, sulfur-, and oxygen-containing ligands, facilitating functionalization. These unique properties make zinc an ideal choice for designing biomimetic catalytic materials. Therefore, zinc-based nanozymes hold promise for achieving efficient, stable, and safe catalytic performance by mimicking the active site of natural zinc enzymes.

[0005] Patent CN119386044A discloses a method for preparing zinc-dihydromyricetin nanozyme and its application in preparing drugs to promote the healing of diabetic wounds. The zinc-dihydromyricetin nanozyme has antioxidant properties, can promote cell proliferation and migration, and can accelerate the early healing of diabetic wounds.

[0006] Patent CN119528122A discloses a method for preparing and applying a zinc-manganese bimetallic organic framework-derived composite nanozyme. The zinc-manganese bimetallic organic framework-derived composite nanozyme is obtained by calcining zinc-manganese bimetallic organic framework nanoparticles in a nitrogen atmosphere. It has peroxidase-like activity, glutathione peroxidase-like activity, and catalase-like activity.

[0007] Patent CN114042470A discloses a boron-doped zinc-based single-atom nanozyme, its preparation method, and its application. The boron-doped zinc-based single-atom nanozyme changes the coordination environment by introducing boron atoms, improves the hydrophilicity of the nanozyme, significantly enhances its peroxidase-like activity, and enables the monitoring of trace amounts of p-phenylenediamine in hair after dyeing.

[0008] However, the above-mentioned methods for preparing zinc-based nanozymes generally suffer from problems such as complex processes and poor environmental friendliness. They often require reactions under high temperature and high pressure conditions, the use of toxic and harmful chemical reagents, or long-term dialysis, which greatly restricts their large-scale production and further practical application.

[0009] Therefore, there is an urgent need to develop a novel zinc-based nanozyme and its simple and controllable preparation method. Summary of the Invention

[0010] The purpose of this invention is to overcome at least one of the defects of existing technologies, such as complex processes, unfriendly environment, and long preparation time, and to provide a zinc-based nanozyme, its preparation method, and its application.

[0011] The objective of this invention can be achieved through the following technical solutions:

[0012] This invention provides a zinc-based nanozyme, which is prepared by coordination complexation of a zinc-containing compound and a polyphenolic compound as precursors.

[0013] Further, the zinc-containing compound includes one or more of zinc pyrrolidone carboxylate, zinc gluconate, zinc lactate, zinc aspartate, zinc glycine, zinc hydrolyzed hyaluronic acid, zinc hydrolyzed collagen, zinc DNA, zinc acetate, zinc sulfate, or zinc chloride, preferably zinc pyrrolidone carboxylate (PCA-Zn).

[0014] Furthermore, the polyphenolic compound includes one or more of epigallocatechin galloglycoside, tannic acid, epigallocatechin gallate, tea polyphenols or catechins, preferably epigallocatechin galloglycoside (EGCG).

[0015] Furthermore, the average particle size of the zinc-based nanozyme ranges from 100 to 400 nm.

[0016] This invention also provides a method for preparing zinc-based nanozymes, the method comprising the following steps:

[0017] S1: Mix the zinc-containing compound and the polyphenol compound in water and stir until homogeneous to obtain a uniformly dispersed solution;

[0018] S2: Adjust the pH of the solution to 5.0-7.4, and then carry out the heating reaction;

[0019] S3: After cooling, a zinc-based nanozyme solution is obtained, which is then separated by centrifugation and dried to obtain zinc-based nanozyme powder.

[0020] Further, in step S1, the mass ratio of the zinc-containing compound to the polyphenol compound is 1:(0.02-0.2).

[0021] Further, in step S1, the concentration of the zinc-containing compound in the solution is 0.1-0.5 g / mL.

[0022] Furthermore, in step S2, the temperature of the heating reaction is 30-60°C, more preferably 30-45°C.

[0023] Furthermore, in step S2, the heating reaction time is 3-6 hours to increase the reaction rate of coordination complexation.

[0024] Furthermore, in step S3, the centrifugation speed is 8000-12000 rpm. When the speed is too low, the zinc-based nanozymes in the solution cannot be effectively separated.

[0025] Furthermore, in step S3, the drying temperature is 50-70°C.

[0026] This invention also provides the application of zinc-based nanozymes in the preparation of skin and scalp care products.

[0027] Furthermore, the care products include, but are not limited to, face creams, lotions, serums, cleansing gels, shampoos, and conditioning oils.

[0028] Furthermore, the zinc-based nanozyme can scavenge hydroxyl radicals and hydrogen peroxide, exhibiting antioxidant properties.

[0029] Furthermore, the zinc-based nanozyme can inhibit lipid synthesis in sebaceous gland cells and has anti-oil properties.

[0030] Furthermore, the zinc-based nanozyme exhibits antibacterial properties against lipophilic fungi and Propionibacterium.

[0031] Furthermore, the lipophilic fungi include Malassezia.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) This invention achieves the controllable preparation of zinc-based nanozymes by using a low-temperature, template-free one-step synthesis method with zinc-containing compounds and polyphenolic compounds as precursors through coordination complexation. The preparation process is simple and efficient, requiring only one step of aqueous phase reaction, and is suitable for the large-scale preparation of zinc-based nanozymes.

[0034] (2) The zinc-based nanozyme preparation method of the present invention is simple, does not require high temperature and high pressure conditions, and is efficient and fast; it does not require the introduction of organic solvents, is green and mild; and has a wide range of materials to choose from, which is suitable for the large-scale production of zinc-based nanozyme materials and helps to further broaden the application field of zinc-based nanozymes.

[0035] (3) The zinc-based nanozyme prepared by this invention has peroxidase-like activity and catalase-like activity, which can effectively scavenge hydroxyl radicals and hydrogen peroxide, and has excellent antioxidant properties; it can inhibit lipid synthesis in sebaceous gland cells, and has excellent anti-oil properties; it also has good bactericidal activity against lipophilic fungi and Propionibacterium. The above-mentioned excellent properties enable the zinc-based nanozyme of this invention to be further used in the preparation of skin and scalp care products, and has broad application prospects in the fields of skin care, scalp and hair health, and biomedicine. Attached Figure Description

[0036] Figure 1 This is the photoelectron spectrum of the zinc-based nanozyme of Example 1 of the present invention.

[0037] Figure 2 This is the UV-Vis spectrum of the zinc-based nanozyme of Example 1 of the present invention.

[0038] Figure 3 This is the Fourier transform infrared spectrum of the zinc-based nanozyme of Example 1 of the present invention.

[0039] Figure 4 This is a transmission electron microscope image of the zinc-based nanozyme of Example 1 of the present invention.

[0040] Figure 5 This is a dynamic light scattering diagram of the zinc-based nanozyme of Example 1 of the present invention.

[0041] Figure 6The value represents the hydroxyl radical scavenging rate of the zinc-based nanozyme in Example 1 of this invention.

[0042] Figure 7 The hydrogen peroxide scavenging rate of the zinc-based nanozyme in Example 1 of this invention.

[0043] Figure 8 The cytotoxicity of the zinc-based nanozyme of Example 1 of this invention.

[0044] Figure 9 The results show the lipid synthesis inhibition of the zinc-based nanozyme in Example 1 of this invention.

[0045] Figure 10 This is a schematic diagram of the growth of the corresponding colonies after co-culturing the zinc-based nanozyme with Malassezia in Example 1 of the present invention.

[0046] Figure 11 This is a schematic diagram of the growth of the corresponding colonies after co-culturing the zinc-based nanozyme with Propionibacterium acnes in Example 1 of the present invention. Detailed Implementation

[0047] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0048] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0049] Example 1:

[0050] This embodiment provides a zinc-based nanozyme, which is prepared by coordination complexation of zinc pyrrolidone carboxylate (PCA-Zn, purity ≥99%, Guangzhou Nuoyan) and epigallocatechin gallate glucoside (EGCG, purity ≥98%, Shanghai Dibai Biotechnology) as precursors, and is denoted as NanoZn NMs.

[0051] The specific preparation method of NanoZn NMs is as follows:

[0052] 20g of EGCG was dissolved in 100mL of deionized water, and 100g of PCA-Zn was dissolved in 400mL of deionized water. The aqueous solutions were mixed and stirred at 500rpm to obtain a uniformly dispersed solution. The pH was adjusted to approximately 5.5 with sodium hydroxide solution, and the reaction mixture was stirred at 30℃ for 3 hours until the reaction was complete. After the reaction, a NanoZn NMs solution was obtained. After centrifugation at 10000rpm, the solution was thoroughly dried in a vacuum drying oven at 60℃ to obtain NanoZn NMs powder.

[0053] In this embodiment, the prepared NanoZn NMs were characterized by the following tests:

[0054] (1) Photoelectron spectroscopy characterization: The photoelectron spectrum of NanoZn NMs is as follows Figure 1 As shown, the distribution of C, O, and Zn can be clearly observed through XPS full-spectrum analysis. The binding energy of the C1s peak is 284.4 eV, which is relatively stable and often used to calibrate XPS spectra. The binding energy of the O1s peak is 531.3 eV, higher than that of typical metal oxides, indicating that oxygen may exist in the form of hydroxyl groups, carbonates, or adsorbed water molecules. The binding energy of the Zn2p peak is 1021.6 eV. The two main peaks at 1022.2 eV and 1045.2 eV in the high-resolution Zn2p spectrum are attributed to Zn2p3 / 2 and Zn2p1 / 2, respectively, indicating that zinc exists as Zn. 2+ It exists in its natural form and forms coordination bonds with oxygen-containing ligands. Meanwhile, the Zn LMM Auger parameter of approximately 2011.8 eV confirms that Zn exists in its natural form. 2+ Coordination with EGCG.

[0055] (2) Ultraviolet-visible spectral characterization: The ultraviolet-visible spectrum of NanoZn NMs is shown in the figure. Figure 2 As shown, the π→π* transition of the catechol structure in EGCG exhibits a characteristic absorption peak at 274 nm, while the UV characteristic absorption peak of NanoZn NMs is red-shifted to 324 nm. This indicates that the polyphenol structure of EGCG coordinates with zinc ions through hydroxyl groups to form a stable metal-polyphenol complex, which leads to a change in the π→π* transition of the catechol structure.

[0056] (3) Fourier transform infrared spectroscopy characterization: The Fourier transform infrared spectrum of NanoZn NMs is shown below. Figure 3 As shown, 1299cm -1 The intensity of the CN stretching vibration peak of the pyrrolidone ring was significantly weakened, at 1631 cm⁻¹. -1 The shift in the C=C stretching vibration peak in the benzene ring indicates that Zn coordinates with EGCG.

[0057] (4) Morphological characterization: Transmission electron microscopy images of NanoZn NMs are shown below. Figure 4 As shown, the nanozymes appear as sheets and aggregate, which may be due to the drying process during TEM sample preparation.

[0058] Example 2:

[0059] This embodiment provides a zinc-based nanozyme and a specific preparation method. The difference from Example 1 is that the zinc-containing compound used in this embodiment is zinc gluconate, while other compounds and preparation methods can refer to Example 1.

[0060] Example 3:

[0061] This embodiment provides a zinc-based nanozyme and a specific preparation method. The difference from Example 1 is that the polyphenol compound used in this embodiment is tannic acid, while other compounds and preparation methods can refer to Example 1.

[0062] Example 4:

[0063] This embodiment provides a zinc-based nanozyme and a specific preparation method. The difference from Embodiment 1 is that the ratio of EGCG to PCA-Zn is different in this embodiment.

[0064] The specific preparation method in this embodiment is as follows:

[0065] 20g of EGCG was dissolved in 100mL of deionized water, and 200g of PCA-Zn was dissolved in 400mL of deionized water. The mixture was stirred to obtain a homogeneous dispersion. The pH of the solution was adjusted to approximately 5.5 using sodium hydroxide solution. The reaction mixture was then stirred at 30℃ for 3 hours until the reaction was complete. After the reaction, the solution was allowed to cool naturally to room temperature to obtain the NanoZn NMs solution. After centrifugation at 10000rpm, the solution was thoroughly dried in a vacuum drying oven at 60℃ to obtain NanoZn NMs powder.

[0066] Comparative Example 1:

[0067] This comparative example provides a pure PCA-Zn powder, prepared according to the preparation method in Example 1. The specific preparation method is as follows:

[0068] 200g of PCA-Zn was weighed and dissolved in 400mL of deionized water. The mixture was stirred to obtain a homogeneous dispersion. The pH of the solution was adjusted to approximately 5.5 using sodium hydroxide solution. The reaction mixture was then stirred at 30℃ for 3 hours until the reaction was complete. After the reaction, the solution was allowed to cool naturally to room temperature to obtain the final solution. After centrifugation at 10000rpm, the solution was thoroughly dried in a vacuum drying oven at 60℃ to obtain the PCA-Zn powder of Comparative Example 1.

[0069] Comparative Example 2:

[0070] This comparative example provides a pure ECCG powder, prepared according to the preparation method in Example 1. The specific preparation method is as follows:

[0071] Weigh 20g of EGCG and dissolve it in 100mL of deionized water. Mix and stir to obtain a homogeneous dispersion. Adjust the pH of the solution to approximately 5.5 using sodium hydroxide solution. Then, stir the reaction solution at 30℃ for 3 hours until the reaction is complete. After the reaction is complete, allow the solution to cool naturally to room temperature to obtain the final solution. Centrifuge at 10000rpm and then dry thoroughly in a vacuum drying oven at 60℃ to obtain the powder of Comparative Example 2.

[0072] Comparative Example 3:

[0073] This comparative example provides a zinc-based nanozyme and a specific preparation method. The difference from Example 1 is that the ratio of EGCG to PCA-Zn is different in this example.

[0074] The specific preparation method for this comparative example is as follows:

[0075] 200g of EGCG (excess) was dissolved in 100mL of deionized water, and 200g of PCA-Zn was dissolved in 400mL of deionized water. The mixtures were stirred to obtain a homogeneous dispersion. The pH of the solution was adjusted to approximately 5.5 using sodium hydroxide solution. The reaction mixture was then stirred at 30℃ for 3 hours until the reaction was complete. After the reaction, the solution was allowed to cool naturally to room temperature to obtain the NanoZnNMs solution. After centrifugation at 10000rpm, the solution was thoroughly dried in a vacuum drying oven at 60℃ to obtain the NanoZnNMs powder of Comparative Example 3.

[0076] Comparative Example 4:

[0077] This comparative example uses dihydromyricetin as a raw material to attempt to prepare zinc-dihydromyricetin nanozyme according to the method of the present invention. The specific preparation method is as follows:

[0078] 20g of dihydromyricetin was dissolved in 100mL of deionized water, and 100g of PCA-Zn was dissolved in 400mL of deionized water. The mixture was stirred to obtain a homogeneous dispersion. The pH of the solution was adjusted to approximately 5.5 using sodium hydroxide solution. The reaction mixture was then stirred at 30℃ for 3 hours until the reaction was complete. After the reaction, the solution was allowed to cool naturally to room temperature to obtain the zinc-dihydromyricetin nanozyme solution. After centrifugation at 10000rpm, the solution was thoroughly dried in a vacuum drying oven at 60℃ to obtain the sample powder of Comparative Example 4.

[0079] The particle size of the above-described embodiments and comparative examples was characterized, and the test results are shown in Table 1.

[0080] Table 1 Summary of sample particle size test results

[0081] Serial Number Sample Name Average particle size PDI 1 Example 1 164nm 0.2 2 Example 2 137.5nm 0.493 3 Example 3 228.9nm 0.488 4 Example 4 383.5nm 0.468 6 Comparative Example 1 231.9nm 0.504 7 Comparative Example 2 793nm 0.793 8 Comparative Example 3 246.8nm 0.714 9 Comparative Example 4 1308nm 1

[0082] The dynamic light scattering pattern of NanoZn NMs obtained in Example 1 is shown below. Figure 5 As shown, the average particle size of NanoZn NMs is 164 nm, and the polydispersity index (PDI) is 0.2, indicating that Example 1 successfully prepared a uniformly dispersed zinc-based nanozyme. Similarly, the zinc-based nanozymes prepared in Examples 2-4 have average particle sizes in the range of 100-400 nm, and PDIs are all no greater than 0.5, demonstrating that the method of this invention can achieve low-temperature, template-free one-step synthesis of zinc-based nanozymes, producing nanoscale and uniformly dispersed zinc-based nanozymes. In contrast, in Comparative Example 3, the excessive addition of polyphenolic compounds resulted in a polydispersity index as high as 0.714, indicating that the entire zinc-based nanozyme was not a uniformly dispersed system and was prone to aggregation and precipitation, undoubtedly limiting its further application in solution systems. The zinc-based nanozymes prepared by this invention have good aqueous dispersibility, therefore they can be dispersed in liquids such as water, physiological saline, PBS buffer, and cell culture medium during use.

[0083] In view of the successful preparation of zinc-based nanozymes of the present invention, the present invention further explores the application of the prepared zinc-based nanozymes in the fields of skin care, scalp and hair health, and biomedicine. Taking the zinc-based nanozymes prepared in Example 1 as an example, the following antioxidant, oil-controlling, and antibacterial properties were tested:

[0084] (1) Hydroxyl radical scavenging rate test:

[0085] 1 mL of sample solutions of different concentrations were mixed thoroughly with 1 mL of FeSO4 solution (9 mM), 1 mL of H2O2 solution (8.8 mM), and 1 mL of ethanol-salicylic acid solution (9 mM). A control group without H2O2 and a blank group without sample solutions were also included. The mixture was reacted at 37°C for 15 min, then cooled, and the absorbance of the mixed solution was measured at 510 nm. The formula for calculating the hydroxyl radical scavenging rate is as follows:

[0086]

[0087] In the formula, A X It is the absorbance of the sample, A x0 A1 is the absorbance of the control group (excluding H2O2), and A0 is the absorbance of the blank group (excluding sample).

[0088] (2) Hydrogen peroxide scavenging rate test:

[0089] H2O2 indicator was prepared by dissolving 25 mg of Ti(SO4)2 in 25 mL of 3 M H2SO4 solution. 200 μL of the indicator was added to 100 μL of H2O2 solutions of different concentrations (0.2, 0.4, 0.6, 1, 2, 4 mM), and the absorbance of these solutions at 407 nm was measured to establish a standard curve for different concentrations of hydrogen peroxide. Samples of different masses were mixed thoroughly with 4 mL of 1 mM H2O2 and incubated with shaking at 37 °C. After 1 hour of incubation, 100 μL of the mixed solution was mixed with 200 μL of the indicator, and the absorbance of the solution at 407 nm was measured using a microplate reader. The H2O2 concentration was calculated based on the standard curve. The formula for calculating the H2O2 scavenging rate is as follows:

[0090]

[0091] In the formula, C0 is the initial concentration of H2O2, C t It is the concentration of H2O2 at time t.

[0092] (3) Cytotoxicity test:

[0093] The cytotoxicity of NanoZn NMs prepared in Example 1 against human sebaceous gland SZ95 cells was detected using the CCK-8 assay. The human sebaceous gland cells used in the test were SZ95 cells, and the specific test steps are as follows:

[0094] Human sebaceous gland cells (SZ95) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-drug antibiotics at 37°C in a 5% CO2 incubator until the logarithmic growth phase. The cells were then cultured at a rate of 6 × 10⁻⁶ cells / year. 3 Cells were seeded at a density of [number] cells / well in 96-well plates. After reaching 70%–80% confluence, different concentrations of NanoZn NMs were added and co-cultured for 24 h. The culture medium was removed, and the cells were washed three times with PBS. 200 μL of culture medium and 20 μL of CCK-8 solution were added, and the cells were incubated at 37°C in a CO2 incubator for 4 h. The absorbance of each well was measured at 450 nm using a microplate reader. The cytotoxicity of PCA-Zn and EGCG was tested using the same method.

[0095] The method for calculating cell viability is as follows:

[0096]

[0097] Where A 样品 It is the OD value of the well containing cells, CCK-8 solution, and sample, A 对照 It is the OD value of a well containing cells and CCK-8 solution but no sample solution, A 空白 It is the OD value of a cell-free pore containing CCK-8 solution.

[0098] (4) Oil control performance test:

[0099] Human sebaceous gland cells (SZ95) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-drug antibiotics at 37°C in a 5% CO2 incubator until the logarithmic growth phase. The cells were then cultured at a rate of 1×10⁻⁶ cells / year. 4 Cells were seeded at a density of 100 cells / well in 12-well plates. After the cells reached a confluence of 70%-80%, they were divided into control, model, and sample groups. The model group was treated with 100 μmol / L dihydrotestosterone, while the sample groups were treated with dihydrotestosterone followed by different concentrations of sample. After incubation for 24 h, the supernatant was aspirated, the cells were washed three times with PBS, and incubated with Nile Red staining for 20 min. The supernatant was discarded, and the cells were centrifuged in 1.5 mL centrifuge tubes at 2000 rpm for 5 min. After discarding the supernatant, the cells were resuspended in 4% paraformaldehyde solution, and the mean fluorescence intensity of the cells was detected by flow cytometry.

[0100] (5) Antibacterial performance test:

[0101] 5.1 Malassezia Antibacterial Experiment

[0102] The inhibitory effect of NanoZn NMs prepared in Example 1 on Malassezia was tested using the plate coating method. The Malassezia strain used in the test was Malassezia ATCC14521, and the specific test steps are as follows:

[0103] 1. Culture medium preparation: Accurately weigh 20g of LB broth, dissolve it in 1000mL of distilled water, heat to dissolve, and adjust the pH to between 7.2 and 7.4 with 2mol / L NaOH and 2mol / L HCl to prepare liquid culture medium. For solid culture medium, add 20g of agar powder to the liquid culture medium and heat to boiling. All culture media should be autoclaved for later use.

[0104] 2. Recovery and culture of Malassezia furfur: Under aseptic conditions, use an inoculation loop to pick up Malassezia bacterial suspension and streak it on a solid culture plate. Place the plate flat in a biochemical incubator and incubate at 28°C for 24 hours. Then, pick a single colony and place it in liquid culture medium and incubate at 28°C and 180 r / min for 24 hours for later use.

[0105] 3. Determination of the antibacterial effect of NanoZn NMs on Malassezia: Take the test bacterial suspension of Malassezia furfur and dilute it with PBS to approximately 10. 6 -10 7 CFU / mL was prepared for use. The nanozyme was diluted with liquid culture medium to five final concentration gradients: 25 mg / mL, 12.5 mg / mL, 6.25 mg / mL, 3.12 mg / mL, and 1.56 mg / mL, using a two-fold dilution method. 0.2 mL was added to each test tube, and 10 mg / mL of the solution was added to each. 60.2 mL of bacterial suspension at CFU / mL. After incubating all test tubes at 28°C for 24 h in a biochemical incubator, 0.2 mL of culture from each test tube was aspirated and evenly spread onto a solid culture plate. The plate was then incubated flat in the biochemical incubator at 28°C for 24 h. The presence or absence of colonies was observed. The concentration at which no colonies grew was the minimum bactericidal concentration (MBC) of the nanozyme against Malassezia.

[0106] 5.2 Propionibacterium inhibition test

[0107] The inhibitory effect of NanoZn NMs prepared in Example 1 on Propionibacterium acnes was tested using the plate coating method. The Propionibacterium acnes used in the test was Propionibacterium acnes BNCC252396, and the specific test steps are as follows:

[0108] 1. Culture medium preparation: Accurately weigh 25g of LB broth (Haibo Biotechnology: Product No. HB0128), dissolve in 1000mL of distilled water, dispense into containers, and autoclave at 121℃ for 15 minutes. Accurately weigh 40g of LB nutrient agar (Haibo Biotechnology: Product No. HB0129), dissolve in 1000mL of distilled water, and autoclave at 121℃ for 15 minutes.

[0109] 2. Resuscitation and culture of Propionibacterium acnes: Under aseptic conditions, use an inoculation loop to pick up Propionibacterium acnes and streak it on a solid culture plate. Place the plate flat in a biochemical incubator and anaerobically culture at 30°C for 24 hours. Then, pick a single colony and culture it in liquid culture medium at 30°C and 180 rpm for 24 hours for later use.

[0110] 3. Antibacterial activity assay: Take the test bacterium Propionibacterium acnes and dilute it with PBS to approximately 10. 6 -10 7 CFU / mL was prepared for use. Using the microdilution method, 100 μL of sterile LB medium was added to columns 2–11 of a 96-well plate. 200 μL of the diluted sample was added to column 1, and serially diluted to column 10. 100 μL of bacterial suspension was added to each well in columns 1–11, with column 11 serving as a growth control. 200 μL of LB liquid medium was added to column 12 as a blank control. The plate was incubated anaerobically at 30°C for 24 hours. Afterward, 100 μL of bacterial suspension from each well was evenly spread onto LB solid agar plates and incubated anaerobically at 30°C for 24 hours. Colonies were observed and counted. The concentration corresponding to no colony growth was the MBC of the nanozyme against Propionibacterium acnes.

[0111] The test results are as follows:

[0112] The hydroxyl radical scavenging rate and H2O2 scavenging rate of the NanoZn NMs prepared in Example 1 are as follows: Figure 6-7 As shown in the figure, at concentrations above 0.5 mg / mL, the hydroxyl radical scavenging rate of NanoZn NMs was significantly higher than that of EGCG and PCA-Zn, reaching approximately 90% at a concentration of 2 mg / mL. At concentrations above 1 mg / mL, the H2O2 scavenging rate of NanoZn NMs was significantly higher than that of EGCG and PCA-Zn, approaching 100% at a concentration of 4 mg / mL. These experiments all demonstrate that NanoZn NMs possess excellent antioxidant properties.

[0113] Cytotoxicity test results as follows Figure 8 As shown, the viability of SZ95 cells gradually decreased with increasing sample concentration. The viability of SZ95 cells was >90% with NanoZn NMs at 320 μg / mL, >90% with PCA-Zn at 8 μg / mL, and >90% with EGCG at 32 μg / mL. These results indicate that NanoZn NMs have good cell compatibility, and the final NanoZn NMs significantly reduced the toxicity of single-component PCA-Zn.

[0114] The test results for lipid synthesis inhibition are as follows: Figure 9 As shown in Table 2, the results of the dihydrotestosterone (DHT) model of sebaceous gland cells indicate that the addition of DHT significantly increased intracellular lipid synthesis in SZ95 cells (average fluorescence intensity greater than 3000), demonstrating successful modeling. NanoZn NMs can effectively inhibit intracellular lipid synthesis in human sebaceous gland cells (SZ95) of the male pattern baldness model. In particular, when the concentration of NanoZn NMs reaches 100 μg / mL, the inhibition rate of lipid synthesis in sebaceous gland cells can be increased to 27.9%. The above data indicate that the NanoZn NMs prepared in this invention have excellent oil-controlling effects.

[0115] Table 2. Experimental data on lipid synthesis inhibition (human sebaceous gland cells)

[0116] Group concentration Average fluorescence intensity Inhibition rate % NanoZn NMs 50μg / ml 2826.7±43.5 5.94％ NanoZn NMs 100μg / ml 2166.3±28.9 27.9％ Model group DHT / 3005.3±34.8 / control group / 2561.7±37.6 /

[0117] Antibacterial test results are as follows Figure 10-11As shown. The results indicated that only a very small number of Malassezia colonies grew in the 3.12 mg / mL NanoZn NMs group, indicating that the MBC of the NanoZn NMs prepared in Example 1 was 3.12 mg / mL, demonstrating its highly effective bactericidal effect against lipophilic fungi. This suggests potential for treating folliculitis and seborrheic dermatitis caused by Malassezia, and helps avoid the damage to the skin barrier caused by traditional antifungal drugs. For Propionibacterium, only a very small number of Propionibacterium colonies grew in the 6.25 mg / mL NanoZn NMs group, indicating that the MBC of the NanoZn NMs prepared in Example 1 was 6.25 mg / mL. This suggests that although the thick peptidoglycan layer of Propionibacterium may partially weaken the penetration of nanozymes, NanoZn NMs still possess certain bactericidal properties, and hold promise for treating acne inflammation, redness, and nodules caused by Propionibacterium.

[0118] In summary, this invention achieves the controllable preparation of zinc-based nanozymes through a developed low-temperature, template-free one-step synthesis method. The prepared zinc-based nanozymes exhibit peroxidase-like and catalase-like activities, and can significantly scavenge hydroxyl radicals and hydrogen peroxide, showing broad application prospects in skin care, scalp and hair health, and biomedicine.

[0119] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A zinc-based nanozyme, characterized in that, The zinc-based nanozyme is prepared by coordination complexation of zinc-containing compounds and polyphenolic compounds as precursors.

2. The zinc-based nanozyme according to claim 1, characterized in that, The zinc-containing compounds include one or more of zinc pyrrolidone carboxylate, zinc gluconate, zinc lactate, zinc aspartate, zinc glycine, hydrolyzed zinc hyaluronic acid, hydrolyzed collagen zinc, DNA zinc, zinc acetate, zinc sulfate, or zinc chloride.

3. The zinc-based nanozyme according to claim 1, characterized in that, The polyphenolic compounds include one or more of epigallocatechin galloglycoside, tannic acid, epigallocatechin gallate, tea polyphenols, or catechins.

4. The zinc-based nanozyme according to claim 1, characterized in that, The average particle size of the zinc-based nanozyme ranges from 100 to 400 nm.

5. A method for preparing the zinc-based nanozyme according to any one of claims 1-4, characterized in that, Includes the following steps: S1: Mix the zinc-containing compound and the polyphenol compound in water and stir until homogeneous to obtain a uniformly dispersed solution; S2: Adjust the pH of the solution to 5.0-7.4, and then carry out the heating reaction; S3: After cooling, a zinc-based nanozyme solution is obtained, which is then separated by centrifugation and dried to obtain zinc-based nanozyme powder.

6. The method for preparing zinc-based nanozymes according to claim 5, characterized in that, In step S1, the mass ratio of the zinc-containing compound to the polyphenol compound is 1:(0.02-0.2); The concentration of the zinc-containing compound in the solution is 0.1-0.5 g / mL.

7. The method for preparing zinc-based nanozymes according to claim 5, characterized in that, In step S2, the temperature of the heating reaction is 30-60℃; The heating reaction takes 3-6 hours.

8. The method for preparing zinc-based nanozymes according to claim 5, characterized in that, In step S3, the centrifugal separation speed is 8000-12000 rpm; The drying temperature is 50-70℃.

9. The use of the zinc-based nanozyme according to any one of claims 1-4 in the preparation of skin and scalp care products.

10. The application of the zinc-based nanozyme according to claim 9 in the preparation of skin and scalp care products, characterized in that, The zinc-based nanozyme can scavenge hydroxyl radicals and hydrogen peroxide, and has antioxidant properties. The zinc-based nanozyme can inhibit lipid synthesis in sebaceous gland cells and has anti-oil properties; The zinc-based nanozyme exhibits antibacterial properties against lipophilic fungi and Propionibacterium.

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