Antioxidant material and application thereof

Abstract

The invention belongs to the technical field of nano materials, and particularly relates to an antioxidant material and application thereof. The antioxidant material takes carboxylated cellulose or a nano-cellulose composite material obtained by coating the carboxylated cellulose with cerium as an active ingredient; the carboxylated cellulose is obtained by oxidizing cellulose through a TEMPO method, and CeNPs are uniformly coated on the fiber surface of TO-CNF through an in-situ synthesis method, so that agglomeration of CeNPs in a physiological environment can be effectively prevented, the physiological stability and biocompatibility of CeNPs are remarkably improved, and the long-lasting and stable exertion of the antioxidant effect of CeNPs is ensured.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, specifically relating to an antioxidant material and its applications. Background Technology

[0002] In recent years, the deepening of the concept of sustainable development has prompted researchers to focus on the functionalization of natural polymer materials. As one of the most promising bio-based nanomaterials, cellulose nanoparticles, with their unique structural characteristics such as high aspect ratio, excellent biocompatibility, and tunable surface chemistry, have shown great application potential in biomedicine, food packaging, and environmental remediation. Particularly in the biomedical field, with a deeper understanding of the mechanisms of oxidative stress-related diseases, the demand for antioxidant materials is increasing daily. These materials play a crucial role in wound repair, sustained-release drug delivery systems, and regenerative medicine engineering. However, unmodified cellulose nanoparticles have insufficient antioxidant activity, severely limiting their practical application value in this field. Currently widely used antioxidant systems, such as vitamin E derivatives, plant polyphenols, and metal oxide nanoparticles, while possessing certain free radical scavenging capabilities, suffer from poor stability, short duration of action, and limited biocompatibility. Therefore, developing a novel antioxidant composite material based on cellulose nanoparticles is of great significance.

[0003] Currently, the main methods for converting inert nanocellulose into functional active materials include physical adsorption and chemical grafting modification to load natural antioxidants (such as polyphenolic compounds) or metal nanoparticles. For example, adsorbing a natural extract rich in polyphenols onto the surface of nanocellulose yields a nanocomposite that exhibits high antioxidant capacity and can be used as a food additive. However, loading drugs or natural antioxidants onto the surface of nanocellulose presents problems of instability and easy deactivation. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides an antioxidant material and its applications.

[0005] An antioxidant material, wherein the antioxidant material uses carboxylated cellulose or a nanocellulose composite material obtained by coating the carboxylated cellulose with cerium as the active ingredient; The carboxylated cellulose was obtained by oxidizing cellulose using the TEMPO method.

[0006] The total antioxidant activity of the carboxylated cellulose of the present invention is much higher than that of cellulose. Furthermore, the carboxylated cellulose provides sufficient surface area and active sites to form a mesh film that uniformly coats CeNPs on the surface of the carboxylated cellulose. This effectively prevents CeNPs from agglomerating in the physiological environment and prevents small-sized CeNPs from being phagocytosed by cells, significantly improving its physiological stability, ensuring survival rate, and ensuring that its antioxidant effect is sustained and stable.

[0007] Preferably, the preparation steps of the carboxylated cellulose are as follows: 2,2,6,6-Tetramethylpiperidine and sodium bromide were added to a cellulose solution and mixed to obtain a mixed solution. A quenching product was obtained. The quenching product was centrifuged to obtain a nanocellulose precipitate. The precipitate was resuspended and washed until neutral to obtain the carboxylated cellulose. The mass ratio of cellulose, 2,2,6,6-tetramethylpiperidine, and sodium bromide is 1:0.01~0.02:0.1~0.2. Preferably, the preparation steps of the nanocellulose composite material are as follows: Carboxylated cellulose was added to a cerium nitrate solution and incubated for 10-15 hours to obtain a mixture of nanocellulose and cerium nitrate; the mass concentration ratio of carboxylated cellulose to cerium nitrate was 1:1-4. A mixture of nanocellulose and cerium nitrate was added to a solution of 6-aminohexanoic acid to obtain a reaction solution. The mixture was stirred for 1 min to 60 min, cooled, and then a pre-cooled non-polar organic solvent was added. The mixture was kept at -20°C for 20 to 30 h and then centrifuged to collect the precipitate. The precipitate was dried to obtain the nanocellulose-CeNPs composite material, i.e., the nanocellulose composite material. The mass ratio of the mixture of nanocellulose and cerium nitrate to 6-aminohexanoic acid is 1:1~2.

[0008] Preferably, the pH of the 6-aminohexanoic acid solution is 5-6.

[0009] Preferably, the nonpolar organic solvent is ethanol.

[0010] Preferably, the volume ratio of ethanol to the reaction solution is 9~12:1.

[0011] The application of the antioxidant material in the preparation of drugs for treating photodamage.

[0012] Preferably, the photodamage includes erythema caused by ultraviolet radiation.

[0013] Preferably, the photodamage includes the formation of skin wrinkles, epidermal thickening, and hardening caused by ultraviolet radiation.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention uses an in-situ synthesis method to uniformly coat CeNPs onto the surface of TO-CNF fibers, which can effectively prevent CeNPs from agglomerating in the physiological environment and prevent small-sized CeNPs from being phagocytosed by cells, significantly improving its physiological stability and biocompatibility, and ensuring its long-lasting and stable antioxidant effect.

[0015] The cerium oxide nanoparticles (CeNPs) synthesized in this invention are uniformly dispersed on nanocellulose, and the composite material exhibits excellent antioxidant properties, capable of scavenging O2. - It is resistant to free radicals such as H2O2; and has good biocompatibility, making it suitable for biological applications such as wound dressings and tissue engineering. Attached Figure Description

[0016] Figure 1 The results show the morphology and structure. In this image, A is the TEM image of nanocellulose, B is the TEM image of nanocellulose-CeNPs, C is the SEM image and mapping image of nanocellulose-CeNPs, and D is the EDS energy dispersive spectroscopy analysis of nanocellulose-CeNPs.

[0017] Figure 2 The results represent the in vitro scavenging capacity of reactive oxygen species, where A represents simulated superoxide dismutase activity and B represents simulated catalase activity.

[0018] Figure 3 The results represent biocompatibility, where A represents cell survival rates after direct contact between TO-CNF, CeNPs, and TO-CNF-CeNPs and cells, and B represents the cytotoxicity test results of TO-CNF-CeNPs at different concentrations.

[0019] Figure 4 The results show the ROS scavenging ability, where A is the ROS scavenging fluorescence image and B is the ROS scavenging fluorescence intensity.

[0020] Figure 5 A comparison of the morphology of the composite materials prepared in Example 1 and Comparative Example 1.

[0021] Figure 6 This is a comparison of the antioxidant activity of the composite materials prepared in Example 1 and Comparative Example 1.

[0022] Figure 7 The therapeutic effect of the composite material in Example 1 on photodamage.

[0023] Figure 8 The composite material of Example 1 was subjected to hematoxylin-eosin (HE) staining for pathological analysis of photodamaged skin tissue.

[0024] Figure 9To illustrate the scavenging effect of superanion oxide, A represents the control group for the superanion oxide generation system, and B represents the experimental group with the sample to be tested added.

[0025] Figure 10 To illustrate the scavenging effect of hydrogen peroxide, A is a hydrogen peroxide control solution, and B is the experimental group in which the sample to be tested was added.

[0026] Figure 11 The total antioxidant activity of TO-CNFs. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0028] The cerium oxide / cellulose nanofiber composite material with antioxidant properties of the present invention is synthesized by in-situ method by uniformly coating cerium oxide nanoparticles on the surface of cellulose nanofibers. Firstly, TO-CNF containing carboxyl groups is prepared by TEMPO oxidation, and then cerium oxide nanoparticles are uniformly coated onto the surface of cellulose nanofibers through coordination bonds. 4+ CeNPs were synthesized in situ on the surface of TO-CNF by using 6-aminohexanoic acid as a complexing and dispersing agent, attached to the carboxyl group of TO-CNF.

[0029] Existing antioxidant materials are prone to inactivation mainly due to the weak binding characteristics of physical adsorption (such as hydrogen bonds, van der Waals forces, etc.), which easily leads to a decrease in loading efficiency and loss of activity, thus limiting their practical application. The TO-CNF-CeNPs composite material prepared in this invention has CeNPs coated on its surface with small particle size, regular morphology, and uniform distribution, which can simulate the activity of superoxide dismutase and catalase, and has excellent antioxidant effect.

[0030] This invention utilizes an in-situ synthesis method to uniformly coat CeNPs onto the surface of TO-CNF fibers. This effectively prevents CeNPs from agglomerating in physiological environments and prevents small-particle CeNPs from being phagocytosed by cells, significantly improving its physiological stability and biocompatibility, and ensuring its long-lasting and stable antioxidant effect. Furthermore, TO-CNF, as a matrix material, can spontaneously form a mesh film through methods such as spraying and filtration, exhibiting good air permeability and antibacterial properties. Its high porosity also facilitates the binding of CeNPs with free substrates, thereby exerting its antioxidant effect.

[0031] In addition to its durable, stable, and excellent antioxidant activity, the TO-CNF-CeNPs composite material of this invention can also exert air permeability and antibacterial effects through the formed mesh film, which is of great significance for the care and health maintenance of skin, oral cavity, ocular surface, intestines, mucous membrane tissues, etc.

[0032] A method for preparing nanocellulose includes the following steps: Preparation of TEMPO-oxidized cellulose nanoparticles: 0.0156 g of TEMPO (2,2,6,6-tetramethylpiperidine) and 0.103 g of NaBr were added to 100 mL of a 1% (w / w) cellulose solution and stirred until homogeneous using a magnetic stirrer. Then, 10 mL of a 10% (w / w) NaClO solution was added to the mixture and stirred again using a magnetic stirrer. The pH of the reaction system was adjusted in real-time using 0.5 mol / L NaOH solution to maintain it between 10.3 and 10.5. The reaction was terminated when the pH remained constant or changed very little, and 10 mL of anhydrous ethanol was added to quench the reaction. The product was centrifuged to obtain a cellulose nanoparticle precipitate, which was resuspended and washed in an 8-14 KD dialysis bag until neutral to obtain TEMPO-oxidized cellulose nanoparticles, denoted as TO-CNF, for later use.

[0033] Preparation of nanocellulose-CeNPs composite materials: Measure 50 mL of 2% TO-CNF and add 50 mL of 2%-8% cerium nitrate solution. Incubate the two solutions together for 10-15 hours to obtain a mixed suspension. Centrifuge the mixed suspension to obtain a precipitate. Wash the precipitate three times with ultrapure water for later use to obtain a mixture of nanocellulose and cerium nitrate.

[0034] Weigh 1.31 g of 6-aminohexanoic acid and dissolve it in 60 mL of water. Heat the solution to 95 °C and then add 70 μL of HCl (36.46% by volume) to adjust the pH of the solution to 5.5, thus obtaining a 6-aminohexanoic acid solution.

[0035] Weigh 1.09 g of a mixture of nanocellulose and cerium nitrate, dissolve it in 50 mL of water, and then add it to a 6-aminocaproic acid solution. Stir vigorously for 1 min, cool to room temperature, add 10 times the volume of pre-cooled ethanol, place at -20 °C overnight, centrifuge at 8000 rpm for 5 min, collect the precipitate, wash it 3 times with ethanol, and dry the obtained product at 60 °C to obtain the nanocellulose-CeNPs composite material, denoted as TO-CNF-CeNPs.

[0036] Example 1 Preparation of TEMPO-oxidized cellulose nanoparticles: 0.0156 g of TEMPO and 0.103 g of NaBr were added to 100 mL of a 1% (w / w) cellulose solution and stirred evenly with a magnetic stirrer to obtain a mixed solution. Then, 10 mL of a 10% (w / w) NaClO solution was added to the mixed solution and stirred with a magnetic stirrer. The pH of the reaction system was adjusted in real time using 0.5 mol / L NaOH solution to maintain it between 10.3 and 10.5. When the pH remained constant or changed very little, the reaction was terminated, and 10 mL of anhydrous ethanol was added to quench the reaction. The product was centrifuged to obtain a cellulose nanoparticle precipitate, which was resuspended and washed in a 12 KD dialysis bag until neutral to obtain TEMPO-oxidized cellulose nanoparticles, denoted as TO-CNF, for later use.

[0037] TO-CNF-1%CeNPs: Measure 50 mL of 2% TO-CNF and add 50 mL of 2% cerium nitrate solution. Incubate the two solutions together for 12 h to obtain a mixed suspension. Centrifuge the mixed suspension to obtain a precipitate. Wash the precipitate three times with ultrapure water for later use to obtain a mixture of nanocellulose and cerium nitrate.

[0038] Weigh 1.31 g of 6-aminohexanoic acid and dissolve it in 60 mL of water. Heat the solution to 95 °C and then add 70 μL of HCl (36.46% by volume) to adjust the pH of the solution to 5.5, thus obtaining a 6-aminohexanoic acid solution.

[0039] Weigh 1.09 g of a mixture of nanocellulose and cerium nitrate, dissolve it in 50 mL of water, and then add it to a 6-aminocaproic acid solution. Stir vigorously for 1 min, cool to room temperature, add 10 times the volume of pre-cooled ethanol, place at -20 °C overnight, centrifuge at 8000 rpm for 5 min, collect the precipitate, wash it 3 times with ethanol, and dry the obtained product at 60 °C to obtain the nanocellulose-CeNPs composite material, denoted as TO-CNF-1%CeNPs.

[0040] Example 2 The difference between Example 2 and Example 1 is that TO-CNF-2%CeNPs: 50 mL of 2% TO-CNF was measured and 50 mL of 4% cerium nitrate solution was added. The remaining steps and conditions were the same as in Example 1.

[0041] Example 3 The difference between Example 3 and Example 1 is that TO-CNF-3%CeNPs: 50 mL of 2% TO-CNF was measured and 50 mL of 6% cerium nitrate solution was added. The remaining steps and conditions were the same as in Example 1.

[0042] Example 4 The difference between Example 4 and Example 1 is that: TO-CNF-4%CeNPs: 50 mL of 2% TO-CNF was measured and added to 50 mL of 8% cerium nitrate solution. The remaining steps and conditions were the same as in Example 1.

[0043] Comparative Example 1 Ethylenediamine synthesis method: (1) Weigh 0.5g Tempo cellulose and 0.5g cerium nitrate, add 50mL of water, and mix at 25℃ with the magnetic stirrer set to 500rpm for 12h. After the reaction is complete, wash three times with pure water by centrifugation;

[0044] (2) Weigh 0.234g of Tempo cellulose-cerium nitrate mixture, add 32mL of ethylenediamine, and stir the mixture at 25℃ with the magnetic stirrer set to 500rpm for 24h. After the reaction is complete, wash the mixture three times with water by centrifugation.

[0045] The SEM morphology characterization of the composite material synthesized using this comparative example is as follows: Figure 5 As shown, the antioxidant activity is as follows Figure 6 As shown, Figure 5 and Figure 6 It can be seen that the composite material has poor dispersibility and weak antioxidant activity.

[0046] Effect verification 1. Morphology and Structure: Cerium oxide was successfully modified onto TO-CNF via in-situ synthesis. Transmission electron microscopy (TEM) revealed that… Figure 1 Figure A shows that TO-CNF exhibits a slender fibrous structure with a diameter of approximately 11.12 ± 2.58 nm. From Figure 1 In Figure B, cerium oxide nanoparticles are uniformly attached to the surface of nanocellulose, increasing the diameter of the TO-CNF to approximately 21.88 ± 6.25 nm. Scanning electron microscopy (SEM) also revealed a rougher surface to the TO-CNF, confirming that cerium oxide successfully bonded to the surface of TO-CNF as a template. Furthermore, to further observe the cerium oxide content on the TO-CNF surface, energy-dispersive spectroscopy (EDS) analysis revealed the presence of C, O, and Ce elements in the nanocellulose-CeNPs composite material, uniformly distributed on the surface of the TO-CNF. Figure 1 The C content was calculated to be 37.5% and O content to be 8.1%, while Ce content was as high as 54.3%. Figure 1The "D" in the figure indicates that a high content of cerium oxide is bound to the surface of TO-CNF. This is because the high aspect ratio of TO-CNF provides sufficient surface area and active sites, which facilitates the binding of cerium ions to the nanocellulose through coordination bonds and electrostatic interactions.

[0047] 2. Ability to scavenge reactive oxygen species in vitro: (1) The activity of the superoxide dismutase (SOD) activity assay kit was determined using a superoxide dismutase (SOD) activity assay kit. The superoxide dismutase (SOD) activity assay kit was purchased from Solarbio Beijing, model BC5165. Reagents 1, 2, 3 and 4 in Table 1 are reagents 1, 2, 3 and 4 of the superoxide dismutase (SOD) activity assay kit.

[0048] First, prepare 1 mL sample solutions of composite materials prepared by incubating carboxylated cellulose and cerium nitrate at different ratios with a concentration of 50 mg / mL. Then, prepare the working solutions: measure reagent 1 according to the number of samples; dilute reagent 2 100 times with ultrapure water before use; measure reagent 3 according to the number of samples; and dilute reagent 4 10 times with ultrapure water before use. Before adding the samples for incubation, incubate reagents 3 and 4 in a 37°C water bath for 5 min, and then add them to the centrifuge tubes in the order shown in Table 1.

[0049] Table 1: Sample Addition Table After sample addition, mix thoroughly, place in a 37℃ water bath for 30 min, then add to a 96-well plate. Measure absorbance at 450 nm and calculate superoxide anion (O2) using the following formula 1. - The clearance rate and enzyme activity of ) [(∆A blank - ∆A measurement) / ∆A blank] × 100% (Formula 1) Where ΔAblank = A1blank tube - A2blank tube, and ΔAdetermination = Adetection - Acontrol.

[0050] (2) The activity of nanocellulose-CeNPs mimicking CAT enzyme was determined using Amplex Red dye.

[0051] First, prepare 1 mL of different sample solutions with a concentration of 10 mg / mL for later use.

[0052] Then prepare the Amplex Red working solution: Tris-HCl solution (50 mM): Weigh 0.18 g Tris and dissolve it in 30 mL of pure water. After complete dissolution, slowly adjust the pH value to 7.5 with 1 mol / L HCl solution.

[0053] Amplex Red (10 mM): Weigh 5 g of Amplex Red and dissolve it in 1.948 mL of DMSO. Aliquot the solution into light-protected EP tubes and store at -20°C.

[0054] HRP (10 U / mL): Take 200 μL of HRP with a concentration of 1 μg / μL and then add 5.12 mL of water to dilute it.

[0055] Amplex Red working solution (5 mL): 4.85 mL Tris-HCl solution (50 mM), 50 μL Amplex Red (10 mM), 100 μL HRP (10 U / mL).

[0056] The specific measurement steps are as follows: Take 500 μL of the sample solution of the composite material prepared by incubating carboxylated cellulose and cerium nitrate at different ratios (10 mg / mL) into a centrifuge tube, then add 500 μL of 400 μM hydrogen peroxide solution. Mix the two solutions thoroughly and incubate for 4 h before detection. Add 50 μL of the sample and hydrogen peroxide mixture to a 96-well plate, then add 50 μL of Amplex Red working solution. Incubate at room temperature in the dark for 30 min, and then perform detection using a microplate reader. Set the excitation wavelength to 545 nm and the emission wavelength to 590 nm. The specific calculation formula is as follows:

[0057] [(A control - A assay) / A control] × 100%" (Formula 2) The results are as follows Figure 2 , Figure 9 and Figure 10 , Figure 2 In this context, A represents the TO-CNF-CeNPs pair for O2. - The removal rate can reach 97%, where the Ce³⁺ sites can provide electrons to combine with O₂⁻ and reduce it to H₂O₂. For example... Figure 2 As shown in Figure B, at an H2O2 concentration of 200 μM, TO-CNF-CeNPs achieved a 91% scavenging rate for H2O2, further decomposing H2O2 into O2 and H2O. Furthermore, at the same concentration, TO-CNF-CeNPs also showed a significant reduction in O2 scavenging rate. - The removal rate of Ce is higher than that of H2O2, which is due to the higher removal rate of Ce. 3+ The content of Ce³⁺ is higher than that of Ce⁴⁺. During this process, the ratio of Ce³⁺ to Ce⁴⁺... 4The redox cycle of ⁺ can both reduce O⁻ to H₂O₂ and catalyze the decomposition of H₂O₂ into H₂O and O₂, thereby achieving a cascade scavenging of reactive oxygen species. Furthermore, TO-CNF, as a support, not only improves the dispersibility and stability of CeNPs but also increases the exposure of active sites, further enhancing catalytic efficiency.

[0058] The data above indicate that the composite material TO-CNF-CeNPs exhibits good antioxidant activity, mimicking the activities of superoxide dismutase (SOD) and catalase (CAT), thereby effectively scavenging excess reactive oxygen species (ROS). Furthermore, we observe no significant difference in free radical scavenging rates with increasing cerium content. This may be due to the uneven distribution and aggregation of cerium on the TO-CNF as the cerium content increases, yet it still maintains good antioxidant activity.

[0059] 3. Biocompatibility: First, sterilize the experimental samples under a UV lamp for 2 hours. Then, seed the cell suspension into 96-well plates at a volume of 100 µL per well (cell density 5 × 10⁶ cells / well). 3 Cells were cultured overnight in a CO2 incubator to ensure full cell adhesion. After 24 hours, the culture medium was discarded, and the cells were gently washed 2-3 times with PBS. 100 µL of culture medium containing the sample was added to each well in the experimental group, while an equal volume of complete culture medium was added to the control group. Each group had three replicates. After another 24 hours of culture, the culture medium and sample were discarded, and the cells were washed 2-3 times with PBS. Then, 100 µL of fresh culture medium and 10 µL of CCK-8 solution were added to each well (for blank control wells, only culture medium and CCK-8 were added), and the cells were incubated for 1-2 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader. Each experiment was repeated three times, and the average value was taken. Cell viability was calculated using formula 3 as follows:

[0060] Cell viability = [(Experimental group - Blank group) / (Control group - Blank group)] × 100% (Formula 3) Results Explanation: Figure 3In the figure, A represents the cell survival rate after direct contact with TO-CNF, CeNPs, and TO-CNF-CeNPs. The results showed that, compared with the untreated control group, there was no significant difference in cell survival rate between the TO-CNF and TO-CNF-CeNPs treatment groups at the same concentration, with survival rates all above 90%. This indicates that TO-CNF-CeNPs did not exhibit cytotoxic effects, demonstrating excellent biocompatibility. However, after treatment with CeNPs alone, the cell survival rate was only 54%, below 70%, indicating cytotoxicity. This may be because the positively charged surface of cerium nanoparticles combines with the negatively charged cell membrane, disrupting the integrity of the cell membrane structure, and may also be phagocytosed into the cell, leading to a decrease in cell survival. Using TO-CNF as a template can prevent TO-CNF-CeNPs from entering the cell, significantly reducing cytotoxicity. Figure 3 B in the figure represents the cytotoxicity assay of TO-CNF-CeNPs at different concentrations. It can be seen that at a concentration of 5 mg / mL, the cell survival rate is still above 90%.

[0061] 4. ROS Scavenging Capacity: The ROS scavenging capacity of TO-CNF-CeNPs cells was detected using a reactive oxygen species (ROS) assay kit. When the cells at the bottom of the culture flask reached 80% confluence, they were digested into a suspension using 0.25% trypsin. 1 mL of the cell suspension (cell density 5 × 10⁶ cells / well) was added to each well of a 24-well plate. 4 The cells were incubated overnight in an incubator with 5 mg / mL TO-CNF-CeNPs. After 24 h, the old culture medium was discarded, and the cells were washed with PBS, repeated 2-3 times. Then, hydrogen peroxide solution and samples (5 mg / mL TO-CNF-CeNPs) were added. For the negative control, 1 mL of complete culture medium was added, and for the positive control, 1 mL of 500 μM hydrogen peroxide solution was added. For the experimental group, 500 μM hydrogen peroxide solution containing TO-CNF-CeNPs was added. After culturing for another 24 h, the old culture medium and samples were discarded, and 300 μL of 10 μM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was added. The cells were incubated in the dark for 20 min, and the probes were discarded. The cells were then gently washed 2-3 times with PBS buffer to completely remove the free probes. Immediately afterwards, fluorescence microscopy was performed, and the fluorescence intensity of the acquired images was analyzed using ImageJ software to assess the intracellular reactive oxygen species (ROS) levels.

[0062] Results: To confirm the ROS scavenging capacity of NCM-460 cells under oxidative stress, intracellular ROS-sensitive fluorescent dye DCFH-DA was used for staining, followed by qualitative analysis of intracellular ROS levels using fluorescence microscopy. The results are as follows: Figure 4As shown in the figure, fluorescence detection results indicated that under the action of 500 μM hydrogen peroxide, the cell fluorescence signal was significantly enhanced compared to the untreated control group, and the ROS level was increased. However, treatment with TO-CNF-CeNPs significantly reduced the fluorescence signal. These results demonstrate that TO-CNF-CeNPs can effectively scavenge ROS and protect cells from oxidative stress damage.

[0063] The total antioxidant activity of TO-CNFs was determined using the Total Antioxidant Capacity Assay Kit (ABTS method). (1) Preparation of test samples: Prepare 2 mg / mL CNF and TO-CNF respectively.

[0064] (2) Preparation of ABTS working stock solution: Calculate the volume of ABTS working stock solution required before dilution. Mix ABTS and oxidant evenly at a volume ratio of 1:1 and let stand for 16 h to obtain working stock solution. Dilute the working stock solution so that its A734 is 1.4±0.1.

[0065] (3) Determination of total antioxidant activity: The diluted stock solution and the sample to be tested were mixed 1:1 to make the final concentration of the sample 1 mg / mL. The reaction system was 210 µL, and a control group was set up at the same time. After 1 h, the supernatant was centrifuged and its absorbance was measured under A734 light. The total antioxidant capacity was calculated as shown in equation (3.1).

[0066] (3.1) in A This is the absorbance value for the control. B It is the absorbance value of the sample to be tested.

[0067] See results Figure 11 .

[0068] Example 2 Research on the application of composite materials in the treatment of photodamage to the skin This embodiment aims to provide evidence of the application of the antioxidant material in the prevention and / or treatment of photoaging and photodamage of the skin. By establishing a standard animal model, the significant improvement effect of the material on various photodamage phenotypes such as UV-induced skin erythema, wrinkles, and loss of elasticity after topical administration was verified, and its potential antioxidant and anti-inflammatory mechanisms were elucidated.

[0069] Twelve healthy female mice (SKH-1 strain), aged 8 weeks and weighing 20±2g, were selected. The animals were housed separately in SPF-grade environments with free access to food and water, and the experiment began one week after acclimatization. The mice were randomly divided into four groups of three: a positive control group, a negative control group, a TO-CNFs treatment group, and a TO-CNF-CeNPs treatment group. Diabetic mice were anesthetized with 6% chloral hydrate at a dose of 400 mg / kg. After anesthesia, the skin on the back of the mice was shaved, and a hair removal cream was applied. After 5 minutes, the cream was wiped off, and the mice were cleaned. The skin on the back of the mice was disinfected with alcohol swabs. All mice except the negative control group were irradiated with UVB at an intensity of 200 mJ / cm² for three consecutive days. Treatment began after the first irradiation, once daily, with the appropriate preparation evenly applied to the irradiated area on the back of the mice at a dose of 200 µL / cm². The negative and positive control groups were treated with purified water. High-resolution digital cameras were used to photograph the skin on the backs of mice under fixed lighting conditions. Skin roughness, wrinkle formation, erythema, and scaling were observed and recorded. After the experiment, the mice were euthanized, and back skin tissue was collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Hematoxylin-eosin staining was used to observe epidermal thickness, stratum corneum integrity, and dermal inflammatory cell infiltration.

[0070] In an animal model of ultraviolet radiation-induced photodamage to the skin, the skin of the experimental animals was continuously observed. The results are as follows: Figure 7 The results showed that by day 5 of UV irradiation, the skin of animals in the positive control group exhibited significant erythema, wrinkling, epidermal thickening, and hardening, displaying typical photodamage pathological features. In contrast, the skin of animals in the experimental groups treated with TO-CNFs and TO-CNF-CeNPs showed only mild erythema, with the erythema in the TO-CNF-CeNPs-treated group being relatively milder. By day 10, the skin damage in the positive control group further aggravated, with obvious inflammatory exudate and crust formation; the material-treated groups showed only mild exudate and localized crusting, with the crusting in the TO-CNF-CeNPs-treated group being milder than that in the TO-CNFs-treated group. By day 15 of the experiment, the positive control group still had obvious and persistent photodamage wounds, while the skin damage in the material-treated groups had basically healed.

[0071] Skin tissue collected on day 15 was subjected to hematoxylin-eosin (HE) staining for pathological analysis. Results are as follows: Figure 8The results showed that the positive control group exhibited significant epidermal thickening, hyperkeratosis, acanthosis, and disordered cell arrangement; the superficial dermis showed extensive inflammatory cell infiltration. These changes are consistent with typical pathological features of severe photodamage. In contrast, tissue sections treated with TO-CNFs and TO-CNF-CeNPs showed clear epidermal structures, near-normal thickness, and regular cell morphology and arrangement; inflammatory cell infiltration in the dermis was significantly reduced. These results indicate that this antioxidant material can effectively reverse UV-induced skin pathological changes and promote the restoration of normal tissue structure.

[0072] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0073] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0074] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An antioxidant material, characterized in that, The antioxidant material uses carboxylated cellulose or a nano-cellulose composite material obtained by coating the carboxylated cellulose with cerium as the active ingredient; The carboxylated cellulose was obtained by oxidizing cellulose using the TEMPO method.

2. The antioxidant material according to claim 1, characterized in that, The preparation steps of the carboxylated cellulose are as follows: 2,2,6,6-Tetramethylpiperidine and sodium bromide were added to a cellulose solution and mixed to obtain a mixed solution. NaClO was added to obtain a quenching product. The quenching product was centrifuged to obtain a nanocellulose precipitate. The precipitate was resuspended and washed until neutral to obtain the carboxylated cellulose. The mass ratio of cellulose, 2,2,6,6-tetramethylpiperidine, and sodium bromide is 1:0.01~0.02:0.1~0.

2.

3. The antioxidant material according to claim 1, characterized in that, The preparation steps of the nanocellulose composite material are as follows: Carboxylated cellulose was added to a cerium nitrate solution and incubated for 10-15 hours to obtain a mixture of nanocellulose and cerium nitrate; the mass concentration ratio of carboxylated cellulose to cerium nitrate was 1:1-4. A mixture of nanocellulose and cerium nitrate was added to a solution of 6-aminohexanoic acid to obtain a reaction solution. The mixture was stirred for 1 min to 60 min, cooled, and then a pre-cooled non-polar organic solvent was added. The mixture was kept at -20°C for 20 to 30 h and then centrifuged to collect the precipitate. The precipitate was dried to obtain the nanocellulose-CeNPs composite material, i.e., the nanocellulose composite material. The mass ratio of the mixture of nanocellulose and cerium nitrate to 6-aminohexanoic acid is 1:1~2.

4. The antioxidant material according to claim 3, characterized in that, The pH of the 6-aminohexanoic acid solution is 5-6.

5. The antioxidant material according to claim 3, characterized in that, The nonpolar organic solvent is ethanol.

6. The antioxidant material according to claim 5, characterized in that, The volume ratio of ethanol to the reaction solution is 9~12:

1.

7. The use of the antioxidant material of claim 1 in the preparation of a medicament for treating photodamage.

8. The application according to claim 7, characterized in that, The photodamage includes erythema caused by ultraviolet radiation.

9. The application according to claim 7, characterized in that, The photodamage includes the formation of skin wrinkles, epidermal thickening, and hardening caused by ultraviolet radiation.

Images