An artificial antioxidant enzyme, its preparation method and application

Abstract

The present invention belongs to the technical field of biocatalysts, and particularly relates to an artificial antioxidant enzyme and its preparation method and application. In the process of preparing titanium oxide clusters by solvothermal reaction, Mn is incorporated to prepare an MnTi6 artificial antioxidant enzyme with an amorphous nanoporous structure. The incorporation of Mn endows the MnTi6 artificial antioxidant enzyme prepared by the present invention with excellent efficacy and broad-spectrum antioxidant properties, and has good application prospects in the field of biocatalysis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biocatalysts, and particularly relates to an artificial antioxidant enzyme, a preparation method thereof, and an application thereof. Background Art

[0002] In biological systems, endogenous antioxidant enzymes, including superoxide dismutase (SOD) and catalase (CAT), contribute to maintaining cellular redox balance by neutralizing ROS and converting them into harmless by-products. However, these natural antioxidants have limitations such as high antigenicity, instability, and delivery challenges, which hinder their direct application in the treatment of oxidative stress-related diseases. Therefore, chemists and materials scientists have actively participated in mimicking the mechanisms of natural antioxidant enzymes to create new biocatalytic reactive oxygen species (ROS) scavenging materials for antioxidant treatment. In recent years, inspired by the structure and function of natural enzymes, much attention has been paid to using catalytic materials containing metal oxides and metal-coordination structures to scavenge ROS and protect cells. Titanium (Ti) has advantages such as low density, good compatibility with human tissues, and low risk of immune rejection in the application of biomaterials, and is a biocompatible metal material. However, there are few reports on artificial antioxidant enzymes with Ti as the active center. In addition, preparing artificial antioxidant enzymes by mimicking the active center of natural antioxidant enzyme manganese superoxide dismutase (MnSOD) has also become a research hotspot. However, the ROS scavenging ability of these single-metal-based artificial enzymes is limited, and the broad-spectrum property is insufficient to solve various ROS commonly present in organisms, which emphasizes the necessity of designing broad-spectrum ROS scavenging materials. Summary of the Invention

[0003] To solve the above problems, the present invention prepared an artificial antioxidant enzyme with a dual-metal active site having an electronically regulated active center by integrating Mn / Ti catalytic sites, which is referred to as MnTi 6 artificial antioxidant enzyme herein. It has excellent efficacy and broad-spectrum antioxidant characteristics and has good application prospects in the field of biocatalysis.

[0004] In the first aspect, the present invention provides a MnTi 6 artificial antioxidant enzyme. The MnTi 6 artificial antioxidant enzyme is a titanium-oxygen cluster doped with Mn formed by a solvothermal reaction of a titanium source, a manganese source, and 4-aminobenzoic acid. It has an amorphous nanoporous structure, and there are Mn-O and Mn-Ti coordination structures in the MnTi 6 artificial antioxidant enzyme.

[0005] Furthermore, the MnTi 6 artificial antioxidant enzyme has cascade SOD-CAT catalytic activity.

[0006] As used herein, the cascade SOD-CAT catalytic activity involves first converting superoxide anions (•O 2 - ⁻) into hydrogen peroxide (H 2 ₂ 2 O 2 ₂ 2 ) through superoxide dismutase (SOD)-like activity, and then converting H 2 ₂ 2 O

[0007] ₂ 6 into oxygen (O 6 ₂ 6 ) and water (H

[0008] ₂ 6 O) through catalase (CAT)-like activity.

[0009] Generally speaking, the mass ratio of Mn to Ti and the incorporation amount of Mn in the MnTi 6 artificial antioxidant enzyme are not particularly limited, as long as the MnTi 6 artificial antioxidant enzyme has the structure described herein. In some embodiments, the mass percentage of Ti in the MnTi

[0010] artificial antioxidant enzyme can be 10 - 25%, and the incorporation amount of Mn can be 5 - 10% by mass percentage. 6

[0011] As used herein, titanium-oxo clusters are a type of metal-oxo clusters formed by titanium (Ti) atoms connected to oxygen (O) atoms through bridging oxygen (O²⁻) or hydroxyl (OH⁻). They usually consist of multiple titanium atom centers connected by oxygen bridges to form a polynuclear structure, and can be further modified by organic or inorganic ligands. The synthesis of titanium-oxo clusters generally involves a hydrolysis-condensation reaction, i.e., the titanium precursor undergoes hydrolysis under appropriate conditions and forms Ti-O-Ti bridging bonds through hydroxyl condensation, and finally assembles into titanium-oxo clusters. The titanium-oxo clusters formed by solvothermal reaction under the regulation of the ligand 4-aminobenzoic acid in the present invention are abbreviated as Ti

[0012] -

[0013] - oxo clusters.

[0009] In a second aspect, the present invention provides a method for preparing the MnTi 6 artificial antioxidant enzyme as described herein, wherein the MnTi 6 artificial antioxidant enzyme is prepared by a solvothermal reaction of a titanium source, a manganese source, and 4-aminobenzoic acid.

[0010] Specifically, the preparation method is as follows: Add the manganese source to a solvent containing the titanium source and 4-aminobenzoic acid, react at 90 - 120 °C for 60 - 90 hours, collect the reaction product, and after washing and drying, obtain the MnTi 6 artificial antioxidant enzyme.

[0011] Furthermore, the titanium source includes titanium tetraisopropoxide, titanium tetrachloride, or titanate.

[0012] Preferably, the titanium source is titanium tetraisopropoxide.

[0013] Further, the manganese source includes manganese nitrate tetrahydrate, manganese chloride hexahydrate or manganese sulfate hexahydrate.

[0014] Preferably, the manganese source is manganese nitrate tetrahydrate.

[0015] Further, the solvent includes isopropanol, methanol, ethanol, toluene, acetonitrile or N,N-dimethylformamide.

[0016] Preferably, the solvent is isopropanol.

[0017] In a third aspect, the present invention provides the use of the MnTi 6 artificial antioxidant enzyme as described herein in the preparation of a preparation for scavenging reactive oxygen species.

[0018] Further, the scavenging of reactive oxygen species includes scavenging reactive oxygen species through cascade SOD-CAT catalytic activity.

[0019] Advantages of the invention

[0020] In the present invention, a MnTi 6 artificial antioxidant enzyme with an amorphous nanoporous structure is prepared by incorporating Mn during the preparation of titanium oxide clusters using a solvothermal reaction. The incorporation of Mn can form coordination with oxygen in the titanium oxide cluster by replacing the position of Ti, thereby leading to the formation of an amorphous nanoporous structure. In addition, it is also found that there is an electron transfer from the Mn site to the Ti site, which enables MnTi 6 to overcome the challenges associated with multi-electron reactions during the scavenging of reactive oxygen species (ROS). The MnTi 6 artificial antioxidant enzyme prepared by the present invention has excellent efficacy and broad-spectrum antioxidant properties, and has good application prospects in the field of biocatalysis. Description of the drawings

[0021] Figure 1 Shows the structural characterization of the MnTi 6 biocatalyst with an amorphous nanoporous structure prepared in the examples: (a) SEM images of Ti 6 and MnTi 6 ; (b) and (c) atomic-level high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images of MnTi 6 ; (d) energy-dispersive X-ray spectroscopy (EDS) elemental mapping diagrams of O, Ti, and Mn; and (e) EDS spectrum of MnTi 6 ; (f) MnTi 6Mass ratio of Mn and Ti; (g) Zeta potential and hydrodynamic size distribution; (h) Ti 6 and MnTi 6 Raman spectra; (i) Ti 6 and MnTi 6 XPS full spectrum scans; (j-l) Ti 6 and MnTi 6 High-resolution XPS spectra of O1s, Ti2p and Mn2p; (m) MnTi 6 , manganese foil, manganese oxide (MnO) and manganese dioxide (MnO 2 ) K-edge XANES spectra of manganese (Mn); (n) MnTi 6 , manganese oxide (MnO) and manganese dioxide (MnO 2 ) Average valence state of manganese (Mn); (o) Fourier transform (FT) of the extended EXAFS spectrum in R space.

[0022] Figure 2 Shows the ·O 2 - and H 2 O 2 Scavenging performance tests of: (a) TiO 2 , Ti 6 , CeTi 6 , CuTi 6 , MnTi 6 and VTi 6 ·O 2 - Scavenging performance; (b) TiO 2 , Ti 6 , CeTi 6 , CuTi 6 , MnTi 6 and VTi 6 H 2 O 2 Scavenging performance;

[0023] Figure 3 Shows the reactive oxygen species scavenging activity and kinetics of MnTi 6 : (a) Schematic diagram of the catalytic activity of the cascade superoxide dismutase-catalase (SOD-CAT) of MnTi 6 ; (b) Scavenging rates of Ti 6 and MnTi 6 on the 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) cation radical (ABTS·+) at different times; (c) Ti 6 and MnTi 6Superoxide anion (·O 2 - )-scavenging superoxide dismutase (SOD) activity; (d) Dynamic scavenging activity of hydrogen peroxide (H 2 O 2 ) at different times; (e) Dynamic generation characteristics of oxygen (O 2 ) at different times; (f) Recycling catalytic ability of MnTi 2 O 2 after addition of hydrogen peroxide (H 6 ); (g) Typical Michaelis curve and double-reciprocal plot for determining the kinetic constants of MnTi 2 O 2 with hydrogen peroxide (H 6 ) as the substrate; (h) Comparison of the maximum reaction rate (V 6 ) and turnover number (TON) values of MnTi max with other recently reported nano-bio-catalysts for scavenging reactive oxygen species; (i) V 6 and Michaelis constant (K max ) values of MnTi m ; (j) In-situ Fourier transform infrared spectroscopy (FTIR) of MnTi 6 during the catalysis of catalase (CAT), and (k) corresponding contour plots. Detailed implementation mode

[0024] The present invention synthesizes MnTi 6 with an amorphous and nanoporous structure through a simple solvothermal method. By uniformly dissolving metal salts in an isopropanol solution and allowing them to react and precipitate rapidly under high temperature and pressure, the formation and growth of crystal nuclei are inhibited, thus achieving co-precipitation of a uniform amorphous structure. In the process of creating the present invention, first, the inventor successfully synthesized a titanium material without manganese through exploration and named it Ti 6 . Commercial TiO 2 was also purchased and its performance was tested as a control group for Ti 6 . Meanwhile, metal salts of cerium (Ce) / copper (Cu) / manganese (Mn) / vanadium (V) were used to prepare MTi 6 (M represents the metal Ce / Cu / Mn / V) materials, and their antioxidant enzyme-like properties were tested for comparison. It was found that MnTi 6 had the best antioxidant enzyme-like properties. Since MnTi 6 exhibited the most excellent multiple free radical scavenging abilities, the inventor also attempted to synthesize a control group without titanium using a similar method, but the yield was extremely low.

[0025] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0026] Materials and Reagents

[0027] Manganese(II) nitrate tetrahydrate (Mn(NO 3 ) 2 •4H 2 O), titanium(IV) isopropoxide, titanium(IV) sulfate (Ti(SO 4 ) 2 ), and 4-aminobenzoic acid were purchased from Aladdin Reagent Co., Ltd. The ultrapure water (18.2 MΩ•cm) used in the experiment was prepared by a Milli-Q Academic system (Millipore Corporation, Billerica, Massachusetts, USA). All chemical reagents were used as received without further purification.

[0028] Example: Synthesis of MnTi 6 Artificial Antioxidant Enzyme

[0029] MnTi with an amorphous and nanoporous structure was synthesized by a facile solvothermal method. 6 Briefly, manganese(II) nitrate tetrahydrate (3514.14 mg, 14 mmol) was added to an isopropanol solution (24 mL) containing 4-aminobenzoic acid (768.4 mg, 5.60 mmol) and titanium(IV) isopropoxide (103.6 μL, 0.35 mmol). After stirring at room temperature for 30 minutes, the orange slurry was transferred to a Teflon-lined autoclave and heated to 100 °C for 72 hours. The brown solid was collected, washed with isopropanol, and then dried overnight under vacuum.

[0030] Comparative Example 1: Synthesis of Ti 6

[0031] In this comparative example, a titanium oxo cluster material without Mn doping was prepared. 6 Briefly, titanium(IV) isopropoxide (103.6 μL, 0.35 mmol) was added to an isopropanol solution (6.0 mL) containing 4-aminobenzoic acid (192.1 mg, 1.40 mmol). After stirring at room temperature for 30 minutes, the orange slurry was transferred to a sealed glass tube and heated to 100 °C for 77 hours. The bright yellow crystalline product was collected, washed with isopropanol, and then dried overnight under vacuum.

[0032] Comparative Example 2: Synthesis of CeTi 6

[0033] Cerium(IV) sulfate tetrahydrate was used to replace manganese(II) nitrate tetrahydrate, and CeTi 6 materials were synthesized as described in the examples.

[0034] Comparative Example 3: CuTi 6 Synthesis

[0035] Anhydrous copper chloride was used to replace manganese(II) nitrate tetrahydrate, and CuTi 6 materials were synthesized as described in the examples.

[0036] Comparative Example 4: VTi 6 Synthesis

[0037] Vanadyl acetylacetonate was used to replace manganese(II) nitrate tetrahydrate, and VTi 6 materials were synthesized as described in the examples.

[0038] Performance testing

[0039] 1. Structure characterization

[0040] Scanning electron microscope (SEM) images were obtained using an Apreo S HiVoc from Thermo Fisher Scientific (FEI). A gold coating approximately 1 nm thick was deposited on the sample surface. Transmission electron microscope (TEM) tests were performed using a Tecnai G2 F20S-TWIN, operating at a voltage of 200 kV. Raman spectra were measured using an XploRA PLUS (Horiba, France) to evaluate molecular vibrations and crystal defects within the samples. X-ray photoelectron spectroscopy (XPS) was carried out on a K-Alpha™+ X-ray photoelectron spectrometer system (Thermo Fisher Scientific), using a 180° hemispherical double-focusing analyzer equipped with a 128-channel detector for detecting the valence state and electronic structure of MnTi 6 . Inductively coupled plasma optical emission spectrometer (ICP-OES, Thermo Fisher ICAPPRO) was used to analyze the element content. Scanning transmission electron microscope (STEM) images and energy-dispersive X-ray spectroscopy (EDX) elemental mapping were obtained using a Cs-corrected STEM (FEI Titan Cubed Themis G2 300). X-ray absorption (XAS) spectra at the K-edge were collected at the BL07A1 beamline station in Taiwan, China. The radiation was generated by scanning a silicon (111) double-crystal monochromator. Data processing and analysis of X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure spectra (EXAFS) were performed using Athena software. In this study, in-situ Fourier transform infrared (FTIR) measurements were carried out using an infrared spectrometer (Thermo Fisher Scientific, iS50 FTIR) equipped with an in-situ spectral cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-III).

[0041] 2. ABTS + · Scavenging test

[0042] Mix 7.4 mmol of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2.6 mmol of potassium persulfate (K 2 S 2 O 8 ) in a volume ratio of 1:1 and let the reaction stand overnight to oxidize ABTS to ABTS + ·. Then add the above solution to phosphate buffer solution (PBS, pH = 7.4) to make the total volume reach 1 mL and the material concentration be 5 μg / mL. After reacting for 5 minutes, measure the absorbance of the solution at 734 nm to evaluate the scavenging ability of the biocatalyst for ABTS + ·.

[0043] 3. •O 2 - Scavenging test

[0044] Dissolve 1 mg of potassium superoxide (KO 2 ) in 1 mL of dimethyl sulfoxide solution (DMSO, containing 3 mg / mL of 18-crown-6-ether) to generate and stabilize •O 2 - . Then disperse Ti 6 and MnTi 6 into the above KO 2 / DMSO solution with a final concentration of 50 μg / mL. After reacting for 5 minutes, the remaining •O 2 - will be captured by nitroblue tetrazolium (NBT)-DMSO solution (10 μL, 10 mg / mL). Measure the absorbance of the solution at 680 nm and then compare it with the initial concentration of •O 2 - to determine its scavenging ability for •O 2 - .

[0045] 4. Catalase (CAT) test - Hydrogen peroxide (H 2 O 2 ) scavenging

[0046] Dissolve 10 mmol of hydrogen peroxide (H 2 O 2)( ) and 50 μg / mL of the biocatalyst were mixed in phosphate buffer solution (PBS, pH = 7.4) to make the total volume reach 2 mL. Then, 100 μL of the above solution was mixed with titanium(IV) sulfate solution (100 μL, 13.9 mmol), and the absorbance value at 405 nm was recorded every 10 minutes until 40 minutes. After the reaction reached 40 minutes, the absorbance of the test solution at 405 nm was measured to evaluate the scavenging ability of the biocatalyst for H 2 O 2 .

[0047] 5. Catalase-like (CAT) test - Oxygen (O 2 ) generation determination

[0048] 200 mmol of hydrogen peroxide (H 2 O 2 ) and 25 μg / mL of the biocatalyst were mixed in phosphate buffer solution (PBS, pH = 7.4) to make the total volume reach 20 mL, and then the oxygen concentration was measured every 5 seconds using a dissolved oxygen meter (Shanghai Yidian Scientific Instrument Co., Ltd., JPSJ - 605F) until 300 seconds. To analyze the biocatalytic kinetics of O 2 generation, 25 μg / mL of the biocatalyst was mixed with different concentrations of hydrogen peroxide (H 2 O 2 ), which were 50, 100, 150, 200, 250, and 300 mmol respectively, in PBS to prepare 20 mL of solution, and then the O 2 concentration was measured every 5 seconds until 100 seconds. The reaction rate was plotted against the corresponding H 2 O 2 concentration, and then fitted with the Michaelis - Menten equation (Equation (1)). In addition, the double - reciprocal plotting method (Lineweaver–Burk plot, Equation (2)) was used to determine the maximum reaction rate (V max ) and the Michaelis constant (K m ). In addition, the turnover number (TON, that is, the maximum number of substrate conversions per unit active catalytic center) was calculated according to Equation (3). Where [S] is the concentration of hydrogen peroxide (H 2 O 2 ), and [E 0 represents the molar concentration of the metal in the material.

[0049] (S1)

[0050] (S2)

[0051] (S3)

[0052] Among them, Vmax is the maximum reaction rate in the reactive oxygen catalytic reaction; K m represents the affinity between the catalyst and the substrate (the smaller the value, the better the affinity); TON reflects the maximum number of substrate conversions per unit of active catalytic atoms, reflecting the intrinsic activity independent of the number of active centers.

[0053] 6. Details of in-situ Fourier transform infrared (FTIR) measurement

[0054] In this study, in-situ FTIR measurements were carried out using an infrared spectrometer (Thermo Fisher Scientific, iS50 FTIR) equipped with an in-situ spectral cell (Shanghai Yuanfang Technology Co., Ltd., SPECEL-III). We usually prepared a Nafion solution consisting of 210 μL of isopropanol, 750 μL of deionized water, and 40 μL of Nafion (perfluorosulfonic acid ion exchange resin, Yuanye Bio, 5% mass fraction, dissolved in water and 1-propanol). Subsequently, a catalyst / Nafion solution with a concentration of 10 mg / mL was prepared. During the experiment, 40 μL of the catalyst solution was drop-coated on the surface of a zinc selenide (ZnSe) crystal, dried, and then the crystal was installed in the in-situ cell. Then, 5 mL of 0.5 M hydrogen peroxide (H 2 O 2 ) solution was added to the in-situ cell, and in-situ FTIR spectra were collected at specific time intervals, with the reaction time maintained at 10 minutes.

[0055] Test results

[0056] 1. Structural characterization

[0057] The nanoparticle morphology of MnTi 6 was confirmed by scanning electron microscopy (SEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images, as shown in Figure 1 a - b. The images show that compared with the poreless dense structure of Ti 6 , MnTi 6 contains many nanopores, which are marked with yellow circles. These nanopores can increase the specific surface area, which is considered beneficial to improving the catalytic performance. As shown in Figure 1 c, no obvious long-range ordered atomic arrangement was observed, demonstrating the structural disorder of MnTi 6 . Scanning transmission electron microscopy - energy dispersive X-ray spectroscopy (STEM-EDS) showed that in the nanostructure, oxygen (O), titanium (Ti), and manganese (Mn) were well-distributed and uniform, without local aggregation of manganese, which confirmed the successful synthesis of MnTi 6 ( Figure 1 d, e). Dynamic light scattering (DLS) and ζ potential indicated that MnTi6 It has good water dispersibility (ζ potential: -6.04 mV), and the average hydrodynamic size is about 900 nanometers ( Figure 1 g).

[0058] In addition, we deeply studied the detailed chemical structure of MnTi 6 First, using Raman spectroscopy, the Ti-O-Ti structure of MnTi was determined based on the ν(Ti-O-Ti) peak at 614 cm⁻¹, as 6 shown in Figure 1 h. The Raman peaks of coordinated 4-aminobenzoic acid [A1g and B2g modes at ν(COO) at 1394 cm -1 , 1524 cm -1 and 1600 cm -1 and isopropanol [ν(C-C-O) at 857 cm⁻¹] were observed in both materials, which confirmed that MnTi 6 has a coordination structure similar to that of Ti 6 . Notably, a new Raman peak located at 536 cm⁻¹ was observed, which is attributed to the stretching vibration of Mn-O, indicating the formation of Mn-O bonds. The surface composition and electronic structure of MnTi 6 were studied by X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum ( Figure 1 i) showed that manganese (Mn), titanium (Ti), nitrogen (N) and oxygen (O) exist in MnTi 6 , which is consistent with the results of STEM-EDS. The specific contents of manganese and titanium are 7.19% and 17.94% respectively, which are consistent with the results of inductively coupled plasma optical emission spectrometer (ICP-OES) ( Figure 1 f). As Figure 1 shown in 6 j, the O 1s spectra of both MnTi 6 and Ti 6 showed different signals related to organic oxygen and O-M (metal). Compared with Ti 6 , the binding energies of the O 1s peaks of MnTi 6 decreased by 0.14 eV and 0.22 eV. These results indicate the formation of atomic-level Mn-O sites in MnTi 6 . At the same time, compared with Ti 6 , the binding energy level of the Ti 2p peak in MnTi Figure 1 k) decreased. These observations indicate the existence of electron transfer from electron-donating manganese to the titanium oxygen substrate. The results of high-resolution Mn 2p spectroscopy ( Figure 1l) The existence of such electron transfer was demonstrated. The spectrum could be deconvoluted into 2p1 / 2 and 2p3 / 2 regions. The two main components of Mn³⁺ at 652.26 eV and 641.26 eV and Mn²⁺ at 651.42 eV and 639.74 eV in the spectrum were well fitted, indicating a higher valence of manganese compared with the initial salt Mn(NO 3 ) 2 ·4H 2 O, and suggesting the formation of atomic-level Mn-O sites in MnTi 6 . Subsequently, we performed X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) analyses to explore the local coordination environment of MnTi 6 . The K-edge spectrum of manganese (Mn) indicated that the pre-edge peak of MnTi 6 was located between the pre-edge peak positions of manganese oxide (MnO) and manganese dioxide (MnO 2 ) ( Figure 1 m), and the average valence of manganese was +2.22 ( Figure 1 n), indicating the existence of electron transfer from low-valence manganese atoms to titanium (Ti) atoms, which was consistent with the results of our XPS analysis. According to the k³-weighted Fourier transform extended X-ray absorption fine structure (EXAFS) spectrum, the main peaks of MnTi 6 appeared at 1.66 Å and 2.31 Å, indicating the existence of manganese-oxygen (Mn-O) and manganese-titanium (Mn-Ti) structures ( Figure 1 o). Overall, the data of STEM, XPS, and XAS corroborated each other, confirming that the incorporation of Mn might lead to the formation of an amorphous nanoporous structure by substituting the position of Ti and forming coordination with oxygen in the titanium oxygen cluster. In addition, we also found the existence of electron transfer from the Mn site to the Ti site, which might enable MnTi 6 to overcome the challenges associated with multi-electron reactions during the scavenging of reactive oxygen species (ROS).

[0059] 2. Evaluation of reactive oxygen species scavenging activity

[0060] After verifying the chemical and electronic structure of MnTi 6 , the performance of its biocatalytic scavenging of ROS was systematically studied next. First, all the materials obtained from the examples and comparative examples were tested for ·O 2 - scavenging performance, as shown in Figure 2 a. Commercial TiO 2 did not show the performance of scavenging free radicals, while Ti 6 showed excellent ·O 2 -Scavenging performance. At the same time, other MTi 6 (where M represents the metals Ce / Cu / Mn / V) materials also all exhibited ·O 2 - scavenging performance. It is worth noting that Cu and Mn are the metal active sites of natural SOD enzymes. However, Ti 6 , as a material without doping these active metals (Cu, Mn), still showed excellent SOD-like enzyme performance. There are few reports on the high-efficiency SOD-like enzyme performance of titanium metal-related materials at present. Secondly, as Figure 2 shown in Fig. b, we monitored the H 2 O 2 scavenging ability of the obtained materials. Within 30 minutes, only MnTi 6 had an H 2 O 2 scavenging efficiency as high as 91%. The H 2 O 2 scavenging, as the first step of CAT performance, indicated the existence of CAT performance of MnTi 6 .

[0061] After the preliminary screening of the performance of the obtained materials, the performance of MnTi 6 was investigated in detail. The general anti-reactive oxygen species cascade reaction of the biocatalyst (MnTi 6 ) is shown in Fig. a. First, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging experiment was used to test the total radical scavenging ability of the material. The results showed that, compared with Ti Figure 3 , the scavenging ability of MnTi 6 for ABTS⁺· was improved ( 6 Fig. b). Since scavenging superoxide anion (·O Figure 3 ) is the initial step of the anti-reactive oxygen species cascade reaction, thus, using nitro blue tetrazolium chloride (NBT) as a specific indicator, the scavenging rate of superoxide anion (·O 2 - ) was monitored to study its superoxide dismutase (SOD)-like activity (converting ·O 2 - into hydrogen peroxide (H 2 - ). As shown in Fig. c, both Ti 2 O 2 and MnTi Figure 3 showed strong superoxide dismutase (SOD)-like activity, which indicated that the titanium (Ti) site plays a key role in SOD-like activity. At the same time, both samples had a strong scavenging ability for •O 6 and MnTi 6 2 - ​The clearance of ·O is dose-dependent, and at extremely low concentrations (25 μg / mL), the clearance rate of ·O 2 - can reach approximately 90%.

[0062] Thereafter, the activities of catalase (CAT)-like enzymes were monitored for Ti 6 and MnTi 6 [which convert hydrogen peroxide (H 2 O 2 2) into oxygen (O 2 2) and water (H 2 2 2 O)]. This is the second key step in the reactive oxygen species scavenging cascade system. In the time-course hydrogen peroxide (H 2 O 2 2) scavenging experiment, compared with the original Ti 6 (94.39%), the H 6 2 2 O 2 content of MnTi Figure 3 decreased significantly (6.54%) ( 2 O 2 d), indicating that the Mn site plays a key role in CAT-like activity. The generated oxygen was also tested, and it was correlated with the decomposition of H Figure 3 2 6 O 2 2 6 ( 2 O 2 e). The oxygen (O 6 2) concentration increased significantly in MnTi Figure 3 within 5 minutes, being 27.26 mg / mL higher than that of Ti 2 O 2 2 max ). Importantly, after repeated addition of hydrogen peroxide (H Figure 3 2 6 O max ), MnTi -1 exhibited good cycling durability (clearance rate greater than 90% after 5 cycles) ( -1 f). Using hydrogen peroxide (H Figure 3 2 6 O maxThe TON value was compared with that of transition metal-based reactive oxygen species scavenging materials (Table 1). MnTi 6 The biocatalyst showed significant differences from the most advanced reported reactive oxygen species scavenging nanobiocatalysts, including most manganese-based biocatalysts (Mn 3 O 4 cubes, Mn 3 O 4 hexagonal plates, etc.), as well as other metal-based biocatalysts (ultra-small Cu 5.4 O clusters, Co 3 O 4 nanoparticles, Fe Nzy, RuSA-CN, etc.) ( Figure 3 h and Table 1). In-situ Fourier transform infrared spectroscopy (FTIR) was used to study the structural changes of intermediates during the simulation of catalase (CAT), and the results showed the formation of * OOH and •O 2 - intermediates, as well as the reaction of hydrogen peroxide (H 2 O 2 ), being converted into oxygen (O 2 ). ( Figure 3 j, k). Therefore, we demonstrated that the MnTi 6 artificial antioxidant enzyme exhibited cascaded SOD-CAT catalytic activity due to the presence of Mn-O-Ti dual active sites.

[0063] Table 1: Comparison of the maximum reaction rate (V max ) and turnover number (TON) with those of the most advanced reported ROS scavenging biocatalysts recently. Turnover number (TON) = maximum reaction rate (V max ) / [E 0 , where [E 0 is the molar concentration of the metal in the whole nanomaterial.

[0064] Biocatalyst <![CDATA[V max (μM·s -1 )]]> <![CDATA[[E 0 (μM)]]> <![CDATA[TON (s -1 )]]> References <![CDATA[MnTi 6 > 44.30 35.16 1.26 The present invention RuTeNRs 0.98 196340 5.00E-06 1 <![CDATA[MnO 2 > 2.4 4965.52 4.83E-04 2 OxgeMCC-r 0.2 29.28 6.83E-03 3 <![CDATA[Mn 3 O 4 > 10.73 198.7 5.40E-02 4 <![CDATA[Mn 3 O 4 Cb]]> 5.3 65.45 0.08 5 <![CDATA[Mn 3 O 4 Ph]]> 5.8 65.45 8.86E-02 5 MC-1.0 24.82 118.19 0.21 4 Cu NCs 418.41 1820 0.23 6 Fe Nzy 1.22 4.82 0.25 7 <![CDATA[Cu 5.4 O]]> 3.92 15.04 0.26 8 Mfk 21.75 65.45 0.33 5 <![CDATA[Ru SA -CN]]> 10.02 13.01 0.77 9 <![CDATA[Cu x O]]> 109.2 125.81 0.87 10

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[0076] It should be noted that the specification of the present invention and its accompanying drawings provide preferred embodiments of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments do not serve as additional limitations to the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. Furthermore, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as within the scope described in the specification of the present invention; further, for those of ordinary skill in the art, improvements or modifications can be made according to the above description, and all such improvements and modifications should fall within the protection scope of the appended claims of the present invention.

Claims

1. A MnTi6 artificial antioxidant enzyme, characterized in that: The MnTi6 artificial antioxidant enzyme is a Mn-doped titanium oxide cluster formed by a solvothermal reaction of a titanium source, a manganese source and 4-aminobenzoic acid, and has an amorphous nanoporous structure; Furthermore, there are Mn-O and Mn-Ti coordination structures in the MnTi6 artificial antioxidant enzyme.

2. The MnTi6 artificial antioxidant enzyme according to claim 1, characterized in that The MnTi6 artificial antioxidant enzyme has cascade SOD-CAT catalytic activity.

3. A method for preparing the MnTi6 artificial antioxidant enzyme as claimed in claim 1 or 2, characterized in that: The preparation method comprises the following steps: adding a manganese source to a solvent containing a titanium source and 4-aminobenzoic acid, reacting at 90-120° C. for 60-90 hours, collecting the reaction product, washing it, and drying it to obtain the MnTi6 artificial antioxidant enzyme.

4. The preparation method according to claim 3, characterized in that: The titanium source includes titanium tetraisopropoxide, titanium tetrachloride or titanate.

5. The preparation method according to claim 4, characterized in that: The titanium source is titanium tetraisopropoxide.

6. The preparation method according to claim 3, characterized in that: The manganese source includes manganese nitrate tetrahydrate, manganese chloride hexahydrate or manganese sulfate hexahydrate.

7. The preparation method according to claim 6, characterized in that: The manganese source is manganese nitrate tetrahydrate.

8. The preparation method according to claim 3, characterized in that: The solvent includes isopropanol, methanol, ethanol, toluene, acetonitrile or N,N-dimethylformamide.

9. The preparation method according to claim 8, characterized in that: The solvent is isopropanol.

10. Use of the MnTi6 artificial antioxidant enzyme as claimed in claim 1 or 2 in the preparation of a preparation for removing active oxygen.

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