Nano enzyme as well as preparation method and application thereof

Abstract

The invention discloses a nano-enzyme as well as a preparation method and application thereof, and relates to the technical field of nano-enzymes. According to the preparation method, natural polyphenol, a cerium source and polyethylene glycol are mixed to prepare the novel polyphenol nano-enzyme, the preparation method is simple and easy to implement, the preparation cost is low, the prepared nano-enzyme has good biocompatibility, and a new way is provided for effective treatment of inflammatory diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of nanozymes, and in particular to a nanozyme and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) produced by organisms play an important role in various cell signaling pathways and are associated with many diseases (such as inflammatory bowel disease and cancer). For example, stress, environmental pollutants, radiation, viral infection, and bacterial infection can interact with cells through the production of ROS, leading to inflammatory responses, cell transformation, cancer cell survival, proliferation, and invasion. Therefore, regulating ROS in biological organisms has become a key factor in maintaining the body's balance.

[0003] Current studies have shown that inflammation and oxidative stress are closely related. During excessive inflammatory reactions, the presence of reactive oxygen species will further aggravate local tissue damage and lead to the continued occurrence of chronic inflammation. Chronic inflammation is one of the common and difficult diseases. As a disease with a low cure rate, according to statistics, the number of deaths among 6.8 million patients in 2017 was as high as 38,000. WHO calls chronic inflammation a huge threat to human health. Taking the United States as an example, about 125 million people suffer from chronic inflammation-related diseases. According to statistics in 2014, 42% of people in the United States have more than one chronic inflammatory disease, and 12% have 5 or more chronic inflammation-related diseases. Three out of every five people in the world suffer from chronic inflammation. These chronic inflammatory diseases include inflammatory bowel disease, rheumatoid arthritis, psoriasis, Alzheimer's disease, Parkinson's disease, depression, osteomyelitis, systemic lupus erythematosus, chronic nephritis, non-alcoholic fatty liver disease, cirrhosis, autoimmune oophoritis, autoimmune orchitis, cancer, chronic respiratory disease, stroke, cardiovascular disease, obesity and diabetes.

[0004] Although the direct mortality rate of chronic inflammation is not high, it has a significant impact on the patient's quality of life. The long-term development of the disease will cause some serious lesions, such as cancer. At present, small molecule drugs, antibodies and antibiotics are used as the main therapeutic drugs in clinical practice, but the overall treatment effect is not ideal. In addition, since patients with chronic inflammation need to take medication for a long time, it is easy to cause a series of complications such as antibiotic efficacy and immune response. Studies have shown that the lack of drug specificity, high-dose medication and side effects caused by extensive systemic contact are the main reasons for the poor treatment effect. Therefore, scientists have tried to develop a targeted delivery system that is dependent on pH, pressure and time. However, most delivery systems do not have a targeted target, and these drugs rely on the blood system for delivery. There is still no clear drug system that only treats the site of onset, that is, the site of inflammation. Therefore, it is urgent to develop a new targeted strategy that can locate and treat the site of inflammation.

[0005] Through the investigation of the microenvironment of inflammatory disease lesions, it was found that the inflammatory site is characterized by the enrichment of positively charged proteins and the overexpression of highly reactive oxygen free radicals. Therefore, negatively charged drugs or drug delivery systems can be used to target the lesion site, and these drugs can be used to eliminate local excessive ROS to reduce inflammation. This treatment strategy may be an effective treatment for inflammatory diseases. However, among the many known antioxidants, ROS scavengers or negatively charged substances, few can alleviate inflammatory diseases. Although some antioxidant substances have been developed as inflammatory disease therapeutics, their effects and various limitations in application are not satisfactory.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a nanozyme and a preparation method and application thereof.

[0008] The present invention is achieved in that:

[0009] In a first aspect, an embodiment of the present invention provides a method for preparing a nanozyme, comprising the following steps: mixing natural polyphenols, a cerium source and polyethylene glycol to obtain the nanozyme.

[0010] In a second aspect, an embodiment of the present invention provides a nanozyme, which is prepared by the preparation method described in the preceding embodiment.

[0011] In a third aspect, an embodiment of the present invention provides a product for removing active oxygen, which includes: the nanozyme described in the above embodiment.

[0012] In a fourth aspect, embodiments of the present invention provide the use of the nanozymes described in the preceding embodiments in scavenging reactive oxygen species or preparing products for scavenging reactive oxygen species.

[0013] In a fifth aspect, the embodiments of the present invention provide the use of the nanozymes described in the above embodiments in the preparation of products for treating or assisting in the treatment of inflammatory diseases. The present invention has the following beneficial effects:

[0014] The present invention provides a novel polyphenol nanozyme with combined anti-inflammatory activity of natural polyphenols, which is prepared by mixing natural polyphenols, a cerium source and polyethylene glycol. The preparation method is simple and easy to implement, and the preparation cost is low. The prepared nanozyme has good biocompatibility, providing a new approach for the effective treatment of inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 This is a transmission electron microscopy (TEM) image of polyphenol nanozyme;

[0017] Figure 2 The distribution of polyphenol nanozymes elements;

[0018] Figure 3 is the UV-visible spectrum of polyphenol nanozymes and their corresponding polyphenols;

[0019] Figure 4 is the free radical scavenging activity of polyphenol nanozymes at different concentrations on DPPH·;

[0020] Figure 5 is the free radical scavenging activity of polyphenol nanozymes at different concentrations towards ·OH;

[0021] Figure 6 The effects of different concentrations of polyphenol nanozymes on ·O 2 - Free radical scavenging activity;

[0022] Figure 7 The effects of different concentrations of polyphenol nanozymes on H 2 O 2 Free radical scavenging activity;

[0023] Figure 8 The viability of HEK-293T cells after treatment with different concentrations of polyphenol nanozymes;

[0024] Fig. 9 Fluorescence images of RAW264.7 cells after different treatments;

[0025] Fig.10Different natural polyphenols, Ce 3+ Transmission electron microscopy (TEM) images of polyphenol nanozymes with different mass ratios of 1:1 and 1:1;

[0026] Fig.11 Transmission electron microscopy (TEM) images of polyphenol nanozymes with PEG of different molecular weights;

[0027] Fig.12 Transmission electron microscopy (TEM) images of polyphenol nanozymes at different mixing times;

[0028] Fig.13 Polyphenol nanozymes react with natural catalase and the currently common CeO 2 Nanoparticles for H 2 O 2 free radical scavenging activity. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0030] Explanation of terms

[0031] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0032] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0033] Technical Solution

[0034] Some studies have shown that natural enzymes that can eliminate ROS, such as superoxide dismutase (SOD) and catalase (CAT), can be encapsulated into liposomes to treat inflammatory diseases. However, natural enzymes have poor stability, high cost, easy to cause immune response, and high specificity (eliminating a single type of ROS), which hinder the effective application of natural enzymes. The present invention provides a novel polyphenol nanozyme with natural antioxidant activity of polyphenols. The nanozyme has high stability, low preparation cost, simple and easy process, and good biocompatibility, and can effectively treat or assist in the treatment of inflammatory diseases.

[0035] On the one hand, an embodiment of the present invention provides a method for preparing a nanozyme, which comprises the following steps: mixing natural polyphenols, a cerium source and polyethylene glycol to obtain the nanozyme.

[0036] In some embodiments, the mixing mass ratio of the natural polyphenol, the cerium source and the polyethylene glycol is 1-200:11-220:10-600. Specifically, 40-200 in the mass ratio is specifically selected from any one or any two of 1, 5, 10, 20, 30, 40, 60, 80, 100, 120, 140, 160, 180, 200. 11-220 in the mass ratio can be selected from any one or any two of 11, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220. The mass ratio of 10 to 600 can be selected from any one of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, and 600, or a range between any two of the ranges.

[0037] In some embodiments, the natural polyphenols include: any one or more of quercetin (Que), kaempferol (Kae), luteolin (Lut), apigenin (Api), baicalein (Bai), epigallocatechin gallate (EGCG), proanthocyanidins (Pro), theaflavins (The), tannic acid (TA), arbutin (Arb), hesperidin (Hes), morin (Mor), fisetin (FIT), licorice (Liq), chrysin (Chr), galangin (Gal), myricetin (Myr), rutin (Rut), eriodictyol (Eri), naringenin (Nar), citronellol (Dih), dimethicone (DMY), safflower (Pru) and silybin (Sil).

[0038] In some embodiments, before the mixing, the preparation method further comprises: dissolving the natural polyphenol in a first solvent, adjusting the pH to 4-7, and obtaining a natural polyphenol solution for mixing with the natural polyphenol and the cerium source.

[0039] Specifically, the pH value may be adjusted to a range between 4 and 7, specifically, to any one of 4, 4.5, 5, 5.5, 6, 6.5, and 7, or to a range between any two of them.

[0040] In some embodiments, the first solvent is water or an organic solvent.

[0041] In some embodiments, the first solvent includes any one or more of water, DMSO solution, DMF solution, methanol solution and ethanol solution.

[0042] In some embodiments, in the natural polyphenol solution, the concentration of the natural polyphenol is 2-10 mg / mL, and the concentration can be any one of 2, 4, 6, 8 and 10 mg / mL or a range between any two of them.

[0043] In some embodiments, before the mixing, the preparation method further comprises: dissolving the cerium source and the polyethylene glycol in a second solvent to obtain a cerium source solution and a polyethylene glycol solution, and then mixing them with the natural polyphenols.

[0044] In some embodiments, the second solvent comprises water.

[0045] In some embodiments, the cerium source comprises Ce(NO 3 ) 3 6H 2 O.Ce 2 (CO 3 ) 3 and (NH 4 ) 2 Ce(NO 3 ) 6 Any one or more of .

[0046] In some embodiments, in the cerium source solution, the concentration of the cerium source is 1 to 20 mg / mL; the concentration can specifically be any one of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 mg / mL or a range between any two thereof.

[0047] In some embodiments, in the polyethylene glycol solution, the concentration of the polyethylene glycol is 15 to 30 mg / mL; the concentration can specifically be any one of 15, 16, 18, 20, 22, 24, 26, 28, 30 mg / mL or a range between any two of them.

[0048] In some embodiments, the molecular weight of the polyethylene glycol is 2000-10000; the molecular weight can specifically be any one of 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or a range between any two of them.

[0049] In some embodiments, the mixing is performed in a third solvent; the third solvent comprises water;

[0050] In some embodiments, 200 mL of the third solvent is added for every 40-200 mg of the natural polyphenol. The 40-200 mg specifically refers to any one of 40, 60, 80, 100, 120, 140, 160, 180, 200 mg or a range between any two thereof.

[0051] In some embodiments, the mixing time is 1 to 5 hours. The time can be any one of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 hours or a range between any two of them. There is no special requirement for the mixing temperature, which can be room temperature, which can be 0 to 30°C, and can be any one of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30°C or a range between any two of them.

[0052] In some embodiments, after the mixing, the preparation method further comprises centrifuging the mixed product and taking the precipitate to obtain the nanozyme.

[0053] In some embodiments, the centrifugal conditions include: 8000-15000 rpm, 10-20 min. The centrifugal speed can be any one of 8000, 10000, 12000, 15000 rpm or a range between any two of them. The centrifugal time can be any one of 10, 12, 14, 16, 18, 20 min or a range between any two of them.

[0054] On the other hand, an embodiment of the present invention provides a nanozyme prepared by the preparation method described in any of the preceding embodiments.

[0055] On the other hand, an embodiment of the present invention provides a product for removing active oxygen, which includes: the nanozyme described in any of the preceding embodiments.

[0056] In some embodiments, a product for scavenging reactive oxygen species comprises a reagent set or kit.

[0057] On the other hand, an embodiment of the present invention also provides the use of the nanozyme as described in any of the preceding embodiments in scavenging reactive oxygen or preparing a product for scavenging reactive oxygen.

[0058] In some embodiments, the reactive oxygen species include: ·OH, DPPH·, ·O 2 -、H 2 O 2 Any one or more of .

[0059] In addition, an embodiment of the present invention also provides the use of the nanozyme as described in any of the preceding embodiments in the preparation of a product for treating or assisting in the treatment of inflammatory diseases.

[0060] In some embodiments, the inflammatory disease comprises any one or more of inflammatory bowel disease, rheumatoid arthritis, psoriasis, Alzheimer's disease, Parkinson's disease, depression, osteomyelitis, systemic lupus erythematosus, chronic nephritis, non-alcoholic fatty liver disease, cirrhosis, autoimmune oophoritis, autoimmune orchitis, cancer, chronic respiratory disease, stroke, cardiovascular disease, obesity and diabetes.

[0061] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0062] Example 1

[0063] This embodiment provides a polyphenol nanozyme and a preparation method thereof, which comprises the following steps.

[0064] (1) Preparation of cerium source solution

[0065] Will Ce(NO 3 ) 3 6H 2 O was dissolved in ultrapure water and ultrasonically dissolved to obtain Ce(NO 3 ) 3 6H 2 O solution, Ce(NO 3 ) 3 6H 2 The concentration of O was 1 mg / mL;

[0066] (2) Preparation of polyethylene glycol solution

[0067] Dissolve polyethylene glycol in ultrapure water and dissolve by ultrasonication to obtain a polyethylene glycol solution; wherein the molecular weight of the polyethylene glycol is 8000 and the concentration of the solution is 20 mg / mL;

[0068] (3) Preparation of natural polyphenol solution

[0069] Dissolve various natural polyphenols in DMSO solution respectively, dissolve by ultrasonication, adjust the pH to 6, and obtain a natural polyphenol solution, wherein the concentration of the natural polyphenol is 5 mg / mL;

[0070] The various natural polyphenols are quercetin (Que), kaempferol (Kae), luteolin (Lut), apigenin (Api), baicalein (Bai), EGCG, proanthocyanidins (Pro) and theaflavins (The).

[0071] (4) Mixing

[0072] The cerium source solution, polyethylene glycol solution and natural polyphenol solution prepared in the above steps are added to ultrapure water for mixing, the mixing volume ratio of the cerium source solution, polyethylene glycol solution and natural polyphenol solution to ultrapure water is 11:20:20:200, the mixing time is 2 hours, and the mixing method is stirring;

[0073] (5) Centrifugation

[0074] The mixed product was centrifuged at 12000 rpm for 15 min, and the precipitate was collected to obtain polyphenol nanozyme.

[0075] Example 2

[0076] The polyphenol nanozyme prepared in Example 1 was characterized.

[0077] Through TEM transmission electron microscopy, polyphenol nanozymes with roughly spherical shapes were obtained, such as Figure 1 As shown, nanozymes with a particle size of about 50 nm were obtained using a simple self-assembly method.

[0078] Typical high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images and energy-dispersive X-ray spectroscopy show that there are uniformly distributed C, O, and Ce in the nanozyme, as shown in Figure 2. Figure 2 shown.

[0079] The UV-visible spectroscopic determination of polyphenol-cerium-based nanozymes and their corresponding polyphenols, e.g. Figure 3 As shown, polyphenols were deprotonated under the coordination of metal ions during the synthesis of nanozymes, resulting in a red shift of the characteristic peaks, indicating the successful synthesis of nanozymes.

[0080] Example 3

[0081] The free radical scavenging ability of the polyphenol nanozyme prepared in Example 1 was measured.

[0082] Polyphenol nanozymes of different concentrations (0, 25, 50, 100 μg / mL) were added to the free radical scavenging reaction reagent according to the instructions of the Solebao DPPH free radical scavenging kit, the Nanjing Jiancheng Bioengineering Institute hydroxyl radical test kit, the Beyotime active oxygen detection kit, and the Beyotime hydrogen peroxide detection kit. Figure 4 to Figure 7As shown in the figure, the UV-visible spectra show the nanozymes' effects on DPPH·, ·OH, and ·O at different concentrations. 2 - and H 2 O 2 free radical scavenging activity.

[0083] Example 4

[0084] The polyphenol nanozyme prepared in Example 1 was subjected to cell proliferation-toxicity assay.

[0085] Through the cell proliferation experiment (CCK8 method was used to determine its effect on the cell proliferation of HEK-293T cells), HEK-293T cells were cultured in a 96-well plate for 24 hours. Then the polyphenol nanozymes prepared in Example 1 were added, and the concentrations were 0μg / mL, 3.125μg / mL, 6.25μg / mL, 12.5μg / mL, 15μg / mL, 25μg / mL, 50μg / mL, 75μg / mL and 100μg / mL, respectively, and incubated for 24 hours. 110μL of a 1:10 mixture of CCK8 and DMEM culture medium was added to each well. After incubation for 2 hours, the absorbance wavelength at 450nm was measured with a microplate reader. The cell viability rate is defined as: OD (sample) / OD (control) × 100%, where OD (control) and OD (sample) represent the absorbance values ​​at 450nm with and without samples, respectively), and the results are as follows Figure 8 shown.

[0086] Example 5

[0087] The intracellular free radical scavenging ability of the polyphenol nanozyme prepared in Example 1 was measured.

[0088] The intracellular ROS scavenging ability of polyphenol nanozymes was studied using CLSM (confocal laser scanning microscopy). The cells were seeded in 24-well plates and cultured for 24 hours. The cells were stimulated with LPS (10 μg / mL) for 24 hours, and then cultured with polyphenol nanozymes (25 μg / mL, 50 μg / mL, 100 μg / mL) prepared in Example 1 in equal amounts for 24 hours. The DAFH-DA reactive oxygen species fluorescent probe was added and incubated for 20 minutes, then washed 3 times, and the fluorescence was observed by CLSM. Fig. 9 It can be seen that nanozymes have a certain ability to clear intracellular ROS.

[0089] Example 6

[0090] Based on the polyphenol nanozyme and its preparation method provided in Example 1, 4 experimental groups were set up to prepare polyphenol nanozymes. Group 1 had the same preparation process as Example 1 (natural polyphenols, Ce 3+and polyethylene glycol in a mass ratio of 100:11:400), the preparation process of groups 2 to 4 is substantially the same as that of Example 1, except that natural polyphenols, Ce 3+ The mass ratios of PEG and PEG were (20:11:80, 10:11:40, 5:11:20) respectively. The morphology of the polyphenol nanozymes prepared in the four groups was characterized. Fig.10 .

[0091] From the results, it can be seen that changing the mass ratio of different cerium ions has no significant effect on the morphological characterization of cerium-based polyphenol nanozymes.

[0092] Example 7

[0093] Based on the polyphenol nanozyme and its preparation method provided in Example 1, two experimental groups were set up to prepare polyphenol nanozymes. The preparation process of group 1 was the same as that of Example 1 (PEF8000), and the preparation process of group 2 was roughly the same as that of Example 1, except that the polyethylene glycol used was PEF6000. The morphology of the polyphenol nanozymes prepared in the two groups was characterized, and the results are shown in FIG. Fig.11 .

[0094] The results show that polyethylene glycol has no significant effect on the morphology of polyphenol nanozymes (CeLutNCs).

[0095] Example 8

[0096] Based on the polyphenol nanozyme and its preparation method provided in Example 1, three experimental groups were set up to prepare polyphenol nanozymes. The preparation process of group 1 was the same as that of Example 1 (in the mixing step (4), the mixing time was 2 h). The preparation processes of groups 2 to 3 were roughly the same as those of Example 1, except that in the mixing step (4), the mixing time was 1 h and 12 h. The morphology of the polyphenol nanozymes prepared in the three groups was characterized, and the results are shown in Table 1. Fig.12 .

[0097] From the results, it can be seen that there are very few successfully assembled nanoparticles when stirring for 1 hour, the nanoparticles are over-assembled and have no fixed morphology when stirring for 12 hours, and the nanoparticles are completely assembled and have a clear morphology when stirring for 2 hours, which is the optimal stirring time.

[0098] Example 9

[0099] The same concentration of 25 μg / mL polyphenol nanozymes (Example 1, CeLutNCs), natural catalase and the currently common CeO 2 Nanoparticles for H 2 O 2 The cleaning ability was tested in the same manner as in Example 3. The results are shown in Fig.13 .

[0100] The results show that the H of CeLutNCs nanozymes at the same concentration2 O 2 Stronger clearing ability.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a nanozyme, characterized in that: It includes the following steps: Natural polyphenols, a cerium source and polyethylene glycol are mixed to obtain the nanozyme.

2. The preparation method according to claim 1, characterized in that: The mixing mass ratio of the natural polyphenol, the cerium source and the polyethylene glycol is 1-200:11-220:10-600.

3. The preparation method according to claim 1, characterized in that: The natural polyphenols include any one or more of quercetin, kaempferol, luteolin, apigenin, baicalein, epigallocatechin gallate, proanthocyanidins, theaflavins, tannic acid, arbutin, hesperidin, morin, fisetin, licorice, chrysin, galangin, myricetin, rutin, eriodictyol, naringenin, citronellol, scutellarin, silybin and silybin; Optionally, before the mixing, the preparation method further comprises: dissolving the natural polyphenol in a first solvent, adjusting the pH to 4 to 7, to obtain a natural polyphenol solution; Optionally, the first solvent is water or an organic solvent; Optionally, the first solvent includes any one or more of water, DMSO solution, DMF solution, methanol solution and ethanol solution; Optionally, in the natural polyphenol solution, the concentration of the natural polyphenol is 2 to 10 mg / mL.

4. The preparation method according to claim 1, characterized in that: Before the mixing, the preparation method further comprises: dissolving the cerium source and the polyethylene glycol in a second solvent to obtain a cerium source solution and a polyethylene glycol solution, and then mixing the cerium source solution and the polyethylene glycol solution with the natural polyphenol; Optionally, the second solvent comprises water; Optionally, the cerium source includes any one or more of Ce(NO3)3·6H2O, Ce2(CO3)3 and (NH4)2Ce(NO3)6; Optionally, in the cerium source solution, the concentration of the cerium source is 1 to 20 mg / mL; Optionally, in the polyethylene glycol solution, the concentration of the polyethylene glycol is 15 to 30 mg / mL; Optionally, the molecular weight of the polyethylene glycol is 2000 to 10000; Optionally, the molecular weight of the polyethylene glycol is 3000-8000.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The mixing is performed in a third solvent; the third solvent comprises water; Optionally, 200 mL of the third solvent is added for every 40 to 200 mg of the natural polyphenol.

6. The preparation method according to any one of claims 1 to 4, characterized in that: The mixing time is 1 to 5 hours; Optionally, after the mixing, the preparation method further comprises centrifuging the mixed product and taking a precipitate to obtain the nanozyme; Optionally, the centrifugal conditions include: 8000-15000 rpm, 10-20 min.

7. A nanozyme, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 6.

8. A product for removing active oxygen, characterized in that: It includes: The nanozyme according to claim 7.

9. Use of the nanozyme according to claim 7 in scavenging active oxygen or preparing products for scavenging active oxygen.

10. Use of the nanozyme according to claim 7 in preparing a product for treating or assisting in treating inflammatory diseases; Optionally, the inflammatory disease comprises: Any one or more of inflammatory bowel disease, rheumatoid arthritis, psoriasis, Alzheimer's disease, Parkinson's disease, depression, osteomyelitis, systemic lupus erythematosus, chronic nephritis, non-alcoholic fatty liver disease, cirrhosis, autoimmune oophoritis, autoimmune orchitis, cancer, chronic respiratory disease, stroke, cardiovascular disease, obesity and diabetes.

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