Metal-based antioxidant with antibacterial activity as well as preparation method and application of metal-based antioxidant

Abstract

The invention discloses a metal-based antioxidant with antibacterial activity and a preparation method and application thereof, and belongs to the technical field of biomedicine. The metal-based antioxidant disclosed by the invention is prepared by taking phenolic compounds and metal salts such as copper, iron and cerium as raw materials through a hydrothermal method, and shows excellent free radical scavenging efficiency. In the invention, due to the existence of a metal matrix, under the catalytic action of hydrogen peroxide, the antioxidant shows unique antibacterial ability, and can effectively kill super-drug-resistant bacteria; further experiments prove that the metal-based antioxidant disclosed by the invention has excellent biological safety, and the biological application capability of the metal-based antioxidant can be guaranteed. The metal-based antioxidant not only has a remarkable anti-oxidation effect, but also can effectively kill super-drug-resistant bacteria, so that the metal-based antioxidant has good application prospects and advantages in the aspect of bacterial infection treatment.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and specifically relates to a metal-based antioxidant with antibacterial activity and a preparation method and application thereof. Background Art

[0002] Bacterial infection has become one of the major problems threatening human health. Antibiotic resistance caused 4.95 million deaths in 2019. Therefore, it is of great significance to find and develop new antibacterial materials that can replace antibiotics.

[0003] Metal-based materials have attracted great attention from researchers because they can release metal ions with antibacterial activity across bacterial membranes and their biofilms, thereby exerting bactericidal properties without developing drug resistance. Metal-based antibacterial materials mainly achieve bactericidal effects by releasing metal ions to produce reactive oxygen species. However, long-term accumulation of excessive reactive oxygen species and metal ions can promote excessive secretion of inflammatory factors, aggravating inflammatory reactions and related diseases caused by excessive metal ions. As a countermeasure, reducing the metal-based content and introducing antioxidant properties are effective strategies to remove excess reactive oxygen species and alleviate inflammatory effects, but it is difficult to achieve a temporal and spatial balance between antioxidant activity and the generation of reactive oxygen species, which will lead to a significant decrease in antibacterial ability. Therefore, it is urgent to develop a metal-based antioxidant with high antibacterial activity to regulate the contradiction between bactericidal and reactive oxygen removal, so as to achieve an effective antibacterial effect. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a metal-based antioxidant with antibacterial activity and a preparation method thereof, wherein the metal-based antioxidant not only has excellent free radical scavenging ability, but also can effectively kill super-resistant bacteria.

[0005] Another object of the present invention is to provide application of metal-based antioxidants.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A method for preparing a metal-based antioxidant with antibacterial activity comprises the following steps: dissolving a metal salt and a phenolic compound in deionized water, transferring the obtained reaction solution to a reaction kettle, reacting in a muffle furnace for 0.5-12 hours, and then subjecting the obtained product to centrifugal washing and freeze drying to obtain the metal-based antioxidant with antibacterial activity.

[0008] Preferably, the metal salt is one or more of copper, iron, cerium or magnesium metal salts; the copper, iron, cerium or magnesium metal salt is its nitrate or chloride.

[0009] Preferably, the phenolic compound is one or more of catechin, protocatechuic acid or quercetin.

[0010] Preferably, the molar ratio of the metal salt to the phenolic compound is 1:10-5:1; the content of the metal salt in deionized water is 0.5-10wt%; and the reaction temperature is 50-150°C.

[0011] The present invention also provides a metal-based antioxidant prepared by the above preparation method.

[0012] Furthermore, the present invention also provides application of the above-mentioned metal-based antioxidant in the field of anti-oxidation.

[0013] Preferably, the metal-based antioxidant can remove more than 80% of ABTs within a working concentration range of 1-20 μg / mL. + ·It can resist free radicals such as hydroxyl radicals and exhibit excellent antioxidant capacity.

[0014] Furthermore, the present invention also provides the use of the above-mentioned metal-based antioxidant in combating super bacteria.

[0015] Preferably, the anti-super bacteria target is methicillin-resistant Staphylococcus aureus.

[0016] Preferably, the metal-based antioxidant has a slightly better antibacterial effect under the catalytic action of hydrogen peroxide than itself, and can kill more than 90% of bacteria at a concentration of about 30 μg / mL and a concentration of 5×10 8 CFU / mL of methicillin-resistant Staphylococcus aureus.

[0017] Furthermore, the metal-based antioxidant has excellent biosafety, ensuring its further application in the treatment of superbug infections.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The metal-based antioxidant of the present invention has the characteristics of simple and easy preparation process, excellent antioxidant performance and excellent biosafety; it has good anti-super bacteria effect and shows significant killing ability against methicillin-resistant Staphylococcus aureus. Therefore, the metal-based antioxidant with antibacterial activity of the present invention shows great application prospects in the treatment of super bacteria infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a transmission electron microscope image of the metal-based antioxidant of Example 1 of the present invention.

[0021] Figure 2 This is the photoelectron spectrum XPS spectrum of the metal-based antioxidant of Example 1 of the present invention.

[0022] Figure 3ABTs removal by metal-based antioxidants in Example 1 of the present invention + · The ability to destroy free radicals such as hydroxyl radicals.

[0023] Figure 4 This is a colony plate diagram of Example 1 of the present invention after the metal-based antioxidant is co-cultured with methicillin-resistant Staphylococcus aureus in the presence or absence of hydrogen peroxide. DETAILED DESCRIPTION

[0024] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0025] Example 1

[0026] Weigh 0.5 g of copper nitrate trihydrate solid and dissolve it in 100 mL of deionized water. Then slowly drop 3.02 g of quercetin and 1.54 g of protocatechuic acid pre-dissolved in an appropriate amount of ethanol (3 mL) into the copper nitrate aqueous solution. After the reactants are completely mixed, transfer them to a reactor, seal them, place them in a muffle furnace, and react at 90 ° C for 6 hours. When the temperature drops to room temperature, centrifuge and wash the reaction product, collect the solid, and finally freeze-dry to obtain a copper-based antioxidant with antibacterial activity.

[0027] The transmission electron microscopy image of the copper-based antioxidant prepared in this embodiment is shown in Figure 1 , photoelectron spectroscopy XPS spectrum see Figure 2 .

[0028] Example 2

[0029] 5 g of copper nitrate trihydrate and 5 g of solid ferric nitrate were weighed and dissolved in 100 mL of deionized water, and then 0.62 g of protocatechuic acid and 1.16 g of catechins pre-dissolved in an appropriate amount of ethanol (1 mL) were slowly added dropwise to the aqueous solution of the metal salt, and after the reactants were completely mixed, they were transferred to a reactor, sealed and placed in a muffle furnace, and reacted at 150° C. for 0.5 h. When the temperature dropped to room temperature, the reaction product was centrifuged and washed, the solid was collected, and finally freeze-dried to obtain a copper-iron-based antioxidant with antibacterial activity.

[0030] Example 3

[0031] 1g of copper nitrate trihydrate, 2g of cerium chloride heptahydrate and 1g of magnesium chloride solid were weighed and dissolved in 100mL of deionized water, and then 4.35g of catechins and 1.51g of quercetin pre-dissolved in an appropriate amount of ethanol (3mL) were slowly added dropwise to the metal salt aqueous solution, and the reactants were transferred to a reactor after being completely mixed, sealed and placed in a muffle furnace, and reacted at 50°C for 12h. When the temperature dropped to room temperature, the reaction product was centrifuged and washed, the solid was collected, and finally freeze-dried to obtain a copper-cerium-magnesium-based antioxidant with antibacterial activity.

[0032] Example 4

[0033] 1g of iron nitrate trihydrate, 2g of cerium chloride heptahydrate and 1g of magnesium chloride solid were weighed and dissolved in 100mL of deionized water, and then 3.02g of quercetin, 1.54g of protocatechuic acid and 2.90g of catechins pre-dissolved in an appropriate amount of ethanol (4mL) were slowly added dropwise to the metal salt aqueous solution, and the reactants were transferred to a reactor after being completely mixed, sealed and placed in a muffle furnace, and reacted at 120°C for 8h. When the temperature dropped to room temperature, the reaction product was centrifuged and washed, the solid was collected, and finally the iron cerium magnesium-based antioxidant with antibacterial activity was obtained by freeze drying.

[0034] Example 5

[0035] ABTs + Free radical scavenging:

[0036] Weigh 200 mg ABTS and 34.4 mg potassium persulfate, dissolve in 50 mL ultrapure water, shake well, and place at room temperature away from light for 18-24 hours to prepare the ABTS stock solution. Then take an appropriate amount of the ABTS stock solution and dilute it with PBS until the absorbance is 0.7 ± 0.2 (OD 734 ) for subsequent antioxidant assay (prepared and used immediately).

[0037] Then, an appropriate amount of the copper-based antioxidant prepared in Example 1 was weighed and dissolved in deionized water to prepare a copper-based antioxidant aqueous solution with a concentration of 500 μg / mL. Then, 0, 0.8, 1.6, 2.4, 3.2 and 6 μL of the copper-based antioxidant solution (final concentration: 2, 4, 6, 8, 10, 15 μg / mL) were respectively weighed to 180 μL with an absorbance value of 0.7±0.2 (OD 734 ) ABTS solution, add PBS to 200 μL, place at room temperature in the dark for 5 min, and use an enzyme reader to measure the absorbance change of the sample at 734 nm. Each concentration is repeated 3 times. 734 The antioxidant capacity of copper-based antioxidants was calculated and evaluated based on the change in absorbance [scavenging rate % = (OD 734-0 -OD 734-抗氧化剂 ) / OD 734-0×100%], the corresponding results are shown in Figure 3 .

[0038] Hydroxyl (·OH) free radical scavenging:

[0039] An appropriate amount of the copper-based antioxidant prepared in Example 1 was weighed, and a copper-based antioxidant solution with a concentration of 1 mg / mL was prepared using 0.2 mol / L NaH2PO4-Na2HPO4 buffer (pH=7.4) as solvent. 0, 1.2, 2.4, 3.6, 4.8, 6 and 9 μL of copper-based antioxidant solution (final concentration of 0, 2, 4, 6, 8, 10 and 15 μg / mL) were measured in 2 mL centrifuge tubes, and NaH2PO4-Na2HPO4 buffer was added to make up to 300 μL. Then 100 μL of EDTA (5 mmol / L) solution, 50 μL of FeCl3 solution (0.3125 mmol / L), 50 μL of hydrogen peroxide (33.6 mmol / L), 50 μL of deoxyribose solution (33.6 mmol / L) and 50 μL of ascorbic acid (1.2 mmol / L) were added respectively. After mixing, the mixture was incubated in a 50°C water bath for 20 min. Take out the sample, add 0.5mL trichloroacetic acid solution (50mg / mL) and 0.5mL thiobarbituric acid solution (1%, W / V), mix well, and place in a 105℃ oil bath for incubation for 15min. After the sample solution cools to room temperature, measure 200μL of the solution in a 96-well plate, and use an ELISA reader to measure the change in its absorbance at 532nm. Each sample concentration is repeated 3 times. The scavenging rate of copper-based antioxidants on hydroxyl free radicals [(OD 532-0 -OD 532-抗氧化剂 ) / OD 532-0 ×100%], the corresponding results are shown in Figure 3 .

[0040] The free radical scavenging capabilities of the metal-based antioxidants prepared in Examples 1-4 are shown in Table 1.

[0041] Table 1 Effect of metal-based antioxidants prepared in Examples 1-4 on ABTs + ·Scavenging ability of hydroxyl free radicals

[0042]

[0043] Example 6

[0044] Evaluation of antimicrobial activity:

[0045] This example uses the copper-based antioxidant prepared in Example 1 of the present invention as an example to evaluate its ability to kill the super bacteria methicillin-resistant Staphylococcus aureus: fresh methicillin-resistant Staphylococcus aureus bacterial solution in the logarithmic growth period is centrifuged and washed with PBS, the bacterial cells are collected and resuspended with PBS, and the concentration of the bacterial solution is adjusted to an OD of 600 ≈1.0(1.0×10 9 CFU / mL). Subsequently, part of the bacterial solution was mixed with equal volumes of copper-based antioxidants at concentrations of 60 and 120 μg / mL, and the other part of the bacterial solution was mixed with equal volumes of copper-based antioxidants at concentrations of 60 and 120 μg / mL containing 200 μmol / L hydrogen peroxide, mixed evenly, and placed in a 37°C incubator for incubation for 5 hours. 100 μL of the incubated bacterial solution was evenly applied to LB solid culture medium plates, placed in a 37°C incubator for incubation for 24 hours, and the number of colonies was checked and analyzed to evaluate the bactericidal ability of copper-based antioxidants. The corresponding results are shown in Figure 4 .

[0046] The corresponding sterilization rates of the metal-based antioxidants prepared in Examples 1-4 against methicillin-resistant Staphylococcus aureus are shown in Table 2.

[0047] Table 2 Killing ability of metal-based antioxidants prepared in Examples 1-4 against methicillin-resistant Staphylococcus aureus

[0048]

[0049]

[0050] In summary, the metal-based antioxidant of the present invention has excellent free radical scavenging and super bacteria resistance capabilities.

[0051] The above description is only a preferred embodiment of the present invention and is used to limit the present invention. Any modification, equivalent substitution, improvement, etc. made within the principle and content of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a metal-based antioxidant having antibacterial activity, characterized in that: The following steps are involved: The metal salt and the phenolic compound are dissolved in deionized water, the obtained reaction solution is transferred to a reactor, and the reaction is carried out in a muffle furnace for 0.5-12 hours. The obtained product is then centrifuged, washed and freeze-dried to obtain a metal-based antioxidant with antibacterial activity; the metal salt is one or more of copper, iron, cerium or magnesium metal salts; the phenolic compound is one or more of catechin, protocatechuic acid or quercetin.

2. The preparation method according to claim 1, characterized in that: The copper, iron, cerium or magnesium metal salt is a nitrate or chloride thereof.

3. The preparation method according to claim 1, characterized in that: The molar ratio of the metal salt to the phenolic compound is 1:10-5:1; the content of the metal salt in deionized water is 0.5-10wt%; and the reaction temperature is 50-150°C.

4. The metal-based antioxidant prepared according to the preparation method according to any one of claims 1 to 3.

5. Use of the metal-based antioxidant according to claim 4 in the field of anti-oxidation.

6. The use according to claim 5, characterized in that: The antioxidant activity includes scavenging ABTs + Free radicals and hydroxyl radicals.

7. Use of the metal-based antioxidant according to claim 4 in combating super bacteria.

8. The use according to claim 7, characterized in that: The anti-super bacteria target is methicillin-resistant Staphylococcus aureus.

9. The use according to claim 7, characterized in that: The metal-based antioxidant has a slightly better antibacterial effect under the catalytic action of hydrogen peroxide than itself, and can kill more than 90% of the bacteria at a concentration of 30 μg / mL and a concentration of 5×10 8 CFU / mL of methicillin-resistant Staphylococcus aureus.

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