Nickel-based plant polyphenol coordination polymer artificial enzyme as well as preparation method and application thereof

Abstract

The invention discloses a nickel-based plant polyphenol coordination polymer artificial enzyme as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: dissolving salvianolic acid B in an organic solvent, adding dodecanol, and carrying out heating reaction by taking sulfuric acid as a catalyst to obtain a salvianolic acid B ester; the preparation method comprises the following steps: dissolving salvianolic acid B ester in an organic solvent, adding a nickel acetate aqueous solution, uniformly mixing to obtain a mixed solution, carrying out ultrasonic degassing on the mixed solution in a protective atmosphere, then adding N-methyl pyrrolidone, and carrying out a heating reaction to obtain black powdery solid Ni-SalB, namely the nickel-based plant polyphenol coordination polymer artificial enzyme. The metal-organic coordination polymer Ni-SalB is prepared by taking salvianolic acid B ester and nickel as structural units. The Ni-SalB can reduce apoptosis by inhibiting oxidative stress and inhibiting release of inflammatory factors, can effectively relieve lipopolysaccharide-induced sepsis cardiomyopathy, and is good in biocompatibility and free of toxic and side effects.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a nickel-based plant polyphenol coordination polymer artificial enzyme and a preparation method and application thereof. Background Art

[0002] Sepsis, a life-threatening systemic inflammatory response syndrome characterized by host immune dysregulation, remains a leading cause of mortality in intensive care units. Of particular concern is sepsis-induced myocardial dysfunction (SIMD), which increases mortality from 20% in septic patients without cardiac involvement to 70-90% in SIMD cases. Despite its clinical significance, effective pharmacological intervention remains elusive, and innovative therapeutic strategies are needed.

[0003] The pathogenesis of SIMD involves the generation of excessive reactive oxygen species (ROS), driving a vicious cycle of oxidative stress, lipid peroxidation, and inflammatory cascades. Although endogenous antioxidant systems (SOD, CAT, GPx) maintain redox homeostasis, their reduced activity during sepsis allows the accumulation of ROS. Specifically, excessive accumulation of ROS outcompetes endogenous antioxidant systems, triggering a pathological cascade including lipid peroxidation, genomic instability, and proteotoxic stress, ultimately leading to regulatory imbalance. This oxidative cascade further activates redox-sensitive transcription factors and upregulates adhesion molecules and proinflammatory cytokines, thereby amplifying leukocyte infiltration, establishing a self-sustaining inflammatory microenvironment, and accelerating myocardial remodeling. Given this dual pathogenic mechanism that simultaneously targets oxidative and inflammatory pathways, current approaches to treat myocardial injury in sepsis face significant limitations. Therefore, the development of innovative therapeutic agents that can neutralize excessive ROS production and modulate pathological changes is an important frontier in SIMD treatment.

[0004] Conventional treatment options include free radical scavengers (e.g., ascorbic acid, α-tocopherol) and broad-spectrum COX inhibitors (e.g., nonsteroidal anti-inflammatory drugs), which face severe pharmacological limitations in terms of biocompatibility and pharmacokinetics. Despite their remarkable catalytic activity and substrate specificity, natural enzymes encounter several limitations that hinder their clinical application, including high production and storage costs, bioinstability, limited recyclability, and potential immunogenicity. To address these challenges, recent advances in artificial catalysts have demonstrated great potential in mitigating excess reactive oxygen species (ROS) in the field of cardiac regenerative medicine. Among them, artificial multifunctional materials with enzyme-like properties, known as artificial enzymes, have emerged as efficient ROS scavengers. These synthetic catalysts have several advantages, such as cost-effective production, straightforward synthesis, tunable structural properties, and enhanced biostability, making them viable alternatives for treating SIMD. Notably, metal-organic coordination polymers (MOCPs) have attracted extensive attention in biomedical research due to their unique properties. MOCPs consist of metal-based nodes interconnected by organic ligands, which are able to mimic the structural and functional properties of natural enzymes. Their unique features, including multiple catalytic sites, optimal size, inherent biocompatibility, and biodegradability, make MOCPs broadly applicable for therapeutic interventions in various diseases. While coordination of metal ions or clusters has been widely used to induce catalytic activity, the development of cascade reaction systems that mimic multiple enzyme functions has largely relied on multicomponent strategies. These approaches typically involve the integration of different functional elements into a single nanoparticle, which often results in complex design and synthesis processes, hampering scalability and large-scale production.

[0005] To date, various nanomaterials with enzyme-like activity have been developed, including metal oxides, metal nanoparticles and polymers, which have become the focus of research due to their excellent physicochemical properties, biosafety and ability to inhibit ROS. Nanozymes are more stable and cheaper than natural enzymes. At present, various nanostructures with the ability to catalyze the elimination or neutralization of ROS have been reported; for example, ceria-zirconia nanozymes and cobalt-doped carbon nanozymes (Co / PMCS) have successfully simulated antioxidant enzymes such as SOD, catalase and glutathione peroxidase, and showed effective therapeutic effects in sepsis. Therefore, it is of great significance to develop nanomaterials with simple preparation methods and enzyme-like activity. Polyphenols have attracted much attention in the biomedical field due to their antioxidant, anti-inflammatory, metabolic regulation and neuroprotective properties, but their phenolic hydroxyl group has a weak coordination ability and is prone to depolymerization, resulting in rapid metabolism of active molecules, short action time and functional attenuation, as well as low biological effects after metabolism in the body. Improving the low bioavailability of polyphenols in the body has become the main limitation of its application. In order to solve these limitations, it is necessary to design a safe and multifunctional nanozyme based on metal-organic coordination polymers to improve the catalytic performance of artificial nanozymes and enhance their bioavailability, thereby effectively reducing myocardial damage and effectively treating septic myocardial damage by inhibiting oxidative stress, inhibiting the release of inflammatory factors, and reducing cell apoptosis. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a nickel-based plant polyphenol coordination polymer artificial enzyme and its preparation method and application. The present invention uses tannic acid B ester and nickel as structural units to prepare a metal-organic coordination polymer Ni-SalB. Ni-SalB can effectively alleviate lipopolysaccharide-induced septic cardiomyopathy by inhibiting oxidative stress, inhibiting the release of inflammatory factors, and reducing cell apoptosis, and has good biocompatibility and no toxic side effects.

[0007] To achieve the above object, the present invention adopts the following technical solution: The first aspect of the present invention provides a method for preparing a nickel-based plant polyphenol coordination polymer artificial enzyme, comprising the following steps: (1) dissolving salvianolic acid B in an organic solvent, adding dodecanol, using sulfuric acid as a catalyst, and heating to react to obtain salvianolic acid B ester; (2) The ester of salvia salviae nitrile B is dissolved in an organic solvent, and a nickel acetate aqueous solution is added and mixed uniformly to obtain a mixed solution. The mixed solution is ultrasonically degassed in a protective atmosphere, and then N-methylpyrrolidone is added and heated to react to obtain a black powder solid Ni-SalB, which is a nickel-based plant polyphenol coordination polymer artificial enzyme.

[0008] Preferably, in step (1), the organic solvent is tetrahydrofuran; the molar ratio of the salvianolic acid B to dodecanol is 1:3; the volume of the sulfuric acid accounts for 4% of the volume of the organic solvent; and the concentration of the sulfuric acid is 98 wt%.

[0009] Preferably, in step (1), the heating reaction temperature is 60-65° C., and the heating reaction time is 5-10 days.

[0010] Preferably, in step (2), the organic solvent is dimethyl sulfoxide; and the ratio of the added amount of the salvianolic acid B ester, nickel acetate, and N-methylpyrrolidone is 1 mmol:2 mmol:1.6 mL.

[0011] Preferably, in step (2), the protective atmosphere is nitrogen; and the ultrasonic degassing is performed 3 times, each time for 3 minutes.

[0012] Preferably, in step (2), the heating reaction temperature is 85° C. and the heating reaction time is 48 h.

[0013] The second aspect of the present invention provides the nickel-based plant polyphenol coordination polymer artificial enzyme obtained by the above preparation method.

[0014] The third aspect of the present invention provides the use of a nickel-based plant polyphenol coordination polymer artificial enzyme in the preparation of a drug for treating septic cardiomyopathy.

[0015] Preferably, the septic cardiomyopathy is septic cardiomyopathy induced by lipopolysaccharide.

[0016] Preferably, the concentration of the nickel-based plant polyphenol coordination polymer artificial enzyme is 100 μg / mL.

[0017] Beneficial effects of the present invention: (1) The metal organic coordination polymer with salvianolic acid B ester and nickel as structural units has a simple preparation method and reduces the preparation cost. Although the material contains heavy metals, the hemolysis rate is less than 2%, the biocompatibility is good, and there is no toxic side effect.

[0018] (2) The nickel-based plant polyphenol coordination polymer artificial enzyme Ni-SalB prepared by the present invention has superoxide dismutase (SOD)-like, catalase-like (CAT)-like and glutathione peroxidase (GSH-Px)-like activities. Ni-SalB can catalyze the conversion of reactive oxygen species (ROS) into hydrogen peroxide (H2O2), which is then converted into oxygen (O2). Ni-SalB is a promising ROS scavenger (including H2O2, O2 •-The nanoplatform of LPS-induced septic cardiomyopathy can be alleviated by inhibiting oxidative stress, inhibiting the release of inflammatory factors, and reducing cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Characterization spectra of Ni-SalB, including (a) XPS spectrum of Ni-SalB; (b) spectrum of Ni 2p; (c) spectrum of O1s; (d) spectrum of C 1s; (e) X-ray diffraction spectrum of Ni-SalB; Figure 2 : SEM and TEM of Ni-SalB, including (a) SEM of Ni-SalB at 1μm scale; (b) SEM of Ni-SalB at 500nm scale; (c) SEM of Ni-SalB at 200nm scale; (d) TEM of Ni-SalB at 1μm scale; (e) TEM of Ni-SalB at 50nm scale; (f) HR-TEM of Ni-SalB at 10 nm scale; (g) element distribution map of Ni-SalB at 500nm scale; (h) C element distribution map of Ni-SalB; (i) O element distribution map of Ni-SalB; (j) Ni element distribution map of Ni-SalB; Figure 3 : Spectra of Ni-SalB scavenging superoxide anions, hydroxyl radicals, hydrogen peroxide, and reactive nitrogen species, where (a) O2 generated by the AP-TEMED system was treated with different concentrations of Ni-SalB (0, 25, 50, 75, 100, and 200 μg mL-1). •- UV-visible spectra of Ni-SalB; (b) Ni-SalB to O2 •- scavenging effect of Ni-SalB on H2O2; (g) UV-visible spectrum of DPPH after Ni-SalB treatment; (h) scavenging effect of Ni-SalB on DPPH; (i) O2 •- and Ni-SalB+O2 •- ESR spectra of OH and Ni-SalB+·OH; (k) ESR spectra of DPPH and Ni-SalB+DPPH; Figure 4: In vitro anti-oxidative stress and protective effects of Ni-SalB on cardiomyocytes, where (a) representative ROS staining (green fluorescence) of LPS-induced H9C2 cells using DCFH-DA probe, scale bar 200 μm; (b) live / dead staining of H9C2 cells treated with PBS, H2O2, Ni-SalB, or both, scale bar 200 μm; Figure 5 : Biocompatibility of Ni-SalB, including (a) hemolysis of Ni-SalB at different concentrations; (b) hemolysis of Ni-SalB-NE at different concentrations; (c) cell viability of Ni-SalB at different concentrations; (d) H&E staining images of the heart, liver, spleen, lung and kidney of mice in the PBS group and the Ni-SalB group after 14 days; Figure 6 :(a) Transmission electron microscopy images of mitochondria in different groups of mice;(b) Echocardiograms of different groups of mice;(c) H&E staining images of different groups of mice;(d) Masson images of different groups of mice;(e) Ejection fractions of different groups of mice;(f) Short axis shortening rates of different groups of mice;(g) Left ventricular end-systolic volumes of different groups of mice;(h) Left ventricular end-diastolic volumes of different groups of mice;(i) Phosphokinase isozymes of different groups of mice;(j) Lactate dehydrogenase of different groups of mice;(k) Tumor necrosis factor-α of different groups of mice;(l) Interleukin-1-β of different groups of mice;(m) Interleukin-6 of different groups of mice;(n=3, error bars indicate standard deviation); Figure 7 :(a)SOD levels of mice in each group;(b)MDA levels of mice in each group;(c)Tunel staining images of mice in different groups;(d)DHE staining images of mice in different groups; Figure 8 :Ni-SalB synthesis route map. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0021] As introduced in the background technology section, polyphenols have attracted much attention in the biomedical field due to their antioxidant, anti-inflammatory, metabolic regulation and neuroprotective properties. However, their phenolic hydroxyl groups have weak coordination ability and are prone to depolymerization, resulting in rapid metabolism of active molecules, short action time and functional attenuation, as well as lower biological effects after metabolism in the body. Improving the low bioavailability of polyphenols in the body has become the main limitation of their application.

[0022] Based on this, the purpose of the present invention is to provide a method for preparing a nickel-based plant polyphenol coordination polymer artificial enzyme. The present invention first uses salvianolic acid B and dodecanol to prepare salvianolic acid B ester by esterification reaction; then uses salvianolic acid B ester and nickel as structural units to prepare metal organic coordination polymer Ni-SalB by one-step hydrothermal reaction.

[0023] Ni-SalB has integrated and efficient catalytic properties, exhibiting both superoxide dismutase (SOD)-like and glutathione peroxidase (GSH-Px)-like activities. Specifically, it catalyzes the conversion of reactive oxygen species (ROS) into hydrogen peroxide (H2O2), which is subsequently converted into water (H2O), while enhancing its transmembrane absorption capacity, lipid solubility and bioavailability. This ability can enable long-term ventricular remodeling, promote the recovery of cardiac function, and protect myocardial tissue from damage. In addition, Ni-SalB significantly alleviates oxidative stress in vitro and shows potential as a treatment for sepsis-induced myocardial injury through its antioxidant and anti-inflammatory properties. In a lipopolysaccharide-induced sepsis mouse model, Ni-SalB effectively alleviated myocardial injury by inhibiting oxidative stress, suppressing the release of inflammatory factors, and reducing cell apoptosis. In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0024] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels.

[0025] Example 1: Preparation of Ni-SalB (1) Salvianolic acid B (1 mmol) was dissolved in tetrahydrofuran (3 mL), then (3 mmol) dodecanol was added, sulfuric acid (98 wt%, 0.12 mL) was used as a catalyst, and heated at 65°C for 5 days. The progress of the reaction was monitored by reverse phase HPLC, and the reaction was terminated by adding 2 mL of sodium carbonate (1 M). The mixture was then dissolved in 100 mL of heated acetonitrile and filtered and then stored at 4°C for purification to obtain salvianolic acid B ester.

[0026] (2) Dissolve the ester of salvia salviae B (36 mg) in 8 mL of DMSO to obtain an ester solution, and dissolve 25 mg of Ni(OAc)2·4H2O in 16 mL of deionized water to obtain a Ni(OAc)2·4H2O solution. Add the Ni(OAc)2·4H2O solution to the ester solution and stir evenly to obtain a mixed solution. The mixed solution was degassed by ultrasonication (40 kHz, 300 W) under nitrogen protection for 10 minutes. Subsequently, 1.6 mL of N-methylpyrrolidone (NMP) was added to the mixed solution and stirred continuously at 85°C for 48 hours. After cooling, the crude product was collected by centrifugation, soaked in DMF overnight to remove residual impurities, thoroughly washed with deionized water, and finally freeze-dried to obtain Ni-SalB. For the specific synthesis route, see Figure 8 .

[0027] Comparative Example 1: Preparation of Ni-SalB-NE Salvianolic acid B (36 mg) was dissolved in 8 mL of DMSO to obtain a Salvianolic acid B solution, and 25 mg of Ni(OAc)2·4H2O was dissolved in 16 mL of deionized water to obtain a Ni(OAc)2·4H2O solution. The Ni(OAc)2·4H2O solution was added to the Salvianolic acid B solution and stirred evenly to obtain a mixed solution. The mixed solution was degassed by ultrasonication (40 kHz, 300 W) under nitrogen protection for 10 minutes. Subsequently, 1.6 mL of N-methylpyrrolidone (NMP) was added to the mixed solution and stirred continuously at 85°C for 48 hours. After cooling, the crude product was collected by centrifugation, soaked in DMF overnight to remove residual impurities, thoroughly washed with deionized water, and finally freeze-dried to obtain Ni-SalB-NE.

[0028] Example 2: Characterization of Ni-SalB prepared in Example 1 (1) X-ray photoelectron spectroscopy of Ni-SalB: The composition of Ni-SalB was studied using X-ray photoelectron spectroscopy. Figure 1 As shown in (a), the full scan XPS spectrum of Ni-SalB confirms the presence of carbon (C), oxygen (O), and nickel (Ni). Figure 1 (b) High-resolution analysis of the Ni 2p spectrum shows that at 873.2 eV (Ni 2p 1 / 2 ) and 856.1 eV (Ni 2p 3 / 2 ) and two characteristic peaks at 861.1 eV and 879.7 eV, indicating the presence of Ni(II). Figure 1 The O1s spectrum of (c) has four obvious peaks at 531.0 eV, 531.8 eV, 532.6 eV and 533.5 eV, corresponding to Ni-O, CO, OH and C=O bonds, respectively. In addition, Figure 1The C 1s spectrum of (d) has three peaks at 284.8 eV, 286.4 eV, and 288.5 eV, which are assigned to CC, CO, and C=O bonds, respectively, further confirming the successful coordination between Ni and SalB. Based on these results, it can be concluded that SalB and nickel metal ions are successfully coordinated to form a stable Ni-SalB metal organic coordination polymer.

[0029] (2) The morphology of Ni-SalB is understood through X-ray diffraction patterns, such as Figure 1 (e) X-ray diffraction shows only a broad peak at about 25°, reflecting the amorphous structure of the synthesized Ni-SalB.

[0030] (3) The morphology of Ni-SalB was observed by SEM and TEM. Figure 2 (a) ~ Figure 2 (d) It can be seen that Ni-SalB exhibits an irregular structure consisting of irregular stacking of nearly spherical particles with mesopores and macropores interconnected. Figure 2 High-resolution transmission electron microscopy (HR-TEM) analysis of (e) shows the absence of metal-based nanoparticle aggregation or lattice fringes, further confirming the amorphous nature of the synthesized material. Figure 2 (f) Energy dispersive X-ray spectroscopy and elemental mapping results show that carbon (C 56.69%), oxygen (O 33.68%), and nickel (Ni 9.63%) are uniformly distributed throughout the carbon-dominated porous framework.

[0031] (4) Detection of the scavenging ability of Ni-SalB for reactive oxygen and reactive nitrogen. The superoxide anion scavenging ability test kit (Slorbio, BC1410) was used to detect the O2 •- According to the instructions of the superoxide anion scavenging ability test kit, the AP-TEMED system was used to generate superoxide anions, which then reacted with hydroxylamine hydrochloride to produce nitrite ions (NO2 - ). These nitrite ions interact with p-aminobenzenesulfonamide and α-naphthylamine to form a red azo compound. This compound has a characteristic absorption peak at 530 nm. Follow the instructions of the kit and measure the absorbance of the blank solution and the Ni-SalB solution at 530 nm and record them as AB and AT. O2 •- The calculation formula for clearance rate is: D%=(AB-AT)÷AB×100%. Using AP-TEMED / O2 •- Hydroxylamine colorimetric system was used to explore SOD mimetic activity. Figure 3 (a) and Figure 3As shown in (b), Ni-SalB exhibited a dose-dependent superoxide dismutase-like activity at 200 μg mL -1 82.4 ± 4.4% O2 •− Removal efficiency.

[0032] (5) The ·OH scavenging ability of Ni-SalB at different concentrations (0, 25, 50, 75, 100 and 200 μg / mL) was detected by 3,3′,5,5′-tetramethylbisphenylmethane (TMB) colorimetric method. PBS buffer (0.5 M, pH 4.5) solutions containing TMB (1.5 mM), hydrogen peroxide (10 mM), ferrous sulfate (0.5 mM) and different concentrations of Ni-SalB were prepared in the dark and allowed to stand for 5 min. The solution was then centrifuged (10,000 rpm, 2 min) and the absorbance at 652 nm was measured. The ·OH scavenging rate was D% = (AB -AT) ÷ AB × 100%. The absorbance values ​​of the blank solution and the solution containing Ni-SalB were measured at 652 nm and recorded as AB and AT.

[0033] Figure 3 (c) and Figure 3 (d) The hydroxyl radical scavenging ability was studied using the Fenton reaction / TMB oxidation system. At 200 μg / mL, Ni-SalB showed ·OH scavenging activity with an inhibition rate of 39.9±2.0%, showing a clear concentration-dependent behavior.

[0034] (6) The glutathione peroxidase (GPx)-like activity of Ni-SalB was evaluated at different concentrations (0, 25, 50, 75, 100, and 200 μg / mL) using a glutathione peroxidase activity assay kit (Shanghai Yuanye Biotechnology Co., Ltd., R21877-50T). Ni-SalB and benzoic acid colorimetric solution were added to the GSH working solution. The concentration of yellow anions produced in the GSH and benzoic acid colorimetric solution was measured using a UV-spectrophotometer at 422 nm.

[0035] Then, the GPx-like cascade catalytic ability was investigated by hydrogen peroxide decomposition method. Figure 3 (e) and Figure 3 As shown in (f), Ni-SalB also possesses glutathione-dependent peroxidase activity, with a hydrogen peroxide clearance of 49.9 ± 3.9% at 200 μg / mL in the presence of 1 mM GSH, indicating efficient regeneration of the catalytic cycle.

[0036] (7) The scavenging ability of Ni-SalB against reactive nitrogen species (RNS) was evaluated at different concentrations (0, 25, 50, 75, 100, and 200 μg / mL). DPPH solution (50 μg / ml) was mixed with different concentrations of Ni-SalB and incubated for 20 min. Subsequently, the supernatant was collected by centrifugation (10,000 rpm, 5 min), and the absorbance was measured at 517 nm. The DPPH scavenging rate was calculated as D% = (AB -AT) ÷ AB × 100%, and the absorbance values ​​of the blank solution and the Ni-SalB-containing solution were measured at 517 nm and recorded as AB and AT.

[0037] The RNS neutralization ability of Ni-SalB was evaluated by DPPH free radical scavenging assay. Figure 3 (g) and Figure 3 As shown in (h), Ni-SalB also showed a dose-responsive RNS elimination ability, reaching 42.6 ± 9.8% at 200 μg / mL.

[0038] (8) The free radical scavenging mechanism of Ni-SalB was verified by electron spin resonance (ESR) spectroscopy. Hydroxyl radical (•OH) detection: Hydroxyl radicals were generated by Fenton reaction system (containing FeSO4 (1 mg / mL, 75 μL) and H2O2 (10 M, 75 μL)) in phosphate buffer (0.5 M, 600 μL) at pH 4.5, and DMPO spin trap (3% v / v, 20 μL) was added. The ESR spectrum showed characteristic quartet signals (intensity ratio 1:2:2:1), indicating that DMPO formed a stable spin adduct with •OH; superoxide anion (O2 •- ) Detection: O2 was generated by photolysis (4000 lux) of riboflavin (20 μM) and L-methionine (13 mM) in phosphate buffer at pH 7.4. •- , a unique sextet signal was formed after DMPO capture, indicating the presence of superoxide radicals; DPPH free radical scavenging experiment, using DPPH ethanol solution as the free radical source; after treatment with Ni-SalB (200 μg / mL), the ESR signal attenuation quantitatively indicated its ability to scavenge ROS and RNS.

[0039] To further evaluate the scavenging ability of Ni-SalB against reactive oxygen and nitrogen species (RONS), electron spin resonance (ESR) spectroscopy was employed, which provides direct visualization of the reduction of free radical signals. Figure 3As shown in (j), DMPO spin trapping shows a 1:2:2:1 multiplex pattern caused by ·OH. It is worth noting that in the presence of Ni-SalB, the signal intensity of ·OH is significantly reduced (the intensity is reduced by 74.4% at 200 μg / mL), indicating the high sensitivity of Ni-SalB to ·OH scavenging. Similarly, Figure 3 (i) The addition of Ni-SalB leads to O2 •- The peak intensity of DEPMPO-O2 •- The adduct signal decreased by 78.2%, confirming its effect on O2 •- Effective consumption of species. In addition, Figure 3 (k) shows that in the presence of Ni-SalB, the ESR signal peak of DPPH radical was significantly weakened (the signal was reduced by 79.8% at 200 μg / mL), indicating its strong DPPH scavenging ability. These results collectively indicate that Ni-SalB has multiple antioxidant properties and can effectively remove ROS and RNS.

[0040] Experimental Example 1: In vitro anti-oxidative stress and protective effect of Ni-SalB on cardiomyocytes (1) LPS-induced ROS staining of H9C2 cardiomyocytes: H9C2 cardiomyocytes (rat cardiomyocytes, cell bank of the Chinese Academy of Sciences) were seeded in 6-well plates and incubated for 24 h. Then, the cells were treated with LPS (10 µg / mL), Ni-SalB (100 µg / mL), and LPS+Ni-SalB, respectively. After incubation, DCFH-DA solution was added to the cells and incubated for another 30 min. After washing with PBS, the stained cells were observed under a fluorescence microscope.

[0041] Given the good enzymatic activity of Ni-SalB in scavenging reactive species, its intracellular antioxidant capacity and cytoprotective effects were further investigated. Lipopolysaccharide (LPS), a bacterial endotoxin known to stimulate intracellular reactive oxygen species (ROS) production, was used to induce oxidative stress in H9C2 cardiomyocytes. After LPS treatment, ROS levels in H9C2 cells were significantly increased, as evidenced by the increase in green fluorescence intensity detected using the ROS-sensitive probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). Notably, Ni-SalB treatment (100 μg / mL) effectively scavenged the overproduced ROS, reducing the fluorescence intensity to a level comparable to that of untreated cells, see Figure 3. Figure 4 (a) These results indicate that Ni-SalB can effectively eliminate intracellular ROS, highlighting its potential as a novel therapeutic agent for ROS-related diseases.

[0042] (2) Live / dead staining of H2O2-induced cardiomyocyte oxidative stress: H9C2 cells were seeded in 6-well plates and incubated for 24 h. Cells were treated with H2O2 (700 μM), Ni-SalB (100 µg / mL), and H2O2+Ni-SalB, respectively. After incubation, Calcein / PI solution was added to the cardiomyocytes and incubated for another 30 min. The stained cells were observed using a fluorescence microscope. In addition, the protective effect of Ni-SalB on H9C2 cardiomyocytes was evaluated under hydrogen peroxide (H2O2)-induced oxidative stress. Live / dead cell staining assay showed that hydrogen peroxide (H2O2) treatment significantly increased cell death, as indicated by the presence of PI-positive cells (red fluorescence). Figure 4 (b) Ni-SalB pretreatment completely prevented H2O2-induced cell death. These results highlight that Ni-SalB has a special antioxidant activity, which can effectively protect cells from oxidative stress-induced damage by neutralizing intracellular ROS.

[0043] Experimental Example 2: In vitro biocompatibility study of Ni-SalB (1) Hemolysis test Fresh blood from C57BL / 6 mice (8 weeks old, purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd.) was centrifuged at 1500 rpm for 10 min, the supernatant was taken, rinsed with PBS, and centrifuged again. This was repeated 3 times, and finally red blood cells were taken to form a 5% red blood cell suspension. Red blood cells were mixed with different concentrations of Ni-SalB (25μg / mL, 50μg / mL, 75μg / mL, 100μg / mL, 150μg / mL) at a ratio of 1:1 (ratio of the volume of red blood cell suspension to different concentrations of Ni-SalB) and incubated at 37°C for 3h. After centrifugation, the supernatant was taken to determine the OD value (540nm). 0.5 mL of diluted red blood cell suspension and 0.5 mL of PBS were used as negative controls, and 0.5 mL of diluted red blood cell suspension and 0.5 mL of distilled water were used as positive controls. The hemolysis rate was calculated using the formula: Hemolysis amount (%) = (As-An) / (Ap-An) × 100%; Where "As" is the absorbance obtained by taking the supernatant after adding Ni-SalB to erythrocytes. "An" is the absorbance obtained by taking the supernatant after adding PBS to erythrocytes (negative control). "Ap" is the absorbance obtained by taking the supernatant after adding distilled water to erythrocytes (positive control).

[0044] like Figure 5As shown in (a) and 5(b), Ni-SalB showed only a small amount (less than 2%) or no hemolytic activity within the concentration range showing antioxidant activity. The hemolysis rate of Ni-SalB varied with the concentration of Ni-SalB. As the concentration increased from 0 to 150 μg / mL, the hemolysis rate was less than 2%, while the hemolysis rate of Ni-SalB-NE prepared without esterification reaction was higher than that of Ni-SalB, indicating that the prepared Ni-SalB-NE had poor compatibility, while Ni-SalB had good blood compatibility and no damage to the red blood cell membrane.

[0045] (2) Cytotoxicity assay The cytotoxicity of Ni-SalB was studied using rat cardiomyocytes H9C2 (from the Cell Bank of the Chinese Academy of Sciences). H9C2 cells were plated at 5 × 10 3 The cells were inoculated in a 96-well plate at a density of 10 cells / mL, and then incubated with different concentrations of Ni-SalB solution for 24 h. 10 μL CCK-8 working solution was added, and after incubation for 30 min, the OD value at 450 nm was measured using a microplate reader. Each group of experiments was repeated three times.

[0046] like Figure 5 (c) shows the cell viability of H9C2 rat cardiomyocytes cultured for 2 days with different concentrations of Ni-SalB (0-150 μg / mL). It can be clearly seen that as the concentration of the material decreases, the relative cell viability gradually increases, and its value can be maintained at more than 90% at all experimental concentrations, indicating that it has no toxicity to H9C2 rat cardiomyocytes. All these results confirm that it has good in vitro biocompatibility.

[0047] Test Example 3: In vivo biocompatibility study Ni-SalB (100 μg / mL) was intraperitoneally injected into C57BL / 6 male mice (8 weeks old, purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd.) to evaluate its in vivo biocompatibility; an equal amount of PBS was injected as a control. After 14 days of administration, major organs, including heart, liver, spleen, lung, and kidney, were collected for histopathological examination using hematoxylin and eosin (H&E) staining. Figure 5 As shown in (d), no signs of tissue inflammation, lesions, or other abnormalities were observed in any organ of the Ni-SalB-treated group, confirming its good in vivo biocompatibility. Taken together, these findings indicate that Ni-SalB is a highly biocompatible material suitable for biomedical applications.

[0048] Test Example 4 (1) In vivo study of lipopolysaccharide (LPS)-induced septic cardiomyopathy: Based on the multi-enzyme mimetic ability and good biocompatibility of Ni-SalB, its therapeutic potential for LPS-induced septic cardiomyopathy was systematically studied through multimodal evaluation. Eight-week-old male C57BL / 6 mice were used and divided into five groups (n=6), namely PBS group, Ni-SalB group, LPS group, LPS+Ni-SalB group, and LPS+Ni-SalB-NE. Ni-SalB and LPS+Ni-SalB groups were injected with Ni-SalB in advance to verify its protective effect on septic cardiomyopathy. Ni-SalB-NE was injected with Ni-SalB-NE in advance to verify its protective effect on septic cardiomyopathy. The injection volume was 100 μg / mL. The PBS group was injected with an equal amount of PBS in advance, and the LPS group was not injected. The drug was administered (injected) continuously for 14 days. Then, LPS (12 mg / kg) was injected intraperitoneally in the LPS group, LPS+Ni-SalB group, and LPS+Ni-SalB-NE group to establish the LPS-induced septic cardiomyopathy model. After 24 hours, cardiac ultrasound examination was performed to determine the establishment of the septic cardiomyopathy model, and then the mouse heart blood and heart, liver, spleen, lung, and kidney were taken for biochemical and H&E staining to verify its in vivo effect.

[0049] like Figure 6 As shown in (a), electron microscopy showed that the mice in the LPS group had mitochondrial cristae rupture and myofibril disorder, while the mice in the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group had alleviated the above changes, and the LPS+Ni-SalB group had a better effect than the LPS+Ni-SalB-NE group. Figure 6 (b) and Figure 6 (e) ~ Figure 6 (h) shows that compared with the PBS group, echocardiography showed that the mice in the LPS group had signs of cardiac dysfunction. Among them, the shortening fraction (FS) and ejection fraction (EF) decreased by 19.8% and 41.4%, respectively, while the left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV) increased to 29.5 μl and 50.6 μl, respectively. These changes indicate LPS-induced cardiac damage and dysfunction in vivo. In contrast, the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group showed significant improvements in FS and EF compared with the LPS group. And the improvement of cardiac dysfunction in the LPS+Ni-SalB group was more obvious. These results indicate that Ni-SalB can effectively alleviate LPS-induced myocardial dysfunction. In addition, the potential to alleviate LPS-induced effects can also be demonstrated from the improvement of histopathology. As Figure 6As shown in (c) and 6 (d), H&E and Masson trichrome staining showed that compared with the LPS group, mice in the LPS+Ni-SalB group and the LPS+Ni-SalB-NE group showed milder inflammatory response, bleeding, structural disorder and fibrosis symptoms. At the same time, compared with the LPS+Ni-SalB-NE group, the effect of LPS+Ni-SalB was more obvious. These results indicate that Ni-SalB can effectively improve septic myocardial injury and has a superior therapeutic effect. In addition, Figure 6 (i) and Figure 6 (j) showed that the serum creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels in the LPS+Ni-SalB group and LPS+Ni-SalB-NE group were significantly lower than those in the LPS group, which further indicated the superiority of Ni-SalB in protecting LPS-induced cardiac injury compared with the LPS+Ni-SalB-NE group. The inflammatory response of cardiomyocytes is also one of the most important pathological changes in myocardial injury. This response, in turn, drives the production of mitochondrial reactive oxygen species (ROS), leading to cardiomyocyte injury and aggravated cardiac dysfunction.

[0050] To further explore its anti-inflammatory effects, cardiac blood was extracted from mice, and ELISA kits (Servicebio GEM0002-96T / GEM0004-96T / GEM0001-96T) were used to quantify the release of inflammatory factors. Figure 6 (k) ~ Figure 6 (m) The results showed that compared with the LPS group, the levels of interleukin 1-β (IL-1β), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) in the serum of mice in the LPS+Ni-SalB group and LPS+Ni-SalB-NE group were significantly downregulated, and the effect was more obvious in the LPS+Ni-SalB group, indicating that Ni-SalB can effectively reduce the inflammatory response by inhibiting the production of inflammatory cytokines IL-1β, TNF-α, and IL-6 induced by LPS. Therefore, Ni-SalB shows good anti-inflammatory potential in the treatment of septic cardiomyopathy.

[0051] As previously reported, septic cardiomyopathy is also associated with increased oxidative stress. To assess this, SOD (superoxide dismutase) activity and MDA (malondialdehyde) levels in the heart blood of mice were measured using specific kits (Servicebio G4306-96T / G4302-96T). Figure 7 (a) ~ Figure 7(b) showed that Ni-SalB and Ni-SalB-NE significantly increased SOD activity and decreased MDA levels compared with the LPS group. In addition, DHE (dihydroethidium) staining of cardiac tissue showed that although there was no significant difference in myocardial tissue between the Ni-SalB group and the PBS control group, the LPS group showed significant red fluorescence compared with the control group, indicating that the generation of ROS (reactive oxygen species) was significantly increased. However, according to Figure 7 (c) It can be seen that in the LPS+Ni-SalB and LPS+Ni-SalB-NE groups, the intensity of the red fluorescence signal was significantly reduced compared with the LPS group, confirming that Ni-SalB can effectively inhibit the production of ROS in the mouse myocardium. These results are consistent with the results of in vitro experiments. In addition, Figure 7 Tunel staining in (d) showed that LPS+Ni-SalB and LPS+Ni-SalB-NE significantly inhibited LPS-induced cardiomyocyte apoptosis compared with the LPS group. Ni-SalB was superior to Ni-SalB-NE, which indicated that Ni-SalB inhibited LPS-induced oxidative stress and cardiomyocyte apoptosis by increasing the level of antioxidant enzymes and reducing ROS generation indicators.

[0052] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a nickel-based plant polyphenol coordination polymer artificial enzyme, characterized in that: The following steps are involved: (1) dissolving salvianolic acid B in an organic solvent, adding dodecanol, using sulfuric acid as a catalyst, and heating to react to obtain salvianolic acid B ester; (2) The ester of salvia salviae nitrile B is dissolved in an organic solvent, and a nickel acetate aqueous solution is added and mixed uniformly to obtain a mixed solution. The mixed solution is ultrasonically degassed in a protective atmosphere, and then N-methylpyrrolidone is added and heated to react to obtain a black powder solid Ni-SalB, which is a nickel-based plant polyphenol coordination polymer artificial enzyme.

2. The preparation method according to claim 1, characterized in that: In step (1), the organic solvent is tetrahydrofuran; the molar ratio of the salvianolic acid B to dodecanol is 1:3; the volume of the sulfuric acid accounts for 4% of the volume of the organic solvent; and the concentration of the sulfuric acid is 98wt%.

3. The preparation method according to claim 1, characterized in that: In step (1), the heating reaction temperature is 60-65° C., and the heating reaction time is 5-10 days.

4. The preparation method according to claim 1, characterized in that: In step (2), the organic solvent is dimethyl sulfoxide; the ratio of the added amount of the ester of salvianolic acid B, nickel acetate and N-methylpyrrolidone is 1 mmol:2 mmol:1.6 mL.

5. The preparation method according to claim 1, characterized in that: In step (2), the protective atmosphere is nitrogen; the ultrasonic degassing is performed 3 times, each time for 3 minutes.

6. The preparation method according to claim 1, characterized in that: In step (2), the heating reaction temperature is 85° C. and the heating reaction time is 48 h.

7. A nickel-based plant polyphenol coordination polymer artificial enzyme obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the nickel-based plant polyphenol coordination polymer artificial enzyme according to claim 7 in the preparation of a drug for treating septic cardiomyopathy.

9. The use according to claim 8, characterized in that: The septic cardiomyopathy is septic cardiomyopathy induced by lipopolysaccharide.

10. The use according to claim 8, characterized in that: The concentration of the nickel-based plant polyphenol coordination polymer artificial enzyme is 100 μg / mL.

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